RNA vaccine

Abstract

The present invention relates to a vaccine composition comprising RNA for use in inducing an immune response to Porphyromonas gingivalis (Porphyromonas gingivalis) in a subject, and to the use of the vaccine composition comprising RNA for use in inducing an immune response to Porphyromonas gingivalis in a subject.

Description

Technical Field

[0001] This invention relates to RNA-containing vaccine compositions for inducing an immune response against Porphyromonas gingivalis in subjects, and their use.

[0002] Related applications

[0003] This application claims priority to Australian Provisional Application AU 2023902379, the entire contents of which are hereby incorporated by reference. Background Technology

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

[0005] Chronic periodontitis is an inflammatory disease of the supporting tissues of the teeth, causing resorption of the alveolar bone and ultimately leading to tooth loss. This disease is a major public health problem in all communities and is estimated to affect up to 30% of the adult population, with the severe form affecting 12-15% of the adult population.

[0006] One-third of adults have moderate to severe periodontitis. Epidemiological studies have linked periodontitis to an increased risk of inflammatory diseases, including cardiovascular disease, certain cancers, premature birth, rheumatoid arthritis, and dementia. Recent research has linked chronic infection caused by *Porphyromonas gingivalis* to dementia and rheumatoid arthritis. For example, in one study, *Porphyromonas gingivalis* was found in 96% of brain samples from Alzheimer's disease (AD). Another study showed that chronic oral infection in mice with *Porphyromonas gingivalis* caused AD-associated brain plaques in humans, and that *Porphyromonas gingivalis* proteases can cleave amyloid precursors and tau proteins to form AD-associated plaques and tangles.

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

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

[0009] One problem is that, given the abundance of virulence agents available, it remains unclear how to obtain a strong protective response against Porphyromonas gingivalis infection.

[0010] There are currently no commercially approved vaccines for the prevention or reduction of the incidence and / or severity of Porphyromonas gingivalis infection, or for the treatment of Porphyromonas gingivalis infection and disease in subjects.

[0011] Therefore, there is a need for alternative and / or improved methods for designing and preparing Porphyromonas gingivalis vaccines, as well as alternative and / or improved vaccines produced by Porphyromonas gingivalis.

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

[0013] In a first aspect, the present invention provides an RNA polynucleotide encoding a protein comprising or composed of the following:

[0014] - One or more amino acid sequences of the active site of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or sequences that are at least 80% identical thereto; and / or

[0015] - An amino acid sequence of one or more adhesin-binding motifs (ABMs) of the adhesin domain of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto.

[0016] The polynucleotides mentioned therein can be translated in mammalian cells.

[0017] In any embodiment, the protein encoded by the RNA polynucleotide may further comprise:

[0018] - The amino acid sequence of the Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical to it.

[0019] Optionally, the protein encoded by RNA may contain the aforementioned domains in any order: for example, one or more amino acid sequences of the active site of *Porphyromonas gingivalis* Arg gingivalis protease or Lys gingivalis protease may be located at one or more amino acid sequences of the adhesin binding motif (ABM) of the adhesin domain of *Porphyromonas gingivalis* Arg gingivalis protease or Lys gingivalis protease and / or the DUF2 of *Porphyromonas gingivalis* Arg gingivalis protease or Lys gingivalis protease. The N-terminus of the amino acid sequence of the DUF2436 domain; or the amino acid sequence of one or more adhesin-binding motifs (ABMs) of the adhesin domain of *Porphyromonas gingivalis* Arg gingivalis or Lys gingivalis may be located at the N-terminus of the active site of *Porphyromonas gingivalis* Arg gingivalis or Lys gingivalis and / or the N-terminus of the amino acid sequence of the DUF2436 domain of *Porphyromonas gingivalis* Arg gingivalis or Lys gingivalis. The domains may bind directly in the context of a chimeric or fusion protein, or may bind via a linker region of one or more amino acid residues, as further defined herein.

[0020] In other words, the protein from the N-terminus to the C-terminus may contain or consist of the following: active site (K)-ABM (A); or active site (R)-ABM (A); or active site (K)-DUF 2436 (D); or active site (R)-DUF2436 (D); or AMB (A)-active site (K); or ABM (A)-active site (R); or DUF2436 (D)-active site (K); or DUF2436 (D)-active site (R); or active site (R)-ABM (A)-active site (K); or active site (R)-ABM (A)-active site (R); or active site (K)-ABM (A)-active site (R); or DUF2436 (D)-ABM (A); or DUF2436 (D)-ABM (A)-active site (R) or (K).

[0021] In any embodiment herein, the RNA polynucleotide is in the form of a messenger RNA (mRNA) molecule. However, it should be understood that the RNA polynucleotide may be in any suitable form for translation in mammalian cells and enabling the synthesis of proteins encoded by the RNA.

[0022] In some embodiments, the RNA polynucleotide may be formed entirely of a ribose-containing nucleotide, or alternatively, may comprise a combination of a ribose-containing nucleoside and a nucleotide containing 2'-deoxyribose.

[0023] In any embodiment, the RNA polynucleotide may be a synthetic RNA molecule.

[0024] In any embodiment, the RNA polynucleotide may be a circular RNA (circRNA) molecule.

[0025] In any embodiment, the RNA polynucleotide may be a complementary RNA (cRNA) molecule.

[0026] In any embodiment, the RNA polynucleotide may be a self-amplifying RA (saRNA) molecule or a trans-amplifying (taRNA) molecule.

[0027] Further examples of various RNA molecular forms are described in Fang et al., (2022) Signal Transduction and Targeted Therapy, 7: Paper 94, which is incorporated herein by reference.

[0028] In any embodiment herein, the RNA may further encode an N-terminal signal peptide to enable the secretion of the protein post-translation. The N-terminal signal peptide may comprise any amino acid sequence capable of enabling the RNA-encoded protein to be processed by ribosomes that bind to the cell's rough endoplasmic reticulum (ER), thereby introducing the protein into the ER. From the ER, the protein can be transported to the plasma membrane and secreted from the mammalian cell. N-terminal secretory peptides are known to those skilled in the art and are further described herein.

[0029] In a preferred embodiment, the RNA may further comprise a 5' untranslated region (UTR) and a 3' UTR. The RNA may also comprise a 5' cap analogue, such as 7mG(5')ppp(5')NlmpNp. The RNA may also comprise a poly(A) tail. The poly(A) tail may be unsegmented or segmented using short spacer elements.

[0030] The RNA may contain chemical modifications. Examples of suitable chemical modifications include N1-methylpseuuridine modification or N1-ethylpseuuridine modification, or may contain any of the chemical modifications described herein.

[0031] Preferably, the uridine content of the polynucleotide is 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 a preferred embodiment, the uridine content of the polynucleotide is from about 15% to about 35%, preferably from about 15% to about 25%.

[0032] In a preferred embodiment, the uridine in the polynucleotide is replaced by a chemical modification such as N1-methyl-pseudouridine. Preferably, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uridine nucleoside is replaced by N1-methyl-pseudouridine.

[0033] In any embodiment of the invention, the RNA polynucleotide is in the form of a codon-optimized RNA molecule, optionally depleting uridine nucleosides. In any embodiment, codon optimization involves changing the codon encoding serine to UCG.

[0034] In any embodiment, the protein encoded by the RNA polynucleotide is a chimeric or fusion protein comprising or composed of the following:

[0035] - One or more amino acid sequences of the active site of Arg gingivase or Lys gingivase from *Porphyromonas gingivalis*, or sequences that are at least 80% identical thereto; and

[0036] - An amino acid sequence of one or more adhesin-binding motifs (ABMs) of the adhesin domain of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto.

[0037] It should be understood that the chimeric or fusion protein encoded by the RNA may contain the aforementioned domains in any order: for example, one or more amino acid sequences of the active site of the Arg gingivalase or Lys gingivalase of *Porphyromonas gingivalis* may be located at the N-terminus of one or more amino acid sequences of the adhesin binding motif (ABM) of the adhesin domain of the Arg gingivalase or Lys gingivalase of *Porphyromonas gingivalis*; or the amino acid sequences of one or more adhesin binding motifs (ABM) of the adhesin domain of the Arg gingivalase or Lys gingivalase of *Porphyromonas gingivalis* may be located at the N-terminus of one or more amino acid sequences of the active site of the Arg gingivalase or Lys gingivalase of *Porphyromonas gingivalis*. The domains may bind directly in the context of the chimeric or fusion protein, or may bind via a linker region of one or more amino acid residues, as further defined herein. (In other words, the chimeric or fusion protein may contain or consist of the following from the N-terminus to the C-terminus: active site (K) - ABM (A); or active site (R) - ABM (A); or ABM (A) - active site (K); or ABM (A) - active site (R); or active site (R) - ABM (A) - active site (K); or active site (R) - ABM (A) - active site (R); or active site (K) - ABM (A) - active site (R)).

[0038] This document further describes exemplary amino acid sequences (and RNA sequences encoding said amino acid sequences) of Arg gingival protease or Lys gingival protease from *Porphyromonas gingivalis*. Preferably, the amino acid sequence of the active site of the Arg gingival protease or Lys gingival protease from *Porphyromonas gingivalis* comprises an amino acid sequence of KAS or RAS (lysine or arginine active site histidine sequence), i.e., a peptide including the active site histidine and the surrounding region of the active site.

[0039] In some embodiments, the amino acid sequence of the active site of Arg gingivase from *Porphyromonas gingivalis* (also referred to herein as “R”) comprises the amino acid sequence of SEQ ID NO: 38 (encoded by the RNA sequence shown in SEQ ID NO: 50) or is identical to 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%.

[0040] In some embodiments, the amino acid sequence of the active site of Lys gingivase from Porphyromonas gingivalis (also referred to herein as "K") comprises the amino acid sequence of SEQ ID NO: 8 (encoded by the RNA sequence shown in SEQ ID NO: 43) or is identical to 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%.

[0041] Preferably, the amino acid sequence of the active site of Arg gingivase or Lys gingivase does not contain the entire catalytic domain of gingivase.

[0042] In some embodiments, the chimeric or fusion protein encoded by the RNA polynucleotide comprises: i) an amino acid sequence containing the active site of Arg gingival protease from Porphyromonas gingivalis, or an amino acid sequence that is at least 80% identical to or composed thereof; and ii) an amino acid sequence containing the active site of Lys gingival protease from Porphyromonas gingivalis, or an amino acid sequence that is at least 80% identical to or composed thereof.

[0043] Optionally, the chimeric or fusion protein encoded by the RNA polynucleotide comprises at least two amino acid sequences, which comprise or consist of the amino acid sequence of the active site of Arg gingivalase or Lys gingivalase of *Porphyromonas gingivalis*, or a sequence that is at least 80% identical thereto. The at least two amino acid sequences may be located consecutively within the chimeric or fusion protein, or may be located at different positions 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 either the N-terminus or the 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 (i.e., neither at the N-terminus nor the C-terminus). Optionally, the at least two amino acid sequences may be located at the N-terminus of the chimeric or fusion protein, or the at least two amino acid sequences may be located at the C-terminus of the chimeric or fusion protein.

[0044] In any embodiment, the chimeric or fusion protein encoded by the RNA polynucleotide may further comprise:

[0045] - The amino acid sequence of the Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical to it.

[0046] Optionally, the amino acid sequence containing the DUF2436 domain is located between the amino acid sequence of the active site of the gingival protease of *Porphyromonas gingivalis* and the amino acid sequence of one or more adhesin-binding motifs (ABMs). (In other words, this makes the chimeric or fusion protein comprise, from the N-terminus to the C-terminus or from the C-terminus to the N-terminus: active site (K) or (R) - DUF domain (D) - ABM (A); or active site (K) or (R) - DUF domain (D) - ABM (A) - active site (K) or (R)).

[0047] In some embodiments, the amino acid sequence of the Arg gingival protease or Lys gingival protease DUF2436 domain of Porphyromonas gingivalis comprises or consists of the following: the amino acid sequence of SEQ ID NO: 35 or 76 (encoded by the RNA sequence as shown in SEQ ID NO: 51) or the sequence that is 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 to it.

[0048] In any embodiment, the cysteine ​​residues in the DUF2436 domain may be substituted with serine or valine residues, preferably with serine residues (as shown in SEQ ID NO: 36).

[0049] In any embodiment, one or more adhesin-binding motifs (ABMs) comprise or consist of the amino acid sequences of ABM2 and / or ABM1 (e.g., as shown in SEQ ID NO: 22 and SEQ ID NO: 21, respectively), or comprise the amino acid sequence (ABM2+1) shown in SEQ ID NO: 24, or are 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 to it. In this document, such amino acid sequences may be encoded by RNA comprising the sequences shown in SEQ ID NO: 52, 53, and 55.

[0050] ABM may further include ABM3 (e.g., SEQ ID NO: 73 or encoded by SEQ ID NO: 54).

[0051] ABMs can be provided in any order, but are preferably provided in the order ABM2 - ABM1 - ABM3.

[0052] Optionally, one or more adhesin-binding motifs (ABMs) may comprise or consist of the amino acid sequences of ABM2, ABM1, and ABM3 (e.g., as shown in SEQ ID NO: 25, 72, or 73 and encoded by RNA containing the sequence of SEQ ID NO: 56, 77, or 78) or the amino acid sequences described therein.

[0053] In any embodiment, one or more adhesin-binding motifs may include one or more modifications selected from:

[0054] a) In the corresponding region, one or more cysteine ​​amino acids are substituted compared to the naturally occurring Arg gingivase or Lys gingivase sequence;

[0055] b) Substitution of proline and / or asparagine residues in sequence PxxN corresponding to residues 6 to 9 of the sequence of SEQ ID NO: 21 (ABM1) or located at positions equivalent to said residues;

[0056] c) Substitution of motif NxFA to SxYQ in the sequence corresponding to residues 2 to 5 of SEQ ID NO: 21 (ABM1) or located at positions equivalent to said residues;

[0057] d) The second tyrosine residue corresponding to or located at a position equivalent to the residue at position 5 of SEQ ID NO: 22 (ABM2) and the tryptophan residue corresponding to or located at a position equivalent to the residue at position 23 of SEQ ID NO: 21 (ABM1) are replaced by an alanine residue.

[0058] The one or more cysteine ​​amino acid substitutions may be substitutions that change to serine residues or valine residues. Preferably, the one or more cysteine ​​substitutions may include one or more substitutions that change to serine residues.

[0059] In some embodiments, only one cysteine ​​residue may be substituted. In other embodiments, two or three cysteine ​​residues may be substituted. In a particularly preferred embodiment, the cysteine ​​residues are substituted with a combination of valine and serine residues. In other embodiments, all substituted cysteine ​​residues are substituted with serine or all substituted cysteine ​​residues are substituted with valine.

[0060] The motif PxxN (e.g., PVQN, SEQ ID NO: 106) corresponding to residues 6 to 9 of SEQ ID NO: 21 or located at positions equivalent to said residues may contain substitutions of proline residues and asparagine residues.

[0061] The proline amino acid substitution is preferably a substitution of alanine residues.

[0062] The asparagine amino acid substitution can be a substitution of a proline residue or an alanine residue. Preferably, the asparagine residue is substituted with a proline residue. In other embodiments, the asparagine residue is not substituted.

[0063] Preferably, the substitution is PxxN to AxxP (e.g., AVQP, SEQ ID NO: 107) (as illustrated in the amino acid sequences of SEQ ID NO: 30 to 32).

[0064] In some other embodiments, the one or more adhesin-binding motifs comprise an amino acid sequence as shown in any of SEQ ID NO: 21 to 25, and include:

[0065] a) In the corresponding region, compared with the sequence of naturally occurring Arg gingivase or Lys gingivase, one or more cysteine ​​amino acids are substituted, preferably all cysteine ​​residues are substituted; and

[0066] b) Substitution of motif PxxN to AxxP corresponding to residues 6 to 9 of the sequence of SEQ ID NO: 21 (ABM1) or located at positions equivalent to said residues.

[0067] Therefore, in such embodiments, the one or more adhesin-binding motifs comprise or consist of the following: an amino acid sequence as shown in any of SEQ ID NO: 26 to 34 or the same sequence as 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%, provided that the sequence comprises the above-described substitutions of cysteine ​​residues, proline residues, and asparagine residues.

[0068] In a particularly preferred embodiment of the invention, the RNA (preferably mRNA) encodes a chimeric or fusion protein comprising or consisting of the following:

[0069] a)

[0070] - One or more amino acid sequences of the active site of Arg gingivase or Lys gingivase from *Porphyromonas gingivalis*, or sequences that are at least 80% identical thereto; and

[0071] - An amino acid sequence of one or more adhesin-binding motifs (ABMs) of the adhesin domain of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto.

[0072] Preferably, the chimeric or fusion protein comprises any one of SEQ ID NO: 18, SEQ ID NO: 39, or SEQ ID NO: 58 to 63; or the amino acid sequence of SEQ ID NO: 81 to 87; or

[0073] b)

[0074] - One or more amino acid sequences of the active site of Arg gingivase or Lys gingivase of Porphyromonas gingivalis, or sequences that are at least 80% identical to them.

[0075] - The amino acid sequence of the DUF2436 domain of Arg gingival protease or Lys gingival protease of *Porphyromonas gingivalis*, or a sequence that is at least 80% identical thereto, and

[0076] - An amino acid sequence of one or more adhesin-binding motifs (ABMs) of the adhesin domain of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto.

[0077] - Preferably, the chimeric or fusion protein comprises the amino acid sequence of SEQ ID NO: 4, 12, 16 or 20;

[0078] -More preferably, the chimeric or fusion protein comprises the amino acid sequence of SEQ ID NO: 4; or

[0079] c)

[0080] - The amino acid sequence of the DUF2436 domain of Arg gingival protease or Lys gingival protease of *Porphyromonas gingivalis*, or a sequence that is at least 80% identical thereto, and

[0081] - An amino acid sequence of one or more adhesin-binding motifs (ABMs) of the adhesin domain of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto.

[0082] - Preferably, the chimeric or fusion protein comprises the amino acid sequence of SEQ ID NO: 14; or

[0083] d)

[0084] - One or more amino acid sequences of the active site of Arg gingivase or Lys gingivase from *Porphyromonas gingivalis*, or sequences that are at least 80% identical thereto; and

[0085] - The amino acid sequence of the Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical to it.

[0086] - Preferably, the chimeric or fusion protein comprises the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 108; or

[0087] e)

[0088] - One or more amino acid sequences of the active site of Arg gingivase or Lys gingivase of Porphyromonas gingivalis, or sequences that are at least 80% identical to them.

[0089] - The amino acid sequence of the DUF2436 domain of Arg gingival protease or Lys gingival protease of *Porphyromonas gingivalis*, or a sequence that is at least 80% identical thereto, and

[0090] - An amino acid sequence of one or more adhesin-binding motifs (ABMs) of the adhesin domain of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto.

[0091] - Preferably, the chimeric or fusion protein comprises the amino acid sequence of SEQ ID NO: 6.

[0092] In some embodiments, the linker region may be included between an amino acid sequence of the DUF2436 domain and an amino acid sequence of one or more adhesin-binding motifs (ABMs) of the adhesin domain. Those skilled in the art will be familiar with suitable linkers that can be used to conjugate any of the aforementioned domains. In some embodiments, and as illustrated in the tables herein, the linker comprises or is composed of the sequence EVEDDSP (SEQ ID NO: 109). In some embodiments, the chimeric or fusion protein encoded by the RNA does not include the linker sequence between the DUF2436 domain and one or more adhesin domains.

[0093] In some embodiments, the amino acid sequence of the AMB2 domain as defined herein (e.g., in SEQ ID NO:22 or SEQ ID NO 24 to 34) may further include the amino acid sequence of EVEDDSP (SEQ ID NO: 109) at its N-terminus, said amino acid sequence being derived from the original Porphyromonas gingivalis gingival protease polyprotein sequence.

[0094] It will be understood that, in some cases, after mRNA is translated into a protein, the N-terminal methionine residue of the polypeptide is cleaved. Therefore, this disclosure provides a basis for generating chimeric or fusion proteins from RNA molecules, wherein said chimeric or fusion proteins do not contain an N-terminal methionine residue.

[0095] In other embodiments of the invention, the RNA molecule comprises or is composed of a nucleotide sequence encoding a protein, said nucleotide sequence comprising or is composed of an amino acid sequence of any of the following: SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 38.

[0096] In a particularly preferred embodiment of the invention, the RNA molecule comprises or consists of a nucleotide sequence of any of the following:

[0097] a) SEQ ID NO: 48 or 57;

[0098] b) SEQ ID NO: 45, 47, or 49, or SEQ ID NO: 41;

[0099] c) SEQ ID NO: 46;

[0100] d) SEQ ID NO: 44;

[0101] e) SEQ ID NO: 42;

[0102] Or a sequence that is 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 to it.

[0103] In other embodiments of the invention, the RNA comprises or consists of the nucleotide sequence of any of the following: SEQ ID NO: 40, SEQ ID NO: 43, or SEQ ID NO: 50.

[0104] The present invention further provides the use of any DNA polynucleotide described herein (and specifically any DNA polynucleotide comprising or composed of the sequences illustrated in Table 1). Optionally, the use of the DNA polynucleotide can be used to obtain the RNA polynucleotide of the present invention.

[0105] The present invention also provides a composition comprising a pharmaceutical composition containing RNA as described herein. Preferably, the composition comprises one or more pharmaceutically acceptable excipients. Optionally, the RNA may contain one or more agents for enabling delivery of the RNA to mammalian cells and thereby enabling translation of the RNA in the cells. In any embodiment, the composition may comprise a combination of one or more of the RNA molecules described herein.

[0106] The present invention further provides compositions comprising RNA molecules as described herein, wherein the compositions also comprise lipid components. The RNA (e.g., RNA) vaccines disclosed herein can be formulated using one or more liposomes, lipid vesicles, lipid complexes (such as lipid-multicationic complexes), or lipid nanoparticles.

[0107] In a preferred embodiment, RNA is formulated within lipid nanoparticles.

[0108] In one embodiment, RNA, as described herein, is the only polynucleotide species present in the composition or in liposomes, lipid vesicles, lipid complexes (such as lipid-polycationic complexes), or lipid nanoparticles. Preferably, the polynucleotide, as described herein, is the only active ingredient present in the composition or in liposomes, lipid vesicles, lipid complexes (such as lipid-polycationic complexes), or lipid nanoparticles.

[0109] In other instances, the composition or liposome, lipid vesicle, lipid complex (such as a lipid-multicationic complex), or lipid nanoparticle may comprise more than one RNA (polynucleotide) species, as described herein (e.g., thereby providing an RNA molecule encoding more than one chimeric or fusion protein amino acid sequence). Furthermore, the composition or liposome, lipid vesicle, lipid complex (such as a lipid-multicationic complex), or lipid nanoparticle may comprise a single polynucleotide construct containing one or more RNA sequences as described herein (and thus also encoding more than one chimeric or fusion protein amino acid sequence). Therefore, the present invention contemplates providing compositions, liposomes, lipid vesicles, lipid complexes (such as lipid-multicationic complexes), or lipid nanoparticles for delivering combinations of two or more of any of the RNA molecules described herein.

[0110] In any embodiment, the present invention provides lipid nanoparticles or other nanomedia, such as nanopolymers, for delivering polynucleotides to subjects in need.

[0111] Lipid nanoparticles are well known in the art and are further described herein. Preferably, the lipid nanoparticles comprise cationic lipids and / or ionizable lipids, phospholipids, PEG (or PEGylated) lipids, and structural lipids.

[0112] In any embodiment, the lipid nanoparticles may comprise:

[0113] - Cationic lipids and / or ionizable lipids, wherein the cationic lipids and / or ionizable lipids constitute approximately 25% to approximately 75 mol% of the total lipids present in the nanoparticles;

[0114] - Sterols (structural lipids), said sterols comprising about 5 mol% to about 60 mol% of the total lipids present in said nanoparticles;

[0115] - Phospholipids, wherein the phospholipids comprise approximately 5 mol% to approximately 50 mol% of the total lipids present in the nanoparticles;

[0116] -PEGylated lipids, wherein the PEGylated lipids account for approximately 0.5 mol% to 20 mol% of the total lipids present in the nanoparticles.

[0117] In a non-limiting example, the lipid nanoparticles comprise:

[0118] - An ionizable lipid in the form of [(4-hydroxybutyl)azanidinediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315),

[0119] - Sterols in the form of cholesterol,

[0120] - Phospholipids in the form of distearate phosphatidylcholine (DSPC), and

[0121] - A PEGylated lipid in the form of 2-[(polyethylene glycol)-2000]-N,N-bis(tetradecyl)acetamide (ALC-0159).

[0122] Preferably, the lipids are present in lipid nanoparticles at a molar lipid ratio (%) of 46.3 ALC-0315: 42.7 cholesterol: 9.4 DSPC: 1.6 ALC-0159, and optionally in Tris / sucrose buffer (25 mM Tris, 8.8% sucrose w / v, pH 7.4).

[0123] The present invention also provides a method for generating lipid nanoparticles comprising RNA encoding a protein or a chimeric or fusion protein as described herein. Preferably, the method comprises adapting any RNA molecule of the present invention, and one or more lipids that can be used to generate lipid nanoparticles. Preferably, the lipid component comprises phospholipids, PEG lipids, and structural lipids.

[0124] The present invention also provides a nucleic acid construct or vector comprising polynucleotides as described herein.

[0125] The vector can be any vector suitable for generating RNA from a DNA template. The vector may additionally contain a 3'UTR and a 5'UTR, as well as a polyadenosine fragment.

[0126] Examples of such vectors include IVT RNA vectors or similar vectors containing T7, T3, and SP6 signals for expression. These vectors may be derived from plasmids or generated via PCR or Phi29 DNA polymerase (e.g., GenomiPhi™ V2 DNA) or other bacterial constructs.

[0127] The vector may be a self-amplifying RNA replicon, such as, but not limited to, self-amplifying RNA vectors derived from alpha viruses, optionally Venezuelan Equine Encephalitis Virus (VEEV), diploid VEEV, or variants thereof (including the TC83 mutant variant). Examples of self-amplifying mRNA platforms are known to those skilled in the art and are described, for example, in Maruggi et al., (2017), Vaccines 35: 361-368, which is incorporated herein by reference.

[0128] 5' capping of polynucleotides can be performed using any commercially available capping reagent. Such reagents are known to skilled technicians, such as Cap1, a commercial capping reagent from TriLink Biotechnologies Inc. Other capping reagents may also be used, including but not limited to Cap 0 and Cap 2.

[0129] The present invention provides a method for inducing an immune response against Porphyromonas gingivalis in a subject in need, the method comprising administering to the subject a polynucleotide, carrier, nanoparticle or composition described herein.

[0130] The present invention provides a method for inducing an immune response against *Porphyromonas gingivalis* in a subject in need, the method comprising administering a composition to the subject, the composition comprising:

[0131] - RNA as described herein, wherein the RNA is capable of being translated into a polypeptide encoded by the polynucleotide in the cells of a mammalian subject;

[0132] - Optionally, a drug that enables the delivery of the RNA into mammalian cells.

[0133] Agents used to deliver RNA into mammalian cells can be any suitable agent known to a skilled technician for RNA delivery. Such agents can include: cell-penetrating peptides, lipid-based formulations.

[0134] This invention provides a method for inducing an immune response against *Porphyromonas gingivalis* in a subject in need, the method comprising administering a nanoparticle composition to the subject, the nanoparticle composition comprising:

[0135] -Lipid components; and

[0136] - RNA as described herein, wherein the RNA can be translated in the subject's cells to produce a polypeptide encoded by a polynucleotide.

[0137] The present invention also provides a method for producing chimeric or fusion proteins as described herein in mammalian cells, the method comprising contacting the mammalian cells with a composition comprising:

[0138] - RNA as described herein, wherein the RNA can be translated into the protein in the mammalian cell;

[0139] - Optionally, a drug that enables the delivery of the RNA into mammalian cells.

[0140] Agents used to deliver RNA into mammalian cells can be any suitable agent known to a skilled technician for RNA delivery. Such agents can include: cell-penetrating peptides, lipid-based formulations.

[0141] The present invention also provides a method for producing chimeric or fusion proteins as described herein in mammalian cells, the method comprising contacting the mammalian cells with a nanoparticle composition comprising:

[0142] -Lipid components; and

[0143] - RNA as described herein, wherein the RNA is capable of being translated into proteins in the mammalian cells.

[0144] Preferably, the lipid component comprises cationic lipids and / or ionizable lipids, phospholipids, PEG lipids, and structural lipids.

[0145] The present invention also provides a method for delivering RNA to mammalian cells in a subject in need, the method comprising administering a nanoparticle composition to the subject in need, the composition comprising:

[0146] -Lipid components; and

[0147] - RNA comprising a polynucleotide sequence as described herein, wherein the RNA is capable of being translated in the mammalian cell to produce the chimeric or fusion protein described herein;

[0148] The application involves contacting the mammalian cells with the nanoparticle composition, thereby enabling the delivery of the RNA to the mammalian cells.

[0149] Preferably, the lipid component comprises cationic lipids and / or ionizable lipids, phospholipids, PEG lipids, and structural lipids.

[0150] In any embodiment, the ionizable lipids may be replaced by or combined with adjuvant lipids to enhance RNA delivery.

[0151] The present invention also provides the use of the polynucleotide, carrier or nanoparticle described herein for the preparation of compositions for inducing an immune response against Porphyromonas gingivalis in subjects.

[0152] The present invention also provides i) the use of lipid components as described herein and ii) the use of RNA as described herein for preparing compositions for delivering said RNA to mammalian cells in subjects in need.

[0153] The present invention also provides a polynucleotide, carrier, nanoparticle or composition as described herein for inducing an immune response against Porphyromonas gingivalis in subjects.

[0154] As used herein, “at least 80% identity” is applied to provide a 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”.

[0155] 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” with a particular SEQ ID NO may also be referred to as a “substitute variant”.

[0156] As used herein, unless the context otherwise requires, the term “comprise” and variations thereof, such as “comprising,” “comprises,” and “comprised,” are not intended to exclude additional additions, components, wholes, or steps.

[0157] Further aspects of the invention and further embodiments of the aspects described in the foregoing paragraphs will become apparent from the following description, which is given by way of example and with reference to the accompanying drawings. Attached Figure Description

[0158] Figure 1 The schematic diagram illustrates the domain architecture of the Kgp multiprotein and its derived chimeric proteins. (KDcAK1n: K = Kgp active site, Dc = N-terminal truncated DUF2436 domain, A = ABM2, ABM1, and ABM3, K1n = C-terminal truncated CAD domain; KDAK: K = Kgp active site, D = DUF2436 domain, A = ABM2, ABM1, and ABM3; KDAK-3S-AVQP: K = Kgp active site, D = DUF2436 domain, A = ABM2, ABM1, and ABM3, 3S-AVQP = substitution of PVQN (SEQ ID NO: 106) in the ABM1 domain for AVQP (SEQ ID NO: 107) and substitution of cysteine ​​residues in the D and A domains for serine).

[0159] Figure 2 : Exemplary structure of mRNA. Cap = 5' cap to maximize RNA stability; 5'UTR and 3'UTR = 5' and 3' untranslated sequences; SP = signal peptide; antigen sequence = mRNA sequence encoding a protein antigen to be expressed in the cell during mRNA translation; Poly(A) tail: a polyadenosine tail used to provide RNA stability and maximize translation.

[0160] Figure 3Expression and secretion of chimeric proteins in HeLa cells via mRNA constructs. A. HeLa supernatant. Lane 1 = SEAP-KDcAK1n, Lane 2 = KDcAK1n (no secreted peptide), Lane 3 = mimic, Lane 4 = protein size marker. B. HeLa supernatant: Lane 1: SEAP-KDAK, Lane 2 = SEAP-KDAK-3S-AVQP, Lane 3 = mimic, Lane 5 = protein size marker. C. HeLa supernatant: Lane 1 = SEAP-KDAK-3S-AVQP, Lane 2 = KDAK-3S-AVQP (no secreted peptide), Lane 3 = mimic, Lane 4 = protein size marker. SEAP = SEAP secreted peptide derived from secreted embryonic alkaline phosphatase. Mimic = negative control (dummy transfection).

[0161] Figure 4 : A schematic diagram of a vaccination regimen used to evaluate the immunogenicity of a candidate vaccine.

[0162] Figure 5 Immunogenicity of mRNA vaccines. A. Total IgG titer of KDAK-3S-AVQP. B. IgG1 titer of KDAK-3S-AVQP. C. IgG2a titer of KDAK-3S-AVQP. D. Total IgG titer of heat-inactivated Porphyromonas gingivalis. E. IgG1 titer of heat-inactivated Porphyromonas gingivalis. F. IgG2a titer of heat-inactivated Porphyromonas gingivalis. G. Total IgG Kgpcat titer. H. Kgpcat IgG titer. I. KAS2 titer. Original = unvaccinated; mΨ1-Neg = KDAK-3S-AVQP encoding mRNA but uncapped; Chimera = KDcAK1n encoding mRNA (SEQ ID NO: 2); KDAK = KDAK protein encoding mRNA (SEQ ID NO: 4); KDAK-3S-AVQP = KDAK-3S-AVQP protein encoding RNA (SEQ ID NO: 6). m1Ψ = mRNA sequence modified with N1-methylpseudouridine. Protein control: 200 μg of KDAK-3S-AVQP protein adjuvanted with alum. * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001.

[0163] Figure 6 : A schematic diagram (treatment model) of a research protocol used to determine the in vivo efficacy of a vaccine.

[0164] Figure 7Alveolar bone loss (mm) induced by *Porphyromonas gingivalis* after vaccination with different mRNA constructs. Primary = Uninfected. Infected = Unvaccinated control. Positive control: 200 μg of protein KDAK-3S-AVQP adjuvanted with alum. m1Ψ = Modified mRNA sequence. * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001.

[0165] Figure 8 Antibody response in mice after alveolar bone loss experiment. A. Total IgG titer of KDAK-3S-AVQP. B. KDAK-3S-AVQP IgG1 titer. C. KDAK-3S-AVQP IgG2a titer. D. Total IgG titer of heat-inactivated Porphyromonas gingivalis. E. IgG1 titer of heat-inactivated Porphyromonas gingivalis. F. IgG2a titer of heat-inactivated Porphyromonas gingivalis. G. Total IgG titer of Kgpcat. H. Kgpcat IgG1 titer. I. Kgpcat IgG2a titer. J. KAS2 titer. Primary = unvaccinated; Infected = no vaccine control; KDAK = KDAK protein encoding mRNA (SEQ ID NO: 4); KDAK-3S-AVQP = KDAK-3S-AVQP protein encoding mRNA (SEQ ID NO: 6). m1Ψ = mRNA sequence modified with N1-methylpseuuridine. Protein control: 200 μg of protein KDAK-3S-AVQP adjuvanted with alum. * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001.

[0166] Figure 9 : Constructs encoding truncated antigens for expression and secretion. A. Western blot of lysate 24 hours post-transfection. Left inset: using anti-KDAK-3S-AVQP antibody. Right inset: using anti-KAS antibody. B. Western blot of supernatant 24 hours post-transfection. Left inset: using anti-KAS antibody. Right inset: using anti-KDAK-3S-AVQP antibody. A and B: Lanes: L = sequence ladder; 1 = K; 2 = KA; 3 = KD; 4 = DA; 5 = KDA ΔABM36 = KDA; 7 = RDA; 8 = KDA-3S-AVQP (positive control); 9 = simulated transfection (negative control). All constructs included the SEAP secretory peptide. C. K expression over time compared to KDAK-3S-AVQP: delay in K expression at 6, 24, and 48 hours post-transfection. Lanes: L = sequence ladder; 1 = K; 2 = KDAK-3S-AVQP at 6 hours; 3 = simulated transfection at 6 hours; 4 = K; 5 = KDAK-3S-AVQP at 24 hours; 6 = simulated transfection at 24 hours; 7 = K at 48 hours; 8 = KDA-3S-AVQP at 48 hours; 9 = simulated transfection at 48 hours. All constructs included the SEAP secretory peptide.

[0167] Figure 10 Alveolar bone loss (mm) induced by *Porphyromonas gingivalis* after vaccination with different mRNA constructs. Primary = Uninfected. Infected = Unvaccinated control. Positive control: 200 μg of protein KDAK-3S-AVQP adjuvanted with alum. mΨ1 = Modified mRNA sequence. * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001.

[0168] Figure 11Antibody response in mice after alveolar bone loss assays using an mRNA vaccine construct encoding a truncated antigen. A. Total IgG titer of KDAK-3S-AVQP. B. KDAK-3S-AVQP IgG1 titer. C. KDAK-3S-AVQP IgG2a titer. D. Total IgG titer of heat-inactivated *Porphyromonas gingivalis*. E. Heat-inactivated *Porphyromonas gingivalis* IgG1 titer. F. Heat-inactivated *Porphyromonas gingivalis* IgG2a titer. G. Total IgG titer of Kgpcat. H. Kgpcat IgG1 titer. I. Kgpcat IgG2a titer. J. KAS2 titer. Primary = unvaccinated; Infected = no vaccine control; KDAK-3S-AVQP = KDAK-3S-AVQP protein encoding mRNA (SEQ ID NO: 6). K. Total IgG titer of Kgp-RgpA complex. L. IgG1 titer of Kgp-RgpA complex. M. IgG2a titer of Kgp-RpgA complex. m1Ψ = mRNA sequence modified with N1-methylpseudouridine. KA = KA protein encoding mRNA (SEQ ID NO: 18). KD = KD protein encoding mRNA (SEQ ID NO: 10). DA = DA protein encoding mRNA (SEQ ID NO: 14). KDA21 = KDA protein encoding mRNA. ΔAMB3 Protein (SEQ ID NO: 16). Protein control: 200 μg of protein KDAK-3S-AVQP adjuvanted with alum. * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001.

[0169] Figure 12Immunogenicity of truncated antigens RDA and K. A. Total IgG titer of KDAK-3S-AVQP. B. KDAK-3S-AVQP IgG1 titer. C. KDAK-3S-AVQP IgG2a titer. D. Total IgG titer of heat-inactivated *Porphyromonas gingivalis*. E. IgG1 titer of heat-inactivated *Porphyromonas gingivalis*. F. IgG2a titer of heat-inactivated *Porphyromonas gingivalis*. G. Total IgG titer of Kgpcat. H. IgG1 titer of Kgpcat. I. IgG2a titer of Kgpcat. J. KAS2 titer. K. Total IgG titer of RgpA-Kgp complex. L. IgG1 titer of RgpA-Kgp complex. M. IgG2a titer of RgpA-Kgp complex. Original = Unvaccinated; K = K protein encoding mRNA (SEQ ID NO: 8); KD = KD protein encoding mRNA (SEQ ID NO: 10); RDA = RDA protein encoding mRNA (SEQ ID NO: 20); * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001.

[0170] Figure 13 : A schematic diagram of a research protocol used to determine the in vivo efficacy of a vaccine in a prevention model.

[0171] Figure 14 : The number of KDAK-3S-AVQP-specific interferon-γ-secreting spleen cells from a prophylactic vaccine-fed mouse model, as determined by in vitro ELISpot assay. Primary = Uninfected. Infected = Unvaccinated control. Positive control: 200 μg of KDAK-3S-AVQP protein adjuvanted with alum. m1Ψ = Modified mRNA sequence. * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001.

[0172] Figure 15 Alveolar bone loss (mm) induced by *Porphyromonas gingivalis* after prophylactic vaccination with different mRNA constructs prior to challenge with *Porphyromonas gingivalis*. Primary = Uninfected. Infected = Unvaccinated control. Positive control: 200 μg of protein KDAK-3S-AVQP adjuvanted with alum. m1Ψ = Modified mRNA sequence. * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001.

[0173] Figure 16Antibody response in mice after alveolar bone loss experiment in a prevention model. A. Total IgG titer. B. IgG1 titer. C. IgG2a titer. D. Total IgG titer of heat-inactivated *Porphyromonas gingivalis*. E. IgG1 titer of heat-inactivated *Porphyromonas gingivalis*. F. IgG2a titer of heat-inactivated *Porphyromonas gingivalis*. G. Total IgG titer of RgpA-Kgp complex. H. IgG1 titer of RgpA-Kgp complex. I. IgG2a titer of RgpA-Kgp complex. *Primitive* = unvaccinated; *Infected* = no vaccine control; KDAK-3S-AVQP = KDAK-3S-AVQP protein encoding mRNA (SEQ ID NO: 6). m1Ψ = mRNA sequence modified with N1-methylpseuuridine. * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001.

[0174] Sequence information

[0175] Table 1: DNA and amino acid sequences

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192] Table 2: Exemplary RNA sequences of the present invention

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201] Detailed Implementation

[0202] It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more features mentioned or obviously present in the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0203] Reference will now be made to certain embodiments of the invention. Although the invention will be described in conjunction with embodiments, it should be understood that it is not intended to limit the invention to those embodiments. Rather, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the invention as defined in the claims.

[0204] Those skilled in the art will recognize that many methods and materials can be similar to or equivalent to those described herein, and can be used to practice this invention. This invention is by no means limited to the methods and materials described. It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more features mentioned or apparent in the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0205] All patents and publications mentioned in this article are incorporated herein by reference in their entirety.

[0206] For the purposes of interpreting this specification, terms used in the singular will also include the plural form, and vice versa.

[0207] In the work that led to this invention, the inventors investigated various chimeric or fusion proteins for inducing an immune response against Porphyromonas gingivalis, and methods for the large-scale production of such chimeras for use as vaccine candidates.

[0208] One such candidate fusion protein (called KDcAK1n, as further described herein) has been found to induce a robust immune response against Porphyromonas gingivalis, but there are problems with its large-scale preparation and production.

[0209] The inventors initially considered whether the problem of protein preparation could be overcome by providing the protein to the subject while it is encoded by an RNA vaccine. Surprisingly, the inventors found that KDcAK1n, which encodes RNA vaccines, is poorly expressed, and therefore KDcAK1n is not a preferred candidate for RNA vaccines.

[0210] Surprisingly, the inventors discovered that robust immune responses were obtained when alternative chimeric proteins containing encoding RNAs of various domains derived from Porphyromonas gingivalis Arg gingivalis protein or Lys gingivalis protein were provided.

[0211] Therefore, the present invention relates to the design of novel RNA vaccines encoding protein antigens for inducing an immune response against Porphyromonas gingivalis, or the design of chimeric or fusion proteins containing protein antigens, as well as methods and uses of said designs.

[0212] Gingival protease

[0213] The pathogenicity of *Porphyromonas gingivalis* is attributed to numerous surface-associated virulence factors, including cysteine ​​proteases (gingival proteases), pili, heme-binding proteins, and outer membrane transporters. Specifically, the extracellular Arg-specific and Lys-specific proteases of *Porphyromonas gingivalis*, 'gingival proteases' (RgpA / B and Kgp), are considered key virulence factors, crucial for colonization, penetration into host tissues, dysregulation of immune responses, ecological imbalances, and disease.

[0214] Gingival proteases, particularly the Lys-specific protease Kgp, are crucial for *Porphyromonas gingivalis* to induce alveolar bone resorption in a mouse model of periodontitis. High concentrations of gingival proteases have also been found in gingival tissue near subgingival plaques and in distal sites deep within the gingival tissue at sites of severe periodontitis. Lys-specific and Arg-specific proteases have been shown to degrade a variety of host proteins in vitro, such as fibrinogen, fibronectin, and laminin. Plasma host defense and regulatory protease inhibitors α-trypsin, α2-macroglobulin, antichymotrypsin, antithrombin III, and antifibrinolysin have also been degraded by Lys and Arg proteases from *Porphyromonas gingivalis*. This leads to the development of a compelling mechanism to explain the key role *Porphyromonas gingivalis* plays in the development of chronic periodontitis.

[0215] The RgpA, RgpB, and Kgp genes all encode an N-terminal signal peptide of approximately 22 amino acids, an unusually long propeptide of approximately 200 amino acids, and a catalytic domain of approximately 480 amino acids. The C-terminus of the catalytic domain is a large hemagglutinin-adhesin (HA) domain, which includes an adhesin-binding domain (ABM, of which five distinct sequences have been described), a "domain of unknown function" (designated DUF2436, defined as a conserved Pfam domain of unknown function; IPR018832), and a C-terminal adhesin domain or a cleaved adhesin domain (or CAD). The specific arrangement of ABM, DUF, and CAD varies between naturally occurring Kgp and RgpA / B genes.

[0216] The architecture of domains in Kgp multiproteins Figure 1 As shown in the diagram. For example, Kgp contains (from N-terminus to C-terminus): a catalytic domain, a first ABM (ABM1), DUF2436, a domain containing ABM2, ABM1, and ABM3, two CAD domains (referred to as K1 and K2), another domain containing ABM1 and ABM2, another CAD domain (referred to as K3), ABM2, and a C-terminal domain.

[0217] As used herein, references to ABM 1, 2, and 3 will be understood to generally refer to the ABMs found in the sequence ABM2, ABM1, and ABM3 in the C-terminus of DUF2436 immediately following Kgp, such as... Figure 1 What is depicted.

[0218] The catalytic domains of RgpB and RgpA share a high degree of sequence homology. However, RgpB lacks the HA domain and is located on the outer membrane in monomeric form. Some of the HA domains have been alternatively described as C-terminal adhesin domains or cleaved adhesin domains (CAD), and some are DUF (“domain of unknown function”) 2436 domains (conserved Pfam domain of unknown function; IPR018832).

[0219] RgpA and Kgp precursor proteins are cleaved into multiple domains that remain non-covalently associated, forming a large outer membrane protein complex. Thus, in vivo, Arg-specific and Lys-specific proteases have been found in cell-associated complexes of non-covalently associated proteases and adhesins. One such complex has been named the RgpA-Kgp protease-adhesin complex (previously known as the PrtR-PrtK protease-adhesin complex). This complex consists of a 45 kDa Arg-specific calcium-stabilized cysteine ​​protease and seven sequence-associated adhesin domains.

[0220] As used herein, the Lys gingivase catalytic domain can also be referred to as the KAS domain or PAS domain. Similarly, the Arg gingivase catalytic domain can be referred to as the RAS domain or PAS domain. Typically, the catalytic domains of Lys or Arg gingivase are located approximately 480 amino acids from the N-terminus of the protein. The active sites within the catalytic domain are usually located at amino acid residues 426-446 (for RgpA) and 432-453 (for Kgp).

[0221] As used herein, the adhesin domain of Arg gingivase or Lys gingivase from *Porphyromonas gingivalis* will be understood to generally refer to the region of Arg gingivase or Lys gingivase located at the C-terminus of the catalytic or active site domain. The adhesin domain (also known as the HA domain) typically contains a domain of unknown function (DUF) (particularly the conserved unfunctional Pfam domain of DUF 2436; IPR018832), several adhesin-binding motif (ABM) domains, and a cleaved adhesin domain (CAD).

[0222] Nucleic acid

[0223] The term "nucleic acid" in its broadest sense includes any compound and / or substance comprising a polymer of nucleotides. These polymers are commonly referred to as polynucleotides. Typically, the polynucleotides of this invention are in the form of RNA molecules, preferably mRNA. As used herein, the term "messenger RNA" (RNA) refers to any polynucleotide that encodes the polypeptide of interest and is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ, or ex vivo. Those skilled in the art will understand that, unless otherwise indicated, the polynucleotide sequences presented in this application will denote a "T" in a representative DNA sequence, but where the sequence represents RNA (e.g., RNA), the "T" will replace a "U". Thus, any RNA polynucleotide encoded by DNA identified by a specific sequence identifier may also comprise a corresponding RNA (e.g., RNA) sequence encoded by said DNA, wherein each "T" in the DNA sequence is replaced by a "U".

[0224] The basic building blocks of an RNA molecule include at least one coding region, a 5' UTR, a 3' UTR, a 5' cap, and a poly-A tail. The polynucleotides disclosed herein can function as RNA but are distinguishable from wild-type RNA in terms of their functional and / or structural design features, which is intended to overcome existing problems with efficient peptide expression using nucleic acid-based therapeutic agents.

[0225] The “5' untranslated region” (5'UTR) is the region of RNA that does not encode a polypeptide, located directly upstream (i.e., 5') of the start codon (i.e., the first codon of the RNA transcript translated by the ribosome).

[0226] The “3' untranslated region” (3'UTR) is the region of RNA that does not encode a polypeptide, located directly downstream (i.e., 3') of the stop codon (i.e., the codon of the RNA transcript that signals the termination of translation).

[0227] An "open reading frame" is a continuous DNA extension that begins with a start codon (e.g., methionine (ATG)) and ends with a stop codon (e.g., TAA, TAG, or TGA) and encodes a polypeptide.

[0228] A "polyA tail" is a region of RNA (usually mRNA) containing multiple (sometimes consecutive) monophosphates located downstream of the 3' UTR, for example, directly downstream (i.e., 3'). A polyA tail can contain from 10 to 300 monophosphates. For example, a polyA tail can 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 monophosphates. In some embodiments, a polyA tail contains 50 to 250 monophosphates. In some embodiments, a segmented poly(A) tail (typically a continuous segment of adenosine monophosphate separated by short spacer subregions between segments) can be used. In the relevant biological environment (e.g., in cells, in vivo), the poly(A) tail serves to protect mRNA from enzymatic degradation, for example, in the cytoplasm, and facilitates transcription termination, export of mRNA from the nucleus, and translation.

[0229] In some embodiments, the polynucleotide comprises 200 to 3,000 nucleotides. For example, the polynucleotide may comprise 200 to 500, 200 to 1,000, 200 to 1,500, 200 to 3,000, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 3,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 3,000, 1,500 to 3,000, or 2,000 to 3,000 nucleotides.

[0230] The present invention also contemplates the use of one or more structural and / or chemical modifications or alterations that impart useful properties to polynucleotides, including, in some embodiments, a lack of significant induction of the innate immune response that introduces the polynucleotide into the cell. Therefore, the modified RNA molecules of the present invention may also be referred to as “mmRNA”. As used herein, a “structural” feature or modification refers to the insertion, deletion, duplication, inversion, or randomization of two or more linked nucleotides in a polynucleotide, primary construct, or mRNA without significant chemical modification to the nucleotides themselves. Structural modifications are chemical in nature and are therefore chemical modifications because chemical bonds must be broken and reformed to achieve the structural modification. However, structural modifications result in different nucleotide sequences. For example, the polynucleotide “ATCG” can be chemically modified to “AT-5meC-G”. The same polynucleotide can be structurally modified from “ATCG” to “ATCCCG”. Here, the dinucleotide “CC” is inserted, thereby causing a structural modification to the polynucleotide.

[0231] The RNA molecule of the present invention may also contain a 5' end cap. In some embodiments, the 5' end cap is 7mG(5')ppp(5')NlmpNp, although it should be understood that any number of different 5' end caps commonly used in the art may also be used.

[0232] In some embodiments, the RNA molecule contains at least one chemical modification. The terms “chemical modification” and “chemically modified” refer to a modification at least at one of the following: adenosine (A), guanosine (G), uridine (U), thymidine (T), or cytidine (C) ribonucleoside or deoxyribonucleoside. Generally, these terms do not refer to ribonucleotide modifications in the naturally occurring 5' end RNA cap portion. In the case of polypeptides, the term “modification” refers to a modification relative to the canonical set of 20 amino acids. As provided herein, polypeptides are also considered “modified” because they contain amino acid substitutions, insertions, or combinations of substitutions and insertions.

[0233] In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) contains a variety (more than one) different modifications. In some embodiments, a specific region of the polynucleotide contains one, two or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, the modified RNA polynucleotide (e.g., modified RNA polynucleotide) introduced into a cell or organism exhibits reduced degradation in the cell or organism, respectively, compared to the unmodified polynucleotide. In some embodiments, the modified RNA polynucleotide (e.g., modified RNA polynucleotide) introduced into a cell or organism may exhibit reduced immunogenicity (e.g., reduced innate response) in the cell or organism, respectively.

[0234] Modifications to polynucleotides include, but are not limited to, the modifications described herein. Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) may contain naturally occurring modifications, non-naturally occurring modifications, or a combination of naturally occurring and non-naturally occurring modifications. Polynucleotides may include, for example, any useful modifications to sugars, nucleobases, or nucleoside bonds (e.g., to linked phosphate esters, to phosphodiester bonds, or to the phosphodiester backbone).

[0235] In some embodiments, a polynucleotide (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises a non-natural modified nucleotide introduced during or after the synthesis of the polynucleotide to achieve a desired function or property. Modifications may be present at nucleotide internucleotide bonds, purine or pyrimidine bases, or sugars. Modifications may be introduced chemically or by polymerase at the ends of the chain or at any other location in the chain. Any region of the polynucleotide may be chemically modified.

[0236] This disclosure provides modified nucleosides and nucleotides of polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides). "Nucleoside" refers to a compound containing a sugar molecule (e.g., pentose or ribose) or a derivative thereof combined with an organic base (e.g., purine or pyrimidine) or a derivative thereof (also referred to herein as a "nucleobase"). "Nucleotide" refers to a nucleoside comprising a phosphate ester group. Modified nucleotides can be synthesized by any useful method, such as chemical, enzymatic, or recombinant methods, to comprise one or more modified or non-natural nucleosides. Polynucleotides may contain one or more regions of linked nucleosides. Such regions may have variable backbone bonds. The bond may be a standard phosphodiester bond, in which case the polynucleotide will contain the nucleotide region.

[0237] Modified nucleotide base pairings encompass not only standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and / or modified nucleotides that include non-standard or modified bases, wherein the arrangement of hydrogen bond donors and acceptors allows hydrogen bonding between non-standard bases and standard bases or between two complementary non-standard base structures. An example of such non-standard base pairings is the base pairing between modified nucleotide inosine and adenine, cytosine, or uracil. Any combination of base / sugar or linker can be incorporated into the polynucleotides of this disclosure.

[0238] At least one chemical modification may be selected from pseudouridine, N1-methylpseuuridine, N1-ethylpseuuridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deazo-pseuuridine, 2-thio-1-methylpseuuridine, 2-thio-5-aza-uridine, 2-thio-dihydropseuuridine, 2-thio-dihydrouridine, 2-thio-pseuuridine, 4-methoxy-2-thio-pseuuridine, 4-methoxy-pseuuridine, 4-thio-1-methylpseuuridine, 4-thio-pseuuridine, 5-aza-uridine, dihydropseuuridine, 5-methoxyuridine, and 2'-O-methyluridine. In some embodiments, the chemical modification is at the 5-position of uracil. In some embodiments, the chemical modification is N1-methylpseuuridine. In some embodiments, the chemical modification is N1-ethylpseuuridine. In some embodiments, the polynucleotide includes a combination of at least two (e.g., two, three, four or more) of the aforementioned modified nucleobases.

[0239] In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) are uniformly modified (e.g., completely modified, modified throughout the sequence) to achieve a specific modification. For example, a polynucleotide may 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 may be uniformly modified by replacing any type of nucleoside residue present in the sequence with modified residues (as shown above).

[0240] Exemplary nucleobases and nucleosides having 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-pseudo-cytidine, 2-thio-cytidine (s2C), and 2-thio-5-methyl-cytidine.

[0241] In some embodiments, the modified nucleobase is a modified uridine. Exemplary nucleobases, and in some embodiments the modified nucleobase, are modified cytosine. Nucleosides having modified uridine include 5-cyanoururidine and 4'-thiouridine.

[0242] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having modified adenine include 7-deazo-adenine, 1-methyl-adenine (m1A), 2-methyl-adenine (m2A), and N6-methyl-adenine (m6A).

[0243] In some embodiments, the modified nucleobase is modified guanine. Exemplary nucleobases and nucleosides having modified guanine include inosine (I), 1-methyl-inosine (m1I), wyoside (imG), methyl wyoside (mimG), 7-denitroguanosine, 7-cyano-7-denitroguanosine (preQO), 7-aminomethyl-7-denitroguanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (mlG), 8-oxo-guanosine, and 7-methyl-8-oxo-guanosine.

[0244] The polynucleotides of this disclosure can be partially or completely modified along the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) can be uniformly modified in the polynucleotides of this disclosure or in a given predetermined sequence region (e.g., in mRNA including or excluding a polyA tail). In some embodiments, all nucleotides X in the polynucleotides of this disclosure (or in a given sequence region thereof) are modified nucleotides, wherein X can be any or a combination of nucleotides A, G, U, C, A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C, or A+G+C.

[0245] (Relative to the total nucleotide content, or relative to one or more types of nucleotides, i.e., any one or more of A, G, U, or C), polynucleotides may contain about 1% to about 100% or any intermediate percentage (e.g., 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% to 100%, 2). Modified nucleotides (0% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100%). Any remaining percentages explain the presence of unmodified A, G, U, or C.

[0246] The polynucleotide may contain at least 1% and at most 100% or any intermediate percentage of modified nucleotides, 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 polynucleotide may contain modified pyrimidines, such as 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 by modified uracil (e.g., 5-substituted uracil). The modified uracil may be replaced by a compound having a single unique structure or by multiple compounds having different structures (e.g., two, three, four, or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the cytosine in the polynucleotide is replaced with a modified cytosine (e.g., a 5-substituted cytosine). The modified cytosine may be replaced by a compound having a single unique structure, or by multiple compounds having different structures (e.g., two, three, four, or more unique structures).

[0247] In some embodiments, the modified nucleobase is modified uracil.Exemplary nucleobases and nucleosides having modified uracil include pseudouridine (Ψ), pyridine-4-ketoribonucleotide, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4-thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine, 5-methoxy-uridine, 5-oxyacetic acid uridine, 5-oxyacetic acid methyl ester uridine, 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-taurylmethyl-uridine, 1-taurylmethyl-pseudouridine, 5-taurylmethyl-2-thio-uridine, 1-taurylmethyl-4-thio-pseudouridine, 5-methyl-uridine (i.e., with nucleobase deoxythymidine), 1-methyl-pseudouridine, 5-methyl-2-thio-uridine, 1-methyl-4-thio-pseudouridine, 4-thio-1-methyl 3-Methyl-pseudouridine, 2-Thio-1-methyl-pseudouridine, 1-Methyl-1-denitro-pseudouridine, 2-Thio-1-methyl-1-denitro-pseudouridine, dihydrouridine (D), dihydrouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine, 2-thio-dihydrouridine, 2-thio-dihydrouridine, 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, α-thio-uridine, 2'-O-methyl-uridine, 5,2'-O-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-arasu-uridine, 2'-F-uridine, 2'-OH-arasu-uridine, 5-(2-carbonmethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)]uridine.

[0248] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having modified cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-methylcytidine, 5-halo-cytidine (e.g., 5-iodocytidine), 5-hydroxymethylcytidine, 1-methyl-pseudocytidine, pyrrolo-cytidine, pyrrolo-pseudocytidine, 2-thiocytidine, 2-thio-5-methylcytidine, 4-thio-pseudocytidine, 4-thio-1-methyl-pseudocytidine, 4-thio-1-methyl-1-deazo-pseudocytidine, 1-methyl-1-deazo-pseudocytidine, and zebularine. 5-aza-zabraline, 5-methylzabraline, 5-aza-2-thio-zabraline, 2-thio-zabraline, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudo-cytidine, 4-methoxy-1-methyl-pseudo-cytidine, lysine, α-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'-trimethyl-cytidine, 1-thio-cytidine, 2'-F-arasacchar-cytidine, 2'-F-cytidine, and 2'-OH-arasacchar-cytidine.

[0249] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having modified adenine include 2-amino-purine, 2,6-diamino-purine, 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-deadenine, 7-deadenine-8-aza-adenosine, 7-deadenine-2-amino-purine, and 7-deadenine-8-aza-2-amino-purine. 7-Denitro-2,6-diaminopurine, 7-Denitro-8-aza-2,6-diaminopurine, 1-methyl-adenosine, 2-methyl-adenosine, 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-glycylcarbamoyl-adenosine, N6 -Threonylcarbamoyl-adenosine, N6-methyl-N6-threonylcarbamoyl-adenosine, 2-methylthio-N6-threonylcarbamoyl-adenosine, N6,N6-dimethyl-adenosine, N6-hydroxyn-valinecarbamoyl-adenosine, 2-methylthio-N6-hydroxyn-valinecarbamoyl-adenosine, N6-acetyl-adenosine, 7-methyl-adenosine, 2-methylthio-adenosine, 2-methoxy-adenosine, α-thio-adenosine, 2'- O-methyl-adenosine, N6,2'-O-dimethyl-adenosine, N6,N6,2'-O-trimethyl-adenosine, 1,2'-O-dimethyl-adenosine, 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-arasyl-adenosine, 2'-F-adenosine, 2'-OH-arasyl-adenosine, and N6-(19-amino-pentaenodecyl)-adenosine.

[0250] In some embodiments, the modified nucleobase is modified guanine. Exemplary nucleobases and nucleosides having modified guanine include inosine, 1-methyl-inosine, wyoside, methyl wyoside, 4-demethyl-wyoside, isowyoside (imG2), huaistin, peroxyhuaistin, hydroxyhuaistin, undermodified hydroxyhuaistin, 7-deazo-guanosine, piracene, epoxypiracene, galactosylpiracene (galQ), mannosylpiracene, 7-cyano-7- Denitro-guanosine, 7-aminomethyl-7-denitro-guanosine, archapurin, 7-denitro-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-denitro-guanosine, 6-thio-7-denitro-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-di Methyl-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, α-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, O6-methyl-guanosine, 2'-F-arose-guanosine and 2'-F-guanosine.

[0251] In some embodiments, an RNA (e.g., RNA) vaccine comprises a 5' UTR element, optionally codon-optimized open reading frames and a 3' UTR element, a poly(A) sequence and / or a polyadenylation signal, wherein the RNA is unmodified.

[0252] In some embodiments, the polynucleotides disclosed herein are codon-optimized. Codon optimization methods are known in the art and can be used as provided herein. In some embodiments, codon optimization can 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 transport sequences; remove / add post-translational modification sites (e.g., glycosylation sites) in encoded proteins; add, remove, or reorganize protein domains; insert or delete restriction sites; modify ribosome binding sites and RNA degradation sites; adjust translation rates to allow proper folding of individual protein domains; or reduce or eliminate problematic secondary structures within polynucleotides. Codon optimization tools, algorithms, and services are known in the art—non-limiting examples include services and / or proprietary methods from GeneArt (Life Technologies), DNA2.0 (Menlo Park, California). In some embodiments, optimization algorithms are used to optimize open reading frame (ORF) sequences.

[0253] In some embodiments, the codon-optimized sequence shares less than 95%, less than 90%, less than 85%, less than 80%, or less than 75% sequence identity with 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)).

[0254] In some embodiments, the codon-optimized sequence shares 65% to 85% (e.g., about 67% to about 85% or about 67% to about 80%) sequence identity with 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 antigen protein or polypeptide)). In some embodiments, the codon-optimized sequence shares 65% to 75% or about 80% sequence identity with 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 antigen protein or polypeptide)).

[0255] In some embodiments, codon-optimized RNA (e.g., mRNA) may be, for example, RNA in which the level of G / C is enhanced. The G / C content of a nucleic acid molecule can affect the stability of RNA. RNA with increased amounts of guanine (G) and / or cytosine (C) residues may be functionally more stable than nucleic acids containing large amounts of adenine (A) and thymine (T) or uracil (U) nucleotides. WO02 / 098443 discloses pharmaceutical compositions containing RNA stabilized by sequence modifications in the translation region. Due to the degeneracy of the genetic code, these modifications work by replacing existing codons with codons that promote greater RNA stability without altering the resulting amino acids. The method is limited to the coding region of RNA.

[0256] It has been found that, in addition to other structural features such as a 5' cap or a 3'-poly(A) tail, naturally occurring eukaryotic RNA molecules also contain stabilizing elements, including but not limited to untranslated regions (UTRs) located at their 5' end (5'UTR) and / or at their 3' end (3'UTR). Both the 5'UTR and 3'UTR are typically transcribed from genomic DNA and are elements of precocious RNA. During RNA processing, characteristic structural features of mature RNA, such as the 5'-cap and 3'-poly(A) tail, are typically added to the transcribed (precocious) RNA. The 3'-poly(A) tail is typically an extension of adenine nucleotides added to the 3' end of the transcribed RNA. It can contain up to about 400 adenine nucleotides. In some embodiments, the length of the 3'-poly(A) tail can be a fundamental element related to the stability of the RNA alone.

[0257] In some embodiments, RNA (e.g., mRNA) vaccines may include one or more stabilizing elements. Stabilizing elements may include, for example, histone stem-loops. A stem-loop binding protein (SLBP) has been identified as a 32 kDa protein. The SLBP associates with the histone stem-loop at the 3' end of the histone message in both the nucleus and cytoplasm. The expression level of the SLBP is regulated by the cell cycle; it peaks during S phase when histone RNA levels are also elevated. This protein has been shown to be essential for efficient 3' end treatment of histone precursor RNA via U7 snRNP. After treatment, the SLBP remains associated with the stem-loop and then stimulates the translation of mature histone RNA into histones in the cytoplasm. The RNA-binding domain of the SLBP is conserved in metazoans and protozoa; the binding of this domain to the histone stem-loop depends on the loop's structure. Minimal binding sites include at least three nucleotides at the 5' end relative to the stem-loop and two nucleotides at the 3' end.

[0258] In some embodiments, an RNA (e.g., mRNA) vaccine includes 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 should generally enhance the expression level of the encoded protein. In some embodiments, the encoded protein is not a histone, reporter protein (e.g., luciferase, GFP, EGFP, β-galactosidase, EGFP), or a marker or selector protein (e.g., α-globulin, galactokinase, and xanthine:guanine phosphoribosyltransferase (GPT)).

[0259] In some embodiments, the combination of a poly(A) sequence or polyadenylation signal and at least one histone stem-loop, even if both represent alternative mechanisms in nature, works synergistically to increase protein expression to levels observed with either element alone. It has been found that the synergistic effect of the combination of poly(A) and at least one histone stem-loop is independent of the element order or the length of the poly(A) sequence.

[0260] In some embodiments, RNA (e.g., mRNA) vaccines do not contain a histone downstream element (HDE). A “histone downstream element” (HDE) comprises a purine-rich polynucleotide extension of approximately 15 to 20 nucleotides located at the 3' of a naturally occurring stem-loop, representing a binding site for U7 snRNA, which participates in processing histone precursor RNA into mature histone RNA. Ideally, the nucleic acids of the present invention do not include introns.

[0261] In some embodiments, RNA (e.g., mRNA) vaccines may or may not contain enhancer and / or promoter sequences, which may be modified or unmodified, or activated or inactivated. In some embodiments, histone stem-loops are typically derived from histone genes and comprise intramolecular base pairings of two adjacent partial or completely inverse complementary sequences separated by spacers that form the loop, the spacers comprising short sequences (e.g., composed of short sequences). Unpaired loop regions typically cannot pair with any of the stem-loop elements. This is more common in RNA, as are many key components of RNA secondary structures, but can also occur in single-stranded DNA. The stability of stem-loop structures typically depends on the length of the paired regions, the number of mismatches or protrusions, and the base composition. In some embodiments, wobbly base pairings (non-Watson-Crick base pairings) may occur. In some embodiments, at least one histone stem-loop sequence comprises 15 to 45 nucleotides in length.

[0262] In other embodiments, RNA (e.g., mRNA) vaccines may remove one or more AU-rich sequences. These sequences, sometimes referred to as AURES, are unstable sequences present in the 3' UTR. AURES can be removed from RNA (e.g., mRNA) vaccines. Alternatively, AURES may be retained in RNA (e.g., mRNA) vaccines.

[0263] In other embodiments, the RNA (e.g., mRNA) of the present invention may contain ribosomal jumping sequences, such as 2A jumping sequences. The use of such sequences in RNA coding sequences is known to those skilled in the art and makes it possible to express multiple proteins or peptides from a single mRNA. Therefore, in any embodiment, the mRNA of the present invention may encode two or more of the domains K, D, A (including AΔAMB3), or R as defined elsewhere herein and also in Table 1, or may encode two or more of the proteins exemplified in Table 1 as proteins that can be encoded by the RNA sequence of the present invention. In some non-limiting examples, the mRNA of the present invention may encode one or more or combinations of KA chimeric proteins, DA chimeric proteins, RA chimeric proteins, AR chimeric proteins, AK chimeric proteins, AD chimeric proteins, KDA chimeric proteins, RDA chimeric proteins, DAR chimeric proteins, and DAK chimeric proteins.

[0264] Non-limiting examples of 2A peptide sequences used to introduce ribosome jumping include T2A or T2A-like sequences derived from Thosea asigna virus and Porcine teschovirus-1 2A.

[0265] polypeptide

[0266] It should be understood that the polynucleotides of the present invention encode chimeric or fusion proteins. Proteins encoded by the RNA molecules of the present invention may also be referred to as "antigenic polypeptides" or simply "antigens".

[0267] As used herein, “polypeptide” refers to a polymer of (natural or non-natural) amino acid residues typically linked together by peptide bonds. The term as used herein refers to proteins, polypeptides, and peptides of any size, structure, or function. In some cases, the encoded polypeptide is less than about 50 amino acids, and then the polypeptide is called a peptide. If a polypeptide is a peptide, then 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. Polypeptides can be monomolecules or multi-molecule complexes, such as dimers, trimers, or tetramers. Polypeptides can also comprise single-chain or multi-chain polypeptides, such as antibodies or insulin, and can associate or link together. Disulfide bonds are most commonly found in multi-chain polypeptides. The term polypeptide can also be applied to amino acid polymers, where one or more amino acid residues are artificial chemical analogs of corresponding naturally occurring amino acids.

[0268] As used herein, a chimeric or fusion protein refers to a polypeptide comprising an amino acid sequence arranged in a spatial configuration different from that found in the natural state. For example, and in the context of this invention, the chimeric or fusion protein encoded by the polynucleotides of this invention comprises a portion of Arg gingival protease or Lys gingival protease from Porphyromonas gingivalis, said portion being in a spatial arrangement different from that of the full-length gingival protease.

[0269] connector

[0270] In the context of this invention, the RNA preferably encodes a chimeric or fusion protein comprising various domains (as defined herein) derived from *Porphyromonas gingivalis* gingival protease. The domains may be directly bound within the chimeric or fusion protein, or the chimeric or fusion protein may contain a linker for binding the domains.

[0271] Suitable adapters for joining amino acid sequences are well known to those skilled in the art. Preferably, the adapter is non-immunogenic. Typically, the adapter contains an amino acid and is therefore referred to as a peptide adapter.

[0272] Linkers are typically peptides of up to 20 amino acids in length, although they may be longer. The terms “linked with” or “fused with” refer to a covalent bond (e.g., a peptide bond) formed between two parts. Therefore, in the context of this invention, the length of a linker can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 or more amino acids. For example, the chimeric or fusion protein encoded by the RNA of this invention may comprise a linker between the amino acid sequence of the active site of *Porphyromonas gingivalis* gingivalis protease and the amino acid sequence of the adhesin domain of *Porphyromonas gingivalis* gingivalis protease. The advantage of such a linker is that it makes it more likely that the different polypeptides of the fusion protein will fold independently and exhibit the expected behavior. Suitable linker lengths can be up to 50 amino acids, although less than 20, less than 15, or less than five amino acids are preferred. Connectors can serve to bring the domains closer together than the spatial arrangement typically observed in *Porphyromonas gingivalis* trypsin-like enzymes. Alternatively, the connectors can space the domains apart.

[0273] Suitable linkers used in protein constructs (including linkers with minimal impact on solubility) are known in the art. Linkers can be any linker known to those skilled in the art and can be flexible linkers (e.g., linkers containing repeating sequences of glycine and serine residues), rigid linkers (e.g., linkers containing glutamic and lysine residues flanking alanine repeating sequences), and / or cleavable linkers (e.g., sequences susceptible to protease cleavage). Examples of such linkers are known to those skilled in the art and are described, for example, in Chen et al., (2013) *Advanced Drug Delivery Reviews*, 65: 1357-1369.

[0274] Useful linkers include glycine-serine (GlySer) linkers, which are well known in the art, and comprise glycine and serine units in various combinations in different sequences. Examples include, but are not limited to, (GS), (GSGGS)n (SEQ ID NO: 88), (GGGS)n (SEQ ID NO: 89), and (GGGGS)n (SEQ ID NO: 90), where n is an integer of at least one, typically from 1 to 10, such as from 1 to 8, from 1 to 6, or from 1 to 5.

[0275] In some embodiments, the peptide linker may comprise glycine and serine amino acids of various lengths and combinations. In some aspects, the peptide linker may comprise the sequences Gly-Gly-Ser (GGS), Gly-Gly-Gly-Ser (GGGS, SEQ ID NO: 89), or Gly-Gly-Gly-Gly-Ser (GGGGS, SEQ ID NO: 90), as well as variants or repeating sequences thereof. In some aspects, the peptide linker may comprise the amino acid sequence GGGGS (a linker of 6 amino acids in length, SEQ ID NO: 90) or even longer. The linker may be a series of repeating glycine and serine residues (GS) of varying lengths, i.e., (GS)n, where n is any number from 1 to 15 or greater. For example, the linker may be (GS)3 (i.e., GSGSGS, SEQ ID NO: 91) or longer (GS)11 or longer. It should be understood that n may be any number including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or greater. Fusion proteins with linkers of this length are included within the scope of this invention. Similarly, the linker can be a series of repeating glycine residues separated by serine residues. For example, (GGGGS)3 (i.e., the linker can contain the amino acid sequence GGGGSGGGGSGGGGS, (G4S)3, SEQ ID NO: 92) and its variants.

[0276] In one embodiment, the peptide linker may comprise the amino acid sequence GGGGS (a linker of 6 amino acids in length) or even longer. The linker may be a series of repeating glycine and serine residues (GS) of varying lengths, i.e., (GS)n, where n is any number from 1 to 15 or greater. For example, the linker may be (GS)3 (i.e., GSGSGS, SEQ ID NO: 91) or longer (GS)11 or longer. It should be understood that n may be any number including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or greater.

[0277] Other useful adapters include DSSG (SEQ ID NO: 93), DSSGAS (SEQ ID NO: 94), KLDSSG (SEQ ID NO: 95), and their variants. Examples of other suitable adapters are described in Chen et al., (2013) Advanced Drug Delivery Review, 65: 1357-1369.

[0278] The term "peptide variant" refers to a molecule whose amino acid sequence differs from that of the native or reference sequence. Compared to the native or reference sequence, the amino acid sequence variant may have substitutions, deletions, and / or insertions at certain positions within the amino acid sequence. Typically, the variant will have at least about 50% identity (homology) with the native or reference sequence, and preferably, the variant will be at least about 80%, more preferably at least about 90% identical (homologous) to the native or reference sequence.

[0279] This invention envisions several types of compositions encoding polypeptides, including variants and derivatives. The compositions include substituted, inserted, deleted, and covalent variants and derivatives. The term "derivative" is used synonymously with the term "variant," but generally refers to a molecule that is modified and / or altered in any way relative to a reference molecule or starting molecule.

[0280] Therefore, RNAs of the present invention encoding polypeptides containing substitutions, insertions and / or additions, deletions, and covalent modifications relative to a reference sequence (specifically, the polypeptide sequences disclosed herein) are included within the scope of the present invention. For example, sequence tags or amino acids, such as one or more lysine residues, may be added to the peptide sequences of the present invention (e.g., at the N-terminus or C-terminus). Sequence tags can be used for peptide purification or localization. Lysine residues can be used to increase peptide solubility or allow biotinylation. Alternatively, amino acid residues located in the carboxyl and amino-terminal regions of the amino acid sequence of a peptide or protein may be optionally deleted, thereby providing a truncated sequence. Certain amino acids (e.g., C-terminal or N-terminal residues) may alternatively be deleted depending on the use of the sequence, for example, as part of the expression of a larger sequence that is soluble or linked to a solid-phase carrier.

[0281] When referring to polypeptides, a “substitutional variant” is a variant in which at least one amino acid residue is removed from the native or starting sequence and a different amino acid is inserted at the same position. Substitution can be single, in which case only one amino acid in the molecule is substituted, or substitution can be multiple, in which case two or more (e.g., three, four, or five) amino acids in the same molecule are substituted. In some embodiments, substitution can be a conserved amino acid substitution.

[0282] As used herein, the term "conservative amino acid substitution" refers to the substitution of a normally present amino acid in a sequence with a different amino acid having similar size, charge, or polarity. Examples of conservative substitution include the substitution of one nonpolar (hydrophobic) residue for another nonpolar residue, such as isoleucine, valine, or leucine. Similarly, examples of conservative substitution include the substitution of one polar (hydrophilic) residue for another polar residue, such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Additionally, the substitution of one basic residue for another basic residue with a basic residue, such as lysine, arginine, or histidine, or the substitution of one acidic residue for another acidic residue with one acidic residue, such as aspartic acid or glutamic acid, are further examples of conservative substitution. Examples of nonconservative substitution include the substitution of polar (hydrophilic) residues such as cysteine, glutamine, glutamic acid, or lysine with nonpolar (hydrophobic) amino acid residues, such as isoleucine, valine, leucine, alanine, or methionine, and / or the substitution of nonpolar residues with polar residues.

[0283] Amino acid deletions or insertions can also occur relative to the native sequence of *Porphyromonas gingivalis* proteins. Thus, for example, amino acids that do not substantially affect the activity of the peptide, or at least do not eliminate such activity, can be deleted.

[0284] As used herein, when referring to polypeptides, the term "domain" refers to a motif of a polypeptide that has one or more identifiable structural or functional features or properties (e.g., binding capacity, acting as a site for protein-protein interactions).

[0285] In any embodiment of the invention, those skilled in the art can modify the RNA molecule of the invention to include codons encoding additional amino acid residues encoding a naturally occurring domain sequence derived from gingivase. For example, when the RNA of the invention encodes a chimeric protein (such as RA, KA, KDA, KDAK, etc.), and wherein the sequences of K, R, D, and A are as defined in the invention, including additional codons encoding additional amino acids at the N-terminus or C-terminus of each domain, for example, to further stabilize the encoded protein, will be within the scope of the skill of the art. Typically, these additional amino acids correspond to the naturally occurring gingivase sequence. In a non-limiting example, methionine may be encoded at the N-terminal region of the A domain. It should be understood that the RNA may encode 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more additional amino acid residues. In another example, the D domain may contain a C-terminal methionine residue (or alternatively, this residue may be omitted from the sequence of the D domain).

[0286] As used herein, when referring to polypeptides or polynucleotides, the terms "termini" or "terminus" refer to the end of the polypeptide or polynucleotide, respectively. Such ends are not limited to the first or final site of the polypeptide or polynucleotide and may also include additional amino acids or nucleotides in the terminal region. Polypeptide-based molecules may be characterized by having both an N-terminus (terminated with an amino acid having a free amino group (NH2)) and a C-terminus (terminated with an amino acid having a free carboxyl group (COOH)). In some cases, proteins consist of multiple polypeptide chains bonded together by disulfide bonds or by non-covalent forces (polymers, oligomers). These proteins have multiple N-termini and C-termini. Alternatively, the ends of polypeptides may be modified to begin or end with a non-polypeptide-based portion, such as an organic conjugate, where appropriate.

[0287] As will be recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of the polypeptide of interest. For example, this document provides any protein fragment of a reference protein of lengths of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or longer than 100 amino acids (meaning a polypeptide sequence that is at least one amino acid residue shorter than the reference polypeptide sequence but otherwise identical). In another instance, according to this disclosure, any protein may be used comprising an extension of 20, 30, 40, 50, or 100 (continuous) amino acids that is 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% identical to any sequence described herein. In some embodiments, the polypeptide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations, as shown in any sequence provided herein or referenced herein. In another instance, according to this disclosure, any protein may be used comprising an extension having 20, 30, 40, 50, or 100 amino acids that is more than 80%, 90%, 95%, or 100% identical to any sequence described herein, wherein said protein has an extension having 5, 10, 15, 20, 25, or 30 amino acids that is less than 80%, 75%, 70%, 65% to 60% identical to any sequence described herein.

[0288] The polypeptide or polynucleotide molecules disclosed herein may share a degree of sequence similarity or identity with the mentioned SEQ ID NO. As is known in the art, the term "identity" refers to the relationship between the sequences of two or more polypeptides or polynucleotides, as determined by sequence comparison. In the art, identity also means the degree of sequence relevance 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 percentage of identical matches between the smaller of two or more sequences, where gap alignment (if any) is resolved by a specific mathematical model or computer program (e.g., an "algorithm"). The identity of related peptides can be readily calculated by known methods. "Identity %" when applied to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in a candidate amino acid or nucleic acid sequence that are identical to residues in the amino acid sequence or nucleic acid sequence of a second sequence after aligning the sequences and introducing vacancies where necessary to achieve the maximum identity percentage. Methods and computer programs used for alignment are well known in the art. Identity depends on the calculation of the identity percentage, but its value may vary due to vacancies and penalties introduced in the calculation. Typically, a variant of a specific polynucleotide or polypeptide has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% but less than 100% sequence identity with the specific reference polynucleotide or polypeptide, as determined by sequence alignment procedures and parameters described herein and known to those skilled in the art. Such tools for alignment include BLAST kits (Stephen F. Altschul et al. (1997). “Gapped BLAST and PSI-BLAST: a new generation of protein databasesearch programs”, Nucleic Acids Res. 25:3389-3402). Another popular local alignment technique is based on the Smith-Waterman algorithm (Smith, TF and Waterman, MS (1981) “Identification of common molecular subsequences.” J. Mol. Biol. 147:195-197).The general global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, SB and Wunsch, CD (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins" *Journal of Molecular Biology* 48:443-453). Recently, the Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed, which is claimed to produce global alignments of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm. Other tools are specifically described in the definition of "identity" below.

[0289] The term "identity" refers to the overall relevance between polymer molecules, such as polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. The percentage of identity between two polynucleotide sequences can be calculated, for example, by aligning the two sequences for optimal comparison purposes (e.g., for optimal alignment, vacancies can be introduced in one or both of the first and second nucleic acid sequences, and dissimilar sequences can be discarded for comparison purposes). In some embodiments, the length of the 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. Nucleotides located 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, the molecules are identical at that position. The percentage of identity between two sequences is a function of the number of common positions shared by the sequences, taking into account the number of vacancies and the length of each vacancy, which requires the introduction of such vacancies for optimal alignment of the two sequences. The comparison of sequences and the determination of the percentage of identity between two sequences can be accomplished using mathematical algorithms.For example, the percentage of identity between two nucleic acid sequences can be determined using methods described in the following literature: *Computational Molecular Biology*, edited by Lesk, AM, Oxford University Press, New York, 1988; *Biocomputing: Informatics and Genome Projects*, edited by Smith, DW, Academic Press, New York, 1993; *Sequence Analysis in Molecular Biology*, by von Heinje, G., Academic Press, 1987; *Computer Analysis of Sequence Data, Part I*, edited by Griffin, AM and Griffin, HG, Humana Press, New Jersey, 1994; and *Sequence Analysis Primer*, by Gribskov, M. and Devereux. J., ed., M Stockton Press, New York, 1991; each of the cited references is incorporated herein by reference. For example, the Meyers and Miller algorithm (CABIOS, 1989, 4:11-17) which has been incorporated into the ALIGN program (version 2.0) can be used to determine the percentage of identity between two nucleic acid sequences using a PAM 120 weighted residue table, a vacancy length penalty of 12, and a vacancy penalty of 4. Alternatively, the GAP program in the GCG software package can be used to determine the percentage of identity between two nucleic acid sequences using the NWSgapdna.CMP matrix. Methods commonly used to determine the percentage of identity between sequences include, but are not limited to, the method disclosed in Carillo, H. and Lipman, D., SIAM J Applied Math, 48:1073 (1988); the cited reference is incorporated herein by reference. Methods for determining identity have been incorporated into publicly available computer programs.Exemplary computer software for determining homology between two sequences includes, but is not limited to, the GCG package (Devereux, J. et al., Nucleic Acid Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul et al., Journal of Molecular Biology, 215, 403 (1990)).

[0290] signal peptide

[0291] The polypeptides encoded by the polynucleotides of this invention typically contain an N-terminal signal peptide. The signal peptide, comprising 15-60 amino acids at the N-terminus of a protein, is generally required for transmembrane translocation in the secretory pathway and thus universally controls the entry of most proteins into the secretory pathway in eukaryotes and prokaryotes. The signal peptide typically comprises three regions: an N-terminal region of varying lengths, which typically contains positively charged amino acids; a hydrophobic region; and a short C-terminal peptide region. In eukaryotes, the signal peptide of a nascent precursor protein (preprotein) guides the ribosome to the rough endoplasmic reticulum (ER) membrane and induces the transport of the growing peptide chain across the membrane for processing. ER processing yields a mature protein, wherein the signal peptide is typically cleaved from the precursor protein by a host cell ER-resident signal peptidase, or the signal peptide remains uncleaned and acts as a membrane anchor. The signal peptide can also promote protein targeting to the cell membrane. However, the signal peptide is not responsible for the final destination of the mature protein. Secretory proteins lacking additional address tags in their sequence are secreted into the external environment by default. In recent years, a more advanced view of signal peptides has emerged, revealing that the functions and immunogenicity of some signal peptides are far more diverse than previously thought.

[0292] In any embodiment, the N-terminal secretory signal peptide may comprise ribosomes capable of processing the chimeric or fusion protein by binding to the rough endoplasmic reticulum (ER) of the cell, thereby introducing the chimeric or fusion protein into any amino acid sequence in the ER.

[0293] Preferably, the N-terminal secretion signal peptide is any peptide that enables the cell expressing or translating RNA to secrete a protein encoded therein.

[0294] In some embodiments, the signal peptide fused to the antigenic polypeptide is an artificial signal peptide. In some embodiments, the artificial signal peptide fused to the antigenic polypeptide encoded by an RNA (e.g., mRNA) vaccine is obtained from an immunoglobulin (e.g., an IgE signal peptide or an IgG signal peptide). In some embodiments, the signal peptide fused to the antigenic polypeptide encoded by an RNA (e.g., mRNA) vaccine is an Ig heavy chain ε-1 signal peptide (IgE HC SP) having the following sequence: MDWTWILFLVAAATRVHS (SEQ ID NO: 96).

[0295] In some embodiments, the signal peptide fused with the antigenic polypeptide encoded by an RNA (e.g., mRNA) vaccine is the IgGk chain V-III region HAH signal peptide (IgGk SP) having the following sequence: METPAQLLFLLLLWLPDTTG (SEQ ID NO: 97). In some embodiments, the signal peptide is selected from: the Japanese encephalitis PRM signal sequence (MLGSNSGQRVVFTILLLLVAPAYS, SEQ ID NO: 98), the VSVg protein signal sequence (MKCLLYLAFLFIGVNCA, SEQ ID NO: 99), and the Japanese encephalitis JEV signal sequence (MWLVSLAIVTACAGA, SEQ ID NO: 100).

[0296] Other examples of suitable signal peptides include sequences derived from the following: tPA (tissue plasminogen activator): MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 101); variants such as: tPA(VSA): MDAMKRGLCCVLLLCGAVFVSA (SEQ ID NO: 102), tPA(VSAR): MDAMKRGLCCVLLLCGAVFVSAR (SEQ ID NO: 103), tPA(VSP): MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 104), tPA(VSPS): MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 101).

[0297] In some embodiments, the amino acid sequence of the signal peptide includes the sequence SEAP (secretory embryonic alkaline phosphatase): MLLLLLLLGLRLQLSLG[A] (SEQ ID NO: 105), such that the expressed RNA product includes the sequence MLLLLLLLGLRLQLSLG[A] (SEQ ID NO: 105) located at the N-terminus of the sequence defined herein (including the sequences in Table 1).

[0298] In a preferred embodiment of the present invention, the signal peptide comprises a sequence as shown in any of these examples or is 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 to the sequence shown in any of these examples.

[0299] The examples disclosed herein are not intended to be limiting, and any signaling peptides known in the art for facilitating the targeting of proteins to the ER for treatment and / or the targeting of proteins to the cell membrane may be used in accordance with this disclosure.

[0300] The length of a signal peptide can range from 15 to 60 amino acids. For example, the length of a signal peptide can be 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, the length of the signal peptide is 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, 4 0-50, 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.

[0301] Signal peptides are typically cleaved from nascent polypeptides at the cleavage junction during ER treatment. Mature antigenic polypeptides generated from the RNA vaccines of this disclosure typically do not contain signal peptides.

[0302] Designing polypeptides encoded by the RNA of this invention to facilitate their expression and translation in vivo is entirely within the scope of the skill of the person skilled in the art. For example, in some non-limiting instances, the RNA of this invention may include sequences encoding N-terminal methionine and / or other residues (such as alanine) to enable the expression, secretion, and / or cleavage of signal peptides.

[0303] Therefore, in any embodiment, the RNA of the present invention may encode one, two, three, four or more N-terminal amino acids of the sequences defined herein in Table 1. For example, RNA encoding an amino acid sequence as shown in any of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 38 or 39 may encode one or more additional N-terminal amino acids, optionally an N-terminal alanine residue and / or optionally an N-terminal methionine residue.

[0304] In other instances, RNA encoding an amino acid sequence of the D domain (e.g., in SEQ ID NO: 35 and 36) may contain a C-terminal methionine residue (as shown in these sequences) or the C-terminal methionine residue may be omitted from the D domain sequence.

[0305] Composition and lipid nanoparticles

[0306] The present invention envisions providing a polynucleotide (preferably RNA) encoding a chimeric or fusion protein for inducing an immune response against *Porphyromonas gingivalis*, preferably formulated in lipid nanoparticles. Therefore, the present invention also provides lipid nanoparticles comprising the polynucleotides described herein. It should be understood that, in any embodiment, the nanoparticles of the present invention may also be described as a "vaccine" composition or an "immunostimulatory" composition.

[0307] In some embodiments, the RNA of the present invention is formulated in a lipid-multicationic complex referred to as cationic lipid nanoparticles. As a non-limiting example, the multicationic complex may include cationic peptides or polypeptides, such as, but not limited to, polylysine, polyornithine, and / or polyarginine. In some embodiments, the RNA may be formulated in lipid nanoparticles comprising noncationic lipids, such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

[0308] Lipid nanoparticle formulations typically contain lipids, specifically ionizable cationic lipids, and further contain non-cationic lipids, sterols, and molecules that can reduce particle aggregation, such as PEG or PEG-modified lipids.

[0309] In some embodiments, the cationic lipid is an ionizable cationic lipid, and the non-cationic lipid is a neutral lipid, and the sterol is cholesterol. In some embodiments, the cationic lipid is selected from the group consisting of: 2,2-dilinoleoyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleoyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butyryl)oxy)heptadecanoic acid ester (L319), (12Z,15Z)-N,N-dimethyl-2-nonyltecosano-12,15-dien-1-amine (L608) and N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecano-8-amine (L530).

[0310] In some embodiments, the lipid nanoparticle formulation comprises, by molar weight, 25-75% of a cationic lipid, 0.5-15% of a neutral lipid, 5-50% of a sterol, and 0.5-20% of a PEG or PEG-modified lipid: 2,2-dilinoleoyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleoyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butyryl)oxy)heptadecanoate (L319).

[0311] In some embodiments, the lipid nanoparticle formulation is substantially composed of a lipid mixture with a molar ratio of 20-70% cationic lipids: 5-45% neutral lipids: 20-55% cholesterol: 0.5-15% PEG-modified lipids. In some embodiments, the lipid nanoparticle formulation is substantially composed of a lipid mixture with a molar ratio of 20-60% cationic lipids: 5-25% neutral lipids: 25-55% cholesterol: 0.5-15% PEG-modified lipids.

[0312] In some embodiments, the molar lipid ratio is 50 / 10 / 38.5 / 1.5 (mol% cationic lipids / neutral lipids, e.g., DSPC / Chol / PEG-modified lipids, e.g., PEG-DMG, PEG-DSG, or PEG-DPG), 57.2 / 7.1 / 134.3 / 1.4 (mol% cationic lipids / neutral lipids, e.g., DPPC / Chol / PEG-modified lipids, e.g., PEG-cDMA), 40 / 15 / 40 / 5 (mol% cationic lipids / neutral lipids, e.g., DSPC / Chol / PEG-modified lipids, e.g., PEG-DMG), and 50 / 10 / 35 / 4.5 / 0.5 (mol% cationic lipids / neutral lipids, e.g., DSPC / Chol / PEG-modified lipids). For example, PEG-DSG), 50 / 10 / 35 / 5 (cationic lipids / neutral lipids, e.g., DSPC / Chol / PEG-modified lipids, e.g., PEG-DMG), 40 / 10 / 40 / 10 (mol% cationic lipids / neutral lipids, e.g., DSPC / Chol / PEG-modified lipids, e.g., PEG-DMG or PEG-cDMA), 35 / 15 / 40 / 10 (mol% cationic lipids / neutral lipids, e.g., DSPC / Chol / PEG-modified lipids, e.g., PEG-DMG or PEG-cDMA), or 52 / 13 / 30 / 5 (mol% cationic lipids / neutral lipids, e.g., DSPC / Chol / PEG-modified lipids, e.g., PEG-DMG or PEG-cDMA).

[0313] Lipid nanoparticle formulations can be influenced by, but are not limited to, the selection of cationic lipid components, the degree of cationic lipid saturation, the nature of PEGylation, the 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 consisted of 57.1% cationic lipids, 7.1% dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA.

[0314] In some embodiments, the lipid nanoparticles comprise a molar ratio of about 20-60% cationic lipids, 0.5-15% PEG-modified lipids, 25-55% sterols, and 25% non-cationic lipids. In some embodiments, the cationic lipids are ionizable cationic lipids, the non-cationic lipids are neutral lipids, and the sterols are cholesterol. In some embodiments, the cationic lipids are selected from 2,2-dilinoleoyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleoyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butyryl)oxy)heptadecanoate (L319).

[0315] In some embodiments, the lipid nanoparticle formulation may comprise 35% to 45% cationic lipids, 40% to 50% cationic lipids, 50% to 60% cationic lipids, and / or 55% to 65% cationic lipids.

[0316] In some embodiments, the ratio of lipids to RNA (e.g., mRNA) in the lipid nanoparticles can be 5:1 to 20:1, 10:1 to 25:1, 15:1 to 30:1 and / or at least 30:1.

[0317] In some embodiments, the ratio of PEG in the lipid nanoparticle formulation may be increased or decreased, and / or the carbon chain length of the PEG lipid may be changed from C14 to C18 to alter the pharmacokinetics and / or biodistribution of the lipid nanoparticle formulation. As a non-limiting example, compared to cationic lipids, DSPC, and cholesterol, the lipid nanoparticle formulation may contain PEG-c-DOMG (R-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl)]-1,2-dimyristyloxypropyl-3-amine) (also referred to herein as PEG-DOMG) in a lipid molar ratio of 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%. In some embodiments, PEG-c-DOMG may be replaced with PEG lipids such as, but not limited to, PEG-DSG (1,2-distearyl-sn-glycerol, methoxy polyethylene glycol), PEG-DMG (1,2-dimyristoyl-sn-glycerol), and / or PEG-DPG (1,2-dispalmitoyl-sn-glycerol, methoxy polyethylene 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.

[0318] Amino alcohol cationic lipids can be lipids described in U.S. Patent Publication No. US20130150625 and / or lipids prepared by the methods described in that patent, which is incorporated herein by reference in its entirety. As a non-limiting example, cationic lipids can be 2-amino-3-[(9Z,12Z)-octadecane-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadecane-9,12-dien-1-yloxy]methyl}prop-1-ol (compound 1 in US20130150625); 2-amino-3-[(9Z)-octadecane-9-en-1-yloxy]-2-{[(9Z)-octadecane-9-en-1-yloxy]methyl}prop-1-ol (compound 2 in US20130150625); 2- Amino-3-[(9Z,12Z)-octadecane-9,12-dien-1-yloxy]-2-[(octoxy)methyl]prop-1-ol (compound 3 in US20130150625); and 2-(dimethylamino)-3-[(9Z,12Z)-octadecane-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadecane-9,12-dien-1-yloxy]methyl}prop-1-ol (compound 4 in US20130150625); or any pharmaceutically acceptable salt or stereoisomer thereof.

[0319] Cationic lipids can be any of the following: N,N-dioleno-N,N-dimethylammonium chloride (DODAC), 1,2-diolenoyloxy-3-(dimethylamino)propane (DODAP), 1,2-diolenoyloxy-N,N-dimethylaminopropane (DODMA), 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), N-(1-(2,3-diolenoyloxy)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-dimyristyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 2,3-dioleoyloxy-N-[2(glycero-carboxamide)ethyl]-N,N-dimethyl-1-propanediamine trifluoroacetate (DOSPA), bis(octadecylamide-glycyl)sperylamine (D... OGS), 3-dimethylamino-2-(cholest-5-en-3-β-oxybut-4-oxy)-1-(cis,cis-9,12-octadedienoxy)propane (CLinDMA), 2-(5'-(cholest-5-en-3β-oxy)-3'-oxaproloxy)-3-dimethyl-1-(cis,cis-9',1-2'-octadedienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleoyloxybenzylamine (DMOBA), 1,2-N,N'-dioleoylcarbamoyl-3-dimethyl Aminopropane (DOcarbDAP), 1,2-N,N'-dilinoleoylcarbamoyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleoylcarbamoyl-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.

[0320] Cationic lipids can be cationic lipids of formula I:

[0321]

[0322] Where R 1 and R 2 Independently selected, and is H or C1-C3 alkyl, R 3 and R 4 Independently selected, and being an alkyl group having about 10 to about 20 carbon atoms, and R 3 and R4 At least one of them contains at least two unsaturated sites, and preferably the cationic lipid of formula I is 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA) or 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA).

[0323] Cationic lipids can be cationic lipids of formula II:

[0324]

[0325] Where R 1 and R 2 Independently selected, and is H or C1-C3 alkyl, R 3 and R 4 Independently selected, and being an alkyl group having about 10 to about 20 carbon atoms, and R 3 and R 4 At least one of them contains at least two unsaturated sites.

[0326] Cationic lipids can be cationic lipids of formula III:

[0327]

[0328] Where R 1 and R 2 Same or different, and independently for optional substitution of C 12 -C 24 Alkyl, optionally substituted C 12 -C 24 alkenyl, optionally substituted C 12 -C 24 alkynyl or optionally substituted C 12 -C 24 Acyl group; R 3 and R 4 The same or different, and independently of an optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkenyl, or optionally substituted C1-C5 ynyl, or R 3 and R 4 They can be joined to form a heterocycle having 4 to 6 carbon atoms and 1 or 2 optionally substituted heteroatoms selected from nitrogen and oxygen; R 5 It is absent, or is hydrogen or C1-C6 alkyl to provide quaternary ammonium; m, n and p are the same or different, and are independently 0 or 1, provided that m, n and p are not simultaneously 0; q is 0, 1, 2, 3 or 4; and Y and Z are the same or different, and are independently O, S or NH.

[0329] The cationic lipid of Formula III can be 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA; "XTC2"), 2,2-dilinoleoyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleoyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), 2,2-dilinoleoyl-5- Dimethylaminomethyl-[1,3]-dioxolane (DLin-K6-DMA), 2,2-dilinoleoyl-4-N-methylpiperazino-[1,3]-dioxolane (DLin-K-MPZ), 2,2-dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-dilinoleoylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleoyloxy-3-(dimethylamino)acetoxypropane ( DLin-DAC), 1,2-dilinoleoyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleoylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleoyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleoyloxy-3-trimethylaminopropane chloride (DLin-TMA.C1), 1,2- Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.C1), 1,2-dilinoleoyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleoylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleoylamino)-1,2-propanediol (DOAP), 1,2-dilinoleoyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), or mixtures thereof.

[0330] Phospholipids can be expressed according to formula (IV):

[0331]

[0332] R represents a phospholipid moiety, and R and R' represent unsaturated or saturated fatty acid moieties that may be the same or different.

[0333] The phospholipid fraction can be selected from the following groups: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin.

[0334] The fatty acid portion of phospholipids can be selected from the non-restricted group consisting of: lauric acid, myristic acid, myristenoic acid, palmitic acid, palmenoic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, docosanoic acid, docosanopentaenoic acid, and docosanohexaenoic acid.

[0335] Phospholipids can be lecithin, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lecithin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, diceryl phosphate, distearate, distearate, dioleoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoylphosphatidylcholine (POPC), palmitoylphosphatidylethanolamine (POPC), and palmitoylphosphatidylethanolamine (POPC). Alkylamine (POPE), palmitoyl oleoyl phosphatidylglycerol (POPG), 4-(N-maleimidemethyl)-cyclohexane-1-carboxylic acid dioleoyl phosphatidylethanolamine (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearate phosphatidylethanolamine (DSPE), monomethyl phosphatidylethanolamine, dimethyl phosphatidylethanolamine, ditrans-oleoyl phosphatidylethanolamine (DEPE), stearoyl oleoyl phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoyl phosphatidylcholine, and mixtures thereof.

[0336] Phospholipids can be distearylphosphatidylcholine (DSPC).

[0337] Phospholipids may account for about 5 mol% to about 20 mol%, about 5 mol% to about 15 mol%, about 5 mol% to about 10 mol%, about 10 mol% to about 20 mol%, and about 15 mol% to about 20 mol% of the total lipids present in the particles.

[0338] Phospholipids may account for 5 mol% to 20 mol%, 5 mol% to 15 mol%, 5 mol% to 10 mol%, 10 mol% to 20 mol%, or 15 mol% to 20 mol% of the total lipids present in the particles.

[0339] Structural lipids can be selected from the group consisting of: cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassinosteroidol, lycopene, tomatine, ursolic acid, α-tocopherol, and mixtures thereof.

[0340] The structural lipid may be cholesterol. In some embodiments, the structural lipid includes cholesterol and corticosteroids (such as prednisolone, dexamethasone, prednisone, and hydrocortisone) or combinations thereof. Furthermore, the structural lipid may be squalene, squalene, or combinations thereof.

[0341] Structural lipids may include lipids containing geraniol acetate, farnesyl acetate, or digeraniol, or ethers, esters, or other derivatives.

[0342] Structural lipids comprise about 30 mol% to about 50 mol%, about 30 mol% to about 45 mol%, about 30 mol% to about 40 mol%, about 30 mol% to about 35 mol%, about 35 mol% to about 50 mol%, about 40 mol% to about 50 mol%, or about 45 mol% to about 50 mol% of the total lipids present in the particles.

[0343] Examples of lipid nanoparticle compositions and methods for their preparation are described, for example, in Semple et al. (2010) Nature Biotechnol. 28:172-176; Jayarama et al. (2012) Angewandte Chemie International Edition, 51:8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578 (each of which is incorporated herein by reference in its entirety).

[0344] In some embodiments, the lipid nanoparticle formulation comprises 0.5% to 15% by mole of neutral lipids, such as 3% to 12%, 5% to 10%, 15%, 10%, or 7.5% by mole. Examples of neutral lipids include, but are not limited to, DSPC, POPC, DPPC, DOPE, and SM. In some embodiments, the formulation comprises 5% to 50% by mole of sterols (e.g., 15% to 45%, 20% to 40%, 40%, 38.5%, 35%, or 31% by mole). A non-limiting example of a sterol is cholesterol. In some embodiments, the lipid nanoparticle formulation comprises 0.5% to 20% by mole of PEG or PEG-modified lipids (e.g., 0.5% to 10%, 0.5% to 5%, 1.5%, 0.5%, 1.5%, 3.5%, or 5% by mole). In some embodiments, the PEG or PEG-modified lipids comprise PEG molecules with an average molecular weight of 2,000 Da. In some embodiments, the PEG or PEG-modified lipid comprises PEG molecules with an average molecular weight of less than 2,000, such as about 1,500 Da, about 1,000 Da, or about 500 Da. Non-limiting examples of PEG-modified lipids include PEG-distearate (PEG-DMG) (also referred to herein as PEG-C14 or C14-PEG) and PEG-cDMA (further discussed in Reyes et al., J. Controlled Release, 107, 276-287 (2005), the contents of which are incorporated herein by reference in their entirety).

[0345] In any embodiment, the PEGylated lipids 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 lipids present in the particles.

[0346] PEGylated lipids may be selected from the group consisting of: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof.

[0347] PEGylated lipids can be selected from the following groups: PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipids.

[0348] The molecular weight of the PEG component of the PEGylated lipid can be from about 100 Da to about 100,000 Da, from about 100 Da to about 100,000 Da, from about 1,000 Da to 9,000 Da, from about 1,000 Da to 8,000 Da, from about 1,000 Da to 7,000 Da, from about 1,000 Da to 6,000 Da, from about 1,000 Da to 5,000 Da, from about 1,000 Da to 4,000 Da, from about 1,000 Da to 3,000 Da, or from about 1,000 Da to 2,000 Da.

[0349] PEGylated lipids can be DSPE-PEG, where the molecular weight of PEG is 2000 Da.

[0350] In some embodiments, the pharmaceutical composition of an RNA (e.g., mRNA) vaccine may include at least one of the PEGylated lipids described in International Publication No. WO2012099755, the contents of which are incorporated herein by reference in their entirety.

[0351] PEGylated lipids can be ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-bis(tetradecyl)acetamide). ALC-0159 is a PEG / lipid conjugate (i.e., a PEGylated lipid), specifically, it is N,N-dimyristicamide of 2-hydroxyacetic acid, O-PEGylated to a PEG chain with a mass of approximately 2 kilodaltons (corresponding to approximately 45-46 ethylene oxide units per molecule of N,N-dimyristic hydroxyacetamide). It is essentially a nonionic surfactant.

[0352] Non-limiting examples of lipid nanoparticle compositions and methods for their preparation are described, for example, in Semple et al. (2010) Nature Biotechnology 28:172-176; Jayarama et al. (2012) Angewandte Chemie, 51:8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578 (each of which is incorporated herein by reference in its entirety).

[0353] The relative amounts of the active ingredient, pharmaceutically acceptable excipients, and / or any other components in the vaccine composition may vary depending on the identity, size, and / or condition of the subject being treated and, more importantly, on the route of administration of the composition. For example, the composition may contain 0.1% to 99% (w / w) of the active ingredient. For instance, the composition may contain 0.1% to 100%, such as 0.5% to 50%, 1% to 30%, 5% to 80%, or at least 80% (w / w) of the active ingredient.

[0354] In some embodiments, the RNA vaccine composition of the present invention may contain the polynucleotide described herein formulated in lipid nanoparticles comprising ALC-0315 ([(4-hydroxybutyl)azanidinediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), cholesterol, DSPC, and ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-bis(tetradecyl)acetamide), buffer Tris-sucrose, and water for injection.

[0355] As a non-limiting example, the composition comprises: 0.6 mg / mL of a drug (e.g., a polynucleotide encoding the chimeric or fusion protein described herein and comprising a component of the *Porphyromonas gingivalis* gingival protease polyprotein 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-bis(tetradecyl)acetamide), 3.03 mg / mL of Tris (tris(hydroxymethyl)aminomethane), and 88 mg / mL of sucrose-containing water, with a typical injection volume of 50 μL.

[0356] In alternative embodiments, the RNA vaccine composition of the present invention may comprise four lipids, DLin-MC3-DMA, cholesterol, DSPC, and DMG-PEG 2000, in a ratio (molar ratio) of 50:39.8:10:0.2.

[0357] In some embodiments, the average diameter of the nanoparticles (e.g., lipid nanoparticles) is 10-500 nm, 20-400 nm, 30-300 nm, or 40-200 nm. In some embodiments, the average diameter of the nanoparticles (e.g., lipid nanoparticles) is 50-150 nm, 50-200 nm, 80-100 nm, or 80-200 nm.

[0358] In some embodiments, the RNA (e.g., RNA) vaccine of this disclosure can have a diameter of about 10 nm to about 100 nm, such as, but not limited to, about 10 nm to about 20 nm, about 10 nm to about 30 nm, about 10 nm to about 40 nm, about 10 nm to about 50 nm, about 10 nm to about 60 nm, about 10 nm to about 70 nm, about 10 nm to about 80 nm, about 10 nm to about 90 nm, about 20 nm to about 30 nm, about 20 nm to about 40 nm, about 20 nm to about 50 nm, about 20 nm to about 60 nm, about 20 nm to about 70 nm, about 20 nm to about 80 nm, about 20 nm to about 90 nm, about 20 nm to about 100 nm, about 30 nm to about 40 nm, about 30 nm to about 50 nm, about 30 nm to about 60 nm, about 30 nm to about 70 nm, about 30 nm to about 80 nm, about 30 nm to about 90 nm, about 30 nm to about 100 nm, about 40 nm to about 50 nm. Formulated in lipid nanoparticles of about 40 nm to about 60 nm, about 40 nm to about 70 nm, about 40 nm to about 80 nm, about 40 nm to about 90 nm, about 40 nm to about 100 nm, about 50 nm to about 60 nm, about 50 nm to about 70 nm, about 50 nm to about 80 nm, about 50 nm to about 90 nm, about 50 nm to about 100 nm, about 60 nm to about 70 nm, about 60 nm to about 80 nm, about 60 nm to about 90 nm, about 60 nm to about 100 nm, about 70 nm to about 80 nm, about 70 nm to about 90 nm, about 70 nm to about 100 nm, about 80 nm to about 90 nm, about 80 nm to about 100 nm and / or about 90 nm to about 100 nm.

[0359] In some embodiments, the diameter of the lipid nanoparticles can be from about 10 nm to 500 nm.

[0360] In some embodiments, the diameter of the lipid nanoparticles can be 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.

[0361] In some embodiments, the lipid nanoparticles may be the lipid nanoparticles of the extreme size described in International Patent Publication No. WO2013059922, the contents of which are incorporated herein by reference in their entirety. The extreme size lipid nanoparticles may comprise a lipid bilayer surrounding an aqueous or hydrophobic core; wherein the lipid bilayer may comprise phospholipids, such as, but not limited to, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelin, dihydrosphingomyelin, cephalin, cerebrosides, C8-C20 fatty acid diacylphosphatidylcholine, and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC). In some embodiments, the extreme size lipid nanoparticles may comprise polyethylene glycol lipids, such as, but not limited to, DLPE-PEG, DMPE-PEG, DPPC-PEG, and DSPE-PEG.

[0362] In some embodiments, RNA (e.g., RNA) vaccines may be associated with cationic or multi-cationic compounds, including protamine, nucleolin, 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, penetrantin, VP-derived peptides or analogues, Pestivirus Erns, HSV, VP (herpes simplex virus), MAP, KALA or protein transduction domain (PTD), PpT620, proline-rich peptides, arginine-rich peptides, lysine-rich peptides, MPG peptides, Pep-1, L-oligomers, calcitonin peptides, and Antennapedia-derived peptides. peptides (especially those derived from Drosophila antennal foot mutations), pAntp, pIsl, FGF, lactoferrin, transport proteins, toad antimicrobial peptide-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, histones, cationic polysaccharides (e.g., chitosan), polygluconine, cationic polymers (e.g., polyethyleneimine (PEI)), cationic lipids (e.g., DOTMA: [1-(2,3-dioleoxy)propyl)]-N,N,N-trimethylammonium chloride, DMRI E, Di-C14-amidinium, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE: Dioleoylphosphatidylethanolamine, DOSPA, DODAB, DOIC, DMEPC, DOGS: Dioctadecylamide glycyl spermine, DIMRI: Dimyroxypropyl dimethylhydroxyethyl ammonium bromide, DOTAP: Dioleoyloxy-3-(trimethylammonium)propane, DC-6-14: O,O-Di(tetradecanoyl)-N-α-trimethylammonium acetyl)diethanolamine chloride, CLIP 1: Racemic-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium chloride, CLIP 6: Racemic-[2(2,3-dihexadecimaloxypropyloxymethyloxy)ethyl]-trimethylammonium, CLIP 9: Racemic-[2(2,3-dihexadecimaloxypropyloxymethyloxy)ethyl]-trimethylammonium3-Dihexadecyloxypropyloxysuccinyloxy)ethyl]-trimethylammonium, oligofectamine, or cationic or polycationic polymers, such as modified polyamino acids (e.g., β-amino acid polymers or reverse polyamides), modified polyethylene (e.g., PVP (poly(N-ethyl-4-vinylpyridine bromide)), modified acrylates (e.g., pDMAEMA (poly(dimethylaminoethyl methacrylate)), modified amide amines (e.g., pAMAM (poly(amide amine)), modified polyβ-amino esters (PBAE)) (e.g., diamine-terminated 1,4-butanediol diacrylate-co-5- Polymers such as amino-1-pentanol polymers, dendritic macromolecules (e.g., polypropylamine dendritic macromolecules or pAMAM-based dendritic macromolecules), polyimides (e.g., PEI: poly(ethyleneimine), poly(propyleneimine)), polyallylamines, sugar-based polymers (e.g., cyclodextrin-based polymers, dextran-based polymers, chitosan), silane-based polymers (e.g., PMOXA-PDMS copolymers), and block polymers composed of one or more cationic blocks (e.g., selected from the cationic polymers mentioned above) and one or more hydrophilic or hydrophobic blocks (e.g., polyethylene glycol).

[0363] In other embodiments, the RNA (e.g., RNA) vaccine does not associate with cationic or polycationic compounds.

[0364] Other examples of suitable lipid nanoparticle formulations are provided in US 10,702,600, the contents of which are incorporated herein by reference.

[0365] The lipid nanoparticles described in this article can be prepared in a sterile environment.

[0366] Nanoparticle formulations may contain phosphate conjugates. Phosphate conjugates can increase in vivo circulation time and / or enhance targeted delivery of nanoparticles. As a non-limiting example, phosphate conjugates may include compounds having any of the chemical formulas described in International Application No. WO2013033438, the contents of which are incorporated herein by reference in their entirety.

[0367] Nanoparticle formulations may comprise polymer conjugates. The polymer conjugates may be water-soluble conjugates. The polymer conjugates may have the structure described in U.S. Patent Application No. 20130059360, the contents of which are incorporated herein by reference in their entirety. In some embodiments, polymer conjugates of the polynucleotides disclosed herein may be prepared using the methods and / or segmented polymer reagents described in U.S. Patent Application No. 20130072709, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the polymer conjugates may have side groups comprising ring moieties, such as, but not limited to, the polymer conjugates described in U.S. Patent Publication No. US20130196948, the contents of which are incorporated herein by reference in their entirety.

[0368] The nanoparticle formulation may include a conjugate for enhancing the delivery of the nanoparticles of this disclosure in a subject. Further, the conjugate may inhibit phagocytic clearance of the nanoparticles in the subject. In one embodiment, the conjugate may be a “self” peptide designed from the human membrane protein CD47 (e.g., “self” particles described by Rodriguez et al. (Science 2013 339, 971-975), which is incorporated herein by reference in its entirety). As shown by Rodriguez et al., self peptides delay macrophage-mediated clearance of nanoparticles, which enhances nanoparticle delivery. In another embodiment, the conjugate may be the membrane protein CD47 (e.g., see Rodriguez et al., Science 2013 339, 971-975, which is incorporated herein by reference in its entirety). Rodriguez et al. demonstrated that, similar to “self” peptides, CD47 can increase the ratio of circulating particles in a subject, compared to disordered peptides and PEG-coated nanoparticles.

[0369] In some embodiments, the RNA (e.g., RNA) vaccine of this disclosure is formulated in nanoparticles comprising conjugates for enhancing delivery of the nanoparticles of this disclosure in a subject. The conjugate may be a CD47 membrane, or the conjugate may be derived from the CD47 membrane protein, such as the previously described “autologous” peptide. In some embodiments, the nanoparticles may comprise PEG and a CD47 conjugate or a derivative thereof. In some embodiments, the nanoparticles may comprise both the aforementioned “autologous” peptide and the membrane protein CD47.

[0370] In some embodiments, the RNA (e.g., RNA) vaccine pharmaceutical composition comprises the polynucleotide of this disclosure and a conjugate that may have degradable bonds. Non-limiting examples of conjugates include an aromatic moiety comprising ionizable hydrogen atoms, a spacer moiety, and a water-soluble polymer. As a non-limiting example, pharmaceutical compositions comprising conjugates having degradable bonds and methods for delivering such pharmaceutical compositions are described in U.S. Patent Publication No. US20130184443, the contents of which are incorporated herein by reference in their entirety.

[0371] The nanoparticle formulation may be carbohydrate nanoparticles comprising a carbohydrate carrier and an RNA (e.g., RNA) vaccine. As a non-limiting example, the carbohydrate carrier may include, but is not limited to, anhydride-modified plant glycogen or glycogenotype material, octenyl succinate plant glycogen, plant glycogen β-dextrin, and anhydride-modified plant glycogen β-dextrin. (See, for example, International Publication WO2012109121; the contents of which are incorporated herein by reference in their entirety).

[0372] The nanoparticle formulations disclosed herein may be coated with surfactants or polymers to improve particle delivery. In some embodiments, the nanoparticles may be coated with a hydrophilic coating, such as, but not limited to, a PEG coating and / or a coating having a neutral surface charge. Hydrophilic coatings may facilitate the delivery of nanoparticles with large payloads within the central nervous system, such as, but not limited to, RNA (e.g., RNA) vaccines. As a non-limiting example, nanoparticles comprising a hydrophilic coating and methods for preparing such nanoparticles are described in U.S. Patent Publication No. US20130183244, the contents of which are incorporated herein by reference in their entirety.

[0373] In some embodiments, the lipid nanoparticles of this disclosure may be hydrophilic polymer particles. Non-limiting examples of hydrophilic polymer particles and methods for preparing hydrophilic polymer particles are described in U.S. Patent Publication No. US20130210991, the contents of which are incorporated herein by reference in their entirety.

[0374] In some embodiments, the lipid nanoparticles disclosed herein may be hydrophobic polymer particles.

[0375] Lipid nanoparticle formulations can be improved by replacing cationic lipids with biodegradable cationic lipids, known as rapidly clearing lipid nanoparticles (reLNPs). 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 rapidly clearing lipids can improve the tolerability and therapeutic index of lipid nanoparticles by increasing the dose from 1 mg / kg to the order of 10 mg / kg in rats. Including enzymatically degradable ester bonds can improve the degradation and metabolic properties of the cationic component while maintaining the activity of the reLNP formulation. The ester bond can be located inside the lipid chain or at the end of the lipid chain. The internal ester bond can replace any carbon in the lipid chain.

[0376] In some embodiments, the internal ester bond can be located on either side of the saturated carbon.

[0377] In some embodiments, an immune response can be induced by delivering lipid nanoparticles, which may include nanospecies, polymers, and immunogens. (US Publication No. 20120189700 and International Publication No. WO2012099805; each of these patents is incorporated herein by reference in its entirety). The polymer may encapsulate or partially encapsulate the nanospecies. The immunogen may be a recombinant protein, modified RNA, and / or polynucleotide as described herein. In some embodiments, the lipid nanoparticles may be formulated for use in vaccines, such as, but not limited to, those against pathogens.

[0378] Lipid nanoparticles can be engineered to modify their surface properties, enabling them to penetrate mucosal barriers. Mucus is located on mucosal tissues, such as, but not limited to, the oral cavity (e.g., buccal and esophageal membranes and tonsils), eyes, gastrointestinal tract (e.g., stomach, small intestine, large intestine, colon, rectum), nose, respiratory tract (e.g., nasal, pharyngeal, tracheal, and bronchial membranes), and genitals (e.g., vaginal, cervical, and urethral membranes). Nanoparticles larger than 10–200 nm are preferred for high drug encapsulation efficiency and the ability to provide sustained delivery of multiple drugs and are considered too large to diffuse rapidly across the mucosal barrier. Mucus is constantly secreted, shed, discarded, or dissolved and circulated, so most of the captured particles can be removed from the mucosal tissue within seconds or hours. Large polymer nanoparticles (200 nm–500 nm in diameter) densely coated with low molecular weight polyethylene glycol (PEG) diffuse through mucus, which are only 1 / 6 to 1 / 4 the size of the same particles that diffuse in water (Lai et al., Proceedings of the National Academy of Sciences (PNAS) (2007) 104(5):1482–487; Lai et al., Advanced Drug Delivery Review 2009 61(2): 158–171; each of these references is incorporated herein by reference in its entirety). The delivery of nanoparticles can be determined using permeation rate and / or fluorescence microscopy techniques, including but not limited to fluorescence recovery after photobleaching (FRAP) and high-resolution multi-particle tracking (MPT). As a non-limiting example, compositions capable of penetrating mucosal barriers can be prepared as described in U.S. Patent No. 8,241,670 or International Patent Publication No. WO2013110028, the contents of each of these patents are incorporated herein by reference in their entirety.

[0379] Lipid nanoparticles engineered to penetrate mucus may comprise a polymeric material (i.e., a polymeric core) and / or a polymer-vitamin conjugate and / or a triblock copolymer. The polymeric material may include, but is not limited to, polyamines, polyethers, polyamides, polyesters, polyurethanes, polyureas, polycarbonates, poly(styrene), polyimides, polysulfones, polyurethanes, polyacetylene, polyethylene, polyethyleneimine, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitrile, 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 incorporated herein by reference in their entirety. The polymeric material may also be irradiated. As a non-limiting example, the polymeric material may be γ-irradiated (see, for example, International Application No. WO201282165, which is incorporated herein 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-glycolic acid), and poly(D,L-lactide-co-P EO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), alkyl cyanoacrylate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, poly(hydroxy acid), polyanhydride, polyorthoester, poly(esteramide), polyamide, poly(ether ester), polycarbonate, polyalkylene, such as polyethylene and polypropylene, polyalkylene glycols, such as poly(ethylene glycol) (PEG), polyalkylene oxide (PEO), polyalkylene terephthalate, such as poly(ethylene terephthalate). Polyvinyl alcohol (PVA), polyvinyl ether, polyethylene esters such as poly(vinyl acetate), polyethylene halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxane, polystyrene (PS), polyurethane, derived cellulose (such as alkyl cellulose, hydroxyalkyl cellulose, cellulose ether, cellulose ester, nitrocellulose, hydroxypropyl cellulose, carboxymethyl cellulose), acrylic polymers (such as poly((meth)acrylate) methyl methacrylate (PMMA), poly((((meth)acrylate) ethyl acrylate), poly(((((meth)acrylate) butyl acrylate), poly(((((meth)acrylate) isobutyl acrylate), poly((((meth)acrylate) propylene) Hexyl acrylate), poly(((meth)acrylate) isodecyl acrylate), poly(((meth)acrylate) lauryl acrylate), poly(((meth)acrylate) phenyl acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) and their copolymers and mixtures, polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamer, poly(orthocyanin), poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), PEG-PLGA-PEG and trimethylene carbonate, polyvinylpyrrolidone.Lipid nanoparticles may be coated with or associated with copolymers, such as, but not limited to, block copolymers (e.g., branched polyether-polyamide block copolymers as described in International Publication No. WO2013012476, which is incorporated herein by reference in its entirety) and (poly(ethylene glycol))-(poly(propylene oxide))-(poly(ethylene glycol)) triblock copolymers (see, for example, U.S. Publications 20120121718 and 20100003337 and U.S. Patent No. 8,263,665, the contents of each of which are incorporated herein by reference in their entirety). The copolymers may be generally considered safe polymers (GRAS), and the formation of lipid nanoparticles may be carried out in a manner that does not create new chemical entities. For example, lipid nanoparticles can contain PLGA nanoparticles coated with polyoxides without forming new chemical entities, while still being able to rapidly penetrate human mucus (Yang et al., Angewandte Chemie 2011 50:2597-2600; the contents of the cited literature are incorporated herein by reference in their entirety). Xu et al. describe a non-limiting scalable method for generating nanoparticles that can penetrate human mucus. (See, for example, Controlled Release Journal 2013, 170(2):279-86; the contents of the cited literature are incorporated herein by reference in their entirety).

[0380] The vitamin in the polymer-vitamin conjugate may be vitamin E. The vitamin portion of the conjugate may be replaced by other suitable components (such as, but not limited to, vitamin A, vitamin E, other vitamins, cholesterol, hydrophobic portions, or hydrophobic components of other surfactants (e.g., sterol chains, fatty acids, hydrocarbon chains, and oxidized olefin chains)).

[0381] Lipid nanoparticles engineered to penetrate mucus can include surface modifiers such as, but not limited to, polynucleotides, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyl dioctadecyl ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), and mucolytic agents (e.g., N-acetylcysteine, Artemisia argyi, bromelain, papain, Clerodendrum, acetylcysteine, bromhexine, carbocysteine). Surface modifiers include sterene, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin 34, alfa streptococcus, neltenexine, erdosteine, and various DNases, including rhDNase. Surface modifiers can be embedded or incorporated into the surface of the particles or placed (e.g., by coating, adsorption, covalent bonding, or other processes) on the surface of lipid nanoparticles. (See, for example, US Publication 20100215580 and US Publication 20080166414 and US20130164343; the contents of each of these publications are incorporated herein by reference in their entirety).

[0382] In some embodiments, the mucus-penetrating lipid nanoparticles may comprise at least one polynucleotide as described herein. The polynucleotide may be encapsulated within the lipid nanoparticles and / or disposed on the surface of the particles. The polynucleotide may be covalently coupled to the lipid nanoparticles. Formulations of mucus-penetrating lipid nanoparticles may comprise multiple nanoparticles. Furthermore, the formulation may include particles that can interact with mucus and alter the structure and / or adhesive properties of the surrounding mucus to reduce adhesion, which may increase the delivery of mucus-penetrating lipid nanoparticles to mucosal tissue.

[0383] In some embodiments, the mucus-penetrating lipid nanoparticles may be hypotonic formulations comprising a mucosal penetration-enhancing coating. The formulations may be hypotonic to the epithelial tissue to which they are delivered. Non-limiting examples of hypotonic formulations are described in International Patent Publication No. WO2013110028, the contents of which are incorporated herein by reference in their entirety.

[0384] In some embodiments, to enhance delivery across the mucosal barrier, RNA (e.g., mRNA) vaccine formulations may contain a hypotonic solution or a hypotonic solution.

[0385] It has been found that hypotonic solutions increase the rate at which mucus-inert particles (such as, but not limited to, mucus-penetrating particles) reach the vaginal epithelial surface (see, for example, Ensign et al., Biomaterials 2013 34(28):6922-9, the contents of which are incorporated herein by reference in their entirety).

[0386] In some embodiments, RNA (e.g., mRNA) vaccines are formulated as lipid complexes, such as, but not limited to, the ATUPLEX™ system, DACC system, DBTC system, and other siRNA-lipid complex technologies from Silence Therapeutics (London, United Kingdom), STEMFECT™ from STEMGENT® (Cambridge, Massachusetts), and targeted and non-targeted delivery of nucleic acids based on polyethyleneimine (PEI) or protamine (Aleku et al., Cancer Research, 2008, 68:9788-9798; Strumberg et al., International Journal of Clinical Pharmacology and Therapeutics, 2012, 50:76-78; Santel et al., Gene Therapy, 2006, 13:1222-1234; Santel et al., Gene Therapy, 2006). 13:1360-1370; Gutbier et al., *Pulm Pharmacol. Ther*. 2010 23:334-344; Kaufmann et al., *Microvasc Research*. 2010 80:286-293; Weide et al., *Journal of Immunotherapy*. 2009 32:498-507; Weide et al., *Journal of Immunotherapy*. 2008 31:180-188; Pascolo, *Expert Opinion on Biotherapy*. 4:1285-1294; Fotin-Mleczek et al., *Journal of Immunotherapy*. 2011 34:1-15; Song et al., *Nature Biotechnology*. 2005, 23:709-717; Peer et al. Proceedings of the National Academy of Sciences of the United States of America 2007 6; 104:4095-4100; de Fougerolles Human Gene Therapy. 2008 19:125-132; The contents of each of these references are incorporated herein by reference in their entirety.

[0387] In some embodiments, RNA (e.g., mRNA) vaccines are formulated as solid lipid nanoparticles. Solid lipid nanoparticles (SLNs) may be spherical with an average diameter of 10 nm to 1000 nm. SLNs have a solid lipid core matrix that can dissolve lipophilic molecules and can be stabilized by surfactants and / or emulsifiers. In some embodiments, lipid nanoparticles may be self-assembled lipid polymer nanoparticles (see Zhang et al., ACS Nano, 2008, 2(8), pp. 1696-1702; the contents of which are incorporated herein by reference in their entirety). As a non-limiting example, SLNs may be those described in International Patent Publication WO2013105101, the contents of which are incorporated herein by reference in their entirety. As another non-limiting example, SLNs may be prepared by the method or process described in International Patent Publication WO2013105101, the contents of which are incorporated herein by reference in their entirety.

[0388] In some embodiments, the RNA (e.g., mRNA) vaccine of this disclosure may 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 specific release profile to achieve a therapeutic outcome. In some embodiments, the RNA (e.g., mRNA) vaccine may be encapsulated in delivery agents described herein and / or known in the art for controlled release and / or targeted delivery. As used herein, the term “encapsulation” means enclosure, surrounding, or encapsulation. Encapsulation can be substantially, completely, or partially because it involves formulations of compounds of this disclosure. The term “substantially encapsulated” means that at least 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 this disclosure may be encapsulated, surrounded, or encapsulated within a delivery agent. "Partial encapsulation" means that less than 10, 10, 20, 30, 40, 50, or fewer of the pharmaceutical compositions or compounds of this disclosure may be encapsulated, surrounded, or sealed within a delivery agent. Advantageously, encapsulation can be determined by measuring the efflux or activity of the pharmaceutical compositions or compounds of this disclosure using fluorescence and / or electron microscopy. 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 compositions or compounds of this disclosure are encapsulated in a delivery agent.

[0389] In some embodiments, the controlled-release formulation may include, but is not limited to, triblock copolymers. As a non-limiting example, the formulation may include two different types of triblock copolymers (International Publications WO2012131104 and WO2012131106, the contents of each of which are incorporated herein by reference in their entirety).

[0390] In some embodiments, an RNA (e.g., mRNA) vaccine may be encapsulated in lipid nanoparticles or rapidly eliminated lipid nanoparticles, and the lipid nanoparticles or rapidly eliminated lipid nanoparticles may then be encapsulated in polymers, hydrogels and / or surgical sealants described herein and / or known in the art. As non-limiting examples, polymers, hydrogels, or surgical sealants may be PLGA, ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, Fla.), HYLENEX® (Halozyme Therapeutics, San Diego, California.), surgical sealants (such as fibrinogen polymers (Ethicon Inc. Cornelia, Georgia.), TISSELL® (Baxter International, Inc. Deerfield, Illinois.), PEG-based sealants, and COSEAL® (Baxter International, Inc. Deerfield, Illinois.).

[0391] In some embodiments, lipid nanoparticles may be encapsulated in any polymer known in the art, which may form a gel when injected into a subject. As another non-limiting example, lipid nanoparticles may be encapsulated in a polymer matrix that may be biodegradable.

[0392] In some embodiments, RNA (e.g., mRNA) vaccine formulations 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 aqueous dispersions of ethyl cellulose (AQUACOAT® and SURELEASE®).

[0393] In some embodiments, RNA (e.g., mRNA) vaccine controlled release and / or targeted delivery formulations may comprise at least one biodegradable polyester, said at least one biodegradable polyester may contain multi-cationic side chains. Biodegradable 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 biodegradable polyester may include PEG conjugation to form a PEGylated polymer.

[0394] In some embodiments, RNA (e.g., mRNA) vaccine formulations comprising at least one polynucleotide may comprise at least one PEG and / or PEG-related polymer derivatives, as described in U.S. Patent No. 8,404,222, the contents of which are incorporated herein by reference in their entirety.

[0395] In some embodiments, a controlled-release delivery formulation of an RNA (e.g., mRNA) vaccine containing at least one polynucleotide can be a controlled-release polymer system as described in US20130130348, the contents of which are incorporated herein by reference in their entirety.

[0396] In some embodiments, the RNA (e.g., mRNA) vaccine of this disclosure may be encapsulated in therapeutic nanoparticles, referred to herein as "therapeutic nanoparticle RNA (e.g., mRNA) vaccines". Therapeutic nanoparticles may be as described herein and in the art, such as, but not limited to, International Publications WO2010005740, WO2010030763, WO2010005721, WO2010005723, WO2012054923, and U.S. Publications US20110262491 and US20100... The therapeutic polymer nanoparticles are formulated using methods known in US Publication No. 104645, US20100087337, US20100068285, US20110274759, US20100068286, US20120288541, US20130123351, and US20130230567, as well as US Patent Nos. 8,206,747, 8,293,276, 8,318,208, and 8,318,211, the contents of each of which are incorporated herein by reference in their entirety. In some embodiments, therapeutic polymer nanoparticles can be identified by the method described in US Publication No. US20120140790, the contents of which are incorporated herein by reference in their entirety.

[0397] In some embodiments, therapeutic nanoparticle RNA (e.g., mRNA) vaccines can be formulated for sustained release. As used herein, “sustained release” means a pharmaceutical composition or compound that conforms to a certain release rate over a specific time period. The time period may include, but is not limited to, hours, days, weeks, months, and years. As a non-limiting example, sustained-release nanoparticles may comprise polymers and therapeutic agents (such as, but not limited to, polynucleotides of this disclosure) (see International Publication No. 2010075072 and U.S. Publications Nos. US20100216804, US20110217377, and US20120201859, the contents of each of which are incorporated herein by reference in their entirety). In another non-limiting example, sustained-release formulations may comprise agents that allow for sustained 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).

[0398] In some embodiments, therapeutic nanoparticle RNA (e.g., mRNA) vaccines may be formulated to have target specificity. As a non-limiting example, therapeutic nanoparticles may include corticosteroids (see International Publication WO2011084518, the contents of which are incorporated herein by reference in their entirety). As a non-limiting example, therapeutic nanoparticles may be formulated in nanoparticles described in International Publications WO2008121949, WO2010005726, WO2010005725, WO2011084521 and U.S. Publications US20100069426, US20120004293, and US20100104655, the contents of which are incorporated herein by reference in their entirety.

[0399] In some embodiments, the nanoparticles of this disclosure may comprise a polymer matrix. As a non-limiting example, the nanoparticles may comprise two or more polymers, such as, but not limited to, polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropyl fumarate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polylysine, poly(ethyleneimine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), or combinations thereof.

[0400] In some embodiments, the therapeutic nanoparticles comprise a diblock copolymer. In some embodiments, the diblock copolymer may comprise a combination of PEG and a polymer, such as, but not limited to, polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropyl fumarate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polylysine, poly(ethyleneimine), 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 the diblock copolymer described in International Patent Publication No. WO2013120052, the contents of which are incorporated herein by reference in their entirety.

[0401] As a non-limiting example, therapeutic nanoparticles comprise PLGA-PEG block copolymers (see US Publication No. US20120004293 and US Patent No. 8,236,330, each of which is incorporated herein by reference in its entirety). In another non-limiting example, therapeutic nanoparticles are cloaking nanoparticles comprising diblock copolymers of PEG and PLA or PEG and PLGA (see US Patent No. 8,246,968 and International Publication No. WO2012166923, each of which is incorporated herein by reference in its entirety). In yet another non-limiting example, therapeutic nanoparticles are cloaking nanoparticles or target-specific cloaking nanoparticles as described in US Patent Publication No. US20130172406, the contents of which are incorporated herein by reference in their entirety.

[0402] In some embodiments, therapeutic nanoparticles may comprise multiblock copolymers (see, for example, U.S. Patent Nos. 8,263,665 and 8,287,910 and U.S. Patent Publication No. US20130195987, the contents of each of which are incorporated herein by reference in their entirety).

[0403] In yet another non-limiting example, lipid nanoparticles comprise the block copolymer PEG-PLGA-PEG (see, for example, Thermosensitive Hydrogel (PEG-PLGA-PEG) used as a TGF-β1 gene delivery medium, in Lee et al. Thermosensitive Hydrogel as a Tgf-β1 Gene Delivery Vehicle Enhances Diabetic Wound Healing. Pharmaceutical Research, 2003 20(12): 1995-2000; used 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 PEG-PLGA-PEG copolymer enhances gene delivery efficiency in rat skeletal muscle). (The biodegradable PEG-PLGA-PEG copolymer enhances gene delivery efficiency in rat skeletal muscle. *Controlled Release Journal* 2007118:245-253, the contents of each of the aforementioned documents are incorporated herein by reference in their entirety.) The RNA (e.g., mRNA) vaccine disclosed herein can be formulated in lipid nanoparticles comprising PEG-PLGA-PEG block copolymers.

[0404] In some embodiments, therapeutic nanoparticles may comprise multiblock copolymers (see, for example, U.S. Patent Nos. 8,263,665 and 8,287,910 and U.S. Patent Publication No. US20130195987, the contents of each of which are incorporated herein by reference in their entirety).

[0405] In some embodiments, the block copolymers described herein may be included in a polyionic complex comprising nonpolymer micelles and block copolymers. (See, for example, U.S. Publication No. 20120076836, the contents of which are incorporated herein by reference in their entirety).

[0406] In some embodiments, the therapeutic nanoparticles 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 methacrylate, cyanoethyl methacrylate, alkyl methacrylate copolymers, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylate, and combinations thereof.

[0407] 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 the structure described in International Application No. WO2013032829 or U.S. Patent Publication No. US20130121954, the contents of each of which are incorporated herein by reference in their entirety. In some embodiments, the poly(vinyl ester) polymer may be conjugated with the polynucleotides described herein.

[0408] In some embodiments, the therapeutic nanoparticles may comprise at least one diblock copolymer. The diblock copolymer may be, but is not limited to, a poly(lactic acid)-poly(vinyl alcohol) copolymer (see, for example, International Patent Publication No. WO2013044219, the contents of which are incorporated herein by reference in their entirety).

[0409] As a non-limiting example, therapeutic nanoparticles can be used to treat cancer (see International Publication No. WO2013044219, the contents of which are incorporated herein by reference in their entirety).

[0410] In some embodiments, therapeutic nanoparticles may comprise at least one cationic polymer described herein and / or known in the art.

[0411] In some embodiments, the therapeutic nanoparticles may comprise at least one amine-containing polymer, such as, but not limited to, polylysine, polyethyleneimine, poly(amidoamine) dendritic polymers, poly(β-amino esters) (see, for example, U.S. Patent No. 8,287,849, the contents of which are incorporated herein by reference in their entirety) and combinations thereof.

[0412] In some embodiments, the nanoparticles described herein may comprise amine cationic lipids, such as those described in International Patent Application No. WO2013059496, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the cationic lipids may have an amino-amine or amino-amide moiety.

[0413] In some embodiments, the therapeutic nanoparticles may comprise at least one biodegradable polyester, which may contain multi-cationic side chains. Biodegradable 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 biodegradable polyester may include PEG conjugation to form a PEGylated polymer.

[0414] In some embodiments, synthetic nanocarriers can be formulated for targeted release. In some embodiments, synthetic nanocarriers are formulated to release polynucleotides at a specified pH and / or after a desired time interval. As a non-limiting example, synthetic nanoparticles can be formulated to release RNA (e.g., mRNA) vaccines after 24 hours and / or at pH 4.5 (see International Publications WO2010138193 and WO2010138194 and US Publications US20110020388 and US20110027217, each of which is incorporated herein by reference in its entirety).

[0415] In some embodiments, synthetic nanocarriers can be formulated for the controlled and / or sustained release of the polynucleotides described herein. As a non-limiting example, synthetic nanocarriers for sustained release can be formulated using methods known in the art, described herein, and / or International Publication No. WO2010138192 and U.S. Publication No. 20100303850, each of which is incorporated herein by reference in its entirety.

[0416] In some embodiments, RNA (e.g., mRNA) vaccines can be formulated for controlled and / or sustained release, wherein the formulation comprises at least one polymer, said at least one polymer being a crystal side-chain (CYSC) polymer. CYSC polymers are described in U.S. Patent No. 8,399,007, which is incorporated herein by reference in its entirety.

[0417] In some embodiments, synthetic nanocarriers can be formulated for use as vaccines. In some embodiments, synthetic nanocarriers can encapsulate at least one polynucleotide encoding at least one antigen. As a non-limiting example, synthetic nanocarriers may include at least one antigen and excipient for a vaccine formulation (see International Publication WO2011150264 and US Publication US20110293723, the contents of each of which are incorporated herein by reference in their entirety). As another non-limiting example, a vaccine formulation may include at least two synthetic nanocarriers having the same or different antigens and excipients (see International Publication WO2011150249 and US Publication US20110293701, the contents of each of which are incorporated herein by reference in their entirety). Vaccine formulations may be selected by methods described herein, known in the art, and / or methods described in International Publication WO2011150258 and US Publication US20120027806, the contents of each of which are incorporated herein by reference in their entirety.

[0418] In some embodiments, the synthetic nanocarrier may comprise at least one polynucleotide encoding at least one adjuvant. As a non-limiting example, the adjuvant may comprise dimethyl dioctadecyl ammonium bromide, dimethyl dioctadecyl ammonium chloride, dimethyl dioctadecyl ammonium phosphate, or dimethyl dioctadecyl ammonium acetate (DDA), and a nonpolar component or a portion of a total lipid extract of mycobacterium (see, for example, U.S. Patent No. 8,241,610, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the synthetic nanocarrier may comprise at least one polynucleotide and an adjuvant. As a non-limiting example, synthetic nanocarriers comprising adjuvants may be formulated using the methods described in International Publication No. WO2011150240 and U.S. Publication No. US20110293700, the contents of each of which are incorporated herein by reference in their entirety.

[0419] In some embodiments, the synthetic nanocarrier may encapsulate at least one polynucleotide encoding a peptide, fragment, or region derived from a virus. As a non-limiting example, the synthetic nanocarrier may include, but is not limited to, any nanocarrier described in International Publications WO2012024621, WO201202629, WO2012024632 and U.S. Publications US20120064110, US20120058153, and US20120058154, the contents of each of which are incorporated herein by reference in their entirety.

[0420] In some embodiments, the synthetic nanocarrier may be conjugated with a polynucleotide that is capable of triggering humoral and / or cytotoxic T lymphocyte (CTL) responses (see, for example, International Publication No. WO2013019669; the contents of which are incorporated herein by reference in their entirety).

[0421] In some embodiments, RNA (e.g., mRNA) vaccines may be encapsulated in zwitterionic lipids, linked to and / or associated with the zwitterionic lipids. Non-limiting examples of zwitterionic lipids and methods using zwitterionic lipids are described in U.S. Patent Publication No. US20130216607, the contents of which are incorporated herein by reference in their entirety.

[0422] In some embodiments, zwitterionic lipids can be used in the liposomes and lipid nanoparticles described herein.

[0423] In some embodiments, RNA (e.g., mRNA) vaccines may be formulated in colloidal nanocarriers, as described in U.S. Patent Publication No. US20130197100, the contents of which are incorporated herein by reference in their entirety.

[0424] In some embodiments, the nanoparticles may be optimized for oral administration. The nanoparticles may comprise at least one cationic biopolymer, such as, but not limited to, chitosan or a derivative thereof. As a non-limiting example, the nanoparticles may be formulated using the method described in U.S. Publication No. 20120282343, the contents of which are incorporated herein by reference in their entirety.

[0425] In some embodiments, the LNP comprises the lipid KL52 (an aminolipid disclosed in U.S. Patent Application Publication No. 2012 / 0295832, the contents of which are incorporated herein by reference in their entirety). The activity and / or safety of LNP administration (as measured by examining one or more of, for example, ALT / AST, white blood cell count, and cytokine induction) can be enhanced by incorporation of such lipids. LNPs containing KL52 can be administered intravenously and / or in one or more doses. In some embodiments, administration of LNPs containing KL52 produces equal or enhanced mRNA and / or protein expression compared to administration of LNPs containing MC3.

[0426] In some embodiments, RNA (e.g., mRNA) vaccines may be delivered using smaller LNPs. These particles can include diameters from less than 0.1 μm up to 100 nm, such as, but not limited to, less than 0.1 μm, less than 1.0 μm, less than 5 μm, less than 10 μm, less than 15 μm, less than 20 μm, less than 25 μm, less than 30 μm, less than 35 μm, less than 40 μm, less than 50 μm, less than 55 μm, less than 60 μm, less than 65 μm, less than 70 μm, less than 75 μm, less than 80 μm, less than 85 μm, less than 90 μm, less than 95 μm, less than 100 μm, less than 125 μm, less than 150 μm, less than 175 μm, less than 200 μm, less than 225 μm, less than 250 μm, less than 275 μm, less than 300 μm, less than 325 μm, less than 350 μm, less than 375 μm, less than 400 μm, less than 425 μm, less than 450 μm. <1 um, <475 um, <500 um, <525 um, <550 um, <575 um, <600 um, <625 um, <650 um, <675 um, <700 um, <725 um, <750 um, <775 um, <800 um, <825 um, <850 um, <875 um, <900 um, <925 um, <950 um, <975 um or <1000 um.

[0427] In some embodiments, RNA (e.g., mRNA) vaccines may be delivered using smaller LNPs, which may include diameters of approximately 1 nm to 100 nm, approximately 1 nm to 10 nm, approximately 1 nm to 20 nm, approximately 1 nm to 30 nm, approximately 1 nm to 40 nm, approximately 1 nm to 50 nm, approximately 1 nm to 60 nm, approximately 1 nm to 70 nm, approximately 1 nm to 80 nm, approximately 1 nm to 90 nm, approximately 5 nm to 100 nm, approximately 5 nm to 10 nm, approximately 5 nm to 20 nm, approximately 5 nm to 30 nm, approximately 5 nm to 40 nm, approximately 5 nm to 50 nm, approximately 5 nm to 60 nm, approximately 5 nm to 70 nm, approximately 5 nm to 80 nm, approximately 5 nm to 90 nm, approximately 10 nm to 50 nm, approximately 20 nm to 50 nm, approximately 30 nm to 50 nm, approximately 40 nm to 50 nm, approximately 20 nm to 60 nm, approximately 30 nm to 60 nm, approximately 40 nm to 50 nm, approximately 20 nm to 60 nm, approximately 40 nm to 50 nm, approximately 30 nm to 60 nm, approximately 40 nm to 50 nm, approximately 40 nm to 60 nm, approximately 40 nm to 50 nm, approximately 20 nm to 60 nm, approximately 30 nm to 60 nm, approximately 40 nm to 5 ... nm to about 60 nm, about 20 nm to about 70 nm, about 30 nm to about 70 nm, about 40 nm to about 70 nm, about 50 nm to about 70 nm, about 60 nm to about 70 nm, about 20 nm to about 80 nm, about 30 nm to about 80 nm, about 40 nm to about 80 nm, about 50 nm to about 80 nm, about 60 nm to about 80 nm, about 20 nm to about 90 nm, about 30 nm to about 90 nm, about 40 nm to about 90 nm, about 50 nm to about 90 nm, about 60 nm to about 90 nm and / or about 70 nm to about 90 nm.

[0428] In some embodiments, such LNPs are synthesized using a method that includes a microfluidic mixer. Examples of microfluidic mixers may include, but are not limited to, slit-finger micromixers, including but not limited to mixers and / or staggered herringbone micromixers (SHM) fabricated by Microinnova (Allerheiligen bei Wildon, Austria) (Zhigaltsev, IV 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 Journal, 2012, 28:3633-40); Belliveau, NM 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 these references are incorporated herein by reference in their entirety). In some embodiments, the method for generating LNPs containing SHM further comprises mixing at least two input streams, wherein mixing occurs via microstructure-induced chaotic convection (MICA). According to this method, fluid flows through channels existing in a herringbone pattern, thereby causing rotational flow and causing the fluids to fold around each other.This method may also include a surface for fluid mixing, wherein the surface changes orientation during fluid circulation. Methods for generating LNPs using SHM include those disclosed in U.S. Application Publications No. 2004 / 0262223 and No. 2012 / 0276209, the contents of each of which are incorporated herein by reference in their entirety.

[0429] In some embodiments, the RNA (e.g., mRNA) vaccine disclosed herein may be formulated in lipid nanoparticles produced using a micromixer, such as, but not limited to, a slit-interdigital microstructure mixer (SIMM-V2) or a standard slit-interdigital micromixer (SSIMM) or a Caterpillar (CPMM) or impingement jet (IJMM) from the Institut für Mikrotechnik Mainz GmbH, Mainz Germany.

[0430] In some embodiments, the RNA (e.g., mRNA) vaccines disclosed herein can be formulated in lipid nanoparticles generated using microfluidic technology (see, for example, 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 these documents is incorporated herein by reference in its entirety). As a non-limiting example, controlled microfluidic formulations include passive methods for mixing stable pressure-driven flows in microchannels at low Reynolds numbers (see, for example, Abraham et al. Chaotic Mixer for Microchannels. Science, 2002 295: 647-651, the contents of which are incorporated herein by reference in their entirety).

[0431] In some embodiments, the RNA (e.g., mRNA) vaccine disclosed herein can be formulated in lipid nanoparticles generated using a micromixer chip, such as, but not limited to, micromixer chips from Harvard Apparatus (Holliston, Massachusetts) or Dolomite Microfluidics (Royston, UK). The micromixer chip can be used for the rapid mixing of two or more fluid flows with splitting and recombination mechanisms.

[0432] In some embodiments, the RNA (e.g., mRNA) vaccine of this disclosure may be formulated for delivery using drug-encapsulated microspheres as described in International Patent Publication No. WO2013063468 or U.S. Patent No. 8,440,614, the contents of each of which are incorporated herein by reference in their entirety. The microspheres may comprise compounds of formulas (I), (II), (III), (IV), (V), or (VI) as described in International Patent Publication No. WO2013063468, the contents of which are incorporated herein by reference in their entirety. In some embodiments, amino acids, peptides, polypeptides, and lipids (APPL) may be used to deliver the RNA (e.g., mRNA) vaccine of this disclosure into cells (see International Patent Publication No. WO2013063468, the contents of which are incorporated herein by reference in their entirety).

[0433] In any embodiment, lipid-based formulations comprising any LNP of this disclosure may further include one or more adjuvants. For example, in any embodiment, ionizable lipids present in the nanoparticle formulation may be used for adjuvant lipid substitution or binding to said adjuvant lipids to enhance RNA delivery. Examples of such methods are described in the prior art, such as in Han et al., (2023) Nature Nanotechnology, https: / / doi.org / 10.1038 / s41565-023-01404-4, and Salleh et al., (2022) Peer Journal, 10:e13083; these references are incorporated herein by reference.

[0434] In some embodiments, the antibody titer generated by the mRNA vaccine of the present invention is a neutralizing antibody titer. In some embodiments, the neutralizing antibody titer is higher than that of a protein vaccine. In other embodiments, the neutralizing antibody titer generated by the mRNA vaccine of the present invention is greater than that of a protein vaccine adjuvant. In other embodiments, the neutralizing antibody titer generated by the mRNA vaccine of the present 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, 2,000-10,000, 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. Neutralization titer is typically expressed as the highest serum dilution required to achieve a 50% reduction in the number of spots.

[0435] In some embodiments, this disclosure features a pharmaceutical composition comprising a nanoparticle composition according to the foregoing embodiments and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be as described herein and may also include one or more agents that facilitate storage of the composition at low temperatures. For example, the pharmaceutical composition may be refrigerated or frozen for storage and / or transport (e.g., stored at temperatures of 4°C or lower, such as about -150°C to about 0°C or about -80°C to 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 transport at temperatures such as about -20°C, -30°C, -40°C, -50°C, -60°C, -70°C, or -80°C. Therefore, it should be understood that the compositions described herein may further comprise one or more cryoprotectants or cryopreservatives. Optionally, the cryopreservative or cryoprotectant may comprise sugars, such as sucrose, glucose, or related glycosyl cryoprotectants.

[0436] Liposomes and lipid complexes as well as lipid nanoparticles

[0437] The RNA (e.g., mRNA) vaccines disclosed herein can be formulated using one or more liposomes, lipid complexes, or lipid nanoparticles. In some embodiments, the pharmaceutical composition of the RNA (e.g., mRNA) vaccine includes liposomes. Liposomes are artificially prepared vesicles that may consist primarily of a lipid bilayer and can be used as delivery media for administering nutrients and pharmaceutical formulations. Liposomes can have different sizes, such as, but not limited to, multilayer vesicles (MLVs) with diameters of hundreds of nanometers and containing a series of concentric bilayers separated by narrow aqueous compartments; small single-cell vesicles (SUVs) with diameters of less than 50 nm; and large single-layer vesicles (LUVs) with diameters of 50 nm to 500 nm. Liposome design may include, but is not limited to, opsonins or ligands, to improve liposome attachment to or activation of unhealthy tissues, such as, but not limited to, endocytosis. Liposomes may contain low or high pH to improve the delivery of pharmaceutical formulations.

[0438] The formation of liposomes can depend on physicochemical characteristics, such as, but not limited to, the drug formulation and liposome components captured, the nature of the medium in which the liposomes are dispersed, the effective concentration and potential toxicity of the captured substance, any additional processes involved in the application and / or delivery of the vesicles, optimized size, polydispersity and shelf life of the vesicles for the intended application, and batch-to-batch reproducibility and the possibility of large-scale production of safe and efficient liposome products.

[0439] In some embodiments, the pharmaceutical compositions described herein may include, but are not limited to, liposomes, such as 1,2-dioleoyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, Wash.), 1,2-dilinoleoyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA) and MC3 (US20100324120; the literature described herein is incorporated herein by reference in its entirety), and liposomes capable of delivering small molecule drugs, such as, but not limited to, liposomes formed from DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.).

[0440] In some embodiments, the pharmaceutical compositions described herein may include, but are not limited to, liposomes, such as liposomes formed from the synthesis of stable plasmid-lipid particles (SPLPs) or stable nucleic acid-lipid particles (SNALPs), which have been previously described and shown to be suitable for in vitro and in vivo oligonucleotide delivery (see Wheeler et al., Gene Therapy, 1999 6:271-281; ​​Zhang et al., Gene Therapy, 1999 6:1438-1447; Jeffs et al., Pharmaceutical Research, 2005 22:362-372; Morrissey et al., Nature Biotechnology, 2005 2:1002-1007; Zimmermann et al., Nature, 2006 441:111-114; Heyes et al., Controlled Release Journal, 2005 107:276-287; Semple et al., Nature Biotechnology, 2010). 28:172-176; Judge et al., *Journal of Clinical Research (J Clin Invest)*, 2009, 119:661-673; de Fougerolles, *Human Gene Therapy*, 2008, 19:125-132; US Patent Publication No. US20130122104 (the entire contents of the cited literature are incorporated herein by reference). Wheeler et al.'s initial preparation method was a detergent dialysis method, which was later improved by Jeffs et al. and referred to as the spontaneous vesicle formation method. In addition to polynucleotides, the liposome formulation consists of three to four lipid components. As an example, liposomes may contain, but are not limited to, 55% cholesterol, 20% distearate phosphatidylcholine (DSPC), 10% PEG-S-DSG, and 15% 1,2-dioleoxy-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 lipids, as described by Heyes et al., wherein the cationic lipids may be 1,2-distearateoxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or 1,2-dilinolenicooxy-3-dimethylaminopropane (DLenDMA).

[0441] In some embodiments, the liposome formulation may comprise about 25.0% to about 40.0% cholesterol, about 30.0% to about 45.0% cholesterol, about 35.0% to about 50.0% cholesterol, and / or about 48.5% to about 60% cholesterol. In some embodiments, the formulation 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, the formulation may comprise about 5.0% to about 10.0% DSPC and / or about 7.0% to about 15.0% DSPC.

[0442] In some embodiments, RNA (e.g., mRNA) vaccine pharmaceutical compositions may be formulated in liposomes such as, but not limited to, DiLa2 liposomes (Marina Biotechnology, Bosell, WA), SMARTICLES® (Marina Biotechnology, Bosell, WA), neutral DOPC (1,2-dioleoyl-sn-glycerol-3-phosphocholine)-based liposomes (e.g., siRNA delivery for ovarian cancer (Landen et al., Cancer Biology & Therapy, 2006, 5(12): 1708-1713; the literature is incorporated herein by reference in its entirety), and hyaluronic acid-coated liposomes (Quiet Therapeutics, Israel).

[0443] In some embodiments, the cationic lipid may be a low molecular weight cationic lipid, such as the low molecular weight cationic lipid described in U.S. Patent Application No. 20130090372, the contents of which are incorporated herein by reference in their entirety.

[0444] In some embodiments, RNA (e.g., mRNA) vaccines may be formulated in lipid vesicles that may have cross-links between functionalized lipid bilayers.

[0445] In some embodiments, RNA (e.g., mRNA) vaccines may be formulated in a lipid-polycationic complex. The formation of the lipid-polycationic complex can be accomplished by methods known in the art and / or those described in U.S. Publication No. 20120178702, which is incorporated herein by reference in its entirety. As a non-limiting example, the polycations may include cationic peptides or polypeptides, such as, but not limited to, polylysine, polyornithine, and / or polyarginine. In some embodiments, RNA (e.g., mRNA) vaccines may be formulated in a lipid-polycationic complex, which may further include non-cationic lipids, such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

[0446] 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; all of which are incorporated herein by reference in their entirety.

[0447] Subjects and Administration

[0448] The present invention also provides the use of the polynucleotides and compositions of the invention for generating an antigen-specific immune response in subjects. Such methods typically involve administering the polynucleotides of the invention (preferably formulated in lipid nanoparticles as described herein) to subjects in need.

[0449] Therefore, the present invention further provides compositions comprising polynucleotides (RNA) as defined herein, and the use of such RNA in immunogenic or vaccine compositions for the treatment or prevention of Porphyromonas gingivalis infection.

[0450] As used herein, the term "vaccine composition" is defined as a composition intended to induce an immune response against an antigen (immunogen) encoded by RNA within the composition in order to protect or treat an organism from disease.

[0451] As used herein, the terms “immunostimulatory composition,” “vaccine composition,” and “immunogenic composition” are generally used interchangeably.

[0452] The present invention provides methods and compositions for treating or preventing Porphyromonas gingivalis infection in individuals in need, or for minimizing the likelihood of Porphyromonas gingivalis infection, the methods comprising administering the vaccine composition of the present invention.

[0453] Therefore, the present invention includes methods and compositions for preventing Porphyromonas gingivalis infection in subjects, minimizing the likelihood of infection, and / or reducing the severity and duration of Porphyromonas gingivalis infection.

[0454] The present invention also provides a method for obtaining antibodies targeting *Porphyromonas gingivalis*, the method comprising administering the chimeric or fusion protein, composition, vaccine, or immunostimulatory composition of the present invention to a non-human animal, thereby generating antibodies targeting *Porphyromonas gingivalis* in the animal. Preferably, the method further comprises isolating the antibodies from the animal (e.g., from the animal's blood) or from the animal's eggs (e.g., in the case of generating IgY antibodies from chickens).

[0455] The present invention also provides an antibody preparation comprising an antibody targeting *Porphyromonas gingivalis*, wherein the antibody preparation is obtained by administering the composition of the present invention, a vaccine, or an immunostimulatory composition to a non-human animal, thereby generating an antibody targeting *Porphyromonas gingivalis* in the animal, and isolating the antibody from the animal or its eggs.

[0456] Antibodies targeting *Porphyromonas gingivalis* can be used therapeutically to eliminate or reduce *Porphyromonas gingivalis* infection, or prophylactically to prevent or reduce the severity of *Porphyromonas gingivalis* infection.

[0457] As used herein, the term "treatment" or "treating" refers to administering or applying the compositions of the present invention (or applying or applying compounds of the present invention to cells or tissues derived from the subject) to a subject with the aim of delaying, slowing, stabilizing, curing, inhibiting, healing, alleviating, relieving, altering, remedying, reducing the worsening of, improving, enhancing, or influencing the disease or condition, symptoms of the disease or condition, or the risk (or susceptibility) to the disease or condition. The term "treatment" means any indicator of successful treatment or improvement of an injury, pathology, or condition, including any objective or subjective parameter such as reduction; relief; reduction of the rate of worsening; reduction of the severity of the disease; stabilization, reduction of symptoms, or making the injury, pathology, or condition more tolerable for the subject; slowing the rate of worsening or decline; making the late stages of worsening less debilitating; or improving the physical or mental health of the subject.

[0458] As used herein, “preventing” or “prevention” means at least a reduction in the likelihood of acquiring a disease or condition (i.e., causing a subject who may be exposed to or susceptible to a disease but has not yet experienced or displayed symptoms of the disease to not develop at least one of the clinical symptoms of the disease). This article provides biological and physiological parameters for identifying such subjects, and these parameters are also well known to physicians.

[0459] The vaccine compositions of the present invention can be administered to subjects who need them most, such as in human patients, children or the elderly, or individuals at risk of exposure to *Porphyromonas gingivalis*. The vaccine compositions of the present invention can also be administered to subjects suspected of having or diagnosed with *Porphyromonas gingivalis* infection.

[0460] The compositions and methods of the present invention are equally extended to use in both human medicine and / or veterinary medicine, in the production of diagnostic agents or other therapeutic agents.

[0461] As used herein, the term "subject" should be understood to mean any animal, including humans, such as mammals. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, a subject can be a human. In other instances, a subject can be a veterinary subject, such as a companion animal (cat, dog, guinea pig, etc.).

[0462] As used herein, the terms “subject,” “individual,” and “patient” are used interchangeably.

[0463] Those skilled in the art will be familiar with methods for determining successful vaccination / immunization with chimeric or fusion proteins or compositions as described herein. For example, those skilled in the art will be familiar with methods for quantifying antibodies generated after immunization and / or for quantifying the extent of humoral (Th2) responses induced after immunization or for quantifying the extent of Th1 responses generated.

[0464] In some embodiments, after administration of the polynucleotide or composition of the present invention, subjects exhibit a seroconversion rate of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) following a first or second (booster) dose of the vaccine. Seroconversion refers to the period during which specific antibodies are produced and are detectable in the blood. During infection or immunization, an antigen enters the bloodstream, and the immune system begins to produce antibodies in response. Prior to seroconversion, the antigen itself may or may not be detectable, but antibodies are considered to be absent. During seroconversion, antibodies are present but not yet detected. At any time after seroconversion, antibodies can be detected in the blood, indicating a previous or current infection.

[0465] In some embodiments, a polynucleotide (e.g., mRNA) vaccine is administered to a subject via intradermal or intramuscular injection, subcutaneous, intravenous, or intranasal route, or any other suitable route for delivering an RNA-based vaccine.

[0466] In some embodiments of this disclosure, a method for inducing an antigen-specific immune response in a subject is provided, the method comprising administering to the subject an effective amount of an RNA (e.g., mRNA) vaccine, as described herein, to generate an antigen-specific immune response in the subject. In some embodiments, the antigen-specific immune response in the subject can be determined by measuring antibody titers after administration of any polynucleotide (e.g., mRNA) vaccine of this disclosure to the subject. In some embodiments, the titer of anti-antigenic peptide antibodies generated in the subject is increased by at least one log relative to a control. In some embodiments, the titer of anti-antigenic peptide antibodies generated in the subject is increased by 1-3 logs relative to a control.

[0467] In some embodiments, the titer of anti-antigenic peptide antibodies produced in the subject is increased at least 2-fold compared to the control. In some embodiments, the titer of anti-antigenic peptide antibodies produced in the subject is increased at least 5-fold compared to the control. In some embodiments, the titer of anti-antigenic peptide antibodies produced in the subject is increased at least 10-fold compared to the control. In some embodiments, the titer of anti-antigenic peptide antibodies produced in the subject is increased 2-10-fold compared to the control.

[0468] In some embodiments, the control is the titer of anti-antigenic peptide antibodies generated in subjects who have not been administered an RNA (e.g., mRNA) vaccine of this disclosure. In some embodiments, the control is the titer of anti-antigenic peptide antibodies generated in subjects who have been administered a live, attenuated, or inactivated *Porphyromonas gingivalis* vaccine (see, for example, Ren J. et al., *Journal of General Virology* 2015; 96: 1515-1520), or wherein the control is the titer of anti-antigenic peptide antibodies generated in subjects who have been administered a recombinant or purified *Porphyromonas gingivalis* protein vaccine.

[0469] The polynucleotide (e.g., mRNA) vaccine of this disclosure is administered to subjects at an effective amount (an amount that effectively induces an immune response). In some embodiments, the effective amount is a dose equivalent to a standard therapeutic dose of a recombinant *Porphyromonas gingivalis* protein vaccine reduced by at least 2, at least 4, at least 10, at least 100, or at least 1000 times, wherein the titer of antiantigenic polypeptide antibodies produced in the subject is equivalent to the titer of antiantigenic polypeptide antibodies produced in a control subject who has received a standard therapeutic dose of a recombinant *Porphyromonas gingivalis* protein vaccine, a purified *Porphyromonas gingivalis* protein vaccine, a live attenuated *Porphyromonas gingivalis* vaccine, or an inactivated *Porphyromonas gingivalis* vaccine. In some embodiments, the effective dose is a dose equivalent to a standard therapeutic dose of recombinant Porphyromonas gingivalis protein vaccine reduced by 2 to 1000 times, wherein the titer of antiantigenic polypeptide antibodies produced in the subject is equivalent to the titer of antiantigenic polypeptide antibodies produced in a control subject who received a standard therapeutic dose of recombinant Porphyromonas gingivalis protein vaccine, purified Porphyromonas gingivalis protein vaccine, live attenuated Porphyromonas gingivalis vaccine, or inactivated Porphyromonas gingivalis vaccine.

[0470] In some embodiments, a polynucleotide (e.g., mRNA) vaccine is formulated in an effective amount to generate an antigen-specific immune response in a subject.

[0471] The dosage of the vaccine composition of the present invention can vary within a range, depending on the age and condition of the individual to be treated, and the physician will ultimately determine the appropriate dosage to be used.

[0472] This dose can be repeated at an appropriate frequency. For example, an initial dose of vaccine can be administered, followed by a booster dose at a later date.

[0473] In some embodiments, the effective amount is a total dose of 25 μg to 1000 μg or 50 μg to 1000 μg. In some embodiments, the effective amount is a total dose of 100 μg. In some embodiments, the effective amount is a dose of 25 μg administered to the subject twice. In some embodiments, the effective amount is a dose of 100 μg administered to the subject twice. In some embodiments, the effective amount is a dose of 400 μg administered to the subject twice. In some embodiments, the effective amount is a dose of 500 μg administered to the subject twice.

[0474] In some embodiments, the efficacy (or effectiveness) of the polynucleotide (e.g., mRNA) vaccine is greater than 60%. In some embodiments, the efficacy (or effectiveness) of the 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%.

[0475] Vaccine efficacy can be assessed using standard analyses (see, for example, Weinberg et al., J Infect Dis. 2010 June 1; 201(11):1607-10). Vaccine efficacy can be measured, for example, through double-blind, randomized, clinically controlled trials. Vaccine efficacy can be expressed as a reduction in the proportion of incidence (AR) between unvaccinated (ARU) and vaccinated (ARV) study cohorts, and can be calculated using the following formula based on the relative risk (RR) of disease in the vaccinated group:

[0476] Therapeutic effect = (ARU-ARV) / ARU × 100; and

[0477] Therapeutic effect = (1-RR) × 100.

[0478] Similarly, standard analyses can be used to assess vaccine efficacy (see, for example, Weinberg et al., Journal of Infectious Diseases, June 1, 2010; 201(11):1607-10). Vaccine efficacy is an assessment of how a vaccine (which may have demonstrated high efficacy) reduces disease in a population. This metric assesses 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 efficacy is directly proportional to vaccine efficacy (potency) but is also influenced by the immunization status of the target group in the population and other vaccine-independent factors affecting “real-world” outcomes such as hospitalization, outpatient visits, or costs. For example, a retrospective case-control analysis can be used, where vaccination rates are compared in a cohort of infected cases and in appropriate controls. Vaccine efficacy can be expressed as a ratio difference, i.e., the odds ratio (OR) of infection occurring despite vaccination:

[0479] Validity = (1-OR) × 100.

[0480] Vaccine effectiveness can also be assessed by evidence of treatment for *Porphyromonas gingivalis* infection, as defined herein. Evidence of treatment may include reducing the severity or duration of *Porphyromonas gingivalis* infection in subjects, such as reducing inflammation caused by the infection.

[0481] In other embodiments, the present invention is a composition or method for vaccinating a subject, the method comprising administering a nucleic acid vaccine to the subject, the nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, wherein the subject is administered a dose of the nucleic acid vaccine from 10 μg / kg to 400 μg / kg. In some embodiments, the dosage of RNA polynucleotide is 1-5 μg, 5-10 μg, 10-15 μg, 15-20 μg, 10-25 μg, 20-25 μg, 20-50 μg, 30-50 μg, 40-50 μg, 40-60 μg, 60-80 μg, 60-100 μg, 50-100 μg, 80-120 μg, 40-120 μg, 40-150 μg, 50-150 μg, 50-200 μg, 80-200 μg, 100-200 μg, 120-250 μg, 150-250 μg, 180-280 μg, 200-300 μg, 50-300 μg, 80-300 μg, 100-300 μg per dose. The doses are 40-300 μg, 50-350 μg, 100-350 μg, 200-350 μg, 300-350 μg, 320-400 μg, 40-380 μg, 40-100 μg, 100-400 μg, 200-400 μg, or 300-400 μg. In some embodiments, the nucleic acid vaccine is administered to the subject via 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.

[0482] In some embodiments, the nucleic acid vaccine administered to the subject comprises at least about 2 micrograms (μg), or at least about 10 μg, or at least about 20 μg, or at least about 30 μg of RNA polynucleotide. In some embodiments, the nucleic acid vaccine administered to the subject comprises 100 micrograms (μg) of RNA polynucleotide. In some embodiments, the nucleic acid vaccine administered to the subject comprises 50 micrograms (μg) of RNA polynucleotide. In some embodiments, the nucleic acid vaccine administered to the subject comprises 75 micrograms (μg) of RNA polynucleotide. In some embodiments, the nucleic acid vaccine administered to the subject comprises 150 micrograms (μg) of RNA polynucleotide. In some embodiments, the nucleic acid vaccine administered to the subject comprises 400 micrograms of RNA polynucleotide. In some embodiments, the nucleic acid vaccine administered to the subject comprises 200 micrograms (μg) of RNA polynucleotide. In some embodiments, the accumulation level of RNA polynucleotide in local lymph nodes is 100 times the accumulation level in distal lymph nodes.

[0483] Embodiments of the present invention provide a method for creating, maintaining, or restoring antigenic memory against *Porphyromonas gingivalis* in an individual or a population of individuals, the method comprising administering an antigenic memory booster nucleic acid vaccine to the individual or population, the antigenic memory booster nucleic acid vaccine comprising: (a) at least one RNA polynucleotide, the polynucleotide comprising at least one chemical modification or optionally not comprising a nucleotide modification and two or more codon-optimized 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, intradermal, and subcutaneous administration. In some embodiments, the administration step comprises contacting the subject's muscle tissue with a device suitable for injecting the composition. In some embodiments, the administration step comprises contacting the subject's muscle tissue with a device suitable for injecting the composition, combined with electroporation.

[0484] Embodiments of the present invention provide a method for vaccinating a subject, the method comprising administering a single dose of a nucleic acid vaccine to the subject at a dose of 25 μg / kg to 400 μg / kg, the nucleic acid vaccine comprising one or more RNA polynucleotides as described herein for vaccinating the subject.

[0485] In other embodiments, the present invention covers a method for treating elderly subjects aged 60 years or older, the method comprising administering a nucleic acid vaccine to the subject, the 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 for vaccinating the subject.

[0486] In other embodiments, the present invention covers a method of treating a young subject aged 17 years or younger, the method comprising administering a nucleic acid vaccine to the subject, the 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 for vaccinating the subject.

[0487] In other embodiments, the present invention covers a method of treating an adult subject, the method comprising administering a nucleic acid vaccine to the subject, the 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 for vaccinating the subject.

[0488] In a preferred embodiment, the vaccine of the present invention (e.g., an LNP-encapsulated mRNA vaccine) produces a preventive and / or therapeutic level, concentration, and / or titer of antigen-specific antibodies in the blood or serum of the vaccinated subject.

[0489] As defined herein, the term antibody titer refers to the amount of antigen-specific antibodies produced in the body of a subject (e.g., a human subject). In an exemplary embodiment, antibody titer is expressed as the reciprocal of the maximum dilution (in a series of dilutions) that still produces a positive result.

[0490] In exemplary embodiments, antibody titers are determined or measured by enzyme-linked immunosorbent assay (ELISA). In exemplary embodiments, antibody titers are determined or measured by neutralization assays (e.g., by micro-neutralization assays). In some embodiments, antibody titer measurements are expressed as ratios, such as 1:40, 1:100, etc. In exemplary embodiments of the invention, the antibody titers produced by an effective vaccine are greater than 1:40, greater than 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, and greater than 1:10000. In exemplary embodiments, antibody titers are generated or reached 10 days, 20 days, 30 days, 40 days, or 50 days or longer after vaccination. In exemplary embodiments, antibody titers are generated or reached after a single dose of the vaccine is administered to the subject. In other embodiments, the titer is generated or reached after multiple doses, for example, after a first dose and a second dose (e.g., a booster dose). In exemplary embodiments of the invention, antigen-specific antibodies are measured in μg / ml, or in IU / L (International Units per Liter) or mIU / ml (milli-International Units per ml). In exemplary embodiments of the invention, an effective vaccine produces >0.5 μg / ml, >0.1 μg / ml, >0.2 μg / ml, >0.35 μg / ml, >0.5 μg / ml, >1 μg / ml, >2 μg / ml, >5 μg / ml, or >10 μg / ml. In exemplary embodiments of the invention, an effective 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, antibody levels or concentrations are produced or reached 10 days, 20 days, 30 days, 40 days, or 50 days or longer after vaccination. In exemplary embodiments, levels or concentrations are produced or reached after administration of a single dose of the vaccine to the subject. In other embodiments, levels or concentrations are produced or reached after multiple doses, for example, after a first dose and a second dose (e.g., a booster dose). In exemplary embodiments, antibody levels or concentrations are determined or measured by enzyme-linked immunosorbent assay (ELISA).

[0491] In an exemplary embodiment, antibody levels or concentrations are determined or measured by a neutralization assay (e.g., by a micro-neutralization assay).

[0492] Example

[0493] Example 1: Materials and Methods

[0494] mRNA-LNP production

[0495] Following the manufacturer's instructions, mRNA was transcribed in vitro using the T7 In Vitro Transcription Kit (NEB Corporation (NEB)) with a linearized DNA template encoding 5' and 3' UTRs, a signal peptide, a candidate sequence, and a 125-nucleotide poly(A) tail. Capping was then performed using Clean Cap mRNA Capping Technology (TriLink Biotechnology). Where explicitly stated, during mRNA production, the UTP was replaced by N1-methylpseuuridine (N1-methylpseuuridine, m1Ψ). DNA was removed using DNase I (NEB), and double-stranded RNA (dsRNA) was removed using cellulose binding, as previously described [Baiersdörfer et al. (2019) Molecular Ther Nucleic Acids. April 15; 15:26-35], and was formulated in lipid nanoparticles (LNPs) having the following lipid composition: ALC-0315, cholesterol, DSPC, and ALC-0159 in a molar lipid ratio (%) of 46.3:42.7:9.4:1.6, in Tris / sucrose buffer (25 mM Tris, 8.8% sucrose w / v, pH 7.4).

[0496] Expression of mRNA vaccine candidates in cell culture in vitro

[0497] HeLa cells were cultured in DMEM medium (Thermo Fisher Scientific, catalog number 10569010) containing 10% FBS, high glucose, GlutaMAX™ supplement, and pyruvate at 37°C and 5% CO2 according to standard protocol. The mRNA candidate (before LNP preparation) was prepared using Lipofectamine MessengerMAX (Thermo Fisher Scientific) and used for cell transfection according to the manufacturer's instructions. 1.8 μg of mRNA was used to transfect 35,000 cells / well in a six-well plate. To collect the supernatant, cells were pelleted by centrifugation at 14,000 g for 15 minutes. The supernatant was transferred to new tubes and frozen at -20°C until Western blotting was performed. To collect whole-cell lysis, cells in each well were washed with approximately 2 mL of DPBS. Remove the liquid and add 250 μL of RIPA lysis buffer containing a protease inhibitor (Thermo Fisher Scientific) to each well, and gently vortex to mix for 10 seconds. Transfer the lysis buffer to a test tube using a cell scraper and centrifuge at 16,000 g for 15 minutes at 4 °C to precipitate the fragments. Collect the clarified lysis buffer into a new test tube and store at -20 °C until Western blotting is performed.

[0498] Protein blot

[0499] The sample was mixed with 4X Laemmli sample buffer (10% v / v) supplemented with 2-mercaptoethanol and 100 mM DTT, centrifuged at 13,000 g for 30 seconds, and incubated at room temperature for 20 minutes, followed by denaturation at 95 °C for 10 minutes. The sample was loaded into Any kD™ Mini-PROTEAN® TGX™ pre-prepared protein gel (BioRad, catalog 4569036) and the proteins were separated at 150 V by SDS-PAGE (BioRad, catalog 1610732) with Tris / glycine / SDS electrophoresis buffer for 45 minutes. Precision Plus Protein™ WesternC™ blot standards were used as protein biomarkers (BioRad, catalog 161-0376). Proteins were transferred to a PDV membrane (Life Technologies, catalog LC2002) pre-activated in methanol for 30 seconds and washed in transfer buffer (10x Tris / glycine buffer for protein blotting and native gels) (Bio-Bio, catalog 1610734) using a Trans-Blot® Turbo™ transfer system (Bio-Bio) according to standard protocol: 25 V and 1.0 A for 30 minutes.

[0500] Proteins were transferred onto a PVDF membrane and blocked on a shaker in blocking buffer (5% skim milk in PBS-T) for 2 hours. The membrane was then probed overnight at 4°C on a shaker with a self-made primary antibody bound to either KAS2 (1:2500 in blocking buffer) or KDAK-3S-AVQP (1:3500 in blocking buffer). The membrane was washed five times with PBS-T and incubated on a shaker at room temperature for one hour with goat anti-mouse HRP (1:2500 in blocking buffer, supplemented with a 1:10,000 solution of PrecisionProtein StrepTactin-HRP conjugate, Bio-Rad Laboratories, catalog 1610381). The membrane was washed five times with PBS-T, developed using Clarity™ Western ECL substrate (Bio-Rad Laboratories, catalog 170-5060), and imaged using a ChemiDoc™ Touch imaging system with chemiluminescence detection.

[0501] Bacterial culture in a mouse model of periodontitis

[0502] *Porphyromonas gingivalis* strain; W50 (serotype C); obtained from the Oral Health Cooperative Research Centre, The Melbourne Dental School, University of Melbourne, Australia. *Porphyromonas gingivalis* W50 was grown on horse blood agar (HBA) supplemented with 10% v / v lysed horse blood (37°C) in an anaerobic N2 atmosphere containing 5% CO2 at the MK3 anaerobic workstation (Don Whitley Scientific Ltd., Adelaide, Australia) (20 g / L HBA; Oxoid Ltd., Hampshire, UK). Colonies were inoculated into a starting culture consisting of 20 mL of sterile brain heart extract (37 g / L BHI; Oxoid, Hampshire, UK) supplemented with 5 mg / L heme and 0.5 mg / L cysteine, and incubated anaerobically (24 h, 37 °C). The absorbance of batches of cultures at OD 650 nm was monitored using a spectrophotometer (Model 295E, Perkin-Elmer, Germany). Bacterial cells were harvested during the late exponential growth phase by centrifugation (7,000 g, 20 min, 4 °C). Bacterial purity is usually confirmed by Gram staining [Slots (1982). In: Host-Parasite Interaction in Periodontal Disease, Genco, RJ and Merganhagan, SE (eds.). Washington DC: American Society for Microbiology. pp. 27-45].

[0503] Preparation of heat-inactivated bacteria

[0504] Cultures of *Porphyromonas gingivalis* W50 were collected (6,500 g, 4°C) and washed once with phosphate-buffered saline (PBS) (pH 7.4: 0.01 M Na₂HPO₄, 1.5 mM KH₂PO₄, and 0.15 M NaCl), followed by centrifugation (7,000 g, 20 min, 4°C) to precipitate the cells. The bacterial cells were resuspended in PBS and heated to 65°C for 15 min. The suspension was centrifuged (7,000 g, 20 min, 4°C) and resuspended in sterile PBS, and this was repeated once. After the second wash, the supernatant was discarded, and the cell pellet was resuspended in sterile PBS to obtain 2 × 10⁶ cells / mL. 10 Cell density was determined by cells / mL, and protein concentration was determined using Biorad protein assay dye reagent concentrate (Life Science, NSW, Australia).

[0505] Mouse periodontitis model

[0506] A periodontitis experiment was conducted in mice as previously described by O'Brien-Simpson et al. (JImmunol 175: 3980-3989, 2005). From day 0, mice (female BALB / c; 6–8 weeks old, 10 mice / group) were intraorally inoculated with *Porphyromonas gingivalis*, which consisted of four doses of *Porphyromonas gingivalis* W50 [suspended in 20 μL of PG buffer (pH 7.4, 50 mM Tris-HCl, 150 mM NaCl, 10 mM MgSO4, and 14.3 mM β-mercaptoethanol) containing 2% w / v carboxymethyl cellulose (CMC, Sigma, New South Wales, Australia)], administered two days apart. Inoculum was prepared anaerobically and then immediately applied to the gingival margin of the maxillary molars. The number of viable bacteria in each inoculum was verified by flow cytometry and CFU counting on blood agar. The animal groups consisted of: an oral inoculation group with *Porphyromonas gingivalis* W50 (infection control), a non-bacterial inoculation control group, and an immunization group. For therapeutic vaccination of periodontitis models ( Figure 6Mice were immunized on day 19 after their first oral administration of either a 200 μg protein vaccine control in saline / alum (Alhydrogel; 10 mg / mL w / v aluminum hydroxide wet gel suspension; Invivogen) or mRNA formulated in LNP in Tris / sucrose buffer (25 mM Tris, 8.7% sucrose). On the same day as the recombinant protein vaccine, mice receiving the mRNA vaccine received an intramuscular injection of 30 μg or 3 μg of formulated mRNA, for a total volume of 50 μL. The injection was administered intramuscularly in the semitendinosus or semimembranosus muscle of the right leg using a 27G needle. Mice received a second immunization on day 40 via subcutaneous (for the alum-adjuvanted protein vaccine) or intramuscular (for the mRNA vaccine candidate, the semitendinosus or semimembranosus muscle of the left leg). On day 62, mice were euthanized by exsanguination via cardiac puncture. The maxilla was removed and bisected along the midline, with 10 halves used to determine alveolar bone loss. Serum was used to determine antibody profiles using ELISA.

[0507] Measurement results of alveolar bone loss in the maxilla of mice

[0508] The maxillae with bone loss to be examined were boiled in deionized water (1 minute), mechanically demineralized, and immersed in 2% w / v potassium hydroxide (16 hours, 25°C). The maxillae were washed twice with deionized water (25°C), dried (1 hour, 37°C), and stained with 0.5% w / v methylene blue aqueous solution. Digital images of the buccal side of the maxillae were taken using Olympus DP12 digital cameras mounted on a dissecting microscope to assess horizontal bone loss, using OLYSIA BioReport software version 3.2 (Olympus Australia Pty Ltd, New South Wales, Australia). The maxillae were oriented so that the buccal and lingual molar cusps overlapped. Images were taken in micrometers per frame to allow measurements to be standardized for each image. Horizontal bone loss was defined as loss occurring in a horizontal plane perpendicular to the alveolar ridge, resulting in a decrease in ridge height. The visible area from the cementum-enamel junction (CEJ) to the alveolar ridge (ABC) of each molar was measured using OLYSIA BioReport software version 3.2 imaging software, and the results were obtained in mm. 2Total visible CEJ-ABC area in mm². *Porphyromonas gingivalis*-induced alveolar bone loss in mm² was calculated by subtracting the total visible CEJ-ABC area of ​​the uninoculated (NC) group from the total visible CEJ-ABC area of ​​each experimental group. Alveolar bone loss measurements were determined twice in a randomized and blinded protocol. Data are expressed as mean + / - standard deviation in mm² and analyzed using one-way ANOVA or the Kruskal-Wallis test.

[0509] Use ELISA to determine subclass antibodies in serum.

[0510] ELISA was performed to evaluate subclass antibodies in serum, as described by Pathirana et al. (2007). Infect Immun 75: 1436-1442, using heat-inactivated W50 cells (10 μg / mL) and domain subunits or epitopes (1 μg / mL) in 0.1 M PBS (pH 7.4) to coat wells of flat-bottomed polyethylene microplates (Microtiter; Dynatech Laboratories, McLean, VA, US) for 16 hours at 4°C. In these experiments, the following antibody dilutions were used: a 1 / 4000 dilution of goat anti-mouse antibody; and IgG (M8642), IgG1 (M8770), and IgG2a (M4434) antibodies (Sigma, New South Wales, Australia). A 1 / 4000 dilution of horseradish peroxidase-conjugated porcine anti-goat IgG antibody (M5420; Sigma, NSW, Australia) was used to develop ELISA assays. For epitope ELISA, biotinylated peptides were conjugated to pre-blocked streptavidin-coated flat plates (Pierce; Thermo Fisher Scientific) at 10 μg / mL. After incubation with serum, ELISAs were developed using 1 / 4000 goat anti-mouse IgG and 1 / 4000 horseradish peroxidase-conjugated porcine anti-goat IgG antibodies. In all ELISA assays, three washes with 220 μL of PBS-Tween 20 (0.1% v / v) were performed between steps. All densitometric measurements were performed at 405 nm on a Wallac VICTOR3 1420 multi-tag counter (PerkinElmer) and data were analyzed using one-way ANOVA or Kruskal-Wallis test.

[0511] Preventive vaccination model

[0512] Mice (female C57BL6) were orally inoculated with *Porphyromonas gingivalis* [suspended in 20 μL PG buffer containing 2% (w / v) carboxymethyl cellulose [pH 7.4, 50 mM Tris-HCl, 150 mM NaCl, 10 mM MgSO4, and 14.3 mM mercaptoethanol] at a dose of 1 x 10 10 [Viable *Porphyromonas gingivalis* W50 cells]. Inoculation began on day 42 and was repeated three times a week for three weeks. The inoculum was prepared anaerobically and then immediately applied to the gingival margin of the maxillary molars. The number of viable bacteria in each inoculum was verified by CFU counting on blood agar.

[0513] The animal groups consisted of: an oral inoculation group with *Porphyromonas gingivalis* W50 (infection control), a non-bacterial inoculation control (primary), and an inoculation group. On day 84, mice were euthanized and bled by cardiac puncture. The maxilla was removed and bisected along the midline, with 10 halves used to determine alveolar bone loss. Serum was used to determine antibody profiles using ELISA. Spleens were removed from the mice and stored in pre-prepared DuPont modified Eagle medium (DMEM) supplemented with 10% (v / v) heat-inactivated FBS (56°C, 30 min), 2 mM L-glutamine, 2 mM sodium pyruvate, and 100 IU / mL penicillin / streptomycin before tissue lysis for ELISPOT assay.

[0514] Spleen cell collection and ELISPOT assay

[0515] Female mice (C57BL6) were orally inoculated with *Porphyromonas gingivalis* [suspended in 20 μL PG buffer [50 mM Tris-HCl, 150 mM NaCl, 10 mM MgSO4] at a dose of 1 x 10 10 [Live *Porphyromonas gingivalis* W50 cells]. Single-cell suspensions of spleen cells were prepared from the spleen using the GentleMACS system according to the manufacturer's instructions (Miltenyi Biotec) and then treated with erythrocyte lysis buffer (Sigma-Aldrich Pty. Ltd., NSW, Australia) for 5 min at room temperature, followed by washing twice in DuPont PBS and centrifugation at 800 g for 5 min. The cell suspension was then filtered through a 0.45 μM filter and used for analysis. ELISPOT assays were performed using the BD® ELISPOT kit for IL-4 and IFNγ according to the manufacturer's instructions. 3 x 10 5Cells were added to each well of an ELSIPOT plate, either with or without KDAK-3S-AVQP protein (10 μg / mL). Concanavalin A was used as a positive control mitogen (10 μg / mL).

[0516] The plates were incubated in a humidified incubator at 37°C in air under a 5% CO2 atmosphere for 48 hours. Then, before terminating the reaction by washing with water, the plates were washed and developed according to the manufacturer's instructions, allowing the spots to develop for 20–30 minutes. Spots were counted using an EliSpot Lite plate reader (Version 2.9, Autoimmun Diagnostika GmbH, Ebinger Strasse 4, Strassberg, Germany). Data are expressed as cells per million spots (SFC / million) and statistically analyzed using one-way ANOVA and Dunningt's 3T test (Graphpad Prism).

[0517] Example 2: Expression of mRNA constructs encoding chimeric proteins

[0518] The inventors have obtained an mRNA construct encoding a chimeric protein containing a putative antigenic domain derived from the Kgp gingival protease protein. The mRNA construct comprises, as follows: Figure 2 The general architecture shown, and the chimeric protein (antigen) encoded by the mRNA expression construct, contains, as follows: Figure 1 The structures named KDcAK1n, KDAK, and KDAK-3S-AVQP are shown (amino acid sequences as shown in SEQ ID NO: 2, 4, and 6, respectively, and mRNA sequences as shown in SEQ ID NO: 40, 41, and 42, respectively).

[0519] In vitro antigen expression and secretion of mRNA in 293T (not shown) or HeLa cells were tested using a variety of secreted peptides, including tPA-derived signal peptides and SEAP signal peptides. Cells were transfected with mRNA constructs using a lipid transfection agent, as outlined in Example 1. After 48 hours, the supernatant was collected and Western blot analysis was performed to test the secreted peptides.

[0520] Figure 3 Western blots were displayed, showing expression and secretion in HeLa cells using mRNA constructs encoding KDcAK1n, KDAK-3S-AVQP, and KDAK. Various signal peptides were tested. Results of peptide secretion using SEAP are shown in the figure.

[0521] The results showed that the construct encoding KDcAK1n was poorly expressed, while the constructs encoding KDAK and KDAK-3S-AVQP expressed large amounts of protein, which was secreted into the supernatant of HeLa cells.

[0522] Example 3: Determining the immunogenicity of candidate mRNA vaccines

[0523] Figure 4 A schematic diagram of a vaccination regimen used to evaluate the immunogenicity of a candidate vaccine is shown.

[0524] In summary, the mRNA constructs encoding the chimeric KDcAK1n, KDAK, and KDAK-3S-AVQP described above were formulated into lipid nanoparticles (LNPs) using standard techniques. The LNPs used in these experiments contained ALC-0315, cholesterol, distearate phosphatidylcholine, and ALC-0159 in a molar percentage (%) ratio of 46.3: 42.7: 9.4: 1.6.

[0525] For each antigen, an mRNA construct is generated using either a natural RNA sequence (unmodified) or a sequence modified with N1-methyl-pseudouridine (M1Ψ).

[0526] Two doses of the mRNA vaccine were tested: 30 μg or 3 μg of mRNA formulated in LNP. The positive control used in the experiment was protein KDAK-3S-AVQP with alum as an adjuvant.

[0527] according to Figure 4 The timeline shown indicates that mice were immunized intramuscularly with mRNA-LNP.

[0528] As outlined in Example 1, serum was collected on day 35 post-immunization and tested using ELISA to determine serum antibody subclass responses in immunized mice. Antiserum was used to detect the following adsorbed antigens: KDAK-3S-AVQP (purified recombinant protein), heat-inactivated *Porphyromonas gingivalis* strain W50 (HKPg), and Kgp. cat (The catalytic domain of Kgp gingival protease) and the biotinylated linear peptide corresponding to the sequence encoded by the tested RNA vaccine candidate. The sequence of the biotinylated KAS2 peptide is shown below:

[0529]

[0530] Antibody response is expressed as the obtained ELISA titer minus three times the background level, where each titer represents the mean ± standard deviation of 5 individual mice. Figure 5Both m1Ψ-KDAK and m1Ψ-KDAK-3S-AVQP induced strong responses to KDAK-3S-AVQP at both 30 μg and 3 μg doses, resulting in strong total IgG, IgG1, and IgG2 titers. Natural RNA KDAK and KDAK-3S-AVQP produced strong responses at 30 μg, but weaker responses (especially KDAK-3S-AVQP) at 3 μg. KDcAK1n and m1Ψ-negative RNA controls did not produce significant responses.

[0531] At 30 μg, m1Ψ-KDAK, m1Ψ-KDAK-3S-AVQP, and KDAK produced a strong IgG1 response against HKPg, and only m1Ψ-KDAK 30 μg induced a significant IgG2 titer.

[0532] m1Ψ-KDAK 30 μg, m1Ψ-KDAK-3S-AVQP 30 μg, KDAK 30 μg and m1Ψ-KDAK 3 μg induced significant IgG titers against Kgpcat, and m1Ψ-KDAK 30 μg, m1Ψ-KDAK-3S-AVQP 30 μg and KDAK 30 μg also induced significant IgG1.

[0533] This finding is surprising given that existing techniques describe the efficacy of the protein KDcAK1n in generating robust immune responses against Porphyromonas gingivalis antigens, including heat-inactivated Porphyromonas gingivalis and recombinant Kgpcat protein. In this use case, the efficacy of the protein vaccine was not reproduced when using an mRNA vaccine encoding the same protein sequence, likely due to poor expression and insufficient secretion of the KDcAK1n protein from the mRNA construct.

[0534] The results also showed that vaccines containing 30 μg mRNA generally had greater immunogenicity than vaccines containing 3 μg mRNA. Similarly, modified (M1Ψ) mRNA had greater immunogenicity than native (unmodified) mRNA.

[0535] Example 4: Determining the in vivo efficacy of mRNA vaccines in a periodontitis model

[0536] Figure 6 A schematic diagram of a vaccination regimen is shown for determining the in vivo efficacy (e.g., assessed by preventing alveolar bone loss) and immunogenicity (e.g., determined by antibody levels in serum) of a candidate mRNA vaccine.

[0537] Figure 7Bone loss (mm) induced by *Porphyromonas gingivalis* in different treatment groups is shown. KDAK-3S-AVQP protein was used as a positive control, and "infected" mice (unvaccinated with RNA or protein) were used as negative controls.

[0538] Prepare and formulate the mRNA vaccine as outlined above and as used in Example 3.

[0539] The results showed that vaccination with m1Ψ-KDAK 30 μg and m1Ψ-KDAK-3S-AVQP 30 μg mRNA vaccines provided significant protection against bone loss induced by Porphyromonas gingivalis, similar to that of the alum-adjuvanted peptide KDAK-3S-AVQP.

[0540] KDAK 30 μg and KDAK-3S-AVQP provided partial protection.

[0541] Antiserum was used to detect the following adsorbed antigens: recombinant protein KDAK-3S-AVQP, heat-inactivated Porphyromonas gingivalis strain W50 (HKPg), and Kgp. cat And the biotinylated linear peptide corresponding to the sequence encoded by the tested mRNA vaccine candidate. Antibody response is expressed as the obtained ELISA titer minus three times the background level, where each titer represents the mean ± standard deviation of 10 individual mice. Figure 8 ).

[0542] All constructs tested—m1Ψ-KDAK, m1Ψ-KDAK-3S-AVQP, KDAK, and KDAK-3S-AVQP—provided strong total IgG, IgG1, and IgG2a responses against the recombinant protein KDAK-3S-AVQP. m1Ψ-KDAK, m1Ψ-KDAK-3S-AVQP, and KDAK induced strong total IgG and IgG1 titers against HKPg. Only m1Ψ-KDAK induced a weak IgG2a response.

[0543] All constructs induce KGP-related behavior. cat The IgG (mainly IgG1) response was significant, but only m1Ψ-KDAK, m1Ψ-KDAK-3S-AVQP and KDAK were statistically significant.

[0544] All constructs induced antibodies against the KAS2 peptide.

[0545] Example 5: Expression and secretion of truncated antigens encoded by alternative mRNA constructs

[0546] Various truncated mRNA constructs were evaluated, each expressing a truncated variant of the protein antigen encoded by the mRNA tested in the examples above. All mRNAs were modified with m1Ψ.

[0547] The tested mRNA construct encodes the following:

[0548] -K (also referred to in this paper as the KAS or KAS2 active site domain; amino acid sequence SEQ ID NO: 8)

[0549] -KD (amino acid sequence: SEQ ID NO: 10)

[0550] -KA (amino acid sequence SEQ ID NO: 18)

[0551] -DA (amino acid sequence SEQ ID NO: 14)

[0552] -KDA (amino acid sequence SEQ ID NO: 12)

[0553] -KDA ΔAMB3 (That is, the adhesin domain contains ABM 2 and 1, but not ABM3; amino acid sequence SEQ ID NO: 16)

[0554] -RDA (replacing the arginine-dependent gingivase active site KAS with the active site from the lysine-dependent gingivase RAS) (amino acid sequence: SEQ ID NO: 20)

[0555] - Positive control: KDAK-3S-AVQP.

[0556] Western blotting was used to test the in vitro expression and secretion of mRNA encoding target antigens in HeLa cells. Figure 9 The results of these studies are shown. The SEAP signal peptide is encoded at the N-terminus of each antigen.

[0557] Except for the construct encoding K (m1Ψ-K), which showed poor expression (possibly due to post-expression protein degradation), all constructs expressed well. In samples transfected at 24 hours, the m1Ψ-K construct was initially not detected in either the lysis buffer or the supernatant. Figure 9 A). Further time-history analysis over 6–48 hours, along with loading of additional m1Ψ-K samples (x 3 volumes), showed poor expression and rapid degradation of the construct. Figure 9 B). All other constructs: m1Ψ-KD, m1Ψ-KA, m1Ψ-DA, ​​m1Ψ-KDA, and m1Ψ-KDA21 showed good expression and secretion. Figure 9 A).

[0558] RDA antigens are mainly found in whole-cell lysates, not in the supernatant (indicating insufficient secretion).

[0559] Example 6: In vivo efficacy and immunogenicity of truncated constructs

[0560] In this next study, the encoded KDA and KDA were evaluated. ΔABM3 The mRNAs of DA, KA, and KD (refined in LNP) were compared with KDAK-3S-AVQP mRNA and protein vaccines. All mRNAs were modified with m1Ψ and administered at a dose of 30 μg mRNA.

[0561] Figure 10 The results shown indicate the codes KA, DA, and KDA. ΔABM3 The mRNA vaccine from KDA provides strong protection against alveolar bone loss induced by Porphyromonas gingivalis.

[0562] Vaccines containing m1Ψ-KDAK-3S-AVQP and m1Ψ-KDA21 prevented bone loss in animal models, similar to the recombinant protein KDAK-3S-AVQP with alum as an adjuvant. m1Ψ-DA and m1Ψ-KA provided partial protection. Figure 10 This indicates the importance of the A and K domains for protection.

[0563] Antiserum was used to detect the following adsorbed antigens: recombinant protein KDAK-3S-AVQP, heat-inactivated Porphyromonas gingivalis strain W50 (HKPg), and Kgpcat corresponding to the sequence encoded by the mRNA vaccine candidate being tested.

[0564] Antibody response was expressed as the obtained ELISA titer minus three times the background level, where each titer represents the mean ± standard deviation of 10 individual mice. All constructs induced strong total IgG, IgG1, and IgG2 responses against KDAK-3S-AVQP. Figure 11 ).

[0565] All constructs induced strong total IgG and IgG1 titers against HKPg. Only m1Ψ-DA and m1Ψ-KDA21 induced an IgG2a response.

[0566] Except for m1Ψ-DA (because it does not contain the K domain as part of Kgpcat), all constructs induced an IgG (primarily IgG1) response against Kgpcat, but the response of m1Ψ-KDAK-3S-AVQP was weaker and did not show statistical significance for IgG1. Only m1Ψ-KDA21 and m1Ψ-KDAK induced IgG2 titers.

[0567] Except for m1Ψ-KDA21 (because it does not contain a KAS2 epitope), all constructs induce antibodies against the KAS2 peptide.

[0568] Example 7: Additional assessment of the immunogenicity of truncated constructs

[0569] Further immunogenicity assessments were performed using the following constructs: m1Ψ-K 30 μg and m1Ψ-RDA 30 μg.

[0570] according to Figure 4 The timeline shown in the figure describes the intramuscular immunization of mice with mRNA-LNP encoding the target antigen. Serum antibody subclass responses in immunized mice were examined by ELISA. Antiserum was used to detect the following adsorbed antigens: KDAK-3S-AVQP peptide, heat-inactivated *Porphyromonas gingivalis* strain W50 (HKPg), and *Kgpcat*.

[0571] Antibody response is expressed as the obtained ELISA titer minus three times the background level, where each titer represents the mean ± standard deviation of 5 individual mice. Figure 12 Only m1Ψ-RDA induced a strong response to KDAK-3S-AVQP, resulting in strong total IgG, IgG1, and IgG2 titers. It also induced significant total IgG (primarily IgG1) but not IgG2 titers against HKPg, and significant IgG1 titers against Kgpcat. It also induced antibody titers against KAS2 and ABM3 peptides. As mentioned above, m1Ψ-K did not show immunogenicity, possibly due to poor expression / stability.

[0572] Example 8: In vivo efficacy and immunogenicity in a prophylactic model

[0573] In a prophylactic vaccination model, the mRNA encoding KDAK-3S-AVQP (modulated in LNP) was evaluated. Figure 13 The mRNA was modified with m1Ψ and administered at a dose of 30 μg mRNA.

[0574] Figure 15 The results shown in the study indicate that, in a preventative vaccination model, the mRNA vaccine provides robust protection against alveolar bone loss induced by Porphyromonas gingivalis.

[0575] mRNA vaccines containing m1Ψ-KDAK-3S-AVQP prevented bone loss in animal models, similar to recombinant protein KDAK-3S-AVQP adjuvanted with alum. Figure 15 ).

[0576] Antiserum was used to detect the following adsorbed antigens: recombinant protein KDAK-3S-AVQP, heat-inactivated Porphyromonas gingivalis strain W50 (HKPg), and RgpA-Kgp complex.

[0577] Antibody response was expressed as the obtained ELISA titer minus three times the background level, where each titer represents the mean ± standard deviation of 10 individual mice. The tested mRNA construct induced strong total IgG, IgG1, and IgG2 responses against the KDAK-3S-AVQP, HK-Pg, and RgpA-Kgp complexes. Figure 16 ).

[0578] In the ELISpot assay, in vitro restimulation of recovered spleen cells with KDAK-3S-AVQP protein revealed the presence of CD4+ T cells that secrete interferon-γ with KDAK-3S-AVQP specificity. Figure 14 ).

[0579] It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more features mentioned or obviously present in the text or drawings. All these different combinations constitute various alternative aspects of the invention.

Claims

1. An RNA polynucleotide encoding a protein comprising or composed of the following: - One or more amino acid sequences of the active site of Arg gingivalase or Lys gingivalase of Porphyromonas gingivalis, or sequences that are at least 80% identical thereto; and / or - An amino acid sequence of one or more adhesin-binding motifs (ABMs) of the adhesin domain of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto. The polynucleotides mentioned therein can be translated in mammalian cells.

2. The RNA of claim 1, wherein the protein encoded by the RNA comprises or is composed of the following: - One or more amino acid sequences of the active site of Arg gingival protease or Lys gingival protease of *Porphyromonas gingivalis*, or sequences that are at least 80% identical thereto; and - An amino acid sequence of one or more adhesin-binding motifs (ABMs) of the adhesin domain of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto.

3. The RNA according to claim 1 or 2, wherein the protein encoded by the RNA polynucleotide further comprises: - The amino acid sequence of the Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical to it.

4. The RNA according to any one of the preceding claims, wherein the protein encoded by the RNA polynucleotide is a chimeric or fusion protein comprising or composed of the following: - One or more amino acid sequences of the active site of Arg gingival protease or Lys gingival protease of *Porphyromonas gingivalis*, or sequences that are at least 80% identical thereto; and - An amino acid sequence of one or more adhesin-binding motifs (ABMs) of the adhesin domain of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto.

5. The RNA according to any one of the preceding claims, wherein the RNA encodes an amino acid sequence of the active site of Arg gingival protease of Porphyromonas gingivalis, the amino acid sequence comprising the amino acid sequence of SEQ ID NO: 38 (e.g., encoded by the RNA sequence shown in SEQ ID NO: 50) or the same sequence as 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%.

6. The RNA according to any one of the preceding claims, wherein the RNA encodes an amino acid sequence of the active site of Lys gingival protease of Porphyromonas gingivalis, the amino acid sequence comprising the amino acid sequence of SEQ ID NO: 8 (e.g., encoded by the RNA sequence shown in SEQ ID NO: 43) or the same sequence as 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%.

7. The RNA according to any one of the preceding claims, wherein the RNA encodes a chimeric or fusion protein comprising: i) Contains or consists of an amino acid sequence comprising: the amino acid sequence of the active site of Arg gingivase from *Porphyromonas gingivalis*, or a sequence that is at least 80% identical thereto; and ii) Contains or consists of the following amino acid sequence: the amino acid sequence of the active site of Lys gingival protease of Porphyromonas gingivalis or a sequence that is at least 80% identical to it.

8. The RNA according to any one of the preceding claims, wherein the RNA encodes a chimeric or fusion protein comprising at least two amino acid sequences, said at least two amino acid sequences comprising or consisting of the following: The amino acid sequence of the active site of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical to it.

9. The RNA of claim 8, wherein the at least two amino acid sequences are located sequentially 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-terminus 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 (both) are located at the N-terminus of the chimeric or fusion protein, or the at least two amino acid sequences (both) are located at the C-terminus of the chimeric or fusion protein.

13. The RNA according to 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 the Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical to it.

14. The RNA of claim 13, wherein the RNA encodes: - One or more amino acid sequences of the active site of Arg gingival protease or Lys gingival protease of *Porphyromonas gingivalis*, or sequences that are at least 80% identical thereto; and - The amino acid sequence of one or more adhesin-binding motifs (ABMs) of the adhesin domain of Arg gingival protease or Lys gingival protease of *Porphyromonas gingivalis*, or a sequence that is at least 80% identical thereto; and The amino acid sequence of the DUF2436 domain is located between the amino acid sequence of the active site of the gingival protease of *Porphyromonas gingivalis* and the amino acid sequence of one or more adhesin-binding motifs (ABMs).

15. The RNA according to claim 13 or 14, wherein the RNA encodes an amino acid sequence of the DUF2436 domain of Arg gingival protease or Lys gingival protease of *Porphyromonas gingivalis*, said amino acid sequence comprising or consisting of the following: The amino acid sequence of SEQ ID NO: 35 (e.g., encoded by an RNA sequence as shown in SEQ ID NO: 50) or the sequence that is 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 to it.

16. The RNA according to any one of claims 13 to 15, wherein the RNA encodes a substitution of a cysteine ​​residue in the DUF2436 domain, such as a substitution of a serine or valine residue.

17. The RNA of claim 16, wherein the RNA encodes an amino acid sequence of the DUF2436 domain, the amino acid sequence comprising a substitution of cysteine ​​for serine (as shown in SEQ ID NO: 36).

18. The RNA according to any one of the preceding claims, wherein the RNA encodes one or more adhesin-binding motifs (ABMs), the one or more ABMs comprising or consisting of the amino acid sequences of ABM2 and ABM1.

19. The RNA of claim 18, wherein the RNA encodes an amino acid sequence of a polypeptide, the amino acid sequence comprising ABM2 and ABM1 as shown in SEQ ID NO: 22 and SEQ ID NO: 21, respectively, or comprising an amino acid sequence (ABM2+1) as shown in SEQ ID NO: 24, or a sequence that is 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 to it.

20. The RNA of claim 19, wherein the RNA comprises a nucleotide sequence as shown in any one of SEQ ID NO: 52 to 55.

21. The RNA according to any one of claims 18 to 20, wherein the RNA encodes one or more adhesin-binding motifs (ABMs), the one or more ABMs comprising or consisting of the amino acid sequences of ABM2, ABM1, and ABM3.

22. The RNA of claim 21, wherein the RNA encodes an amino acid sequence as shown in SEQ ID NO: 25 (e.g., encoded by an RNA comprising the sequence of SEQ ID NO: 56).

23. The RNA according to any one of claims 18 to 22, wherein the RNA encodes one or more adhesin-binding motifs, the one or more adhesin-binding motifs comprising one or more modifications selected from: a) In the corresponding region, one or more cysteine ​​amino acids are substituted compared to the naturally occurring Arg gingivase or Lys gingivase sequence; b) Substitution of proline and / or asparagine residues in sequence PxxN corresponding to residues 6 to 9 of the sequence of SEQ ID NO: 21 (ABM1) or located at positions equivalent to said residues; c) Substitution of motif NxFA to SxYQ in the sequence corresponding to residues 2 to 5 of SEQ ID NO: 21 (ABM1) or located at positions equivalent to said residues; d) The second tyrosine residue corresponding to or located at a position equivalent to the residue at position 5 of SEQ ID NO: 22 (ABM2) and the tryptophan residue corresponding to or located at a position equivalent to the residue at position 23 of SEQ ID NO: 21 (ABM1) are replaced by an alanine residue.

24. The RNA of claim 23, wherein the RNA encodes one or more adhesin-binding motifs, the one or more adhesin-binding motifs comprising substitutions of one or more cysteine ​​residues for serine residues or for valine residues.

25. The RNA of claim 24, wherein the RNA encodes one or more adhesin-binding motifs, the one or more adhesin-binding motifs comprising substitutions of all cysteine ​​residues for serine or valine residues.

26. The RNA according to any one of claims 23 to 25, wherein the RNA encodes one or more adhesin-binding motifs, the one or more adhesin-binding motifs comprising a proline and / or asparagine substitution of motif PxxN (e.g., PVQN, SEQ ID NO: 106) corresponding to residues 6 to 9 of SEQ ID NO: 21 or located at a position equivalent to said residues.

27. The RNA according to claim 26, wherein the proline amino acid substitution is a substitution of an alanine residue.

28. The RNA according to claim 26 or 27, wherein the asparagine amino acid substitution is replaced by a proline residue or an alanine residue.

29. The RNA according to any one of claims 23 to 28, wherein the RNA encodes one or more adhesin-binding motifs, wherein the one or more adhesin-binding motifs comprise a substitution of PxxN in the region encoding ABM1 (as illustrated in the amino acid sequences of SEQ ID NO: 30 to 32) for AxxP (e.g., AVQP, SEQ ID NO: 107).

30. The RNA according to any one of claims 23 to 29, wherein the RNA encodes one or more adhesin-binding motifs, the one or more adhesin-binding motifs comprising an amino acid sequence as shown in any one of SEQ ID NO: 21 to 25, and comprising: a) In the corresponding region, compared with the sequence of naturally occurring Arg gingivase or Lys gingivase, one or more cysteine ​​amino acids are substituted, preferably all cysteine ​​residues are substituted; and b) Substitution of motif PxxN to AxxP corresponding to residues 6 to 9 of the sequence of SEQ ID NO: 21 (ABM1) or located at positions equivalent to said residues.

31. The RNA of claim 30, wherein the RNA encodes one or more adhesin-binding motifs, the one or more adhesin-binding motifs comprising or consisting of the following: The amino acid sequence shown in any of SEQ ID NO: 26 to 34, or the sequence that is 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 to the sequence, provided that the sequence contains the above-described substitutions of cysteine ​​residues, proline residues, and asparagine residues.

32. An RNA polynucleotide encoding a chimeric or fusion protein comprising or consisting of the following: - One or more amino acid sequences of the active site of Arg gingival protease or Lys gingival protease of *Porphyromonas gingivalis*, or sequences that are at least 80% identical thereto; and - An amino acid sequence of one or more adhesin-binding motifs (ABMs) of the adhesin domain of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto.

33. The RNA of claim 32, wherein the RNA encodes a chimeric or fusion protein comprising or consisting of the following amino acid sequences: SEQ ID NO: 18 or 83, or SEQ ID NO: 39 or 81, or SEQ ID NO: 58 to 63, or SEQ ID NO: 82 to 87.

34. An RNA polynucleotide encoding a chimeric or fusion protein comprising or composed of the following: - One or more amino acid sequences of the active site of Arg gingivase or Lys gingivase of Porphyromonas gingivalis, or sequences that are at least 80% identical to them. - The amino acid sequence of the DUF2436 domain of Arg gingival protease or Lys gingival protease of *Porphyromonas gingivalis*, or a sequence that is at least 80% identical thereto, and - An amino acid sequence of one or more adhesin-binding motifs (ABMs) of the adhesin domain of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto.

35. The RNA of claim 34, wherein the RNA encodes a chimeric or fusion protein comprising or consisting of the amino acid sequence of SEQ ID NO: 12, 16 or 20.

36. The RNA of claim 34, wherein the RNA encodes a chimeric or fusion protein, said chimeric or fusion protein comprising or consisting of the amino acid sequence of SEQ ID NO:

4.

37. An RNA polynucleotide encoding a chimeric or fusion protein comprising or consisting of the following: - The amino acid sequence of the DUF2436 domain of Arg gingival protease or Lys gingival protease of *Porphyromonas gingivalis*, or a sequence that is at least 80% identical thereto, and - An amino acid sequence of one or more adhesin-binding motifs (ABMs) of the adhesin domain of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto.

38. The RNA of claim 37, wherein the RNA encodes a chimeric or fusion protein, said chimeric or fusion protein comprising or consisting of the amino acid sequence of SEQ ID NO:

14.

39. An RNA polynucleotide encoding a chimeric or fusion protein comprising or consisting of the following: - One or more amino acid sequences of the active site of Arg gingival protease or Lys gingival protease of *Porphyromonas gingivalis*, or sequences that are at least 80% identical thereto; and - The amino acid sequence of the Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical to it.

40. The RNA of claim 39, wherein the RNA encodes a chimeric or fusion protein comprising or consisting of the amino acid sequence of SEQ ID NO: 10 or 108.

41. An RNA polynucleotide encoding a chimeric or fusion protein comprising or consisting of the following: - One or more amino acid sequences of the active site of Arg gingivase or Lys gingivase of Porphyromonas gingivalis, or sequences that are at least 80% identical to them. - The amino acid sequence of the DUF2436 domain of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto, and - An amino acid sequence of one or more adhesin-binding motifs (ABMs) of the adhesin domain of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto.

42. The RNA of claim 41, wherein the RNA encodes a chimeric or fusion protein, said chimeric or fusion protein comprising or consisting of the amino acid sequence of SEQ ID NO:

6.

43. An RNA polynucleotide comprising or consisting of a nucleotide sequence encoding a protein, said nucleotide sequence comprising or consisting of an amino acid sequence of any of the following: SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:

38.

44. An RNA that comprises or consists of a nucleotide sequence of any of the following: a) SEQ ID NO: 48, 57; b) SEQ ID NO: 45, 47, or 49, or SEQ ID NO: 41; c) SEQ ID NO: 46; d) SEQ ID NO: 44; e) SEQ ID NO:

42.

45. An RNA that comprises or consists of a nucleotide sequence of any of the following: SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO:

50.

46. ​​The RNA according to any one of the preceding claims, wherein the RNA is mRNA.

47. The RNA according to any one of the preceding claims, wherein the RNA further encodes an N-terminal signal peptide to enable the secretion of the protein post-translation.

48. The RNA according to any one of the preceding claims, wherein the RNA further comprises a 5' untranslated region (UTR) and / or a 3' UTR.

49. The RNA according to any one of the preceding claims, wherein the RNA further comprises a 5' cap analogue, such as 7mG(5')ppp(5')NlmpNp.

50. The RNA according to any one of the preceding claims, wherein the RNA further comprises a polyA tail.

51. The RNA according to any one of the preceding claims, wherein the RNA comprises a chemical modification, preferably wherein the chemical modification is 1-methylpseuuridine modification or 1-ethylpseuuridine modification.

52. The RNA according to any one of the preceding claims, wherein the uridine content of said RNA is 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%.

53. The RNA according to any one of the preceding claims, wherein the uridine in the RNA is replaced by a chemical modification such as N-methyl-pseudouridine.

54. The RNA of claim 53, 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 nucleoside is replaced by N-methyl-pseudouridine.

55. The RNA according to any one of the preceding claims, wherein the RNA is in the form of a codon-optimized RNA molecule.

56. A composition comprising RNA according to any one of the preceding claims.

57. A lipid nanoparticle composition comprising RNA according to any one of claims 1 to 55.

58. The lipid nanoparticle composition according to claim 57, comprising: - Cationic lipids and / or ionizable lipids, wherein the cationic lipids and / or ionizable lipids constitute approximately 25% to approximately 75 mol% of the total lipids present in the nanoparticles; - Sterols (structural lipids), said sterols comprising about 5 mol% to about 60 mol% of the total lipids present in said nanoparticles; - Phospholipids, wherein the phospholipids comprise approximately 5 mol% to approximately 50 mol% of the total lipids present in the nanoparticles; -PEGylated lipids, wherein the PEGylated lipids account for approximately 0.5 mol% to 20 mol% of the total lipids present in the nanoparticles.

59. The lipid nanoparticle composition according to claim 57 or 58, comprising: - An ionizable lipid in the form of [(4-hydroxybutyl)azanidinediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), - Sterols in the form of cholesterol, - Phospholipids in the form of distearate phosphatidylcholine (DSPC), and - A PEGylated lipid in the form of 2-[(polyethylene glycol)-2000]-N,N-bis(tetradecyl)acetamide (ALC-0159).

60. The lipid nanoparticle composition of claim 59, wherein the lipids are present in the lipid nanoparticles at a molar lipid ratio (%) of 46.3 ALC-0315:42.7 cholesterol:9.4 DSPC:1.6 ALC-0159, optionally in Tris / sucrose buffer (25 mM Tris, 8.8% sucrose w / v, pH 7.4).

61. A method for producing lipid nanoparticles, the lipid nanoparticles comprising RNA according to any one of claims 1 to 55, wherein preferably the method comprises adapting any RNA molecule of any one of claims 1 to 55, and one or more lipids that can be used to produce lipid nanoparticles.

62. The method of claim 61, wherein the lipid component comprises phospholipids, PEG lipids, and structural lipids.

63. A nucleic acid construct or vector comprising an RNA polynucleotide according to any one of claims 1 to 55.

64. A method for inducing an immune response in a subject in need of *Porphyromonas gingivalis*, the method comprising administering to the subject RNA according to any one of claims 1 to 55 or nanoparticles or compositions according to any one of claims 56 to 60.

65. A method for inducing an immune response against *Porphyromonas gingivalis* in a subject in need, the method comprising administering a nanoparticle composition to the subject, the nanoparticle composition comprising: - RNA according to any one of claims 1 to 55; - A pharmaceutical agent that enables the delivery of the RNA to the subject's cells; - wherein the RNA can be translated into a polypeptide encoded by the polynucleotide in the subject's cells.

66. A method for producing chimeric or fusion proteins in mammalian cells, the method comprising contacting the mammalian cells with a nanoparticle composition, the composition comprising: - RNA according to any one of claims 1 to 55; - A pharmaceutical agent that enables the delivery of the RNA to the subject's cells; - wherein the RNA can be translated into the protein in the mammalian cell.

67. A method for delivering RNA to mammalian cells in a subject in need, the method comprising administering a nanoparticle composition to the subject in need, the composition comprising: -RNA, said RNA comprising the polynucleotide sequence of any one of claims 1 to 55 - A pharmaceutical agent that enables the delivery of the RNA to the subject's cells; - wherein the RNA can be translated in the mammalian cells to produce the chimeric or fusion proteins described herein; The application involves contacting the mammalian cells with the nanoparticle composition, thereby enabling the delivery of the RNA into the mammalian cells.

68. The method according to any one of claims 65 to 67, wherein the agent for enabling the delivery of the RNA to the cells of the subject is a lipid.

69. The method of claim 68, wherein the lipid comprises cationic lipids and / or ionizable lipids, phospholipids, PEG lipids, and structural lipids.

70. Use of RNA or a carrier or nanoparticle comprising the RNA according to any one of claims 1 to 55 for preparing a composition for inducing an immune response against Porphyromonas gingivalis in a subject.

71. An i) lipid component, preferably comprising cationic lipids and / or ionizable lipids, phospholipids, PEG lipids and structural lipids; and ii) use of the RNA according to any one of claims 1 to 55 for preparing a composition for delivering said RNA to mammalian cells in a subject in need.

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