Signal sequences for nucleic acid vaccines

Abstract

Provided herein is a nucleic acid (e.g., messenger RNA) vaccine encoding at least one antigenic prokaryotic polypeptide linked to one or both of a viral secretion signal peptide and a transmembrane domain. Also provided are methods of vaccinating against prokaryotic infections with the nucleic acids described herein.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is related to European priority application No. 22305680.5 filed on May 6, 2022, European 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) represent a major threat to global human health. An estimated 5 million people die each year from antibiotic-resistant bacterial infections, and the burden on healthcare systems is increasing (Antimicrobial Resistance Collaborators. The Lancet. 399(10325):629-655.2022). Although vaccines exist for prokaryotic infections, they are fewer in number than more common antiviral vaccines.

[0003] Nucleic acid-based vaccines, and more particularly mRNA vaccines, have recently become another type of vaccine with a fast, safe and cost-effective production process, especially for viral pathogens. The mRNA vaccines for severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) mainly use spike viral proteins as antigens. Usually combined with a delivery vehicle such as lipid nanoparticles (LNPs), COVID-19 mRNA vaccines can achieve high efficacy. Nevertheless, there is still a need for more effective RNA-based vaccines for prokaryotic infections.

[0004] There are challenges in producing nucleic acid-based vaccines (such as RNA (e.g., mRNA-based vaccines) containing prokaryotic antigens (i.e., antigens derived from antigens in prokaryotic cells) because prokaryotic antigens are not naturally expressed by eukaryotic cells. Specifically, prokaryotic cells and eukaryotic cells have different secretion systems. Without appropriate engineering, the prokaryotic antigens used in these vaccines will accumulate in the intracellular compartments of eukaryotic (e.g., human) cells, which may reduce the immunogenicity of these vaccines.

[0005] The present invention aims to solve this problem by improving the immunogenicity of prokaryotic antigens expressed by nucleic acid-based (e.g., mRNA) vaccines. As described herein, this is achieved by adding secretion signals to allow prokaryotic antigens to be secreted in the extracellular compartment and / or adding transmembrane domains to allow expression at the cell surface. By doing so, immune cells can better access antigens, ultimately improving the immunogenicity of nucleic acid-based (e.g., mRNA) vaccines. Summary of the invention

[0006] The present disclosure provides 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.

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

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

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

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

[0011] In certain embodiments, the HA secretion signal peptide sequence comprises the amino acid sequence MKX1X2LX3VX4LX5TFX6X7X8X9A (SEQ ID NO: 145), wherein X1 is selected from A and V; X2 is selected from I and K; X3 is selected from V and L; X4 is selected from L and M; X5 is selected from Y and C; X6 is selected from T and A; X7 is selected from T and A; X8 is selected from A and T; and X9 is selected from N and Y.

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

[0013] In certain embodiments, the HA secretion signal peptide sequence comprises the amino acid sequence MKX1IIALSX2ILCLVFX3 (SEQ ID NO: 146), wherein X1 is selected from T and A; X2 is selected from Y, N, C and H; and X3 is selected from T and A.

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

[0015] In certain embodiments, the HA secretion signal peptide sequence comprises the amino acid sequence MKAIIVLLMVVTSX1A (SEQ ID NO: 147), wherein X1 is selected from S and N.

[0016] In certain embodiments, the HA secretion signal peptide sequence comprises the amino acid sequence MX1AIIVLLMVVTSNA (SEQ ID NO: 148), wherein X1 is selected from K and E.

[0017] In certain embodiments, the HA secretion signal peptide sequence comprises an amino acid sequence selected from the group consisting of 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); MSPCGYYSKWRNRDRPEYRRNLRFRRFFSSIHPNAAAGSGFNGPGVFITSVTGVWLCF LCIFSMFVTAVVS (SEQ ID NO: 7); MGTVNKPVVGVLMGFGIITGTLRITNPVRA (SEQ ID NO: 8); MFLIQCLISAVIFYIQVTNA (SEQ ID NO: 9); MQALGIKTEHFIIMCLLSGHA (SEQ ID NO: 10); NO: 10); MGLKVNVSAIFMAVLLTLQTPTG (SEQ ID NO: 11); MGAAAALTAVVLQGYNPPAYG (SEQ ID NO: 12); MGAPQAFLAGLLLAAVAVGTARA (SEQ ID NO: 13); MKVFLVTCLGFAVFSSSSVC (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 antigenic prokaryotic polypeptide.

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

[0022] In certain embodiments, the viral secretion signal peptide is attached to the antigenic prokaryotic polypeptide with a linker.

[0023] In certain embodiments, 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% hydrophobic amino acid residues, which are preferably selected from the group consisting of alanine, isoleucine, leucine, tyrosine, 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 membrane 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 antigenic prokaryotic polypeptide is derived from a prokaryotic transmembrane protein, and wherein the TMB is the TMB of the prokaryotic transmembrane protein.

[0027] In certain embodiments, the antigenic 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 virus transmembrane domain sequence, and a non-influenza virus transmembrane domain sequence selected from the group consisting of a SARS CoV-2 transmembrane domain sequence, a varicella-zoster virus (VZV) transmembrane domain sequence, a measles virus transmembrane domain sequence, a rubella virus transmembrane domain sequence, a mumps virus transmembrane domain sequence, an Ebola virus transmembrane domain sequence, and a rabies virus transmembrane domain sequence.

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

[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). NO:27); WWQLTLGAICALLLAGLLACC (SEQ ID NO:28); IVALVLSILSIIISLLLFCCW (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 attached to the antigenic prokaryotic polypeptide with 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 present disclosure provides a nucleic acid comprising an open reading frame (ORF), wherein the ORF comprises: - 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,

[0037] Optionally, the ORF further comprises a polynucleotide sequence encoding at least one secretion signal peptide, preferably a viral secretion signal peptide, more preferably a viral secretion signal peptide as described in any of the preceding claims.

[0038] In another aspect, the present disclosure provides 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 transmembrane domain (TMB).

[0039] In certain embodiments, 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% hydrophobic amino acid residues, which are preferably selected from the group consisting of alanine, isoleucine, leucine, tyrosine, phenylalanine, tryptophan and tyrosine; and / or (c) comprises at least one alpha helix.

[0040] In certain embodiments, the TMB is derived from an integral membrane protein, preferably from a single-pass membrane protein, more preferably from a dipenic protein, and even more preferably from a type I dipenic protein.

[0041] In certain embodiments, the TMB is derived from a non-human sequence.

[0042] In certain embodiments, the antigenic polypeptide is not derived from a transmembrane protein.

[0043] In certain embodiments, the TMB is derived from a viral sequence.

[0044] In certain embodiments, the TMB is derived from a viral transmembrane domain sequence selected from the group consisting of an influenza virus transmembrane domain sequence, and a non-influenza virus transmembrane domain sequence selected from the group consisting of a SARS CoV-2 transmembrane domain sequence, a varicella-zoster virus (VZV) transmembrane domain sequence, a measles virus transmembrane domain sequence, a rubella virus transmembrane domain sequence, a mumps virus transmembrane domain sequence, an Ebola virus transmembrane domain sequence, and a rabies virus transmembrane domain sequence.

[0045] In certain embodiments, the TMB is selected from: influenza virus hemagglutinin (HA) transmembrane domain sequence, SARSCoV-2 spike transmembrane domain sequence, VZV gB transmembrane domain sequence, VZV gE transmembrane domain sequence, VZV gI transmembrane domain sequence, VZV gK transmembrane domain sequence, measles virus F protein transmembrane domain sequence, rubella virus E1 protein transmembrane domain sequence, rubella virus E2 protein transmembrane domain sequence, mumps virus F protein transmembrane domain sequence, Ebola virus GP protein transmembrane domain sequence and rabies virus G protein transmembrane domain sequence, preferably wherein the TMB comprises an HA transmembrane domain sequence from influenza A virus or influenza B virus, more preferably from influenza A virus.

[0046] 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). NO:27); WWQLTLGAICALLLAGLLACC (SEQ ID NO:28); IVAALVLSILSIIISLLLFCCW (SEQ ID NO:29); WIPAGIGVTGVIIAVIALFCI (SEQ ID NO:30); and VLLSAGALTALMLIIFLMTCW (SEQ ID NO:185).

[0047] In certain embodiments, the TMB comprises the amino acid sequence of ILAIYSTVASSLVLLVSLGAISF (SEQ ID NO: 17).

[0048] In certain embodiments, the TMB is attached to the antigenic prokaryotic polypeptide with a linker.

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

[0050] In certain embodiments, the TMB is located at the C-terminus of the antigenic prokaryotic polypeptide.

[0051] In certain embodiments, the antigenic prokaryotic polypeptide is derived from a bacterium of the genus 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 ter), Enterococcus, Escherichia, Francisella, Fusobacterium, Gardnerella, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Legionella, Listeria, Methanobacterium terium), Microbacterium, Micrococcus, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Pasteurella, Pediococcus, 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 a bacterium from the species selected from the group consisting of Acetobacter aurantifolia. 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 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 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,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 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 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 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, Serratiamarcescens, 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 oralis), Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus rattus, Streptococcus salivarius, Streptococcus sanguis, Streptococcus sobrinus, Treponema pallidum, Treponema denticola, Vibrio cholerae, Vibrio comma, Vibrio parahaemolyticus, Vibrio vulnificus, Viridans streptococci, Wolbachia, Yersinia enterocolitica enterocolitica), Yersinia pestis, and Yersinia pseudotuberculosis.

[0052] In certain embodiments, the antigenic prokaryotic polypeptide is derived from a bacterium of the genus Borrelia, preferably selected from the following species: Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii, Borrelia bavariensis, Borrelia mayonii, Borrelia spielmanii, Borrelia lusitaniae, Borrelia bissetii and / or Borrelia valaisiana.

[0053] In certain embodiments, the antigenic prokaryotic polypeptide is OspA or a fragment or variant thereof, wherein the OspA or a fragment or variant thereof comprises at least 5 amino acids, preferably wherein the antigenic prokaryotic polypeptide comprises: (a) an amino acid sequence derived from OspA ST1, which preferably has at least 85% identity with the following sequence: (b) an amino acid sequence derived from OspA ST2, which preferably has at least 85% identity with the following sequence: (c) an amino acid sequence derived from OspAST3, which preferably has at least 85% identity with the following sequence: (d) an amino acid sequence derived from OspAST4, which preferably has at least 85% identity with the following sequence: (e) an amino acid sequence derived from OspA ST5, which preferably has at least 85% identity with the following sequence: (f) an amino acid sequence derived from OspA ST6, which preferably has at least 85% identity with the following sequence: (g) an amino acid sequence derived from OspA ST7, which preferably has at least 85% identity with the following sequence: (h) any combination of (a)-(g); (i) a sequence derived from OspA ST1 according to (a) and a sequence derived from OspA ST2 according to (b); or (j) a sequence derived from OspA ST1 according to (a), a sequence derived from OspA ST2 according to (b), a sequence derived from OspAST3 according to (c), a sequence derived from OspA ST4 according to (d), a sequence derived from OspA ST5 according to (e), a sequence derived from OspA ST6 according to (f), and a sequence derived from OspA ST7 according to (g).

[0054] In certain embodiments, the antigenic prokaryotic polypeptide comprises at least one mutated glycosylation site, preferably at least one mutated N-linked glycosylation site.

[0055] In certain embodiments, the polynucleotide sequence of the nucleic acid is codon optimized.

[0056] In certain embodiments, the polynucleotide sequence of the ORF is codon-optimized.

[0057] In certain embodiments, the polynucleotide sequence encoding the at least one viral secretion signal peptide is codon-optimized.

[0058] In certain embodiments, the polynucleotide sequence encoding the at least one TMB is codon optimized.

[0059] In certain embodiments, the nucleic acid is DNA.

[0060] In certain embodiments, the nucleic acid is a messenger RNA (mRNA), wherein in particular, the mRNA can be a non-replicating mRNA, a self-replicating mRNA, or a trans-replicating mRNA.

[0061] 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.

[0062] In certain embodiments, the mRNA comprises at least one chemical modification.

[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 mRNA are chemically modified.

[0064] 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.

[0065] In certain embodiments, the chemical modification is selected from pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, and 2'-O-methyluridine.

[0066] In certain embodiments, the chemical modification is selected from pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0067] In certain embodiments, the chemical modification is N1-methylpseudouridine.

[0068] In one aspect, the present disclosure provides a composition comprising at least one of the above-mentioned nucleic acids.

[0069] In certain embodiments, the composition further comprises lipid nanoparticles (LNP).In certain embodiments, the nucleic acid is encapsulated in the LNP.

[0070] 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 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.

[0071] In certain embodiments, the LNP further comprises a polyethylene glycol (PEG)-conjugated (PEGylated) lipid, a cholesterol-based lipid, and a helper lipid.

[0072] In certain embodiments, the LNP comprises: - a cationic lipid in a molar ratio of 35% to 55%; - a polyethylene glycol (PEG)-conjugated (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%, wherein all molar ratios are relative to the total lipid content of the LNP.

[0073] In certain embodiments, the LNP comprises: - 40% by molar ratio of cationic lipid; - 1.5% by molar ratio of PEGylated lipid; - 28.5% by molar ratio of cholesterol-based lipid; and - 30% by molar ratio of helper lipid.

[0074] In certain embodiments, the LNP comprises: - a cationic lipid at a molar ratio of 45% to 50%; - a PEGylated lipid at a molar ratio of 1.5% to 1.7%; - a cholesterol-based lipid at a molar ratio of 38% to 43%; and - a helper lipid at a molar ratio of 9% to 10%.

[0075] In certain embodiments, the PEGylated lipid is tetradecanoyl-PEG2000 (DMG-PEG2000) or 2-[(polyethylene glycol)-2000]-N,N-ditetradecanoylacetamide (ALC-0159).

[0076] In certain embodiments, the cholesterol-based lipid is cholesterol.

[0077] In certain embodiments, the helper lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0078] In certain embodiments, the LNP comprises: - a cationic lipid selected from 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%.

[0079] In certain embodiments, the LNP comprises: - 50% SM-102 by mole; - 1.5% DMG-PEG2000 by mole; - 38.5% cholesterol by mole; and - 10% DSPC by mole.

[0080] In certain embodiments, the LNP comprises: - 46.3% ALC-0315 by mole; - 1.6% ALC-0159 by mole; - 42.7% cholesterol by mole; and - 9.4% DSPC by mole.

[0081] In certain embodiments, the LNP comprises: - 47.4% by molar ratio of ALC-0315; - 1.7% by molar ratio of ALC-0159; - 40.9% by molar ratio of cholesterol; and - 10% by molar ratio of DSPC.

[0082] In certain embodiments, the LNPs have an average diameter of 30 nm to 200 nm.

[0083] In certain embodiments, the LNPs have an average diameter of 80 nm to 150 nm.

[0084] In certain embodiments, the composition comprises between 1 mg / mL and 10 mg / mL of the LNP.

[0085] In certain embodiments, the LNP comprises between 1 and 20 nucleic acid molecules, preferably mRNA molecules.

[0086] In certain embodiments, the composition is formulated for intramuscular, intranasal, intravenous, subcutaneous, or intradermal administration.

[0087] In certain embodiments, the composition comprises a phosphate buffer solution.

[0088] In certain embodiments, the composition is a pharmaceutical composition, such as an immunogenic composition or a vaccine, particularly an mRNA vaccine.

[0089] In another aspect, the disclosure provides a nucleic acid or composition for eliciting an immune response in a subject in need thereof.

[0090] In another aspect, the disclosure provides a nucleic acid or composition for treating or preventing a prokaryotic infection in a subject in need thereof.

[0091] In another aspect, the present disclosure provides a method for secreting an antigenic prokaryotic polypeptide in a host cell, the method comprising administering the above-mentioned nucleic acid or composition to the host cell.

[0092] In another aspect, the present disclosure provides a method for displaying an antigenic prokaryotic polypeptide on the surface of a host cell, the method comprising administering the above-mentioned nucleic acid or composition to the host cell.

[0093] In another aspect, the disclosure provides a kit comprising a container comprising a single-use or multi-use dose of the above-described nucleic acid or composition, optionally wherein the container is a vial or a pre-filled syringe or syringe. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 Describe the structure of outer surface protein A (OspA) serotype 1 (ST1) and serotype 2 (ST2) mRNA construct.Use OspA sequence, design different mRNA sequences to guide OspA in intracellular expression, secretion or transmembrane, the OspA sequence and hemagglutinin secretion signal (HASS) and / or HA transmembrane domain (HATMB) do not merge or merge.Use OspA native sequence and have glycosylation site sudden change (Gly-) to avoid the glycosylated sequence of the protein encoded by mRNA.

[0095] Figure 2A-2B Depicted are Western blot images showing in vitro expression of OspA ST1 and ST2 mRNA in HEK293T cell supernatants. Depicted are mRNA-OspA ST1 (without or with HA secretion signal and without or with glycosylation site mutations. Figure 2A ) and mRNA-OspA ST2( Figure 2B ). A negative control (buffer) and a positive control (recombinant OspA) were also used. HEK293 cells were transfected with vaccine mRNA. After 48 hours, the supernatant was collected and subjected to Western blotting. The blotted proteins were detected with a polyclonal rabbit antibody that recognizes OspA.

[0096] Figure 3A-3C Depicted are Western blot images showing in vitro expression of OspA ST1 mRNA containing HA SS and HA TMB and without or with glycosylation site mutations in HEK293 cells. Cell supernatants ( Figure 3A )、Crude extract( Figure 3B ) and intracellular compartments ( Figure 3C ). After 48 hours or 72 hours, the supernatant and cells were collected and subjected to Western blotting. The blotted proteins were detected with a polyclonal rabbit antibody that recognizes OspA.

[0097] Figure 4A-4C Depicted are Western blot images showing in vitro expression of OspA ST2 mRNA containing HA SS and HA TMB and without or with glycosylation site mutations in HEK293 cells. Cell supernatants ( Figure 4A )、Crude extract( Figure 4B ) and intracellular compartments ( Figure 4C). After 48 hours or 72 hours, the supernatant and cells were collected and subjected to Western blotting. The blotted proteins were detected with a polyclonal rabbit antibody that recognizes OspA.

[0098] Figure 5 The antigenicity of OspA ST1 antigen delivered by mRNA in HEK293 cells is depicted. The transfected cell supernatants were used to perform sandwich ELISA with functional monoclonal antibodies LA-2, 857-2 and 221-7.

[0099] Figure 6 The antigenicity of OspA ST2 antigen delivered by mRNA in HEK293 cells is depicted. The transfected cell supernatants were used to perform sandwich ELISA with functional monoclonal antibodies 857-2 and 221-7.

[0100] Figure 7A-7B Depicted are the results from the first dose (day 20) ( Fig. 7A ) and after the second dose (day 35) ( Figure 7B ) IgG titer values ​​of anti-OspA ST1 IgG ELISA. HA-SS=secreted signal hemagglutinin; TMB=transmembrane domain; Gly(-)=glycosylation site mutation; dotted line=quantification limit.

[0101] Figure 8 It is the schematic diagram of the assembly included in the expansion set (panel) of mRNA construct.Described set is made up of three kinds of different prokaryotic antigens: OspA ST1, CAMP2 and PITP as shown in the left side of schematic diagram.The right side of schematic diagram shows that antigen merges with the signal sequence (labeled as " SS ") of the glycoprotein deriving from following virus family: influenza virus (type A or type B subtype), rabies virus, varicella virus (VZV) or Ebola virus.Some set constructs further contain corresponding glycoprotein transmembrane domain (labeled as " TMB "). " Flu " represents " influenza " virus.

[0102] Fig. 9Describe Western blot image, it shows after transfection in HEK Expi293F cell, contain from the SS of the glycoprotein deriving from influenza virus (type A or type B hypotype), rabies virus, varicella virus (VZV) or Ebola virus and have or do not have the in vitro protein expression of the OspA ST1 mRNA of their corresponding glycoprotein TMB domain.Use 1:2000 dilution for the rabbit polyclonal antibody (ABCAM ab106081) of OspA to detect OspA ST1 (expected size about 28-34kDa).Control comprises the OspA ST1 construct (first swimming lane on all gels, is labeled as " without SS ") and the reorganization OspA ST1 (last swimming lane on all gels) without any SS or any lipidation sequence.Collect sample at 48 hours time point from crude extract (total lysate), cell supernatant and the cell sample of fractionation containing intracellular compartment or transmembrane compartment.

[0103] Fig.10 Describe Western blot image, it shows after transfection in HEK Expi293F cell, contain from derive from influenza virus (type A or type B subtype) and rabies virus (left side gel) and varicella virus (VZV) and Ebola virus (right side gel) SS of glycoprotein and have or do not have the in vitro protein expression of OspA ST1mRNA of their corresponding glycoprotein TMB domain.Collect cell supernatant and concentrate 7 times at 48 hours time point.By half deglycosylation (represented by " D " on gel) of gained sample volume, with before and after enzymatic treatment, by Western blot analysis protein.Use 1:2000 dilution for OspA rabbit polyclonal antibody (ABCAM ab106081) to detect OspA ST1 (expected size about 28-34kDa). Controls included transfection control (cells not transfected with mRNA), an OspA ST1 construct without any SS (labeled "No SS"), and recombinant OspA ST1 (last lane on all gels).

[0104] Fig.11Describe Western blot images, it shows in HEK Expi293F cells after transfection containing SS from glycoproteins derived from influenza virus (type A or type B subtype), rabies virus, varicella virus (VZV) or Ebola virus and with or without their corresponding glycoprotein TMB domains of CAMP2 mRNA in vitro protein expression. Use 1: 1500 dilution for CAMP2 rabbit polyclonal antibody detection CAMP2 (expected size of about 26-32kDa). Control includes CAMP2 constructs (the first lane on all gels, marked as "no SS") and recombinant CAMP2 (the last lane on all gels) without any SS. Collect samples from crude extracts (total lysates), cell supernatants and fractionated cell samples containing intracellular compartments or transmembrane compartments at 48 hours.

[0105] Fig.12 Describe Western blot image, it shows after transfection in HEK Expi293F cell, contain SS from the glycoprotein of influenza virus (type A or type B subtype), rabies virus, varicella virus (VZV) or Ebola virus and have or do not have the in vitro protein expression of PITP mRNA of their corresponding glycoprotein TMB domain.Use 1:1000 dilution for PITP mouse polyclonal antibody detection PITP (expected size about 42-48kDa).Control includes the PITP construct (the first swimming lane on all gels, marked as "no SS") and recombinant PITP (the last swimming lane on all gels) without any SS or any TMB.Collect samples from crude extract (total lysate), cell supernatant and the fractionated cell sample containing intracellular compartment or transmembrane compartment at 48 hours time point.

[0106] Fig.13 The left side summarizes Western blot analysis of protein expression and localization of OspA ST1, CAMP2, and PITP mRNA constructs containing SS from glycoproteins derived from influenza virus (subtype A or B), rabies virus, varicella virus (VZV), or Ebola virus with or without their corresponding glycoprotein TMB domains in HEK Expi293F cells after transfection. Fig.13 The right side of the table is the computer signal sequence prediction score derived from SignalP. DETAILED DESCRIPTION The present disclosure relates, inter alia, to nucleic acid (eg, mRNA) compositions encoding antigenic prokaryotic polypeptides linked to one or both of a viral secretory signal peptide sequence and a transmembrane domain (TMB), and methods of vaccination using the compositions. I. Definitions

[0108] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present disclosure should have the meaning commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, but methods and materials similar to or equivalent to those described herein and materials may also be used in the practice or testing of the present disclosure. In the case of a conflict, this specification including the definition will be taken as the standard. Typically, the nomenclature used in conjunction with cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicine and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization as described herein and its technology are well known in the art and those commonly used. Enzymatic reactions and purification techniques are carried out as usually achieved in the art or as described herein according to the manufacturer's instructions. Further, unless the context otherwise requires, singular terms should include plural numbers, and plural terms should include singular numbers. Throughout the present specification and embodiments, the words "have" and "comprise" or variations such as "has", "having", "comprises" or "comprising" should be understood to imply the inclusion of the stated integer or groups of integers but not the exclusion of any other integer or groups of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0109] It should be noted that the term "a" or "an" entity refers to one or more of said entities: for example, "a nucleotide sequence" is understood to represent one or more nucleotide sequences. Therefore, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein.

[0110] In addition, "and / or" as used herein is considered a specific disclosure of each of the two specified features or components with or without the other features or components. Therefore, the term "and / or" as used herein with phrases such as "A and / or B" is intended 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 cover 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).

[0111] It should be understood that regardless of the fact that various aspects are described herein with the language “comprising,” other similar aspects described with the language “consisting of” and / or “consisting essentially of” are also provided.

[0112] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure relates. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised Edition, 2000, Oxford University Press can provide a general dictionary of many of the terms used in the present disclosure for one of ordinary skill in the art.

[0113] Units, prefixes and symbols are all expressed in a form acceptable to the International System of Units (SI). Numerical ranges include numbers that define the range. Unless otherwise indicated, amino acid sequences are written from left to right in the amino to carboxyl direction. The headings provided herein are not limitations on the various aspects of the present disclosure. Therefore, the terms defined immediately below can be more fully defined by reference to the specification as a whole.

[0114] The term "approximately" or "about" is used herein to mean approximately, roughly, roughly or around. When the term "approximately" is used in conjunction with a numerical range, it modifies the range by extending the upper and lower boundaries of the numerical value. Typically, the term "approximately" can modify the numerical value to be higher and lower than the value (higher or lower) by variance (e.g., 10%, upward or downward). In some embodiments, the term indicates 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%. In some embodiments, the deviation of "approximately" indicates a deviation of ±10% from the indicated numerical value. In some embodiments, the deviation of "approximately" indicates a deviation of ±5% from the indicated numerical value. In some embodiments, the deviation of "approximately" indicates a deviation of ±4% from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±3% from the indicated value. In some embodiments, "about" indicates a deviation of ±2% from the indicated value. In some embodiments, "about" indicates a deviation of ±1% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.9% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.8% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.7% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.6% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.5% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.4% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.3% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.1% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.05% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.01% from the indicated value.

[0115] As used herein, the term "messenger RNA" or "mRNA" refers to a polynucleotide encoding at least one polypeptide. As used herein, mRNA encompasses both modified RNA and unmodified RNA. mRNA may contain one or more coding regions and non-coding regions. The coding region may alternatively be referred to as an open reading frame (ORF). The non-coding region in mRNA includes a 5' cap, a 5' untranslated region (UTR), a 3' UTR, and a poly A tail. mRNA can be purified from natural sources, produced using a recombinant expression system (e.g., in vitro transcription), and optionally purified or chemically synthesized.

[0116] As used herein, the term "open reading frame," "ORF," or "coding region" refers to a polynucleotide sequence that begins with a start codon (e.g., ATG) and ends with a stop codon (e.g., TAA, TAG, or TGA) without any other stop codons in between and encodes a protein (e.g., an antigenic prokaryotic polypeptide).

[0117] As used herein, the term "viral secretory signal peptide" or "SS" refers to an amino acid sequence derived from a virus that directs a polypeptide sequence attached thereto through a cellular secretory pathway. A polypeptide having an SS sequence is transported through one or more organelles in a cell until it is secreted outside the cell through a secretory vesicle.

[0118] As used herein, the term "transmembrane domain" or "TMB" refers to an amino acid sequence that has cell membrane spanning properties. TMB triggers the anchoring of a polypeptide attached thereto to the cell membrane.

[0119] The present disclosure also includes fragments or variants of polypeptides and any combination thereof. When referring to the antigenic prokaryotic polypeptides of the present disclosure, the term "fragment" or "variant" includes any polypeptide that retains at least some properties of the reference polypeptide (e.g., the specific antigenic properties of the polypeptide or the ability of the polypeptide to help induce antibody binding). Fragments of polypeptides include fragments of N-terminal and / or C-terminal truncations, such as C-terminal fragments and N-terminal fragments, and deletion fragments, but do not include naturally occurring full-length polypeptides (or mature polypeptides). Deletion fragments refer to polypeptides that lack one or more internal amino acids from a full-length polypeptide. Variants of polypeptides include fragments as described above, as well as polypeptides with altered amino acid sequences due to amino acid substitutions, deletions or insertions. Variants may be naturally occurring or non-naturally occurring. Non-naturally occurring variants may be produced using mutagenesis techniques known in the art. Variant polypeptides may contain conservative or non-conservative amino acid substitutions, deletions or additions. Such variations (i.e., truncations and / or amino acid substitutions, deletions or insertions) may occur at the amino acid level or, correspondingly, at the nucleic acid level.

[0120] A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including 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), non-polar side chains (e.g., alanine, tyrosine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, tyrosine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is replaced by another amino acid from the same side chain family, the substitution is considered conservative. In another embodiment, a string of amino acids can be conservatively substituted with a structurally similar string but with a different order and / or composition of the side chain family members.

[0121] As used herein, the term "connection" or "attachment" refers to the covalent or non-covalent bonding of a first amino acid sequence or nucleotide sequence to a second amino acid sequence or nucleotide sequence, respectively (e.g., a secretion signal amino acid sequence and / or a transmembrane domain amino acid sequence is connected to an antigenic prokaryotic polypeptide amino acid sequence). The first amino acid or nucleotide sequence can be directly bonded or juxtaposed to the second amino acid or nucleotide sequence, or alternatively, a spacer sequence can covalently bond the first sequence to the second sequence. The term "connection" refers not only to the fusion of the first amino acid sequence to the second amino acid sequence at the C-terminus or N-terminus, but also includes inserting the entire first amino acid sequence (or the second amino acid sequence) into any two amino acids in the second amino acid sequence (or, respectively, the first amino acid sequence). In one embodiment, the first amino acid sequence can be connected to the second amino acid sequence by a peptide bond or a linker. The first nucleotide sequence can be connected to the second nucleotide sequence by a phosphodiester bond or a linker. The linker can be a peptide or polypeptide (for a polypeptide chain) or a nucleotide or nucleotide chain (for a nucleotide chain) or any chemical part (for both polypeptides and polynucleotide chains). The term "connection" is also indicated by a hyphen (-).

[0122] As used herein, the term "glycosylation" refers to the addition of sugar units to a protein.

[0123] As used herein, the term "N-glycan" refers to a sugar chain attached to a protein at the amide nitrogen of an N (asparagine) residue of the protein. In this way, an N-glycan is formed by the process of N-glycosylation. This glycan may be a polysaccharide.

[0124] 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 polymorphonuclear cells) to a stimulus (such as an antigen or a vaccine). An immune response can include any cell of the body that participates in the host defense response, including, for example, epithelial cells that secrete interferons or cytokines. An immune response includes, but is not limited to, an innate and / or adaptive immune response.

[0125] As used herein, a "protective immune response" refers to an immune response that protects a subject from infection (e.g., preventing infection or preventing developing a disease associated with infection). Methods for measuring immune responses are well known in the art and include, for example, by measuring proliferation and / or activity of lymphocytes (such as B or T cells), secretion of cytokines or chemokines, inflammation, antibody production, etc.

[0126] As used herein, an "antibody response" is an immune response that produces antibodies.

[0127] As used herein, "antigen" refers to a factor that triggers an immune response; and / or a factor that is bound by a T cell receptor when exposed or administered to an organism (e.g., when presented by an MHC molecule) or a factor that is bound to an antibody (e.g., produced by a B cell). In some embodiments, an antigen triggers a humoral response in an organism (e.g., including the production of antigen-specific antibodies). Alternatively or additionally, in some embodiments, an antigen triggers a cellular response in an organism (e.g., involving T cells whose receptors specifically interact with the antigen). A specific antigen can trigger an immune response in one or several members of a target organism (e.g., mouse, rabbit, primate, human), but not in all members of the target organism species. In some embodiments, antigen causes immune response 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% member of target organism species.In some embodiments, antigen is combined with antibody and / or t cell receptor, and may induce or may not induce specific physiological response in organism.In some embodiments, for example, antigen can be combined with antibody and / or t cell receptor in vitro, no matter whether this interaction occurs in vivo.In some embodiments, antigen reacts with the product of specific body fluid or cellular immunity.Antigen includes prokaryotic antigen polypeptide (for example, OspA ST1 and ST2) encoded by mRNA as described herein." prokaryotic antigen " or " antigenic prokaryotic polypeptide " include any antigenic polypeptide derived from prokaryotic organism that can cause immune response.

[0128] As used herein, "adjuvant" refers to a substance or medium that enhances the immune response to an antigen. An adjuvant may include, but is not limited to, a suspension of a mineral (e.g., alum, aluminum hydroxide, or phosphate) on which an antigen is adsorbed; an oil-in-water or water-in-oil emulsion (e.g., Freund's incomplete adjuvant) emulsified in mineral oil or water. Killed mycobacteria (e.g., Freund's complete adjuvant) are sometimes included to further enhance antigenicity. Immunostimulatory oligonucleotides (e.g., CpG motifs) may also be used as adjuvants (e.g., see 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). An adjuvant may also include biomolecules, such as Toll-like receptor (TLR) agonists and co-stimulatory molecules. 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, e.g., 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 term "individual" or "patient" is used and is intended to be interchangeable with "subject."

[0129] As used herein, the terms "prevent," "preventing," "prevention," or "prophylaxis" (and grammatical variations thereof) refer to the partial or complete inhibition of the onset of one or more symptoms or characteristics of a particular infection, disease, disorder, and / or condition.

[0130] As used herein, the terms "treat," "treating," "treatment," "therapy," or "therapeutic" (and grammatical variations thereof) 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 condition.

[0131] As used herein, the term "effective amount" refers to an amount (e.g., an amount of a nucleic acid or composition) sufficient to achieve beneficial or desired results. 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.

[0132] The term "effective amount" includes, for example, a "therapeutically effective amount" and / or a "prophylactically effective amount".

[0133] As used herein, the phrase "therapeutically effective amount" refers to an amount (e.g., an amount of a nucleic acid or composition) effective to produce some desired therapeutic effect in the treatment of infections, diseases, disorders and / or conditions, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0134] As used herein, the phrase "prophylactically effective amount" refers to an amount (e.g., an amount of a nucleic acid or composition) effective to produce some desired prophylactic effect in the prevention of infection, disease, disorder and / or condition, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0135] As used herein, the term "vaccination" or "vaccinate" refers to the administration of a composition intended to produce an immune response, such as an immune response to a disease-causing agent. Vaccination can be administered before, during, and / or after exposure to a disease-causing agent and / or the onset of one or more symptoms, and in some embodiments, before, during, and / or shortly after exposure to the agent. In some embodiments, vaccination comprises multiple administrations of a vaccine composition at appropriate intervals.

[0136] The present disclosure describes nucleic acid sequences (eg, 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, respectively (reference sequence).

[0137] The "sequence identity" between two nucleic acid sequences indicates the percentage of identical nucleotides between the sequences. The "sequence identity" between two amino acid sequences indicates the percentage of identical amino acids between the sequences.

[0138] The terms "% identical", "% identity" or similar terms are intended to refer specifically to the percentage of identical nucleotides or amino acids in an optimal alignment between the sequences to be compared. The percentage is purely statistical, and the differences between the two sequences may, but are not necessarily, randomly distributed over the entire length of the sequences to be compared. The comparison of two sequences is usually performed in the following manner: after optimal alignment, the sequences are compared with respect to segments or "comparison windows" in order to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually or with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482; the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443; the similarity search method of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444; or a computer program using such an algorithm (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin).

[0139] The percent identity is obtained by determining the number of corresponding identical positions in the sequences being compared, dividing this number by the number of compared positions (eg, the number of positions in the reference sequence), and multiplying this result by 100.

[0140] In some embodiments, the degree of identity is given for a region that is 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 entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given 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 (contiguous nucleotides in some embodiments). In some embodiments, the degree of identity is given for the entire length of the reference sequence.

[0141] A nucleic acid sequence or an amino acid sequence having a specific degree of identity to a given nucleic acid sequence or an amino acid sequence, respectively, can have at least one functional property of the given sequence, for example and in some cases, be functionally equivalent to the given sequence. In some embodiments, a nucleic acid sequence or an amino acid sequence having a specific degree of identity to a given nucleic acid sequence or an amino acid sequence is functionally equivalent to the given sequence.

[0142] As used herein, the term "kit" refers to a packaged set of related components, such as one or more compounds or compositions and one or more related materials, such as solvents, solutions, buffers, instructions, or desiccants. II. Secretion signal sequence

[0143] The use of a viral secretory signal peptide (SS) sequence attached to an antigenic prokaryotic polypeptide can provide many advantages for vaccination. When expressed from mRNA, especially in eukaryotic cells, the SS-prokaryotic antigen fusion protein can have increased extracellular expression relative to prokaryotic antigens without SS sequences. Increased extracellular expression can promote higher immunogenicity and, by extension, better vaccine efficacy.

[0144] Viral SS sequences can be found in publicly available databases (eg, NCBI or UniProt databases), which include annotated viral polypeptide sequences and identify the experimentally verified start and end positions of the SS.

[0145] In certain embodiments, the position of the SS sequence and the SS sequence cleavage site of a given known input polypeptide sequence can be predicted using the SignalP algorithm. The SignalP algorithm (and more specifically, SignalP v6.0) is further described in 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 predicted strength is evaluated based on a cumulative rank score, which takes into account the likelihood of the canonical features of the detection signal sequence (SS likelihood score) and the probability of cutting at the cleavage site (cutting probability score).

[0146] In certain embodiments, the viral secretory signal peptide is derived from a viral sequence in a virus capable of infecting humans. The phrases "influenza virus (influenza)", "SARS CoV-2", "varicella-zoster virus (VZV)", "measles virus (measles)", "rubella virus (rubella)", "rabies virus (rabies)", "Ebola virus (Ebola)" and "smallpox virus (smallpox)" before the phrase "secretion signal peptide sequence" indicate that the secretory signal peptide is derived from the virus corresponding to the name.

[0147] In certain embodiments, the viral secretory signal peptide is derived from a viral sequence selected from the group consisting of an influenza virus secretory signal peptide sequence, a SARS CoV-2 secretory signal peptide sequence, a varicella-zoster virus (VZV) secretory signal peptide sequence, a measles virus secretory signal peptide sequence, a rubella virus secretory signal peptide sequence, a mumps virus secretory signal peptide sequence, an Ebola virus secretory signal peptide sequence, a rabies virus secretory signal peptide sequence, and a smallpox virus secretory signal peptide sequence. These specific signal peptides are derived from viral sequences in viruses that are administered to humans as vaccines (live attenuated, inactivated, or mRNA) with proven strong safety characteristics.

[0148] In certain embodiments, the viral secretion signal peptide is selected from: influenza virus hemagglutinin (HA) secretion signal peptide sequence, SARS CoV-2 spike secretion signal peptide sequence, VZV gB secretion signal peptide sequence, VZV gE secretion signal peptide sequence, VZV gI secretion signal peptide sequence, VZV gK secretion signal peptide sequence, measles virus F protein secretion signal peptide sequence, rubella virus E1 protein secretion signal peptide sequence, rubella virus E2 protein secretion signal peptide sequence, mumps virus F protein secretion signal peptide sequence, Ebola virus GP protein secretion signal peptide sequence, rabies virus glycoprotein (rabies G) secretion signal peptide sequence and smallpox virus 6kDa IC protein secretion signal peptide sequence.

[0149] In certain embodiments, the viral secretion signal peptide comprises an HA secretion signal peptide sequence from influenza A virus or influenza B virus, preferably from influenza A virus.

[0150] 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 viruses of the present disclosure are shown in Table 2 below. Table 1 - Amino acid sequence of viral secretion signal peptide (SS) Table 2 - Amino acid sequences of influenza A and B virus-specific viral secretion signal peptides (SS) III. Transmembrane domain

[0151] The purpose of including a transmembrane domain (TMB) in a construct (especially an mRNA construct used as a vaccine antigen) is to position the antigen on the cell surface by anchoring it to the membrane, and the construct may also include SS, thereby producing an SS-antigen-TMB fusion protein (and possibly subsequently producing an antigen-TMB protein after cutting SS in the mature protein). This can reduce the intracellular localization of the antigen, and further promote higher immunogenicity relative to antigens without TMB sequences. The addition of TMB can especially allow an increase in humoral (B cell) responses to antigens. This may be useful for prokaryotic antigens, but may also be used for other antigens (e.g., viral antigens). The addition of TMB can be used together with an antigen derived from a membrane protein or an antigen derived from a protein that is not a membrane protein (e.g., a secretory protein or an intracellular protein). The addition of any more specific TMB as described herein may be particularly useful for an antigen derived from a protein that is not a membrane protein (i.e., a protein that is not naturally containing TMB (or similar)).

[0152] TMB can be from any known TMB in the art, including but not limited to TMB from eukaryotic transmembrane proteins (e.g., mammalian transmembrane proteins, such as human transmembrane proteins), TMB from prokaryotic transmembrane proteins, and TMB from viral transmembrane proteins.TMB can be further identified by computer prediction algorithms, such as in the TMHMM prediction methods 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 incorporated herein by reference in its entirety. Some features of TMB are further described in detail in Albers et al. (Chapter 2 - cell membrane structures and functions. Basic Neurochemistry 8th Edition. Pages 26-39. 2012), which are incorporated herein by reference. TMB is typically, but not exclusively, mainly composed of non-polar (hydrophobic) amino acid residues, and can cross the lipid bilayer once or several times. Technicians are fully aware of the 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.

[0153] TMB generally comprises alpha helices, each helix containing 18-21 amino acids, which is sufficient to span the lipid bilayer. Thus, in certain embodiments, the transmembrane domain comprises one or more alpha helices.

[0154] In certain embodiments, the transmembrane domain is derived from an integral membrane protein, as further defined below and in Albers et al., "integral membrane proteins" (also referred to as intrinsic membrane proteins) are membrane proteins permanently attached to a lipid membrane. In certain embodiments, the transmembrane domain is derived from an integral multi-pass (polytopic) protein. An integral multi-pass protein is a protein that spans the entire membrane. In certain embodiments, the transmembrane domain is derived from a single-pass (trans) membrane protein, more particularly a two-pass membrane protein, such as type I or type II. A single-pass membrane protein only crosses the membrane once (i.e., a two-pass membrane protein), while a multi-pass membrane protein penetrates in and out, several times across the membrane. A single-pass membrane protein can be classified as type I or type II, with the positioning of type I so that its carboxyl terminus is toward the cytosol, and the amino terminus of type II is toward the cytosol. In certain embodiments, the transmembrane domain is derived from an integral single-pass (monotopic) protein. An integral single-pass protein is a protein that is associated with the membrane only from one side and does not completely cross a lipid bilayer.

[0155] 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.

[0156] In certain embodiments, the transmembrane domain is derived from a viral sequence. The phrases "influenza virus", "SARS CoV-2", "varicella-zoster virus (VZV)", "measles virus", "rubella virus", "rabies virus", "Ebola virus" and "smallpox virus" before the phrase "transmembrane domain sequence" indicate that the transmembrane domain sequence is derived from the virus corresponding to the name.

[0157] In certain embodiments, the transmembrane domain is derived from a viral transmembrane domain sequence selected from the group consisting of an influenza virus transmembrane domain sequence, a SARS CoV-2 transmembrane domain sequence, a varicella-zoster virus (VZV) transmembrane domain sequence, a measles virus transmembrane domain sequence, a rubella virus transmembrane domain sequence, a mumps virus transmembrane domain sequence, a rabies virus transmembrane domain sequence, and an Ebola virus transmembrane domain sequence. These specific transmembrane domains are derived from viral sequences in viruses that are administered to humans as vaccines (live attenuated, inactivated, or mRNA) with proven strong safety characteristics.

[0158] In certain embodiments, the transmembrane domain is selected from: influenza virus hemagglutinin (HA) transmembrane domain sequence, SARS CoV-2 spike transmembrane domain sequence, VZV gB transmembrane domain sequence, VZV gE transmembrane domain sequence, VZV gI transmembrane domain sequence, VZV gK transmembrane domain sequence, measles virus F protein transmembrane domain sequence, rubella virus E1 protein transmembrane domain sequence, rubella virus E2 protein transmembrane domain sequence, mumps virus F protein transmembrane domain sequence, rabies virus glycoprotein (rabies G) transmembrane domain sequence and Ebola virus GP protein transmembrane domain sequence.

[0159] In certain embodiments, the transmembrane domain comprises an HA transmembrane domain sequence from influenza A virus or influenza B virus, preferably from influenza A virus.

[0160] Exemplary viral transmembrane domain amino acid sequences of the present disclosure are shown in Table 3 below. Table 3 - Virus transmembrane domain (TMB) signal amino acid sequence

[0161] In certain embodiments, the SS sequence is located at the N-terminus of the antigenic prokaryotic polypeptide.

[0162] In certain embodiments, the SS sequence is located at the C-terminus of the antigenic prokaryotic polypeptide.

[0163] In certain embodiments, the TMB sequence is located at the N-terminus of the antigenic prokaryotic polypeptide.

[0164] In certain embodiments, the TMB sequence is located at the C-terminus of the antigenic prokaryotic polypeptide.

[0165] In certain embodiments, the SS amino acid sequence is encoded by a codon-optimized polynucleotide sequence.

[0166] In certain embodiments, the TMB amino acid sequence is encoded by a codon-optimized polynucleotide sequence. IV. Connectors

[0167] In certain embodiments of the present disclosure, the viral secretory signal peptide (SS) sequence or transmembrane domain (TMB) is directly fused to the antigenic prokaryotic polypeptide (i.e., there is no linker, such as an amino acid linker, connecting the SS sequence or TMB to the antigenic prokaryotic polypeptide).

[0168] In other embodiments, the SS sequence and TMB of the present disclosure are optionally attached to an antigenic prokaryotic polypeptide with a linker. In certain embodiments, the linker is an amino acid linker. In certain embodiments, the amino acid linker is 1-10 amino acids in length (e.g., the amino acid linker is 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 in length).

[0169] Illustrative examples of linkers include glycine polymers (Gly)n, wherein n is an integer of at least one, two, three, four, five, six, seven, or eight; glycine-serine polymers (GlySer)n, wherein n is an integer of at least one, two, three, four, five, six, seven, or eight; glycine-alanine polymers; alanine-serine polymers; and other flexible linkers known in the art.

[0170] Glycine polymers and glycine-serine polymers are relatively unstructured and flexible, and are therefore able to act as a neutral link between the SS sequence and / or TMB and the antigenic prokaryotic polypeptide. In certain embodiments, the linker is SGS or GSG.

[0171] 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), wherein 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: 83); 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.

[0172] Additional examples of linkers are provided in Chen et al. (Adv Drug Deliv Rev. 65(10): 1357-1369. 2013), which is incorporated herein by reference. V. Antigenic Prokaryotic Peptides

[0173] The viral secretion signal peptide (SS) and / or transmembrane domain (TMB) of the present disclosure is linked to the antigenic prokaryotic polypeptide. A. Prokaryotes

[0174] In certain embodiments, the antigenic prokaryotic polypeptide is derived from a bacterium of the genus Acetobacter, Acinetobacter, Actinomyces, Aeromonas, Agrobacterium, Anaplasma, Azorhizobium, Azotobacter, Bacillus, Bacteroides, Bartonella, Bordetella, Borrelia, Brucella, Burkholderia, Sheathbacter, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Coxiella, Propionibacterium, Ehrlichia, Enterobacter, Enterococcus, Escherichia, Francisella, Fusobacterium, Gardnerella, Haemophilus, Helicobacter, Klebsiella , Lactobacillus, Lactococcus, Legionella, Listeria, Methanobacterium, Microbacterium, Micrococcus, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Pasteurella, Pediococcus, Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, Pseudomonas, Rhizobium, Rickettsia, Rocalima, Roseburia, Salmonella, Serratia, Shigella, Sarcina, Spirillum, Spirochete, Staphylococcus, Stenotrophomonas, Streptobacillus, Streptococcus, Tetragenococcus, Treponema, Vibrio, Viridans, Wolbachia, and Yersinia. In certain embodiments, the antigenic prokaryotic polypeptide is derived from a bacterium of the following species: Acetobacter aurantifolia, Acinetobacter baumannii, Actinomyces israelii, Agrobacterium radiobacterium, Agrobacterium tumefaciens, Anaplasma phagocytophilum, Rhizobium nodosarum, Azotobacter vinelandii, Bacillus anthracis, Bacillus brevis, Bacillus cereus, Bacillus spindle, Bacillus licheniformis, Bacillus megaterium, Bacillus mycoides, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Bacteroides fragilis, Bacteroides gingivalis, Bacteroides melaninogenicus, Bartonella henselae, Bartonella pentaphyllum, The whole body, Bordetella bronchiseptica, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella Malta, Brucella suis, Burkholderia glanders, Burkholderia pseudomallei, Burkholderia cepacia, Granulomatous sheath bacteria, Campylobacter coli, Campylobacter fetus, Campylobacter jejuni, Campylobacter pylori, Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Corynebacterium fusiformis, Coxiella burnetii, Propionibacterium acnes (Cutibacteriumacnes), Propionibacterium vulgaris, Propionibacterium granulosum, Propionibacterium naum, Propionibacterium humosum, Ehrlichia chaffeensis, Enterobacter cloacae, Enterococcus avium, Enterococcus durgentis, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus malorum, 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, Mononuclear cells Listeria monocytogenes, Methanobacterium externum, Microbacterium polymorphum, Micrococcus luteus, Moraxella catarrhalis, Mycobacterium avium, Mycobacterium bovis, Mycobacterium diphtheriae, Mycobacterium intracellulare, Mycobacterium leprae, Mycobacterium leprae murine, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycoplasma fermentans, Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma penetrantis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Pasteurella tularensis, Peptostreptococcus, Porphyromonas gingivalis, Prevotella melaninogenicus, Propionibacterium acnes acnes), Pseudomonas aeruginosa, Rhizobium radiobacterium, Rickettsia prowazekii, Rickettsia psittaci, Rickettsia quinquefasciatus, Rickettsia rickettsii, Rickettsia trachomatis, Henselae henselae, Rickettsia quinquefasciatus, Rosella dentata, Salmonella enteritidis, Salmonella typhi, Salmonella typhimurium, Serratia marcescens, Shigella dysenteriae, Spirillum tetrastichum, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus avium, Streptococcus bovis, Streptococcus hamsteris In certain embodiments, the antigenic prokaryotic polypeptide is derived from the bacterium of Borrelia burgdorferi, preferably selected from species Borrelia burgdorferi, Borrelia elsdenii, Borrelia garinii, Borrelia bavaria, Borrelia mayorii, Borrelia salivarius, Borrelia denticola, Vibrio cholerae, Vibrio comma, Vibrio parahaemolyticus, Vibrio vulnificus, Streptococcus viridans, Wolbachia, Yersinia enterocolitica, Yersinia pestis and Yersinia pseudotuberculosis. In certain embodiments, the antigenic prokaryotic polypeptide is derived from the bacterium of Borrelia burgdorferi, preferably selected from species Borrelia burgdorferi, Borrelia elsdenii, Borrelia garinii, Borrelia bavariae, Borrelia mayorii, Borrelia sparmannii, Borrelia portugalis, Borrelia biseti and / or Borrelia faresii. B. Glycosylation

[0175] Glycosylation can occur in eukaryotic cells (but not in prokaryotes). Specifically, N-linked glycosylation is the attachment of glycans to the amide nitrogen of asparagine (Asn; N) residues of proteins. The attachment process produces glycosylated proteins. Glycosylation can occur at any asparagine residue in a protein that can be accessed and recognized by glycosylases after protein translation, and is most commonly found at accessible asparagine that is part of the NXS / T motif, where the first amino acid residue (X) after asparagine is any amino acid except proline, and the second amino acid residue after asparagine is serine or threonine. Non-human glycosylation patterns can cause polypeptides to have undesirable reactivity when used to elicit antibodies. In addition, glycosylation of polypeptides that are not normally glycosylated (such as antigenic prokaryotic polypeptides) can change their immunogenicity. For example, glycosylation can mask important immunogenic epitopes within a protein. Therefore, in order to reduce or eliminate glycosylation, asparagine residues or serine / threonine residues can be modified, for example by substitution for another amino acid.

[0176] 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, the removal of the N-glycosylation site reduces the glycosylation of the antigenic prokaryotic polypeptide. In some embodiments, the antigenic prokaryotic polypeptide has reduced glycosylation relative to the native antigenic prokaryotic polypeptide. In some embodiments, the removal of the N-glycosylation site eliminates the N-glycosylation of the antigenic prokaryotic polypeptide.

[0177] In certain embodiments, the modification comprises substitution of one or more of N, S and T amino acids in the NXS / T sequence motif, wherein X corresponds to any amino acid except proline (P). In some embodiments, the N, S or T amino acid is substituted by a conservative amino acid substitution. In certain embodiments, the polynucleotide sequence encoding the antigenic prokaryotic polypeptide is codon optimized. C.OspA

[0178] In certain embodiments, the antigenic prokaryotic polypeptide is OspA (outer surface protein A). In certain embodiments, the OspA is preferably derived from OspA serotype (ST) 1, 2, 3, 4, 5, 6 and / or 7, more preferably derived from serotype 1 Borrelia burgdorferi strain B31, serotype 2 Borrelia eldenii strain PKO, serotype 3 Borrelia garinii strain PBr, serotype 4 Bavaria Borrelia, serotype 5 Borrelia garinii, serotype 6 Borrelia garinii or serotype 7 Borrelia garinii.

[0179] Exemplary amino acid sequences encoding OspA proteins of the present disclosure are shown in Table 4 below. D.CAMP2

[0180] In certain embodiments, the antigenic prokaryotic polypeptide is a pore-forming toxin, preferably CAMP2 (Christie-Atkins-Munch-Peterson Factor 2).

[0181] In certain embodiments, the CAMP2 is preferably derived from bacteria of the genus Propionibacterium, more preferably from the species Propionibacterium acnes (formally known as Propionibacterium acnes).

[0182] In certain embodiments, the CAMP2 polypeptide comprises the amino acid sequence

[0183] Exemplary amino acid sequences encoding the P. acnes CAMP2 factor proteins of the present disclosure are shown in Table 10 below. E.PITP

[0184] In certain embodiments, the antigenic prokaryotic polypeptide is a putative iron transport protein (PITP).

[0185] In certain embodiments, the PITP is preferably derived from bacteria of the genus Propionibacterium, more preferably from the species Propionibacterium acnes (formally known as Propionibacterium acnes).

[0186] In certain embodiments, the PITP polypeptide comprises the amino acid sequence

[0187] Exemplary amino acid sequences encoding P. acnes PITP factor proteins of the present disclosure are shown in Table 10 below. VI. Lipid Nanoparticles (LNP)

[0188] The LNPs of the present disclosure comprise four types of lipids: (i) ionizable lipids (eg, cationic lipids); (ii) PEGylated lipids; (iii) cholesterol-based lipids; and (iv) helper lipids. A. Ionizable lipids

[0189] Ionizable lipids facilitate mRNA encapsulation and can be cationic lipids. Cationic lipids provide a positively charged environment at low pH to facilitate efficient encapsulation of negatively charged mRNA drug substances.

[0190] In some embodiments, the cationic lipid is OF-02:

[0191] OF-02 is a non-degradable structural analogue of OF-Deg-Lin. OF-Deg-Lin contains a degradable ester connection to attach a diketopiperazine core and a diunsaturated tail, while OF-02 contains a non-degradable 1,2-amino-alcohol connection to attach the same diketopiperazine core and a diunsaturated tail (Fenton et al., Adv Mater. (2016) 28: 2939; U.S. Patent No. 10,201,618). The exemplary LNP formulation lipid A herein contains OF-2.

[0192] In some embodiments, the cationic lipid is cKK-E10 (Dong et al., PNAS (2014) 111(11):3955-60; U.S. Patent 9,512,073):

[0193] Exemplary LNP formulations herein, Lipid B, contain cKK-E10.

[0194] 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)butyrate), which is a HEPES-based disulfide cationic lipid with a piperazine core having Formula III:

[0195] Exemplary LNP formulation lipid C herein contains GL-HEPES-E3-E10-DS-3-E18-1. In addition to the difference in cationic lipids, lipid C has the same composition as lipid A or lipid B.

[0196] 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)butyrate), which is a HEPES-based disulfide cationic lipid with a piperazine core having Formula IV:

[0197] Exemplary LNP formulations herein, lipid D, contain GL-HEPES-E3-E12-DS-4-E10. In addition to the difference in cationic lipids, lipid D has the same composition as lipid A or lipid B.

[0198] 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)butyrate), which is a HEPES-based disulfide cationic lipid with a piperazine core having Formula V:

[0199] Exemplary LNP formulations herein include lipid E, which contains GL-HEPES-E3-E12-DS-3-E14. In addition to the differences in cationic lipids, lipid E ​​has the same composition as lipid A or lipid B.

[0200] 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 described in Scheme 1:

[0201] Scheme 1: General synthetic scheme for lipids of formula (III), (IV) and (V)

[0202] In some embodiments, the cationic lipid is MC3, having Formula VI:

[0203] In some embodiments, the cationic lipid is SM-102 (9-heptadecyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate) having Formula VII:

[0204] In some embodiments, the cationic lipid is ALC-0315 [(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) having Formula VIII:

[0205] In some embodiments, the cationic lipid is cOrn-EE1, having Formula IX:

[0206] In some embodiments, the cationic lipid can be selected from cKK-E10; OF-02; [(6Z,9Z,28Z,31Z)-triacon ... 9-heptadecyl)[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-methylheptane-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthrene-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); (2-(dioctylammonium)ethyl)decyl phosphate (9A1P9); 5,5-di((Z)-heptadeca-8-en-1-yl)-1-(3-(pyrrolidin-1-yl)propyl)-2,5-dihydro-1H-imidazole -2-carboxylic acid ethyl ester (A2-Iso5-2DC18); bis(2-(dodecyldisulfanyl)ethyl)3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazacosyl)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)tri(azanediyl))tri(propane-3,1-diyl))tri(azanetriyl))hexanonanoate (FTT5); (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetra(ethane-2,1-diyl)(9Z,9'Z,9"Z,9"'Z,12Z,12'Z,12"Z,12"'Z)-tetra(octadec-9,12-dienoate) (OF-Deg-Lin); TT3; N; 1 ,N 3 ,N 5 -Tris(3-(didodecylamido)propyl)benzene-1,3,5-tricarboxamide; N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5); 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoic acid heptadecan-9-yl ester (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.

[0207] In some embodiments, the cationic lipid is biodegradable.

[0208] In some embodiments, the cationic lipid is not biodegradable.

[0209] In some embodiments, the cationic lipid is cleavable.

[0210] In some embodiments, the cationic lipid is not cleavable.

[0211] Cationic lipids are further described in detail in Dong et al. (PNAS. 111(11):3955-60.2014); Fenton et al. (Adv Mater. 28:2939.2016); U.S. Pat. No. 9,512,073; and U.S. Pat. No. 10,201,618, each of which is incorporated herein by reference. B. PEGylated lipids

[0212] The lipid components of PEGylation provide control of the particle size and stability of nanoparticles. The addition of such components can prevent complex aggregation, and provide for increasing the circulation life of lipid-nucleic acid pharmaceutical compositions and increasing the means of their delivery to target tissues (Klibanov et al. FEBS Letters 268 (1): 235-7.1990). These components can be selected to quickly exchange pharmaceutical compositions in vivo (see, e.g., U.S. Patent number 5,885,613).

[0213] Contemplated PEGylated lipids include, but are not limited to, polyethylene glycol (PEG) chains up to 5 kDa in length covalently attached to C6-C 20 (e.g., C8, C 10 , C 12 , C 14 , C 16 or C 18 ) length, such as a derivatized ceramide (e.g., N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)] (C8 PEG ceramide)). In some embodiments, the PEGylated lipid is 1,2-tetradecanoyl-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-dialkoxypropylcarbamate; 2-[(polyethylene glycol)-2000]-N,N-ditetradecanoylacetamide (ALC-0159); and combinations thereof.

[0214] 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 lipid herein is DMG-PEG2000, DSPE-PEG2000, DLPE-PEG2000, DSG-PEG2000, C8 PEG2000, or ALC-0159 (2-[(polyethylene glycol)-2000]-N, N-di-tetradecyl acetamide). In certain embodiments, the PEGylated lipid herein is DMG-PEG2000. C. Cholesterol-based lipids

[0215] The cholesterol component provides stability for the lipid bilayer structure within the nanoparticle. In some embodiments, the LNP comprises one or more cholesterol-based lipids. Suitable cholesterol-based lipids include, for example: DC-Choi (N, N-dimethyl-N-ethylformamidocholesterol), 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"; WO 2011 / 068810), sitosterol (22,23-dihydrostigmasterol), β-sitosterol, sitostanol, fucosterol, stigmasterol (stigmasterane-5,22-dien-3-ol), ergosterol; streptosterol (3β-hydroxy-5,24-cholestadiene); lanosterol (8,24-lanostadiene-3b-ol); 7-dehydrocholesterol (Δ5,7-cholesterol); dihydrolanosterol (24,25-dihydrolanosterol); yeast sterol (5α-cholestadiene-8,24-dien-3β-ol); cholesterol Lathosterol (5α-cholest-7-en-3β-ol); diosgenin ((3β,25R)-spirostan-5-en-3-ol); campesterol (campesterol-5-en-3β-ol); campestanol (5a-campestan-3b-ol); 24-methylene cholesterol (5,24(28)-cholestadiene-24-methylen-3β-ol); cholesterol margarate (cholesteryl margarate) (cholesteryl margarate); cholesterol oleate; cholesterol stearate and other modified forms of cholesterol. In some embodiments, the cholesterol-based lipid used in the LNP is cholesterol. D. Helper lipids

[0216] The helper lipid enhances the structural stability of the LNP and helps the LNP in endosomal escape. It improves the uptake and release of the mRNA drug payload. In some embodiments, the helper lipid is a zwitterionic lipid that has fusogenic properties for enhancing 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-dioleoyl-sn-glycero-3-phosphoethanolamine (DEPE); and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPOC), dipalmitoylphosphatidylcholine (DPPC), DMPC, 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-distearoylphosphatidylethanolamine (DSPE) and 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE).

[0217] Other exemplary helper lipids are dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), phosphatidylserine, sphingolipids, sphingomyelin, ceramides, cerebrosides, gangliosides, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, l-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE) or a combination thereof. In certain embodiments, the helper lipid is DOPE. In certain embodiments, the helper lipid is DSPC.

[0218] In various embodiments, the LNPs of the present invention comprise (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.

[0219] In other embodiments, the LNPs of the invention comprise (i) SM-102; (ii) DMG-PEG2000; (iii) cholesterol; and (iv) DSPC.

[0220] In yet other embodiments, the LNPs of the invention comprise (i) ALC-0315; (ii) ALC-0159; (iii) cholesterol; and (iv) DSPC. E. Molar ratio of lipid components

[0221] The mol ratio of the above-mentioned components is important for the effectiveness of LNP in delivering mRNA. The mol ratio of cationic lipid, PEGylated lipid, lipid based on cholesterol and auxiliary lipid is A: B: C: D, wherein A + B + C + D = 100%. In some embodiments, the mol ratio of cationic lipid in LNP relative to total lipid (i.e., A) is 35%-55%, such as 35%-50% (for example, 38%-42%, such as 40%, or 45%-50%). In some embodiments, the mol ratio of the lipid component of PEGylated relative to total lipid (i.e., B) is 0.25%-2.75% (for example, 1%-2%, such as 1.5%). In some embodiments, the mol ratio of lipid based on cholesterol relative to total lipid (i.e., C) is 20%-50% (for example, 27%-30%, such as 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%, such as 30%, or 8%-12%, such as 10%). In some embodiments, the (PEGylated lipid + cholesterol) component has the same molar amount as the helper lipid. In some embodiments, the molar ratio of the cationic lipid to the helper lipid contained in the LNP is greater than 1.

[0222] In certain embodiments, the LNPs of the present disclosure comprise:

[0223] 35% to 55% or 40% to 50% cationic lipid in a molar ratio (e.g., 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54% or 55% cationic lipid in a molar ratio);

[0224] 0.25% to 2.75% or 1.00% to 2.00% polyethylene glycol (PEG) conjugated (PEGylated) lipids (e.g., 0.25%, 0.50%, 0.75%, 1.00%, 1.25%, 1.50%, 1.75%, 2.00%, 2.25%, 2.50%, or 2.75% PEGylated lipids at a molar ratio);

[0225] 20% to 50%, 25% to 45%, or 28.5% to 43% cholesterol-based lipids (e.g., 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% cholesterol-based lipids); and

[0226] 5% to 35%, 8% to 30%, or 10% to 30% of a helper lipid in a molar ratio (e.g., a 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% helper lipid in a molar ratio),

[0227] All molar ratios are relative to the total lipid content of the LNP.

[0228] In certain embodiments, the LNP comprises: a cationic lipid at a molar ratio of 40%; a PEGylated lipid at a molar ratio of 1.5%; a cholesterol-based lipid at a molar ratio of 28.5%; and a helper lipid at a molar ratio of 30%.

[0229] In certain embodiments, the LNP comprises: a cationic lipid at a molar ratio of 45% to 50%; a PEGylated lipid at a molar ratio of 1.5% to 1.7%; a cholesterol-based lipid at a molar ratio of 38% to 43%; and a helper lipid at a molar ratio of 9% to 10%.

[0230] In certain embodiments, the PEGylated lipid is tetradecanoyl-PEG2000 (DMG-PEG2000).

[0231] In various embodiments, the cholesterol-based lipid is cholesterol.

[0232] In some embodiments, the helper lipid is 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE).

[0233] In certain embodiments, the LNP comprises: OF-02 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%.

[0234] In certain embodiments, the LNP comprises: cKK-E10 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%.

[0235] In certain embodiments, the LNP comprises: GL-HEPES-E3-E10-DS-3-E18-1 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%.

[0236] In certain embodiments, the LNP comprises: GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%.

[0237] In certain embodiments, the LNP comprises: GL-HEPES-E3-E12-DS-3-E14 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%.

[0238] In certain embodiments, the LNP comprises: SM-102 at a molar ratio of 35% to 55%; DMG-PEG2000 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%.

[0239] 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%.

[0240] 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 designated herein as "Lipid A".

[0241] In certain embodiments, the LNP comprises: 40% cKK-E10 at a molar ratio; 1.5% DMG-PEG2000 at a molar ratio; 28.5% cholesterol at a molar ratio; and 30% DOPE at a molar ratio. This LNP formulation is designated herein as "Lipid B".

[0242] In certain embodiments, the LNP comprises: 40% GL-HEPES-E3-E10-DS-3-E18-1 at a molar ratio; 1.5% DMG-PEG2000 at a molar ratio; 28.5% cholesterol at a molar ratio; and 30% DOPE at a molar ratio. This LNP formulation is designated herein as "lipid C".

[0243] In certain embodiments, the LNP comprises: 40% GL-HEPES-E3-E12-DS-4-E10 at a molar ratio; 1.5% DMG-PEG2000 at a molar ratio; 28.5% cholesterol at a molar ratio; and 30% DOPE at a molar ratio. This LNP formulation is designated herein as "lipid D".

[0244] In certain embodiments, the LNP comprises: 40% GL-HEPES-E3-E12-DS-3-E14 at a molar ratio; 1.5% DMG-PEG2000 at a molar ratio; 28.5% cholesterol at a molar ratio; and 30% DOPE at a molar ratio. This LNP formulation is designated herein as "lipid E".

[0245] In certain embodiments, the LNP comprises: 50% molar ratio of 8-{(2-hydroxyethyl)[6-oxo-6-(undecanyloxy)hexyl]amino}octanoic acid 9-heptadecanyl ester (SM-102); 10% molar ratio of 1,2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC); 38.5% molar ratio of cholesterol; and 1.5% molar ratio of 1,2-tetracosanoyl-racemic-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000).

[0246] In certain embodiments, the LNP comprises: (4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) at a molar ratio of 46.3%; 1,2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC) at a molar ratio of 9.4%; cholesterol at a molar ratio of 42.7%; and 2-[(polyethylene glycol)-2000]-N,N-ditetradecanoylacetamide (ALC-0159) at a molar ratio of 1.6%.

[0247] In certain embodiments, the LNP comprises: (4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) at a molar ratio of 47.4%; 1,2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC) at a molar ratio of 10%; cholesterol at a molar ratio of 40.9%; and 2-[(polyethylene glycol)-2000]-N,N-ditetradecanoylacetamide (ALC-0159) at a molar ratio of 1.7%.

[0248] To calculate the actual amount of each lipid to be placed in the LNP formulation, first determine the molar amount of the cationic lipid 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, calculate the molar amount of each other lipid based on the molar amount of the cationic lipid and the selected molar ratio. These molar amounts are then converted to weight using the molecular weight of each lipid. F. Active ingredients of LNP

[0249] The active ingredient of the LNP vaccine composition of the present invention is a nucleic acid (eg, mRNA) encoding an antigenic prokaryotic polypeptide.

[0250] In the case of desired, the LNP can be multivalent. In some embodiments, the LNP can carry nucleic acid, such as mRNA, and the nucleic acid encodes more than one antigenic prokaryotic polypeptide, such as two, three, four, five, six, seven or eight antigens. For example, the LNP can carry multiple nucleic acids (e.g., mRNA), each encoding different antigenic prokaryotic polypeptides; or carry polycistronic mRNA (e.g., each antigen coding sequence is separated by a nucleotide linker encoding a self-cleaving peptide (such as a 2A peptide)) that can be translated into more than one antigenic prokaryotic polypeptide. The LNP carrying different nucleic acids (e.g., mRNA) typically includes (encapsulates) multiple copies of each nucleic acid. For example, the LNP carrying or encapsulating two different nucleic acids typically carries multiple copies of each of the two different nucleic acids.

[0251] In some embodiments, a single LNP formulation can contain multiple species (e.g., two, three, four, five, six, seven, eight, nine, ten, or more species) of LNPs, each species carrying a different nucleic acid (e.g., mRNA).

[0252] When the nucleic acid is mRNA, the mRNA can be unmodified (i.e., containing only natural ribonucleotides A, U, C, and / or G linked by phosphodiester bonds), or can be chemically modified (e.g., including nucleotide analogs such as pseudouridine (e.g., N-1-methylpseudouridine), 2'-fluororibonucleotides and 2'-methoxyribonucleotides; and / or phosphorothioate bonds). The mRNA molecule can include a 5' cap and a poly A tail. G. Buffer and other components

[0253] In order to stabilize nucleic acids and / or LNPs (e.g., to extend the shelf life of vaccine products), to facilitate the administration of LNP pharmaceutical compositions, and / or to enhance the in vivo expression of nucleic acids, nucleic acids and / or LNPs can be formulated in combination with one or more carriers, targeting ligands, stabilizing agents (e.g., preservatives and antioxidants), and / or other pharmaceutically acceptable excipients. Examples of such excipients are parabens, thimerosal, thiomersal sodium, chlorobutanol, benzalkonium chloride, and chelating agents (e.g., EDTA).

[0254] The LNP compositions of the present disclosure can be provided as frozen liquid form or lyophilized form. Various cryoprotectants can be used, including but not limited to sucrose, trehalose, glucose, mannitol, mannose, dextrose, etc. Cryoprotectants can constitute 5%-30% (w / v) of the LNP compositions. In some embodiments, the LNP compositions include trehalose, for example, 5%-30% (e.g., 10%) (w / v). Once prepared with cryoprotectants, the LNP compositions can be frozen (or lyophilized and frozen) at -20°C to -80°C.

[0255] The LNP composition can be provided to the patient in an aqueous buffer solution: thawed if previously frozen, or reconstituted in an aqueous buffer solution at the bedside if previously lyophilized. The buffer solution is preferably isotonic and suitable for, e.g., intramuscular or intradermal injection. In some embodiments, the buffer solution is phosphate buffered saline (PBS). VII. RNA

[0256] The vaccine composition of the present invention of the present disclosure may include an RNA molecule (e.g., mRNA) encoding a target antigen (e.g., an antigenic prokaryotic polypeptide). The RNA molecule of the present disclosure may include at least one ribonucleic acid (RNA) comprising an ORF encoding the target antigen. In certain embodiments, the RNA is a messenger RNA (mRNA) comprising an ORF encoding the target antigen. In certain embodiments, the RNA (e.g., mRNA) further comprises at least one 5'UTR, 3'UTR, poly (A) tail and / or 5' cap. A.5' cap

[0257] The mRNA 5' cap can provide resistance to nucleases found in most eukaryotic cells and promote translation efficiency. Several types of 5' caps are known. The 7-methylguanosine cap (also called the "m 7 G" or "cap-0") comprises a guanosine linked to the first transcribed nucleotide via a 5'-5'-triphosphate bond.

[0258] The 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one terminal phosphate group from the 5' nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphate via a guanylyltransferase, resulting in a 5'5'5 triphosphate linkage; the 7-nitrogen of guanine is then methylated by a methyltransferase. 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. Publication No. US 2016 / 0032356 and U.S. Publication No. US2018 / 0125989, which are incorporated herein by reference.

[0259] 5' capping of polynucleotides can be accomplished concomitantly during an in vitro transcription reaction using the following chemical RNA cap analogs according to the manufacturer's protocol to produce a 5'-guanosine cap structure: 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). 5' capping of the modified RNA can be accomplished post-transcriptionally using a vaccinia virus capping enzyme to produce the Cap 0 structure: m7G(5')ppp(5')G. The Cap 1 structure can be produced using both a vaccinia virus capping enzyme and a 2'-O methyl-transferase to produce m7G(5')ppp(5')G-2'-O-methyl. The Cap 2 structure can be produced from the Cap 1 structure, followed by 2'-O-methylation of the 5'-third to last nucleotide using a 2'-O methyl-transferase. The Cap 3 structure can be produced from the Cap 2 structure, followed by 2'-O-methylation of the 5'-fourth to last nucleotide using a 2'-O methyl-transferase.

[0260] 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.

[0261] In certain embodiments, the mRNA of the present disclosure comprises the following 5' cap: B. Untranslated region (UTR)

[0262] In some embodiments, the mRNA of the present disclosure comprises a 5' and / or 3' untranslated region (UTR). In the 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 until the transcription termination signal.

[0263] In some embodiments, the mRNA disclosed herein may include a 5'UTR that includes one or more components that affect the stability or translation of the mRNA. In some embodiments, the 5'UTR may have a length of about 10 to 5,000 nucleotides. In some embodiments, the 5'UTR may have a length of about 50 to 500 nucleotides. In some embodiments, the 5'UTR has a length of at least about 10 nucleotides, a length of about 20 nucleotides, a length of about 30 nucleotides, a length of about 40 nucleotides, a length of about 50 nucleotides, a length of about 100 nucleotides, a length of about 150 nucleotides, a length of about 200 nucleotides, a length of about 250 nucleotides, a length of about 300 nucleotides, a length of about 350 nucleotides, a length of about 400 nucleotides, a length of about 450 nucleotides, a length of about 500 nucleotides, a length of about 550 nucleotides, a length of about 600 nucleotides, a length of about 6 A length of about 50 nucleotides, a length of about 700 nucleotides, a length of about 750 nucleotides, a length of about 800 nucleotides, a length of about 850 nucleotides, a length of about 900 nucleotides, a length of about 950 nucleotides, a length of about 1,000 nucleotides, a length of about 1,500 nucleotides, a length of about 2,000 nucleotides, a length of about 2,500 nucleotides, a length of about 3,000 nucleotides, a length of about 3,500 nucleotides, a length of about 4,000 nucleotides, a length of about 4,500 nucleotides, or a length of about 5,000 nucleotides.

[0264] In some embodiments, the mRNA disclosed herein may include a 3'UTR, which includes one or more of the following: a polyadenylation signal, a binding site for a protein that affects the stability of the position of the mRNA in the cell, or one or more binding sites for a miRNA. In some embodiments, the 3'UTR may have a length of 50 to 5,000 nucleotides or longer. In some embodiments, the 3'UTR may have a length of 50 to 1,000 nucleotides or longer. In some embodiments, the 3'UTR has a length of at least about 50 nucleotides, a length of about 100 nucleotides, a length of about 150 nucleotides, a length of about 200 nucleotides, a length of about 250 nucleotides, a length of about 300 nucleotides, a length of about 350 nucleotides, a length of about 400 nucleotides, a length of about 450 nucleotides, a length of about 500 nucleotides, a length of about 550 nucleotides, a length of about 600 nucleotides, a length of about 650 nucleotides, a length of about 700 nucleotides, a length of about 800 nucleotides, a length of about 900 nucleotides, a length of about 1000 nucleotides, a length of about 1500 nucleotides, a length of about 2000 nucleotides, a length of about 2500 nucleotides, a length of about 3000 nucleotides, a length of about 3500 nucleotides, a length of about 4000 nucleotides, a length of about 4500 nucleotides, a length of about 5000 nucleotides, a length of about 5500 nucleotides, a length of about 6000 nucleotides, a length of about 6500 nucleotides, a length of about 7000 nucleotides, A length of about 750 nucleotides, a length of about 800 nucleotides, a length of about 850 nucleotides, a length of about 900 nucleotides, a length of about 950 nucleotides, a length of about 1,000 nucleotides, a length of about 1,500 nucleotides, a length of about 2,000 nucleotides, a length of about 2,500 nucleotides, a length of about 3,000 nucleotides, a length of about 3,500 nucleotides, a length of about 4,000 nucleotides, a length of about 4,500 nucleotides, or a length of about 5,000 nucleotides.

[0265] In some embodiments, an mRNA disclosed herein may comprise a 5' or 3' UTR that is derived from a gene different from the gene encoded by the mRNA transcript (ie, the UTR is a heterologous UTR).

[0266] In certain embodiments, 5' and / or 3'UTR sequences can be derived from stable mRNA (e.g., globin, actin, GAPDH, tubulin, histone or citric acid cycle enzymes) to increase the stability of mRNA. For example, 5'UTR sequences can include a partial sequence or a fragment thereof of CMV immediate early 1 (IE1) gene to improve the nuclease resistance of mRNA and / or improve the half-life of mRNA. It is also envisioned that a sequence encoding human growth hormone (hGH) or a fragment thereof is included in the 3' end or untranslated region of mRNA. Typically, these modifications improve the stability and / or pharmacokinetic properties (e.g., half-life) of mRNA relative to its unmodified counterpart, and include, for example, modifications made to improve the resistance of mRNA to nuclease digestion in vivo.

[0267] Exemplary 5'UTRs include a sequence derived from the CMV immediate early 1 (IE1) gene (U.S. 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. Publication No. 2016 / 0151409, which is incorporated herein by reference).

[0268] In various embodiments, 5'UTR can be derived from the 5'UTR of TOP gene. TOP gene is typically characterized by the presence of 5' terminal oligopyrimidine (TOP) bundle. In addition, most TOP genes are characterized by growth-related translation regulation. However, TOP genes with tissue-specific translation regulation are also known. In certain embodiments, the 5'UTR derived from the 5'UTR of TOP gene lacks 5'TOP motif (oligopyrimidine bundle) (e.g., U.S. Publication Nos. 2017 / 0029847, 2016 / 0304883, 2016 / 0235864 and 2016 / 0166710, each of which is incorporated herein by reference).

[0269] In certain embodiments, the 5'UTR is derived from the ribosomal protein large 32 (L32) gene (U.S. Publication No. 2017 / 0029847, supra).

[0270] In certain embodiments, the 5'UTR is derived from the 5'UTR of the hydroxysteroid (17-b) dehydrogenase 4 gene (HSD17B4) (U.S. Publication No. 2016 / 0166710, supra).

[0271] In certain embodiments, the 5'UTR is derived from the 5'UTR of the ATP5A1 gene (U.S. Publication No. 2016 / 0166710, supra). In some embodiments, an internal ribosome entry site (IRES) is used in place of the 5'UTR.

[0272] In some embodiments, the 5'UTR comprises the nucleic acid sequence shown in SEQ ID NO:89 and reproduced below: GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG (SEQ ID NO: 89).

[0273] In some embodiments, the 3'UTR comprises the nucleic acid sequence shown in SEQ ID NO:90 and reproduced below: CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC (SEQ ID NO: 90).

[0274] The 5'UTR and 3'UTR are described in further detail in WO2012 / 075040, which is incorporated herein by reference. C. Polyadenylated tail

[0275] 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 impart stability to the mRNA and protect it from exonuclease degradation. The poly (A) tail can enhance translation. In some embodiments, the poly (A) tail is substantially homopolymeric. For example, a poly (A) tail of 100 adenosine nucleotides can have a length of substantially 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 (comprising 100 adenosine nucleotides and at least one nucleotide or a stretch of nucleotides different from an adenosine nucleotide). In certain embodiments, the poly (A) tail comprises the sequence AAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 91).

[0276] As used herein, a "poly(A) tail" typically relates to RNA. However, in the context of the present disclosure, the term also relates to the corresponding sequence in a DNA molecule (eg, a "poly(T) sequence").

[0277] The poly(A) tail may comprise about 10 to about 500 adenosine nucleotides, about 10 to about 200 adenosine nucleotides, about 40 to about 200 adenosine nucleotides, or about 40 to about 150 adenosine nucleotides. The length of the poly(A) tail may be at least about 10, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 adenosine nucleotides.

[0278] 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 conventional chemical synthesis methods without transcription from a DNA template. In various embodiments, the poly(A) tail is produced by enzymatic polyadenylation of the RNA (after in vitro transcription of the RNA) using a commercially available polyadenylation kit and corresponding protocols, or alternatively, by using an immobilized poly(A) polymerase, for example using methods and means as described in WO 2016 / 174271.

[0279] The nucleic acid may comprise a poly(A) tail obtained by enzymatic polyadenylation, wherein a majority of the nucleic acid molecules comprise about 100 (+ / - 20) to about 500 (+ / - 50) or about 250 (+ / - 20) adenosine nucleotides.

[0280] In some embodiments, the nucleic acid may comprise a poly(A) tail derived from a template DNA, and may further comprise at least one additional poly(A) tail produced by enzymatic polyadenylation, for example, as described in WO2016 / 091391.

[0281] In certain embodiments, the nucleic acid comprises at least one polyadenylation signal.

[0282] In various embodiments, the nucleic acid may comprise at least one poly(C) sequence.

[0283] As used herein, the term "poly (C) sequence" is intended to be a sequence of cytosine nucleotides up to about 200 cytosine nucleotides. In some embodiments, the poly (C) sequence comprises about 10 to about 200 cytosine nucleotides, about 10 to about 100 cytosine nucleotides, about 20 to about 70 cytosine nucleotides, about 20 to about 60 cytosine nucleotides, or about 10 to about 40 cytosine nucleotides. In some embodiments, the poly (C) sequence comprises about 30 cytosine nucleotides. D. Chemical modification

[0284] mRNA disclosed herein can be modified or unmodified. In some embodiments, the mRNA may include at least one chemical modification. In some embodiments, mRNA disclosed herein may contain one or more modifications that typically enhance RNA stability. Exemplary modifications may include backbone modifications, sugar modifications, or base modifications. In some embodiments, the disclosed mRNA may be synthesized by naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), and the naturally occurring nucleotides and / or nucleotide analogs 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 can be synthesized from modified nucleotide analogs or derivatives of purines and pyrimidines, such as, for example, 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-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), dihydrouracil, 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, quercetin, β-D-mannosyl-quercetin, phosphoramidate, phosphorothioate, peptide nucleotide, methylphosphonate, 7-deazaguanosine, 5-methylcytosine and inosine.

[0285] In some embodiments, the disclosed mRNA may contain at least one chemical modification, including but not limited to pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine and 2'-O-methyluridine.

[0286] In some embodiments, the chemical modification is selected from pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0287] In some embodiments, the chemical modification comprises N1-methylpseudouridine.

[0288] 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.

[0289] 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.

[0290] The preparation of such analogs is described, for example, in U.S. Pat. No. 4,373,071, U.S. Pat. No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat. No. 4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418, U.S. Pat. No. 5,153,319, U.S. Pat. No. 5,262,530, and U.S. Pat. No. 5,700,642. E. mRNA synthesis The mRNA disclosed herein can be synthesized according to any of a variety of methods. For example, 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. In brief, IVT is typically performed with a linear or circular DNA template containing a promoter, a ribonucleoside triphosphate pool, a buffer system that can include DTT and magnesium ions, an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNase I, pyrophosphatase and / or RNase inhibitors. The exact conditions can vary depending on the specific application. The presence of these agents is generally undesirable in the final mRNA product, and these agents can be considered impurities or contaminants that can be purified or removed to provide clean and / or homogeneous mRNA suitable for therapeutic use. While in some embodiments mRNA provided from an in vitro transcription reaction may be desirable, other sources of mRNA may be used in accordance with the present disclosure, including wild-type mRNA produced from bacteria, fungi, plants, and / or animals. VIII. Methods for preparing LNP vaccines of the present invention

[0291] The LNP of the present invention can be prepared by various techniques currently known in the art. For example, multilamellar vesicles (MLVs) can be prepared according to conventional techniques, such as by depositing the selected lipid on the inner wall of a suitable container or vessel (by dissolving the lipid in a suitable solvent and then evaporating the solvent to leave a thin film inside the vessel) or by spray drying. The aqueous phase can then be added to the vessel with a vortex motion, which leads to the formation of MLVs. Unilamellar vesicles (ULVs) can then be formed by homogenization, ultrasonic treatment or extrusion of multilamellar vesicles. In addition, unilamellar vesicles can be formed by detergent removal techniques.

[0292] Various methods are described in US2011 / 0244026, US2016 / 0038432, US2018 / 0153822, US 2018 / 0125989 and PCT / US2020 / 043223 (submitted on July 23, 2020), and can be used to practice the present disclosure. An exemplary method needs to be encapsulated mRNA by mixing it with a mixture of lipids, without first preforming lipid nanoparticles by lipids, as described in US2016 / 0038432. Another exemplary method needs to be encapsulated mRNA by mixing preformed LNP with mRNA, as described in US2018 / 0153822.

[0293] In some embodiments, the method for preparing the LNP loaded with mRNA includes the step of heating one or more solutions to a temperature higher than ambient temperature, wherein the one or more solutions are solutions comprising preformed lipid nanoparticles, solutions comprising mRNA, and mixed solutions comprising mRNA encapsulated by LNP. In some embodiments, the method includes the step of heating one or both of the mRNA solution and the preformed LNP solution before the mixing step. In some embodiments, the method includes heating one or more of the solutions comprising preformed LNP, solutions comprising mRNA, and solutions comprising mRNA encapsulated by LNP during the mixing step. In some embodiments, the method includes the step of heating the mRNA encapsulated by LNP after the mixing step. In some embodiments, the temperature to which one or more solutions are heated is or is higher than about 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C. In some embodiments, the temperature to which the one or more solutions are heated ranges from about 25° C.-70° C., about 30° C.-70° C., about 35° C.-70° C., about 40° C.-70° C., about 45° C.-70° C., about 50° C.-70° C., or about 60° C.-70° C. In some embodiments, the temperature is about 65° C.

[0294] Various methods can be used to prepare mRNA solutions suitable for the present disclosure. In some embodiments, mRNA can be directly dissolved in a buffer solution as described herein. In some embodiments, mRNA solution can be produced by mixing an mRNA stock solution with a buffer solution, and then mixing with a lipid solution for encapsulation. In some embodiments, mRNA solution can be produced by mixing an mRNA stock solution with a buffer solution, and then mixing with a lipid solution for encapsulation immediately. In some embodiments, suitable mRNA stock solutions can contain an mRNA concentration of or greater than about 0.2mg / ml, 0.4mg / ml, 0.5mg / ml, 0.6mg / ml, 0.8mg / ml, 1.0mg / ml, 1.2mg / ml, 1.4mg / ml, 1.5mg / ml, or 1.6mg / ml, 2.0mg / ml, 2.5mg / ml, 3.0mg / ml, 3.5mg / ml, 4.0mg / ml, 4.5mg / ml or 5.0mg / ml in water or buffer.

[0295] In some embodiments, the mRNA stock solution is mixed with a 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 rate greater than the rate of the mRNA stock solution. For example, the buffer solution can be mixed at a rate of at least 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x or 20x of the rate of the mRNA stock solution. In some embodiments, the buffer solution is mixed with a flow rate ranging from about 100-6000ml / minute (for example, about 100-300ml / minute, 300-600ml / minute, 600-1200ml / minute, 1200-2400ml / minute, 2400-3600ml / minute, 3600-4800ml / minute, 4800-6000ml / minute or 60-420ml / minute). 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.

[0296] In some embodiments, the mRNA stock solution is mixed at a flow rate ranging between 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.

[0297] 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. The nucleic acid incorporated into the LNP can be completely or partially located in 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. Incorporating mRNA into lipid nanoparticles is also referred to as "encapsulation" herein, wherein the nucleic acid is completely or substantially contained in the internal space of the lipid nanoparticle.

[0298] Suitable LNP can be prepared in various sizes. In some embodiments, the lipid nanoparticle size that reduces is relevant to the more effective delivery of mRNA. The selection of suitable LNP size can consider the position of target cell or tissue and consider the application that the lipid nanoparticle will be used for to some extent.

[0299] Various methods known in the art can be used to change the size of lipid nanoparticle populations. The preferred method herein utilizes Zetasizer Nano ZS (Malvern Panalytical) to measure LNP particle size. In one approach, 10 μl of LNP sample is mixed with 990 μl of 10% trehalose. This solution is loaded into a cuvette and then placed in a Zetasizer machine. The z-average diameter (nm) or cumulative mean is considered to be the average size of the LNP in the sample. The Zetasizer machine can also be used to measure the polydispersity index (PDI) by using dynamic light scattering (DLS) and cumulative analysis of the autocorrelation function. The average LNP diameter can be reduced by ultrasonic treatment of the formed LNP. Intermittent ultrasonic treatment cycles can be alternated with quasi-elastic light scattering (QELS) evaluations to guide effective lipid nanoparticle synthesis.

[0300] In some embodiments, a majority of the purified LNPs (i.e., greater 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., greater 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).

[0301] In some embodiments, the average size of the LNPs in the compositions of the invention is 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.

[0302] In some embodiments, greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the LNPs in the compositions of the invention range in size from 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., 55-65 nm), which is particularly suitable for pulmonary delivery via aerosolization.

[0303] In some embodiments, the dispersibility or molecular size heterogeneity measure (PDI) of the LNPs in the pharmaceutical compositions provided by the present disclosure is less than about 0.5. In some embodiments, the PDI of the LNPs is 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.

[0304] In some embodiments, the pharmaceutical compositions provided herein are greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the purified LNP in each separate particle encapsulates mRNA. In some embodiments, substantially all (for example, greater than 80% or 90%) of the purified lipid nanoparticles in the pharmaceutical compositions are encapsulated mRNA in each separate particle. In some embodiments, the encapsulation efficiency of lipid nanoparticles is between 50% and 99%; or greater than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 98% or 99%. Typically, the encapsulation efficiency for the lipid nanoparticles used in this article is at least 90 (for example, at least 91%, 92%, 93%, 94% or 95%).

[0305] In some embodiments, the N / P ratio of the LNP is between 1 and 10. In some embodiments, the N / P ratio of the lipid nanoparticle is greater than 1, about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8. In further embodiments, the N / P ratio of a typical LNP herein is 4.

[0306] In some embodiments, the pharmaceutical composition according to the present disclosure contains 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 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.

[0307] In some embodiments, mRNA can be prepared by chemical synthesis or by in vitro transcription (IVT) of a DNA template. An exemplary method for preparing and purifying mRNA is described in Embodiment 1. In this method, during the IVT process, a cDNA template is used to generate mRNA transcripts, and the DNA template is degraded by DNase. The transcripts are purified by depth filtration and tangential flow filtration (TFF). The purified transcripts are further modified by adding caps and tails, and the modified RNA is purified again by depth filtration and TFF.

[0308] The mRNA is then prepared in an aqueous buffer, and mixed with the amphipathic solution of the lipid component containing LNP. The amphipathic solution for dissolving four kinds of lipid components of LNP can be an alcoholic solution. In some embodiments, the alcohol is ethanol. The aqueous buffer can be, for example, citrate, phosphate, acetate or succinate buffer, and can have a pH of about 3.0-7.0 (e.g., about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0 or about 6.5). The buffer may contain other components, such as salts (eg, sodium, potassium and / or calcium salts). In a particular embodiment, the aqueous buffer has 1 mM citrate, 150 mM NaCl, pH 4.5.

[0309] An exemplary non-limiting method for preparing mRNA-LNP compositions is described in Embodiment 1. The method involves mixing a buffered mRNA solution with a lipid solution in ethanol in a controlled uniform manner, wherein the lipid:mRNA ratio is maintained throughout the mixing process. In this illustrative example, mRNA is presented in an aqueous buffer containing citric acid monohydrate, trisodium citrate dihydrate and sodium chloride. The mRNA solution is added to a solution (1mM citrate buffer, 150mM NaCl, pH 4.5). A lipid mixture of four lipids (e.g., cationic lipids, PEGylated lipids, cholesterol-based lipids and auxiliary lipids) is dissolved in ethanol. Aqueous mRNA solution and ethanol lipid solution are mixed in a "T" mixer with an almost "pulse-free" pump system at a volume ratio of 4:1. Then downstream purification and buffer exchange are carried out to the resulting mixture. Buffer exchange can be achieved using a dialysis cassette or a TFF system. TFF can be used immediately after formation via the T mixing process to concentrate and buffer exchange the resulting nascent LNP. The diafiltration process is a continuous operation where the volume is kept constant by adding an appropriate buffer at the same rate as the permeate flow. IX. Packaging and use of mRNA-LNP vaccines

[0310] The mRNA-LNP vaccine can be formulated or packaged for parenteral (e.g., intramuscular, intradermal or subcutaneous) administration or nasopharyngeal (e.g., intranasal) administration. The vaccine composition can be in the form of a ready-to-use formulation, wherein the LNP composition is lyophilized and reconstituted with a physiological buffer (e.g., PBS) immediately before use. The vaccine composition can also be shipped and provided in the form of an aqueous solution or frozen aqueous solution, and can be directly administered to a subject without reconstitution (if previously frozen, administered after thawing).

[0311] Thus, the present disclosure provides an article of manufacture (such as 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 one or more containers may contain a single-use dose or multiple-use doses. The container may be a pre-treated glass vial or ampoule. The article of manufacture may also include instructions for use.

[0312] In certain embodiments, mRNA-LNP vaccines are provided for intramuscular (IM) injection. The vaccine can be injected into the subject, for example, in the deltoid muscle of the upper arm. In some embodiments, the vaccine is provided in a pre-filled syringe or syringe (e.g., single-chamber or multi-chamber). In some embodiments, the vaccine is provided for inhalation and is provided in a pre-filled pump, nebulizer, or inhaler.

[0313] The mRNA-LNP vaccine can be administered to a subject in need in a prophylactic effective amount, i.e., an amount that provides sufficient immune protection against the target pathogen for a sufficient amount of time (e.g., one year, two years, five years, ten years, or a lifetime). Sufficient immune protection can be, for example, the prevention or alleviation of symptoms associated with pathogen infection. In some embodiments, multiple doses (e.g., two doses) of the vaccine are injected into a subject in need to achieve the desired prophylactic effect. Doses (e.g., a primary immunization dose and a booster dose) can be separated by, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, one month, two months, three months, four months, five months, six months, one year, two years, five years, or ten years.

[0314] In some embodiments, a single dose of mRNA-LNP vaccine contains 1-50 μg of mRNA (e.g., monovalent or multivalent). For example, a single dose can contain about 2.5 μg, about 5 μg, about 7.5 μg, about 10 μg, about 12.5 μg, or about 15 μg of mRNA for intramuscular (IM) injection. In further embodiments, a multivalent single dose of LNP vaccine contains multiple (e.g., 2, 3, or 4) species of LNP, each species for a different antigen, and each species of LNP has, for example, 2.5 μg, about 5 μg, about 7.5 μg, about 10 μg, about 12.5 μg, or about 15 μg of mRNA.

[0315] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present disclosure should have the meaning commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, but methods and materials similar to or equivalent to those described herein and materials may also be used in the practice or testing of the present disclosure. In the case of a conflict, this specification including the definition will be taken as the standard. Typically, the nomenclature used in conjunction with cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicine and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization as described herein and its technology are well known in the art and those commonly used. Enzymatic reactions and purification techniques are carried out as usually achieved in the art or as described herein according to the manufacturer's instructions. Further, unless the context otherwise requires, singular terms should include plural numbers, and plural terms should include singular numbers. Throughout the present specification and embodiments, the words "have" and "comprise" or variations such as "has", "having", "comprises" or "comprising" should be understood to imply the inclusion of the stated integer or group of integers, but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents form part of the common general knowledge in the art. As used herein, the term "about" or "approximately" as applied to one or more target values ​​refers to values ​​similar to the reference value stated. In certain embodiments, unless otherwise stated or otherwise obvious from the context, the term refers to a range of values ​​that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater or less) of the stated reference value. X.Carrier

[0316] In one aspect, disclosed herein are vectors comprising mRNA compositions disclosed herein. RNA sequences encoding target proteins (e.g., mRNA encoding antigenic prokaryotic polypeptides) can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest can include expression vectors, replication vectors, probe generation vectors, sequencing vectors, and vectors optimized for in vitro transcription.

[0317] In certain embodiments, the vector can be used to express 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.

[0318] In some embodiments, the vector disclosed herein may include 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 vector disclosed herein may include a polynucleotide sequence encoding a poly(A) sequence and / or a polyadenylation signal.

[0319] A variety of RNA polymerase promoters are known. In some embodiments, the promoter may be a T7 RNA polymerase promoter. Other useful promoters may include, but are not limited to, T3 and SP6 RNA polymerase promoters. The consensus nucleotide sequences for T7, T3, and SP6 promoters are known.

[0320] Also disclosed herein are host cells (eg, mammalian cells, eg, human cells) comprising a vector or RNA composition disclosed herein.

[0321] The polynucleotides can be introduced into the target cells using any of a variety of different methods (e.g., commercially available methods), including but not limited to electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, MA) or Gene Pulser II (BioRad, Denver, CO), Multiporator (Eppendorf, Hamburg, Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, biolistic particle delivery systems such as a "gene gun" (see, e.g., Nishikawa et al. (2001). Hum Gene Ther. 12(8):861-70 or TransIT-RNA transfection kit (Mirus, Madison, WI).

[0322] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles, and liposomes). An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0323] Regardless of the method used to introduce exogenous nucleic acid into host cells or otherwise expose cells to an inhibitor of the disclosure, a variety of assays can be performed to confirm the presence of the mRNA sequence in the host cells. XI. Self-replicating RNA, trans-replicating RNA, and non-replicating RNA

[0324] Self-replicating RNA:

[0325] Self-replicating (or self-amplification) RNA can be produced by using a replication assembly derived from, for example, an alphavirus and replacing structural viral proteins with a nucleotide sequence encoding a target protein (e.g., an antigenic prokaryotic polypeptide). Self-replicating RNA is typically a positive strand molecule that can be directly translated after being delivered to a cell, and this translation provides an RNA-dependent RNA polymerase, which then produces antisense transcripts and sense transcripts from the RNA delivered. Therefore, the RNA delivered causes the production of multiple sub-RNAs. These sub-RNAs and colinear subgenomic transcripts can be self-translated to provide the in situ expression of the encoded antigen, or can be transcribed to provide a further transcript having the same meaning as the RNA delivered, and the transcript is translated to provide the in situ expression of the antigen. The overall result of this series of transcriptions is that the number of the replicon RNA introduced is amplified in large quantities, and the encoded antigen therefore becomes the main polypeptide product of the cell.

[0326] A kind of suitable system realizing self-replication in this way is to use the replicon based on alphavirus.These replicons are positive strand (sense strand) RNA, which causes the translation of replicase (or replicase-transcriptase) after being delivered to the cell.Replicase is translated into polyprotein, and described polyprotein automatically cuts to provide replication complex, and described complex produces the genome chain copy of positive strand delivery RNA.These negative (-) strand transcripts themselves can be transcribed to produce the further copy of positive (-) strand parent RNA, and also produce the subgenomic transcript of encoding antigen.Therefore, the translation of subgenomic transcript causes the in situ expression of described antigen by infected cells.Suitable alphavirus replicon can use the replicase from Sindbis virus (Sindbisvirus), Semliki forest virus (Semliki forest virus), eastern equine encephalitis virus (eastern equine encephalitis virus), Venezuelan equine encephalitis virus (Venezuelan equine encephalitis virus) etc. Mutant or wild-type viral sequences may be used, for example, the attenuated TC83 mutant of VEEV has been used in replicons, see the following reference: WO 2005 / 113782, which is incorporated herein by reference.

[0327] In one embodiment, each self-replicating RNA described herein encodes (i) an RNA-dependent RNA polymerase that can transcribe RNA from a self-replicating RNA molecule, and (ii) a protein antigen. The polymerase can be an alphavirus replicase, for example, comprising one or more of the alphavirus proteins nsP1, nsP2, nsP3, and nsP4. In addition to the non-structural replicase polyprotein, the natural alphavirus genome also encodes structural virion proteins, while in certain embodiments, the self-replicating RNA molecules do not encode alphavirus structural proteins. Therefore, the self-replicating RNA may lead to the production of copies of its own genomic RNA in cells, but does not lead to the production of RNA-containing virions. The inability to produce these virions means that, unlike wild-type alphaviruses, self-replicating RNA molecules cannot perpetuate themselves in an infectious form. The alphavirus structural proteins necessary for perpetuation in wild-type viruses do not exist in the self-replicating RNA of the present disclosure, and their positions are occupied by one or more genes encoding the immunogen of interest, so that the subgenomic transcripts encode the immunogen, rather than the alphavirus structural virion proteins. Self-replicating RNAs are further described in detail in WO 2011005799, which is incorporated herein by reference.

[0328] Trans-replicating RNA:

[0329] Trans-replication (or trans-amplification) RNA has a component similar to the above-mentioned self-replication RNA. However, for trans-replication RNA, two separate RNA molecules are used. The first RNA molecule encodes the above-mentioned RNA replicase (e.g., alphavirus replicase), and the second RNA molecule encodes the target protein (e.g., antigenic prokaryotic polypeptide). RNA replicase can copy one or both of the first RNA molecule and the second RNA molecule, thereby greatly increasing the copy number of the RNA molecule encoding the target protein. Trans-replication RNA is further described in detail in WO 2017162265, which is incorporated herein by reference.

[0330] Non-replicating RNA:

[0331] Non-replicating (or non-amplifying) RNA is RNA that does not have the ability to replicate itself. XI. Pharmaceutical Compositions

[0332] The pharmaceutical composition according to the present disclosure typically comprises a nucleic acid, particularly RNA, and more particularly mRNA, and a pharmaceutically acceptable carrier, or a pharmaceutically acceptable excipient or a pharmaceutically acceptable diluent, which makes the composition particularly suitable for therapeutic use. The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions and / or dosage forms that are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response or other problems or complications within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0333] The pharmaceutical composition may for example be an immunogenic composition, ie a composition that elicits 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 are therefore intended to have equivalent meanings.

[0334] The pharmaceutical composition of the present disclosure may also include one or more other components, such as small molecule immunopotentiators (e.g., TLR agonists). The pharmaceutical composition 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 a nucleic acid molecule encapsulated in the coding antigen in the LNP. XII. Methods of vaccination

[0335] The nucleic acid (e.g., mRNA) vaccines disclosed herein can be administered to a subject to induce an immune response to an antigenic prokaryotic polypeptide, wherein the anti-antigen antibody titer in the subject is increased after vaccination relative to the anti-antigen antibody titer in a subject not vaccinated with the nucleic acid vaccine disclosed herein, or relative to an alternative vaccine against the prokaryotic polypeptide. "Anti-antigen antibodies" are serum antibodies that specifically bind to an antigen.

[0336] In one aspect, the present disclosure provides a method of eliciting 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 described herein, optionally intramuscularly, intranasally, intravenously, subcutaneously, or intradermally.

[0337] 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 described herein, optionally intramuscularly, intranasally, intravenously, subcutaneously or intradermally.

[0338] In another aspect, the present disclosure provides use of a nucleic acid vaccine described herein for the manufacture of a medicament for eliciting an immune response in a subject in need thereof.

[0339] In another aspect, the present disclosure provides use of a nucleic acid vaccine described herein for the manufacture of a medicament for treating or preventing a prokaryotic infection in a subject in need thereof.

[0340] In another aspect, the present disclosure provides a nucleic acid vaccine as described herein for use in eliciting an immune response in a subject in need thereof.

[0341] 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.

[0342] In one aspect, the disclosure provides a method for secreting an antigenic prokaryotic polypeptide in a host cell, the method comprising administering to the host cell a nucleic acid vaccine described herein.

[0343] 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 to the host cell a nucleic acid vaccine described herein.

[0344] The present invention includes the following embodiments:

[0345] 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.

[0346] Embodiment 2. The nucleic acid of embodiment 1, wherein the ORF further comprises a polynucleotide sequence encoding at least one transmembrane domain (TMB).

[0347] 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.

[0348] Embodiment 4. A nucleic acid according to any one of embodiments 1-3, wherein the viral secretory signal peptide is derived from a viral sequence selected from the following: an influenza virus secretory signal peptide sequence, and a non-influenza virus secretory signal peptide sequence selected from the following: a SARS CoV-2 secretory signal peptide sequence, a varicella-zoster virus (VZV) secretory signal peptide sequence, a measles virus secretory signal peptide sequence, a rubella virus secretory signal peptide sequence, a mumps virus secretory signal peptide sequence, an Ebola virus secretory signal peptide sequence, a smallpox virus secretory signal peptide sequence, and a rabies virus secretory signal peptide sequence.

[0349] Embodiment 5. A nucleic acid according to any one of embodiments 1 to 4, wherein the viral secretory signal peptide is selected from: an influenza virus hemagglutinin (HA) secretory signal peptide sequence, a SARS CoV-2 spike secretory signal peptide sequence, a VZV gB secretory signal peptide sequence, a VZV gE secretory signal peptide sequence, a VZV gI secretory signal peptide sequence, a VZV gK secretory signal peptide sequence, a measles virus F protein secretory signal peptide sequence, a rubella virus E1 protein secretory signal peptide sequence, a rubella virus E2 protein secretory signal peptide sequence, a mumps virus F protein secretory signal peptide sequence, an Ebola virus GP protein secretory signal peptide sequence, a smallpox virus 6kDa IC protein secretory signal peptide sequence, and a rabies virus G protein secretory signal peptide sequence, preferably wherein the viral secretory signal peptide comprises an HA secretory signal peptide sequence from influenza A virus or influenza B virus, more preferably from influenza A virus.

[0350] Embodiment 6. The nucleic acid of embodiment 5, wherein the HA secretion signal peptide sequence comprises the amino acid sequence MKX1X2LX3VX4LX5TFX6X7X8X9A (SEQ ID NO: 145), wherein X1 is selected from A and V; X2 is selected from I and K; X3 is selected from V and L; X4 is selected from L and M; X5 is selected from Y and C; X6 is selected from T and A; X7 is selected from T and A; X8 is selected from A and T; and X9 is selected from N and Y.

[0351] Embodiment 7. The nucleic acid of embodiment 5 or 6, wherein the HA secretion signal peptide sequence comprises an amino acid sequence selected from SEQ ID NOs: 95-109.

[0352] Embodiment 8. The nucleic acid of embodiment 5, wherein the HA secretion signal peptide sequence comprises the amino acid sequence MKX1IIALSX2ILCLVFX3 (SEQ ID NO: 146), wherein X1 is selected from T and A; X2 is selected from Y, N, C and H; and X3 is selected from T and A.

[0353] Embodiment 9. The nucleic acid of embodiment 8, wherein the HA secretion signal peptide sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 110-131.

[0354] Embodiment 10. The nucleic acid of embodiment 5, wherein the HA secretion signal peptide sequence comprises the amino acid sequence MKAIIVLLMVVTSX1A (SEQ ID NO: 147), wherein X1 is selected from S and N.

[0355] Embodiment 11. The nucleic acid of embodiment 5, wherein the HA secretion signal peptide sequence comprises the amino acid sequence MX1AIIVLLMVVTSNA (SEQ ID NO: 148), wherein X1 is selected from K and E.

[0356] Embodiment 12. The nucleic acid of embodiment 10 or 11, wherein the HA secretion signal peptide sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 132-144.

[0357] Embodiment 13. The nucleic acid of any one of embodiments 1-12, wherein the viral secretion signal peptide sequence comprises an amino acid sequence selected from the group consisting of:

[0358] MRSLIIFLLFPSIIYS (SEQ ID NO:16); and MVPQALLFVPLLVFPLCFG (SEQ ID NO:184).

[0359] Embodiment 14. The nucleic acid of embodiment 13, wherein the viral secretion signal peptide comprises the amino acid sequence of MKAKLLVLLCTFTATYA (SEQ ID NO: 1).

[0360] Embodiment 15. The nucleic acid of any one of embodiments 1-14, wherein the viral secretion signal peptide is located at the N-terminus of the antigenic prokaryotic polypeptide.

[0361] Embodiment 16. The nucleic acid of any one of embodiments 1-14, wherein the viral secretion signal peptide is located at the C-terminus of the antigenic prokaryotic polypeptide.

[0362] Embodiment 17. The nucleic acid of any one of embodiments 1-16, wherein the viral secretion signal peptide is attached to the antigenic prokaryotic polypeptide with a linker.

[0363] Embodiment 18. The nucleic acid of any one of embodiments 1-17, wherein the viral secretory signal peptide has a SignalP cleavage probability score of at least 0.8, at least 0.85, at least 0.90, or at least 0.95, as determined using SignalP 6.0.

[0364] Embodiment 19. The nucleic acid of any one of embodiments 1-18, wherein the viral secretion signal peptide 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 as determined using SignalP 6.0.

[0365] Embodiment 20. A nucleic acid according to any one of embodiments 2-19, 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: alanine, isoleucine, leucine, tyrosine, phenylalanine, tryptophan and tyrosine; and / or (c) comprises at least one alpha helix.

[0366] Embodiment 21. The nucleic acid according to any one of embodiments 2-20, wherein the TMB is derived from an integral membrane protein, preferably from a single-pass membrane protein, more preferably from a diploid membrane protein, and even more preferably from a type I diploid membrane protein.

[0367] Embodiment 22. A nucleic acid according to any one of embodiments 2-11, wherein the TMB is derived from a non-human sequence.

[0368] Embodiment 23. The nucleic acid of any one of embodiments 18-22, wherein the antigenic prokaryotic polypeptide is derived from a prokaryotic transmembrane protein, and wherein the TMB is the TMB of the prokaryotic transmembrane protein.

[0369] Embodiment 24. The nucleic acid of any one of embodiments 18-23, wherein the antigenic prokaryotic polypeptide is not derived from a prokaryotic transmembrane protein.

[0370] Embodiment 25. The nucleic acid of embodiment 24, wherein the TMB is derived from a viral sequence.

[0371] Embodiment 26. A nucleic acid according to embodiment 25, wherein the TMB is derived from a viral transmembrane domain sequence selected from the group consisting of an influenza virus transmembrane domain sequence, and a non-influenza virus transmembrane domain sequence selected from the group consisting of a SARS CoV-2 transmembrane domain sequence, a varicella-zoster virus (VZV) transmembrane domain sequence, a measles virus transmembrane domain sequence, a rubella virus transmembrane domain sequence, a mumps virus transmembrane domain sequence, an Ebola virus transmembrane domain sequence, and a rabies virus transmembrane domain sequence.

[0372] Embodiment 27. The nucleic acid according to embodiment 26, wherein the TMB is selected from: influenza virus hemagglutinin (HA) transmembrane domain sequence, SARS CoV-2 spike transmembrane domain sequence, VZV gB transmembrane domain sequence, VZV gE transmembrane domain sequence, VZV gI transmembrane domain sequence, VZV gK transmembrane domain sequence, measles virus F protein transmembrane domain sequence, rubella virus E1 protein transmembrane domain sequence, rubella virus E2 protein transmembrane domain sequence, mumps virus F protein transmembrane domain sequence, Ebola virus GP protein transmembrane domain sequence and rabies virus G protein transmembrane domain sequence, preferably wherein the TMB comprises an HA transmembrane domain sequence from influenza A virus or influenza B virus, more preferably from influenza A virus.

[0373] Embodiment 28. A nucleic acid according to any one of embodiments 18-22 and 24-27, wherein 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). NO:26); LDHAFAAFVLLVPWVLIFMVC (SEQ ID NO:27); WWQLTLGAICALLLAGLLACC (SEQ ID NO:28); IVAALVLSILSIIISLLLFCCW (SEQ ID NO:29); WIPAGIGVTGVIIAVIALFCI (SEQ ID NO:30); and VLLSAGALTALMLIIFLMTCW (SEQ ID NO:185).

[0374] Embodiment 29. The nucleic acid of embodiment 28, wherein the TMB comprises the amino acid sequence of ILAIYSTVASSLVLLVSLGAISF (SEQ ID NO: 17).

[0375] Embodiment 30. The nucleic acid of embodiments 2 and 18-29, wherein the TMB is attached to the antigenic prokaryotic polypeptide with a linker.

[0376] Embodiment 31. The nucleic acid of any one of embodiments 2 and 18-30, wherein the TMB is located at the N-terminus of the antigenic prokaryotic polypeptide.

[0377] Embodiment 32. The nucleic acid of any one of embodiments 2 and 18-30, wherein the TMB is located at the C-terminus of the antigenic prokaryotic polypeptide.

[0378] Embodiment 33. A nucleic acid comprising an open reading frame (ORF), wherein the ORF comprises: - 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, wherein optionally, the ORF further comprises a polynucleotide sequence encoding at least one secretion signal peptide, preferably a viral secretion signal peptide, more preferably a viral secretion signal peptide as described in any of the preceding claims.

[0379] Embodiment 34. 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 transmembrane domain (TMB).

[0380] Embodiment 35. A 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: alanine, isoleucine, leucine, tyrosine, phenylalanine, tryptophan and tyrosine; and / or (c) comprises at least one alpha helix.

[0381] Embodiment 36. 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 dipenic protein, and even more preferably from a type I dipenic protein.

[0382] Embodiment 37. A nucleic acid according to Embodiment 36, wherein the TMB is derived from a non-human sequence.

[0383] Embodiment 38. The nucleic acid of any one of embodiments 1-32, wherein the antigenic prokaryotic polypeptide is derived from a prokaryotic transmembrane protein, and wherein the TMB is Heterogeneous The transmembrane domain of a prokaryotic transmembrane protein.

[0384] Embodiment 39. The nucleic acid of any one of embodiments 35-38, wherein the antigenic polypeptide is not derived from a transmembrane protein.

[0385] Embodiment 40. The nucleic acid of any one of Embodiments 35-37 and 39, wherein the TMB is derived from a viral sequence.

[0386] Embodiment 41. The nucleic acid of embodiments 33-35, 39 and 40, wherein the TMB is derived from a viral transmembrane domain sequence selected from the group consisting of an influenza virus transmembrane domain sequence, and a non-influenza virus transmembrane domain sequence selected from the group consisting of a SARS CoV-2 transmembrane domain sequence, a varicella-zoster virus (VZV) transmembrane domain sequence, a measles virus transmembrane domain sequence, a rubella virus transmembrane domain sequence, a mumps virus transmembrane domain sequence, an Ebola virus transmembrane domain sequence, and a rabies virus transmembrane domain sequence.

[0387] Embodiment 42. The nucleic acid according to claim 41, wherein the TMB is selected from: influenza virus hemagglutinin (HA) transmembrane domain sequence, SARS CoV-2 spike transmembrane domain sequence, VZV gB transmembrane domain sequence, VZV gE transmembrane domain sequence, VZV gI transmembrane domain sequence, VZV gK transmembrane domain sequence, measles virus F protein transmembrane domain sequence, rubella virus E1 protein transmembrane domain sequence, rubella virus E2 protein transmembrane domain sequence, mumps virus F protein transmembrane domain sequence, Ebola virus GP protein transmembrane domain sequence and rabies virus G protein transmembrane domain sequence, preferably wherein the TMB comprises an HA transmembrane domain sequence from influenza A virus or influenza B virus, more preferably from influenza A virus.

[0388] Embodiment 43. The nucleic acid of embodiment 42, wherein the TMB comprises an amino acid sequence selected from the group consisting of:

[0389] 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); IVYILIAVCLGLGLIGIPALIC (SEQ ID NO:26); LDHAFAAFVLLVPWVLIFMVC (SEQ ID NO:27); WWQLTLGAICALLLAGLLACC (SEQ ID NO:28); IVALVLSILSIIISLLFCCW (SEQ ID NO:29); WIPAGIGVTGVIIAVIALFCI (SEQ ID NO:30); and

[0390] VLLSAGALTALMLIIFLMTCW (SEQ ID NO: 185).

[0391] Embodiment 44. The nucleic acid of Embodiment 43, wherein the TMB comprises the amino acid sequence of ILAIYSTVASSLVLLVSLGAISF (SEQ ID NO: 17).

[0392] Embodiment 45. The nucleic acid of any one of Embodiments 34-44, wherein the TMB is attached to the antigenic prokaryotic polypeptide with a linker.

[0393] Embodiment 46. The nucleic acid of any one of Embodiments 34-44, wherein the TMB is located at the N-terminus of the antigenic prokaryotic polypeptide.

[0394] Embodiment 47. The nucleic acid of any one of Embodiments 34-44, wherein the TMB is located at the C-terminus of the antigenic prokaryotic polypeptide.

[0395] Embodiment 48. A nucleic acid according to any one of embodiments 34-47, wherein the antigenic prokaryotic polypeptide is derived from a bacterium of the genus Acetobacter, Acinetobacter, Actinomyces, Aeromonas, Agrobacterium, Anaplasma, Azorhizobium, Azotobacter, Bacillus, Bacteroides, Bartonella, Bordetella, Borrelia, Brucella, Burkholderia, Sheathbacter, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Coxiella, Propionibacterium, Ehrlichia, Enterobacter, Enterococcus, Escherichia Bacillus, Francisella, Fusobacterium, Gardnerella, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Legionella, Listeria, Methanobacterium, Microbacterium, Micrococcus, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Pasteurella, Pediococcus, Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, Pseudomonas, Rhizobium, Rickettsia, Rocalima, Roseburia, Salmonella, Serratia, Shigella, Sarcina, Spirillum, Spirochete , Staphylococcus, Stenotrophomonas, Streptobacillus, Streptococcus, Tetragenococcus, Treponema, Vibrio, Viridans, Wolbachia and Yersinia, preferably a bacterium from the species selected from the group consisting of Acetobacter aurantifolia, Acinetobacter baumannii, Actinomyces israelii, Agrobacterium radiobacter, Agrobacterium tumefaciens, Anaplasma phagocytophilum, Rhizobium stem-nodularis, Azotobacter vinelandii, Bacillus anthracis, Bacillus brevis, Bacillus cereus, Bacillus spindle, Bacillus licheniformis, Bacillus megaterium, Bacillus mycoides, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Bacteroides fragilis, Bacillus gingivalis Bacillus, melanin-producing Bacteroides, Bartonella henselae, Bartonella quinquefasciatus, Bordetella bronchiseptica, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella maltese, Brucella suis, Burkholderia glanders, Burkholderia pseudomallei, Burkholderia cepacia, Bacillus granulomatous, Campylobacter coli, Campylobacter fetus, Campylobacter jejuni, Campylobacter pylori, Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Corynebacterium fusiformis, Coxiella burnetii, Propionibacterium acnes (Cutibacteriumacnes), Propionibacterium vulgaris, Propionibacterium granulosum, Propionibacterium naum, Propionibacterium humosum, Ehrlichia chaffeensis, Enterobacter cloacae, Enterococcus avium, Enterococcus durgentis, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus malorum, 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, Mononuclear cells Listeria monocytogenes, Methanobacterium externum, Microbacterium polymorphum, Micrococcus luteus, Moraxella catarrhalis, Mycobacterium avium, Mycobacterium bovis, Mycobacterium diphtheriae, Mycobacterium intracellulare, Mycobacterium leprae, Mycobacterium leprae murine, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycoplasma fermentans, Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma penetrantis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Pasteurella tularensis, Peptostreptococcus, Porphyromonas gingivalis, Prevotella melaninogenicus, Propionibacterium acnes acnes), Pseudomonas aeruginosa, Rhizobium radiobacterium, Rickettsia prowazekii, Rickettsia psittaci, Rickettsia quinquefasciatus, Rickettsia rickettsii, Rickettsia trachomatis, Henselae henselae, Rickettsia quinquefasciatus, Rosella dentata, Salmonella enteritidis, Salmonella typhi, Salmonella typhimurium, Serratia marcescens, Shigella dysenteriae, Spirillum tetrastichum, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus avium, Streptococcus bovis, Streptococcus hamsteris Bacteria, Streptococcus faecalis, Streptococcus faecalis, Streptococcus rats, Streptococcus gallinarum, Streptococcus lactis, Streptococcus mitis, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus rats, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus sobrinus, Treponema pallidum, Treponema denticola, Vibrio cholerae, Vibrio comma, Vibrio parahaemolyticus, Vibrio vulnificus, Streptococcus viridans, Wolbachia, Yersinia enterocolitica, Yersinia pestis and Yersinia pseudotuberculosis.

[0396] Embodiment 49. The nucleic acid according to any one of embodiments 1-48, wherein the antigenic prokaryotic polypeptide is derived from a bacterium of the genus Borrelia, preferably selected from the species Borrelia burgdorferi, Borrelia elsdenii, Borrelia garinii, Borrelia bavaria, Borrelia mayorii, Borrelia sparmannii, Borrelia portugalensis, Borrelia bisettii and / or Borrelia faresi.

[0397] Embodiment 50. The nucleic acid of embodiment 49, wherein the antigenic prokaryotic polypeptide is OspA ST1, OspA ST2, OspA ST3, OspA ST4, OspA ST5, OspA ST6, OspA ST7 or a fragment thereof.

[0398] Embodiment 51. A nucleic acid according to embodiment 50, wherein the antigenic prokaryotic polypeptide is OspA or a fragment or variant thereof, wherein the OspA or a fragment or variant thereof comprises 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 wherein the antigenic prokaryotic polypeptide comprises: (a) an amino acid sequence derived from OspA ST1, which is preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to: (b) an amino acid sequence derived from OspA ST2, which is preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to: (c) an amino acid sequence derived from OspAST3, which preferably has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to: (d) an amino acid sequence derived from OspAST4, which preferably has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to: (e) an amino acid sequence derived from OspA ST5, which preferably has at least 85% identity with the following sequence: (f) an amino acid sequence derived from OspA ST6, which is preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to: (g) an amino acid sequence derived from OspA ST7, which is preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to: (h) any combination of (a)-(g); (i) a sequence derived from OspA ST1 according to (a) and a sequence derived from OspA ST2 according to (b); or (j) a sequence derived from OspA ST1 according to (a), a sequence derived from OspA ST2 according to (b), a sequence derived from OspAST3 according to (c), a sequence derived from OspA ST4 according to (d), a sequence derived from OspA ST5 according to (e), a sequence derived from OspA ST6 according to (f), and a sequence derived from OspA ST7 according to (g).

[0399] Embodiment 52. The nucleic acid according to any one of embodiments 1-48, wherein the antigenic prokaryotic polypeptide is derived from a bacterium of the genus Propionibacterium, preferably selected from the species Propionibacterium acnes, Propionibacterium vulgaris, Propionibacterium granulosum, Propionibacterium namnetense and / or Propionibacterium brachii.

[0400] Embodiment 53. The nucleic acid of embodiment 52, wherein the antigenic prokaryotic polypeptide is CAMP2.

[0401] Embodiment 54. The nucleic acid of embodiment 53, wherein the amino acid sequence encoding the 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 NO: 162-172.

[0402] Embodiment 55. The nucleic acid of embodiment 52, wherein the antigenic prokaryotic polypeptide is PITP.

[0403] Embodiment 56. The nucleic acid of embodiment 54, wherein the amino acid sequence encoding the PITP or 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: 150 or SEQ ID NOs: 173-183.

[0404] Embodiment 57. The nucleic acid of any one of embodiments 1-56, wherein the antigenic prokaryotic polypeptide comprises at least one mutated glycosylation site, preferably at least one mutated N-linked glycosylation site.

[0405] Embodiment 58. The nucleic acid of any one of embodiments 1-57, wherein the polynucleotide sequence of the nucleic acid is codon-optimized.

[0406] Embodiment 59. The nucleic acid of any one of embodiments 1-58, wherein the polynucleotide sequence of the ORF is codon-optimized.

[0407] Embodiment 60. The nucleic acid of any one of embodiments 1-59, wherein the polynucleotide sequence encoding the at least one viral secretory signal peptide is codon-optimized.

[0408] Embodiment 61. The nucleic acid of any one of Embodiments 20-60, wherein the polynucleotide sequence encoding the at least one TMB is codon optimized.

[0409] Embodiment 62. The nucleic acid of any one of embodiments 1-61, wherein the nucleic acid is DNA.

[0410] Embodiment 63. A nucleic acid according to any one of embodiments 1-61, wherein the nucleic acid is a messenger RNA (mRNA), wherein in particular, the mRNA can be a non-replicating mRNA, a self-replicating mRNA or a trans-replicating mRNA.

[0411] Embodiment 64. The nucleic acid of 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.

[0412] Embodiment 65. The nucleic acid of embodiment 63 or 64, wherein the mRNA comprises at least one chemical modification.

[0413] Embodiment 66. A nucleic acid according to any of embodiments 63-65, 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.

[0414] Embodiment 67. A nucleic acid according to any of embodiments 63-66, 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 ORF are chemically modified.

[0415] Embodiment 68. The nucleic acid of any one of embodiments 65-67, wherein the chemical modification is selected from pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine and 2'-O-methyluridine.

[0416] Embodiment 69. The nucleic acid of any one of embodiments 65-68, wherein the chemical modification is selected from pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0417] Embodiment 70. The nucleic acid of any one of Embodiments 65-69, wherein the chemical modification is N1-methylpseudouridine.

[0418] Embodiment 71. A composition comprising at least one nucleic acid according to any one of embodiments 1-70.

[0419] Embodiment 72. The composition of embodiment 71, further comprising lipid nanoparticles (LNP).

[0420] Embodiment 73. A composition according to Embodiment 72, wherein the nucleic acid is encapsulated in the LNP.

[0421] Embodiment 74. A composition according to embodiment 72 or 73, wherein the LNP comprises at least one cationic lipid.

[0422] Embodiment 75. The composition of Embodiment 74, wherein the cationic lipid is biodegradable.

[0423] Embodiment 76. The composition of Embodiment 74, wherein the cationic lipid is not biodegradable.

[0424] Embodiment 77. A composition according to any one of embodiments 74-76, wherein the cationic lipid is cleavable.

[0425] Embodiment 78. A composition according to any one of embodiments 74-76, wherein the cationic lipid is not cleavable.

[0426] Embodiment 79. A composition according to any one of embodiments 74-78, wherein the cationic lipid is selected from 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.

[0427] Embodiment 80. A composition according to any one of embodiments 74-79, wherein the LNP further comprises a polyethylene glycol (PEG) conjugated (PEGylated) lipid, a cholesterol-based lipid, and a helper lipid.

[0428] Embodiment 81. A composition according to any of embodiments 72-80, wherein the LNP comprises: - a cationic lipid in a molar ratio of 35% to 55%; - a polyethylene glycol (PEG)-conjugated (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 - an auxiliary lipid in a molar ratio of 5% to 35%, wherein all molar ratios are relative to the total lipid content of the LNP.

[0429] Embodiment 82. A composition according to any one of embodiments 72-81, wherein the LNP comprises: - a cationic lipid at a molar ratio of 40%; - a PEGylated lipid at a molar ratio of 1.5%; - a cholesterol-based lipid at a molar ratio of 28.5%; and - a helper lipid at a molar ratio of 30%.

[0430] Embodiment 83. A composition according to any one of embodiments 72-82, 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 - an auxiliary lipid in a molar ratio of 9% to 10%.

[0431] Embodiment 84. A composition according to any one of embodiments 80-83, wherein the PEGylated lipid is tetracosanoyl-PEG2000 (DMG-PEG2000) or 2-[(polyethylene glycol)-2000]-N,N-ditetradecanoylacetamide (ALC-0159).

[0432] Embodiment 85. The composition of any one of embodiments 80-84, wherein the cholesterol-based lipid is cholesterol.

[0433] Embodiment 86. A composition according to any one of embodiments 80-85, wherein the helper lipid is 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0434] Embodiment 87. A composition according to any one of embodiments 72-82 and 84-86, wherein the LNP comprises: - a cationic lipid selected from 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%.

[0435] Embodiment 88. A composition according to any one of embodiments 72-81 and 83-86, wherein 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%.

[0436] Embodiment 89. A composition according to any one of embodiments 72-81 and 83-86, wherein the LNP comprises: - 46.3% ALC-0315 at a molar ratio; - 1.6% ALC-0159 at a molar ratio; - 42.7% cholesterol at a molar ratio; and - 9.4% DSPC at a molar ratio.

[0437] Embodiment 90. A composition according to any one of embodiments 72-81 and 83-86, wherein the LNP comprises: - 47.4% ALC-0315 at a molar ratio; - 1.7% ALC-0159 at a molar ratio; - 40.9% cholesterol at a molar ratio; and - 10% DSPC at a molar ratio.

[0438] Embodiment 91. A composition according to any of Embodiments 72-90, wherein the average diameter of the LNPs is 30 nm to 200 nm.

[0439] Embodiment 92. The composition of any one of Embodiments 72-91, wherein the LNPs have an average diameter of 80 nm to 150 nm.

[0440] Embodiment 93. The composition of any one of Embodiments 72-92, comprising between 1 mg / mL and 10 mg / mL of the LNP.

[0441] Embodiment 94. A composition according to any one of embodiments 72-93, wherein the LNP comprises between 1 and 20 nucleic acid molecules, preferably mRNA molecules.

[0442] Embodiment 95. The composition of any one of Embodiments 71-94, formulated for intramuscular, intranasal, intravenous, subcutaneous, or intradermal administration.

[0443] Embodiment 96. The composition of any one of Embodiments 71-95, wherein the composition comprises phosphate buffered saline.

[0444] Embodiment 97. The composition of any one of Embodiments 71-96, wherein the composition is a pharmaceutical composition, such as an immunogenic composition or a vaccine, in particular an mRNA vaccine.

[0445] Embodiment 98. A nucleic acid according to any one of embodiments 1-70 or a composition according to any one of embodiments 71-97 for use in eliciting an immune response in a subject in need thereof.

[0446] Embodiment 99. A method of eliciting an immune response in a subject in need thereof, the method comprising administering to the subject an effective amount of a nucleic acid according to any one of embodiments 1-70 or a composition according to any one of embodiments 71-97, optionally intramuscularly, intranasally, intravenously, subcutaneously or intradermally.

[0447] Embodiment 100. Use of a nucleic acid according to any one of embodiments 1-70 or a composition according to any one of embodiments 71-97 for the manufacture of a medicament for eliciting an immune response in a subject in need thereof.

[0448] Embodiment 101. A nucleic acid according to any one of embodiments 1-70 or a composition according to any one of embodiments 71-97 for use in treating or preventing a prokaryotic infection in a subject in need thereof.

[0449] Embodiment 102. 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 according to any one of embodiments 1-70 or a composition according to any one of embodiments 71-97, optionally intramuscularly, intranasally, intravenously, subcutaneously, or intradermally.

[0450] Embodiment 103. Use of a nucleic acid according to any one of embodiments 1-70 or a composition according to any one of embodiments 71-97 for the manufacture of a medicament for treating or preventing a prokaryotic infection in a subject in need thereof.

[0451] Embodiment 104. A method for secreting an antigenic prokaryotic polypeptide in a host cell, the method comprising administering to the host cell a nucleic acid according to any one of embodiments 1-70 or a composition according to any one of embodiments 71-97.

[0452] Embodiment 105. A method of displaying an antigenic prokaryotic polypeptide on the surface of a host cell, the method comprising administering to the host a nucleic acid according to any one of embodiments 1-70 or a composition according to any one of embodiments 71-97.

[0453] Embodiment 106. A kit comprising a container comprising a single-use or multi-use dose of a nucleic acid according to any one of embodiments 1-70 or a composition according to any one of embodiments 71-97, optionally wherein the container is a vial or a pre-filled syringe.

[0454] In order to better understand the present disclosure, the following embodiments are described. These embodiments are only for illustrative purposes and should not be interpreted as limiting the scope of the present disclosure in any way. Example Example 1: Materials and Methods

[0286] mRNA production

[0455] As previously disclosed, mRNA was produced (Kalnin et al. (2021), NPJ Vaccines 6(1):61 and WO2021226436). In short, mRNA incorporating the coding sequence of OspA ST1 or ST2 was synthesized by in vitro transcription using an RNA polymerase using unmodified nucleotides with a plastid DNA template encoding the desired gene. As determined by gel electrophoresis, the resulting purified precursor mRNA was further reacted via enzymatic addition of a 5' cap structure (cap 1) of approximately 200 nucleotides in length and a 3' poly (A) tail.

[0456] For the preparation of mRNA / lipid nanoparticle (LNP) formulations, an ethanolic solution of a mixture of lipids (cationic / ionizable lipids, phosphatidylethanolamine, cholesterol, and polyethylene glycol-lipids) was combined with an aqueous buffer solution of the target mRNA under controlled conditions at an acidic pH at a fixed lipid and mRNA ratio to obtain a suspension of uniform LNPs. After ultrafiltration and diafiltration into a suitable diluent system, the resulting nanoparticle suspension was diluted to the final concentration, filtered, and stored frozen at -80°C until use.

[0457] Generation of antigens used as benchmark comparators

[0458] OspA-ferritin ST1 and ST2 antigens were produced by the Sanofi Breakthrough Lab in Cambridge, MA, USA according to previously published materials and methods (Kamp et al. (2020), NPJ Vaccines 5(1):33).

[0459] For OspA fusion ST1-ST2, the internal plasmid pSP401+LPP-chimera OspA ST1-OspA ST2 that allows to express OspA fusion ST1-ST2 C-terminal domain is introduced in Escherichia coli expression strain C43-(DE3) (Lucigen).After growing 2 to 3 hours at 37 ℃ in rich culture medium, by adding inducer induction target protein expression, and induction stops cultivation after 3 hours.After processing bacterial mass, on the SDS-Page gel of Coomassie blue staining or by the western blotting that uses specific antibody to carry out, protein visualization is made.Scale up to produce enough biomass required for purification under optimal expression conditions.Handle bacterial mass with lysozyme to extract membrane protein.Then extract OspA ST1-OspA ST2 fusion protein with urea 2M+TritonX114 2%.After carrying out three times cultivation-centrifugation steps at 37 ℃, collect lower layer phase and carry out Q agarose chromatography under the existence of Zwittergent 3.14 detergents (0.5%). Fractions eluted with 400 mM NaCl were subjected to ceramic hydroxyapatite chromatography. OspA ST1-OspA ST2 fusion protein was eluted with Tween 0,05% PO4 NaNa2 180 mM pH 6,7 buffer and replaced with PBS + Tween 20 0.05% pH 7.3 as final buffer.

[0460] Transfection of HEK cells

[0461] Suspended HEK293T cells (5 mL, 2 x 10 6 The mixture was added dropwise to the cells and incubated at 37°C, 100 rpm, 8% CO2 for 48 to 72 hours.

[0462] Western blot analysis of mRNA-transfected cells

[0463] After transfection, cells and culture medium were collected and centrifuged (500 x g) to collect supernatant. Lysozyme (Ready-Lyse Lysozyme Solution-Lucigen ref R1804M) + Benzonase (Sigma-ref E1014) + protease inhibitor cocktail (Sigma-ref P8340) was used to lyse the cell pellet at 800 rpm for 10 minutes at 20°C. The cell pellet lysate was then centrifuged (11,000 x g) to collect supernatant and crude extract.

[0464] The extract of HEK293T cell from mRNA transfection is analyzed by denaturation (95 ℃) PAGE and Western blotting using 4%-12% Bis-Tris / MES gel (Invitrogen).Use semi-dry transfer system (Trans-Blot turbine transfer system, Bio-Rad) to transfer to nitrocellulose membrane (Bio-Rad).Use polyclonal (rabbit) antibody (anti-OspA polyclonal / rabbit-Abcam, ref ab10608-1:2000) and secondary anti-(anti-rabbit IgG goat antibody DyLight800-Rockland, ref 611-145-002-1:2000) to detect the protein of trace with identification OspA.Use Odyssey infrared imager-LICOR to make trace imaging.

[0465] Characterization of mRNA OspAST1 or ST2 antigenicity by sandwich ELISA with functional monoclonal antibodies (mAbs)

[0466] Characterization of LA-2 with ST1-specific functional mAb

[0467] mAb 857-2 (R&D Biotech, in-house order) was coated at 2.5 μg / mL in PBS solution 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.

[0468] The transfection supernatant was diluted 2 times in dilution buffer (PBS-Tween 0.05%-milk 1%) and incubated for 1.5 hours at room temperature. After washing with PBS-0.05% Tween, the protein attached to the coating was detected by incubating with mAb LA-2 (AbsoluteAntibody, catalog number Ab01070-3.0-BT) at 1:1000 for 1.5 hours at room temperature, and then incubated with goat anti-mouse IgG HRP (Jackson Laboratories). After washing, the plate was developed using 3,3,5,5-tetramethylbenzidine (Tebu-bio, catalog number TMB100-1000) and terminated with 1N HCl (VWRProLabo). The optical density (OD) was read at 450nm-650nm.

[0469] Characterization with cross-specific functional mAbs 857-2 or 221-7

[0470] mAb 221-7 or 857-2 (Wang et al. J. Infect Dis. 214(2):205-211.2016) was coated at 5 μg / mL in PBS solution on a microtiter plate (Greiner Bio-one, catalog number 655061). The plate was incubated at 4°C overnight and then blocked with PBS-Tween 0.05%-milk 5% at room temperature for 1 hour.

[0471] The transfection supernatant is serially diluted 2 times in dilution buffer (PBS-Tween 0.05%-milk 1%), and is cultivated for 1.5 hours at room temperature.After PBS-0.05% Tween washing, by cultivating anti-OspA mouse polyclonal serum (inside) for 1.5 hours at room temperature, the detection of the protein attached to coating is carried out.The plate is washed and cultivated together with goat anti-mouse IgGHRP (Jackson Laboratories, catalog number (Cat. No.) 115-036-062) at room temperature for 1.5 hours.After washing, 3,3,5,5-tetramethylbenzidine (Tebu-bio, catalog number (Cat. No.) TMB100-1000) is used to make the plate develop the color for 30 minutes at room temperature in the dark.Use 1N HCl (VWR Prolabo, catalog number (Cat. No.) 30024290) to stop colorimetric reaction.Read optical density (OD) at 450nm-650nm.

[0472] Antigens and mouse immunization

[0473] OF-1 mice (Charles River) were randomly divided into immunization groups with eight animals each. Four different doses of mRNA-OspA-LNPs: 0.2 μg, 1 μg, 5 μg or 10 μg were administered intramuscularly (50 μL) at day 0 (D0) (dose 1) and day 21 (D21) (dose 2). Serum was collected at baseline (D0), on day 19 (D19) before dose 2, and on day 35 (D35).

[0474] 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(-). The mRNA sequence with TMB also contained HA SS, such as Figure 1 as shown in .

[0475] Comparing those mRNA formulations with a benchmark Lyme canine vaccine (Merial) 1μg / dose (50μL), recombinant fusion OspA ST1-ST2 (2μg / dose) + AlOOH adjuvant and OspA-ferritin (ST1 and ST2) 1.7μg / dose + AF03 adjuvant were compared.

[0476] OspA-specific IgG ELISA

[0477] Determine the antibody reaction in mice by ELISA.In brief, 384 hole microtiter plates (PerkinElmer#6007509) are coated with 1 μ g / mL OspA ST1-His diluted in PBS, and cultivate overnight at 4 ℃.Remove OspA ST1-His and use the 5% skim milk blocking plate dissolved in PBS-tween.After removing blocking reagent, add original serum sample after serial dilution 2 times in 1% skim milk-PBS-Tween.After cultivating 1.5 hours together with original serum sample at room temperature, plate is washed with PBS-Tween, and cultivate 1.5 hours together with goat anti-mouse IgG-HRP (Jackson115-036-062) at room temperature.Suction and wash secondary antibody, and plate is cultivated together with TMB substrate (TEBU-TMB100-1000), and cultivated together with isopyknic stop solution (HCl 1N) subsequently.Measure absorbance at 450nm-650nm. OspA specific IgG titers were quantified by an internal anti-OspA mouse serum reference. The titer of this reference was pre-calculated as the reciprocal of the dilution to obtain an OD of 1.

[0478] Statistical analysis

[0479] Two-way ANOVA was performed with vaccine dose and its interaction as factors (one model per time point). Intra-group heterogeneity was taken into account when necessary.

[0480] OspA amino acid and mRNA sequences

[0481] The OspA amino acid sequence and mRNA sequence used in the examples are described in the table below. Table 4 - Amino acid sequence of OspA protein Table 5 - mRNA sequence of OspA mRNA, including 5'UTR and 3'UTR

[0482] The following mRNA sequences are variants encoding the same viral secretion signal peptide MKAKLLVLLCTFTATYA (SEQ ID NO: 1):

[0483] The following mRNA sequences are variants encoding the same transmembrane domain ILAIYSTVASSLVLLVSLGAISF (SEQ ID NO: 17): Example 2: In vitro mRNA expression and antigenicity of antigenic prokaryotic polypeptides containing signal sequences

[0484] Borrelia protein outer surface protein A (OspA) was used as an exemplary antigenic prokaryotic polypeptide to test the effect of linking one or both of the hemagglutinin secretion signal (HA1 SS) and the HA transmembrane domain (TMB) to OspA. OspA serotype 1 (ST1) and serotype 2 (ST2) were used.

[0485] Design and express the mRNA of OspA ST1 or ST2.Use OspA sequence, design different mRNA sequences to guide OspA to express, secrete or cross membrane in cell, and described OspA sequence does not merge or merges with hemagglutinin secretion signal (HA1 SS) and / or HA transmembrane domain (TMB).HA1 SS causes OspA secretion, and TMB guides OspA to film, and OspA without HA1 SS or TMB is present in cell.

[0486] Prior to vaccination of mice, the expression and antigenicity of OspA ST1 and ST2 antigens delivered by mRNA were confirmed in vitro. The methods of transfection and expression of mRNA are described in Example 1 above.

[0487] like Figure 2A and Figure 2BShown in, realized the in vitro expression of OspA ST1 and ST2mRNA in HEK293T cell supernatant.Tested not having or having HA secretion signal and not having or having mRNA-OspAST1 and mRNA-OspA ST2 of glycosylation site mutation.Also used negative control (buffer) and positive control (recombinant OspA).With every kind of mRNA transfection HEK293T cell, and after 48 hours, collect supernatant and carry out western blotting.Detect protein of blot with polyclonal rabbit antibody identifying OspA.Result confirms that adding secretion signal increases the extracellular expression of OspA.The sudden change of glycosylation site effectively avoids the glycosylation of OspA, wherein there is single point of Gly (-) to replace multiple points of natural (glycosylation) protein.

[0488] like Figure 3A-3C As shown in, the in vitro expression of mRNA-TMB-OspA ST1 in HEK293T cells was achieved. The OspA ST1 expression of cell supernatant, crude extract and intracellular compartment was tested. After 48-72 hours, supernatant and cells were collected and western blotted. The protein of blot was detected with the polyclonal rabbit antibody identifying OspA. The result confirmed that the addition of transmembrane domain induced OspA ST1 to be positioned at the cell membrane, and reduced secretion and intracellular localization. The mutation of glycosylation site effectively avoided the glycosylation of OspA ST1.

[0489] like Figure 4A-4C Shown in, realized in HEK293T cell mRNA-TMB-OspA ST2 in vitro expression.Tested the OspA ST2 expression of cell supernatant, crude extract and intracellular compartment.After 48-72 hours, collect supernatant and cell and carry out western blotting.Detect protein of blot with polyclonal rabbit antibody identifying OspA.The same as OspA ST1, result confirms, add transmembrane domain and induce OspA ST2 to be positioned at cell membrane, and reduce secretion and intracellular localization.The sudden change of glycosylation site effectively avoids the glycosylation of OspA ST2.

[0490] like Figure 5 The antigenicity of OspA ST1 antigen delivered by mRNA in HEK293T cells is shown in . The transfected cell supernatants were used to perform sandwich ELISA with functional monoclonal antibodies LA-2, 857-2 and 221-7.

[0491] mAb LA-2 targets only the C-terminus of OspA ST1 and has been shown to be associated with protection in clinical studies (Van Hoecke, supra; Steere, supra; Embers, supra). Based on the borreliocidal activity and protection against tick-mediated Borrelia burgdorferi transmission in mice, Mabs 221-7 and 857-2 were selected as lead candidates, as shown in Wang et al., supra.

[0492] Secreted OspA ST1 produced from mRNA (HA-SS-OspA ST1) was correctly recognized by three functional mAbs, indicating that the antigen was in the correct conformation after in vitro expression in human cells (HEK293T).

[0493] Mutation of the glycosylation site inhibited the binding of mAb LA-2, probably due to the induction of a misaligned conformation in the C-terminal epitope, which was not observed with the 221-7 and 857-2 antibodies.

[0494] like Figure 6 The antigenicity of OspA ST2 antigen delivered by mRNA in HEK293T cells is shown in Figure 2. Transfected cell supernatants were used to perform sandwich ELISA with functional monoclonal antibodies 857-2 and 221-7. mAb LA-2 was not used with OspA ST2 because it only recognizes OspA ST1.

[0495] Secreted OspA ST2 produced from mRNA (HA-SS-OspA ST2) was correctly recognized by functional mAbs 221-7 and 857-2, indicating that the antigen was in the correct conformation after in vitro expression in human cells. Mutation of the glycosylation site improved the binding of both mAbs, probably due to reduced masking of the epitope by glycosylation. Example 3: Immunogenicity of mRNA encoding OspA protein in mice

[0496] The relative immunogenicity of various mRNAs expressing OspA in mice was tested by measuring the IgG titer against OspA, as described above in Example 1. Each mRNA was encapsulated into a LNP consisting of 40% cationic lipid cKK-E10, 30% phospholipid DOPE, 1.5% PEGylated lipid DMGPEG2000 and 28.5% cholesterol. Alternatively, the LNP lipids can be described by a ratio wherein cationic lipid: PEGylated lipid: cholesterol: phospholipid is 40:1.5:28.5:30.

[0497] Each LNP-mRNA composition was administered to mice at a dose of 0.2 μg, 1 μg, 5 μg or 10 μg. A total of 4 groups of 8 mice were used.

[0498] Three benchmark compositions were used: OspA fusion with AlOOH adjuvant ("OspA fusion ST1-ST2") (2 μg (1 μg per serotype) / dose); Lyme canine vaccine (Merial) (1 μg dose); and OspA-ferritin fusion (ST1 or ST2) with AF03 adjuvant (1.7 μg (wherein, 1 μg OspA + 0.7 μg ferritin) / dose). OspA-ferritin fusions are further described in US20210017238A1, which is incorporated herein by reference.

[0499] like Fig. 7A As shown in , anti-OspA ST1 IgG titers increased after the first dose (day 19). A trend of increasing IgG titers with HA-SS was observed. In addition, the addition of the TMB domain significantly increased IgG titers.

[0500] like Figure 7B As shown in , anti-OspA ST1 IgG titers further increased after the second dose (day 35). As can be seen in Table 6, the addition of HA-SS and / or TMB domains significantly improved immunogenicity (p<0.05).

[0501] Table 6 - Statistical analysis of all dose combinations after the second dose (day 35) Comparison Group multiple P-value SS-natural=SS-gly(-) - P>0.05 SS-Natural = TMB-Natural - P>0.05 natural <SS-gly(-) X7.9 0.0102 Natural <SS-Natural X8 0.0088 natural <TMB-gly(-) X66.5 P<0.001 Natural <TMB-Natural X19.8 P<0.001 SS-gly(-)<TMB-gly(-) X8.4 P<0.001 SS-Natural <TMB-gly(-) X8.3 P<0.001 TMB-Natural <TMB-gly(-) X3.4 0.0454

[0502] Conclusions from the mouse study:

[0503] The mRNA of coding OspA ST1 with one or two in HA-SS or TMB has immunogenicity, and in mice, all induces strong anti-OspA IgG titer after the 1st dose and after the 2nd dose.Find that compared with the mRNA OspA without any one in HA-SS or TMB, adding secretion signal (HA-SS) or TMB structural domain significantly improves immunogenicity (p<0.05).Observe dosage effect (that is, along with dosage increase, IgG titer increases). Example 4: Expanded mRNA Design Kit

[0504] In Example 3, it was confirmed that adding hemagglutinin secretion signal (HA-SS) and / or HA transmembrane domain (HA-TMB) enhanced the immunogenicity of OspA ST1 target antigen. Based on these results, a more comprehensive mRNA construct set was designed, which was connected to different target antigens with secretion signal (SS) and transmembrane domain (TMB) of the glycoprotein from various virus families. The schematic diagram of the assembly included in this extended mRNA set is provided in Figure 8In. The set consists of three different prokaryotic antigens: OspA ST1, CAMP2 and PITP (the latter two are derived from Acetobacter acnes). In addition, the mRNA sequences encoding these antigens are engineered to guide the antigens to be localized in the cell, secreted outside the cell, or exposed to the cell membrane. This is achieved by fusing the antigen sequence with or without SS and with or without TMB, and the SS and TMB are derived from glycoproteins of different virus families, such as influenza virus (type A or type B subtype), rabies virus, varicella virus (VZV) or Ebola virus.

[0505] Fifty-seven different constructs were analyzed in silico using SignalP 6.0 to determine the strength of their signal peptides. SignalP is an algorithm for signal sequence prediction and is further described in 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 intensity is evaluated based on a cumulative rank score that takes into account the likelihood of the canonical features of the detection signal sequence (SS likelihood score, also referred to as SP likelihood score) and the probability of cutting at the cleavage site (cutting probability score). The sequences used for this analysis are shown in Table 7, and the results of this analysis are presented in Table 8 below. Table 7 - Sequences analyzed in SignalP 6.0 Table 8 - Results from SignalP 6.0 analysis of all test sequences presented in Table 7

[0506] In addition, the signal peptide and its cleavage site are not as conservative as the mature region of the protein (Nielsen et al. (2019), The Protein Journal, 38: 200-216). Therefore, in order to understand the differences in 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 virus (H1N1 and H3N2 subtypes) and influenza B virus (Victoria and Yamagata spectrum), and the resulting consensus hemagglutinin signal sequence is shown in Table 2.

[0507] Based on these computer results, the set was narrowed down to constructs selected for downstream testing. The final combination of selected signal sequences and antigens is shown in Tables 8 and 9 along with the corresponding SignalP 6.0 scores for each construct. The amino acid sequences corresponding to this mRNA set design are shown in Table 7. In Table 7, the construct identification name provides information about the antigen, as well as a brief representation of the viral glycoprotein signal sequence, and an indication of whether a transmembrane domain is incorporated (denoted as "TMB").

[0508] Subsequently, mRNA was produced from the constructs shown in Table 7. The mRNA production method was the same as described in Example 1. The parameters of the mRNA produced from the constructs in this set were determined, including the efficiency of the capping reaction and the length of the poly(A) tail. All mRNAs were fully capped and poly-A-tailed. Table 9 - Results of SignalP 6.0 analysis of selected signal sequences Table 10 - Amino acid sequence design of the extended mRNA design set Example 5: In vitro antigen expression from an expanded mRNA design panel

[0509] This example outlines the analysis of cell viability, protein expression and localization following transfection of the mRNA kit (described in Example 4) into HEK293T cells. Transfection and Western blot analysis were previously described in Example 1. The antigens in the Western blots analyzed were OspA ST1, CAMP2 and PITP.

[0510] HEK Expi293F cell counts and cell viability percentages were measured after transfection for two expression tests with a panel of mRNA constructs. All cell viability values ​​were above 80% at 24 and 48 hours post-transfection, indicating that conditions were normal and that no mRNA constructs produced off-target cytotoxicity. OspA

[0511] like Fig. 9 Shown in, after transfection 48 hours, realized the in vitro expression of the OspA ST1 fused with the signal sequence (labeled as " SS " on gel) deriving from influenza A virus, influenza B virus, rabies virus, VZV and Ebola virus glycoprotein in HEK Expi293F cell, described OspA ST1 has or does not have their corresponding transmembrane domain (" TMB ").Use 1:2000 dilution for the rabbit polyclonal antibody (ABCAM ab106081) of OspA to detect OspA ST1 (expected size about 28-34kDa).Control comprises the OspA ST1 construct (first swimming lane on all gels, labeled as " without SS ") and restructuring OspA ST1 (last swimming lane on all gels) without any SS or any lipidation sequence.About testing OspA location, collect sample from crude extract (total lysate), cell supernatant and the cell sample containing the fractionation of intracellular compartment or transmembrane compartment. These results demonstrate that the addition of the transmembrane domain induces the localization of OspA ST1 at the cell membrane and reduces secretion and intracellular localization.

[0512] In addition, due to Fig. 9 Representative gels of supernatant fractions showed faint signals (last column in the figure), the supernatant OspA fractions were concentrated 7-fold, and half of the resulting volume was deglycosylated (indicated by "D" on the gel) to analyze proteins by Western blotting before and after enzymatic treatment, as shown in Figure 1. Fig.10 As shown in . For all OspA constructs tested, OspA ST1 was present in the supernatant fraction, but the expression levels were different. For example, the OspA ST1 construct fused to the Ebola virus glycoprotein SS (which also had a lower SignalP cleavage score than the other constructs) had reduced expression in the supernatant fraction relative to the other OspA constructs (see Fig.10 , second gel, lanes 5 and 6). Additionally, fusing the construct to the transmembrane domain reduced (but did not completely eliminate) supernatant detection of OspA in some samples. CAMP2

[0513] like Fig.11As shown in, in vitro expression of CAMP2 fused to signal sequences (marked as "SS" on the gel) derived from influenza A, influenza B, rabies, VZV and Ebola virus glycoproteins was achieved in HEK Expi293F cells 48 hours after transfection, with or without their corresponding transmembrane domains ("TMB"). CAMP2 (expected size of approximately 26-32 kDa) was detected using a rabbit polyclonal antibody (generated in-house) against CAMP2 at a dilution of 1:1500. Controls included a CAMP2 construct without any SS (the first lane on all gels, marked as "no SS") and recombinant CAMP2 (the last lane on all gels). For testing CAMP2 localization, samples were collected from crude extracts (total lysates), cell supernatants, and fractionated cell samples containing intracellular compartments or transmembrane compartments. These results confirm that the addition of the transmembrane domain induces CAMP2 localization at the cell membrane and reduces secretion and intracellular localization (compared to constructs with or without the transmembrane domain, labeled "supernatant" in the last column). Western blot analysis confirmed that CAMP2 was well expressed and localized in the expected fractions depending on the presence of the secretory signal peptide or transmembrane domain. PITP

[0514] like Fig.12 As shown in, in HEK Expi293F cells, 48 ​​hours after transfection, the in vitro expression of PITP fused with the signal sequence (marked as "SS" on the gel) derived from influenza A, influenza B, rabies, VZV and Ebola virus glycoproteins was achieved, with or without their corresponding transmembrane domains ("TMB"). PITP (expected size of about 42-48 kDa) was detected using a mouse polyclonal antibody (produced in-house) against PITP at a dilution of 1:1000. Controls included PITP constructs without any SS or any TMB (the first lane on all gels, marked as "no SS") and recombinant PITP (the last lane on all gels). Samples were collected from crude extracts (total lysates), cell supernatants, and fractionated cell samples containing intracellular compartments or transmembrane compartments. Like OspA and CAMP2 antigen localization analysis, these results confirm that adding transmembrane domain induces PITP localization at cell membrane and reduces secretion and intracellular localization (compared with constructs with or without transmembrane domain, marked as "supernatant" in the last column). Nevertheless, the construct containing PITP transmembrane does show some escape into the supernatant fraction.

[0515] This Western blot analysis confirmed that PITP was well expressed and localized in the expected fractions based on the presence of a secretion signal peptide or transmembrane domain. Summarize

[0516] To compare the Western blot analysis of OspA ST1, CAMP2, and PITP, the protein expression and localization results were tabulated as Fig.13 In the embodiment of the present invention, the construct of OspA ST1 antigen is expressed in the expression of the protein of the present invention.Usually, the construct containing OspA ST1 antigen is expressed poorly compared with the construct containing CAMP2 or PITP.All three protein antigens are expressed at their expected position, but the expression degree is different, and when introducing the transmembrane domain, the display has some escapes to the supernatant to different degrees.The construct OspA ST1_SS-Ebola virus-GP with low SignalP cutting score shows lower expression in the supernatant fraction, indicating that it may accumulate in the intracellular fraction.

[0517] Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice disclosed herein. It is intended that the specification and examples be considered exemplary only, with the following claims indicating the true scope and spirit of the disclosure.

[0518] All patents and publications cited herein are 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 of 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 secretory signal peptide is derived from a viral sequence selected from the group consisting of an influenza virus secretory signal peptide sequence, and a non-influenza virus secretory signal peptide sequence selected from the group consisting of a SARSCoV-2 secretory signal peptide sequence, a varicella-zoster virus (VZV) secretory signal peptide sequence, a measles virus secretory signal peptide sequence, a rubella virus secretory signal peptide sequence, a mumps virus secretory signal peptide sequence, an Ebola virus secretory signal peptide sequence, a smallpox virus secretory signal peptide sequence, and a rabies virus secretory signal peptide sequence.

5. The nucleic acid according to any one of claims 1 to 4, wherein the viral secretion signal peptide is selected from: influenza virus hemagglutinin (HA) secretion signal peptide sequence, SARS CoV-2 spike secretion signal peptide sequence, VZV gB secretion signal peptide sequence, VZVgE secretion signal peptide sequence, VZV gI secretion signal peptide sequence, VZV gK secretion signal peptide sequence, measles virus F protein secretion signal peptide sequence, rubella virus E1 protein secretion signal peptide sequence, rubella virus E2 protein secretion signal peptide sequence, mumps virus F protein secretion signal peptide sequence, Ebola virus GP protein secretion signal peptide sequence, smallpox virus 6kDa IC protein secretion signal peptide sequence and rabies virus G protein secretion signal peptide sequence, preferably wherein the viral secretion signal peptide comprises an HA secretion signal peptide sequence from influenza A virus or influenza B virus, more preferably from influenza A virus.

6. The nucleic acid of claim 5, wherein the HA secretion signal peptide sequence comprises the amino acid sequence MKX1X2LX3VX4LX5TFX6X7X8X9A (SEQ ID NO: 145), wherein X1 is selected from A and V; X2 is selected from I and K; X3 is selected from V and L; X4 is selected from L and M; X5 is selected from Y and C; X6 is selected from T and A; X7 is selected from T and A; X8 is selected from A and T; and X9 is selected from N and Y.

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

8. The nucleic acid of claim 5, wherein the HA secretion signal peptide sequence comprises the amino acid sequence MKX1IIALSX2ILCLVFX3 (SEQ ID NO: 146), wherein X1 is selected from T and A; X2 is selected from Y, N, C and H; and X3 is selected from T and A.

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

10. The nucleic acid of claim 5, wherein the HA secretion signal peptide sequence comprises the amino acid sequence MKAIIVLLMVVTSX1A (SEQ ID NO: 147), wherein X1 is selected from S and N.

11. The nucleic acid of claim 5, wherein the HA secretion signal peptide sequence comprises the amino acid sequence MX1AIIVLLMVVTSNA (SEQ ID NO: 148), wherein X1 is selected from K and E.

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

13. The nucleic acid according to any one of claims 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); MSPCGYYSKWRNRDRPEYRRNLRFRRFFSSIHPNAAAGSGFNGPGVFITSVTGV WLCFLCIFSMFVTAVVS(SEQ ID NO:7); MGTVNKPVVGVLMGFGIITGTLRITNPVRA (SEQ ID NO: 8); MFLIQCLISAVIFYIQVTNA(SEQ ID NO:9); MQALGIKTEHFIIMCLLSGHA(SEQ ID NO:10); MGLKVNVSAIFMAVLLTLQTPTG(SEQ ID NO:11); MGAAALTAVVLQGYNPPAYG(SEQ ID NO:12); MGAPQAFLAGLLLAAVAVGTARA (SEQ ID NO: 13); MKVFLVTCLGFAVFSSSSVC(SEQ ID NO:14); MGVTGILQLPRDRFKRTSFFLWVIILFQRTFS(SEQ ID NO:15); MRSLIIFLLFPSIIYS (SEQ ID NO: 16); and MVPQALLFVPLLVFPLCFG (SEQ ID NO: 184).

14. The nucleic acid of 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 antigenic 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 antigenic prokaryotic polypeptide.

17. The nucleic acid of any one of claims 1-16, wherein the viral secretion signal peptide is attached to the antigenic prokaryotic polypeptide with a linker.

18. The nucleic acid of any one of claims 2-17, 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% hydrophobic amino acid residues, preferably selected from the group consisting of alanine, isoleucine, leucine, tyrosine, phenylalanine, tryptophan and tyrosine; and / or (c) contains at least one alpha helix.

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 membrane protein, more preferably from a diperentiate, even more preferably from a type I diperentiate.

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

21. The nucleic acid of any one of claims 18-20, wherein the antigenic prokaryotic polypeptide is derived from a prokaryotic transmembrane protein, and wherein the TMB is the TMB of the prokaryotic transmembrane protein.

22. The nucleic acid of any one of claims 18-20, wherein the antigenic prokaryotic polypeptide is not derived from a prokaryotic transmembrane protein.

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

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

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

26. The nucleic acid of any one of claims 18-20 and 22-25, wherein the TMB comprises an amino acid sequence selected from the group consisting of: ILAIYSTVASSLVLLVSLGAISF(SEQ ID NO:17); ILAIYSTVASSLVLVVSLGAISF(SEQ ID NO:18); ILWISFAAISCFLLCVVLLGFI (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); IVYILIAVCLGLGLIGIPALIC(SEQ ID NO:26); LDHAFAAFVLLVPWVLIFMVC(SEQ ID NO:27); WWQLTLGAICALLLAGLLACC(SEQ ID NO:28); IVALVLSILSIIISLLFCCW (SEQ ID NO: 29); WIPAGIGVTGVIIAVIALFCI (SEQ ID NO: 30); and VLLSAGALTALMLIIFLMTCW (SEQ ID NO: 185).

27. The nucleic acid of claim 16, wherein the TMB comprises the amino acid sequence of ILAIYSTVASSLVLLVSLGAISF (SEQ ID NO: 17).

28. The nucleic acid of any one of claims 2 and 18-27, wherein the TMB is attached to the antigenic prokaryotic polypeptide with a linker.

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

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

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

32. 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 transmembrane domain (TMB).

33. The nucleic acid of claim 32, 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% hydrophobic amino acid residues, preferably selected from the group consisting of alanine, isoleucine, leucine, tyrosine, phenylalanine, tryptophan and tyrosine; and / or (c) contains at least one alpha helix.

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 diperentiate, even more preferably from a type I diperentiate.

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

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

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

38. The nucleic acid of any one of claims 33-37, wherein the TMB is derived from a viral transmembrane domain sequence selected from the group consisting of an influenza virus transmembrane domain sequence, and a non-influenza virus transmembrane domain sequence selected from the group consisting of a SARS CoV-2 transmembrane domain sequence, a varicella-zoster virus (VZV) transmembrane domain sequence, a measles virus transmembrane domain sequence, a rubella virus transmembrane domain sequence, a mumps virus transmembrane domain sequence, an Ebola virus transmembrane domain sequence, and a rabies virus transmembrane domain sequence.

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

40. The nucleic acid of claim 39, wherein the TMB comprises an amino acid sequence selected from the group consisting of: ILAIYSTVASSLVLLVSLGAISF(SEQ ID NO:17); ILAIYSTVASSLVLVVSLGAISF(SEQ ID NO:18); ILWISFAAISCFLLCVVLLGFI (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); IVYILIAVCLGLGLIGIPALIC(SEQ ID NO:26); LDHAFAAFVLLVPWVLIFMVC(SEQ ID NO:27); WWQLTLGAICALLLAGLLACC(SEQ ID NO:28); IVALVLSILSIIISLLFCCW (SEQ ID NO: 29); WIPAGIGVTGVIIAVIALFCI (SEQ ID NO: 30); and VLLSAGALTALMLIIFLMTCW (SEQ ID NO: 185).

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

42. The nucleic acid of any one of claims 32-41, wherein the TMB is attached to the antigenic prokaryotic polypeptide with a linker.

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

44. The nucleic acid of any one of claims 32-42, wherein the TMB is located at the C-terminus of the antigenic prokaryotic polypeptide.

45. The nucleic acid of any one of claims 32-44, wherein the antigenic prokaryotic polypeptide is derived from a bacterium of the genus selected from the group consisting of Acetobacter, Acinetobacter, Actinomyces, Aerococcus, Agrobacterium, Anaplasma, Azorhizobia, Azotobacter, Bacillus, Bacteroides, Bartonella ), Bordetella, Borrelia, Brucella, Burkkolderia, Calymmatobacterium, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Coxiella, Cutibacterium, Ehrlichia ichia), Enterobacter, Enterococcus, Escherichia, Francisella, Fusobacterium, Gardnerella, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Legionella, Listeria teria), Methanobacterium, Microbacterium, Micrococcus, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Pasteurella, Pediococcus, Peptostreptococcus, Porphyromonas, Prevotella,Propionibacterium, Pseudomonas, Rhizobium, Rickettsia, Rochalimaea, Rothia, Salmonella, Serratia, Shigella, Sarcina, Spirillum, Spirochaetes, Staphylococcus The present invention relates to bacteria of the genera Staphylococcus, Stenotrophomonas, Streptobacillus, Streptococcus, Tetragenococcus, Treponema, Vibrio, Viridans, Walbachia and Yersinia, preferably bacteria from the species selected from the group consisting of Acetobacter aurantifolia, 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, 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, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Corynebacterium fusiforme, Coxiella burnetii, Cutibacterium acnes, Cutibacterium avidum, Cutibacterium granulosum 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 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 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 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, 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 mutans), Streptococcus oralis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus rattus, Streptococcus salivarius, Streptococcus sanguis, Streptococcus sobrinus, Treponema pallidum, Treponema denticola, Vibriocholerae, Vibrio comma, Vibrio parahaemolyticus, Vibrio vulnificus, Viridans streptococci, Wolbachia, Yersinia enterocolitica enterocolitica), Yersinia pestis, and Yersinia pseudotuberculosis.

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

47. according to the nucleic acid described in any one among the claim 1-46, wherein said antigenic prokaryotic polypeptide is OspA or its fragment or variant, wherein said OspA or its fragment or variant comprise at least 5 amino acids, preferably wherein said antigenic prokaryotic polypeptide comprises: (a) an amino acid sequence derived from OspA ST1, which preferably has at least 85% identity with the following sequence: (b) an amino acid sequence derived from OspA ST2, which preferably has at least 85% identity with the following sequence: (c) an amino acid sequence derived from OspA ST3, which preferably has at least 85% identity with the following sequence: (d) an amino acid sequence derived from OspA ST4, which preferably has at least 85% identity with the following sequence: (e) an amino acid sequence derived from OspA ST5, which preferably has at least 85% identity with the following sequence: (f) an amino acid sequence derived from OspA ST6, which preferably has at least 85% identity with the following sequence: DTITVQRYDSAGTNLEGKAVEITTLKELKNALK(SEQ ID NO:189); (g) an amino acid sequence derived from OspA ST7, which preferably has at least 85% identity with the following sequence: KQNVSSLDEKNSVSVDLPGEMKVLVSKEKDKDGKYSLEATVDKLELKGTSDKNN GSGVLEGVKAAKSKAKLTIADDLSQTKFEIFKEDGKTLVSKKVTLKDKSSTEEKFNDKGKLSEKVVTRANGTRLEYTEIQNDGSGKAKEVLKSLTLEGTLTAD GETKLTVEAGTVTLSKNISESGEITVELKDTETTPADKKSGTWDSKTSTLTISKNSQKTKQLVFTKENTITVQKYNTAGTKLEGSPAEIKDLEALKAALK(SEQ ID NO:190); (h) Any combination of (a) to (g); (i) a sequence derived from OspA ST1 according to (a) and a sequence derived from OspA ST2 according to (b); or (j) a sequence derived from OspA ST1 according to (a), a sequence derived from OspA ST2 according to (b), a sequence derived from OspA ST3 according to (c), a sequence derived from OspA ST4 according to (d), a sequence derived from OspA ST5 according to (e), a sequence derived from OspA ST6 according to (f), and a sequence derived from OspA ST7 according to (g).

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

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

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

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

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

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

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

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

57. A nucleic acid according to any one of claims 54-56, 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 of any one of claims 54-57, 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 of any one of claims 56-58, wherein the chemical modification is selected from pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine and 2'-O-methyluridine.

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

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

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

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

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

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

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

67. The composition of claim 65, wherein the cationic lipid is not biodegradable.

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

69. The composition of any one of claims 65-67, wherein the cationic lipid is not cleavable.

70. A composition according to any one of claims 65-69, wherein the cationic lipid is selected from 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 of any one of claims 65-70, wherein the LNP further comprises a polyethylene glycol (PEG)-conjugated (PEGylated) lipid, a cholesterol-based lipid, and a helper lipid.

72. The composition of any one of claims 63-71, wherein the LNP comprises: - 35% to 55% cationic lipids in a molar ratio; - 0.25% to 2.75% polyethylene glycol (PEG) conjugated (PEGylated) lipids in a molar ratio; - 20% to 45% by molar ratio of cholesterol-based lipids; and - helper lipids in a molar ratio of 5% to 35%, All molar ratios are relative to the total lipid content of the LNP.

73. The composition of any one of claims 63-72, wherein the LNP comprises: - 40% cationic lipid in a molar ratio; - 1.5% molar ratio of PEGylated lipids; - 28.5% by molar ratio of cholesterol-based lipids; and - 30% helper lipids in a molar ratio.

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

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

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

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

78. The composition of any one of claims 63-73 and 75-77, wherein the LNP comprises: - a cationic lipid selected from 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%; - 1.5% molar ratio of DMG-PEG2000; - 28.5% by molar ratio of cholesterol; and - 30% DOPE by molar ratio.

79. The composition of any one of claims 63-72 and 74-77, wherein the LNP comprises: - SM-102 at a molar ratio of 50%; - 1.5% molar ratio of DMG-PEG2000; - 38.5% by molar ratio of cholesterol; and - 10% DSPC by mole.

80. The composition of any one of claims 63-72 and 74-77, wherein the LNP comprises: - 46.3% molar ratio of ALC-0315; - 1.6% by molar ratio of ALC-0159; - 42.7% by molar ratio of cholesterol; and - 9.4% DSPC by mole.

81. The composition of any one of claims 63-72 and 74-77, wherein the LNP comprises: - 47.4% by molar ratio of ALC-0315; - 1.7% by molar ratio of ALC-0159; - 40.9% by molar ratio of cholesterol; and - 10% DSPC by mole.

82. A composition according to any one of claims 63-81, wherein the average diameter of the LNPs is 30nm to 200nm.

83. A composition according to any one of claims 63-82, wherein the average diameter of the LNPs is 80nm to 150nm.

84. The composition of any one of claims 63-83, comprising between 1 mg / mL and 10 mg / mL of the LNP.

85. A composition according to any one of claims 63-84, wherein the LNP comprises between 1 and 20 nucleic acid molecules, preferably mRNA molecules.

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

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

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

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

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

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

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

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

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