Signal sequence of nucleic acid vaccine

Abstract

Nucleic acids (e.g., messenger RNA) encoding at least one antigen prokaryotic polypeptide linked to one or both of a viral secretion signal peptide and a transmembrane domain are provided herein. Also provided is a method of vaccinating against prokaryotic infection with the nucleic acids described herein.

Description

Technical Field

[0001] Related Applications This application is related to European Patent Priority Application No. 22305680.5 filed on May 6, 2022, European Patent Priority Application No. 22306227.4 filed on August 16, 2022, and US Application No. 63 / 449,573 filed on March 2, 2023, the contents of each of which are incorporated herein by reference.

Background Art

[0002] Prokaryotic infections (e.g., bacterial infections) are a significant global threat to human health. It is estimated that 5 million people die annually from antimicrobial-resistant bacterial infections, and the burden on the healthcare system is increasing (Antimicrobial Resistance Collaborators. The Lancet. 399(10325):629 - 655. 2022). Vaccines against prokaryotic infections exist, but are fewer in number compared to more common antiviral vaccines.

[0003] Nucleic acid-based vaccines, more specifically mRNA vaccines, have recently emerged as an additional vaccine type with a particularly rapid, safe, and cost-effective production process, especially against viral pathogens. mRNA vaccines against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV-2) most commonly use the spike viral protein as an antigen. Often combined with a delivery vehicle, e.g., lipid nanoparticles (LNPs), COVID-19 mRNA vaccines can achieve high efficacy. In any case, more effective RNA-based vaccines against prokaryotic infections are needed.

[0004] Generating nucleic acid-based vaccines, such as RNA (e.g., mRNA)-based vaccines that contain prokaryotic antigens (i.e., antigens derived from antigens in prokaryotic cells), presents challenges because prokaryotic antigens are not naturally expressed by eukaryotic cells. In particular, prokaryotic and eukaryotic cells have different secretory systems. Without proper engineering, the prokaryotic antigens used in these vaccines can accumulate in the intracellular compartments of eukaryotic (e.g., human) cells, thereby reducing the immunogenicity of these vaccines.

Summary of the Invention

Problems to be Solved by the Invention

[0005] The present invention aims to address this problem by improving the immunogenicity of prokaryotic antigens expressed through nucleic acid (e.g., mRNA)-based vaccines. As described herein, this is achieved by adding a secretory signal in a transmembrane domain to enable the expression of the prokaryotic antigen in the extracellular compartment and / or on the cell surface. By doing so, immune cells can access the antigen better, ultimately improving the immunogenicity of the nucleic acid (e.g., mRNA)-based vaccine.

Means for Solving the Problems

[0006] The present disclosure provides a nucleic acid comprising an open reading frame (ORF), the ORF comprising - a polynucleotide sequence encoding at least one antigenic prokaryotic polypeptide and - a polynucleotide sequence encoding at least one viral secretory signal peptide.

[0007] In certain embodiments, the ORF further comprises a polynucleotide sequence encoding at least one transmembrane domain (TMD).

[0008] In certain embodiments, the viral secretory signal peptide is derived from a viral sequence in a virus capable of infecting humans.

[0009] In certain embodiments, the viral secretion signal peptide is derived from a viral sequence selected from the group consisting of an influenza secretion signal peptide sequence, and a non-influenza secretion signal peptide sequence selected from the group consisting of a SARS CoV-2 secretion signal peptide sequence, a varicella zoster virus (VZV) secretion signal peptide sequence, a measles secretion signal peptide sequence, a rubella secretion signal peptide sequence, a mumps secretion signal peptide sequence, an Ebola secretion signal peptide sequence, a smallpox secretion signal peptide sequence, and a rabies secretion signal peptide sequence.

[0010] In certain embodiments, the viral secretion signal peptide is selected from the group consisting of an influenza hemagglutinin (HA) secretion signal peptide sequence, a SARS CoV-2 spike secretion signal peptide sequence, a VZV gB secretion signal peptide sequence, a VZV gE secretion signal peptide sequence, a VZV gI secretion signal peptide sequence, a VZV gK secretion signal peptide sequence, a measles F-protein secretion signal peptide sequence, a rubella E1 protein secretion signal peptide sequence, a rubella E2 protein secretion signal peptide sequence, a mumps F-protein secretion signal peptide sequence, an Ebola GP protein secretion signal peptide sequence, a smallpox 6 kDa IC protein secretion signal peptide sequence, and a rabies G protein secretion signal peptide sequence, preferably, the viral secretion signal peptide comprises an HA secretion signal peptide sequence derived from influenza A or influenza B, more preferably, an HA secretion signal peptide sequence derived from influenza A.

[0011] In certain embodiments, the HA secretion signal peptide sequence comprises the amino acid sequence MKX 1 X 2 LX 3 VX 4 LX 5 TFX 6 X 7 X 8 X 9 A (SEQ ID NO: 145), wherein X 1 is selected from A and V; X 2is selected from I and K; X 3 is selected from V and L; X 4 is selected from L and M; X 5 is selected from Y and C; X 6 is selected from T and A, X 7 is selected from T and A; X 8 is selected from A and T; X 9 is selected from N and Y.

[0012] In certain embodiments, the HA secretion signal peptide sequence comprises an amino acid sequence selected from SEQ ID NOs: 95 - 109.

[0013] In certain embodiments, the HA secretion signal peptide sequence is the amino acid sequence MKX 1 IIALSX 2 ILCLVFX 3 (SEQ ID NO: 146), and X 1 is selected from T and A; X 2 is selected from Y, N, C, and H; X 3 is selected from T and A.

[0014] In certain embodiments, the HA secretion signal peptide sequence comprises an amino acid sequence selected from SEQ ID NOs: 110 - 131.

[0015] In certain embodiments, the HA secretion signal peptide sequence is the amino acid sequence MKAIIVLLMVVTSX 1 A (SEQ ID NO: 147), and X 1 is selected from S and N.

[0016] In certain embodiments, the HA secretion signal peptide sequence is the amino acid sequence MX 1 AIIVLLMVVTSNA (SEQ ID NO: 148), and X 1 is selected from K and E.

[0017] In certain embodiments, the HA secretion signal peptide sequence comprises an amino acid sequence selected from SEQ ID NOs: 132 - 144.

[0018] In certain embodiments, the viral secretion signal peptide comprises an amino acid sequence selected from the group consisting of MKAKLLVLLCTFTATYA (SEQ ID NO: 1); MKAILVVLLYTFATANA (SEQ ID NO: 2); MKTIIALSYILCLVFA (SEQ ID NO: 3); MKAIIVLLMVVTSNA (SEQ ID NO: 4); MFVFLVLLPLVS (SEQ ID NO: 5); MFLLTTKRTMFVFLVLLPLVS (SEQ ID NO: 6) MSPCGYYSKWRNRDRPEYRRNLRFRRFFSSIHPNAAAGSGFNGPGVFITSVTGVWLCFLCIFSMFVTAVVS (SEQ ID NO: 7); MGTVNKPVVGVLMGFGIITGTLRITNPVRA (SEQ ID NO: 8); MFLIQCLISAVIFYIQVTNA (SEQ ID NO: 9); MQALGIKTEHFIIMCLLSGHA (SEQ ID NO: 10); MGLKVNVSAIFMAVLLTLQTPTG (SEQ ID NO: 11); MGAAAALTAVVLQGYNPPAYG (SEQ ID NO: 12); MGAPQAFLAGLLLAAVAVGTARA (SEQ ID NO: 13); MKVFLVTCLGFAVFSSSVC (SEQ ID NO: 14); MGVTGILQLPRDRFKRTSFFLWVIILFQRTFS (SEQ ID NO: 15); MRSLIIFLLFPSIIYS (SEQ ID NO: 16); and MVPQALLFVPLLVFPLCFG (SEQ ID NO: 184).

[0019] In certain embodiments, the viral secretion signal peptide comprises the amino acid sequence of MKAKLLVLLCTFTATYA (SEQ ID NO: 1).

[0020] In certain embodiments, the viral secretion signal peptide is located at the N-terminus of the antigen prokaryotic polypeptide.

[0021] In certain embodiments, the viral secretion signal peptide is located at the C-terminus of the antigen prokaryotic polypeptide.

[0022] In certain embodiments, the viral secretion signal peptide is linked to the antigen prokaryotic polypeptide by a linker.

[0023] In certain embodiments, the TMB comprises or consists of (a) 15 to 50 amino acid residues, preferably 15 to 30 amino acid residues, more preferably 18 to 25 amino acid residues; and / or (b) comprises at least 50% hydrophobic amino acid residues, preferably selected from the group consisting of alanine, isoleucine, leucine, valine, phenylalanine, tryptophan, and tyrosine; and / or (c) comprises at least one alpha helix.

[0024] In certain embodiments, the TMB is derived from an integral membrane protein, preferably from a single-pass transmembrane protein, more preferably from a bitopic membrane protein, and even more preferably from a type I bitopic membrane protein.

[0025] In certain embodiments, the TMB is derived from a non-human sequence.

[0026] In certain embodiments, the antigen prokaryotic polypeptide is derived from a prokaryotic transmembrane protein, and the TMB is the TMB of the prokaryotic transmembrane protein.

[0027] In certain embodiments, the antigen prokaryotic polypeptide is not derived from a prokaryotic transmembrane protein.

[0028] In certain embodiments, the TMB is derived from a viral sequence.

[0029] In certain embodiments, the TMB is derived from a viral transmembrane domain sequence selected from the group consisting of an influenza transmembrane domain sequence and non-influenza transmembrane domain sequences selected from the group consisting of SARS CoV-2 transmembrane domain sequences, varicella-zoster virus (VZV) transmembrane domain sequences, measles transmembrane domain sequences, rubella transmembrane domain sequences, mumps transmembrane domain sequences, Ebola transmembrane domain sequences, and rabies transmembrane domain sequences.

[0030] In certain embodiments, the TMB is selected from the group consisting of the influenza hemagglutinin (HA) transmembrane domain sequence, the SARS CoV-2 spike transmembrane domain sequence, the VZV gB transmembrane domain sequence, the VZV gE transmembrane domain sequence, the VZV gI transmembrane domain sequence, the VZV gK transmembrane domain sequence, the measles F protein transmembrane domain sequence, the rubella E1 protein transmembrane domain sequence, the rubella E2 protein transmembrane domain sequence, the mumps F protein transmembrane domain sequence, the Ebola GP protein transmembrane domain sequence, and the rabies G protein transmembrane domain sequence, and preferably, the TMB comprises an HA transmembrane domain sequence derived from influenza A or influenza B, more preferably an HA transmembrane domain sequence derived from influenza A.

[0031] In certain embodiments, the TMB comprises an amino acid sequence selected from the group consisting of: ILAIYSTVASSLVLLVSLGAISF (SEQ ID NO: 17); ILAIYSTVASSLVLVVSLGAISF (SEQ ID NO: 18); ILWISFAISCFLLCVVLLGFI (SEQ ID NO: 19); STAASSLAVTLMLAIFIVYMV (SEQ ID NO: 20); WYIWLGFIAGLIAIVMVTIML (SEQ ID NO: 21); FGALAVGLLVLAGLVAAFFAY (SEQ ID NO: 22); AAWTGGLAAVVLLCLVIFLIC (SEQ ID NO: 23); IIIPIVASVMILTAMVIVIVI (SEQ ID NO: 24); YFWCVQLKMIFFAWFVYGMYL (SEQ ID NO: 25); IVYILIAVCLGGLIGIPALIC (SEQ ID NO: 26); LDHAFAAFVLLVPWVLIFMVC (SEQ ID NO: 27); WWQLTLGAICALLLAGLLACC (SEQ ID NO: 28); IVAALVLSILSIIISLLFCCW (SEQ ID NO: 29); WIPAGIGVTGVIIAVIALFCI (SEQ ID NO: 30); and VLLSAGALTALMLIIFLMTCW (SEQ ID NO: 185).

[0032] In certain embodiments, the TMB comprises the amino acid sequence of ILAIYSTVASSLVLLVSLGAISF (SEQ ID NO: 17).

[0033] In certain embodiments, the TMB is linked to the antigenic prokaryotic polypeptide by a linker.

[0034] In certain embodiments, the TMB is located at the N-terminus of the antigenic prokaryotic polypeptide.

[0035] In certain embodiments, the TMB is located at the C-terminus of the antigenic prokaryotic polypeptide.

[0036] In another aspect, the disclosure provides a nucleic acid comprising an open reading frame (ORF), the ORF comprising: - a polynucleotide sequence encoding at least one antigenic polypeptide, preferably an antigenic prokaryotic polypeptide, and - a polynucleotide sequence encoding at least one transmembrane domain (TMB), wherein the TMB is heterologous to the antigenic polypeptide, and optionally, the ORF further comprises a polynucleotide sequence encoding at least one secretion signal peptide as described in any one of the preceding claims, preferably a viral secretion signal peptide, more preferably a polynucleotide sequence encoding a viral secretion signal peptide.

[0037] In another aspect, the disclosure provides a nucleic acid comprising an open reading frame (ORF), the ORF comprising: - a polynucleotide sequence encoding at least one antigenic prokaryotic polypeptide and - a polynucleotide sequence encoding at least one transmembrane domain (TMB).

[0038] In certain embodiments, the TMB comprises or consists of (a) 15 to 50 amino acid residues, preferably 15 to 30 amino acid residues, more preferably 18 to 25 amino acid residues; and / or (b) comprises at least 50% hydrophobic amino acid residues, preferably selected from the group consisting of alanine, isoleucine, leucine, valine, phenylalanine, tryptophan, and tyrosine; and / or (c) comprises at least one alpha helix.

[0039] In certain embodiments, the TMB is derived from an integral membrane protein, preferably from a single-pass transmembrane protein, more preferably from a bitopic membrane protein, and even more preferably from a type I bitopic membrane protein.

[0040] In certain embodiments, the TMB is derived from a non-human sequence.

[0041] In certain embodiments, the antigenic polypeptide is not derived from a transmembrane protein.

[0042] In certain embodiments, the TMB is derived from a viral sequence.

[0043] In certain embodiments, the TMB is derived from a viral transmembrane domain sequence selected from the group consisting of influenza transmembrane domain sequences and non-influenza transmembrane domain sequences selected from the group consisting of SARS CoV-2 transmembrane domain sequences, varicella-zoster virus (VZV) transmembrane domain sequences, measles transmembrane domain sequences, rubella transmembrane domain sequences, mumps transmembrane domain sequences, Ebola transmembrane domain sequences, and rabies transmembrane domain sequences.

[0044] In certain embodiments, the TMB is selected from the group consisting of influenza hemagglutinin (HA) transmembrane domain sequences, SARS CoV-2 spike transmembrane domain sequences, VZV gB transmembrane domain sequences, VZV gE transmembrane domain sequences, VZV gI transmembrane domain sequences, VZV gK transmembrane domain sequences, measles F protein transmembrane domain sequences, rubella E1 protein transmembrane domain sequences, rubella E2 protein transmembrane domain sequences, mumps F protein transmembrane domain sequences, Ebola GP protein transmembrane domain sequences, and rabies G protein transmembrane domain sequences, and preferably, the TMB comprises an HA transmembrane domain sequence derived from influenza A or influenza B, and more preferably an HA transmembrane domain sequence derived from influenza A.

[0045] In certain embodiments, TMB comprises an amino acid sequence selected from the group consisting of: ILAIYSTVASSLVLLVSLGAISF (SEQ ID NO: 17); ILAIYSTVASSLVLVVSLGAISF (SEQ ID NO: 18); ILWISFAISCFLLCVVLLGFI (SEQ ID NO: 19); STAASSLAVTLMLAIFIVYMV (SEQ ID NO: 20); WYIWLGFIAGLIAIVMVTIML (SEQ ID NO: 21); FGALAVGLLVLAGLVAAFFAY (SEQ ID NO: 22); AAWTGGLAAVVLLCLVIFLIC (SEQ ID NO: 23); IIIPIVASVMILTAMVIVIVI (SEQ ID NO: 24); YFWCVQLKMIFFAWFVYGMYL (SEQ ID NO: 25); IVYILIAVCLGGLIGIPALIC (SEQ ID NO: 26); LDHAFAAFVLLVPWVLIFMVC (SEQ ID NO: 27); WWQLTLGAICALLLAGLLACC (SEQ ID NO: 28); IVAALVLSILSIIISLLFCCW (SEQ ID NO: 29); WIPAGIGVTGVIIAVIALFCI (SEQ ID NO: 30); and VLLSAGALTALMLIIFLMTCW (SEQ ID NO: 185).

[0046] In certain embodiments, TMB comprises ILAIYSTVASSLVLLVSLGAISF (SEQ ID NO: 17).

[0047] In certain embodiments, TMB is linked to an antigenic prokaryotic polypeptide by a linker.

[0048] In certain embodiments, TMB is located at the N-terminus of the antigenic prokaryotic polypeptide.

[0049] In certain embodiments, TMB is located at the C-terminus of the antigenic prokaryotic polypeptide.

[0050] In certain embodiments, the antigen prokaryotic polypeptide is from Acetobacter, Acinetobacter, Actinomyces, Aerococcus, Agrobacterium, Anaplasma, Azorhizobia, Azotobacter, Bacillus, Bacteroides, Bartonella, Bordetella, Borrelia, Brucella, Burkholderia, Calymmatobacterium, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Coxiella, Cutibacterium, Ehrlichia, Enterobacter, Enterococcus, Escherichia, Francisella, Fusobacterium, Gardnerella, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Legionella, Listeria, Methanobacterium, Microbacterium, Micrococcus, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Pasteurella, Pediococcus,Bacteria of a species selected from the group consisting of Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, Pseudomonas, Rhizobium, Rickettsia, Rochalimaea, Rothia, Salmonella, Serratia, Shigella, Sarcina, Spirillum, Spirochaetes, Staphylococcus, Stenotrophomonas, Streptobacillus, Streptococcus, Tetragenococcus, Treponema, Vibrio, Viridans, Walbachia, and Yersinia, preferably Acetobacter aurantius, Acinetobacter baumannii, Actinomyces israelii, Agrobacterium radiobacter, Agrobacterium tumefaciens, Anaplasma phagocytophilum, Azorhizobium caulinodans, Azotobacter vinelandii, Bacillus anthracis, Bacillus brevis, Bacillus cereus, Bacillus fusiformis,Bacillus licheniformis, Bacillus megaterium, Bacillus mycoides, Bacillus stearothermophilus, Bacillus subtilis, Bacillus Thuringiensis, Bacteroides fragilis, Bacteroides gingivalis, Bacteroides melaninogenicus, Bartonella henselae, Bartonella Quintana, Bordetella bronchiseptica, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella melitensis, Brucella suis, Burkholderia mallei, Burkholderia pseudomallei, Burkholderia cepacia, Calymmatobacterium granulomatis, Campylobacter coli, Campylobacter fetus, Campylobacter jejuni, Campylobacter pylori, Chlamydia trachomatis, Chlamydophila pneumoniae,Psittacosis Chlamydia (Chlamydophila psittaci), Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Corynebacterium fusiforme, Coxiella burnetii, Cutibacterium acnes, Cutibacterium avidum, Cutibacterium granulosum, Cutibacterium namnetense, Cutibacterium humerusii, Ehrlichia chaffeensis, Enterobacter cloacae, Enterococcus avium, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus galllinarum, Enterococcus maloratus, Escherichia coli Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Haemophilus ducreyi, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus pertussis, Haemophilus vaginalis, Helicobacter pylori, Klebsiella pneumoniae, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactococcus lactis, Legionella pneumophila, Listeria monocytogenes, Methanobacterium extroquens, Microbacterium multiforme, Micrococcus luteus, Moraxella catarrhalis, Mycobacterium avium, Mycobacterium bovis, Mycobacterium diphtheriae, Mycobacterium intracellulare, Mycobacterium leprae, Mycobacterium lepraemurium, MycobacteriumPhlei), Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycoplasma fermentans, Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma penetrans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Pasteurella tularensis, Peptostreptococcus, Porphyromonas gingivalis, Prevotella melaninogenica, Propionibacterium acnes, Pseudomonas aeruginosa, Rhizobium radiobacter, Rickettsia prowazekii, Rickettsia psittaci, Rickettsia quintana, Rickettsia rickettsii, Rickettsia trachomae, Rochalimaea henselae, Rochalimaea quintana, Rothia dentocariosa, Salmonella enteritidis, Salmonella typhi, Salmonella typhimurium (SalmonellaSalmonella typhimurium, Serratia marcescens, Shigella dysenteriae, Spirillum volutans, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus avium, Streptococcus bovis, Streptococcus cricetus, Streptococcus faceium, Streptococcus faecalis, Streptococcus ferus, Streptococcus gallinarum, Streptococcus lactis, Streptococcus mitior, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus rattus, Streptococcus salivarius, Streptococcus sanguis, Streptococcus sobrinus, Treponema pallidum, Treponema denticola, Vibrio choleraederived from bacteria of a genus selected from the group consisting of Vibrio cholerae, Vibrio comma, Vibrio parahaemolyticus, Vibrio vulnificus, Viridans streptococci, Wolbachia, Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis.

[0051] In certain embodiments, the antigenic prokaryotic polypeptide is derived from bacteria of the genus Borrelia, preferably selected from B. burgdorferi, afzelii, garinii, bavariensis, mayonii, spielmanii, lusitaniae, bissettii, and / or valaisiana.

[0052] In certain embodiments, the antigenic prokaryotic polypeptide is OspA or a fragment or variant thereof, and OspA or a fragment or variant thereof comprises at least 5 amino acids. Preferably, the antigenic prokaryotic polypeptide is (a) an amino acid sequence derived from OspA ST1, preferably having at least 85% identity with the sequence

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0053] In certain embodiments, the antigen prokaryotic polypeptide comprises at least one mutated glycosylation site, preferably at least one mutated N-linked glycosylation site.

[0054] In certain embodiments, the polynucleotide sequence of the nucleic acid is codon-optimized.

[0055] In certain embodiments, the ORF is codon-optimized.

[0056] In certain embodiments, the polynucleotide sequence encoding at least one viral secretion signal peptide is codon-optimized.

[0057] In certain embodiments, the polynucleotide sequence encoding at least one TMB is codon-optimized.

[0058] In certain embodiments, the nucleic acid is DNA.

[0059] In certain embodiments, the nucleic acid is messenger RNA (mRNA), and in particular, the mRNA can be non-replicating mRNA, self-replicating mRNA, or trans-replicating mRNA.

[0060] In certain embodiments, the mRNA comprises at least one 5' untranslated region (5'UTR), at least one 3' untranslated region (3'UTR), and / or at least one polyadenylation (poly(A)) sequence.

[0061] In certain embodiments, the mRNA comprises at least one chemical modification.

[0062] In certain embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the mRNA are chemically modified.

[0063] In certain embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the ORF are chemically modified.

[0064] In certain embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-1-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxypseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0065] In certain embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0066] In certain embodiments, the chemical modification is N1-methylpseudouridine.

[0067] In one aspect, the present disclosure provides a composition comprising at least one of the above nucleic acids.

[0068] In certain embodiments, the composition further comprises lipid nanoparticles (LNP). In certain embodiments, the nucleic acid is encapsulated in the LNP.

[0069] In certain embodiments, the LNP comprises at least one cationic lipid. In certain embodiments, the cationic lipid is biodegradable. In certain embodiments, the cationic lipid is not biodegradable. In certain embodiments, the cationic lipid is cleavable. In certain embodiments, the cationic lipid is not cleavable. In certain embodiments, the cationic lipid is selected from the group consisting of OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14, SM-102, and ALC-0315.

[0070] In certain embodiments, the LNP further comprises a polyethylene glycol (PEG) conjugate (PEGylated) lipid, a cholesterol-based lipid, and a helper lipid.

[0071] In certain embodiments, the LNP comprises a cationic lipid in a molar ratio of -35% to 55%; a polyethylene glycol (PEG) conjugate (PEGylated) lipid in a molar ratio of -0.25% to 2.75%; a cholesterol-based lipid in a molar ratio of -20% to 45%; and a helper lipid in a molar ratio of -5% to 35%, where all of the molar ratios are relative to the total lipid content of the LNP.

[0072] In certain embodiments, the LNP comprises a cationic lipid in a molar ratio of -40%; a PEGylated lipid in a molar ratio of -1.5%; a cholesterol-based lipid in a molar ratio of -28.5%; and a helper lipid in a molar ratio of -30%.

[0073] In certain embodiments, the LNP comprises a cationic lipid in a molar ratio of -45 to 50%; a PEGylated lipid in a molar ratio of -1.5 to 1.7%; a cholesterol-based lipid in a molar ratio of -38 to 43%; and a helper lipid in a molar ratio of -9 to 10%.

[0074] In certain embodiments, the PEGylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000) or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0075] In certain embodiments, the cholesterol-based lipid is cholesterol.

[0076] In certain embodiments, the helper lipid is 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0077] In certain embodiments, the LNP comprises a cationic lipid selected from the group consisting of OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, and GL-HEPES-E3-E12-DS-3-E14 at a molar ratio of -40%; DMG-PEG2000 at a molar ratio of -1.5%; cholesterol at a molar ratio of -28.5%; and -DOPE at a molar ratio of -30%.

[0078] In certain embodiments, the LNP comprises SM-102 at a molar ratio of -50%; DMG-PEG2000 at a molar ratio of -1.5%; cholesterol at a molar ratio of -38.5%; and DSPC at a molar ratio of -10%.

[0079] In certain embodiments, the LNP comprises ALC-0315 at a molar ratio of -46.3%; ALC-0159 at a molar ratio of -1.6%; cholesterol at a molar ratio of -42.7%; and DSPC at a molar ratio of -9.4%.

[0080] In certain embodiments, the LNP comprises ALC-0315 at a molar ratio of -47.4%; ALC-0159 at a molar ratio of -1.7%; cholesterol at a molar ratio of -40.9%; and DSPC at a molar ratio of -10%.

[0081] In certain embodiments, the LNPs have an average diameter of 30 nm to 200 nm.

[0082] In certain embodiments, the LNPs have an average diameter of 80 nm to 150 nm.

[0083] In certain embodiments, the composition comprises LNPs at 1 mg / mL to 10 mg / mL.

[0084] In certain embodiments, the LNPs comprise 1 to 20 nucleic acid molecules, preferably mRNA molecules.

[0085] In certain embodiments, the composition is formulated for intramuscular, intranasal, intravenous, subcutaneous, or intradermal administration.

[0086] In certain embodiments, the composition comprises phosphate buffered saline.

[0087] In certain embodiments, the composition is a pharmaceutical composition, such as an immunogenic composition or a vaccine, particularly an mRNA vaccine.

[0088] In another aspect, the disclosure provides a nucleic acid or composition for use in inducing an immune response in a subject in need thereof.

[0089] In another aspect, the disclosure provides a nucleic acid or composition for use in treating or preventing a prokaryotic infection in a subject in need thereof.

[0090] In another aspect, the disclosure provides a method of secreting an antigenic prokaryotic polypeptide in a host cell, the method comprising administering the nucleic acid or composition as described above to the host cell.

[0091] In another aspect, the disclosure provides a method of displaying an antigenic prokaryotic polypeptide on the surface of a host cell, the method comprising administering the nucleic acid or composition as described above to the host cell.

[0092] In another aspect, the present disclosure provides a kit comprising a container containing a single-use or multiple-use dosage of the above nucleic acid or composition, and optionally, the container is a vial or a filled syringe or a syringe.

Brief Description of the Drawings

[0093]

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Mode for Carrying Out the Invention

[0094] The present disclosure is directed, inter alia, to nucleic acid (e.g., mRNA) compositions encoding antigen prokaryotic polypeptides linked to one or both of a viral secretion signal peptide sequence and a transmembrane domain (TMB), and vaccination methods using the same.

[0095] I. Definitions Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Exemplary methods and materials are described below, but methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. In general, the terms and techniques related to cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal chemistry, and pharmaceutical chemistry, as well as protein and nucleic acid chemistry and hybridization, and those used in these techniques described herein are well-known and commonly used in the art. Enzyme reactions and purification techniques are performed according to the manufacturer's specifications, as commonly practiced in the art or as described herein. Further, unless the context dictates otherwise, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and the embodiments, the words "have" and "comprise", or variations thereof, such as "has", "having", "comprises", or "comprising", are understood to mean including the recited integer or group of integers, but not to mean excluding any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although many references are cited herein, this citation does not constitute an admission that any of these references form a part of the common general knowledge in the art.

[0096] It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a nucleotide sequence" is understood to represent one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more", and "at least one" can be used interchangeably herein.

[0097] Furthermore, "and / or" as used herein should be regarded as a specific disclosure of each of two recited features or components, regardless of the presence or absence of the other. Thus, the term "and / or" as used in phrases such as "A and / or B" is intended herein to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0098] It is understood that whenever an aspect is described herein in the language of "comprising", other similar aspects defined in terms of the terms "consisting of" and / or "consisting essentially of" are also provided.

[0099] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press can provide one of ordinary skill in the art with many general dictionaries of the terms used in this disclosure.

[0100] Units, prefixes, and symbols are shown in the form approved by the International System of Units (SI). Numerical ranges include the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in the amino- to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure. Accordingly, the terms defined immediately below are more fully defined by reference to the entire specification.

[0101] The terms "about" or "approximately" are used herein to mean about, roughly, around, or in the range of. When the term "about" is used with a numerical range, it modifies that range by extending the boundaries above and below the recited numerical values. Generally, the term "about" can modify the numerical values above and below the recited value by, for example, a difference of up to or down to (higher or lower) 10 percent. In some embodiments, the term can indicate a deviation of ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, or ±0.01% from the recited value. In some embodiments, "about" indicates a deviation of ±10% from the recited value. In some embodiments, "about" indicates a deviation of ±5% from the recited value. In some embodiments, "about" indicates a deviation of ±4% from the recited value. In some embodiments, "about" indicates a deviation of ±3% from the recited value. In some embodiments, "about" indicates a deviation of ±2% from the recited value. In some embodiments, "about" indicates a deviation of ±1% from the recited value. In some embodiments, "about" indicates a deviation of ±0.9% from the recited value. In some embodiments, "about" indicates a deviation of ±0.8% from the recited value. In some embodiments, "about" indicates a deviation of ±0.7% from the recited value. In some embodiments, "about" indicates a deviation of ±0.6% from the recited value. In some embodiments, "about" indicates a deviation of ±0.5% from the recited value. In some embodiments, "about" indicates a deviation of ±0.4% from the recited value. In some embodiments, "about" indicates a deviation of ±0.3% from the recited value. In some embodiments, "about" indicates a deviation of ±0.1% from the recited value. In some embodiments, "about" indicates a deviation of ±0.05% from the recited value. In some embodiments, "about" indicates a deviation of ±0.01% from the recited value.

[0102] As used herein, the terms "messenger RNA" or "mRNA" refer to a polynucleotide encoding at least one polypeptide. The mRNA used herein encompasses both modified and unmodified RNA. The mRNA may contain one or more coding regions and non-coding regions. The coding region is alternatively referred to as an open reading frame (ORF). Non-coding regions of the mRNA include a 5' cap, 5' untranslated region (UTR), 3' UTR, and polyA tail. The mRNA can be purified from a natural source, produced using a recombinant expression system (e.g., in vitro transcription), and optionally purified or chemically synthesized.

[0103] As used herein, the terms "open reading frame", "ORF", or "coding region" refer to a polynucleotide sequence that starts with a start codon (e.g., ATG), ends with a stop codon (e.g., TAA, TAG, or TGA), and has no other stop codons in between, and encodes a protein (e.g., an antigenic prokaryotic polypeptide).

[0104] As used herein, the terms "viral secretion signal peptide" or "SS" refer to an amino acid sequence derived from a virus that induces a polypeptide sequence to be conjugated through the cell secretion pathway. A polypeptide having an SS sequence passes through one or more organelles in the cell until it is secreted outside the cell through secretory vesicles.

[0105] As used herein, the terms "transmembrane domain" or "TMB" refer to an amino acid sequence having transmembrane characteristics. The TMB induces an anchor for a polypeptide bound to the cell membrane.

[0106] The present disclosure also includes fragments or variants of polypeptides, and any combination thereof. The terms "fragment" or "variant" when referring to an antigenic prokaryotic polypeptide of the present disclosure include any polypeptide that retains at least a portion of the properties of the reference polypeptide (e.g., the specific antigenic properties of the polypeptide or the ability of the polypeptide to induce antibody binding). Fragments of a polypeptide include N-terminal and / or C-terminal truncated fragments, e.g., C-terminal fragments and N-terminal fragments, as well as deletion fragments, but do not include the naturally occurring full-length polypeptide (or mature polypeptide). Deletion fragments refer to polypeptides in which one or more internal amino acids are deleted from the full-length polypeptide. Variants of a polypeptide include the above-mentioned fragments, and further include polypeptides having an altered amino acid sequence resulting from amino acid substitutions, deletions, or insertions. Variants may or may not occur naturally. Non-naturally occurring variants can be produced using mutagenesis techniques known in the art. Variant polypeptides may include conservative or non-conservative amino acid substitutions, deletions, or additions. Such mutations (i.e., truncations and / or amino acid substitutions, deletions, or insertions) can occur at either the amino acid level or, correspondingly, at the nucleic acid level.

[0107] "Conservative amino acid substitutions" are substitutions where an amino acid residue has been replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, when an amino acid in a polypeptide has been replaced with another amino acid of the same side chain family, the substitution is considered conservative. In another embodiment, an amino acid chain can be conservatively replaced with a structurally similar chain having a different order and / or composition of side chain family members.

[0108] As used herein, the terms "linked" or "coupled" each refer to a first amino acid sequence or nucleotide sequence covalently or non-covalently attached to a second amino acid sequence or nucleotide sequence (e.g., a transmembrane domain amino acid sequence linked to a secretory signal amino acid sequence and / or an antigen prokaryotic polypeptide amino acid sequence). The first amino acid sequence or nucleotide sequence can be directly attached or juxtaposed to the second amino acid sequence or nucleotide sequence, or an intervening sequence can covalently attach the first sequence to the second sequence. The term "linked" not only means that the first amino acid sequence is fused to the second amino acid sequence at the C-terminus or N-terminus, but also includes the entire first amino acid sequence (or second amino acid sequence) being inserted within any two amino acids in the second amino acid sequence (or first amino acid sequence, respectively). In one embodiment, the first amino acid sequence can be linked to the second amino acid sequence by a peptide bond or a linker. The first nucleotide sequence can be linked to the second nucleotide sequence by a phosphodiester bond or a linker. The linker can be a peptide or polypeptide (in the case of a polypeptide chain), or a nucleotide or nucleotide chain (in the case of a nucleotide chain), or any chemical moiety (in the case of both a polypeptide chain and a polynucleotide chain). The term "linked" is also denoted by a hyphen (-).

[0109] As used herein, the term "glycosylation" refers to the addition of sugar units to a protein.

[0110] As used herein, the term "N-glycan" refers to a sugar chain attached to a protein at the amide nitrogen of the N (asparagine) residue of the protein. Thus, an N-glycan is formed by the process of N-glycosylation. This glycan can be a polysaccharide.

[0111] As used herein, the term "immune response" refers to the response of cells of the immune system, such as B cells, T cells, dendritic cells, macrophages or polymorphonucleocytes, to a stimulus, such as an antigen or a vaccine. The immune response can include any cell of the body involved in the host defense response, including, for example, epithelial cells that secrete interferons or cytokines. Immune responses include, but are not limited to, natural and / or adaptive immune responses.

[0112] As used herein, "protective immune response" refers to an immune response that protects a subject from infection (e.g., prevents infection or prevents the development of a disease associated with the infection). Methods for measuring immune responses are well known in the art and include, for example, measuring the proliferation and / or activation of lymphocytes (e.g., B cells or T cells), measuring the secretion of cytokines or chemokines, measuring inflammation, and measuring antibody production.

[0113] As used herein, "antibody response" is an immune response in which antibodies are produced.

[0114] As used herein, "antigen" refers to an agent that, when exposed to or administered to an organism, elicits an immune response, and / or an agent that binds to a T cell receptor (e.g., when presented by an MHC molecule) or an antibody (e.g., produced by a B cell). In some embodiments, the antigen elicits a humoral response (e.g., including the production of antigen-specific antibodies) in an organism. Alternatively or additionally, in some embodiments, the antigen elicits a cellular response (e.g., involving T cells whose receptors specifically interact with the antigen) in an organism. A particular antigen may elicit an immune response in one or some members of a target organism (e.g., mouse, rabbit, primate, human), but not in all members of the target species. In some embodiments, the antigen elicits an immune reaction in at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the members of a target species. In some embodiments, the antigen may or may not bind to an antibody and / or a T cell receptor and elicit a specific physiological response in an organism. In some embodiments, for example, the antigen can bind to an antibody and / or a T cell receptor in vitro, regardless of whether such an interaction occurs in vivo. In some embodiments, the antigen reacts with the products of specific humoral or cellular immunity. Examples of antigens include prokaryotic antigen polypeptides encoded by the mRNAs described herein (e.g., OspA ST1 and ST2). "Prokaryotic antigen" or "antigenic prokaryotic polypeptide" includes any antigenic polypeptide derived from a prokaryote that can elicit an immune response.

[0115] As used herein, "adjuvant" refers to a substance or vehicle that enhances the immune response to an antigen. Examples of adjuvants include, but are not limited to, suspensions of minerals (e.g., alum, aluminum hydroxide, or phosphate) to which the antigen adsorbs; water-in-oil or oil-in-water emulsions (e.g., Freund's incomplete adjuvant) in which the antigen solution is emulsified in mineral oil or water. Killed mycobacteria may be included to further enhance antigenicity (e.g., Freund's complete adjuvant). Immunostimulatory oligonucleotides (e.g., CpG motifs) can also be used as adjuvants (see, e.g., U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199). Biological molecules, such as Toll-like receptor (TLR) agonists and costimulatory molecules, can also be mentioned as adjuvants. As used herein, "subject" refers to any member of the animal kingdom. In some embodiments, "subject" refers to a human. In some embodiments, "subject" refers to a non-human animal. In certain embodiments, the non-human subject is a mammal, such as a rodent, mouse, rat, rabbit, monkey, llama, horse, dog, cat, cow, sheep, goat, primate, pig. In some embodiments, when the subject is a human, the terms "individual" or "patient" are used and are intended to be interchangeable with "subject".

[0116] As used herein, the terms "prevent", "preventing", "prevention", or "prophylaxis" (and their grammatical variations) refer to partially or completely inhibiting the development of one or more symptoms or features of a particular infection, disease, disorder, and / or condition.

[0117] As used herein, the terms "treating," "treatment," "treat," "therapy," or "therapeutic" (and their grammatical variations) refer to partially or completely alleviating, ameliorating, improving, relieving, inhibiting the progression of, and / or reducing the severity of one or more symptoms or features of an infection, disease, disorder, and / or medical condition.

[0118] As used herein, the term "effective amount" refers to an amount (e.g., of a nucleic acid or composition) sufficient to produce a beneficial or desired result. An effective amount can be administered in one or more administrations, applications, or dosages and is not intended to be limited to a particular formulation or route of administration.

[0119] The term "effective amount" includes, for example, "therapeutically effective amount" and / or "prophylactically effective amount."

[0120] As used herein, the phrase "therapeutically effective amount" refers to an amount (e.g., of a nucleic acid or composition) effective to produce some desired therapeutic effect in the treatment of an infection, disease, disorder, and / or medical condition at a reasonable benefit / risk ratio applicable to any medical treatment.

[0121] As used herein, the phrase "prophylactically effective amount" refers to an amount (e.g., of a nucleic acid or composition) effective to produce some desired prophylactic effect in the prevention of an infection, disease, disorder, and / or medical condition at a reasonable benefit / risk ratio applicable to any medical treatment.

[0122] As used herein, the terms "vaccinating" or "vaccination" refer to the administration of a composition intended, for example, to generate an immune response against an agent that causes a disease. Vaccination can be administered before, during, and / or after exposure to the agent that causes the disease, and / or before, during, and / or after the occurrence of one or more symptoms, and in some embodiments, before, during, and / or immediately after exposure to the agent. In some embodiments, vaccination includes multiple administrations of the vaccine composition at appropriate time intervals.

[0123] The present disclosure describes nucleic acid sequences (e.g., DNA and RNA sequences) and amino acid sequences that have a certain degree of identity to a given nucleic acid sequence or amino acid sequence (reference sequence), respectively.

[0124] "Sequence identity" between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences.

[0125] The terms "% identical", "% identity" or similar terms are intended to refer specifically to the percentage of nucleotides or amino acids that are identical in the optimal alignment between the sequences to be compared. The percentage is purely statistical and the differences between the two sequences may be randomly distributed over the entire length of the sequences to be compared, but do not necessarily have to be distributed. The comparison of two sequences is usually done by comparing the sequences with respect to segments or "windows of comparison" after optimal alignment to identify local regions of the corresponding sequences. The optimal alignment for comparison can be done manually or using local homology algorithms such as those by Smith and Waterman, 1981, Ads App. Math. 2, 482, Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, similarity search algorithms such as those by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or using computer programs that use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA of the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0126] The percentage of identity is obtained by determining the number of positions at which the sequences to be compared are identical, dividing this number by the number of positions to be compared (e.g., the number of positions in the reference sequence), and multiplying the result by 100.

[0127] In some embodiments, the degree of identity is given for regions that are at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the full length of the reference sequence. For example, if the reference nucleic acid sequence is 200 nucleotides in length, the degree of identity is for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, and in some embodiments is given for contiguous nucleotides. In some embodiments, the degree of identity is given for the full length of the reference sequence.

[0128] A nucleic acid sequence or amino acid sequence having a particular degree of identity to a given nucleic acid sequence or amino acid sequence may have at least one functional property of the given sequence, e.g., in some instances is functionally equivalent to the given sequence. In some embodiments, a nucleic acid sequence or amino acid sequence having a particular degree of identity to a given nucleic acid sequence or amino acid sequence is functionally equivalent to the given sequence.

[0129] As used herein, the term "kit" refers to a packaged set of related components, e.g., one or more compounds or compositions and one or more related materials, e.g., solvents, solutions, buffers, instructions, or desiccants.

[0130] II. Secretion signal sequence The use of a viral secretory signal peptide (SS) sequence conjugated to an antigenic prokaryotic polypeptide can provide numerous advantages for vaccination. From mRNA, especially when expressed in eukaryotic cells, the SS-prokaryotic antigen fusion protein can have increased extracellular expression compared to the prokaryotic antigen without the SS sequence. The increased extracellular expression can promote higher immunogenicity and, by extension, better vaccine efficacy.

[0131] Viral SS sequences can be found in publicly accessible databases (e.g., NCBI or UniProt databases) that contain annotated viral polypeptide sequences and identify experimentally verified start and end positions of the SS.

[0132] In certain embodiments, the location of the SS cleavage site for an SS sequence and a given known input polypeptide sequence can be predicted by using the SinalP algorithm. The SinalP algorithm (more specifically SignalP v6.0) is described in more detail in Armenteros et al. (Nature Biotechnology. 37:420 - 423. 2019), Teufel et al. (Nature Biotechnology. 40:1023 - 1025. 2022) and https: / / services.healthtech.dtu.dk / services / SignalP-6.0 / , each of which is incorporated herein by reference in its entirety. The strength of the prediction is evaluated based on a cumulative rank score that takes into account the likelihood of detecting standard features of the signal sequence (SS likelihood score) and the likelihood of cleavage at the cleavage site (cleavage probability score).

[0133] In certain embodiments, the viral secretion signal peptide is derived from a viral sequence in a virus capable of infecting humans. The phrases "influenza", "SARS CoV-2", "varicella-zoster virus (VZV)", "measles", "rubella", "rabies", "Ebola", and "smallpox" preceding the phrase "secretion signal peptide sequence" indicate that the secretion signal peptide is derived from the virus corresponding to its name.

[0134] In certain embodiments, the viral secretion signal peptide is derived from a viral sequence selected from the group consisting of an influenza secretion signal peptide sequence, a SARS CoV-2 secretion signal peptide sequence, a varicella-zoster virus (VZV) secretion signal peptide sequence, a measles secretion signal peptide sequence, a rubella secretion signal peptide sequence, a mumps secretion signal peptide sequence, an Ebola secretion signal peptide sequence, a rabies secretion signal peptide sequence, and a smallpox secretion signal peptide sequence. These specific signal peptides are derived from viral sequences in viruses administered to humans as vaccines (attenuated, inactivated, or mRNA) and have demonstrated a strong safety profile.

[0135] In certain embodiments, the viral secretion signal peptide is selected from the group consisting of an influenza hemagglutinin (HA) secretion signal peptide sequence, a SARS CoV-2 spike secretion signal peptide sequence, a VZV gB secretion signal peptide sequence, a VZV gE secretion signal peptide sequence, a VZV gI secretion signal peptide sequence, a VZV gK secretion signal peptide sequence, a measles F-protein secretion signal peptide sequence, a rubella E1-protein secretion signal peptide sequence, a rubella E2-protein secretion signal peptide sequence, a mumps F-protein secretion signal peptide sequence, an Ebola GP-protein secretion signal peptide sequence, a rabies virus glycoprotein (rabies G) secretion signal peptide sequence, and a smallpox 6 kDa IC-protein secretion signal peptide sequence.

[0136] In certain embodiments, the viral secretion signal peptide comprises a HA transmembrane domain sequence from influenza A or influenza B virus, preferably a HA secretion signal peptide sequence from influenza A virus.

[0137] Exemplary viral secretion signal peptide amino acid sequences of the present disclosure are shown in Table 1 below. Exemplary viral secretion signal peptide amino acid sequences derived from influenza A or B virus of the present disclosure are shown in Table 2 below.

[0138] [Table 1]

[0139] [Table 2]

[0140] [Table 3]

[0141] [Table 4]

[0142] [Table 5]

[0143] [Table 6]

[0144] [Table 7]

[0145] III. Transmembrane Domain Incorporating a transmembrane domain (TMB) into a construct, particularly an mRNA construct used as a vaccine antigen, may also include an SS, thereby producing an SS-antigen-TMB fusion protein (optionally, after cleavage of the SS in the mature protein, an antigen-TMB protein), and localizing the antigen to the cell surface by immobilizing it on the membrane. This can reduce the intracellular localization of the antigen and further promote higher immunogenicity compared to an antigen that does not contain the TMB sequence. The addition of TMB may particularly enable an increase in the humoral (B cell) response against the antigen. This may be useful for prokaryotic antigens, but can also be used for other antigens (e.g., viral antigens). The addition of TMB can be used in conjunction with an antigen derived from a membrane protein or a protein that is not a membrane protein (e.g., a secreted protein or an intracellular protein). The addition of any of the more specific TMBs described herein may be particularly useful for antigens derived from proteins that are not membrane proteins, i.e., proteins that do not naturally contain TMB (or the like).

[0146] TMB can be derived from any known TMB in the art, including but not limited to TMB derived from eukaryotic transmembrane proteins (such as mammalian transmembrane proteins like human transmembrane proteins), prokaryotic transmembrane proteins, and viral transmembrane proteins. TMB can be further identified through, for example, the in-silico prediction algorithms in the TMHMM prediction method described in Krogh et al. (J Mol Biol. 305(3):567-580. 2001) and https: / / services.healthtech.dtu.dk / services / TMHMM-2.0 / , each of which is hereby incorporated by reference in its entirety. Some features of TMB are described in more detail in Albers et al. (Chapter 2 - cell membrane structures and functions. Basic Neurochemistry eighth edition. Pages 26-39. 2012), which is hereby incorporated by reference. TMB typically, but not exclusively, consists mainly of non-polar (hydrophobic) amino acid residues and can cross the lipid bilayer one or several times. Those skilled in the art are well aware of methods for determining the hydrophobicity of amino acids. See Simm et al. (2016), Biol Res., 49(1):31; Wimlet and White (1996), Nat Struct Biol., 3(10):842-848; https: / / blanco.biomol.uci.edu / hydrophobicity_scales.html; and https: / / www.cgl.ucsf.edu / chimera / docs / UsersGuide / midas / hydrophob.html.

[0147] TMB usually contains alpha helices, and each helix contains 18-21 amino acids sufficient to span the lipid bilayer. Thus, in certain embodiments, the transmembrane domain comprises one or more alpha helices.

[0148] In certain embodiments, the transmembrane domain is derived from an integral membrane protein, as further defined herein. In Albers et al., an "integral membrane protein" (also known as an intrinsic membrane protein) is a membrane protein that is permanently associated with the lipid membrane. In certain embodiments, the transmembrane domain is derived from an integral polytopic protein. An integral polytopic protein spans the entire membrane. In certain embodiments, the transmembrane domain is derived from a single-pass (trans) membrane protein, more specifically, for example, a type I or type II double membrane protein. A single-pass membrane protein crosses the membrane only once (i.e., a double membrane protein), and a multi-pass membrane protein crosses the membrane several times, being woven in and out. Single-pass transmembrane proteins can be classified into type I, where their carboxyl termini are oriented towards the cytosol, or type II, where their amino termini face the cytosol. In certain embodiments, the transmembrane domain is derived from an integral monotopic protein. An integral monotopic protein is only associated from one side of the membrane and does not span the lipid bilayer completely.

[0149] In certain embodiments, the transmembrane domain is derived from a non-human sequence. In certain embodiments, the antigenic prokaryotic polypeptide is derived from a prokaryotic transmembrane protein, and the transmembrane domain is the transmembrane domain of the prokaryotic transmembrane protein.

[0150] In certain embodiments, the transmembrane domain is derived from a viral sequence. The phrases "influenza", "SARS CoV-2", "varicella zoster virus (VZV)", "measles", "rubella", "rabies", "ebola", and "smallpox" preceding the phrase "transmembrane domain" indicate that the transmembrane domain is derived from the virus corresponding to its name.

[0151] In certain embodiments, the transmembrane domain is derived from a viral transmembrane domain sequence selected from the group consisting of an influenza transmembrane domain sequence, a SARS COV-2 transmembrane domain sequence, a varicella-zoster virus (VZV) transmembrane domain sequence, a measles transmembrane domain sequence, a rubella transmembrane domain sequence, a mumps transmembrane domain sequence, a rabies transmembrane domain sequence, and an Ebola transmembrane domain sequence. These particular transmembrane domains are derived from viral sequences in viruses administered to humans as vaccines (attenuated, inactivated, or mRNA) and have demonstrated a strong safety profile.

[0152] In certain embodiments, the transmembrane domain is selected from the group consisting of an influenza hemagglutinin (HA) transmembrane domain sequence, a SARS COV-2 spike transmembrane domain sequence, a VZV gB transmembrane domain sequence, a VZV gE transmembrane domain sequence, a VZV gI transmembrane domain sequence, a VZV gK transmembrane domain sequence, a measles F protein transmembrane domain sequence, a rubella E1 protein transmembrane domain sequence, a rubella E2 protein domain sequence, a mumps F protein transmembrane domain sequence, a rabies virus glycoprotein (rabies G) transmembrane domain sequence, and an Ebola GP protein transmembrane domain sequence.

[0153] In certain embodiments, the transmembrane domain comprises an HA transmembrane domain sequence derived from influenza A or influenza B, preferably an HA transmembrane domain sequence derived from influenza A.

[0154] Exemplary viral transmembrane domain amino acid sequences of the present disclosure are shown in Table 3 below.

[0155]

Table 8

[0156] In certain embodiments, in certain embodiments, the SS sequence is located at the N-terminus of the antigenic prokaryotic polypeptide.

[0157] In certain embodiments, the SS sequence is located at the C-terminus of the antigen prokaryotic polypeptide.

[0158] In certain embodiments, the TMB sequence is located at the N-terminus of the antigen prokaryotic polypeptide.

[0159] In certain embodiments, the TMB sequence is located at the C-terminus of the antigen prokaryotic polypeptide.

[0160] In certain embodiments, the SS amino acid sequence is encoded by a codon-optimized polynucleotide sequence.

[0161] In certain embodiments, the TMB amino acid sequence is encoded by a codon-optimized polynucleotide sequence.

[0162] IV. Linker In certain embodiments of the present disclosure, the viral secretion signal peptide (SS) sequence or transmembrane domain (TMB) is directly fused to the antigen prokaryotic polypeptide (i.e., there is no linker such as an amino acid linker connecting the SS sequence or TMB to the antigen prokaryotic polypeptide).

[0163] In other embodiments, the SS sequence and TMB of the present disclosure are optionally linked to the antigen prokaryotic polypeptide using a linker. In certain embodiments, the linker is an amino acid linker. In certain embodiments, the amino acid linker is 1 to 10 amino acids in length (e.g., the amino acid linker has a length of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids).

[0164] Exemplary examples of linkers include glycine polymers (Gly)n, where n is at least 1, 2, 3, 4, 5, 6, 7, or 8; glycine-serine polymers (GlySer)n, where n is at least 1, 2, 3, 4, 5, 6, 7, or 8; glycine-alanine polymers; alanine-serine polymers; and other flexible linkers known in the art.

[0165] Glycine and glycine-serine polymers are relatively unstructured and flexible and can thus potentially function as neutral tethers between the SS sequence and / or TMB and the antigen prokaryotic polypeptide. In certain embodiments, the linker is SGS or GSG.

[0166] Other exemplary linkers include, but are not limited to, the following amino acid sequences: GGG; DGGGS (SEQ ID NO: 81); TGEKP (SEQ ID NO: 82) (Liu et al. Proc. Natl. Acad. Sci. 94:5525-5530. 1997); GGRR (SEQ ID NO: 92); (GGGGS)n (SEQ ID NO: 93), n = 1, 2, 3, 4, or 5 (Kim et al. Proc. Natl. Acad. Sci. 93:1156-1160. 1996); EGKSSGSGSESKVD (SEQ ID NO: 83) (Chaudhary et al. Proc. Natl. Acad. Sci. 87:1066-1070. 1990); KESGSVSSEQLAQFRSLD (SEQ ID NO: 84) (Bird et al. Science. 242:423-426. 1988), GGRRGGGS (SEQ ID NO: 85); LRQRDGERP (SEQ ID NO: 86); LRQKDGGGSERP (SEQ ID NO: 87); and GSTSGSGKPGSGEGSTKG (SEQ ID NO: 88) (Cooper et al. Blood. 101(4):1637-1644. 2003). Preferred linkers are shorter, for example, consisting of 3, 4, or 5 amino acids.

[0167] Further examples of linkers are provided by Chen et al. (Adv Drug Deliv Rev. 65(10):1357-1369. 2013), which is incorporated herein by reference.

[0168] V. Antigenic prokaryotic polypeptides The viral secretion signal peptide (SS) and / or transmembrane domain (TMD) of the present disclosure are linked to an antigenic prokaryotic polypeptide.

[0169] A. Prokaryotic genera In certain embodiments, the antigen prokaryotic polypeptide is from Acetobacter, Acinetobacter, Actinomyces, Aerococcus, Agrobacterium, Anaplasma, Azorhizobia, Azotobacter, Bacillus, Bacteroides, Bartonella, Bordetella, Borrelia, Brucella, Burkkolderia, Calymmatobacterium, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Coxiella, Cutibacterium, Ehrlichia, Enterobacter, Enterococcus, Escherichia, Francisella, Fusobacterium, Gardnerella, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Legionella, Listeria, Methanobacterium, Microbacterium, Micrococcus, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Pasteurella, Pediococcus,derived from bacteria of a genus selected from the group consisting of Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, Pseudomonas, Rhizobium, Rickettsia, Rochalimaea, Rothia, Salmonella, Serratia, Shigella, Sarcina, Spirillum, Spirochaetes, Staphylococcus, Stenotrophomonas, Streptobacillus, Streptococcus, Tetragenococcus, Treponema, Vibrio, Viridans, Walbachia, and Yersinia., In certain embodiments, the antigen prokaryotic polypeptide is Acetobacter aurantius, Acinetobacter baumannii, Actinomyces israelii, Agrobacterium radiobacter, Agrobacterium tumefaciens, Anaplasma phagocytophilum, Azorhizobium caulinodans, Azotobacter vinelandii, Bacillus anthracis, Bacillus brevis, Bacillus cereus, Bacillus fusiformis, Bacillus licheniformis, Bacillus megaterium, Bacillus mycoides, Bacillus stearothermophilus, Bacillus subtilis, Bacillus Thuringiensis, Bacteroides fragilis, Bacteroides gingivalis, Bacteroides melaninogenicus, Bartonella henselae, Bartonella Quintana, Bordetella bronchiseptica, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus (Brucellaabortus), Brucella melitensis, Brucella suis, Burkholderia mallei, Burkholderia pseudomallei, Burkholderia cepacia, Calymmatobacterium granulomatis, Campylobacter coli, Campylobacter fetus, Campylobacter jejuni, Campylobacter pylori, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Corynebacterium fusiforme, Coxiella burnetii, Cutibacterium acnes, Cutibacterium avidum, Cutibacterium granulosum, Cutibacterium namnetense, Cutibacterium humerusii, Ehrlichia chaffeensis (EhrlichiaChaffeensis), Enterobacter cloacae, Enterococcus avium, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus maloratus, Escherichia coli, Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Haemophilus ducreyi, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus pertussis, Haemophilus vaginalis, Helicobacter pylori, Klebsiella pneumoniae, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactococcus lactis, Legionella pneumophila, Listeria monocytogenes, Methanobacterium extroquens, Microbacterium multiforme, Micrococcus luteus (MicrococcusStaphylococcus aureus, Moraxella catarrhalis, Mycobacterium avium, Mycobacterium bovis, Mycobacterium diphtheriae, Mycobacterium intracellulare, Mycobacterium leprae, Mycobacterium lepraemurium, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycoplasma fermentans, Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma penetrans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Pasteurella tularensis, Peptostreptococcus, Porphyromonas gingivalis, Prevotella melaninogenica, Propionibacterium acnes, Pseudomonas aeruginosa, Rhizobium radiobacter, Rickettsia prowazekii, Rickettsia psittaci, Rickettsia quintana, Rickettsia rickettsii, Rickettsia trachomae, Rochalimaea henselae, Rochalimaea quintana, Rothia dentocariosa, Salmonella enteritidis, Salmonella typhi, Salmonella typhimurium, Serratia marcescens, SerratiaSerratia marcescens, Shigella dysenteriae, Spirillum volutans, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus avium, Streptococcus bovis, Streptococcus cricetus, Streptococcus faceium, Streptococcus faecalis, Streptococcus ferus, Streptococcus gallinarum, Streptococcus lactis, Streptococcus mitior, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus rattus, Streptococcus salivarius, Streptococcus sanguis, Streptococcus sobrinus, Treponema pallidum, Treponema denticola, Vibrio cholerae, Vibrio comma, Vibrio enteritisderived from a bacterium of a species selected from the group consisting of Vibrio parahaemolyticus, Vibrio vulnificus, Viridans streptococci, Wolbachia, Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis. In certain embodiments, the antigenic prokaryotic polypeptide is derived from a bacterium of the genus Borrelia, preferably selected from B. burgdorferi, afzelii, garinii, bavariensis, mayonii, spielmanii, lusitaniae, bissettii, and / or valaisiana.

[0170] B. glycosylation Glycosylation can occur in eukaryotic cells (but not in prokaryotic cells). In particular, N-linked glycosylation is the attachment of a glycan to the amide nitrogen of an asparagine (Asn; N) residue of a protein. The process of attachment results in a glycosylated protein. Glycosylation can occur at any asparagine residue in a protein that is accessible to and recognized by glycosylation enzymes after protein translation, and is most common at accessible asparagines that are part of the NXS / T motif, where the first amino acid residue following asparagine (X) can be any amino acid except proline, and the second amino acid residue following asparagine is serine or threonine. Non-human glycosylation patterns can make a polypeptide immunoreactive when used to induce antibodies. In addition, glycosylation of a polypeptide that is normally not glycosylated (such as an antigenic prokaryotic polypeptide) can alter its immunogenicity. For example, glycosylation can mask important immunogenic epitopes within a protein. Thus, to reduce or eliminate glycosylation, either asparagine residues or serine / threonine residues can be modified, for example, by substitution with another amino acid.

[0171] In certain embodiments, the antigenic prokaryotic polypeptide comprises at least one mutated glycosylation site, preferably at least one mutated N-linked glycosylation site and / or at least one O-linked glycosylation site. In some embodiments, one or more N-glycosylation sites in the antigenic prokaryotic polypeptide are removed. In some embodiments, removal of the N-glycosylation site reduces glycosylation of the antigenic prokaryotic polypeptide. In some embodiments, the antigenic prokaryotic polypeptide has reduced glycosylation compared to the native antigenic prokaryotic polypeptide. In some embodiments, removal of the N-glycosylation site eliminates N-glycosylation of the antigenic prokaryotic polypeptide.

[0172] In certain embodiments, the modification comprises substitution of one or more of the N, S, and T amino acids in the NXS / T sequence motif, where X corresponds to any amino acid other than proline (P). In some embodiments, the N, S, or T amino acid is substituted with a conservative amino acid substitution. In certain embodiments, the polynucleotide sequence encoding the antigenic prokaryotic polypeptide is codon-optimized.

[0173] C.OspA In certain embodiments, the antigenic prokaryotic polypeptide is OspA (outer surface protein A). In certain embodiments, OspA is preferably derived from OspA serotype (ST) 1, 2, 3, 4, 5, 6, and / or 7, more preferably from Borrelia burgdorferi strain B31 of serotype 1, Borrelia afzelii strain PKO of serotype 2, Borrelia garinii strain PBr of serotype 3, Borrelia bavariensis of serotype 4, Borrelia garinii of serotype 5, Borrelia garinii of serotype 6, or Borrelia garinii of serotype 7.

[0174] Exemplary amino acid sequences encoding the OspA protein of the present disclosure are shown in Table 4.

[0175] D.CAMP2 In certain embodiments, the antigenic prokaryotic polypeptide is a pore-forming toxin, preferably CAMP2 (Christie-Atkins-Munch-Peterson factor 2).

[0176] In certain embodiments, CAMP2 is preferably derived from bacteria of the genus Cutibacerium, more preferably from bacteria of the species Cutibacterium acnes (formally known as Propionibacterium acnes).

[0177] In certain embodiments, the CAMP2 polypeptide comprises an amino acid sequence.

Chemical formula

[0178] An exemplary amino acid sequence encoding the C. acnes CAMP2 factor protein of the present disclosure is shown in Table 10 below.

[0179] E.PITP In certain embodiments, the antigen prokaryotic polypeptide is a putative iron transport protein (PITP).

[0180] In certain embodiments, PITP is preferably derived from bacteria of the genus Cutibacerium, more preferably from bacteria of the species Cutibacterium acnes (formally known as Propionibacterium acnes).

[0181] In certain embodiments, the PITP polypeptide comprises an amino acid sequence.

Chemical formula

[0182] An exemplary amino acid sequence encoding the C. acnes PITP factor protein of the present disclosure is shown in Table 10 below.

[0183] VI. Lipid Nanoparticles (LNP) The LNPs of the present disclosure contain lipids of four categories: (i) ionizable lipids (e.g., cationic lipids); (ii) PEGylated lipids; (iii) cholesterol-based lipids, and (iv) helper lipids.

[0184] A. Ionizable lipids Ionizable lipids can be cationic lipids that facilitate mRNA encapsulation. Cationic lipids provide a positively charged environment at low pH to facilitate efficient encapsulation of negatively charged mRNA drug substances.

[0185] In some embodiments, the cationic lipid is OF-02:

Chemical formula

[0186] OF-02 is a non-degradable structural analog of OF-Deg-Lin. OF-Deg-Lin contains a diketopiperazine core and a degradable ester linkage for connecting the double unsaturated tail, while OF-02 contains a non-degradable 1,2-amino-alcohol linkage for connecting the same diketopiperazine core and double unsaturated tail (Fenton et al., Adv Mater. (2016) 28:2939; U.S. Patent No. 10,201,618). Lipid A, an exemplary LNP formulation herein, contains OF-2.

[0187] In some embodiments, the cationic lipid is cKK-E10 (Dong et al., PNAS (2014) 111(11):3955 - 60; U.S. Patent No. 9,512,073):

Chemical formula

[0188] Lipid B, an exemplary LNP formulation herein, contains cKK-E10.

[0189] In some embodiments, the cationic lipid is GL-HEPES-E3-E10-DS-3-E18-1 (2-(4-(2-(3-(bis((Z)-2-hydroxyoctadec-9-en-1-yl)amino)propyl)disulfanyl(ethyl)piperazin-1-yl)ethyl 4-(bis(2-hydroxydecyl)amino)butanoate), which is a HEPES-based disulfide cationic lipid having a piperazine core and has Formula III: [Chemical Formula]

[0190] Lipid C, an exemplary LNP formulation herein, contains GL-HEPES-E3-E10-DS-3-E18-1. Lipid C has the same composition as Lipid A or Lipid B but has a difference in the cationic lipid.

[0191] In some embodiments, the cationic lipid is GL-HEPES-E3-E12-DS-4-E10 (2-(4-(2-((3-(bis(2-hydroxydecyl)amino)butyl)disulfanyl)ethyl)piperazin-1-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butanoate), which is a HEPES-based disulfide cationic lipid having a piperazine core and has Formula IV: [Chemical Formula]

[0192] Lipid D, an exemplary LNP formulation herein, contains GL-HEPES-E3-E12-DS-4-E10. Lipid D has the same composition as Lipid A or Lipid B but has a difference in the cationic lipid.

[0193] In some embodiments, the cationic lipid is GL-HEPES-E3-E12-DS-3-E14 (2-(4-(2-((3-(bis(2-hydroxytetradecyl)amino)propyl)disulfanyl)ethyl)piperazin-1-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butanoate), which is a HEPES-based disulfide cationic lipid having a piperazine core and has the formula V:

Chemical formula

[0194] Lipid E, an exemplary LNP formulation herein, contains GL-HEPES-E3-E12-DS-3-E14. Lipid E has the same composition as Lipid A or Lipid B but has a difference in the cationic lipid.

[0195] The cationic lipids GL-HEPES-E3-E10-DS-3-E18-1 (III), GL-HEPES-E3-E12-DS-4-E10 (IV), and GL-HEPES-E3-E12-DS-3-E14 (V) can be synthesized according to the general procedure shown in Scheme 1.

[0196] Scheme 1: General synthetic scheme for lipids of formula (III), (IV), and (V)

Chemical formula

[0197] In some embodiments, the cationic lipid is MC3 having the formula VI:

Chemical formula

[0198] In some embodiments, the cationic lipid is SM-102 (9-heptadecanoyl 8-{ (2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate), which has the formula VII: [Chem.]

[0199] In some embodiments, the cationic lipid is ALC-0315 [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate), which has Formula VIII: [Chem.]

[0200] In some embodiments, the cationic lipid is cOrn-EE1, which has Formula IX: [Chem.]

[0201] In some embodiments, the cationic lipid is cKK-E10; OF-02; [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-yl] 4-(dimethylamino)butanoate (D-Lin-MC3-DMA); 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA); 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLin-DMA); bis((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); 9-heptadecanoyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102); [(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); [3-(dimethylamino)-2-[(Z)-octadec-9-enoyl]oxypropyl](Z)-octadec-9-enoate (DODAP); 2,5-bis(3-aminopropylamino)-N-[2-[di(heptadecyl)amino]-2-oxoethyl]pentanamide (DOGS); [(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(2R)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-yl] N-[2-(dimethylamino)ethyl]carbamate (DC-Chol); tetrakis(8-methylnonyl) 3,3’,3’’,3’’’-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate (306Oi10); decyl(2-(dioctylammonio)ethyl)phosphate (9A1P9); ethyl 5,5-bis((Z)-heptadec-8-en-1-yl)-1-(3-(pyrrolidin-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate (A2-iso5-2DC18);Bis(2-(dodecylsulfanyl)ethyl) 3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazapentacosyl)azanediyl)dipropionate (BAME-O16B); 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200); 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione (cKK-E12); hexa(octan-3-yl) 9,9',9'',9''',9'''',9''''''-((((benzene-1,3,5-tricarbonyl)tris(azanediyl))tris(propane-3,1-diyl))tris(azantriyl))hexanonanoate (FTT5); ((((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azantriyl))tetrakis(ethane-2,1-diyl) (9Z,9'Z,9''Z,9'''Z,12Z,12'Z,12''Z,12'''Z)-tetrakis(octadeca-9,12-dienoate) (OF-Deg-Lin); TT3; N; 1 ,N 3 ,N 5 -tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide; N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamide)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5); heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (Lipid 5); GL-HEPES-E3-E10-DS-3-E18-1; GL-HEPES-E3-E12-DS-4-E10; GL-HEPES-E3-E12-DS-3-E14; and combinations thereof may be selected from the group consisting of.

[0202] In some embodiments, the cationic lipid is biodegradable.

[0203] In some embodiments, the cationic lipid is not biodegradable.

[0204] In some embodiments, the cationic lipid is cleavable.

[0205] In some embodiments, the cationic lipid is not cleavable.

[0206] The cationic lipid is described in more detail in Dong et al. (PNAS. 111(11):3955 - 60. 2014); Fenton et al. (Adv Mater. 28:2939. 2016); U.S. Patent No. 9,512,073; and U.S. Patent No. 10,201,618, each of which is incorporated herein by reference.

[0207] B. PEGylated Lipids PEGylated lipid components provide control over the particle size and stability of the nanoparticles. Addition of such components can prevent complex aggregation, extend the circulation lifetime, and provide a means to increase delivery of the lipid - nucleic acid pharmaceutical composition to the target tissue (Klibanov et al. FEBS Letters 268(1):235 - 7 1990). These components can be selected to be rapidly exchanged from the pharmaceutical composition in vivo (see, for example, U.S. Patent No. 5,885,613).

[0208] Contemplated PEGylated lipids include C 6 ~C 20 (e.g., C 8 , C 10 , C 12 , C 14 , C 16 , or C 18)A polyethylene glycol (PEG) chain up to 5 kDa in length covalently attached to a lipid having a long alkyl chain, such as derivatized ceramide (e.g., N-octanoyl-sphingosine-1-[succinyl (methoxypolyethylene glycol)] (C8 PEG ceramide)), is exemplified, but not limited thereto. In some embodiments, the PEGylated lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG); 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DLPE-PEG); or 1,2-distearoyl-rac-glycero-polyethylene glycol (DSG-PEG), PEG-DAG; PEG-PE; PEG-S-DAG; PEG-S-DMG; PEG-cer; PEG-dialkyloxypropylcarbamate; 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); and combinations thereof.

[0209] In certain embodiments, the PEG has a high molecular weight, such as 2000 - 2400 g / mol. In certain embodiments, the PEG is PEG2000 (or PEG-2K). In certain embodiments, the PEGylated lipids herein are DMG-PEG2000, DSPE-PEG2000, DLPE-PEG2000, DSG-PEG2000, C8PEG2000, or ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide). In certain embodiments, the PEGylated lipid herein is DMG-PEG2000.

[0210] C. Cholesterol-based lipids Cholesterol components provide stability to the lipid bilayer structure within the nanoparticles. In some embodiments, the LNP contains one or more cholesterol-based lipids. Suitable cholesterol-based lipids include, for example, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), l,4-bis(3-N-oleylamino-propyl)piperazine (Gao et al., Biochem Biophys Res Comm. (1991) 179:280; Wolf et al., BioTechniques (1997) 23:139; U.S. Patent No. 5,744,335), imidazole cholesterol ester ("ICE"; International Publication No. 2011 / 068810 pamphlet), sitosterol (22,23-dihydrostigmasterol), β-sitosterol, sitostanol, fucosterol, stigmasterol (stigmastera-5,22-dien-3-ol), ergosterol; desmosterol (3β-hydroxy-5,24 cholestadiene); lanosterol (8,24 lanostadiene-3b-ol); 7-dehydrocholesterol (Δ5,7-cholesterol); dihydrolanosterol (24,25 dihydrolanosterol); thimosterol (5α-cholesta-8,24 diene-3β-ol); lasosterol (5α-cholesta-7-ene-3β-ol); diosgenin ((3β,25R)-spirost-5-ene-3-ol); campesterol (campest-5-ene-3β-ol); campestanol (5a-campestan-3b-ol); 24-methylenecolesterol (5,24(28)-cholestadiene-24-methylene-3β-ol); cholesteryl margarate (cholesta-5-ene-3β-yl heptadecanoate); cholesteryl oleate; cholesteryl stearate and other modified forms of cholesterol. In some embodiments, the cholesterol-based lipid used in the LNP is cholesterol.

[0211] D. Helper Lipids Helper lipids improve the structural stability of LNPs and assist LNPs in endosomal escape. It improves the uptake and release of the mRNA drug payload. In some embodiments, the helper lipid is an amphiphilic lipid having fusion properties for improving the uptake and release of the drug payload. Examples of helper lipids are 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS); 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE); and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPOC), dipalmitoyl phosphatidylcholine (DPPC), DMPC, 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-distearoyl phosphatidylethanolamine (DSPE), and 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE).

[0212] Other exemplary helper lipids are dioleoyl phosphatidylcholine (DOPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), phosphatidylserine, sphingolipids, sphingomyelin, ceramide, cerebroside, ganglioside, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or combinations thereof. In certain embodiments, the helper lipid is DOPE. In certain embodiments, the helper lipid is DSPC.

[0213] In various embodiments, the present LNP comprises: (i) a cationic lipid selected from OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, or GL-HEPES-E3-E12-DS-3-E14; (ii) DMG-PEG2000; (iii) cholesterol; and (iv) DOPE.

[0214] In other embodiments, the present LNP comprises: (i) SM-102; (ii) DMG-PEG2000; (iii) cholesterol; and (iv) DSPC.

[0215] In yet other embodiments, the present LNP comprises: (i) ALC-0315; (ii) ALC-0159; (iii) cholesterol; and (iv) DSPC.

[0216] E. Molar Ratio of Lipid Components The molar ratio of the above components is important for the effectiveness of the LNP in mRNA delivery. The molar ratio of the cationic lipid, PEGylated lipid, cholesterol-based lipid, and helper lipid is A:B:C:D (where A + B + C + D = 100%). In some embodiments, the molar ratio of the cationic lipid in the LNP to the total lipid (i.e., A) is 35-55%, such as 35-50% (e.g., 38-42%, e.g., 40%, or 45-50%). In some embodiments, the molar ratio of the PEGylated lipid component to the total lipid (i.e., B) is 0.25-2.75% (e.g., 1-2%, e.g., 1.5%). In some embodiments, the molar ratio of the cholesterol-based lipid to the total lipid (i.e., C) is 20-50% (e.g., 27-30%, e.g., 28.5%, or 38-43%). In some embodiments, the molar ratio of the helper lipid to the total lipid (i.e., D) is 5-35% (e.g., 28-32%, e.g., 30%, or 8-12%, e.g., 10%). In some embodiments, the (PEGylated lipid + cholesterol) component has the same molar amount as the helper lipid. In some embodiments, the LNP contains a molar ratio of cationic lipid to helper lipid that is greater than 1.

[0217] In certain embodiments, the LNP of the present disclosure a cationic lipid in a molar ratio of 35% to 55% or 40% to 50% (e.g., a cationic lipid in a molar ratio of 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, or 55%); a polyethylene glycol (PEG) conjugate (PEGylated) lipid in a molar ratio of 0.25% to 2.75% or 1.00% to 2.00% (e.g., a PEGylated lipid in a molar ratio of 0.25%, 0.50%, 0.75%, 1.00%, 1.25%, 1.50%, 1.75%, 2.00%, 2.25%, 2.50%, or 2.75%); Cholesterol-based lipids in a molar ratio of 20% to 50%, 25% to 45%, or 28.5% to 43% (e.g., cholesterol-based lipids in a molar ratio of 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%, or 50%); and Helper lipids in a molar ratio of 5% to 35%, 8% to 30%, or 10% to 30% (e.g., helper lipids in a molar ratio of 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35%), where all molar ratios are relative to the total lipid content of the LNP.

[0218] In certain embodiments, the LNP comprises cationic lipids in a molar ratio of 40%; PEGylated lipids in a molar ratio of 1.5%; cholesterol-based lipids in a molar ratio of 28.5%; and helper lipids in a molar ratio of 30%.

[0219] In certain embodiments, the LNP of the present disclosure comprises cationic lipids in a molar ratio of 45 - 50%; PEGylated lipids in a molar ratio of 1.5 - 1.7%; cholesterol-based lipids in a molar ratio of 38 - 43%; and helper lipids in a molar ratio of -9 - 10%.

[0220] In certain embodiments, the PEGylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000).

[0221] In various embodiments, the cholesterol-based lipid is cholesterol.

[0222] In some embodiments, the helper lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0223] In certain embodiments, the LNP comprises OF-02 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DOPE in a molar ratio of 5% to 35%.

[0224] In certain embodiments, the LNP comprises cKK-E10 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DOPE in a molar ratio of 5% to 35%.

[0225] In certain embodiments, the LNP comprises GL-HEPES-E3-E10-DS-3-E18-1 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DOPE in a molar ratio of 5% to 35%.

[0226] In certain embodiments, the LNP comprises GL-HEPES-E3-E12-DS-4-E10 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DOPE in a molar ratio of 5% to 35%.

[0227] In certain embodiments, the LNP comprises GL-HEPES-E3-E12-DS-3-E14 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DOPE in a molar ratio of 5% to 35%.

[0228] In certain embodiments, the LNP comprises SM-102 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DSPC in a molar ratio of 5% to 35%.

[0229] In certain embodiments, the LNP comprises ALC-0315 at a molar ratio of 35% to 55%; ALC-0159 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DSPC at a molar ratio of 5% to 35%.

[0230] In certain embodiments, the LNP comprises OF-02 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%. This LNP formulation is referred to herein as "Lipid A".

[0231] In certain embodiments, the LNP comprises cKK-E10 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%. This LNP formulation is referred to herein as "Lipid B".

[0232] In certain embodiments, the LNP comprises GL-HEPES-E3-E10-DS-3-E18-1 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%. This LNP formulation is referred to herein as "Lipid C".

[0233] In certain embodiments, the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%. This LNP formulation is referred to herein as "Lipid D".

[0234] In certain embodiments, the LNP comprises GL-HEPES-E3-E12-DS-3-E14 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%. This LNP formulation is referred to herein as "Lipid E".

[0235] In certain embodiments, the LNP comprises 50% molar ratio of 9-heptadecanoyl 8-{ (2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102); 10% molar ratio of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 38.5% molar ratio of cholesterol; and 1.5% molar ratio of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000).

[0236] In certain embodiments, the LNP comprises 46.3% molar ratio of (4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 9.4% molar ratio of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 42.7% molar ratio of cholesterol; and 1.6% molar ratio of 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0237] In certain embodiments, the LNP comprises 47.4% molar ratio of (4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 10% molar ratio of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 40.9% molar ratio of cholesterol; and 1.7% molar ratio of 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0238] To calculate the actual amount of each lipid to be included in the LNP formulation, first the molar amount of the cationic lipid is determined based on the desired N / P ratio (where N is the number of nitrogen atoms in the cationic lipid and P is the number of phosphate groups in the mRNA to be transported by the LNP). Next, based on the molar amount of the cationic lipid and the selected molar ratios, the molar amount of each of the other lipids is calculated. These molar amounts are then converted to weights using the molecular weights of the respective lipids.

[0239] Active ingredient of F.LNP The active ingredient of the present LNP vaccine composition is a nucleic acid (e.g., mRNA) encoding an antigenic prokaryotic polypeptide.

[0240] Optionally, the LNP can be multivalent. In some embodiments, the LNP can carry nucleic acids such as mRNAs encoding two or more antigenic prokaryotic polypeptides, such as two, three, four, five, six, seven, or eight antigens. For example, the LNP can carry multiple nucleic acids (e.g., mRNAs), each encoding a different antigenic prokaryotic polypeptide; or can carry a polycistronic mRNA that can be translated into two or more antigenic prokaryotic polypeptides (e.g., each antigen-encoding sequence is separated by a nucleotide linker encoding a self-cleaving peptide such as a 2A peptide). LNPs carrying different nucleic acids (e.g., mRNAs) typically contain (encapsulate) multiple copies of each nucleic acid. For example, an LNP carrying or encapsulating two different nucleic acids typically carries multiple copies of each of the two different nucleic acids.

[0241] In some embodiments, a single LNP formulation can contain multiple types (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of LNPs, each type carrying a different nucleic acid (e.g., mRNA).

[0242] When the nucleic acid is mRNA, the mRNA may be unmodified (i.e., containing only natural ribonucleotides A, U, C, and / or G linked by phosphodiester bonds), or may be chemically modified (e.g., nucleotide analogs such as pseudouridine (e.g., N-1-methylpseudouridine), 2'-fluororibonucleotides, and 2'-methoxyribonucleotides, and / or phosphorothioate bonds). The mRNA molecule may contain a 5' cap and a polyA tail.

[0243] G. Buffer and other components To stabilize nucleic acids and / or LNPs (e.g., to extend the shelf life of vaccine products), facilitate the administration of LNP pharmaceutical compositions, and / or improve the in vivo expression of nucleic acids, the nucleic acids and / or LNPs can be formulated in combination with one or more carriers, targeting ligands, stabilizing reagents (e.g., preservatives and antioxidants), and / or other pharmaceutically acceptable excipients. Examples of such excipients are parabens, thimerosal, thiomersal, chlorobutanol, benzalkonium chloride, and chelating agents (e.g., EDTA).

[0244] The LNP compositions of the present disclosure can be provided in a frozen liquid form or a lyophilized form. Various cryoprotectants can be used, including but not limited to sucrose, trehalose, glucose, mannitol, mannose, dextrose, etc. The cryoprotectant can constitute 5 - 30% (w / v) of the LNP composition. In some embodiments, the LNP composition contains trehalose, e.g., 5 - 30% (e.g., 10%) (w / v). Once formulated with the cryoprotectant, the LNP composition can be frozen (or lyophilized and stored frozen) at -20 o °C to -80 o °C.

[0245] The LNP composition can be provided to the patient in an aqueous buffer solution (thawed if previously frozen or reconstituted at the bedside in an aqueous buffer solution if previously lyophilized). The buffer is preferably isotonic and suitable for, for example, intramuscular or intradermal injection. In some embodiments, the buffer solution is phosphate buffered saline (PBS).

[0246] VII.RNA The vaccine composition of the present disclosure may include an RNA molecule (e.g., mRNA) encoding an antigen of interest (e.g., an antigenic prokaryotic polypeptide). The RSV molecule of the present disclosure may include at least one ribonucleic acid (RNA) containing an open reading frame (ORF) encoding an antigen of interest. In certain embodiments, the RNA is a messenger RNA (mRNA) containing an ORF encoding an antigen of interest. In certain embodiments, the RNA (e.g., mRNA) further includes at least one 5’ UTR, 3’ UTR, poly(A) tail, and / or 5’ cap.

[0247] A. 5’ Cap The 5’ cap of mRNA can provide resistance to nucleases found in most eukaryotic cells and can promote translation efficiency. Several types of 5’ caps are known. The 7-methylguanosine cap (also referred to as “m 7 G” or “Cap-0”) contains guanosine linked to the first transcribed nucleotide through a 5’-5’-triphosphate bond.

[0248] The 5’ cap is typically added as follows: First, RNA terminal phosphatase removes one of the terminal phosphate groups from the 5’ nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphate via guanylyl transferase to produce a 5’5’5 triphosphate bond; then, the 7-nitrogen of guanine is methylated by methyl transferase. Examples of cap structures include, but are not limited to, m7G(5’)ppp, (5’(A, G(5’)ppp(5’)A, and G(5’)ppp(5’)G. Additional cap structures are described in U.S. Patent Application Publication No. 2016 / 0032356 and U.S. Patent Application Publication No. 2018 / 0125989, which are incorporated herein by reference.

[0249] To generate a 5'-guanosine cap structure according to the manufacturer's protocol, the 5'-capping of polynucleotides can be simultaneously completed during in vitro transcription reactions using the following chemical RNA cap analogs: 3'-O-Me-m7G(5')ppp(5')G (ARCA cap); G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G; m7G(5')ppp(5')(2'OMeA)pG; m7G(5')ppp(5')(2'OMeA)pU; m7G(5')ppp(5')(2'OMeG)pG (New England BioLabs, Ipswich, MA; TriLink Biotechnologies). The 5'-capping of the modified RNA can be completed post-transcriptionally using the vaccinia virus capping enzyme to generate a Cap 0 structure: m7G(5')ppp(5')G. Both the vaccinia virus capping enzyme and 2'-O methyl-transferase can be used to generate a Cap 1 structure to produce m7G(5')ppp(5')G-2'-O-methyl. The Cap2 structure is generated from the Cap1 structure, followed by 2'-O methyl-transferase-mediated 2'-O-methylation of the 5'-penultimate nucleotide. The Cap3 structure is generated from the Cap2 structure, followed by 2'-O methyl-transferase-mediated 2'-O-methylation of the 5'-antepenultimate nucleotide.

[0250] In certain embodiments, the mRNA of the present disclosure comprises a 5' cap selected from the group consisting of 3'-O-Me-m7G(5')ppp(5')G (ARCA cap), G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, m7G(5')ppp(5')G, m7G(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeA)pU, and m7G(5')ppp(5')(2'OMeG)pG.

[0251] In certain embodiments, the mRNA of the present disclosure comprises the following 5' cap:

Chemical formula

[0252] B. Untranslated Region (UTR) In some embodiments, the mRNA of the present disclosure includes a 5' and / or 3' untranslated region (UTR). In mRNA, the 5' UTR starts at the transcription start site and continues to the start codon, but does not include the start codon. The 3' UTR starts immediately after the stop codon and continues to the transcription termination signal.

[0253] In some embodiments, the mRNA disclosed herein may include a 5' UTR that contains one or more elements that affect the stability or translation of the mRNA. In some embodiments, the 5' UTR can be about 10 to 5,000 nucleotides in length. In some embodiments, the 5' UTR can be about 50 to 500 nucleotides in length. In some embodiments, the 5' UTR is at least about 10 nucleotides in length, about 20 nucleotides in length, about 30 nucleotides in length, about 40 nucleotides in length, about 50 nucleotides in length, about 100 nucleotides in length, about 150 nucleotides in length, about 200 nucleotides in length, about 250 nucleotides in length, about 300 nucleotides in length, about 350 nucleotides in length, about 400 nucleotides in length, about 450 nucleotides in length, about 500 nucleotides in length, about 550 nucleotides in length, about 600 nucleotides in length, about 650 nucleotides in length, about 700 nucleotides in length, about 750 nucleotides in length, about 800 nucleotides in length, about 850 nucleotides in length, about 900 nucleotides in length, about 950 nucleotides in length, about 1,000 nucleotides in length, about 1,500 nucleotides in length, about 2,000 nucleotides in length, about 2,500 nucleotides in length, about 3,000 nucleotides in length, about 3,500 nucleotides in length, about 4,000 nucleotides in length, about 4,500 nucleotides in length, or about 5,000 nucleotides in length.

[0254] In some embodiments, the mRNA disclosed herein may include a 3'UTR that contains one or more of a polyadenylation signal, a binding site for a protein that affects the stability of the location of the mRNA in the cell, or a binding site for one or more miRNAs. In some embodiments, the 3'UTR can be 50 to 5,000 nucleotides in length or more. In some embodiments, the 3'UTR can be 50 to 1,000 nucleotides in length or more. In some embodiments, the 3'UTR is at least about 50 nucleotides in length, about 100 nucleotides in length, about 150 nucleotides in length, about 200 nucleotides in length, about 250 nucleotides in length, about 300 nucleotides in length, about 350 nucleotides in length, about 400 nucleotides in length, about 450 nucleotides in length, about 500 nucleotides in length, about 550 nucleotides in length, about 600 nucleotides in length, about 650 nucleotides in length, about 700 nucleotides in length, about 750 nucleotides in length, about 800 nucleotides in length, about 850 nucleotides in length, about 900 nucleotides in length, about 950 nucleotides in length, about 1,000 nucleotides in length, about 1,500 nucleotides in length, about 2,000 nucleotides in length, about 2,500 nucleotides in length, about 3,000 nucleotides in length, about 3,500 nucleotides in length, about 4,000 nucleotides in length, about 4,500 nucleotides in length, or about 5,000 nucleotides in length.

[0255] In some embodiments, the mRNA disclosed herein may include a 5' or 3'UTR derived from a gene that is distinct from the gene encoded by the mRNA transcript (i.e., the UTR is a heterologous UTR).

[0256] In certain embodiments, the 5’ and / or 3’ UTR sequences can be derived from mRNAs that are stable (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes) to enhance mRNA stability. For example, the 5’ UTR sequence can include a partial sequence or fragment of the CMV immediate early 1 (IE1) gene to improve nuclease resistance and / or improve the half-life of the mRNA. Inclusion of a sequence encoding human growth hormone (hGH) or a fragment thereof at the 3’ end or untranslated region of the mRNA is also contemplated. Generally, these modifications improve the stability and / or pharmacokinetic properties (e.g., half-life) of the mRNA compared to the unmodified counterparts, including modifications made to improve such mRNA resistance to in vivo nuclease digestion.

[0257] Exemplary 5’ UTRs include sequences derived from the CMV immediate early 1 (IE1) gene (U.S. Patent Application Publication Nos. 2014 / 0206753 and 2015 / 0157565, each of which is incorporated herein by reference) or the sequence GGGAUCCUACC (SEQ ID NO: 94) (U.S. Patent Application Publication No. 2016 / 0151409, which is incorporated herein by reference).

[0258] In various embodiments, the 5’ UTR can be derived from the 5’ UTR of a TOP gene. TOP genes are typically characterized by the presence of a 5’ terminal oligopyrimidine (TOP) tract. Further, most TOP genes are characterized by growth-related translational regulation. However, TOP genes with tissue-specific translational regulation are also known. In certain embodiments, the 5’ UTR derived from the 5’ UTR of a TOP gene lacks the 5’ TOP motif (oligopyrimidine region) (e.g., U.S. Patent Application Publication Nos. 2017 / 0029847, 2016 / 0304883, 2016 / 0235864, and 2016 / 0166710, each of which is incorporated herein by reference).

[0259] In certain embodiments, the 5’UTR is derived from the ribosomal protein large 32 (L32) gene (U.S. Patent Application Publication No. 2017 / 0029847, supra).

[0260] In certain embodiments, the 5’UTR is derived from the 5’UTR of the hydroxysteroid (17 - b) dehydrogenase 4 gene (HSD17B4) (U.S. Patent Application Publication No. 2016 / 0166710, supra).

[0261] In certain embodiments, the 5’UTR is derived from the 5’UTR of the ATP5A1 gene (U.S. Patent Application Publication No. 2016 / 0166710, supra). In some embodiments, an internal ribosome entry site (IRES) is used in place of the 5’UTR.

[0262] In some embodiments, the 5’UTR comprises the nucleic acid sequence shown in SEQ ID NO: 89 and reproduced below.

Chemical formula

[0263] In some embodiments, the 3’UTR comprises the nucleic acid sequence shown in SEQ ID NO: 90 and reproduced below: CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC (SEQ ID NO: 90).

[0264] The 5’UTR and 3’UTR are described in further detail in International Publication No. WO 2012 / 075040, which is incorporated herein by reference.

[0265] C. Polyadenylation Tail As used herein, the terms “poly(A) sequence,” “poly(A) tail,” and “poly(A) region” refer to the sequence of adenosine nucleotides at the 3′ end of an mRNA molecule. The poly(A) tail can confer stability to the mRNA and protect the mRNA from exonucleolytic degradation. The poly(A) tail can enhance translation. In some embodiments, the poly(A) tail is essentially a homopolymer. For example, a poly(A) tail of 100 adenosine nucleotides can essentially have a length of 100 nucleotides. In certain embodiments, the poly(A) tail can be interrupted by at least one nucleotide different from an adenosine nucleotide (e.g., a nucleotide that is not an adenosine nucleotide). For example, a poly(A) tail of 100 adenosine nucleotides can have a length of more than 100 nucleotides (including 100 adenosine nucleotides and at least one nucleotide or stretch of nucleotides different from an adenosine nucleotide). In certain embodiments, the poly(A) tail comprises a sequence.

Chemical formula

[0266] As used herein, the “poly(A) tail” typically relates to RNA. However, in the context of the present disclosure, this term also relates equally to the corresponding sequence in a DNA molecule (e.g., a “poly(T) sequence”).

[0267] The poly(A) tail can comprise from about 10 to about 500 adenosine nucleotides, from about 10 to about 200 adenosine nucleotides, from about 40 to about 200 adenosine nucleotides, or from about 40 to about 150 adenosine nucleotides. The length of the poly(A) tail can be at least about 10, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 adenosine nucleotides.

[0268] In some embodiments where the nucleic acid is RNA, the poly(A) tail of the nucleic acid is obtained from a DNA template during in vitro transcription of the RNA. In certain embodiments, the poly(A) tail is obtained in vitro by general chemical synthesis methods without being transcribed from a DNA template. In various embodiments, the poly(A) tail is enzymatically polyadenylated on the RNA (after in vitro transcription of the RNA) using a commercially available polyadenylation kit and corresponding protocol or alternatively by using immobilized poly(A) polymerase using methods and means described, for example, in WO 2016 / 174271 pamphlet.

[0269] The nucleic acid may contain a poly(A) tail obtained by enzymatic polyadenylation, and most of the nucleic acid molecule contains from about 100 (+ / -20) to about 500 (+ / -50) or about 250 (+ / -20) adenosine nucleotides.

[0270] In some embodiments, the nucleic acid may contain a poly(A) tail derived from the template DNA as described, for example, in WO 2016 / 091391 pamphlet, and may additionally contain at least one additional poly(A) tail generated by enzymatic polyadenylation.

[0271] In certain embodiments, the nucleic acid contains at least one polyadenylation signal.

[0272] In various embodiments, the nucleic acid may contain at least one poly(C) sequence.

[0273] As used herein, the term "poly(C) sequence" is intended to be a sequence of up to about 200 cytosine nucleotides. In some embodiments, the poly(C) sequence contains from about 10 to about 200 cytosine nucleotides, from about 10 to about 100 cytosine nucleotides, from about 20 to about 70 cytosine nucleotides, from about 20 to about 60 cytosine nucleotides, or from about 10 to about 40 cytosine nucleotides. In some embodiments, the poly(C) sequence contains about 30 cytosine nucleotides.

[0274] D. Chemical modification The mRNA disclosed herein may or may not be modified. In some embodiments, the mRNA may comprise at least one chemical modification. In some embodiments, the mRNA disclosed herein may contain one or more modifications that typically improve RNA stability. Exemplary modifications can include backbone modifications, sugar modifications, or base modifications. In some embodiments, the disclosed mRNA can be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides) that include, but are not limited to, purines (adenine (A) and guanine (G)) or pyrimidines (thymine (T), cytosine (C), and uracil (U)). In certain embodiments, the disclosed mRNA is a modified nucleotide analog or derivative of purines and pyrimidines, such as 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queuosine, β-D-mannosyl-queuosine, phosphoramidate, phosphorothioate, peptide nucleotide, methylphosphonate, 7-deazaguanosine, 5-methylcytosine, and inosine.

[0275] In some embodiments, the disclosed mRNA may comprise at least one chemical modification including, but not limited to, pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-1-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxypseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0276] In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0277] In some embodiments, the chemical modification comprises N1-methylpseudouridine.

[0278] In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the mRNA are chemically modified.

[0279] In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the ORF are chemically modified.

[0280] The preparation of such analogs is described, for example, in U.S. Patent No. 4,373,071, U.S. Patent No. 4,401,796, U.S. Patent No. 4,415,732, U.S. Patent No. 4,458,066, U.S. Patent No. 4,500,707, U.S. Patent No. 4,668,777, U.S. Patent No. 4,973,679, U.S. Patent No. 5,047,524, U.S. Patent No. 5,132,418, U.S. Patent No. 5,153,319, U.S. Patent No. 5,262,530, and U.S. Patent No. 5,700,642.

[0281] E. mRNA synthesis The mRNA disclosed herein can be synthesized according to any of a variety of methods. For example, the mRNA according to the present disclosure can be synthesized via in vitro transcription (IVT). Some methods for in vitro transcription are described, for example, in Geall et al. (2013) Semin. Immunol. 25(2):152-159; Brunelle et al. (2013) Methods Enzymol. 530:101-14. Briefly, IVT is typically performed using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may contain DTT and magnesium ions, an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or an RNase inhibitor. The exact conditions can vary depending on the particular application. The presence of these reagents is generally undesirable in the final mRNA product, and these reagents can be considered impurities or contaminants that can be purified or removed to provide a clean and / or homogeneous mRNA suitable for therapeutic use. In some embodiments, mRNA provided from an in vitro transcription reaction may be desirable, but other mRNA sources, including wild-type mRNA produced from bacteria, fungi, plants, and / or animals, can be used according to the present disclosure.

[0282] VIII. Process for preparing the present LNP vaccine This LNP can be prepared by various techniques currently known in the art. For example, multilamellar vesicles (MLVs) can be prepared according to conventional techniques, e.g., by dissolving lipids in a suitable solvent and depositing the selected lipids on the inner wall of a suitable container or vessel, then evaporating the solvent to leave a thin film on the inside of the vessel or by spray drying. Then an aqueous phase can be added to the vessel by vortexing, which results in the formation of MLVs. Then, unilamellar vesicles (ULVs) can be formed by homogenization, sonication or extrusion of the multilamellar vesicles. In addition, unilamellar vesicles can be formed by detergent removal techniques.

[0283] Various methods are described in U.S. Patent Application Publication No. 2011 / 0244026, U.S. Patent Application Publication No. 2016 / 0038432, U.S. Patent Application Publication No. 2018 / 0153822, U.S. Patent Application Publication No. 2018 / 0125989, and PCT / U.S. Patent Application Publication No. 2020 / 043223 (filed Jul. 23, 2020) and can be used to practice the present disclosure. One exemplary process involves encapsulating mRNA by mixing the mRNA with a mixture of lipids without first pre-forming the lipids into lipid nanoparticles as described in U.S. Patent Application Publication No. 2016 / 0038432. Another exemplary process involves encapsulating mRNA by mixing a pre-formed LNP with the mRNA as described in U.S. Patent Application Publication No. 2018 / 0153822.

[0284] In some embodiments, the process of preparing the mRNA-loaded LNP includes heating one or more of the solutions to a temperature higher than the ambient temperature, and the one or more solutions are a solution containing pre-formed lipid nanoparticles, a solution containing mRNA, and a mixed solution containing mRNA encapsulated in LNP. In some embodiments, the process includes heating one or both of the mRNA solution and the pre-formed LNP solution before the mixing step. In some embodiments, the process includes heating one or more of the solution containing pre-formed LNP, the solution containing mRNA, and the solution containing mRNA encapsulated in LNP during the mixing step. In some embodiments, the process includes heating the mRNA encapsulated in LNP after the mixing step. In some embodiments, the temperature to which one or more of the solutions are heated is about 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C or higher. In some embodiments, the temperature to which one or more of the solutions are heated ranges from about 25 to 70°C, about 30 to 70°C, about 35 to 70°C, about 40 to 70°C, about 45 to 70°C, about 50 to 70°C, or about 60 to 70°C. In some embodiments, the temperature is about 65°C.

[0285] Various methods can be used to prepare an mRNA solution suitable for the present disclosure. In some embodiments, the mRNA can be directly dissolved in the buffer solution described herein. In some embodiments, the mRNA solution can be generated by mixing an mRNA stock solution with a buffer solution before mixing with the lipid solution for encapsulation. In some embodiments, the mRNA solution can be generated by mixing an mRNA stock solution with a buffer solution immediately before mixing with the lipid solution for encapsulation. In some embodiments, a suitable mRNA stock solution can contain mRNA at a concentration of about 0.2 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1.0 mg / ml, 1.2 mg / ml, 1.4 mg / ml, 1.5 mg / ml, or 1.6 mg / ml, 2.0 mg / ml, 2.5 mg / ml, 3.0 mg / ml, 3.5 mg / ml, 4.0 mg / ml, 4.5 mg / ml, or 5.0 mg / ml or higher in water or a buffer.

[0286] In some embodiments, the mRNA stock solution is mixed with the buffer solution using a pump. Exemplary pumps include, but are not limited to, gear pumps, peristaltic pumps, and centrifugal pumps. Typically, the buffer solution is mixed at a faster rate than the mRNA stock solution. For example, the buffer solution can be mixed at a rate that is at least 1 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, or 20 times greater than the rate of the mRNA stock solution. In some embodiments, the buffer solution is mixed at a flow rate in the range of about 100 - 6000 ml / min (e.g., about 100 - 300 ml / min, 300 - 600 ml / min, 600 - 1200 ml / min, 1200 - 2400 ml / min, 2400 - 3600 ml / min, 3600 - 4800 ml / min, 4800 - 6000 ml / min, or 60 - 420 ml / min). In some embodiments, the buffer solution is mixed at a flow rate of about 60 ml / min, 100 ml / min, 140 ml / min, 180 ml / min, 220 ml / min, 260 ml / min, 300 ml / min, 340 ml / min, 380 ml / min, 420 ml / min, 480 ml / min, 540 ml / min, 600 ml / min, 1200 ml / min, 2400 ml / min, 3600 ml / min, 4800 ml / min, or 6000 ml / min or more.

[0287] In some embodiments, the mRNA stock solution is mixed at a flow rate in the range of about 10 - 600 ml / min (e.g., about 5 - 50 ml / min, about 10 - 30 ml / min, about 30 - 60 ml / min, about 60 - 120 ml / min, about 120 - 240 ml / min, about 240 - 360 ml / min, about 360 - 480 ml / min, or about 480 - 600 ml / min). In some embodiments, the mRNA stock solution is mixed at a flow rate of about 5 ml / min, 10 ml / min, 15 ml / min, 20 ml / min, 25 ml / min, 30 ml / min, 35 ml / min, 40 ml / min, 45 ml / min, 50 ml / min, 60 ml / min, 80 ml / min, 100 ml / min, 200 ml / min, 300 ml / min, 400 ml / min, 500 ml / min, or 600 ml / min or more.

[0288] The process of incorporating the desired mRNA into lipid nanoparticles is referred to as "loading". Exemplary methods are described in Lasic et al., FEBS Lett. (1992) 312:255-8. Nucleic acids incorporated into the LNP can be completely or partially within the internal space of the lipid nanoparticle, within the bilayer membrane of the lipid nanoparticle, or associated with the outer surface of the lipid nanoparticle membrane. The incorporation of mRNA into lipid nanoparticles is also referred to herein as "encapsulation", and the nucleic acid is contained completely or substantially within the internal space of the lipid nanoparticle.

[0289] Suitable LNPs can be made in various sizes. In some embodiments, a decrease in the size of the lipid nanoparticles is associated with more efficient delivery of the mRNA. The selection of an appropriate LNP size can take into account to some extent the site of the target cell or tissue and the use for which the lipid nanoparticles are being made.

[0290] A variety of methods known in the art are available for sizing a population of lipid nanoparticles. The preferred method herein is to measure the LNP particle size using a Zetasizer Nano ZS (Malvern Panalytical). In one protocol, 10 μl of the LNP sample is mixed with 990 μl of 10% trehalose. This solution is loaded into a cuvette and then placed into the Zetasizer machine. The z-average diameter (nm), or the cumulant average, is considered the average size of the LNPs in the sample. The Zetasizer machine can also be used to measure the polydispersity index (PDI) by using dynamic light scattering (DLS) and cumulant analysis of the autocorrelation function. The average LNP diameter can be decreased by sonication of the formed LNPs. Intermittent sonication cycles can be alternated with quasi-elastic light scattering (QELS) evaluations to lead to efficient lipid nanoparticle synthesis.

[0291] In some embodiments, most of the purified LNPs, i.e., more than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the LNPs, have a size of about 70 - 150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm). In some embodiments, substantially all (e.g., more than 80 or 90%) of the purified lipid nanoparticles have a size of about 70 - 150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm).

[0292] In some embodiments, the LNPs in the composition have an average size of less than 150 nm, less than 120 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 30 nm, or less than 20 nm.

[0293] In some embodiments, more than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the LNPs in the composition have a size in the range of about 40 - 90 nm (e.g., about 45 - 85 nm, about 50 - 80 nm, about 55 - 75 nm, about 60 - 70 nm) or about 50 - 70 nm (e.g., about 55 - 65 nm), and are particularly suitable for pulmonary delivery by spraying.

[0294] In some embodiments, the dispersity of the LNPs in the pharmaceutical composition provided by the present disclosure, or the polydispersity index (PDI), a measure of the heterogeneity of the molecular size, is less than about 0.5. In some embodiments, the LNPs have a PDI of less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.28, less than about 0.25, less than about 0.23, less than about 0.20, less than about 0.18, less than about 0.16, less than about 0.14, less than about 0.12, less than about 0.10, or less than about 0.08. The PDI can be measured by a Zetasizer machine as described above.

[0295] In some embodiments, greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified LNPs in the pharmaceutical compositions provided herein encapsulate mRNA within each individual particle. In some embodiments, substantially all (e.g., greater than 80% or 90%) of the purified lipid nanoparticles in the pharmaceutical composition encapsulate mRNA within each individual particle. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of 50% - 99%; or greater than about 60, 65, 70, 75, 80, 85, 90, 92, 95, 98, or 99%. Typically, the lipid nanoparticles for use herein have an encapsulation efficiency of at least 90% (e.g., at least 91, 92, 93, 94, or 95%).

[0296] In some embodiments, the LNP has an N / P ratio of 1 - 10. In some embodiments, the lipid nanoparticles have an N / P ratio greater than 1, about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8. In further embodiments, typical LNPs herein have an N / P ratio of 4.

[0297] In some embodiments, the pharmaceutical compositions according to the present disclosure contain at least about 0.5 μg, 1 μg, 5 μg, 10 μg, 100 μg, 500 μg, or 1000 μg of encapsulated mRNA. In some embodiments, the pharmaceutical composition contains from about 0.1 μg to 1000 μg, at least about 0.5 μg, at least about 0.8 μg, at least about 1 μg, at least about 5 μg, at least about 8 μg, at least about 10 μg, at least about 50 μg, at least about 100 μg, at least about 500 μg, or at least about 1000 μg of encapsulated mRNA.

[0298] In some embodiments, the mRNA can be made by chemical synthesis or by in vitro transcription (IVT) of a DNA template. Exemplary processes for making and purifying the mRNA are described in Example 1. In this process, the IVT process, a cDNA template is used to produce an mRNA transcript, and the DNA template is degraded by DNase. The transcript is purified by depth filtration and tangential flow filtration (TFF). The purified transcript is further modified by adding a cap and a tail, and the modified RNA is purified again by depth filtration and TFF.

[0299] The mRNA is then prepared in an aqueous buffer and mixed with an amphiphilic solution containing the lipid components of the LNP. The amphiphilic solution for dissolving the four lipid components of the LNP can be an alcohol solution. In some embodiments, the alcohol is ethanol. The aqueous buffer can be, for example, a citrate, phosphate, acetate, or succinate buffer and can have a pH of about 3.0 to 7.0, such as about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, or about 6.5. The buffer can contain other components, such as salts (e.g., sodium salts, potassium salts, and / or calcium salts). In certain embodiments, the aqueous buffer has 1 mM citrate, 150 mM NaCl, and a pH of 4.5.

[0300] Exemplary non-limiting processes for making mRNA-LNP compositions are described in Example 1. This process involves mixing a buffered mRNA solution with a solution of lipids in ethanol in a controlled and uniform manner, and the lipid:mRNA ratio is maintained throughout the mixing process. In this illustrative example, the mRNA is present in an aqueous buffer containing citric acid monohydrate, trisodium citrate dihydrate, and sodium chloride. The mRNA solution is added to a solution (1 mM citrate buffer, 150 mM NaCl, pH 4.5). A lipid mixture of four lipids (e.g., a cationic lipid, a PEGylated lipid, a cholesterol-based lipid, and a helper lipid) is dissolved in ethanol. The aqueous mRNA solution and the ethanol lipid solution are mixed in a "T" mixer equipped with a substantially "pulse-free" pump system at a volume ratio of 4:1. The resulting mixture is then subjected to downstream purification and buffer exchange. Buffer exchange can be achieved using a dialysis cassette or a TFF system. Using TFF, the nascent LNP obtained immediately after formation via the T mixing process can be concentrated and buffer exchanged. The diafiltration process is a continuous operation that maintains a constant volume by adding an appropriate buffer at the same rate as the permeate flow.

[0301] IX. Packaging and Use of mRNA-LNP Vaccines mRNA-LNP vaccines can be formulated or packaged for parenteral (e.g., intramuscular, intradermal or subcutaneous) administration or for administration to the nasopharynx (e.g., intranasally). The vaccine composition can be in the form of an immediate formulation where the LNP composition is lyophilized and reconstituted with a physiological buffer (e.g., PBS) immediately prior to use. The vaccine composition can also be shipped and provided in the form of an aqueous solution or a frozen aqueous solution and can be administered directly to the subject without reconstitution (if previously frozen, after thawing).

[0302] Accordingly, the present disclosure provides a manufactured product, e.g., a kit, that provides an mRNA-LNP vaccine in a single container or provides an mRNA-LNP vaccine in one container and a physiological buffer for reconstitution in another container. The container can contain a single-use dosage or a multi-use dosage. The container can be a pre-treated glass vial or ampoule. The manufactured product can also include instructions for use.

[0303] In certain embodiments, the mRNA-LNP vaccine is provided for use by intramuscular (IM) injection. The vaccine can be injected into a subject, e.g., into the deltoid muscle of the subject's upper arm. In some embodiments, the vaccine is provided in a filled syringe or injector (e.g., single-chamber type or multi-chamber type). In some embodiments, the vaccine is provided for use by inhalation and is provided in a filled pump, aerosol generator, or inhaler.

[0304] The mRNA-LNP vaccine can be administered to a subject in need thereof in a prophylactically effective amount, i.e., an amount that provides sufficient immune defense against a target pathogen over a sufficient period of time (e.g., 1 year, 2 years, 5 years, 10 years, or for life). Sufficient immune defense can be, e.g., prevention or alleviation of symptoms associated with infection by the pathogen. In some embodiments, multiple doses (e.g., 2 doses) of the vaccine are injected into a subject in need thereof to achieve the desired prophylactic effect. The doses (e.g., the first dose and booster doses) can be spaced, e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5 years, or 10 years apart.

[0305] In some embodiments, a single dose of the mRNA-LNP vaccine contains 1 to 50 μg of mRNA (e.g., monovalent or multivalent). For example, a single dose may contain mRNA for intramuscular (IM) injection of about 2.5 μg, about 5 μg, about 7.5 μg, about 10 μg, about 12.5 μg, or about 15 μg. In further embodiments, a multivalent single dose of the LNP vaccine contains multiple (e.g., 2, 3, or 4) types of LNPs for different antigens, and each type of LNP has an mRNA amount of, for example, 2.5 μg, about 5 μg, about 7.5 μg, about 10 μg, about 12.5 μg, or about 15 μg.

[0306] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Exemplary methods and materials are described below, but methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. In general, the terms and techniques related to cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal chemistry, and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein, and those used in those techniques are well-known and commonly used in the art. Enzyme reactions and purification techniques are performed as generally practiced in the art or as described herein, according to the manufacturer's specifications. Further, unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and the embodiments, the words "have" and "comprise," or variations thereof, such as "has," "having," "comprises," or "comprising," are understood to mean including the recited integer or group of integers, but not meaning to exclude any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although many references are cited herein, this citation does not constitute an admission that any of these references form a part of the common general knowledge in the art. As used herein, the term "about" or "approximately" when applied to one or more values of interest refers to a value similar to the recited reference value. In certain embodiments, the term refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the recited reference value in either direction (greater than or less than), unless otherwise stated or otherwise apparent from the context.

[0307] X. Vector In one aspect, vectors comprising the mRNA compositions disclosed herein are disclosed herein. RNA sequences encoding a protein of interest (e.g., mRNA encoding an antigenic prokaryotic polypeptide) can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Particular vectors of interest can include expression vectors, replication vectors, probe generation vectors, sequencing vectors, and vectors optimized for in vitro transcription.

[0308] In certain embodiments, the vector can be used to express the mRNA in a host cell. In various embodiments, the vector can be used as a template for IVT. The construction of optimally translated IVT mRNA suitable for therapeutic use is disclosed in detail in Sahin, et al. (2014). Nat. Rev. Drug Discov. 13, 759-780; Weissman (2015). Expert Rev. Vaccines 14, 265-281.

[0309] In some embodiments, the vectors disclosed herein can comprise at least the following from 5' to 3': an RNA polymerase promoter; a polynucleotide sequence encoding a 5'UTR; a polynucleotide sequence encoding an ORF; a polynucleotide sequence encoding a 3'UTR; and a polynucleotide sequence encoding at least one RNA aptamer. In some embodiments, the vectors disclosed herein can comprise a poly(A) sequence and / or a polynucleotide sequence encoding a polyadenylation signal.

[0310] A variety of RNA polymerase promoters are known. In some embodiments, the promoter can be a T7 RNA polymerase promoter. Other useful promoters can include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for the T7 promoter, T3 promoter, and SP6 promoter are known.

[0311] Also disclosed herein are host cells (e.g., mammalian cells, e.g., human cells) containing the vectors or RNA compositions disclosed herein.

[0312] The polynucleotide can be introduced into target cells using any of a number of different methods, including, but not limited to, commercially available methods such as electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM830 (BTX) (Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Colo.)), multiporator (Eppendorf, Hamburg, Germany), cationic lipid-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, "gene gun" (see, e.g., Nishikawa, et al. (2001). Hum Gene Ther. 12(8):861-70) or TransIT-RNA transfection kit (Mirus, Madison, WI), or biological particle delivery systems.

[0313] Chemical means for introducing polynucleotides into host cells include lipid-based systems such as colloidal dispersion systems, e.g., macromolecular complexes, nanocapsules, microspheres, beads, and water-in-oil emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro and in vivo delivery vehicle is a liposome (e.g., artificial membrane vesicles).

[0314] Regardless of the method used to introduce exogenous nucleic acid into a host cell or, alternatively, to expose the cell to an inhibitor of the present disclosure, various assays can be performed to confirm the presence of an mRNA sequence in the host cell.

[0315] XI. Self-replicating RNA, trans-replicating RNA, and non-replicating RNA Self-replicating RNA: Self-replicating (or self-propagating) RNA can be produced, for example, by using replication elements derived from alphaviruses and replacing the structural viral proteins with a nucleotide sequence encoding a protein of interest (e.g., an antigenic prokaryotic polypeptide). Self-replicating RNA is typically a plus-strand molecule that can be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase that produces both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA results in the production of multiple daughter RNAs. These daughter RNAs, and collinear subgenomic transcripts, can themselves be translated to provide in situ expression of the encoded antigen, or can be transcribed to provide additional transcripts having the same sense as the delivered RNA, which can then be translated to provide in situ expression of the antigen. The overall result of this series of transcriptions is a large amplification of the number of introduced replicon RNAs, such that the encoded antigen becomes the major polypeptide product of the cell.

[0316] One preferred system for achieving self - replication is to use an alphavirus - based replicon. These replicons are plus - strand (plus - sense strand) RNAs that, upon delivery to cells, result in the translation of a replicase (or replicase - transcriptase). The replicase is translated as a polyprotein that provides a replication complex that self - cleaves to generate a genomic - strand copy of the plus - strand delivery RNA. These minus (-) - strand transcripts can themselves be transcribed to give further copies of the plus - strand parental RNA and, furthermore, sub - genomic transcripts that encode an antigen. Thus, translation of the sub - genomic transcripts results in the in - situ expression of the antigen by the infected cell. Suitable alphavirus replicons can use replicases from Sindbis virus, Semliki Forest virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus, etc. Mutant or wild - type virus sequences can be used; for example, the attenuated TC83 mutant of VEEV has been used in the replicon. See the following references: International Publication No. WO 2005 / 113782 pamphlet, which is incorporated herein by reference.

[0317] In one embodiment, each self-replicating RNA described herein encodes (i) an RNA-dependent RNA polymerase capable of transcribing RNA from a self-replicating RNA molecule and (ii) a protein antigen. The polymerase can be, for example, an alphavirus replicase comprising one or more of the alphavirus proteins nsP1, nsP2, nsP3, and nsP4. While the native alphavirus genome encodes structural virion proteins in addition to the non-structural replicase polyprotein, in certain embodiments, the self-replicating RNA molecule does not encode alphavirus structural proteins. Thus, the self-replicating RNA can result in the production of its own genomic RNA copies in cells, but does not result in the production of RNA-containing virions. This inability to produce these virions means that, unlike wild-type alphaviruses, the self-replicating RNA molecule cannot persist on its own in an infectious form. The alphavirus structural proteins required for persistence in wild-type viruses are not present in the self-replicating RNAs of the present disclosure, and their place is taken by the gene encoding the immunogen of interest, such that the subgenomic transcript encodes that immunogen rather than the structural alphavirus virion protein. The self-replicating RNAs are described in further detail in International Publication No. WO 2011 / 005799, which is incorporated herein by reference.

[0318] Trans-replicating RNA: Trans-replicating (or trans-amplifying) RNA has elements similar to the self-replicating RNAs described above. However, in trans-replicating RNA, two separate RNA molecules are used. The first RNA molecule encodes the above-described RNA replicase (e.g., an alphavirus replicase), and the second RNA molecule encodes the protein of interest (e.g., an antigenic prokaryotic polypeptide). The RNA replicase can replicate one or both of the first and second RNA molecules, thereby significantly increasing the copy number of the RNA molecule encoding the protein of interest. Trans-replicating RNAs are described in further detail in International Publication No. WO 2017 / 162265, which is incorporated herein by reference.

[0319] Non-replicating RNA: Non-replicating (or non-amplifying) RNA is RNA that does not have the ability to replicate itself.

[0320] XI. Pharmaceutical Compositions The pharmaceutical compositions described in this disclosure typically include a nucleic acid, particularly RNA, more specifically mRNA, and a pharmaceutically acceptable carrier, or a pharmaceutically acceptable excipient or a pharmaceutically acceptable diluent, whereby the composition is particularly suitable for therapeutic use. The phrase "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues within the scope of sound medical judgment, without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0321] The pharmaceutical composition can be, for example, an immunogenic composition, i.e., a composition that induces an immune response when administered to a subject. It should be understood that the terms "immunogenic composition", "vaccine composition" and "vaccine" are used interchangeably herein and thus have equivalent meanings.

[0322] The pharmaceutical compositions of the present disclosure may also include one or more additional components such as small molecule immune potentiators (e.g., TLR agonists). The pharmaceutical compositions of the present disclosure may also include a delivery system for RNA such as liposomes, oil-in-water emulsions or microparticles. In some embodiments, the pharmaceutical composition includes lipid nanoparticles (LNP). In certain embodiments, the composition includes an antigen-encoding nucleic acid molecule encapsulated within the LNP.

[0323] XII. Vaccination Methods The nucleic acids (e.g., mRNA) disclosed herein can be administered to a subject to induce an immune response against an antigenic prokaryotic polypeptide, and the antigen-specific antibody titer of the subject increases after vaccination compared to that of a subject not vaccinated with the nucleic acid vaccine disclosed herein or compared to an alternative vaccine against the prokaryotic polypeptide. "Antigen-specific antibody" is a serum antibody that specifically binds to an antigen.

[0324] In one aspect, the present disclosure provides a method of inducing an immune response in a subject in need thereof, the method comprising administering to the subject an effective amount of a nucleic acid (e.g., mRNA) vaccine as described herein, optionally intramuscularly, intranasally, intravenously, subcutaneously, or intradermally.

[0325] In another aspect, the present disclosure provides a method of treating or preventing a prokaryotic infection in a subject in need thereof, the method comprising administering to the subject an effective amount of a nucleic acid vaccine as described herein, optionally intramuscularly, intranasally, intravenously, subcutaneously, or intradermally.

[0326] In another aspect, the present disclosure provides the use of a nucleic acid vaccine as described herein for the manufacture of a medicament for use in inducing an immune response in a subject in need thereof.

[0327] In another aspect, the present disclosure provides the use of a nucleic acid vaccine as described herein for the manufacture of a medicament for use in treating or preventing a prokaryotic infection in a subject in need thereof.

[0328] In another aspect, the present disclosure provides a nucleic acid vaccine as described herein for use in inducing an immune response in a subject in need thereof.

[0329] In another aspect, the present disclosure provides a nucleic acid vaccine as described herein for use in treating or preventing a prokaryotic infection in a subject in need thereof.

[0330] In one aspect, the present disclosure provides a method of secreting an antigenic prokaryotic polypeptide in a host cell, the method comprising administering a nucleic acid vaccine as described herein to the host cell.

[0331] In another aspect, the present disclosure provides a method of displaying an antigenic prokaryotic polypeptide on the surface of a host cell, the method comprising administering a nucleic acid vaccine as described herein to the host cell.

[0332] The present invention includes the following embodiments.

[0333] Embodiment 1. A nucleic acid comprising an open reading frame (ORF), wherein the ORF comprises: - a polynucleotide sequence encoding at least one antigenic prokaryotic polypeptide and - a polynucleotide sequence encoding at least one viral secretion signal peptide.

[0334] Embodiment 2. The nucleic acid according to Embodiment 1, wherein the ORF further comprises a polynucleotide sequence encoding at least one transmembrane domain (TMD).

[0335] Embodiment 3. The nucleic acid according to Embodiment 1 or 2, wherein the viral secretion signal peptide is derived from a viral sequence in a virus capable of infecting humans.

[0336] Embodiment 4. The nucleic acid according to any one of Embodiments 1 to 3, wherein the viral secretion signal peptide is derived from a viral sequence selected from the group consisting of non-influenza secretion signal peptide sequences selected from the group consisting of influenza secretion signal peptide sequences, SARS CoV-2 secretion signal peptide sequences, varicella-zoster virus (VZV) secretion signal peptide sequences, measles secretion signal peptide sequences, rubella secretion signal peptide sequences, mumps secretion signal peptide sequences, Ebola secretion signal peptide sequences, smallpox secretion signal peptide sequences, and rabies secretion signal peptide sequences.

[0337] Embodiment 5. The viral secretion signal peptide is selected from the group consisting of an influenza hemagglutinin (HA) secretion signal peptide sequence, a SARS CoV-2 spike secretion signal peptide sequence, a VZV gB secretion signal peptide sequence, a VZV gE secretion signal peptide sequence, a VZV gI secretion signal peptide sequence, a VZV gK secretion signal peptide sequence, a measles F-protein secretion signal peptide sequence, a rubella E1 protein secretion signal peptide sequence, a rubella E2 protein secretion signal peptide sequence, a mumps F-protein secretion signal peptide sequence, an Ebola GP protein secretion signal peptide sequence, a smallpox 6 kDa IC protein secretion signal peptide sequence, and a rabies G protein secretion signal peptide sequence, preferably, the viral secretion signal peptide contains an HA secretion signal peptide sequence derived from influenza A or influenza B, more preferably, an HA secretion signal peptide sequence derived from influenza A, the nucleic acid according to any one of Embodiments 1 to 4.

[0338] Embodiment 6. The HA secretion signal peptide sequence is the amino acid sequence MKX 1 X 2 LX 3 VX 4 LX 5 TFX 6 X 7 X 8 X 9 including A (SEQ ID NO: 145), and X 1 is selected from A and V; X 2 is selected from I and K; X 3 is selected from V and L; X 4 is selected from L and M; X 5 is selected from Y and C; X 6 is selected from T and A; X 7 is selected from T and A; X 8 is selected from A and T; X 9 is selected from N and Y, the nucleic acid according to Embodiment 5.

[0339] Embodiment 7. The nucleic acid according to Embodiment 5 or 6, wherein the HA secretion signal peptide sequence comprises an amino acid sequence selected from SEQ ID NOs: 95 to 109.

[0340] Embodiment 8. The HA secretion signal peptide sequence is the amino acid sequence MKX 1 IIALSX 2 ILCLVFX 3 (SEQ ID NO: 146), wherein X 1 is selected from T and A; X 2 is selected from Y, N, C, and H; X 3 is selected from T and A, and the nucleic acid according to Embodiment 5.

[0341] Embodiment 9. The nucleic acid according to Embodiment 8, wherein the HA secretion signal peptide sequence comprises an amino acid sequence selected from SEQ ID NOs: 110 to 131.

[0342] Embodiment 10. The HA secretion signal peptide sequence is the amino acid sequence MKAIIVLLMVVTSX 1 A (SEQ ID NO: 147), wherein X 1 is selected from S and N, and the nucleic acid according to Embodiment 5.

[0343] Embodiment 11. The HA secretion signal peptide sequence is the amino acid sequence MX 1 AIIVLLMVVTSNA (SEQ ID NO: 148), wherein X 1 is selected from K and E, and the nucleic acid according to Embodiment 5.

[0344] Embodiment 12. The nucleic acid according to Embodiment 10 or 11, wherein the HA secretion signal peptide sequence comprises an amino acid sequence selected from SEQ ID NOs: 132 to 144.

[0345] Embodiment 13. The nucleic acid according to any one of Embodiments 1 to 12, wherein the viral secretion signal peptide comprises an amino acid sequence selected from the group consisting of MKAKLLVLLCTFTATYA (SEQ ID NO: 1); MKAILVVLLYTFATANA (SEQ ID NO: 2); MKTIIALSYILCLVFA (SEQ ID NO: 3); MKAIIVLLMVVTSNA (SEQ ID NO: 4); MFVFLVLLPLVS (SEQ ID NO: 5); MFLLTTKRTMFVFLVLLPLVS (SEQ ID NO: 6); MSPCGYYSKWRNRDRPEYRRNLRFRRFFSSIHPNAAAGSGFNGPGVFITSVTGVWLCFLCIFSMFVTAVVS (SEQ ID NO: 7); MGTVNKPVVGVLMGFGIITGTLRITNPVRA (SEQ ID NO: 8); MFLIQCLISAVIFYIQVTNA (SEQ ID NO: 9); MQALGIKTEHFIIMCLLSGHA (SEQ ID NO: 10); MGLKVNVSAIFMAVLLTLQTPTG (SEQ ID NO: 11); MGAAAALTAVVLQGYNPPAYG (SEQ ID NO: 12); MGAPQAFLAGLLLAAVAVGTARA (SEQ ID NO: 13); MKVFLVTCLGFAVFSSSVC (SEQ ID NO: 14); MGVTGILQLPRDRFKRTSFFLWVIILFQRTFS (SEQ ID NO: 15); MRSLIIFLFPSIIYS (SEQ ID NO: 16); and MVPQALLLFVPLLVFPLCFG (SEQ ID NO: 184).

[0346] Embodiment 14. The nucleic acid according to Embodiment 13, wherein the viral secretion signal peptide comprises the amino acid sequence of MKAKLLVLLCTFTATYA (SEQ ID NO: 1).

[0347] Embodiment 15. The nucleic acid according to any one of Embodiments 1 to 14, wherein the viral secretion signal peptide is located at the N-terminus of the antigen prokaryotic polypeptide.

[0348] Embodiment 16. The nucleic acid according to any one of Embodiments 1 to 14, wherein the viral secretion signal peptide is located at the C-terminus of the antigen prokaryotic polypeptide.

[0349] Embodiment 17. The nucleic acid according to any one of Embodiments 1 to 16, wherein the viral secretion signal peptide is bound to the antigen prokaryotic polypeptide by a linker.

[0350] Embodiment 18. The nucleic acid according to any one of Embodiments 1 to 17, wherein when the viral secretion signal peptide is determined using SignalP6.0, it has a SignalP cleavage probability score of at least 0.8, at least 0.85, at least 0.90, or at least 0.95.

[0351] Embodiment 19. The nucleic acid according to any one of Embodiments 1 to 18, wherein when the viral secretion signal peptide is determined using SignalP6.0, it has a SignalP signal peptide likelihood score of at least 0.8, at least 0.85, at least 0.90, or at least 0.95.

[0352] Embodiment 20. The TMB (a) comprises or consists of 15 to 50 amino acid residues, preferably 15 to 30 amino acid residues, more preferably 18 to 25 amino acid residues; and / or (b) contains at least 50%, at least 55%, or at least 60% hydrophobic amino acid residues, preferably selected from the group consisting of alanine, isoleucine, leucine, valine, phenylalanine, tryptophan, and tyrosine; and / or (c) contains at least one alpha helix. The nucleic acid according to any one of Embodiments 2 to 19.

[0353] Embodiment 21. The TMB is derived from an integral membrane protein, preferably from a single-pass transmembrane protein, more preferably from a bitopic membrane protein, and even more preferably from a type I bitopic membrane protein. The nucleic acid according to any one of Embodiments 2 to 20.

[0354] Embodiment 22. The nucleic acid according to any one of Embodiments 2 to 11, wherein the TMB is derived from a non-human sequence.

[0355] Embodiment 23. The nucleic acid according to any one of Embodiments 18 to 22, wherein the antigenic prokaryotic polypeptide is derived from a prokaryotic transmembrane protein, and the TMB is the TMB of the prokaryotic transmembrane protein.

[0356] Embodiment 24. The nucleic acid according to any one of Embodiments 18 to 23, wherein the antigenic prokaryotic polypeptide is not derived from a prokaryotic transmembrane protein.

[0357] Embodiment 25. The nucleic acid according to Embodiment 24, wherein the TMB is derived from a viral sequence.

[0358] Embodiment 26. The nucleic acid according to Embodiment 25, wherein the TMB is derived from a viral transmembrane domain sequence selected from the group consisting of non-influenza transmembrane domain sequences selected from the group consisting of influenza transmembrane domain sequences, SARS CoV-2 transmembrane domain sequences, varicella-zoster virus (VZV) transmembrane domain sequences, measles transmembrane domain sequences, rubella transmembrane domain sequences, mumps transmembrane domain sequences, Ebola transmembrane domain sequences, and rabies transmembrane domain sequences.

[0359] Embodiment 27. The TMB is selected from the group consisting of influenza hemagglutinin (HA) transmembrane domain sequences, SARS CoV-2 spike transmembrane domain sequences, VZV gB transmembrane domain sequences, VZV gE transmembrane domain sequences, VZV gI transmembrane domain sequences, VZV gK transmembrane domain sequences, measles F protein transmembrane domain sequences, rubella E1 protein transmembrane domain sequences, rubella E2 protein transmembrane domain sequences, mumps F protein transmembrane domain sequences, Ebola GP protein transmembrane domain sequences, and rabies G protein transmembrane domain sequences, preferably, the TMB comprises an HA transmembrane domain sequence derived from influenza A or influenza B, more preferably an HA transmembrane domain sequence derived from influenza A. The nucleic acid according to Embodiment 26.

[0360] Embodiment 28. The nucleic acid according to any one of Embodiments 18 to 22 and 24 to 27, wherein TMB comprises an amino acid sequence selected from the group consisting of: ILAIYSTVASSLVLLVSLGAISF (SEQ ID NO: 17); ILAIYSTVASSLVLVVSLGAISF (SEQ ID NO: 18); ILWISFAISCFLLCVVLLGFI (SEQ ID NO: 19); STAASSLAVTLMLAIFIVYMV (SEQ ID NO: 20); WYIWLGFIAGLIAIVMVTIML (SEQ ID NO: 21); FGALAVGLLVLAGLVAAFFAY (SEQ ID NO: 22); AAWTGGLAAVVLLCLVIFLIC (SEQ ID NO: 23); IIIPIVASVMILTAMVIVIVI (SEQ ID NO: 24); YFWCVQLKMIFFAWFVYGMYL (SEQ ID NO: 25); IVYILIAVCLGGLIGIPALIC (SEQ ID NO: 26); LDHAFAAFVLLVPWVLIFMVC (SEQ ID NO: 27); WWQLTLGAICALLLAGLLACC (SEQ ID NO: 28); IVAALVLSILSIIISLLFCCW (SEQ ID NO: 29); WIPAGIGVTGVIIAVIALFCI (SEQ ID NO: 30); and VLLSAGALTALMLIIFLMTCW (SEQ ID NO: 185).

[0361] Embodiment 29. The nucleic acid according to Embodiment 28, wherein TMB comprises the amino acid sequence of ILAIYSTVASSLVLVLVLGAISF (SEQ ID NO: 17).

[0362] Embodiment 30. The nucleic acid according to Embodiments 2 and 18 to 29, wherein TMB is bound to an antigen prokaryotic polypeptide via a linker.

[0363] Embodiment 31. The nucleic acid according to any one of Embodiments 2 and 18 to 30, wherein TMB is located at the N-terminus of the antigen prokaryotic polypeptide.

[0364] Embodiment 32. The nucleic acid according to any one of Embodiments 2 and 18 to 30, wherein TMB is located at the C-terminus of the antigen prokaryotic polypeptide.

[0365] Nucleic acid comprising an open reading frame (ORF), wherein the ORF comprises: - a polynucleotide sequence encoding at least one antigen polypeptide, preferably an antigen prokaryotic polypeptide, and - a polynucleotide sequence encoding at least one transmembrane domain (TMB), wherein the TMB is heterologous to the antigen polypeptide, and optionally, the ORF further comprises a polynucleotide sequence encoding at least one secretion signal peptide as described in any one of the preceding claims, preferably a viral secretion signal peptide, more preferably a polynucleotide sequence encoding a viral secretion signal peptide.

[0366] Nucleic acid comprising an open reading frame (ORF), wherein the ORF comprises: - a polynucleotide sequence encoding at least one antigen prokaryotic polypeptide and - a polynucleotide sequence encoding at least one transmembrane domain (TMB).

[0367] The nucleic acid according to embodiment 34, wherein the TMB: (a) comprises or consists of 15 to 50 amino acid residues, preferably 15 to 30 amino acid residues, more preferably 18 to 25 amino acid residues; and / or (b) comprises at least 50%, at least 55%, or at least 60% hydrophobic amino acid residues, preferably selected from the group consisting of alanine, isoleucine, leucine, valine, phenylalanine, tryptophan, and tyrosine; and / or (c) comprises at least one alpha helix.

[0368] The nucleic acid according to embodiment 34 or 35, wherein the TMB is derived from an integral membrane protein, preferably from a single-pass membrane protein, more preferably from a type I bitopic membrane protein.

[0369] The nucleic acid according to embodiment 36, wherein the TMB is derived from a non-human sequence.

[0370] Embodiment 38. The nucleic acid according to any one of Embodiments 1 to 32, wherein the antigenic prokaryotic polypeptide is derived from a prokaryotic membrane protein and the TMB is a transmembrane domain of a heterologous prokaryotic membrane protein.

[0371] Embodiment 39. The nucleic acid according to any one of Embodiments 35 to 38, wherein the antigenic polypeptide is not derived from a transmembrane protein.

[0372] Embodiment 40. The nucleic acid according to any one of Embodiments 35 to 37 and 39, wherein the TMB is derived from a viral sequence.

[0373] Embodiment 41. The nucleic acid according to Embodiments 33 to 35, 39, and 40, wherein the TMB is derived from a viral transmembrane domain sequence selected from the group consisting of non-influenza transmembrane domain sequences selected from the group consisting of influenza transmembrane domain sequences, SARS CoV-2 transmembrane domain sequences, varicella-zoster virus (VZV) transmembrane domain sequences, measles transmembrane domain sequences, rubella transmembrane domain sequences, mumps transmembrane domain sequences, Ebola transmembrane domain sequences, and rabies transmembrane domain sequences.

[0374] Embodiment 42. The TMB is selected from the group consisting of an influenza hemagglutinin (HA) transmembrane domain sequence, a SARS CoV-2 spike transmembrane domain sequence, a VZV gB transmembrane domain sequence, a VZV gE transmembrane domain sequence, a VZV gI transmembrane domain sequence, a VZV gK transmembrane domain sequence, a measles F protein transmembrane domain sequence, a rubella E1 protein transmembrane domain sequence, a rubella E2 protein transmembrane domain sequence, a mumps F protein transmembrane domain sequence, an Ebola GP protein transmembrane domain sequence, and a rabies G protein transmembrane domain sequence. Preferably, the TMB comprises an HA transmembrane domain sequence derived from influenza A or influenza B, more preferably an HA transmembrane domain sequence derived from influenza A. The nucleic acid according to Embodiment 41.

[0375] Embodiment 43. The nucleic acid according to Embodiment 42, wherein TMB comprises an amino acid sequence selected from the group consisting of: ILAIYSTVASSLVLLVSLGAISF (SEQ ID NO: 17); ILAIYSTVASSLVLVVSLGAISF (SEQ ID NO: 18); ILWISFAISCFLLCVVLLGFI (SEQ ID NO: 19); STAASSLAVTLMLAIFIVYMV (SEQ ID NO: 20); WYIWLGFIAGLIAIVMVTIML (SEQ ID NO: 21); FGALAVGLLVLAGLVAAFFAY (SEQ ID NO: 22); AAWTGGLAAVVLLCLVIFLIC (SEQ ID NO: 23); IIIPIVASVMILTAMVIVIVI (SEQ ID NO: 24); YFWCVQLKMIFFAWFVYGMYL (SEQ ID NO: 25); IVYILIAVCLGGLIGIPALIC (SEQ ID NO: 26); LDHAFAAFVLLVPWVLIFMVC (SEQ ID NO: 27); WWQLTLGAICALLLAGLLACC (SEQ ID NO: 28); IVAALVLSILSIIISLLFCCW (SEQ ID NO: 29); WIPAGIGVTGVIIAVIALFCI (SEQ ID NO: 30); and VLLSAGALTALMLIIFLMTCW (SEQ ID NO: 185).

[0376] Embodiment 44. The nucleic acid according to Embodiment 43, wherein TMB comprises the amino acid sequence of ILAIYSTVASLVLVLVLGAISF (SEQ ID NO: 17).

[0377] Embodiment 45. The nucleic acid according to any one of Embodiments 34 to 44, wherein TMB is bound to an antigen prokaryotic polypeptide by a linker.

[0378] Embodiment 46. The nucleic acid according to any one of Embodiments 34 to 44, wherein TMB is located at the N-terminus of the antigen prokaryotic polypeptide.

[0379] Embodiment 47. The nucleic acid according to any one of Embodiments 34 to 44, wherein TMB is located at the C-terminus of the antigen prokaryotic polypeptide.

[0380] Embodiment 48. The antigen prokaryotic polypeptide is Acetobacter, Acinetobacter, Actinomyces, Aerococcus, Agrobacterium, Anaplasma, Azorhizobia, Azotobacter, Bacillus, Bacteroides, Bartonella, Bordetella, Borrelia, Brucella, Burkholderia, Calymmatobacterium, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Coxiella, Cutibacterium, Ehrlichia, Enterobacter, Enterococcus, Escherichia, Francisella, Fusobacterium, Gardnerella, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Legionella, Listeria, Methanobacterium, Microbacterium, Micrococcus, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Pasteurella, Pediococcus,Bacteria of a species selected from the group consisting of Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, Pseudomonas, Rhizobium, Rickettsia, Rochalimaea, Rothia, Salmonella, Serratia, Shigella, Sarcina, Spirillum, Spirochaetes, Staphylococcus, Stenotrophomonas, Streptobacillus, Streptococcus, Tetragenococcus, Treponema, Vibrio, Viridans, Walbachia, and Yersinia, Preferably, Acetobacter aurantius, Acinetobacter baumannii, Actinomyces israelii, Agrobacterium radiobacter, Agrobacterium tumefaciens, Anaplasma phagocytophilum, Azorhizobium caulinodans, Azotobacter vinelandii, Bacillus anthracis, Bacillus brevis, Bacillus cereus, Bacillus fusiformis, Bacillus licheniformis, Bacillus megaterium, Bacillus mycoides, Bacillus stearothermophilus, Bacillus subtilis, Bacillus Thuringiensis, Bacteroides fragilis, Bacteroides gingivalis, Bacteroides melaninogenicus, Bartonella henselae, Bartonella Quintana, Bordetella bronchiseptica, Bordetella pertussis, Borrelia burgdorferi, Brucella abortusabortus), Brucella melitensis, Brucella suis, Burkholderia mallei, Burkholderia pseudomallei, Burkholderia cepacia, Calymmatobacterium granulomatis, Campylobacter coli, Campylobacter fetus, Campylobacter jejuni, Campylobacter pylori, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Corynebacterium fusiforme, Coxiella burnetii, Cutibacterium acnes, Cutibacterium avidum, Cutibacterium granulosum, Cutibacterium namnetense, Cutibacterium humerusii, Ehrlichia chaffeensis (EhrlichiaChaffeensis, Enterobacter cloacae, Enterococcus avium, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus maloratus, Escherichia coli, Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Haemophilus ducreyi, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus pertussis, Haemophilus vaginalis, Helicobacter pylori, Klebsiella pneumoniae, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactococcus lactis, Legionella pneumophila, Listeria monocytogenes, Methanobacterium extroquens, Microbacterium multiforme, Micrococcus luteusStaphylococcus aureus, Moraxella catarrhalis, Mycobacterium avium, Mycobacterium bovis, Mycobacterium diphtheriae, Mycobacterium intracellulare, Mycobacterium leprae, Mycobacterium lepraemurium, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycoplasma fermentans, Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma penetrans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Pasteurella tularensis, Peptostreptococcus, Porphyromonas gingivalis, Prevotella melaninogenica, Propionibacterium acnes, Pseudomonas aeruginosa, Rhizobium radiobacter, Rickettsia prowazekii, Rickettsia psittaci, Rickettsia quintana, Rickettsia rickettsii, Rickettsia trachomae, Rochalimaea henselae, Rochalimaea quintana, Rothia dentocariosa, Salmonella enteritidis, Salmonella typhi, Salmonella typhimurium, Serratia marcescens, SerratiaSerratia marcescens, Shigella dysenteriae, Spirillum volutans, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus avium, Streptococcus bovis, Streptococcus cricetus, Streptococcus faceium, Streptococcus faecalis, Streptococcus ferus, Streptococcus gallinarum, Streptococcus lactis, Streptococcus mitior, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus rattus, Streptococcus salivarius, Streptococcus sanguis, Streptococcus sobrinus, Treponema pallidum, Treponema denticola, Vibrio cholerae, Vibrio comma, Vibrio enteritisThe nucleic acid according to any one of Embodiments 34 to 47, which is derived from a bacterium of a species selected from the group consisting of Vibrio parahaemolyticus, Vibrio vulnificus, Viridans streptococci, Wolbachia, Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis.

[0381] Embodiment 49. The antigen prokaryotic polypeptide is derived from a bacterium of the genus Borrelia, preferably selected from B. burgdorferi, afzelii, garinii, bavariensis, mayonii, spielmanii, lusitaniae, bissettii, and / or valaisiana, and the nucleic acid according to any one of Embodiments 1 to 48.

[0382] Embodiment 50. The antigen prokaryotic polypeptide is OspA ST1, OspA ST2, OspA ST3, OspA ST4, OspA ST5, OspA ST6, OspA ST7, or a fragment thereof, and the nucleic acid according to Embodiment 49.

[0383] Embodiment 51. The antigen prokaryotic polypeptide is OspA or a fragment or variant thereof, and OspA or a fragment or variant thereof contains at least 5, at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 amino acids. Preferably, the antigen prokaryotic polypeptide is (a) Preferably, an amino acid sequence derived from OspA ST1 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0384] Embodiment 52. The antigenic prokaryotic polypeptide is derived from a bacterium of the genus Cutibacerium, preferably selected from the species acnes, avidum, granulosum, namnetense, and / or humerusii, the nucleic acid according to any one of embodiments 1 to 48.

[0385] Embodiment 53. The antigenic prokaryotic polypeptide is CAMP2, the nucleic acid according to embodiment 52.

[0386] Embodiment 54. The amino acid sequence encoding CAMP2 or a fragment thereof is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence comprising SEQ ID NO: 149 or SEQ ID NOs: 162 - 172, the nucleic acid according to embodiment 53.

[0387] Embodiment 55. The nucleic acid according to Embodiment 52, wherein the antigen prokaryotic polypeptide is PITP.

[0388] Embodiment 56. The nucleic acid according to Embodiment 54, wherein the amino acid sequence encoding PITP or a fragment thereof is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence containing SEQ ID NO: 150 or SEQ ID NOs: 173 to 183.

[0389] Embodiment 57. The nucleic acid according to any one of Embodiments 1 to 56, wherein the antigen prokaryotic polypeptide contains at least one mutated glycosylation site, preferably at least one mutated N-linked glycosylation site.

[0390] Embodiment 58. The nucleic acid according to any one of Embodiments 1 to 57, wherein the polynucleotide sequence of the nucleic acid is codon-optimized.

[0391] Embodiment 59. The nucleic acid according to any one of Embodiments 1 to 58, wherein the polynucleotide sequence of the ORF is codon-optimized.

[0392] Embodiment 60. The nucleic acid according to any one of Embodiments 1 to 59, wherein the polynucleotide sequence encoding at least one viral secretion signal peptide is codon-optimized.

[0393] Embodiment 61. The nucleic acid according to any one of Embodiments 20 to 60, wherein the polynucleotide sequence encoding at least one TMB is codon-optimized.

[0394] Embodiment 62. The nucleic acid according to any one of Embodiments 1 to 61, wherein the nucleic acid is DNA.

[0395] Embodiment 63. The nucleic acid according to any one of Embodiments 1 to 61, wherein the nucleic acid is messenger RNA (mRNA), and in particular, the mRNA is non-replicating mRNA, self-replicating mRNA, or trans-replicating mRNA.

[0396] Embodiment 64. The nucleic acid according to Embodiment 63, wherein the mRNA comprises at least one 5'untranslated region (5'UTR), at least one 3'untranslated region (3'UTR), and / or at least one polyadenylation (poly(A)) sequence.

[0397] Embodiment 65. The nucleic acid according to Embodiment 63 or 64, wherein the mRNA comprises at least one chemical modification.

[0398] Embodiment 66. The nucleic acid according to any one of Embodiments 63 to 65, wherein at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the mRNA are chemically modified.

[0399] Embodiment 67. The nucleic acid according to any one of Embodiments 63 to 66, wherein at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the open reading frame (ORF) are chemically modified.

[0400] Embodiment 68. The nucleic acid according to any one of Embodiments 65 to 67, wherein the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-1-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0401] Embodiment 69. The nucleic acid according to any one of Embodiments 65 to 68, wherein the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0402] Embodiment 70. The nucleic acid according to any one of Embodiments 65 to 69, wherein the chemical modification is N1-methylpseudouridine.

[0403] Embodiment 71. A composition comprising at least one nucleic acid according to any one of Embodiments 1 to 70.

[0404] Embodiment 72. The composition according to Embodiment 71, further comprising lipid nanoparticles (LNP).

[0405] Embodiment 73. The composition according to Embodiment 72, wherein the nucleic acid is encapsulated in the LNP.

[0406] Embodiment 74. The composition according to Embodiment 72 or 73, wherein the LNP comprises at least one cationic lipid.

[0407] Embodiment 75. The composition according to Embodiment 74, wherein the cationic lipid is biodegradable.

[0408] Embodiment 76. The composition according to Embodiment 74, wherein the cationic lipid is non-biodegradable.

[0409] Embodiment 77. The composition according to any one of Embodiments 74 to 76, wherein the cationic lipid is cleavable.

[0410] Embodiment 78. The composition according to any one of Embodiments 74 to 76, wherein the cationic lipid is non-cleavable.

[0411] Embodiment 79. The composition according to any one of Embodiments 74 to 78, wherein the cationic lipid is selected from the group consisting of OF-02, cKK-E10, OF-Deg-Lin, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14, SM-102, and ALC-0315.

[0412] Embodiment 80. The composition according to any one of Embodiments 74 to 79, wherein the LNP further comprises a polyethylene glycol (PEG) conjugate (PEGylated) lipid, a cholesterol-based lipid, and a helper lipid.

[0413] Embodiment 81. The LNP comprises a cationic lipid in a molar ratio of -35% to 55%; a polyethylene glycol (PEG) conjugate (PEGylated) lipid in a molar ratio of -0.25% to 2.75%; a cholesterol-based lipid in a molar ratio of -20% to 45%; and a helper lipid in a molar ratio of -5% to 35%, and all of the molar ratios are relative to the total lipid content of the LNP. The composition according to any one of Embodiments 72 to 80.

[0414] Embodiment 82. The LNP comprises a cationic lipid in a molar ratio of -40%; a PEGylated lipid in a molar ratio of -1.5%; a cholesterol-based lipid in a molar ratio of -28.5%; and a helper lipid in a molar ratio of -30%. The composition according to any one of Embodiments 72 to 81.

[0415] Embodiment 83. The composition according to any one of Embodiments 72 to 82, wherein the LNP comprises a cationic lipid in a molar ratio of -45 to 50%; a PEGylated lipid in a molar ratio of -1.5 to 1.7%; a cholesterol-based lipid in a molar ratio of -38 to 43%; and a helper lipid in a molar ratio of -9 to 10%.

[0416] Embodiment 84. The composition according to any one of Embodiments 80 to 83, wherein the PEGylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000) or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0417] Embodiment 85. The composition according to any one of Embodiments 80 to 84, wherein the cholesterol-based lipid is cholesterol.

[0418] Embodiment 86. The composition according to any one of Embodiments 80 to 85, wherein the helper lipid is 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0419] Embodiment 87. The composition according to any one of Embodiments 72 to 82 and 84 to 86, wherein the LNP comprises a cationic lipid in a molar ratio of -40%, selected from the group consisting of OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, and GL-HEPES-E3-E12-DS-3-E14; DMG-PEG2000 in a molar ratio of -1.5%; cholesterol in a molar ratio of -28.5%; and -DOPE in a molar ratio of -30%.

[0420] Embodiment 88. The composition according to any one of Embodiments 72 to 81 and 83 to 86, wherein the LNP comprises SM-102 in a molar ratio of -50%; DMG-PEG2000 in a molar ratio of -1.5%; cholesterol in a molar ratio of -38.5%; and DSPC in a molar ratio of -10%.

[0421] Embodiment 89. The composition according to any one of Embodiments 72 to 81 and 83 to 86, wherein the LNP comprises ALC-0315 at a molar ratio of -46.3%; ALC-0159 at a molar ratio of -1.6%; cholesterol at a molar ratio of -42.7%; and -DSPC at a molar ratio of -9.4%.

[0422] Embodiment 90. The composition according to any one of Embodiments 72 to 81 and 83 to 86, wherein the LNP comprises ALC-0315 at a molar ratio of -47.4%; ALC-0159 at a molar ratio of -1.7%; cholesterol at a molar ratio of -40.9%; and DSPC at a molar ratio of -10%.

[0423] Embodiment 91. The composition according to any one of Embodiments 72 to 90, wherein the LNP has an average diameter of 30 nm to 200 nm.

[0424] Embodiment 92. The composition according to any one of Embodiments 72 to 91, wherein the LNP has an average diameter of 80 nm to 150 nm.

[0425] Embodiment 93. The composition according to any one of Embodiments 72 to 92, comprising LNP at 1 mg / mL to 10 mg / mL.

[0426] Embodiment 94. The composition according to any one of Embodiments 72 to 93, wherein the LNP comprises 1 to 20 nucleic acid molecules, preferably mRNA molecules.

[0427] Embodiment 95. The composition according to any one of Embodiments 71 to 94, formulated for intramuscular, intranasal, intravenous, subcutaneous, or intradermal administration.

[0428] Embodiment 96. The composition according to any one of Embodiments 71 to 95, wherein the composition comprises phosphate buffered saline.

[0429] Embodiment 97. The composition according to any one of Embodiments 71 to 96, wherein the composition is a pharmaceutical composition, such as an immunogenic composition or a vaccine, particularly an mRNA vaccine.

[0430] Embodiment 98. A nucleic acid according to any one of Embodiments 1 to 70 or a composition according to any one of Embodiments 71 to 97 for use in inducing an immune response in a subject in need thereof.

[0431] Embodiment 99. A method of inducing an immune response in a subject in need thereof, the method comprising administering to the subject, optionally intramuscularly, intranasally, intravenously, subcutaneously, or intradermally, an effective amount of a nucleic acid according to any one of Embodiments 1 to 70 or a composition according to any one of Embodiments 71 to 97.

[0432] Embodiment 100. Use of a nucleic acid according to any one of Embodiments 1 to 70 or a composition according to any one of Embodiments 71 to 97 for the manufacture of a medicament for use in inducing an immune response in a subject in need thereof.

[0433] Embodiment 101. A nucleic acid according to any one of Embodiments 1 to 70 or a composition according to any one of Embodiments 71 to 97 for use in treating or preventing a prokaryotic infection in a subject in need thereof.

[0434] Embodiment 102. A method of treating or preventing a prokaryotic infection in a subject in need thereof, the method comprising administering to the subject, optionally intramuscularly, intranasally, intravenously, subcutaneously, or intradermally, an effective amount of a nucleic acid according to any one of Embodiments 1 to 70 or a composition according to any one of Embodiments 71 to 97.

[0435] Embodiment 103. Use of a nucleic acid according to any one of Embodiments 1 to 70 or a composition according to any one of Embodiments 71 to 97 for the manufacture of a medicament for use in treating or preventing a prokaryotic infection in a subject in need thereof.

[0436] Embodiment 104. A method for secreting an antigenic prokaryotic polypeptide in a host cell, the method comprising administering to the host cell the nucleic acid according to any one of Embodiments 1 to 70 or the composition according to any one of Embodiments 71 to 97.

[0437] Embodiment 105. A method for displaying an antigenic prokaryotic polypeptide on the surface of a host cell, the method comprising administering to the host the nucleic acid according to any one of Embodiments 1 to 70 or the composition according to any one of Embodiments 71 to 97.

[0438] Embodiment 106. A kit comprising a container containing a single-dose or multiple-dose of the nucleic acid according to any one of Embodiments 1 to 70 or the composition according to any one of Embodiments 71 to 97, optionally wherein the container is a vial or a filled syringe or a syringe.

[0439] To better understand the present disclosure, the following examples are shown. These examples are for illustrative purposes only and should in no way be construed as limiting the scope of the present disclosure.

Examples

[0440] Example 1: Materials and Methods mRNA production mRNA was produced as previously published (Kalnin et al (2021), NPJ Vaccines 6(1):61 and WO 2021 / 226436 pamphlet). Briefly, mRNA incorporating the coding sequence containing either OspA ST1 or ST2 was synthesized by in vitro transcription using RNA polymerase with a plasmid DNA template encoding the desired gene using unmodified nucleotides. The resulting crude precursor mRNA was further reacted via enzymatic addition of a 5' cap structure (Cap1) and a 3' poly(A) tail of approximately 200 nucleotides in length determined by gel electrophoresis.

[0441] For the preparation of the mRNA / lipid nanoparticle (LNP) formulation, an ethanol solution of a mixture of lipids (cationic / ionizable lipid, phosphatidylethanolamine, cholesterol, and polyethylene glycol-lipid) was combined with an aqueous buffer solution of the target mRNA at acidic pH under controlled conditions at a fixed lipid and mRNA ratio to obtain a homogeneous suspension of LNPs. During ultrafiltration and diafiltration into a suitable dilution system, the resulting nanoparticle suspension was diluted to the final concentration, filtered, and stored frozen at -80 °C until use.

[0442] Production of antigens used as benchmark comparators The OspA-ferritin ST1 and ST2 antigens were produced by the Sanofi Breakthrough Lab in Cambridge, MA, USA, according to the materials and methods already published (Kamp et al (2020), NPJ Vaccines 5(1):33).

[0443] For the OspA fusion ST1-ST2, the in-house plasmid pSP401+LPP-chimer OspA ST1-OspA ST2, which enables the expression of the C-terminal domain of the OspA fusion ST1-ST2, was introduced into the E. coli expression strain C43-(DE3) (Lucigen). After growth for 2-3 hours at 37 °C in rich medium, the expression of the target protein was induced by the addition of an inducer, and the culture was stopped 3 hours after induction. After treating the bacterial pellet, the protein was visualized by Coomassie blue-stained SDS-Page gel or Western blot using specific antibodies. Scale-up was performed under the best expression conditions to produce sufficient biomass for purification. The bacterial pellet was treated with lysozyme to extract membrane proteins. Then, the OspA ST1-OspA ST2 fusion protein was extracted with 2 M urea + 2% Triton X114. After 3 incubation-centrifugation steps at 37 °C, the lower phase was recovered and subjected to Q Sepharose chromatography in the presence of Zwittergent 3.14 detergent (0.5%). The fraction eluted with 400 mM NaCl was subjected to ceramic hydroxyapatite chromatography. The OspA ST1-OspA ST2 fusion protein was eluted with 0.05% Tween NaNa2PO4 180 mM pH 6.7 buffer and replaced with PBS+Tween20 0.05% pH 7.3 as the final buffer.

[0444] Transfection of HEK cells HEK293T cells (2×10 6 cells / mL - 5 mL in a 125 mL shaker) in suspension were transfected for 2-5 minutes with 5 μg / μg of naked mRNA (1 μg / μL) mixed with an equal amount of Transit-mRNA reagent and mRNA Boost reagent - Transit-mRNA transfection kit Milus (reference MIR 2250). The mixture was added dropwise to the cells and incubated at 37 °C, 100 rpm, 8% CO 2 for 48-72 hours.

[0445] Western blot analysis of mRNA-transfected cells After transfection, cells and medium were harvested and centrifuged (500×g) to recover the supernatant. The cell pellet was lysed using lysozyme (Ready-Lyse Lysozyme solution - Lucigen reference R1804M) + benzonase (Sigma - reference E1014) + protease inhibitor cocktail (Sigma - reference P8340) at 20°C for 10 minutes at 800 rpm. The cell pellet lysate was then centrifuged (11,000×g) to recover the supernatant and crude extract.

[0446] Extracts from mRNA-transfected HEK293T cells were analyzed by denaturing (95°C) PAGE using a 4 - 12%BIS-Tris / MES gel (Invitrogen) and Western blot. Transfer to a nitrocellulose membrane (Bio-Rad) was performed using a semi-dry transfer system (Trans-Blot Turbo Transfer System, Bio-Rad). Blotting proteins were detected with a polyclonal (rabbit) antibody recognizing OspA (anti-OspA polyclonal / Rabit-Abcam, reference ab10608-1:2000) and a secondary antibody (anti-rabbit IgG goat antibody DyLit800-Rockland, reference 611-145-002-1:2000). The blot was imaged with an Odyssey infrared imager - LICOR.

[0447] Characterization of mRNA OspA ST1 or ST2 antigenicity by sandwich ELISA using functional monoclonal antibodies (mAbs) Characterization by ST1-specific functional mAb LA-2 mAb 857-2 (R&D Biotech, Internal Order) was coated at 2.5 μg / mL in PBS on a microtiter plate (Greiner Bio-One). The plate was incubated overnight at 4°C and then blocked with PBS-Tween 0.05%-milk 5% for 1 hour at room temperature.

[0448] The transfection supernatant was serially diluted 2-fold in dilution buffer (PBS-Tween 0.05% - milk 1%) and incubated at room temperature for 1.5 hours. After washing with PBS-0.05% Tween, detection of the protein attached to the coating was performed by incubating with mAb LA-2 at 1:1000 for 1.5 hours at room temperature (Absolute Antibody, catalog Ab01070-3.0-BT), and then incubating with goat anti-mouse IgG HRP. After washing, the plates were developed using 3,3,5,5-tetramethylbenzidine (tebu-bio, catalog TMB100-1000) and stopped with 1N HCl (VWR ProLabo). The optical density (OD) was read at 450 nm - 650 nm.

[0449] Characterization by cross-specific functional mAbs 857-2 or 221-7 mAbs 221-7 or 857-2 (Wang et al. J. Infect Dis. 214(2):205-211. 2016) were coated at 5 μg / mL in PBS on microtiter plates (Greiner Bio-One, catalog 655061). The plates were incubated overnight at 4°C and then blocked with PBS-Tween 0.05% - milk 5% for 1 hour at room temperature.

[0450] The transfection supernatant was serially diluted 2-fold in dilution buffer (PBS-Tween 0.05% - milk 1%) and incubated at room temperature for 1.5 hours. After washing with PBS-0.05% Tween, detection of the protein attached to the coating was carried out by incubation with anti-OspA mouse polyclonal serum (in-house) at room temperature for 1.5 hours. The plate was washed and incubated with goat anti-mouse IgG HRP (Jackson Laboratories, catalog 115-036-062) at room temperature for 1.5 hours. After washing, the plate was developed with 3,3,5,5-tetramethylbenzidine (tebu-bio, catalog TMB100-1000) in the dark at room temperature for 30 minutes. The colorimetric reaction was stopped with 1N HCl (VWR Prolo, catalog 30024290). The optical density (OD) was read at 450 nm - 650 nm.

[0451] Antigen and mouse immunization OF-1 mice (Charles River) were randomized into immunization groups of 8 animals each. Four different doses of mRNA-OspA-LNP: 0.2 μg, 1 μg, 5 μg, or 10 μg were administered intramuscularly (50 μL) on day 0 (D0) (dose 1) and day 21 (D21) (dose 2). Serum was collected at baseline (D0), 19 days before dose 2 (D19), and day 35 (D35).

[0452] The following mRNA-OspA sequences were tested: mRNA-OspA ST1-native, mRNA-HA SS-OspA ST1-native, mRNA-HA SS-OspA ST1-Gly(-), mRNA-TMB-OspA ST1-native, mRNA-TMB-OspA ST1-Gly(-). As shown in Figure 1, the mRNA sequences with TMB also contain HA SS.

[0453] These mRNA formulations were compared to the benchmark Lyme dog vaccine Recombitek® (Merial) at 1 μg / dose (50 μL), recombinant fusion OspA ST1-ST2 (2 μg / dose) + AlOOH adjuvant, and OspA-ferritin (ST1 and ST2) at 1.7 μg / dose + AF03 adjuvant.

[0454] OspA-specific IgG ELISA Antibody responses in mice were determined by ELISA. Briefly, 384-well microplates (Perkin Elmer #6007509) were coated with 1 μg / mL of OspA ST1-His diluted in PBS and incubated overnight at 4 °C. OspA ST1-His was removed and the plates were blocked with 5% skim milk dissolved in PBS-tween. After removing the blocking reagent, primary serum samples were added after being serially diluted 2-fold in 1% skim milk-PBS-Tween. After incubating the primary serum samples for 1.5 h at room temperature, the plates were washed with PBS-Tween and incubated with goat anti-mouse IgG-HRP (Jackson 115-036-062) for 1.5 h at room temperature. The secondary antibody was aspirated and washed, and the plates were incubated with TMB substrate (TEBU-TMB100-1000) and then an equal volume of stop solution (HCl 1N). Absorbance was measured at 450 nm - 650 nm. OspA-specific IgG titers were quantified via an internal anti-OspA mouse serum reference. The titer of this reference was pre-calculated as the reciprocal of the dilution rate to obtain an OD of 1.

[0455] Statistical analysis Two-way ANOVA with vaccine dose and their interactions as factors was performed (one model per time point). Where necessary, within-group heterogeneity was taken into account.

[0456] OspA amino acid and mRNA sequences The OspA amino acid sequences and mRNA sequences used in the examples are listed in the following table.

[0457]

Table 9

[0458]

Table 10

[0459]

Table 11

[0460]

Table 12

[0461]

Table 13

[0462]

Table 14

[0463]

Table 15

[0464]

Table 16

[0465]

Table 17

[0466]

Table 18

[0467]

Table 19

[0468]

Table 20

[0469]

Table 21

[0470]

Table 22

[0471]

Table 23

[0472]

Table 24

[0473]

Table 25

[0474]

Table 26

[0475]

Table 27

[0476]

Table 28

[0477]

Table 29

[0478]

Table 30

[0479]

Table 31

[0480] The following mRNA sequences are variants encoding the same viral secretion signal peptide, MKAKLLVLLCTFTATYA (SEQ ID NO: 1): AUGAAGGCCAAGCUGCUGGUCCUGCUCUGUACCUUUACAGCCACUUACGCC (SEQ ID NO: 60); AUGAAGGCCAAACUGCUCGUGCUCUUAUGCACAUUCACAGCAACCUACGCC (SEQ ID NO: 61); AUGAAGGCUAAGCUGCUGGUUCUGCUGUGUACUUUUACCGCCACAUACGCU (SEQ ID NO: 62); AUGAAGGCCAAACUCCUGGUGCUCCUGUGUACCUUCACCGCUACCUACGCC (SEQ ID NO: 63); AUGAAGGCAAAGCUGCUGGUGCUGCUGUGUACCUUCACUGCCACCUACGCC (SEQ ID NO: 64); AUGAAGGCCAAACUGCUGGUGCUGCUGUGCACUUUCACUGCAACUUACGCC (SEQ ID NO: 65); AUGAAAGCCAAACUUCUGGUCCUGCUCUGUACCUUCACUGCAACCUACGCC (SEQ ID NO: 66); AUGAAGGCCAAGCUGCUGGUGCUGCUGUGUACAUUCACAGCAACCUAUGCC (SEQ ID NO: 67); AUGAAAGCAAAGCUGCUGGUGCUGCUGUGCACAUUCACCGCAACAUACGCC (SEQ ID NO: 68); AUGAAAGCAAAGCUGCUGGUCCUGCUGUGUACUUUCACAGCAACUUAUGCA (SEQ ID NO: 69); AUGAAAGCCAAGCUCCUGGUGCUCCUGUGCACAUUCACUGCAACUUACGCC (SEQ ID NO: 70); AUGAAGGCUAAACUGCUGGUCCUGCUGUGUACCUUCACCGCUACAUACGCC (SEQ ID NO: 71); AUGAAGGCAAAACUGCUGGUGCUGCUGUGUACAUUCACAGCUACUUAUGCA (SEQ ID NO: 72).

[0481] The following mRNA sequences are variants encoding the same transmembrane domain, ILAIYSTVASLVLVLVSLGAISF (SEQ ID NO: 17): AUCCUGGCAAUCUAUAGCACAGUCGCCAGCUCCCUGGUUCUCCUGGUGAGCCUGGGGGCAAUUUCCUUC (SEQ ID NO: 73); AUCCUGGCCAUCUAUAGCACCGUCGCCAGCUCUCUGGUGCUGCUGGUGUCCCUCGGGGCUAUCUCAUUC (SEQ ID NO: 74); AUUCUGGCAAUCUACAGCACAGUGGCCUCUUCUCUGGUGCUGCUGGUUUCCCUGGGCGCCAUUAGUUUU (SEQ ID NO: 75); AUCCUCGCCAUCUACUCCACCGUGGCCUCUAGCCUGGUUCUGCUGGUGAGCCUGGGCGCCAUUUCUUUU (SEQ ID NO: 76); AUCCUGGCUAUCUAUAGCACUGUGGCUUCCUCUCUGGUGCUGCUGGUUUCCCUGGGGGCCAUUUCCUUC (SEQ ID NO: 77); AUCCUGGCUAUCUAUAGCACCGUCGCCUCCAGCCUCGUUCUGCUGGUGAGCCUGGGCGCCAUUUCCUUC (SEQ ID NO: 78); AUUCUGGCAAUCUACUCCACAGUGGCUUCAAGCCUGGUGCUGCUCGUGUCCCUCGGGGCAAUCUCCUUC (SEQ ID NO: 79); AUCCUGGCAAUCUACUCUACAGUGGCUUCCUCCCUUGUUCUGCUGGUCAGCCUGGGCGCCAUCAGCUUU (SEQ ID NO: 80).

[0482] Example 2: In Vitro mRNA Expression and Antigenicity of Signal Sequence-Containing Antigenic Prokaryotic Polypeptides Using the Borrelia outer surface protein A (OspA) as an exemplary antigenic prokaryotic polypeptide, the effect of linking one or both of the hemagglutinin secretion signal (HA1 SS) and the HA transmembrane domain (TMB) to OspA was tested. OspA serotype 1 (ST1) and serotype 2 (ST2) were used.

[0483] mRNA expressing either OspA ST1 or ST2 was designed. Different mRNA sequences were designed to induce the expression of OspA intracellularly, secreted, or transmembrane using the OspA sequence without or with fusion to the hemagglutinin secretion signal (HA1 SS) and / or the HA transmembrane domain (TMB). HA1 SS causes OspA secretion, TMB induces OspA to the membrane, and OspA without either HA1 SS or TMB is present intracellularly.

[0484] The expression and antigenicity of the OspA ST1 and ST2 antigens delivered by mRNA were confirmed in vitro prior to vaccination of mice. The methods of mRNA transfection and expression are described above in Example 1.

[0485] As shown in FIGS. 2A and 2B, in vitro expression of OspA ST1 and ST2 mRNAs in HEK293T cell supernatants was achieved. mRNA-OspA ST1 and mRNA-OspA ST2 were tested with or without the HA secretion signal and with or without glycosylation site mutations. Negative controls (buffer) and positive controls (recombinant OspA) were also used. HEK293T cells were transfected with each mRNA, and after 48 hours, the supernatants were collected and run on Western blots. The blotted proteins were detected using a polyclonal rabbit antibody that recognizes OspA. The results demonstrated that extracellular expression of OspA was increased by adding the secretion signal. Mutations in the glycosylation sites effectively avoided glycosylation of OspA, as evidenced by the presence of a single spot of Gly(-) instead of multiple spots of the native (glycosylated) protein.

[0486] As shown in FIGS. 3A-3C, in vitro expression of mRNA-TMB-OspA ST1 in HEK293T cells was achieved. Cell supernatants, crude extracts, and intracellular fractions were tested for OspA ST1 expression. After 48-72 hours, the supernatants and cells were collected and run on Western blots. The blotted proteins were detected using a polyclonal rabbit antibody that recognizes OspA. The results demonstrate that adding the transmembrane domain induces localization of OspA ST1 in the cell membrane and reduces secretion and intracellular localization. Mutations in the glycosylation sites effectively avoided glycosylation of OspA ST1.

[0487] As shown in FIGS. 4A-4C, in vitro expression of mRNA-TMB-OspA ST2 in HEK293T cells was achieved. Cell supernatants, crude extracts, and intracellular fractions were tested for OspA ST2 expression. After 48-72 hours, the supernatants and cells were harvested and run on Western blots. Blotted proteins were detected using a polyclonal rabbit antibody that recognizes OspA. Similar to OspA ST1, these results demonstrate that addition of the transmembrane domain induces localization of OspA ST2 in the cell membrane and reduces secretion and intracellular localization. Mutations in the glycosylation sites effectively circumvented glycosylation of OspA ST2.

[0488] As shown in FIG. 5, the antigenicity of OspA ST1 antigen delivered by mRNA in HEK293T cells was shown. Sandwich ELISA using functional monoclonal antibodies LA-2, 857-2, and 221-7 was performed using transfected cell supernatants.

[0489] mAbLA-2 targets only the C-terminus of OspA ST1 and has been shown to correlate with protection in clinical studies (VanHoecke, supra; Steere, supra; Embers, supra). Mabs 221-7 and 857-2 were selected as lead candidates based on their anticorrosion activity and protection in mice against tick-borne infection with Borrelia burgdorferi as shown by Wang et al. supra.

[0490] Secreted OspA ST1 (HA SS-OspA ST1) produced by mRNA was correctly recognized by the three functional mAbs, indicating that the antigen was in the correct conformation after in vitro expression in human cells (HEK293T).

[0491] Mutations in the glycosylation sites inhibited binding of mAb LA-2, presumably due to induction of an incorrect conformation in the C-terminal epitope. This was not observed with the 221-7 and 857-2 antibodies.

[0492] As shown in FIG. 6, the antigenicity of the OspA ST2 antigen delivered by mRNA in HEK293T cells was shown. Using the transfected cell supernatant, a sandwich ELISA was performed using the functional monoclonal antibodies 857-2 and 221-7. mAb LA-2 recognizes only OspA ST1 and thus was not used with OspA ST2.

[0493] Secreted OspA ST2 (HA SS-OspA ST2) produced by mRNA was correctly recognized by the functional mAbs 221-7 and 857-2, indicating that the antigen was in the correct conformation after in vitro expression in human cells. Mutations at the glycosylation sites improved the binding of both mAbs, probably due to low epitope masking by glycosylation.

[0494] Example 3: Immunogenicity of mRNA Encoding OspA Protein in Mice The relative immunogenicity of various OspA-expressing mRNAs was tested in mice by measuring the IgG titer against OspA as described above in Example 1. Each mRNA was encapsulated in an LNP composed of 40% cationic lipid cKK-E10, 30% phospholipid DOPE, 1.5% PEGylated lipid DMGPEG2000, and 28.5% cholesterol. Alternatively, the LNP lipids are listed as a ratio where cationic lipid:PEGylated lipid:cholesterol:phospholipid is 40:1.5:28.5:30.

[0495] Each LNP-mRNA composition was administered to mice at a dose of 0.2 μg, 1 μg, 5 μg, or 10 μg. In total, four groups with 8 mice / group were used.

[0496] Three benchmark compositions were used: the OspA fusion with AlOOH adjuvant (“OspA fusion ST1-ST2”) (2 μg (1 μg per serotype) / dose); the Lyme canine vaccine RECOMBITEK® (Merial) (1 μg dose), and the OspA-ferritin fusion (ST1 or ST2) with AF03 adjuvant (1.7 μg (1 μg of OspA + 0.7 μg of ferritin) / dose). The OspA-ferritin fusion is further described in U.S. Patent Application Publication No. 20210017238A1, which is incorporated herein by reference.

[0497] As shown in Figure 7A, the anti-OspA ST1 IgG titer increased at 1 (day 19) after dosing. A tendency for HA-SS to increase the IgG titer was observed. Furthermore, when the TMB domain was added, the IgG titer increased significantly.

[0498] As shown in Figure 7B, the anti-OspA ST1 IgG titer further increased at 2 (day 35) after dosing. As seen in Table 6, addition of HA-SS and / or the TMB domain significantly improved immunogenicity (p<0.05).

[0499]

Table 32

[0500] Conclusions regarding the mouse study: mRNA encoding OspA ST1 with one or both of HA-SS or TMB was immunogenic and induced strong anti-OspA IgG titers in mice at both 1 and 2 after dosing. Addition of the secretion signal (HA-SS) or the TMB domain was found to significantly improve immunogenicity compared to mRNA OspA that did not contain either HA-SS or TMB (p<0.05). A dose effect (i.e., an increase in IgG titer with increasing dose) was observed.

[0501] Example 4: Expanded mRNA design panel In Example 3, the addition of the hemagglutinin secretion signal (HA-SS) and / or the HA transmembrane domain (HA-TMB) was demonstrated to enhance the immunogenicity of the OspA ST1 target antigen. Based on these results, a more comprehensive panel of mRNA constructs was designed in which secretion signals (SS) and transmembrane domains (TMB) from glycoproteins of various viral families were linked to different target antigens. A schematic of the elements included in this expanded mRNA panel is shown in FIG. 8. The panel consisted of three different prokaryotic antigens: OspA ST1, CAMP2, and PITP (the latter two are from Cutibacterium acnes). The mRNA sequences encoding these antigens were additionally engineered to either induce intracellular localization of the antigen, secrete the antigen extracellularly, or expose the antigen at the cell membrane. This was achieved by fusing antigen sequences from glycoproteins from different viral families such as influenza (subtype A or B), rabies, varicella (VZV), or Ebola, with or without the SS and with or without the TMB.

[0502] Fifty-seven different constructs were subjected to in silico analysis using SignalP6.0 to determine the strength of their signal peptides. SignalP is an algorithm used for signal sequence prediction and is described in more detail in Armenteros et al. (Nature Biotechnology. 37:420 - 423, 2019), Teufel et al. (Nature Biotechnology. 40:1023 - 1025, 2022) and https: / / services.healthtech.dtu.dk / services / SignalP-6.0 / , each of which is hereby incorporated by reference in its entirety. The strength is evaluated based on a cumulative rank score that takes into account the likelihood of detecting standard features of the signal sequence (SS likelihood score, also known as the SP likelihood score) and the likelihood of cleavage at the cleavage site (cleavage probability score). The sequences used in this analysis are shown in Table 7, and the results of this analysis are presented in Table 8 below.

[0503]

Table 33

[0504]

Table 34

[0505]

Table 35

[0506]

Table 36

[0507]

Table 37

[0508]

Table 38

[0509]

Table 39

[0510]

Table 40

[0511]

Table 41

[0512]

Table 42

[0513] In addition, signal peptides and their cleavage sites are less conserved than the mature regions of proteins (Nielsen et al. (2019), The Protein Journal, 38:200 - 216). Therefore, to understand the differences between signal sequences at the species and subtype levels, the homology of several hemagglutinin signal sequences was analyzed. Table 2 shows several strains tested for influenza A (subtypes H1N1 and H3N2) and influenza B (Victoria lineage and Yamagata lineage), and the resulting consensus hemagglutinin signal sequences are shown in Table 2.

[0514] Based on these in silico results, the panel was narrowed down to the constructs selected for downstream testing. The final combinations of signal sequences with their respective SignalP 6.0 scores and the selected antigens for each construct are shown in Tables 8 and 9. The amino acid sequences corresponding to this mRNA panel design are shown in Table 7. In Table 7, the construct synonyms provide information about the antigen, along with a simple representation of the viral glycoprotein signal sequence, and an indication of whether a transmembrane domain was incorporated (denoted as "TMB").

[0515] Subsequently, mRNA was produced from the constructs shown in Table 7. The mRNA production method was the same as that described in Example 1. The parameters of the mRNA produced from the constructs of this panel, including the efficiency of the capping reaction and the length of the poly(A) tail, were determined. All mRNAs were fully capped and polyadenylated.

[0516]

Table 43

[0517]

Table 44

[0518]

Table 45

[0519]

Table 46

[0520]

Table 47

[0521]

Table 48

[0522]

Table 49

[0523] Example 5: In Vitro Antigen Expression from an Expanded mRNA Design Panel This example shows an overview of the analysis of cell viability, protein expression, and localization after transfection of an mRNA panel (described in Example 4) into HEK293T cells. Transfection and Western blot analysis have already been described in Example 1. The antigens in the Western blots analyzed were OspA ST1, CAMP2, and PITP.

[0524] HEK Expi293F cell counts and cell viability resulting from two expression tests using the mRNA construct panel were measured after transfection. All cell viability values exceeded 80% at 24 and 48 hours after transfection, indicating that the conditions were normal and that none of the mRNA constructs produced off-target cytotoxicity.

[0525] OspA As shown in Fig. 9, in vitro expression of OspA ST1 fused to signal sequences derived from influenza A, influenza B, rabies, VZV, and Ebola glycoprotein (labeled "SS" on the gel) was achieved 48 hours after transfection in HEK Expi293F cells regardless of the presence or absence of each transmembrane domain ("TMB"). OspA ST1 (predicted size approximately 28 - 34 kDa) was detected using a rabbit polyclonal antibody against OspA at a 1:2000 dilution (ABCAM ab106081). Controls included an OspA ST1 construct without any SS or any lipid sequence (labeled "no SS" in the first lane on all gels) and recombinant OspA ST1 (last lane on all gels). For the test of OspA localization, samples were recovered from crude extracts (total lysates), cell supernatants, and fractionated cell samples containing either the intracellular or transmembrane fractions. These results demonstrate that addition of the transmembrane domain induces the localization of OspA ST1 in the cell membrane and reduces secretion and intracellular localization.

[0526] Furthermore, since the representative gel of the supernatant fraction in Fig. 9 exhibited a weak signal (last column of the panel), the supernatant OspA fraction was concentrated 7 - fold and half of the resulting volume was deglycosylated (represented by "D" on the gel), and the proteins were analyzed by Western blot before and after the enzymatic treatment as shown in Fig. 10. For all OspA constructs tested, OspA ST1 was present in the supernatant fraction, but the expression levels varied. For example, the OspA ST1 construct fused to the Ebola glycoprotein SS had a lower SignalP cleavage score compared to other constructs and reduced expression in the supernatant fraction compared to other OspA constructs (see Fig. 10, second gel, lanes 5 and 6). In addition, fusion of the construct to the transmembrane domain decreased OspA supernatant detection in some samples (but did not completely disappear).

[0527] CAMP2 As shown in Fig. 11, in vitro expression of CAMP2 fused to signal sequences derived from influenza A, influenza B, rabies, VZV, and Ebola glycoprotein (labeled "SS" on the gel) was achieved 48 hours after transfection in HEK Expi293F cells regardless of the presence or absence of each transmembrane domain ("TMD"). CAMP2 (predicted size approximately 26 - 32 kDa) was detected using a rabbit polyclonal antibody against CAMP2 (generated in-house) at a 1:1500 dilution. Controls included a CAMP2 construct without any SS (the first lane on all gels, labeled "no SS") and recombinant CAMP2 (the last lane on all gels). For the test of CAMP2 localization, samples were recovered from crude extracts (total lysates), cell supernatants, and fractionated cell samples containing either the intracellular or transmembrane fraction. These results demonstrate that adding a transmembrane domain induces the localization of CAMP2 in the cell membrane and reduces secretion and intracellular localization (compare constructs with or without a transmembrane domain in the last column labeled "supernatant"). Western blot analysis demonstrated that CAMP2 was sufficiently expressed and localized to the fractions predicted according to the presence of a secretion signal peptide or transmembrane domain.

[0528] PITP As shown in Fig. 12, in vitro expression of PITP fused to signal sequences derived from influenza A, influenza B, rabies, VZV, and Ebola glycoprotein (labeled "SS" on the gel) was achieved 48 hours after transfection in HEK Expi293F cells regardless of the presence or absence of each transmembrane domain ("TMB"). PITP (predicted size ~42 - 48 kDa) was detected using a mouse polyclonal antibody against PITP (generated in-house) at a 1:1000 dilution. Controls included a PITP construct without any SS or any TMB (the first lane on all gels, labeled "no SS") and recombinant PITP (the last lane on all gels). For the PITP localization study, samples were recovered from crude extracts (total lysates), cell supernatants, and fractionated cell samples containing either the intracellular or transmembrane fractions. Similar to the OspA and CAMP2 antigen localization analyses, these results demonstrate that adding a transmembrane domain induces the localization of PITP at the cell membrane and reduces secretion and intracellular localization (compare constructs with or without the transmembrane domain in the last column labeled "supernatant"). Nevertheless, the PITP transmembrane construct showed some escape into the supernatant fraction.

[0529] This Western blot analysis demonstrated that PITP was sufficiently expressed and localized to the fractions predicted according to the presence of the secretion signal peptide or transmembrane domain.

[0530] Summary To compare the Western blot analyses of OspA ST1, CAMP2, and PITP, the results of protein expression and localization were tabulated as shown in FIG. 13. Generally, the OspA ST1 antigen-containing constructs were not expressed well compared to the CAMP2- or PITP-containing constructs. All three protein antigens were expressed at their predicted locations, but the levels of expression varied, and some escape into the supernatant was indicated to varying degrees when the transmembrane domain was introduced. OspA ST1_SS-Ebola-GP, a construct with a low SignalP cleavage score, showed low expression in the supernatant fraction, indicating that it may be accumulating in the intracellular fraction.

[0531] Other embodiments of the present disclosure will be apparent to those of ordinary skill in the art from consideration of the specification and practice of the disclosure disclosed herein. The specification and examples are intended to be considered as exemplary only, with the true scope and spirit of the disclosure being indicated by the following claims.

[0532] All patents and publications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A nucleic acid comprising an open reading frame (ORF), wherein the ORF comprises: - a polynucleotide sequence encoding at least one antigenic prokaryotic polypeptide, and - a polynucleotide sequence encoding at least one viral secretion signal peptide.

2. The nucleic acid according to claim 1, wherein the ORF further comprises a polynucleotide sequence encoding at least one transmembrane domain (TMB).

3. The nucleic acid according to claim 1 or 2, wherein the viral secretion signal peptide is derived from a viral sequence in a virus capable of infecting humans.

4. The nucleic acid according to any one of claims 1 to 3, wherein the viral secretion signal peptide is derived from a viral sequence selected from the group consisting of an influenza secretion signal peptide sequence, a SARS-CoV-2 secretion signal peptide sequence, a varicella-zoster virus (VZV) secretion signal peptide sequence, a measles secretion signal peptide sequence, a rubella secretion signal peptide sequence, a mumps secretion signal peptide sequence, an Ebola secretion signal peptide sequence, a smallpox secretion signal peptide sequence, and a rabies secretion signal peptide sequence, and is a non-influenza secretion signal peptide sequence.

5. The viral secretion signal peptide is selected from the group consisting of an influenza hemagglutinin (HA) secretion signal peptide sequence, a SARS-CoV-2 spike secretion signal peptide sequence, a VZV gB secretion signal peptide sequence, a VZV gE secretion signal peptide sequence, a VZV gI secretion signal peptide sequence, a VZV gK secretion signal peptide sequence, a measles F-protein secretion signal peptide sequence, a rubella E1-protein secretion signal peptide sequence, a rubella E2-protein secretion signal peptide sequence, a mumps F-protein secretion signal peptide sequence, an Ebola GP-protein secretion signal peptide sequence, a smallpox 6 kDa IC-protein secretion signal peptide sequence, and a rabies G-protein secretion signal peptide sequence, preferably, the viral secretion signal peptide comprises an HA secretion signal peptide sequence derived from influenza A or influenza B, more preferably, an HA secretion signal peptide sequence derived from influenza A.

6. The HA secretion signal peptide sequence is the amino acid sequence MKX １ X ２ LX ３ VX ４ LX ５ TFX ６ X ７ X ８ X ９ including A (SEQ ID NO: 145), X １ is selected from A and V; X ２ is selected from I and K; X ３ is selected from V and L; X ４ is selected from L and M; X ５ is selected from Y and C; X ６ is selected from T and A; X ７ is selected from T and A; X ８ is selected from A and T; X ９ The nucleic acid according to claim 5, wherein X is selected from N and Y.

7. The nucleic acid according to claim 5 or 6, wherein the HA secretion signal peptide sequence comprises an amino acid sequence selected from SEQ ID NOs: 95 to 109.

8. the HA secretion signal peptide sequence contains the amino acid sequence MKX １ IIALSX ２ ILCLVFXX ３ (SEQ ID NO: 146), X １ is selected from T and A; X ２ is selected from Y, N, C, and H; X ３ The nucleic acid according to claim 5, wherein X is selected from T and A.

9. The nucleic acid according to claim 8, wherein the HA secretion signal peptide sequence comprises an amino acid sequence selected from SEQ ID NOs: 110 to 131.

10. The HA secretion signal peptide sequence contains the amino acid sequence MKAIIVLLMVVTSX １ A (SEQ ID NO: 147), and X １ The nucleic acid according to claim 5, wherein X is selected from S and N.

11. the HA secretion signal peptide sequence contains the amino acid sequence MX １ AIIVLLMVVTSNA (SEQ ID NO: 148), X １ The nucleic acid according to claim 5, wherein X is selected from K and E.

12. The nucleic acid according to claim 10 or 11, wherein the HA secretion signal peptide sequence comprises an amino acid sequence selected from SEQ ID NOs: 132 to 144.

13. The viral secretion signal peptide is MKAKLLVLLCTFTATYA (SEQ ID NO: 1); MKAILVVLLYTFATANA (SEQ ID NO: 2); MKTIIALSYILCLVFA (SEQ ID NO: 3); MKAIIVLLMVVTSNA (SEQ ID NO: 4); MFVFLVLLPLVS (SEQ ID NO: 5); MFLLTTKRTMFVFLVLLPLVS (SEQ ID NO: 6) MSPCGYYSKWRNRDRPEYRRNLRFRRFFSSIHPNAAAAGSGFNGPGVFITSVTGVWLCFLCIFSMFVTAVS (SEQ ID NO: 7); MGTvNKPVVGVLMGFIGITGTLRITNPVRA (SEQ ID NO: 8); MFLIQCLISAVIFYIQVTNA (SEQ ID NO: 9); MQALGIKTEHFIIMCLLSGHA (SEQ ID NO: 10); MGLKVNVSIFMAVLLTLQTPTG (SEQ ID NO: 11); MGAAAAAALTVVLQGYNPAYG (SEQ ID NO: 12); MGAPQAFLLAGLLLAAVAVGTRA (SEQ ID NO: 13); MKVFLVTCLGFAVFSSSVC (SEQ ID NO: 14); MGTGILQLPRDRFKRTFSFFLWVIILFQRTFS (SEQ ID NO: 15); MRSLIIFLLFPSIIYS (SEQ ID NO: 16); and The nucleic acid according to any one of claims 1 to 12, comprising an amino acid sequence selected from the group consisting of MVPQALLFVPLLVFPLLCFG (SEQ ID NO: 184).

14. The nucleic acid according to claim 13, wherein the viral secretion signal peptide comprises the amino acid sequence of MKAKLLVLLCTFTATYA (SEQ ID NO: 1).

15. The nucleic acid according to any one of claims 1 to 14, wherein the viral secretion signal peptide is located at the N-terminus of the antigen prokaryotic polypeptide.

16. The nucleic acid according to any one of claims 1 to 14, wherein the viral secretion signal peptide is located at the C-terminus of the antigen prokaryotic polypeptide.

17. The nucleic acid according to any one of claims 1 to 16, wherein the viral secretion signal peptide is linked to the antigen prokaryotic polypeptide by a linker.

18. The TMB is (a) comprising or consisting of 15 to 50 amino acid residues, preferably 15 to 30 amino acid residues, more preferably 18 to 25 amino acid residues; and / or (b) containing at least 50% hydrophobic amino acid residues, preferably selected from the group consisting of alanine, isoleucine, leucine, valine, phenylalanine, tryptophan, and tyrosine; and / or (c) containing at least one alpha helix, the nucleic acid according to any one of claims 2 to 17.

19. The nucleic acid according to any one of claims 2 to 18, wherein the TMB is derived from an integral membrane protein, preferably from a single-pass transmembrane protein, more preferably from a vitopee membrane protein, and even more preferably from a type I vitopee membrane protein.

20. The nucleic acid according to any one of claims 2 to 19, wherein the TMB is derived from a non-human sequence.

21. The nucleic acid according to any one of claims 18 to 20, wherein the antigen prokaryotic polypeptide is derived from a prokaryotic membrane protein, and the TMB is the TMB of the prokaryotic membrane protein.

22. The nucleic acid according to any one of claims 18 to 20, wherein the antigen prokaryotic polypeptide is not derived from a prokaryotic membrane protein.

23. The nucleic acid according to claim 22, wherein the TMB is derived from a viral sequence.

24. The nucleic acid according to claim 23, wherein the TMB is derived from a viral transmembrane domain sequence selected from the group consisting of an influenza transmembrane domain sequence, a SARS-CoV-2 transmembrane domain sequence, a varicella-zoster virus (VZV) transmembrane domain sequence, a measles transmembrane domain sequence, a rubella transmembrane domain sequence, a mumps transmembrane domain sequence, an Ebola transmembrane domain sequence, and a rabies transmembrane domain sequence, and a non-influenza transmembrane domain sequence selected from the group consisting of non-influenza transmembrane domain sequences.

25. The TMB is selected from the group consisting of an influenza hemagglutinin (HA) transmembrane domain sequence, a SARS-CoV-2 spike transmembrane domain sequence, a VZV gB transmembrane domain sequence, a VZV gE transmembrane domain sequence, a VZV gI transmembrane domain sequence, a VZV gK transmembrane domain sequence, a measles F protein transmembrane domain sequence, a rubella E1 protein transmembrane domain sequence, a rubella E2 protein transmembrane domain sequence, a mumps F protein transmembrane domain sequence, an Ebola GP protein transmembrane domain sequence, and a rabies G protein transmembrane domain sequence; preferably, the TMB comprises an HA transmembrane domain sequence derived from influenza A or influenza B, more preferably an HA transmembrane domain sequence derived from influenza A, the nucleic acid according to claim 24.

26. The TMB is ILA IYS TVA SSL VLL VSL GAISF (SEQ ID NO: 17); ILA IYS TVA SSL VLV VSL GAISF (SEQ ID NO: 18); ILW ISF AIS CFL LLC VLL GF I (SEQ ID NO: 19); STA ASS LAV TLM LAI FIV YMV (SEQ ID NO: 20); WYI WLG FIA GLI AIV MTV TIML (SEQ ID NO: 21); FGA LAV GLL VLA GLV AAF FAY (SEQ ID NO: 22); AAW TGG LAA VVL LCL VIF LIC (SEQ ID NO: 23); III PIV ASV MIL TAM VIV IVI (SEQ ID NO: 24); YFW CVQ LKM IFF AW FVY GMYL (SEQ ID NO: 25); IVY ILI AVC LGG LIG IPA LIC (SEQ ID NO: 26); LDH AFA AFF VLL VPW VLI FMC (SEQ ID NO: 27); WWQ LTL GAI CAL LLA GLL ACC (SEQ ID NO: 28); IVA ALV LSI LSI IIS LLF CCW (SEQ ID NO: 29); WIP AGI GVT GVI IAV IAL FCI (SEQ ID NO: 30); and VLL SAG ALT ALM LII FLM T CW (SEQ ID NO: 185), the nucleic acid according to any one of claims 18 to 20 and 22 to 25, comprising an amino acid sequence selected from the group consisting of.

27. The TMB comprises the amino acid sequence of ILA IYS TVA SSL VLL VSL GAISF (SEQ ID NO: 17), the nucleic acid according to claim 16.

28. The nucleic acid according to any one of claims 2 and 18 to 27, wherein the TMB is linked to the antigen prokaryotic polypeptide by a linker.

29. The nucleic acid according to any one of claims 2 and 18 to 28, wherein the TMB is located at the N-terminus of the antigen prokaryotic polypeptide.

30. The nucleic acid according to any one of claims 2 and 18 to 28, wherein the TMB is located at the C-terminus of the antigen prokaryotic polypeptide.

31. A nucleic acid comprising an open reading frame (ORF), wherein the ORF comprises: - a polynucleotide sequence encoding at least one antigen polypeptide, preferably an antigen prokaryotic polypeptide, and - a polynucleotide sequence encoding at least one transmembrane domain (TMB), wherein the TMB is heterologous to the antigen polypeptide, Optionally, the ORF further comprises a polynucleotide sequence encoding at least one secretion signal peptide according to any one of claims 1 to 30, preferably a viral secretion signal peptide, more preferably a polynucleotide sequence encoding a viral secretion signal peptide.

32. A nucleic acid comprising an open reading frame (ORF), wherein the ORF comprises: - a polynucleotide sequence encoding at least one antigen prokaryotic polypeptide, and - a polynucleotide sequence encoding at least one transmembrane domain (TMB).

33. The TMB is: (a) comprises or consists of 15 to 50 amino acid residues, preferably 15 to 30 amino acid residues, more preferably 18 to 25 amino acid residues; and / or (b) contains at least 50% hydrophobic amino acid residues, preferably selected from the group consisting of alanine, isoleucine, leucine, valine, phenylalanine, tryptophan, and tyrosine; and / or (c) contains at least one alpha helix, the nucleic acid according to claim 32.

34. The nucleic acid according to claim 32 or 33, wherein the TMB is derived from an integral membrane protein, preferably from a single-pass membrane protein, more preferably from a bitopic membrane protein, even more preferably from a type I bitopic membrane protein.

35. The nucleic acid according to claim 34, wherein the TMB is derived from a non-human sequence.

36. The nucleic acid according to any one of claims 33 to 35, wherein the antigen polypeptide is not derived from a transmembrane protein.

37. The nucleic acid according to any one of claims 33 to 36, wherein the TMB is derived from a viral sequence.

38. The nucleic acid according to any one of claims 33 to 37, wherein the TMB is derived from a viral transmembrane domain sequence selected from the group consisting of an influenza transmembrane domain sequence, and a SARS-CoV-2 transmembrane domain sequence, a varicella-zoster virus (VZV) transmembrane domain sequence, a measles transmembrane domain sequence, a rubella transmembrane domain sequence, a mumps transmembrane domain sequence, an Ebola transmembrane domain sequence, and a rabies transmembrane domain sequence, and is not an influenza transmembrane domain sequence.

39. The TMB is selected from the group consisting of an influenza hemagglutinin (HA) transmembrane domain sequence, a SARS-CoV-2 spike transmembrane domain sequence, a VZV gB transmembrane domain sequence, a VZV gE transmembrane domain sequence, a VZV gI transmembrane domain sequence, a VZV gK transmembrane domain sequence, a measles F protein transmembrane domain sequence, a rubella E1 protein transmembrane domain sequence, a rubella E2 protein transmembrane domain sequence, a mumps F protein transmembrane domain sequence, an Ebola GP protein transmembrane domain sequence, and a rabies G protein transmembrane domain sequence, and preferably, the TMB comprises an HA transmembrane domain sequence derived from influenza A or influenza B, more preferably an HA transmembrane domain sequence derived from influenza A. The nucleic acid according to claim 38.

40. The TMB is ILA IYS TVA SSL VLL VSL GAI SF (SEQ ID NO: 17); ILA IYS TVA SSL VLV VSL GAI SF (SEQ ID NO: 18); ILW ISF AIS CFL LLC VLL GFI (SEQ ID NO: 19); STA ASS LAV TLM LAI FIV YMV (SEQ ID NO: 20); WYI WLG FIA GLI AIV MTV TIM L (SEQ ID NO: 21); FGA LAV GLL VLA GLV AAF FAY (SEQ ID NO: 22); AAW TGG LAA VVL LCL VIF LIC (SEQ ID NO: 23); III PIV ASV MIL TAM VIV IVI VI (SEQ ID NO: 24); YFW CVQ LKM IFF AWF VYG MYL (SEQ ID NO: 25); IVY ILI AVC LGG LIG IPA LIC (SEQ ID NO: 26); LDHAFAFVLLVPWVLIFMVC (SEQ ID NO: 27); WWQLTLGAICALLLAGLLACC (SEQ ID NO: 28); IVAAVLVSILSLIIISLLFCCW (SEQ ID NO: 29); WIPAGIGVTGVIIAVIALFC I (SEQ ID NO: 30); and The nucleic acid according to claim 39, comprising an amino acid sequence selected from the group consisting of VLLSAGALTALM LIIFLMTCW (SEQ ID NO: 185).

41. The nucleic acid according to claim 40, wherein the TMB comprises the amino acid sequence of ILAIYSTVASSLLLVSLGAISF (SEQ ID NO: 17).

42. The nucleic acid according to any one of claims 32 to 41, wherein the TMB is bound to the antigen prokaryotic polypeptide by a linker.

43. The nucleic acid according to any one of claims 32 to 42, wherein the TMB is located at the N-terminus of the antigen prokaryotic polypeptide.

44. The nucleic acid according to any one of claims 32 to 42, wherein the TMB is located at the C-terminus of the antigen prokaryotic polypeptide. The antigen prokaryotic polypeptide is Acetobacter, Acinetobacter, Actinomyces, Aerococcus, Agrobacterium, Anaplasma, Azorhizobia, Azotobacter, Bacillus, Bacteroides, Bartonella, Bordetella, Borrelia, Brucella, Burkholderia, Calymmatobacterium, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Coxiella, Cutibacterium, Ehrlichia, Enterobacter, Enterococcus, Escherichia, Francisella, Fusobacterium, Gardnerella, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Legionella, Listeria, Methanobacterium, Microbacterium, Micrococcus, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Pasteurella, Pediococcus,Bacteria of a species selected from the group consisting of Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, Pseudomonas, Rhizobium, Rickettsia, Rochalimaea, Rothia, Salmonella, Serratia, Shigella, Sarcoma, Spirillum, Spirochaetes, Staphylococcus, Stenotrophomonas, Streptobacillus, Streptococcus, Tetragenococcus, Treponema, Vibrio, Viridans, Wolbachia, and Yersinia Preferably, Acetobacter aurantius, Acinetobacter baumannii, Actinomyces israelii, Agrobacterium radiobacter, Agrobacterium tumefaciens, Anaplasma phagocytophilum, Azorhizobium caulinodans, Azotobacter vinelandii, Bacillus anthracis, Bacillus brevis, Bacillus cereus, Bacillus fusiformis, Bacillus licheniformis, Bacillus megaterium, Bacillus mycoides, Bacillus stearothermophilus, Bacillus subtilis, Bacillus Thuringiensis, Bacteroides fragilis, Bacteroides gingivalis, Bacteroides melaninogenicus, Bartonella henselae, Bartonella Quintana, Bordetella bronchiseptica, Bordetella pertussis, Borrelia burgdorferi, Brucella abortusabortus), Brucella melitensis, Brucella suis, Burkholderia mallei, Burkholderia pseudomallei, Burkholderia cepacia, Calymmatobacterium granulomatis, Campylobacter coli, Campylobacter fetus, Campylobacter jejuni, Campylobacter pylori, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Corynebacterium fusiforme, Coxiella burnetii, Cutibacterium acnes, Cutibacterium avidum, Cutibacterium granulosum, Cutibacterium namnetense, Cutibacterium humerusii, Ehrlichia chaffeensisChaffensis), Enterobacter cloacae, Enterococcus avium, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus malolatus, Escherichia coli, Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Haemophilus ducreyi, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus pertussis, Haemophilus vaginalis, Helicobacter pylori, Klebsiella pneumoniae, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactococcus lactis, Legionella pneumophila, Listeria monocytogenes, Methanobacterium extroquens, Microbacterium multiforme, Micrococcus luteus, Moraxella catarrhalis, Mycobacterium avium, Mycobacterium bovis, Mycobacterium diphtheriae, Mycobacterium intracellulare, Mycobacterium leprae, Mycobacterium lepraemurium, Mycobacterium playPhlei), Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycoplasma fermentans, Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma penetrans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Pasteurella tularensis, Peptostreptococcus, Porphyromonas gingivalis, Prevotella melaninogenica, Propionibacterium acnes, Pseudomonas aeruginosa, Rhizobium radiobacter, Rickettsia prowazekii, Rickettsia psittaci, Rickettsia quintana, Rickettsia rickettsii, Rickettsia trachomae, Rochalimaea henselae, Rochalimaea quintana, Rothia dentocarios, Salmonella enteritidis, Salmonella typhi, Salmonella typhimuriumSalmonella typhimurium, Serratia marcescens, Shigella dysenteriae, Spirillum volutans, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus avium, Streptococcus bovis, Streptococcus cricetus, Streptococcus facium, Streptococcus faecalis, Streptococcus ferus, Streptococcus gallinarum, Streptococcus lactis, Streptococcus mitior, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus rattus, Streptococcus salivarius, Streptococcus sanguis, Streptococcus sobrinus, Treponema pallidum, Treponema denticola, Vibrio choleraeThe nucleic acid according to any one of claims 32 to 44, which is derived from a bacterium of a genus selected from the group consisting of cholerae), Vibrio comma, Vibrio parahaemolyticus, Vibrio vulnificus, Viridans streptococci, Wolbachia, Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis.

45.

46. The nucleic acid according to any one of claims 1 to 45, wherein the antigen prokaryotic polypeptide is derived from a bacterium of the genus Borrelia, preferably B. burgdorferi, afzelii, garinii, bavariensis, mayonii, spielmanii, lusitaniae, bissettii, and / or valaisiana.

47. 【Chemical 1】 The antigen prokaryotic polypeptide is OspA or a fragment or variant thereof, and the OspA or a fragment or variant thereof comprises at least 5 amino acids. Preferably, the antigen prokaryotic polypeptide is 【Chemical 2】 a) an amino acid sequence derived from OspA ST1, preferably having at least 85% identity with the sequence 【Chemical 3】 b) an amino acid sequence derived from OspA ST2, preferably having at least 85% identity with the sequence c) an amino acid sequence derived from OspA ST3, preferably having at least 85% identity with the sequence d) an amino acid sequence derived from OspA ST4, preferably having at least 85% identity with the sequence 【Chemical Formula 4】 (e) An amino acid sequence derived from OspA ST5, preferably having at least 85% identity with the sequence 【Chemical Formula 5】 (f) An amino acid sequence derived from OspA ST6, preferably having at least 85% identity with the sequence 【Chemical Formula 6】 (g) An amino acid sequence derived from OspA ST7, preferably having at least 85% identity with the sequence 【Chemical Formula 7】 (h) Any combination of (a) to (g); (i) The sequence derived from OspA ST1 described in (a) and the sequence derived from OspA ST2 described in (b), or (j) The nucleic acid according to any one of claims 1 to 46, comprising the sequence derived from OspA ST1 described in (a), the sequence derived from OspA ST2 described in (b), the sequence derived from OspA ST3 described in (c), the sequence derived from OspA ST4 described in (d), the sequence derived from OspA ST5 described in (e), the sequence derived from OspA ST6 described in (f), and the sequence derived from OspA ST7 described in (g).

48. The nucleic acid according to any one of claims 1 to 47, wherein the antigen prokaryotic polypeptide comprises at least one mutated glycosylation site, preferably at least one mutated N-linked glycosylation site.

49. The nucleic acid according to any one of claims 1 to 48, wherein the polynucleotide sequence of the nucleic acid is codon-optimized.

50. The nucleic acid according to any one of claims 1 to 49, wherein the polynucleotide sequence of the ORF is codon-optimized.

51. The nucleic acid according to any one of claims 1 to 31, wherein the polynucleotide sequence encoding the at least one viral secretion signal peptide is codon-optimized.

52. The nucleic acid according to any one of claims 18 to 51, wherein the polynucleotide sequence encoding the at least one TMB is codon-optimized.

53. The nucleic acid according to any one of claims 1 to 52, wherein the nucleic acid is DNA.

54. The nucleic acid according to any one of claims 1 to 52, wherein the nucleic acid is messenger RNA (mRNA), and in particular, the mRNA can be non-replicating mRNA, self-replicating mRNA, or trans-replicating mRNA.

55. The nucleic acid according to claim 54, wherein the mRNA comprises at least one 5'untranslated region (5'UTR), at least one 3'untranslated region (3'UTR), and / or at least one polyadenylation (poly(A)) sequence.

56. The nucleic acid according to claim 54 or 55, wherein the mRNA comprises at least one chemical modification.

57. The nucleic acid according to any one of claims 54 to 56, wherein at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the mRNA are chemically modified.

58. The nucleic acid according to any one of claims 54 to 57, wherein at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the ORF are chemically modified.

59. The nucleic acid according to any one of claims 56 to 58, wherein the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-1-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

60. The nucleic acid according to any one of claims 56 to 59, wherein the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

61. The nucleic acid according to any one of claims 56 to 60, wherein the chemical modification is N1-methylpseudouridine.

62. A composition comprising at least one nucleic acid according to any one of claims 1 to 61.

63. The composition according to claim 62, further comprising lipid nanoparticles (LNP).

64. The composition according to claim 63, wherein the nucleic acid is encapsulated in the LNP.

65. The composition according to claim 63 or 64, wherein the LNP comprises at least one cationic lipid.

66. The composition according to claim 65, wherein the cationic lipid is biodegradable.

67. The composition according to claim 65, wherein the cationic lipid is non - biodegradable.

68. The composition according to any one of claims 65 to 67, wherein the cationic lipid is cleavable.

69. The composition according to any one of claims 65 to 67, wherein the cationic lipid is non - cleavable.

70. The composition according to any one of claims 65 to 69, wherein the cationic lipid is selected from the group consisting of OF - 02, cKK - E10, OF - Deg - Lin, GL - HEPES - E3 - E10 - DS - 3 - E18 - 1, GL - HEPES - E3 - E12 - DS - 4 - E10, GL - HEPES - E3 - E12 - DS - 3 - E14, SM - 102, and ALC - 0315.

71. The composition according to any one of claims 65 to 70, wherein the LNP further comprises a polyethylene glycol (PEG) conjugate (PEGylated) lipid, a cholesterol - based lipid, and a helper lipid.

72. The LNP is - 35% to 55% molar ratio of cationic lipid; - 0.25% to 2.75% molar ratio of polyethylene glycol (PEG) conjugate (PEGylated) lipid; - 20% to 45% molar ratio of cholesterol - based lipid; and - 5% to 35% molar ratio of helper lipid, wherein all of the molar ratios are relative to the total lipid content of the LNP. The composition according to any one of claims 63 to 71.

73. The LNP is - 40% molar ratio of cationic lipid; - 1.5% molar ratio of PEGylated lipid; - 28.5% molar ratio of cholesterol - based lipid; and - 30% molar ratio of helper lipid. The composition according to any one of claims 63 to 72.

74. The LNP is - 45% to 50% molar ratio of cationic lipid; - 1.5% to 1.7% molar ratio of PEGylated lipid; - Cholesterol-based lipids in a molar ratio of -38% to 43%; and - Helper lipids in a molar ratio of -9% to 10%, the composition according to any one of claims 63 to 73.

75. The composition according to any one of claims 71 to 74, wherein the PEGylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000) or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

76. The composition according to any one of claims 71 to 75, wherein the cholesterol-based lipid is cholesterol.

77. The composition according to any one of claims 71 to 76, wherein the helper lipid is 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

78. The LNP is - Cationic lipids selected from the group consisting of OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, and GL-HEPES-E3-E12-DS-3-E14 in a molar ratio of -40%; - DMG-PEG2000 in a molar ratio of -1.5%; - Cholesterol in a molar ratio of -28.5%; and - DOPE in a molar ratio of -30%, the composition according to any one of claims 63 to 73 and 75 to 77.

79. The LNP is - SM-102 in a molar ratio of -50%; - DMG-PEG2000 in a molar ratio of -1.5%; - Cholesterol in a molar ratio of -38.5%; and - DSPC in a molar ratio of -10%, the composition according to any one of claims 63 to 72 and 74 to 77.

80. The LNP is - ALC-0315 in a molar ratio of -46.3%; - ALC-0159 in a molar ratio of -1.6%; - Cholesterol in a molar ratio of -42.7%; and - DSPC in a molar ratio of -9.4%, the composition according to any one of claims 63 to 72 and 74 to 77.

81. The LNP is - ALC-0315 in a molar ratio of -47.4%; - ALC-0159 in a molar ratio of -1.7%; - Cholesterol in a molar ratio of -40.9%; and - DSPC in a molar ratio of -10%, the composition according to any one of claims 63 to 72 and 74 to 77.

82. The composition according to any one of claims 63 to 81, wherein the LNP has an average diameter of 30 nm to 200 nm.

83. The composition according to any one of claims 63 to 82, wherein the LNP has an average diameter of 80 nm to 150 nm.

84. The composition according to any one of claims 63 to 83, comprising the LNP at 1 mg / mL to 10 mg / mL.

85. The composition according to any one of claims 63 to 84, wherein the LNP contains 1 to 20 nucleic acid molecules, preferably mRNA molecules.

86. The composition according to any one of claims 62 to 85, formulated for intramuscular, intranasal, intravenous, subcutaneous, or intradermal administration.

87. The composition according to any one of claims 62 to 86, wherein the composition contains phosphate-buffered saline.

88. The composition according to any one of claims 62 to 87, wherein the composition is a pharmaceutical composition, such as an immunogenic composition or a vaccine, particularly an mRNA vaccine.

89. The nucleic acid according to any one of claims 1 to 61 or the composition according to any one of claims 62 to 88 for use in inducing an immune response in a subject in need thereof.

90. The nucleic acid according to any one of claims 1 to 61 or the composition according to any one of claims 62 to 88 for use in treating or preventing prokaryotic infection in a subject in need thereof.

91. A method for secreting an antigenic prokaryotic polypeptide in a host cell, comprising administering to the host cell the nucleic acid according to any one of claims 1 to 61 or the composition according to any one of claims 62 to 88.

92. A method for displaying an antigenic prokaryotic polypeptide on the surface of a host cell, comprising administering to the host the nucleic acid according to any one of claims 1 to 61 or the composition according to any one of claims 62 to 88.

93. A kit comprising a container containing a single-use dose or multiple-use doses of the nucleic acid according to any one of claims 1 to 61 or the composition according to any one of claims 62 to 88, optionally wherein the container is a vial or a filled syringe or a syringe.

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