EP4694918A1 - Impfstoffe gegen lyme-krankheit - Google Patents

Impfstoffe gegen lyme-krankheit

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Publication number
EP4694918A1
EP4694918A1 EP24724044.3A EP24724044A EP4694918A1 EP 4694918 A1 EP4694918 A1 EP 4694918A1 EP 24724044 A EP24724044 A EP 24724044A EP 4694918 A1 EP4694918 A1 EP 4694918A1
Authority
EP
European Patent Office
Prior art keywords
mrna
seq
protein
ospa
acid sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24724044.3A
Other languages
English (en)
French (fr)
Inventor
Obadiah PLANTE
Sunny HIMANSU
Guillaume Stewart-Jones
Gwo-yu CHUANG
Meredith FINN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
ModernaTx Inc
Original Assignee
ModernaTx Inc
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Filing date
Publication date
Application filed by ModernaTx Inc filed Critical ModernaTx Inc
Publication of EP4694918A1 publication Critical patent/EP4694918A1/de
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/0225—Spirochetes, e.g. Treponema, Leptospira, Borrelia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Lyme disease is primarily caused by Borrelia afzelii, Borrelia garinii, and/or Borrelia bavariensis. Symptoms of Lyme disease include fever, headache, body aches fatigue, and a skin rash called erythema migrans. Left untreated, the infection can develop into a chronic illness that spreads to joints, the heart, and the nervous system, leading to long-term and serious health problems, such as Bell’s palsy and liver inflammation. There were two Lyme disease vaccines available in the United States, LYMERIX ® and IMULYME ® , but both were withdrawn from the market years ago due to low demand and concerns about possible side effects.
  • mRNA vaccines for the prevention of Lyme disease caused by Borrelia infection.
  • Some aspects provide a messenger ribonucleic acid (mRNA) vaccine, comprising: an mRNA polynucleotide comprising an open reading frame encoding a protein comprising a Borrelia outer surface protein A (OspA) extracellular domain and optionally a heterologous amino terminal transmembrane domain; and a lipid nanoparticle.
  • mRNA messenger ribonucleic acid
  • an mRNA vaccine comprises an mRNA polynucleotide comprising: an open reading frame encoding a nonlipidated Borrelia OspA protein; and a lipid nanoparticle.
  • the mRNA vaccine comprises multiple mRNA polynucleotides each comprising an open reading frame encoding a different nonlipidated Borrelia OspA protein.
  • the mRNA vaccine comprises seven different open reading frames encoding seven different nonlipidated Borrelia OspA proteins.
  • the Borrelia OspA protein(s) comprises a heterologous amino terminal transmembrane domain.
  • the Borrelia OspA protein(s) comprises a heterologous carboxyl terminal transmembrane domain.
  • the heterologous amino terminal transmembrane domain is from an influenza virus neuraminidase protein.
  • the Borrelia OspA protein(s) comprises one or more modified N glycosylation sites, relative to a corresponding naturally occurring Borrelia OspA protein.
  • the Borrelia OspA protein comprises a Borrelia burgdorferi OspA serotype 1 (S1, also referred to herein as SR1) extracellular domain.
  • the Borrelia OspA S1 protein comprises a human leukocyte function-associated antigen (hLFA-1) epitope that is modified or removed relative to a corresponding naturally occurring Borrelia OspA S1 protein.
  • the hLFA-1 epitope of the Borrelia OspA S1 protein comprises the following mutations Y165F, V166I, T170R, and L171F, relative to a naturally occurring Borrelia OspA S1 protein comprising the amino acid sequence of SEQ ID NO: 24.
  • mRNA vaccine further comprises one or more polynucleotides mRNA polynucleotide comprising an open reading frame encoding a nonlipidated Borrelia OspA protein extracellular domain selected from the group consisting of: a Borrelia afzelii OspA serotype 2 (S2, also referred to herein as SR2) extracellular domain, a Borrelia garinii OspA serotype 3 (S3, also referred to herein as SR3) extracellular domain, a Borrelia bavariensis OspA serotype 4 (S4, also referred to herein as SR4) extracellular domain, a Borrelia garinii OspA serotype 5 (S5, also referred to herein as SR5) extracellular domain, a Borrelia garinii OspA serotype 6 (S6, also referred to herein as SR6) extracellular domain, and a Borrelia garinii OspA serotype 7
  • the mRNA vaccine further comprises two, three, four, five or six additional mRNAs, each comprising an open reading frame encoding a nonlipidated Borrelia OspA protein comprising a different Borrelia OspA extracellular domain selected from a Borrelia afzelii OspA S2 extracellular domain, a Borrelia garinii OspA S3 extracellular domain, a Borrelia bavariensis OspA S4 extracellular domain, a Borrelia garinii OspA S5 extracellular domain, a Borrelia garinii S6 protein, and a Borrelia garinii OspA S7 extracellular domain, optionally wherein each of the nonlipidated Borrelia OspA proteins encoded by the additional mRNAs comprises a heterologous amino terminal transmembrane domain and/or N-linked glycan site mutations.
  • the mRNA vaccine further comprises a second mRNA polynucleotide comprising an open reading frame encoding a nonlipidated Borrelia OspA protein comprising a Borrelia afzelii OspA S2 extracellular domain, optionally wherein the nonlipidated Borrelia OspA protein encoded by the second mRNA polynucleotide comprises a heterologous amino terminal transmembrane domain and/or N-linked glycan site mutations.
  • the mRNA vaccine further comprises a third mRNA polynucleotide comprising an open reading frame encoding a nonlipidated Borrelia OspA protein comprising a Borrelia garinii OspA S3 extracellular domain, optionally wherein the nonlipidated Borrelia OspA protein encoded by the third mRNA polynucleotide comprises a heterologous amino terminal transmembrane domain and/or N-linked glycan site mutations.
  • the mRNA vaccine further comprises a fourth mRNA polynucleotide comprising an open reading frame encoding a nonlipidated Borrelia OspA protein comprising a Borrelia bavariensis OspA S4 extracellular domain, optionally wherein the nonlipidated Borrelia OspA protein encoded by the fourth mRNA polynucleotide comprises a heterologous amino terminal transmembrane domain and/or N-linked glycan site mutations.
  • the mRNA vaccine further comprises a fifth mRNA polynucleotide comprising an open reading frame encoding a nonlipidated Borrelia OspA protein comprising a Borrelia garinii OspA S5 extracellular domain, optionally wherein the nonlipidated Borrelia OspA protein encoded by the fifth mRNA polynucleotide comprises a heterologous amino terminal transmembrane domain and/or N-linked glycan site mutations.
  • the mRNA vaccine further comprises a six mRNA polynucleotide comprising an open reading frame encoding a nonlipidated Borrelia OspA protein comprising a Borrelia garinii OspA S6 extracellular domain, optionally wherein the nonlipidated Borrelia OspA protein encoded by the sixth mRNA polynucleotide comprises a heterologous amino terminal transmembrane domain and/or N-linked glycan site mutations.
  • the nonlipidated Borrelia OspA protein comprising a Borrelia garinii OspA S6 extracellular domain comprises one or more amino acid modification(s), relative to a corresponding naturally occurring Borrelia garinii OspA S6 protein, to stabilize expression in mammalian cells.
  • the one or more amino acid modification(s) is a mutation corresponding to a S88L mutation or an A200V mutation of the naturally occurring Borrelia garinii OspA S6 protein.
  • the one or more amino acid modification(s) is a mutation corresponding to a S88L mutation and an A200V mutation of the naturally occurring Borrelia garinii OspA S6 protein.
  • the mRNA vaccine further comprises a seventh mRNA polynucleotide comprising an open reading frame encoding a nonlipidated Borrelia OspA protein comprising a Borrelia garinii OspA S7 extracellular domain, optionally wherein the nonlipidated Borrelia OspA protein encoded by the seventh mRNA polynucleotide comprises a heterologous amino terminal transmembrane domain and/or N-linked glycan site mutations.
  • an mRNA vaccine comprising: (a) an mRNA polynucleotide comprising an open reading frame encoding an outer surface protein A (OspA) protein comprising an extracellular domain of a first serotype of Borrelia; (b) an mRNA polynucleotide comprising an open reading frame encoding an OspA protein comprising an extracellular domain of a second serotype of Borrelia, wherein the second serotype of Borrelia is of a species that is different from that of the first serotype of Borrelia; and a lipid nanoparticle.
  • OspA outer surface protein A
  • an mRNA vaccine comprising: at least two, three, four, five, six, or seven mRNAs, each comprising an open reading frame encoding a Borrelia outer surface protein A (OspA) protein comprising an extracellular domain, wherein the extracellular domain of each of the Borrelia OspA proteins is of a different Borrelia serotype; and a lipid nanoparticle.
  • OspA outer surface protein A
  • an mRNA vaccine comprising at least four mRNAs, optionally seven mRNAs, each encoding a Borrelia OspA protein comprising an extracellular domain of one of four different serotypes of Borrelia, optionally selected from Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii, and Borrelia bavariensis; and a lipid nanoparticle.
  • each of the Borrelia OspA proteins comprises a heterologous amino terminal transmembrane domain, optionally an influenza virus neuraminidase transmembrane domain.
  • an mRNA vaccine comprising: an mRNA polynucleotide comprising an open reading frame encoding a protein comprising an extracellular domain from a Borrelia garinii OspA serotype 6 protein, wherein the extracellular domain comprises one or more amino acid modification(s), optionally selected from a S88L mutation and an A200V mutation, relative to a corresponding naturally occurring Borrelia garinii OspA S6 extracellular domain, to stabilize expression in mammalian cells; and a lipid nanoparticle.
  • the protein further comprises a heterologous amino terminal transmembrane domain and/or one or more N-linked glycan site mutation(s).
  • the one or more mRNA(s) of the vaccine comprises a chemical modification. In some embodiments, 100% of the uracil nucleotides of the one or more mRNA(s) comprise a chemical modification. In some embodiments, the chemical modification is 1- methylpseudouracil.
  • the lipid nanoparticle comprises an ionizable lipid, a neutral lipid, a sterol, and a PEG-modified lipid. In some embodiments, the lipid nanoparticle comprises 40– 50 mol% ionizable lipid, 5–15 mol% neutral lipid, 30–50 mol% sterol, and 0.5–3 mol% PEG- modified lipid.
  • the ionizable lipid comprises a structure of Compound (I): ; the neutral lipid is distearoylphosphatidylcholine (DSPC); the sterol is cholesterol; and/or the PEG-modified lipid is 1,2 dimyristoyl-sn-glycerol, methoxypolyethyleneglycol (PEG-DMG).
  • DSPC distearoylphosphatidylcholine
  • PEG-DMG 1,2 dimyristoyl-sn-glycerol, methoxypolyethyleneglycol
  • Some aspects relate to a method of preventing Lyme disease in a subject in need thereof, the method comprising administering to the subject one or more doses of the vaccine of any one of the preceding claims in an effective amount to produce an immune response to a Borrelia bacterial infection.
  • the vaccine is administered intramuscularly.
  • the method comprises administering a single dose of the vaccine to the subject. In some embodiments, the method comprises administering more than one dose of the vaccine to the subject.
  • an Borrelia burgdorferi OspA S1 protein described herein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to an amino acid sequence consisting of amino acid residues 40-287 of SEQ ID NO: 1. In some embodiments, the Borrelia burgdorferi OspA S1 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 1.
  • the amino acid sequence of the Borrelia burgdorferi OspA S1 protein (e.g., an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to an amino acid sequence consisting of amino acid residues 40-287 of SEQ ID NO: 1 and/or at least at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 1) comprises the conserved OspA amino acids depicted with an asterisk (*) in FIG.16.
  • the amino acid sequence of the Borrelia burgdorferi OspA S1 protein (e.g., an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to an amino acid sequence consisting of amino acid residues 40-287 of SEQ ID NO: 1 and/or at least at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 1) comprises: the amino acid sequence EKNSXSVDLPGXMXVLVSKEKXKDGKYXLXATVXKXELKGTSDKXNGXGXLEGXKX XKSKXKXTIXXDLXXTXXEXFKEDGKTLVSXKVXXKDKXSXXEXFNXKGXXSXKXXX XRXGTXLEYTXXXXDGXGKAKEVLKXXXLEG (SEQ ID NO: 67), wherein
  • an Borrelia afzelii OspA S2 protein described herein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to an amino acid sequence consisting of amino acid residues 40-287 of SEQ ID NO: 2.
  • the Borrelia afzelii OspA S2 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 2.
  • the amino acid sequence of the Borrelia afzelii OspA S2 protein (e.g., an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to an amino acid sequence consisting of amino acid residues 40-287 of SEQ ID NO: 2 and/or at least at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 2) comprises the conserved OspA amino acids depicted with an asterisk (*) in FIG.16.
  • the amino acid sequence of the Borrelia afzelii OspA S2 protein (e.g., an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to an amino acid sequence consisting of amino acid residues 40-287 of SEQ ID NO: 2 and/or at least at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 2) comprises: the amino acid sequence EKNSXSVDLPGXMXVLVSKEKXKDGKYXLXATVXKXELKGTSDKXNGXGXLEGXKX XKSKXKXTIXXDLXXTXXEXFKEDGKTLVSXKVXXKDKXSXXEXFNXKGXXSXKXXXRXGTXLEYTXXXXDGXGKAKEVLKXXXLEG (SEQ ID NO: 67), wherein
  • an Borrelia garinii OspA S3 protein described herein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to an amino acid sequence consisting of amino acid residues 40-288 of SEQ ID NO: 3.
  • the Borrelia garinii OspA S3 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 3.
  • the amino acid sequence of the Borrelia garinii OspA S3 protein (e.g., an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to an amino acid sequence consisting of amino acid residues 40-288 of SEQ ID NO: 3 and/or at least at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 3) comprises the conserved OspA amino acids depicted with an asterisk (*) in FIG.16.
  • the amino acid sequence of the Borrelia garinii OspA S3 protein (e.g., an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to an amino acid sequence consisting of amino acid residues 40-288 of SEQ ID NO: 3 and/or at least at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 3) comprises: the amino acid sequence EKNSXSVDLPGXMXVLVSKEKXKDGKYXLXATVXKXELKGTSDKXNGXGXLEGXKX XKSKXKXTIXXDLXXTXXEXFKEDGKTLVSXKVXXKDKXSXXEXFNXKGXXSXKXXX XRXGTXLEYTXXXXDGXGKAKEVLKXXXLEG (SEQ ID NO: 67), wherein
  • an Borrelia bavariensis OspA S4 protein described herein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to an amino acid sequence consisting of amino acid residues 40-287 of SEQ ID NO: 4.
  • the Borrelia bavariensis OspA S4 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 4.
  • the amino acid sequence of the Borrelia bavariensis OspA S4 protein (e.g., an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to an amino acid sequence consisting of amino acid residues 40-287 of SEQ ID NO: 4 and/or at least at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 4) comprises the conserved OspA amino acids depicted with an asterisk (*) in FIG.16.
  • the amino acid sequence of the Borrelia bavariensis OspA S4 protein (e.g., an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to an amino acid sequence consisting of amino acid residues 40-287 of SEQ ID NO: 4 and/or at least at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 4) comprises: the amino acid sequence EKNSXSVDLPGXMXVLVSKEKXKDGKYXLXATVXKXELKGTSDKXNGXGXLEGXKX XKSKXKXTIXXDLXXTXXEXFKEDGKTLVSXKVXXKDKXSXXEXFNXKGXXSXKXXX XRXGTXLEYTXXXXDGXGKAKEVLKXXXLEG (SEQ ID NO: 67), wherein
  • an Borrelia garinii OspA S5 protein described herein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to an amino acid sequence consisting of amino acid residues 40-288 of SEQ ID NO: 5.
  • the Borrelia garinii OspA S5 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 5.
  • the amino acid sequence of the Borrelia garinii OspA S5 protein (e.g., an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to an amino acid sequence consisting of amino acid residues 40-288 of SEQ ID NO: 5 and/or at least at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 5) comprises the conserved OspA amino acids depicted with an asterisk (*) in FIG.16.
  • the amino acid sequence of the Borrelia garinii OspA S5 protein (e.g., an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to an amino acid sequence consisting of amino acid residues 40-288 of SEQ ID NO: 5 and/or at least at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 5) comprises: the amino acid sequence EKNSXSVDLPGXMXVLVSKEKXKDGKYXLXATVXKXELKGTSDKXNGXGXLEGXKX XKSKXKXTIXXDLXXTXXEXFKEDGKTLVSXKVXXKDKXSXXEXFNXKGXXSXKXXX XRXGTXLEYTXXXXDGXGKAKEVLKXXXLEG (SEQ ID NO: 67), wherein
  • an Borrelia garinii OspA S6 protein described herein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to an amino acid sequence consisting of amino acid residues 40-288 of SEQ ID NO: 6.
  • the Borrelia garinii OspA S6 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 6.
  • the amino acid sequence of the Borrelia garinii OspA S6 protein (e.g., an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to an amino acid sequence consisting of amino acid residues 40-288 of SEQ ID NO: 6 and/or at least at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 6) comprises the conserved OspA amino acids depicted with an asterisk (*) in FIG.16.
  • the amino acid sequence of the Borrelia garinii OspA S6 protein (e.g., an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to an amino acid sequence consisting of amino acid residues 40-288 of SEQ ID NO: 6 and/or at least at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 6) comprises: the amino acid sequence EKNSXSVDLPGXMXVLVSKEKXKDGKYXLXATVXKXELKGTSDKXNGXGXLEGXKX XKSKXKXTIXXDLXXTXXEXFKEDGKTLVSXKVXXKDKXSXXEXFNXKGXXSXKXXX XRXGTXLEYTXXXXDGXGKAKEVLKXXXLEG (SEQ ID NO: 67), wherein
  • an Borrelia garinii OspA S7 protein described herein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to an amino acid sequence consisting of amino acid residues 40-288 of SEQ ID NO: 7.
  • the Borrelia garinii OspA S7 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 7.
  • the amino acid sequence of the Borrelia garinii OspA S7 protein (e.g., an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to an amino acid sequence consisting of amino acid residues 40-288 of SEQ ID NO: 7 and/or at least at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 7) comprises the conserved OspA amino acids depicted with an asterisk (*) in FIG.16.
  • the amino acid sequence of the Borrelia garinii OspA S7 protein (e.g., an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to an amino acid sequence consisting of amino acid residues 40-288 of SEQ ID NO: 7 and/or at least at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 7) comprises: the amino acid sequence EKNSXSVDLPGXMXVLVSKEKXKDGKYXLXATVXKXELKGTSDKXNGXGXLEGXKX XKSKXKXTIXXDLXXTXXEXFKEDGKTLVSXKVXXKDKXSXXEXFNXKGXXSXKXXX XRXGTXLEYTXXXXDGXGKAKEVLKXXXLEG (SEQ ID NO: 67), wherein
  • the Borrelia burgdorferi OspA S1 protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1;
  • the Borrelia afzelii OspA S2 protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 2;
  • the Borrelia garinii OspA S3 protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 3;
  • the Borrelia bavariensis OspA S4 protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 4;
  • the Borrelia garinii OspA S5 protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 5;
  • the Borrelia garinii OspA S6 protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 6;
  • the Borrelia burgdorferi OspA S1 protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 1;
  • the Borrelia afzelii OspA S2 protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 2;
  • the Borrelia garinii OspA S3 protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 3;
  • the Borrelia bavariensis OspA S4 protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 4;
  • the Borrelia garinii OspA S5 protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 5;
  • the Borrelia garinii OspA S6 protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:
  • the Borrelia burgdorferi OspA S1 protein comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 1;
  • the Borrelia afzelii OspA S2 protein comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 2;
  • the Borrelia garinii OspA S3 protein comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 3;
  • the Borrelia bavariensis OspA S4 protein comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 4;
  • the Borrelia garinii OspA S5 protein comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 5;
  • the Borrelia garinii OspA S6 protein comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO:
  • the Borrelia burgdorferi OspA S1 protein comprises the amino acid sequence of SEQ ID NO: 1;
  • the Borrelia afzelii OspA S2 protein comprises the amino acid sequence of SEQ ID NO: 2;
  • the Borrelia garinii OspA S3 protein comprises the amino acid sequence of SEQ ID NO: 3;
  • the Borrelia bavariensis OspA S4 protein comprises the amino acid sequence of SEQ ID NO: 4;
  • the Borrelia garinii OspA S5 protein comprises the amino acid sequence of SEQ ID NO: 5;
  • the Borrelia garinii OspA S6 protein comprises the amino acid sequence of SEQ ID NO: 6; and
  • the Borrelia garinii OspA S7 protein comprises the amino acid sequence of SEQ ID NO: 7.
  • Some aspects of the present disclosure relate to a method of vaccinating a subject, the method comprising administering to the subject one or more doses of an mRNA vaccine of the present disclosure in an effective amount to produce an immune response to a Borrelia bacterial infection.
  • the vaccine is administered intramuscularly.
  • the method comprises administering a dose of 12.5-150 ⁇ g of the mRNA comprised in the mRNA vaccine.
  • the method comprises administering a single dose of the vaccine to the subject.
  • the method comprises administering a first dose of the vaccine, a second dose of the vaccine, and a third dose of the vaccine.
  • the second dose is administered two months following the first dose, the third dose is administered six months following the first dose, or a combination thereof.
  • the first dose, the second dose, and the third dose is 50 ⁇ g of the mRNA, and wherein the second dose is administered 2 months after the first dose, and the third dose is administered 4 months after the second dose.
  • the first dose, the second dose, and the third dose is 100 ⁇ g of the mRNA, and wherein the second dose is administered 2 months after the first dose, and the third dose is administered 4 months after the second dose.
  • the first dose, the second dose, and the third dose is 150 ⁇ g of the mRNA, and wherein the second dose is administered 2 months after the first dose, and the third dose is administered 4 months after the second dose.
  • the lipid nanoparticle comprises a mixture of lipids that comprises 20-60 mol% ionizable lipid heptadecan-9-yl 8 ((2 hydroxyethyl)(6 oxo 6- (undecyloxy)hexyl)amino)octanoate (Compound 1); 5-25 mol% 1,2 distearoyl sn glycero-3 phosphocholine (DSPC); 25-55 mol% cholesterol; and 0.5-15 mol% 1- monomethoxypolyethyleneglycol-2,3-dimyristylglycerol with polyethylene glycol of average molecular weight 2000 (PEG2000 DMG).
  • the lipid nanoparticle comprises: 47 mol% ionizable cationic lipid; 11.5 mol% neutral lipid; 38.5 mol% sterol; and 3.0 mol% PEG-modified lipid; 48 mol% ionizable cationic lipid; 11 mol% neutral lipid; 38.5 mol% sterol; and 2.5 mol% PEG-modified lipid; 49 mol% ionizable cationic lipid; 10.5 mol% neutral lipid; 38.5 mol% sterol; and 2.0 mol% PEG-modified lipid; 50 mol% ionizable cationic lipid; 10 mol% neutral lipid; 38.5 mol% sterol; and 1.5 mol% PEG-modified lipid; or 51 mol% ionizable cationic lipid; 9.5 mol% neutral lipid; 38.5 mol% sterol; and 1.0 mol% PEG-modified lipid.
  • the mRNA vaccine further comprises Tris buffer, sucrose, and sodium acetate, or any combination thereof.
  • the population of neutralizing antibodies comprises antibodies that bind to OspA S1, antibodies that bind to OspA S2, antibodies that bind to OspA S3, antibodies that bind to OspA S4, antibodies that bind to OspA S5, antibodies that bind to OspA S6, antibodies that bind to OspA S7, or any combination thereof.
  • the population of neutralizing antibodies comprises: IgG antibodies.
  • the polynucleotide comprises the nucleic acid sequence of any one of SEQ ID NOs: 8-14.
  • Some aspects of the present disclosure relate to mRNA vaccine compositions comprising a plurality of polynucleotides, wherein the plurality comprises two or more of: a first polynucleotide comprising a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 8; a second polynucleotide comprising a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 9; a third polynucleotide comprising a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 10; a fourth polynucleotide comprising a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence
  • the first polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 8; the second polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 9; the third polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 10; the fourth polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 11; the fifth polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 12; the sixth polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 13; and the seventh polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 14.
  • FIG.1 shows a graph of in vitro cellular expression levels of protein encoded by mRNA OspA S1 and S6 variants (OspA-S1-ND165-173-NtermTM and OspA-S6-S88L-A200V- NTerm-TM) compared to mock-transfected cells and non-transfected cells. Two concentrations (500 ng/ ⁇ L and 125 ng/ ⁇ L) were tested and measured for mean fluorescence intensity (MFI) x Frequency (Freq).
  • MFI mean fluorescence intensity
  • FIGs.2A-2B show graphs of total OspA-specific IgG titers (FIG.2A) and LA-2 Competition ELISAs (FIG.2B).
  • the groups tested were non-translating mRNA (NTFIX), and mRNA having an ORF encoding a wild-type (WT) (i.e., naturally occurring) full-length, soluble, OspA Serotype 1 (S1) (WT-Soluble), or an OspA S1 with the LFA epitope removed (Soluble), or an OspA S1 with the LFA epitope removed and an N-terminal transmembrane domain (Nterm-TM), or an OspA S1 with the LFA epitope removed and a C-terminal transmembrane domain (Cterm-TM).
  • WT wild-type
  • S1 OspA Serotype 1
  • Soluble Soluble
  • Nterm-TM N-terminal transmembrane domain
  • FIGs.3A-3B show graphs of the serum bactericidal activity of OspA S1 with the LFA epitope removed and an N-terminal transmembrane domain (OspA S1 Nterm-TM).
  • the OspA S1 Nterm-TM construct was compared to non-translating mRNA (NTFIX) and LA-2, measured by the Survival Index (FIG.3A). Additionally, the OspA S1 Nterm-TM (Pool 2) was compared to an OspA S1 with the LFA epitope removed and a C-terminal transmembrane domain (OspA S1 Cterm-TM; Pool 3) and NTFIX (Pool 1), measured by the Survival Index (FIG.3B).
  • FIGs.4A-4B show graphs of total OspA-specific IgG titers (FIG.4A) and LA-2 Competition ELISAs (FIG.4B).
  • NTFIX non-translating mRNA
  • Soluble an OspA S1 with the LFA epitope removed
  • Nterm- TM an OspA S1 with the LFA epitope removed and an N-terminal transmembrane domain
  • Cterm-TM C-terminal transmembrane domain
  • Samples were tested on days 21 (d21), 44 (d44), 56 (d56) and Pre-Challenge and Post-Challenge for total OspA-specific IgG titers and Pre-Challenge and Post-Challenge for LA- 2 Competition ELISA.
  • FIGs.5A-5C show graphs of the total and functional antibody titers to recombinant antibodies.
  • FIG.5A Shown are the total OspA-specific IgG titers (FIG.5A) and functional equivalents, LA-2 (only bactericidal against S1) (FIG.5B) and a positive control antibody (bactericidal against all 7 serotypes) (FIG.5C).
  • the groups tested were non-translating mRNA (NTFIX), and mRNA having an ORF encoding either an OspA S1 with the LFA epitope removed and an N- terminal transmembrane domain (OspA S1 Nterm-TM), or mRNA having an ORF(s) encoding OspA S2-S5, S7, S1 with the LFA epitope removed and an N-terminal transmembrane domain and S6 with amino acid mutations S88L and A200V (OspA S1-S7 NtermTM) or mRNA having an ORF(s) encoding OspA S1 with a C-terminal transmembrane domain and ferritin stabilization (OspA S1 Cterm ferritin stabilized), and a purified Recombitek® (i.e., lipidated OspA protein)+Alum.
  • NTFIX non-translating mRNA
  • OspA S1 Nterm-TM m
  • the total OspA-specific IgG titers were measured using mean fluorescence intensity (MFI) (FIG.5A) and LA-2 competition ELISA and positive control antibodies were measured using signal reduction (1:20) (FIGs.5B-5C).
  • FIG.6 shows graphs of antibodies generated by administration of either OspA S1 with the LFA epitope removed and an N-terminal transmembrane domain (OspA S1 NtermTM; mRNA-1982), or mRNA having an ORF(s) encoding OspA S2-S5, S7, S1 with the LFA epitope removed and an N-terminal transmembrane domain and S6 with amino acid mutations S88L and A200V (OspA S1-S7 NtermTM; mRNA-1975).
  • OspA S1 NtermTM was administered at either 0.5 ⁇ g or 1 ⁇ g and OspA S1-S7 NtermTM was administered at either 3.5 ⁇ g or 7 ⁇ g.
  • FIGs.7A-7B show the ability of antibodies generated in response to OspA S1 NtermTM and OspA S1-S7 NtermTM to bind to Borrelia bacteria using a Surface binding assay.
  • Groups tested include OspA S1 with the LFA epitope removed and an N-terminal transmembrane domain (S1), or mRNA having an ORF(s) encoding OspA S2-S5, S7, S1 with the LFA epitope removed and an N-terminal transmembrane domain and S6 with amino acid mutations S88L and A200V (S1-S7).
  • the mRNA vaccines were compared to mice administered PBS or Recombitek® or S1-S7 protein, LA-2 or S1 Hyper+. The results are measured by percent (%) positive for Alexa-647 fluorescence (FIG.7A) or the geometric mean (MFI) (FIG.7B).
  • FIGs.8A-8B show the ability of antibodies generated in response to OspA S1 NtermTM and OspA S1-S7 NtermTM to agglutinate (FIG.8A) and compromise the membranes of Borrelia bacteria (FIG.8B). These were measured using an Agglutination assay (FIG.8A) and a Membrane Integrity assay (FIG.8B).
  • Groups tested include OspA S1 with the LFA epitope removed and an N-terminal transmembrane domain (S1), or mRNA having an ORF(s) encoding OspA S2-S5, S7, S1 with the LFA epitope removed and an N-terminal transmembrane domain and S6 with amino acid mutations S88L and A200V (S1-S7).
  • the mRNA vaccines were compared to mice administered PBS or Recombitek® or S1-S7 protein, LA-2 or S1 Hyper+. The results are measured by percent (%) agglutination (FIG.8A) and percent (%) propidium iodide positive (FIG.8B).
  • FIG.9 shows the serum bactericidal activity of antibodies generated in response to OspA S1 NtermTM and OspA S1-S7 NtermTM to kill Borrelia bacteria. This was measured using a serum bactericidal assay (SBA). Groups tested include OspA S1 with the LFA epitope removed and an N-terminal transmembrane domain (S1 mRNA), or mRNA having an ORF(s) encoding OspA S2-S5, S7, S1 with the LFA epitope removed and an N-terminal transmembrane domain and S6 with amino acid mutations S88L and A200V (S1-S7 mRNA).
  • SBA serum bactericidal assay
  • S1 mRNA was administered at either 0.5 ⁇ g or 1 ⁇ g and S1-S7 mRNA was administered at either 3.5 ⁇ g or 7 ⁇ g.
  • the mRNA vaccines were compared to untreated IPTG, positive control antibody (10 ⁇ g/ml), LA-2 (10 ⁇ g/ml), PBS, S1 Hyper Immune or Recombitek® (1 ⁇ g, 2 ⁇ g or 5 ⁇ g) or S1-S7 (7 ⁇ g or 14 ⁇ g) protein. Results were measured based on mScarlet fluorescence for serum dilutions of 1:400, 1:800, 1:1600 or 1:3200.
  • FIGs.10A-10B show the ability of either 2 or 3 doses of an mRNA vaccine delivered at different dosage amounts to protect mice from Borrelia infection in a tick bite model pre- challenge (FIG.10A) and post-challenge (FIG.10B).
  • Mice were received either 2 or 3 doses of OspA S1 with the LFA epitope removed and an N-terminal transmembrane domain (0.2 ⁇ g, 0.5 ⁇ g or 1 ⁇ g) and compared to Recombitek®+Alum and non-translating mRNA (NTFIX). Results were measured using mean fluorescence intensity (MFI).
  • MFI mean fluorescence intensity
  • FIG.11 shows the ability of 2 doses of the indicated mRNA or protein vaccines to protect mice from Borrelia infection in a tick bite model challenge (see also Example 2).
  • FIG.12 shows graphs of antibodies generated by administration of mRNA having an ORF(s) encoding OspA S1-S7 NtermTM at a variety of doses with either two or three administrations (see also Example 9).
  • FIG.13 shows the ability of 2 or 3 doses of the indicated mRNA or protein vaccines to protect mice from Borrelia infection at a variety of doses in a tick bite model challenge (see also Example 9).
  • FIG.14 shows OspA S1 IgG amounts in blood samples isolated at 42, 84, 126, 168, and 189 days following initial administration of the indicated mRNA or protein vaccines (see also Example 10).
  • FIGs.15A-15D are graphs depicting the results of studies involving C3H/HeN mice administered mRNA having an ORF(s) encoding Lyme OspA antigens from Serotype 1, Serotypes 1-7 or Serotypes 2-7, as described in Table 13, to test the cross-protection of the vaccine (see also Example 11).
  • Antigen specific IgG titers, represented by mean fluorescence intensity (MFI), from vaccinated mice directed towards OspA S1 are shown in FIG.15A.
  • MFI mean fluorescence intensity
  • FIG.15B shows a multiple sequence alignment of the OspA proteins of SEQ ID NOs: 1-7 and 24-30. The multiple sequence alignment was performed using Clustal Omega.
  • FIG.17 shows a percent identity matrix comparing the amino acid sequences of the OspA proteins of SEQ ID NOs: 24-30 (which include OspA S1-S7 proteins) compared using multiple sequence alignment performed with Clustal Omega.
  • Lyme disease is an infectious disease caused by various species of Borrelia bacteria which are transmitted to humans through the bite of infected black-legged ticks. The disease is most commonly found in the northeastern, mid-Atlantic, and north-central regions of the United States, as well as in some parts of Europe and Asia. Lyme disease can cause a range of symptoms, including fever, fatigue, headache, and a characteristic "bull's-eye" rash. If left untreated, Lyme disease can lead to more serious complications. Vaccines have been developed to prevent Lyme disease, although currently there are no vaccines approved for use in the United States.
  • Lyme disease vaccines In the past, two vaccines were available in the U.S., but they were withdrawn from the market primarily due to concerns about side effects, such as autoantibody production.
  • the development of effective Lyme disease vaccines remains an important area of research, as tick- borne diseases continue to be a major public health concern in many parts of the world.
  • the development of a safe and effective vaccine against Lyme disease has been met with several challenges, including a limited understanding of the disease, complexity of the bacterium that causes the disease, low prevalence of the disease, and limited animal models.
  • bacterial antigens are more complex and diverse. Borrelia burgdorferi (B.
  • burgdorferi a pathogenic spirochete responsible for Lyme disease, for example, has the ability to change its surface proteins, making it difficult for the immune system to recognize and respond to the bacterium. This antigenic variability also makes it challenging to develop a vaccine that will be effective against more than one strain of the bacterium.
  • Borrelia burgdorferi is the predominant strain that causes Lyme disease in the U.S.
  • Borrelia afzelii, Borrelia garinii, and Borrelia bavariensis are the predominant strains to cause Lyme disease outside of the U.S.
  • bacterial lipidation is thought to be essential for the proper function of Borrelia proteins (see, e.g., Zuckert WR Biochim Biophys Acta.2014 Aug;1843(8):1509-16), but is challenging to reproduce in mammalian cells.
  • Borrelia bacterial proteins are associated with high reactogenicity, which refers to their ability to cause adverse reactions or side effects in the body, that can lead to inflammation, fever, or other adverse reactions.
  • the Borrelia proteins encoded by the mRNA vaccines provided herein elicit low reactogenicity and high immunogenicity, in some embodiments in response to multiple strains/serotypes of Borrelia, despite the absence of bacterial lipidation thought to be essential for proper functioning of the bacterial proteins.
  • these bacterial proteins are designed such that, when expressed in vivo by mammalian cells, they are still capable of folding into protein antigens having conformationally correct extracellular domains that preserve important epitopes critical for these properties.
  • the administration of these Borrelia proteins to a subject in the form of an mRNA vaccine stimulates the production of antibodies that exhibit high bactericidal and agglutination activity, properties that are not shared by identical Borrelia proteins when administered in the form of a protein vaccine.
  • the mRNA vaccines encode Borrelia OspA proteins that have conformationally correct extracellular domains.
  • a conformationally correct protein domain is a protein domain that has the correct three-dimensional structure (i.e., conformation) that is necessary for its proper function.
  • the mRNA vaccines are capable of eliciting the production of bactericidal antibodies. Bactericidal activity simply refers to the ability of the vaccine, or more precisely the antibodies produced by the body in response to the vaccine, to kill the bacteria.
  • the mRNA vaccines are capable of eliciting the production of antibodies that exhibit high agglutination activity. Agglutination activity refers to the ability of those antibodies to cause agglutination (the clumping together) of the bacterial cells expressing the specific epitopes to which the antibodies bind.
  • Borrelia Proteins Borrelia is a genus of bacteria that includes several species, some of which are known to cause Lyme disease in humans. The most common species associated with Lyme disease is Borrelia burgdorferi, which is primarily transmitted to humans through the bite of infected blacklegged ticks. Borrelia bacteria are spiral-shaped, Gram-negative bacteria that are capable of surviving in a variety of environments, including inside the bodies of animals and insects. The bacteria have a complex life cycle that involves both ticks and mammals, and they are capable of adapting to different hosts and environments. Borrelia bacteria are known for their ability to evade the immune system and cause persistent infections. The bacteria are able to change their surface proteins, making it difficult for the immune system to recognize and respond to the infection.
  • Lyme disease can become a chronic condition in some individuals.
  • both the innate and adaptive immune systems respond to Borrelia infection.
  • Most manifestations and symptoms of Lyme disease are caused by inflammation generated by the immune response because Borrelia does not produce toxins or extracellular matrix-degrading proteases.
  • the host immune response can reduce bacterial infection and decrease symptoms within several weeks to months, even without antibiotic intervention. Nonetheless, persistent bacteria can survive for years in untreated patients resulting in chronic illness.
  • Borrelia infection is characterized by several pathogenic processes. Such processes include adherence of bacteria to host target cells, local multiplication, and migration to distant sites.
  • OspA Outer Surface Protein A
  • OspC Outer Surface Protein C
  • OspA is downregulated and OspC is upregulated.
  • B. burgdorferi selectively produces OspA in ticks and OspC in mammals, after the bacteria has been transmitted to a mammalian host.
  • OspC is thought to be critical for the establishment of the bacteria in the mammalian host and plays an important role in the early stages of the disease.
  • OspC is involved in a number of processes that are important for the bacteria's survival in the mammalian host, including evasion of the host immune system and adhesion to host cells.
  • Borrelia OspA protein is a 31 kilodalton lipoprotein having two globular domains that are connected via a unique single-layer beta-sheet.
  • the C terminus of the protein contains a receptor binding domain and is variable among different OspA serotypes, while the N terminus attaches to the bacterial cell membrane via post-translational palmitoylation on the signal sequence.
  • the OspA protein also contains human lymphocyte function associated antigen-1 (hLFA-1) epitope regions localized within OspA amino acid residues 165–173 (OspA 165–173 ). In some embodiments, the hLFA-1 epitopes are modified or removed.
  • the OspA165–173 epitope is removed.
  • the Borrelia protein encoded by an mRNA of the present disclosure is a Borrelia OspA protein.
  • the Borrelia protein is an OspA protein variant, relative to a naturally occurring Borrelia OspA protein.
  • the terms “naturally occurring” and “wild type” are used interchangeably herein.
  • a naturally occurring Borrelia OspA protein is an Borrelia OspA protein (e.g., one of Borrelia serotypes 1- 7) that occurs in nature, i.e., which is a naturally occurring isolate. As is known in the art, a naturally occurring protein is not genetically engineered.
  • a naturally occurring protein is not genetically (or otherwise) modified to substitute, remove, or add any amino acids.
  • At least seven naturally occurring Borrelia OspA serotypes have been identified with eight monoclonal antibodies against different epitopes of the Borrelia OspA protein. As determined by 16S rRNA sequence analysis, these serotypes correlated well with recently delineated genospecies: serotype 1 (S1) corresponds to B. burgdorferi, serotype 2 (S2) corresponds to Borrelia afzelii, serotype 4 corresponds to Borrelia bavariensis, and serotypes 3 (S3) and 5-7 (S5-S7) correspond to Borrelia garinii.
  • the mRNA vaccines provided herein may encode any one or more Borrelia OspA proteins selected from Borrelia burgdorferi OspA serotype 1 (S1); Borrelia afzelii OspA serotype 2 (S2); Borrelia garinii OspA serotype 3 (S3); Borrelia bavariensis OspA serotype 4 (S4); Borrelia garinii OspA serotype 5 (S5); Borrelia garinii OspA serotype 6 (S6); and Borrelia garinii OspA serotype 7 (S7).
  • S1 Borrelia burgdorferi OspA serotype 1
  • S2 Borrelia afzelii OspA serotype 2
  • S3 Borrelia garinii OspA serotype 3
  • S4 Borrelia bavariensis OspA serotype 4
  • S5 Borrelia garinii
  • a vaccine of the present disclosure comprises an mRNA polynucleotide comprising an open reading frame encoding Borrelia burgdorferi OspA S1. In some embodiments, a vaccine of the present disclosure comprises an mRNA polynucleotide comprising an open reading frame encoding Borrelia afzelii OspA S2. In some embodiments, a vaccine of the present disclosure comprises an mRNA polynucleotide comprising an open reading frame encoding Borrelia garinii OspA S3. In some embodiments, a vaccine of the present disclosure comprises an mRNA polynucleotide comprising an open reading frame encoding Borrelia bavariensis OspA S4.
  • a vaccine of the present disclosure comprises an mRNA polynucleotide comprising an open reading frame encoding Borrelia garinii OspA S5. In some embodiments, a vaccine of the present disclosure comprises an mRNA polynucleotide comprising an open reading frame encoding Borrelia garinii OspA S6. In some embodiments, a vaccine of the present disclosure comprises an mRNA polynucleotide comprising an open reading frame encoding Borrelia garinii OspA S7.
  • a vaccine of the present disclosure comprises an mRNA polynucleotide comprising an open reading frame encoding Borrelia burgdorferi OspA S1 (e.g., SEQ ID NO: 1), an mRNA polynucleotide comprising an open reading frame encoding Borrelia afzelii OspA S2 (e.g., SEQ ID NO: 2), an mRNA polynucleotide comprising an open reading frame encoding Borrelia garinii OspA S3 (e.g., SEQ ID NO: 3), an mRNA polynucleotide comprising an open reading frame encoding Borrelia bavariensis OspA S4 (e.g., SEQ ID NO: 4), an mRNA polynucleotide comprising an open reading frame encoding Borrelia garinii OspA S5 (e.g., SEQ ID NO: 5), an mRNA polynucleotide compris
  • a vaccine of the present disclosure comprises an mRNA polynucleotide comprising an open reading frame encoding Borrelia afzelii OspA S2 (e.g., SEQ ID NO: 2), an mRNA polynucleotide comprising an open reading frame encoding Borrelia garinii OspA S3 (e.g., SEQ ID NO: 3), an mRNA polynucleotide comprising an open reading frame encoding Borrelia bavariensis OspA S4 (e.g., SEQ ID NO: 4), an mRNA polynucleotide comprising an open reading frame encoding Borrelia garinii OspA S5 (e.g., SEQ ID NO: 5), an mRNA polynucleotide comprising an open reading frame encoding Borrelia garinii OspA S6 (e.g., SEQ ID NO: 6), and an mRNA polynucleotide compris
  • the bacterial proteins encoded by the mRNA vaccines comprises a heterologous transmembrane domain.
  • a transmembrane domain is a region of a protein that spans the lipid bilayer of a biological membrane, such as a cell membrane.
  • Transmembrane domains are composed of hydrophobic amino acids that are able to interact with the hydrophobic core of the membrane, anchoring the protein to the membrane and allowing it to interact with other proteins or molecules on either side of the membrane.
  • a transmembrane domain is “heterologous” to a protein if the protein does not naturally occur with the transmembrane domain.
  • viral transmembrane domains may also be used to anchor bacterial proteins to a host cell membrane, including without limitation, the transmembrane domain from any of the following: human immunodeficiency virus (HIV) envelope glycoprotein (Env), hepatitis C virus (HCV) envelope glycoproteins E1 and E2, herpes simplex virus (HSV) glycoprotein D (gD), and other influenza virus proteins, such as hemagglutinin and M2 protein.
  • HCV human immunodeficiency virus
  • HCV hepatitis C virus
  • HCV herpes simplex virus
  • gD herpes simplex virus
  • influenza virus proteins such as hemagglutinin and M2 protein.
  • the bacterial proteins are naturally anchored to the cell surface via lipidation at the N terminus of the protein.
  • the mRNA described herein encodes bacterial proteins wherein residues prone to N-linked glycosylation (e.g., asparagine) have been removed, modified, or substituted in order to prevent glycosylation. Modifications to Increase Stability/Expression
  • the bacterial proteins encoded by the mRNA vaccines comprises one or more stabilizing mutation.
  • a vaccine of the present disclosure comprises an mRNA polynucleotide comprising an open reading frame encoding a Borrelia OspA S1 protein, wherein the OspA S1 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 1.
  • a vaccine of the present disclosure comprises an mRNA polynucleotide comprising an open reading frame encoding a Borrelia OspA S2 protein, wherein the OspA S2 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 2.
  • a vaccine of the present disclosure comprises an mRNA polynucleotide comprising an open reading frame encoding a Borrelia OspA S3 protein, wherein the OspA S3 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 3.
  • a vaccine of the present disclosure comprises an mRNA polynucleotide comprising an open reading frame encoding a Borrelia OspA S5 protein, wherein the OspA S5 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 5.
  • a vaccine of the present disclosure comprises an mRNA polynucleotide comprising an open reading frame encoding a Borrelia OspA S6 protein, wherein the OspA S6 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 6.
  • a vaccine of the present disclosure comprises an mRNA polynucleotide comprising an open reading frame encoding a Borrelia OspA S7 protein, wherein the OspA S7 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 7.
  • a vaccine of the present disclosure comprises an mRNA polynucleotide comprising an open reading frame encoding a Borrelia OspA S1 protein, wherein the OspA S1 protein comprises the amino acid sequence of SEQ ID NO: 1.
  • a vaccine of the present disclosure comprises an mRNA polynucleotide comprising an open reading frame encoding a Borrelia OspA S2 protein, wherein the OspA S2 protein comprises the amino acid sequence of SEQ ID NO: 2.
  • a vaccine of the present disclosure comprises an mRNA polynucleotide comprising an open reading frame encoding a Borrelia OspA S3 protein, wherein the OspA S3 protein comprises the amino acid sequence of SEQ ID NO: 3.
  • a vaccine of the present disclosure comprises an mRNA polynucleotide comprising an open reading frame encoding a Borrelia OspA S4 protein, wherein the OspA S4 protein comprises the amino acid sequence of SEQ ID NO: 4.
  • a vaccine of the present disclosure comprises an mRNA polynucleotide comprising an open reading frame encoding a Borrelia OspA S5 protein, wherein the OspA S5 protein comprises the amino acid sequence of SEQ ID NO: 5.
  • a vaccine of the present disclosure comprises an mRNA polynucleotide comprising an open reading frame encoding a Borrelia OspA S6 protein, wherein the OspA S6 protein comprises the amino acid sequence of SEQ ID NO: 6.
  • a vaccine of the present disclosure comprises an mRNA polynucleotide comprising an open reading frame encoding a Borrelia OspA S7 protein, wherein the OspA S7 protein comprises the amino acid sequence of SEQ ID NO: 7.
  • the mRNA polynucleotides of the present disclosure encode at least one Borrelia protein of interest, intended to raise an immune response which is protective against subsequent Borrelia infection in mammalian subjects.
  • the mRNA vaccines lead to expression of Borrelia proteins of the present disclosure which are antigenic, i.e., they provoke a protective immune response. Antigenicity can be measured by the ability to generate cellular and/or humoral immune responses. Delivery of the mRNA polynucleotides of the present disclosure is achieved by formulating the mRNA in appropriate carriers or delivery vehicles (e.g., lipid nanoparticles) such that upon administration to cells, tissues or subjects, the mRNA is taken up by cells which, in turn, express the protein(s) encoded by the mRNA.
  • appropriate carriers or delivery vehicles e.g., lipid nanoparticles
  • the vaccines of the present disclosure provide a unique advantage over traditional protein-based vaccination approaches in which protein antigens are purified or produced in vitro, e.g., recombinant protein production technologies.
  • the vaccines of the present disclosure comprise mRNA encoding the desired Borrelia antigen(s), which when introduced into the body, i.e., administered to a mammalian subject (for example a human) in vivo, cause the cells of the body to express the desired antigen(s).
  • the mRNA is formulated (e.g., encapsulated) in a lipid nanoparticle.
  • the mRNA Upon delivery and uptake by cells of the body, the mRNA is translated in the cytosol and the antigens are generated by the host cell machinery.
  • the antigens are expressed and presented by the host cells and elicit humoral and/or cellular immune responses.
  • Neutralizing antibodies are directed against the expressed antigens, and hence the antigens are considered relevant target antigens for vaccine development.
  • Many proteins have a quaternary or three-dimensional structure, which includes more than one polypeptide or several polypeptide chains that associate into an oligomeric molecule.
  • subunit refers to a single protein molecule, for example, a polypeptide or polypeptide chain resulting from processing of a nascent protein molecule, which subunit assembles (or “coassembles”) with other protein molecules (e.g., subunits or chains) to form a protein complex.
  • Proteins can have a relatively small number of subunits and therefore be described as “oligomeric” or can consist of a large number of subunits and therefore be described as “multimeric”.
  • the subunits of an oligomeric or multimeric protein may be identical, homologous or totally dissimilar and dedicated to disparate tasks. Proteins or protein subunits can further comprise domains.
  • domain refers to a distinct functional and/or structural unit within a protein. Typically, a “domain” is responsible for a particular function or interaction, contributing to the overall role of a protein. Domains can exist in a variety of biological contexts. Similar domains (i.e., domains sharing structural, functional and/or sequence homology) can exist within a single protein or can exist within distinct proteins having similar or different functions. A protein domain is often a conserved part of a given protein tertiary structure or sequence that can function and exist independently of the rest of the protein or subunit thereof. An extracellular protein domain is a part of a protein molecule that is located outside of a cell.
  • a transmembrane domain is a structural component of a protein that spans the lipid bilayer of a cell membrane. Transmembrane domains typically include one or more alpha helices or beta strands that cross the hydrophobic lipid bilayer of the cell membrane.
  • the term “antigen” is distinct from the term “epitope,” which is a substructure of an antigen.
  • An epitope may be a peptide, for example, a 7-10 amino acid peptide, or a carbohydrate structure.
  • mRNA technology is amenable to rapid design and testing of mRNA encoding a variety of antigens.
  • rapid production of mRNA coupled with formulation in appropriate delivery vehicles can proceed quickly and can rapidly produce mRNA vaccines at large scale.
  • antigens encoded by the mRNAs of the present disclosure are expressed by the cells of the subject, e.g., are expressed by the human body, and thus the subject, e.g., the human body, serves as the “factory” to produce the antigens which, in turn, elicits the desired immune response.
  • the vaccines may include an mRNA polynucleotide or multiple mRNA polynucleotides encoding two or more antigens of the same or different Borrelia strains.
  • multiple OspA antigens e.g., S1, S2, S3, S4, S5, S6, and S7 can be included in the subject vaccines.
  • combination vaccines that include mRNA encoding one or more Borrelia antigens and one or more antigen(s) of a different organism.
  • the vaccines of the present disclosure may be combination vaccines that target one or more antigens of the same strain/species, or one or more antigens of different strains/species, e.g., antigens that induce immunity to organisms that are found in the same geographic areas where the risk of Borrelia infection is high or organisms to which an individual is likely to be exposed to when exposed to Borrelia.
  • Percent Identity In some embodiments, the compositions of the present disclosure include mRNA that encodes a Borrelia protein variant.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence of any one of the sequences provided herein or comprises a nucleotide sequence that has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to a nucleotide sequence of any one of the sequences provided herein. See, e.g., SEQ ID NOs: 8-14.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 70% identity to the nucleotide sequence of SEQ ID NO: 8.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 70% identity to the nucleotide sequence of SEQ ID NO: 9. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 70% identity to the nucleotide sequence of SEQ ID NO: 10. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 70% identity to the nucleotide sequence of SEQ ID NO: 11.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 70% identity to the nucleotide sequence of SEQ ID NO: 12. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 70% identity to the nucleotide sequence of SEQ ID NO: 13. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 70% identity to the nucleotide sequence of SEQ ID NO: 14.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 75% identity to the nucleotide sequence of SEQ ID NO: 8. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 75% identity to the nucleotide sequence of SEQ ID NO: 9. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 75% identity to the nucleotide sequence of SEQ ID NO: 10.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 75% identity to the nucleotide sequence of SEQ ID NO: 11. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 75% identity to the nucleotide sequence of SEQ ID NO: 12. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 75% identity to the nucleotide sequence of SEQ ID NO: 13.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 75% identity to the nucleotide sequence of SEQ ID NO: 14. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 80% identity to the nucleotide sequence of SEQ ID NO: 8. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 80% identity to the nucleotide sequence of SEQ ID NO: 9.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 80% identity to the nucleotide sequence of SEQ ID NO: 10. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 80% identity to the nucleotide sequence of SEQ ID NO: 11. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 80% identity to the nucleotide sequence of SEQ ID NO: 12.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 85% identity to the nucleotide sequence of SEQ ID NO: 9. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 85% identity to the nucleotide sequence of SEQ ID NO: 10. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 85% identity to the nucleotide sequence of SEQ ID NO: 11.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 85% identity to the nucleotide sequence of SEQ ID NO: 12. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 85% identity to the nucleotide sequence of SEQ ID NO: 13. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 85% identity to the nucleotide sequence of SEQ ID NO: 14.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 90% identity to the nucleotide sequence of SEQ ID NO: 8. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 90% identity to the nucleotide sequence of SEQ ID NO: 9. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 90% identity to the nucleotide sequence of SEQ ID NO: 10.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 90% identity to the nucleotide sequence of SEQ ID NO: 11. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 90% identity to the nucleotide sequence of SEQ ID NO: 12. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 90% identity to the nucleotide sequence of SEQ ID NO: 13.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 90% identity to the nucleotide sequence of SEQ ID NO: 14. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 95% identity to the nucleotide sequence of SEQ ID NO: 8. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 95% identity to the nucleotide sequence of SEQ ID NO: 9.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 95% identity to the nucleotide sequence of SEQ ID NO: 10. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 95% identity to the nucleotide sequence of SEQ ID NO: 11. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 95% identity to the nucleotide sequence of SEQ ID NO: 12.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 95% identity to the nucleotide sequence of SEQ ID NO: 13. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 95% identity to the nucleotide sequence of SEQ ID NO: 14. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises the nucleotide sequence of SEQ ID NO: 8. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises the nucleotide sequence of SEQ ID NO: 9.
  • an mRNA polynucleotide comprises an open reading frame that comprises the nucleotide sequence of SEQ ID NO: 10. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises the nucleotide sequence of SEQ ID NO: 11. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises the nucleotide sequence of SEQ ID NO: 12. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises the nucleotide sequence of SEQ ID NO: 13. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises the nucleotide sequence of SEQ ID NO: 14.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 70% identity to the nucleotide sequence of SEQ ID NO: 15. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 70% identity to the nucleotide sequence of SEQ ID NO: 16. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 70% identity to the nucleotide sequence of SEQ ID NO: 17.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 70% identity to the nucleotide sequence of SEQ ID NO: 18. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 70% identity to the nucleotide sequence of SEQ ID NO: 19. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 70% identity to the nucleotide sequence of SEQ ID NO: 20.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 70% identity to the nucleotide sequence of SEQ ID NO: 21. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 75% identity to the nucleotide sequence of SEQ ID NO: 15. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 75% identity to the nucleotide sequence of SEQ ID NO: 16.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 75% identity to the nucleotide sequence of SEQ ID NO: 17. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 75% identity to the nucleotide sequence of SEQ ID NO: 18. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 75% identity to the nucleotide sequence of SEQ ID NO: 19.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 75% identity to the nucleotide sequence of SEQ ID NO: 20. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 75% identity to the nucleotide sequence of SEQ ID NO: 21. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 80% identity to the nucleotide sequence of SEQ ID NO: 15.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 80% identity to the nucleotide sequence of SEQ ID NO: 16. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 80% identity to the nucleotide sequence of SEQ ID NO: 17. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 80% identity to the nucleotide sequence of SEQ ID NO: 18.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 80% identity to the nucleotide sequence of SEQ ID NO: 19. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 80% identity to the nucleotide sequence of SEQ ID NO: 20. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 80% identity to the nucleotide sequence of SEQ ID NO: 21.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 85% identity to the nucleotide sequence of SEQ ID NO: 15. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 85% identity to the nucleotide sequence of SEQ ID NO: 16. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 85% identity to the nucleotide sequence of SEQ ID NO: 17.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 85% identity to the nucleotide sequence of SEQ ID NO: 18. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 85% identity to the nucleotide sequence of SEQ ID NO: 19. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 85% identity to the nucleotide sequence of SEQ ID NO: 20.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 85% identity to the nucleotide sequence of SEQ ID NO: 21. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 90% identity to the nucleotide sequence of SEQ ID NO: 15. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 90% identity to the nucleotide sequence of SEQ ID NO: 16.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 90% identity to the nucleotide sequence of SEQ ID NO: 17. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 90% identity to the nucleotide sequence of SEQ ID NO: 18. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 90% identity to the nucleotide sequence of SEQ ID NO: 19.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 90% identity to the nucleotide sequence of SEQ ID NO: 20. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 90% identity to the nucleotide sequence of SEQ ID NO: 21. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 95% identity to the nucleotide sequence of SEQ ID NO: 15.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 95% identity to the nucleotide sequence of SEQ ID NO: 16. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 95% identity to the nucleotide sequence of SEQ ID NO: 17. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 95% identity to the nucleotide sequence of SEQ ID NO: 18.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 95% identity to the nucleotide sequence of SEQ ID NO: 19. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 95% identity to the nucleotide sequence of SEQ ID NO: 20. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has at least 95% identity to the nucleotide sequence of SEQ ID NO: 21.
  • an mRNA polynucleotide comprises an open reading frame that comprises a nucleotide sequence that has 100% identity to the nucleotide sequence of SEQ ID NO: 15 (i.e., comprises the nucleotide sequence of SEQ ID NO: 15). In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises the nucleotide sequence of SEQ ID NO: 16. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises the nucleotide sequence of SEQ ID NO: 17. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises the nucleotide sequence of SEQ ID NO: 18.
  • an mRNA polynucleotide comprises an open reading frame that comprises the nucleotide sequence of SEQ ID NO: 19. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises a the nucleotide sequence of SEQ ID NO: 20. In some embodiments, an mRNA polynucleotide comprises an open reading frame that comprises the nucleotide sequence of SEQ ID NO: 21.
  • an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence of any one of the sequences provided herein or comprises an amino acid sequence that has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the amino acid sequence of any one of the sequences provided herein. See, e.g., SEQ ID NOs: 1-7.
  • an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has at least 80% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has at least 80% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has at least 80% identity to the amino acid sequence of SEQ ID NO: 4.
  • an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has at least 80% identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has at least 80% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has at least 80% identity to the amino acid sequence of SEQ ID NO: 7.
  • an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has at least 85% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has at least 85% identity to the amino acid sequence of SEQ ID NO: 3.
  • an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has at least 85% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has at least 85% identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has at least 85% identity to the amino acid sequence of SEQ ID NO: 6.
  • an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has at least 85% identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has at least 90% identity to the amino acid sequence of SEQ ID NO: 2.
  • an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has at least 90% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has at least 90% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has at least 90% identity to the amino acid sequence of SEQ ID NO: 5.
  • an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has at least 90% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has at least 90% identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has at least 95% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has at least 95% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has at least 95% identity to the amino acid sequence of SEQ ID NO: 4.
  • an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has at least 95% identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has at least 95% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has at least 95% identity to the amino acid sequence of SEQ ID NO: 7.
  • an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has 95% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has 95% identity to the amino acid sequence of SEQ ID NO: 3.
  • an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising an amino acid sequence that has 95% identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, an mRNA polynucleotide comprises an open reading frame that encodes a protein comprising the amino acid sequence of SEQ ID NO: 3.
  • Identity refers to a relationship between two or among three or more sequences (e.g., amino acid sequences or nucleotide sequences) as determined by comparing the sequences to each other. Identity also refers to the degree of sequence relatedness between or among sequences as determined by the number of matches between or among strings of amino acids (polypeptides) or strings of nucleotides (polynucleotides). Identity is a measure of the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (e.g., “algorithms”). Identity of related polypeptides and polynucleotides can be readily calculated by known methods.
  • Percent (%) identity as it applies to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid or nucleic acid residues) in the candidate (first) polypeptide or polynucleotide sequence that are identical with the residues in a second polypeptide or polynucleotide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity.
  • percent (%) identity between two mRNA polynucleotides or between two proteins refers to percent (%) identity determined using a global sequence alignment, comparing the length of entire sequences (e.g., entire mRNA polynucleotide, entire open reading frame of an mRNA, or entire protein encoded by an mRNA, as described herein). Methods and computer programs for the alignment are well known in the art. It is understood that identity depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation.
  • variants of a particular polynucleotide or polypeptide have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular naturally occurring or reference sequence as determined by sequence alignment programs and parameters described herein and known to those skilled in the art.
  • tools for alignment include but are not limited to those of the BLAST suite (Altschul, S.F., et al. Nucleic Acids Res.1997;25:3389-3402); and those based on the Smith-Waterman algorithm (Smith, T.F. & Waterman, M.S. J. Mol. Biol.1981;147:195- 197).
  • a general global alignment technique based on dynamic programming is the Needleman– Wunsch algorithm (Needleman, S.B. & Wunsch, C.D. J. Mol. Biol.1920;48:443-453).
  • a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) also has been developed that purportedly produces global alignment of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman–Wunsch algorithm.
  • Signal peptides comprising the N-terminal 15-60 amino acids of proteins, are typically needed for the translocation across the membrane on the secretory pathway and, thus, universally control the entry of most proteins both in eukaryotes and prokaryotes to the secretory pathway.
  • the signal peptide of a nascent precursor protein directs the ribosome to the rough endoplasmic reticulum (ER) membrane and initiates the transport of the growing peptide chain across it for processing.
  • ER processing produces mature proteins, wherein the signal peptide is cleaved from precursor proteins, typically by an ER-resident signal peptidase of the host cell, or they remain uncleaved and function as a membrane anchor.
  • a signal peptide may also facilitate the targeting of the protein to the cell membrane.
  • a signal peptide may have a length of 15-60 amino acids.
  • a signal peptide may have a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids.
  • a signal peptide has a length of 20-60, 25-60, 30-60, 35- 60, 40-60, 45- 60, 50-60, 55-60, 15-55, 20-55, 25-55, 30-55, 35-55, 40-55, 45-55, 50-55, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 15-45, 20-45, 25-45, 30-45, 35-45, 40-45, 15-40, 20- 40, 25-40, 30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30, 20-30, 25-30, 15-25, 20-25, or 15- 20 amino acids.
  • an mRNA polynucleotide comprises an ORF that encodes a fusion protein.
  • an encoded protein may include two or more proteins (e.g., protein and/or protein fragment) joined together with or without a linker. Fusion proteins, in some embodiments, retain the functional property of each independent (nonfusion) protein.
  • the linker is selected from the group consisting of F2A linker, P2A linker, T2A linker, E2A linker, and combinations thereof (see, e.g., WO 2017/127750).
  • This family of self-cleaving peptide linkers referred to as 2A peptides, has been described in the art (see, e.g., Kim, J.H. et al. PLoS ONE 2011;6:e18556).
  • the linker is an F2A linker.
  • the linker is a GS linker.
  • GS linkers are polypeptide linkers that include glycine and serine amino acids repeats.
  • a GS linker is (or is at least) 4 amino acid long (e.g., GSGG (SEQ ID NO: 36)).
  • the GS linker comprises (GSGG)n (SEQ ID NO: 37), where n is any integer from 1-5.
  • a linker is a glycine linker, for example having a length of (or a length of at least) 3 amino acids (e.g., GGG).
  • a protein encoded by an mRNA includes two or more linkers, which may be the same or different from each other.
  • Protein stabilization domains are protein sequences or structures that can enhance the stability of a protein to various environmental stresses, such as temperature, pH, and proteolysis.
  • Non-limiting examples of protein stabilization domains for use to stabilize a Borrelia protein expressed by an mRNA include: lumazine synthase, ferritin, and thioredoxin.
  • a Borrelia OspA protein e.g., an OspA S1 protein is fused to a lumazine synthase.
  • Lumazine synthase is protein from bacteria and plants that can stabilize fusion partners by forming homodimers or oligomers, which can enhance the solubility and stability of the target protein.
  • a Borrelia OspA protein e.g., an OspA S1 protein is fused to ferritin.
  • Ferritin is a protein found in animals, plants, and bacteria that can form a cage-like structure that can store and sequester iron ions, protecting the cell from oxidative damage.
  • a Borrelia OspA protein e.g., an OspA S1 protein is fused to thioredoxin.
  • Thioredoxin is small protein found in bacteria and eukaryotes that can act as a reducing agent and stabilize proteins by forming disulfide bonds.
  • Nucleic Acids Encoding Lyme Disease Proteins Nucleic acids comprise a polymer of nucleotides (nucleotide monomers). Thus, nucleic acids are also referred to as polynucleotides.
  • Nucleic acids may be or may include, for example, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA having a ⁇ -D-ribo configuration, ⁇ -LNA having an ⁇ -L-ribo configuration (a diastereomer of LNA), 2′-amino-LNA having a 2′-amino functionalization, and 2′-amino- ⁇ -LNA having a 2′- amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA) and/or chimeras and/or combinations thereof.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • TAA glycol nucleic acid
  • PNA peptide nucleic acid
  • LNA locked nucleic
  • mRNA polynucleotides of the present disclosure comprises an open reading frame (ORF) encoding a Borrelia protein.
  • the mRNA polynucleotides further comprises a 5 ⁇ untranslated region (UTR), 3 ⁇ UTR, a poly(A) tail and/or a 5 ⁇ cap analog.
  • the polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 9. In some embodiments, the polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 10.
  • the polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 12. In some embodiments, the polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 13.
  • the polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 14. In some embodiments, the polynucleotide comprises the nucleic acid sequence of any one of SEQ ID NOs: 8-14. In some embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 9. In some embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 10. In some embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 11.
  • the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 12. In some embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 14.
  • compositions comprising a plurality of polynucleotides, wherein the plurality comprises two or more of, three or more of, four or more of, five or more of, six or more of, or all seven of: a first polynucleotide comprising a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 8; a second polynucleotide comprising a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 9; a third polynucleotide comprising a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 10; a fourth polynucleotide comprising a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 11
  • the plurality comprises two or more of, three or more of, four or more of, five or more of, six or more of, or all seven of: a first polynucleotide comprising a sequence having at least 85% identity to the nucleic acid sequence of SEQ ID NO: 8; a second polynucleotide comprising a sequence having at least 85% identity to the nucleic acid sequence of SEQ ID NO: 9; a third polynucleotide comprising a sequence having at least 85% identity to the nucleic acid sequence of SEQ ID NO: 10; a fourth polynucleotide comprising a sequence having at least 85% identity to the nucleic acid sequence of SEQ ID NO: 11; a fifth polynucleotide comprising a sequence having at least 85% identity to the nucleic acid sequence of SEQ ID NO: 12; a sixth polynucleotide comprising a sequence having at least 85% identity to the nucleic acid sequence of SEQ ID NO: 13;
  • the plurality comprises two or more of, three or more of, four or more of, five or more of, six or more of, or all seven of: a first polynucleotide comprising a sequence having at least 95% identity to the nucleic acid sequence of SEQ ID NO: 8; a second polynucleotide comprising a sequence having at least 95% identity to the nucleic acid sequence of SEQ ID NO: 9; a third polynucleotide comprising a sequence having at least 95% identity to the nucleic acid sequence of SEQ ID NO: 10; a fourth polynucleotide comprising a sequence having at least 95% identity to the nucleic acid sequence of SEQ ID NO: 11; a fifth polynucleotide comprising a sequence having at least 95% identity to the nucleic acid sequence of SEQ ID NO: 12; a sixth polynucleotide comprising a sequence having at least 95% identity to the nucleic acid sequence of SEQ ID NO: 13;
  • the plurality comprises two or more of, three or more of, four or more of, five or more of, six or more of, or all seven of: a first polynucleotide comprising a sequence having at least 98% identity to the nucleic acid sequence of SEQ ID NO: 8; a second polynucleotide comprising a sequence having at least 98% identity to the nucleic acid sequence of SEQ ID NO: 9; a third polynucleotide comprising a sequence having at least 98% identity to the nucleic acid sequence of SEQ ID NO: 10; a fourth polynucleotide comprising a sequence having at least 98% identity to the nucleic acid sequence of SEQ ID NO: 11; a fifth polynucleotide comprising a sequence having at least 98% identity to the nucleic acid sequence of SEQ ID NO: 12; a sixth polynucleotide comprising a sequence having at least 98% identity to the nucleic acid sequence of SEQ ID NO: 13;
  • the first polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 8; the second polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 9; the third polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 10; the fourth polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 11; the fifth polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 12; the sixth polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 13; and the seventh polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 14.
  • the second polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 9; the third polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 10; the fourth polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 11; the fifth polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 12; the sixth polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 13; and the seventh polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 14.
  • RNA Messenger RNA Messenger RNA is RNA that encodes a (at least one) protein (a naturally- occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded protein in vitro, in vivo, in situ, or ex vivo. It is understood that mRNA is not self-amplifying RNA (saRNA) (see, e.g., Bloom K et al. Gene Therapy 2021; 28: 117–129 for a comparison of mRNA and saRNA). saRNAs include alphavirus replicase sequences that encode an RNA-dependent RNA polymerase. mRNA does not include alphavirus replicase sequences.
  • nucleic acid sequences set forth in the instant application may recite “T”s in a representative DNA sequence but where the sequence represents mRNA, the “T”s would be substituted for “U”s.
  • any of the DNAs disclosed and identified by a particular sequence identification number herein also disclose the corresponding mRNA sequence complementary to the DNA, where each “T” of the DNA sequence is substituted with “U.”
  • Naturally-occurring eukaryotic mRNA molecules can contain stabilizing elements, including, but not limited to, UTRs at their 5′-end (5′ UTR) and/or at their 3′-end (3′ UTR), in addition to other structural features, such as a 5′-cap structure or a 3′-poly(A) tail.
  • Both the 5′ UTR and the 3′ UTR are typically transcribed from the genomic DNA and are elements of the premature mRNA. Characteristic structural features of mature mRNA, such as the 5′-cap and the 3′-poly(A) tail are usually added to the transcribed (premature) mRNA during mRNA processing. Exemplary sequences of mRNA that encode Borrelia proteins of the present disclosure are provided in Table 1.
  • the mRNA polynucleotide comprises an ORF that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or identical to a sequence selected from SEQ ID NOs: 8-14 and 15-21.
  • the mRNA polynucleotide comprises a nucleotide sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or identical to a sequence selected from SEQ ID NOs: 8-14, 15-21, 40, 55, 57, 59, 61, 63, and 65.
  • the mRNA polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 15.
  • the mRNA polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 40. In some embodiments, the mRNA polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 16. In some embodiments, the mRNA polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 55.
  • the mRNA polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 17. In some embodiments, the mRNA polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 57. In some embodiments, the mRNA polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 18.
  • the mRNA polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 59. In some embodiments, the mRNA polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 19. In some embodiments, the mRNA polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 61.
  • the mRNA polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 20. In some embodiments, the mRNA polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 63. In some embodiments, the mRNA polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 21.
  • the mRNA polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 65. In some embodiments, the mRNA polynucleotide comprises the nucleic acid sequence of Table 1. In some embodiments, the mRNA polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 15. In some embodiments, the mRNA polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 40. In some embodiments, the mRNA polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 16.
  • the mRNA polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 55. In some embodiments, the mRNA polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 17. In some embodiments, the mRNA polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 57. In some embodiments, the mRNA polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 18. In some embodiments, the mRNA polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 59. In some embodiments, the mRNA polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 19.
  • the mRNA polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 61. In some embodiments, the mRNA polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 20. In some embodiments, the mRNA polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 63. In some embodiments, the mRNA polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 21. In some embodiments, the mRNA polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 65.
  • the mRNAs of the present disclosure may comprise one or more regions or parts which act or function as an untranslated region.
  • a “5′ untranslated region” refers to a region of an mRNA that is directly upstream (i.e., 5′) from the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome) that does not encode a polypeptide.
  • a “3′ untranslated region” (UTR) refers to a region of an mRNA that is directly downstream (i.e., 3′) from the stop codon (i.e., the codon of an mRNA transcript that signals a termination of translation) that does not encode a polypeptide.
  • the 5’ UTR may comprise a promoter sequence.
  • promoter sequences are known in the art. It should be understood that such promoter sequences will not be present in a vaccine of the disclosure.
  • the mRNA may comprise a 5’ UTR and/or 3’ UTR.
  • UTRs of an mRNA are transcribed but not translated. In mRNA, the 5′ UTR starts at the transcription start site and continues to the start codon but does not include the start codon; the 3′ UTR starts immediately following the stop codon and continues until the transcriptional termination signal.
  • UTR nucleic acid molecule
  • the regulatory features of a UTR can be incorporated into the polynucleotides of the present disclosure to, among other things, enhance the stability of the molecule.
  • the specific features can also be incorporated to ensure controlled down-regulation of the transcript in case they are misdirected to undesired organs sites.
  • a variety of 5’ UTR and 3’ UTR sequences are known. It should also be understood that the mRNA of the present disclosure may include any 5’ UTR and/or any 3’ UTR.
  • Exemplary UTR sequences include SEQ ID NOs: 22, 23, 38, 39, 41- 50, 53, 56, 58, 60, 62, 64, and 66; however, other UTR sequences may be used or exchanged for any of the UTR sequences described herein.
  • a 5' UTR of the present disclosure comprises a sequence selected from: GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC (SEQ ID NO: 38), GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCGCCGCC ACC (SEQ ID NO: 39), GAGGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAGUUUUCUCGCAA CUAGCAAGCUUUUGUUCUCGCC (SEQ ID NO: 22), GGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAGUUUUCUCGCAACU AGCAAGCUUUUUGUUCUCGCC (SEQ ID NO: 41), AGGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUAGUUUUCUCGCAAC UAGCAAGCUUUUUGUUCUCGCC (SEQ ID NO: 50), and
  • a 3' UTR of the present disclosure comprises a sequence selected from UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGA GUGGGCGGC (SEQ ID NO: 42), UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGA GUGGGCGGC (SEQ ID NO: 43), UAAAGCUCCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCAGGAGAUUGAGUGUAGUGACUAGUGA AUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 44), UAAAGCUCCCCGGGGGCCUCGGUGGC
  • a 3′ UTR comprises, in 5′-to-3′ order: (a) the nucleic acid sequence UAAAGCUCCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCAG (SEQ ID NO: 51), (b) an identification and ratio determination (IDR) sequence, and (c) the nucleic acid sequence UGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 52). IDR sequences are described herein in the section entitled “Identification and Ratio Determination (IDR) Sequences.” UTRs may also be omitted from the mRNA provided herein.
  • a 5 ⁇ UTR does not encode a protein (is non-coding).
  • Natural 5′ UTRs have features that play roles in translation initiation. They harbor signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A/G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another 'G'.5′UTR also have been known to form secondary structures which are involved in elongation factor binding.
  • a 5’ UTR is a heterologous UTR, i.e., is a UTR found in nature associated with a different ORF.
  • a 5’ UTR is a synthetic UTR, i.e., does not occur in nature.
  • Synthetic UTRs include UTRs that have been mutated to improve their properties, e.g., which increase gene expression as well as those which are completely synthetic.
  • Exemplary 5’ UTRs include Xenopus or human derived a-globin or b- globin (8278063; 9012219), human cytochrome b-245 a polypeptide, and hydroxysteroid (17b) dehydrogenase, and Tobacco etch virus (US8278063, US9012219).
  • CMV immediate-early 1 (IE1) gene (US2014/0206753, WO2013/185069), the sequence GGGAUCCUACC (SEQ ID NO: 54) (WO2014/144196) may also be used.
  • a 5' UTR is a 5' UTR of a TOP gene lacking the 5' TOP motif (the oligopyrimidine tract) (e.g., WO2015/101414, WO2015/101415, WO2015/062738, WO2015/024667, WO2015/024667); 5' UTR element derived from ribosomal protein Large 32 (L32) gene (WO/2015101414, WO2015101415, WO/2015/062738), 5' UTR element derived from the 5' UTR of an hydroxysteroid (17- ⁇ ) dehydrogenase 4 gene (HSD17B4) (WO2015024667), or a 5' UTR element derived from the 5' UTR of ATP5A1 (WO2015/024667) can be used.
  • L32 ribosomal protein Large 32
  • HSD17B4 hydroxysteroid
  • HSD17B4 hydroxysteroid
  • WO2015024667 or a 5' UTR element derived from
  • an internal ribosome entry site is used instead of a 5' UTR.
  • a 3 ⁇ UTR does not encode a protein (is non-coding).
  • Natural or wild type 3′ UTRs are known to have stretches of adenosines and uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes (Chen et al, 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs.
  • AREs possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of AREs include GM-CSF and TNF-a. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes.
  • AREs 3′ UTR AU rich elements
  • AREs 3′ UTR AU rich elements
  • one or more copies of an ARE can be introduced to make nucleic acids of the disclosure less stable and thereby curtail translation and decrease production of the resultant protein.
  • AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein. Transfection experiments can be conducted in relevant cell lines, using nucleic acids of the disclosure and protein production can be assayed at various time points post-transfection.
  • cells can be transfected with different ARE-engineering molecules and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hours, 12 hours, 1 day, 2 days, and 7 days post-transfection.
  • 5’ UTRs that are heterologous or synthetic may be used with any desired 3’ UTR sequence.
  • a heterologous or synthetic 5’ UTR may be used with a synthetic 3’ UTR or with a heterologous 3’ UTR.
  • Non-UTR sequences may also be used as regions or subregions within a nucleic acid.
  • introns or portions of introns sequences may be incorporated into regions of nucleic acid of the disclosure.
  • the ORF may be flanked by a 5' UTR which may contain a strong Kozak translational initiation signal and/or a 3' UTR which may include an oligo(dT) sequence for templated addition of a poly-A tail.
  • 5′ UTR may comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and/or different genes such as the 5′ UTRs described in US2010/0293625 and WO2015/085318A2, each of which is herein incorporated by reference.
  • any UTR from any gene may be incorporated into the regions of a nucleic acid.
  • multiple wild-type UTRs of any known gene may be utilized. It is also within the scope of the present disclosure to provide artificial UTRs which are not variants of wild type regions. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5′ or 3′ UTR may be inverted, shortened, lengthened, made with one or more other 5′ UTRs or 3′ UTRs.
  • the term “altered” as it relates to a UTR sequence means that the UTR has been changed in some way in relation to a reference sequence.
  • a 3′ UTR or 5′ UTR may be altered relative to a wild-type/native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. Any of these changes producing an “altered” UTR (whether 3′ or 5′) comprise a variant UTR.
  • a double, triple or quadruple UTR such as a 5′ UTR or 3′ UTR may be used.
  • a “double” UTR is one in which two copies of the same UTR are encoded either in series or substantially in series.
  • a double beta-globin 3′ UTR may be used as described in US2010/0129877, which is incorporated herein by reference. It is also within the scope of the present disclosure to have patterned UTRs. As used herein “patterned UTRs” are those UTRs which reflect a repeating or alternating pattern, such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than 3 times. In these patterns, each letter, A, B, or C represent a different UTR at the nucleotide level. In some embodiments, flanking regions are selected from a family of transcripts whose proteins share a common function, structure, feature, or property.
  • polypeptides of interest may belong to a family of proteins which are expressed in a particular cell, tissue or at some time during development.
  • the UTRs from any of these genes may be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide.
  • a “family of proteins” is used in the broadest sense to refer to a group of two or more polypeptides of interest which share at least one function, structure, feature, localization, origin, or expression pattern.
  • the untranslated region may also include translation enhancer elements (TEE).
  • TEE translation enhancer elements
  • the TEE may include those described in US 2009/0226470, herein incorporated by reference, and those known in the art.
  • Open Reading Frames An open reading frame (ORF) is a continuous stretch of DNA or RNA beginning with a start codon (e.g., methionine (ATG or AUG)) and ending with a stop codon (e.g., TAA, TAG or TGA, or UAA, UAG or UGA).
  • An ORF typically encodes a protein. It will be understood that the sequences disclosed herein may further comprise additional elements, e.g., 5′ and/or 3′ UTRs, but that those elements, unlike the ORF, need not necessarily be present in an mRNA of the present disclosure.
  • an mRNA polynucleotide comprises a 5′ terminal cap.
  • 5′-capping of polynucleotides may be completed concomitantly during an in vitro transcription reaction using, for example, the following chemical RNA cap analogs to generate the 5′-guanosine cap structure according to manufacturer protocols: 3 ⁇ -O-Me-m7G(5')ppp(5') G [the ARCA cap];G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA).
  • 5′-capping of modified mRNA may be completed post-transcriptionally using, for example, a Vaccinia Virus Capping Enzyme to generate the “Cap 0” structure: m7G(5')ppp(5')G (New England BioLab
  • Cap 1 structure may be generated using both Vaccinia Virus Capping Enzyme and a 2′-O methyl-transferase to generate: m7G(5')ppp(5')G-2′-O-methyl.
  • Cap 2 structure may be generated from the Cap 1 structure followed by the 2′-O-methylation of the 5′-antepenultimate nucleotide using a 2′-O methyl- transferase.
  • Cap 3 structure may be generated from the Cap 2 structure followed by the 2′-O- methylation of the 5′-preantepenultimate nucleotide using a 2′-O methyl-transferase.
  • Enzymes may be derived from a recombinant source. Other cap analogs may be used.
  • poly(A) tail is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3′), from the 3′ UTR that contains multiple, consecutive adenosine monophosphates.
  • a poly(A) tail may contain 10 to 300 adenosine monophosphates. It can, in some instances, comprise up to about 400 adenine nucleotides.
  • a poly(A) tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates.
  • a poly(A) tail contains 50 to 250 adenosine monophosphates.
  • the poly(A) tail functions to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, and/or export of the mRNA from the nucleus and translation.
  • the length of the 3′-poly(A) tail may be an essential element with respect to the stability of the individual mRNA.
  • a poly(A) tail has a length of about 50, about 100, about 150, about 200, about 250, about 300, about 350, or about 400 nucleotides.
  • a poly(A) tail has a length of 100 nucleotides.
  • Stabilizing elements may include, for example, a histone stem-loop. A stem-loop binding protein (SLBP), a 32 kDa protein has been identified.
  • SLBP stem-loop binding protein
  • SLBP RNA binding domain of SLBP is conserved through metazoa and protozoa; its binding to the histone stem-loop depends on the structure of the loop.
  • the minimum binding site includes at least three nucleotides 5’ and two nucleotides 3′ relative to the stem-loop.
  • an mRNA includes an open reading frame (coding region), a histone stem-loop, and optionally, a poly(A) sequence or polyadenylation signal.
  • the poly(A) sequence or polyadenylation signal generally should enhance the expression level of the encoded protein.
  • the encoded protein in some embodiments, is not a histone protein, a reporter protein (e.g., Luciferase, GFP, EGFP, ⁇ -Galactosidase, EGFP), or a marker or selection protein (e.g., alpha-Globin, Galactokinase and Xanthine:guanine phosphoribosyl transferase (GPT)).
  • a reporter protein e.g., Luciferase, GFP, EGFP, ⁇ -Galactosidase, EGFP
  • a marker or selection protein e.g., alpha-Globin, Galactokinase and Xanthine:guanine phosphoribosyl transferase (GPT)
  • GPT galanine phosphoribosyl transferase
  • an mRNA includes the combination of a poly(A) sequence or polyadenylation signal and at least one histone stem-loop
  • an mRNA does not include a histone downstream element (HDE).
  • Histone downstream element includes a purine-rich polynucleotide stretch of approximately 15 to 20 nucleotides 3′ of naturally-occurring stem-loops, representing the binding site for the U7 snRNA, which is involved in processing of histone pre-mRNA into mature histone mRNA.
  • the nucleic acid does not include an intron.
  • an mRNA may or may not contain an enhancer and/or promoter sequence, which may be modified or unmodified or which may be activated or inactivated.
  • the histone stem-loop is generally derived from histone genes and includes an intramolecular base pairing of two neighbored partially or entirely reverse complementary sequences separated by a spacer, consisting of a short sequence, which forms the loop of the structure.
  • the unpaired loop region is typically unable to base pair with either of the stem loop elements. It occurs more often in RNA, as is a key component of many RNA secondary structures but may be present in single- stranded DNA as well. Stability of the stem-loop structure generally depends on the length, number of mismatches or bulges, and base composition of the paired region.
  • wobble base pairing (non-Watson-Crick base pairing) may result.
  • the at least one histone stem-loop sequence comprises a length of 15 to 45 nucleotides.
  • an mRNA has one or more AU-rich sequences removed. These sequences, sometimes referred to as AURES are destabilizing sequences found in the 3 ’UTR. The AURES may be removed from the mRNA. Alternatively, the AURES may remain in the mRNA. Sequence Optimization In some embodiments, an open reading frame encoding a protein of the disclosure is codon optimized. Codon optimization methods are known in the art. An open reading frame of any one or more of the sequences provided herein may be codon optimized.
  • Codon optimization may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove/add post translation modification sites in encoded protein (e.g., glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or reduce or eliminate problem secondary structures within the polynucleotide.
  • encoded protein e.g., glycosylation sites
  • add, remove or shuffle protein domains add or delete restriction sites
  • modify ribosome binding sites and mRNA degradation sites adjust translational rates to allow the various domains of the protein to fold properly; or reduce or eliminate problem secondary structures within the polynucleotide.
  • Codon optimization tools, algorithms and services are known in the art – non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and/or proprietary methods.
  • the open reading frame sequence is optimized using optimization algorithms.
  • a codon optimized sequence shares less than 95% sequence identity to a naturally-occurring or wild-type sequence open reading frame (e.g., a naturally- occurring or wild-type mRNA sequence encoding a Borrelia protein antigen).
  • a codon optimized sequence shares less than 90% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a Borrelia protein).
  • a codon optimized sequence shares less than 85% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a Borrelia protein). In some embodiments, a codon optimized sequence shares less than 80% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a Borrelia protein). In some embodiments, a codon optimized sequence shares less than 75% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a Borrelia protein).
  • a codon optimized sequence shares between 65% and 85% (e.g., between about 67% and about 85% or between about 67% and about 80%) sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a Borrelia protein). In some embodiments, a codon optimized sequence shares between 65% and 75% or about 80% sequence identity to a naturally-occurring or wild- type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a Borrelia protein).
  • a codon-optimized sequence encodes an antigen that is as immunogenic as, or more immunogenic than (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 200% more), than a Borrelia protein encoded by a non-codon-optimized sequence.
  • the modified mRNAs When transfected into mammalian host cells, the modified mRNAs have a stability of between 12-18 hours, or greater than 18 hours, e.g., 24, 36, 48, 60, 72, or greater than 72 hours and are capable of being expressed by the mammalian host cells.
  • a codon optimized mRNA may be one in which the levels of G/C are enhanced.
  • the G/C-content of nucleic acid molecules may influence the stability of the RNA.
  • mRNA having an increased amount of guanine (G) and/or cytosine (C) residues may be functionally more stable than RNA containing a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides.
  • WO02/098443 discloses a pharmaceutical composition containing an mRNA stabilized by sequence modifications in the translated region. Due to the degeneracy of the genetic code, the modifications work by substituting existing codons for those that promote greater RNA stability without changing the resulting amino acid. The approach is limited to coding regions of the mRNA.
  • an mRNA is not chemically modified and comprises the standard ribonucleotides consisting of adenosine, guanosine, cytosine and uridine.
  • nucleotides and nucleosides of the present disclosure comprise standard nucleoside residues such as those present in transcribed RNA (e.g., A, G, C, or U).
  • nucleotides and nucleosides of the present disclosure comprise standard deoxyribonucleosides such as those present in DNA (e.g., dA, dG, dC, or dT).
  • compositions of the present disclosure comprise, in some embodiments, an RNA having an open reading frame encoding a Borrelia protein, wherein the nucleic acid comprises nucleotides and/or nucleosides that can be standard (unmodified) or modified as is known in the art.
  • nucleotides and nucleosides of the present disclosure comprise modified nucleotides or nucleosides.
  • modified nucleotides and nucleosides can be naturally-occurring modified nucleotides and nucleosides or non-naturally-occurring modified nucleotides and nucleosides.
  • Such modifications can include those at the sugar, backbone, or nucleobase portion of the nucleotide and/or nucleoside as are recognized in the art.
  • a naturally-occurring modified nucleotide or nucleotide of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such naturally-occurring modified nucleotides and nucleotides can be found, inter alia, in the widely recognized MODOMICS database.
  • a non-naturally-occurring modified nucleotide or nucleoside of the disclosure is one as is generally known or recognized in the art.
  • nucleic acids of the disclosure can comprise standard nucleotides and nucleosides, naturally-occurring nucleotides and nucleosides, non-naturally-occurring nucleotides and nucleosides, or any combination thereof.
  • Nucleic acids of the disclosure comprise various (more than one) different types of standard and/or modified nucleotides and nucleosides.
  • a particular region of a nucleic acid contains one, two or more (optionally different) types of standard and/or modified nucleotides and nucleosides.
  • a modified mRNA introduced to a cell or organism exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.
  • a modified mRNA introduced into a cell or organism may exhibit reduced immunogenicity in the cell or organism, respectively (e.g., a reduced innate response) relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.
  • Nucleic acids e.g., RNA, such as mRNA
  • Nucleic acids comprise non-natural modified nucleotides that are introduced during synthesis or post-synthesis of the nucleic acids to achieve desired functions or properties.
  • the modifications may be present on internucleotide linkages, purine or pyrimidine bases, or sugars.
  • the modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain.
  • nucleic acid e.g., RNA, such as mRNA
  • a “nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”).
  • a “nucleotide” refers to a nucleoside, including a phosphate group.
  • Modified nucleotides may by synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides.
  • Nucleic acids can comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the nucleic acids would comprise regions of nucleotides.
  • Modified nucleotide base pairing encompasses not only the standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and/or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures, such as, for example, in those nucleic acids having at least one chemical modification.
  • non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine or uracil.
  • modified nucleobases in nucleic acids comprise 1-methyl-pseudouridine (m1 ⁇ ), 1-ethyl-pseudouridine (e1 ⁇ ), 5-methoxy- uridine (mo5U), 5-methyl-cytidine (m5C), and/or pseudouridine ( ⁇ ).
  • modified nucleobases in nucleic acids comprise 5-methoxymethyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, and/or 5- methoxy cytidine.
  • the polyribonucleotide includes a combination of at least two (e.g., 2, 3, 4 or more) of any of the aforementioned modified nucleobases, including but not limited to chemical modifications.
  • an mRNA of the disclosure comprises 1-methyl-pseudouridine (m1 ⁇ ) substitutions at one or more or all uridine positions of the nucleic acid.
  • an mRNA of the disclosure comprises 1-methyl-pseudouridine (m1 ⁇ ) substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the nucleic acid.
  • an mRNA of the disclosure comprises pseudouridine ( ⁇ ) substitutions at one or more or all uridine positions of the nucleic acid.
  • an mRNA of the disclosure comprises pseudouridine ( ⁇ ) substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the nucleic acid.
  • an mRNA includes N1-methylpseudouridine. In some embodiments, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of uracil nucleotides in an mRNA comprise N1- methylpseudouridine. In some embodiments, each uracil nucleotide of an mRNA transcript comprises N1-methylpseudouridine. In some embodiments, an mRNA includes 5- methylcytidine.
  • At least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of cytosine nucleotides in an mRNA comprise 5-methylcytidine.
  • each cytosine nucleotide of an mRNA transcript comprises 5-methylcytidine.
  • an mRNA includes 5- methyluridine.
  • at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of uracil nucleotides in an mRNA comprise 5-methyluridine.
  • each uracil nucleotide of an mRNA transcript comprises 5-methyluridine.
  • an mRNA includes 5-methylcytidine and 5- methyluridine.
  • at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of uracil nucleotides in an mRNA comprise 5-methyluridine and at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of cytosine nucleotides in an mRNA comprise 5-methylcytidine.
  • each cytosine nucleotide of an mRNA transcript comprises 5-methylcytidine and each uracil nucleotide of an mRNA transcript comprises 5- methyluridine.
  • an mRNA of the disclosure comprises uridine at one or more or all uridine positions of the nucleic acid.
  • RNAs e.g., mRNAs
  • a nucleic acid can be uniformly modified with 1-methyl-pseudouridine, meaning that all uridine residues in the mRNA sequence are replaced with 1-methyl-pseudouridine.
  • nucleic acid can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as those set forth above.
  • the nucleic acids of the present disclosure may be partially or fully modified along the entire length of the molecule.
  • one or more or all or a given type of nucleotide e.g., purine or pyrimidine, or any one or more or all of A, G, U, C
  • nucleotides X in a nucleic acid of the present disclosure are modified nucleotides, wherein X may be any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.
  • the nucleic acid may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to
  • the mRNA may contain at a minimum 1% and at maximum 100% modified nucleotides, or any intervening percentage, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides.
  • the nucleic acids may contain a modified pyrimidine such as a modified uracil or cytosine.
  • At least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in the nucleic acid is replaced with a modified uracil (e.g., a 5-substituted uracil).
  • the modified uracil can be replaced by a compound having a single unique structure or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).
  • cytosine in the nucleic acid is replaced with a modified cytosine (e.g., a 5-substituted cytosine).
  • the modified cytosine can be replaced by a compound having a single unique structure or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).
  • an Identification and Ratio Determination (IDR) sequence is a sequence of a biological molecule (e.g., nucleic acid or protein) that, when combined with the sequence of a target biological molecule, serves to identify the target biological molecule.
  • an IDR sequence is a heterologous sequence that is incorporated within or appended to a sequence of a target biological molecule and can be used as a reference to identify the target molecule.
  • a nucleic acid e.g., mRNA
  • a target sequence of interest e.g., a coding sequence encoding an antigenic peptide or protein
  • a unique IDR sequence e.g., a unique IDR sequence.
  • RNA species may comprise an IDR sequence that differs from the IDR sequence of other RNA species (e.g., RNA(s) having different coding sequence(s)).
  • Each IDR sequence thus identifies a particular RNA species, and so the abundance of IDR sequences may be measured to determine the abundance of each RNA species in a composition.
  • Use of distinct IDR sequences to identify RNA species allows for analysis of multivalent RNA compositions (e.g., containing multiple RNA species) containing RNA species with similar coding sequences and/or lengths, which could otherwise be difficult to distinguish using PCR- or chromatography-based analysis of full-length RNAs.
  • Each RNA species in a multivalent RNA composition may comprise an IDR sequence that is not a sequence isomer of an IDR sequence of another RNA species in a multivalent RNA composition (e.g., the IDR sequence does not have the same number of adenosine nucleotides, the same number of cytosine nucleotides, the same number of guanine nucleotides, and the same number of uracil nucleotides, as another IDR sequence in the composition, even if those sequences have different sequences).
  • Having identical nucleotide compositions causes sequence isomers to have the same mass, presenting a challenge to distinguishing sequence isomers using mass-based identification methods (e.g., mass spectrometry).
  • Each RNA species in a multivalent RNA composition may comprise an IDR sequence having a mass that differs from the mass of IDR sequences of each other RNA species in a multivalent RNA composition.
  • the mass of each IDR sequence may differ from the mass of other IDR sequences by at least 9 Da, at least 25 Da, at least 25 Da, or at least 50 Da.
  • Use of IDR sequences with distinct masses allows RNA fragments comprising different IDR sequences to be distinguished using mass-based analysis methods (e.g., mass spectrometry), which do not require reverse transcription, amplification, or sequencing of RNAs.
  • Each RNA species in an RNA composition may comprises an IDR sequence with a different length.
  • each IDR sequence may have a length independently selected from 0 to 25 nucleotides.
  • the length of a nucleic acid influences the rate at which the nucleic acid traverses a chromatography column, and so the use of IDR sequences of different lengths on different RNA species allows RNA fragments having different IDR sequences to be distinguished using chromatography-based methods (e.g., LC-UV).
  • IDR sequences may be chosen such that no IDR sequence comprises a start codon, ‘AUG’. Lack of a start codon in an IDR sequence prevents undesired translation of nucleotide sequences within and/or downstream from the IDR sequence.
  • IDR sequences may be chosen such that no IDR sequence comprises a recognition site for a restriction enzyme.
  • no IDR sequence comprises a recognition site for XbaI, ‘UCUAG’.
  • Lack of a recognition site for a restriction enzyme e.g., XbaI recognition site ‘UCUAG’) allows the restriction enzyme to be used in generating and modifying a DNA template for in vitro transcription, without affecting the IDR sequence or sequence of the transcribed RNA.
  • Nucleic Acid Production Chemical Synthesis Solid-phase chemical synthesis. Nucleic acids the present disclosure may be manufactured in whole or in part using solid phase techniques. Solid-phase chemical synthesis of nucleic acids is an automated method wherein molecules are immobilized on a solid support and synthesized step by step in a reactant solution.
  • Solid-phase synthesis is useful in site-specific introduction of chemical modifications in the nucleic acid sequences.
  • the synthesis of nucleic acids of the present disclosure by the sequential addition of monomer building blocks may be carried out in a liquid phase.
  • the synthetic methods discussed above each has its own advantages and limitations. Attempts have been conducted to combine these methods to overcome the limitations. Such combinations of methods are within the scope of the present disclosure.
  • the use of solid-phase or liquid-phase chemical synthesis in combination with enzymatic ligation provides an efficient way to generate long chain nucleic acids that cannot be obtained by chemical synthesis alone. Ligation Assembling nucleic acids by a ligase may also be used.
  • DNA or RNA ligases promote intermolecular ligation of the 5’ and 3’ ends of polynucleotide chains through the formation of a phosphodiester bond.
  • Nucleic acids such as chimeric polynucleotides and/or circular nucleic acids may be prepared by ligation of one or more regions or subregions. DNA fragments can be joined by a ligase catalyzed reaction to create recombinant DNA with different functions. Two oligodeoxynucleotides, one with a 5’ phosphoryl group and another with a free 3’ hydroxyl group, serve as substrates for a DNA ligase.
  • nucleic acid clean-up may include, but is not limited to, nucleic acid clean-up, quality assurance and quality control. Clean-up may be performed by methods known in the arts such as, but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNATM oligo-T capture probes (EXIQON® Inc, Vedbaek, Denmark) or HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC).
  • AGENCOURT® beads Beckman Coulter Genomics, Danvers, MA
  • poly-T beads poly-T beads
  • LNATM oligo-T capture probes EXIQON® Inc, Vedbaek, Denmark
  • HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (
  • purified when used in relation to a nucleic acid such as a “purified nucleic acid” refers to one that is separated from at least one contaminant.
  • a “contaminant” is any substance that makes another unfit, impure or inferior.
  • a purified nucleic acid e.g., DNA and RNA
  • a quality assurance and/or quality control check may be conducted using methods such as, but not limited to, gel electrophoresis, UV absorbance, or analytical HPLC.
  • the nucleic acids may be sequenced by methods including, but not limited to reverse-transcriptase-PCR. Quantification In some embodiments, the nucleic acids of the present disclosure may be quantified in exosomes or when derived from one or more bodily fluid.
  • Bodily fluids include peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood.
  • CSF cerebrospinal fluid
  • saliva aqueous humor
  • amniotic fluid cerumen
  • breast milk broncheoalveolar lavage fluid
  • exosomes may be retrieved from an organ selected from the group consisting of lung, heart, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colon, breast, prostate, brain, esophagus, liver, and placenta.
  • Assays may be performed using construct specific probes, cytometry, qRT-PCR, real- time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or combinations thereof while the exosomes may be isolated using immunohistochemical methods such as enzyme linked immunosorbent assay (ELISA) methods.
  • ELISA enzyme linked immunosorbent assay
  • Exosomes may also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, or combinations thereof. These methods afford the investigator the ability to monitor, in real time, the level of nucleic acids remaining or delivered. This is possible because the nucleic acids of the present disclosure, in some embodiments, differ from the endogenous forms due to the structural or chemical modifications. In some embodiments, the nucleic acid may be quantified using methods such as, but not limited to, ultraviolet visible spectroscopy (UV/Vis).
  • UV/Vis ultraviolet visible spectroscopy
  • a non-limiting example of a UV/Vis spectrometer is a NANODROP® spectrometer (ThermoFisher, Waltham, MA).
  • the quantified nucleic acid may be analyzed in order to determine if the nucleic acid may be of proper size, check that no degradation of the nucleic acid has occurred.
  • Degradation of the nucleic acid may be checked by methods such as, but not limited to, agarose gel electrophoresis, HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC- HPLC), liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis (CGE).
  • IVTT in vitro transcription
  • RNA of the present disclosure is prepared in accordance with any one or more of the methods described in WO 2018/053209 or WO 2019/036682, each of which is incorporated by reference herein.
  • the mRNA transcript is generated using a non-amplified, linearized DNA template in an in vitro transcription reaction to generate the RNA transcript.
  • the template DNA is isolated DNA.
  • the template DNA is cDNA.
  • the cDNA is formed by reverse transcription of an RNA, for example, but not limited to Borrelia mRNA.
  • cells e.g., bacterial cells, e.g., E. coli, e.g., DH-1 cells are transfected with the plasmid DNA template.
  • the transfected cells are cultured to replicate the plasmid DNA which is then isolated and purified.
  • the DNA template includes an RNA polymerase promoter, e.g., a T7 promoter located 5 ' to and operably linked to the gene of interest.
  • an in vitro transcription template encodes a 5′ untranslated (UTR) region, contains an open reading frame, and encodes a 3′ UTR and a poly(A) tail.
  • UTR untranslated
  • a nucleic acid (e.g., template DNA and/or RNA) includes 200 to 3,000 nucleotides.
  • a nucleic acid may include 200 to 500, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to 3000 nucleotides.
  • An in vitro transcription system typically comprises a transcription buffer (e.g., with magnesium), nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase (e.g., T7 RNA polymerase).
  • NTPs nucleotide triphosphates
  • an RNase inhibitor e.g., T7 RNA polymerase
  • a polymerase e.g., T7 RNA polymerase
  • one or more of the NTPs is a chemically modified NTP (e.g., with 1-methylpseudouridine or other chemical modifications described herein and/or known in the art).
  • the NTPs comprise adenosine triphosphate (ATP), cytidine triphosphate (CTP), uridine triphosphate (UTP), and guanosine triphosphate (GTP), or an analog of each respective NTP.
  • the ratio of NTPs may vary.
  • the ratio of GTP:ATP:CTP:UTP is 1:1:1:1. In some embodiments, the amount of the GTP or an analogue thereof is greater than an amount of the UTP or an analogue thereof. In some embodiments, the amount of the GTP is greater than the amount of the UTP. In some embodiments, the amount of ATP is greater than the amount of UTP, and the amount of CTP is greater than the amount of UTP. In some embodiments, the amount of the GTP or an analogue thereof is greater than an amount of the UTP or an analogue thereof.
  • an IVT system comprises an at least 2:1 ratio of GTP concentration to ATP concentration, an at least 2:1 ratio of GTP concentration to CTP concentration, and an at least 4:1 ratio of GTP concentration to UTP concentration.
  • an IVT system comprises a 2:1 ratio of GTP concentration to ATP concentration, a 2:1 ratio of GTP concentration to CTP concentration, and a 4:1 ratio of GTP concentration to UTP concentration.
  • an IVT system comprises guanosine diphosphate (GDP).
  • GDP guanosine diphosphate
  • an IVT system comprises an at least 3:1 ratio of GTP plus GDP concentration to ATP concentration, an at least 6:1 ratio of GTP plus GDP concentration to CTP concentration, and an at least 6:1 ratio of GTP plus GDP concentration to UTP concentration.
  • the NTPs may be manufactured in house, may be selected from a supplier, or may be synthesized as described herein.
  • the NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified) NTPs. Any number of RNA polymerases or variants may be used in the method of the present disclosure.
  • the polymerase may be selected from, but is not limited to, a phage RNA polymerase, e.g., a T7 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase, and/or mutant polymerases such as, but not limited to, polymerases able to incorporate modified nucleic acids and/or modified nucleotides, including chemically modified nucleic acids and/or nucleotides. Some embodiments exclude the use of DNase.
  • An IVT system in some embodiments, comprises magnesium buffer, dithiothreitol (DTT) spermidine, pyrophosphatase, and/or RNase inhibitor. In some embodiments, an IVT system omits an RNase inhibitor.
  • RNA transcript is capped via enzymatic capping.
  • the RNA comprises 5' terminal cap, for example, 7mG(5’)ppp(5’)NlmpNp.
  • the nucleic acids of in e.g., formulated as) a lipid composition, such as a composition comprising a lipid nanoparticle, a liposome, and/or a lipoplex.
  • nucleic acids of the present disclosure are in (e.g., formulated as) lipid nanoparticle (LNP) compositions.
  • LNP lipid nanoparticle
  • Lipid nanoparticles typically comprise amino lipid, non- cationic lipid, structural lipid, and PEG lipid components along with the nucleic acid cargo (e.g., RNA, such as mRNA) of interest.
  • a lipid nanoparticles of the present disclosure can be generated using components, compositions, and methods as are generally known in the art, see for example PCT/US2016/052352; PCT/US2016/068300; PCT/US2017/037551; PCT/US2015/027400; PCT/US2016/047406; PCT/US2016/000129; PCT/US2016/014280; PCT/US2017/038426; PCT/US2014/027077; PCT/US2014/055394; PCT/US2016/052117; PCT/US2012/069610; PCT/US2017/027492; PCT/US2016/059575; PCT/US2016/069491; PCT/US2016/069493; and PCT/US2014/066242, all of which are incorporated by reference herein in their entirety.
  • a lipid nanoparticle comprises at least one ionizable amino lipid, at least one non-cationic lipid, at least one sterol, and/or at least one polyethylene glycol (PEG)- modified lipid.
  • a lipid nanoparticle comprises 20-60 mole percent (mol%) ionizable amino lipid, 5-25 mol% non-cationic lipid, 25-55 mol% structural lipid, and 0.5-15 mol% PEG-modified lipid.
  • a lipid nanoparticle comprises 20-60 mol% ionizable amino lipid, 5-30 mol% non-cationic lipid, 10-55 mol% structural lipid, and 0.5-15 mol% PEG-modified lipid.
  • a lipid nanoparticle comprises 40-50 mol% ionizable lipid, optionally 45-50 mol%, for example, 45-46 mol%, 46-47 mol%, 47-48 mol%, 48-49 mol%, or 49-50 mol% for example about 45 mol%, 45.5 mol%, 46 mol%, 46.5 mol%, 47 mol%, 47.5 mol%, 48 mol%, 48.5 mol%, 49 mol%, or 49.5 mol%.
  • a lipid nanoparticle comprises 20-60 mol% ionizable amino lipid.
  • a lipid nanoparticle may comprise 20-50 mol%, 20-40 mol%, 20-30 mol%, 30-60 mol%, 30-50 mol%, 30-40 mol%, 40-60 mol%, 40-50 mol%, or 50-60 mol% ionizable amino lipid.
  • a lipid nanoparticle comprises 20 mol%, 30 mol%, 40 mol%, 50 mol%, or 60 mol% ionizable amino lipid.
  • a lipid nanoparticle comprises 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, or 55 mol% ionizable amino lipid. In some embodiments, a lipid nanoparticle comprises 45-55 mol% ionizable amino lipid.
  • lipid nanoparticle may comprise 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mol% ionizable amino lipid.
  • Ionizable amino lipids in some embodiments, the ionizable amino lipid of the present disclosure is a compound of Formula (AI): its N-oxide, or a salt or isomer thereof, wherein R’ a is R’ branched ; wherein R’ branched is: ; wherein denotes a point of attachment; wherein R a ⁇ , R a ⁇ , R a ⁇ , and R a ⁇ are each independently selected from the group consisting of H, C 2-12 alkyl, and C 2-12 alkenyl; R 2 and R 3 are each independently selected from the group consisting of C 1-14 alkyl and C2-14 alkenyl; R 4 is selected from the group consisting of -(CH 2 ) n OH, wherein n is selected from the group consisting , wherein denotes
  • R’ a is R’ branched ;
  • R’ branched is a point of attachment;
  • R a ⁇ , R a ⁇ , R a ⁇ , and R a ⁇ are each H;
  • R 2 are 14
  • R 4 is -(CH2)nOH; n is 2;
  • each R 5 is H;
  • each R 6 is H;
  • M and M’ are each -C(O)O-;
  • R’ is a C 1-12 alkyl; l is 5; and m is 7.
  • R’ a is R’ branched ;
  • R’ branched is attach a ⁇ a ⁇ a ⁇ a ⁇ 2 ment;
  • R , R , R , and R are each H;
  • R and R 3 are each C1-14 alkyl;
  • R 4 is -(CH2)nOH; n is 2;
  • each R 5 is H;
  • each R 6 is H;
  • M and M’ are each - C(O)O-;
  • R’ is a C1-12 alkyl; l is 3; and m is 7.
  • R’ a is R’ branched ; R’ branched is a point of attachment; R a ⁇ is C2-12 alkyl; R a ⁇ , R a ⁇ , and R a ⁇ are each H; R 2 and R 3 are each C 1-14 alkyl; ; R 10 NH(C 1-6 alkyl); n2 is 2; R 5 is H; each R 6 is H; M and M’ are each -C(O)O-; R’ is a C 1-12 alkyl; l is 5; and m is 7.
  • R’ a is R’ branched ; R’ branched is ; a point of attachment; R a ⁇ , R a ⁇ , and R a ⁇ are each H; R a ⁇ is C 2- 12 alkyl; R 2 and R 3 are each C1-14 alkyl; R 4 is -(CH2)nOH; n is 2; each R 5 is H; each R 6 is H; M and M’ are each -C(O)O-; R’ is a C1-12 alkyl; l is 5; and m is 7.
  • the ionizable amino lipid is a compound of Formula (AIa): its N-oxide, or a salt or isomer thereof, wherein R’ a is R’ branched ; wherein denotes a point of attachment; wherein R a ⁇ , R a ⁇ , and R a ⁇ are each independently selected from the group consisting of H, C2-12 alkyl, and C2-12 alkenyl; R 2 and R 3 are each independently selected from the group consisting of C1-14 alkyl and C 2-14 alkenyl; R 4 is selected from the group wherein n is selected from the group consisting of 1, 2, 3, 4, and 5, and , wherein denotes a point of attachment; wherein R 10 is N(R) 2 ; each R is independently selected from the group consisting of C1-6 alkyl, C2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10
  • the ionizable amino lipid is a compound of Formula (AIb): its N-oxide, or a salt or isomer thereof, wherein R’ a is R’ branched ; wherein denotes a point of attachment; wherein R a ⁇ , R a ⁇ , R a ⁇ , and R a ⁇ are each independently selected from the group consisting of H, C 2-12 alkyl, and C 2-12 alkenyl; R 2 and R 3 are each independently selected from the group consisting of C 1-14 alkyl and C2-14 alkenyl; R 4 is -(CH2)nOH, wherein n is selected from the group consisting of 1, 2, 3, 4, and 5; each R 5 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H; each R 6 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; M and M’ are each independently selected from the group consisting of -C(AIb):
  • R’ a is R’ branched ; R’ branched is a point of attachment; R a ⁇ , R a ⁇ , and R a ⁇ are each H; R 2 and R 3 -(CH2)nOH; n is 2; each R 5 is H; each R 6 is H; M and M’ are each - C(O)O-; R’ is a C 1-12 alkyl; l is 5; and m is 7.
  • R’ a is R’ branched ; R’ branched is a point of attachment; R a ⁇ , R a ⁇ , and R a ⁇ are each H; R 2 and R 3 are each C 1-14 alkyl; R 4 is -(CH 2 ) n OH; n is 2; each R 5 is H; each R 6 is H; M and M’ are each - C(O)O-; R’ is a C1-12 alkyl; l is 3; and m is 7.
  • R’ a is R’ branched ; is a point of attachment; R a ⁇ and R a ⁇ are each H; R a ⁇ is C2-12 alkyl; R 2 and R 3 are each C 1-14 alkyl; R 4 is -(CH 2 ) n OH; n is 2; each R 5 is H; each R 6 is H; M and M’ are each -C(O)O-; R’ is a C1-12 alkyl; l is 5; and m is 7.
  • the ionizable amino lipid is a compound of Formula (AIc): its N-oxide, or a salt or isomer thereof, wherein R’ a is: ; wherein denotes a point of attachment; wherein R a ⁇ , R a ⁇ , R a ⁇ , and R a ⁇ are each independently selected from the group consisting of H, C2-12 alkyl, and C2-12 alkenyl; R 2 and R 3 are each independently selected from the group consisting of C 1-14 alkyl and C 2-14 alkenyl; denotes a point of attachment; wherein R 10 is N(R)2; group consisting of C1-6 alkyl, C2-3 alkenyl, and H; n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R 5 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H; each R 6 is independently selected from the group consisting of C1-3 alkyl
  • R a ⁇ , R a ⁇ , and R a ⁇ are each H; R a ⁇ is C 2-12 alkyl; R 2 and R 3 are each C 1-14 alkyl; a point of attachment; R 10 is NH(C1-6 alkyl); n2 is 2; each R 5 is H; each R 6 is H; M and M’ are each -C(O)O-; R’ is a C 1-12 alkyl; l is 5; and m is 7.
  • the of Formula (AIc) is: .
  • the ionizable amino lipid is a compound of Formula (AII): of attachment; R a ⁇ and R a ⁇ are each independently selected from the group consisting of H, C 1-12 alkyl, and C2-12 alkenyl, wherein at least one of R a ⁇ and R a ⁇ is selected from the group consisting of C 1-12 alkyl and C 2-12 alkenyl; R b ⁇ and R b ⁇ are each independently selected from the group consisting of H, C 1-12 alkyl, and C2-12 alkenyl, wherein at least one of R b ⁇ and R b ⁇ is selected from the group consisting of C1-12 alkyl and C2-12 alkenyl; R 2 and R 3 are each independently selected from the group consisting of C 1-14 alkyl and C2-14 alkenyl; R 4 is selected from the group consisting of -(CH2)nOH wherein n is selected from the group consisting , wherein denotes a point of attachment; wherein R 10 is N
  • the ionizable amino lipid is a compound of Formula (AII-a): ; wherein denotes a point of attachment; R a ⁇ and R a ⁇ are each independently selected from the group consisting of H, C 1-12 alkyl, and C 2-12 alkenyl, wherein at least one of R a ⁇ and R a ⁇ is selected from the group consisting of C1-12 alkyl and C2-12 alkenyl; R b ⁇ and R b ⁇ are each independently selected from the group consisting of H, C1-12 alkyl, and C 2-12 alkenyl, wherein at least one of R b ⁇ and R b ⁇ is selected from the group consisting of C1-12 alkyl and C2-12 alkenyl; R 2 and R 3 are each independently selected from the group consisting of C1-14 alkyl and C 2-14 alkenyl; R 4 is selected from the group consisting of -(CH2)nOH wherein n is selected from the group consisting , wherein a point of attachment; R
  • the ionizable amino lipid is a compound of Formula (AII-b): wherein denotes a point of attachment; R a ⁇ and R b ⁇ are each independently selected from the group consisting of C1-12 alkyl and C 2-12 alkenyl; R 2 and R 3 are each independently selected from the group consisting of C1-14 alkyl and C2-14 alkenyl; R 4 is selected from the group consisting of -(CH 2 ) n OH wherein n is selected from the group consisting , wherein denotes a point of attachment; wherein R 10 is N(R)2; each R is independently selected from the group consisting of C 1-6 alkyl, C 2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R’ independently is a C1-12 alkyl or C2-12 alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2,
  • R’ branched is: and R’ b is: ; wherein denotes a point of attachment; wherein from the group consisting of C1-12 alkyl and C2-12 alkenyl; R 2 and R 3 are each independently selected from the group consisting of C1-14 alkyl and C 2-14 alkenyl; R 4 is selected from the group consisting of -(CH2)nOH wherein n is selected from the group consisting , wherein denotes a point of attachment; 2 ; from the group consisting of C1-6 alkyl, C2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; R’ is a C 1-12 alkyl or C 2-12 alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.
  • the ionizable amino lipid is a compound of Formula (AII-d): its N-oxide, or a salt or isomer thereof, wherein R’ a is R’ branched or R’ cyclic ; wherein ; wherein denotes a point of attachment; wherein R a ⁇ and R b ⁇ are each independently selected from the group consisting of C 1-12 alkyl and C2-12 alkenyl; R 4 is wherein n is selected from the group consisting of 1, 2, 3, 4, and 5, and , wherein denotes a point of attachment; wherein R 10 is N(R)2; each R is independently selected from the group consisting of C 1-6 alkyl, C 2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R’ independently is a C 1-12 alkyl or C 2-12 alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from
  • the ionizable amino lipid is a compound of Formula (AII-e): its N-oxide, or a salt or isomer thereof, wherein R’ a is or wherein ; wherein denotes a point of attachment; wherein R a ⁇ is selected from the group consisting of C 1-12 alkyl and C 2-12 alkenyl; R 2 and R 3 are each independently selected from the group consisting of C 1-14 alkyl and C2-14 alkenyl; R 4 is -(CH 2 ) n OH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5; R’ is a C 1-12 alkyl or C 2-12 alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.
  • AII-e N-oxide, or a salt or isomer thereof, wherein R’ a is or wherein ; wherein denotes a point of attachment; wherein R a ⁇
  • m and l are each independently selected from 4, 5, and 6. In some embodiments of the compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), m and l are each 5. In some embodiments of the compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e), each R’ independently is a C 1-12 alkyl.
  • each R’ independently is a C2-5 alkyl.
  • R’ b is: and R 2 and R 3 are each independently a C1-14 alkyl.
  • R’ b is: and R 2 and R 3 are each independently a C6-10 alkyl.
  • R’ b is: and R 2 and R 3 are each a C8 alkyl.
  • R’ branched is: and R 3 are each is: , R a ⁇ is a C2-6 alkyl and R 2 and R 3 are each independently a C6-10 alkyl.
  • R a ⁇ is a C2-6 alkyl
  • R 2 and R 3 are each independently a C6-10 alkyl.
  • 6 alkyl and R 2 and R 3 are each a C 8 alkyl.
  • the compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII- d) are each a C1-12 alkyl. In some embodiments of the compound of Formula (AII), (AII-a), (AII-b), , , , , and R b ⁇ are each a C 2-6 alkyl. In some embodiments of the compound of Formula (AII), (AII-a), (AII-b), (AII-c), (AII- d), or (AII-e), m and l are each independently selected from 4, 5, and 6 and each R’ independently is a C 1-12 alkyl.
  • m and l are each 5 and each R’ independently is a C 2-5 alkyl.
  • R’ branched is:
  • R’ b is:
  • m and l are each independently selected from 4, 5, and 6, each R’ independently is a C 1-12 alkyl, and R a ⁇ and R b ⁇ are each a C 1-12 alkyl.
  • each a 12 a 12 and R 2 and R 3 are each independently a C6-10 alkyl.
  • R’ is a C2-5 alkyl
  • R a ⁇ is a C2-6 alkyl
  • R 2 and R 3 are each a C8 alkyl.
  • R 10 is NH(C 1-6 alkyl) and n2 is 2.
  • R 4 is R 10 is NH(CH 3 ) and n2 is 2.
  • R’ branched is: , R’ b is: , m and l are each 5, each R’ independently is a C 2-5 alkyl, R a ⁇ and R b ⁇ are each a C 2-6 , wherein R 10 is NH(CH3) and n2 is 2.
  • R 10 is NH(CH3) and n2 is 2.
  • a 12 are a C6-10 alkyl, R a ⁇ is a C1-12 alkyl, and R 4 is , wherein R 10 is NH(C1-6 alkyl) and n2 is 2.
  • R’ is a C2-5 alkyl
  • R a ⁇ is a C2-6 alkyl
  • R 2 and R 3 are each a C8 alkyl
  • R 4 is , wherein R 10 is NH(CH3) and n2 is 2.
  • R 4 is -(CH2)nOH and n is 2, 3, or 4.
  • R 4 is -(CH2)nOH and n is 2.
  • each R’ independently is a C 1-12 alkyl
  • R a ⁇ and R b ⁇ are each a C1-12 - of Formula (AII), (AII-a), (AII-b), (AII-c), (AII-d), or (AII-e)
  • R’ b is:
  • m and l are each 5, each R’ independently is a C2-5 alkyl, R a ⁇ and R b ⁇ are each a C2-6 alkyl, R 4 is -(CH2)nOH, and n is 2.
  • the ionizable amino lipid is a compound of Formula (AII-f): ; wherein denotes a point of attachment; R a ⁇ is a C 1-12 alkyl; R 2 and R 3 are each independently a C1-14 alkyl; R 4 is -(CH 2 ) n OH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5; R’ is a C 1-12 alkyl; m is selected from 4, 5, and 6; and l is selected from 4, 5, and 6.
  • m and l are each 5, and n is 2, 3, or 4.
  • R’ is a C2-5 alkyl, R a ⁇ is a C2-6 alkyl, and R 2 and R 3 are each a C 6-10 alkyl.
  • m and l are each 5, n is 2, 3, or 4
  • R’ is a C2-5 alkyl, R a ⁇ is a C2-6 alkyl, and R 2 and R 3 are each a C6-10 alkyl.
  • the ionizable amino lipid is a compound of Formula (AII-g): , R a ⁇ is a C 2-6 alkyl; R’ is a C2-5 alkyl; and R 4 is selected from the group consisting of -(CH 2 ) n OH wherein n is selected from the group consisting wherein denotes a point of attachment, R 10 is , the group consisting of 1, 2, and 3.
  • m is 5, 7, or 9.
  • Q is OH, -NHC(S)N(R) 2 , or -NHC(O)N(R) 2 .
  • Q is -N(R)C(O)R, or -N(R)S(O) 2 R.
  • a subset of compounds of Formula (VI) includes those of Formula (VI-B): (VI-B), or its N-oxide, or a salt or isomer thereof in which all variables are as defined herein.
  • m is selected from 5, 6, 7, 8, and 9;
  • M and M’ are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M”-C(O)O-, -C(O)N(R’)-, -P(O)(OR’)O-, -S-S-, an aryl group,
  • m is 5, 7, or 9.
  • Q is OH, -NHC(S)N(R) 2 , or -NHC(O)N(R) 2 .
  • Q is -N(R)C(O)R, or -N(R)S(O) 2 R.
  • (VI) are of Formula (VIIa), (VIIa), or their N-oxides, or salts or isomers thereof, wherein R4 is as described herein.
  • the compounds of Formula (VI) are of Formula (VIIb), (VIIb), or their N-oxides, or salts or isomers as
  • the compounds of Formula (VI) are of Formula (VIIc) or (VIIe): their N-oxides, or salts or isomers thereof, wherein R 4 is as described herein.
  • the compounds of Formula (VI) are of Formula (VIIf): (VIIf) or their N-oxides, or salts or isomers thereof, wherein M is -C(O)O- or –OC(O)-, M” is C 1-6 alkyl or C 2-6 alkenyl, R 2 and R 3 are independently selected from the group consisting of C5-14 alkyl and C5-14 alkenyl, and n is selected from 2, 3, and 4.
  • the of Formula (VI) are of Formula (VIId), (VIId), or their N-oxides, or salts or isomers thereof, wherein n is 2, 3, or 4; and m, R’, R”, and R2 through R6 are as described herein.
  • each of R 2 and R 3 may be independently selected from the group consisting of C 5-14 alkyl and C 5-14 alkenyl.
  • an ionizable amino lipid of the disclosure comprises a compound having structure: .
  • an a compound (Compound II) In some an a compound (Compound II).
  • the compounds of Formula (VI) are of Formula (VIIg), (VIIg), or their N-oxides, or salts or isomers thereof, wherein l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; M1 is a bond or M’; M and M’ are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M”-C(O)O-, -C(O)N(R’)-, -P(O)(OR’)O-, -S-S-, an aryl group, and a heteroaryl group; and R 2 and R 3 are independently selected from the group consisting of H, C1-14 alkyl, and C2-14 alkenyl.
  • M is C1-6 alkyl (e.g., C1-4 alkyl) or C2-6 alkenyl (e.g. C2-4 alkenyl).
  • R2 and R 3 are independently selected from the group consisting of C 5-14 alkyl and C 5-14 alkenyl.
  • the ionizable amino lipids are one or more of the compounds described in U.S. Application Nos.
  • the central amine moiety of a lipid according to Formula (VI), (VI-A), (VI-B), (VII), (VIIa), (VIIb), (VIIc), (VIId), (VIIe), (VIIf), or (VIIg) may be protonated at a physiological pH.
  • a lipid may have a positive or partial positive charge at physiological pH.
  • Such amino lipids may be referred to as cationic lipids, ionizable lipids, cationic amino lipids, or ionizable amino lipids.
  • Amino lipids may also be zwitterionic, i.e., neutral molecules having both a positive and a negative charge.
  • the ionizable amino lipids of the present disclosure may be one or more of compounds of formula (VIII), , ; t is 1 or 2; A 1 and A 2 are each independently selected from CH or N; Z is CH2 or absent wherein when Z is CH2, the dashed lines (1) and (2) each represent a single bond; and when Z is absent, the dashed lines (1) and (2) are both absent; R 1 , R 2 , R 3 , R 4 , and R 5 are independently selected from the group consisting of C 5-20 alkyl, C5-20 alkenyl, -R”MR’, -R*YR”, -YR”, and -R*OR”; RX1 and RX2 are each independently H or C1-3 alkyl; each M is independently selected from the group consisting of -C(O)O-, -OC(O)-, -OC(O)O-, -C(O)N(R’)-, -N(R’)C(O)-,
  • the compound is of any of formulae (VIIIa1)-(VIIIa8): (VIIIa3),
  • the ionizable amino lipid is , a salt thereof.
  • the central amine moiety of a lipid according to Formula (VIII), (VIIIa1), (VIIIa2), (VIIIa3), (VIIIa4), (VIIIa5), (VIIIa6), (VIIIa7), or (VIIIa8) may be protonated at a physiological pH.
  • a lipid may have a positive or partial positive charge at physiological pH.
  • R 1 is optionally substituted C1-C24 alkyl or optionally substituted C2-C24 alkenyl
  • R 2 and R 3 are each independently optionally substituted C 1 -C 36 alkyl
  • R 4 and R 5 are each independently optionally substituted C 1 -C 6 alkyl, or R 4 and R 5 join, along with the N to which they are attached, to form a heterocyclyl or heteroaryl
  • L 1 , L 2 , and L 3 are each independently optionally substituted C 1 -C 18 alkylene
  • n is an integer
  • each R 1a is independently hydrogen, R 1c , or R 1d ; each R 1b is independently R 1c or R 1d ; each R 1c is independently –[CH 2 ] 2 C(O)X 1 R 3 ; each R 1d Is independently -C(O)R 4 ; each R 2 is independently -[C(R 2a )2]cR 2b ; each R 2a is independently hydrogen or C 1 -C 6 alkyl; R 2b is -N(L 1 -B) 2 ; -(OCH 2 CH 2 ) 6 OH; or -(OCH 2 CH 2 ) b OCH 3 ; each R 3 and R 4 is independently C6-C30 aliphatic; each I.3 is independently C1-C10 alkylene; each B is independently hydrogen or an ionizable nitrogen-containing group; each X 1 is independently a covalent
  • G 1a and G 2b are each independently C 2 -C 12 alkylene or C 2 -C 12 alkenylene;
  • G 1b and G 2b are each independently C 1 -C 12 alkylene or C 2 -C 12 alkenylene;
  • G 3 is C1-C24 alkylene, C2-C24 alkenylene, C3-C8 cycloalkylene or C3-C8 cycloalkenylene;
  • R a , R b , R d and R e are each independently H
  • R1 and R2 are the same or different, each a linear or branched alkyl with 1-9 carbons, or as alkenyl or alkynyl with 2 to 11 carbon atoms
  • L 1 and L 2
  • a lipid nanoparticle comprises an ionizable lipid having the structure: , or a pharmaceutically acceptable salt thereof. In some embodiments, a lipid nanoparticle comprises a lipid having the structure: a pharmaceutically acceptable salt thereof. In some embodiments, a lipid nanoparticle comprises a lipid having the structure: , or a pharmaceutically acceptable salt thereof. In some embodiments, a a a the structure: (XX-L), or a pharmaceutically acceptable salt thereof. In some embodiments, a lipid nanoparticle comprises a lipid having the structure: a In some embodiments, a lipid nanoparticle comprises a lipid having the structure: a pharmaceutically acceptable salt thereof.
  • a lipid nanoparticle comprises a lipid having the structure: (XXIII-L), or a pharmaceutically acceptable salt thereof. In some embodiments, a lipid nanoparticle comprises a lipid having the structure: , or a pharmaceutically acceptable salt thereof. having the structure: (XXV-L), or a pharmaceutically acceptable salt thereof. In some embodiments, a lipid nanoparticle comprises a lipid having the structure: (XXVI-L), or a pharmaceutically acceptable salt thereof. In some embodiments, a lipid nanoparticle comprises a lipid having the structure: a pharmaceutically acceptable salt thereof.
  • R 1 is NR N -C4-10 cycloalkenyl optionally substituted with one or more oxo or -N(R
  • ionizable lipid is of Formula (IL*-I): (IL*-Ia) or a salt thereof, wherein: R 1 , o, m, n, M, M’, R 2c , and R 3c are as defined for variable IL*; and R 3a is C 1-8 alkyl.
  • ionizable lipid is of Formula (IL*-Ia): (IL*-Ia) or a salt thereof, wherein: R 1 , o, m, n, M, M’, R 2c , and R 3c are as defined for Formula IL*; and R 3a is C1-8 alkyl.
  • the ionizable lipid is of Formula (IL*-Ia’): or a salt thereof, wherein: o, M, M’, R 2c and R 3c are as defined for variable IL*; and R 3a is C1-8 alkyl.
  • the ionizable lipid is of Formula (IL*-IIa): (IL*-IIa) or a salt thereof, wherein: R 1 , o, m, n, M, M’, R 2c , and R 3c are as defined for Formula IL*; and R 3a is C1-8 alkyl.
  • the ionizable is of Formula (IL*-II’): (IL*-II’) or a salt thereof, wherein: o, M, M’, R 2c and R 3c are as defined for variable IL*; and R 3a is C1-8 alkyl.
  • the ionizable lipid is of Formula (IL*-III): (IL*-III) or a salt thereof, wherein: R 1 , o, m, n, M, M’, R 2c , and R 3c are as defined for variable IL*; R 2a is a C 1-8 alkyl; and R 3a is C1-8 alkyl.
  • the ionizable lipid is of Formula (IL*-IIIa): (IL*-IIIa) or a salt thereof, wherein: R 1 , o, m, n, M, M’, R 2c , and R 3c are as defined for variable IL*; R 2b is a C 1-8 alkyl; and R 3a is C1-8 alkyl.
  • the ionizable lipid is of Formula (IL*-IIIa): (IL*-IIIa) or a salt thereof, wherein: R 1 , o, M, M’, R 2c , and R 3c are as defined for variable IL*; R 2a is a C1-8 alkyl; and R 3a is C 1-8 alkyl.
  • the ionizable lipid is of Formula (IL*-IIIb): (IL*-IIIb) or a salt thereof, wherein: R 1 , o, M, M’, R 2c , and R 3c are as defined for variable IL*; R 2a is a C1-8 alkyl; and R 3a is C 1-8 alkyl.
  • the ionizable lipid is of Formula (IL*-IIIb’): or a salt thereof, wherein: R 1 , o, M, M’, R 2c , and R 3c are as defined for variable IL*; R 2a is a C 1-8 alkyl; and R 3a is C1-8 alkyl.
  • the ionizable lipid is of Formula (IL*-IV): (IL*-IV) or a salt thereof, wherein: R 1 , o, m, n, M, M’, R 2c , and R 3c are as defined for variable IL*; R 2b is a C1-8 alkyl; and R 3a is C1-8 alkyl.
  • the ionizable lipid is of Formula (IL*-IVa): (IL*-IVa) or a salt thereof, wherein: R 1 , o, m, n, M, M’, R 2c , and R 3c are as defined for variable IL*; R 2b is a C 1-8 alkyl; and R 3a is C1-8 alkyl.
  • the ionizable lipid is of Formula (IL*-Iva’): (IL*-IVa) or a salt thereof, wherein: o, M, M’, R 2c , and R 3c are as defined for variable IL*; R 2a is a C1-8 alkyl; and R 3a is C 1-8 alkyl.
  • Variables o, R 1 , R N , R N’ , R N’’ of Ionizable Lipid In some embodiments of the ionizable lipid, o is 1. In some embodiments of the ionizable lipid, o is 2. In some embodiments of the ionizable lipid, o is 3.
  • o is 4. In some embodiments of the ionizable lipid, R 1 is -OH. In some embodiments of the ionizable lipid, R N is H. In some embodiments of the ionizable lipid, R N is methyl. In some embodiments of the ionizable lipid, R N is ethyl. In some embodiments of the ionizable lipid, R 1 is -NR N -cyclobutenyl, wherein the cyclobutenyl is optionally substituted with one or more oxo or -N(R N’ R N’’ ). In some embodiments of the ionizable lipid, R N’ is H.
  • R N’ is methyl. In some embodiments of the ionizable lipid, R N’ is ethyl. In some embodiments of the ionizable lipid, R N’’ is H. In some embodiments of the ionizable lipid, R N’’ is methyl. In some embodiments of the ionizable lipid, R N’’ is ethyl. In some embodiments of the ionizable lipid, R N’ is H and R N’’ is methyl. O 3 In some embodiments of the ionizable lipid, . In some embodiments of the ionizable lipid, .
  • Variables m and n of the Ionizable Lipid In some embodiments of the ionizable lipid, m is 4. In some embodiments of the ionizable lipid, m is 5. In some embodiments of the ionizable lipid, m is 6. In some embodiments of the ionizable lipid, m is 7. In some embodiments of the ionizable lipid, m is 8. In some embodiments of the ionizable lipid, m is 4. In some embodiments of the ionizable lipid, n is 5. In some embodiments of the ionizable lipid, n is 6. In some embodiments of the ionizable lipid, n is 7.
  • Variables R 2 , R 2a , R 2b , R 2c In some embodiments of the ionizable lipid, R 2 .
  • R 2a is hydrogen. In some embodiments of the ionizable lipid, R 2a is methyl. In some embodiments of the ionizable lipid, R 2a is ethyl. In some embodiments of the ionizable lipid, R 2a is propyl. In some embodiments of the ionizable lipid, R 2a is butyl. In some embodiments of the ionizable lipid, R 2a is pentyl. In some embodiments of the ionizable lipid, R 2a is hexyl. In some embodiments of the ionizable lipid, R 2a is heptyl.
  • R 2a is octyl.
  • R 2b is hydrogen.
  • R 2b is methyl.
  • R 2b is ethyl.
  • R 2b is propyl.
  • R 2b is butyl.
  • R 2b is pentyl.
  • R 2b is hexyl.
  • R 2b is heptyl. In some embodiments of the ionizable lipid, R 2b is octyl. In some embodiments of the ionizable lipid, R 2a is hydrogen and R 2b is hydrogen. In some embodiments of the ionizable lipid, R 2a is hexyl and R 2b is hydrogen. In some embodiments of the ionizable lipid, R 2a is octyl and R 2b is hydrogen. In some embodiments of the ionizable lipid, R 2a is hydrogen and R 2b is butyl. In some embodiments of the ionizable lipid, R 2c is methyl.
  • R 2c is ethyl. In some embodiments of the ionizable lipid, R 2c is propyl. In some embodiments of the ionizable lipid, R 2c is butyl. In some embodiments of the ionizable lipid, R 2c is pentyl. In some embodiments of the ionizable lipid, R 2c is hexyl. In some embodiments of the ionizable lipid, R 2c is heptyl. In some embodiments of the ionizable lipid, R 2c is octyl.
  • R 2 is –(C 1-6 alkylene)-(C 3-8 cycloalkyl)-C 1-6 alkyl. In some embodiments of the ionizable lipid, R 2 is –(C 1-6 alkylene)-(cyclohexyl)-C 1-6 alkyl. In some embodiments of the ionizable lipid, R 2 is –(C1-6 alkylene)-(cyclopentyl)-C1-6 alkyl.
  • Variables R 3 , R 3a , R 3b , and R 3c In some embodiments of the ionizable lipid, R 3 . In some embodiments of the ionizable lipid, R 3a is hydrogen.
  • R 3a is methyl. In some embodiments of the ionizable lipid, R 3a is ethyl. In some embodiments of the ionizable lipid, R 3a is propyl. In some embodiments of the ionizable lipid, R 3a is butyl. In some embodiments of the ionizable lipid, R 3a is pentyl. In some embodiments of the ionizable lipid, R 3a is hexyl. In some embodiments of the ionizable lipid, R 3a is heptyl. In some embodiments of the ionizable lipid, R 3a is octyl.
  • R 3b is hydrogen. In some embodiments of the ionizable lipid, R 3b is methyl. In some embodiments of the ionizable lipid, R 3b is ethyl. In some embodiments of the ionizable lipid, R 3b is propyl. In some embodiments of the ionizable lipid, R 3b is butyl. In some embodiments of the ionizable lipid, R 3b is pentyl. In some embodiments of the ionizable lipid, R 3b is hexyl. In some embodiments of the ionizable lipid, R 3b is heptyl.
  • R 3b is octyl. In some embodiments of the ionizable lipid, R 3a is octyl and R 3b is hydrogen. In some embodiments of the ionizable lipid, R 3a is ethyl and R 3b is hydrogen. In some embodiments of the ionizable lipid, R 3a is hexyl and R 3b is hydrogen. In some embodiments of the ionizable lipid, R 3c is methyl. In some embodiments of the ionizable lipid, R 3c is ethyl. In some embodiments of the ionizable lipid, R 3c is propyl.
  • R 3c is butyl. In some embodiments of the ionizable lipid, R 3c is pentyl. In some embodiments of the ionizable lipid, R 3c is hexyl. In some embodiments of the ionizable lipid, R 3c is heptyl. In some embodiments of the ionizable lipid, R 3c is octyl.
  • variables o, R 1 , R N , R N’ , R N’ , m, n, M, M’, R 2 , R 2a , R 2b , R 2c , R 3 , R 3a , R 3b , and R 3c can each be, where applicable, selected from the groups described herein, and any group described herein for any of variables o,.R 1 , R N , R N’ , R N’ , m, n, M, M’, R 2 , R 2a , R 2b , R 2c , R 3 , R 3a , R 3b , and R 3c can be combined, where applicable, with any group described herein for one or more of the remainder of variables o, R 1 , R N , R N’ , R N’ , m, n, M, M’, R 2 , R 2a , R 2b , R 2c , R 3
  • the ionizable lipid is a compound selected from: , . In (I-18). In (I-25). In some embodiments, the ionizable lipid is . In Without wishing to be bound by theory, it is understood that an ionizable lipid may have a positive or partial positive charge at physiological pH. Such lipids may be referred to as cationic or ionizable (amino)lipids. Lipids may also be zwitterionic, i.e., neutral molecules having both a positive and a negative charge. mRNA-Lipid Adduct It has been determined that certain ionizable lipids are susceptible to the formation of lipid-polynucleotide adducts.
  • ionizable lipids that comprise a tertiary amine group may decompose into one or both of a secondary amine and a reactive aldehyde species capable of interacting with polynucleotides (such as mRNA) to form an ionizable lipid-polynucleotide adduct impurity that can be detected by reverse phase ion pair chromatography (RP-IP HPLC).
  • RP-IP HPLC reverse phase ion pair chromatography
  • oxidation of the tertiary amine may lead to N-oxide formation that can undergo acid/base-catalyzed hydrolysis at the amine to generate aldehydes and secondary amines which may form adducts with mRNA.
  • the ionizable lipid-polynucleotide adduct impurity is an aldehyde-mRNA adduct impurity. It also has been determined that such adducts may disrupt mRNA translation and impact the activity of lipid nanoparticle (LNP) formulated mRNA products.
  • LNP lipid nanoparticle
  • LNP compositions with a reduced content of ionizable lipid- polynucleotide adduct impurity such as wherein less than about 20%, less than about 10%, less than about 5%, or less than about 1%, of the mRNA is in the form of ionizable lipid- polynucleotide adduct impurity, as may be measured by RP-IP HPLC.
  • an LNP composition wherein less than about 10%, less than about 5%, or less than about 1%, of the mRNA is in the form of ionizable lipid-polynucleotide adduct impurity, including less than 10%, less than 5%, or less than 1%, as may be measured by RP-IP HPLC.
  • an amount of lipid aldehydes in the composition is less than about 50 ppm, including less than 50 ppm.
  • an amount of N- oxide compounds in the composition is less than about 50 ppm, including less than 50 ppm.
  • an amount of transition metals, such as Fe, in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some aspects an amount of alkyl halide compounds in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some aspects an amount of anhydride compounds in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some aspects an amount of ketone compounds in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some aspects an amount of conjugated diene compounds in the composition is less than about 50 ppm, including less than 50 ppm.
  • the composition is stable against the formation of ionizable lipid- polynucleotide adduct impurity.
  • an amount of ionizable lipid-polynucleotide adduct impurity in the composition increases at an average rate of less than about 2% per day when stored at a temperature of about 25 °C or below, including at an average rate of less than 2% per day.
  • an amount of ionizable lipid-polynucleotide adduct impurity in the composition increases at an average rate of less than about 0.5% per day when stored at a temperature of about 5 °C or below, including at an average rate of less than 0.5% per day.
  • an amount of ionizable lipid-polynucleotide adduct impurity in the composition increases at an average rate of less than about 0.5% per day when stored at a refrigerated temperature, optionally wherein the refrigerated temperature is about 5 °C.
  • Lipid vehicle (e.g., LNP) compositions with a reduced content of ionizable lipid- polynucleotide adduct impurity can be prepared by methods that inhibit formation of one or both of N-oxides and aldehydes.
  • Such methods may comprise treating a composition comprising an ionizable lipid comprising a tertiary amine group to inhibit formation of one or both of N-oxides and aldehydes, such as by treating the composition with a reducing agent; treating the composition with a chelating agent; adjusting the pH of the composition; adjusting the temperature of the composition; and adjusting the buffer in the composition.
  • Such methods may comprise, prior to combining the ionizable lipid with a polynucleotide, one or more of treating the ionizable lipid with a scavenging agent; treating the ionizable lipid with a reductive treatment agent; treating the ionizable lipid with a reducing agent; treating the ionizable lipid with a chelating agent; treating the polynucleotide with a reducing agent; and treating the polynucleotide with a chelating agent.
  • the scavenging agent, reductive treatment agent, and/or reducing agent may be an agent that reacts with aldehyde, ketone, anhydride and/or diene compounds.
  • a scavenging agent may comprise one or more selected from (O-(2,3,4,5,6- Pentafluorobenzyl)hydroxylamine hydrochloride) (PFBHA), methoxyamine (e.g., methoxyamine hydrochloride), benzyloxyamine (e.g., benzyloxyamine hydrochloride), ethoxyamine (e.g., ethoxyamine hydrochloride), 4-[2-(aminooxy)ethyl]morpholine dihydrochloride, butoxyamine (e.g., tert-butoxyamine hydrochloride), 4-Dimethylaminopyridine (DMAP), 1,4-diazabicyclo[2.2.2]octane (DABCO), Triethyl
  • DMAP 1,4-d
  • a reductive treatment agent may comprise a boron compound (e.g., sodium borohydride and/or bis(pinacolato)diboron).
  • a reductive treatment agent may comprise a boron compound, such as one or both of sodium borohydride and bis(pinacolato)diboron).
  • a chelating agent may comprise immobilized iminodiacetic acid.
  • a reducing agent may comprise an immobilized reducing agent, such as immobilized diphenylphosphine on silica (Si-DPP), immobilized thiol on agarose (Ag-Thiol), immobilized cysteine on silica (Si-Cysteine), immobilized thiol on silica (Si-Thiol), or a combination thereof.
  • an immobilized reducing agent such as immobilized diphenylphosphine on silica (Si-DPP), immobilized thiol on agarose (Ag-Thiol), immobilized cysteine on silica (Si-Cysteine), immobilized thiol on silica (Si-Thiol), or a combination thereof.
  • a reducing agent may comprise a free reducing agent, such as potassium metabisulfite, sodium thioglycolate, tris(2-carboxyethyl)phosphine (TCEP), sodium thiosulfate, N-acetyl cysteine, glutathione, dithiothreitol (DTT), cystamine, dithioerythritol (DTE), dichlorodiphenyltrichloroethane (DDT), homocysteine, lipoic acid, or a combination thereof.
  • the pH may be, or adjusted to be, a pH of from about 7 to about 9 (for example, about 7, about 7.5, about 8, about 8.5, or about 9).
  • a buffer may be selected from sodium phosphate, sodium citrate, sodium succinate, histidine, histidine-HCl, sodium malate, sodium carbonate, and TRIS (tris(hydroxymethyl)aminomethane).
  • a buffer may be TRIS and may be, or adjusted to be, from about 20 mM to about 150 mM TRIS.
  • the temperature of the composition may be, or adjusted to be, 25 0C or less.
  • the composition may also comprise a free reducing agent or antioxidant.
  • Non-cationic lipids In certain embodiments, a lipid nanoparticle described herein comprise one or more non- cationic lipids.
  • Non-cationic lipids may be phospholipids.
  • a lipid nanoparticle comprises 5-25 mol% non-cationic lipid.
  • a lipid nanoparticle may comprise 5-20 mol%, 5-15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-25 mol%, 15-25 mol%, 15-20 mol%, or 20-25 mol% non-cationic lipid.
  • a lipid nanoparticle comprises 5 mol%, 10 mol%, 15 mol%, 20 mol%, or 25 mol% non-cationic lipid.
  • a non-cationic lipid of the disclosure comprises 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero- phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phospho
  • a lipid nanoparticle comprises 5 – 15 mol%, 5 – 10 mol%, or 10 – 15 mol% DSPC.
  • a lipid nanoparticle may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mol% DSPC.
  • the lipid composition of a lipid nanoparticle composition disclosed herein can comprise one or more phospholipids, for example, one or more saturated or (poly)unsaturated phospholipids or a combination thereof.
  • phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.
  • a phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.
  • a fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.
  • Particular phospholipids can facilitate fusion to a membrane.
  • a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., an mRNA) of a lipid-containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue.
  • a membrane e.g., a cellular or intracellular membrane.
  • elements e.g., an mRNA
  • a lipid-containing composition e.g., LNPs
  • Non-natural phospholipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated.
  • a phospholipid can be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond).
  • alkynes e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond.
  • an alkyne group can undergo a copper-catalyzed cycloaddition upon exposure to an azide.
  • Such reactions can be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye).
  • Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidylglycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin.
  • a phospholipid of the present disclosure comprises 1,2-distearoyl- sn-glycero-3-phosphocholine (DSPC), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3- phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero-phosphocholine (DMPC), 1,2-dioleoyl-sn- glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC),
  • a phospholipid useful or potentially useful in the present disclosure is an analog or variant of DSPC.
  • a phospholipid useful or potentially useful in the present disclosure is a compound of Formula (IX): or each R 1 is independently optionally substituted alkyl; or optionally two R 1 are joined together with the intervening atoms to form optionally substituted monocyclic carbocyclyl or optionally substituted monocyclic heterocyclyl; or optionally three R 1 are joined together with the intervening atoms to form optionally substituted bicyclic carbocyclyl or optionally substitute bicyclic heterocyclyl; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is of the formula: ; each instance of L 2 is independently a bond or optionally substituted C 1-6 alkylene, wherein one methylene unit of the optionally substituted C1-6 alkylene is optionally replaced with O, N(R N ), S, C(O), C
  • the phospholipids may be one or more of the phospholipids described in PCT Application No. PCT/US2018/037922.
  • a lipid nanoparticle comprises 5-25 mol% non-cationic lipid relative to the other lipid components.
  • a lipid nanoparticle may comprise 5-30 mol%, 5-15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-25 mol%, 15-25 mol%, 15-20 mol%, 20-25 mol%, or 25-30 mol% non-cationic lipid.
  • a lipid nanoparticle comprises a 5 mol%, 10 mol%, 15 mol%, 20 mol%, 25 mol%, or 30 mol% non- cationic lipid. In some embodiments, a lipid nanoparticle comprises 5-25 mol% phospholipid relative to the other lipid components. For example, the lipid nanoparticle may comprise 5-30 mol%, 5- 15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-25 mol%, 15-25 mol%, 15-20 mol%, 20-25 mol%, or 25-30 mol% phospholipid.
  • the lipid nanoparticle 5 mol%, 10 mol%, 15 mol%, 20 mol%, 25 mol%, or 30 mol% phospholipid lipid.
  • Structural Lipids The lipid composition of a pharmaceutical composition disclosed herein can comprise one or more structural lipids.
  • structural lipid includes sterols and also to lipids containing sterol moieties. Incorporation of structural lipids in a lipid nanoparticle may help mitigate aggregation of other lipids in the particle.
  • Structural lipids can be selected from the group including but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof.
  • the structural lipid is a sterol.
  • “sterols” are a subgroup of steroids consisting of steroid alcohols.
  • the structural lipid is a steroid.
  • the structural lipid is cholesterol.
  • the structural lipid is an analog of cholesterol.
  • the structural lipid is alpha-tocopherol.
  • the structural lipids may be one or more of the structural lipids described in U.S. Application No.16/493,814.
  • a lipid nanoparticle comprises 25-55 mol% structural lipid relative to the other lipid components.
  • a lipid nanoparticle may comprise 10-55 mol%, 25-50 mol%, 25-45 mol%, 25-40 mol%, 25-35 mol%, 25-30 mol%, 30-55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35-40 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 45-55 mol%, 45-50 mol%, or 50-55 mol% structural lipid.
  • a lipid nanoparticle comprises 10 mol%, 15 mol%, 20 mol%, 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, or 55 mol% structural lipid.
  • a lipid nanoparticle comprises 30-45 mol% sterol, optionally 35- 40 mol%, for example, 30-31 mol%, 31-32 mol%, 32-33 mol%, 33-34 mol%, 34-35 mol%, 35- 36 mol%, 36-37 mol%, 37-38 mol%, 38-39 mol%, or 39-40 mol%.
  • a lipid nanoparticle comprises 25-55 mol% sterol.
  • a lipid nanoparticle may comprise 25-50 mol%, 25-45 mol%, 25-40 mol%, 25-35 mol%, 25-30 mol%, 30-55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35-40 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 45-55 mol%, 45-50 mol%, or 50-55 mol% sterol.
  • a lipid nanoparticle comprises 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, or 55 mol% sterol. In some embodiments, a lipid nanoparticle comprises 35-40 mol% cholesterol. For example, a lipid nanoparticle may comprise 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, or 40 mol% cholesterol.
  • PEG polyethylene Glycol
  • the lipid composition of a pharmaceutical composition disclosed herein can comprise one or more polyethylene glycol (PEG) lipids.
  • PEG-lipid or “PEG-modified lipid” refers to polyethylene glycol (PEG)-modified lipids.
  • PEG-lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropan-3- amines.
  • PEG-lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropan-3- amines.
  • PEGylated lipids PEGylated lipids.
  • a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.
  • the PEG-lipid includes, but not limited to 1,2-dimyristoyl-sn- glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG- DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-l,2- dimyristyloxlpropyl-3-amine
  • the PEG-lipid is selected from the group consisting of a PEG- modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof.
  • the PEG-modified lipid is PEG- DMG, PEG-c-DOMG (also referred to as PEG-DOMG), PEG-DSG, and/or PEG-DPG.
  • the lipid moiety of the PEG-lipids includes those having lengths of from about C14 to about C22, preferably from about C14 to about C16.
  • a PEG moiety for example an mPEG-NH 2 , has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons.
  • the PEG-lipid is PEG2k-DMG.
  • a lipid nanoparticles described herein can comprise a PEG lipid which is a non-diffusible PEG.
  • Non-limiting examples of non-diffusible PEGs include PEG- DSG and PEG-DSPE.
  • PEG-lipids are known in the art, such as those described in U.S. Patent No. 8158601 and International Publ. No. WO 2015/130584 A2, which are incorporated herein by reference in their entirety.
  • some of the other lipid components (e.g., PEG lipids) of various formulae described herein may be synthesized as described International Patent Application No. PCT/US2016/000129, filed December 10, 2016, entitled “Compositions and Methods for Delivery of Therapeutic Agents,” which is incorporated by reference in its entirety.
  • the lipid component of a lipid nanoparticle composition may include one or more molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids.
  • a PEG lipid is a lipid modified with polyethylene glycol.
  • a PEG lipid may be selected from the non-limiting group including PEG- modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof.
  • a PEG lipid may be PEG-c-DOMG, PEG- DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.
  • the PEG-modified lipids are a modified form of PEG DMG.
  • PEG lipids useful in the present disclosure can be PEGylated lipids described in International Publication No. WO2012099755, the contents of which is herein incorporated by reference in its entirety. Any of these exemplary PEG lipids described herein may be modified to comprise a hydroxyl group on the PEG chain.
  • the PEG lipid is a PEG-OH lipid.
  • a “PEG-OH lipid” (also referred to herein as “hydroxy-PEGylated lipid”) is a PEGylated lipid having one or more hydroxyl (–OH) groups on the lipid.
  • the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain.
  • a PEG-OH or hydroxy- PEGylated lipid comprises an –OH group at the terminus of the PEG chain.
  • a PEG lipid useful in the present disclosure is a compound of Formula (X): or salts thereof, wherein: R 3 is –OR O ; R O is hydrogen, optionally substituted alkyl, or an oxygen protecting group; r is an integer between 1 and 100, inclusive; L 1 is optionally substituted C 1-10 alkylene, wherein at least one methylene of the optionally substituted C 1-10 alkylene is independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, O, N(R N ), S, C(O), C(O)N(R N ), NR N C(O), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), NR N C(O)O, or NR N C(O)N(R N ); D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions;
  • the compound of Fomula (X) is a PEG-OH lipid (i.e., R 3 is – OR O , and R O is hydrogen).
  • the compound of Formula (X) is of Formula (X-OH): or a salt thereof.
  • a PEG lipid useful in the present disclosure is a PEGylated fatty acid.
  • a PEG lipid useful in the present disclosure is a compound of Formula (XI).
  • R 3 is–OR O ;
  • R O is hydrogen, optionally substituted alkyl or an oxygen protecting group;
  • r is an integer between 1 and 100, inclusive;
  • the compound of Formula (XI) is of Formula (XI-OH): or a some r is 40-50. In yet other embodiments the compound of Formula (XI) is: . or a salt thereof. In some embodiments, the compound of Formula (XI) is .
  • the lipid composition of the pharmaceutical compositions disclosed herein does not comprise a PEG-lipid. In some embodiments, the PEG-lipids may be one or more of the PEG lipids described in U.S. Application No. US15/674,872. In some embodiments, a lipid nanoparticle comprises 0.5-15 mol% PEG lipid relative to the other lipid components.
  • a lipid nanoparticle may comprise 0.5-10 mol%, 0.5-5 mol%, 1-15 mol%, 1-10 mol%, 1-5 mol%, 2-15 mol%, 2-10 mol%, 2-5 mol%, 5-15 mol%, 5-10 mol%, or 10-15 mol% PEG lipid.
  • a lipid nanoparticle comprises 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol% PEG- lipid.
  • a lipid nanoparticle comprises 1-5% PEG-modified lipid, optionally 1-3 mol%, for example, 1.5 to 2.5 mol%, 1-2 mol%, 2-3 mol%, 3-4 mol%, or 4-5 mol%.
  • a lipid nanoparticle comprises 0.5-15 mol% PEG-modified lipid.
  • a lipid nanoparticle may comprise 0.5-10 mol%, 0.5-5 mol%, 1-15 mol%, 1-10 mol%, 1-5 mol%, 2-15 mol%, 2-10 mol%, 2-5 mol%, 5-15 mol%, 5-10 mol%, or 10-15 mol%.
  • a lipid nanoparticle comprises 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol% PEG-modified lipid.
  • Some embodiments comprise adding PEG to a composition comprising an LNP encapsulating a nucleic acid (e.g., which already includes PEG in the amounts listed above). Without being bound by theory, it is believed that spiking an LNP composition with additional PEG can provide benefits during lyophilization.
  • a lipid nanoparticle comprises 20-60 mol% ionizable amino lipid, 5-25 mol% non-cationic lipid, 25-55 mol% sterol, and 0.5-15 mol% PEG-modified lipid.
  • an LNP of the disclosure comprises an ionizable amino lipid of Compound 1, wherein the non-cationic lipid is DSPC, the structural lipid that is cholesterol, and the PEG lipid is DMG-PEG.
  • an LNP of the present disclosure comprises an ionizable amino lipid of any of Formula VI, VII or VIIII, a phospholipid comprising DSPC, a structural lipid, and a PEG lipid comprising PEG-DMG.
  • an LNP of the present disclosure comprises an ionizable amino lipid of any of Formula VI, VII or VIII, a phospholipid comprising DSPC, a structural lipid, and a PEG lipid comprising a compound having Formula XI.
  • an LNP of the present disclosure comprises an ionizable amino lipid of Formula VI, VII or VIII, a phospholipid comprising a compound having Formula VIII, a structural lipid, and the PEG lipid comprising a compound having Formula X or XI.
  • an LNP of the present disclosure comprises an ionizable amino lipid of Formula VI, VII or VIII, a phospholipid comprising a compound having Formula IX, a structural lipid, and the PEG lipid comprising a compound having Formula X or XI.
  • an LNP of the present disclosure comprises an ionizable amino lipid of Formula VI, VII or VIII, a phospholipid having Formula IX, a structural lipid, and a PEG lipid comprising a compound having Formula XI.
  • a lipid nanoparticle comprises 49 mol% ionizable amino lipid, 10 mol% DSPC, 38.5 mol% cholesterol, and 2.5 mol% DMG-PEG.
  • a lipid nanoparticle comprises 49 mol% ionizable amino lipid, 11 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% DMG-PEG.
  • a lipid nanoparticle comprises 48 mol% ionizable amino lipid, 11 mol% DSPC, 38.5 mol% cholesterol, and 2.5 mol% DMG-PEG.
  • an LNP of the present disclosure comprises an N:P ratio of from about 2:1 to about 30:1.
  • an LNP of the present disclosure comprises an N:P ratio of about 6:1.
  • an LNP of the present disclosure comprises an N:P ratio of about 3:1, 4:1, or 5:1.
  • an LNP of the present disclosure comprises a wt/wt ratio of the ionizable amino lipid component to the RNA of from about 10:1 to about 100:1.
  • an LNP of the present disclosure comprises a wt/wt ratio of the ionizable amino lipid component to the RNA of about 20:1. In some embodiments, an LNP of the present disclosure comprises a wt/wt ratio of the ionizable amino lipid component to the RNA of about 10:1. Some embodiments comprise a composition having one or more LNPs having a diameter of about 150 nm or less, such as about 140 nm, 130 nm, 120 nm, 110 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, or 20 nm or less.
  • Some embodiments comprise a composition having a mean LNP diameter of about 150 nm or less, such as about 140 nm, 130 nm, 120 nm, 110 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, or 20 nm or less.
  • the composition has a mean LNP diameter from about 30nm to about 150nm, or a mean diameter from about 60nm to about 120nm.
  • An LNP may comprise or one or more types of lipids, including but not limited to amino lipids (e.g., ionizable amino lipids), neutral lipids, non-cationic lipids, charged lipids, PEG- modified lipids, phospholipids, structural lipids and sterols.
  • an LNP may further comprise one or more cargo molecules, including but not limited to nucleic acids (e.g., mRNA, plasmid DNA, DNA or RNA oligonucleotides, siRNA, shRNA, snRNA, snoRNA, lncRNA, etc.), small molecules, proteins and peptides.
  • the composition comprises a liposome.
  • a liposome is a lipid particle comprising lipids arranged into one or more concentric lipid bilayers around a central region.
  • the central region of a liposome may comprise an aqueous solution, suspension, or other aqueous composition.
  • a lipid nanoparticle may comprise two or more components (e.g., amino lipid and nucleic acid, PEG-lipid, phospholipid, structural lipid).
  • a lipid nanoparticle may comprise an amino lipid and a nucleic acid.
  • Compositions comprising lipid nanoparticles, such as those described herein, may be used for a wide variety of applications, including the stealth delivery of mRNA with minimal adverse innate immune response.
  • nucleic acids i.e., originating from outside of a cell or organism
  • a particulate carrier e.g., lipid nanoparticles
  • the particulate carrier should be formulated to have minimal particle aggregation, be relatively stable prior to intracellular delivery, effectively deliver nucleic acids intracellularly, and illicit no or minimal immune response.
  • many conventional particulate carriers have relied on the presence and/or concentration of certain components (e.g., PEG-lipid).
  • nucleic acid e.g., mRNA molecules
  • a lipid nanoparticles comprise one or more of ionizable molecules, polynucleotides, and optional components, such as structural lipids, sterols, neutral lipids, phospholipids and a molecule capable of reducing particle aggregation (e.g., polyethylene glycol (PEG), PEG-modified lipid), such as those described above.
  • an LNP described herein may include one or more ionizable molecules (e.g., amino lipids or ionizable lipids). The ionizable molecule may comprise a charged group and may have a certain pKa.
  • the pKa of the ionizable molecule may be greater than or equal to about 6, greater than or equal to about 6.2, greater than or equal to about 6.5, greater than or equal to about 6.8, greater than or equal to about 7, greater than or equal to about 7.2, greater than or equal to about 7.5, greater than or equal to about 7.8, greater than or equal to about 8.
  • the pKa of the ionizable molecule may be less than or equal to about 10, less than or equal to about 9.8, less than or equal to about 9.5, less than or equal to about 9.2, less than or equal to about 9.0, less than or equal to about 8.8, or less than or equal to about 8.5.
  • each type of ionizable molecule may independently have a pKa in one or more of the ranges described above.
  • an ionizable molecule comprises one or more charged groups.
  • an ionizable molecule may be positively charged or negatively charged.
  • an ionizable molecule may be positively charged.
  • an ionizable molecule may comprise an amine group.
  • the term “ionizable molecule” has its ordinary meaning in the art and may refer to a molecule or matrix comprising one or more charged moiety.
  • a “charged moiety” is a chemical moiety that carries a formal electronic charge, e.g., monovalent (+1, or -1), divalent (+2, or -2), trivalent (+3, or -3), etc.
  • the charged moiety may be anionic (i.e., negatively charged) or cationic (i.e., positively charged).
  • Examples of positively-charged moieties include amine groups (e.g., primary, secondary, and/or tertiary amines), ammonium groups, pyridinium group, guanidine groups, and imidizolium groups.
  • the charged moieties comprise amine groups.
  • Examples of negatively- charged groups or precursors thereof include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups, and the like.
  • the charge of the charged moiety may vary, in some cases, with the environmental conditions, for example, changes in pH may alter the charge of the moiety, and/or cause the moiety to become charged or uncharged.
  • an ionizable molecule e.g., an amino lipid or ionizable lipid
  • an ionizable molecule may include one or more precursor moieties that can be converted to charged moieties.
  • the ionizable molecule may include a neutral moiety that can be hydrolyzed to form a charged moiety, such as those described above.
  • the molecule or matrix may include an amide, which can be hydrolyzed to form an amine, respectively.
  • a given chemical moiety carries a formal electronic charge (for example, by inspection, pH titration, ionic conductivity measurements, etc.), and/or whether a given chemical moiety can be reacted (e.g., hydrolyzed) to form a chemical moiety that carries a formal electronic charge.
  • the ionizable molecule e.g., amino lipid or ionizable lipid
  • the molecular weight of an ionizable molecule is less than or equal to about 2,500 g/mol, less than or equal to about 2,000 g/mol, less than or equal to about 1,500 g/mol, less than or equal to about 1,250 g/mol, less than or equal to about 1,000 g/mol, less than or equal to about 900 g/mol, less than or equal to about 800 g/mol, less than or equal to about 700 g/mol, less than or equal to about 600 g/mol, less than or equal to about 500 g/mol, less than or equal to about 400 g/mol, less than or equal to about 300 g/mol, less than or equal to about 200 g/mol, or less than or equal to about 100 g/mol.
  • the molecular weight of an ionizable molecule is greater than or equal to about 100 g/mol, greater than or equal to about 200 g/mol, greater than or equal to about 300 g/mol, greater than or equal to about 400 g/mol, greater than or equal to about 500 g/mol, greater than or equal to about 600 g/mol, greater than or equal to about 700 g/mol, greater than or equal to about 1000 g/mol, greater than or equal to about 1,250 g/mol, greater than or equal to about 1,500 g/mol, greater than or equal to about 1,750 g/mol, greater than or equal to about 2,000 g/mol, or greater than or equal to about 2,250 g/mol.
  • each type of ionizable molecule may independently have a molecular weight in one or more of the ranges described above.
  • the percentage (e.g., by weight, or by mole) of a single type of ionizable molecule (e.g., amino lipid or ionizable lipid) and/or of all the ionizable molecules within a particle may be greater than or equal to about 15%, greater than or equal to about 16%, greater than or equal to about 17%, greater than or equal to about 18%, greater than or equal to about 19%, greater than or equal to about 20%, greater than or equal to about 21%, greater than or equal to about 22%, greater than or equal to about 23%, greater than or equal to about 24%, greater than or equal to about 25%, greater than or equal to about 30%, greater than or equal to about 35%, greater than or equal to about 40%, greater than or equal to about 42%, greater than or equal to about 45%, greater than or equal to about 48%, greater than or equal to about 50%, greater than or equal to about 52%, greater than or equal to about 55%, greater than or equal to about 58%, greater than
  • the percentage (e.g., by weight, or by mole) may be less than or equal to about 70%, less than or equal to about 68%, less than or equal to about 65%, less than or equal to about 62%, less than or equal to about 60%, less than or equal to about 58%, less than or equal to about 55%, less than or equal to about 52%, less than or equal to about 50%, or less than or equal to about 48%. Combinations of the above referenced ranges are also possible (e.g., greater than or equal to 20% and less than or equal to about 60%, greater than or equal to 40% and less than or equal to about 55%, etc.).
  • each type of ionizable molecule may independently have a percentage (e.g., by weight, or by mole) in one or more of the ranges described above.
  • the percentage e.g., by weight, or by mole
  • the percentage may be determined by extracting the ionizable molecule(s) from the dried particles using, e.g., organic solvents, and measuring the quantity of the agent using high pressure liquid chromatography (i.e., HPLC), liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR), or mass spectrometry (MS).
  • HPLC may be used to quantify the amount of a component, by, e.g., comparing the area under the curve of a HPLC chromatogram to a standard curve.
  • charge or “charged moiety” does not refer to a “partial negative charge” or “partial positive charge” on a molecule.
  • partial negative charge and “partial positive charge” are given their ordinary meaning in the art.
  • a “partial negative charge” may result when a functional group comprises a bond that becomes polarized such that electron density is pulled toward one atom of the bond, creating a partial negative charge on the atom.
  • a lipid composition may comprise one or more lipids as described herein. Such lipids may include those useful in the preparation of lipid nanoparticle formulations as described above or as known in the art.
  • Multivalent Vaccines As described herein, the term “monovalent” refers to an mRNA polynucleotide having an ORF encoding one Borrelia proteins. As described herein, the term “multivalent” refers to an mRNA polynucleotide (or an mRNA vaccine) encoding multiple Borrelia proteins (on one or more mRNAs).
  • the term “hexavalent” refers to six mRNA polynucleotides, each mRNA comprising an ORF encoding one or more Borrelia proteins.
  • the term “heptavalent” refers to seven mRNA polynucleotides, each mRNA comprising an ORF encoding one or more Borrelia proteins.
  • the compositions, as provided herein may include one mRNA polynucleotide or multiple RNA polynucleotides (e.g., mRNAs) collectively encoding two or more antigens of the same or different species.
  • composition includes an mRNA polynucleotide or multiple mRNA polynucleotides collectively encoding two or more Borrelia proteins.
  • a single mRNA polynucleotde encodes 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more Borrelia proteins.
  • the mRNA polynucleotide comprises an ORF that encodes 2 or more Borrelia proteins (e.g., 2 or more of OspA S1-S7).
  • the mRNA polynucleotide comprises an ORF that encodes 3 or more Borrelia proteins (e.g., 3 or more of OspA S1-S7).
  • the mRNA polynucleotide comprises an ORF that encodes 4 or more Borrelia proteins (e.g., 4 or more of OspA S1-S7). In some embodiments, the mRNA polynucleotide comprises an ORF that encodes 5 or more Borrelia proteins (e.g., 5 or more of OspA S1-S7). In some embodiments, the mRNA polynucleotide comprises an ORF that encodes 6 or more Borrelia proteins (e.g., 6 or more of OspA S1-S7).
  • the mRNA polynucleotide comprises an ORF that encodes 7 or more Borrelia proteins (e.g., each of OspA S1-S7).
  • a composition comprises multiple mRNA polynucleotides each encoding a distinct Borrelia protein.
  • a composition comprises a first mRNA polynucleotide comprising an ORF encoding a first Borrelia protein (e.g., one of OspA S1-S7) and a second mRNA polynucleotide comprising an ORFencoding a second Borrelia protein (e.g., one of OspA S1-S7), wherein the first Borrelia protein and the second Borrelia protein are distinct proteins.
  • a composition comprises a first mRNA polynucleotide encoding a first Borrelia protein (e.g., one of OspA S1-S7), a second RNA polynucleotide encoding a second Borrelia protein (e.g., one of OspA S1-S7), and a third RNA polynucleotide encoding a third Borrelia protein (e.g., one of OspA S1-S7), wherein the first Borrelia protein, the second Borrelia protein, and the third Borrelia protein are distinct proteins.
  • a composition comprises a first mRNA polynucleotide encoding a first Borrelia protein (e.g., one of OspA S1-S7), a second mRNA polynucleotide encoding a second Borrelia protein (e.g., one of OspA S1-S7), a third mRNA polynucleotide encoding a third Borrelia protein (e.g., one of OspA S1-S7), and a fourth mRNA polynucleotide encoding a fourth Borrelia protein (e.g., one of OspA S1-S7), wherein the first Borrelia protein, the second Borrelia protein, the third Borrelia protein, and the fourth Borrelia protein are distinct proteins.
  • a composition comprises a first mRNA polynucleotide encoding a first Borrelia protein (e.g., one of OspA S1-S7), a second mRNA polynucleotide encoding a second Borrelia protein (e.g., one of OspA S1-S7), a third mRNA polynucleotide encoding a third Borrelia protein (e.g., one of OspA S1-S7), a fourth mRNA polynucleotide encoding a fourth Borrelia protein (e.g., one of OspA S1-S7), and a fifth mRNA polynucleotide encoding a fifth Borrelia protein (e.g., one of OspA S1-S7), wherein the first Borrelia protein, the second Borrelia protein, the third Borrelia protein, the fourth Borrelia protein, and the fifth Borrelia protein are distinct proteins
  • a composition comprises a first mRNA polynucleotide encoding a first Borrelia protein (e.g., one of OspA S1-S7), a second mRNA polynucleotide encoding a second Borrelia protein (e.g., one of OspA S1-S7), a third mRNA polynucleotide encoding a third Borrelia protein (e.g., one of OspA S1-S7), a fourth RNA encoding a fourth Borrelia protein (e.g., one of OspA S1-S7), a fifth mRNA polynucleotide encoding a fifth Borrelia proteins (e.g., one of OspA S1-S7), and a sixth mRNA polynucleotide encoding a sixth Borrelia protein (e.g., one of OspA S1-S7), wherein the first Borrelia
  • a composition comprises a first mRNA polynucleotide encoding a first Borrelia protein (e.g., OspA S1), a second mRNA polynucleotide encoding a second Borrelia protein (e.g., OspA S2), a third mRNA polynucleotide encoding a third Borrelia protein (e.g., OspA S3), a fourth mRNA polynucleotide encoding a fourth Borrelia protein (e.g., OspA S4), a fifth mRNA polynucleotide encoding a fifth Borrelia proteins (e.g., OspA S4), a sixth mRNA polynucleotide encoding a sixth Borrelia protein (e.g., OspA S6), and a seventh mRNA polynucleotide encoding a seventh Borrelia protein (e.g., OspA
  • two or more different mRNA polynucleotides encoding antigens may be formulated in the same lipid nanoparticle.
  • two or more different mRNA encoding antigens may be formulated in separate lipid nanoparticles (each mRNA formulated in a single lipid nanoparticle). Lipid nanoparticles may then be combined and administered as a single vaccine composition (e.g., comprising multiple RNAs (e.g., mRNAs) encoding multiple antigens) or may be administered separately.
  • compositions e.g., pharmaceutical compositions, such as vaccines
  • methods, kits and reagents for prevention of Lyme disease and other conditions directly or indirectly cause by Borrelia infection in humans and other mammals for example.
  • the compositions provided herein can be used as a prophylactic agent to prevent a Borrelia infection, and thus Lyme disease, cause by a Borrelia infection.
  • the compositions containing mRNA polynucleotide(s) as described herein can be administered to a subject (e.g., a mammalian subject, such as a human subject), and the mRNA polynucleotides are translated in vivo to produce an antigenic polypeptide (antigen).
  • an “effective amount” of a composition is based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the mRNA (e.g., length, nucleotide composition, and/or extent of modified nucleosides), other components of the vaccine, and other determinants, such as age, body weight, height, sex and general health of the subject.
  • an effective amount of a composition induces or boosts an immune response as a function of antigen production in the cells of the subject.
  • an effective amount of the composition containing mRNA polynucleotide(s) having at least one chemical modification are more efficient than a composition containing a corresponding unmodified polynucleotide encoding the same antigen or a peptide antigen.
  • Increased antigen production may be demonstrated by increased cell transfection (the percentage of cells transfected with the mRNA vaccine), increased protein translation and/or expression from the polynucleotide, decreased nucleic acid degradation (as demonstrated, for example, by increased duration of protein translation from a modified polynucleotide), or altered antigen specific immune response of the host cell.
  • composition refers to the combination of an active agent (e.g., mRNA polynucleotide) with a carrier (e.g., lipid composition, e.g., LNP)), inert or active, making the composition especially suitable for prophylactic use in vivo or ex vivo.
  • a carrier e.g., lipid composition, e.g., LNP
  • the carrier in the pharmaceutical composition must be “acceptable” also in the sense that it is compatible with the active ingredient and can be capable of stabilizing it.
  • One or more solubilizing agents can be utilized as pharmaceutical carriers for delivery of an active agent.
  • compositions comprising polynucleotides and their encoded polypeptides in accordance with the present disclosure may be used for the prevention of a Borrelia infection.
  • a composition may be administered prophylactically as part of an active immunization scheme to healthy individuals or early in infection during the incubation phase.
  • the amount of mRNA provided to a cell, a tissue or a subject may be an amount effective for immune prophylaxis.
  • the compositions (comprising polynucleotides and their encoded polypeptides) in accordance with the present disclosure may be used in a method of vaccinating a subject.
  • vaccinating refers to a method of inducing an immune response in a subject to a particular antigen or pathogen.
  • a prophylactic composition such as a vaccine
  • the term “booster” refers to administration of the prophylactic (vaccine) composition to a subject that has had a previous infection or has had a previous vaccine.
  • a booster (or booster vaccine) may be given after an earlier administration of the prophylactic composition.
  • the time of administration between the initial administration of the prophylactic composition and the booster may be, but is not limited to, 12 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or more.
  • the time of administration between the initial administration of the prophylactic composition and the booster may be, but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, or 6 months.
  • the booster may comprise the same or different mRNA as compared to the earlier administration of the prophylactic composition.
  • the booster in some embodiments is monovalent (e.g., the mRNA polynucleotide comprises an ORF that encodes a single antigen).
  • the booster is multivalent (e.g., the mRNA polynucleotide comprises an ORF that encodes more than one antigen and/or the booster comprises two or more mRNApolynucleotides, collectively encoding more than one antigen).
  • “administering” or “administration” means providing a material to a subject in a manner that is pharmacologically useful.
  • a composition disclosed herein is administered to the subject parenterally.
  • a composition disclosed herein is administered to a subject subcutaneously or, intramuscularly.
  • a composition may be utilized in various settings depending on the prevalence of the infection or the degree or level of unmet medical need.
  • the mRNA vaccines may be utilized to prevent Lyme disease.
  • mRNA vaccines have superior properties in that they produce much larger antibody titers, better neutralizing immunity, produce more durable immune responses, and/or produce responses earlier than commercially available vaccines.
  • pharmaceutical compositions including mRNA and/or complexes optionally in combination with one or more pharmaceutically acceptable excipients.
  • the mRNA may be formulated or administered alone or in conjunction with one or more other components.
  • a vaccine may comprise other components including, but not limited to, adjuvants.
  • a vaccine does not include an adjuvant (they are adjuvant free).
  • An mRNA may be formulated or administered in combination with one or more pharmaceutically-acceptable excipients.
  • vaccines comprise at least one additional active substance, such as, for example, a therapeutically-active substance, a prophylactically-active substance, or a combination of both.
  • Vaccine compositions may be sterile, pyrogen-free or both sterile and pyrogen-free. General considerations in the formulation and/or manufacture of pharmaceutical agents, such as vaccines, may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).
  • a vaccine is administered to humans, human patients or subjects.
  • the phrase “active ingredient” generally refers to the mRNA contained therein, for example, mRNA encoding Borrelia protein antigens.
  • Formulations of the vaccines described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient (e.g., mRNA) into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.
  • compositions e.g., vaccines
  • methods, kits and reagents for the prevention of a Borrelia infection in humans and other mammals.
  • the compositions can be used as prophylactic agents, for example.
  • the compositions are used to provide prophylactic protection from a Borrelia infection.
  • a subject may be any mammal, including non-human primate and human subjects. Typically, a subject is a human subject.
  • a composition is administered to a subject (e.g., a mammalian subject, such as a human subject) in an effective amount to induce an antigen-specific immune response.
  • a subject e.g., a mammalian subject, such as a human subject
  • the RNA encoding the Borrelia protein is expressed and translated in vivo to produce the antigen, which then stimulates an immune response in the subject.
  • Prophylactic protection from Lyme disease or other condition cause directly or as a result of Borrelia infection can be achieved following administration of a composition of the present disclosure.
  • the compositions can be administered once, twice, three times, four times or more but it is likely sufficient to administer the composition once (optionally followed by repeated administration). Dosing may need to be adjusted accordingly.
  • a method involves administering to the subject a vaccine comprising a mRNA polynucleotide having an open reading frame encoding a Borrelia protein (or multiple Borrelia antigens), thereby inducing in the subject an immune response specific to the Borrelia protein (or multiple Borrelia antigens), wherein anti- Borrelia antibody titer in the subject is increased following vaccination relative to a subject that has not been vaccinated.
  • Some aspects of the disclosure relate to methods and compositions of eliciting an immune response in a subject against a Borrelia protein (or multiple Borrelia antigens) that is not encoded by an mRNA of a vaccine.
  • the term “cross-reactivity” refers to an immune response against an antigen that is not encoded by an mRNA of the subject vaccines.
  • the terms “cross-protect” and “cross-protection” refer to clinical protection against related antigens not encoded by the mRNA polynucleotide(s) of the vaccine (e.g., cross- protection as measured according to methods described in Example 11).
  • the immune response in the subject is induced 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 5 weeks, or 10 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine.
  • methods of eliciting an immune response in a subject against Borrelia by administering to the subject an mRNA having an open reading frame encoding at least one Borrelia protein, wherein the mRNA does not include a stabilization element, and wherein an adjuvant is not co-formulated or co-administered with the vaccine.
  • a composition may be administered by any route that results in a prophylactically effective outcome.
  • mRNA vaccines are administered to a subject in need thereof.
  • the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like.
  • the mRNA is typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the mRNA may be decided by the attending physician within the scope of sound medical judgment.
  • the specific prophylactically effective dose level for any particular patient will depend upon a variety of factors including the disorder being addressed and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
  • the effective amount of the mRNA e.g., an effective dose
  • the first effective vaccine dose and the second effective vaccine dose are the same amount. In some embodiments, the first effective vaccine dose and the second effective vaccine dose are different amounts. In some embodiments, the effective amount is a total dose of 5 ⁇ g-30 ⁇ g, 5 ⁇ g -25 ⁇ g, 5 ⁇ g -20 ⁇ g, 5 ⁇ g -15 ⁇ g, 5 ⁇ g -10 ⁇ g, 10 ⁇ g -30 ⁇ g, 10 ⁇ g -25 ⁇ g, 10 ⁇ g-20 ⁇ g, 10 ⁇ g -15 ⁇ g, 15 ⁇ g -30 ⁇ g, 15 ⁇ g -25 ⁇ g, 15 ⁇ g -20 ⁇ g, 20 ⁇ g -30 ⁇ g, 25 ⁇ g -30 ⁇ g, or 25 ⁇ g-300 ⁇ g.
  • the effective dose (e.g., effective amount) is at least 10 ⁇ g and less than 25 ⁇ g of the composition. In some embodiments, the effective dose (e.g., effective amount) is at least 5 ⁇ g and less than 25 ⁇ g of the composition.
  • the effective amount may be a total dose of 5 ⁇ g, 10 ⁇ g, 15 ⁇ g, 20 ⁇ g, 25 ⁇ g, 30 ⁇ g, 35 ⁇ g, 40 ⁇ g, 45 ⁇ g, 50 ⁇ g, 55 ⁇ g, 60 ⁇ g, 65 ⁇ g, 70 ⁇ g, 75 ⁇ g, 80 ⁇ g, 85 ⁇ g, 90 ⁇ g, 95 ⁇ g, 100 ⁇ g, 110 ⁇ g, 120 ⁇ g, 130 ⁇ g, 140 ⁇ g, 150 ⁇ g, 160 ⁇ g, 170 ⁇ g, 180 ⁇ g, 190 ⁇ g, 200 ⁇ g, 250 ⁇ g, or 300 ⁇ g.
  • the effective amount (e.g., effective dose) is a total dose of 10 ⁇ g. In some embodiments, the effective amount is a total dose of 20 ⁇ g (e.g., two 10 ⁇ g doses). In some embodiments, the effective amount is a total dose of 25 ⁇ g. In some embodiments, the effective amount is a total dose of 30 ⁇ g. In some embodiments, the effective amount is a total dose of 50 ⁇ g. In some embodiments, the effective amount is a total dose of 60 ⁇ g (e.g., two 30 ⁇ g doses). In some embodiments, the effective amount is a total dose of 75 ⁇ g. In some embodiments, the effective amount is a total dose of 100 ⁇ g.
  • an mRNA vaccine is a monovalent mRNA vaccine comprising a single mRNA polynucleotide comprising an ORF that encodes one OspA serotype (i.e., one of OspA S1-S7), such as a construct comprising any one of SEQ ID NOs: 8-14, including the constructs of Table 1).
  • a monovalent mRNA vaccine comprises an mRNA having an ORF that encodes for OspA S1 (e.g., comprises the sequence of SEQ ID NO: 8), but lacks a mRNA(s) comprising one or more ORFs encoding for any of OspA S2-S7.
  • the effective dose (e.g., effective amount) of monovalent mRNA vaccine is between about 10 ⁇ g and about 150 ⁇ g (e.g., between about 12.5 ⁇ g and about 50 ⁇ g).
  • the effective dose of monovalent mRNA vaccine is about 10 ⁇ g, about 12.5 ⁇ g, about 15 ⁇ g, about 20 ⁇ g, about 25 ⁇ g, about 30 ⁇ g, about 35 ⁇ g, about 40 ⁇ g, about 45 ⁇ g, about 50 ⁇ g, about 55 ⁇ g, about 60 ⁇ g, about 65 ⁇ g, about 70 ⁇ g, about 75 ⁇ g, about 80 ⁇ g, about 85 ⁇ g, about 90 ⁇ g, about 95 ⁇ g, about 100 ⁇ g, about 110 ⁇ g, about 120 ⁇ g, about 130 ⁇ g, about 140 ⁇ g, or about 150 ⁇ g.
  • the effective dose of monovalent mRNA vaccine is 10 ⁇ g, 12.5 ⁇ g, 15 ⁇ g, 20 ⁇ g, 25 ⁇ g, 30 ⁇ g, 35 ⁇ g, 40 ⁇ g, 45 ⁇ g, 50 ⁇ g, 55 ⁇ g, 60 ⁇ g, 65 ⁇ g, 70 ⁇ g, 75 ⁇ g, 80 ⁇ g, 85 ⁇ g, 90 ⁇ g, 95 ⁇ g, 100 ⁇ g, 110 ⁇ g, 120 ⁇ g, 130 ⁇ g, 140 ⁇ g, or 150 ⁇ g.
  • the effective dose of monovalent mRNA vaccine is about 12.5 ⁇ g. In some embodiments, the effective dose of monovalent mRNA vaccine (e.g., a construct comprising the sequence of SEQ ID NO: 8) is about 25 ⁇ g. In some embodiments, the effective dose of monovalent mRNA vaccine (e.g., a construct comprising the sequence of SEQ ID NO: 8) is about 50 ⁇ g.
  • the effective dose (e.g., effective amount) of monovalent mRNA vaccine may be administered for example as a single dose.
  • the effective does of monovalent vaccine may be administered as multiple doses (e.g., a first effective vaccine dose, a second effective vaccine dose, a third effective vaccine dose).
  • a first effective vaccine dose, a second effective vaccine dose, a third effective vaccine dose may be administered as multiple doses (e.g., a first effective vaccine dose, a second effective vaccine dose, a third effective vaccine dose).
  • the first effective vaccine dose, the second effective vaccine dose, and/or the third effective vaccine dose are the same amount (e.g., each 25 ⁇ g, 50 ⁇ g, 100 ⁇ g, or 150 ⁇ g).
  • first effective vaccine dose, the second effective vaccine dose, and/or the third effective vaccine dose are different amounts.
  • the effective vaccine doses are administered at least about 2 months (e.g., 2 weeks, 3 weeks, 4 weeks, 1 month, 1.5 months, 2 months, 3 months, 4 months, 5 months, 6 months) apart. In some embodiments, the effective vaccine doses are administered at least about 2 weeks, 3 weeks, 4 weeks, 1 month, 1.5 months, 2 months, 3 months, 4 months, 5 months, 6 months apart. In some embodiments, the effective vaccine doses are administered at least about 2 months apart (e.g., a second does at 2 months and a third does at 3 months).
  • an mRNA vaccine is a multivalent mRNA vaccine comprising an mRNA polynucleotide comprising at least two ORF (e.g., encoding for two or more of OspA S1-S7, such as two or more of an ORF comprising the sequence of SEQ ID NO: 8, an ORF comprising the sequence of SEQ ID NO: 9, an ORF comprising the sequence of SEQ ID NO: 10, an ORF comprising the sequence of SEQ ID NO: 11, an ORF comprising the sequence of SEQ ID NO: 12, an ORF comprising the sequence of SEQ ID NO: 13, and an ORF comprising the sequence of SEQ ID NO: 14).
  • ORF e.g., encoding for two or more of OspA S1-S7, such as two or more of an ORF comprising the sequence of SEQ ID NO: 8, an ORF comprising the sequence of SEQ ID NO: 9, an ORF comprising the sequence of SEQ ID NO: 10, an ORF comprising the sequence of SEQ ID NO: 11,
  • the effective dose (e.g., effective amount) of multivalent mRNA vaccine is between about 10 ⁇ g and about 150 ⁇ g (e.g., between about 12.5 ⁇ g and about 50 ⁇ g). In some embodiments, the effective dose of multivalent mRNA vaccine is about 10 ⁇ g, about 12.5 ⁇ g, about 15 ⁇ g, about 20 ⁇ g, about 25 ⁇ g, about 30 ⁇ g, about 35 ⁇ g, about 40 ⁇ g, about 45 ⁇ g, about 50 ⁇ g, about 55 ⁇ g, about 60 ⁇ g, about 65 ⁇ g, about 70 ⁇ g, about 75 ⁇ g, about 80 ⁇ g, about 85 ⁇ g, about 90 ⁇ g, about 95 ⁇ g, about 100 ⁇ g, about 110 ⁇ g, about 120 ⁇ g, about 130 ⁇ g, about 140 ⁇ g, or about 150 ⁇ g.
  • the effective dose of multivalent mRNA vaccine is 10 ⁇ g, 12.5 ⁇ g, 15 ⁇ g, 20 ⁇ g, 25 ⁇ g, 30 ⁇ g, 35 ⁇ g, 40 ⁇ g, 45 ⁇ g, 50 ⁇ g, 55 ⁇ g, 60 ⁇ g, 65 ⁇ g, 70 ⁇ g, 75 ⁇ g, 80 ⁇ g, 85 ⁇ g, 90 ⁇ g, 95 ⁇ g, 100 ⁇ g, 110 ⁇ g, 120 ⁇ g, 130 ⁇ g, 140 ⁇ g, or 150 ⁇ g. In some embodiments, the effective dose of multivalent mRNA vaccine is about 12.5 ⁇ g. In some embodiments, the effective dose of multivalent mRNA vaccine is about 25 ⁇ g.
  • the effective dose of multivalent mRNA vaccine is about 50 ⁇ g.
  • the effective dose (e.g., effective amount) of multivalent mRNA vaccine may be administered for example as a single dose.
  • the effective does of multivalent vaccine may be administered as multiple doses (e.g., a first effective vaccine dose, a second effective vaccine dose, a third effective vaccine dose).
  • the first effective vaccine dose, the second effective vaccine dose, and/or the third effective vaccine dose are the same amount (e.g., each 25 ⁇ g, 50 ⁇ g, 100 ⁇ g, or 150 ⁇ g).
  • first effective vaccine dose, the second effective vaccine dose, and/or the third effective vaccine dose are different amounts.
  • the effective vaccine doses are administered at least about 2 months (e.g., 2 weeks, 3 weeks, 4 weeks, 1 month, 1.5 months, 2 months, 3 months, 4 months, 5 months, 6 months) apart. In some embodiments, the effective vaccine doses are administered at least about 2 weeks, 3 weeks, 4 weeks, 1 month, 1.5 months, 2 months, 3 months, 4 months, 5 months, 6 months apart. In some embodiments, the effective vaccine doses are administered at least about 2 months apart (e.g., a second does at 2 months and a third does at 3 months).
  • an mRNA vaccine is a multivalent mRNA vaccine comprising two or more mRNA constructs, each comprising one or more ORF encoding for a distinct OspA serotype (e.g., a mRNA vaccine comprising two, three, four, five, six, seven, or more of the constructs provided in Table 1).
  • an mRNA vaccine is a multivalent mRNA vaccine comprising at least one mRNA construct(s), each comprising an ORF, collectively encoding for OspA S1-S7 (e.g., an ORF comprising the sequence of SEQ ID NO: 8, an ORF comprising the sequence of SEQ ID NO: 9, an ORF comprising the sequence of SEQ ID NO: 10, an ORF comprising the sequence of SEQ ID NO: 11, an ORF comprising the sequence of SEQ ID NO: 12, an ORF comprising the sequence of SEQ ID NO: 13, and an ORF comprising the sequence of SEQ ID NO: 14).
  • an ORF comprising the sequence of SEQ ID NO: 8 an ORF comprising the sequence of SEQ ID NO: 9
  • an ORF comprising the sequence of SEQ ID NO: 10 an ORF comprising the sequence of SEQ ID NO: 11
  • an ORF comprising the sequence of SEQ ID NO: 12 an ORF comprising the sequence of SEQ ID NO: 13
  • the effective dose of multivalent mRNA vaccine (e.g., comprising an ORF comprising the sequence of SEQ ID NO: 8, an ORF comprising the sequence of SEQ ID NO: 9, an ORF comprising the sequence of SEQ ID NO: 10, an ORF comprising the sequence of SEQ ID NO: 11, an ORF comprising the sequence of SEQ ID NO: 12, an ORF comprising the sequence of SEQ ID NO: 13, and an ORF comprising the sequence of SEQ ID NO: 14) is about 20 ⁇ g, about 25 ⁇ g, about 30 ⁇ g, about 35 ⁇ g, about 40 ⁇ g, about 45 ⁇ g, about 50 ⁇ g, about 55 ⁇ g, about 60 ⁇ g, about 65 ⁇ g, about 70 ⁇ g, about 75 ⁇ g, about 80 ⁇ g, about 85 ⁇ g, about 90 ⁇ g, about 95 ⁇ g, about 100 ⁇ g, about 110 ⁇ g, about 120 ⁇ g, about 130 ⁇ g, about 140 ⁇ g, about 150 ⁇ g
  • the effective dose of multivalent mRNA vaccine (e.g., comprising an ORF comprising the sequence of SEQ ID NO: 8, an ORF comprising the sequence of SEQ ID NO: 9, an ORF comprising the sequence of SEQ ID NO: 10, an ORF comprising the sequence of SEQ ID NO: 11, an ORF comprising the sequence of SEQ ID NO: 12, an ORF comprising the sequence of SEQ ID NO: 13, and an ORF comprising the sequence of SEQ ID NO: 14), is 20 ⁇ g, 25 ⁇ g, 30 ⁇ g, 35 ⁇ g, 40 ⁇ g, 45 ⁇ g, 50 ⁇ g, 55 ⁇ g, 60 ⁇ g, 65 ⁇ g, 70 ⁇ g, 75 ⁇ g, 80 ⁇ g, 85 ⁇ g, 90 ⁇ g, 95 ⁇ g, 100 ⁇ g, 110 ⁇ g, 120 ⁇ g, 130 ⁇ g, 140 ⁇ g, 150 ⁇ g, 160 ⁇ g, 170 ⁇ g, 180 ⁇ g, 190 ⁇ g
  • the effective dose of multivalent mRNA vaccine (e.g., comprising an ORF comprising the sequence of SEQ ID NO: 8, an ORF comprising the sequence of SEQ ID NO: 9, an ORF comprising the sequence of SEQ ID NO: 10, an ORF comprising the sequence of SEQ ID NO: 11, an ORF comprising the sequence of SEQ ID NO: 12, an ORF comprising the sequence of SEQ ID NO: 13, and an ORF comprising the sequence of SEQ ID NO: 14) is about 25 ⁇ g.
  • the effective dose of multivalent mRNA vaccine (e.g., comprising an ORF comprising the sequence of SEQ ID NO: 8, an ORF comprising the sequence of SEQ ID NO: 9, an ORF comprising the sequence of SEQ ID NO: 10, an ORF comprising the sequence of SEQ ID NO: 11, an ORF comprising the sequence of SEQ ID NO: 12, an ORF comprising the sequence of SEQ ID NO: 13, and an ORF comprising the sequence of SEQ ID NO: 14) is about 50 ⁇ g.
  • the effective dose of multivalent mRNA vaccine (e.g., comprising an ORF comprising the sequence of SEQ ID NO: 8, an ORF comprising the sequence of SEQ ID NO: 9, an ORF comprising the sequence of SEQ ID NO: 10, an ORF comprising the sequence of SEQ ID NO: 11, an ORF comprising the sequence of SEQ ID NO: 12, an ORF comprising the sequence of SEQ ID NO: 13, and an ORF comprising the sequence of SEQ ID NO: 14) is about 100 ⁇ g.
  • the effective dose of multivalent mRNA vaccine (e.g., comprising an ORF comprising the sequence of SEQ ID NO: 8, an ORF comprising the sequence of SEQ ID NO: 9, an ORF comprising the sequence of SEQ ID NO: 10, an ORF comprising the sequence of SEQ ID NO: 11, an ORF comprising the sequence of SEQ ID NO: 12, an ORF comprising the sequence of SEQ ID No: 13, and an ORF comprising the sequence of SEQ ID NO: 14) is about 150 ⁇ g.
  • the effective dose (e.g., effective amount) of multivalent mRNA vaccine may be administered for example as a single dose.
  • multivalent mRNA vaccine e.g., comprising an ORF comprising the sequence of SEQ ID NO: 8, an ORF comprising the sequence of SEQ ID NO: 9, an ORF comprising the sequence of SEQ ID NO: 10, an ORF comprising the sequence of SEQ ID NO: 11, an ORF comprising the sequence of SEQ ID NO: 12, an ORF comprising the sequence of SEQ ID No: 13, and an ORF comprising the sequence of SEQ ID NO: 14
  • the effective does of multivalent mRNA vaccine may be administered as multiple doses (e.g., a first effective vaccine dose, a second effective vaccine dose, a third effective vaccine dose).
  • the first effective vaccine dose, the second effective vaccine dose, and/or the third effective vaccine dose are the same amount (e.g., each 12.5 ⁇ g, 25, or 50 ⁇ g). In some embodiments, first effective vaccine dose, the second effective vaccine dose, and/or the third effective vaccine dose are different amounts. In some embodiments, the effective vaccine doses are administered at least about 2 months (e.g., 2 weeks, 3 weeks, 4 weeks, 1 month, 1.5 months, 2 months, 3 months, 4 months, 5 months, 6 months) apart. In some embodiments, the effective vaccine doses are administered at least about 2 weeks, 3 weeks, 4 weeks, 1 month, 1.5 months, 2 months, 3 months, 4 months, 5 months, 6 months apart.
  • an mRNA vaccine is a heptavalent mRNA vaccine comprising seven mRNA constructs, each comprising an ORF, collectively encoding for OspA S1-S7 (e.g., a first construct comprising the sequence of SEQ ID NO: 8, a second construct comprising the sequence of SEQ ID NO: 9, a third construct comprising the sequence of SEQ ID NO: 10, a fourth construct comprising the sequence of SEQ ID NO: 11, a fifth construct comprising the sequence of SEQ ID NO: 12, a sixth construct comprising the sequence of SEQ ID NO: 13, and a seventh construct comprising the sequence of SEQ ID NO: 14).
  • a first construct comprising the sequence of SEQ ID NO: 8
  • a second construct comprising the sequence of SEQ ID NO: 9
  • a third construct comprising the sequence of SEQ ID NO: 10
  • a fourth construct comprising the sequence of SEQ ID NO: 11
  • a fifth construct comprising the sequence of SEQ ID NO: 12
  • a sixth construct comprising
  • the effective dose (e.g., effective amount) of heptavalent mRNA vaccine e.g., comprising a first construct comprising the sequence of SEQ ID NO: 8, a second construct comprising the sequence of SEQ ID NO: 9, a third construct comprising the sequence of SEQ ID NO: 10, a fourth construct comprising the sequence of SEQ ID NO: 11, a fifth construct comprising the sequence of SEQ ID NO: 12, a sixth construct comprising the sequence of SEQ ID NO: 13, and a seventh construct comprising the sequence of SEQ ID NO: 14).
  • heptavalent mRNA vaccine e.g., comprising a first construct comprising the sequence of SEQ ID NO: 8, a second construct comprising the sequence of SEQ ID NO: 9, a third construct comprising the sequence of SEQ ID NO: 10, a fourth construct comprising the sequence of SEQ ID NO: 11, a fifth construct comprising the sequence of SEQ ID NO: 12, a sixth construct comprising the sequence of SEQ ID NO: 13, and a seventh construct comprising the
  • the effective dose of heptavalent mRNA vaccine (e.g., comprising a first construct comprising the sequence of SEQ ID NO: 8, a second construct comprising the sequence of SEQ ID NO: 9, a third construct comprising the sequence of SEQ ID NO: 10, a fourth construct comprising the sequence of SEQ ID NO: 11, a fifth construct comprising the sequence of SEQ ID NO: 12, a sixth construct comprising the sequence of SEQ ID NO: 13, and a seventh construct comprising the sequence of SEQ ID NO: 14) is about 20 ⁇ g, about 25 ⁇ g, about 30 ⁇ g, about 35 ⁇ g, about 40 ⁇ g, about 45 ⁇ g, about 50 ⁇ g, about 55 ⁇ g, about 60 ⁇ g, about 65 ⁇ g, about 70 ⁇ g, about 75 ⁇ g, about 80 ⁇ g, about 85 ⁇ g, about 90 ⁇ g, about 95 ⁇ g, about 100 ⁇ g, about 110 ⁇ g, about 120 ⁇ g, about 130 ⁇ g, about
  • the effective dose of heptavalent mRNA vaccine (e.g., comprising a first construct comprising the sequence of SEQ ID NO: 8, a second construct comprising the sequence of SEQ ID NO: 9, a third construct comprising the sequence of SEQ ID NO: 10, a fourth construct comprising the sequence of SEQ ID NO: 11, a fifth construct comprising the sequence of SEQ ID NO: 12, a sixth construct comprising the sequence of SEQ ID NO: 13, and a seventh construct comprising the sequence of SEQ ID NO: 14), is 20 ⁇ g, 25 ⁇ g, 30 ⁇ g, 35 ⁇ g, 40 ⁇ g, 45 ⁇ g, 50 ⁇ g, 55 ⁇ g, 60 ⁇ g, 65 ⁇ g, 70 ⁇ g, 75 ⁇ g, 80 ⁇ g, 85 ⁇ g, 90 ⁇ g, 95 ⁇ g, 100 ⁇ g, 110 ⁇ g, 120 ⁇ g, 130 ⁇ g, 140 ⁇ g, 150 ⁇ g, 160 ⁇ g, 170 ⁇ g,
  • the effective dose of heptavalent mRNA vaccine (e.g., comprising a first construct comprising the sequence of SEQ ID NO: 8, a second construct comprising the sequence of SEQ ID NO: 9, a third construct comprising the sequence of SEQ ID NO: 10, a fourth construct comprising the sequence of SEQ ID NO: 11, a fifth construct comprising the sequence of SEQ ID NO: 12, a sixth construct comprising the sequence of SEQ ID NO: 13, and a seventh construct comprising the sequence of SEQ ID NO: 14) is about 25 ⁇ g.
  • the effective dose of heptavalent mRNA vaccine (e.g., comprising a first construct comprising the sequence of SEQ ID NO: 8, a second construct comprising the sequence of SEQ ID NO: 9, a third construct comprising the sequence of SEQ ID NO: 10, a fourth construct comprising the sequence of SEQ ID NO: 11, a fifth construct comprising the sequence of SEQ ID NO: 12, a sixth construct comprising the sequence of SEQ ID NO: 13, and a seventh construct comprising the sequence of SEQ ID NO: 14) is about 50 ⁇ g.
  • the effective dose of heptavalent mRNA vaccine (e.g., comprising a first construct comprising the sequence of SEQ ID NO: 8, a second construct comprising the sequence of SEQ ID NO: 9, a third construct comprising the sequence of SEQ ID NO: 10, a fourth construct comprising the sequence of SEQ ID NO: 11, a fifth construct comprising the sequence of SEQ ID NO: 12, a sixth construct comprising the sequence of SEQ ID NO: 13, and a seventh construct comprising the sequence of SEQ ID NO: 14) is about 100 ⁇ g.
  • the effective dose of heptavalent mRNA vaccine (e.g., comprising a first construct comprising the sequence of SEQ ID NO: 8, a second construct comprising the sequence of SEQ ID NO: 9, a third construct comprising the sequence of SEQ ID NO: 10, a fourth construct comprising the sequence of SEQ ID NO: 11, a fifth construct comprising the sequence of SEQ ID NO: 12, a sixth construct comprising the sequence of SEQ ID No: 13, and a seventh construct comprising the sequence of SEQ ID NO: 14) is about 150 ⁇ g.
  • the effective dose (e.g., effective amount) of heptavalent mRNA vaccine may be administered for example as a single dose.
  • heptavalent mRNA vaccine e.g., comprising a first construct comprising the sequence of SEQ ID NO: 8, a second construct comprising the sequence of SEQ ID NO: 9, a third construct comprising the sequence of SEQ ID NO: 10, a fourth construct comprising the sequence of SEQ ID NO: 11, a fifth construct comprising the sequence of SEQ ID NO: 12, a sixth construct comprising the sequence of SEQ ID No: 13, and a seventh construct comprising the sequence of SEQ ID NO: 14
  • a single dose e.g., comprising a single dose.
  • the effective does of heptavalent mRNA vaccine may be administered as multiple doses (e.g., a first effective vaccine dose, a second effective vaccine dose, a third effective vaccine dose).
  • first effective vaccine dose, the second effective vaccine dose, and/or the third effective vaccine dose are the same amount (e.g., each 12.5 ⁇ g, 25 ⁇ g, or 50 ⁇ g). In some embodiments, first effective vaccine dose, the second effective vaccine dose, and/or the third effective vaccine dose are different amounts. In some embodiments, the effective vaccine doses are administered at least about 2 months (e.g., 2 weeks, 3 weeks, 4 weeks, 1 month, 1.5 months, 2 months, 3 months, 4 months, 5 months, 6 months) apart. In some embodiments, the effective vaccine doses are administered at least about 2 weeks, 3 weeks, 4 weeks, 1 month, 1.5 months, 2 months, 3 months, 4 months, 5 months, 6 months apart.
  • the effective vaccine doses are administered at least about 2 months apart (e.g., a second does at 2 months and a third does at 3 months).
  • the ratio of mRNA comprising an ORF encoding OspA S1: OspA S2: OspA S3: OspA S4: OspA S5: OspA S6: OspA S7 is 1:1:1:1:1:1:1:1.
  • the ratio of mRNA comprising an ORF encoding OspA S1: OspA S2: OspA S3: OspA S4: OspA S5: OspA S6: OspA S7 is 1:2:1:1:1:1:1. In some embodiments, the ratio of mRNA comprising an ORF encoding OspA S1: OspA S2: OspA S3: OspA S4: OspA S5: OspA S6: OspA S7 is 1:1:2:1:1:1:1:1.
  • the ratio of mRNA comprising an ORF encoding OspA S1: OspA S2: OspA S3: OspA S4: OspA S5: OspA S6: OspA S7 is 1:1:1:2:1:1:1. In some embodiments, the ratio of mRNA comprising an ORF encoding OspA S1: OspA S2: OspA S3: OspA S4: OspA S5: OspA S6: OspA S7 is 1:1:1:1:2:1:1.
  • the ratio of mRNA comprising an ORF encoding OspA S1: OspA S2: OspA S3: OspA S4: OspA S5: OspA S6: OspA S7 is 1:1:1:1:1:2:1. In some embodiments, the ratio of mRNA comprising an ORF encoding OspA S1: OspA S2: OspA S3: OspA S4: OspA S5: OspA S6: OspA S7 is 1:1:1:1:1:1:1:1:2.
  • compositions e.g., RNA vaccines
  • the mRNA is formulated in an effective amount to produce an antigen specific immune response in a subject (e.g., production of antibodies specific to a Borrelia antigen).
  • an effective amount is a dose of the mRNA effective to produce a protective antigen-specific immune response in a subject.
  • an immune response to a vaccine of the present disclosure is the development in a subject of a protective humoral and/or a cellular immune response to a (one or more) Borrelia protein(s) encoded by the mRNA present in the vaccine.
  • a “humoral” immune response refers to an immune response mediated by antibody molecules, including, e.g., secretory (IgA) or IgG molecules, while a “cellular” immune response is one mediated by T-lymphocytes (e.g., CD4+ helper and/or CD8+ T cells (e.g., CTLs) and/or other white blood cells.
  • T-lymphocytes e.g., CD4+ helper and/or CD8+ T cells (e.g., CTLs) and/or other white blood cells.
  • CTLs cytolytic T-cells
  • CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells.
  • MHC major histocompatibility complex
  • helper T-cells help induce and promote the destruction of intracellular microbes or the lysis of cells infected with such microbes.
  • Another aspect of cellular immunity involves and antigen-specific response by helper T-cells.
  • Helper T- cells act to help stimulate the function and focus the activity nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface.
  • a cellular immune response also leads to the production of cytokines, chemokines, and other such molecules produced by activated T-cells and/or other white blood cells including those derived from CD4+ and CD8+ T-cells. Immune responses may be further divided into Th1 and Th2 responses, resulting the production of Th1-type cytokines and Th2-type cytokines, respectively.
  • Th1-type cytokines tend to produce the proinflammatory responses responsible for killing intracellular parasites and for perpetuating autoimmune responses.
  • the main Th1 cytokine is interferon gamma.
  • Excessive proinflammatory responses e.g., Th1-based responses
  • the Th2-type cytokines include interleukins 4, 5, and 13, which are associated with the promotion of IgE and eosinophilic responses in atopy, and also interleukin-10, which is anti- inflammatory. In excess, Th2 responses will counteract the Th1 mediated microbicidal action.
  • the vaccines provided herein elicit a balanced Th1 and Th2 response.
  • administration of the vaccines provided herein may result in a Th17 response.
  • T helper 17 cells are a subset of pro-inflammatory T helper cells defined by their production of interleukin 17. Th17 cells maintain mucosal barriers and contribute to pathogen clearance at the mucosal surfaces.
  • the Th17-type cytokines target innate immune cells and epithelial cells to produce G-CSF and Il-8, leading to neutrophil production and recruitment.
  • the compositions (e.g., vaccines) of the present disclosure produce a Th1 response.
  • compositions (e.g., vaccines) of the present disclosure produce a Th2 response. In some embodiments, the compositions (e.g., vaccines) of the present disclosure produce a Th17 response. In some embodiments, the compositions (e.g., vaccines) of the present disclosure produce Th1 and Th2 responses, Th1 and Th17 responses, Th2 and Th17 responses, or Th1, Th2, and Th17 responses.
  • serological tests can be used to measure antibody against encoded antigen of interest, for example, a Borrelia antigen. These tests include the hemagglutination-inhibition test, complement fixation test, fluorescent antibody test, enzyme-linked immunosorbent assay (ELISA), and plaque reduction neutralization test (PRNT).
  • a plaque reduction neutralization test or PRNT (e.g., PRNT50 or PRNT90) is used as a serological correlate of protection.
  • PRNT measures the biological parameter of in vitro virus neutralization and is the most serologically virus-specific test among certain classes of viruses, correlating well to serum levels of protection from virus infection.
  • the basic design of the PRNT allows for virus-antibody interaction to occur in a test tube or microtiter plate, and then measuring antibody effects on viral infectivity by plating the mixture on virus-susceptible cells, preferably cells of mammalian origin. The cells are overlaid with a semi-solid media that restricts spread of progeny virus.
  • Each virus that initiates a productive infection produces a localized area of infection (a plaque), that can be detected in a variety of ways. Plaques are counted and compared back to the starting concentration of virus to determine the percent reduction in total virus infectivity.
  • PRNT the serum sample being tested is usually subjected to serial dilutions prior to mixing with a standardized amount of virus. The concentration of virus is held constant such that, when added to susceptible cells and overlaid with semi-solid media, individual plaques can be discerned and counted. In this way, PRNT end-point titers can be calculated for each serum sample at any selected percent reduction of virus activity.
  • the serum sample dilution series for antibody titration should ideally start below the “seroprotective” threshold titer.
  • a seropositivity threshold of 1:10 can be considered a seroprotection threshold in certain embodiments.
  • PRNT end-point titers are expressed as the reciprocal of the last serum dilution showing the desired percent reduction in plaque counts.
  • the PRNT titer can be calculated based on a 50% or greater reduction in plaque counts (PRNT50).
  • a PRNT50 titer is preferred over titers using higher cut-offs (e.g., PRNT90) for vaccine sera, providing more accurate results from the linear portion of the titration curve.
  • PRNT titers There are several ways to calculate PRNT titers. The simplest and most widely used way to calculate titers is to count plaques and report the titer as the reciprocal of the last serum dilution to show >50% reduction of the input plaque count as based on the back-titration of input plaques. Use of curve fitting methods from several serum dilutions may permit calculation of a more precise result. There are a variety of computer analysis programs available for this (e.g., SPSS or GraphPad Prism). In some embodiments, an antibody titer is used to assess whether a subject has had an infection or to determine whether an immunizations are required.
  • an antibody titer is used to determine the strength of an autoimmune response, to determine whether a booster immunization is needed, to determine whether a previous vaccine was effective, and to identify any recent or prior infections.
  • an antibody titer may be used to determine the strength of an immune response induced in a subject by an RNA vaccine.
  • the anti-Borrelia antigen antibody titer, produced in a subject is increased by at least 1 log relative to a control that has not been vaccinated.
  • the anti-Borrelia antigen antibody titer produced in a subject may be increased by at least 1.5, at least 2, at least 2.5, or at least 3 log relative to a control.
  • the anti-Borrelia antigen antibody titer produced in the subject is increased by 1, 1.5, 2, 2.5 or 3 log relative to a control. In some embodiments, the anti-Borrelia antigen antibody titer produced in the subject is increased by 1-3 log relative to a control. For example, the anti-Borrelia antigen antibody titer produced in a subject may be increased by 1-1.5, 1-2, 1-2.5, 1-3, 1.5-2, 1.5-2.5, 1.5-3, 2-2.5, 2-3, or 2.5-3 log relative to a control. In some embodiments, the anti-Borrelia antigen antibody titer produced in a subject is increased at least 2 times relative to a control that has not been vaccinated.
  • the anti-Borrelia antigen antibody titer produced in a subject may be increased at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times relative to a control.
  • the anti-Borrelia antigen antibody titer produced in the subject is increased 2, 3, 4, 5, 6, 7, 8, 9, or 10 times relative to a control.
  • the anti-Borrelia antigen antibody titer produced in a subject is increased 2- 10 times relative to a control.
  • the anti-Borrelia antigen antibody titer produced in a subject may be increased 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8- 9, or 9-10 times relative to a control.
  • an antigen-specific immune response is measured as a ratio of geometric mean titer (GMT), referred to as a geometric mean ratio (GMR), of serum neutralizing antibody titers to LD.
  • GTT geometric mean titer
  • a geometric mean titer (GMT) is the average antibody titer for a group of subjects calculated by multiplying all values and taking the nth root of the number, where n is the number of subjects with available data.
  • a control in some embodiments, is an anti-Borrelia antigen antibody titer produced in a subject who has not been administered an mRNA vaccine. In some embodiments, a control is an anti-Borrelia antigen antibody titer produced in a subject administered a recombinant or purified protein vaccine.
  • Recombinant protein vaccines typically include protein antigens that either have been produced in a heterologous expression system (e.g., bacteria or yeast) or purified from large amounts of the pathogenic organism.
  • Vaccine efficacy may be assessed using standard analyses (see, e.g., Weinberg et al., J Infect Dis.2010 Jun 1;201(11):1607-10). For example, vaccine efficacy may be measured by double-blind, randomized, clinical controlled trials.
  • AR disease attack rate
  • RR relative risk
  • vaccine effectiveness may be assessed using standard analyses (see, e.g., Weinberg et al., J Infect Dis.2010 Jun 1;201(11):1607-10).
  • Vaccine effectiveness is an assessment of how a vaccine (which may have already proven to have high vaccine efficacy) reduces disease in a population.
  • Vaccine effectiveness is proportional to vaccine efficacy (potency) but is also affected by how well target groups in the population are immunized, as well as by other non-vaccine-related factors that influence the ‘real-world’ outcomes of hospitalizations, ambulatory visits, or costs.
  • a retrospective case control analysis may be used, in which the rates of vaccination among a set of infected cases and appropriate controls are compared.
  • efficacy of an mRNA vaccine is at least 60% relative to unvaccinated control subjects.
  • efficacy of the composition may be at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95%, at least 98%, or 100% relative to unvaccinated control subjects.
  • An antibody titer is a measurement of the number of antibodies within a subject, for example, antibodies that are specific to a particular antigen (e.g., an anti-Borrelia antigen). Antibody titer is typically expressed as the inverse of the greatest dilution that provides a positive result.
  • Enzyme-linked immunosorbent assay is a common assay for determining antibody titers, for example.
  • the effective amount of a composition of the present disclosure is sufficient to produce a 1:1,000-10,000 neutralizing antibody titer produced by neutralizing antibody against the specific antigen as measured in serum of the subject at 1-72 hours post administration.
  • the effective amount is sufficient to produce a 1:1,000- 5,000 neutralizing antibody titer produced by neutralizing antibody against the specific antigen as measured in serum of the subject at 1-72 hours post administration.
  • the effective amount is sufficient to produce a 1:5,000-10,000 neutralizing antibody titer produced by neutralizing antibody against the specific antigen as measured in serum of the subject at 1-72 hours post administration.
  • the neutralizing antibody titer is at least 100 NT 50 .
  • the neutralizing antibody titer may be at least 200, 300, 400, 500, 600, 700, 800, 900 or 1000 NT 50 .
  • the neutralizing antibody titer is at least 10,000 NT 50 .
  • the neutralizing antibody titer is at least 100 neutralizing units per milliliter (NU/mL).
  • the neutralizing antibody titer may be at least 200, 300, 400, 500, 600, 700, 800, 900 or 1000 NU/mL.
  • the neutralizing antibody titer is at least 10,000 NU/mL.
  • an anti-Borrelia antigen antibody titer produced in the subject is increased by at least 1 log relative to an unvaccinated control subject.
  • an anti- Borrelia antigen antibody titer produced in the subject may be increased by at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 log relative to an unvaccinated control subject.
  • an anti-Borrelia antigen antibody titer produced in the subject is increased at least 2 times relative to an unvaccinated control subject.
  • an anti- Borrelia antigen antibody titer produced in the subject is increased by at least 3, 4, 5, 6, 7, 8, 9 or 10 times relative to an unvaccinated control subject.
  • the anti-Borrelia antigen antibody titer produced in the subject is a population of neutralizing antibodies.
  • the population of neutralizing antibodies comprises antibodies that bind to OspA S1, antibodies that bind to OspA S2, antibodies that bind to OspA S3, antibodies that bind to OspA S4, antibodies that bind to OspA S5, antibodies that bind to OspA S6, and/or antibodies that bind to OspA S7.
  • the method comprises administering to the subject a mRNA vaccine described herein in an amount effective at inducing in the subject a population of neutralizing antibodies.
  • the population of neutralizing antibodies comprises IgG antibodies.
  • the population of neutralizing antibodies comprises antibodies that bind to OspA S1, antibodies that bind to OspA S2, antibodies that bind to OspA S3, antibodies that bind to OspA S4, antibodies that bind to OspA S5, antibodies that bind to OspA S6, antibodies that bind to OspA S7, or any combination thereof.
  • the manufacture of polynucleotides and/or parts or regions thereof may be accomplished utilizing the methods taught in International Publication WO2014/152027, entitled “Manufacturing Methods for Production of RNA Transcripts,” the content of which is incorporated herein by reference in its entirety.
  • Purification methods may include those taught in International Publication WO2014/152030 and International Publication WO2014/152031, each of which is incorporated herein by reference in its entirety.
  • Detection and characterization methods of the polynucleotides may be performed as taught in International Publication WO2014/144039, which is incorporated herein by reference in its entirety.
  • Characterization of the polynucleotides of the disclosure may be accomplished using polynucleotide mapping, reverse transcriptase sequencing, charge distribution analysis, detection of RNA impurities, or any combination of two or more of the foregoing.
  • “Characterizing” comprises determining the RNA transcript sequence, determining the purity of the RNA transcript, or determining the charge heterogeneity of the RNA transcript, for example. Such methods are taught in, for example, International Publication WO2014/144711 and International Publication WO2014/144767, the contents of each of which are incorporated herein by reference in their entirety.
  • LNP Lyme Disease
  • the formulation includes 0.5-15% PEG-modified lipid, 5-25% non-cationic lipid, 25-55% sterol, and 20-60% ionizable amino lipid.
  • the PEG-modified lipid is 1,2 dimyristoyl-sn-glycerol, methoxypolyethyleneglycol (PEG2000 DMG), the non-cationic lipid is 1,2 distearoyl-sn- glycero-3-phosphocholine (DSPC), the sterol is cholesterol, and the ionizable amino lipid is .
  • Example 1 Expression of mRNA Encoding Borrelia OspA S1-S7 Proteins Seven different serotypes (S) of OspA (S1, S2, S3, S4, S5, S6, and S7) were tested for their ability to be expressed in vitro.
  • mRNA having an ORF encoding one of the OspA S1-S7 (seven mRNAs, each comprising an ORF encoding one of the OspA serotypes) were introduced to cells.
  • the mRNA having ORF(s) encoding each serotype (S) of OspA (S1, S2, S3, S4, S5, S6 and S7) was modified to include an N-terminal transmembrane (TM) domain (SEQ ID NOs: 8- 12 and 14).
  • TM transmembrane domain
  • the transmembrane constructs were analyzed for protein expression using flow cytometry. The results indicate that OspA S6 had markedly less expression than the other OspA serotypes (S1-S5, and S7) (data not shown).
  • OspA S6 amino acid sequence of OspA S6 was aligned with amino acid sequences of the other OspA serotypes (S1-S5, and S7). Based on these alignments it was determined that there were two amino acids in OspA S6 that were different from other OspA serotypes (S1-S5, and S7) and may cause destabilization. At amino acid position 88 of OspA S6 there was a serine, whereas other OspA serotypes (S1-S5, and S7) had a leucine at this position.
  • OspA S6 At amino acid position 200 of OspA S6 there was an alanine, whereas other OspA serotypes (S1-S5, and S7) had a valine at this position.
  • SEQ ID NO: 13 mRNA constructs expressing a variant of OspA S6 was expressed in vitro.
  • S88L serine to leucine mutation
  • A200V amino acid position 200
  • OspA S1 variant was engineered and expressed in vitro.
  • OspA S1 The modifications to OspA S1 include removal of the leukocyte function-associated antigen (LFA) epitope (amino acid residues 165-173) and the N- terminus (Nterm) was modified to include a transmembrane (TM) domain from neuraminidase.
  • LFA leukocyte function-associated antigen
  • Nterm N-terminus
  • TM transmembrane domain from neuraminidase.
  • the modified OspA S1 and S6 variant constructs (SEQ ID NO: 8 and SEQ ID NO: 13) were expressed in cells at a concentration of 500 ng/ ⁇ L and 125 ng/ ⁇ L.
  • Example 2 Total IgG, LA-2 Competition ELISAs and In vivo Challenge Study A screen was conducted using vaccines comprising lipid nanoparticles encapsulating an mRNA having an ORF encoding OspA S1 wild-type or OspA S1 variants to test the immunogenicity of the vaccines. Additionally, the ability of the mRNA vaccine compositions to elicit antibodies that recognize the same epitope as a known bactericidal antibody (LA-2) was assessed. Construct designs of immunogens encoded by the mRNA are indicated in Table 3.
  • NTFIX non-translating mRNA
  • mice that were immunized with mRNA having an ORF encoding an OspA S1 antigen or OspA S1 variant did not enhance OspA IgG beyond levels measured on d44 (FIG.2A). Therefore, several OspA S1 constructs demonstrate high induction of total IgG.
  • LA2 competition ELISA assays were performed on serum samples from d44 and d56. Compared to NTFIX treated mice, mice administered vaccines comprising mRNA having an ORF encoding an OspA S1 antigen or OspA S1 variant showed significant LA2 signal reduction at d44 and a further decrease at d56 (FIG.2B).
  • results from the two ELISA assays indicate that treatment with OspA-S1-A results in high total IgG and LA2 signal reduction. Therefore, the mRNA OspA S1 constructs result in high levels of functional antibodies.
  • Example 3 Serum Bactericidal Activity of Borrelia OspA S1 Immunized Mouse Sera To determine the bactericidal activity of OspA-S1-ND165-173-Nterm-TM (Nterm-TM) (SEQ ID NO: 8), a serum bactericidal assay was performed.
  • the bactericidal activity of the OspA-S1-ND165-173-Nterm-TM (Nterm-TM) (SEQ ID NO: 8) (Pool 2) was compared to the OspA-S1-ND165-173-Cterm-TM (Pool 3) and non- translating mRNA (NTFIX) controls (Pool 1) using a serum bactericidal assay.
  • the results indicate that the survival index of N-terminal transmembrane constructs (Pool 2) was much less than the C-terminal transmembrane constructs (Pool 3).
  • mice administered a non-translating mRNA (NTFIX), an mRNA having an ORF encoding an OspA S1 with the LFA epitope removed (Soluble), an mRNA having an ORF encoding OspA S1 with the LFA epitope removed and an N-terminal transmembrane domain (Nterm-TM) (SEQ ID NO: 8), or an mRNA having an ORF encoding OspA S1 with the LFA epitope removed and a C-terminal transmembrane domain (Cterm-TM).
  • NTFIX non-translating mRNA
  • Soluble Soluble
  • Nterm-TM N-terminal transmembrane domain
  • Cterm-TM C-terminal transmembrane domain
  • tick bite challenge was performed using Borrelia burgdorferi strain B31. Mice were challenged with five ticks, resulting in an unknown bacterial load administered, and 2 weeks post tick feed mice were sacrificed, and skin, hearts and bladders were collected. These tissues were then cultured and monitored for the presence of spirochetes for 3 weeks. The tick bite challenge readout was performed 2 weeks post tick feed and the presence of spirochetes on skin, heart and bladder was observed for 3 weeks (no quantification).
  • LA2 ELISA competition assays were performed on blood samples pre-tick bite and post- tick bite challenge. Groups of mice with low LA2 competition titers were considered protected in the challenge. Therefore, in this model protection is also provided by an LA-2 independent mechanism.
  • mice administered vaccine compositions comprising mRNA having an ORF encoding an OspA-S1-ND165-173-Nterm-TM (Nterm-TM) (SEQ ID NO: 8) showed significant LA2 signal reduction (FIG.4B).
  • Results from the two ELISA assays indicate that OspA-S1-ND165-173-Nterm-TM (Nterm-TM) (SEQ ID NO: 8) treatment results in high total IgG and LA2 signal reduction.
  • results of the tick bite challenge indicate that several constructs, including OspA-S1- ND165-173-Nterm-TM (Nterm-TM) (SEQ ID NO: 8), resulted in high induction of total IgG. Also, the results of the LA-2 competition ELISAs suggest high levels of functional antibodies produced in response to several OspA S1 constructs, notably, the OspA-S1-ND165-173-Nterm- TM (Nterm-TM) (SEQ ID NO: 8). These results are reproducible and demonstrate variation in ability of constructs to protect mice from challenge. Example 5.
  • mice immunized twice with a lower dose of OspA S1 NtermTM mRNA, or with six additional mRNAs, OspA S1-S7 NtermTM, are still protected in a tick bite challenge model the following experiments were performed, or if the six additional mRNAs would interfere with the ability to elicit a protective response with the serotype 1 mRNA.
  • the total and functional antibody titers to recombinant antibodies were compared using LA-2 (only bactericidal against S1) and a positive control antibody (bactericidal against all 7 serotypes).
  • mice were administered vaccines comprising lipid nanoparticles encapsulating an mRNA having one ORF encoding OspA S1 (e.g., monovalent) (SEQ ID NO: 8) or seven mRNAs collectively encoding OspA S1-S7 (e.g., heptavalent; seven mRNAs, each mRNA having an ORF encoding one of the seven OspA serotypes) (SEQ ID NOs: 8-14).
  • Study design and construct designs of immunogens encoded by the mRNA are indicated in TABLE 5.
  • OspA S1 e.g., monovalent
  • OspA S1-S7 e.g., heptavalent
  • LFA leukocyte function-associated antigen
  • Nterm N-terminus
  • the S6 was engineered to include a serine to leucine mutation at amino acid position 88 (S88L) and an alanine to valine mutation at amino acid position 200 (A200V) and the N- terminus (Nterm) was modified to include a transmembrane (TM) domain (SEQ ID NO: 13).
  • S88L serine to leucine mutation at amino acid position 88
  • A200V alanine to valine mutation at amino acid position 200
  • Nterm transmembrane domain
  • Some mice were immunized with non-translating mRNA (NTFIX) as a negative control.
  • Other mice were immunized with OspA S1 Cterm ferritin stabilized or Recombitek® (Santa Cruz Biotechnology) with alum.
  • Table 5 Study design to compare monovalent and heptavalent constructs.
  • GR N Dose Immunogen Dose Bleeding Challeng Readout 1 2 3 4 5 Mice (n 5 animals/group) were immunized with two doses of one of the vaccines listed in Table 5.
  • the dosing regimen was as follows: the first dose was administered on day 1 (d1), and the second dose on day 21 (d21). Blood samples were collected for all groups on d1, day 20 (d20), and pre-challenge and post-challenge.
  • the readouts were measuring OspA antibody response and a tick bite challenge. The tick bite challenge was performed using Borrelia burgdorferi. Mice were challenged with five ticks. Mice were monitored for 3-4 weeks comprising of combined cultures from multiple tissues.
  • OspA S1 IgG levels were measured. Compared to NTFIX treated mice, all groups immunized with mRNA having an ORF(s) encoding OspA S1 (e.g., monovalent) (SEQ ID NO: 8) or OspA S1-S7 (e.g., heptavalent) (SEQ ID NOs: 8-14) showed enhanced production of OspA S1 IgG (FIG.5A).
  • LA2 ELISA competition assays were performed. Groups of mice with low LA2 competition titers were considered protected in the challenge.
  • mice administered vaccines comprising mRNA having an ORF(s) encoding OspA S1 (e.g., monovalent) (SEQ ID NO: 8) or OspA S1-S7 (e.g., heptavalent) (SEQ ID NOs: 8-14) showed significant LA2 signal reduction (FIG.5B).
  • a positive control antibody was also used in an ELISA competition assay that was performed. The positive control antibody is an additional monoclonal antibody to OspA that is bactericidal. In contrast to LA-2, which only recognizes an epitope on OspA S1, this positive control antibody recognizes all seven OspA serotypes.
  • mice administered vaccines comprising mRNA having an ORF(s) encoding OspA S1 (e.g., monovalent) (SEQ ID NO: 8) or OspA S1-S7 (e.g., heptavalent) (SEQ ID NOs: 8-14) showed significant signal reduction compared to positive control antibody (FIG.5C).
  • OspA S1 e.g., monovalent
  • OspA S1-S7 e.g., heptavalent
  • mice were administered vaccines comprising lipid nanoparticles encapsulating an mRNA having an ORF(s) encoding OspA-S1- ND165-173-Nterm-TM (OspA S1 NtermTM; mRNA-1982) (SEQ ID NO: 8) and OspA S1- S7(OspA S1-S7 NtermTM; mRNA-1975; seven mRNAs, each mRNA having an ORF encoding one of the seven OspA serotypes) (SEQ ID NOs: 8-14) constructs.
  • mice were immunized with S1-S7 soluble protein with alum or Recombitek® (Santa Cruz Biotechnology) with Alum or PBS control.
  • OspA S1 NtermTM was administered at either 0.5 ⁇ g or 1 ⁇ g and OspA S1-S7 NtermTM was administered at either 3.5 ⁇ g or 7 ⁇ g.
  • the total OspA IgG levels were measured on d20 and d36 using Luminex.
  • the mean fluorescence intensity (MFI) measured across all serotypes indicate that higher titers were observed in OspA NtermTM S1 and OspA NtermTM S1-S7 compared to mice that were administered protein or PBS. These results indicate that the modified OspA S1 and S6 or monovalent OspA S1 induce potent antibody responses (FIG.6).
  • OspA Antibodies The ability of antibodies generated in response to OspA NtermTM S1 (SEQ ID NO: 8) and OspA NtermTM S1-S7 (SEQ ID NOs: 8-14) to bind to Borrelia bacteria were tested using a surface binding assay.
  • the mRNA vaccines were compared to mice administered PBS or Recombitek® or S1-S7 protein, LA-2 or S1 Hyper+ (hyperimmune sera generated by immunizing mice with 2 doses of 25 ⁇ g of the OspA S1 mRNA).
  • the results of the surface binding assay demonstrate that antibodies generated by OspA NtermTM S1 (SEQ ID NO: 8) and OspA NtermTM S1-S7 (SEQ ID NOs: 8-14) mRNA immunization bind to the surface of Borrelia bacteria (FIGs.7A-7B).
  • Agglutination/Membrane Integrity Study The ability of antibodies generated in response to OspA NtermTM S1 (SEQ ID NO: 8) and OspA NtermTM S1-S7 (SEQ ID NOs: 8-14) to agglutinate Borrelia bacteria was tested using an Agglutination assay.
  • Dosing Study The following dosing study was conducted for OspA S1 NtermTM (SEQ ID NO: 8) to determine the lower limit of dosing amount to result in breakthrough infection in a tick bite model and whether 2 doses is as effective at protecting as 3 doses in a tick bite model.
  • the experimental design of the dosing study is indicated in Table 8.
  • Control animals were administered 1 ⁇ g/animal of either NTFIX or Recombitek® (Santa Cruz Biotechnology) +Alum.
  • the first dose was administered on day 1 (d1)
  • the second dose was administered on day 21 (d21)
  • the third dose was administered on day 42 (d42).
  • blood samples were collected on d1, day 20 (d20), day 41 (d41), pre-challenge on day 56 (d56) and post-challenge day 74 (d74).
  • For the 2 dose regime blood samples were collected on d1, day 20 (d20), pre-challenge on day 35 (d35) and post-challenge day 54 (d54).
  • 5 ticks were administered per mouse.
  • the readout assays performed were Total OspA ELISA, LA-2 competition, positive control antibody competition, 4 week monitoring of cultured tissues.
  • the results, as measured by total IgG levels demonstrate that 2 doses of OspA S1 NtermTM (SEQ ID NO: 8) resulted in 1 mouse per group getting infected and that antibody titers were not predictive of infection. Also, 3 doses of OspA S1 NtermTM (SEQ ID NO: 8) provided full protection to all mice, even at 0.2 ⁇ g dose.
  • Heptavalent mRNA vaccine (SEQ ID NOs: 8-14) and monovalent mRNA vaccine (SEQ ID NO: 8) as 3-dose prophylactic vaccines against Borrelia infection by eliciting anti-OspA antibody responses, which had been shown to block the transmission of Borrelia spirochetes from the bite of an infected tick and prevent Lyme disease.
  • Heptavalent mRNA vaccine (SEQ ID NOs: 8-14) encodes for 7 different serotype OspA for 4 major Borrelia species causing disease in the US and Europe: Borrelia burgdorferi (SR1), afzelii (SR2), bavariensis (SR4), and garinii (SR3, 5-7).
  • monovalent mRNA vaccine encodes for OspA from SR1 that is specific for B. burgdorferi, the dominant species circulating in the US that causes Lyme disease.
  • the primary objective of the study is to evaluate the safety and reactogenicity of heptavalent mRNA vaccine (SEQ ID NOs: 8-14) and monovalent mRNA vaccine (SEQ ID NO: 8).
  • the primary endpoints are: solicited local and systemic adverse reactions (ARs) through 7 days after each study injection; unsolicited AEs through 28 days after each study injection; medically attended adverse events (MAAEs) from Day 1 to 6 months after last study injection; adverse event of special interests (AESIs) from Day 1 to end-of-study (EoS); serious adverse events (SAEs) from Day 1 to EoS; AEs leading to discontinuation of study injection or study participation from Day 1 to EoS; and safety laboratory abnormalities through 7 days after each study injection.
  • ARs local and systemic adverse reactions
  • MAAEs medically attended adverse events
  • AESIs adverse event of special interests
  • EoS end-of-study
  • SAEs serious adverse events
  • the secondary objective of the study is to evaluate the humoral immunogenicity of heptavalent mRNA vaccine (SEQ ID NOs: 8-14) and monovalent mRNA vaccine (SEQ ID NO: 8) at Days 1, 29, 85, and 197.
  • GTT geometric mean titer
  • GMFR geometric mean fold rise
  • the exploratory objectives of the study are to evaluate the humoral immunogenicity of heptavalent mRNA vaccine (SEQ ID NOs: 8-14) and monovalent mRNA vaccine (SEQ ID NO: 8); to further characterize antibody responses of heptavalent mRNA vaccine (SEQ ID NOs: 8- 14) and monovalent mRNA vaccine (SEQ ID NO: 8); and to evaluate procedures in determining cases of Borrelia infection using a combination of clinical assessments and Lyme infection serology test results during the study.
  • Exploratory endpoints include: GMT, GMFR, and seroresponse of anti-OspA-binding IgG antibodies at all evaluable timepoints; GMT and GMFR of serum bactericidal activity (SBA) elicited against Borrelia species expressing each of the SR1-7 OspA for a subset of participants; inhibition of LA-2 or other bactericidal epitope- specific antibodies when competing with elicited anti-OspA antibodies post study injection; and frequency of Borrelia infection assessed by combination of serological tests and clinical assessments.
  • SBA serum bactericidal activity
  • mice were administered vaccines comprising lipid nanoparticles encapsulating an mRNA having an ORF(s) encoding OspA S1-S7 (e.g., heptavalent; seven mRNAs, each mRNA having an ORF encoding one of the seven OspA serotypes) (SEQ ID NOs: 8-14).
  • OspA S1-S7 e.g., heptavalent
  • seven mRNAs e.g., heptavalent
  • SEQ ID NOs: 8-14 were designed as previously described in Example 5.
  • mice were immunized with non-translating mRNA (NTFIX) as a negative control.
  • Other mice were immunized with Recombitek® (Santa Cruz Biotechnology) with alum.
  • the three dose regimen was as follows: the first dose was administered on day 1 (d1), the second dose on day 21 (d21), and the third does on day 42 (d42). Blood samples were collected for all groups on d- 1, day 20 (d20), day 41 (d41), and pre-challenge and post-challenge. The readouts were measuring OspA antibody response and a tick bite challenge. The tick bite challenge was performed using Borrelia burgdorferi. Mice were challenged with five ticks. Two weeks after tick feeding, mice were sacrificed and skin samples, ankles, bladders, and hearts were collected. These tissues were cultures and monitored for 3-4 weeks to monitor for the presence of spirochetes.
  • OspA S1-S7 IgG levels were measured pre-challenge.
  • all groups immunized with mRNA having an ORFs encoding OspA S1-S7 (e.g., heptavalent) (SEQ ID NOs: 8-14) showed enhanced production of OspA S1-S7 IgGs (FIG.12).
  • OspA S1-S7 e.g., heptavalent
  • OspA S1-S7 IgG Longevity Study Experiments were performed to test OspA SR1-S7 IgG longevity in mice immunized twice with a OspA S1 NtermTM mRNA or a OspA S1-S7 NtermTM mRNA or protein.
  • mice were administered vaccines comprising lipid nanoparticles encapsulating an mRNA having an ORF(s) encoding OspA S1 (e.g., monovalent) (SEQ ID NO: 8), OspA S1-S7 (e.g., heptavalent; seven mRNAs, each mRNA having an ORF encoding one of the seven OspA serotypes) (SEQ ID NOs: 8-14), lipidated purified OspA with alum, or purified OspA proteins of serotypes 1-7 with alum. Study design and construct designs of immunogens encoded by the mRNA are indicated in TABLE 11.
  • OspA S1 e.g., monovalent
  • OspA S1-S7 e.g., heptavalent; seven mRNAs, each mRNA having an ORF encoding one of the seven OspA serotypes
  • SEQ ID NOs: 8-14 lipidated purified
  • mRNA having an ORF(s) encoding OspA S1 e.g., monovalent
  • OspA S1-S7 e.g., heptavalent
  • SEQ ID NOs: 8-14 were designed as previously described in Example 5.
  • Some mice were immunized with PBS as a negative control.
  • Other mice were immunized with OspA S1-S7 protein or Recombitek® (Santa Cruz Biotechnology) with alum.
  • Table 11 Study design to compare OspA S1 IgG longevity.
  • the dosage regimen was as follows: the first dose was administered on day 1 (d1), and the second dose on day 29 (d29). Blood samples were collected for all groups on day 29 (d29), day 42 (d42), day 84 (d84), day 126 (d126), day 168 (d168), and day 189 (d189).
  • the total OspA S1 IgG levels were measured in the serum samples using Luminex (readout in mean fluorescence intensity (MFI)) (FIG.14).
  • Total OspA S1-S7 IgG levels were also measured in serum samples collected from the OspA S1-S7 NtermTM (SEQ ID NOs: 8-14) injected mice and the OspA S1-S7 protein+Alum injected mice (data not shown). T50 for these IgGs are provided in Table 12.
  • Table 12 Comparison of OspA S1-S7 IgG T50s. I S S S S S S 7 173 100
  • OspA S1-S7 IgGs produced following administration of the mRNA constructs have increased half-lives relative to OspA S1-S7 IgGs produced following administration of the protein constructs.
  • Example 11 Cross-Protection Study Experiments were performed to test for potential cross-protection activity among OspA antigens.
  • C3H/HeN mice were administered vaccines comprising lipid nanoparticles encapsulating an mRNA having an ORF(s) encoding OspA S1 (monovalent) (SEQ ID NO: 8), OspA S1-S7 (heptavalent; seven mRNAs, each mRNA having an ORF encoding one of the seven OspA serotypes) (SEQ ID NOs: 8-14), OspA S2-S7 (hexavalent; six mRNAs, each mRNA having an ORF encoding one of the six OspA serotypes) (SEQ ID NOs: 9-14), non-translating mRNA (NTFIX), or Recombitek® (Santa Cruz Biotechnology) with alum.
  • OspA S1 monoovalent
  • OspA S1-S7 heptavalent; seven mRNAs, each mRNA having an ORF encoding one of the seven OspA serotypes
  • mRNA having an ORF(s) encoding OspA S1 (monovalent) (SEQ ID NO: 8), OspA S1-S7 (heptavalent) (SEQ ID NOs: 8-14), or OspA S2-S7 (hexavalent) (SEQ ID NOs: 9- 14) were designed as previously described in Example 5. Some mice were immunized with NTFIX as a negative control. Other mice were immunized with Recombitek® with alum. Table 13: Study design to assess cross-protection.
  • Control animals were administered 3.5 ⁇ g/animal of non- translating mRNA (NTFIX), or 1 ⁇ g/animal of Recombitek®+Alum.
  • the dosage regimen was as follows: the first dose was administered on Day 1 (D1), and the second dose on day 21 (D21). Blood samples were collected for all groups on D1, and Day 20 (D20), pre-challenge on day 35 (D35) and post-challenge day 53 (D53).
  • 5 ticks S1 Borrelia burgdorferi
  • the readout assays performed were Total OspA ELISA, LA-2 competition, positive control antibody competition, and 4 week monitoring of cultured tissues.
  • the total OspA S1 IgG levels were measured.
  • all groups immunized with mRNA having an ORF(s) encoding OspA S1 (monovalent) (SEQ ID NO: 8), OspA S1-S7 (heptavalent) (SEQ ID NOs: 8-14), or OspA S2-S7 (hexavalent) (SEQ ID NOs: 9-14) showed enhanced production of OspA S1 IgG relative to NTFIX treated controls (FIG.15A).
  • LA2 ELISA competition assays were performed.
  • mice administered vaccines comprising mRNA having an ORF(s) encoding OspA S1 (monovalent) (SEQ ID NO: 8), OspA S1-S7 (heptavalent) (SEQ ID NOs: 8-14), or OspA S2-S7 (hexavalent) (SEQ ID NOs: 9-14) showed significant LA2 signal reduction (FIG.15B).
  • a positive control antibody which is a known bactericidal monoclonal antibody that recognizes all 7 OspA serotypes, was also used in an ELISA competition assay.
  • mice administered vaccines comprising mRNA having an ORF(s) encoding OspA S1 (monovalent) (SEQ ID NO: 8), OspA S1-S7 (heptavalent) (SEQ ID NOs: 8- 14), or OspA S2-S7 (hexavalent) (SEQ ID NOs: 9-14) showed significant positive control antibody signal reduction (FIG.15C). The percent of infected mice was also measured.
  • mice There was 100% infection in mice that received NTFIX treatment, and 0% infection in mice administered vaccines comprising mRNA having an ORF encoding OspA S1 (monovalent) (SEQ ID NO: 8), OspA S1-S7 (heptavalent) (SEQ ID NOs: 8-14), or OspA S2-S7 (hexavalent) (SEQ ID NOs: 9-14) (FIG.15D).
  • a messenger ribonucleic acid (mRNA) vaccine comprising: an mRNA polynucleotide comprising an open reading frame encoding a protein comprising a Borrelia outer surface protein A (OspA) extracellular domain and optionally a heterologous amino terminal transmembrane domain; and a lipid nanoparticle.
  • a messenger ribonucleic acid (mRNA) vaccine comprising: an mRNA polynucleotide comprising an open reading frame encoding a nonlipidated Borrelia outer surface protein A (OspA) protein; and a lipid nanoparticle.
  • the mRNA vaccine of embodiment 1 or 2 wherein the Borrelia OspA protein comprises a heterologous amino terminal transmembrane domain.
  • hLFA-1 epitope of the Borrelia OspA S1 protein comprises the following mutations Y165F, V166I, T170R, and L171F, relative to a naturally occurring Borrelia OspA S1 protein comprising the amino acid sequence of SEQ ID NO: 24.
  • the mRNA vaccine of embodiment 9, further comprising two, three, four, five or six additional mRNAs, each comprising an open reading frame encoding a nonlipidated Borrelia OspA protein comprising a different Borrelia OspA extracellular domain selected from a Borrelia afzelii OspA S2 extracellular domain, a Borrelia garinii OspA S3 extracellular domain, a Borrelia bavariensis OspA S4 extracellular domain, a Borrelia garinii OspA S5 extracellular domain, a Borrelia garinii S6 protein, and a Borrelia garinii OspA S7 extracellular domain, optionally wherein each of the nonlipidated Borrelia OspA proteins encoded by the additional mRNAs comprises a heterologous amino terminal transmembrane domain and/or N-linked glycan site mutations.
  • the one or more amino acid modification(s) is a mutation corresponding to a S88L mutation or an A200V mutation of the naturally occurring Borrelia garinii OspA S6 protein. 18.
  • a messenger ribonucleic acid (mRNA) vaccine comprising: (a) an mRNA polynucleotide comprising an open reading frame encoding an outer surface protein A (OspA) protein comprising an extracellular domain of a first serotype of Borrelia; (b) an mRNA polynucleotide comprising an open reading frame encoding an OspA protein comprising an extracellular domain of a second serotype of Borrelia, wherein the second serotype of Borrelia is of a species that is different from that of the first serotype of Borrelia; and a lipid nanoparticle. 21.
  • a messenger ribonucleic acid (mRNA) vaccine comprising: at least two, three, four, five, six, or seven mRNAs, each comprising an open reading frame encoding a Borrelia outer surface protein A (OspA) protein comprising an extracellular domain, wherein the extracellular domain of each of the Borrelia OspA proteins is of a different Borrelia serotype; and a lipid nanoparticle. 22.
  • mRNA messenger ribonucleic acid
  • a messenger ribonucleic acid (mRNA) vaccine comprising at least four mRNAs, optionally seven mRNAs, each encoding a Borrelia OspA protein comprising an extracellular domain of one of four different serotypes of Borrelia, optionally selected from Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii, and Borrelia bavariensis; and a lipid nanoparticle.
  • a messenger ribonucleic acid (mRNA) vaccine comprising: an mRNA polynucleotide comprising an open reading frame encoding a protein comprising an extracellular domain from a Borrelia garinii OspA serotype 6 protein, wherein the extracellular domain comprises one or more amino acid modification(s), optionally selected from a S88L mutation and an A200V mutation, relative to a corresponding naturally occurring Borrelia garinii OspA S6 extracellular domain, to stabilize expression in mammalian cells; and a lipid nanoparticle.
  • the protein further comprises a heterologous amino terminal transmembrane domain and/or one or more N-linked glycan site mutation(s).
  • 29. The mRNA vaccine of any one of the preceding embodiments, wherein the lipid nanoparticle comprises an ionizable lipid, a neutral lipid, a sterol, and a PEG-modified lipid.
  • the ionizable lipid comprises a structure of Compound (I): ; the ; the sterol is cholesterol; and/or the PEG-modified lipid is 1,2 dimyristoyl-sn-glycerol, methoxypolyethyleneglycol (PEG-DMG).
  • the Borrelia afzelii OspA S2 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 2. 34.
  • the Borrelia garinii OspA S6 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 6.
  • the Borrelia burgdorferi OspA S1 protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1;
  • the Borrelia afzelii OspA S2 protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 2;
  • the Borrelia garinii OspA S3 protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 3;
  • the Borrelia bavariensis OspA S4 protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 4;
  • the Borrelia garinii OspA S5 protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 5;
  • the Borrelia garinii OspA S6 protein comprises an amino acid sequence having at least 90% identity
  • the Borrelia burgdorferi OspA S1 protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 1;
  • the Borrelia afzelii OspA S2 protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 2;
  • the Borrelia garinii OspA S3 protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 3;
  • the Borrelia bavariensis OspA S4 protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 4;
  • the Borrelia garinii OspA S5 protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 5;
  • the Borrelia garinii OspA S6 protein comprises an amino amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 5;
  • the Borrelia burgdorferi OspA S1 protein comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 1;
  • the Borrelia afzelii OspA S2 protein comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 2;
  • the Borrelia garinii OspA S3 protein comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 3;
  • the Borrelia bavariensis OspA S4 protein comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 4;
  • the Borrelia garinii OspA S5 protein comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 5;
  • the Borrelia garinii OspA S6 protein comprises an amino amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 5;
  • the Borrelia burgdorferi OspA S1 protein comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 1;
  • the Borrelia afzelii OspA S2 protein comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 2;
  • the Borrelia garinii OspA S3 protein comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 3;
  • the Borrelia bavariensis OspA S4 protein comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 4;
  • the Borrelia garinii OspA S5 protein comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 5;
  • the Borrelia garinii OspA S6 protein comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO:
  • a method of preventing Lyme disease in a subject in need thereof comprising administering to the subject one or more doses of the vaccine of any one of the preceding embodiments in an effective amount to produce an immune response to a Borrelia bacterial infection.
  • 44. The method of embodiment 43, wherein the vaccine is administered intramuscularly.
  • 45. The method of embodiment 43 or embodiment 44, comprising administering a dose of 12.5-150 ⁇ g of the mRNA.
  • the method of any one of embodiments 43-45 comprising administering a single dose of the vaccine to the subject.
  • the method of any one of embodiments 43-45 comprising administering a prime dose and a booster dose of the vaccine to the subject. 48.
  • the method of any one of embodiments 43-45 comprising administering a first dose of the vaccine, a second dose of the vaccine, and a third dose of the vaccine. 49.
  • the method embodiment 48, wherein in second dose is administered two months following the first dose.
  • the method of embodiment 48 or embodiment 49, wherein the third dose is administered six months following the first dose.
  • the method of any one of embodiments 48-50, wherein the first dose, the second dose, and the third dose each comprise 12.5-150 ⁇ g of the RNA.
  • the method of embodiment 51, wherein the first dose, the second dose, and the third dose each comprise 12.5, 25, 50, 100, 150 ⁇ g of the RNA. 53.
  • the mRNA vaccine comprises a first mRNA polynucleotide comprising an open reading frame encoding a protein comprising a Borrelia burgdorferi outer surface protein A serotype 1 (OspA S1 protein), a second mRNA polynucleotide comprising an open reading frame encoding a protein comprising a Borrelia afzelii outer surface protein A serotype 2 (OspA S2 protein), a third mRNA polynucleotide comprising an open reading frame encoding a protein comprising a Borrelia garinii outer surface protein A serotype 3 (OspA S3 protein), a fourth mRNA polynucleotide comprising an open reading frame encoding a protein comprising a Borrelia bavariensis outer surface protein A serotype 4 (OspA S4 protein), a fifth mRNA polynucleotide comprising an open reading frame encoding
  • the mRNA vaccine comprises a mRNA polynucleotide comprising an open reading frame encoding a protein comprising a Borrelia burgdorferi outer surface protein A serotype 1 (OspA S1 protein), wherein the Borrelia burgdorferi OspA S1 protein comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 1.
  • the lipid nanoparticle comprises a mixture of lipids that comprises 20-60 mol% ionizable lipid heptadecan-9-yl 8 ((2 hydroxyethyl)(6 oxo 6-(undecyloxy)hexyl)amino)octanoate (Compound 1); 5-25 mol% 1,2 distearoyl sn glycero-3 phosphocholine (DSPC); 25-55 mol% cholesterol; and 0.5-15 mol% 1- monomethoxypolyethyleneglycol-2,3-dimyristylglycerol with polyethylene glycol of average molecular weight 2000 (PEG2000 DMG). 57.
  • the lipid nanoparticle comprises: 47 mol% ionizable cationic lipid; 11.5 mol% neutral lipid; 38.5 mol% sterol; and 3.0 mol% PEG-modified lipid; 48 mol% ionizable cationic lipid; 11 mol% neutral lipid; 38.5 mol% sterol; and 2.5 mol% PEG-modified lipid; 49 mol% ionizable cationic lipid; 10.5 mol% neutral lipid; 38.5 mol% sterol; and 2.0 mol% PEG-modified lipid; 50 mol% ionizable cationic lipid; 10 mol% neutral lipid; 38.5 mol% sterol; and 1.5 mol% PEG-modified lipid; or 51 mol% ionizable cationic lipid; 9.5 mol% neutral lipid; 38.5 mol% sterol; and 1.0 mol% PEG-modified lipid.
  • mRNA vaccine further comprises Tris buffer, sucrose, and sodium acetate, or any combination thereof.
  • mRNA vaccine further comprises 30-40 mM Tris buffer, 80-95 mg/mL sucrose, and 5-15 mM sodium acetate.
  • 60. The method of any one of embodiments 43-59, wherein the mRNA vaccine has a pH value of 6-8, optionally 7.5. 61.
  • any one of embodiments 54-60 wherein the first dose, the second dose, and the third dose is 12.5 ⁇ g of the mRNA, and wherein the second dose is administered 2 months after the first dose, and the third dose is administered 4 months after the second dose.
  • the first dose, the second dose, and the third dose is 50 ⁇ g of the mRNA, and wherein the second dose is administered 2 months after the first dose, and the third dose is administered 4 months after the second dose.
  • any one of embodiments 54-60 wherein the first dose, the second dose, and the third dose is 100 ⁇ g of the mRNA, and wherein the second dose is administered 2 months after the first dose, and the third dose is administered 4 months after the second dose.
  • 64. The method of any one of embodiments 54-60, wherein the first dose, the second dose, and the third dose is 150 ⁇ g of the mRNA, and wherein the second dose is administered 2 months after the first dose, and the third dose is administered 4 months after the second dose. 65.
  • a method of inducing in a subject an immune response to a Borrelia bacterial infection comprising: administering to the subject the mRNA vaccine of any one of embodiments 1-42 in an amount effective at inducing in the subject a population of neutralizing antibodies.
  • the population of neutralizing antibodies comprises IgG antibodies.
  • the population of neutralizing antibodies comprises antibodies that bind to OspA S1, antibodies that bind to OspA S2, antibodies that bind to OspA S3, antibodies that bind to OspA S4, antibodies that bind to OspA S5, antibodies that bind to OspA S6, antibodies that bind to OspA S7, or any combination thereof.
  • a composition comprising a plurality of polynucleotides, wherein the plurality comprises two or more of: a first polynucleotide comprising a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 8; a second polynucleotide comprising a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 9; a third polynucleotide comprising a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 10; a fourth polynucleotide comprising a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 11; a fifth polynucleotide comprising a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to
  • composition of embodiment 70 wherein: the first polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 8; the second polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 9; the third polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 10; the fourth polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 11; the fifth polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 12; the sixth polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 13; and the seventh polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 14. 72.
  • OspA Borrelia outer surface protein A
  • a messenger ribonucleic acid (mRNA) vaccine comprising: an mRNA polynucleotide comprising an open reading frame encoding a Borrelia outer surface protein A (OspA) extracellular domain and optionally a heterologous amino terminal transmembrane domain, optionally wherein the heterologous amino terminal transmembrane domain is from an influenza virus neuraminidase protein; and a lipid nanoparticle.
  • OspA Borrelia outer surface protein A
  • the mRNA vaccine of embodiment 75 wherein the mRNA polynucleotide comprises an open reading frame encoding a nonlipidated Borrelia OspA protein comprising a Borrelia burgdorferi OspA serotype 1 (S1) extracellular domain, optionally wherein the nonlipidated Borrelia OspA mRNA polynucleotide comprises a heterologous amino terminal transmembrane domain and/or N-linked glycan site mutations. 77.
  • the mRNA vaccine of embodiment 76 comprising: a first mRNA polynucleotide comprising an open reading frame encoding a nonlipidated Borrelia OspA protein comprising a Borrelia burgdorferi OspA serotype 1 (S1) extracellular domain, optionally wherein the nonlipidated Borrelia OspA protein encoded by the first mRNA polynucleotide comprises a heterologous amino terminal transmembrane domain and/or N- linked glycan site mutations; a second mRNA polynucleotide comprising an open reading frame encoding a nonlipidated Borrelia OspA protein comprising a Borrelia afzelii OspA S2 extracellular domain, optionally wherein the nonlipidated Borrelia OspA protein encoded by the second mRNA polynucleotide comprises a heterologous amino terminal transmembrane domain and/or N- linked glycan site mutations; a third
  • mRNA vaccine of embodiment 75 or embodiment 76 wherein the mRNA polynucleotide comprises at least two open reading frames, each encoding a Borrelia OspA extracellular domain and optionally a heterologous amino terminal transmembrane domain.
  • a messenger ribonucleic acid (mRNA) vaccine comprising: at least two, three, four, five, six, or seven mRNAs, each comprising an open reading frame encoding a Borrelia outer surface protein A (OspA) protein comprising an extracellular domain, wherein the extracellular domain of each of the Borrelia OspA proteins is of a different Borrelia serotype; and a lipid nanoparticle. 80.
  • mRNA messenger ribonucleic acid
  • the mRNA vaccine of embodiment 79 wherein the mRNA vaccine, when administered to a subject, cross-protects against a Borrelia outer surface protein A (OspA) protein that is not encoded by the mRNA vaccine, optionally wherein the cross-protection comprises generation of cross-reactive antibodies against the Borrelia outer surface protein A (OspA) protein that is not encoded by the mRNA vaccine.
  • OspA Borrelia outer surface protein A
  • a messenger ribonucleic acid (mRNA) vaccine comprising an mRNA polynucleotide comprising an open reading frame encoding a protein comprising an extracellular domain from a Borrelia garinii OspA serotype 6 protein, wherein the extracellular domain comprises one or more amino acid modification(s), optionally selected from a S88L mutation and an A200V mutation, relative to a corresponding naturally occurring Borrelia garinii OspA S6 extracellular domain, to stabilize expression in mammalian cells.
  • the mRNA vaccine of embodiment 82 wherein 100% of the uracil nucleotides of the one or more mRNA(s) comprise a chemical modification, wherein the chemical modification is 1-methylpseudouracil.
  • the mRNA vaccine of any one of the preceding embodiments comprising a lipid nanoparticle, wherein the lipid nanoparticle comprises an ionizable lipid, a neutral lipid, a sterol, and a PEG-modified lipid, optionally wherein the lipid nanoparticle comprises 40–50 mol% ionizable lipid, 5–15 mol% neutral lipid, 30–50 mol% sterol, and 0.5–3 mol% PEG-modified lipid.
  • the lipid nanoparticle comprises 40–50 mol% ionizable lipid, 5–15 mol% neutral lipid, 30–50 mol% sterol, and 0.5–3 mol% PEG-modified lipid.
  • the mRNA vaccine of embodiment 84 wherein the ionizable lipid comprises a structure of Compound (I): ; the ; the sterol is cholesterol; and/or the PEG-modified lipid is 1,2 dimyristoyl-sn-glycerol, methoxypolyethyleneglycol (PEG-DMG). 86.
  • the mRNA vaccine of any one of embodiments 75-85 comprising: (a) an mRNA polynucleotide comprising an open reading frame encoding a protein comprising a Borrelia burgdorferi OspA S1 protein comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 98% identity to the amino acid sequence of SEQ ID NO: 1; (b) an mRNA polynucleotide comprising an open reading frame encoding a protein comprising a Borrelia afzelii OspA S2 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 98% identity to the amino acid sequence of SEQ ID NO: 2; (c) an mRNA polynucleotide comprising an open reading frame encoding a protein comprising a Borrelia garinii OspA S3 protein comprises an amino acid sequence having at least 80%, at least 85%, at
  • the Borrelia burgdorferi OspA S1 protein comprises the amino acid sequence of SEQ ID NO: 1; the Borrelia afzelii OspA S2 protein comprises the amino acid sequence of SEQ ID NO: 2; the Borrelia garinii OspA S3 protein comprises the amino acid sequence of SEQ ID NO: 3; the Borrelia bavariensis OspA S4 protein comprises the amino acid sequence of SEQ ID NO: 4; the Borrelia garinii OspA S5 protein comprises the amino acid sequence of SEQ ID NO: 5; the Borrelia garinii OspA S6 protein comprises the amino acid sequence of SEQ ID NO: 6; and the Borrelia garinii OspA S7 protein comprises the amino acid sequence of SEQ ID NO: 7.
  • a method of inducing an immune response to a Borrelia antigen in a subject comprising administering to the subject one or more doses of the mRNA vaccine of any one of the preceding embodiments in an effective amount to produce an immune response to a Borrelia antigen in the subject.
  • the method of any one of embodiments 88-90 comprising administering a single dose of the vaccine to the subject. 92.
  • any one of embodiments 88-90 comprising administering a first dose of the vaccine, a second dose of the vaccine, and a third dose of the vaccine.
  • the method of embodiment 92 wherein the second dose is administered two months following the first dose, the third dose is administered six months following the first dose, or a combination thereof.
  • first dose, the second dose, and the third dose is 100 ⁇ g of the mRNA, and wherein the second dose is administered 2 months after the first dose, and the third dose is administered 4 months after the second dose.
  • first dose, the second dose, and the third dose is 150 ⁇ g of the mRNA, and wherein the second dose is administered 2 months after the first dose, and the third dose is administered 4 months after the second dose.
  • the lipid nanoparticle comprises a mixture of lipids that comprises 20-60 mol% ionizable lipid heptadecan-9-yl 8 ((2 hydroxyethyl)(6 oxo 6-(undecyloxy)hexyl)amino)octanoate (Compound 1); 5-25 mol% 1,2 distearoyl sn glycero-3 phosphocholine (DSPC); 25-55 mol% cholesterol; and 0.5-15 mol% 1- monomethoxypolyethyleneglycol-2,3-dimyristylglycerol with polyethylene glycol of average molecular weight 2000 (PEG2000 DMG). 98.
  • the lipid nanoparticle comprises: 47 mol% ionizable cationic lipid; 11.5 mol% neutral lipid; 38.5 mol% sterol; and 3.0 mol% PEG-modified lipid; 48 mol% ionizable cationic lipid; 11 mol% neutral lipid; 38.5 mol% sterol; and 2.5 mol% PEG-modified lipid; 49 mol% ionizable cationic lipid; 10.5 mol% neutral lipid; 38.5 mol% sterol; and 2.0 mol% PEG-modified lipid; 50 mol% ionizable cationic lipid; 10 mol% neutral lipid; 38.5 mol% sterol; and 1.5 mol% PEG-modified lipid; or 51 mol% ionizable cationic lipid; 9.5 mol% neutral lipid; 38.5 mol% sterol; and 1.0 mol% PEG-modified lipid.
  • mRNA vaccine further comprises Tris buffer, sucrose, and sodium acetate, or any combination thereof.
  • 100. A method of inducing in a subject an immune response to a Borrelia bacterial infection, the method comprising: administering to the subject the mRNA vaccine of any one of embodiments 77-89 in an amount effective at inducing in the subject a population of neutralizing antibodies.
  • 101. A polynucleotide comprising a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of any one of SEQ ID NOs: 8-14. 102.
  • a composition comprising a plurality of polynucleotides, wherein the plurality comprises two or more of: a first polynucleotide comprising a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 8; a second polynucleotide comprising a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 9; a third polynucleotide comprising a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 10; a fourth polynucleotide comprising a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to the nucleic acid sequence of SEQ ID NO: 11; a fifth polynucleotide comprising a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to
  • composition of embodiment 102 wherein: the first polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 8; the second polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 9; the third polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 10; the fourth polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 11; the fifth polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 12; the sixth polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 13; and the seventh polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 14.

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