WO2025019903A1 - Rna vaccines for use in animal health - Google Patents

Rna vaccines for use in animal health Download PDF

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Publication number
WO2025019903A1
WO2025019903A1 PCT/AU2024/050797 AU2024050797W WO2025019903A1 WO 2025019903 A1 WO2025019903 A1 WO 2025019903A1 AU 2024050797 W AU2024050797 W AU 2024050797W WO 2025019903 A1 WO2025019903 A1 WO 2025019903A1
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Prior art keywords
rna
amino acid
sequence
seq
gulae
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PCT/AU2024/050797
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French (fr)
Inventor
Christopher Smith
Eric Reynolds
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Denteric Pty Ltd
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Priority claimed from AU2023902376A external-priority patent/AU2023902376A0/en
Application filed by Denteric Pty Ltd filed Critical Denteric Pty Ltd
Publication of WO2025019903A1 publication Critical patent/WO2025019903A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/0216—Bacteriodetes, e.g. Bacteroides, Ornithobacter, Porphyromonas
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K2039/53—DNA (RNA) vaccination
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K2039/55505—Inorganic adjuvants
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
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    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/22—Cysteine endopeptidases (3.4.22)
    • C12Y304/22037—Gingipain R (3.4.22.37)
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    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/22—Cysteine endopeptidases (3.4.22)
    • C12Y304/22047—Gingipain K (3.4.22.47)

Definitions

  • the present invention relates to RNA-containing vaccine compositions for inducing an immune response to Porphyromonas gulae in a subject, and uses thereof.
  • gingival inflammation gingival inflammation
  • Periodontitis a periodontal pathogen of the Porphyromonas sp. 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.
  • Periodontitis is an inflammatory disease of the supporting tissues of the teeth associated with a dysbiotic subgingival plaque which results in destruction of those tissues and loss of tooth attachment in humans and in companion animals. More than 80% of dogs show signs of periodontitis by age three and 70% of cats by the same age. Consequently there is a significant disease burden from periodontitis in companion animal populations.
  • the invention provides an RNA polynucleotide encoding a protein comprising or consisting of:
  • ABSMs adhesin binding motifs
  • the protein encoded by the RNA polynucleotide may further comprise:
  • the protein encoded by the RNA may comprise the afore-mentioned domains in any order: for example: the one or more amino acid sequences of an active site of an Arg- or Lys-gingipain homologue of P.
  • gulae may be located N-terminally to the amino acid sequence of one or more adhesin binding motifs (ABMs) and/or the amino acid sequence of a DUF2436 domain; or the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain may be located N-terminally to the one or more amino acid sequences of an active site of an Arg- or Lys-gingipain homologue of P.
  • ABSMs adhesin binding motifs
  • 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.
  • the protein may comprises 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); or DUF2436 (D) — ABMs (A); or DUF2436 (D) — ABMs (A
  • the RNA polynucleotide is in the form of a messenger RNA (mRNA) molecule.
  • mRNA messenger RNA
  • 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.
  • 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.
  • the RNA polynucleotide may be a synthetic RNA molecule.
  • the RNA polynucleotide may be a circular RNA (circRNA) molecule.
  • RNA polynucleotide may be a complementary RNA (cRNA) molecule.
  • cRNA complementary RNA
  • the RNA polynucleotide made be a self-amplifying RA (saRNA) molecule or trans-amplifying (taRNA) molecule.
  • saRNA self-amplifying RA
  • taRNA trans-amplifying
  • RNA molecule forms are described in Fang et al., (2022) Signal Transduction and Targeted Therapy, 7: article 94, incorporated herein by reference.
  • the RNA further encodes 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 of the mammalian cell and secreted therefrom.
  • ER endoplasmic reticulum
  • N-terminal signal peptides are known to the skilled person and are further described herein.
  • 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.
  • the RNA may comprise a chemical modification.
  • suitable chemical modification include a N1-methylpseudouridine modification or a N1-ethylpseudouridine modification or may comprise any chemical modification described herein.
  • 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%.
  • the uridines in the polynucleotide are replaced with a chemical modification such as N-methyl-pseudouridine.
  • a chemical modification such as N-methyl-pseudouridine.
  • 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.
  • the RNA polynucleotide is in the form of a codon optimised RNA molecule, optionally depleted of uridine nucleosides.
  • the codon optimisations comprises conversion of codons encoding serine to UCG.
  • the protein encoded by the RNA polynucleotide is a chimeric or fusion protein comprising or consisting of:
  • the chimeric or fusion protein encoded by the RNA may comprise the afore-mentioned domains in any order: for example: the one or more amino acid sequences of an active site of an Arg- or Lys-gingipain homologue of P.
  • gulae may be located N-terminally to the amino acid sequence of one or more adhesin binding motifs (ABMs), or the amino acid sequence of one or more adhesin binding motifs (ABMs) may be located N-terminally to the one or more amino acid sequences of an active site of an Arg- or Lys-gingipain homologue 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.
  • the chimeric or fusion protein may comprises or consist of, N to C terminus: active site (K) - ABMs (A); or active site (R) - ABMs (A); or AMBs (A) - active site (K); or ABMs (A)-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) .)
  • amino acid sequences of the active sites of Arg- or Lys-gingipain homologues of P. gulae are further described herein.
  • the amino acid sequence of the active of an Arg- or Lys- gingipain homologue of P. gulae 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.
  • the amino acid sequence of an active site of the Arg- gingipain homologue of P. gulae comprises the amino acid sequence of SEQ ID NO: 4, encoded by the RNA sequence as set forth in SEQ ID NO: 69, 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.
  • amino acid sequence of an Arg-gingipain homologue of P. gulae may comprise a substitution of the cysteine residue (position 5 of SEQ ID NO: 4) optionally to a valine, serine or alanine residue.
  • the amino acid sequence of the active site of the Lys- gingipain homologue of P. gulae (also designated “K” herein) comprises the amino acid sequence of SEQ ID NO: 2, encoded by the RNA sequence as set forth in SEQ ID NO: 59, 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.
  • the amino acid sequence of the active site of an Arg- or Lys- gingipain homologue does not comprise the entire catalytic domain of the gingipain homologue.
  • 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 homologue of P. gulae, 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 homologue of P. gulae, or sequences that are at least 80% identical thereto.
  • 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 homologue of P. gulae, or sequences that are 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.
  • 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.
  • 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).
  • 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.
  • the chimeric or fusion protein encoded by the RNA polynucleotide may further comprise: - the amino acid sequence of a DUF2436 domain of the Arg- and Lys-gingipain surface complexes of P. gulae, or a sequence that is at least 80% identical thereto.
  • 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. gulae and the amino acid sequence of the or more adhesin binding motifs (ABMs).
  • ABSMs adhesin binding motifs
  • the amino acid sequence of a DUF2436 domain of the Arg and Lys gingipain surface complexes of P. gulae comprises or consists of the amino acid sequence of SEQ ID NO: 22, encoded by the RNA sequence as set forth in SEQ ID NO: 70, 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.
  • the amino acid sequence of a DUF2436 domain of the Arg and Lys gingipain surface complexes of P. gulae comprises or consists of the amino acid sequence of SEQ ID NO: 19, encoded by the RNA sequence as set forth in SEQ ID NO: 71 , 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.
  • one or more cysteine residues in the DUF2436 domain may be substituted to a serine or valine residue or alanine residue, preferably to a serine residue (such as shown in any of SEQ ID NOs: 20 and 21).
  • the one or more cysteine residues are substituted to one or more serine residues.
  • at least two cysteine residues in the DUF2436 domain are substituted, optionally at least three of the cysteine residues.
  • all four cysteine residues in this variant of the DUF2436 domain are substituted.
  • the one or more adhesin binding motifs also found in surface complexes of the Arg and Lys gingipain homologues of P. gulae, comprise or consist of the amino acid sequence of ABM2 and ABM 1 (for example as set forth in SEQ ID NO: 6 and SEQ ID NO: 5, respectively, or comprising the amino acid sequence as set forth in SEQ ID NO: 8 (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: 72, 73 and 75, herein
  • the one or more adhesin binding motifs may comprise or consist of the amino acid sequence of ABM2, ABM1 and ABM3 (for example as set forth in SEQ ID NO: 9 and encoded by an RNA comprising the sequence of SEQ ID NO: 76).
  • the one or more adhesin binding motifs may comprise one or more modifications selected from: a) one or more cysteine amino acid substitutions in the adhesin A domain compared to the A domain found naturally occurring in the Arg- Lys-gingipain homologue protein complex 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: 5 (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: 5 (ABM1); d) substitution of the second tyrosine residue, corresponding to or at a position equivalent to residues at position 10 of SEQ ID NO: 6 (ABM2), and of the tryptophan residue, corresponding to or at a position equivalent to residue at position 23 of SEQ
  • the one or more cysteine amino acid substitutions may be a substitution to a serine residue or to a valine residue.
  • the one or more cysteine substitutions may comprise one or more substitutions to a serine residue.
  • only one cysteine residue may be substituted.
  • two or three cysteine residues may be substituted.
  • the cysteine residues are substituted to a combination of valine and serine residues.
  • all substituted cysteine residues are substituted to serine or all substitute cysteine residues are substituted to valine.
  • 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: 5, may comprise a substitution of the proline and asparagine residues.
  • the proline amino acid substitution is preferably a substitution to an alanine residue.
  • the asparagine amino acid substitution may be a substitution to a proline residue or an alanine residue.
  • the asparagine residue is substituted to a proline residue. In other embodiments, the asparagine residue is not substituted.
  • 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: 14 to 16).
  • the one or more adhesin binding motifs comprise the amino acid sequence as set forth in any one of SEQ ID NOs: 8 or 9, and comprising: a) one or more cysteine amino acid substitutions compared to the naturally occurring Arg- or Lys-gingipain homologue sequences in corresponding regions, preferably substitution of all cysteine residues; and b) substitution of the motif PxxN corresponding to, or at a position equivalent to, residues 6 to 9 of the sequence of SEQ ID NO: 5 (ABM1), to AxxP.
  • the one or more adhesin binding motifs comprise or consist the amino acid sequence as set forth in any one of SEQ ID NOs: 17 or 18, 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.
  • the RNA encodes a chimeric or fusion protein comprising or consisting of:
  • ABSMs adhesin binding motifs
  • the chimeric or fusion protein comprises the amino acid sequence of SEQ ID NO: 24, SEQ ID NO: 46 or any one of SEQ ID NOs: 78 to 82; or
  • ABSMs adhesin binding motifs
  • the chimeric or fusion protein comprises the amino acid sequence of SEQ ID NO: 30, 32, 38, 40, 48, 50, 90, 92, 94, 96, 98, 100, 102, or 104;
  • chimeric or fusion protein comprises the amino acid sequence of SEQ ID NO: 34 or 36;
  • ABSMs adhesin binding motifs
  • the chimeric or fusion protein comprises the amino acid sequence of SEQ ID NO: 42 or 44; or
  • the chimeric or fusion protein comprises the amino acid sequence of SEQ ID NO: 26 or 28; or
  • the RNA 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 or SEQ ID NO: 4.
  • the RNA comprises or consists of a nucleotide sequence of any one of: a) SEQ ID NO: 67 or 68 b) SEQ ID NO: 62, 65, 66, 77or SEQ ID NO: 55 or 56; c) SEQ ID NO: 63 or 64; d) SEQ ID NO: 60 or 61 ; e) SEQ ID NO: 57 or 58, 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.
  • the RNA comprises or consists of a nucleotide sequence of any one of: SEQ ID NO: 59 or SEQ ID NO: 69.
  • the invention further provides for the use of any DNA polynucleotide described herein (and particularly any DNA polynucleotide comprising or consisting of a sequence exemplified in Table 1).
  • the use of the DNA polynucleotide may be for obtaining an RNA polynucleotide of the invention.
  • the present invention also provides a composition, including a pharmaceutical composition comprising an RNA as described herein.
  • the composition comprises one or more pharmaceutically acceptable excipients.
  • the RNA 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.
  • the composition may comprise a combination of one or more of the RNA molecules described herein.
  • the present invention therefore further provides compositions comprising an RNA molecule as described herein, wherein the composition also comprises a lipid component.
  • the RNA (e.g., RNA) vaccines of the disclosure can be formulated using one or more liposomes, lipid vesicle, lipoplexes (such as a lipid-polycation complex), or lipid nanoparticles
  • the RNA is formulated in a lipid nanoparticle.
  • the RNA as described herein is the only polynucleotide present in the composition or in the liposomes, lipid vesicle, lipoplexes (such as a lipid- polycation complex), or lipid nanoparticles.
  • the polynucleotide as described herein is the only active ingredient present in the composition or in the liposomes, lipid vesicle, lipoplexes (such as a lipid-polycation complex), or lipid nanoparticles.
  • the invention provides a lipid nanoparticle or other nanovehicle, such as nanopolymer, for delivery of the polynucleotide to a subject in need thereof.
  • Lipid nanoparticles are well known in the art and are further described herein.
  • the lipid nanoparticle comprises a cationic and/or ionisable lipid, a phospholipid, a PEG (or PEGylated) lipid, and a structural lipid.
  • 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;
  • 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;
  • PEGylated lipid comprising from about 0.5 mol % to 20 mol % of the total lipid present in the nanoparticle.
  • the lipid nanoparticle comprises:
  • DSPC distearoylphosphatidylcholine
  • 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).
  • the present invention also provides a method for producing a lipid nanoparticle comprising an RNA, encoding a protein, or a chimeric or fusion protein as described herein.
  • the method comprises formulating any RNA molecule of the invention, with one or more lipids useful for producing a lipid nanoparticle.
  • the lipid components comprise a phospholipid, a PEG lipid, and a structural lipid.
  • the present invention also provides a nucleic acid construct or vector, comprising a polynucleotide as described herein.
  • 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.
  • 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. GenomiPhiTM V2 DNA) or other bacterial constructs.
  • 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).
  • VEEV Venezuelan Equine Encephalitis Virus
  • 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.
  • capping of the polynucleotide may be performed using any commercially available capping reagent.
  • capping 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.
  • the present invention provides a method for eliciting an immune response P. gulae in a subject in need thereof, the method comprising administering to the subject, a polynucleotide, vector, nanoparticle or composition described herein.
  • the invention provides a method for eliciting an immune response to P. gulae in a subject in need thereof, the method comprising administering to the subject, a composition comprising:
  • RNA as described herein, wherein said RNA is capable of being translated in a cell of a mammalian subject to produce the polypeptide encoded by the polynucleotide;
  • RNA optionally, an agent for enabling delivery of the RNA into mammalian cells.
  • the agent for delivery of the RNA into mammalian cells may be any suitable agent known to the skilled person for delivery of RNA.
  • agents may include: cell penetrating peptides, lipid-based formulations.
  • the invention provides a method for eliciting an immune response to P. gulae in a subject in need thereof, the method comprising administering to the subject, a nanoparticle composition comprising:
  • RNA as described herein, wherein said RNA is capable of being translated in a cell of the subject to produce the polypeptide encoded by the polynucleotide.
  • the invention also provides a method for producing a chimeric or fusion protein as described herein in a mammalian cell, the method comprising contacting the mammalian cell with a composition comprising:
  • RNA as described herein, wherein said RNA is capable of being translated in the mammalian cell to produce the protein
  • an agent for enabling delivery of the RNA into mammalian cells may be any suitable agent known to the skilled person for delivery of RNA.
  • agents may include: cell penetrating peptides, lipid-based formulations.
  • the invention also provides a method for producing a chimeric or fusion protein as described herein in a mammalian cell, the method comprising contacting the mammalian cell with a nanoparticle composition, the composition comprising:
  • RNA as described herein, wherein said RNA is capable of being translated in the mammalian cell to produce the polynucleotide.
  • the lipid component comprises a cationic and/or ionisable lipid, a phospholipid, a PEG lipid, and a structural lipid.
  • the invention also provides a method for delivering an RNA to a mammalian cell in a subject in need thereof, said method comprising administering to a subject in need thereof, a nanoparticle composition, the composition comprising:
  • RNA comprising a polynucleotide sequence as described herein, wherein said RNA is capable of being translated in the mammalian cell to produce the chimeric or fusion protein described herein; wherein the administering comprises contacting said mammalian cell with the nanoparticle composition, thereby enabling delivery of the RNA to the mammalian cell.
  • the lipid component comprises a cationic and/or ionisable lipid, a phospholipid, a PEG lipid, and a structural lipid.
  • the ionisable lipid may be substituted or combined with an adjuvant lipidoid for enhancing RNA delivery.
  • the present invention also provides the use of a polynucleotide, vector, or nanoparticle described herein, in the manufacture of a composition for eliciting an immune response to P. gulae in a subject.
  • the present invention also provides the use of i) a lipid component as described herein, and ii) an RNA as described herein, in the manufacture of a composition for delivering the RNA to a mammalian cell in a subject in need thereof.
  • the present invention also provides a polynucleotide, vector, nanoparticle or composition as described herein, for use in eliciting an immune response to P. gulae in a subject, preferably in a non-human animal subject.
  • the RNA vaccines of the invention may be administered in combination with one or more additional therapeutic agents, or other agents for eliciting an immune response to P. gulae in the subject.
  • the one or more therapeutic agents may comprise an anti-microbial compound or an antiinflammatory agent.
  • the other agents for elicing an immune response to P. gulae may comprise one or more additional protein, RNA or whole-cell based vaccine compositions or immunogens for generating an immune response to P. gulae.
  • 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”.
  • Figure 1 Expression and Solubility of P. gulae chimeric proteins KDFAK- 2S-AVQP and KDAAK-2S-AVQP.
  • A Lysis fractions;
  • B Small scale purification tests with Ni-NTA spin column: 1. clarified lysate before loaded to the column; 2. flow through; 3. column wash; 4. eluted protein fraction.
  • Figure 2 SDS-PAGE analysis of expressed P. gulae antigens and the cell lysis fractions.
  • A Expression of KDFAK-2S-AVQP and KDAAK-2S-AVQP in LB and TB media for 2-3 hours.
  • B Lysis under non-reducing conditions.
  • C (i & ii) Attempt to solubilise KDFAK-2S-AVQP from the insoluble fraction of non-reducing lysis with reducing buffer (5 mM and 100 mM DTT); (iii) Lysis under reducing conditions (10 mM DTT) for proteins KDFAK-2S-AVQP.
  • M protein standards
  • TC total cell lysate
  • Sup clarified lysate.
  • Figure 3 Ni-affinity chromatography.
  • A Elution profile of KDAAK-2S-AVQP under normal non-reducing conditions and reducing SDS-PAGE of the column fractions.
  • B Elution profile of antigen-F under reducing conditions (R) and reducing SDS-PAGE of the column fractions.
  • C Elution profile of KDFAK-2S-AVQP under normal non-reducing conditions (NR) and reducing SDS-PAGE of the column fractions.
  • TC total cell lysate
  • Sp clarified supernatant before loaded to the column
  • FT flow through
  • W column wash.
  • M protein standards.
  • Figure 4 Purification with anion exchange chromatography (AIEX).
  • AIEX Purification with anion exchange chromatography
  • B Elution profile of KDFAK-2S-AVQP under reducing conditions (R) and reducing SDS-PAGE of the column fractions.
  • C Elution profile of KDFAK-2S-AVQP under normal non-reducing conditions (NR) and reducing SDS-PAGE of the column fractions.
  • BL before loading
  • FT flow through
  • M protein standards.
  • Figure 5 Size exclusion chromatography under non-reducing conditions (NR).
  • A Elution profile KDAAK-2S-AVQP and reducing SDS-PAGE of the column fractions.
  • B Elution profile of KDFAK-2S-AVQP being purified under reducing conditions (R) before this step and reducing SDS-PAGE of the column fractions.
  • C Elution profile of KDFAK-2S-AVQP being purified under non-reducing conditions (NR) before this step and reducing SDS-PAGE of the column fractions.
  • BL before loading
  • M protein standards.
  • Figure 6 PAGE analysis of the P. gulae antigen final products.
  • A SDS- PAGE;
  • B Native PAGE or on native gels.
  • A-version KDAAK-2S-AVQP;
  • F-R KDFAK-2S- AVQP purified under reducing conditions until the final size exclusion step;
  • F-NR KDFAK- 2S-AVQP purified under non-reducing conditions.
  • R reducing;
  • Figure 7 Mouse periodontitis model: therapeutic vaccination.
  • Figure 8 P. gulae induced bone loss.
  • Statistical analysis One-way ANOVA and post-hoc Dunnet’s T3. # (p ⁇ 0.05, compared to Naive control); ## (p ⁇ 0.05, compared to infected control).
  • Figure 9. Anti-P. gulae antibody isotype response. Antibody titres in mouse sera (individual) towards heat-killed P. gulae whole cells.
  • A Total IgG Titres;
  • B lgG1 Subtype titres;
  • C lgG2a Subtype titres.
  • Figure 10 Antibody IgG response against P. gulae protease complex. Antibody titres in mouse sera (individual) towards purified P. gulae RgpA/Kgp protease complex. (A) Total IgG Titres; (B) lgG1 Subtype titres; (C) lgG2a Subtype titres.
  • Figure 11 Antibody IgG anti-vaccine antigen immune response. Antibody titres in mouse sera (Individual sera) towards vaccine antigen used to immunise mice.
  • Figure 12 Schematic of experimental protocol for canine serology studies.
  • Figure 13 IgG response to immunising antigen (KDAAK-2S-AVQP-6His). A) pooled sera; b) individual sera; C) analysis of individual sera; d) tittering of individual sera.
  • Figure 14 Serological endpoint titres (2xbaseline) vs P. gulae whole cells.
  • Figure 15 Comparison of titre responses to P. gulae KAS2 protein following immunisation with KDAAK-2S-AVQP-6His.
  • Figure 16 Serological midpoint titres for each serum collection time point.
  • the midpoint titre values were plotted for each serum collection time point. Mean and standard error of the mean (SEM) are shown for each serum collection time point. The ordinate axis represents the serum dilution required to achieve the titration midpoint. Pairs of serum time points that showed a statistical significant difference (p ⁇ 0.05) between the means are represented by the bars above the graph.
  • Figure 17 Serological endpoint titres for each serum collection time point.
  • Figure 18 Schematic of vaccination protocol for assessing immunogenicity of RNA candidate vaccines.
  • Figure 19 Schematic of study protocol for determining in vivo efficacy of RNA vaccines.
  • Table 2 exemplary RNA sequences of the invention
  • RNA vaccines encoding chimeric or fusion proteins comprising components of P. gulae virulence genes, for use in inducing an immune response to P. gulae, and methods and uses comprising the same.
  • the pathogenicity of P. gulae is attributed to a number of surface-associated virulence factors that include cysteine proteinases (gingipain-homologues), fimbriae, haem-binding proteins, and outer membrane transport proteins amongst others.
  • cysteine proteinases e.g., cysteine proteinases (gingipain-homologues), fimbriae, haem-binding proteins, and outer membrane transport proteins amongst others.
  • the extracellular Arg- and Lys-specific proteinases ‘gingipain-homologues’ (RgpA/B and Kgp) of P. gingivalis, a related pathogen have been implicated as major virulence factors that are critical for colonisation, penetration into host tissue, dysregulation of the immune response, dysbiosis and disease.
  • the gingipains in particular the Lys-specific proteinase Kgp are essential for the ability of a related pathogen, 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 high concentrations proximal to the subgingival plaque and at lower concentrations at distal sites deeper into the gingival tissue.
  • Lys-specific and Argspecific 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 a-trypsin, a2-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.
  • the RgpA, and Kgp genes of P. gingivalis 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 a “Domain of Unknown Function” (termed DUF2436 which is defined as conserved Pfam Domain of Unknown Function; IPR018832) and an adhesin domain (comprising adhesin binding domains).
  • HA hemagglutinin-adhesin
  • DUF2436 which is defined as conserved Pfam Domain of Unknown Function; IPR018832
  • adhesin domain comprising adhesin binding domains.
  • the particular arrangement of the adhesin domains and DUF2436 varies between Kgp and RgpA/
  • the RgpA and Kgp precursor proteins are cleaved into multiple domains that remain non-covalently associated forming large outer membrane protein complexes.
  • 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 and domains from the hemagglutinin genes.
  • a Lys-gingipain catalytic domain sequence around the active site Histidine may also be referred to a KAS or K domain.
  • an Arg-gingipain catalytic domain sequence around the active site Histidine may also be referred to as a RAS or R domain.
  • the catalytic domain of the Lys-gingipain or Arg-gingipains is located in the N-terminal region of the protein.
  • Exemplary Histidine active site peptides, as found within the catalytic domains are set out in in Table 1 as SEQ ID NOs: 2 and 4
  • an HA domain of an Arg- or Lys-gingipain of P. gulae will be understood to either refer to the region of an Arg- or Lys-gingipain homologue that is C- terminal to the catalytic or active site domain or to an homologous HA domain sequence encoded by a separate polyadhesin gene such as Hag.
  • the HA domain typically comprise a Domain of Unknown Function (DUF) domain (especially DUF 2436 conserved Pfam Domain of Unknown Function; IPR018832) and an adhesin domain comprising adhesin binding domains (ABMs).
  • DUF Domain of Unknown Function
  • IPR018832 conserved Pfam Domain of Unknown Function
  • ABSMs adhesin domain comprising adhesin binding domains
  • 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.
  • the polynucleotides of the invention are in the form of an RNA molecule, preferably an mRNA.
  • messenger RNA mRNA 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.
  • RNA 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.
  • 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 “U.”
  • RNA molecules 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.
  • a “5' untranslated region” (5'IITR) 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.
  • a “3' untranslated region” 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.
  • 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.
  • a start codon e.g., methionine (ATG)
  • a stop codon e.g., TAA, TAG or TGA
  • 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, consecutive adenosine monophosphates.
  • a polyA tail may contain 10 to 300 adenosine monophosphates.
  • 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.
  • a polyA tail contains 50 to 250 adenosine monophosphates.
  • the polyA tail may be segments (eg comprising segments of consecutive adenosine monophosphates joined by short spacers).
  • the poly(A) tail functions to protect RNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, export of the RNA from the nucleus and translation.
  • a polynucleotide includes 200 to 3,000 nucleotides.
  • 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.
  • 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.
  • modified mRNA molecules of the present invention may also be termed “mmRNA.”
  • a “structural” feature or modification is one in which two or more linked nucleotides are inserted, deleted, duplicated, inverted or randomized in a polynucleotide, primary construct or mmRNA without significant chemical modification to the nucleotides themselves.
  • the polynucleotide “ATCG” may be chemically modified to “AT-5meC-G”.
  • the same polynucleotide may be structurally modified from “ATCG” to “ATCCCG”.
  • the dinucleotide “CO” has been inserted, resulting in a structural modification to the polynucleotide.
  • the RNA molecules of the invention may also comprise a 5’ terminal cap.
  • 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.
  • the RNA molecule comprises at least one chemical modification.
  • chemical modification and “chemically modified” refer to modification with respect to adenosine (A), guanosine (G), uridine (II), thymidine (T) or cytidine (C) ribonucleosides or deoxyribnucleosides 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.
  • modification refers to a modification relative to the canonical set 20 amino acids. Polypeptides, as provided herein, are also considered “modified” of they contain amino acid substitutions, insertions or a combination of substitutions and insertions.
  • Polynucleotides e.g., RNA polynucleotides, such as RNA polynucleotides
  • RNA polynucleotides comprise various (more than one) different modifications.
  • a particular region of a polynucleotide contains one, two or more (optionally different) nucleoside or nucleotide modifications.
  • 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.
  • 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).
  • Modifications of polynucleotides include, without limitation, those described herein.
  • Polynucleotides e.g., RNA polynucleotides, such as RNA polynucleotides
  • 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).
  • an internucleoside linkage e.g., to a linking phosphate, to a phosphodiester linkage or to the phosphodiester backbone.
  • Polynucleotides e.g., RNA polynucleotides, such as RNA polynucleotides
  • RNA 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 an internucleotide linkages, purine or pyrimidine bases, or sugars.
  • the modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a polynucleotide may be chemically modified.
  • nucleosides and nucleotides of a polynucleotide e.g., RNA polynucleotides, such as RNA 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 phosphdioester linkages, in which case the polynucleotides would comprise regions of nucleotides.
  • 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.
  • 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.
  • 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.
  • 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.
  • polynucleotides e.g., RNA polynucleotides, such as RNA polynucleotides
  • RNA polynucleotides are uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification.
  • 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).
  • m5C 5-methyl-cytidine
  • 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.
  • 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.
  • ac4C N4-acetyl-cytidine
  • m5C 5-methyl-cytidine
  • 5-halo-cytidine e.g., 5-iodo- cytidine
  • 5-hydroxymethyl-cytidine hm5C
  • 1-methyl-pseudoisocytidine 2-thio-cytidine (s2C)
  • 2-thio-5-methyl-cytidine 2-thio-5-methyl-
  • a modified nucleobase is a modified uridine.
  • exemplary nucleobases and in some embodiments, a modified nucleobase is a modified cytosine, nucleosides having a modified uridine include 5-cyano uridine, and 4'-thio uridine.
  • 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).
  • a modified nucleobase is a modified guanine.
  • nucleobases and nucleosides having a modified guanine include inosine (I), 1 -methylinosine (mi l), 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.
  • the polynucleotides of the present disclosure may be partially or fully modified along the entire length of the molecule.
  • 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, II, C
  • nucleotides X in a polynucleotide of the present disclosure are modified nucleotides, wherein X may any one of nucleotides A, G, II, C, or any one of the combinations A+G, A+ll, A+C, G+ll, G+C, ll+C, A+G+ll, A+G+C, G+U+C or A+G+C.
  • 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, II 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 20% to 95%, from 20%
  • 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.
  • the polynucleotides may contain a modified pyrimidine such as a modified uracil or cytosine.
  • 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 polynucleotide is replaced with a modified uracil (e.g., a 5-substituted uracil).
  • 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), n 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).
  • the modified nucleobase is a modified uracil.
  • exemplary nucleobases and nucleosides having a modified uracil include pseudouridine (ip), pyridin-
  • 4-one ribonucleoside 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4- thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine, 5-aminoallyl- uridine, 5-halo-uridine (e.g., 5-iodo-uridineor 5-bromo-uridine), 3-methyl-uridine, 5- methoxy-uridine, uridine 5-oxyacetic acid, uridine 5-oxyacetic acid methyl ester, 5- carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl- uridine, 5-carboxyhydroxymethyl-uridine methyl ester, 5-methoxycarbonylmethyl-uridine,
  • 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 sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or antigenic polypeptide)).
  • a naturally- occurring or 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 antigenic polypeptide)
  • 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)).
  • 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)
  • 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)).
  • 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)
  • a codon-optimized RNA 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 (II) nucleotides.
  • WO02/098443 discloses a pharmaceutical composition containing an RNA stabilized by sequence modifications in the translated region.
  • 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'IITR) and/or at their 3'-end (3'IITR), in addition to other structural features, such as a 5'-cap structure or a 3'-poly(A) tail. Both the 5'IITR and the 3'IITR are typically transcribed from the genomic DNA and are elements of the premature RNA.
  • UTR untranslated regions
  • 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.
  • the length of the 3'-poly(A) tail may be an essential element with respect to the stability of the individual RNA.
  • the RNA (e.g., RNA) 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.
  • the combination of a poly(A) sequence or polyadenylation signal and at least one histone stem-loop 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.
  • the RNA (e.g., RNA) vaccine does not comprise a histone downstream element (HDE).
  • Histone downstream element 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.
  • the inventive nucleic acid does not include an intron.
  • the RNA (e.g., RNA) vaccine may or may not contain a enhancer and/or promoter sequence, which may be modified or unmodified or which may be activated or inactivated.
  • 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.
  • the Stability of the stem-loop structure generally depends on the length, number of mismatches or bulges, and base composition of the paired region.
  • wobble base pairing non- Watson-Crick base pairing
  • the at least one histone stem-loop sequence comprises a length of 15 to 45 nucleotides.
  • the RNA (e.g., RNA) vaccine may have one or more Allrich 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., RNA) vaccines. Alternatively the AURES may remain in the RNA (e.g., RNA) vaccine.
  • the RNA of the invention may comprise a ribosome skipping sequence, such as a 2A skipping sequence.
  • a ribosome skipping sequence such as a 2A skipping sequence.
  • 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.
  • polynucleotides 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”.
  • 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.
  • the chimeric or fusion proteins encoded by the RNAs of the invention may comprise a linker between the amino acid sequence of a P. gulae gingipain active site, and the amino acid sequence of the adhesin domain of a P.
  • 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. gulae trypsin-like enzyme. Alternatively, it may space domains apart.
  • the peptide linker may include the amino acids glycine and serine in various lengths and combinations.
  • the peptide linker can include the sequence Gly-Gly-Ser (GGS), Gly-Gly-Gly-Ser (GGGS, SEQ ID NO: 109) or Gly-Gly-Gly-Gly-Ser (GGGGS, SEQ ID NO: 110) and variations or repeats thereof.
  • the peptide linker can include the amino acid sequence GGGGS (SEQ ID NO: 110, 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.
  • the linker may be (GS)3 (i.e., GSGSGS, SEQ ID NO: 111) 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.
  • the linker may be a series of repeating glycine residues separated by serine residues.
  • (GGGGS)3 i.e., the linker may comprise the amino acid sequence GGGGSGGGGSGGGGS, (G4S)3, SEQ ID NO: 112) and variations thereof.
  • the peptide linker can include the amino acid sequence GGGGS (SEQ ID NO: 110, 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.
  • the linker may be (GS)3 (i.e., GSGSGS, SEQ ID NO: 111) 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.
  • DSSG SEQ ID NO: 113
  • DSSGAS SEQ ID NO: 114
  • KLDSSG SEQ ID NO: 115
  • Examples of other suitable linkers are described in Chen et al., (2013) Advanced Drug Delivery Reviews, 65: 1357- 1369.
  • 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.
  • 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.
  • 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.
  • non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and/or a polar residue for a non-polar residue.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two nucleic acid sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M.
  • the percent identity between two nucleic acid sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two nucleic acid sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.
  • the signal peptide of a nascent precursor 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 a ER- 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 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.
  • 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.
  • a signal peptide fused to the antigenic polypeptide encoded by the (e.g., RNA) RNA (e.g., RNA) vaccine is an IgGk chain V-lll region HAH signal peptide (IgGk SP) having the sequence of METPAQLLFLLLLWLPDTTG (SEQ ID NO: 117).
  • the signal peptide is selected from: Japanese encephalitis PRM signal sequence (MLGSNSGQRVVFTILLLLVAPAYS, SEQ ID NO: 118), VSVg protein signal sequence (MKCLLYLAFLFIGVNCA, SEQ ID NO: 119) and Japanese encephalitis JEV signal sequence (MWLVSLAIVTACAGA, SEQ ID NO: 120).
  • variants such as: tPA (VSA): MDAMKRGLCCVLLLCGAVFVSA (SEQ ID NO:
  • tPA VSAR: MDAMKRGLCCVLLLCGAVFVSAR (SEQ ID NO: 123), tPA (VSP): MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 124), tPA (VSPS).
  • the signal peptide comprises the sequence of the SEAP (secreted embryonic alkaline phosphatase) secretion signal, having the amino acid sequence MLLLLLLLGLRLQLSLG (SEQ ID NO: 125).
  • the amino acid sequence of the signal peptide comprises the sequence MLLLLLLLGLRLQLSLG[A] (SEQ ID NO: 126), such that the expressed RNA product comprises the sequence MLLLLLLLGLRLQLSLG[A] (SEQ ID NO: 126) N terminal to the sequences defined herein including in Table 1.
  • a signal peptide may have a length of 15-60 amino acids.
  • 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.
  • 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.
  • 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.
  • the RNA of the invention may encode one, two, three, four or more N terminal amino acids to the sequences defined herein in Table 1.
  • an RNA encoding an amino acid sequence as set forth in any of SEQ ID NOs: 2, 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 78 to 82 may encode one or more additional N terminal amino acids, optionally an N terminal alanine residue.
  • the present invention contemplates the provision of a polynucleotide (preferably an RNA) encoding a chimeric or fusion protein for inducing an immune response to P. gulae, 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.
  • 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.
  • a cationic lipid is an ionizable cationic lipid and the noncationic lipid is a neutral lipid, and the sterol is a cholesterol.
  • a cationic lipid is selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl- [1 ,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3- DMA), di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), (12Z.15Z) — N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine (L608), and N,N- dimethyl-1-[(1S,2R)-2
  • lipid nanoparticle formulations include 25-75% of a cationic lipid optionally selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1 ,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 0.5- 15% of the neutral lipid, 5-50% of the sterol, and 0.5-20% of the PEG or PEG-modified lipid on a molar basis.
  • a cationic lipid optionally selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1 ,3]-dioxolane (DLin-KC2-DMA), dilino
  • the molar lipid ratio is 50/10/38.5/1.5 (mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG, PEG-DSG or PEG-DPG), 57.2/7.1134.3/1.4 (mol % cationic lipid/neutral lipid, e.g., DPPC/Chol/PEG- modified lipid, e.g., PEG-cDMA), 40/15/40/5 (mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG), 50/10/35/4.5/0.5 (mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DSG), 50/10/35/5 (cationic lipid/neutral lipid
  • 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.
  • the lipid nanoparticle formulation is composed of 57.1 % cationic lipid, 7.1 % dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA.
  • 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.
  • the cationic lipid is an ionizable cationic lipid and the noncationic lipid is a neutral lipid, and the sterol is a cholesterol.
  • the cationic lipid is selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1 ,3]-dioxolane (DLin- KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non- 2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).
  • 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.
  • the ratio of lipid to RNA (e.g., RNA) 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.
  • lipid nanoparticle formulations may contain 0.5% to 3.0%, 1.0% to 3.5%, 1.5% to 4.0%, 2.0% to 4.5%, 2.5% to 5.0% and/or 3.0% to 6.0% of the lipid molar ratio of PEG-c-DOMG (R-3-[(w-methoxy- poly(ethyleneglycol)2000)carbamoyl)]-1 ,2-dimyristyloxypropyl-3-amine) (also referred to herein as PEG-DOMG) as compared to the cationic lipid, DSPC and cholesterol.
  • PEG-c-DOMG R-3-[(w-methoxy- poly(ethyleneglycol)2000)carbamoyl)]-1 ,2-dimyristyloxypropyl-3-amine
  • the cationic lipid may be of Formula III
  • 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.
  • the phospholipid may comprise from 5 mol % to 20 mol %, from 5 mol % to 15 mol %, from 5 mol % to 10 mol %, from 10 mol % to 20 mol %, or from 15 mol % to 20 mol % of the total lipid present in the particle.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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- 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).
  • 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.
  • 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.
  • the RNA (e.g., mRNA) vaccines of the disclosure may be formulated in lipid nanoparticles having a diameter from about 10 to about 100 nm such as, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about 90 nm, about 20 to about 100
  • the lipid nanoparticle may have a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than
  • the lipid nanoparticle may be a limit size lipid nanoparticle described in International Patent Publication No. WO2013059922, the contents of which are herein incorporated by reference in their entirety.
  • the limit size lipid nanoparticle may comprise a lipid bilayer surrounding an aqueous core or a hydrophobic core; where the lipid bilayer may comprise a phospholipid such as, but not limited to, diacylphosphatidylcholine, a diacylphosphatidylethanolamine, a ceramide, a sphingomyelin, a dihydrosphingomyelin, a cephalin, a cerebroside, a C8-C20 fatty acid diacylphophatidylcholine, and 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC).
  • POPC 1-palmitoyl-2-oleoyl phosphatidylcholine
  • POPC 1-palmitoyl-2-o
  • the RNA (e.g., mRNA) 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), including HIV-binding peptides, HIV-1 Tat (HIV), Tat-derived peptides, Penetratin, VP derived or analog peptides, Pestivirus Erns, HSV, VP (Herpes simplex), MAP, KALA or protein transduction domains (PTDs), PpT620, prolin-rich peptides, arginine-rich peptides, lysine-rich peptides, MPG-peptide(s), Pep-1 , L-oligomers, Calcitonin peptide(s), Antennapedia-
  • PLL poly-L
  • PEI polyethyleneimine
  • DOTMA [1-(2,3- sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride
  • DMRIE di-C14-amidine
  • DOTIM DOTIM
  • SAINT DC-Chol
  • BGTC CTAP
  • DOPC DODAP
  • DOPE Dioleyl phosphatidylethanolamine
  • DOSPA DODAB
  • DOIC DOIC
  • DMEPC DOGS: Dioctadecylamidoglicylspermin
  • DIMRI Dimyristooxypropyl dimethyl hydroxyethyl ammonium bromide
  • DOTAP dioleoyloxy-3-(trimethylammonio)propane
  • DC-6-14 O,O-ditetradecanoyl-N-.
  • modified polyaminoacids such as beta-aminoacid- polymers or reversed polyamides, etc.
  • modified polyethylenes such as PVP (poly(N- ethyl-4-vinylpyridinium bromide)), etc.
  • modified acrylates such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc.
  • modified amidoamines such as pAMAM (poly(amidoamine)), etc.
  • modified polybetaminoester (PBAE) such as diamine end modified 1 ,4 butanediol diacrylate-co-5-amino-1-pentanol polymers, etc.
  • dendrimers such as polypropylamine dendrimers or pAMAM based dendrimers, etc.
  • polyimine(s) such as PEI: poly(ethyleneimine), poly(propyleneimine), etc.
  • polyallylamine sugar backbone based polymers,
  • RNA e.g., mRNA
  • the RNA vaccine is not associated with a cationic or polycationic compounds.
  • lipid nanoparticles described herein may be made in a sterile environment.
  • 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.
  • 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.
  • 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.
  • polymer conjugates with the polynucleotides of the present disclosure may be made using the methods and/or segmented polymeric reagents described in U.S. Patent Application No. 20130072709, the contents of which are herein incorporated by reference in its entirety.
  • the polymer conjugate may have pendant side groups comprising ring moieties such as, but not limited to, the polymer conjugates described in U.S. Patent Publication No. US20130196948, the contents which are herein incorporated by reference in its entirety.
  • the nanoparticle formulations may comprise a conjugate to enhance the delivery of nanoparticles of the present disclosure in a subject. Further, the conjugate may inhibit phagocytic clearance of the nanoparticles in a subject.
  • the conjugate may be a “self” peptide designed from the human membrane protein CD47 (e.g., the “self” particles described by Rodriguez et al. (Science 2013 339, 971-975), herein incorporated by reference in its entirety). As shown by Rodriguez et al., the self peptides delayed macrophage-mediated clearance of nanoparticles which enhanced delivery of the nanoparticles.
  • the conjugate may be the membrane protein CD47 (e.g., see Rodriguez et al.
  • CD47 can increase the circulating particle ratio in a subject as compared to scrambled peptides and PEG coated nanoparticles.
  • the RNA (e.g., mRNA) vaccines of the present disclosure are formulated in nanoparticles which comprise a conjugate to enhance the delivery of the nanoparticles of the present disclosure in a subject.
  • the conjugate may be the CD47 membrane or the conjugate may be derived from the CD47 membrane protein, such as the “self” peptide described previously.
  • the nanoparticle may comprise PEG and a conjugate of CD47 or a derivative thereof.
  • the nanoparticle may comprise both the “self” peptide described above and the membrane protein CD47.
  • RNA vaccine pharmaceutical compositions comprising the polynucleotides of the present disclosure and a conjugate that may have a degradable linkage.
  • conjugates include an aromatic moiety comprising an ionizable hydrogen atom, a spacer moiety, and a water- soluble polymer.
  • pharmaceutical compositions comprising a conjugate with a degradable linkage and methods for delivering such pharmaceutical compositions are described in U.S. Patent Publication No. US20130184443, the contents of which are herein incorporated by reference in their entirety.
  • the nanoparticle formulations may be a carbohydrate nanoparticle comprising a carbohydrate carrier and a RNA (e.g., mRNA) vaccine.
  • the carbohydrate carrier may include, but is not limited to, an anhydride-modified phytoglycogen or glycogen-type material, phtoglycogen octenyl succinate, phytoglycogen beta-dextrin, anhydride-modified phytoglycogen beta-dextrin. (See e.g., International Publication No. W02012109121 ; the contents of which are herein incorporated by reference in their entirety).
  • Nanoparticle formulations of the present disclosure may be coated with a surfactant or polymer in order to improve the delivery of the particle.
  • 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.
  • the hydrophilic coatings may help to deliver nanoparticles with larger payloads such as, but not limited to, RNA (e.g., mRNA) vaccines within the central nervous system.
  • RNA e.g., mRNA
  • 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.
  • the lipid nanoparticles of the present disclosure may be hydrophilic polymer particles.
  • hydrophilic polymer particles and methods of making hydrophilic polymer particles are described in U.S. Patent Publication No. US20130210991 , the contents of which are herein incorporated by reference in their entirety.
  • the lipid nanoparticles of the present disclosure may be hydrophobic polymer particles.
  • Lipid nanoparticle formulations may be improved by replacing the cationic lipid with a biodegradable cationic lipid which is known as a rapidly eliminated lipid nanoparticle (reLNP).
  • Ionizable cationic lipids such as, but not limited to, DLinDMA, DLin- KC2-DMA, and DLin-MC3-DMA, have been shown to accumulate in plasma and tissues over time and may be a potential source of toxicity.
  • the rapid metabolism of the rapidly eliminated lipids can improve the tolerability and therapeutic index of the lipid nanoparticles by an order of magnitude from a 1 mg/kg dose to a 10 mg/kg dose in rat.
  • Inclusion of an enzymatically degraded ester linkage can improve the degradation and metabolism profile of the cationic component, while still maintaining the activity of the reLNP formulation.
  • the ester linkage can be internally located within the lipid chain or it may be terminally located at the terminal end of the lipid chain. The internal ester linkage may replace any carbon in the lipid chain.
  • the internal ester linkage may be located on either side of the saturated carbon.
  • an immune response may be elicited by delivering a lipid nanoparticle which may include a nanospecies, a polymer and an immunogen.
  • a lipid nanoparticle which may include a nanospecies, a polymer and an immunogen.
  • the polymer may encapsulate the nanospecies or partially encapsulate the nanospecies.
  • the immunogen may be a recombinant protein, a modified RNA and/or a polynucleotide described herein.
  • the lipid nanoparticle may be formulated for use in a vaccine such as, but not limited to, against a pathogen.
  • Lipid nanoparticles may be engineered to alter the surface properties of particles so the lipid nanoparticles may penetrate the mucosal barrier.
  • Mucus is located on mucosal tissue such as, but not limited to, oral (e.g., the buccal and esophageal membranes and tonsil tissue), ophthalmic, gastrointestinal (e.g., stomach, small intestine, large intestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal, tracheal and bronchial membranes), genital (e.g., vaginal, cervical and urethral membranes).
  • oral e.g., the buccal and esophageal membranes and tonsil tissue
  • ophthalmic e.g., gastrointestinal (e.g., stomach, small intestine, large intestine, colon, rectum)
  • nasal, respiratory e.g., nasal, pharyngeal, tracheal and bronchial
  • Nanoparticles larger than 10-200 nm which are preferred for higher drug encapsulation efficiency and the ability to provide the sustained delivery of a wide array of drugs have been thought to be too large to rapidly diffuse through mucosal barriers. Mucus is continuously secreted, shed, discarded or digested and recycled so most of the trapped particles may be removed from the mucosa tissue within seconds or within a few hours. Large polymeric nanoparticles (200 nm-500 nm in diameter) which have been coated densely with a low molecular weight polyethylene glycol (PEG) diffused through mucus only 4 to 6-fold lower than the same particles diffusing in water (Lai et al. PNAS 2007 104(5): 1482-487; Lai et al.
  • PEG polyethylene glycol
  • the transport of nanoparticles may be determined using rates of permeation and/or fluorescent microscopy techniques including, but not limited to, fluorescence recovery after photobleaching (FRAP) and high resolution multiple particle tracking (MPT).
  • FRAP fluorescence recovery after photobleaching
  • MPT high resolution multiple particle tracking
  • compositions which can penetrate a mucosal barrier may be made as described in U.S. Pat. No. 8,241 ,670 or International Patent Publication No. WO2013110028, the contents of each of which are herein incorporated by reference in its entirety.
  • the lipid nanoparticle engineered to penetrate mucus may comprise a polymeric material (i.e. a polymeric core) and/or a polymer-vitamin conjugate and/or a tri-block copolymer.
  • the polymeric material may include, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes), polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates.
  • the polymeric material may be biodegradable and/or biocompatible.
  • biocompatible polymers are described in International Patent Publication No. WO2013116804, the contents of which are herein incorporated by reference in their entirety.
  • the polymeric material may additionally be irradiated.
  • the polymeric material may be gamma irradiated (see e.g., International App. No. WO201282165, herein incorporated by reference in its entirety).
  • Non-limiting examples of specific polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co- glycolic acid) (PLLGA), poly(D.L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide- co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co- PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA
  • the lipid nanoparticle may be coated or associated with a co-polymer such as, but not limited to, a block copolymer (such as a branched polyether-polyamide block copolymer described in International Publication No. WO2013012476, herein incorporated by reference in its entirety), and (poly(ethylene glycol))-(poly(propylene oxide))-(poly(ethylene glycol)) triblock copolymer (see e.g., U.S. Publication 20120121718 and U.S. Publication 20100003337 and U.S. Pat. No. 8,263,665, the contents of each of which is herein incorporated by reference in their entirety).
  • a block copolymer such as a branched polyether-polyamide block copolymer described in International Publication No. WO2013012476, herein incorporated by reference in its entirety
  • the co-polymer may be a polymer that is generally regarded as safe (GRAS) and the formation of the lipid nanoparticle may be in such a way that no new chemical entities are created.
  • the lipid nanoparticle may comprise poloxamers coating PLGA nanoparticles without forming new chemical entities which are still able to rapidly penetrate human mucus (Yang et al. Angew. Chem. Int. Ed. 2011 50:2597-2600; the contents of which are herein incorporated by reference in their entirety).
  • a non-limiting scalable method to produce nanoparticles which can penetrate human mucus is described by Xu et al. (see, e.g., J Control Release 2013, 170(2):279-86; the contents of which are herein incorporated by reference in their entirety).
  • the vitamin of the polymer-vitamin conjugate may be vitamin E.
  • the vitamin portion of the conjugate may be substituted with other suitable components such as, but not limited to, vitamin A, vitamin E, other vitamins, cholesterol, a hydrophobic moiety, or a hydrophobic component of other surfactants (e.g., sterol chains, fatty acids, hydrocarbon chains and alkylene oxide chains).
  • the lipid nanoparticle engineered to penetrate mucus may include surface altering agents such as, but not limited to, polynucleotides, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as for example dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol and poloxamer), mucolytic agents (e.g., N-acetylcysteine, mugwort, bromelain, papain, clerodendrum, acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thy
  • the surface altering agent may be embedded or enmeshed in the particle's surface or disposed (e.g., by coating, adsorption, covalent linkage, or other process) on the surface of the lipid nanoparticle, (see e.g., U.S. Publication 20100215580 and U.S. Publication 20080166414 and US20130164343; the contents of each of which are herein incorporated by reference in their entirety).
  • the mucus penetrating lipid nanoparticles may comprise at least one polynucleotide described herein.
  • the polynucleotide may be encapsulated in the lipid nanoparticle and/or disposed on the surface of the particle.
  • the polynucleotide may be covalently coupled to the lipid nanoparticle.
  • Formulations of mucus penetrating lipid nanoparticles may comprise a plurality of nanoparticles. Further, the formulations may contain particles which may interact with the mucus and alter the structural and/or adhesive properties of the surrounding mucus to decrease mucoadhesion, which may increase the delivery of the mucus penetrating lipid nanoparticles to the mucosal tissue.
  • the mucus penetrating lipid nanoparticles may be a hypotonic formulation comprising a mucosal penetration enhancing coating.
  • the formulation may be hypotonice for the epithelium to which it is being delivered.
  • hypotonic formulations may be found in International Patent Publication No. WO2013110028, the contents of which are herein incorporated by reference in their entirety.
  • RNA vaccine formulation in order to enhance the delivery through the mucosal barrier may comprise or be a hypotonic solution.
  • the RNA (e.g., mRNA) vaccine is formulated as a lipoplex, such as, without limitation, the ATUPLEXTM system, the DACC system, the DBTC system and other siRNA-lipoplex technology from Silence Therapeutics (London, United Kingdom), STEMFECTTM from STEMGENT® (Cambridge, Mass.), and polyethylenimine (PEI) or protamine-based targeted and non-targeted delivery of nucleic acids acids (Aleku et al. Cancer Res. 2008 68:9788-9798; Strumberg et al.
  • a lipoplex such as, without limitation, the ATUPLEXTM system, the DACC system, the DBTC system and other siRNA-lipoplex technology from Silence Therapeutics (London, United Kingdom), STEMFECTTM from STEMGENT® (Cambridge, Mass.), and polyethylenimine (PEI) or protamine-based targeted and non-targeted delivery of nucleic acids acids (Aleku et al.
  • the RNA (e.g., mRNA) vaccine is formulated as a solid lipid nanoparticle.
  • a solid lipid nanoparticle may be spherical with an average diameter between 10 to 1000 nm. SLN possess a solid lipid core matrix that can solubilize lipophilic molecules and may be stabilized with surfactants and/or emulsifiers.
  • the lipid nanoparticle may be a self-assembly lipid-polymer nanoparticle (see Zhang et al., ACS Nano, 2008, 2 (8), pp 1696-1702; the contents of which are herein incorporated by reference in their entirety).
  • the SLN may be the SLN described in International Patent Publication No. W02013105101 , the contents of which are herein incorporated by reference in their entirety.
  • the SLN may be made by the methods or processes described in International Patent Publication No. W02013105101 , the contents of which are herein incorporated by reference in their entirety.
  • the RNA (e.g., mRNA) vaccines of the present disclosure can be formulated for controlled release and/or targeted delivery.
  • controlled release refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to effect a therapeutic outcome.
  • the RNA (e.g., RNA) vaccines may be encapsulated into a delivery agent described herein and/or known in the art for controlled release and/or targeted delivery.
  • encapsulate means to enclose, surround or encase. As it relates to the formulation of the compounds of the disclosure, encapsulation may be substantial, complete or partial.
  • substantially encapsulated means that at least greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9 or greater than 99.999% of the pharmaceutical composition or compound of the disclosure may be enclosed, surrounded or encased within the delivery agent.
  • Partially encapsulation means that less than 10, 10, 20, 30, 4050 or less of the pharmaceutical composition or compound of the disclosure may be enclosed, surrounded or encased within the delivery agent.
  • encapsulation may be determined by measuring the escape or the activity of the pharmaceutical composition or compound of the disclosure using fluorescence and/or electron micrograph.
  • At least 1 , 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the pharmaceutical composition or compound of the disclosure are encapsulated in the delivery agent.
  • the controlled release formulation may include, but is not limited to, tri-block co-polymers.
  • the formulation may include two different types of tri-block co-polymers (International Pub. No. W02012131104 and W02012131106, the contents of each of which are incorporated herein by reference in their entirety).
  • the RNA (e.g., mRNA) vaccines may be encapsulated into a lipid nanoparticle or a rapidly eliminated lipid nanoparticle and the lipid nanoparticles or a rapidly eliminated lipid nanoparticle may then be encapsulated into a polymer, hydrogel and/or surgical sealant described herein and/or known in the art.
  • the polymer, hydrogel or surgical sealant may be PLGA, ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc.
  • HYLENEX® Hazyme Therapeutics, San Diego Calif.
  • surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, Ga.), TISSELL® (Baxter International, Inc Deerfield, III.), PEG-based sealants, and COSEAL® (Baxter International, Inc Deerfield, III.).
  • the lipid nanoparticle may be encapsulated into any polymer known in the art which may form a gel when injected into a subject.
  • the lipid nanoparticle may be encapsulated into a polymer matrix which may be biodegradable.
  • the RNA (e.g., mRNA) vaccine formulation for controlled release and/or targeted delivery may also include at least one controlled release coating.
  • Controlled release coatings include, but are not limited to, OPADRY®, polyvinylpyrrolidone/vinyl acetate copolymer, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, EUDRAGIT RL®, EUDRAGIT RS® and cellulose derivatives such as ethylcellulose aqueous dispersions (AQUACOAT® and SURELEASE®).
  • the RNA (e.g., mRNA) vaccine controlled release and/or targeted delivery formulation may comprise at least one degradable polyester which may contain polycationic side chains.
  • Degradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof.
  • the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.
  • the RNA (e.g., RNA) vaccine controlled release and/or targeted delivery formulation comprising at least one polynucleotide may comprise at least one PEG and/or PEG related polymer derivatives as described in U.S. Pat. No. 8,404,222, the contents of which are incorporated herein by reference in their entirety.
  • the RNA (e.g., mRNA) vaccine controlled release delivery formulation comprising at least one polynucleotide may be the controlled release polymer system described in US20130130348, the contents of which are incorporated herein by reference in their entirety.
  • the RNA (e.g., mRNA) vaccines of the present disclosure may be encapsulated in a therapeutic nanoparticle, referred to herein as “therapeutic nanoparticle RNA (e.g., mRNA) vaccines.”
  • Therapeutic nanoparticles may be formulated by methods described herein and known in the art such as, but not limited to, International Pub Nos. WQ2010005740, WQ2010030763, WQ2010005721 , WQ2010005723, WQ2012054923, U.S. Publication Nos. US20110262491 ,
  • therapeutic polymer nanoparticles may be identified by the methods described in US Pub No. US20120140790, the contents of which are herein incorporated by reference in their entirety.
  • the therapeutic nanoparticle RNA (e.g., mRNA) vaccine may be formulated for sustained release.
  • sustained release refers to a pharmaceutical composition or compound that conforms to a release rate over a specific period of time. The period of time may include, but is not limited to, hours, days, weeks, months and years.
  • the sustained release nanoparticle may comprise a polymer and a therapeutic agent such as, but not limited to, the polynucleotides of the present disclosure (see International Pub No. 2010075072 and US Pub No. US20100216804, US20110217377 and US20120201859, the contents of each of which are incorporated herein by reference in their entirety).
  • the sustained release formulation may comprise agents which permit persistent bioavailability such as, but not limited to, crystals, macromolecular gels and/or particulate suspensions (see U.S. Patent Publication No US20130150295, the contents of each of which are incorporated herein by reference in their entirety).
  • the therapeutic nanoparticle RNA (e.g., mRNA) vaccines may be formulated to be target specific.
  • the therapeutic nanoparticles may include a corticosteroid (see International Pub. No. WO2011084518, the contents of which are incorporated herein by reference in their entirety).
  • the therapeutic nanoparticles may be formulated in nanoparticles described in International Pub No. WO2008121949, W02010005726, W02010005725, WO2011084521 and US Pub No. US20100069426, US20120004293 and US20100104655, the contents of each of which are incorporated herein by reference in their entirety.
  • the nanoparticles of the present disclosure may comprise a polymeric matrix.
  • the nanoparticle may comprise two or more polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly (orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) or combinations thereof.
  • the therapeutic nanoparticle comprises a diblock copolymer.
  • the diblock copolymer may include PEG in combination with a polymer such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly (orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) or combinations thereof.
  • the diblock copolymer may be a high- X diblock copolymer such as those described
  • the therapeutic nanoparticle comprises a PLGA-PEG block copolymer (see U.S. Publication No. US20120004293 and U.S. Pat. No. 8,236,330, each of which is herein incorporated by reference in their entirety).
  • the therapeutic nanoparticle is a stealth nanoparticle comprising a diblock copolymer of PEG and PLA or PEG and PLGA (see U.S. Pat. No. 8,246,968 and International Publication No. WO2012166923, the contents of each of which are herein incorporated by reference in their entirety).
  • the therapeutic nanoparticle is a stealth nanoparticle or a target-specific stealth nanoparticle as described in U.S. Patent Publication No. US20130172406, the contents of which are herein incorporated by reference in their entirety.
  • the therapeutic nanoparticle may comprise a multiblock copolymer (see e.g., U.S. Pat. Nos. 8,263,665 and 8,287,910 and U.S. Patent Pub. No. US20130195987, the contents of each of which are herein incorporated by reference in their entirety).
  • the lipid nanoparticle comprises the block copolymer PEG-PLGA-PEG (see e.g., the thermosensitive hydrogel (PEG-PLGA-PEG) was used as a TGF-beta1 gene delivery vehicle in Lee et al.
  • Thermosensitive Hydrogel as a Tgf-pi Gene Delivery Vehicle Enhances Diabetic Wound Healing. Pharmaceutical Research, 2003 20(12): 1995-2000; as a controlled gene delivery system in Li et al. Controlled Gene Delivery System Based on Thermosensitive Biodegradable Hydrogel.
  • RNA vaccines of the present disclosure may be formulated in lipid nanoparticles comprising the PEG- PLGA-PEG block copolymer.
  • the therapeutic nanoparticle may comprise a multiblock copolymer (see e.g., U.S. Pat. Nos. 8,263,665 and 8,287,910 and U.S. Patent Pub. No. US20130195987, the contents of each of which are herein incorporated by reference in their entirety).
  • the block copolymers described herein may be included in a polyion complex comprising a non-polymeric micelle and the block copolymer, (see e.g., U.S. Publication No. 20120076836, the contents of which are herein incorporated by reference in their entirety).
  • the therapeutic nanoparticle may comprise at least one acrylic polymer.
  • Acrylic polymers include but are not limited to, acrylic acid, methacrylic acid, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates and combinations thereof.
  • the therapeutic nanoparticles may comprise at least one poly(vinyl ester) polymer.
  • the poly(vinyl ester) polymer may be a copolymer such as a random copolymer.
  • the random copolymer may have a structure such as those described in International Application No. WO2013032829 or U.S. Patent Publication No US20130121954, the contents of each of which are herein incorporated by reference in their entirety.
  • the poly(vinyl ester) polymers may be conjugated to the polynucleotides described herein.
  • the therapeutic nanoparticle may comprise at least one diblock copolymer.
  • the diblock copolymer may be, but it not limited to, a poly(lactic) acid- poly(ethylene)glycol copolymer (see, e.g., International Patent Publication No. WO2013044219, the contents of which are herein incorporated by reference in their entirety).
  • the therapeutic nanoparticle may be used to treat cancer (see International publication No. WO2013044219, the contents of which are herein incorporated by reference in their entirety).
  • the therapeutic nanoparticles may comprise at least one cationic polymer described herein and/or known in the art.
  • the therapeutic nanoparticles may comprise at least one amine-containing polymer such as, but not limited to polylysine, polyethylene imine, poly(amidoamine) dendrimers, poly(beta-amino esters) (see, e.g., U.S. Pat. No. 8,287,849, the contents of which are herein incorporated by reference in their entirety) and combinations thereof.
  • amine-containing polymer such as, but not limited to polylysine, polyethylene imine, poly(amidoamine) dendrimers, poly(beta-amino esters) (see, e.g., U.S. Pat. No. 8,287,849, the contents of which are herein incorporated by reference in their entirety) and combinations thereof.
  • the nanoparticles described herein may comprise an amine cationic lipid such as those described in International Patent Application No. WO2013059496, the contents of which are herein incorporated by reference in their entirety.
  • the cationic lipids may have an amino-amine or an aminoamide moiety.
  • the therapeutic nanoparticles may comprise at least one degradable polyester which may contain polycationic side chains.
  • Degradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof.
  • the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.
  • the synthetic nanocarriers may be formulated for targeted release.
  • the synthetic nanocarrier is formulated to release the polynucleotides at a specified pH and/or after a desired time interval.
  • the synthetic nanoparticle may be formulated to release the RNA (e.g., mRNA) vaccines after 24 hours and/or at a pH of 4.5 (see International Publication Nos. W02010138193 and W02010138194 and US Pub Nos. US20110020388 and US20110027217, each of which is herein incorporated by reference in their entireties).
  • the synthetic nanocarriers may be formulated for controlled and/or sustained release of the polynucleotides described herein.
  • the synthetic nanocarriers for sustained release may be formulated by methods known in the art, described herein and/or as described in International Pub No. W02010138192 and US Pub No. 20100303850, each of which is herein incorporated by reference in their entirety.
  • the RNA (e.g., mRNA) vaccine may be formulated for controlled and/or sustained release wherein the formulation comprises at least one polymer that is a crystalline side chain (CYSC) polymer.
  • CYSC polymers are described in U.S. Pat. No. 8,399,007, herein incorporated by reference in its entirety.
  • the synthetic nanocarrier may be formulated for use as a vaccine.
  • the synthetic nanocarrier may encapsulate at least one polynucleotide which encode at least one antigen.
  • the synthetic nanocarrier may include at least one antigen and an excipient for a vaccine dosage form (see International Publication No. WO2011150264 and U.S. Publication No. US20110293723, the contents of each of which are herein incorporated by reference in their entirety).
  • a vaccine dosage form may include at least two synthetic nanocarriers with the same or different antigens and an excipient (see International Publication No. WO2011150249 and U.S. Publication No.
  • the vaccine dosage form may be selected by methods described herein, known in the art and/or described in International Publication No. WO2011150258 and U.S. Publication No. US20120027806, the contents of each of which are herein incorporated by reference in their entirety).
  • the synthetic nanocarrier may comprise at least one polynucleotide which encodes at least one adjuvant.
  • the adjuvant may comprise dimethyldioctadecylammonium-bromide, dimethyldioctadecylammonium-chloride, dimethyldioctadecylammonium-phosphate or dimethyldioctadecylammonium-acetate (DDA) and an apolar fraction or part of said apolar fraction of a total lipid extract of a mycobacterium (see, e.g., U.S. Pat. No.
  • the synthetic nanocarrier may comprise at least one polynucleotide and an adjuvant.
  • the synthetic nanocarrier comprising and adjuvant may be formulated by the methods described in International Publication No. WO2011150240 and U.S. Publication No. US20110293700, the contents of each of which are herein incorporated by reference in their entirety.
  • the synthetic nanocarrier may encapsulate at least one polynucleotide that encodes a peptide, fragment or region from a virus.
  • the synthetic nanocarrier may include, but is not limited to, any of the nanocarriers described in International Publication No. WO2012024621, WO201202629, WO2012024632 and U.S. Publication No. US20120064110, US20120058153 and US20120058154, the contents of each of which are herein incorporated by reference in their entirety.
  • the synthetic nanocarrier may be coupled to a polynucleotide which may be able to trigger a humoral and/or cytotoxic T lymphocyte (CTL) response (see, e.g., International Publication No. WO2013019669, the contents of which are herein incorporated by reference in their entirety).
  • CTL cytotoxic T lymphocyte
  • the RNA (e.g., mRNA) vaccine may be encapsulated in, linked to and/or associated with zwitterionic lipids.
  • zwitterionic lipids and methods of using zwitterionic lipids are described in U.S. Patent Publication No. US20130216607, the contents of which are herein incorporated by reference in their entirety.
  • the zwitterionic lipids may be used in the liposomes and lipid nanoparticles described herein.
  • the RNA (e.g., mRNA) vaccine may be formulated in colloid nanocarriers as described in U.S. Patent Publication No. US20130197100, the contents of which are herein incorporated by reference in their entirety.
  • the nanoparticle may be optimized for oral administration.
  • the nanoparticle may comprise at least one cationic biopolymer such as, but not limited to, chitosan or a derivative thereof.
  • the nanoparticle may be formulated by the methods described in U.S. Publication No. 20120282343, the contents of which are herein incorporated by reference in their entirety.
  • LNPs comprise the lipid KL52 (an amino-lipid disclosed in U.S. Application Publication No. 2012/0295832, the contents of which are herein incorporated by reference in their entirety. Activity and/or safety (as measured by examining one or more of ALT/AST, white blood cell count and cytokine induction, for example) of LNP administration may be improved by incorporation of such lipids.
  • LNPs comprising KL52 may be administered intravenously and/or in one or more doses. In some embodiments, administration of LNPs comprising KL52 results in equal or improved mRNA and/or protein expression as compared to LNPs comprising MC3.
  • RNA (e.g., mRNA) vaccine may be delivered using smaller LNPs.
  • Such particles may comprise a diameter from below 0.1 urn up to 100 nm such as, but not limited to, less than 0.1 urn, less than 1.0 urn, less than 5 urn, less than 10 urn, less than 15 urn, less than 20 urn, less than 25 urn, less than 30 urn, less than 35 urn, less than 40 urn, less than 50 urn, less than 55 urn, less than 60 urn, less than 65 urn, less than 70 urn, less than 75 urn, less than 80 urn, less than 85 urn, less than 90 urn, less than 95 urn, less than 100 urn, less than 125 urn, less than 150 urn, less than 175 urn, less than 200 urn, less than 225 urn, less than 250 urn, less than
  • RNA (e.g., mRNA RNA) vaccines may be delivered using smaller LNPs, which may comprise a diameter from about 1 nm to about 100 nm, from about 1 nm to about 10 nm, about 1 nm to about 20 nm, from about 1 nm to about 30 nm, from about 1 nm to about 40 nm, from about 1 nm to about 50 nm, from about 1 nm to about 60 nm, from about 1 nm to about 70 nm, from about 1 nm to about 80 nm, from about 1 nm to about 90 nm, from about 5 nm to about from 100 nm, from about 5 nm to about 10 nm, about 5 nm to about 20 nm, from about 5 nm to about 30 nm, from about 5 nm to about 40 nm, from about 5 nm to about 50 nm, from about 5 nm to about
  • microfluidic mixers may include, but are not limited to, a slit interdigital micromixer including, but not limited to those manufactured by Microinnova (Allerheiligen bei Wildon, Austria) and/or a staggered herringbone micromixer (SHM) (Zhigaltsev, I. V. et al., Bottom-up design and synthesis of limit size lipid nanoparticle systems with aqueous and triglyceride cores using millisecond microfluidic mixing have been published (Langmuir. 2012. 28:3633-40; Belliveau, N. M.
  • a slit interdigital micromixer including, but not limited to those manufactured by Microinnova (Allerheiligen bei Wildon, Austria) and/or a staggered herringbone micromixer (SHM) (Zhigaltsev, I. V. et al., Bottom-up design and synthesis of limit size lipid nanoparticle systems with aqueous and trigly
  • methods of LNP generation comprising SHM, further comprise the mixing of at least two input streams wherein mixing occurs by microstructure-induced chaotic advection (MICA).
  • MICA microstructure-induced chaotic advection
  • fluid streams flow through channels present in a herringbone pattern causing rotational flow and folding the fluids around each other.
  • This method may also comprise a surface for fluid mixing wherein the surface changes orientations during fluid cycling.
  • Methods of generating LNPs using SHM include those disclosed in U.S. Application Publication Nos. 2004/0262223 and 2012/0276209, the contents of each of which are herein incorporated by reference in their entirety.
  • the RNA (e.g., mRNA) vaccine of the present disclosure may be formulated in lipid nanoparticles created using a micromixer such as, but not limited to, a Slit Interdigital Microstructured Mixer (SIMM-V2) or a Standard Slit Interdigital Micro Mixer (SSIMM) or Caterpillar (CPMM) or Impinging-jet (IJMM) from the Institut fiir Mikrotechnik Mainz GmbH, Mainz Germany).
  • a micromixer such as, but not limited to, a Slit Interdigital Microstructured Mixer (SIMM-V2) or a Standard Slit Interdigital Micro Mixer (SSIMM) or Caterpillar (CPMM) or Impinging-jet (IJMM) from the Institut fiir Mikrotechnik Mainz GmbH, Mainz Germany).
  • the RNA (e.g., mRNA) vaccines of the present disclosure may be formulated in lipid nanoparticles created using microfluidic technology (see, e.g., Whitesides, George M. The Origins and the Future of Microfluidics. Nature, 2006 442: 368-373; and Abraham et al. Chaotic Mixer for Microchannels. Science, 2002 295: 647-651 ; each of which is herein incorporated by reference in its entirety).
  • controlled microfluidic formulation includes a passive method for mixing streams of steady pressure-driven flows in micro channels at a low Reynolds number (see, e.g., Abraham et al. Chaotic Mixer for Microchannels. Science, 2002 295: 647-651 , the contents of which are herein incorporated by reference in their entirety).
  • a lipid-based formulation including any LNP disclosed herein may further comprise one or more adjuvants.
  • 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), PeerJ, 10:e13083; incorporated herein by reference.
  • the RNA (e.g., mRNA) vaccines of the present disclosure may be formulated in lipid nanoparticles created using a micromixer chip such as, but not limited to, those from Harvard Apparatus (Holliston, Mass.) or Dolomite Microfluidics (Royston, UK).
  • a micromixer chip can be used for rapid mixing of two or more fluid streams with a split and recombine mechanism.
  • the RNA (e.g., mRNA) vaccines of the disclosure may be formulated for delivery using the drug encapsulating microspheres described in International Patent Publication No. WO2013063468 or U.S. Pat. No. 8,440,614, the contents of each of which are herein incorporated by reference in their entirety.
  • the microspheres may comprise a compound of the formula (I), (II), (III), (IV), (V) or (VI) as described in International Patent Publication No. WO2013063468, the contents of which are herein incorporated by reference in their entirety.
  • the amino acid, peptide, polypeptide, lipids are useful in delivering the RNA (e.g., RNA) vaccines of the disclosure to cells (see International Patent Publication No. WO2013063468, the contents of which are herein incorporated by reference in their entirety).
  • the antibody titre produced by the RNA vaccines of the invention is a neutralizing antibody titre.
  • the neutralizing antibody titre is greater than a protein vaccine.
  • the neutralizing antibody titre produced by the RNA vaccines of the invention is greater than an adjuvanted protein vaccine.
  • the neutralizing antibody titre produced by the RNA vaccines of the invention is 1 ,000-10,000, 1 ,200-10,000, 1 ,400-10,000, 1 ,500-10,000, 1 ,000-5,000, 1 ,000-4,000, 1 ,800-10,000, 2000-10,000, 2,000-5,000, 2,000-3,000, 2,000- 4,000, 3,000-5,000, 3,000-4,000, or 2,000-2,500.
  • a neutralization titre is typically expressed as the highest serum dilution required to achieve a 50% reduction in the number of plaques.
  • 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.
  • 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.
  • 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, -80° C, -90° C, -130° C or -150° C.).
  • 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.
  • compositions described herein may further comprise one or more cryoprotectants or cryopreservatives.
  • cryopreservative or cryoprotectant may comprise a sugar such as sucrose, glucose or related sugar-based cryoprotectant.
  • RNA (e.g., mRNA) vaccines of the disclosure can be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles.
  • pharmaceutical compositions of RNA (e.g., mRNA) vaccines include liposomes. Liposomes are artificially-prepared vesicles which may primarily be composed of a lipid bilayer and may be used as a delivery vehicle for the administration of nutrients and pharmaceutical formulations.
  • Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 and 500 nm in diameter.
  • MLV multilamellar vesicle
  • SUV small unicellular vesicle
  • LUV large unilamellar vesicle
  • Liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis.
  • Liposomes may contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations.
  • liposomes may depend on the physicochemical characteristics such as, but not limited to, the pharmaceutical formulation entrapped and the liposomal ingredients, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the entrapped substance and its potential toxicity, any additional processes involved during the application and/or delivery of the vesicles, the optimization size, polydispersity and the shelf-life of the vesicles for the intended application, and the batch-to-batch reproducibility and possibility of large-scale production of safe and efficient liposomal products.
  • 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.).
  • DOXIL® from Janssen Biotech, Inc.
  • 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. 19996:271-281 ; Zhang et al. Gene Therapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372; Morrissey et al., Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al., Nature.
  • 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
  • a liposome can contain, but is not limited to, 55% cholesterol, 20% disteroylphosphatidyl choline (DSPC), 10% PEG-S-DSG, and 15% 1 ,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), as described by Jeffs et al.
  • DSPC disteroylphosphatidyl choline
  • PEG-S-DSG 10%
  • DODMA 1,2-dioleyloxy-N,N-dimethylaminopropane
  • certain liposome formulations may contain, but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, where the cationic lipid can be 1 ,2-distearloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or 1 ,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA), as described by Heyes et al.
  • DSDMA 1,2-distearloxy-N,N-dimethylaminopropane
  • DODMA DODMA
  • DLin-DMA 1,2-dilinolenyloxy-3-dimethylaminopropane
  • 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.
  • 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%.
  • formulations may comprise from about 5.0% to about 10.0% DSPC and/or from about 7.0% to about 15.0% DSPC.
  • the RNA (e.g., mRNA) vaccine pharmaceutical compositions may be formulated in liposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell, Wash.), neutral DOPC (1 ,2-dioleoyl-sn-glycero-3-phosphocholine) based liposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. Cancer Biology & Therapy 2006 5(12)1708- 1713); herein incorporated by reference in its entirety) and hyaluronan-coated liposomes (Quiet Therapeutics, Israel).
  • liposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell, Wash.), neutral DOPC (1 ,2-dioleoyl-sn-glycero
  • the cationic lipid may be a low molecular weight cationic lipid such as those described in U.S. Patent Application No. 20130090372, the contents of which are herein incorporated by reference in their entirety.
  • the RNA (e.g., mRNA) vaccines may be formulated in a lipid vesicle, which may have crosslinks between functionalized lipid bilayers.
  • 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. 20120178702, herein incorporated by reference in its entirety.
  • the polycation may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyornithine and/or polyarginine.
  • 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).
  • a non-cationic lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).
  • DOPE dioleoyl phosphatidylethanolamine
  • the present invention also provides uses of the polynucleotides and compositions of the invention for producing an antigen-specific immune response in a subject.
  • Such methods typically comprise administering a polynucleotide of the invention, preferably formulated in a lipid nanoparticle as described herein, to a subject in need thereof.
  • the invention further provides compositions comprising the polynucleotides (RNA) defined herein, and the use of such RNA in immunogenic or vaccine compositions in the treatment or prevention of P. gulae infection.
  • RNA polynucleotides
  • 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.
  • the terms “immunostimulating composition”, “vaccine composition” and “immunogenic composition” may generally be used interchangeably.
  • the present invention provides methods and compositions for treating or preventing infection or minimising the likelihood of infection with P. gulae, in an individual in need thereof, the methods comprising administering a vaccine composition of the invention.
  • the present invention includes methods and compositions for preventing infection with P. gulae, minimising the likelihood of infection and/or reducing the severity and duration of P. gulae infection in a subject.
  • the present invention also provides a method for obtaining an antibody directed to P. gulae, the method comprising administering a chimeric or fusion protein, composition, vaccine or immune stimulating composition of the invention, to a non-human animal, thereby generating antibodies directed to P. gulae in the animal.
  • the method further comprises isolating the antibody from the animal (eg from the blood of the animal) or from an egg of the animal (eg in the case of generating IgY antibodies from chickens).
  • the present invention also provides an antibody preparation comprising an antibody directed to P. gulae, wherein the antibody preparation is obtained by administering a composition, vaccine or immune stimulating composition of the invention, to a non-human animal, thereby generating antibodies directed to P. gulae in the animal, and isolating the antibodies from the animal or egg thereof.
  • the antibody directed to P. gulae may be used therapeutically to eliminate or reduce P. gulae infection or prophylactically, to prevent or reduce the severity of P. gulae infection.
  • treatment 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, 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.
  • 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.
  • 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.
  • the vaccine compositions of the invention can be administered to subjects felt to be in greatest need thereof, for example in the context of human patients, to children or the elderly or individuals at risk of exposure to P. gulae.
  • the vaccine compositions of the invention can also be administered to subjects suspected of having or diagnosed with having infection with P. gulae.
  • compositions and methods of the present invention extend equally to uses in both human and/or veterinary medicine, generation of diagnostic agents or the generation of other treatment reagents.
  • the term “subject” shall be taken to mean any animal including humans, for example a mammal, and in particular, non-human animals.
  • the subject may be a veterinary subject, such as a companion animal (cat, dog, guinea pig, and the like).
  • the skilled person will be familiar with methods for determining successful vaccination/immunisation with a chimeric or fusion protein or composition as described herein. For example, the skilled person will be familiar with methods for quantifying the antibodies generated following immunisation and/or for quantifying the extent of the humoural (Th2) response induced following immunisation or for quantifying the extent of a Th1 response generated.
  • the subject following administration of a polynucleotide or composition of the invention, the subject exhibits a seroconversion rate of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) following the first dose or the second (booster) dose of the vaccine.
  • Seroconversion is the time period during which a specific antibody develops and becomes detectable in the blood.
  • antigens enter the blood, and the immune system begins to produce antibodies in response.
  • the antigen itself may or may not be detectable, but antibodies are considered absent.
  • antibodies are present but not yet detectable. Any time after seroconversion, the antibodies can be detected in the blood, indicating a prior or current infection.
  • a polynucleotide (e.g., RNA) 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.
  • RNA e.g., mRNA
  • Antigen-specific immune responses in a subject may be determined, in some embodiments, by assaying for antibody titre following administration to the subject of any of the polynucleotide (e.g., mRNA) vaccines of the present disclosure.
  • the anti-antigenic polypeptide antibody titre produced in the subject is increased by at least 1 log relative to a control. In some embodiments, the anti-antigenic polypeptide antibody titre produced in the subject is increased by 1-3 log relative to a control.
  • the anti-antigenic polypeptide antibody titre produced in a subject is increased at least 2 times relative to a control. In some embodiments, the anti-antigenic polypeptide antibody titre produced in the subject is increased at least 5 times relative to a control. In some embodiments, the anti-antigenic polypeptide antibody titre produced in the subject is increased at least 10 times relative to a control. In some embodiments, the anti-antigenic polypeptide antibody titre produced in the subject is increased 2-10 times relative to a control.
  • the control is an anti-antigenic polypeptide antibody titre produced in a subject who has not been administered a RNA (e.g., mRNA) vaccine of the present disclosure.
  • the control is an anti-antigenic polypeptide antibody titre produced in a subject who has been administered a live attenuated or inactivated P. gulae vaccine (see, e.g., Ren J. et al. J of Gen. Virol. 2015; 96: 1515-1520), or wherein the control is an anti-antigenic polypeptide antibody titre produced in a subject who has been administered a recombinant or purified P. gulae protein vaccine.
  • a polynucleotide (e.g., mRNA) vaccine of the present disclosure is administered to a subject in an effective amount (an amount effective to induce an immune response).
  • the effective amount is a dose equivalent to an at least 2-fold, at least 4-fold, at least 10-fold, at least 100-fold, at least 1000-fold reduction in the standard of care dose of a recombinant P. gulae protein vaccine, wherein the anti-antigenic polypeptide antibody titre produced in the subject is equivalent to an anti-antigenic polypeptide antibody titre produced in a control subject administered the standard of care dose of a recombinant P. gulae protein vaccine, a purified P. gulae protein vaccine, a live attenuated P.
  • the effective amount is a dose equivalent to 2-1000-fold reduction in the standard of care dose of a recombinant P. gulae protein vaccine, wherein the anti-antigenic polypeptide antibody titre produced in the subject is equivalent to an anti-antigenic polypeptide antibody titre produced in a control subject administered the standard of care dose of a recombinant P. gulae protein vaccine, a purified P. gulae protein vaccine, a live attenuated P. gulae vaccine, or an inactivated P. gulae vaccine.
  • the polynucleotide (e.g., mRNA) vaccine is formulated in an effective amount to produce an antigen specific immune response in a subject.
  • Dosages of the vaccine composition of the present invention 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.
  • 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.
  • the effective amount is a total dose of 25 pg to 1000 pg, or 50 pg to 1000 pg. In some embodiments, the effective amount is a total dose of 100 pg. In some embodiments, the effective amount is a dose of 25 pg administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 100 pg administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 400 pg administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 500 pg administered to the subject a total of two times.
  • the efficacy (or effectiveness) of a polynucleotide (e.g., mRNA) vaccine is greater than 60%. In some embodiments, the efficacy (or effectiveness) of a polynucleotide (e.g., mRNA) vaccine is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%.
  • Vaccine efficacy may be assessed using standard analyses (see, e.g., Weinberg et al., J Infect Dis. 2010 Jun. 1 ; 201 (11):1607-10). For example, vaccine efficacy may be measured by double-blind, randomized, clinical controlled trials. Vaccine efficacy may be expressed as a proportionate reduction in disease attack rate (AR) between the unvaccinated (ARU) and vaccinated (ARV) study cohorts and can be calculated from the relative risk (RR) of disease among the vaccinated group with use of the following formulas:
  • AR disease attack rate
  • vaccine effectiveness may be assessed using standard analyses (see, e.g., Weinberg et al., J Infect Dis. 2010 Jun. 1 ; 201(11):1607-10).
  • Vaccine effectiveness is an assessment of how a vaccine (which may have already proven to have high vaccine efficacy) reduces disease in a population. This measure can assess the net balance of benefits and adverse effects of a vaccination program, not just the vaccine itself, under natural field conditions rather than in a controlled clinical trial.
  • Vaccine effectiveness is proportional to vaccine efficacy (potency) but is also affected by how well target groups in the population are immunized, as well as by other non-vaccine-related factors that influence the ‘real-world’ outcomes of hospitalizations, ambulatory visits, or costs.
  • a retrospective case control analysis may be used, in which the rates of vaccination among a set of infected cases and appropriate controls are compared.
  • the invention is a composition for or method of vaccinating a subject comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide wherein a dosage of between 10 pg/kg and 400 pg /kg of the nucleic acid vaccine is administered to the subject.
  • the dosage of the RNA polynucleotide is 1-5 pg, 5-10 pg, 10-15 pg, 15-20 pg, 10-25 pg, 20-25 pg, 20- 50 pg, 30-50 pg, 40-50 pg, 40-60 pg, 60-80 pg, 60-100 pg, 50-100 pg, 80-120 pg, 40-120 pg, 40-150 pg, 50-150 pg, 50-200 pg, 80-200 pg, 100-200 pg, 120-250 pg, 150-250 pg, 180-280 pg, 200-300 pg, 50-300 pg, 80-300 pg, 100-300 pg, 40-300 pg, 50-350 pg, 100- 350 pg, 200-350 pg, 300-350 pg, 320-400 pg, 40-380 pg, 40-100 pg, 100-
  • the nucleic acid vaccine is administered to the subject by intradermal or intramuscular injection. In some embodiments, the nucleic acid vaccine is administered to the subject on day zero. In some embodiments, a second dose of the nucleic acid vaccine is administered to the subject on day twenty one.
  • a dosage of at least about 2 micrograms (pg) or at least about 10 pg or at least about 20 pg or at least about 30 pg of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 100 micrograms (pg) of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 50 micrograms (pg) of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject.
  • a dosage of 75 micrograms (pg) of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 150 micrograms (pg) of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 400 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 200 micrograms (pg) of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, the RNA polynucleotide accumulates at a 100 fold higher level in the local lymph node in comparison with the distal lymph node.
  • Embodiments of the invention provide methods of creating, maintaining or restoring antigenic memory to a P. gulae in an individual or population of individuals comprising administering to said individual or population an antigenic memory booster nucleic acid vaccine comprising (a) at least one RNA polynucleotide, said polynucleotide comprising at least one chemical modification or optionally no nucleotide modification and two or more codon-optimized open reading frames, said open reading frames encoding a set of reference antigenic polypeptides, and (b) optionally a pharmaceutically acceptable carrier or excipient.
  • the vaccine is administered to the individual via a route selected from the group consisting of intramuscular administration, intradermal administration and subcutaneous administration.
  • the administering step comprises contacting a muscle tissue of the subject with a device suitable for injection of the composition. In some embodiments, the administering step comprises contacting a muscle tissue of the subject with a device suitable for injection of the composition in combination with electroporation.
  • Embodiments of the invention provide methods of vaccinating a subject comprising administering to the subject a single dosage of between 25 pg/kg and 400 pg/kg of a nucleic acid vaccine comprising one or more RNA polynucleotides as described herein, in an effective amount to vaccinate the subject.
  • the invention encompasses a method of treating an elderly subject age 60 years or older comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding an antigenic polypeptide as described herein in an effective amount to vaccinate the subject.
  • the invention encompasses a method of treating a young subject age 17 years or younger comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding an antigenic polypeptide as described hereinin an effective amount to vaccinate the subject.
  • the invention encompasses a method of treating an adult subject comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding an antigenic polypeptide as described herein in an effective amount to vaccinate the subject.
  • vaccines of the invention e.g., LNP-encapsulated RNA vaccines
  • antibody titre refers to the amount of antigen-specific antibody produces in s subject, e.g., a human subject.
  • antibody titre is expressed as the inverse of the greatest dilution (in a serial dilution) that still gives a positive result.
  • antibody titre is determined or measured by enzyme-linked immunosorbent assay (ELISA).
  • antibody titre is determined or measured by neutralization assay, e.g., by microneutralization assay.
  • antibody titre measurement is expressed as a ratio, such as 1 :40, 1 :100, etc.
  • an efficacious vaccine produces an antibody titre of greater than 1 :40, greater that 1 : 100, greater than 1 :400, greater than 1 :1000, greater than 1 :2000, greater than 1 :3000, greater than 1 :4000, greater than 1 :500, greater than 1 :6000, greater than 1 :7500, greater than 1 :10000.
  • the antibody titre is produced or reached by 10 days following vaccination, by 20 days following vaccination, by 30 days following vaccination, by 40 days following vaccination, or by 50 or more days following vaccination.
  • the titre is produced or reached following a single dose of vaccine administered to the subject.
  • the titre is produced or reached following multiple doses, e.g., following a first and a second dose (e.g., a booster dose.)
  • antigen-specific antibodies are measured in units of pg /ml or are measured in units of I U/L (International Units per liter) or mIU/ml (milli International Units per ml).
  • an efficacious vaccine produces >0.5 pg/ml, >0.1 pg /ml, >0.2 pg /ml, >0.35 pg /ml, >0.5 pg /ml, >1 pg /ml, >2 pg /ml, >5 pg /ml or >10 pg /ml.
  • an efficacious vaccine produces >10 mIU/ml, >20 mIU/ml, >50 mIU/ml, >100 mIU/ml, >200 mIU/ml, >500 mIU/ml or >1000 mIU/ml.
  • the antibody level or concentration is produced or reached by 10 days following vaccination, by 20 days following vaccination, by 30 days following vaccination, by 40 days following vaccination, or by 50 or more days following vaccination.
  • the level or concentration is produced or reached following a single dose of vaccine administered to the subject.
  • the level or concentration is produced or reached following multiple doses, e.g., following a first and a second dose (e.g., a booster dose.)
  • antibody level or concentration is determined or measured by enzyme- linked immunosorbent assay (ELISA).
  • antibody level or concentration is determined or measured by neutralization assay, e.g., by microneutralization assay.
  • Example 1 describes in vitro materials and methods.
  • Example 2 describes the results of in vitro studies.
  • Examples 3 describes materials and methods for in vivo mouse studies and
  • Example 4 describes the results of those in vivo studies.
  • Example 5 describes materials and methods for canine serology studies and
  • Example 6 describes the results of those studies.
  • Examples 7 to 11 describe generation and use of RNA vaccines encoding such chimeric or fusion proteins.
  • Example 1 In vitro materials and methods
  • DNA fragments encoding KDFAK-2S-AVQP (DUF2436 F variant; SEQ ID NO: 54) and KDAAK-2S-AVQP (DUF2436 A variant, SEQ ID NO: 52) containing Ncol and Xhol restriction sequences at the 5’ and 3’ ends were synthesized and ligated into transport vector, pBHA, by Bioneer Pacific (Aus.) to generate pBHA-gulA and pBHA-gulF. The cloned inserts were verified by DNA sequencing (Bioneer Pacific). pBHA-F and pBHA-A vectors were introduced into E.
  • coli ABLE-K (Agilent Technologies) chemically competent cells following manufacturer’s protocol and recombinant strains were selected on LB containing Amp (100 pg/mL). Plasmid DNA was purified from positive clones and digested with Ncol and Xhol and ligated to Ncol/Xhol digested pET28b plasmid vector (Novagen). Ligation products were introduced into E. coli ABLE-K chemically competent cells and recombinant strains were selected on LB containing Kanamycin (30 pg /mL).
  • Plasmid DNA from positive clones was purified, digested with Ncol/Xhol and plasmid digests were subject to agarose gel electrophoresis to verify the presence of correctly sized insert. Confirmed recombinant pET-gulA and pET-gulF variant constructs were then introduced into the E. coli expression host, BL21 (DE3) (Invitrogen) for recombinant protein expression. Select positive clones were grown in LB-Kan (30 pg/mL) to approximately mid log phase and glycerol stocks (25 % glycerol) were snap frozen for storage at -70°C.
  • the His-tagged vaccine candidates for the animal model were expressed as recombinant C-terminally His-tagged fusion proteins (SEQ ID NO: 30 and 31 , plus His tag) in E. coli BL21(DE3) as previously described.
  • the cells were grown at 37°C in LB medium or Terrific broth supplemented with 50 pg/mL kanamycin.
  • culture OD600 1.0-1.2
  • protein expression was induced with 0.2 mM IPTG at 32°C for 2-3 hours.
  • the cells were harvested at 8000 g by centrifugation at 4°C and stored at -80°C for later use.
  • the cells were lysed on ice by sonication for 15 min with 35% power output after thawed and resuspended in the lysis buffer [10 mM imidazole, 50 mM Tris, 300 mM NaCI, pH 7.5, 1x EDTA free protease inhibitor cocktail (Sigma) (for the reducing lysis buffer for the four Cys antigen-F: supplemented with 10 mM DTT)].
  • Cell lysate was clarified by centrifugation for 40 min at 25,000 g at 4°C.
  • the insoluble pellet was washed twice with buffer PBS500 (20 mM NaPi, 500 mM NaCI, pH 7.4).
  • the protein expressed as inclusion bodies was solubilised with 8 M urea at room temperature on a rolling platform for 1 hour in PBS500.
  • the protein extract was centrifuged at 20,000 g.
  • the supernatant was further filtered through a 0.22 pm filter unit and then mixed with the nickel affinity resin (Thermofisher) with gentle stirring for 2 hours in the urea containing buffer PBSLI (8 M urea, 20 mM NaPi pH 7.8, 500 mM NaCI, plus 20 mM imidazole).
  • the bound target protein was eluted off the resin with 500 mM imidazole in the same buffer.
  • the eluted protein was stepwise dialysed into 6 M, 4 M and then 2 M urea phosphate buffers in a dialysis tube with molecular weight cutoff of 3.5 kDa (Fisher Biotec, Australia).
  • Target protein in the 2 M urea buffer was buffer exchanged using a PD10 gel filtration column to remove urea before animal model experiments.
  • the clarified cell lysates were filtered through a 0.22 pm filter unit and loaded to a HisTrap or HisPrep Ni-affinity column (GE Healthcare) in the loading buffer TBS300 (50 mM Tris Ci, 300 mM NaCI, pH 7.5), plus 10 mM imidazole.
  • TBS300 50 mM Tris Ci, 300 mM NaCI, pH 7.5
  • cell lysate was diluted 4 fold with the imidazole containing TBS300 and then filtered before loaded to the column in the reducing loading buffer with inclusion of 2 mM DTT.
  • Columns were washed extensively with appropriate loading buffer and then 20 mM imidazole in TBS300 or plus 2 mM DTT for reducing conditions.
  • Bound proteins were eluted with a 20-350 mM imidazole gradient in TBS300 with the absorbance being monitored at 280 nm. Peak fractions were analysed with SDS-PAGE. Eluted target proteins were concentrated using the Amicon filter units with a 10 kDa molecular weight cut-off. The resulting protein solutions were stored on ice for further purification.
  • the proteins were eluted with a NaCI gradient from 50 to 350 mM and then from 350 to 700 mM. The absorbance at 280 nm was monitored in the process. Target proteins in the peak fractions were verified with SDS-PAGE and concentrated using the Amicon filter units. The protein solutions were stored on ice for further purification and buffer exchange using size exclusion chromatography.
  • Intact protein mass spectrometric analysis was performed on an electrospray ionisation time-of-flight mass spectrometer (ESI-TOF) coupled with liquid chromatography.
  • EI-TOF electrospray ionisation time-of-flight mass spectrometer
  • protein samples passed through a C4 HPLC column (Phenomenex) with 0.1% formic acid aqueous solution as buffer A and acetonitrile + 0.1% formic acid as buffer B.
  • Acetonitrile gradient for elution was set to be 5-60% and then 60-95% buffer B for a total of 15 minutes.
  • MS data were collected from 400 to 3200 m/z on the Agilent 6520 QTOF mass spectrometer operated in positive mode, with in-situ internal mass reference standards. Mass spectra were deconvoluted to obtain the intact protein molar masses using Agilent Mass Hunter Qualitative Analysis software (B.05) with maximum entropy algorithm.
  • SEC-MALS were run in buffer TBS150 at room temperature on a SEC-MALS system (Wyatt Technology). Species were resolved on a HPLC size exclusion column (Shim-Pack Bio Diol-300) and passed to the Wyatt 18-angle light scattering detector and Wyatt refractive index monitor. The concentrations of the samples were 2 mg/mL for the antigens and 5 mg/mL for the control protein BSA which was used to set up the method. Measurements were started following the stepwise instructions on screen of the control station with auto-injected volume set to be 10 pL for each run. Data was processed with Wyatt Technology ASTRA software for the information about sample homogeneity, protein aggregation and molar masses of the species in the solution.
  • A260/A280 ratio was determined on a NanoDrop lite UV spectrophotometer (Thermo Scientific) to assess nucleic acid contamination.
  • P. gulae was obtained from the culture collection of the Oral Health Cooperative Research Centre, The Melbourne Dental School, University of Melbourne, Australia. P. gulae was cultivated (5% CO2, 10% H2, and 85% N2) in an anaerobic chamber (Whitley MG500 anaerobic workstation) at 37°C in brain-heart infusion (BHI) broth (BD Bacto Laboratories, USA) supplemented with cysteine (1 g/L; Sigma-Aldrich, Australia), tryptic soy broth (5% w/v, BD Bacto Laboratories, USA), hemin (5 pg/mL, Calbiochem, Netherlands), and Vitamin K (10 pg/mL, Sigma-Aldrich, USA).
  • P. gulae culture was harvested (6,500 g, 4°C), washed once with phosphate buffered saline (PBS) (0.01 M Na 2 HPO 4 , 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.
  • PBS phosphate buffered saline
  • the cell pellet was resuspended in sterile PBS to obtain a cell density of 2 x 10 10 cells/mL, and protein concentration determined using Biorad Protein Assay Dye Reagent Concentrate (Life Science, NSW, Australia).
  • the chimeric protein comprising the DUF2436 variant F exhibited a high level of total recombinant protein expression in the insoluble fraction. Nevertheless, despite the relatively high level of KDFAK-2S-AVQP recombinant protein as insoluble inclusion bodies, there was still a relatively high level of soluble protein expressed in the soluble fraction due to the extremely high overall level of total recombinant protein expressed by this strain under the conditions tested.
  • KDFAK-2S-AVQP and KDAAK-2S-AVQP proteins were expressed well at a similar level in either LB or TB medium (Fig. 4).
  • antigen- A had a high solubility as evidenced by SDS-PAGE analysis of the lysis fractions (Fig. 2).
  • the KDFAK-2S-AVQP protein was much less soluble with its major portion being found in the precipitate after lysis under the same non-reducing conditions (Fig. 2).
  • reducing buffer containing either low (5 mM) or high (100 mM) concentration of DTT failed to extract antigen-F from the precipitate fraction (Fig. 2).
  • KDFAK-2S-AVQP was highly soluble with most protein being in the soluble fraction (Fig. 2).
  • the low solubility of KDFAK-2S-AVQP was likely due to the presence of disulfide bonds formed by wrongly paired Cys residues which caused the protein to have a misfolded structure. Once this abnormal structure formed, it was irreversible and its disulfide bonds were not accessible to the reductant if without assistance of unfolding force such as SDS and heating.
  • purification of KDFAK-2S-AVQP from soluble fractions under non-reducing conditions was not an ideal approach although low temperature expression may improve soluble expression levels for this protein in the beginning as seen from small scale trial.
  • the samples of this protein for the animal model experiments were prepared from lysis and purification under reducing conditions until the last step for removal of the included reductant DTT.
  • KDcAKI n was stable in 2M urea after stepwise dialysed into 2 M urea phosphate buffer and thus stored at -80°C after quantitated for further use. No precipitation was observed after buffer exchange with gel filtration before the animal model experiments.
  • KDAAK-2S-AVQP was purified under non-reducing conditions and KDFAK-2S-AVQP was purified under reducing conditions until the final buffer exchange and size exclusion purification step (Figs. 3 to 5).
  • Non-reducing conditions for the purification was also trialed for KDFAK- 2S-AVQP.
  • KDAAK-2S-AVQP expressed in LB medium was used for purification under non-reducing conditions.
  • KDFAK-2S-AVQP expressed in TB medium was used for purification under reducing conditions and the protein expressed in LB was used for nonreducing purification.
  • KDAAK-2S-AVQP had the highest yield (78 mg/L culture, 18.6 mg/g wet cells) (Table 1).
  • KDFAK-2S-AVQP also had a high yield of its final product when purified from lysis supernatant under reducing conditions (34 mg/L culture, 7.6 mg/g wet cells), however when purified under non-reducing conditions its yield was much lower (3.5 mg/L culture, 1.1 mg/g wet cells) (Table 1). Identities of both proteins were confirmed with the first Met residue missing by intact protein MS spectrometry (Table 3).
  • Table 3 Final products of P. gulae antigens purified from 1 L culture in LB or TB Broth. 2 The MWs in red were detected as minor peaks with additional 1 or 2x 183 Da to the measured target monomeric molar masses.
  • TBS buffer 50 mM Tris, 150 mM NaCI, pH 7.5.
  • KDFAK-2S-AVQP and KDAAK-2S-AVQP were concentrated up to 20 and 18 mg/mL, respectively, in TBS150 buffer and these concentrations were not the maximum that could be achieved (Table 1).
  • the majority of expressed KDFAK-2S-AVQP precipitated into the insoluble fraction from non-reducing lysis. Once it was purified under reducing conditions to a high quality, it stayed in solution without reversion to precipitation even when the reducing factor was removed. This solution stability was also resistant against freeze-thaw cycles in air. Thus, it was possible to prepare enough material from one round of purification for the animal model.
  • the host molecular chaperone protein DnaK of ⁇ 70 kDa appeared to contaminate the purification of the P. gulae antigens. Due to its interactions with target proteins, DnaK eluted into the fractions with most overlapping the target protein fractions in the Ni affinity purification step. This contamination, including other impurities, was well separated from target proteins by anion exchange chromatography (Fig. 4). The interactions between DnaK and the target proteins did not seem to be dependent on reducing conditions. Nevertheless, under reducing conditions, KDFAK-2S-AVQP eluted into two peaks possibly due to the presence of two forms (Fig. 4). Essentially, almost all the fractions in the second peak had DnaK contamination.
  • F-NR seemed to have a band shift slightly more than F-R on the SDS gel, whether the samples were heated or not (Fig. 6A). It was hypothesized that F-NR formed intramolecular disulfide bonds as early as at the stage of lysis and F-R formed the disulfide bonds only after removal of the reducing agent at the final purification stage. This difference may have a subtle impact on the protein structure. However, F-R purified from the reducing agent was found to have a similar overall structure to F-NR with minor difference in compactness.
  • SEC-MALS data showed some signs of minor or minimal multimerisation of the two antigens.
  • the species with estimated molar masses of 71.2 kDa for antigen-A and 84.7 kDa for F-R (reduction involved purification) may be assigned to the dimeric forms of the two proteins.
  • F-NR purified under non-reducing conditions
  • a peak of 394.2 kDa may be due to the presence of a small amount of decamer of this protein, although this species was not detected by SDS-PAGE.
  • Cys residues in KDFAK-2S-AVQP may have made this protein prone to form a higher level of multimerisation when it was in nonreducing conditions from the stage of lysis. Therefore, the trace amount of decamer may be intermolecular disulfide linked.
  • these mutimerised species were less than 1% of the total protein amount as estimated by integration of the SEC-MALS liquid chromatograms (Table 4).
  • Endotoxin was determined to have similarly low levels in the two antigens. At a protein concentration of 0.5 mg/mL, both proteins had endotoxin contamination at 2.24 and 2.36 EU/rnL, respectively (Table 5). In addition, the A260/A280 value was lower than 0.6 for both antigens, indicating minimal contamination with nucleic acids (Table 5). [0439] Table 5: Assessment of endotoxin and nucleic acid contamination in P. gulae antigens.
  • Example 3 materials and methods for mouse in vivo studies
  • mice female BALB/c; 6-8 weeks old, 10 mice/group
  • P. gulae consisting of four doses of P. gulae [1 x 10 10 viable P. gulae cells per dose suspended in 20 pL PG buffer (50 mM Tris-HCL, 150 mM NaCI, 10 mM MgSO 4 and 14.3 mM mercaptoethanol, pH 7.4) containing 2% w/v carboxymethylcellulose (CMC, Sigma, New South Wales, Australia)]
  • CMC carboxymethylcellulose
  • mice were immunised on day 19 after the first oral inoculation with 100 pg of vaccine candidate in saline/alum (Alhydrogel; 2% aluminium hydroxide wet gel suspension; Invivogen) via the intraperitoneal route.
  • Mice received a second immunisation (100 pg in saline/alum) on day 40 via the subcutaneous route.
  • mice were bled by cardiac puncture and killed. Maxillae were removed and halved through the midline, with 10 halves used to determine alveolar bone loss.
  • Coded digital images of the buccal aspect of the maxillae were captured with an Olympus DP12 digital camera mounted on a dissecting microscope, using Imaged imaging software for analysis (https://imagej.nih.gov/ij/index.html) to assess horizontal bone loss. Maxillae were oriented so that the buccal and lingual molar cusps were superimposed. Images were captured with a micrometre in frame, so that measurements could be normalised for each image. Horizontal bone loss was defined as the loss occurring in a horizontal plane, perpendicular to the alveolar bone crest that resulted in a reduction of the crest height.
  • the visible area from the cemento-enamel junction (CEJ) to the alveolar bone crest (ABC) for each molar was measured using Imaged version 1.3k imaging software to give the total visible CEd-ABC area in mm 2 .
  • P. gu/ae-induced alveolar bone loss in mm2 was calculated by subtracting the total visible CEd-ABC area of the uninoculated (N-C) group from the total visible CEd-ABC area of each experimental group.
  • Alveolar bone loss measurements were determined twice in a random and blinded protocol. Data are expressed as the mean +/- standard deviation in mm 2 and were analysed using a oneway ANOVA and Dunnetts T3 post-hoc test.
  • ELISAs were performed to evaluate subclass antibody in sera using a solution (1 pg/mL) heat-killed (HK) P. gulae cells in 0.1 M PBS (pH 7.4) to coat wells (16 h, 4 °C) of flat-bottom polyvinyl microtitre plates (Microtitre; Dynatech Laboratories, McLean, VA, US).
  • 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. All optical density measurements were conducted on a Wallac VICTORS 1420 Multilabel counter (Perkin Elmer) at 405nm.
  • the animal model used was the therapeutic vaccine mouse periodontitis model (Fig. 7) developed by O’Brien-Simpson et al (2016, supra). Compared to the naive control, the infected control animals developed significant levels (P ⁇ 0.001) of alveolar bone loss (Fig. 8).
  • a P. gingivalis chimeric protein known in the prior art (KDcAKIn) was used as a positive vaccine control in this study. New vaccines based on the P. gulae virulence domain sequence, KDFAK-2S-AVQP and KDAAK-2S-AVQP provided significant levels of protection compared to the infected control animals (P ⁇ 0.01) (Fig. 8).
  • KDFAK-2S-AVQP and KDAAK-2S-AVQP proteins generated significant antibodies against the RgpA/Kgp protease complex, with KDFAK-2S-AVQP generating higher titres against the RgpA/Kgp protease complex.
  • Fig. 11 shows that the two P. gulae vaccines generated high titre antibody responses against themselves.
  • pre-immune serum aka pre-treatment, i.e., immediately prior to receiving the prime dose.
  • - day 21 primed serum, aka pre-second treatment, i.e., 21 days after receiving the prime dose and immediately prior to receiving the booster).
  • ELISA plate wells were coated with 100 pL/well of Porphyromonas gulae KDAAK- 2S-avqp-6His recombinant protein at concentrations of 10, 2.5, 1 , 0.5 and 0.1 pg/mL (dilutions made in 1xPBS). A PBS-only control was also included. After overnight incubation at 4 °C, the recombinant protein solution was discarded, wells were washed twice with distilled water, and free protein binding sites were blocked by the addition of 200 pL/well of Block Solution. After incubation at 4 °C for 6 hours, the Block Solution was discarded, wells were rinsed twice with Wash Solution, and serum dilutions were applied as described below.
  • ELISA plate wells were coated with 100 pL/well of 10 pg/mL streptavidin solution (dilution made in 1 xPBS). After overnight incubation at 4 °C, the streptavidin solution was discarded, wells were washed twice with distilled water, and free protein binding sites were blocked by the addition of 200 pL/well of Block Solution (10% (w/v) non-fat milk powder in 1 xPBS). After incubation at 4 °C for 6 hours, the Block Solution was discarded, wells were rinsed twice with Wash Solution, and aliquots of 100 pL of biotinylated synthetic peptides (Pgul_KAS2 (SEQ ID NO: 1) and P.
  • Pgul_KAS2 biotinylated synthetic peptides
  • gul_KAS2_scrambled Biotin- KYKGWTNNSSTVLLQTNATLGVETFTHSPDSASDAK, SEQ ID NO: 127) prepared in Dilution Buffer at 10, 2.5, 1 , 0.5 and 0.1 pg/mL were added to the blocked streptavidin- coated wells. A Dilution Buffer-only control was also included. After overnight incubation at 4 °C, the peptide solutions were discarded, wells were washed six times with Wash Solution, and serum dilutions applied as described below.
  • ELISA plate wells were coated with 100 pL/well of whole heat-killed Porphyromonas gulae cells at concentrations of 10, 2.5, 1 , 0.5 and 0.1 pg/mL (dilutions made in 1 xPBS). A PBS-only control was also included. After overnight incubation at 4 °C, the P. gulae cells were discarded, wells were washed twice with distilled water, and free protein binding sites were blocked by the addition of 200 pL/well of Block Solution. After incubation at 4 °C for 6 hours, the Block Solution was discarded, wells were rinsed twice with Wash Solution, and serum dilutions were applied as described below.
  • Dilution Buffer Dilution Buffer. Similarly, the day 35 serum samples were pooled and diluted 1/10 in Dilution Buffer. Aliquots (100 pL) of the 1/10 diluted serum samples were added to the blocked ELISA plates and 4-fold dilution series of the 1/10 serum samples were constructed by serially transferring 25 pL into 75 pL of Dilution Buffer. Control wells that received only Dilution Buffer (i.e. , no serum) were also included. Plates were incubated overnight at 4 °C and detection antibody was applied as described belpw.
  • TMB Substrate Buffer Solution (90 pL) was added to the washed ELISA plates.
  • colour development was followed spectrophotometrical ly at 370 nm in kinetic mode using a SpectraMax i D5 spectrophotometer.
  • titreing of individual sera the colour was allowed to develop for 20 minutes, the reaction stopped by the addition of 50 pL of 1 M H2SO4 to each well, and the plate read in stopped ELISA mode (at 450 nm) in the SpectraMax i D5 spectrophotometer. Data was analysed as described below.
  • ELISA plate wells were coated with 100 pL/well of 0.1 pg/mL Porphyromonas gulae KDAAK-2S-avqp-6His recombinant protein (dilution made in 1 xPBS). After overnight incubation at 4 °C, the recombinant protein solution was discarded, the wells were washed twice with distilled water and free protein binding sites were blocked by the addition of 200 pL/well of Block Solution. After incubation at 4 °C for 6 hours, the Block Solution was discarded, and the wells were rinsed twice with Wash Solution.
  • ELISA plate wells were coated with 100 pL/well of 10 pg/mL streptavidin solution (dilution made in 1 xPBS). After overnight incubation at 4 °C, the streptavidin solution was discarded, the wells were washed twice with distilled water and free protein binding sites were blocked by the addition of 200 pL/well of Block Solution. After incubation at 4 °C for 6 hours, the Block Solution was discarded, wells were rinsed twice with Wash Solution, and 100 pL aliquots of 0.1 pg/mL biotinylated Pgul_KAS2 peptide (dilution made in Dilution Buffer) were added to each well of the streptavidin-coated plates.
  • ELISA plate wells were coated with 100 pL/well of 1 pg/mL whole heat-killed Porphyromonas gulae cells (dilution made in 1 xPBS). After overnight incubation at 4 °C, the P. gulae cells were discarded, the wells were washed twice with distilled water and free protein binding sites were blocked by the addition of 200 pL/well of Block Solution. After incubation at 4 °C for 6 hours, the Block Solution was discarded, and wells were rinsed twice with Wash Solution.
  • ELISA assays based on peroxi dase-TMB systems can be monitored in two ways; continuously recording absorbance at 370 nm; or acid-stopping the reaction following a set time and measuring absorbance at 450 nm. Continuous assays tend to be more accurate with a wider dynamic range than stopped assays but become impractical with multiple plates. Both assay types were deployed during this trial.
  • Equation 1 Equation 1 :
  • x concentration of serum
  • f(x) absorbance response
  • a maximum response
  • b minimum response
  • c slope (Hill coefficient)
  • d Inflection point (midpoint)
  • Example 6 results from initial canine serology studies
  • Figures 13 to 15 show preliminary results from the experimental protocol outlined in Example 5. Briefly, following immunisation, sera were collected at the time points indicated and ELISA was used to determine immune response to the immunising antigen (KDAAK-2S-avqp-6His), P. gulae KAS2 peptide or P. gulae whole cells. [0486] As shown in Figure 13, all dogs showed a robust immune response to antigen following prime and boost immunisation.
  • a further canine study similar to the one discussed at Examples 5 and 6, was conducted. Briefly, 20 dogs were organised into “treatment” or “control” groups (10 dogs in each group). Dogs in the treatment group were immunised 3 times with chimeric protein antigen KDAAK-2S-avqp as herein described, beginning on day 0 (prime), followed by a booster immunisation on day 21 (boost 1) and again on day 42 (boost 2). The control group received no treatment.
  • Serum IgG responses against the P. gulae KAS2 peptide were measured using ELISA, similarly to the methods described in Example 5.
  • Example 8 generation of RNA-LNPs for delivery of chimeric or fusion proteins
  • RNAs are 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 are capped co-transcriptionally using Clean Cap RNA capping technology (TriLink Biotech).
  • RNA vaccine candidates are replaced by N1-methyl pseudouridine (N1-methyl pseudo- UTP, m1Y) during RNA production.
  • DNA is removed using DNAse I (NEB), and doublestranded RNA (dsRNA) is removed using cellulose binding using standard techniques 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).
  • LNPs lipid nanoparticles
  • HeLa cells are cultured in culture media DMEM, high glucose, GlutaMAXTM Supplement, pyruvate (ThermoFisher, CAT#10569010) with 10% FBS at 37°C, 5% CO2, according to standard protocols.
  • RNA candidates (prior to LNP formulation), are formulated with Lipofectamine MessengerMAX (IThermoFisher) and used to transfect cells according to manufacturer’s instructions. 1.8 pg of an RNA is used to transfect 35,000 cells/well on a six well plate. For supernatant collection, cells are pelleted by centrifuging at 14,000 g for 15 mins. Supernatants are transferred to a fresh tube and frozen at -20°C until Western Blotting.
  • cells are rinsed in each well with ⁇ 2 mL DPBS.
  • the liquid is removed and 250 pL of RIPA lysis buffer with protease inhibitors (ThermoFisher) is added to each well, and gently swirled to mix for 10s.
  • ThermoFisher the lysates are transferred into tubes and centrifuged at 4°C at 16,000 g for 15 mins to pellet debris.
  • the clarified lysates are collected into new tubes and stored at -20°C until Western Blotting to confirm expression of protein.
  • P. gulae is grown on Horse Blood Agar (HBA) (20 g/L HBA; Oxoid Ltd., Hampshire, UK) supplemented with 10% v/v lysed horse blood (37°C) in an anaerobic N2 atmosphere containing 5% CO2 in a MK3 Anaerobic Workstation (Don Whitley Scientific Ltd., Sydney, Australia). Colonies are inoculated into starter culture comprised of 20 mL sterilised brain heart infusion (37 g/L BHI; Oxoid Ltd., Hamsphire, UK) medium supplemented with 5 mg/L hemin and 0.5 mg/L cysteine and incubated anaerobically (24 h, 37 °C).
  • HBA Horse Blood Agar
  • BHI sterilised brain heart infusion
  • Oxoid Ltd., Hamsphire, UK medium supplemented with 5 mg/L hemin and 0.5 mg/L cysteine and incubated anaerobically (24 h,
  • P. gulae are 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 is resuspended in PBS and heated to 65°C for 15 minutes. The suspension is centrifuged (7,000 g, 20 min 4°C) and resuspended in sterile PBS and this is repeated once.
  • PBS phosphate buffered saline
  • mice periodontitis experiments to assess protection by the vaccines and immune responses generated are performed as described herein, including methods for assessing bone loss from maxille.
  • Example 9 Expression of RNA constructs encoding chimeric proteins
  • RNA-LNPs are tested for in vitro antigen expression and secretion in HeLa cells.
  • HeLa cells are transfected with the RNA constructs using lipofectants as outlined above. After 48 hours, supernatants are collected and subjected to Western Blot analysis to test for secreted polypeptide. The results will indicate that the RNAs, particularly those encoding chimeric or fusion proteins defined in Table 1 herein, express well and are secreted.
  • Example 10 determining immunogenicity of candidate RNA vaccines
  • Figure 18 shows a schematic of the proposed vaccination protocol for use to assess the immunogenicity of candidate RNA vaccines.
  • RNA constructs as described above and encoding chimeric or fusion proteins defined in Table 1 are formulated into lipid nanoparticles (LNP) using standard techniques.
  • the LNPs used in these experiments comprises: ALC-0315, cholesterol, distearoylphosphatidylcholine and ALC-0159 in mole percent (%) ratio of: 46.3: 42.7: 9.4: 1.6.
  • RNA constructs are generated using either native RNA sequence (unmodified) or N1-methyl-pseudouridine modified (M1 ip) sequence.
  • RNA vaccine Two doses of RNA vaccine are tested: 30 pg or 3 pg of RNA formulated in LNP.
  • the positive control used in experiments is the protein KDAK-3S-AVQP adjuvanted with alum.
  • mice are intramuscularly immunised with RNA-LNPs according to the schedule shown in Figure 18.
  • Example 11 determining in vivo efficacy of RNA vaccines in periodontitis model
  • Figure 19 shows a schematic of the proposed vaccination protocol for determining in vivo efficacy of candidate RNA vaccines (as assessed by protection from alveolar bone loss), and immunogenicity (as determined by antibody levels in sera).
  • RNA vaccines are prepared and formulated as outlined above.
  • Example 12 truncated antigens encoded by alternative RNA constructs
  • RNA constructs are assessed, each expressing truncated variations of the protein antigens encoded by the RNAs tested in the above examples. All RNAs are ml ip-modified.
  • adhesin domain comprises ABMs 2 and 1 but not ABM3; amino acid sequence SEQ ID NO: 38 or 40
  • RNAs encoding the target antigens are tested for in vitro expression and secretion in HeLa cells by Western Blot.

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Abstract

The present invention relates to RNA-containing vaccine compositions for inducing an immune response to Porphyromonas gulae in a subject, and uses thereof.

Description

RNA vaccines for use in animal health
Field of the invention
[0001] The present invention relates to RNA-containing vaccine compositions for inducing an immune response to Porphyromonas gulae in a subject, and uses thereof.
Related application
[0002] The present application claims priority from Australian provisional application AU 2023902376, 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 of the Porphyromonas sp. 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] Periodontitis is an inflammatory disease of the supporting tissues of the teeth associated with a dysbiotic subgingival plaque which results in destruction of those tissues and loss of tooth attachment in humans and in companion animals. More than 80% of dogs show signs of periodontitis by age three and 70% of cats by the same age. Consequently there is a significant disease burden from periodontitis in companion animal populations.
[0005] The predominant periodontal pathogen in companion animals, particularly dogs, is Porphyromonas gulae (P. gulae).
[0006] There is currently no commercially approved treatment for use in preventing or reducing the incidence and/or severity of P. gulae infection or for treating P. gulae infection and disease in companion animals.
[0007] There is therefore a need for new and/or improved approaches for the design and manufacture of agents for treating, preventing or reducing severity of P. gulae infection. [0008] 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
[0009] In a first aspect, the invention 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 homologue of P. gulae, 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 found in surface complexes of the Arg- and Lys-gingipain homologues of P. gulae, or a sequence that is at least 80% identical thereto. wherein the polynucleotide is capable of being translated in a mammalian cell.
[0010] In any embodiment, the protein encoded by the RNA polynucleotide may further comprise:
- the amino acid sequence of a DUF2436 domain of the Arg- and Lys-gingipain homologue surface complexes of P. gulae, or a sequence that is at least 80% identical thereto.
[0011] Optionally, the protein encoded by the RNA may comprise the afore-mentioned domains in any order: for example: the one or more amino acid sequences of an active site of an Arg- or Lys-gingipain homologue of P. gulae may be located N-terminally to the amino acid sequence of one or more adhesin binding motifs (ABMs) and/or the amino acid sequence of a DUF2436 domain; or the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain may be located N-terminally to the one or more amino acid sequences of an active site of an Arg- or Lys-gingipain homologue of P. gulae and/or the amino acid sequence of a DUF2436 domain, 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. (In other words, the protein may comprises 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).
[0012] 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.
[0013] 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.
[0014] In any embodiment, the RNA polynucleotide may be a synthetic RNA molecule.
[0015] In any embodiment, the RNA polynucleotide may be a circular RNA (circRNA) molecule.
[0016] In any embodiment, the RNA polynucleotide may be a complementary RNA (cRNA) molecule.
[0017] In any embodiment, the RNA polynucleotide made be a self-amplifying RA (saRNA) molecule or trans-amplifying (taRNA) molecule.
[0018] 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.
[0019] Preferably, the RNA further encodes 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 of the mammalian cell and secreted therefrom. N-terminal signal peptides are known to the skilled person and are further described herein. [0020] 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.
[0021] 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.
[0022] 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%.
[0023] In preferred embodiments, the uridines in the polynucleotide are replaced with a chemical modification such as N-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 N-methyl- pseudouridine.
[0024] 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.
[0025] In any embodiment, 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 homologue of P. gulae, 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 found in surface complexes of the Arg- and Lys-gingipain homologues of P. gulae, or a sequence that is at least 80% identical thereto. [0026] It will be appreciated that the chimeric or fusion protein encoded by the RNA may comprise the afore-mentioned domains in any order: for example: the one or more amino acid sequences of an active site of an Arg- or Lys-gingipain homologue of P. gulae may be located N-terminally to the amino acid sequence of one or more adhesin binding motifs (ABMs), or the amino acid sequence of one or more adhesin binding motifs (ABMs) may be located N-terminally to the one or more amino acid sequences of an active site of an Arg- or Lys-gingipain homologue 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. (In other words, the chimeric or fusion protein may comprises or consist of, N to C terminus: active site (K) - ABMs (A); or active site (R) - ABMs (A); or AMBs (A) - active site (K); or ABMs (A)-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) .)
[0027] Exemplary amino acid sequences of the active sites of Arg- or Lys-gingipain homologues of P. gulae, (and RNA sequences encoding the same) are further described herein. Preferably, the amino acid sequence of the active of an Arg- or Lys- gingipain homologue of P. gulae 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.
[0028] In certain embodiments, the amino acid sequence of an active site of the Arg- gingipain homologue of P. gulae (also designated “R” herein), comprises the amino acid sequence of SEQ ID NO: 4, encoded by the RNA sequence as set forth in SEQ ID NO: 69, 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. In any embodiment, the amino acid sequence of an Arg-gingipain homologue of P. gulae, (also designated “R” herein), may comprise a substitution of the cysteine residue (position 5 of SEQ ID NO: 4) optionally to a valine, serine or alanine residue.
[0029] In certain embodiments, the amino acid sequence of the active site of the Lys- gingipain homologue of P. gulae, (also designated “K” herein) comprises the amino acid sequence of SEQ ID NO: 2, encoded by the RNA sequence as set forth in SEQ ID NO: 59, 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.
[0030] Preferably, the amino acid sequence of the active site of an Arg- or Lys- gingipain homologue does not comprise the entire catalytic domain of the gingipain homologue.
[0031] 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 homologue of P. gulae, 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 homologue of P. gulae, or sequences that are at least 80% identical thereto.
[0032] 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 homologue of P. gulae, or sequences that are 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.
[0033] In further embodiments, the chimeric or fusion protein encoded by the RNA polynucleotide may further comprise: - the amino acid sequence of a DUF2436 domain of the Arg- and Lys-gingipain surface complexes of P. gulae, or a sequence that is at least 80% identical thereto.
[0034] 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. gulae and the amino acid sequence of the 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) - DUF2436 domain (D) - ABMs (A) or active site (K) or (R) - DUF domain (D) - ABMs (A) - active site (K) or (R)).).
[0035] In certain embodiments, the amino acid sequence of a DUF2436 domain of the Arg and Lys gingipain surface complexes of P. gulae comprises or consists of the amino acid sequence of SEQ ID NO: 22, encoded by the RNA sequence as set forth in SEQ ID NO: 70, 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.
[0036] In certain embodiments, the amino acid sequence of a DUF2436 domain of the Arg and Lys gingipain surface complexes of P. gulae comprises or consists of the amino acid sequence of SEQ ID NO: 19, encoded by the RNA sequence as set forth in SEQ ID NO: 71 , 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.
[0037] In any embodiment, one or more cysteine residues in the DUF2436 domain (having the amino acid sequence of SEQ ID NO: 19) may be substituted to a serine or valine residue or alanine residue, preferably to a serine residue (such as shown in any of SEQ ID NOs: 20 and 21). Preferably, the one or more cysteine residues are substituted to one or more serine residues. Optionally at least two cysteine residues in the DUF2436 domain are substituted, optionally at least three of the cysteine residues. Optionally, all four cysteine residues in this variant of the DUF2436 domain are substituted. [0038] In any embodiment, the one or more adhesin binding motifs (ABMs) also found in surface complexes of the Arg and Lys gingipain homologues of P. gulae, comprise or consist of the amino acid sequence of ABM2 and ABM 1 (for example as set forth in SEQ ID NO: 6 and SEQ ID NO: 5, respectively, or comprising the amino acid sequence as set forth in SEQ ID NO: 8 (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: 72, 73 and 75, herein.
[0039] 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: 9 and encoded by an RNA comprising the sequence of SEQ ID NO: 76).
[0040] 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 in the adhesin A domain compared to the A domain found naturally occurring in the Arg- Lys-gingipain homologue protein complex 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: 5 (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: 5 (ABM1); d) substitution of the second tyrosine residue, corresponding to or at a position equivalent to residues at position 10 of SEQ ID NO: 6 (ABM2), and of the tryptophan residue, corresponding to or at a position equivalent to residue at position 23 of SEQ ID NO: 5 (ABM1) to alanine residues.
[0041] 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. [0042] 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.
[0043] 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: 5, may comprise a substitution of the proline and asparagine residues.
[0044] The proline amino acid substitution is preferably a substitution to an alanine residue.
[0045] 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.
[0046] 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: 14 to 16).
[0047] 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: 8 or 9, and comprising: a) one or more cysteine amino acid substitutions compared to the naturally occurring Arg- or Lys-gingipain homologue sequences in corresponding regions, preferably substitution of all cysteine residues; and b) substitution of the motif PxxN corresponding to, or at a position equivalent to, residues 6 to 9 of the sequence of SEQ ID NO: 5 (ABM1), to AxxP.
[0048] 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: 17 or 18, 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. [0049] In particularly preferred embodiments of the invention, the RNA encodes a chimeric or fusion protein comprising or consisting of:
[0050] a)
- one or more amino acid sequences of active site of an Arg- or Lys-gingipain homologue of P. gulae, 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 found in surface complexes of an Arg- or Lys-gingipain homologues of P. gulae, 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: 24, SEQ ID NO: 46 or any one of SEQ ID NOs: 78 to 82; or
[0051] b)
- one or more amino acid sequences of active site of an Arg- or Lys-gingipain homologues of P. gulae, or a sequence that is at least 80% identical thereto;
- the amino acid sequence of a DUF2436 domain of an Arg- or Lys-gingipain surface complexes of P. gulae, 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 surface complexes of Arg- and Lys-gingipain homologues of P. gulae, 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: 30, 32, 38, 40, 48, 50, 90, 92, 94, 96, 98, 100, 102, or 104;
- more preferably wherein the chimeric or fusion protein comprises the amino acid sequence of SEQ ID NO: 34 or 36; or
[0052] c) - the amino acid sequence of a DUF2436 domain of Arg- or Lys-gingipain homologue surface complexes of P. gulae, 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 surface complex of Arg- and Lys-gingipain homologues of P. gulae, 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: 42 or 44; or
[0053] d)
- one or more amino acid sequences of active site of an Arg- or Lys-gingipain homologue of P. gulae, 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 homologue of P. gulae, 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: 26 or 28; or
[0054] e)
- one or more amino acid sequences of active site of an Arg- or Lys-gingipain homologue of P. gulae, or a sequence that is at least 80% identical thereto;
- the amino acid sequence of a DUF2436 domain of an Arg- or Lys-gingipain homologue surface complex of P. gulae , 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 surface complex of an Arg- or Lys-gingipain homologue of P. gulae, 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: 52 or 54. [0055] In other embodiments of the invention, the RNA 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 or SEQ ID NO: 4.
[0056] In particularly preferred embodiments of the invention, the RNA comprises or consists of a nucleotide sequence of any one of: a) SEQ ID NO: 67 or 68 b) SEQ ID NO: 62, 65, 66, 77or SEQ ID NO: 55 or 56; c) SEQ ID NO: 63 or 64; d) SEQ ID NO: 60 or 61 ; e) SEQ ID NO: 57 or 58, 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.
[0057] In other embodiments of the invention, the RNA comprises or consists of a nucleotide sequence of any one of: SEQ ID NO: 59 or SEQ ID NO: 69.
[0058] The invention further provides for the use of any DNA polynucleotide described herein (and particularly any DNA polynucleotide comprising or consisting of a sequence exemplified in Table 1). Optionally, the use of the DNA polynucleotide may be for obtaining an RNA polynucleotide of the invention.
[0059] The present invention also provides a composition, including a pharmaceutical composition comprising an RNA as described herein. Preferably, the composition comprises one or more pharmaceutically acceptable excipients. Optionally, the RNA 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.
[0060] The present invention therefore further provides compositions comprising an RNA molecule as described herein, wherein the composition also comprises a lipid component. The RNA (e.g., RNA) vaccines of the disclosure can be formulated using one or more liposomes, lipid vesicle, lipoplexes (such as a lipid-polycation complex), or lipid nanoparticles
[0061] In preferred embodiments, the RNA is formulated in a lipid nanoparticle.
[0062] In one embodiment, the RNA as described herein is the only polynucleotide 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 the liposomes, lipid vesicle, lipoplexes (such as a lipid-polycation complex), or lipid nanoparticles.
[0063] In any embodiment, the invention provides a lipid nanoparticle or other nanovehicle, such as nanopolymer, for delivery of the polynucleotide to a subject in need thereof.
[0064] 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.
[0065] 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.
[0066] 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,
- 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).
[0067] 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).
[0068] The present invention also provides a method for producing a lipid nanoparticle comprising an RNA, encoding a protein, or a chimeric or fusion protein as described herein. Preferably the method comprises formulating any RNA molecule of the invention, with one or more lipids useful for producing a lipid nanoparticle. Preferably the lipid components comprise a phospholipid, a PEG lipid, and a structural lipid.
[0069] The present invention also provides a nucleic acid construct or vector, comprising a polynucleotide as described herein.
[0070] 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.
[0071] 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.
[0072] 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.
[0073] 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.
[0074] The present invention provides a method for eliciting an immune response P. gulae in a subject in need thereof, the method comprising administering to the subject, a polynucleotide, vector, nanoparticle or composition described herein.
[0075] The invention provides a method for eliciting an immune response to P. gulae in a subject in need thereof, the method comprising administering to the subject, a composition comprising:
- an RNA as described herein, wherein said RNA is capable of being translated in a cell of a mammalian subject to produce the polypeptide encoded by the polynucleotide;
- optionally, an agent for enabling delivery of the RNA into mammalian cells.
[0076] 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.
[0077] The invention provides a method for eliciting an immune response to P. gulae in a subject in need thereof, the method comprising administering to the subject, a nanoparticle composition comprising:
- a lipid component; and
- an RNA as described herein, wherein said RNA is capable of being translated in a cell of the subject to produce the polypeptide encoded by the polynucleotide.
[0078] The invention also provides a method for producing a chimeric or fusion protein as described herein in a mammalian cell, the method comprising contacting the mammalian cell with a composition comprising:
- an RNA as described herein, wherein said RNA is capable of being translated in the mammalian cell to produce the protein;
- optionally, an agent for enabling delivery of the RNA into mammalian cells. [0079] 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.
[0080] The invention also provides a method for producing a chimeric or fusion protein as described herein in a mammalian cell, the method comprising contacting the mammalian cell with a nanoparticle composition, the composition comprising:
- a lipid component; and
- an RNA as described herein, wherein said RNA is capable of being translated in the mammalian cell to produce the polynucleotide.
[0081] Preferably the lipid component comprises a cationic and/or ionisable lipid, a phospholipid, a PEG lipid, and a structural lipid.
[0082] The invention also provides a method for delivering an RNA to a mammalian cell in a subject in need thereof, said method comprising administering to a subject in need thereof, a nanoparticle composition, the composition comprising:
- a lipid component; and
- an RNA comprising a polynucleotide sequence as described herein, wherein said RNA is capable of being translated in the mammalian cell to produce the chimeric or fusion protein described herein; wherein the administering comprises contacting said mammalian cell with the nanoparticle composition, thereby enabling delivery of the RNA to the mammalian cell.
[0083] Preferably the lipid component comprises a cationic and/or ionisable lipid, a phospholipid, a PEG lipid, and a structural lipid.
[0084] In any embodiment, the ionisable lipid may be substituted or combined with an adjuvant lipidoid for enhancing RNA delivery. [0085] The present invention also provides the use of a polynucleotide, vector, or nanoparticle described herein, in the manufacture of a composition for eliciting an immune response to P. gulae in a subject.
[0086] The present invention also provides the use of i) a lipid component as described herein, and ii) an RNA as described herein, in the manufacture of a composition for delivering the RNA to a mammalian cell in a subject in need thereof.
[0087] The present invention also provides a polynucleotide, vector, nanoparticle or composition as described herein, for use in eliciting an immune response to P. gulae in a subject, preferably in a non-human animal subject.
[0088] In any method or use described herein, the RNA vaccines of the invention may be administered in combination with one or more additional therapeutic agents, or other agents for eliciting an immune response to P. gulae in the subject. Optionally, the one or more therapeutic agents may comprise an anti-microbial compound or an antiinflammatory agent. The other agents for elicing an immune response to P. gulae may comprise one or more additional protein, RNA or whole-cell based vaccine compositions or immunogens for generating an immune response to P. gulae.
[0089] 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”.
[0090] 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”.
[0091] As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps. [0092] 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
[0093] Figure 1 : Expression and Solubility of P. gulae chimeric proteins KDFAK- 2S-AVQP and KDAAK-2S-AVQP. (A) Lysis fractions; (B) Small scale purification tests with Ni-NTA spin column: 1. clarified lysate before loaded to the column; 2. flow through; 3. column wash; 4. eluted protein fraction. Left side panel: KDFAK-2S-AVQP and right side panel: KDAAK-2S-AVQP in both A) and B).
[0094] Figure 2: SDS-PAGE analysis of expressed P. gulae antigens and the cell lysis fractions. (A) Expression of KDFAK-2S-AVQP and KDAAK-2S-AVQP in LB and TB media for 2-3 hours. (B) Lysis under non-reducing conditions. (C) (i & ii) Attempt to solubilise KDFAK-2S-AVQP from the insoluble fraction of non-reducing lysis with reducing buffer (5 mM and 100 mM DTT); (iii) Lysis under reducing conditions (10 mM DTT) for proteins KDFAK-2S-AVQP. M: protein standards; TC: total cell lysate; Sup: clarified lysate.
[0095] Figure 3: Ni-affinity chromatography. (A) Elution profile of KDAAK-2S-AVQP under normal non-reducing conditions and reducing SDS-PAGE of the column fractions. (B) Elution profile of antigen-F under reducing conditions (R) and reducing SDS-PAGE of the column fractions. (C) Elution profile of KDFAK-2S-AVQP under normal non-reducing conditions (NR) and reducing SDS-PAGE of the column fractions. TC: total cell lysate; Sp: clarified supernatant before loaded to the column; FT : flow through; W: column wash. M: protein standards.
[0096] Figure 4: Purification with anion exchange chromatography (AIEX). (A) Elution profile of KDAAK-2S-AVQP under normal non-reducing conditions and reducing SDS-PAGE of the column fractions. (B) Elution profile of KDFAK-2S-AVQP under reducing conditions (R) and reducing SDS-PAGE of the column fractions. (C) Elution profile of KDFAK-2S-AVQP under normal non-reducing conditions (NR) and reducing SDS-PAGE of the column fractions. BL: before loading; FT: flow through; M: protein standards. [0097] Figure 5: Size exclusion chromatography under non-reducing conditions (NR). (A) Elution profile KDAAK-2S-AVQP and reducing SDS-PAGE of the column fractions. (B) Elution profile of KDFAK-2S-AVQP being purified under reducing conditions (R) before this step and reducing SDS-PAGE of the column fractions. (C) Elution profile of KDFAK-2S-AVQP being purified under non-reducing conditions (NR) before this step and reducing SDS-PAGE of the column fractions. BL: before loading; M: protein standards.
[0098] Figure 6: PAGE analysis of the P. gulae antigen final products. (A) SDS- PAGE; (B) Native PAGE or on native gels. A-version: KDAAK-2S-AVQP; F-R: KDFAK-2S- AVQP purified under reducing conditions until the final size exclusion step; F-NR: KDFAK- 2S-AVQP purified under non-reducing conditions. R: reducing; NR: non-reducing; M: protein standards.
[0099] Figure 7: Mouse periodontitis model: therapeutic vaccination.
[0100] Figure 8: P. gulae induced bone loss. Statistical analysis - One-way ANOVA and post-hoc Dunnet’s T3. # (p<0.05, compared to Naive control); ## (p<0.05, compared to infected control).
[0101] Figure 9.- Anti-P. gulae antibody isotype response. Antibody titres in mouse sera (individual) towards heat-killed P. gulae whole cells. (A) Total IgG Titres; (B) lgG1 Subtype titres; (C) lgG2a Subtype titres.
[0102] Figure 10: Antibody IgG response against P. gulae protease complex. Antibody titres in mouse sera (individual) towards purified P. gulae RgpA/Kgp protease complex. (A) Total IgG Titres; (B) lgG1 Subtype titres; (C) lgG2a Subtype titres.
[0103] Figure 11 : Antibody IgG anti-vaccine antigen immune response. Antibody titres in mouse sera (Individual sera) towards vaccine antigen used to immunise mice.
[0104] Figure 12: Schematic of experimental protocol for canine serology studies.
[0105] Figure 13: IgG response to immunising antigen (KDAAK-2S-AVQP-6His). A) pooled sera; b) individual sera; C) analysis of individual sera; d) tittering of individual sera.
[0106] Figure 14: Serological endpoint titres (2xbaseline) vs P. gulae whole cells. [0107] Figure 15: Comparison of titre responses to P. gulae KAS2 protein following immunisation with KDAAK-2S-AVQP-6His.
[0108] Figure 16: Serological midpoint titres for each serum collection time point.
For the 10 dogs in each group, the midpoint titre values were plotted for each serum collection time point. Mean and standard error of the mean (SEM) are shown for each serum collection time point. The ordinate axis represents the serum dilution required to achieve the titration midpoint. Pairs of serum time points that showed a statistical significant difference (p < 0.05) between the means are represented by the bars above the graph.
[0109] Figure 17: Serological endpoint titres for each serum collection time point.
For the 10 dogs in each group, the endpoint titre values were plotted for each serum collection time point. Mean and standard error of the mean (SEM) are shown for each serum collection time point. The ordinate axis represents the serum dilution required to achieve the titration endpoint. Pairs of serum time points that showed a statistical significant difference (p < 0.05) between the means are represented by the bars above the graph.
[0110] Figure 18: Schematic of vaccination protocol for assessing immunogenicity of RNA candidate vaccines.
[0111] Figure 19: Schematic of study protocol for determining in vivo efficacy of RNA vaccines.
Sequence information
[0112] Table 1 : DNA and amino acid sequences
Figure imgf000021_0001
Figure imgf000022_0001
Figure imgf000023_0001
Figure imgf000024_0002
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
I I | | | | | | | | | i i | I | I I | | | |
Figure imgf000047_0001
[0113] Table 2: exemplary RNA sequences of the invention | § J § § J § §
Figure imgf000047_0002
J | § § J § § J § § J § § J § § J | § | § § | § § | § § J § § J § § J § § J § § J § | § | § § | § § |
Figure imgf000048_0001
! | § § J § § J § § J § § J § § | § § | § § | § § | § ^ J § § J § § J § § J § § J | § § § J § § J
Figure imgf000049_0001
! | § § J § § J | § § ! § § ! § § ! § § ^ § § | | § J § § J § § § § § § § § § ! § § § § | | § J § § J
Figure imgf000050_0001
! | § § J § § J § § J § § J | § ! § § | § § | § § | § § J § § J § § | § § ! § § ! § § | § § | § § |
Figure imgf000051_0001
! ! § § | § § § § § ! § § ! § ^ ! § § | § § | § § | | § J § § § § § § § | § J ^ § J § § J § | § |
Figure imgf000052_0001
! | § § J § § J § § J § § | § § § § § § § ! § § ! § | J ! | J § § | § § ! § § | § J § § J §
Figure imgf000053_0001
! I | | | | | I I | | | | | | | I | | | | | |
Figure imgf000054_0001
Detailed description of the embodiments
[0114] 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] 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, and equivalents, which may be included within the scope of the present invention as defined by the claims. [0116] 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.
[0117] All of the patents and publications referred to herein are incorporated by reference in their entirety.
[0118] For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.
[0119] 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.
[0120] Recent studies in dogs and cats suggest that companion animals host similar periodontal pathogens, with a species Porphyromonas gulae closely related to the human keystone pathogen P. gingivalis commonly found in cats and dogs with severe disease. Although P. gulae is a known causative agent for periodontitis in companion animals, no preventative or therapeutic treatment currently exists.
[0121] The inventors describe herein, RNA vaccines encoding chimeric or fusion proteins comprising components of P. gulae virulence genes, for use in inducing an immune response to P. gulae, and methods and uses comprising the same.
Gingipains
[0122] The pathogenicity of P. gulae is attributed to a number of surface-associated virulence factors that include cysteine proteinases (gingipain-homologues), fimbriae, haem-binding proteins, and outer membrane transport proteins amongst others. In particular, the extracellular Arg- and Lys-specific proteinases ‘gingipain-homologues’ (RgpA/B and Kgp) of P. gingivalis, a related pathogen, have been implicated as major virulence factors that are critical for colonisation, penetration into host tissue, dysregulation of the immune response, dysbiosis and disease. [0123] The gingipains, in particular the Lys-specific proteinase Kgp are essential for the ability of a related pathogen, 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 high concentrations proximal to the subgingival plaque and at lower concentrations at distal sites deeper into the gingival tissue. Lys-specific and Argspecific 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 a-trypsin, a2-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.
[0124] The RgpA, and Kgp genes of P. gingivalis 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 a “Domain of Unknown Function” (termed DUF2436 which is defined as conserved Pfam Domain of Unknown Function; IPR018832) and an adhesin domain (comprising adhesin binding domains). The particular arrangement of the adhesin domains and DUF2436 varies between Kgp and RgpA/B and in particular between Kgp and RgpA gingipains from different Porphyromonas species.
[0125] In vivo, 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 and domains from the hemagglutinin genes.
[0126] As used herein a Lys-gingipain catalytic domain sequence around the active site Histidine may also be referred to a KAS or K domain. As used herein an Arg-gingipain catalytic domain sequence around the active site Histidine may also be referred to as a RAS or R domain. Typically, the catalytic domain of the Lys-gingipain or Arg-gingipains is located in the N-terminal region of the protein. Exemplary Histidine active site peptides, as found within the catalytic domains are set out in in Table 1 as SEQ ID NOs: 2 and 4
[0127] As used herein an HA domain of an Arg- or Lys-gingipain of P. gulae will be understood to either refer to the region of an Arg- or Lys-gingipain homologue that is C- terminal to the catalytic or active site domain or to an homologous HA domain sequence encoded by a separate polyadhesin gene such as Hag. The HA domain typically comprise a Domain of Unknown Function (DUF) domain (especially DUF 2436 conserved Pfam Domain of Unknown Function; IPR018832) and an adhesin domain comprising adhesin binding domains (ABMs). Once these domains are expressed and secreted to the cell surface they form non-covalent complexes of the Arg- and Lys-specific proteinases catalytic domains together with the DUF and ABM domains to form a virulence coat.
Nucleic acids
[0128] 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” (mRNA) 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 “U.”
[0129] 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. [0130] A “5' untranslated region” (5'IITR) 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.
[0131] 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.
[0132] 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.
[0133] 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, 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, the polyA tail may be segments (eg comprising segments of consecutive adenosine monophosphates joined by short spacers). In a relevant biological setting (e.g., in cells, in vivo) the poly(A) tail functions to protect RNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, export of the RNA from the nucleus and translation.
[0134] 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.
[0135] 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 mRNA 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 nucleotides are inserted, deleted, duplicated, inverted or randomized in a polynucleotide, primary construct or mmRNA 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.
[0136] 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.
[0137] 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 (II), thymidine (T) or cytidine (C) ribonucleosides or deoxyribnucleosides 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. Polypeptides, as provided herein, are also considered “modified” of they contain amino acid substitutions, insertions or a combination of substitutions and insertions.
[0138] Polynucleotides (e.g., RNA polynucleotides, such as RNA 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). [0139] Modifications of polynucleotides include, without limitation, those described herein. Polynucleotides (e.g., RNA polynucleotides, such as RNA polynucleotides) may comprise 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).
[0140] Polynucleotides (e.g., RNA polynucleotides, such as RNA 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 an internucleotide linkages, purine or pyrimidine bases, or sugars. The modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a polynucleotide may be chemically modified.
[0141] The present disclosure provides for modified nucleosides and nucleotides of a polynucleotide (e.g., RNA polynucleotides, such as RNA 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 phosphdioester linkages, in which case the polynucleotides would comprise regions of nucleotides.
[0142] 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.
[0143] 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.
[0144] In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as RNA 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.
[0145] 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.
[0146] In some embodiments, a modified nucleobase is a modified uridine. Exemplary nucleobases and In some embodiments, a modified nucleobase is a modified cytosine, nucleosides having a modified uridine include 5-cyano uridine, and 4'-thio uridine.
[0147] 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). [0148] In some embodiments, a modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1 -methylinosine (mi l), 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.
[0149] 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, II, C) may be uniformly modified in a polynucleotide of the disclosure, or in a given predetermined sequence region thereof (e.g., in the RNA 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, II, C, or any one of the combinations A+G, A+ll, A+C, G+ll, G+C, ll+C, A+G+ll, A+G+C, G+U+C or A+G+C.
[0150] 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, II 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, II, or C.
[0151] 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 polynucleotide is 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), n 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).
[0152] In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having a modified uracil include pseudouridine (ip), pyridin-
4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4- thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine, 5-aminoallyl- uridine, 5-halo-uridine (e.g., 5-iodo-uridineor 5-bromo-uridine), 3-methyl-uridine, 5- methoxy-uridine, uridine 5-oxyacetic acid, uridine 5-oxyacetic acid methyl ester, 5- carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl- uridine, 5-carboxyhydroxymethyl-uridine methyl ester, 5-methoxycarbonylmethyl-uridine,
5-methoxycarbonylmethyl-2-thio-uridine, 5-aminomethyl-2-thio-uridine, 5- methylaminomethyl-uridine, 5-methylaminomethyl-2-thio-uridine, 5-methylaminomethyl- 2-seleno-uridine, 5-carbamoylmethyl-uridine, 5-carboxymethylaminomethyl-uridine, 5- carboxymethylaminomethyl-2-thio-uridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine, 1 -taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine, 5-methyl-2-thio-uridine, 1-methyl-4-thio- pseudouridine, 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine, 2-thio-1-methyl- pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza- pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl- dihydrouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2- methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1- methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine, 1-methyl-3-(3-amino-3- carboxypropyl)pseudouridine, 5-(isopentenylaminomethyl)uridine, 5-
(isopentenylaminomethyl)-2-thio-uridine, a-thio-uridine, 2'-O-methyl-uridine, 5, 2 -0- dimethyl-uridine, 2'-O-methyl-pseudouridine (Wm), 2-thio-2'-O-methyl-uridine, 5- methoxycarbonylmethyl-2'-O-methyl-uridine, 5-carbamoylmethyl-2'-O-methyl-uridine, 5- carboxymethylaminomethyl-2'-O-methyl-uridine, 3,2'-O-dimethyl-uridine, and 5- (isopentenylaminomethyl)-2'-O-methyl-uridine, 1-thio-uridine, deoxythymidine, 2'-F-ara- uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E- propenylamino)]uridine.
[0153] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include 5-aza- cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetyl-cytidine, 5- formyl-cytidine, N4-methyl-cytidine, 5-methyl-cytidine, 5-halo-cytidine (e.g., 5-iodo- cytidine), 5-hydroxymethyl-cytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza- pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2- methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy- 1-methyl- pseudoisocytidine, lysidine, a-thio-cytidine, 2'-O-methyl-cytidine, 5,2'-O-dimethyl- cytidine, N4-acetyl-2'-O-methyl-cytidine, N4,2'-O-dimethyl-cytidine, 5-formyl-2'-O-methyl- cytidine, N4,N4,2'-O-trimethyl-cytidine, 1 -thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.
[0154] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include 2-amino- purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6- halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7- deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2- amino-purine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1 -methyladenosine, 2-methyl-adenine, N6-methyl-adenosine, 2-methylthio-N6-methyl-adenosine, N6-isopentenyl-adenosine, 2-methylthio-N6-isopentenyl-adenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6- glycinylcarbamoyl-adenosine, N6-threonylcarbamoyl-adenosine, N6-methyl-N6- threonylcarbamoyl-adenosine, 2-methylthio-N6-threonylcarbamoyl-adenosine, N6,N6- dimethyl-adenosine, N6-hydroxynorvalylcarbamoyl-adenosine, 2-methylthio-N6- hydroxynorvalylcarbamoyl-adenosine, N6-acetyl-adenosine, 7-methyl-adenine, 2- methylthio-adenine, 2-methoxy-adenine, a-thio-adenosine, 2'-O-methyl-adenosine, N6,2'-O-dimethyl-adenosine, N6,N6,2'-O-trimethyl-adenosine, 1 ,2'-0-dimethyl- adenosine, 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1 -thio- adenosine, 8-azido-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara- adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.
[0155] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include inosine, 1- methyl-inosine, wyosine, methylwyosine, 4-demethyl-wyosine, isowyosine (imG2), wybutosine, peroxywybutosine, hydroxywybutosine, undermodified hydroxywybutosine, 7-deaza-guanosine, queuosine, epoxyqueuosine, galactosyl-queuosine (galQ), mannosyl-queuosine, 7-cyano-7-deaza-guanosine, 7-aminomethyl-7-deaza-guanosine, archaeosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6- thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7- methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine, N2-methyl-guanosine, N2,N2-dimethyl-guanosine, N2,7-dimethyl-guanosine, N2, N2,7-dimethyl-guanosine, 8- oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6- thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, a-thio-guanosine, 2'-O-methyl- guanosine, N2-methyl-2'-O-methyl-guanosine, N2,N2-dimethyl-2'-O-methyl-guanosine, 1-methyl-2'-O-methyl-guanosine, N2,7-dimethyl-2'-O-methyl-guanosine, 2'-O-methyl- inosine, 1 ,2'-O-dimethyl-inosine, 2'-O-ribosylguanosine (phosphate) (Gr(p)), 1-thio- guanosine, 06-methyl-guanosine, 2'-F-ara-guanosine, and 2'-F-guanosine.
[0156] In some embodiments, the RNA (e.g., RNA) vaccines comprise a 5'IITR element, an optionally codon optimized open reading frame, and a 3'IITR element, a poly(A) sequence and/or a polyadenylation signal wherein the RNA is not chemically modified.
[0157] 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.
[0158] 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 sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or antigenic polypeptide)).
[0159] 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)).
[0160] In some embodiments a codon-optimized RNA (e.g., RNA) 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 (II) 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. [0161] 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'IITR) and/or at their 3'-end (3'IITR), in addition to other structural features, such as a 5'-cap structure or a 3'-poly(A) tail. Both the 5'IITR and the 3'IITR 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.
[0162] In some embodiments the RNA (e.g., RNA) 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.
[0163] In some embodiments, the RNA (e.g., RNA) 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)).
[0164] 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.
[0165] In some embodiments, the RNA (e.g., RNA) 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.
[0166] In some embodiments, the RNA (e.g., RNA) vaccine may or may not contain a 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.
[0167] In other embodiments the RNA (e.g., RNA) vaccine may have one or more Allrich 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., RNA) vaccines. Alternatively the AURES may remain in the RNA (e.g., RNA) vaccine.
[0168] 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, AK chimeric protein, AD chimeric protein, a KDA chimeric protein, an RDA chimeric protein, a DAR chimeric protein, a DAK chimeric protein or combinations thereof.
[0169] Non-limiting examples of 2A peptide sequence for use to introduce ribosome skiping include the T2A or T2A-like sequences derived from Thosea asigna virus and from Porcine teschovirus-1 2A.
Polypeptides
[0170] It will be appreciated that the polynucleotides 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”.
[0171] 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.
[0172] 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 homologues from P. gulae, which are in a different spatial arrangement to full length proteins.
Linkers
[0173] 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. gulae 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. The linker regions may be derived from the native P. gulae gingipain polyprotein sequence, or may comprise artificial (ie non-naturally occurring) linker sequence.
[0174] 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.
[0175] 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. gulae gingipain active site, and the amino acid sequence of the adhesin domain of a P. gulae 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. gulae trypsin-like enzyme. Alternatively, it may space domains apart.
[0176] 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.
[0177] 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: 108), (GGGS)n (SEQ ID NO: 109) and (GGGGS)n (SEQ ID NO: 110), 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.
[0178] 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 can include the sequence Gly-Gly-Ser (GGS), Gly-Gly-Gly-Ser (GGGS, SEQ ID NO: 109) or Gly-Gly-Gly-Gly-Ser (GGGGS, SEQ ID NO: 110) and variations or repeats thereof. In some aspects, the peptide linker can include the amino acid sequence GGGGS (SEQ ID NO: 110, 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: 111) 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: 112) and variations thereof.
[0179] In one embodiment, the peptide linker can include the amino acid sequence GGGGS (SEQ ID NO: 110, 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: 111) 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.
[0180] Other useful linkers include DSSG (SEQ ID NO: 113), DSSGAS (SEQ ID NO: 114), KLDSSG (SEQ ID NO: 115) and variations thereof. Examples of other suitable linkers are described in Chen et al., (2013) Advanced Drug Delivery Reviews, 65: 1357- 1369.
[0181] 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.
[0182] 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 used synonymously 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.
[0183] 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.
[0184] “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.
[0185] 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, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and/or a polar residue for a non-polar residue.
[0186] Amino acid deletions or insertions can also be made relative to the native sequence of the P. gulae 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.
[0187] 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).
[0188] 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 homologue. 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.
[0189] 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 by non-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.
[0190] 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. [0191] 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 acid sequence 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. Other tools are described herein, specifically in the definition of “identity” below.
[0192] The term “identity” refers to the overall relatedness between polymeric molecules, for example, between polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of the percent identity of two polynucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and nonidentical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleic acid sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991 ; each of which is incorporated herein by reference. For example, the percent identity between two nucleic acid sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleic acid sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).
Signal peptides
[0193] 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 a ER- 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.
[0194] 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. [0195] 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.
[0196] 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., RNA) 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., RNA) vaccine is an Ig heavy chain epsilon-1 signal peptide (IgE HC SP) having the sequence of: MDWTWILFLVAAATRVHS (SEQ ID NO: 116). In some embodiments, a signal peptide fused to the antigenic polypeptide encoded by the (e.g., RNA) RNA (e.g., RNA) vaccine is an IgGk chain V-lll region HAH signal peptide (IgGk SP) having the sequence of METPAQLLFLLLLWLPDTTG (SEQ ID NO: 117). In some embodiments, the signal peptide is selected from: Japanese encephalitis PRM signal sequence (MLGSNSGQRVVFTILLLLVAPAYS, SEQ ID NO: 118), VSVg protein signal sequence (MKCLLYLAFLFIGVNCA, SEQ ID NO: 119) and Japanese encephalitis JEV signal sequence (MWLVSLAIVTACAGA, SEQ ID NO: 120).
[0197] Further examples of suitable signal peptides include sequences derived from tPA (tissue plasminogen activator): MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO:
121) variants, such as: tPA (VSA): MDAMKRGLCCVLLLCGAVFVSA (SEQ ID NO:
122), tPA (VSAR): MDAMKRGLCCVLLLCGAVFVSAR (SEQ ID NO: 123), tPA (VSP): MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 124), tPA (VSPS).
[0198] In certain embodiments, the signal peptide comprises the sequence of the SEAP (secreted embryonic alkaline phosphatase) secretion signal, having the amino acid sequence MLLLLLLLGLRLQLSLG (SEQ ID NO: 125).
[0199] In certain embodiments, the amino acid sequence of the signal peptide comprises the sequence MLLLLLLLGLRLQLSLG[A] (SEQ ID NO: 126), such that the expressed RNA product comprises the sequence MLLLLLLLGLRLQLSLG[A] (SEQ ID NO: 126) N terminal to the sequences defined herein including in Table 1.
[0200] 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.
[0201] 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.
[0202] 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.
[0203] 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.
[0204] It will be well within the purview of the skilled person to design a polypeptide encoded by an RNA of the invention, to facilitate expression and translation thereof in vivo. For example, in certain non-limiting examples, the RNA of the invention may 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.
[0205] Accordingly, in any embodiment, the RNA 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, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 78 to 82 may encode one or more additional N terminal amino acids, optionally an N terminal alanine residue. Compositions and lipid nanoparticles
[0206] The present invention contemplates the provision of a polynucleotide (preferably an RNA) encoding a chimeric or fusion protein for inducing an immune response to P. gulae, 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.
[0207] In some embodiments, the RNA of the invention is formulated in a lipidpolycation 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).
[0208] 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.
[0209] 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. In some embodiments, a cationic lipid is selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl- [1 ,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3- DMA), di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), (12Z.15Z) — N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine (L608), and N,N- dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine (L530).
[0210] In some embodiments, lipid nanoparticle formulations include 25-75% of a cationic lipid optionally selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1 ,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 0.5- 15% of the neutral lipid, 5-50% of the sterol, and 0.5-20% of the PEG or PEG-modified lipid on a molar basis. [0211] In some embodiments, lipid nanoparticle formulations consists 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 consists 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.
[0212] In some embodiments, the molar lipid ratio is 50/10/38.5/1.5 (mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG, PEG-DSG or PEG-DPG), 57.2/7.1134.3/1.4 (mol % cationic lipid/neutral lipid, e.g., DPPC/Chol/PEG- modified lipid, e.g., PEG-cDMA), 40/15/40/5 (mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG), 50/10/35/4.5/0.5 (mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DSG), 50/10/35/5 (cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG), 40/10/40/10 (mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA), 35/15/40/10 (mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA) or 52/13/30/5 (mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG- cDMA).
[0213] 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. 2010 28:172-176), the lipid nanoparticle formulation is composed of 57.1 % cationic lipid, 7.1 % dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA.
[0214] 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. In some embodiments, the cationic lipid is selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1 ,3]-dioxolane (DLin- KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non- 2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319). [0215] 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.
[0216] In some embodiments, the ratio of lipid to RNA (e.g., RNA) 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.
[0217] 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. As a non-limiting example, lipid nanoparticle formulations may contain 0.5% to 3.0%, 1.0% to 3.5%, 1.5% to 4.0%, 2.0% to 4.5%, 2.5% to 5.0% and/or 3.0% to 6.0% of the lipid molar ratio of PEG-c-DOMG (R-3-[(w-methoxy- poly(ethyleneglycol)2000)carbamoyl)]-1 ,2-dimyristyloxypropyl-3-amine) (also referred to herein as PEG-DOMG) as compared to the cationic lipid, DSPC and cholesterol. In some embodiments, the PEG-c-DOMG may be replaced with a PEG lipid such as, but not limited to, PEG-DSG (1 ,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG- DMG (1 ,2-Dimyristoyl-sn-glycerol) and/or PEG-DPG (1 ,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationic lipid may be selected from any lipid known in the art such as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2- DMA.
[0218] The amino alcohol cationic lipid may be a lipids described in and/or made by the methods described in U.S. Patent Publication No. US20130150625, herein incorporated by reference in its entirety. As a non-limiting example, the cationic lipid may be 2-amino- 3-[(9Z, 12Z)-octadeca-9, 12-dien-1 -yloxy]-2-{[(9Z,2Z)-octadeca-9, 12-dien-1 -yloxy] methyl}propan-1-ol (Compound 1 in US20130150625); 2-amino-3-[(9Z)-octadec-9-en-1- yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol (Compound 2 in US20130150625); 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-
[(octyloxy)methyl]propan-1-ol (Compound 3 in US20130150625); and 2-(dimethylamino)- 3-[(9Z, 12Z)-octadeca-9, 12-dien-1 -yloxy]-2-{[(9Z, 12Z)-octadeca-9, 12-dien-1 - yloxy]methyl}propan-1-ol (Compound 4 in US20130150625); or any pharmaceutically acceptable salt or stereoisomer thereof.
[0219] 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.
[0220] The cationic lipid may be of Formula I
Figure imgf000083_0001
[0221] wherein R1 and R2 are independently selected and are H or C1-C3 alkyls, R3 and R4 are independently selected and are alkyl groups having from about 10 to about 20 carbon atoms, and at least one of R3 and R4 comprises at least two sites of unsaturation, preferably the cationic lipid of Formula I is 1 ,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA) or 1 ,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).
[0222] The cationic lipid may be of Formula II (II)
Figure imgf000084_0001
[0223] wherein R1 and R2 are independently selected and are H or C1-C3 alkyls, R3 and R4 are independently selected and are alkyl groups having from about 10 to about 20 carbon atoms, and at least one of R3 and R4 comprises at least two sites of unsaturation.
[0224] The cationic lipid may be of Formula III
Figure imgf000084_0002
[0225] wherein R1 and R2 are either the same or different and independently optionally substituted C12-C24 alkyl, optionally substituted C12-C24 alkenyl, optionally substituted C12- C24 alkynyl, or optionally substituted Ci2-C24 acyl; R3 and R4 are either the same or different and independently optionally substituted Ci-Ce alkyl, optionally substituted C1- Ce alkenyl, or optionally substituted Ci-Cs alkynyl or R3 and R4 may join to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and oxygen; R5 is either absent or hydrogen or C1-C6 alkyl to provide a quaternary amine; m, n, and p are either the same or different and independently either 0 or 1 with the proviso that m, n, and p are not simultaneously 0; q is 0, 1 , 2, 3, or 4; and Y and Z are either the same or different and independently O, S, or NH.
[0226] The cationic lipid of Formula III may be 2,2-dilinoleyl-4-(2-dimethylaminoethyl)- [1 ,3]-dioxolane (DLin-K-C2-DMA; “XTC2”), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1 ,3]- dioxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1 ,3]-dioxolane (DLin-K-C4-DMA), 2,2-dilinoleyl-5-dimethylaminomethyl-[1 ,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[1 ,3]-dioxolane (DLin-K-MPZ), 2,2-dilinoleyl-4- dimethylaminomethyl-[1 ,3]-dioxolane (DLin-K-DMA), 1 ,2-dilinoleylcarbamoyloxy-3- dimethylaminopropane (DLin-C-DAP), 1 ,2-dilinoleyoxy-3-
(dimethylamino)acetoxypropane (DLin-DAC), 1 ,2-dilinoleyoxy-3-morpholinopropane (DLin-MA), 1 ,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1 ,2-dilinoleylthio-3- dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1 ,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.C1), 1 ,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.C1), 1 ,2-dilinoleyloxy-3- (N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1 ,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1 ,2-propanedio (DOAP), 1 ,2-dilinoleyloxo-3-(2-N,N- dimethylamino)ethoxypropane (DLin-EG-DMA), or mixtures thereof.
[0227] The phospholipid may be according to Formula (IV):
Figure imgf000085_0001
in which represents a phospholipid moiety and R and R’ represent fatty acid moieties with or without unsaturation that may be the same or different.
[0228] 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.
[0229] 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, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.
[0230] The phosopholipid may be lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, 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.
[0231] The phosopholipid may be distearoylphosphatidylcholine (DSPC).
[0232] 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.
[0233] The phospholipid may comprise from 5 mol % to 20 mol %, from 5 mol % to 15 mol %, from 5 mol % to 10 mol %, from 10 mol % to 20 mol %, or from 15 mol % to 20 mol % of the total lipid present in the particle.
[0234] 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.
[0235] 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.
[0236] The structural lipid may include lipids containing geranyl acetate, farnesyl acetate or geranyl-geranyl, or ether, ester, or other derivatives.
[0237] 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.
[0238] 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).
[0239] In some embodiments, a lipid nanoparticle formulation includes 0.5% to 15% on a molar basis of the neutral lipid, e.g., 3 to 12%, 5 to 10% or 15%, 10%, or 7.5% on a molar basis. Examples of neutral lipids include, without limitation, DSPC, POPC, DPPC, DOPE and SM. In some embodiments, the formulation includes 5% to 50% on a molar basis of the sterol (e.g., 15 to 45%, 20 to 40%, 40%, 38.5%, 35%, or 31 % on a molar basis. A non-limiting example of a sterol is cholesterol. In some embodiments, a lipid nanoparticle formulation includes 0.5% to 20% on a molar basis of the PEG or PEG- modified lipid (e.g., 0.5 to 10%, 0.5 to 5%, 1.5%, 0.5%, 1.5%, 3.5%, or 5% on a molar basis. In some embodiments, a PEG or PEG modified lipid comprises a PEG molecule of an average molecular weight of 2,000 Da. In some embodiments, a PEG or PEG modified lipid comprises a PEG molecule of an average molecular weight of less than 2,000, for example around 1 ,500 Da, around 1 ,000 Da, or around 500 Da. Non-limiting examples of PEG-modified lipids include PEG-distearoyl glycerol (PEG-DMG) (also referred herein as PEG-014 or C14-PEG), PEG-cDMA (further discussed in Reyes et al. J. Controlled Release, 107, 276-287 (2005) the contents of which are herein incorporated by reference in their entirety).
[0240] 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.
[0241] 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. [0242] The PEGylated lipid may be selected from the group consisting of PEG-c- DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.
[0243] 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.
[0244] The PEGylated lipid may be DSPE-PEG, wherein the PEG has a molecular weight of 2000 Da.
[0245] In some embodiments, the pharmaceutical compositions of the RNA (e.g., RNA) 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.
[0246] 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.
[0247] 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- 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).
[0248] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a vaccine composition may vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered. For example, the composition may comprise between 0.1% and 99% (w/w) of the active ingredient. By way of example, the composition may comprise between 0.1 % and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient. [0249] 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.
[0250] 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. gulae gingipain polyprotein homologue complex), 8.58 mg/mL of ALC-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 pLIn 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).
[0251] 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.
[0252] In some embodiments, the RNA (e.g., mRNA) vaccines of the disclosure may be formulated in lipid nanoparticles having a diameter from about 10 to about 100 nm such as, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 to about 60 nm, about 50 to about 70 nm about 50 to about 80 nm, about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100 nm, about 80 to about 90 nm, about 80 to about 100 nm and/or about 90 to about 100 nm.
[0253] In some embodiments, the lipid nanoparticles may have a diameter from about 10 to 500 nm.
[0254] In some embodiments, the lipid nanoparticle may have a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than
300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than
500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than
700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than
900 nm, greater than 950 nm or greater than 1000 nm.
[0255] In some embodiments, the lipid nanoparticle may be a limit size lipid nanoparticle described in International Patent Publication No. WO2013059922, the contents of which are herein incorporated by reference in their entirety. The limit size lipid nanoparticle may comprise a lipid bilayer surrounding an aqueous core or a hydrophobic core; where the lipid bilayer may comprise a phospholipid such as, but not limited to, diacylphosphatidylcholine, a diacylphosphatidylethanolamine, a ceramide, a sphingomyelin, a dihydrosphingomyelin, a cephalin, a cerebroside, a C8-C20 fatty acid diacylphophatidylcholine, and 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC). In some embodiments, the limit size lipid nanoparticle may comprise a polyethylene glycol-lipid such as, but not limited to, DLPE-PEG, DMPE-PEG, DPPC-PEG and DSPE-PEG.
[0256] In some embodiments the RNA (e.g., mRNA) 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), including HIV-binding peptides, HIV-1 Tat (HIV), Tat-derived peptides, Penetratin, VP derived or analog peptides, Pestivirus Erns, HSV, VP (Herpes simplex), MAP, KALA or protein transduction domains (PTDs), PpT620, prolin-rich peptides, arginine-rich peptides, lysine-rich peptides, MPG-peptide(s), Pep-1 , L-oligomers, Calcitonin peptide(s), Antennapedia- derived peptides (particularly from Drosophila antennapedia), pAntp, plsl, FGF, Lactoferrin, Transportan, Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, histones, cationic polysaccharides, for example chitosan, polybrene, cationic polymers, e.g. polyethyleneimine (PEI), cationic lipids, e.g. DOTMA: [1-(2,3- sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE: Dioleyl phosphatidylethanolamine, DOSPA, DODAB, DOIC, DMEPC, DOGS: Dioctadecylamidoglicylspermin, DIMRI: Dimyristooxypropyl dimethyl hydroxyethyl ammonium bromide, DOTAP: dioleoyloxy-3-(trimethylammonio)propane, DC-6-14: O,O-ditetradecanoyl-N-. alpha. - trimethylammonioacetyl)diethanolamine chloride, CLIP 1 : rac-[(2,3- dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium chloride, CLIP6: rac-[2(2,3- dihexadecyloxypropyloxymethyloxy)ethyl]-trimethylammonium, CLIP9: rac-[2(2,3- dihexadecyloxypropyloxysuccinyloxy)ethyl]-trimethylammonium, oligofectamine, or cationic or polycationic polymers, e.g. modified polyaminoacids, such as beta-aminoacid- polymers or reversed polyamides, etc., modified polyethylenes, such as PVP (poly(N- ethyl-4-vinylpyridinium bromide)), etc., modified acrylates, such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc., modified amidoamines such as pAMAM (poly(amidoamine)), etc., modified polybetaminoester (PBAE), such as diamine end modified 1 ,4 butanediol diacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such as polypropylamine dendrimers or pAMAM based dendrimers, etc., polyimine(s), such as PEI: poly(ethyleneimine), poly(propyleneimine), etc., polyallylamine, sugar backbone based polymers, such as cyclodextrin based polymers, dextran based polymers, chitosan, etc., silan backbone based polymers, such as PMOXA-PDMS copolymers, etc., blockpolymers consisting of a combination of one or more cationic blocks (e.g. selected from a cationic polymer as mentioned above) and of one or more hydrophilic or hydrophobic blocks (e.g. polyethyleneglycole), etc.
[0257] In other embodiments the RNA (e.g., mRNA) vaccine is not associated with a cationic or polycationic compounds.
[0258] Other examples of suitable lipid nanoparticle formulations are provided in US 10,702,600, the contents of which are hereby incorporated by reference.
[0259] The lipid nanoparticles described herein may be made in a sterile environment.
[0260] 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.
[0261] 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 some embodiments, polymer conjugates with the polynucleotides of the present disclosure may be made using the methods and/or segmented polymeric reagents described in U.S. Patent Application No. 20130072709, the contents of which are herein incorporated by reference in its entirety. In some embodiments, the polymer conjugate may have pendant side groups comprising ring moieties such as, but not limited to, the polymer conjugates described in U.S. Patent Publication No. US20130196948, the contents which are herein incorporated by reference in its entirety.
[0262] The nanoparticle formulations may comprise a conjugate to enhance the delivery of nanoparticles of the present disclosure in a subject. Further, the conjugate may inhibit phagocytic clearance of the nanoparticles in a subject. In one embodiment, the conjugate may be a “self” peptide designed from the human membrane protein CD47 (e.g., the “self” particles described by Rodriguez et al. (Science 2013 339, 971-975), herein incorporated by reference in its entirety). As shown by Rodriguez et al., the self peptides delayed macrophage-mediated clearance of nanoparticles which enhanced delivery of the nanoparticles. In another embodiment, the conjugate may be the membrane protein CD47 (e.g., see Rodriguez et al. Science 2013 339, 971-975, herein incorporated by reference in its entirety). Rodriguez et al. showed that, similarly to “self” peptides, CD47 can increase the circulating particle ratio in a subject as compared to scrambled peptides and PEG coated nanoparticles.
[0263] In some embodiments, the RNA (e.g., mRNA) vaccines of the present disclosure are formulated in nanoparticles which comprise a conjugate to enhance the delivery of the nanoparticles of the present disclosure in a subject. The conjugate may be the CD47 membrane or the conjugate may be derived from the CD47 membrane protein, such as the “self” peptide described previously. In some embodiments, the nanoparticle may comprise PEG and a conjugate of CD47 or a derivative thereof. In some embodiments, the nanoparticle may comprise both the “self” peptide described above and the membrane protein CD47. [0264] In some embodiments, RNA (e.g., mRNA) vaccine pharmaceutical compositions comprising the polynucleotides of the present disclosure and a conjugate that may have a degradable linkage. Non-limiting examples of conjugates include an aromatic moiety comprising an ionizable hydrogen atom, a spacer moiety, and a water- soluble polymer. As a non-limiting example, pharmaceutical compositions comprising a conjugate with a degradable linkage and methods for delivering such pharmaceutical compositions are described in U.S. Patent Publication No. US20130184443, the contents of which are herein incorporated by reference in their entirety.
[0265] 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, phtoglycogen octenyl succinate, phytoglycogen beta-dextrin, anhydride-modified phytoglycogen beta-dextrin. (See e.g., International Publication No. W02012109121 ; the contents of which are herein incorporated by reference in their entirety).
[0266] 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. The hydrophilic coatings may help to deliver nanoparticles with larger payloads such as, but not limited to, RNA (e.g., mRNA) vaccines within the central nervous system. 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.
[0267] 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 Publication No. US20130210991 , the contents of which are herein incorporated by reference in their entirety.
[0268] In some embodiments, the lipid nanoparticles of the present disclosure may be hydrophobic polymer particles. [0269] Lipid nanoparticle formulations may be improved by replacing the cationic lipid with a biodegradable cationic lipid which is known as a rapidly eliminated lipid nanoparticle (reLNP). Ionizable cationic lipids, such as, but not limited to, DLinDMA, DLin- KC2-DMA, and DLin-MC3-DMA, have been shown to accumulate in plasma and tissues over time and may be a potential source of toxicity. The rapid metabolism of the rapidly eliminated lipids can improve the tolerability and therapeutic index of the lipid nanoparticles by an order of magnitude from a 1 mg/kg dose to a 10 mg/kg dose in rat. Inclusion of an enzymatically degraded ester linkage can improve the degradation and metabolism profile of the cationic component, while still maintaining the activity of the reLNP formulation. The ester linkage can be internally located within the lipid chain or it may be terminally located at the terminal end of the lipid chain. The internal ester linkage may replace any carbon in the lipid chain.
[0270] In some embodiments, the internal ester linkage may be located on either side of the saturated carbon.
[0271] In some embodiments, an immune response may be elicited by delivering a lipid nanoparticle which may include a nanospecies, a polymer and an immunogen. (U.S. Publication No. 20120189700 and International Publication No. W02012099805; each of which is herein incorporated by reference in their entirety). The polymer may encapsulate the nanospecies or partially encapsulate the nanospecies. The immunogen may be a recombinant protein, a modified RNA and/or a polynucleotide described herein. In some embodiments, the lipid nanoparticle may be formulated for use in a vaccine such as, but not limited to, against a pathogen.
[0272] Lipid nanoparticles may be engineered to alter the surface properties of particles so the lipid nanoparticles may penetrate the mucosal barrier. Mucus is located on mucosal tissue such as, but not limited to, oral (e.g., the buccal and esophageal membranes and tonsil tissue), ophthalmic, gastrointestinal (e.g., stomach, small intestine, large intestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal, tracheal and bronchial membranes), genital (e.g., vaginal, cervical and urethral membranes). Nanoparticles larger than 10-200 nm which are preferred for higher drug encapsulation efficiency and the ability to provide the sustained delivery of a wide array of drugs have been thought to be too large to rapidly diffuse through mucosal barriers. Mucus is continuously secreted, shed, discarded or digested and recycled so most of the trapped particles may be removed from the mucosa tissue within seconds or within a few hours. Large polymeric nanoparticles (200 nm-500 nm in diameter) which have been coated densely with a low molecular weight polyethylene glycol (PEG) diffused through mucus only 4 to 6-fold lower than the same particles diffusing in water (Lai et al. PNAS 2007 104(5): 1482-487; Lai et al. Adv Drug Deliv Rev. 2009 61 (2): 158-171 ; each of which is herein incorporated by reference in their entirety). The transport of nanoparticles may be determined using rates of permeation and/or fluorescent microscopy techniques including, but not limited to, fluorescence recovery after photobleaching (FRAP) and high resolution multiple particle tracking (MPT). As a non-limiting example, compositions which can penetrate a mucosal barrier may be made as described in U.S. Pat. No. 8,241 ,670 or International Patent Publication No. WO2013110028, the contents of each of which are herein incorporated by reference in its entirety.
[0273] The lipid nanoparticle engineered to penetrate mucus may comprise a polymeric material (i.e. a polymeric core) and/or a polymer-vitamin conjugate and/or a tri-block copolymer. The polymeric material may include, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes), polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. The polymeric material may be biodegradable and/or biocompatible. Non-limiting examples of biocompatible polymers are described in International Patent Publication No. WO2013116804, the contents of which are herein incorporated by reference in their entirety. The polymeric material may additionally be irradiated. As a non-limiting example, the polymeric material may be gamma irradiated (see e.g., International App. No. WO201282165, herein incorporated by reference in its entirety). Non-limiting examples of specific polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co- glycolic acid) (PLLGA), poly(D.L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide- co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co- PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxides (PEG), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose, polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) and copolymers and mixtures thereof, polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), PEG-PLGA-PEG and trimethylene carbonate, polyvinylpyrrolidone. The lipid nanoparticle may be coated or associated with a co-polymer such as, but not limited to, a block copolymer (such as a branched polyether-polyamide block copolymer described in International Publication No. WO2013012476, herein incorporated by reference in its entirety), and (poly(ethylene glycol))-(poly(propylene oxide))-(poly(ethylene glycol)) triblock copolymer (see e.g., U.S. Publication 20120121718 and U.S. Publication 20100003337 and U.S. Pat. No. 8,263,665, the contents of each of which is herein incorporated by reference in their entirety). The co-polymer may be a polymer that is generally regarded as safe (GRAS) and the formation of the lipid nanoparticle may be in such a way that no new chemical entities are created. For example, the lipid nanoparticle may comprise poloxamers coating PLGA nanoparticles without forming new chemical entities which are still able to rapidly penetrate human mucus (Yang et al. Angew. Chem. Int. Ed. 2011 50:2597-2600; the contents of which are herein incorporated by reference in their entirety). A non-limiting scalable method to produce nanoparticles which can penetrate human mucus is described by Xu et al. (see, e.g., J Control Release 2013, 170(2):279-86; the contents of which are herein incorporated by reference in their entirety).
[0274] The vitamin of the polymer-vitamin conjugate may be vitamin E. The vitamin portion of the conjugate may be substituted with other suitable components such as, but not limited to, vitamin A, vitamin E, other vitamins, cholesterol, a hydrophobic moiety, or a hydrophobic component of other surfactants (e.g., sterol chains, fatty acids, hydrocarbon chains and alkylene oxide chains). [0275] The lipid nanoparticle engineered to penetrate mucus may include surface altering agents such as, but not limited to, polynucleotides, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as for example dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol and poloxamer), mucolytic agents (e.g., N-acetylcysteine, mugwort, bromelain, papain, clerodendrum, acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin 34 dornase alfa, neltenexine, erdosteine) and various DNases including rhDNase. The surface altering agent may be embedded or enmeshed in the particle's surface or disposed (e.g., by coating, adsorption, covalent linkage, or other process) on the surface of the lipid nanoparticle, (see e.g., U.S. Publication 20100215580 and U.S. Publication 20080166414 and US20130164343; the contents of each of which are herein incorporated by reference in their entirety).
[0276] In some embodiments, the mucus penetrating lipid nanoparticles may comprise at least one polynucleotide described herein. The polynucleotide may be encapsulated in the lipid nanoparticle and/or disposed on the surface of the particle. The polynucleotide may be covalently coupled to the lipid nanoparticle. Formulations of mucus penetrating lipid nanoparticles may comprise a plurality of nanoparticles. Further, the formulations may contain particles which may interact with the mucus and alter the structural and/or adhesive properties of the surrounding mucus to decrease mucoadhesion, which may increase the delivery of the mucus penetrating lipid nanoparticles to the mucosal tissue.
[0277] In some embodiments, the mucus penetrating lipid nanoparticles may be a hypotonic formulation comprising a mucosal penetration enhancing coating. The formulation may be hypotonice for the epithelium to which it is being delivered. Nonlimiting examples of hypotonic formulations may be found in International Patent Publication No. WO2013110028, the contents of which are herein incorporated by reference in their entirety.
[0278] In some embodiments, in order to enhance the delivery through the mucosal barrier the RNA (e.g., mRNA) vaccine formulation may comprise or be a hypotonic solution.
[0279] Hypotonic solutions were found to increase the rate at which mucoinert particles such as, but not limited to, mucus-penetrating particles, were able to reach the vaginal epithelial surface (see e.g., Ensign et al. Biomaterials 2013 34(28) :6922-9, the contents of which are herein incorporated by reference in their entirety).
[0280] In some embodiments, the RNA (e.g., mRNA) vaccine is formulated as a lipoplex, such as, without limitation, the ATUPLEX™ system, the DACC system, the DBTC system and other siRNA-lipoplex technology from Silence Therapeutics (London, United Kingdom), STEMFECT™ from STEMGENT® (Cambridge, Mass.), and polyethylenimine (PEI) or protamine-based targeted and non-targeted delivery of nucleic acids acids (Aleku et al. Cancer Res. 2008 68:9788-9798; Strumberg et al. Int J Clin Pharmacol Ther 2012 50:76-78; Santel et al., Gene Ther 2006 13:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther. 2010 23:334- 344; Kaufmann et al. Microvasc Res 2010 80:286-293Weide et al. J Immunother. 2009 32:498-507; Weide et al. J Immunother. 2008 31 :180-188; Pascolo Expert Opin. Biol. Ther. 4:1285-1294; Fotin-Mleczek et al., 2011 J. Immunother. 34:1-15; Song et al., Nature Biotechnol. 2005, 23:709-717; Peer et al., Proc Natl Acad Sci USA. 2007 6; 104:4095- 4100; deFougerolles Hum Gene Ther. 2008 19:125-132, the contents of each of which are incorporated herein by reference in their entirety).
[0281] In some embodiments, the RNA (e.g., mRNA) vaccine is formulated as a solid lipid nanoparticle. A solid lipid nanoparticle (SLN) may be spherical with an average diameter between 10 to 1000 nm. SLN possess a solid lipid core matrix that can solubilize lipophilic molecules and may be stabilized with surfactants and/or emulsifiers. In some embodiments, the lipid nanoparticle may be a self-assembly lipid-polymer nanoparticle (see Zhang et al., ACS Nano, 2008, 2 (8), pp 1696-1702; the contents of which are herein incorporated by reference in their entirety). As a non-limiting example, the SLN may be the SLN described in International Patent Publication No. W02013105101 , the contents of which are herein incorporated by reference in their entirety. As another non-limiting example, the SLN may be made by the methods or processes described in International Patent Publication No. W02013105101 , the contents of which are herein incorporated by reference in their entirety.
[0282] In some embodiments, the RNA (e.g., mRNA) vaccines of the present disclosure can be formulated for controlled release and/or targeted delivery. As used herein, “controlled release” refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to effect a therapeutic outcome. In some embodiments, the RNA (e.g., RNA) vaccines may be encapsulated into a delivery agent described herein and/or known in the art for controlled release and/or targeted delivery. As used herein, the term “encapsulate” means to enclose, surround or encase. As it relates to the formulation of the compounds of the disclosure, encapsulation may be substantial, complete or partial. The term “substantially encapsulated” means that at least greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9 or greater than 99.999% of the pharmaceutical composition or compound of the disclosure may be enclosed, surrounded or encased within the delivery agent. “Partially encapsulation” means that less than 10, 10, 20, 30, 4050 or less of the pharmaceutical composition or compound of the disclosure may be enclosed, surrounded or encased within the delivery agent. Advantageously, encapsulation may be determined by measuring the escape or the activity of the pharmaceutical composition or compound of the disclosure using fluorescence and/or electron micrograph. For example, at least 1 , 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the pharmaceutical composition or compound of the disclosure are encapsulated in the delivery agent.
[0283] In some embodiments, the controlled release formulation may include, but is not limited to, tri-block co-polymers. As a non-limiting example, the formulation may include two different types of tri-block co-polymers (International Pub. No. W02012131104 and W02012131106, the contents of each of which are incorporated herein by reference in their entirety).
[0284] In some embodiments, the RNA (e.g., mRNA) vaccines may be encapsulated into a lipid nanoparticle or a rapidly eliminated lipid nanoparticle and the lipid nanoparticles or a rapidly eliminated lipid nanoparticle may then be encapsulated into a polymer, hydrogel and/or surgical sealant described herein and/or known in the art. As a non-limiting example, the polymer, hydrogel or surgical sealant may be PLGA, ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, Fla.), HYLENEX® (Halozyme Therapeutics, San Diego Calif.), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, Ga.), TISSELL® (Baxter International, Inc Deerfield, III.), PEG-based sealants, and COSEAL® (Baxter International, Inc Deerfield, III.).
[0285] In some embodiments, the lipid nanoparticle may be encapsulated into any polymer known in the art which may form a gel when injected into a subject. As another non-limiting example, the lipid nanoparticle may be encapsulated into a polymer matrix which may be biodegradable.
[0286] In some embodiments, the RNA (e.g., mRNA) vaccine formulation for controlled release and/or targeted delivery may also include at least one controlled release coating. Controlled release coatings include, but are not limited to, OPADRY®, polyvinylpyrrolidone/vinyl acetate copolymer, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, EUDRAGIT RL®, EUDRAGIT RS® and cellulose derivatives such as ethylcellulose aqueous dispersions (AQUACOAT® and SURELEASE®).
[0287] In some embodiments, the RNA (e.g., mRNA) vaccine controlled release and/or targeted delivery formulation may comprise at least one degradable polyester which may contain polycationic side chains. Degradeable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof. In some embodiments, the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.
[0288] In some embodiments, the RNA (e.g., RNA) vaccine controlled release and/or targeted delivery formulation comprising at least one polynucleotide may comprise at least one PEG and/or PEG related polymer derivatives as described in U.S. Pat. No. 8,404,222, the contents of which are incorporated herein by reference in their entirety.
[0289] In some embodiments, the RNA (e.g., mRNA) vaccine controlled release delivery formulation comprising at least one polynucleotide may be the controlled release polymer system described in US20130130348, the contents of which are incorporated herein by reference in their entirety.
[0290] In some embodiments, the RNA (e.g., mRNA) vaccines of the present disclosure may be encapsulated in a therapeutic nanoparticle, referred to herein as “therapeutic nanoparticle RNA (e.g., mRNA) vaccines.” Therapeutic nanoparticles may be formulated by methods described herein and known in the art such as, but not limited to, International Pub Nos. WQ2010005740, WQ2010030763, WQ2010005721 , WQ2010005723, WQ2012054923, U.S. Publication Nos. US20110262491 ,
US20100104645, US20100087337, US20100068285, US20110274759,
US20100068286, US20120288541 , US20130123351 and US20130230567 and U.S. Pat. Nos. 8,206,747, 8,293,276, 8,318,208 and 8,318,211 ; the contents of each of which are herein incorporated by reference in their entirety. In some embodiments, therapeutic polymer nanoparticles may be identified by the methods described in US Pub No. US20120140790, the contents of which are herein incorporated by reference in their entirety.
[0291] In some embodiments, the therapeutic nanoparticle RNA (e.g., mRNA) vaccine may be formulated for sustained release. As used herein, “sustained release” refers to a pharmaceutical composition or compound that conforms to a release rate over a specific period of time. The period of time may include, but is not limited to, hours, days, weeks, months and years. As a non-limiting example, the sustained release nanoparticle may comprise a polymer and a therapeutic agent such as, but not limited to, the polynucleotides of the present disclosure (see International Pub No. 2010075072 and US Pub No. US20100216804, US20110217377 and US20120201859, the contents of each of which are incorporated herein by reference in their entirety). In another non-limiting example, the sustained release formulation may comprise agents which permit persistent bioavailability such as, but not limited to, crystals, macromolecular gels and/or particulate suspensions (see U.S. Patent Publication No US20130150295, the contents of each of which are incorporated herein by reference in their entirety).
[0292] In some embodiments, the therapeutic nanoparticle RNA (e.g., mRNA) vaccines may be formulated to be target specific. As a non-limiting example, the therapeutic nanoparticles may include a corticosteroid (see International Pub. No. WO2011084518, the contents of which are incorporated herein by reference in their entirety). As a nonlimiting example, the therapeutic nanoparticles may be formulated in nanoparticles described in International Pub No. WO2008121949, W02010005726, W02010005725, WO2011084521 and US Pub No. US20100069426, US20120004293 and US20100104655, the contents of each of which are incorporated herein by reference in their entirety.
[0293] In some embodiments, the nanoparticles of the present disclosure may comprise a polymeric matrix. As a non-limiting example, the nanoparticle may comprise two or more polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly (orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) or combinations thereof.
[0294] In some embodiments, the therapeutic nanoparticle comprises a diblock copolymer. In some embodiments, the diblock copolymer may include PEG in combination with a polymer such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly (orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) or combinations thereof. In yet another embodiment, the diblock copolymer may be a high- X diblock copolymer such as those described in International Patent Publication No. W02013120052, the contents of which are incorporated herein by reference in their entirety.
[0295] As a non-limiting example the therapeutic nanoparticle comprises a PLGA-PEG block copolymer (see U.S. Publication No. US20120004293 and U.S. Pat. No. 8,236,330, each of which is herein incorporated by reference in their entirety). In another non-limiting example, the therapeutic nanoparticle is a stealth nanoparticle comprising a diblock copolymer of PEG and PLA or PEG and PLGA (see U.S. Pat. No. 8,246,968 and International Publication No. WO2012166923, the contents of each of which are herein incorporated by reference in their entirety). In yet another non-limiting example, the therapeutic nanoparticle is a stealth nanoparticle or a target-specific stealth nanoparticle as described in U.S. Patent Publication No. US20130172406, the contents of which are herein incorporated by reference in their entirety.
[0296] In some embodiments, the therapeutic nanoparticle may comprise a multiblock copolymer (see e.g., U.S. Pat. Nos. 8,263,665 and 8,287,910 and U.S. Patent Pub. No. US20130195987, the contents of each of which are herein incorporated by reference in their entirety).
[0297] In yet another non-limiting example, the lipid nanoparticle comprises the block copolymer PEG-PLGA-PEG (see e.g., the thermosensitive hydrogel (PEG-PLGA-PEG) was used as a TGF-beta1 gene delivery vehicle in Lee et al. Thermosensitive Hydrogel as a Tgf-pi Gene Delivery Vehicle Enhances Diabetic Wound Healing. Pharmaceutical Research, 2003 20(12): 1995-2000; as a controlled gene delivery system in Li et al. Controlled Gene Delivery System Based on Thermosensitive Biodegradable Hydrogel. Pharmaceutical Research 2003 20(6): 884-888; and Chang et al., Non-ionic amphiphilic biodegradable PEG-PLGA-PEG copolymer enhances gene delivery efficiency in rat skeletal muscle. J Controlled Release. 2007 118:245-253, the contents of each of which are herein incorporated by reference in their entirety). The RNA (e.g., mRNA) vaccines of the present disclosure may be formulated in lipid nanoparticles comprising the PEG- PLGA-PEG block copolymer.
[0298] In some embodiments, the therapeutic nanoparticle may comprise a multiblock copolymer (see e.g., U.S. Pat. Nos. 8,263,665 and 8,287,910 and U.S. Patent Pub. No. US20130195987, the contents of each of which are herein incorporated by reference in their entirety).
[0299] In some embodiments, the block copolymers described herein may be included in a polyion complex comprising a non-polymeric micelle and the block copolymer, (see e.g., U.S. Publication No. 20120076836, the contents of which are herein incorporated by reference in their entirety).
[0300] In some embodiments, the therapeutic nanoparticle may comprise at least one acrylic polymer. Acrylic polymers include but are not limited to, acrylic acid, methacrylic acid, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates and combinations thereof.
[0301] In some embodiments, the therapeutic nanoparticles may comprise at least one poly(vinyl ester) polymer. The poly(vinyl ester) polymer may be a copolymer such as a random copolymer. As a non-limiting example, the random copolymer may have a structure such as those described in International Application No. WO2013032829 or U.S. Patent Publication No US20130121954, the contents of each of which are herein incorporated by reference in their entirety. In some embodiments, the poly(vinyl ester) polymers may be conjugated to the polynucleotides described herein. [0302] In some embodiments, the therapeutic nanoparticle may comprise at least one diblock copolymer. The diblock copolymer may be, but it not limited to, a poly(lactic) acid- poly(ethylene)glycol copolymer (see, e.g., International Patent Publication No. WO2013044219, the contents of which are herein incorporated by reference in their entirety).
[0303] As a non-limiting example, the therapeutic nanoparticle may be used to treat cancer (see International publication No. WO2013044219, the contents of which are herein incorporated by reference in their entirety).
[0304] In some embodiments, the therapeutic nanoparticles may comprise at least one cationic polymer described herein and/or known in the art.
[0305] In some embodiments, the therapeutic nanoparticles may comprise at least one amine-containing polymer such as, but not limited to polylysine, polyethylene imine, poly(amidoamine) dendrimers, poly(beta-amino esters) (see, e.g., U.S. Pat. No. 8,287,849, the contents of which are herein incorporated by reference in their entirety) and combinations thereof.
[0306] In some embodiments, the nanoparticles described herein may comprise an amine cationic lipid such as those described in International Patent Application No. WO2013059496, the contents of which are herein incorporated by reference in their entirety. In some embodiments, the cationic lipids may have an amino-amine or an aminoamide moiety.
[0307] In some embodiments, the therapeutic nanoparticles may comprise at least one degradable polyester which may contain polycationic side chains. Degradeable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof. In some embodiments, the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.
[0308] In some embodiments, the synthetic nanocarriers may be formulated for targeted release. In some embodiments, the synthetic nanocarrier is formulated to release the polynucleotides at a specified pH and/or after a desired time interval. As a non-limiting example, the synthetic nanoparticle may be formulated to release the RNA (e.g., mRNA) vaccines after 24 hours and/or at a pH of 4.5 (see International Publication Nos. W02010138193 and W02010138194 and US Pub Nos. US20110020388 and US20110027217, each of which is herein incorporated by reference in their entireties).
[0309] In some embodiments, the synthetic nanocarriers may be formulated for controlled and/or sustained release of the polynucleotides described herein. As a nonlimiting example, the synthetic nanocarriers for sustained release may be formulated by methods known in the art, described herein and/or as described in International Pub No. W02010138192 and US Pub No. 20100303850, each of which is herein incorporated by reference in their entirety.
[0310] In some embodiments, the RNA (e.g., mRNA) vaccine may be formulated for controlled and/or sustained release wherein the formulation comprises at least one polymer that is a crystalline side chain (CYSC) polymer. CYSC polymers are described in U.S. Pat. No. 8,399,007, herein incorporated by reference in its entirety.
[0311] In some embodiments, the synthetic nanocarrier may be formulated for use as a vaccine. In some embodiments, the synthetic nanocarrier may encapsulate at least one polynucleotide which encode at least one antigen. As a non-limiting example, the synthetic nanocarrier may include at least one antigen and an excipient for a vaccine dosage form (see International Publication No. WO2011150264 and U.S. Publication No. US20110293723, the contents of each of which are herein incorporated by reference in their entirety). As another non-limiting example, a vaccine dosage form may include at least two synthetic nanocarriers with the same or different antigens and an excipient (see International Publication No. WO2011150249 and U.S. Publication No. US20110293701 , the contents of each of which are herein incorporated by reference in their entirety). The vaccine dosage form may be selected by methods described herein, known in the art and/or described in International Publication No. WO2011150258 and U.S. Publication No. US20120027806, the contents of each of which are herein incorporated by reference in their entirety).
[0312] In some embodiments, the synthetic nanocarrier may comprise at least one polynucleotide which encodes at least one adjuvant. As non-limiting example, the adjuvant may comprise dimethyldioctadecylammonium-bromide, dimethyldioctadecylammonium-chloride, dimethyldioctadecylammonium-phosphate or dimethyldioctadecylammonium-acetate (DDA) and an apolar fraction or part of said apolar fraction of a total lipid extract of a mycobacterium (see, e.g., U.S. Pat. No. 8,241 ,610, the content of which is herein incorporated by reference in its entirety). In some embodiments, the synthetic nanocarrier may comprise at least one polynucleotide and an adjuvant. As a non-limiting example, the synthetic nanocarrier comprising and adjuvant may be formulated by the methods described in International Publication No. WO2011150240 and U.S. Publication No. US20110293700, the contents of each of which are herein incorporated by reference in their entirety.
[0313] In some embodiments, the synthetic nanocarrier may encapsulate at least one polynucleotide that encodes a peptide, fragment or region from a virus. As a non-limiting example, the synthetic nanocarrier may include, but is not limited to, any of the nanocarriers described in International Publication No. WO2012024621, WO201202629, WO2012024632 and U.S. Publication No. US20120064110, US20120058153 and US20120058154, the contents of each of which are herein incorporated by reference in their entirety.
[0314] In some embodiments, the synthetic nanocarrier may be coupled to a polynucleotide which may be able to trigger a humoral and/or cytotoxic T lymphocyte (CTL) response (see, e.g., International Publication No. WO2013019669, the contents of which are herein incorporated by reference in their entirety).
[0315] In some embodiments, the RNA (e.g., mRNA) vaccine may be encapsulated in, linked to and/or associated with zwitterionic lipids. Non-limiting examples of zwitterionic lipids and methods of using zwitterionic lipids are described in U.S. Patent Publication No. US20130216607, the contents of which are herein incorporated by reference in their entirety.
[0316] In some embodiment, the zwitterionic lipids may be used in the liposomes and lipid nanoparticles described herein.
[0317] In some embodiments, the RNA (e.g., mRNA) vaccine may be formulated in colloid nanocarriers as described in U.S. Patent Publication No. US20130197100, the contents of which are herein incorporated by reference in their entirety.
[0318] In some embodiments, the nanoparticle may be optimized for oral administration. The nanoparticle may comprise at least one cationic biopolymer such as, but not limited to, chitosan or a derivative thereof. As a non-limiting example, the nanoparticle may be formulated by the methods described in U.S. Publication No. 20120282343, the contents of which are herein incorporated by reference in their entirety.
[0319] In some embodiments, LNPs comprise the lipid KL52 (an amino-lipid disclosed in U.S. Application Publication No. 2012/0295832, the contents of which are herein incorporated by reference in their entirety. Activity and/or safety (as measured by examining one or more of ALT/AST, white blood cell count and cytokine induction, for example) of LNP administration may be improved by incorporation of such lipids. LNPs comprising KL52 may be administered intravenously and/or in one or more doses. In some embodiments, administration of LNPs comprising KL52 results in equal or improved mRNA and/or protein expression as compared to LNPs comprising MC3.
[0320] In some embodiments, RNA (e.g., mRNA) vaccine may be delivered using smaller LNPs. Such particles may comprise a diameter from below 0.1 urn up to 100 nm such as, but not limited to, less than 0.1 urn, less than 1.0 urn, less than 5 urn, less than 10 urn, less than 15 urn, less than 20 urn, less than 25 urn, less than 30 urn, less than 35 urn, less than 40 urn, less than 50 urn, less than 55 urn, less than 60 urn, less than 65 urn, less than 70 urn, less than 75 urn, less than 80 urn, less than 85 urn, less than 90 urn, less than 95 urn, less than 100 urn, less than 125 urn, less than 150 urn, less than 175 urn, less than 200 urn, less than 225 urn, less than 250 urn, less than 275 urn, less than 300 urn, less than 325 urn, less than 350 urn, less than 375 urn, less than 400 urn, less than 425 urn, less than 450 urn, less than 475 urn, less than 500 urn, less than 525 urn, less than 550 urn, less than 575 urn, less than 600 urn, less than 625 urn, less than 650 urn, less than 675 urn, less than 700 urn, less than 725 urn, less than 750 urn, less than 775 urn, less than 800 urn, less than 825 urn, less than 850 urn, less than 875 urn, less than 900 urn, less than 925 urn, less than 950 urn, less than 975 urn, or less than 1000 urn.
[0321] In some embodiments, RNA (e.g., mRNA RNA) vaccines may be delivered using smaller LNPs, which may comprise a diameter from about 1 nm to about 100 nm, from about 1 nm to about 10 nm, about 1 nm to about 20 nm, from about 1 nm to about 30 nm, from about 1 nm to about 40 nm, from about 1 nm to about 50 nm, from about 1 nm to about 60 nm, from about 1 nm to about 70 nm, from about 1 nm to about 80 nm, from about 1 nm to about 90 nm, from about 5 nm to about from 100 nm, from about 5 nm to about 10 nm, about 5 nm to about 20 nm, from about 5 nm to about 30 nm, from about 5 nm to about 40 nm, from about 5 nm to about 50 nm, from about 5 nm to about 60 nm, from about 5 nm to about 70 nm, from about 5 nm to about 80 nm, from about 5 nm to about 90 nm, about 10 to about 50 nm, from about 20 to about 50 nm, from about 30 to about 50 nm, from about 40 to about 50 nm, from about 20 to about 60 nm, from about 30 to about 60 nm, from about 40 to about 60 nm, from about 20 to about 70 nm, from about 30 to about 70 nm, from about 40 to about 70 nm, from about 50 to about 70 nm, from about 60 to about 70 nm, from about 20 to about 80 nm, from about 30 to about 80 nm, from about 40 to about 80 nm, from about 50 to about 80 nm, from about 60 to about 80 nm, from about 20 to about 90 nm, from about 30 to about 90 nm, from about 40 to about 90 nm, from about 50 to about 90 nm, from about 60 to about 90 nm and/or from about 70 to about 90 nm.
[0322] In some embodiments, such LNPs are synthesized using methods comprising microfluidic mixers. Examples of microfluidic mixers may include, but are not limited to, a slit interdigital micromixer including, but not limited to those manufactured by Microinnova (Allerheiligen bei Wildon, Austria) and/or a staggered herringbone micromixer (SHM) (Zhigaltsev, I. V. et al., Bottom-up design and synthesis of limit size lipid nanoparticle systems with aqueous and triglyceride cores using millisecond microfluidic mixing have been published (Langmuir. 2012. 28:3633-40; Belliveau, N. M. et al., Microfluidic synthesis of highly potent limit-size lipid nanoparticles for in vivo delivery of siRNA. Molecular Therapy-Nucleic Acids. 2012. 1 :e37; Chen, D. et al., Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation. J Am Chem Soc. 2012. 134(16):6948-51 , the contents of each of which are herein incorporated by reference in their entirety). In some embodiments, methods of LNP generation comprising SHM, further comprise the mixing of at least two input streams wherein mixing occurs by microstructure-induced chaotic advection (MICA). According to this method, fluid streams flow through channels present in a herringbone pattern causing rotational flow and folding the fluids around each other. This method may also comprise a surface for fluid mixing wherein the surface changes orientations during fluid cycling. Methods of generating LNPs using SHM include those disclosed in U.S. Application Publication Nos. 2004/0262223 and 2012/0276209, the contents of each of which are herein incorporated by reference in their entirety.
[0323] In some embodiments, the RNA (e.g., mRNA) vaccine of the present disclosure may be formulated in lipid nanoparticles created using a micromixer such as, but not limited to, a Slit Interdigital Microstructured Mixer (SIMM-V2) or a Standard Slit Interdigital Micro Mixer (SSIMM) or Caterpillar (CPMM) or Impinging-jet (IJMM) from the Institut fiir Mikrotechnik Mainz GmbH, Mainz Germany).
[0324] In some embodiments, the RNA (e.g., mRNA) vaccines of the present disclosure may be formulated in lipid nanoparticles created using microfluidic technology (see, e.g., Whitesides, George M. The Origins and the Future of Microfluidics. Nature, 2006 442: 368-373; and Abraham et al. Chaotic Mixer for Microchannels. Science, 2002 295: 647-651 ; each of which is herein incorporated by reference in its entirety). As a nonlimiting example, controlled microfluidic formulation includes a passive method for mixing streams of steady pressure-driven flows in micro channels at a low Reynolds number (see, e.g., Abraham et al. Chaotic Mixer for Microchannels. Science, 2002 295: 647-651 , the contents of which are herein incorporated by reference in their entirety).
[0325] 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), PeerJ, 10:e13083; incorporated herein by reference.
[0326] In some embodiments, the RNA (e.g., mRNA) vaccines of the present disclosure may be formulated in lipid nanoparticles created using a micromixer chip such as, but not limited to, those from Harvard Apparatus (Holliston, Mass.) or Dolomite Microfluidics (Royston, UK). A micromixer chip can be used for rapid mixing of two or more fluid streams with a split and recombine mechanism.
[0327] In some embodiments, the RNA (e.g., mRNA) vaccines of the disclosure may be formulated for delivery using the drug encapsulating microspheres described in International Patent Publication No. WO2013063468 or U.S. Pat. No. 8,440,614, the contents of each of which are herein incorporated by reference in their entirety. The microspheres may comprise a compound of the formula (I), (II), (III), (IV), (V) or (VI) as described in International Patent Publication No. WO2013063468, the contents of which are herein incorporated by reference in their entirety. In some embodiments, the amino acid, peptide, polypeptide, lipids (APPL) are useful in delivering the RNA (e.g., RNA) vaccines of the disclosure to cells (see International Patent Publication No. WO2013063468, the contents of which are herein incorporated by reference in their entirety).
[0328] In some embodiments, the antibody titre produced by the RNA vaccines of the invention is a neutralizing antibody titre. In some embodiments the neutralizing antibody titre is greater than a protein vaccine. In other embodiments the neutralizing antibody titre produced by the RNA vaccines of the invention is greater than an adjuvanted protein vaccine. In yet other embodiments the neutralizing antibody titre produced by the RNA vaccines of the invention is 1 ,000-10,000, 1 ,200-10,000, 1 ,400-10,000, 1 ,500-10,000, 1 ,000-5,000, 1 ,000-4,000, 1 ,800-10,000, 2000-10,000, 2,000-5,000, 2,000-3,000, 2,000- 4,000, 3,000-5,000, 3,000-4,000, or 2,000-2,500. A neutralization titre is typically expressed as the highest serum dilution required to achieve a 50% reduction in the number of plaques.
[0329] 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
[0330] 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. Liposomes are artificially-prepared vesicles which may primarily be composed of a lipid bilayer and may be used as a delivery vehicle for the administration of nutrients and pharmaceutical formulations. Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 and 500 nm in diameter. Liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis. Liposomes may contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations.
[0331] The formation of liposomes may depend on the physicochemical characteristics such as, but not limited to, the pharmaceutical formulation entrapped and the liposomal ingredients, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the entrapped substance and its potential toxicity, any additional processes involved during the application and/or delivery of the vesicles, the optimization size, polydispersity and the shelf-life of the vesicles for the intended application, and the batch-to-batch reproducibility and possibility of large-scale production of safe and efficient liposomal products.
[0332] 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.).
[0333] 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. 19996:271-281 ; Zhang et al. Gene Therapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372; Morrissey et al., Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al., Nature. 2006 441 :111-114; Heyes et al. J Contr Rel. 2005 107:276-287; Semple et al. Nature Biotech. 2010 28:172- 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). The original manufacture method by Wheeler et al. was a detergent dialysis method, which was later improved by Jeffs et al. and is referred to as the spontaneous vesicle formation method. The liposome formulations are composed of 3 to 4 lipid components in addition to the polynucleotide. As an example a liposome can contain, but is not limited to, 55% cholesterol, 20% disteroylphosphatidyl choline (DSPC), 10% PEG-S-DSG, and 15% 1 ,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), as described by Jeffs et al. As another example, certain liposome formulations may contain, but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, where the cationic lipid can be 1 ,2-distearloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or 1 ,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA), as described by Heyes et al.
[0334] 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.
[0335] In some embodiments, the RNA (e.g., mRNA) vaccine pharmaceutical compositions may be formulated in liposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell, Wash.), neutral DOPC (1 ,2-dioleoyl-sn-glycero-3-phosphocholine) based liposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. Cancer Biology & Therapy 2006 5(12)1708- 1713); herein incorporated by reference in its entirety) and hyaluronan-coated liposomes (Quiet Therapeutics, Israel).
[0336] In some embodiments, the cationic lipid may be a low molecular weight cationic lipid such as those described in U.S. Patent Application No. 20130090372, the contents of which are herein incorporated by reference in their entirety. [0337] In some embodiments, the RNA (e.g., mRNA) vaccines may be formulated in a lipid vesicle, which may have crosslinks between functionalized lipid bilayers.
[0338] 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. 20120178702, 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).
[0339] 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.
Subjects and methods of administration
[0340] The present invention also provides uses of the polynucleotides and compositions of the invention for producing an antigen-specific immune response in a subject. Such methods typically comprise administering a polynucleotide of the invention, preferably formulated in a lipid nanoparticle as described herein, to a subject in need thereof.
[0341] Accordingly, the invention further provides compositions comprising the polynucleotides (RNA) defined herein, and the use of such RNA in immunogenic or vaccine compositions in the treatment or prevention of P. gulae infection.
[0342] 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.
[0343] As used herein, the terms “immunostimulating composition”, “vaccine composition” and “immunogenic composition” may generally be used interchangeably. [0344] The present invention provides methods and compositions for treating or preventing infection or minimising the likelihood of infection with P. gulae, in an individual in need thereof, the methods comprising administering a vaccine composition of the invention.
[0345] As such, the present invention includes methods and compositions for preventing infection with P. gulae, minimising the likelihood of infection and/or reducing the severity and duration of P. gulae infection in a subject.
[0346] The present invention also provides a method for obtaining an antibody directed to P. gulae, the method comprising administering a chimeric or fusion protein, composition, vaccine or immune stimulating composition of the invention, to a non-human animal, thereby generating antibodies directed to P. gulae in the animal. Preferably the method further comprises isolating the antibody from the animal (eg from the blood of the animal) or from an egg of the animal (eg in the case of generating IgY antibodies from chickens).
[0347] The present invention also provides an antibody preparation comprising an antibody directed to P. gulae, wherein the antibody preparation is obtained by administering a composition, vaccine or immune stimulating composition of the invention, to a non-human animal, thereby generating antibodies directed to P. gulae in the animal, and isolating the antibodies from the animal or egg thereof.
[0348] The antibody directed to P. gulae may be used therapeutically to eliminate or reduce P. gulae infection or prophylactically, to prevent or reduce the severity of P. gulae infection.
[0349] 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, 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.
[0350] 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.
[0351] The vaccine compositions of the invention can be administered to subjects felt to be in greatest need thereof, for example in the context of human patients, to children or the elderly or individuals at risk of exposure to P. gulae. The vaccine compositions of the invention can also be administered to subjects suspected of having or diagnosed with having infection with P. gulae.
[0352] The compositions and methods of the present invention extend equally to uses in both human and/or veterinary medicine, generation of diagnostic agents or the generation of other treatment reagents.
[0353] As used herein, the term “subject” shall be taken to mean any animal including humans, for example a mammal, and in particular, non-human animals. In further examples, the subject may be a veterinary subject, such as a companion animal (cat, dog, guinea pig, and the like).
[0354] As used herein, the terms “subject”, “individual” and “patient” may be used interchangeably.
[0355] The skilled person will be familiar with methods for determining successful vaccination/immunisation with a chimeric or fusion protein or composition as described herein. For example, the skilled person will be familiar with methods for quantifying the antibodies generated following immunisation and/or for quantifying the extent of the humoural (Th2) response induced following immunisation or for quantifying the extent of a Th1 response generated. [0356] In some embodiments, following administration of a polynucleotide or composition of the invention, the subject exhibits a seroconversion rate of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) following the first dose or the second (booster) dose of the vaccine. Seroconversion is the time period during which a specific antibody develops and becomes detectable in the blood. During an infection or immunization, antigens enter the blood, and the immune system begins to produce antibodies in response. Before seroconversion, the antigen itself may or may not be detectable, but antibodies are considered absent. During seroconversion, antibodies are present but not yet detectable. Any time after seroconversion, the antibodies can be detected in the blood, indicating a prior or current infection.
[0357] In some embodiments, a polynucleotide (e.g., RNA) 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.
[0358] In some embodiments of the present disclosure, there is provided methods of inducing an antigen specific immune response in a subject, including administering to a subject a RNA (e.g., mRNA) vaccine as described herein, in an effective amount to produce an antigen specific immune response in a subject. Antigen-specific immune responses in a subject may be determined, in some embodiments, by assaying for antibody titre following administration to the subject of any of the polynucleotide (e.g., mRNA) vaccines of the present disclosure. In some embodiments, the anti-antigenic polypeptide antibody titre produced in the subject is increased by at least 1 log relative to a control. In some embodiments, the anti-antigenic polypeptide antibody titre produced in the subject is increased by 1-3 log relative to a control.
[0359] In some embodiments, the anti-antigenic polypeptide antibody titre produced in a subject is increased at least 2 times relative to a control. In some embodiments, the anti-antigenic polypeptide antibody titre produced in the subject is increased at least 5 times relative to a control. In some embodiments, the anti-antigenic polypeptide antibody titre produced in the subject is increased at least 10 times relative to a control. In some embodiments, the anti-antigenic polypeptide antibody titre produced in the subject is increased 2-10 times relative to a control.
[0360] In some embodiments, the control is an anti-antigenic polypeptide antibody titre produced in a subject who has not been administered a RNA (e.g., mRNA) vaccine of the present disclosure. In some embodiments, the control is an anti-antigenic polypeptide antibody titre produced in a subject who has been administered a live attenuated or inactivated P. gulae vaccine (see, e.g., Ren J. et al. J of Gen. Virol. 2015; 96: 1515-1520), or wherein the control is an anti-antigenic polypeptide antibody titre produced in a subject who has been administered a recombinant or purified P. gulae protein vaccine.
[0361] A polynucleotide (e.g., mRNA) vaccine of the present disclosure is administered to a subject in an effective amount (an amount effective to induce an immune response). In some embodiments, the effective amount is a dose equivalent to an at least 2-fold, at least 4-fold, at least 10-fold, at least 100-fold, at least 1000-fold reduction in the standard of care dose of a recombinant P. gulae protein vaccine, wherein the anti-antigenic polypeptide antibody titre produced in the subject is equivalent to an anti-antigenic polypeptide antibody titre produced in a control subject administered the standard of care dose of a recombinant P. gulae protein vaccine, a purified P. gulae protein vaccine, a live attenuated P. gulae vaccine, an inactivated P. gulae vaccine. In some embodiments, the effective amount is a dose equivalent to 2-1000-fold reduction in the standard of care dose of a recombinant P. gulae protein vaccine, wherein the anti-antigenic polypeptide antibody titre produced in the subject is equivalent to an anti-antigenic polypeptide antibody titre produced in a control subject administered the standard of care dose of a recombinant P. gulae protein vaccine, a purified P. gulae protein vaccine, a live attenuated P. gulae vaccine, or an inactivated P. gulae vaccine.
[0362] 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.
[0363] Dosages of the vaccine composition of the present invention 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.
[0364] 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.
[0365] In some embodiments, the effective amount is a total dose of 25 pg to 1000 pg, or 50 pg to 1000 pg. In some embodiments, the effective amount is a total dose of 100 pg. In some embodiments, the effective amount is a dose of 25 pg administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 100 pg administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 400 pg administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 500 pg administered to the subject a total of two times.
[0366] In some embodiments, the efficacy (or effectiveness) of a polynucleotide (e.g., mRNA) vaccine is greater than 60%. In some embodiments, the efficacy (or effectiveness) of a polynucleotide (e.g., mRNA) vaccine is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%.
[0367] Vaccine efficacy may be assessed using standard analyses (see, e.g., Weinberg et al., J Infect Dis. 2010 Jun. 1 ; 201 (11):1607-10). For example, vaccine efficacy may be measured by double-blind, randomized, clinical controlled trials. Vaccine efficacy may be expressed as a proportionate reduction in disease attack rate (AR) between the unvaccinated (ARU) and vaccinated (ARV) study cohorts and can be calculated from the relative risk (RR) of disease among the vaccinated group with use of the following formulas:
Efficacy = (ARU-ARV)/ARU x 100; and
Efficacy = (1-RR) x 100.
[0368] Similarly, vaccine effectiveness may be assessed using standard analyses (see, e.g., Weinberg et al., J Infect Dis. 2010 Jun. 1 ; 201(11):1607-10). Vaccine effectiveness is an assessment of how a vaccine (which may have already proven to have high vaccine efficacy) reduces disease in a population. This measure can assess the net balance of benefits and adverse effects of a vaccination program, not just the vaccine itself, under natural field conditions rather than in a controlled clinical trial. Vaccine effectiveness is proportional to vaccine efficacy (potency) but is also affected by how well target groups in the population are immunized, as well as by other non-vaccine-related factors that influence the ‘real-world’ outcomes of hospitalizations, ambulatory visits, or costs. For example, a retrospective case control analysis may be used, in which the rates of vaccination among a set of infected cases and appropriate controls are compared. Vaccine effectiveness may be expressed as a rate difference, with use of the odds ratio (OR) for developing infection despite vaccination: Effectiveness = (1-OR) * 100.
[0369] In other embodiments the invention is a composition for or method of vaccinating a subject comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide wherein a dosage of between 10 pg/kg and 400 pg /kg of the nucleic acid vaccine is administered to the subject. In some embodiments the dosage of the RNA polynucleotide is 1-5 pg, 5-10 pg, 10-15 pg, 15-20 pg, 10-25 pg, 20-25 pg, 20- 50 pg, 30-50 pg, 40-50 pg, 40-60 pg, 60-80 pg, 60-100 pg, 50-100 pg, 80-120 pg, 40-120 pg, 40-150 pg, 50-150 pg, 50-200 pg, 80-200 pg, 100-200 pg, 120-250 pg, 150-250 pg, 180-280 pg, 200-300 pg, 50-300 pg, 80-300 pg, 100-300 pg, 40-300 pg, 50-350 pg, 100- 350 pg, 200-350 pg, 300-350 pg, 320-400 pg, 40-380 pg, 40-100 pg, 100-400 pg, 200- 400 pg, or 300-400 pg per dose. In some embodiments, the nucleic acid vaccine is administered to the subject by intradermal or intramuscular injection. In some embodiments, the nucleic acid vaccine is administered to the subject on day zero. In some embodiments, a second dose of the nucleic acid vaccine is administered to the subject on day twenty one.
[0370] In some embodiments, a dosage of at least about 2 micrograms (pg) or at least about 10 pg or at least about 20 pg or at least about 30 pg of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 100 micrograms (pg) of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 50 micrograms (pg) of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 75 micrograms (pg) of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 150 micrograms (pg) of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 400 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 200 micrograms (pg) of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, the RNA polynucleotide accumulates at a 100 fold higher level in the local lymph node in comparison with the distal lymph node.
[0371] Embodiments of the invention provide methods of creating, maintaining or restoring antigenic memory to a P. gulae in an individual or population of individuals comprising administering to said individual or population an antigenic memory booster nucleic acid vaccine comprising (a) at least one RNA polynucleotide, said polynucleotide comprising at least one chemical modification or optionally no nucleotide modification and two or more codon-optimized open reading frames, said open reading frames encoding a set of reference antigenic polypeptides, and (b) optionally a pharmaceutically acceptable carrier or excipient. In some embodiments, the vaccine is administered to the individual via a route selected from the group consisting of intramuscular administration, intradermal administration and subcutaneous administration. In some embodiments, the administering step comprises contacting a muscle tissue of the subject with a device suitable for injection of the composition. In some embodiments, the administering step comprises contacting a muscle tissue of the subject with a device suitable for injection of the composition in combination with electroporation.
[0372] Embodiments of the invention provide methods of vaccinating a subject comprising administering to the subject a single dosage of between 25 pg/kg and 400 pg/kg of a nucleic acid vaccine comprising one or more RNA polynucleotides as described herein, in an effective amount to vaccinate the subject.
[0373] In other embodiments the invention encompasses a method of treating an elderly subject age 60 years or older comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding an antigenic polypeptide as described herein in an effective amount to vaccinate the subject.
[0374] In other embodiments the invention encompasses a method of treating a young subject age 17 years or younger comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding an antigenic polypeptide as described hereinin an effective amount to vaccinate the subject.
[0375] In other embodiments the invention encompasses a method of treating an adult subject comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding an antigenic polypeptide as described herein in an effective amount to vaccinate the subject. [0376] In preferred embodiments, vaccines of the invention (e.g., LNP-encapsulated RNA 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.
[0377] As defined herein, the term antibody titre refers to the amount of antigen-specific antibody produces in s subject, e.g., a human subject. In exemplary embodiments, antibody titre is expressed as the inverse of the greatest dilution (in a serial dilution) that still gives a positive result.
[0378] In exemplary embodiments, antibody titre is determined or measured by enzyme-linked immunosorbent assay (ELISA). In exemplary embodiments, antibody titre is determined or measured by neutralization assay, e.g., by microneutralization assay. In certain embodiments, antibody titre measurement is expressed as a ratio, such as 1 :40, 1 :100, etc. In exemplary embodiments of the invention, an efficacious vaccine produces an antibody titre of greater than 1 :40, greater that 1 : 100, greater than 1 :400, greater than 1 :1000, greater than 1 :2000, greater than 1 :3000, greater than 1 :4000, greater than 1 :500, greater than 1 :6000, greater than 1 :7500, greater than 1 :10000. In exemplary embodiments, the antibody titre is produced or reached by 10 days following vaccination, by 20 days following vaccination, by 30 days following vaccination, by 40 days following vaccination, or by 50 or more days following vaccination. In exemplary embodiments, the titre is produced or reached following a single dose of vaccine administered to the subject. In other embodiments, the titre is produced or reached following multiple doses, e.g., following a first and a second dose (e.g., a booster dose.) In exemplary embodiments of the invention, antigen-specific antibodies are measured in units of pg /ml or are measured in units of I U/L (International Units per liter) or mIU/ml (milli International Units per ml). In exemplary embodiments of the invention, an efficacious vaccine produces >0.5 pg/ml, >0.1 pg /ml, >0.2 pg /ml, >0.35 pg /ml, >0.5 pg /ml, >1 pg /ml, >2 pg /ml, >5 pg /ml or >10 pg /ml. In exemplary embodiments of the invention, an efficacious vaccine produces >10 mIU/ml, >20 mIU/ml, >50 mIU/ml, >100 mIU/ml, >200 mIU/ml, >500 mIU/ml or >1000 mIU/ml. In exemplary embodiments, the antibody level or concentration is produced or reached by 10 days following vaccination, by 20 days following vaccination, by 30 days following vaccination, by 40 days following vaccination, or by 50 or more days following vaccination. In exemplary embodiments, the level or concentration is produced or reached following a single dose of vaccine administered to the subject. In other embodiments, the level or concentration is produced or reached following multiple doses, e.g., following a first and a second dose (e.g., a booster dose.) In exemplary embodiments, antibody level or concentration is determined or measured by enzyme- linked immunosorbent assay (ELISA).
[0379] In exemplary embodiments, antibody level or concentration is determined or measured by neutralization assay, e.g., by microneutralization assay.
Examples
[0380] The following examples describe a series of in vitro and in vivo studies relating to the immunogenicity of chimeric or fusion proteins, including of chimeric or fusion proteins encoded by the RNA constructs of the invention.
[0381] Example 1 describes in vitro materials and methods. Example 2 describes the results of in vitro studies. Examples 3 describes materials and methods for in vivo mouse studies and Example 4 describes the results of those in vivo studies. Example 5 describes materials and methods for canine serology studies and Example 6 describes the results of those studies.
[0382] Examples 7 to 11 describe generation and use of RNA vaccines encoding such chimeric or fusion proteins.
Example 1 : In vitro materials and methods
[0383] Cloning of Porphyromonas c/ulae constructs
[0384] DNA fragments encoding KDFAK-2S-AVQP (DUF2436 F variant; SEQ ID NO: 54) and KDAAK-2S-AVQP (DUF2436 A variant, SEQ ID NO: 52) containing Ncol and Xhol restriction sequences at the 5’ and 3’ ends were synthesized and ligated into transport vector, pBHA, by Bioneer Pacific (Aus.) to generate pBHA-gulA and pBHA-gulF. The cloned inserts were verified by DNA sequencing (Bioneer Pacific). pBHA-F and pBHA-A vectors were introduced into E. coli ABLE-K (Agilent Technologies) chemically competent cells following manufacturer’s protocol and recombinant strains were selected on LB containing Amp (100 pg/mL). Plasmid DNA was purified from positive clones and digested with Ncol and Xhol and ligated to Ncol/Xhol digested pET28b plasmid vector (Novagen). Ligation products were introduced into E. coli ABLE-K chemically competent cells and recombinant strains were selected on LB containing Kanamycin (30 pg /mL). Plasmid DNA from positive clones was purified, digested with Ncol/Xhol and plasmid digests were subject to agarose gel electrophoresis to verify the presence of correctly sized insert. Confirmed recombinant pET-gulA and pET-gulF variant constructs were then introduced into the E. coli expression host, BL21 (DE3) (Invitrogen) for recombinant protein expression. Select positive clones were grown in LB-Kan (30 pg/mL) to approximately mid log phase and glycerol stocks (25 % glycerol) were snap frozen for storage at -70°C.
[0385] Small scale expression test of expression constructs
[0386] Small scale expression was performed to check for soluble expression levels as follows. A single bacterial colony from freshly transformed LB-Kan plates was transferred to 5 mL LB-Kan media and grow O/N at 37°C with shaking at 200 rpm. These starter cultures were then used to inoculate fresh LB-Kan media at a 1 :100 ratio of 5 mL culture in a 50 mL falcon tube. Cell cultures were grown at RT (approximately 24-26°C) until the cell density reached OD600 » 0.6 - 0.8, then induced with 0.5 mM IPTG at RT for 16 hours. Two mL of the cell culture was centrifuged in an Eppendorf tube and resuspended in 0.5 mL of lysis buffer [20 mM Na-phosphate, 500 mM NaCI, 0.5% (v/v) TritonX-100, 20 mM Imidazole, 1x proteinase inhibitor, pH7.8] followed by brief sonication. A sample of total cell lysate was collected for electrophoretic analysis and the remainder was centrifuged at full speed using a bench top Eppendorf centrifuge. The soluble fraction was collected and the insoluble fraction was resuspended in Lysis Buffer. Total cells, soluble cell fraction and Insoluble cell fraction equivalent to 4 pL of induced cell culture were then subjected to SDS-PAGE to assess expression and solubility.
[0387] Mini purification of soluble recombinant proteins by Ni-NTA spin column
[0388] A mini-scale purification was performed on the two recombinant P. gulae constructs to assess for His-Tag integrity/availability for Nickel chromatography purification as follows. Soluble protein fraction (0.5 mL taken from the 2 mL induced culture above) was loaded onto Ni-NTA spin column according to manufacturers’ instructions and the eluate representing a crude Nickel purification was collected, the concentration measured and approximately 3 to 5 pg of purified r-protein (alongside samples of the flow through and washed column fractions) were resolved on SDS-PAGE analysis. [0389] Protein expression and cell lysis of His-tagged candidates
[0390] The His-tagged vaccine candidates for the animal model were expressed as recombinant C-terminally His-tagged fusion proteins (SEQ ID NO: 30 and 31 , plus His tag) in E. coli BL21(DE3) as previously described. The cells were grown at 37°C in LB medium or Terrific broth supplemented with 50 pg/mL kanamycin. At culture OD600 = 1.0-1.2, protein expression was induced with 0.2 mM IPTG at 32°C for 2-3 hours. The cells were harvested at 8000 g by centrifugation at 4°C and stored at -80°C for later use. The cells were lysed on ice by sonication for 15 min with 35% power output after thawed and resuspended in the lysis buffer [10 mM imidazole, 50 mM Tris, 300 mM NaCI, pH 7.5, 1x EDTA free protease inhibitor cocktail (Sigma) (for the reducing lysis buffer for the four Cys antigen-F: supplemented with 10 mM DTT)]. Cell lysate was clarified by centrifugation for 40 min at 25,000 g at 4°C.
[0391] Purification of a His-tagged candidate from inclusion bodies
[0392] Following lysis of cells, the insoluble pellet was washed twice with buffer PBS500 (20 mM NaPi, 500 mM NaCI, pH 7.4). The protein expressed as inclusion bodies was solubilised with 8 M urea at room temperature on a rolling platform for 1 hour in PBS500. The protein extract was centrifuged at 20,000 g. The supernatant was further filtered through a 0.22 pm filter unit and then mixed with the nickel affinity resin (Thermofisher) with gentle stirring for 2 hours in the urea containing buffer PBSLI (8 M urea, 20 mM NaPi pH 7.8, 500 mM NaCI, plus 20 mM imidazole). After extensive wash with PBSLI (pH 7.8 and then pH 6.5), the bound target protein was eluted off the resin with 500 mM imidazole in the same buffer. The eluted protein was stepwise dialysed into 6 M, 4 M and then 2 M urea phosphate buffers in a dialysis tube with molecular weight cutoff of 3.5 kDa (Fisher Biotec, Australia). Target protein in the 2 M urea buffer was buffer exchanged using a PD10 gel filtration column to remove urea before animal model experiments.
[0393] Purification of His-tagged candidates from soluble fractions
[0394] Ni-affinity chromatography
[0395] This is the first chromatographic step for purification of His-tagged proteins. The clarified cell lysates were filtered through a 0.22 pm filter unit and loaded to a HisTrap or HisPrep Ni-affinity column (GE Healthcare) in the loading buffer TBS300 (50 mM Tris Ci, 300 mM NaCI, pH 7.5), plus 10 mM imidazole. For reducing purification, cell lysate was diluted 4 fold with the imidazole containing TBS300 and then filtered before loaded to the column in the reducing loading buffer with inclusion of 2 mM DTT. Columns were washed extensively with appropriate loading buffer and then 20 mM imidazole in TBS300 or plus 2 mM DTT for reducing conditions. Bound proteins were eluted with a 20-350 mM imidazole gradient in TBS300 with the absorbance being monitored at 280 nm. Peak fractions were analysed with SDS-PAGE. Eluted target proteins were concentrated using the Amicon filter units with a 10 kDa molecular weight cut-off. The resulting protein solutions were stored on ice for further purification.
[0396] Anion exchange chromatography
[0397] The online ExPASy ProtParam tool (https://web.expasy.org/protparam/) predicted that the antigens had an acidic isoelectric point (pl), 5.07 for antigen-A and 4.87 for antigen-F. Thus, anion exchange chromatography was applied following the Ni-affinity chromatography step. Briefly, the concentrated proteins from Ni-affinity purification were diluted 10 folds into Buffer A (50 mM Tris. Cl, 20 mM NaCI, pH 7.5; 2 mm DTT for reducing buffer) to reduce ionic strength and then loaded to an anion exchange HiTrap Q column (GE Healthcare) in Buffer A. The proteins were eluted with a NaCI gradient from 50 to 350 mM and then from 350 to 700 mM. The absorbance at 280 nm was monitored in the process. Target proteins in the peak fractions were verified with SDS-PAGE and concentrated using the Amicon filter units. The protein solutions were stored on ice for further purification and buffer exchange using size exclusion chromatography.
[0398] Size exclusion chromatography
[0399] Size exclusion chromatography was performed on a HiLoad Superdex 200 column (GE Healthcare). The target protein solutions from anion exchange purification were concentrated and loaded to the size exclusion column, eluting in the buffer of TBS150 (50 mM Tris, 150 mM NaCI, pH 7.5) with absorbance at 280 nm being monitored. Target proteins in the peak fractions were verified with SDS-PAGE. The fractions of the best protein purity were pooled and concentrated. After quantitated by determination of absorbance at 280 nm on a Cary UV-vis spectrometer and calculation with theoretical extinction coefficients, target proteins were stored at -80°C in aliquots for future use. [0400] ESI-LC-MS intact protein analysis
[0401] Intact protein mass spectrometric analysis was performed on an electrospray ionisation time-of-flight mass spectrometer (ESI-TOF) coupled with liquid chromatography. With programmed automation, protein samples passed through a C4 HPLC column (Phenomenex) with 0.1% formic acid aqueous solution as buffer A and acetonitrile + 0.1% formic acid as buffer B. Acetonitrile gradient for elution was set to be 5-60% and then 60-95% buffer B for a total of 15 minutes. MS data were collected from 400 to 3200 m/z on the Agilent 6520 QTOF mass spectrometer operated in positive mode, with in-situ internal mass reference standards. Mass spectra were deconvoluted to obtain the intact protein molar masses using Agilent Mass Hunter Qualitative Analysis software (B.05) with maximum entropy algorithm.
[0402] SEC-MALS
[0403] SEC-MALS were run in buffer TBS150 at room temperature on a SEC-MALS system (Wyatt Technology). Species were resolved on a HPLC size exclusion column (Shim-Pack Bio Diol-300) and passed to the Wyatt 18-angle light scattering detector and Wyatt refractive index monitor. The concentrations of the samples were 2 mg/mL for the antigens and 5 mg/mL for the control protein BSA which was used to set up the method. Measurements were started following the stepwise instructions on screen of the control station with auto-injected volume set to be 10 pL for each run. Data was processed with Wyatt Technology ASTRA software for the information about sample homogeneity, protein aggregation and molar masses of the species in the solution.
[0404] Determination of endotoxin
[0405] The protein samples were diluted to 0.5 mg/mL using freshly prepared storage buffer TBS150. Endotoxin content was determined using the commercial Pierce Chromogenic Endotoxin Quantitation kit (Thermo Scientific) as per manufacturer instructions.
[0406] Assessment of nucleic acid contamination
[0407] A260/A280 ratio was determined on a NanoDrop lite UV spectrophotometer (Thermo Scientific) to assess nucleic acid contamination. [0408] Culture of Bacteria for Mouse Model of Periodontitis
[0409] P. gulae was obtained from the culture collection of the Oral Health Cooperative Research Centre, The Melbourne Dental School, University of Melbourne, Australia. P. gulae was cultivated (5% CO2, 10% H2, and 85% N2) in an anaerobic chamber (Whitley MG500 anaerobic workstation) at 37°C in brain-heart infusion (BHI) broth (BD Bacto Laboratories, USA) supplemented with cysteine (1 g/L; Sigma-Aldrich, Australia), tryptic soy broth (5% w/v, BD Bacto Laboratories, USA), hemin (5 pg/mL, Calbiochem, Netherlands), and Vitamin K (10 pg/mL, Sigma-Aldrich, USA).
[0410] 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).
[0411] Preparation of Heat Killed Bacteria
[0412] P. gulae 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 1010 cells/mL, and protein concentration determined using Biorad Protein Assay Dye Reagent Concentrate (Life Science, NSW, Australia).
[0413] Animal Ethics
[0414] All animal experimental procedures were carried out in strict accordance with the recommendations in the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.
Example 2: Results of in vitro studies
[0415] Antigen expression and solubility- small scale
[0416] Small scale expression tests of recombinant proteins comprising either DUF2436 variants A or F, induced with 0.5 mM IPTG at RT, showed that both variants expressed relatively high levels of soluble recombinant protein. Fig. 1 shows recombinant proteins of the expected size in the soluble fractions that are very prominent relative to the background of E. coli soluble proteins, suggestive of high levels of soluble expression. At RT, the chimeric protein comprising DUF2436 variant A exhibited very high levels of overall solubility with little or no recombinant protein present in the insoluble fraction that represents inclusion bodies. By comparison the chimeric protein comprising the DUF2436 variant F exhibited a high level of total recombinant protein expression in the insoluble fraction. Nevertheless, despite the relatively high level of KDFAK-2S-AVQP recombinant protein as insoluble inclusion bodies, there was still a relatively high level of soluble protein expressed in the soluble fraction due to the extremely high overall level of total recombinant protein expressed by this strain under the conditions tested.
[0417] Antigen expression and solubility- large scale
[0418] Both KDFAK-2S-AVQP and KDAAK-2S-AVQP proteins were expressed well at a similar level in either LB or TB medium (Fig. 4). Under non-reducing conditions, antigen- A had a high solubility as evidenced by SDS-PAGE analysis of the lysis fractions (Fig. 2). In contrast, the KDFAK-2S-AVQP protein was much less soluble with its major portion being found in the precipitate after lysis under the same non-reducing conditions (Fig. 2). In addition, reducing buffer containing either low (5 mM) or high (100 mM) concentration of DTT failed to extract antigen-F from the precipitate fraction (Fig. 2). However, when the cells were lysed under reducing conditions, KDFAK-2S-AVQP was highly soluble with most protein being in the soluble fraction (Fig. 2). The low solubility of KDFAK-2S-AVQP was likely due to the presence of disulfide bonds formed by wrongly paired Cys residues which caused the protein to have a misfolded structure. Once this abnormal structure formed, it was irreversible and its disulfide bonds were not accessible to the reductant if without assistance of unfolding force such as SDS and heating. Thus, purification of KDFAK-2S-AVQP from soluble fractions under non-reducing conditions was not an ideal approach although low temperature expression may improve soluble expression levels for this protein in the beginning as seen from small scale trial. In the end, the samples of this protein for the animal model experiments were prepared from lysis and purification under reducing conditions until the last step for removal of the included reductant DTT. [0419] Purification of the His-tagged control antigen from inclusion bodies
[0420] This procedure was only applied to purification of a prior art P. gingivalis chimera (termed KDcAKIn) which was expressed in the inclusion bodies as described previously (O'Brien-Simpson et al. 2016, NPJ Vaccines 1 :16022). KDcAKI n was stable in 2M urea after stepwise dialysed into 2 M urea phosphate buffer and thus stored at -80°C after quantitated for further use. No precipitation was observed after buffer exchange with gel filtration before the animal model experiments.
[0421] P. gulae antigen purification
[0422] For the animal model experiments described in examples 3 and 4, KDAAK-2S- AVQP was purified under non-reducing conditions and KDFAK-2S-AVQP was purified under reducing conditions until the final buffer exchange and size exclusion purification step (Figs. 3 to 5). Non-reducing conditions for the purification was also trialed for KDFAK- 2S-AVQP. KDAAK-2S-AVQP expressed in LB medium was used for purification under non-reducing conditions. KDFAK-2S-AVQP expressed in TB medium was used for purification under reducing conditions and the protein expressed in LB was used for nonreducing purification. KDAAK-2S-AVQP had the highest yield (78 mg/L culture, 18.6 mg/g wet cells) (Table 1). KDFAK-2S-AVQP also had a high yield of its final product when purified from lysis supernatant under reducing conditions (34 mg/L culture, 7.6 mg/g wet cells), however when purified under non-reducing conditions its yield was much lower (3.5 mg/L culture, 1.1 mg/g wet cells) (Table 1). Identities of both proteins were confirmed with the first Met residue missing by intact protein MS spectrometry (Table 3).
[0423] Table 3: Final products of P. gulae antigens purified from 1 L culture in LB or TB Broth.
Figure imgf000129_0001
2 The MWs in red were detected as minor peaks with additional 1 or 2x 183 Da to the measured target monomeric molar masses.
3 The listed concentrations were determined based on the absorbance and theoretical extinction coefficients at 280 nm.
NB: All the final products of these antigens were stored in TBS buffer (50 mM Tris, 150 mM NaCI, pH 7.5).
[0424] KDFAK-2S-AVQP and KDAAK-2S-AVQP were concentrated up to 20 and 18 mg/mL, respectively, in TBS150 buffer and these concentrations were not the maximum that could be achieved (Table 1). The majority of expressed KDFAK-2S-AVQP precipitated into the insoluble fraction from non-reducing lysis. Once it was purified under reducing conditions to a high quality, it stayed in solution without reversion to precipitation even when the reducing factor was removed. This solution stability was also resistant against freeze-thaw cycles in air. Thus, it was possible to prepare enough material from one round of purification for the animal model.
[0425] Removal of DnaK
[0426] The host molecular chaperone protein DnaK of ~70 kDa appeared to contaminate the purification of the P. gulae antigens. Due to its interactions with target proteins, DnaK eluted into the fractions with most overlapping the target protein fractions in the Ni affinity purification step. This contamination, including other impurities, was well separated from target proteins by anion exchange chromatography (Fig. 4). The interactions between DnaK and the target proteins did not seem to be dependent on reducing conditions. Nevertheless, under reducing conditions, KDFAK-2S-AVQP eluted into two peaks possibly due to the presence of two forms (Fig. 4). Essentially, almost all the fractions in the second peak had DnaK contamination. This was possibly due to a small portion of misfolded antigen-F that had higher interactions with reduced DnaK. Therefore, the second peak was excluded and the best fractions in the early major peak were used for further purification with size exclusion chromatography (Fig. 5).
[0427] Degradation and site prediction
[0428] Some minor bands could be seen below each major full-length target protein on the SDS gels of the anion exchange fractions including the doublet bands in some fractions for KDFAK-2S-AVQP, suggesting the occurrence of degradation (Fig. 6). Most degradants were essentially removed by size exclusion chromatography (Fig. 5) and a high quality of final product for each protein was thus achieved despite existence of minor degraded species (Fig. 6). The high purity and high homogeneity of the final products were also supported by SEC-MALS analysis, which showed the predominant existence of monomer in solution at 2 mg/mL with high stability for both proteins (Table 4).
[0429] Table 4: Peak area percentages in SEC-MALS chromatograms of the P. gulae antigens
Figure imgf000131_0001
[0430] Intramolecular disulfide bonds in antigen-F
[0431] Slight band shift exhibited in the SDS-PAGE profile of the F-version antigen (KDFAK-2S-AVQP) in response to the reducing conditions, suggesting the presence of intramolecular disulfide bonds (Fig. 9A). Even for KDFAK-2S-AVQP purified involving reducing purification procedures (F-R), it had a similar band shift to the protein purified under non-reducing conditions (F-NR). F-R also formed intramolecular disulfide bonds upon removal of reducing factors at the size exclusion purification step. This intramolecular disulfide bridge may play a role in maintaining the protein structure and make the protein more compact, resulting in a faster mobility in the SDS gel under nonreducing denaturing conditions (Fig. 6A). There was no such band shift for KDAAK-2S- AVQP (Fig. 6A). The slight reduction dependent band shift for KDFAK-2S-AVQP was not resolved in native PAGE of unheated samples (Fig. 6B). In contrast to the results from SDS-PAGE, for heated samples on native gel without SDS, the negative charge on thiol ions of reduced KDFAK-2S-AVQP appeared to take effects on the protein mobility and make the protein move faster than the unreduced sample (Fig. 6B).
[0432] F-NR seemed to have a band shift slightly more than F-R on the SDS gel, whether the samples were heated or not (Fig. 6A). It was hypothesized that F-NR formed intramolecular disulfide bonds as early as at the stage of lysis and F-R formed the disulfide bonds only after removal of the reducing agent at the final purification stage. This difference may have a subtle impact on the protein structure. However, F-R purified from the reducing agent was found to have a similar overall structure to F-NR with minor difference in compactness.
[0433] Oligomeric state
[0434] A faint band at around 90 kDa appearing in the SDS gel lanes of heated nonreduced samples of F-R and F-NR suggested the presence of a small amount of dimer due to intermolecular disulfide formation (Fig. 6A). It is possible that this was an artifact from heating denaturation to temporarily expose the Cys residues for intermolecular disulfide formation as this was not the case for the samples unheated (Fig. 6A).
[0435] Since no effects of reducing conditions were seen on the PAGE profiles of KDFAK-2S-AVQP in native gels, whether the samples were either heated or not (Fig. 6B), a possibility of progressive formation of multimers bridged by intermolecular disulfide bonds was excluded for this containing protein.
[0436] SEC-MALS data showed some signs of minor or minimal multimerisation of the two antigens. The species with estimated molar masses of 71.2 kDa for antigen-A and 84.7 kDa for F-R (reduction involved purification) may be assigned to the dimeric forms of the two proteins. As for F-NR (purified under non-reducing conditions), a peak of 394.2 kDa may be due to the presence of a small amount of decamer of this protein, although this species was not detected by SDS-PAGE. Cys residues in KDFAK-2S-AVQP may have made this protein prone to form a higher level of multimerisation when it was in nonreducing conditions from the stage of lysis. Therefore, the trace amount of decamer may be intermolecular disulfide linked. Importantly, these mutimerised species were less than 1% of the total protein amount as estimated by integration of the SEC-MALS liquid chromatograms (Table 4).
[0437] Assessment of endotoxin and nucleic acid contaminations
[0438] Endotoxin was determined to have similarly low levels in the two antigens. At a protein concentration of 0.5 mg/mL, both proteins had endotoxin contamination at 2.24 and 2.36 EU/rnL, respectively (Table 5). In addition, the A260/A280 value was lower than 0.6 for both antigens, indicating minimal contamination with nucleic acids (Table 5). [0439] Table 5: Assessment of endotoxin and nucleic acid contamination in P. gulae antigens.
Figure imgf000133_0001
Example 3: materials and methods for mouse in vivo studies
[0440] Mouse Periodontitis Model
[0441] Mice (female BALB/c; 6-8 weeks old, 10 mice/group), on Day 0 were intra-orally inoculated with P. gulae consisting of four doses of P. gulae [1 x 1010 viable P. gulae 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 (CMC, Sigma, New South Wales, Australia)], with each dose given two days apart. The inoculum was 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 flow cytometry and CFU counts on blood agar. Groups of animals consisted of: P. gulae orally inoculated (infected control), a non-bacterial inoculated control, and immunised groups. For the therapeutic vaccination periodontitis model 21 mice were immunised on day 19 after the first oral inoculation with 100 pg of vaccine candidate in saline/alum (Alhydrogel; 2% aluminium hydroxide wet gel suspension; Invivogen) via the intraperitoneal route. Mice received a second immunisation (100 pg in saline/alum) on day 40 via the subcutaneous route. On Day 62, mice were bled by cardiac puncture and killed. Maxillae were removed and halved through the midline, with 10 halves used to determine alveolar bone loss.
[0442] Measurement of alveolar bone loss in mouse maxillae [0443] Maxillae to be examined for bone loss were boiled (1 min) in deionised water, mechanically defleshed, and immersed in 2% w/v potassium hydroxide (16 h, 25°C). Maxillae were washed twice with deionised water (25 °C), dried (1 h, 37 °C) and stained with 0.5% w/v aqueous methylene blue. Coded digital images of the buccal aspect of the maxillae were captured with an Olympus DP12 digital camera mounted on a dissecting microscope, using Imaged imaging software for analysis (https://imagej.nih.gov/ij/index.html) to assess horizontal bone loss. Maxillae were oriented so that the buccal and lingual molar cusps were superimposed. Images were captured with a micrometre in frame, so that measurements could be normalised for each image. Horizontal bone loss was defined as the loss occurring in a horizontal plane, perpendicular to the alveolar bone crest that resulted in a reduction of the crest height. The visible area from the cemento-enamel junction (CEJ) to the alveolar bone crest (ABC) for each molar was measured using Imaged version 1.3k imaging software to give the total visible CEd-ABC area in mm2. P. gu/ae-induced alveolar bone loss in mm2 was calculated by subtracting the total visible CEd-ABC area of the uninoculated (N-C) group from the total visible CEd-ABC area of each experimental group. Alveolar bone loss measurements were determined twice in a random and blinded protocol. Data are expressed as the mean +/- standard deviation in mm2 and were analysed using a oneway ANOVA and Dunnetts T3 post-hoc test.
[0444] Enzyme-Linked Immunosorbent Assay (ELISA)
[0445] ELISAs were performed to evaluate subclass antibody in sera using a solution (1 pg/mL) heat-killed (HK) P. gulae cells in 0.1 M PBS (pH 7.4) to coat wells (16 h, 4 °C) of flat-bottom polyvinyl microtitre plates (Microtitre; Dynatech Laboratories, McLean, VA, US).
[0446] In these experiments the following antibody dilutions were used; a dilution of 1/4000 dilution of goat anti-mouse; IgG (M8642), lgG1 (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. All optical density measurements were conducted on a Wallac VICTORS 1420 Multilabel counter (Perkin Elmer) at 405nm.
Example 4: results of mouse in vivo studies
[0447] P. qu/ae- induced Alveolar bone loss in mouse maxillae.
[0448] The animal model used was the therapeutic vaccine mouse periodontitis model (Fig. 7) developed by O’Brien-Simpson et al (2016, supra). Compared to the naive control, the infected control animals developed significant levels (P<0.001) of alveolar bone loss (Fig. 8). A P. gingivalis chimeric protein known in the prior art (KDcAKIn) was used as a positive vaccine control in this study. New vaccines based on the P. gulae virulence domain sequence, KDFAK-2S-AVQP and KDAAK-2S-AVQP provided significant levels of protection compared to the infected control animals (P<0.01) (Fig. 8).
[0449] Antibody response
[0450] Serum antibody subclass responses of immunised mice in the periodontitis model were examined by ELISA. Antisera were used to probe heat killed P. gulae as the adsorbed antigen (Fig. 9). Antibody responses are expressed as the ELISA titre obtained minus double the background level, with each titre representing the mean ± s.d. of the 10 individual mice. The two P. gulae vaccines, KDFAK-2S-AVQP and KDAAK-2S-AVQP, generated significant levels of antibodies against whole cells compared to the negative control (Fig. 9).
[0451] Antibody titres against P. gulae purified RgpA/Kgp protease complex (Fig. 10) were also measured.
[0452] Both the KDFAK-2S-AVQP and KDAAK-2S-AVQP proteins generated significant antibodies against the RgpA/Kgp protease complex, with KDFAK-2S-AVQP generating higher titres against the RgpA/Kgp protease complex.
[0453] Finally, Fig. 11 shows that the two P. gulae vaccines generated high titre antibody responses against themselves.
[0454] In conclusion the inventors have generated P. gulae vaccines using P. gulae specific sequences derived from a complex polyprotein gingipain. Both proteins tested protected as vaccines in the mouse animal model. Example 5: materials and methods for canine in vivo studies
[0455] A series of experiments were planned for determining the ability of a chimeric fusion protein to raise an immune response in dogs.
[0456] 9 dogs were divided into 3 groups as follows:
- Group 1 - low dose: (Jake 3962, Phoebe 0346 and Rachael 8562) were immunised with a low dose (100 pg) of Porphyromonas gulae KDAAK-2S-avqp- 6His recombinant protein.
- Group 2 - mid-dose: (Forest 5635, Milan 3711 and Moo 5498) were immunised with a medium dose (200 pg) of Porphyromonas gulae KDAAK-2S-avqp-6His recombinant protein.
- Group 3 - high dose: (Bruno 8086, Kale 5636 and Lachie 5634) were immunised with a high dose (400 pg) of Porphyromonas gulae KDAAK-2S-avqp-6His recombinant protein.
[0457] Dogs were immunised twice, first on Day 0 (“prime”) and again on Day 21 (“boost”), according to the protocol in Fig. 12. For each dog, serum samples were collected on:
- day 0 (= pre-immune serum, aka pre-treatment, i.e., immediately prior to receiving the prime dose).
- day 21 (= primed serum, aka pre-second treatment, i.e., 21 days after receiving the prime dose and immediately prior to receiving the booster).
- day 35 (= boosted serum, i.e., 14 days after receiving the booster).
[0458] Optimization ELISAs
[0459] Purified Porphyromonas gulae KDAAK-2S-avqp-6His recombinant protein
[0460] ELISA plate wells were coated with 100 pL/well of Porphyromonas gulae KDAAK- 2S-avqp-6His recombinant protein at concentrations of 10, 2.5, 1 , 0.5 and 0.1 pg/mL (dilutions made in 1xPBS). A PBS-only control was also included. After overnight incubation at 4 °C, the recombinant protein solution was discarded, wells were washed twice with distilled water, and free protein binding sites were blocked by the addition of 200 pL/well of Block Solution. After incubation at 4 °C for 6 hours, the Block Solution was discarded, wells were rinsed twice with Wash Solution, and serum dilutions were applied as described below.
[0461] Biotinylated peptides
[0462] ELISA plate wells were coated with 100 pL/well of 10 pg/mL streptavidin solution (dilution made in 1 xPBS). After overnight incubation at 4 °C, the streptavidin solution was discarded, wells were washed twice with distilled water, and free protein binding sites were blocked by the addition of 200 pL/well of Block Solution (10% (w/v) non-fat milk powder in 1 xPBS). After incubation at 4 °C for 6 hours, the Block Solution was discarded, wells were rinsed twice with Wash Solution, and aliquots of 100 pL of biotinylated synthetic peptides (Pgul_KAS2 (SEQ ID NO: 1) and P. gul_KAS2_scrambled: Biotin- KYKGWTNNSSTVLLQTNATLGVETFTHSPDSASDAK, SEQ ID NO: 127) prepared in Dilution Buffer at 10, 2.5, 1 , 0.5 and 0.1 pg/mL were added to the blocked streptavidin- coated wells. A Dilution Buffer-only control was also included. After overnight incubation at 4 °C, the peptide solutions were discarded, wells were washed six times with Wash Solution, and serum dilutions applied as described below.
[0463] Whole heat-killed Porphyromonas gulae cells
[0464] ELISA plate wells were coated with 100 pL/well of whole heat-killed Porphyromonas gulae cells at concentrations of 10, 2.5, 1 , 0.5 and 0.1 pg/mL (dilutions made in 1 xPBS). A PBS-only control was also included. After overnight incubation at 4 °C, the P. gulae cells were discarded, wells were washed twice with distilled water, and free protein binding sites were blocked by the addition of 200 pL/well of Block Solution. After incubation at 4 °C for 6 hours, the Block Solution was discarded, wells were rinsed twice with Wash Solution, and serum dilutions were applied as described below.
[0465] Optimization ELISAs: Serum application
[0466] Aliquots of each of the Day 0 serum samples were pooled and diluted 1/10 in
Dilution Buffer. Similarly, the day 35 serum samples were pooled and diluted 1/10 in Dilution Buffer. Aliquots (100 pL) of the 1/10 diluted serum samples were added to the blocked ELISA plates and 4-fold dilution series of the 1/10 serum samples were constructed by serially transferring 25 pL into 75 pL of Dilution Buffer. Control wells that received only Dilution Buffer (i.e. , no serum) were also included. Plates were incubated overnight at 4 °C and detection antibody was applied as described belpw.
[0467] Detection Antibody
[0468] After overnight incubation, serum samples were discarded, and the wells were rinsed six times with Wash Solution. Aliquots (100 pL) of horseradish peroxidase (HRP)- conjugated Goat anti-Dog IgG (Fc specific) antiserum (10 mg/mL), diluted 1/5,000 in Dilution Buffer, were added to each well. After incubation at room temperature (22 °C) for 2 hours, the labelled antibody was discarded, wells were rinsed six times with Wash Solution and HRP substrate added as described below.
[0469] HRP substrate
[0470] TMB Substrate Buffer Solution (90 pL) was added to the washed ELISA plates. For optimization ELISAs, colour development was followed spectrophotometrical ly at 370 nm in kinetic mode using a SpectraMax i D5 spectrophotometer. For titreing of individual sera, the colour was allowed to develop for 20 minutes, the reaction stopped by the addition of 50 pL of 1 M H2SO4 to each well, and the plate read in stopped ELISA mode (at 450 nm) in the SpectraMax i D5 spectrophotometer. Data was analysed as described below.
[0471] ELISAs for titration of individual dog serum
[0472] Purified Porphyromonas gulae KDAAK-2S-avqp-6His recombinant protein
[0473] ELISA plate wells were coated with 100 pL/well of 0.1 pg/mL Porphyromonas gulae KDAAK-2S-avqp-6His recombinant protein (dilution made in 1 xPBS). After overnight incubation at 4 °C, the recombinant protein solution was discarded, the wells were washed twice with distilled water and free protein binding sites were blocked by the addition of 200 pL/well of Block Solution. After incubation at 4 °C for 6 hours, the Block Solution was discarded, and the wells were rinsed twice with Wash Solution. Aliquots (100 pL) of pre-immune, primed and boosted sera from each dog were diluted 1/10, 1/10 and 1/500, respectively, in Dilution Buffer, added to the ELISA plate, and a 1/5 dilution series was constructed by serially transferring 20 pL into 80 pL of Dilution Buffer. Dilution Buffer-only control wells (i.e. , no serum) were also included. After overnight incubation at 4 °C, detection antibody was applied as described above.
[0474] Biotinylated Pgul_KAS2 peptide
[0475] ELISA plate wells were coated with 100 pL/well of 10 pg/mL streptavidin solution (dilution made in 1 xPBS). After overnight incubation at 4 °C, the streptavidin solution was discarded, the wells were washed twice with distilled water and free protein binding sites were blocked by the addition of 200 pL/well of Block Solution. After incubation at 4 °C for 6 hours, the Block Solution was discarded, wells were rinsed twice with Wash Solution, and 100 pL aliquots of 0.1 pg/mL biotinylated Pgul_KAS2 peptide (dilution made in Dilution Buffer) were added to each well of the streptavidin-coated plates. After overnight incubation at 4 °C, the peptide solution was discarded, and wells were rinsed six times with Wash Solution. Aliquots (100 pL) of pre-immune, primed and boosted sera from each dog were diluted 1/10 in Dilution Buffer, added to the ELISA plate, and a 1/5 dilution series was constructed by serially transferring 20 pL into 80 pL of Dilution Buffer. Dilution Buffer- only control wells (i.e., no serum) were also included. After overnight incubation at 4 °C, detection antibody was applied as described above.
[0476] Whole heat-killed Porphyromonas gulae cells
[0477] ELISA plate wells were coated with 100 pL/well of 1 pg/mL whole heat-killed Porphyromonas gulae cells (dilution made in 1 xPBS). After overnight incubation at 4 °C, the P. gulae cells were discarded, the wells were washed twice with distilled water and free protein binding sites were blocked by the addition of 200 pL/well of Block Solution. After incubation at 4 °C for 6 hours, the Block Solution was discarded, and wells were rinsed twice with Wash Solution. Aliquots (100 pL) of pre-immune, primed and boosted sera from each dog were diluted 1/10 in Dilution Buffer, added to the ELISA plate, and a 1/5 dilution series was constructed by serially transferring 20 pL into 80 pL of Dilution Buffer. Dilution Buffer-only control wells (i.e., no serum) were also included. After overnight incubation at 4 °C, detection antibody was applied as described above.
[0478] Data Analysis: Determination of Antibody Titres
[0479] ELISA assays based on peroxi dase-TMB systems can be monitored in two ways; continuously recording absorbance at 370 nm; or acid-stopping the reaction following a set time and measuring absorbance at 450 nm. Continuous assays tend to be more accurate with a wider dynamic range than stopped assays but become impractical with multiple plates. Both assay types were deployed during this trial.
[0480] All analyses were performed using scripts written for R-4.2.1 (R Foundation). Initial slopes of continuously read ELISA assays were calculated by quadratic regression of the absorbance response to account for curvature. The linear parameter of the quadratic equation corresponds to the slope at time = 0 (i.e., initial slope). Dose response curves for both continuous and acid-stopped assays were calculated using the drc-library of functions for R. A 4-parameter log-logistic equation (LL.4) was used for all samples (Equation 1 , below). To cope with samples with low responses which did not cover the full range, the maximum and minimum parameters were determined at a plate level, the slope parameter was set at 1 .0, while the inflection point parameter was unique for each serum sample.
[0481] Two antibody titre values were calculated and reported: midpoint titres are equivalent to the inflection point parameter in the LL.4 model. Endpoint titres were calculated as the predicted point on the LL.4 curve corresponding to twice the minimum response.
[0482] Equation 1 :
Figure imgf000140_0001
[0483] where : x = concentration of serum; f(x) = absorbance response; a = maximum response; b = minimum response; c = slope (Hill coefficient); d = Inflection point (midpoint)
[0484] Example 6: results from initial canine serology studies
[0485] Figures 13 to 15 show preliminary results from the experimental protocol outlined in Example 5. Briefly, following immunisation, sera were collected at the time points indicated and ELISA was used to determine immune response to the immunising antigen (KDAAK-2S-avqp-6His), P. gulae KAS2 peptide or P. gulae whole cells. [0486] As shown in Figure 13, all dogs showed a robust immune response to antigen following prime and boost immunisation.
[0487] The results shown in Figure 14 indicate that the response to P. gulae whole cells correlates strongly with the response to immunising antigen.
[0488] The results shown in Figure 15 indicate that the response to P. gulae KAS2 peptide correlates strongly with the response to immunising antigen.
[0489] Overall, the results of this study indicate that the vaccines were well tolerated by the animals and generated a strong immune response in the canine study.
Example 7: canine study proof of concept study
[0490] A further canine study, similar to the one discussed at Examples 5 and 6, was conducted. Briefly, 20 dogs were organised into “treatment” or “control” groups (10 dogs in each group). Dogs in the treatment group were immunised 3 times with chimeric protein antigen KDAAK-2S-avqp as herein described, beginning on day 0 (prime), followed by a booster immunisation on day 21 (boost 1) and again on day 42 (boost 2). The control group received no treatment.
[0491] Sera were collected at the following time points:
• Day 0 = pre-immune response (immediately prior to receiving the prime dose)
• Day 21 = prime response (21 days after receiving the prime dose and immediately prior to receiving the boost-1 dose)
• Day 42 = boost-1 response (21 days after receiving the boost-1 dose and immediately prior to receiving the boost-2 dose)
• Day 56 = boost-2 response (i.e. , 14 days after receiving the boost-2 dose).
[0492] Serum IgG responses against the P. gulae KAS2 peptide (SEQ ID NO: 1) were measured using ELISA, similarly to the methods described in Example 5.
[0493] The results, summarised in Figures 16 and 17, show a robust immune response was generated following the immunisation protocol. [0494] Briefly, when either mean midpoint or mean endpoint titres were used as a measure of immune responses to the KAS2 peptide, serum collected from the “control” group dogs at day 21 , day 42 or day 56 showed less than a 1 .3-fold increase relative to the mean pre-immune (day 0) titre. This finding is consistent with this group being comprised of control animals that received no treatment.
[0495] In contrast, all dogs in the treatment group produced titratable boosted (day 42 and day 56) peptide-specific IgG responses that were greater than their pre-immune (day 0) responses across most of the serum dilutions. A side-by-side comparison of the midpoint and endpoint titres for all treatment group dogs showed a progressive increase in titre following administration of successive doses, which appears to reach a maximum in the day 42 serum samples.
[0496] When the peptide-specific serum IgG responses of all ten dogs in the treatment group were analysed collectively, serum harvested at days 21 , 42 and 56 showed statistically significant (p < 0.05) higher mean midpoint and mean endpoint titre values compared to the mean values for the pre-immune (day 0) serum samples. In addition, titres measured at days 42 and 56 were statistically significantly higher than day 21 titres (p < 0.05). When using mean midpoint titres as a measure of response, day 21 , day 42 and day 56 serum samples were, respectively, 5x, 94* and 96* greater than the pre-immune titres.
Example 8: generation of RNA-LNPs for delivery of chimeric or fusion proteins
[0497] Production of RNA-LNPs
[0498] RNAs are 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 are capped co-transcriptionally using Clean Cap RNA capping technology (TriLink Biotech).
[0499] Typically, the UTP is replaced by N1-methyl pseudouridine (N1-methyl pseudo- UTP, m1Y) during RNA production. DNA is removed using DNAse I (NEB), and doublestranded RNA (dsRNA) is removed using cellulose binding using standard techniques 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). [0500] Expression of RNA vaccine candidates in cell culture in vitro
[0501] HeLa cells are cultured in culture media DMEM, high glucose, GlutaMAX™ Supplement, pyruvate (ThermoFisher, CAT#10569010) with 10% FBS at 37°C, 5% CO2, according to standard protocols. RNA candidates (prior to LNP formulation), are formulated with Lipofectamine MessengerMAX (IThermoFisher) and used to transfect cells according to manufacturer’s instructions. 1.8 pg of an RNA is used to transfect 35,000 cells/well on a six well plate. For supernatant collection, cells are pelleted by centrifuging at 14,000 g for 15 mins. Supernatants are transferred to a fresh tube and frozen at -20°C until Western Blotting. For whole cell lysate collection, cells are rinsed in each well with ~2 mL DPBS. The liquid is removed and 250 pL of RIPA lysis buffer with protease inhibitors (ThermoFisher) is added to each well, and gently swirled to mix for 10s. Using a cell scraper the lysates are transferred into tubes and centrifuged at 4°C at 16,000 g for 15 mins to pellet debris. The clarified lysates are collected into new tubes and stored at -20°C until Western Blotting to confirm expression of protein.
[0502] Culture of bacteria for mouse model of periodontitis
[0503] P. gulae is grown on Horse Blood Agar (HBA) (20 g/L HBA; Oxoid Ltd., Hampshire, UK) supplemented with 10% v/v lysed horse blood (37°C) in an anaerobic N2 atmosphere containing 5% CO2 in a MK3 Anaerobic Workstation (Don Whitley Scientific Ltd., Adelaide, Australia). Colonies are inoculated into starter culture comprised of 20 mL sterilised brain heart infusion (37 g/L BHI; Oxoid Ltd., Hamsphire, UK) medium supplemented with 5 mg/L hemin and 0.5 mg/L cysteine and incubated anaerobically (24 h, 37 °C).
[0504] Preparation of heat-killed bacteria
[0505] P. gulae are 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 is resuspended in PBS and heated to 65°C for 15 minutes. The suspension is centrifuged (7,000 g, 20 min 4°C) and resuspended in sterile PBS and this is repeated once. After the second wash, the supernatant is discarded and the cell pellet is resuspended in sterile PBS to obtain a cell density of 2 x 1010 cells/mL, and protein concentration determined using Biorad Protein Assay Dye Reagent Concentrate (Life Science, NSW, Australia). [0506] Mouse periodontitis model
[0507] The mouse periodontitis experiments to assess protection by the vaccines and immune responses generated, are performed as described herein, including methods for assessing bone loss from maxille.
[0508] Example 9: Expression of RNA constructs encoding chimeric proteins
[0509] RNA-LNPs are tested for in vitro antigen expression and secretion in HeLa cells. HeLa cells are transfected with the RNA constructs using lipofectants as outlined above. After 48 hours, supernatants are collected and subjected to Western Blot analysis to test for secreted polypeptide. The results will indicate that the RNAs, particularly those encoding chimeric or fusion proteins defined in Table 1 herein, express well and are secreted.
[0510] Example 10: determining immunogenicity of candidate RNA vaccines
[0511] Figure 18 shows a schematic of the proposed vaccination protocol for use to assess the immunogenicity of candidate RNA vaccines.
[0512] Briefly, RNA constructs as described above and encoding chimeric or fusion proteins defined in Table 1 are formulated into lipid nanoparticles (LNP) using standard techniques. The LNPs used in these experiments comprises: ALC-0315, cholesterol, distearoylphosphatidylcholine and ALC-0159 in mole percent (%) ratio of: 46.3: 42.7: 9.4: 1.6.
[0513] For each antigen, RNA constructs are generated using either native RNA sequence (unmodified) or N1-methyl-pseudouridine modified (M1 ip) sequence.
[0514] Two doses of RNA vaccine are tested: 30 pg or 3 pg of RNA formulated in LNP. The positive control used in experiments is the protein KDAK-3S-AVQP adjuvanted with alum.
[0515] Mice are intramuscularly immunised with RNA-LNPs according to the schedule shown in Figure 18.
[0516] Following immunisation, sera are collected at Day 35 and are tested using ELISA to determine serum antibody subclasses responses of the immunised mice. [0517] m1'+,-KDAK 30 pg, m14’-KDAK-3S-AVQP 30 pg, KDAK 30 pg and m14’-KDAK 3 pg will induce significant IgG titres against Kgpcat and ml^P-KDAK 30 pg, ml^P-KDAK- 3S-AVQP 30 pg, KDAK 30 pg will also induce significant lgG1.
[0518] Example 11 : determining in vivo efficacy of RNA vaccines in periodontitis model
[0519] Figure 19 shows a schematic of the proposed vaccination protocol for determining in vivo efficacy of candidate RNA vaccines (as assessed by protection from alveolar bone loss), and immunogenicity (as determined by antibody levels in sera).
[0520] RNA vaccines are prepared and formulated as outlined above.
[0521] The results will show that vaccination with ml^P-KDAK 30 pg, ml^P-KDAK-SS- AVQP 30 pg RNA vaccines provided for significant protection against P. gulae induced bone loss, similarly to the alum-adjuvanted polypeptide KDAK-3S-AVQP.
[0522] Example 12: truncated antigens encoded by alternative RNA constructs
[0523] Various truncated RNA constructs are assessed, each expressing truncated variations of the protein antigens encoded by the RNAs tested in the above examples. All RNAs are ml ip-modified.
[0524] The RNA constructs for testing encoded:
- K (also referred to herein as Kas 2 active site domain; amino acid sequence SEQ ID NO: 2)
- KD (amino acid sequence: SEQ ID NO: 26 or 28)
- KA (amino acid sequence SEQ ID NO: 24)
- DA (amino acid sequence SEQ ID NO: 42 or 44)
- KDA (amino acid sequence SEQ ID NO: 30 or 32)
- KDAAAMBS (ie wherein the adhesin domain comprises ABMs 2 and 1 but not ABM3; amino acid sequence SEQ ID NO: 38 or 40)
- RDA (replacing Arginine-dependent gingipain active site KAS, with active site from a Lysine-dependent gingipain RAS) (amino acid sequence: SEQ ID NO: 48 or 50)
- positive control: KDAK-3S-AVQP (SEQ ID NO: 52 or 54) [0525] The RNAs encoding the target antigens are tested for in vitro expression and secretion in HeLa cells by Western Blot.
[0526] RNAs (formulated in LNPs) encoding KDA, KDAAABMS, DA, KA and KD are assessed in this next study and compared to KDAK-3S-AVQP RNA and protein vaccines. All RNAs are ml ip-modified and are administered at a dose of 30 pg RNA.
[0527] The results will indicate that the RNA vaccine encoding KA, DA, KDAAABMS and KDA provides robust protection against P. gu/ae-induced alveolar bone loss.
[0528] 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.

Claims

1. 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 homologue of P. gulae, 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 found in surface complexes of the Arg- and Lys-gingipain homologues of P. gulae, or a sequence that is at least 80% identical thereto. wherein the polynucleotide is capable of being translated in a mammalian cell.
2. The RNA of claim 1, 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 homologue of P. gulae, 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 found in surface complexes of the Arg- and Lys-gingipain homologues of P. gulae, or a sequence that is at least 80% identical thereto.
3. The RNA of claim 1 or claim 2 wherein the protein encoded by the RNA polynucleotide further comprises:
- the amino acid sequence of a DUF2436 domain of the Arg- and Lys-gingipain homologue surface complexes of P. gulae, or a sequence that is at least 80% identical thereto.
4. The RNA 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 homologue of P. gulae, 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 found in surface complexes of the Arg- and Lys-gingipain homologues of P. gulae, or a sequence that is at least 80% identical thereto.
5. The RNA of any one of the preceding claims, wherein the RNA encodes the amino acid sequence of an Arg- gingipain homologue of P. gulae, comprising the amino acid sequence of SEQ ID NO: 4, (eg encoded by the RNA sequence as set forth in SEQ ID NO: 69), 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.
6. The RNA of any one of the preceding claims, wherein the RNA encodes the amino acid sequence of an active site of the Lys-gingipain homologue of P. gulae, comprising the amino acid sequence of SEQ ID NO: 2, (eg encoded by the RNA sequence as set forth in SEQ ID NO: 59), 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.
7. The RNA 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 homologue of P. gulae, 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 homologue of P. gulae, or sequences that are at least 80% identical thereto.
8. The RNA of any one of the preceding claims, wherein the RNA encodes a chimeric or fusion protein encoded 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 homologue of P. gulae, or sequences that are at least 80% identical thereto.
9. The RNA of claim 8, wherein the at least two amino acid sequences are located contiguously in the chimeric or fusion protein.
10. The RNA of claim 8, 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.
11. The RNA of claim 8, 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.
12. The RNA of claim 8, 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.
13. The RNA of any one of claims 4 to 12, wherein the chimeric or fusion protein encoded by the RNA polynucleotide further comprises:
- the amino acid sequence of a DUF2436 domain of the Arg- and Lys-gingipain surface complexes of P. gulae, or a sequence that is at least 80% identical thereto.
14. The RNA of claim 13, wherein the RNA encodes:
- one or more amino acid sequences of an active site of an Arg- or Lys-gingipain homologue of P. gulae, 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 found in surface complexes of the Arg- and Lys-gingipain homologues of P. gulae, or a sequence that is at least 80% identical thereto; and wherein the amino acid sequence of a DUF2436 domain is located between an amino acid sequence of an active site of the gingipain of P. gulae and the amino acid sequence of the or more adhesin binding motifs (ABMs).
15. The RNA of any one of claims 13 or 14, wherein the RNA encodes a chimeric or fusion protein comprises an amino acid sequence of a DUF2436 domain of the Arg and Lys gingipain surface complexes of P. gulae, that comprises or consists of the amino acid sequence of SEQ ID NO: 22, (eg) encoded by the RNA sequence as set forth in SEQ ID NO: 70, 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.
16. The RNA of any one of claims 13 or 14, wherein the RNA encodes a chimeric or fusion protein comprises an amino acid sequence of a DUF2436 domain of the Arg and Lys gingipain surface complexes of P. gulae, that comprises or consists of the amino acid sequence of SEQ ID NO: 19, encoded by the RNA sequence as set forth in SEQ ID NO: 71 , 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.
17. The RNA of claim 16, wherein one or more cysteine residues in the DUF2436 domain are substituted to a serine or valine residue or alanine residue, preferably to a serine residue (such as shown in any of SEQ ID NOs: 20 and 21).
18. The RNA of claim 17, wherein at least two cysteine residues in the DUF2436 domain are substituted, optionally at least three of the cysteine residues or all four cysteine residues are substituted.
19. The RNA 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 ABM 1.
20. The RNA of claim 19, wherein RNA encodes the amino acid sequence of a polypeptide comprising ABM2 and ABM1 as set forth in SEQ ID NO: 6 and SEQ ID NO: 6, respectively, or comprising the amino acid sequence as set forth in SEQ ID NO: 8 (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.
21. The RNA of claim 20, wherein the RNA comprises the nucleotide sequence as set forth in any one of SEQ ID NOs: 72 to 75.
22 The RNA of any one of claims 19 to 21, wherein the RNA encodes one or more adhesin binding motifs (ABMs) comprising or consisting of the amino acid sequence of ABM2, ABM1 and ABM3.
23. The RNA of claim 22, wherein the RNA encodies the amino acid sequence set forth in SEQ ID NO: 9 and encoded by an RNA comprising the sequence of SEQ ID NO: 76).
24. The RNA of any one of claims 19 to 23, 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 in the adhesin A domain compared to the A domain found naturally occurring in the Arg- Lys-gingipain homologue protein complex 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: 5 (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: 5 (ABM1); d) substitution of the second tyrosine residue, corresponding to or at a position equivalent to residues at position 10 of SEQ ID NO: 6 (ABM2), and of the tryptophan residue, corresponding to or at a position equivalent to residue at position 23 of SEQ ID NO: 5 (ABM1) to alanine residues.
25. The RNA of claim 24, 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.
26. The RNA of claim 225, wherein the RNA encodes one or more adhesin binding motifs that comprise substitution of all cysteine residues to serine or valine residues.
27. The RNA of any one of claims 23 to 26, 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: 5.
28. The RNA of claim 27, wherein the proline amino acid substitution is a substitution to an alanine residue.
29. The RNA of claim 27 or 28, wherein the asparagine amino acid substitution is a substitution to a proline residue or an alanine residue.
30. The RNA of any one of claims 24 to 29, wherein the RNA encodes one or more adhesin binding motifs comprising a substitution from PxxN to AxxP, (eg AVQP, SEQ ID NO: 107) (such as exemplified in the amino acid sequences of SEQ ID NOs: 14 to 16).
31. The RNA of any one of claims 24 to 30, 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: 8 or 9, and comprising: a) one or more cysteine amino acid substitutions compared to the naturally occurring Arg- or Lys-gingipain homologue sequences in corresponding regions, preferably substitution of all cysteine residues; and b) substitution of the motif PxxN corresponding to, or at a position equivalent to, residues 6 to 9 of the sequence of SEQ ID NO: 5 (ABM1), to AxxP.
32. The RNA of claim 31, wherein the RNA one or more adhesin binding motifs comprising or consisting of the amino acid sequence as set forth in any one of SEQ ID NOs: 17 or 18, 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.
33. An RNA encoding a chimeric or fusion protein comprising or consisting of:
- one or more amino acid sequences of active site of an Arg- or Lys-gingipain homologue of P. gulae, 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 found in surface complexes of an Arg- or Lys-gingipain homologues of P. gulae, or a sequence that is at least 80% identical thereto.
34. The RNA of claim 33, wherein the chimeric or fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 46 or any one of SEQ ID NOs: 78 to 82, or 85 to 89.
35. An RNA encoding a chimeric or fusion protein comprising or consisting of:
- one or more amino acid sequences of active site of an Arg- or Lys-gingipain homologues of P. gulae, or a sequence that is at least 80% identical thereto;
- the amino acid sequence of a DUF2436 domain of an Arg- or Lys-gingipain surface complexes of P. gulae, 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 surface complexes of Arg- and Lys-gingipain homologues of P. gulae, or a sequence that is at least 80% identical thereto.
36. The RNA of claim 35, wherein the RNA encodes a chimeric or fusion protein comprising or consisting of the amino acid sequence of SEQ ID NO: 30, 32, 38, 40, 48, or 50.
37. The RNA of claim 35, wherein the RNA encodes a chimeric or fusion protein comprising or consisting of the amino acid sequence of SEQ ID NO: 34 or 36.
38. An RNA encoding a chimeric or fusion protein comprising or consisting of:
- the amino acid sequence of a DUF2436 domain of Arg- or Lys-gingipain homologue surface complexes of P. gulae, 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 surface complex of Arg- and Lys-gingipain homologues of P. gulae, or a sequence that is at least 80% identical thereto.
39. The RNA of claim 38, wherein the RNA encodes a chimeric or fusion protein comprising or consisting of the amino acid sequence of SEQ ID NO: 42 or 44.
40. An RNA encoding a chimeric or fusion protein comprising or consisting of: - one or more amino acid sequences of active site of an Arg- or Lys-gingipain homologue of P. gulae, 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 homologue of P. gulae, or a sequence that is at least 80% identical thereto.
41. The RNA of claim 40, wherein the RNA encodes a chimeric or fusion protein comprising or consisting of the amino acid sequence of SEQ ID NO: 26 or 28.
42. An RNA encoding a chimeric or fusion protein comprising or consisting of:
- one or more amino acid sequences of active site of an Arg- or Lys-gingipain homologue of P. gulae, or a sequence that is at least 80% identical thereto;
- the amino acid sequence of a DUF2436 domain of an Arg- or Lys-gingipain homologue surface complex of P. gulae , 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 surface complex of an Arg- or Lys-gingipain homologue of P. gulae, or a sequence that is at least 80% identical thereto.
43. The RNA of claim 42, wherein the RNA encodes a chimeric or fusion protein comprising or consisting of the amino acid sequence of SEQ ID NO: 52 or 54.
44. An RNA comprising or consisting of a nucleotide sequence encoding a protein comprising or consisting of the amino acid sequence of any one of: SEQ ID NO: 2 or SEQ ID NO: 4.
45. An RNA comprising or consisting of a nucleotide sequence of any one of: a) SEQ ID NO: 67 or 68 b) SEQ ID NO: 62, 65, 66, 77or SEQ ID NO: 55 or 56; c) SEQ ID NO: 63 or 64 ; d) SEQ ID NO: 60 or 61 ; e) SEQ ID NO: 57 or 58.
46. An RNA comprising or consisting of a nucleotide sequence of SEQ ID NO: 59 or SEQ ID NO: 69.
47. The RNA of any one of the preceding claims, wherein the RNA is an mRNA.
48. The RNA 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.
49. The RNA of any one of the preceding claims, wherein the RNA further comprises a 5’ untranslated region (UTR) and/or a 3’ UTR.
50. The RNA of any one of the preceding claims, wherein the RNA also comprises a 5’ cap analog, such as 7mG(5')ppp(5')NlmpNp.
51. The RNA of any one of the preceding claims wherein the RNA also comprises a polyadenine (polyA) tail.
52. The RNA of any one of the preceding claims, wherein the RNA comprises a chemical modification, preferably wherein the chemical modification is a 1- methylpseudouridine modification or a 1 -ethylpseudouridine modification.
53. The RNA 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%.
54. The RNA of any one of the preceding claims, wherein the uridines in the RNA are replaced with a chemical modification such as N-methyl-pseudouridine.
55. The RNA of claim 54, 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.
56. The RNA of any one of the preceding claims wherein the RNA is in the form of a codon optimised RNA molecule.
57. A composition comprising an RNA of any one of the preceding claims.
58. A lipid nanoparticle composition comprising an RNA of any one of claims 1 to 56.
59. The lipid nanoparticle composition of claim 58, comprising:
- 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.
60. The lipid nanoparticle composition of claim 58 or 59, comprising:
- 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).
61. The lipid nanoparticle composition of claim 60, 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 T ris/sucrose buffer (25 mM T ris pH 7.4, 8.8% sucrose w/v).
62. A method for producing a lipid nanoparticle comprising an RNA of any one of claims 1 to 56, wherein preferably the method comprises formulating any RNA molecule of any one of claims 1 to 56, with one or more lipids useful for producing a lipid nanoparticle.
63. The method of claim 62, wherein the lipid components comprise a phospholipid, a PEG lipid, and a structural lipid.
64. A nucleic acid construct or vector, comprising a polynucleotide of any one of claims 1 to 56.
65. A method for eliciting an immune response P. gulae in a subject in need thereof, the method comprising administering to the subject, an RNA of any one of claims 1 to 56 or a nanoparticle or composition of any one of claims 57 to 61.
66. A method for eliciting an immune response to P. gulae in a subject in need thereof, the method comprising administering to the subject, a nanoparticle composition comprising:
- an RNA of any one of claims 1 to 56,
- an agent for enabling delivery of the RNA to a cell of the subject;
- wherein said RNA is capable of being translated in a cell of the subject to produce the polypeptide encoded by the polynucleotide.
67. A method for producing a chimeric or fusion protein in a mammalian cell, the method comprising contacting the mammalian cell with a nanoparticle composition, the composition comprising:
- an RNA of any one of claims 1 to 56,
- an agent for enabling delivery of the RNA to a cell of the subject;
- wherein said RNA is capable of being translated in the mammalian cell to produce the protein.
68. A method for delivering an RNA to a mammalian cell in a subject in need thereof, said method comprising administering to a subject in need thereof, a nanoparticle composition, the composition comprising:
- an RNA comprising a polynucleotide sequence of any one of claims 1 to 56,
- an agent for enabling delivery of the RNA to a cell of the subject; - wherein said RNA is capable of being translated in the mammalian cell to produce the chimeric or fusion protein described herein; wherein the administering comprises contacting said mammalian cell with the nanoparticle composition, thereby enabling delivery of the RNA to the mammalian cell.
69. The method of any one of claims 66 to 68, wherein the agent for enabling delivery of the RNA to a cell of the subject is a lipid.
70. The method of claim 69, wherein the lipid component comprises a cationic and/or ionisable lipid, a phospholipid, a PEG lipid, and a structural lipid.
71. Use of an RNA of any one of claims 1 to 56, or a vector, or nanoparticle comprising the same, in the manufacture of a composition for eliciting an immune response to P. gulae in a subject.
72. Use of i) a lipid component, preferably comprising a cationic and/or ionisable lipid, a phospholipid, a PEG lipid, and a structural lipid., and ii) an RNA of any one of claims 1 to 56, in the manufacture of a composition for delivering the RNA to a mammalian cell in a subject in need thereof.
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