Lyme Disease RNA Vaccine

Abstract

The present disclosure provides a Lyme disease vaccine comprising a messenger RNA (mRNA) that includes an open reading frame (ORF) encoding at least one antigenic polypeptide derived from at least one bacterium of the genus Borrelia, and a method of eliciting an immune response by administering the vaccine.

Description

[Technical field]

[0001] Related Applications This application is related to European Patent Priority Application No. 21315283.8, filed December 17, 2021, and European Patent Priority Application No. 21315291.1, filed December 23, 2021, the contents of each of which are incorporated by reference in their entirety for all purposes. [Background technology]

[0002] Lyme borreliosis (i.e., Lyme disease) is a zoonotic infectious disease caused by several bacterial species in the genus Borrelia and transmitted to humans and other mammals by the bite of infected Ixodes spp. ticks. Lyme disease is a global public health problem, with cases reported from temperate climates across Europe, North America, and Asia. Outer surface protein A (OspA) is an abundant immunogenic lipoprotein of Borrelia. There are at least seven different OspA serotypes (serotypes 1-7) found in Borrelia worldwide, and there are different genospecies of Borrelia worldwide that can cause Lyme borreliosis. Furthermore, the localized range of Borrelia-carrying ticks means that an OspA serotype associated with Lyme disease in patients from one geographic region may not be associated with Lyme disease in patients from another geographic region.

[0003] RNA-based vaccines (e.g., mRNA vaccines) have emerged in recent years as an additional vaccine type with a rapid, safe, and cost-effective manufacturing process, especially against viral pathogens. mRNA vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) primarily use the spike viral protein as the antigen. Often combined with a delivery vehicle, e.g., lipid nanoparticles (LNPs), COVID-19 mRNA vaccines can achieve high efficacy. Due to the lack of available effective Lyme disease vaccines, there is a need for an RNA-based Lyme disease vaccine that induces a strong immune response for potent neutralization of Lyme disease infection. Summary of the Invention [Means for solving the problem]

[0004] In one aspect, the disclosure provides a Lyme disease vaccine comprising a messenger RNA (mRNA) comprising an open reading frame (ORF) encoding at least one antigenic polypeptide derived from at least one bacterium of the genus Borrelia.

[0005] In certain embodiments, the at least one bacterium is selected from the species B. burgdorferi, afzelii, garinii, bavariensis, mayonii, spielmanii, lusitaniae, bissettii, and / or valaisiana, or any strain or isolate thereof.

[0006] In certain embodiments, the at least one antigenic polypeptide comprises at least one lipoprotein of the genus Borrelia.

[0007] In one embodiment, at least one lipoprotein is OspA or a fragment or variant thereof. Preferably, the fragment or variant comprises at least 5 amino acids.

[0008] In certain embodiments, at least one OspA is from OspA serotype (ST) 1, 2, 3, 4, 5, 6, and / or 7.

[0009] In certain embodiments, at least one OspA serotype 1-7 is from serotype 1 Borrelia burgdorferi strain B31, serotype 2 Borrelia afzelii strain PKO, serotype 3 Borrelia garinii strain PBr, serotype 4 Borrelia bavariensis, serotype 5 Borrelia garinii, serotype 6 Borrelia garinii, or serotype 7 Borrelia garinii.

[0010] In one embodiment, at least one OspA polypeptide comprises an amino acid sequence having at least 85% identity to any one of SEQ ID NOs:1-7.

[0011] In one embodiment, the mRNA of the Lyme disease vaccine disclosed herein comprises a nucleotide sequence that is at least 85% identical to any one of SEQ ID NOs: 10-13 and 16-19.

[0012] In one embodiment, the mRNA of the Lyme disease vaccine disclosed herein encodes at least two different OspA serotypes, or fragments or variants thereof, preferably each fragment or variant comprising at least five amino acids.

[0013] In one embodiment, OspA, or a fragment or variant thereof, of one serotype is fused to OspA, or a fragment or variant thereof, of a different serotype.

[0014] In one embodiment, the fused OspAs, or fragments or variants thereof, of different serotypes are separated by a linker sequence.

[0015] In one embodiment, the linker sequence is derived from P66.

[0016] In one embodiment, the linker sequence comprises an amino acid sequence having at least 85% identity to SEQ ID NO:8.

[0017] In one embodiment, the linker sequence comprises an amino acid sequence having at least 85% identity to SEQ ID NO:9.

[0018] In certain embodiments, the mRNA is a non-replicating mRNA.

[0019] In certain embodiments, the mRNA is an autonomously replicating or trans-replicating mRNA.

[0020] In certain embodiments, the mRNA comprises at least one chemical modification.

[0021] In certain embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytidine, 2-thio-l-methyl-1-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0022] In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 5-methylcytidine, 5-methoxyuridine, and combinations thereof. It is N1-methylpseudouridine.

[0023] In certain embodiments, the mRNA is formulated in a non-viral delivery system.

[0024] In certain embodiments, the mRNA is formulated in a lipid nanoparticle (LNP).

[0025] In certain embodiments, the LNP comprises at least one cationic lipid.

[0026] In some embodiments, the cationic lipids are biodegradable. In some embodiments, the cationic lipids are not biodegradable.

[0027] In some embodiments, the cationic lipid is cleavable. In some embodiments, the cationic lipid is not cleavable.

[0028] In certain embodiments, the cationic lipid is selected from the group consisting of ML7 / OF-02; cKK-E10; GL-HEPES-E3-E10-DS-3-E18-1; GL-HEPES-E3-E12-DS-4-E10; GL-HEPES-E3-E12-DS-3-E14; 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102); and (4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315).

[0029] In an embodiment, the cationic lipid is cKK-E10.

[0030] In certain embodiments, the LNP further comprises a polyethylene glycol (PEG)-conjugated (PEGylated) lipid, a cholesterol-based lipid, and a helper lipid.

[0031] In one embodiment, the LNP comprises a cationic lipid in a molar ratio of 35% to 55%; a polyethylene glycol (PEG)-conjugated (PEGylated) lipid in a molar ratio of 0.25% to 2.75%, a cholesterol-based lipid in a molar ratio of 20% to 45%, and a helper lipid in a molar ratio of 5% to 35%, all of which are relative to the total lipid content of the LNP.

[0032] In one embodiment, the LNP comprises a 40% molar ratio of cationic lipid, a 1.5% molar ratio of PEGylated lipid, a 28.5% molar ratio of cholesterol-based lipid, and a 30% molar ratio of helper lipid.

[0033] In one embodiment, the PEGylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000) or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0034] In certain embodiments, the cholesterol-based lipid is cholesterol.

[0035] In one embodiment, the helper lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0036] In some embodiments, the LNPs comprise cKK-E10 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30%.

[0037] In certain embodiments, the LNPs have an average diameter of 30-200 nm, hi certain embodiments, the LNPs have an average diameter of 80-150 nm.

[0038] In one embodiment, the mRNA of the Lyme disease vaccine disclosed herein comprises at least one 5' untranslated region (5'UTR), at least one 3' untranslated region (3'UTR), and at least one polyadenylation (polyA) sequence.

[0039] In one embodiment, the mRNA contains at least the following structural elements: (i) a structure: [ka] having a 5' cap; (ii) a 5' untranslated region (5'UTR) having the nucleic acid sequence of SEQ ID NO:14; (iii) at least one OspA open reading frame having any one of the nucleic acid sequences of SEQ ID NOs: 16-19; (iv) a 3' untranslated region (3' UTR) having the nucleic acid sequence of SEQ ID NO: 15; and (v) Poly(A) tail Includes.

[0040] In one aspect, the disclosure provides a Lyme disease vaccine comprising a messenger RNA (mRNA) comprising an open reading frame (ORF) encoding at least one antigenic polypeptide derived from at least one bacterium of the genus Borrelia, wherein the mRNA comprises at least the following structural elements: (i) a structure: [ka] having a 5' cap; (ii) a 5' untranslated region (5'UTR) having the nucleic acid sequence of SEQ ID NO:14; (iii) at least one OspA open reading frame having any one of the nucleic acid sequences of SEQ ID NOs: 16-19; (iv) a 3' untranslated region (3' UTR) having the nucleic acid sequence of SEQ ID NO: 15; and (v) Poly(A) tail Includes; A Lyme disease vaccine is provided in which the mRNA is formulated in lipid nanoparticles (LNPs) containing 40% molar ratio of cKK-E10, 1.5% molar ratio of DMG-PEG2000, 28.5% molar ratio of cholesterol, and 30% molar ratio of DOPE.

[0041] In another aspect, the Lyme disease vaccines disclosed herein are for use in eliciting an immune response, preferably a humoral immune response, in a subject in need of such elicitation, and / or in treating or preventing Lyme disease in a subject in need of such treatment or prevention.

[0042] In another aspect, the present disclosure provides a method of inducing an immune response, preferably a humoral immune response, in a subject in need of such induction, and / or a method of treating or preventing Lyme disease in a subject in need of such treatment or prevention, comprising administering to the subject, optionally intramuscularly, intranasally, intravenously, subcutaneously, or intradermally, an effective amount of a Lyme disease vaccine disclosed herein.

[0043] In another aspect, the present disclosure provides for the use of a Lyme disease vaccine disclosed herein for the manufacture of a medicament for use in inducing an immune response, preferably a humoral immune response, in a subject in need of such induction, and / or for use in treating or preventing Lyme disease in a subject in need of such treatment or prevention.

[0044] In one embodiment, a subject has higher serum concentrations of antibodies to OspA following administration of the Lyme disease vaccine relative to subjects administered a Lyme disease vaccine comprising an OspA recombinant protein vaccine.

[0045] In certain embodiments, the subject is a mammal, optionally a human, dog, cat, llama, cow, sheep, goat, horse, rodent, mouse, rat, rabbit, monkey, primate, or pig. In particularly exemplary embodiments, the subject is a human.

[0046] The foregoing and other features and advantages of the present disclosure will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0047] [Figure 1A-1B] Figures 1A-1B show IgG titers in mice from anti-OspA ST1 IgG ELISA at post-dose 1 (day 20) (Figure 1A) and post-dose 2 (day 35) (Figure 1B). Dotted line = limit of quantification. [Figure 2A-2B] Figures 2A-B show IgG titers in mice from anti-OspA ST2 IgG ELISA at post-dose 1 (day 20) (Figure 2A) and post-dose 2 (day 35) (Figure 2B). Dotted line = limit of quantification. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] The present disclosure relates, inter alia, to novel RNA (e.g., mRNA) compositions encoding antigenic polypeptides, such as OspA protein, derived from Borrelia, and vaccination methods therewith. In particular, the disclosure relates to mRNA encoding OspA protein that is formulated in a non-viral delivery system, in particular a lipid nanoparticle (LNP).

[0049] I. Definition Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure. In the event of a conflict, the present specification, including definitions, will control. In general, the terminology used in connection with and in the techniques of cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein is that well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as commonly accomplished in the art or as described herein. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Throughout the specification and embodiments, the words "have" and "comprise", or variations such as "has", "having", "comprises", or "comprising", are understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents form part of the common general knowledge in the art.

[0050] It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a nucleotide sequence" is understood to refer to one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0051] Furthermore, "and / or" as used herein should be considered as a specific disclosure of each of the two defined features or components with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" is intended herein to include "A and B," "A or B," "A" (single), and "B" (single). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0052] Whenever an embodiment is described herein with the language "comprising," it is understood that otherwise similar embodiments are also provided that are described with the terms "consisting of" and / or "consisting essentially of."

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press can provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0054] Units, prefixes, and symbols are shown in the format accepted by the International System of Units (SI). Numerical ranges include the numbers that define the range. Unless otherwise indicated, amino acid sequences are written from left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure. Thus, the terms defined immediately below are more fully defined by reference to the entire specification.

[0055] The term "approximately" or "about" is used herein to mean approximately, roughly, about, or in the range of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" may modify a numerical value above and below the stated value by, for example, a variance of 10 percent above or below (higher or lower). In some embodiments, the term indicates a deviation from the stated numerical value by only ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, or ±0.01%. In some embodiments, "about" indicates a deviation from the stated numerical value by only ±10%. In some embodiments, "about" indicates a deviation from the stated numerical value by only ±5%. In some embodiments, "about" refers to a deviation of only ±4% from the indicated numerical value. In some embodiments, "about" refers to a deviation of only ±3% from the indicated numerical value. In some embodiments, "about" refers to a deviation of only ±2% from the indicated numerical value. In some embodiments, "about" refers to a deviation of only ±1% from the indicated numerical value. In some embodiments, "about" refers to a deviation of only ±0.9% from the indicated numerical value. In some embodiments, "about" refers to a deviation of only ±0.8% from the indicated numerical value. In some embodiments, "about" refers to a deviation of only ±0.7% from the indicated numerical value. In some embodiments, "about" refers to a deviation of only ±0.6% from the indicated numerical value. In some embodiments, "about" refers to a deviation of only ±0.5% from the indicated numerical value. In some embodiments, "about" refers to a deviation of only ±0.4% from the indicated numerical value. In some embodiments, "about" refers to a deviation of only ±0.3% from the indicated numerical value. In some embodiments, "about" refers to a deviation of only ±0.1% from the indicated numerical value. In some embodiments, "about" refers to a deviation of only ±0.05% from the indicated numerical value. In some embodiments, "about" refers to deviations from the indicated numerical value by no more than ±0.01%.

[0056] As used herein, the term "messenger RNA" or "mRNA" refers to a polynucleotide that encodes at least one polypeptide. As used herein, mRNA encompasses both modified and unmodified RNA. An mRNA may contain one or more coding and non-coding regions. The coding region is alternatively referred to as an open reading frame (ORF). The non-coding regions in an mRNA include the 5' cap, 5' untranslated region (UTR), 3'UTR, and polyA tail. An mRNA can be purified from a natural source, produced using a recombinant expression system (e.g., in vitro transcription), optionally purified, or chemically synthesized.

[0057] As used herein, the term "open reading frame," "ORF," or "coding region" refers to a polynucleotide sequence beginning with a start codon (e.g., ATG), ending with a stop codon (e.g., TAA, TAG, or TGA), with no other stop codons in between, that encodes a protein (e.g., an antigenic polypeptide derived from a bacterium of the genus Borrelia).

[0058] The present disclosure also includes fragments or variants of a polypeptide, and any combination thereof. The term "fragment" or "variant" when referring to an OspA polypeptide molecule of the present disclosure includes any polypeptide that retains at least some of the properties of the reference polypeptide (e.g., the specific antigenic properties of the polypeptide or the ability of the polypeptide to contribute to inducing antibody binding). A fragment of a polypeptide includes N-terminally and / or C-terminally truncated fragments, such as C-terminal and N-terminal fragments, and deletion fragments, but does not include a naturally occurring full-length polypeptide (or a mature polypeptide). A deletion fragment refers to a polypeptide in which one or more internal amino acids have been deleted from the full-length polypeptide. A variant of a polypeptide includes the above fragments, and also polypeptides having altered amino acid sequences resulting from amino acid substitutions, deletions, or insertions. A variant may be naturally or non-naturally occurring. Non-naturally occurring variants can be produced using mutagenesis techniques known in the art. A variant polypeptide may include conservative or non-conservative amino acid substitutions, deletions, or additions. Such mutations (ie truncations and / or amino acid substitutions, deletions or insertions) can occur at the amino acid level or, accordingly, at the nucleic acid level.

[0059] "Conservative amino acid substitution" refers to a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Amino acid residue families with similar side chains have been defined in the art, and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered conservative. In another embodiment, a string of amino acids can be conservatively replaced with a structurally similar string that differs in the order and / or composition of side chain family members.

[0060] The term "linked" as used herein refers to a first amino acid sequence or nucleotide sequence that is covalently or non-covalently linked to a second amino acid sequence or nucleotide sequence, respectively. The first amino acid sequence or nucleotide sequence can be directly linked or juxtaposed to the second amino acid sequence or nucleotide sequence, or alternatively, an intervening sequence can covalently link the first sequence to the second sequence. The term "linked" not only means that the first amino acid sequence is fused to the second amino acid sequence at the C-terminus or N-terminus, but also includes that the entire first amino acid sequence (or second amino acid sequence) is inserted within any two amino acids of the second amino acid sequence (or first amino acid sequence, respectively). In one embodiment, the first amino acid sequence can be linked to the second amino acid sequence by a peptide bond or a linker. The first nucleotide sequence can be linked to the second nucleotide sequence by a phosphodiester bond or a linker. A linker can be a peptide or polypeptide (in the case of a polypeptide chain), or a nucleotide or a chain of nucleotides (in the case of a nucleotide chain), or any chemical moiety (in the case of both polypeptide and polynucleotide chains). The term "linked" can also be indicated by a hyphen (-).

[0061] As used herein, the term "immune response" refers to the response of a cell of the immune system, e.g., a B cell, a T cell, a dendritic cell, a macrophage, or a polymorphonucleocyte, to a stimulus, e.g., an antigen or a vaccine. An immune response can include any cell of the body that is involved in a host defense response, including, e.g., epithelial cells that secrete interferons or cytokines. Immune responses include, but are not limited to, innate and / or adaptive immune responses.

[0062] As used herein, a "protective immune response" refers to an immune response that protects a subject from infection (e.g., prevents infection or prevents the development of a disease associated with infection). Methods of measuring immune responses are well known in the art and include, for example, by measuring the proliferation and / or activity of lymphocytes (e.g., B cells or T cells), measuring the secretion of cytokines or chemokines, measuring inflammation, measuring antibody production, etc.

[0063] As used herein, an "antibody response" is an immune response in which antibodies are produced.

[0064] As used herein, "antigen" refers to an agent that elicits an immune response when exposed to or administered to an organism, and / or that binds to a T cell receptor (e.g., when presented by an MHC molecule) or an antibody (e.g., produced by a B cell). In some embodiments, the antigen elicits a humoral response in the organism (e.g., including production of antigen-specific antibodies). Alternatively or additionally, in some embodiments, the antigen elicits a cellular response in the organism (e.g., involving T cells whose receptors specifically interact with the antigen). A particular antigen may elicit an immune response in one or several members of a target organism (e.g., mice, rabbits, primates, humans), but not in all members of the target organism's species. In some embodiments, the antigen elicits an immune response in at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of members of the target species. In some embodiments, the antigen binds to an antibody and / or a T cell receptor and may or may not elicit a specific physiological response in the organism. In some embodiments, for example, the antigen may bind to an antibody and / or a T cell receptor in vitro, whether or not such interactions occur in vivo. In some embodiments, the antigen reacts with the products of specific humoral or cellular immunity. Antigens include OspA polypeptides (e.g., OspA ST1 and ST2) encoded by the mRNAs described herein.

[0065] As used herein, "adjuvant" refers to a substance or vehicle that enhances the immune response to an antigen. Adjuvants can include, but are not limited to, suspensions of minerals (e.g., alum, aluminum hydroxide, or phosphates) to which antigens are adsorbed; water-in-oil or oil-in-water emulsions in which antigen solutions are emulsified in mineral oil or water (e.g., Freund's incomplete adjuvant). Killed mycobacteria may be included to further enhance antigenicity (e.g., Freund's complete adjuvant). Immunostimulatory oligonucleotides (e.g., CpG motifs) can also be used as adjuvants (see, e.g., U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199). Adjuvants can also include biological molecules, such as Toll-like receptor (TLR) agonists and costimulatory molecules.

[0066] As used herein, an "antigenic OspA polypeptide" refers to a polypeptide that contains all or a portion of the OspA amino acid sequence of sufficient length that the molecule is antigenic to Lyme disease and the OspA polypeptide.

[0067] As used herein, "subject" refers to any member of the animal kingdom. In some embodiments, "subject" refers to a human. In some embodiments, "subject" refers to a non-human animal. In certain embodiments, the non-human subject is a mammal, e.g., a rodent, mouse, rat, rabbit, monkey, llama, horse, dog, cat, cow, sheep, goat, primate, or pig). In some embodiments where the subject is a human, the terms "individual" or "patient" are used and are intended to be interchangeable with "subject."

[0068] As used herein, the terms "prevent," "preventing," "prevention," or "prophylaxis" (and grammatical variations thereof) refer to partially or completely inhibiting the onset of one or more symptoms or characteristics of a particular infection, disease, disorder, and / or condition.

[0069] As used herein, the terms "treat," "treating," "treatment," "therapy" or "therapeutic" (and grammatical variations thereof) refer to partially or completely alleviating, ameliorating, improving, relieving, inhibiting the progression of, and / or reducing the severity of one or more symptoms or characteristics of an infection, disease, disorder, and / or condition.

[0070] As used herein, the term "effective amount" refers to an amount (e.g., of a nucleic acid or composition) sufficient to produce a beneficial or desired result. An effective amount can be administered in one or more administrations, applications or dosages, and is not intended to be limited to a particular formulation or route of administration.

[0071] The term "effective amount" includes, for example, a "therapeutically effective amount" and / or a "prophylactically effective amount."

[0072] As used herein, the phrase "therapeutically effective amount" refers to an amount (e.g., of a nucleic acid or composition) effective to produce some desired therapeutic effect in the treatment of an infection, disease, disorder, and / or condition, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0073] As used herein, the phrase "prophylactically effective amount" refers to an amount (e.g., of a nucleic acid or composition) effective to produce some desired prophylactic effect in the prevention of an infection, disease, disorder and / or condition, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0074] As used herein, the term "vaccination" or "vaccinate" refers to the administration of a composition intended to generate an immune response against, for example, a disease-causing agent. Vaccination can be administered before, during, and / or after exposure to the disease-causing agent, and / or before, during, and / or after the onset of one or more symptoms, in some embodiments before, during, and / or immediately after exposure to the agent. In some embodiments, vaccination involves multiple administrations of the vaccine composition at appropriate time intervals.

[0075] This disclosure describes nucleic acid sequences (eg, DNA and RNA sequences) and amino acid sequences that have a degree of identity to a given nucleic acid sequence or amino acid sequence, respectively (a reference sequence).

[0076] "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are identical between the sequences. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences.

[0077] The terms "% identical", "% identity" or similar terms are intended to refer in particular to the percentage of nucleotides or amino acids that are identical in optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may, but do not necessarily, be randomly distributed over the entire length of the sequences to be compared. Comparison of two sequences is usually performed by comparing said sequences over segments or "windows of comparison" after optimal alignment in order to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually or using the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, using the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, using the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or using computer programs which use said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0078] The percentage of identity is obtained by determining the number of corresponding identical positions in the sequences to be compared, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100.

[0079] In some embodiments, the degree of identity is given for a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments, in consecutive nucleotides. In some embodiments, the degree of identity is given for the entire length of the reference sequence.

[0080] A nucleic acid sequence or amino acid sequence that has a particular degree of identity to a given nucleic acid sequence or amino acid sequence, respectively, may have at least one functional characteristic of the given sequence, e.g., in some instances is functionally equivalent to the given sequence. In some embodiments, a nucleic acid sequence or amino acid sequence that has a particular degree of identity to a given nucleic acid sequence or amino acid sequence is functionally equivalent to the given sequence.

[0081] As used herein, the term "kit" refers to a packaged set of related components, e.g., one or more compounds or compositions and one or more associated materials, e.g., solvents, solutions, buffers, instructions, or desiccants.

[0082] II. RNA The Lyme disease vaccines of the present disclosure comprise at least one ribonucleic acid (RNA) comprising an ORF encoding an antigenic polypeptide from the genus Borrelia, such as an OspA protein antigen (e.g., OspA ST1 or ST2). In some embodiments, the RNA is a messenger RNA (mRNA) comprising an open reading frame encoding the OspA protein antigen. In some embodiments, the RNA (e.g., mRNA) further comprises at least one of a 5' UTR, a 3' UTR, a polyA tail, and / or a 5' cap.

[0083] II.A.5' Cap The 5' cap of an mRNA provides resistance to nucleases found in most eukaryotic cells and may promote translation efficiency. Several types of 5' caps are known. 7 The second nucleotide (also referred to as "Cap-G" or "Cap-0") contains a guanosine linked to the first transcribed nucleotide via a 5'-5'-triphosphate linkage.

[0084] A 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphate via guanylyltransferase, producing a 5'5'5 triphosphate linkage; and then, the 7-nitrogen of guanine is methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp, (5'(A,G(5')ppp(5')A, and G(5')ppp(5')G. Additional cap structures are described in U.S. Patent Application Publication Nos. 2016 / 0032356 and 2018 / 0125989, which are incorporated herein by reference.

[0085] 5'-capping of polynucleotides can be completed simultaneously during the in vitro transcription reaction using the following chemical RNA cap analogs to generate a 5'-guanosine cap structure according to the manufacturer's protocol: 3'-O-Me-m7G(5')ppp(5')G (ARCA cap); G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G; m7G(5')ppp(5')(2'OMeA)pG; m7G(5')ppp(5')(2'OMeA)pU; m7G(5')ppp(5')(2'OMeG)pG (New England BioLabs, Ipswich, MA; TriLink Biotechnologies). 5'-capping of modified RNA can be completed post-transcriptionally using vaccinia virus capping enzyme to generate the Cap0 structure: m7G(5')ppp(5')G. Cap1 structures can be generated using both vaccinia virus capping enzyme and 2'-O methyl-transferase to generate m7G(5')ppp(5')G-2'-O-methyl. Cap2 structures can be generated from the Cap1 structure, followed by 2'-O-methylation of the third nucleotide from the 5' end using 2'-O methyl-transferase. Cap3 structures can be generated from the Cap2 structure, followed by 2'-O-methylation of the fourth nucleotide from the 5' end using 2'-O methyl-transferase.

[0086] In some embodiments, an mRNA of the disclosure comprises a 5' cap selected from the group consisting of 3'-O-Me-m7G(5')ppp(5')G (ARCA cap), G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, m7G(5')ppp(5')G, m7G(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeA)pU, and m7G(5')ppp(5')(2'OMeG)pG.

[0087] In one embodiment, the mRNA of the present disclosure comprises: [ka] Contains the 5' cap.

[0088] II.B. Untranslated Regions (UTRs) In some embodiments, an mRNA of the disclosure comprises a 5' and / or 3' untranslated region (UTR). In an mRNA, the 5' UTR begins at the transcription initiation site and continues up to but not including the start codon. The 3' UTR begins immediately after the stop codon and continues to the transcription termination signal.

[0089] In some embodiments, the mRNA disclosed herein may comprise a 5'UTR that comprises one or more elements that affect mRNA stability or translation. In some embodiments, the 5'UTR may be about 10-5,000 nucleotides in length. In some embodiments, the 5'UTR may be about 50-500 nucleotides in length. In some embodiments, the 5'UTR may be at least about 10 nucleotides in length, about 20 nucleotides in length, about 30 nucleotides in length, about 40 nucleotides in length, about 50 nucleotides in length, about 100 nucleotides in length, about 150 nucleotides in length, about 200 nucleotides in length, about 250 nucleotides in length, about 300 nucleotides in length, about 350 nucleotides in length, about 400 nucleotides in length, about 450 nucleotides in length, about 500 nucleotides in length, about 550 nucleotides in length, about 600 nucleotides in length, about The length is 650 nucleotides, about 700 nucleotides, about 750 nucleotides, about 800 nucleotides, about 850 nucleotides, about 900 nucleotides, about 950 nucleotides, about 1,000 nucleotides, about 1,500 nucleotides, about 2,000 nucleotides, about 2,500 nucleotides, about 3,000 nucleotides, about 3,500 nucleotides, about 4,000 nucleotides, about 4,500 nucleotides, or about 5,000 nucleotides.

[0090] In some embodiments, the mRNA disclosed herein comprises a 3'UTR that includes one or more of a polyadenylation signal, a binding site for a protein that affects the stability of the mRNA's location in a cell, or one or more binding sites for an miRNA. In some embodiments, the 3'UTR can be 50-5,000 or more nucleotides in length. In some embodiments, the 3'UTR can be 50-1,000 or more nucleotides in length. In some embodiments, the 3'UTR is about 50 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 350 nucleotides, about 400 nucleotides, about 450 nucleotides, about 500 nucleotides, about 550 nucleotides, about 600 nucleotides, about 650 nucleotides, about 700 nucleotides, about 750 nucleotides, about 800 nucleotides, about 850 nucleotides, about 900 nucleotides, about 950 nucleotides, about 1,000 nucleotides, about 1,500 nucleotides, about 2,000 nucleotides, about 2,500 nucleotides, about 3,000 nucleotides, about 3,500 nucleotides, about 4,000 nucleotides, about 4,500 nucleotides, or about 5,000 nucleotides in length. In some embodiments, the mRNAs disclosed herein may contain a 5' or 3' UTR that is derived from a gene that is distinct from the gene encoded by the mRNA transcript (i.e., the UTR is a heterologous UTR).

[0091] In some embodiments, the mRNAs disclosed herein may contain a 5' or 3' UTR that is derived from a gene that is distinct from the gene encoded by the mRNA transcript (i.e., the UTR is a heterologous UTR).

[0092] In certain embodiments, the 5' and / or 3' UTR sequences are derived from stable mRNAs (e.g., globin, actin, GAPDH, tubulin, histones, or citric acid cycle enzymes) to increase the stability of the mRNA. For example, the 5' UTR sequence may include a subsequence of the CMV immediate early 1 (IE1) gene, or a fragment thereof, to improve nuclease resistance and / or improve half-life of the mRNA. Inclusion of a sequence encoding human growth hormone (hGH), or a fragment thereof, at the 3' end or untranslated region of the mRNA is also contemplated. Generally, these modifications improve the stability and / or pharmacokinetic properties (e.g., half-life) of the mRNA relative to its unmodified counterpart, including, for example, modifications made to improve such mRNA resistance to in vivo nuclease digestion.

[0093] Exemplary 5'UTRs include sequences from the CMV immediate early 1 (IE1) gene (U.S. Patent Application Publication Nos. 2014 / 0206753 and 2015 / 0157565, each of which is incorporated herein by reference), or the sequence GGGAUCCUACC (SEQ ID NO:20) (U.S. Patent Application Publication No. 2016 / 0151409, which is incorporated herein by reference).

[0094] In various embodiments, the 5'UTR is derived from the 5'UTR of a TOP gene. TOP genes are typically characterized by the presence of a 5' terminal oligopyrimidine (TOP) tract. In addition, most TOP genes are characterized by growth-related translational regulation. However, TOP genes with tissue-specific translational regulation are also known. In an embodiment, the 5'UTR derived from the 5'UTR of a TOP gene lacks a 5'TOP motif (oligopyrimidine tract) (e.g., U.S. Patent Application Publication Nos. 2017 / 0029847, 2016 / 0304883, 2016 / 0235864, and 2016 / 0166710, each of which is incorporated herein by reference).

[0095] In one embodiment, the 5'UTR is derived from the ribosomal protein large 32 (L32) gene (US Patent Application Publication No. 2017 / 0029847, supra).

[0096] In one embodiment, the 5'UTR is derived from the 5'UTR of the hydroxysteroid (17-b) dehydrogenase 4 gene (HSD17B4) (US Patent Application Publication No. 2016 / 0166710, supra).

[0097] In one embodiment, the 5'UTR is derived from the 5'UTR of the ATP5A1 gene (US Patent Application Publication No. 2016 / 0166710, supra).

[0098] In some embodiments, an internal ribosome entry site (IRES) is used in place of the 5'UTR.

[0099] In some embodiments, the 5'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 14. In some embodiments, the 3'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 15. The 5'UTR and 3'UTR are described in further detail in WO2012075040, which is incorporated herein by reference.

[0100] II.C. Polyadenylation Tail As used herein, the term "poly(A) sequence" or "poly(A) tail" or "poly(A) region" refers to a sequence of adenosine nucleotides at the 3' end of an mRNA molecule. The poly(A) tail may confer stability to the mRNA and protect it from exonuclease degradation, and is also believed to improve translation. In some embodiments, the poly(A) tail is essentially homopolymeric, e.g., a poly(A) tail of 100 adenosine nucleotides has a length of essentially 100 nucleotides. In other embodiments, the poly(A) tail may be interrupted by at least one nucleotide that is different from adenosine nucleotides, e.g., a poly(A) tail of 100 adenosine nucleotides may have a length of more than 100 nucleotides (comprising 100 adenosine nucleotides and in addition said at least one nucleotide (or stretch of nucleotides) that is different from adenosine nucleotides). In some embodiments, the poly(A) tail may be interrupted by at least one nucleotide that is different from adenosine nucleotides, e.g., a poly(A) tail of 100 adenosine nucleotides has ... [ka] Includes.

[0101] A "poly(A) tail" as defined herein typically relates to RNA, however, in the context of the present disclosure, the term also relates to the corresponding sequence in a DNA molecule (e.g., a "poly(T) sequence").

[0102] The poly(A) tail can comprise from about 10 to about 500 adenosine nucleotides, from about 10 to about 200 adenosine nucleotides, from about 40 to about 200 adenosine nucleotides, or from about 40 to about 150 adenosine nucleotides. The length of the poly(A) tail can be at least about 10, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 adenosine nucleotides.

[0103] In some embodiments, where the nucleic acid is RNA, the poly(A) tail of the nucleic acid is obtained from a DNA template during in vitro transcription of the RNA. In other embodiments, the poly(A) tail is obtained in vitro by common chemical synthesis methods without being transcribed from a DNA template. In other embodiments, the poly(A) tail is generated by enzymatic polyadenylation of the RNA (after RNA in vitro transcription) using commercially available polyadenylation kits and corresponding protocols known in the art, or alternatively by using immobilized poly(A) polymerase, for example, using the methods and means described in WO 2016 / 174271.

[0104] The nucleic acid may include a poly(A) tail obtained by enzymatic polyadenylation, and the majority of the nucleic acid molecules include from about 100 (+ / -20) to about 500 (+ / -50), or about 250 (+ / -20) adenosine nucleotides.

[0105] In other embodiments, the nucleic acid may comprise a poly(A) tail derived from the template DNA and may additionally comprise at least one additional poly(A) tail generated by enzymatic polyadenylation, e.g., as described in WO 2016 / 091391.

[0106] In a further embodiment, the nucleic acid comprises at least one polyadenylation signal.

[0107] In other embodiments, the nucleic acid may comprise at least one poly(C) sequence.

[0108] As used herein, the term "poly(C) sequence" is intended to mean a sequence of cytosine nucleotides of up to about 200 cytosine nucleotides. In some embodiments, the poly(C) sequence comprises about 10 to about 200 cytosine nucleotides, about 10 to about 100 cytosine nucleotides, about 20 to about 70 cytosine nucleotides, about 20 to about 60 cytosine nucleotides, or about 10 to about 40 cytosine nucleotides. In particularly exemplary embodiments, the poly(C) sequence comprises about 30 cytosine nucleotides.

[0109] II.D. Chemical modification The mRNA disclosed herein may be modified or unmodified.In some embodiments, the mRNA disclosed herein may contain one or more modifications that typically improve RNA stability.Exemplary modifications include backbone modifications, sugar modifications, or base modifications. In some embodiments, the disclosed mRNAs are composed of naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including, but not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), as well as modified nucleotide analogs or derivatives of purines and pyrimidines, such as 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2- It can be synthesized as thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, β-D-mannosyl-queosine, phosphoramidate, phosphorothioate, peptide nucleotide, methylphosphonate, 7-deazaguanosine, 5-methylcytosine, and inosine.

[0110] In some embodiments, the disclosed mRNA comprises at least one chemical modification, including, but not limited to, pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-1-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0111] In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0112] In some embodiments, the chemical modification comprises N1-methylpseudouridine.

[0113] In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the mRNA are chemically modified.

[0114] In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the ORF are chemically modified.

[0115] The preparation of such analogs is known to those skilled in the art, for example from U.S. Pat. Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530, and 5,700,642.

[0116] II.E. mRNA synthesis The mRNA disclosed herein can be synthesized according to any of a variety of known methods. For example, the mRNA according to the present disclosure can be synthesized via in vitro transcription (IVT). Methods of in vitro transcription are known in the art. See, for example, Geall et al. (2013) Semin. Immunol. 25(2): 152-159; Brunelle et al. (2013) Methods Enzymol. 530: 101-14, the contents of which are incorporated by reference. Briefly, IVT is typically performed using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or RNase inhibitor. The exact conditions will vary according to the specific application. The presence of these reagents is undesirable in the final mRNA product and is considered an impurity or contaminant, which must be purified to provide clean, homogenous mRNA suitable for therapeutic use. While mRNA provided from an in vitro transcription reaction may be desirable in some embodiments, other sources of mRNA can be used in accordance with the present disclosure, including wild-type mRNA produced from bacteria, fungi, plants, and / or animals.

[0117] In one embodiment, the mRNA contains at least the following structural elements: (i) a structure: [ka] having a 5' cap; (ii) a 5' untranslated region (5'UTR) having the nucleic acid sequence of SEQ ID NO:14; (iii) at least one OspA open reading frame having any one of the nucleic acid sequences of SEQ ID NOs: 16-19; (iv) a 3' untranslated region (3' UTR) having the nucleic acid sequence of SEQ ID NO: 15; and (v) Poly(A) tail Includes.

[0118] In one embodiment, the poly(A) tail has a length of about 10 to about 500 adenosine nucleotides.

[0119] III. OspA Protein The causative agent of Lyme disease is a bacterium of the genus Borrelia. Four species from the genus Borrelia cause most of the human disease: B. burgdorferi, B. afzelii, B. garinii, and B. bavariensis. Each Borrelia species has surface expression of outer surface protein A (OspA), a useful protein target for vaccination in the treatment of Lyme disease. There are several serotypes of OspA, defined by reactivity with monoclonal antibodies against different epitopes of OspA (see Wilske et al., J Clin Mic orbiol 31(2):340-350 (1993)). These serotypes correlate with different genotypes of Borrelia bacteria. In some embodiments, the OspA is any one of serotypes 1-7 (ST1, ST2, ST3, ST4, ST5, ST6, or ST7). In some embodiments, the OspA is from Borrelia burgdorferi, Borrelia mayonii, Borrelia afzelii, Borrelia garinii, Borrelia bavariensis, Borrelia spielmanni, Borrelia lusitaniae, Borrelia bissettii, and / or Borrelia valaisiana. In some embodiments, the OspA is Borrelia burgdorferi OspA. In some embodiments, the Borrelia can be carried by ticks of the genus Ixodes.In some embodiments, the Borrelia genus is Borrelia burgdorferi, Borrelia mayonii, Borrelia afzelii, Borrelia garinii, or Borrelia bavariensis.

[0120] In one aspect, the disclosure provides a Lyme disease vaccine comprising a messenger RNA (mRNA) comprising an open reading frame (ORF) encoding at least one antigenic polypeptide derived from at least one bacterium of the genus Borrelia.

[0121] In certain embodiments, the at least one antigenic polypeptide comprises at least one lipoprotein of the genus Borrelia.

[0122] In one embodiment, at least one lipoprotein is OspA or a fragment or variant thereof. Preferably, the fragment or variant comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 amino acids.

[0123] In certain embodiments, at least one OspA is from OspA serotype (ST) 1, 2, 3, 4, 5, 6, and / or 7.

[0124] In certain embodiments, at least one OspA serotype 1-7 is from serotype 1 Borrelia burgdorferi strain B31, serotype 2 Borrelia afzelii strain PKO, serotype 3 Borrelia garinii strain PBr, serotype 4 Borrelia bavariensis, serotype 5 Borrelia garinii, serotype 6 Borrelia garinii, or serotype 7 Borrelia garinii.

[0125] In some embodiments, the OspA polypeptide is OspA serotype 1 (ST1). In some embodiments, the OspA ST1 polypeptide comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 1. It may be recited in a ratio that is 1. In some embodiments, the OspA polypeptide is OspA serotype 1 (ST1). In some embodiments, the OspA ST1 polypeptide comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1. It may be recited in a ratio that is 1. SEQ ID NO: 1 corresponds to OspA from Borrelia burgdorferi strain B31 (serotype 1) NCBI sequence ID WP_010890378.1 without the signal sequence and the N-terminal methionine amino acid.

[0126] In one embodiment, the OspA ST1 polypeptide is encoded by a nucleotide sequence set forth in any one of SEQ ID NOs: 10, 11, 16, or 17. 10:11-16,17).

[0127] In some embodiments, the OspA polypeptide is OspA serotype 2 (ST2). In some embodiments, the OspA ST2 polypeptide comprises an amino acid sequence having at least 85% identity to SEQ ID NO:2. In some embodiments, the OspA polypeptide is OspA serotype 2 (ST2). In some embodiments, the OspA ST2 polypeptide comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:2. SEQ ID NO:2 corresponds to OspA from Borrelia afzelii strain PKO (serotype 2) NCBI sequence:WP_011703777.1 without the signal sequence and the N-terminal methionine amino acid.

[0128] In one embodiment, the OspA ST2 polypeptide is encoded by a nucleotide sequence set forth in any one of SEQ ID NOs:12, 13, 18 or 19.

[0129] In some embodiments, the OspA polypeptide is OspA serotype 3 (ST3). In some embodiments, the OspA ST3 polypeptide comprises an amino acid sequence having at least 85% identity to SEQ ID NO:3. The ratios may be recited as 1 to 3. In some embodiments, the OspA polypeptide is OspA serotype 3 (ST3). In some embodiments, the OspA ST3 polypeptide comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:3. SEQ ID NO:3 corresponds to OspA from Borrelia garinii strain PBr (serotype 3) GenBank: CAA56549.1 without the signal sequence and the N-terminal methionine amino acid.

[0130] In some embodiments, the OspA polypeptide is OspA serotype 4 (ST4). In some embodiments, the OspA ST4 polypeptide comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 4. In some embodiments, the OspA polypeptide is OspA serotype 4 (ST4). In some embodiments, the OspA ST4 polypeptide comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 4. SEQ ID NO: 4 corresponds to OspA from Borrelia bavariensis (serotype 4) NCBI sequence WP_011187157.1 without the signal sequence and the N-terminal methionine amino acid.

[0131] In some embodiments, the OspA polypeptide is OspA serotype 5 (ST5). In some embodiments, the OspA ST5 polypeptide comprises an amino acid sequence having at least 85% identity to SEQ ID NO:5. In some embodiments, the OspA polypeptide is OspA serotype 5 (ST5). In some embodiments, the OspA ST5 polypeptide comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:5. SEQ ID NO:5 corresponds to OspA from Borrelia garinii (serotype 5) GenBank CAA59727.1 without the signal sequence and the N-terminal methionine amino acid.

[0132] In some embodiments, the OspA polypeptide is OspA serotype 6 (ST6). In some embodiments, the OspA ST6 polypeptide comprises an amino acid sequence having at least 85% identity to SEQ ID NO:6. In some embodiments, the OspA polypeptide is OspA serotype 6 (ST6). In some embodiments, the OspA ST6 polypeptide comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:6. SEQ ID NO:6 corresponds to OspA from Borrelia garinii (serotype 6) GenBank: CAA45010.1 without the signal sequence and the N-terminal methionine amino acid.

[0133] In some embodiments, the OspA polypeptide is OspA serotype 7 (ST7). In some embodiments, the OspA ST7 polypeptide comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 7. In some embodiments, the OspA polypeptide is OspA serotype 7 (ST7). In some embodiments, the OspA ST7 polypeptide comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 7. SEQ ID NO: 7 corresponds to OspA from Borrelia garinii (serotype 7) GenBank CAA56547.1 without the signal sequence and the N-terminal methionine amino acid.

[0134] IV. Lipid Nanoparticles (LNPs) The LNPs of the present disclosure include lipids from four categories of lipids: (i) ionized lipids (e.g., cationic lipids); (ii) PEGylated lipids; (iii) cholesterol-based lipids (e.g., cholesterol), and (iv) helper lipids.

[0135] A. Ionized lipids Ionizable lipids facilitate mRNA encapsulation and can be cationic lipids, which provide a positively charged environment at low pH to facilitate efficient encapsulation of the negatively charged mRNA drug substance.

[0136] In some embodiments, the cationic lipid is OF-02: [ka] It is.

[0137] OF-02 is a non-degradable structural analog of OF-Deg-Lin. OF-Deg-Lin contains a diketopiperazine core and a degradable ester linkage for attaching a double unsaturated tail, while OF-02 contains the same diketopiperazine core and a non-degradable 1,2-amino-alcohol linkage for attaching a double unsaturated tail (Fenton et al., Ave Mater. (2016) 28: 2939; U.S. Patent No. 10,201,618). The exemplary LNP formulation herein, lipid A, contains OF-2.

[0138] In some embodiments, the cationic lipid is cKK-E10 (Dong et al., PNAS (2014) 111(11):3955-60; U.S. Pat. No. 9,512,073): [ka] It is.

[0139] An exemplary LNP formulation herein, lipid B, contains cKK-E10.

[0140] In some embodiments, the cationic lipid is GL-HEPES-E3-E10-DS-3-E18-1 (2-(4-(2-(3-(bis((Z)-2-hydroxyoctadec-9-en-1-yl)amino)propyl)disulfanayl(ethyl)piperazin-1-yl)ethyl 4-(bis(2-hydroxydecyl)amino)butanoate), which has formula III: [ka] It is a HEPES-based disulfide cationic lipid having a piperazine core.

[0141] An exemplary LNP formulation herein, Lipid C, contains GL-HEPES-E3-E10-DS-3-E18-1. Lipid C has the same composition as Lipid A or Lipid B, with the difference being the cationic lipid.

[0142] In some embodiments, the cationic lipid is GL-HEPES-E3-E12-DS-4-E10 (2-(4-(2-((3-(bis(2-hydroxydecyl)amino)butyl)disulfanayl)ethyl)piperazin-1-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butanoate), which has formula IV: [ka] It is a HEPES-based disulfide cationic lipid having a piperazine core.

[0143] An exemplary LNP formulation herein, lipid D, contains GL-HEPES-E3-E12-DS-4-E10. Lipid D has the same composition as lipid A or lipid B, with the difference being the cationic lipid.

[0144] In some embodiments, the cationic lipid is GL-HEPES-E3-E12-DS-3-E14 (2-(4-(2-((3-(bis(2-hydroxytetradecyl)amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butanoate), which has formula V: [ka] It is a HEPES-based disulfide cationic lipid having a piperazine core.

[0145] An exemplary LNP formulation herein, lipid E, contains GL-HEPES-E3-E12-DS-3-E14. Lipid E ​​has the same composition as lipid A or lipid B, with the difference being the cationic lipid.

[0146] The cationic lipids GL-HEPES-E3-E10-DS-3-E18-1 (III), GL-HEPES-E3-E12-DS-4-E10 (IV), and GL-HEPES-E3-E12-DS-3-E14 (V) can be synthesized according to the general procedure shown in Scheme 1.

[0147] Scheme 1: General synthetic scheme for lipids of formulae (III), (IV) and (V) [ka]

[0148] In some embodiments, the cationic lipid has formula VI: [ka] MC3 has the following.

[0149] In some embodiments, the cationic lipid is SM-102 (9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate), represented by formula VII: [ka] has.

[0150] In some embodiments, the cationic lipid is ALC-0315 [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate), represented by formula VIII: [ka] has.

[0151] In some embodiments, the cationic lipid is cOrn-EE1 and has Formula IX: [ka] has.

[0152] In some embodiments, the cationic lipid is cKK-E10; OF-02; [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl]4-(dimethylamino)butanoate (D-Lin-MC3-DMA); 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA); 1,2-Dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLin-DMA); Di((Z)-non-2-en-1-yl )9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319);9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102);[(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315);[3-(dimethylamino)-2-[(Z)-octadec-9-enoyl]oxypropyl](Z)-octadec-9-enoate 2,5-bis(3-aminopropylamino)-N-[2-[di(heptadecyl)amino]-2-oxoethyl]pentanamide (DOGS);[(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(2R)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-yl]N-[2-(dimethylamino)ethyl]carbamate (DC- Chol;Tetrakis(8-methylnonyl) 3,3',3'',3'''-(((methylazanediyl)bis(propane-3,1diyl))bis(azanetriyl))tetrapropionate (306Oi10);Decyl(2-(dioctylammonio)ethyl)phosphate (9A1P9);Ethyl 5,5-di((Z)-heptadec-8-en-1-yl)-1-(3-(pyrrolidin-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate (A2-Iso5-2DC18);Bis(2-(dodecyldisulfanyl)ethyl)3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexacosyl)azanediyl)dipropionate (BAME-O16B);1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200);3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione (cKK-E12);Hexa(octa) 9,9',9'',9''',9'''',9'''''-((((benzene-1,3,5-tricarbonyl)iris(azanediyl))tris(propane-3,1-diyl))tris(azanetriyl))hexanoate (FTT5);(((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrakis(ethane-2,1-diyl) (9Z,9'Z,9''Z,9''Z,12Z,12'Z,12''Z,12''Z)-tetrakis(octadeca-9,12-dienoate) (OF-Deg-Lin);TT3;N; 1 ,N 3 ,N 5 -tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide; N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamide)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5); heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (Lipid 5); GL-HEPES-E3-E10-DS-3-E18-1; GL-HEPES-E3-E12-DS-4-E10; GL-HEPES-E3-E12-DS-3-E14; and combinations thereof.

[0153] In some embodiments, the cationic lipid is biodegradable.

[0154] In some embodiments, the cationic lipid is not biodegradable.

[0155] In some embodiments, the cationic lipid is cleavable.

[0156] In some embodiments, the cationic lipid is not cleavable.

[0157] Cationic lipids are described in further detail in Dong et al. (PNAS. 111(11):3955-60. 2014); Fenton et al. (Adv Mater. 28:2939. 2016); U.S. Patent No. 9,512,073; and U.S. Patent No. 10,201,618, each of which is incorporated herein by reference.

[0158] B. PEGylated lipids PEGylated lipid components provide control of nanoparticle size and stability. The addition of such components can prevent complex aggregation, increase circulation life, and provide a means to increase delivery of lipid-nucleic acid pharmaceutical compositions to target tissues (Klibanov et al. FEBS Letters 268(1):235-71990). These components can be selected to be rapidly exchanged from the pharmaceutical composition in vivo (see, for example, U.S. Pat. No. 5,885,613).

[0159] Contemplated PEGylated lipids include, but are not limited to, C6-C 20 (For example, C8, C 10 , C 12 , C 14 , C 16 Or C 18)-long alkyl chains, such as derivatized ceramides (e.g., N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)] (C8 PEG ceramide)). In some embodiments, the PEGylated lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG); 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DLPE-PEG); 1,2-distearoyl-rac-glycero-polyethylene glycol (DSG-PEG), PEG-DAG; PEG-PE; PEG-S-DAG; PEG-S-DMG; PEG-cer; PEG-dialkyloxypropylcarbamate; 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); and combinations thereof.

[0160] In some embodiments, the PEG has a high molecular weight, for example, 2000-2400 g / mol. In some embodiments, the PEG is PEG2000 (or PEG-2K). In some embodiments, the PEGylated lipid herein is DMG-PEG2000, DSPE-PEG2000, DLPE-PEG2000, DSG-PEG2000, C8PEG2000, or ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide). In some embodiments, the PEGylated lipid herein is DMG-PEG2000.

[0161] C. Cholesterol-based lipids The cholesterol component provides stability to the lipid bilayer structure within the nanoparticle. In some embodiments, the LNP comprises one or more cholesterol-based lipids. Suitable cholesterol-based lipids include, for example, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao et al., Biochem Biophys Res Comm. (1991) 179:280; Wolf ...), and / or 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao et al., Biochem Biophys Res Comm. (1991) 179:280). al., BioTechniques (1997) 23:139; U.S. Patent No. 5,744,335), imidazole cholesterol esters ("ICE"; WO 2011 / 068810), sitosterol (22,23-dihydrostigmasterol), β-sitosterol, sitostanol, fucosterol, stigmasterol (stigmasta-5,22-dien-3-ol), ergosterol; desmosterol (3β-hydroxy-5,24 cholestadiene); lanosterol (8,24 lanostadien-3b-ol); 7-dehydrocholesterol (Δ5,7-cholesterol); dihydrolanosterol (24 ,25 dihydrolanosterol); zymosterol (5α-cholest-8,24-dien-3β-ol); lathosterol (5α-cholest-7-en-3β-ol); diosgenin ((3β,25R)-spirost-5-en-3-ol); campesterol (campest-5-en-3β-ol); campestanol (5a-campestan-3b-ol); 24 methylenecholesterol (5,24(28)-cholestadien-24-methylene-3β-ol); cholesteryl margarate (cholest-5-en-3β-yl heptadecanoate); cholesteryl oleate; cholesteryl stearate and other modified forms of cholesterol. In some embodiments, the cholesterol-based lipid used in the LNP is cholesterol.

[0162] D. Helper lipids Helper lipids improve the structural stability of LNPs and aid LNPs in endosomal escape, which improves uptake and release of mRNA drug payloads. In some embodiments, the helper lipids are zwitterionic lipids with fusogenic properties to improve uptake and release of drug payloads. Examples of helper lipids are 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS); 1,2-dielideyl-sn-glycero-3-phosphoethanolamine (DEPE); and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPOC), dipalmitoylphosphatidylcholine (DPPC), DMPC, 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-distearoylphosphatidylethanolamine (DSPE), and 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE).

[0163] Other exemplary helper lipids are dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), phosphatidylserine, sphingolipids, sphingomyelin, ceramide, cerebrosides, gangliosides, 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or combinations thereof. In some embodiments, the helper lipid is DOPE. In some embodiments, the helper lipid is DSPC.

[0164] In other embodiments, the LNP comprises: (i) SM-102; (ii) DMG-PEG2000; (iii) cholesterol; and (iv) DSPC.

[0165] In yet other embodiments, the LNP comprises: (i) ALC-0315; (ii) ALC-0159; (iii) cholesterol; and (iv) DSPC.

[0166] In yet other embodiments, the LNP comprises: (i) OF-02; (ii) DMG-PEG2000; cholesterol; and (iv) DOPE.

[0167] In yet other embodiments, the LNP comprises: (i) cKK-E10; (ii) DMG-PEG2000; (iii) cholesterol; and (iv) DOPE.

[0168] In yet other embodiments, the LNP comprises: (i) GL-HEPES-E3-E10-DS-3-E18-1; (ii) DMG-PEG2000; (iii) cholesterol; and (iv) DOPE.

[0169] In yet other embodiments, the LNP comprises: (i) GL-HEPES-E3-E12-DS-4-E10; (ii) DMG-PEG2000; (iii) cholesterol; and (iv) DOPE.

[0170] In yet other embodiments, the LNP comprises: (i) GL-HEPES-E3-E12-DS-3-E14; (ii) DMG-PEG2000; (iii) cholesterol; and (iv) DOPE.

[0171] E. Molar ratio of lipid components The molar ratio of the above components is important for the effectiveness of the LNP in delivering mRNA. The molar ratio of cationic lipid, PEGylated lipid, cholesterol-based lipid and helper lipid is A:B:C:D (where A+B+C+D=100%). In some embodiments, the molar ratio of cationic lipid in the LNP to total lipid (i.e., A) is 35-55%, e.g., 35-50% (e.g., 38-42%, e.g., 40%, or 45-50%). In some embodiments, the molar ratio of PEGylated lipid component to total lipid (i.e., B) is 0.25-2.75% (e.g., 1-2%, e.g., 1.5%). In some embodiments, the molar ratio of cholesterol-based lipid to total lipid (i.e., C) is 20-50% (e.g., 27-30%, e.g., 28.5%, or 38-43%). In some embodiments, the molar ratio of helper lipid to total lipid (i.e., D) is 5-35% (e.g., 28-32%, e.g., 30%, or 8-12%, e.g., 10%). In some embodiments, the (PEGylated lipid + cholesterol) components have the same molar amount as the helper lipid. In some embodiments, the LNPs contain a molar ratio of cationic lipid to helper lipid that is greater than 1.

[0172] In certain embodiments, the LNPs of the disclosure comprise: 35%-55% or 40%-50% molar ratio of cationic lipid (e.g., 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, or 55% molar ratio of cationic lipid); Polyethylene glycol (PEG) conjugated (PEGylated) lipids in a molar ratio of 0.25% to 2.75% or 1.00% to 2.00% (e.g., PEGylated lipids in a molar ratio of 0.25%, 0.50%, 0.75%, 1.00%, 1.25%, 1.50%, 1.75%, 2.00%, 2.25%, 2.50%, or 2.75%); Cholesterol-based lipids in a molar ratio of 20% to 50%, 25% to 45%, or 28.5% to 43% (e.g., cholesterol-based lipids in a molar ratio of 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50%); and 5% to 35%, 8% to 30%, or 10% to 30% molar ratio of helper lipid (e.g., 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% molar ratio of helper lipid), All molar ratios are relative to the total lipid content of the LNPs.

[0173] In one embodiment, the LNP comprises a cationic lipid at a molar ratio of 40%; a PEGylated lipid at a molar ratio of 1.5%; a cholesterol-based lipid at a molar ratio of 28.5%; and a helper lipid at a molar ratio of 30%.

[0174] In one embodiment, the LNPs of the present disclosure comprise a molar ratio of 45-50% cationic lipid; a molar ratio of 1.5-1.7% PEGylated lipid; a molar ratio of 38-43% cholesterol-based lipid; and a molar ratio of 9-10% helper lipid.

[0175] In one embodiment, the PEGylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000).

[0176] In certain embodiments, the cholesterol-based lipid is cholesterol.

[0177] In one embodiment, the helper lipid is 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE).

[0178] In one embodiment, the LNP comprises OF-02 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DOPE in a molar ratio of 5% to 35%.

[0179] In one embodiment, the LNP comprises cKK-E10 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DOPE in a molar ratio of 5% to 35%.

[0180] In one embodiment, the LNP comprises GL-HEPES-E3-E10-DS-3-E18-1 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DOPE in a molar ratio of 5% to 35%.

[0181] In one embodiment, the LNP comprises GL-HEPES-E3-E12-DS-4-E10 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DOPE in a molar ratio of 5% to 35%.

[0182] In one embodiment, the LNP comprises GL-HEPES-E3-E12-DS-3-E14 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DOPE in a molar ratio of 5% to 35%.

[0183] In one embodiment, the LNP comprises SM-102 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DSPC in a molar ratio of 5% to 35%.

[0184] In one embodiment, the LNPs comprise ALC-0315 in a molar ratio of 35% to 55%; ALC-0159 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DSPC in a molar ratio of 5% to 35%.

[0185] In one embodiment, the LNP comprises OF-02 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30%. This LNP formulation is referred to herein as "Lipid A."

[0186] In one embodiment, the LNP comprises cKK-E10 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30%. This LNP formulation is referred to herein as "Lipid B."

[0187] In one embodiment, the LNP comprises GL-HEPES-E3-E10-DS-3-E18-1 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30%. This LNP formulation is referred to herein as "Lipid C."

[0188] In one embodiment, the LNPs comprise GL-HEPES-E3-E12-DS-4-E10 (40% molar ratio; DMG-PEG2000 at 1.5% molar ratio; cholesterol at 28.5% molar ratio; and DOPE at 30% molar ratio. This LNP formulation is referred to herein as "Lipid D."

[0189] In one embodiment, the LNP comprises GL-HEPES-E3-E12-DS-3-E14 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30%. This LNP formulation is referred to herein as "Lipid E."

[0190] In one embodiment, the LNP comprises 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102) at a molar ratio of 50%; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) at a molar ratio of 10%; cholesterol at a molar ratio of 38.5%; and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) at a molar ratio of 1.5%.

[0191] In one embodiment, the LNPs comprise (4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) in a molar ratio of 46.3%; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in a molar ratio of 9.4%; cholesterol in a molar ratio of 42.7%; and 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) in a molar ratio of 1.6%.

[0192] In one embodiment, the LNP comprises (4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) in a molar ratio of 47.4%; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in a molar ratio of 10%; cholesterol in a molar ratio of 40.9%; and 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) in a molar ratio of 1.7%.

[0193] To calculate the actual amount of each lipid to be put into the LNP formulation, the molar amount of the cationic lipid is first determined based on the desired N / P ratio (where N is the number of nitrogen atoms in the cationic lipid and P is the number of phosphate groups in the mRNA to be transported by the LNP). Next, based on the molar amount of the cationic lipid and the selected molar ratio, the molar amount of each of the other lipids is calculated. These molar amounts are then converted to weight using the molecular weight of each lipid.

[0194] F. Active ingredient of LNP The active ingredient of the present LNP vaccine composition is a nucleic acid (eg, mRNA) encoding an antigenic polypeptide derived from at least one bacterium of the genus Borrelia.

[0195] Optionally, the LNPs can be multivalent. In some embodiments, the LNPs can carry nucleic acids, e.g., mRNAs, encoding two or more antigenic polypeptides, e.g., 2, 3, 4, 5, 6, 7, or 8 antigens, from at least one bacterium of the genus Borrelia. For example, the LNPs can carry multiple nucleic acids (e.g., mRNAs), each encoding a different antigenic polypeptide from at least one bacterium of the genus Borrelia; or carry a polycistronic mRNA that can be translated into two or more antigenic polypeptides from at least one bacterium of the genus Borrelia (e.g., each antigen-encoding sequence is separated by a nucleotide linker that encodes a self-cleaving peptide, e.g., a 2A peptide). LNPs carrying different nucleic acids (e.g., mRNAs) typically contain (encapsulate) multiple copies of each nucleic acid. For example, an LNP that carries or encapsulates two different nucleic acids typically carries multiple copies of each of the two different nucleic acids.

[0196] In some embodiments, a single LNP formulation can include multiple types of LNPs (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more), each type carrying a different nucleic acid (eg, mRNA).

[0197] When the nucleic acid is an mRNA, the mRNA can be unmodified (i.e., containing only natural ribonucleotides A, U, C, and / or G linked by phosphodiester bonds) or can be chemically modified (e.g., containing nucleotide analogs such as pseudouridines (e.g., N-1-methylpseudouridine), 2'-fluororibonucleotides, and 2'-methoxyribonucleotides, and / or phosphorothioate bonds). The mRNA molecule can include a 5' cap and a poly-A tail.

[0198] G. Buffers and Other Components The nucleic acids and / or LNPs can be formulated in combination with one or more carriers, targeting ligands, stabilizing reagents (e.g., preservatives and antioxidants), and / or other pharma- ceutical acceptable excipients to stabilize the nucleic acids and / or LNPs (e.g., to extend the shelf life of a vaccine product), facilitate administration of the LNP pharmaceutical composition, and / or enhance in vivo expression of the nucleic acids. Examples of such excipients are parabens, thimerosal, thiomersal, chlorobutanol, benzalkonium chloride, and chelating agents (e.g., EDTA).

[0199] The LNP compositions of the present disclosure can be provided in a frozen liquid form or in a lyophilized form. A variety of cryoprotectants can be used, including, but not limited to, sucrose, trehalose, glucose, mannitol, mannose, dextrose, and the like. The cryoprotectant can comprise 5-30% (w / v) of the LNP composition. In some embodiments, the LNP composition comprises, for example, 5-30% (e.g., 10%) (w / v) trehalose. Once formulated with the cryoprotectant, the LNP composition can be stored at -20 o C~-80 o The mixture can be frozen (or lyophilized and stored frozen) at 3°C.

[0200] The LNP compositions can be provided to the patient in an aqueous buffer solution (if previously frozen, thawed, or if previously lyophilized, reconstituted in the aqueous buffer solution at the bedside). The buffer solution is typically isotonic and suitable for, e.g., intramuscular or intradermal injection. In some embodiments, the buffer solution is phosphate buffered saline (PBS).

[0201] V. Process for Producing LNP Vaccines The LNPs can be prepared by various techniques currently known in the art. For example, multilamellar vesicles (MLVs) can be prepared according to conventional techniques, for example, by depositing selected lipids on the inner wall of a suitable container or vessel by dissolving the lipids in a suitable solvent, then evaporating the solvent to leave a thin film on the inside of the vessel, or by spray drying. An aqueous phase can then be added to the vessel with a vortexing action, which results in the formation of MLVs. Unilamellar vesicles (ULVs) can then be formed by homogenization, sonication, or extrusion of the multilamellar vesicles. In addition, unilamellar vesicles can be formed by detergent removal techniques.

[0202] Various methods are described in US Patent Application Publication No. 2011 / 0244026, US Patent Application Publication No. 2016 / 0038432, US Patent Application Publication No. 2018 / 0153822, US Patent Application Publication No. 2018 / 0125989, and US Patent Application Publication No. 2021 / 0046192 and may be used to practice the present disclosure. One exemplary process involves encapsulating the mRNA by mixing the mRNA with a mixture of lipids, without first preforming the lipids into lipid nanoparticles, as described in US Patent Application Publication No. 2016 / 0038432. Another exemplary process involves encapsulating the mRNA by mixing preformed LNPs with the mRNA, as described in US Patent Application Publication No. 2018 / 0153822.

[0203] In some embodiments, the process of preparing mRNA-loaded LNPs comprises heating one or more of the solutions to a temperature above ambient temperature, the one or more solutions being a solution containing preformed lipid nanoparticles, a solution containing mRNA, and a mixed solution containing LNP-encapsulated mRNA. In some embodiments, the process comprises heating one or both of the mRNA solution and the preformed LNP solution prior to the mixing step. In some embodiments, the process comprises heating one or more of the preformed LNP-containing solution, the solution containing mRNA, and the solution containing LNP-encapsulated mRNA during the mixing step. In some embodiments, the process comprises heating the LNP-encapsulated mRNA after the mixing step. In some embodiments, the temperature to which one or more of the solutions are heated is greater than or equal to about 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C. In some embodiments, the temperature to which one or more of the solutions is heated ranges from about 25-70° C., about 30-70° C., about 35-70° C., about 40-70° C., about 45-70° C., about 50-70° C., or about 60-70° C. In some embodiments, the temperature is about 65° C.

[0204] Various methods may be used to prepare an mRNA solution suitable for the present disclosure. In some embodiments, the mRNA may be dissolved directly in a buffer solution as described herein. In some embodiments, the mRNA solution may be made by mixing an mRNA stock solution with a buffer solution before mixing with a lipid solution for encapsulation. In some embodiments, the mRNA solution may be made by mixing an mRNA stock solution with a buffer solution just before mixing with a lipid solution for encapsulation. In some embodiments, a suitable mRNA stock solution may contain mRNA in water or buffer at a concentration of about 0.2 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1.0 mg / ml, 1.2 mg / ml, 1.4 mg / ml, 1.5 mg / ml, or 1.6 mg / ml, 2.0 mg / ml, 2.5 mg / ml, 3.0 mg / ml, 3.5 mg / ml, 4.0 mg / ml, 4.5 mg / ml, or 5.0 mg / ml or more.

[0205] In some embodiments, the mRNA stock solution is mixed with the buffer solution using a pump. Exemplary pumps include, but are not limited to, gear pumps, peristaltic pumps, and centrifugal pumps. Typically, the buffer solution is mixed at a faster rate than the mRNA stock solution. For example, the buffer solution may be mixed at a rate that is at least 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, or 20x greater than the rate of the mRNA stock solution. In some embodiments, the buffer solution is mixed at a flow rate in the range of about 100-6000 ml / min (e.g., about 100-300 ml / min, 300-600 ml / min, 600-1200 ml / min, 1200-2400 ml / min, 2400-3600 ml / min, 3600-4800 ml / min, 4800-6000 ml / min, or 60-420 ml / min). In some embodiments, the buffer solution is mixed at a flow rate of about 60 ml / min, 100 ml / min, 140 ml / min, 180 ml / min, 220 ml / min, 260 ml / min, 300 ml / min, 340 ml / min, 380 ml / min, 420 ml / min, 480 ml / min, 540 ml / min, 600 ml / min, 1200 ml / min, 2400 ml / min, 3600 ml / min, 4800 ml / min, or 6000 ml / min or greater.

[0206] In some embodiments, the mRNA stock solution is mixed at a flow rate in the range of about 10-600 ml / min (e.g., about 5-50 ml / min, about 10-30 ml / min, about 30-60 ml / min, about 60-120 ml / min, about 120-240 ml / min, about 240-360 ml / min, about 360-480 ml / min, or about 480-600 ml / min). In some embodiments, the mRNA stock solution is mixed at a flow rate of about 5 ml / min, 10 ml / min, 15 ml / min, 20 ml / min, 25 ml / min, 30 ml / min, 35 ml / min, 40 ml / min, 45 ml / min, 50 ml / min, 60 ml / min, 80 ml / min, 100 ml / min, 200 ml / min, 300 ml / min, 400 ml / min, 500 ml / min, or 600 ml / min or greater.

[0207] The process of incorporating desired mRNA into lipid nanoparticles is referred to as "loading". Exemplary methods are described in Lasic et al., FEBS Lett. (1992) 312: 255-8. Nucleic acid incorporated into LNPs can be located completely or partially within the inner space of lipid nanoparticles, within the bilayer membrane of lipid nanoparticles, or associated with the outer surface of lipid nanoparticle membrane. The incorporation of mRNA into lipid nanoparticles is also referred to herein as "encapsulation", and the nucleic acid is completely or substantially contained within the inner space of lipid nanoparticles.

[0208] Suitable LNPs can be made in various sizes. In some embodiments, the reduction in size of lipid nanoparticles is associated with more efficient delivery of mRNA. The selection of suitable LNP size can take into account the target cell or tissue site and the application for which lipid nanoparticles are made.

[0209] Various methods known in the art are available for sizing lipid nanoparticle populations. An exemplary method herein utilizes a Zetasizer Nano ZS (Malvern Panalytical) to measure LNP particle size. In one protocol, 10 μl of LNP sample is mixed with 990 μl of 10% trehalose. This solution is loaded into a cuvette and then placed into the Zetasizer machine. The z-average diameter (nm), or cumulant average, is taken as the average size of the LNPs in the sample. The Zetasizer machine can also be used to measure the polydispersity index (PDI) by using dynamic light scattering (DLS) and cumulant analysis of the autocorrelation function. The average LNP diameter can be reduced by sonication of the formed LNPs. Intermittent sonication cycles can be alternated with quasi-elastic light scattering (QELS) evaluation to guide efficient lipid nanoparticle synthesis.

[0210] In some embodiments, the majority of the purified LNPs, i.e., greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the LNPs, have a size of about 70 to 150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm). In some embodiments, substantially all (e.g., greater than 80% or 90%) of the purified lipid nanoparticles have a size of about 70 to 150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm).

[0211] In one embodiment, the LNPs have an average diameter between 30 and 200 nm.

[0212] In one embodiment, the LNPs have an average diameter of between 80 and 150 nm.

[0213] In some embodiments, the LNPs in the composition have an average size of less than 150 nm, less than 120 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 30 nm, or less than 20 nm.

[0214] In some embodiments, about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or greater than 99% of the LNPs in the composition have a size in the range of about 40-90 nm (e.g., about 45-85 nm, about 50-80 nm, about 55-75 nm, or about 60-70 nm), about 40-90 nm (e.g., about 45-85 nm, about 50-80 nm, about 55-75 nm, or about 60-70 nm), or about 50-70 nm (e.g., about 55-65 nm), making them particularly suitable for pulmonary delivery via nebulization.

[0215] In some embodiments, the dispersity, or molecular size heterogeneity measure (PDI), of the LNPs in the pharmaceutical compositions provided by the present disclosure is less than about 0.5. In some embodiments, the LNPs have a PDI of less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.28, less than about 0.25, less than about 0.23, less than about 0.20, less than about 0.18, less than about 0.16, less than about 0.14, less than about 0.12, less than about 0.10, or less than about 0.08. The PDI can be measured by a Zetasizer machine as described above.

[0216] In some embodiments, greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified LNPs in the pharmaceutical compositions provided herein encapsulate mRNA within each individual particle. In some embodiments, substantially all (e.g., greater than 80% or 90%) of the purified lipid nanoparticles in the pharmaceutical composition encapsulate mRNA within each individual particle. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of 50% to 99%; or greater than about 60, 65, 70, 75, 80, 85, 90, 92, 95, 98, or 99%. Typically, lipid nanoparticles for use herein have an encapsulation efficiency of at least 90% (e.g., at least 91, 92, 93, 94, or 95%).

[0217] In some embodiments, the LNPs have an N / P ratio of 1 to 10. In some embodiments, the lipid nanoparticles have an N / P ratio of greater than 1, about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8. In further embodiments, exemplary LNPs herein have an N / P ratio of 4.

[0218] In some embodiments, a pharmaceutical composition according to the present disclosure contains at least about 0.5 μg, 1 μg, 5 μg, 10 μg, 100 μg, 500 μg, or 1000 μg of encapsulated mRNA. In some embodiments, a pharmaceutical composition contains about 0.1 μg-1000 μg, at least about 0.5 μg, at least about 0.8 μg, at least about 1 μg, at least about 5 μg, at least about 8 μg, at least about 10 μg, at least about 50 μg, at least about 100 μg, at least about 500 μg, or at least about 1000 μg of encapsulated mRNA.

[0219] In some embodiments, mRNA can be produced by chemical synthesis or by in vitro transcription (IVT) of DNA template. An exemplary process for producing and purifying mRNA is described in Example 1. In this process, the IVT process, a cDNA template is used to produce mRNA transcripts, and the DNA template is degraded by DNase. The transcripts are purified by depth filtration and tangential flow filtration (TFF). The purified transcripts are further modified by adding caps and tails, and the modified RNA is purified again by depth filtration and TFF.

[0220] The mRNA is then prepared in an aqueous buffer and mixed with an amphipathic solution containing the lipid components of the LNP. The amphipathic solution for dissolving the four lipid components of the LNP can be an alcohol solution. In some embodiments, the alcohol is ethanol. The aqueous buffer can be, for example, a citrate, phosphate, acetate, or succinate buffer and can have a pH of about 3.0 to 7.0, e.g., about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, or about 6.5. The buffer can contain other components, e.g., salts (e.g., sodium salts, potassium salts, and / or calcium salts). In certain embodiments, the aqueous buffer has 1 mM citrate, 150 mM NaCl, pH 4.5.

[0221] An exemplary, non-limiting process for making mRNA-LNP compositions includes mixing a buffered mRNA solution with a solution of lipids in ethanol in a controlled, homogenous manner, maintaining the lipid:mRNA ratio throughout the mixing process. In this illustrative example, the mRNA is present in an aqueous buffer containing citric acid monohydrate, trisodium citrate dihydrate, and sodium chloride. The mRNA solution is added to a solution (1 mM citrate buffer, 150 mM NaCl, pH 4.5). A lipid mixture of four lipids (e.g., cationic lipid, PEGylated lipid, cholesterol-based lipid, and helper lipid) is dissolved in ethanol. The aqueous mRNA solution and the ethanolic lipid solution are mixed in a 4:1 volume ratio in a "T" mixer equipped with a nearly "pulseless" pump system. The resulting mixture is then subjected to downstream purification and buffer exchange. Buffer exchange can be achieved using a dialysis cassette or a TFF system. TFF can be used to concentrate and buffer exchange the nascent LNPs obtained immediately after formation via the T mixing process. The diafiltration process is a continuous operation in which the volume is kept constant by adding an appropriate buffer at the same rate as the permeate flow.

[0222] VI. Packaging and Use of mRNA-LNP OspA Vaccine The mRNA-LNP vaccines can be formulated or packaged for parenteral (e.g., intramuscular, intradermal, or subcutaneous) or nasopharyngeal (e.g., intranasal) administration. The vaccine composition can be in the form of an extemporaneous formulation, in which the LNP composition is lyophilized and reconstituted with a physiological buffer (e.g., PBS) immediately prior to use. The vaccine composition can also be shipped and provided in the form of an aqueous or frozen aqueous solution, and can be administered directly to a subject without reconstitution (after thawing if previously frozen).

[0223] Thus, the present disclosure provides an article of manufacture, e.g., a kit, that provides an mRNA-LNP vaccine in a single container, or that provides an mRNA-LNP vaccine in one container and a physiological buffer for reconstitution in another container. The container may contain a single-use dose or a multi-use dose. The container may be a pre-processed glass vial or ampoule. The article of manufacture may also include instructions for use.

[0224] In certain embodiments, the mRNA-LNP vaccine is provided for use in intramuscular (IM) injection. The vaccine can be injected into a subject, for example, in the deltoid muscle of the subject's upper arm. In some embodiments, the vaccine is provided in a pre-filled syringe or injector (e.g., single-chamber or multi-chamber). In some embodiments, the vaccine is provided for use in inhalation, and is provided in a pre-filled pump, aerosol generator, or inhaler.

[0225] The mRNA-LNP vaccine can be administered to a subject in need thereof in a prophylactically effective amount, i.e., an amount that provides sufficient immune protection against the target pathogen for a sufficient time (e.g., 1 year, 2 years, 5 years, 10 years, or a lifetime). Sufficient immune protection can be, for example, prevention or alleviation of symptoms associated with infection by the pathogen. In some embodiments, multiple doses (e.g., 2 doses) of the vaccine are administered (e.g., injected) to a subject in need thereof to achieve a desired prophylactic effect. The doses (e.g., a prime dose and a booster dose) can be spaced apart, for example, by 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 1 year, 2 years, 3 years, 5 years, or 10 years.

[0226] In some embodiments, a single dose of an mRNA-LNP vaccine contains 1-50 μg of mRNA (e.g., monovalent or multivalent). For example, a single dose may contain about 2.5 μg, about 5 μg, about 7.5 μg, about 10 μg, about 12.5 μg, or about 15 μg of mRNA for intramuscular (IM) injection. In further embodiments, a multivalent single dose of an LNP vaccine contains multiple (e.g., 2, 3, or 4) types of LNPs, each for a different antigen, with each type of LNP having an mRNA amount of, for example, 2.5 μg, about 5 μg, about 7.5 μg, about 10 μg, about 12.5 μg, or about 15 μg.

[0227] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure. In the event of a conflict, the present specification, including definitions, will control. In general, the terminology used in connection with and in the techniques of cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein is that well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as commonly accomplished in the art or as described herein. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Throughout the specification and embodiments, the words "have" and "comprise", or variations such as "has", "having", "comprises", or "comprising" are understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although many documents are cited herein, this citation does not constitute an admission that any of these documents form part of the common general knowledge in the art. As used herein, the term "approximately" or "about", when applied to one or more values ​​of interest, refers to a value similar to the stated reference value. In certain embodiments, the term refers to a range of values ​​that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less) of a stated reference value, unless otherwise stated or apparent from the context.

[0228] VII. Vector In one aspect, a vector is disclosed herein that comprises the mRNA composition disclosed herein. The RNA sequence encoding the protein of interest (e.g., the mRNA encoding the OspA protein) can be cloned into many types of vectors. For example, the nucleic acid can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, sequencing vectors, and vectors optimized for in vitro transcription.

[0229] In some embodiments, the vector can be used to express mRNA in a host cell. In various embodiments, the vector is used as a template for IVT. The construction of optimally translated IVT mRNA suitable for therapeutic use is disclosed in detail in Sahin, et al. (2014). Nat. Rev. Drug Discov. 13, 759-780; Weissman (2015). Expert Rev. Vaccines 14, 265-281.

[0230] In some embodiments, the vectors disclosed herein may include, from 5' to 3', at least the following: an RNA polymerase promoter; a polynucleotide sequence encoding a 5' UTR; a polynucleotide sequence encoding an ORF; a polynucleotide sequence encoding a 3' UTR; and a polynucleotide sequence encoding at least one RNA aptamer. In some embodiments, the vectors disclosed herein may also include a polynucleotide sequence encoding a polyA sequence and / or a polyadenylation signal.

[0231] A variety of RNA polymerase promoters are known in the art. In some embodiments, the promoter may be a T7 RNA polymerase promoter. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.

[0232] Also disclosed herein are host cells (eg, mammalian cells, eg, human cells) that contain the vectors or RNA compositions disclosed herein.

[0233] Polynucleotides can be introduced into target cells using any of a number of different methods, such as commercially available methods including, but not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendorf, Hamburg, Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, biolistic particle delivery systems, such as a "gene gun" (see, e.g., Nishikawa, et al. (2001). Hum Gene Ther. 12(8):861-70), or the TransIT-RNA transfection kit (Mirus, Madison, Wis.).

[0234] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro and in vivo delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0235] Regardless of the method used to introduce exogenous nucleic acid into a host cell or otherwise expose the cell to an inhibitor of the present disclosure, a variety of assays can be performed to confirm the presence of the mRNA sequence in the host cell.

[0236] VIII. Self-replicating, trans-replicating, and non-replicating RNA Self-replicating RNA: Self-replicating (or self-propagating) RNA can be produced, for example, by using replication elements derived from alphaviruses and replacing structural viral proteins with nucleotide sequences encoding a protein of interest (e.g., an antigenic prokaryotic polypeptide). Self-replicating RNAs are typically positive-stranded molecules that can be directly translated after delivery to a cell, and this translation then provides an RNA-dependent RNA polymerase that produces both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA results in the production of multiple daughter RNAs. These daughter RNAs, and collinear subgenomic transcripts, can themselves be translated to provide in situ expression of the encoded antigen, or can be transcribed to provide additional transcripts with the same sense as the delivered RNA that can be translated to provide in situ expression of the antigen. The overall result of this series of transcriptions is a large amplification of the number of introduced replicon RNAs, so that the encoded antigen becomes the major polypeptide product of the cell.

[0237] One suitable system for achieving self-replication in this way is to use alphavirus-based replicons. These replicons are positive-stranded (positive-sense) RNAs that result in the translation of a replicase (or replicase transcriptase) after delivery to the cell. The replicase is translated as a polyprotein that provides a replication complex that self-cleaves to create a genomic strand copy of the positive-stranded delivered RNA. These negative (-) strand transcripts can themselves be transcribed to provide further copies of the positive-stranded parent RNA and even subgenomic transcripts that code for antigens. Translation of the subgenomic transcripts thus results in in situ expression of the antigen by the infected cell. Suitable alphavirus replicons may use replicases from Sindbis virus, Semliki Forest virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus, and the like. Mutant or wild-type virus sequences may be used, for example, the attenuated TC83 mutant of VEEV has been used in the replicon. See the following references: WO 2005 / 113782, incorporated herein by reference.

[0238] In one embodiment, each self-replicating RNA described herein encodes (i) an RNA-dependent RNA polymerase capable of transcribing RNA from the self-replicating RNA molecule and (ii) an influenza protein antigen. The polymerase may be, for example, an alphavirus replicase, including one or more of the alphavirus proteins nsP1, nsP2, nsP3, and nsP4. While native alphavirus genomes encode structural virion proteins in addition to nonstructural replicase polyproteins, in certain embodiments, the self-replicating RNA molecule does not encode alphavirus structural proteins. Thus, a self-replicating RNA may result in the production of its own genomic RNA copies in a cell, but not in the production of RNA-containing virions. The inability to produce these virions means that, unlike wild-type alphaviruses, the self-replicating RNA molecule cannot persist by itself in an infectious form. The alphavirus structural proteins necessary for persistence in wild-type viruses are absent in the self-replicating RNA of the present disclosure, and their place is taken by a gene encoding an immunogen of interest, such that the subgenomic transcript encodes that immunogen rather than the structural alphavirus virion proteins. Self-replicating RNA is described in further detail in WO2011005799, which is incorporated herein by reference.

[0239] Trans-replicating RNA: Trans-replicating (or trans-amplifying) RNA has similar elements to the self-replicating RNA described above. However, in trans-replicating RNA, two separate RNA molecules are used. A first RNA molecule encodes the RNA replicase described above (e.g., an alphavirus replicase), and a second RNA molecule encodes a protein of interest (e.g., an antigenic prokaryotic polypeptide). The RNA replicase can replicate one or both of the first and second RNA molecules, thereby greatly increasing the copy number of the RNA molecule encoding the protein of interest. Trans-replicating RNA is described in more detail in WO2017162265, which is incorporated herein by reference.

[0240] Non-replicating RNA: Non-replicating (or non-amplifying) RNA is RNA that does not have the ability to replicate itself.

[0241] IX. Pharmaceutical Compositions The pharmaceutical compositions described in this disclosure typically comprise a nucleic acid, particularly RNA, more particularly mRNA, and a pharma- ceutically acceptable carrier, or a pharma- ceutically acceptable excipient, or a pharma- ceutically acceptable diluent, making the composition particularly suitable for therapeutic use. The phrase "pharma- ceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, and commensurate with a reasonable benefit / risk ratio.

[0242] A pharmaceutical composition can be, for example, an immunogenic composition, i.e., a composition that induces an immune response when administered to a subject. It should be understood that the terms "immunogenic composition," "vaccine composition," and "vaccine" are used interchangeably herein and are therefore meant to have equivalent meanings.

[0243] The pharmaceutical composition of the present disclosure may also include one or more additional components, such as a small molecule immunostimulant (e.g., a TLR agonist). The pharmaceutical composition of the present disclosure may also include a delivery system for RNA, such as a liposome, an oil-in-water emulsion, or a microparticle. In some embodiments, the pharmaceutical composition includes a lipid nanoparticle (LNP). In one embodiment, the composition includes an antigen-encoding nucleic acid molecule encapsulated within the LNP.

[0244] X. Vaccination Methods The Lyme disease vaccines disclosed herein can be administered to a subject to induce an immune response directed against an antigenic protein from the genus Borrelia, for example, the OspA protein expressed on the surface of bacteria of the genus Borrelia, and the anti-antigen antibody titer in the subject is increased following vaccination relative to the anti-antigen antibody titer of a subject not vaccinated with the Lyme disease vaccine disclosed herein, or against an alternative vaccine against Lyme disease. An "anti-antigen antibody" is a serum antibody that specifically binds to an antigen.

[0245] In one aspect, the present disclosure provides a method of inducing an immune response, preferably a humoral immune response, in a subject in need of such induction, and / or a method of treating or preventing Lyme disease in a subject in need of such treatment or prevention, comprising administering to the subject a Lyme disease vaccine disclosed herein.

[0246] The present disclosure also provides the Lyme disease vaccines described herein for use in inducing an immune response, preferably a humoral immune response, in a subject in need of such induction, and / or for use in treating or preventing Lyme disease in a subject in need of such treatment or prevention.

[0247] The present disclosure also provides a Lyme disease vaccine as described herein for use in inducing an immune response, preferably a humoral immune response, in a subject in need of such induction, and / or for use in the manufacture of a medicament for use in treating or preventing Lyme disease in a subject in need of such treatment or prevention.

[0248] In one embodiment, a subject has similar or higher serum concentrations of antibodies to OspA after administration of a Lyme disease vaccine relative to a subject administered a Lyme disease vaccine comprising an OspA recombinant protein (Recombitek, OspA fusion ST1-ST2, OspA-ferritin).

[0249] The present invention includes the following embodiments.

[0250] Embodiment 1. A nucleic acid comprising an open reading frame (ORF) encoding at least one antigenic polypeptide derived from at least one bacterium of the genus Borrelia, preferably selected from the species B. burgdorferi, afzelii, garinii, bavariensis, mayonii, spielmanii, lusitaniae, bissettii and / or valaisiana, or any strain or isolate thereof.

[0251] Embodiment 2. The nucleic acid of embodiment 1, wherein the at least one antigenic polypeptide comprises at least one lipoprotein of the genus Borrelia.

[0252] Embodiment 3. The nucleic acid of embodiment 1 or 2, wherein at least one antigenic polypeptide or lipoprotein is OspA or a fragment or variant thereof, preferably comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 amino acids.

[0253] Embodiment 4. The nucleic acid of any one of embodiments 1-3, wherein the OspA is from OspA serotype (ST) 1, 2, 3, 4, 5, 6, and / or 7, preferably from serotype 1 Borrelia burgdorferi strain B31, serotype 2 Borrelia afzelii strain PKO, serotype 3 Borrelia garinii strain PBr, serotype 4 Borrelia bavariensis, serotype 5 Borrelia garinii, serotype 6 Borrelia garinii, or serotype 7 Borrelia garinii.

[0254] Embodiment 5. The nucleic acid of any one of embodiments 1 to 4, wherein at least one OspA polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 1 to 7.

[0255] Embodiment 6. The nucleic acid of any one of embodiments 1 to 5, comprising a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 10 to 13 and 16 to 19.

[0256] Embodiment 7. The nucleic acid of any one of embodiments 1 to 6, encoding at least two different OspA serotypes or fragments or variants thereof, each of the at least two different OspA serotypes or fragments or variants thereof preferably comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 amino acids.

[0257] Embodiment 8. The nucleic acid of embodiment 7, wherein OspA of one serotype, or a fragment or variant thereof, is fused to OspA of a different serotype, or a fragment or variant thereof.

[0258] Embodiment 9. The nucleic acid of embodiment 8, wherein the fusion OspAs, or fragments or variants thereof, of different serotypes are separated by a linker sequence, the linker sequence preferably being derived from P66 or comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:8 or to SEQ ID NO:9.

[0259] Embodiment 10. The nucleic acid of any one of embodiments 1 to 9, which is a non-replicating nucleic acid.

[0260] Embodiment 11. The nucleic acid of any one of embodiments 1 to 9, which is an auto-replicating or trans-replicating nucleic acid.

[0261] Embodiment 12. The nucleic acid of any one of embodiments 1 to 11, which is DNA.

[0262] Embodiment 13. The nucleic acid of any one of embodiments 1 to 11, which is messenger RNA (mRNA).

[0263] Embodiment 14. The mRNA comprises at least one 5' cap, at least one 5' untranslated region (5' UTR), at least one 3' untranslated region (3' UTR), and / or at least one polyadenylation (poly A) sequence, e.g., the mRNA comprises at least one 5' cap, or at least one 5'UTR, or at least one 3'UTR, or at least one 5'UTR and at least one 3'UTR, or - at least one polyA sequence, or - at least one 5' cap and at least one polyA sequence, or - at least one 5' cap, at least one 5' UTR, at least one 3' UTR and at least one poly A sequence The nucleic acid of embodiment 13, which may comprise:

[0264] The nucleic acid of embodiment 14, wherein the 5' cap is selected from the group consisting of 3'-O-Me-m7G(5')ppp(5')G (ARCA cap), G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, m7G(5')ppp(5')G, m7G(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeA)pU, and m7G(5')ppp(5')(2'OMeG)pG.

[0265] The embodiment 16.5' cap is [ka] 15. The nucleic acid of embodiment 14, comprising:

[0266] In embodiment 17, the 5'UTR is about 10 to 5,000 nucleotides in length (e.g., about 50 to 500 nucleotides in length or at least about 10 nucleotides in length, about 20 nucleotides in length, about 30 nucleotides in length, about 40 nucleotides in length, about 50 nucleotides in length, about 100 nucleotides in length, about 150 nucleotides in length, about 200 nucleotides in length, about 250 nucleotides in length, about 300 nucleotides in length, about 350 nucleotides in length, about 400 nucleotides in length, about 450 nucleotides in length, about 500 nucleotides in length, about 550 nucleotides in length, about 600 nucleotides in length, or at least about 600 nucleotides in length). 17. The nucleic acid of any of embodiments 14 to 16, wherein the nucleic acid is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 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, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 75, 80, 85, 900, 950, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or 5,000 nucleotides in length.

[0267] In embodiment 18, the 3'UTR is 50 to 5,000 nucleotides or more in length (e.g., 50 to 1,000 nucleotides or more in length or about 50 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 350 nucleotides, about 400 nucleotides, about 450 nucleotides, about 500 nucleotides, about 550 nucleotides, about 600 nucleotides, about 650 nucleotides, about 700 nucleotides, or more in length). 18. The nucleic acid of any one of embodiments 14 to 17, wherein the nucleic acid is about 750 nucleotides long, about 800 nucleotides long, about 850 nucleotides long, about 900 nucleotides long, about 950 nucleotides long, about 1,000 nucleotides long, about 1,500 nucleotides long, about 2,000 nucleotides long, about 2,500 nucleotides long, about 3,000 nucleotides long, about 3,500 nucleotides long, about 4,000 nucleotides long, about 4,500 nucleotides long, or about 5,000 nucleotides long.

[0268] Embodiment 19. The nucleic acid of any of embodiments 14-18, wherein the 5' and / or 3' UTR is derived from a gene that is distinct from the gene encoded by the mRNA transcript (i.e., the UTR is a heterologous UTR).

[0269] Embodiment 20. The nucleic acid of any of embodiments 14-19, wherein the 5' and / or 3' UTR sequences are derived from a stable mRNA (e.g., globin, actin, GAPDH, tubulin, histones, or citric acid cycle enzymes) to increase stability of the mRNA.

[0270] Embodiment 21. The nucleic acid of any of embodiments 14-19, wherein the 5'UTR is derived from the CMV immediate early 1 (IE1) gene.

[0271] Embodiment 22. The nucleic acid of any of embodiments 14 to 19, wherein the 5'UTR comprises the sequence GGGAUCCUACC (SEQ ID NO: 20).

[0272] Embodiment 23. The nucleic acid of any of embodiments 14 to 19, wherein the 5'UTR is derived from the 5'UTR of the TOP gene.

[0273] Embodiment 24. The nucleic acid of any of embodiments 14 to 19, wherein the 5'UTR is derived from the ribosomal protein large 32 (L32) gene.

[0274] Embodiment 25. The nucleic acid of any of embodiments 14-19, wherein the 5'UTR is derived from the 5'UTR of the hydroxysteroid (17-b) dehydrogenase 4 gene (HSD17B4).

[0275] Embodiment 26. The nucleic acid of any of embodiments 14 to 19, wherein the 5'UTR is derived from the 5'UTR of the ATP5A1 gene.

[0276] Embodiment 27. The nucleic acid of any of embodiments 14 to 19, wherein an internal ribosome entry site (IRES) is used instead of the 5'UTR.

[0277] The nucleic acid of any of embodiments 14 to 19, wherein the 5'UTR comprises the nucleic acid sequence set forth in SEQ ID NO:14.

[0278] Embodiment 29. The nucleic acid of any of embodiments 14 to 28, wherein the 3'UTR comprises the nucleic acid sequence set forth in SEQ ID NO:15.

[0279] Embodiment 30. The nucleic acid of any of embodiments 14 to 29, wherein at least one polyadenylation (polyA) sequence comprises from about 10 to about 500 adenosine nucleotides, from about 10 to about 200 adenosine nucleotides, from about 40 to about 200 adenosine nucleotides, or from about 40 to about 150 adenosine nucleotides.

[0280] Embodiment 31. The nucleic acid of any of embodiments 14 to 30, wherein at least one polyadenylation (polyA) sequence comprises at least about 10, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 adenosine nucleotides.

[0281] Embodiment 32. At least one polyadenylation (polyA) sequence is selected from the group consisting of the sequence [ka] The nucleic acid of any one of embodiments 14 to 31, comprising:

[0282] In embodiment 33, the 5'UTR is about 10 to 5,000 nucleotides in length (e.g., about 50 to 500 nucleotides in length or at least about 10 nucleotides in length, about 20 nucleotides in length, about 30 nucleotides in length, about 40 nucleotides in length, about 50 nucleotides in length, about 100 nucleotides in length, about 150 nucleotides in length, about 200 nucleotides in length, about 250 nucleotides in length, about 300 nucleotides in length, about 350 nucleotides in length, about 400 nucleotides in length, about 450 nucleotides in length, about 500 nucleotides in length, about 550 nucleotides in length, about 600 nucleotides in length, or at least about 600 nucleotides in length). 33. The nucleic acid of any of embodiments 30 to 32, wherein the nucleic acid is about 600 nucleotides long, about 650 nucleotides long, about 700 nucleotides long, about 750 nucleotides long, about 800 nucleotides long, about 850 nucleotides long, about 900 nucleotides long, about 950 nucleotides long, about 1,000 nucleotides long, about 1,500 nucleotides long, about 2,000 nucleotides long, about 2,500 nucleotides long, about 3,000 nucleotides long, about 3,500 nucleotides long, about 4,000 nucleotides long, about 4,500 nucleotides long, or about 5,000 nucleotides long.

[0283] In embodiment 34, the 3'UTR is 50 to 5,000 nucleotides or more in length (e.g., 50 to 1,000 nucleotides or more in length or about 50 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 350 nucleotides, about 400 nucleotides, about 450 nucleotides, about 500 nucleotides, about 550 nucleotides, about 600 nucleotides, about 650 nucleotides, about 700 nucleotides, or more in length). 34. The nucleic acid of any of embodiments 30 to 33, wherein the nucleic acid is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 85, 90, 950, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or 5,000 nucleotides in length.

[0284] Embodiment 35. The nucleic acid of any of embodiments 30-34, wherein the 5' and / or 3' UTR is derived from a gene that is distinct from the gene encoded by the mRNA transcript (i.e., the UTR is a heterologous UTR).

[0285] Embodiment 36. The nucleic acid of any of embodiments 30-35, wherein the 5' and / or 3' UTR sequences are derived from a stable mRNA (e.g., globin, actin, GAPDH, tubulin, histones, or citric acid cycle enzymes) to increase stability of the mRNA.

[0286] Embodiment 37. The nucleic acid of any of embodiments 30 to 35, wherein the 5'UTR is derived from the CMV immediate early 1 (IE1) gene.

[0287] Embodiment 38. The nucleic acid of any of embodiments 30 to 35, wherein the 5'UTR comprises the sequence GGGAUCCUACC (SEQ ID NO: 20).

[0288] Embodiment 39. The nucleic acid of any of embodiments 30 to 35, wherein the 5'UTR is derived from the 5'UTR of the TOP gene.

[0289] Embodiment 40. The nucleic acid of any of embodiments 30 to 35, wherein the 5'UTR is derived from the ribosomal protein large 32 (L32) gene.

[0290] Embodiment 41. The nucleic acid of any of embodiments 30-35, wherein the 5'UTR is derived from the 5'UTR of the hydroxysteroid (17-b) dehydrogenase 4 gene (HSD17B4).

[0291] Embodiment 42. The nucleic acid of any of embodiments 30 to 35, wherein the 5'UTR is derived from the 5'UTR of the ATP5A1 gene.

[0292] Embodiment 43. The nucleic acid of any of embodiments 30 to 35, wherein an internal ribosome entry site (IRES) is used instead of the 5'UTR.

[0293] Embodiment 44. The nucleic acid of any of embodiments 30 to 35, wherein the 5'UTR comprises the nucleic acid sequence set forth in SEQ ID NO:14.

[0294] The nucleic acid of any one of embodiments 30 to 35, wherein the 3'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 15.

[0295] Embodiment 46. The mRNA comprises at least the following structural elements: (i) a structure: [ka] having a 5' cap; (ii) a 5' untranslated region (5'UTR) having the nucleic acid sequence of SEQ ID NO:14; (iii) at least one OspA open reading frame having any one of the nucleic acid sequences of SEQ ID NOs: 16-19; (iv) a 3' untranslated region (3' UTR) having the nucleic acid sequence of SEQ ID NO: 15; and (v) Poly(A) tail The nucleic acid of any one of embodiments 13 to 45, comprising:

[0296] Embodiment 47. A nucleic acid comprising an mRNA comprising an open reading frame (ORF) encoding at least one antigenic polypeptide from at least one bacterium of the genus Borrelia, the mRNA comprising at least the following structural elements: (i) a structure: [ka] having a 5' cap; (ii) a 5' untranslated region (5'UTR) having the nucleic acid sequence of SEQ ID NO:14; (iii) at least one OspA open reading frame having any one of the nucleic acid sequences of SEQ ID NOs: 16-19; (iv) a 3' untranslated region (3' UTR) having the nucleic acid sequence of SEQ ID NO: 15; and (v) Poly(A) tail Includes; The mRNA is a nucleic acid formulated in a lipid nanoparticle (LNP) comprising: cKK-E10 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, Cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30%; or OF-02 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; Cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%; or GL-HEPES-E3-E10-DS-3-E18-1 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; Cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%; or GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; Cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%; or GL-HEPES-E3-E12-DS-3-E14 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; Cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%; or 50% molar ratio of 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102); 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) at a molar ratio of 10%; Cholesterol at a molar ratio of 38.5%; and 1.5% molar ratio of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000); or (4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) at a molar ratio of 46.3%; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) at a molar ratio of 9.4%; Cholesterol at a molar ratio of 42.7%; and 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) at a molar ratio of 1.6%; or (4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) at a molar ratio of 47.4%; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) at a molar ratio of 10%; Cholesterol at a molar ratio of 40.9%; and 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) at a molar ratio of 1.7%.

[0297] Embodiment 48. The nucleic acid of any one of embodiments 1 to 47, comprising at least one chemical modification.

[0298] Embodiment 49. The nucleic acid of embodiment 48, wherein at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides are chemically modified.

[0299] Embodiment 50. The nucleic acid of embodiment 48 or 49, wherein the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytidine, 2-thio-l-methyl-1-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0300] Embodiment 51. The nucleic acid of any one of embodiments 48 to 50, wherein the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 5-methylcytidine, 5-methoxyuridine, and combinations thereof.

[0301] Embodiment 52. The nucleic acid of any one of embodiments 48 to 51, wherein the chemical modification is N1-methylpseudouridine.

[0302] Embodiment 53. A composition comprising at least one nucleic acid of any one of embodiments 1 to 52.

[0303] Embodiment 54 The composition of embodiment 53, wherein the nucleic acid is formulated in a non-viral delivery system.

[0304] Embodiment 55. The composition of embodiment 53 or 54, comprising a lipid nanoparticle (LNP).

[0305] Embodiment 56 The composition of embodiment 55, wherein the nucleic acid is encapsulated in an LNP.

[0306] Embodiment 57. The LNP comprises at least one cationic lipid, which may or may not be biodegradable, cleavable or not, and the cationic lipid is preferably selected from the group consisting of cKK-E10; OF-02; [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl]4-(dimethylamino)butanoate (D-Lin-MC3-DMA); 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA); 1,2-Dilinoleyl o Di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)yloxy)heptadecanedioate (L319); 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102); [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); [3-(dimethylamino)-2-[(Z)-o 2,5-bis(3-aminopropylamino)-N-[2-[di(heptadecyl)amino]-2-oxoethyl]pentanamide (DOGS);[(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(2R)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-yl]N-[2-(dimethylamino)-1H-cyclopenta[a]phenanthren-3-yl] ethyl 5,5-di((Z)-heptadec-8-en-1-yl)-1-(3-(pyrrolidin-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate (A2-Iso5-2DC18);Bis(2-(dodecyldisulfanyl)ethyl)3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexacosyl)azanediyl)dipropionate (BAME-O16B);1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200);3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione (cKK-E12);Hexa(octan-3-yl) 9,9',9'',9''',9'''',9''''''-((((benzene-1,3,5-tricarbonyl)iris(azanediyl))tris(propane-3,1-diyl))tris(azanetriyl))hexanoate (FTT5);(((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrakis(ethane-2,1-diyl) (9Z,9'Z,9''Z,9''Z,12Z,12'Z,12''Z,12''Z)-tetrakis(octadeca-9,12-dienoate) (OF-Deg-Lin);TT3;N; 1 ,N 3 ,N 5 57. The composition of embodiment 55 or 56, wherein the compound is selected from the group consisting of: tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide; N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamide)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5); heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (Lipid 5); GL-HEPES-E3-E10-DS-3-E18-1; GL-HEPES-E3-E12-DS-4-E10; GL-HEPES-E3-E12-DS-3-E14; and combinations thereof.

[0307] Embodiment 58. The composition of embodiment 57, wherein the LNP further comprises a polyethylene glycol (PEG)-conjugated (PEGylated) lipid, a cholesterol-based lipid, and / or a helper lipid.

[0308] Embodiment 59. The composition of any one of embodiments 55 to 58, wherein the LNP comprises: - a cationic lipid in a molar ratio of 35% to 55%; - a polyethylene glycol (PEG)-conjugated (PEGylated) lipid in a molar ratio of 0.25% to 2.75%; - a cholesterol-based lipid in a molar ratio of 20% to 45%; and - a helper lipid in a molar ratio of 5% to 35%, all of the molar ratios being relative to the total lipid content of the LNP.

[0309] Embodiment 60. The composition of any one of embodiments 55 to 59, wherein the LNP comprises: - a 40% molar ratio of cationic lipid; - a 1.5% molar ratio of PEGylated lipid; - a 28.5% molar ratio of cholesterol-based lipid; and - a 30% molar ratio of helper lipid.

[0310] Embodiment 61. The composition of any one of embodiments 55 to 60, wherein the LNP comprises: - a molar ratio of 45 to 50% cationic lipid; - a molar ratio of 1.5 to 1.7% PEGylated lipid; - a molar ratio of 38 to 43% cholesterol-based lipid; and - a molar ratio of 9 to 10% helper lipid.

[0311] Embodiment 62. The composition of any one of embodiments 58 to 61, wherein the PEGylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000) or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0312] Embodiment 63 The composition of any one of embodiments 58-62, wherein the cholesterol-based lipid is cholesterol.

[0313] Embodiment 64. The composition of any one of embodiments 58 to 63, wherein the helper lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0314] Embodiment 65. The composition of any one of embodiments 55 to 64, wherein the LNP comprises: a cationic lipid selected from the group consisting of OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, and GL-HEPES-E3-E12-DS-3-E14 in a molar ratio of 40%; DMG-PEG2000 in a molar ratio of 1.5%; cholesterol in a molar ratio of 28.5%; and -DOPE in a molar ratio of 30%.

[0315] Embodiment 66. The composition of any one of embodiments 55 to 65, wherein the LNP comprises: - SM-102 in a molar ratio of 50%; - DMG-PEG2000 in a molar ratio of 1.5%; - cholesterol in a molar ratio of 38.5%; and - DSPC in a molar ratio of 10%.

[0316] Embodiment 67. The composition of any one of embodiments 55 to 65, wherein the LNP comprises: - ALC-0315 in a molar ratio of 46.3%; - ALC-0159 in a molar ratio of 1.6%; - cholesterol in a molar ratio of 42.7%; and - DSPC in a molar ratio of 9.4%.

[0317] Embodiment 68. The composition of any one of embodiments 55 to 65, wherein the LNP comprises: - ALC-0315 in a molar ratio of 47.4%; - ALC-0159 in a molar ratio of 1.7%; - cholesterol in a molar ratio of 40.9%; and - DSPC in a molar ratio of 10%.

[0318] Embodiment 69. The composition of any one of embodiments 55 to 68, wherein the LNPs have an average diameter of 30 nm to 200 nm.

[0319] Embodiment 70. The composition of any one of embodiments 55-68, wherein the LNPs have an average diameter of 80 nm to 150 nm.

[0320] Embodiment 71. The composition of any one of embodiments 55 to 70, comprising 1 mg / mL to 10 mg / mL of LNP.

[0321] Embodiment 72. The composition of any one of embodiments 55 to 71, wherein the LNP comprises 1 to 20 nucleic acid molecules, preferably mRNA molecules.

[0322] Embodiment 73. The composition of any one of embodiments 53-72, which is formulated for intramuscular, intranasal, intravenous, subcutaneous, or intradermal administration.

[0323] Embodiment 74. The composition of any one of embodiments 53 to 73, comprising phosphate buffered saline.

[0324] Embodiment 75. The composition of any one of embodiments 53 to 74, which is a pharmaceutical composition, such as an immunogenic composition or vaccine, in particular a Lyme disease vaccine.

[0325] Embodiment 76. The nucleic acid of any one of embodiments 1 to 52 or the composition of any one of embodiments 53 to 75 for use in inducing an immune response, preferably a humoral immune response, in a subject in need thereof and / or in treating or preventing Lyme disease in a subject in need thereof, wherein preferably the subject has, after administration of the nucleic acid or composition, a high serum concentration of antibodies to OspA relative to a subject administered a Lyme disease vaccine comprising an OspA recombinant protein vaccine, and / or preferably the subject is a mammal, more preferably a human, dog, cat, llama, cow, sheep, goat, horse, rodent, mouse, rat, rabbit, monkey, primate or pig, even more preferably a human.

[0326] Embodiment 77. A method of inducing an immune response, preferably a humoral immune response, in a subject in need thereof and / or a method of treating or preventing Lyme disease in a subject in need thereof, comprising administering to the subject an effective amount of a nucleic acid of any one of embodiments 1-52 or a composition of any one of embodiments 53-75, optionally intramuscularly, intranasally, intravenously, subcutaneously or intradermally, and preferably the subject has, after administration of the nucleic acid or composition, a high serum concentration of antibodies to OspA for subjects to which a Lyme disease vaccine, including an OspA recombinant protein vaccine, is administered, and / or preferably the subject is a mammal, more preferably a human, dog, cat, llama, cow, sheep, goat, horse, rodent, mouse, rat, rabbit, monkey, primate or pig, even more preferably a human.

[0327] Embodiment 78. Use of the nucleic acid of any one of embodiments 1 to 52 or the composition of any one of embodiments 53 to 75 for the manufacture of a medicament for use in inducing an immune response, preferably a humoral immune response, in a subject in need thereof and / or for use in the treatment or prevention of Lyme disease in a subject in need thereof, wherein preferably the subject has, after administration of the nucleic acid or composition, a high serum concentration of antibodies against OspA for a subject to which a Lyme disease vaccine, including an OspA recombinant protein vaccine, is administered, and / or preferably the subject is a mammal, more preferably a human, dog, cat, llama, cow, sheep, goat, horse, rodent, mouse, rat, rabbit, monkey, primate or pig, even more preferably a human.

[0328] In order that this disclosure may be better understood, the following examples are set forth. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the disclosure in any way. EXAMPLES

[0329] The foregoing description of specific embodiments fully reveals the general nature of the present disclosure, so that others, by applying knowledge within the skill of the art, can easily modify and / or adapt such specific embodiments to various applications without departing from the general concept of the present disclosure and without undue experimentation. Such adaptations and modifications are therefore intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It should be understood that the expressions or terms in this specification are intended to be descriptive rather than limiting, as the terms or terms in this specification would be interpreted by one of ordinary skill in the art in light of the teaching and guidance.

[0330] Example 1: Materials and Methods mRNA-OspA production mRNA was produced as previously published (Kalnin et al. (2021), NPJ Vaccines 6(1):61 and WO2021226436). Briefly, mRNA incorporating coding sequences containing either OspA ST1 or ST2 was synthesized by in vitro transcription with RNA polymerase using a plasmid DNA template encoding the desired gene using unmodified nucleotides. The resulting purified precursor mRNA was further reacted via enzymatic addition of a 5' cap structure (Cap1) and a 3' poly(A) tail of approximately 200 nucleotides in length as determined by gel electrophoresis.

[0331] For preparation of mRNA / lipid nanoparticle (LNP) formulations, an ethanolic solution of a mixture of lipids (cationic / ionizable lipids, phosphatidylethanolamine, cholesterol and polyethylene glycol-lipids) at a fixed lipid and mRNA ratio was combined with an aqueous buffer solution of target mRNA at acidic pH under controlled conditions to obtain a homogenous suspension of LNPs. Upon ultrafiltration and diafiltration into a suitable dilution series, the resulting nanoparticle suspension was diluted to the final concentration, filtered and stored frozen at -80°C until use.

[0332] Production of antigens used as benchmark comparisons OspA-ferritin ST1 and ST2 antigens were produced by the Sanofi Breakthrough Lab in Cambridge, MA, USA according to previously published materials and methods (Kamp et al. (2020), NPJ Vaccines 5(1):33).

[0333] For the OspA fusion ST1-ST2, the in-house plasmid pSP401+LPP-chimer OspA1-OspA2, which allows expression of the OspA fusion ST1-ST2 C-terminal domain, was introduced into the E. coli expression strain C43-(DE3) (Lucigen). After 2-3 hours of growth at 37°C in rich medium, expression of the protein of interest was induced by addition of inducer, and the culture was stopped 3 hours after induction. After processing the bacterial pellet, the proteins were visualized on Coomassie blue stained SDS-Page gels or by Western blot using specific antibodies. Scale-up was performed with the best expression conditions to produce sufficient biomass for purification. The bacterial pellet was treated with lysozyme to extract the membrane proteins. The OspA1-OspA2 fusion protein was then extracted with urea 2M+Triton X114 2%. After three incubation-centrifugation steps at 37°C, the lower phase was collected and subjected to Q Sepharose chromatography in the presence of Zwittergent 3.14 detergent (0.5%). The fraction eluted with 400 mM NaCl was subjected to ceramic hydroxyapatite chromatography. The OspA1-OspA2 fusion protein was eluted with Tween 0.05% PO4 NaNa2 180 mM pH 6.7 buffer and replaced with PBS+Tween20 0.05% pH 7.3 as the final buffer.

[0334] Antigen and mouse immunization OF-1 mice (Charles River) were randomized into immunization groups of 8 animals each. Four different doses of mRNA-OspA-LNP were administered intramuscularly (50 μL) on day 0 (D0) (Dose 1) and day 21 (D21) (Dose 2): 0.2 μg, 1 μg, 5 μg, or 10 μg. Serum was collected at baseline (D0), day 20 (D20), and day 35 (D35).

[0335] Two mRNA-OspA sequences were tested: mRNA-OspA-ST1-native and mRNA-OspA-ST2-native.

[0336] The mRNA formulation was compared to the negative control LNP alone and the benchmark Lyme canine vaccine RECOMBITEK® (Merial) at 1 μg / dose (50 μL).

[0337] OspA-specific IgG ELISA Antibody responses in mice were determined by ELISA. Briefly, 384-well microplates (Perkin Elmer #6007509) were coated with 1 μg / mL OspA-His of the determined serotype (ST1 or ST2) diluted in PBS and incubated overnight at 4° C. OspA-His was removed and the plates were blocked with 5% skim milk dissolved in PBS-tween. After removing the blocking reagent, primary serum samples were added after serial two-fold dilutions in 1% skim milk-PBS-Tween. After 1.5 hours of incubation at room temperature with the primary serum samples, the plates were washed with PBS-Tween and incubated with goat anti-mouse IgG-HRP (Jackson 115-036-062) for 1.5 hours at room temperature. The secondary antibody was aspirated and washed and the plates were incubated with TMB substrate (TEBU-TMB100-1000) followed by an equal volume of stop solution (HCl 1N). Absorbance was measured from 450 nm to 650 nm. OspA-specific IgG titers were quantified via an internal anti-OspA mouse serum standard, the titer of which was pre-calculated as the reciprocal of the dilution to give an OD of 1.

[0338] The mRNA encoding the OspA protein The causative agent of Lyme disease is a bacterium of the genus Borrelia. Four species from the genus Borrelia cause most human disease: B. burgdorferi, B. afzelii, B. garinii, and B. bavariensis. Each Borrelia species has surface expression of outer surface protein A (OspA), and seven OspA serotypes (ST1-ST7) are particularly prevalent in the United States and Europe, with ST1 accounting for approximately 98% of OspA serotypes in the United States, while ST2 accounts for more than 50% of OspA serotypes in Europe. To that end, mRNAs expressing either OspA ST1 or ST2 were engineered.

[0339] The OspA native sequence was used. Each of the OspA polypeptide sequences cited below lacks the N-terminal methionine, which is typically removed in eukaryotic cells. In certain embodiments, the amino acid sequence set forth in any one of SEQ ID NOs: 1-7 further comprises an N-terminal methionine amino acid.

[0340] Each nucleic acid cited in Table 3 corresponds to an mRNA sequence. The corresponding DNA sequence can be used as a template to generate mRNA via in vitro transcription. The DNA sequence is identical to the mRNA sequence, except for the substitution of U nucleotides in the mRNA sequence with T nucleotides.

[0341] The amino acid and nucleic acid sequences of the OspA protein and the nucleic acid sequence encoding it are set out in Tables 2 and 3 below (as well as the amino acid sequence and nucleic acid sequences of the 5'UTR and 3'UTR for the linker).

[0342] [Table 1]

[0343] [Table 2]

[0344] [Table 3]

[0345] [Table 4]

[0346] [Table 5]

[0347] [Table 6]

[0348] [Table 7]

[0349] Example 2: Immunogenicity of mRNA encoding OspA protein in mice The relative immunogenicity of the various OspA-expressing mRNAs was tested in mice by measuring IgG titers against OspA as described above in Example 1. Each mRNA was encapsulated in LNPs composed of 40% cationic lipid cKK-E10, 30% phospholipid DOPE, 1.5% PEGylated lipid DMGPEG2000, and 28.5% cholesterol. Alternatively, the LNP lipids can be cited as a ratio of 40:1.5:28.5:30 cationic lipid:PEGylated lipid:cholesterol:phospholipid.

[0350] Each LNP-mRNA composition was administered to mice at a dose of 0.2 μg, 1 μg, 5 μg, or 10 μg. In total, four groups with eight mice / group were used.

[0351] Three benchmark compositions were used: OspA fusion with AlOOH adjuvant ("OspA fusion ST1-ST2") (2 μg (1 μg per serotype) / dose); Lyme canine vaccine RECOMBITEK® (Merial) (1 μg dose), and OspA-ferritin fusion with AF03 adjuvant (ST1 or ST2) (1.7 μg (of which 1 μg OspA + 0.7 μg ferritin) / dose). OspA-ferritin fusions are further described in U.S. Patent Application Publication No. 20210017238A1, which is incorporated herein by reference.

[0352] As a negative control, mice were administered LNPs without mRNA.

[0353] As shown in Figure 1A, anti-OspA ST1 IgG titers increased 1 day post-administration (day 20).

[0354] As shown in Figure 1B, anti-OspA ST1 IgG titers further increased 2 days post-dose (day 35).

[0355] The LNP alone negative control induced non-specific IgG titers that remained low (not shown).

[0356] As shown in Figure 2A, anti-OspA ST2 IgG titers increased 1 day post-administration (day 20).

[0357] As shown in Figure 2B, anti-OspA ST2 IgG titers further increased 2 days post-dose (day 35).

[0358] Conclusions from mouse studies: The mRNA encoding OspA ST1 and ST2 was immunogenic and induced strong anti-OspA IgG titers in both post-dose 1 and post-dose 2 mice.

[0359] A dose effect was observed (i.e., increasing IgG titers with increasing dose).

[0360] The mRNA encoding OspA ST1 induced higher allogeneic IgG titers than the mRNA encoding OspA ST2.

[0361] IgG titers induced by mRNA-OspA ST1 were equivalent to or higher than those induced by the benchmark OspA fusions ST1-ST2 and Recombitek (ST1).

[0362] Other embodiments of the present disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the present disclosure being indicated by the following claims.

[0363] All patents and publications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A Lyme disease vaccine comprising a messenger RNA (mRNA) comprising an open reading frame (ORF) encoding at least one antigenic polypeptide derived from at least one bacterium of the genus Borrelia.

2. 2. The Lyme disease vaccine of claim 1, wherein the mRNA is formulated in a lipid nanoparticle (LNP).

3. 2. The Lyme disease vaccine of claim 1, wherein the mRNA is a non-replicating mRNA.

4. 3. The Lyme disease vaccine of claim 2, wherein the mRNA comprises at least one chemical modification.

5. 5. The Lyme disease vaccine of claim 4, wherein the bacterium of the genus Borrelia is selected from the species B. burgdorferi, afzelii, garinii, bavariensis, mayonii, spielmanii, lusitaniae, bissettii and / or valaisiana, or any strain or isolate thereof.

6. 3. The Lyme disease vaccine of claim 2, wherein the at least one antigenic polypeptide comprises at least one lipoprotein of the genus Borrelia.

7. 3. The Lyme disease vaccine of claim 2, wherein the at least one antigenic polypeptide or lipoprotein is OspA or a fragment or variant thereof.

8. 8. The Lyme disease vaccine of claim 7, wherein the OspA or fragment or variant thereof comprises at least five amino acids.

9. The OspA or a fragment or variant thereof is OspA serotype (ST) 1, 2, 3 8. The Lyme disease vaccine of claim 7, derived from serogroups 1, 2, 3, 4, 5, 6, and / or 7.

10. The OspA or a fragment or variant thereof is effective against serotype 1 Borrelia burgdorferi strain B31, serotype 2 Borrelia afzelii strain PKO, serotype 3 Borrelia garinii strain PBr, serotype 4 Borrelia bavariensis, serotype 5 Borrelia garinii, serotype 6 Borrelia garinii, and the like.

8. The Lyme disease vaccine of claim 7, derived from Borrelia garinii, or serotype 7 Borrelia garinii.

11. 8. The Lyme disease vaccine of claim 7, wherein the at least one OspA polypeptide comprises an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 1-7.

12. 3. The Lyme disease vaccine of claim 2, wherein the mRNA comprises a nucleotide sequence that is at least 85% identical to any one of SEQ ID NOs: 10-13 and 16-19.

13. 3. The Lyme disease vaccine of claim 2, wherein the mRNA encodes at least two different OspA serotypes or fragments or variants thereof.

14. 14. The Lyme disease vaccine of claim 13, wherein the OspA or fragment or variant thereof of one serotype, preferably comprising at least five amino acids, is fused to an OspA or fragment or variant thereof of a different serotype, preferably comprising at least five amino acids.

15. 15. The Lyme disease vaccine of claim 14, wherein the fused OspAs or fragments or variants thereof of different serotypes are separated by a linker sequence.

16. 16. The Lyme disease vaccine of claim 15, wherein the linker sequence is derived from P66 or comprises an amino acid sequence having at least 85% identity to SEQ ID NO:8 or to SEQ ID NO:

9.

17. 3. The Lyme disease vaccine of claim 2, wherein the mRNA comprises at least one chemical modification.

18. 18. The Lyme disease vaccine of claim 17, wherein the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytidine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

19. 19. The Lyme disease vaccine of claim 18, wherein the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 5-methylcytidine, 5-methoxyuridine, and combinations thereof.

20. 20. The Lyme disease vaccine of claim 19, wherein the chemical modification is N1-methylpseudouridine.

21. 3. The Lyme disease vaccine of claim 2, wherein the LNP comprises at least one cationic lipid.

22. 3. The Lyme disease vaccine of claim 2, wherein the cationic lipid is biodegradable or the cationic lipid is not biodegradable.

23. 23. The Lyme disease vaccine of claim 22, wherein the cationic lipid is cleavable or the cationic lipid is not cleavable.

24. 3. The Lyme disease vaccine of claim 2, wherein the cationic lipid is selected from the group consisting of ML7 / OF-02; cKK-E10; GL-HEPES-E3-E10-DS-3-E18-1; GL-HEPES-E3-E12-DS-4-E10; GL-HEPES-E3-E12-DS-3-E14; 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102); and (4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315).

25. 22. The Lyme disease vaccine of claim 21, wherein the LNP further comprises a polyethylene glycol (PEG) conjugated (PEGylated) lipid.

26. 26. The Lyme disease vaccine of claim 25, wherein the PEGylated lipid comprises dimyristoyl-PEG2000 (DMG-PEG2000) or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

27. 22. The Lyme disease vaccine of claim 21, wherein the LNP further comprises a cholesterol-based lipid, optionally wherein the cholesterol-based lipid comprises cholesterol.

28. 22. The Lyme disease vaccine of claim 21, wherein the LNP further comprises a helper lipid, optionally wherein the helper lipid comprises 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

29. The LNP is a molar ratio of 35% to 55% cationic lipid; polyethylene glycol (PEG)-conjugated (PEGylated) lipids at a molar ratio of 0.25% to 2.75%; Cholesterol-based lipids in a molar ratio of 20% to 45%, and Helper lipids at a molar ratio of 5% to 35% Including, 22. The Lyme disease vaccine of claim 21, wherein all of said molar ratios are relative to the total lipid content of said LNP.

30. The LNP is 40% molar ratio of cationic lipid, PEGylated lipid at a molar ratio of 1.5%, Cholesterol-based lipids at a molar ratio of 28.5%, and 30% molar ratio of helper lipid 30. The Lyme disease vaccine of claim 29, comprising:

31. The LNP is cKK-E10 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, Cholesterol at a molar ratio of 28.5%, and 30% molar ratio of DOPE 30. The Lyme disease vaccine of claim 29, comprising:

32. 3. The Lyme disease vaccine of claim 2, wherein the LNPs have an average diameter of 30 to 200 nm.

33. 33. The Lyme disease vaccine of claim 32, wherein the LNPs have an average diameter of 80 to 150 nm.

34. 3. The Lyme disease vaccine of claim 2, wherein the mRNA comprises at least one 5' cap, at least one 5' untranslated region (5'UTR), at least one 3' untranslated region (3'UTR), and / or at least one polyadenylation (polyA) sequence.

35. 3. The Lyme disease vaccine of claim 2, wherein the mRNA comprises at least one 5' untranslated region (5'UTR), at least one 3' untranslated region (3'UTR), and at least one polyadenylation (polyA) sequence.

36. The mRNA contains at least the following structural elements: (i) a molecule having the following structure: 【Chemistry 1】 a 5' cap having (ii) a 5' untranslated region (5'UTR) having the nucleic acid sequence of SEQ ID NO: 14; (iii) at least one OspA open reading frame having any one of the nucleic acid sequences of SEQ ID NOs: 16-19; (iv) a 3' untranslated region (3'UTR) having the nucleic acid sequence of SEQ ID NO: 15; and (v) polyA tail 3. The Lyme disease vaccine of claim 2, comprising:

37. The present invention relates to a method for producing a Borrelia virus comprising administering to a mammalian animal or mammalian cell line comprising administering to said mammalian animal or mammalian cell line a method for producing a Borrelia virus ... (i) a molecule having the following structure: 【Chemistry 2】 a 5' cap having (ii) a 5' untranslated region (5'UTR) having the nucleic acid sequence of SEQ ID NO: 14; (iii) at least one OspA open reading frame having any one of the nucleic acid sequences of SEQ ID NOs: 16-19; (iv) a 3' untranslated region (3'UTR) having the nucleic acid sequence of SEQ ID NO: 15; and (v) polyA tail Including, The mRNA is cKK-E10 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, Cholesterol at a molar ratio of 28.5%, and 30% molar ratio of DOPE 1. A Lyme disease vaccine formulated in lipid nanoparticles (LNPs) comprising:

38. 3. The Lyme disease vaccine of claim 2 for use in eliciting an immune response in a subject.

39. 39. The Lyme disease vaccine of claim 38, wherein the immune response is a humoral immune response.

40. 3. The Lyme disease vaccine of claim 2 for use in treating or preventing Lyme disease in a subject in need thereof.

41. 39. The Lyme disease vaccine of claim 38, wherein the subject has a higher serum concentration of antibodies to OspA after administration of the Lyme disease vaccine relative to subjects administered a Lyme disease vaccine comprising an OspA recombinant protein vaccine.

42. 39. The Lyme disease vaccine of claim 38, wherein the subject is a mammal.

43. The subject is a human, dog, cat, llama, cow, sheep, goat, horse, rodent, mouse, rat, rabbit, monkey, primate or pig, more preferably a human.

39. The Lyme disease vaccine of claim 38.

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