Self-amplifying RNA vaccine compositions and methods for prevention and treatment of LYME disease

Abstract

Provided herein are monovalent and multivalent self-amplifying RNA (saRNA) constructs encoding one or more Borrelia antigens, including antigens from distinct genospecies, and pharmaceutical compositions comprising the same. Also provided are methods of using such saRNA constructs and pharmaceutical compositions, and methods of manufacturing such compositions.

Description

[0001] Attorney Docket No.: KEYL-001WO

[0002] SELF-AMPLIFYING RNA VACCINE COMPOSITIONS AND METHODS FOR PREVENTION AND TREATMENT OF LYME DISEASE

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004]

[0001] This application claims the benefit of and priority to U. S. Provisional Patent Application No. 63 / 734,348, filed on December 16, 2024, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

[0005] SEQUENCE LISTING

[0006]

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on December 11, 2025, is named KEYL-001WO_SL.xml and is 123,775 bytes in size.

[0007] FIELD OF THE INVENTION

[0008]

[0003] The present disclosure relates to compositions and methods for preventing and treating Lyme disease. More particularly, the disclosure provides monovalent and multivalent selfamplifying RNA (saRNA) constructs encoding one or more Borrelia antigens, including antigens from distinct genospecies, and pharmaceutical compositions comprising the same, as well as methods for their use and manufacture. In certain embodiments, the disclosed compositions and methods of use enable induction of protective immunity against Lyme disease using a single RNA construct.

[0009] BACKGROUND

[0010]

[0004] Lyme disease is the most common vector-borne disease in the United States (Pine et al., 2023, PMID: 37533256). It is caused by spirochete bacteria of the Borrelia burgdorferi sensu lato group when a tick bite results in transmission of the bacteria to a host. Symptoms include flu-like illness, skin rashes, and may lead to downstream complications involving the joints, nervous system, or heart if treated late (Pine et al., 2023, PMID: 37533256).

[0011]

[0005] Borrelia burgdorferi sensu lato now comprises twenty (20) different genospecies. Of these, three species, Borrelia burgdorferi, Borrelia afzelii, and Borrelia garinii, are known to be primarily responsible for Lyme disease in humans. Geographic variation in the prevalence of these genospecies complicates the development of broadly protective vaccines. Attorney Docket No.: KEYL-001WO

[0012]

[0006] Prior vaccine strategies have frequently targeted outer surface proteins such as OspA or OspC; however, achieving broad strain coverage and durable antibody responses has proven challenging.

[0013]

[0007] There remains a need for novel strategies for the prevention and / or treatment of Lyme disease.

[0014] SUMMARY

[0015]

[0008] Described herein are compositions and methods for the prevention and / or treatment of Lyme disease, and more particularly, self-amplifying RNA (saRNA) constructs encoding one or more / Lnvc / m-de rived antigens, as well as pharmaceutical composition comprising the same. Also disclosed are methods of manufacturing and using such compositions.

[0016]

[0009] In one aspect, a self-amplifying RNA construct is provided comprising: (a) a 5’ cap structure, (b) a nucleic acid sequence encoding, 5’ to 3’: (i) a nucleotide sequence encoding nonstructural proteins nspl-nsp4 or functional variants thereof; (ii) at least one subgenomic promoter (SGP); (iii) a nucleotide sequence encoding one or more antigens (e.g., lipid antigens) derived from a Borrelia species operably linked to the subgenomic promoter, wherein optionally, the one or more antigens are engineered to comprise a transmembrane domain; and (iv) a poly(A) tail.

[0017]

[0010] In certain embodiments, the saRNA is configured to support sustained in vivo antigen expression.

[0018]

[0011] The one or more antigens (e.g., lipid antigens) may vary. In one embodiment, the one or more antigens are selected from the group consisting of outer surface protein A (OspA), outer surface protein B (OspB), outer surface protein C (OspC), outer surface protein D (OspD), outer surface protein E (OspE), outer surface protein F (OspF), decorin binding protein A (DbpA), decorin binding protein B (DbpB), Erp proteins, VlsE, Ag45, P66 protein, BmpA (P39), CspA, BB0405, BptA, P13, RevA, RevB, Lmpl, BBK07, BBK12, or combinations, fragments, variants or derivatives thereof.

[0019]

[0012] The Borrelia species may vary. In one embodiment, the Borrelia species is selected from the group consisting of Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii, Borrelia mayonii, Borrelia spielmanii, Borrelia bavariensis, Borrelia lusitaniae or combinations thereof.

[0020]

[0013] In a particular embodiment, the Borrelia species is Borrelia burgdorferi.

[0021]

[0014] In one embodiment, the at least one antigen comprises outer surface protein A (OspA) or a fragment, variant, or a derivative thereof. In a particular embodiment, the at least one Attorney Docket No.: KEYL-001WO

[0022] bacterial antigen, fragment or derivative thereof is derived from OspA serotype 1 (Borrelia burgdorferi).

[0023]

[0015] In one embodiment, the at least one bacterial antigen comprises outer surface protein B (OspB) or a fragment, variant, or derivative thereof.

[0024]

[0016] In one embodiment, the at least one bacterial antigen comprises outer surface protein C (OspC) or a fragment, variant, or derivative thereof.

[0025]

[0017] In one embodiment, the at least one bacterial antigen comprises outer surface protein D, E, or F or a fragment, variant, or derivative thereof.

[0026]

[0018] In one embodiment, the at least one bacterial antigen comprises decorin binding protein A (DbpA) or a fragment, variant, or derivative thereof.

[0027]

[0019] In one embodiment, the at least one bacterial antigen comprises decorin binding protein B (DbpB) or a fragment, variant, or derivative thereof.

[0028]

[0020] In one embodiment, the sequence of the at least one antigen sequence is codon-optimized. The codon optimization may be selected from a C maximized coding sequence, CAI maximized coding sequence, a human codon usage adapted coding sequence, G / C content modified coding sequence, a G / C optimized coding sequence, a lowest mean free energy (MFE) RNA structure (e.g. LinearDesign)or any combination thereof.

[0029]

[0021] In one embodiment, the at least one antigen is chimeric, comprising joined or fused domains from two or more Borrelia antigens. In a particular embodiment, the chimeric antigen comprises portions selected to preserve key immunogenic epitopes and may include sequences derived from the same or different Borrelia species. In a particular embodiment, the chimeric antigen comprises consensus or COBRA (Computationally Optimized Broadly Reactive Antigen) design features, in which sequences from two or more Borrelia antigens or strains are computationally aligned and combined to preserve conserved immunogenic epitopes while minimizing sequence regions associated with strain-specific variability.

[0030]

[0022] The self-replicating RNA virus from which the nonstructural proteins, subgenomic promoters and UTRs are derived from may vary. In one embodiment, the self-replicating RNA virus is a positive-sense single-stranded RNA virus.

[0031]

[0023] In a particular embodiment, the self- replicating RNA virus is an alphavirus.

[0032]

[0024] In another particular embodiment, the self-replicating RNA virus is selected from the group consisting of: Venezuelan Equine Encephalitis Virus (VEEV), Semliki Forest Virus (SFV), Sindbis Virus (SIN), Chikungunya Virus (CHIKV), Eastern Equine Encephalitis Virus (EEEV), Mayaro Virus (MAYV), Getah Virus (GETV), Ross River Virus (RRV), Una Virus (UNAV), Middleburg Virus (MIDV), O’nyong nyong virus (ONNV), Barmah Forest Virus Attorney Docket No.: KEYL-001WO

[0033] (BFV), Mucambo Virus (MUCV), Tonate Virus (TONV), Everglades Virus (EVEV), Rio Negro Virus (RNV), Highlands J Virus (HJV), Western Equine Encephalitis Virus (WEEV), and Aura Virus (AURAV), or engineered variants thereof.

[0034]

[0025] In one embodiment, the nucleic acid sequence further encodes an adjuvant. The adjuvant may vary. In one embodiment, the adjuvant is interleukin- 12 (IL-12), In certain embodiments, the IL- 12 is expressed as a single-chain or heterodimeric form, and may be engineered to enhance stability, secretion, or localization. In some embodiments, the IL- 12 comprises one or more mutations to increase half-life or reduce off target immune activation. In certain embodiments, the IL-12 is an IL-12 fusion protein, e.g., comprising a domain that extends half-life (e.g., an Fc domain, albumin domain, Fc binding domain, albumin-binding domain, transferrin domain, hybrid cytokine fusion such as IL-12 / IL-15) or permits targeting (e.g., an antibody or antibody fragment or to a localizing domain such as a transmembrane domain). In one embodiment, the encoded IL-12 adjuvant is membrane-tethered following expression from the construct, i.e., IL- 12 is anchored to the cell surface rather than secreted. In certain embodiments, membrane tethering is achieved by fusion to a transmembrane domain, signal peptide, and / or GPI-anchor sequence, which localizes IL- 12 activity to the site of antigen expression and reduces systemic cytokine exposure. In one embodiment, the nucleotide sequence comprises (i) a sequence encoding IL-12B (p40) that is operably linked to a sequence encoding IL-12A (p35) through a linker domain (e.g., a GGGS repeat linker (SEQ ID NO: 12)) and (ii) a sequence encoding IL-12A operably linked through a linker domain to a sequence encoding a transmembrane domain (TMD), such that expression of the sequences provides a single-chain IL- 12 polypeptide that is membrane-anchored through a C-terminal transmembrane domain (TMD). In certain embodiments, the domain order is: signal peptide -IL-12B - linker - IL-12A - linker - TMD. In certain embodiments, the sa-RNA construct is engineered to reduce ectodomain shedding and to maintain surface-associated cytokine, thereby increasing localized cytokine signaling relative to soluble cytokine expression.

[0035]

[0026] The nucleotide sequence may vary. In one embodiment, the nucleic acid sequence is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or a variant thereof.

[0036]

[0027] In a particular embodiment, the saRNA sequence is transcribed from the DNA template encoding SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or a variant thereof. Attorney Docket No.: KEYL-001WO

[0037]

[0028] In one embodiment, the variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to SEQ ID NO: 1, including codon-optimized or chemically modified nucleotide variants. Sequence identity can be determined by sequence alignment programs known to those skilled in the art.

[0038]

[0029] In one embodiment, the variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to SEQ ID NO: 2, including codon-optimized or chemically modified nucleotide variants. Sequence identity can be determined by sequence alignment programs known to those skilled in the art

[0039]

[0030] In one embodiment, the nucleic acid sequence encodes two or more Borrelia antigens, i.e., a multivalent saRNA.

[0040]

[0031] The two or more Borrelia antigens may vary. In one embodiment, the two or more Borrelia antigens are selected from the group consisting of outer surface protein A (OspA), outer surface protein B (OspB), outer surface protein C (OspC), outer surface protein D (OspD), outer surface protein E (OspE), outer surface protein F (OspF), decorin binding protein A (DbpA), decorin binding protein B (DbpB), Erp proteins, VlsE, Ag45, P66 protein, BmpA (P39), CspA, BB0405, BptA, P13, RevA, RevB, Lmpl, BBK07, BBK12, or any combinations, fragments, variants or derivatives thereof.

[0041]

[0032] In a particular embodiment, the two or more Borrelia antigens are selected from the group consisting of OspA, OspC, DbpA, DbpB, VlsE, or any combination, fragments, variants or derivatives thereof.

[0042]

[0033] In a particular embodiment, the two or more Borrelia antigens are OspA, OspC or fragments, variants or derivatives thereof. In one embodiment, the OspA and OspC or fragments thereof are derived from Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii, Borrelia mayonii, Borrelia spielmanii, Borrelia bavariensis, Borrelia lusitaniae or combinations thereof. In another particular embodiment, the OspA and OspC are derived from Borrelia burgdorferi or variants thereof.

[0043]

[0034] In one embodiment, the two or more antigens are derived from different Borrelia species wherein the antigens may be identical or distinct, to broaden protective coverage across geographic strain variation.

[0044]

[0035] In one embodiment, the two or more antigens derived from different strains of the same Borrelia species, optionally selected to cover known antigenic diversity within that species.

[0045]

[0036] In certain embodiments, the sequence of the at least one of the at least two antigens is codon optimized.

[0046]

[0037] In certain embodiments, at least one of the at least two antigens is chimeric. Attorney Docket No.: KEYL-001WO

[0047]

[0038] The construct may comprise one or more regulatory elements. In one embodiment, the first antigen is operably linked to the nucleotide encoding the second antigen by a nucleotide sequence selected from the group consisting of a 2A sequence, an IRES sequence or a sequence encoding a second subgenomic promoter.

[0048]

[0039] In a particular embodiment, the two or more antigens are separated by a 2A peptide (for example, P2A), wherein the 2A peptide is encoded within the same open reading frame (ORF) as the two or more antigens. In a particular embodiment, the saRNA sequence further encodes a non-limiting furin cleavage site upstream of the 2A peptide.

[0049]

[0040] In a particular embodiment, the two or more antigens are separated by ribosomal 2A elements, within the same open reading frame (ORF).

[0050]

[0041] In one embodiment, two or more antigens are separated by IRES sequences, including viral or synthetic internal ribosome entry elements.

[0051]

[0042] In certain embodiments, the internal ribosome entry site (IRES) is positioned upstream of the at least one antigen to enable polycistronic expression.

[0052]

[0043] In one embodiment, the two or more antigens are separated by subgenomic promoters.

[0053]

[0044] In a particular embodiment, the self-amplifying RNA encodes two or more subgenomic promoters, each operably linked to a distinct antigen coding region, such that each antigen is transcribed as an independent subgenomic RNA.

[0054]

[0045] In one embodiment, the saRNA sequence further encodes at least one transmembrane domain linked to the at least one of the two antigens.

[0055]

[0046] In one embodiment, the nucleic acid sequence encodes three or more Borrelia antigens.

[0056]

[0047] The three or more Borrelia antigens may vary. In one embodiment, the three or more Borrelia antigens are selected from the group consisting of outer surface protein A (OspA), outer surface protein B (OspB), outer surface protein C (OspC), outer surface protein D (OspD), outer surface protein E (OspE), outer surface protein F (OspF), decorin binding protein A (DbpA), decorin binding protein B (DbpB), Erp proteins, VlsE, Ag45, P66 protein, BmpA (P39), CspA, BB0405, BptA, P13, RevA, RevB, Lmpl, BBK07, BBK12, or any combinations thereof.

[0057]

[0048] In one embodiment, the three or more antigens are derived from different Borrelia species wherein the antigens may be identical or distinct, to broaden protective coverage across geographic strain variation.

[0058]

[0049] In one embodiment, the three or more antigens derived from different strains of the same Borrelia species, optionally selected to cover known antigenic diversity within that species. Attorney Docket No.: KEYL-001WO

[0059]

[0050] In certain embodiments, at least one of the three or more antigens is codon optimized.

[0060]

[0051] In certain embodiments, at least one of the three or more antigens is chimeric.

[0061]

[0052] In one embodiment, the nucleic acid sequence selected from the group consisting of a 2A sequence, an IRES sequence or a sequence encoding a second or third subgenomic promoter operably links (i) the nucleotide sequence encoding the first and second antigen and (ii) the nucleotide sequence encoding the second and third antigen.

[0062]

[0053] In certain embodiments, the least one antigen of the self-amplifying RNA constructs disclosed herein is engineered to comprise a transmembrane domain, for example to increase cell-surface presentation and support sustained antigen exposure to the immune system. In a particular embodiment, the nucleotide sequence encoding the at least one antigen is operably linked to the nucleic acid sequence encoding a transmembrane domain. Put another way, the saRNA construct encodes a fusion protein comprising the antigen and the transmembrane domain.

[0063]

[0054] The transmembrane domain may vary. In one embodiment, the transmembrane domain is selected from the group consisting of domains derived from Hemagglutinin (HA), SARS-CoV-2 Spike, ICAM-1, CD8 alpha, CD4, CD28, platelet derived growth factor receptor (PDGFR), T-cell receptor alpha, MHC class I, B7-1 / CD80, GP64, the vesicular stomatitis virus glycoprotein (VSV-G), human erythropoietin receptor, IL-2 receptor alpha, ICOS, 4-1BB (CD137), 0X40 (CD134), Fas, HVEM (TNFRSF14), CD3 zeta, CTLA-4, CD27, SLAMF7, CD2, CD19, CD22. CD33, CD44, CD74, CD99, CD150, CD200, CCR5, CXCR4, CD40, CD83, Notch, LFA-1 (GDI la), integrin beta-1 (ITGB1), integrin alpha-L (ITGAL), integrin alpha-V (ITGAV), transferrin receptor (CD71), E-cadherin, N-cadherin, CD7, CD10, CD45, CD123, CCR7, CX3CR1, CD47, SIRP-alpha, TROP2, TNFR1, TNFR2, RANK (TNFRSF11 A), CD137L (4-1BBL), LIGHT (TNFSF14), TRAILR1 (DR4), TRAILR2 (DR5), TLR2, TLR4, TLR9, CR2 (CD21), CD79a, CD79b, CD86, CD23, Fc receptor gamma (FcRy), or CD18.

[0064]

[0055] In certain embodiments, the at least one antigen of the self-amplifying RNA constructs disclosed herein is one antigen comprises a signal peptide sequence derived from a protein of human or mouse origin. In a particular embodiment, the signal peptide is derived from human tissue plasminogen activator (tPA) signal peptide, human interleukin-2 (IL-2) signal peptide, human immunoglobulin kappa (IgK) light chain signal peptide, mouse immunoglobulin heavy chain signal peptide, Hemagglutinin (HA), SARS-CoV-2 Spike, or CD8a signal peptide. Attorney Docket No.: KEYL-001WO

[0065]

[0056] The replicase of the self-amplifying RNA construct disclosed herein may vary. In one embodiment, the replicase comprises four nonstructural proteins, nsPl through nsP4, which together form an RNA-dependent RNA polymerase (RdRp) complex.

[0066]

[0057] In certain embodiments, the replicase is derived from an alphavirus, such as Venezuelan equine encephalitis virus (VEEV), Semliki Forest vims (SFV), or Sindbis vims (SINV), and enables intracellular amplification of the RNA, thereby supporting sustained antigen expression.

[0067]

[0058] In certain embodiments, the replicase is derived from an alphavirus, such as Venezuelan equine encephalitis virus (VEEV), Semliki Forest vims (SFV), or Sindbis vims (SINV), and enables intracellular amplification of the RNA, thereby supporting sustained antigen expression.

[0068]

[0059] In one embodiment, one or more of the 5' UTR, 3' UTR, replicase coding region, and / or subgenomic promoter (SGP) sequences of the self-amplifying RNA constmct disclosed herein are derived from a positive-sense single-stranded RNA vims, such as an alphavims.

[0069]

[0060] In one embodiment, the saRNA sequence of the self-amplifying RNA contrast disclosed herein comprises one or more optional chemically modified nucleotides. The chemical modifications may vary. In a particular embodiment, the chemical modifications are selected from the group consisting of 5-hydroxymethylcytidine, 5 -methylcytidine, 5-fluorocytidine, 5-hydroxymethyluridine, 5-methyluridine or combinations thereof. In a particular embodiment, between about 10% and 100% of cytidines and / or uridines are modified.

[0070]

[0061] In certain embodiments, the nucleic acid sequence of the self-amplifying RNA further comprises at least one 5’ untranslated region (UTR) positioned 5’ to the nucleotide sequence encoding nonstmctural proteins nspl-nsp4 or functional variants thereof. In one embodiment, the 5’ UTR is derived from a 5’ UTR of a self-replicating RNA virus. In another embodiment, the 5’ UTR is functionally equivalent to a 5’ UTR of a self-replicating RNA virus.

[0071]

[0062] In certain embodiments, the nucleic acid sequence of the self-amplifying RNA further comprises at least one 3’ untranslated region (UTR) positioned between the nucleotide sequence encoding one or more antigens derived from a Borrelia species and the poly(A) tail. In one embodiment, the 3’ UTR is derived from a 3’ UTR of a self-replicating RNA virus. In another embodiment, the 3’ UTR is functionally equivalent to a 3’ UTR of a self-replicating RNA virus. Attorney Docket No.: KEYL-001WO

[0072]

[0063] In a second aspect, a plasmid is provided comprising a nucleotide sequence corresponding to the nucleic acid sequence of the self-amplifying RNA construct disclosed herein.

[0073]

[0064] In a third aspect, a pharmaceutical composition is provided comprising the selfamplifying RNA construct or plasmid disclosed herein and at least one excipient or carrier, such as at least one pharmaceutically acceptable excipient or carrier.

[0074]

[0065] In one embodiment, the at least one excipient is selected from the group consisting of buffers, stabilizers, cryoprotectants, tonicity agents, salts, sugars, or other formulation excipients, and is configured to preserve saRNA integrity and in vivo expression activity. In certain embodiments, the excipient system is optimized to minimize chemical and physical degradation pathways of the saRNA, including hydrolysis, oxidation, depurination, and mechanical or thermal stress-induced fragmentation. For example, cryoprotectants and stabilizers may be included in amounts effective to prevent or significantly reduce RNA degradation during freeze-thaw cycles, lyophilization, refrigerated storage, or elevatedtemperature excursions. Buffers and salts can be selected to maintain pH values that favor maximal structural integrity of the saRNA and prevent acid- or base-catalyzed cleavage. In additional embodiments, the excipients are formulated to improve or preserve the functional performance of the lipid nanoparticle (LNP) delivery system. Such excipients may enhance LNP colloidal stability, control particle size distribution, prevent aggregation or fusion during storage, and maintain the physicochemical characteristics required for efficient endosomal escape and robust in vivo expression of the saRNA payload. Sugars, polyols, amino acids, and polymeric stabilizers can, for example, be added to support LNP membrane packing, preserve bilayer integrity upon freezing or drying, and maintain optimal release kinetics following administration. In certain embodiments, the excipient composition is selected to synergistically enhance translational efficiency, biodistribution, and overall potency of the saRNA-LNP formulation.

[0075]

[0066] In one embodiment, the pharmaceutical composition comprises two self-amplifying RNA constructs disclosed herein, wherein the sequence of the first and second self-amplifying RNA constructs are different. In a particular embodiment, the first and second self-amplifying RNA constructs encode at least one different antigen.

[0076]

[0067] In certain embodiments, the self-amplifying RNA construct is provided in association with a delivery system, comprises a delivery system, such as a viral delivery system or a non-viral delivery system, including but not limited to lipid nanoparticles (LNPs), liposomes, polymeric nanoparticles, emulsions, virus-like particles (VLPs), or protein- or peptide-based Attorney Docket No.: KEYL-001WO

[0077] carrier systems, configured to enhance cellular uptake and in vivo expression of the saRNA. In certain embodiments, LNPs are engineered to optimize physicochemical parameters such as particle size, polydispersity, surface charge, encapsulation efficiency, and ionizable lipid pKa to maximize intracellular delivery and amplify translational output of the saRNA. Liposomal or polymeric carriers may similarly be modified to enhance membrane fusion or endosomal destabilization. Viral delivery systems, such as alphavirus replicon particles or other replication-defective vectors, may also be used to deliver saRNA constructs while maintaining controlled expression and favorable safety profiles. In additional embodiments, virus-like particles (VLPs) or engineered protein cages facilitate targeted delivery by presenting surface ligands, receptor-binding motifs, or cell-penetrating peptides that increase uptake by relevant cell types or tissues. Any such delivery system may further be designed to modulate biodistribution, circulation time, immune activation, or tissue-specific delivery to achieve robust and sustained in vivo amplification and expression of the saRNA payload.

[0078]

[0068] In one embodiment, the delivery system comprises a lipid nanoparticle (LNP). The LNP may comprise an ionizable lipid, a cationic lipid, or mixtures thereof, cholesterol or a cholesterol derivative, a phospholipid, and a polyethylene glycol (PEG)-lipid, or functional equivalents, analogs, derivatives, or substitutes thereof. In certain embodiments, the sterol component may further comprise cholesterol mimics or sterol analogs, including sitosterol (flsitosterol), campesterol, stigmasterol, brassicasterol, ergosterol, desmosterol, lanosterol, dihydrocholesterol, cholesteryl hemisuccinate, cholesteryl oleate, cholesteryl stearate, cholesteryl carbonate, cholesteryl sulfate, cholesteryl ethers, hydrogenated sterols, oxidized sterols, sterol esters, or synthetic sterol analogs engineered to modulate membrane rigidity, packing density, fusogenicity, or structural stability. The LNP may additionally comprise helper lipids, stabilizing lipids, biodegradable lipids, adjuvant lipids, targeting lipids, or modified PEG-lipids configured to influence circulation time, complement activation, or cellular uptake. The ionizable or cationic lipid may be selected to facilitate complexation with the saRNA under acidic conditions while remaining substantially neutral at physiological pH to reduce reactogenicity, and may include pH-responsive, protonatable, permanently cationic, or biodegradable lipids capable of promoting endosomal destabilization and cytosolic release. Phospholipids such as DSPC, DOPE, DOPC, synthetic phosphatidylcholines, or functional equivalents may contribute to bilayer structure, membrane fusion, or colloidal stability, while PEG-lipids of various chain lengths and linker chemistries — including cleavable or slowly detachable PEG species, or zwitterionic polymer-lipids — may control pharmacokinetics, aggregation resistance, and biodistribution. In certain embodiments, the LNP further comprises Attorney Docket No.: KEYL-001WO

[0079] polymer-lipid hybrids, ligand-functionalized lipids, ionizable or cationic sterols, fusogenic or pH-responsive helper lipids, or immunomodulatory lipids that adjust immune sensing or reduce inflammatory responses. The self-amplifying RNA construct may be fully encapsulated, partially encapsulated, surface-associated, adsorbed, or otherwise integrated within or onto the LNP, which may itself adopt monolamellar, multilamellar, core-shell, hybrid, or compartmentalized architectures to improve loading, stability, manufacturability, and in vivo expression potency. Any such lipid composition or combination thereof may be selected or tuned to protect the saRNA from degradation, promote endosomal escape, minimize reactogenicity, optimize biodistribution, and enhance the magnitude and duration of in vivo expression. In a fourth aspect, a method of eliciting an immune response is provided, comprising administering a self-amplifying RNA construct, plasmid or pharmaceutical composition disclosed herein to a subject, thereby eliciting an immune response.

[0080]

[0069] In one embodiment, the method comprises administering two or more self-amplifying RNA constructs to the subject, wherein the nucleotide sequences of the two or more constructs are different, and the administration is sequential.

[0081]

[0070] In a fifth aspect, a method of preventing Lyme disease is provided, comprising administering a self-amplifying RNA construct, plasmid or pharmaceutical composition disclosed herein to subject in need thereof, thereby preventing Lyme disease.

[0082]

[0071] In one embodiment, the method comprises administering two or more self-amplifying RNA, wherein the nucleotide sequences of the two or more constructs are different and wherein the administration is sequential.

[0083]

[0072] In a sixth aspect, a method of treating Lyme disease is disclosed, comprising administering a self-amplifying RNA construct, the plasmid or pharmaceutical composition disclosed herein to a subject in need thereof, thereby treating Lyme disease.

[0084]

[0073] In one embodiment, the method comprises administering two or more self-amplifying RNA, wherein the nucleotide sequences of the two or more constructs are different and wherein the administration is sequential.

[0085]

[0074] The subject may vary. In one embodiment, the subject has been diagnosed with Stage 1, 2 or 3 Lyme disease.

[0086]

[0075] In one embodiment, treatment comprises a reduction or elimination of one or more symptoms of Lyme disease.

[0087]

[0076] In one embodiment, treatment comprises an eradication of persistent infection.

[0088]

[0077] In certain embodiments, the pharmaceutical composition disclosed herein comprises a lipid nanoparticle (LNP) formulation. Attorney Docket No.: KEYL-001WO

[0089]

[0078] In certain embodiments, the self-amplifying RNA construct or pharmaceutical composition is disposed within or on a microneedle patch.

[0090]

[0079] In a seventh aspect, a method of producing the self-amplifying RNA construct of any of claims 1-22 comprising: (a) synthesizing the RNA using in vitro transcription (IVT) from a linearized DNA template; (b) capping the RNA co-transcriptionally or post-transcriptionally with a cap analog; and (c) purifying the RNA using chromatography techniques to remove contaminants.

[0091] BRIEF DESCRIPTION OF THE DRAWINGS

[0092]

[0080] While OspA and OspC are depicted in the illustrative embodiments of FIGS. 1-6, the self-amplifying RNA constructs, expression profiles, immunogenicity data, and resulting antibody responses are likewise applicable to any Lyme disease-associated antigen or combination of antigens disclosed herein. Thus, the figures are provided as non-limiting examples and are not restricted to any specific antigen pair, antigen sequence, or antigen valency.

[0093]

[0081] FIG 1. depicts plasmid DNA encoding a self-amplifying RNA expressing multiple antigens associated with Lyme disease according to one embodiment disclosed herein.

[0094]

[0082] FIG.2 depicts the results of a Western blot to detect the expression of OspA and OspC after the transfection of HEK293 cells with an saRNA encoding OspA and OspC.

[0095]

[0083] FIG. 3 shows the measurement of antibody titers over time against OspA in the serum of mice vaccinated with 1000 ng of the saRNA encoding both OspA and OspC.

[0096]

[0084] FIG. 4 shows the measurement of antibody titers over time against OspA in the serum of mice vaccinated with 10 ng of the saRNA encoding both OspA and OspC.

[0097]

[0085] FIG. 5 shows the measurement of antibody titers over time against OspC in the serum of mice vaccinated with 1000 ng of the saRNA encoding both OspA and OspC.

[0098]

[0086] FIG. 6 shows the measurement of antibody titers over time against OspC in the serum of mice vaccinated with 10 ng of the saRNA encoding both OspA and OspC.

[0099] DETAILED DESCRIPTION

[0100] Definitions

[0101]

[0087] “Adjuvant” as used herein refers to a pharmacological and / or immunological agent that modifies, enhances, prolongs, or otherwise modulates an immune response to an antigen. The term includes nucleic acid adjuvants (e.g., immunostimulatory RNA, immunostimulatory CpG DNA, or expression vectors encoding cytokines or chemokines), as well as protein-based, Attorney Docket No.: KEYL-001WO

[0102] peptide-based, cell death-promoting (e.g., herpes simplex virus thymidine kinase), and chemical adjuvants that can enhance, prolong, or otherwise modulate antigen-specific immune responses when administered with a vaccine antigen. In certain embodiments, the adjuvant is encoded within the same self-amplifying RN A construct as the antigen. In other embodiments, the adjuvant is co-formulated in the pharmaceutical composition or co-administered as a separate agent.

[0103]

[0088] “Antigen” as used herein refers to a molecule, polypeptide, protein, peptide, epitope, or immunogenic fragment thereof that is capable of eliciting an immune response in a subject. The term encompasses full-length proteins, truncated proteins, fusion proteins, chimeric proteins, variants, derivatives, and immunogenic fragments, including but not limited to linear epitopes, conformational epitopes, and surface-exposed domains. In certain embodiments herein, the antigen is derived from a Lyme disease-associated Borrelia species. In other embodiments, the antigen may be engineered, modified, codon-optimized, or membrane- tethered to enhance stability, expression, presentation, or immunogenicity. Unless expressly stated otherwise, the term “antigen” includes any form capable of eliciting or boosting an immune response when expressed in vivo from the disclosed self-amplifying RNA constructs.

[0104]

[0089] “Borrelia” as used herein refers to a genus of bacteria comprising more than fifty ( 50) known species, which are broadly divided according to whether they cause Lyme disease (e.g. the Borrelia burgdorferi sensu lato complex) or relapsing fever. They are members of the family Spirochaetaceae and exhibit the characteristic helical (spirochete) morphology. Borrelia species possess an outer membrane that contains lipoproteins, an inner membrane, and a peptidoglycan layer within the periplasmic space, classifying them as Gram-negative-like bacteria. In certain embodiments, the saRNA compositions and pharmaceutical compositions disclosed herein are useful for preventing, reducing, suppressing, or treating infection with one or more Borrelia species associated with Lyme disease, including, but not limited to, B. burgdorferi, B. afzelii, B. garinii, B. mayonii, B. spielmanii, and B. bavariensis. Strains of B. burgdorferi include, for example, B31, N40, JD1.

[0105]

[0090] “C lamer” as used herein refers to a compound that facilitates transport and / or complexation of another compound. According to the embodiments herein, a carrier is suitable for facilitating transport of nucleic acid molecules, e.g. for mediating dissolution in physiological acceptable liquids, transport and cellular uptake of the nucleic acid molecules or a vector. Representative non-limiting carriers include buffer substances, stabilizers, or further active ingredients, especially ingredients known in connection with pharmaceutical compositions and / or vaccine production. Attorney Docket No.: KEYL-001WO

[0106]

[0091] “C ’.ellular immunity” as used herein refers to activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an antigen. In more general terms, cellular immunity is not based on antibodies, but on the activation of cells of the immune system. Typically, a cellular immune response may be characterized for example by activating antigen-specific cytotoxic T-lymphocytes that are able to induce apoptosis in cells, e.g. specific immune cells like dendritic cells or other cells, displaying epitopes of foreign antigens on their surface. Such cells may be virus-infected or infected with intracellular bacteria, or cancer cells displaying tumor antigens Further characteristics may be activation of macrophages and natural killer cells, enabling them to destroy pathogens and stimulation of cells to secrete a variety of cytokines that influence the function of other cells involved in adaptive immune responses and innate immune responses.

[0107]

[0092] “Chimeric" as used herein with reference, e.g., to a nucleic acid, protein, or vector, indicates that the nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein.

[0108]

[0093] “C Combination” or “combination therapy” as used herein refers to at least two therapeutically active agents or compositions which may be administered or co-administered”, simultaneously, in either separate or combined formulations, or sequentially at different times separated by minutes, hours or days. In general, each agent will be administered at a dose and / or on a time schedule determined for that agent, in certain embodiments, the saRNA construct disclosed herein may be used in combination with one or more additional therapeutic agents (e.g., cytokines, such as IL-10, IFN-y, TNF-a, IL-6, IL-17, and GM-CSF or the like, or antibiotics, such as azlocillin, amoxicillin, Penicillin G, tetracycline, ceftriaxone, cefotaxime or cefuroxime axetil, or enzymatic therapy) in the treatment of Lyme Disease.

[0109]

[0094] “( 'ytokine” as used herein refers to interleukins, interferons (IFN), chemokines, hematopoietic growth factors, tumor necrosis factors (TNF), and transforming growth factors. In general, these are small molecular weight proteins that regulate maturation, activation, proliferation, and differentiation of cells of the immune system.

[0110]

[0095] “Effective amount” or “therapeutically effective amount” as used herein refers to the amount of a nucleic acid such as saRNA sufficient to produce the desired effect, e.g.. an inhibition of expression of a target sequence in comparison to the normal expression level detected in the absence of an interfering RNA; or mRNA-directed expression of an amount of an antigen or protein that causes a desirable biological effect in the subject within which the antigen or protein is expressed. Attorney Docket No.: KEYL-001WO

[0111]

[0096] “Epitope" as used herein refers to that portion of an antigen that determines its immune specificity.

[0112]

[0097] “Expression” as used herein refers to the process by which a nucleic acid sequence or polynucleotide is transcribed from a DNA template (such as into mRNA or another RNA transcript) and / or the process by which a transcribed RNA is subsequently translated into a peptide, polypeptide, or protein. The resulting RNA transcripts and encoded peptides, polypeptides, or proteins may be collectively referred to as a “gene product.” In certain embodiments herein, expression refers to in vivo antigen expression following administration of the self-amplifying RNA compositions disclosed.

[0113]

[0098] “Fragment" as used herein refers to a sequence that is shorter than a full-length sequence of e.g. a nucleic acid molecule or an amino acid sequence. In certain embodiments, the fragment comprises at least 5%, 10%, 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, and most preferably at least 80% of the total (i.e., full-length) molecule from which the fragment is derived. In certain embodiments, the fragment may be an antigen fragment that has a length of 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids. In certain embodiments, the fragment of a protein comprises at least one epitope of the protein, i.e., an immunogenic fragment.

[0114]

[0099] “Fused" or “fusion" as used herein with respect to two polypeptide sequences refers to the joining of the two polypeptide sequences through a backbone peptide bond. Two polypeptides may be fused directly or through a peptide linker that is one or more amino acids long. A fusion polypeptide may be made by recombinant technology from a coding sequence containing the respective nucleic acid coding sequences for the two fusion partners, with or without a nucleic acid coding sequence for a peptide linker in between. In some embodiments, fusion encompasses chemical conjugation.

[0115]

[0100] “Furin cleavage site” as used herein refers to an amino acid sequence that is specifically recognized and cleaved by the host furin protease, which is typically located in the Golgi apparatus and secretory pathway. Furin cleavage sites generally conform to the consensus motif R-X-(K / R)-R],, where cleavage occurs at the indicated position. In certain embodiments herein, inclusion of a furin cleavage site between antigens and / or between an antigen and a 2A peptide allows post-translational separation of expressed polypeptides, enhances proper folding, and / or improves antigen presentation. Non-limiting examples include the sequences RRRR (SEQ ID NO: 13), RQRR (SEQ ID NO: 14), and RRKR (SEQ ID NO: 15). Attorney Docket No.: KEYL-001WO

[0116]

[0101] “Humoral immunity” as used herein refers to antibody production and, optionally, to the accessory immune processes that accompany and support antibody production. A humoral immune response may be characterized, for example, by B cell activation, T helper (Th2 and / or T follicular helper, Tfh) cell signaling, germinal center formation, isotype class switching, affinity maturation, and memory B cell generation. Humoral immunity also typically refers to the effector functions of antibodies, which include pathogen and toxin neutralization, classical complement activation, opsonization, and Fc receptor-mediated clearance.

[0117]

[0102] “Genotype” as used herein to refers to a classification based on DNA sequence differences in a specific gene or set of genes among bacterium, and is considered more precise than strain.

[0118]

[0103] “Immune response” as used herein refers to a cellular, humoral, innate, or adaptive reaction of the immune system to an antigen. An immune response may include antibody production, B cell activation, T cell activation (including CD4+ and / or CD8+ responses), innate immune signaling, and / or cytokine or chemokine expression. In certain embodiments, the immune response comprises both humoral and cellular components. In certain embodiments herein, the immune response results from in vivo expression of one or more Borrelia-associated antigens encoded by the self-amplifying RNA compositions disclosed.

[0119]

[0104] “Internal Ribosome Entry Site” or “IRES” as used herein refers to an RNA sequence that enables cap-independent initiation of translation. An IRES allows ribosomes to bind and initiate protein synthesis at an internal position within an mRNA rather than exclusively at the 5' end. In certain embodiments herein, an IRES is included within the self-amplifying RNA construct to enable polycistronic expression of two or more antigens, adjuvants, or accessory proteins from a single RNA transcript. Suitable IRES elements may be derived from viral genomes (e.g., encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV)) or may be synthetic. Use of an IRES may facilitate coordinated co-expression, controlled stoichiometry, and / or improved antigen presentation.

[0120]

[0105] “LNP” as used herein refers to a lipid nanoparticle, including a lipid-nucleic acid particle or nucleic acid-lipid particle (e.g., a stable nucleic acid-lipid particle). An LNP is composed of one or more lipids and a nucleic acid cargo (e.g., saRNA), wherein the nucleic acid is encapsulated within the lipid particle or, in some embodiments, complexed to the particle surface. In one embodiment, the nucleic acid is at least 50% encapsulated within the lipid; in another embodiment, at least 75%, 90%, or substantially 100% encapsulated. Typical LNP formulations comprise an ionizable or cationic lipid, a helper phospholipid, cholesterol, Attorney Docket No.: KEYL-001WO

[0121] and a lipid conjugate such as a polyethylene glycol (PEG)-lipid to modulate particle stability, size, and circulation properties.

[0122]

[0106] “Membrane-anchored” or “membrane-tethered” as used herein with respect to a antigen refers to an antigen that is tethered to a cell membrane rather than freely floating inside the cell or being secreted. The “anchoring” is achieved through specific structural features that keep the antigen embedded in or attached to the lipid bilayer. Such features include, for example, transmembrane domains that span the lipid bilayer, covalent lipidation such as palmitoyl ati on or bacterial lipoprotein modification, glycosylphosphatidylinositol (GPI) anchors that tether proteins to the outer leaflet of the membrane, membrane localization sequences, signal anchors or the like. In certain embodiments, the antigens expressed by the self-amplifying RNA disclosed herein are membrane-anchored. In certain embodiments, membrane-tethering enhances antigen presentation, stability, immune recognition, or a combination thereof. In certain embodiments, the saRNA construct disclosed herein encodes one or more membrane- tethered proteins or fragments thereof.

[0123]

[0107] “Modified” as used herein refers to a molecule (including but not limited to a nucleic acid, nucleotide, nucleobase, sugar, peptide, polypeptide, protein, lipid, or linker) that differs from its canonical or naturally occurring form, whether the modification is naturally occurring or non-naturally occurring. Modifications may include, but are not limited to, methylation, acetylation, pseudouridylation, phosphorylation, glycosylation, alkylation, conjugation, substitution, addition, deletion, or altered abundance or distribution of chemical groups or residues. A modified species (also referred to as an “altered” species) may be chemically, structurally, or functionally distinct from its unmodified counterpart. In certain embodiments, modified nucleic acids (e.g., modified RNA or DNA) comprise one or more altered nucleotides, nucleobases, sugars, linkages, or backbone chemistries, whether naturally occurring or synthetic. In certain embodiments, modified proteins comprise variant amino acids, fusion domains, signal peptides, or post-translational modifications.

[0124]

[0108] “Modified nucleotide” as used herein refers to a nucleotide in which the nucleobase, sugar, and / or phosphate backbone differs from the canonical form found in unmodified RNA or DNA. Modified nucleotides include both naturally occurring and non-naturally occurring modifications. Non-limiting examples of naturally occurring modifications include, but are not limited to, N1-methylpseudouridine (m1Ψ), pseudouridine (Ψ), 5-fluorocytidine (5FC), 5-methylcytidine (m5C), 5-hydroxymethylcytidine (hm5C), N6-methyladenosine (m6A), inosine (I), and 2'-O-methylated nucleotides. Non-limiting examples of non-naturally occurring modifications include 5-fluorocytidine, 5-hydroxymethylcytidine, 5-methyluridine, 5- Attorney Docket No.: KEYL-001WO

[0125] hydroxymethyluridine and phosphorothioate backbone modifications. In certain embodiments herein, modified nucleotides are incorporated into self-amplifying RNA to modulate stability, translation efficiency, innate immune sensing, or antigen expression.

[0126]

[0109] “Multi-valent” as used herein to refer to the saRNA compositions an RNA or multiple RNAs encoding two or more antigens of the same or different species (e.g., different strains, subtypes or epitopes). In certain embodiments, the saRNA may encode 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more bacterial antigens, such as Borrelia antigens. The multivalent vaccines or therapeutics permit protection against multiple Borrelia species, strains, serotypes or genotypes (e.g., OspC) at the same time, broadening coverage and reducing the risk of immune escape.

[0127] [HO] “Nucleic acid” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, or analogs thereof. This term refers to the primary structure of the molecule, and thus includes double- and single-stranded DNA, as well as double- and single-stranded RNA. It also includes modified nucleic acids such as methylated and / or capped nucleic acids, nucleic acids containing modified bases, backbone modifications, and the like. The terms “nucleic acid” and “nucleotide sequence" are used interchangeably.

[0128]

[0111] “Operably linked” as used herein refers to a first molecule joined to a second molecule, wherein the molecules are so arranged that the first molecule affects the function of the second molecule. The two molecules may or may not be part of a single contiguous molecule and may or may not be adjacent. In certain embodiments, the saRNA disclosed herein comprises one or more regulatory elements operably linked to one or more coding elements, e.g., a nucleic acid sequence encoding one or more antigens.

[0129]

[0112] “Open reading frame” or “ORF” as used herein refers to a sequence of nucleotide triplets that can be translated into a peptide or protein. An ORF typically comprises a start codon (e.g., AUG) at its 5' end and a subsequent coding region of length that is a multiple of three nucleotides and is generally terminated by a stop codon (e.g., UAA, UAG, UGA). As used herein, reference to an ORF includes ORFs present on RNA molecules (such as the selfamplifying RNA constructs described) and ORFs present on DNA templates used to generate such RNA. In certain embodiments, the saRNA disclosed herein contains at least two ORFs, for example, a replicase ORF and a second ORF encoding one or more antigens.

[0130]

[0113] “Pharmaceutically acceptable” as used herein refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Attorney Docket No.: KEYL-001WO

[0131]

[0114] “Protein” or “peptide” as used herein refers to at least two or more amino acid residues linked together by peptide bond. The amino acid sequence in a protein or peptide is shown in the standard format, i.e., from amino terminus (N-terminus) to carboxyl terminus (C-terminus).

[0132]

[0115] “Regulatory’ element” refers to a nucleotide sequence that controls, at least in part, the transcription of a gene or genes of interest. Regulatory elements may include promoters, enhancers, and other nucleic acid sequences (e.g., polyadenylation signals) that control or help to control nucleic acid transcription or translation. In certain embodiments herein, the saRNA disclosed herein comprises one or more regulatory elements.

[0133]

[0116] “RNA dependent RNA polymerase” or “RdRp” as used herein refers to an enzyme that catalyzes the synthesis of RNA from an RNA template. RdRp is an essential protein encoded in the genomes of most RNA-containing viruses, which lack a DNA stage. Despite sequence variations among different viruses, the core structural features of RdRp are highly conserved. As used herein, “RdRp” may refer to a single polypeptide or to a multi-protein replicase complex that together performs RNA-dependent RNA synthesis. In certain embodiments, the saRNA disclosed herein comprises four non-structural proteins (nsPl-nsP4), which assemble into a functional replicase complex that provides RdRp activity.

[0134]

[0117] “Self-amplifying RNA” or “saRNA” as used herein refers to RNAs that contain the basic elements of mRNA, i.e., a cap, 5'UTR, 3'UTR and a poly(A) tail. Unlike conventional (non -replicating) mRNA, saRNA further encodes a replicase (e.g., comprising nonstructural proteins nsPl-nsP4) that provides RNA-dependent RNA polymerase (RdRp) activity. The replicase complex enables intracellular amplification of the RNA and transcription of one or more subgenomic RNAs, resulting in higher and more sustained antigen expression relative to non-replicating mRNA. In some embodiments, saRNA can achieve antigen expression levels up to 10-100 fold greater and for longer durations than conventional mRNA.

[0135]

[0118] “Signal peptide” as used herein refers to a short sequence of amino acids found at the beginning of a newly synthesized proteins important for directing the protein to its correct location within the cell. Typically, signal peptides are about 16-30 amino acids long and are located at the N-terminus of the protein.

[0136]

[0119] “Serotype” as used here refers subgroup of a species defined by antigenic differences on the surface (e.g., proteins, polysaccharides, lipids). The basis of the classification is immune recognition, i.e., antibodies bind to specific antigens, so different serotypes are immunologically distinct. Not all serotypes are species-specific, e.g., OspC genotypes show diversity within a single species. In certain embodiments, the saRNA construct disclosed herein comprises two or more serotypes of a given antigen, from the same or different species. Attorney Docket No.: KEYL-001WO

[0137]

[0120] “Strain" as used herein refers to a member bacterial species with a genetic signature such that it may be differentiated from closely related members of the same bacterial species. The genetic signature may be the absence of all or part of at least one gene, the absence of all or part of at least one regulatory region ( for example, a promoter, a terminator, a riboswitch, a ribosome binding site), the absence (“curing”) of at least one native plasmid, the presence of at least one recombinant gene, the presence of at least one mutated gene, the presence of at least one foreign gene (a gene derived from another species), the presence at least one mutated regulatory region (e.g., a promoter, a terminator, a riboswitch, a ribosome binding site), the presence of at least one non- native plasmid, the presence of at least one antibiotic resistance cassette, or a combination thereof. Genetic signatures between different strains may be identified by PCR amplification optionally followed by DNA sequencing of the genomic region(s) of interest or of the whole genome. In the case in which one strain (compared with another of the same species) has gained or lost antibiotic resistance or gained or lost a biosynthetic capability (such as an auxotrophic strain), strains can be differentiated by selection or counter-selection, using an antibiotic or nutrient / metabolite, respectively. Put another way, strain refers to a specific isolate or lineage, not necessarily antigenically distinct.

[0138]

[0121] “Subject” as used herein refers to any organism to which a composition in accordance ■with the present disclosure may be administered, e.g,, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants. Preferably “patient” refers to a human subject who may seek or need treatment, requires treatment, is receiving treatment, will receive treatment, or a subject who is under care by a trained professional for a particular disease or condition

[0139]

[0122] “Subgenomic promoter” or “SGP” as used herein refers to those RNA sequences capable of being recognized by viral -encoded RNA dependent RN polymerase to direct the production of subgenomic RNAs. Subgenomic promoters have been mapped and characterized in several viruses.

[0140]

[0123] “Sustained antigen expression” or “durable immune response” as used herein refers to in vivo antigen expression and / or induced immune activation that persists for a period longer than that typically observed for non-replicating mRNA. In certain embodiments, antigen expression persists for at least one week, two weeks, three weeks, or four weeks, and in some embodiments for eight or more weeks following administration.

[0141]

[0124] “Transcription” as used herein refers to a process during which a nucleic acid molecule with a particular nucleic acid sequence (the "nucleic acid template") is read by an RNA Attorney Docket No.: KEYL-001WO

[0142] polymerase so that the RNA polymerase produces a single-stranded RNA molecule. During transcription, the genetic information in a nucleic acid template is transcribed.

[0143]

[0125] “Treat”, “treating” or “treatment” as used herein refers to alleviating, abating or ameliorating disease or condition symptoms (for example, Lyme disease), preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition. The terms "treat," "treating" or "treatment'', may include, but are not limited to, prophylactic, diagnostic and / or therapeutic treatments.

[0144]

[0126] “ Vaccine” as used herein refer to a biological preparation that improves immunity to a particular disease (e.g.. Lyme disease or Borrelia infection). A vaccine typically contains an agent that stimulates the hosts immune system to recognize the agent as foreign, destroy it, and "remember” it, so that the immune system can more easily recognize and destroy any of these microorganisms that it later encounters. Vaccines, in various aspects, are prophylactic (prevent or ameliorate the effects of a future infection by any natural or "wild" pathogen), or therapeutic (vaccines against present infection). As set forth above, such vaccine compositions include formulations comprising pharmaceutically acceptable excipients or carriers. In certain embodiments, the self -amplifying RNA constructs disclosed herein function as vaccines.

[0145]

[0127] “V ariant” as used herein in the context of a nucleic acid sequence refer to a nucleic acid sequence that may exhibit one or more nucleotide deletions, insertions, additions, and / or substitutions compared to the nucleic acid sequence from which the variant is derived. A variant of a nucleic acid sequence may at least 40%, 50%, 60%, 70%, 80%, 81 %, 82%, 83%, 84%;, 85%, 86%, 87%. 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% identical to the nucleic acid sequence the variant is derived from. The variant is a functional variant in the sense that the variant has retained at least 40%>, 50%, 60%>, 70%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97'%, 98%;, 99%, 99.5% or more of the function of the sequence where it is derived from. In one embodiment a “variant” of a nucleic acid sequence may have at least 40%, 50%, 60%, 70%, 80%, 81 %, 82%. 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%. 91 %, 92%, 93%, 94%, 95%, 96%?, 97%, 98%, 99%?, 99.5%? nucleotide identity over a stretch of at least 10, 20, 30, 50, 75 or 100 nucleotide of such nucleic acid sequence.

[0146]

[0128] “Variant” as used herein in the context of proteins or peptides refers to a variant having an amino acid sequence which differs from the original sequence in one or more Attorney Docket No.: KEYL-001WO

[0147] mutation(s) / substitution(s), such as one or more substituted, inserted and / or deleted amino acid(s). For example. in some aspects an insertion in a protein sequence comprises an insertion of 1 to 10 amino acids, such as 1, 2, 3, 4, 5, 6,78, 9 or 10 consecutive amino acids. Preferably, these fragments and / or variants may have the same, or a comparable specific antigenic property (immunogenic variants, antigenic variants). Insertions and substitutions are possible, in particular, at those sequence positions which cause no modification to the three-dimensional structure or do not affect the binding region. Modifications to a three-dimensional structure by insertion(s) or deletion(s) can easily be determined e.g. using CD spectra (circular dichroism spectra). A “variant” of a protein or peptide may have at least 40%. 50%, 60%, 70%, 80%. 81 %, 82%, 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%. 98%, 99%, 99.5% amino acid identity over a stretch of at least 10, 20, 30, 50, 75 or 100 amino acids or over the entire length of such protein or peptide. Preferably, a variant of a protein may comprise a functional variant of the protein, which means, in the context of the invention, that the variant exerts essentially the same, or at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more of the immunogenicity as the protein it is deri ved from,

[0148]

[0129] “2A peptide” or “ribosomal slapping peptide” as used herein refers to a short selfcleaving peptide sequence that mediates ribosome skipping during translation, resulting in the generation of two or more separate polypeptides from a single open reading frame. Non¬ limiting examples include P2A, T2A, E2A, and F2A peptides. In certain embodiments herein, two or more Borrelia antigens are encoded in a single open reading frame and are separated by one or more 2A peptides to allow multi-antigen expression from a single saRNA molecule.

[0149] Compositions

[0150]

[0130] Disclosed herein are compositions useful for treating (including preventing) Lyme disease and more particularly, self-amplifying RNA (saRNA) constructs useful for treating Lyme disease or other Borrelia infections. In certain embodiments, the compositions provide antigenic diversity, localization requirements, and structural constraints required for effective vaccination against Borreli and lacking in the prior art.

[0151]

[0131] In one embodiment, an saRNA construct is disclosed comprising an open reading frame, wherein the open reading frame encodes at least one bacterial antigen associated with Lyme disease. According to this embodiment, the self-amplifying RNA molecule also encodes an RNA-dependent RNA polymerase that may transcribe RNA from the self- amplifying RNA construct.

[0152]

[0132] Lyme disease is caused by infection with a pathogenic Borrelia bacterium (a spirochete) and is transmitted by the bite of various species of Borrelia-infected Ixodes ticks. Attorney Docket No.: KEYL-001WO

[0153] Species differences are observed geographically, i.e., Borrelia burgdorferi is common in North American, including strains known as Borrelia burgdorferi B31 and Borrelia Burgdorferi 297. In Europe and Asia, two other Borrelia species dominate, Borrelia garinii and Borrelia afzeli.

[0154]

[0133] In another embodiment, an saRNA construct is disclosed comprising an ORF, wherein the ORF encodes at least two bacterial antigens associated with Lyme disease. The two bacterial antigens may be the same or different. The two bacterial antigens may be from different species of bacteria or different strains of the same species.

[0155]

[0134] Numerous surface proteins from Borrelia burgdorferi have been identified and fall into two main categories - lipid -modified outer surface proteins that are anchored to the outer leaflet of the outer membrane through their lipid moieties (e.g., OspA, OspB, OspC, OspD, OspE, OspF, DbpA. DbpB, CspA, VlsE, BptA, and several others with no known function) and outer surface proteins that have one or more transmembrane domains that anchor them into the outer membrane (e.g., P13, P66, BesC, BaraA, Lmpl and BB0405). The saRNA disclosed herein may encode any of these proteins or fragments thereof, either alone or in combination. In certain embodiments, the saRNA disclosed herein encodes a Borrelia burgdorferi antigen, and more particularly a lipidated Borrelia burgdorferi antigen that maintains its proper (wild-type) folding following expression from the saRNA. In certain embodiments, the lipidated antigen does not aggregate, misfold or create ER stress (i.e., unfolded protein response).

[0156]

[0135] OspA is a ~31-kDa lipoprotein expressed during habitation of the tick midgut then downregulated during transmission. (Bouchon et al., Anal. Biochem. 246: 52-61, 1997). Multiple; serotypes have been identified. The primary disease-causing species of Borrelia in North America and Europe belong to OspA serotypes 1-7. (Piesman J, et al. Parasitology 2004;129 Suppl: S191-220; Wilske et al., J. Clin. Microbiol. 31:340-50, 1993). OspA serotypes of Borrelia correlate with species; serotype 1 corresponds to Borrelia burgdorferi, serotype 2 corresponds to Borrelia afzelii and serotypes 3 to 7 correspond to Borrelia garinii.

[0157]

[0136] In one embodiment, the at least one antigen encoded by the saRNA disclosed herein comprises OspA or a fragment thereof from any suitable Borrelia species, e.g., Borrelia burgdorferi, Borrelia afzelii or Borrelia garinii. In certain embodiments, the saRNA comprises two or more antigens, wherein the two or more antigens comprise OspA (or a fragment thereof) from different Borrelia species or different strains of the same Borrelia species. In other embodiments, the at least one antigen is a chimeric antigen comprising fragments of OspA from different Borrelia species or different strains of the same Borrelia species. In a particular embodiment, the saRNA encodes OspA serotype 1, serotype 2, serotype 3, serotype 4, serotype 5, serotype 6, serotype 7 or a combination thereof. Attorney Docket No.: KEYL-001WO

[0158]

[0137] In one embodiment, expression of the OspA results in a protein that comprises a elongated, rod-like p-sheet-rich structure, composed mainly of antiparallel p-strands arranged in a flattened P-sheet ribbon, with a small globular domain at one end.

[0159]

[0138] OspB is a 34kDa lipoprotein that shares a high degree of sequence and similarity (~50% sequence identity), as well as structural similarity to OspA. Like OspA it is expressed in the tick midgut and downregulated during feeding.

[0160]

[0139] In one embodiment, the at least one antigen of the saRNA disclosed herein comprises OspB or a fragment thereof from any suitable Borrelia species, e.g., Borreliei burgdorferi, Borrelia afzelli or Borrelia garinii. In certain embodiments, the saRNA comprises two or more antigens, wherein the two or more antigens comprise OspB (or a fragment thereof) from different Borrelia species or different strains of the same Borrelia pecies. In other embodiments, the at least one antigen is a chimeric antigen comprising fragments of OspB from different Borrelia species or different strains of the same Borrelia species.

[0161]

[0140] In one embodiment, expression of the OspB result is a protein that comprises a long, flattened P-sheet ribbon with a small globular domain

[0162]

[0141] OspC is a 22 kDa lipoprotein for which a biological role remains unclear. Within 36-48 hours of a blood meal, spirochetes in the engorged tick downregulate their production of OspA and OspB, and OspC production is induced. It is downregulated shortly after spirochetes disseminate into the bloodstream. Key OspC epitopes have been mapped to the hypervariable C-terminal region with the use of protein arrays and sera from mice and humans. (Baum E, et al. PLoS ONE. 2013; 8:e67445). The OspC protein exhibits a high degree of inter- and intraspecies variation, with >20 distinct OspC types having been delineated. In a particular embodiment, the saRNA encodes an Osp protein from genotype A, B, D, E, F, I, K, N, T, or U.

[0163]

[0142] In one embodiment, the at least one antigen of the saRNA disclosed herein comprises OspC or a fragment thereof from any suitable Borrelia species, e.g., Borrelia burgdorferi, Borrelia afzelli or Borrelia garinii. In certain embodiments, the saRNA comprises two or more antigens, wherein the two or more antigens comprise OspC (or a fragment thereof) from different Borrelia species or different strains of the same Borrelia pecies. In other embodiments, the at least one antigen is a chimeric antigen comprising fragments of OspC from different Borrelia species or different strains of the same Borrelia species. In one embodiment, the OpsC is a STI strain.

[0164]

[0143] In one embodiment, expression of the saRNA results in a protein that comprises a compact o / [3 protein that forms a stable homodimer, wherein each monomer contains a central Attorney Docket No.: KEYL-001WO

[0165] p-sheet flanked by a-helices, and the two monomers associate to create a dimer with a distinctive “V”-shaped architecture.

[0166]

[0144] OspD, OspE, and OspF are outer surface proteins that have been implicated in Borrelia’ s evasion of the complement system. These proteins interact with host complement inhibitors such as Factor H and C4-binding protein, preventing complement-mediated destruction of Borrelia.

[0167]

[0145] In one embodiment, the at least one antigen encoded by the saRNA disclosed herein comprises OspD or a fragment thereof from any suitable Borrelia species, e.g., Borrelia burgdorferi, Borrelia afzelli or Borrelia garinii. f. In certain embodiments, the saRNA comprises a nucleic acid sequence encoding two or more antigens, wherein the two or more antigens comprise OspD (or a fragment thereof) from different Borrelia species or different strains of the same Borrelia species. In other embodiments, the at least one antigen is a chimeric antigen comprising fragments of OspD from different Borrelia species or different strains of the same Borrelia species.

[0168]

[0146] In one embodiment, the at least one antigen of the saRNA disclosed herein comprises a nucleic acid sequence encoding OspE or a fragment thereof from any suitable Borrelia species, e.g., Borrelia burgdorferi, Borrelia afzelli or Borrelia garinii. In certain embodiments, the saRNA comprises a nucleic acid sequence encoding two or more antigens, wherein the two or more antigens comprise OspD (or a fragment thereof) from different Borrelia species or different strains of the same Borrelia species. In other embodiments, the at least one antigen is a chimeric antigen comprising fragments of OspE from different Borrelia species or different strains of the same Borrelia species.

[0169]

[0147] In one embodiment, the at least one antigen encoded by the saRNA disclosed herein comprises OspF or a fragment thereof from any suitable Borrelia species, e.g., Borrelia burgdorferi, Borrelia afzelli or Borrelia garinii. In certain embodiments, the saRNA comprises a nucleic acid sequence encoding two or more antigens, wherein the two or more antigens comprise OspF (or a fragment thereof) from different Borrelia species or different strains of the same Borrelia species. In other embodiments, the at least one antigen is a chimeric antigen comprising fragments of OspF from different Borrelia species or different strains of the same Borrelia species.

[0170]

[0148] DbpA and DbpB are surface lipoproteins upregulated on the surface of 5. burgdorferi organisms grown at reduced pH and by a temperature shift from 23° to 37°C, which suggests an important role for these proteins in the mammalian environment. Attorney Docket No.: KEYL-001WO

[0171]

[0149] In one embodiment, the at least one antigen encoded by the saRNA disclosed herein comprises DbpA or a fragment thereof from any suitable Borrelia species, e.g., Borrelia burgdorferi, Borrelia afzelli or Borrelia garinii. In certain embodiments, the saRNA comprises a nucleic acid sequence that encodes a two or more antigens, wherein the two or more antigens comprise DbpA (or a fragment thereof) from different Borrelia species or different strains of the same Borrelia species. In other embodiments, the at least one antigen is a chimeric antigen comprising fragments of DbpA from different Borrelia species or different strains of the same Borrelia species.

[0172]

[0150] In one embodiment, the at least one antigen encoded by the saRNA disclosed herein comprises DbpB or a fragment thereof from any suitable Borrelia species, e.g., Borrelia burgdorferi, Borrelia afzelli or Borrelia garinii. In certain embodiments, the saRNA comprises a nucleic acid sequence encoding two or more antigens, wherein the two or more antigens comprise DbpB (or a fragment thereof) from different Borrelia species or different strains of the same Borrelia species. In other embodiments, the at least one antigen is a chimeric antigen comprising fragments of DbpB from different Borrelia species or different strains of the same Borrelia species.

[0173]

[0151] In certain embodiments, the saRNA is multi-valent, i.e., encodes more than one antigen. In certain embodiments, the saRNA encodes at least two, at least three, at least four, at least five, at least six or more than six antigens. The antigens may be the same antigens or different antigens. The multi-valent saRNA-encoded antigens may be from the same species or different species of Borrelia. In certain embodiments, the multi-valent saRNA-encoded antigens are the same antigen, but from different species of Borrelia or serotypes of the same species. In certain embodiments, the multi-valent saRNA-encoded antigens are different antigens from the same species of Borrelia, e.g., the same or different serotypes of the same species. In certain embodiments, the antigens may be chimeric, i.e,, from more than one species of Borrelia.

[0174]

[0152] In one embodiment, the saRNA encodes at least two bacterial antigens comprising outer surface protein A (OspA) (or a fragment thereof) and outer surface protein C (OspC) (or a fragment thereof). The OspA and OspC (or fragments thereof) may be from the same or different Borrelia species, or different strains of a single Borrelia species.

[0175]

[0153] In certain embodiments, the compositions described herein permit immune response, prevention, and / or treatment of a Borrelia infection, including but not limited to Lyme disease. Representative, non-limiting bacterial species include Borrelia afzelii, Borrelia americana, Borrelia andersonii, Borrelia anserina, Borrelia bahazardii, Borrelia bavariensis, Borrelia bisseltii, Borrelia brasiliensis, Borrelia burgdorferi, Borrelia califomiensis, Borrelia Attorney Docket No.: KEYL-001WO

[0176] caroli nensis, Borrelia caucasica, Borrelia coriaceae, Borrelia crocidurae, Borrelia dugesii, Borrelia dultonii, Borrelia garmii, Borrelia graingeri, Borrelia harveyi, Borrelia hermsii, Borrelia hispanica, Borrelia japonica, Borrelia kurieribachii, Borrelia latyxchewii, Borrelia lonestari, Borrelia lusitaniae, Borrelia mazzotii, Borrelia merionesi, Borrelia microti, Borrelia miyamotoi, Borrelia parke ri, Borrelia persica, Borrelia recurrentis, Borrelia sinica, Borrelia spielmanii, Borrelia ianukii, Borrelia lexasensis, Borrelia lheileri, Borrelia lillae, Borrelia turcica, Borrelia turdi, Borrelia turicatae, Borrelia valaisianu, Borrelia venezuelensis, Borrelia vincentii, Borrelia burgdorferi B31, Borrelia burgdorferi N40, Borrelia burgdorferi JD1, Borrelia burgdorferi 297 or combinations thereof.

[0177]

[0154] In one embodiment, the saRNA encodes two bacterial antigens derived from different Borrelia species. The antigens may be the same or different.

[0178]

[0155] In other embodiments, the saRNA encodes at least three, at least four, at least five or at least six or more bacterial antigens derived from different Borrelia species. The antigens may be the same or different.

[0179]

[0156] In a particular embodiment, the saRNA encodes two bacterial antigens derived from different strains of the same Borrelia species, e.g.. two strains of Borrelia burgdorferi, Borrelia garmii or Borrelia ajzeli. The antigens may be the same or different.

[0180]

[0157] In other embodiments, the saRNA encodes at least three, at least four, at least five or at least six or more bacterial antigens derived from different strains of the same Borrelia species. The antigens may be the same or different.

[0181]

[0158] In a particular embodiment, the saRNA encodes at least three, at least four, at least five or at least six or more bacterial antigens derived from different Borrelia burgdorferi strains. The antigens may be the same or different.

[0182]

[0159] In a particular embodiment, the saRNA encodes at least three, at least four, at least five or at least six or more bacterial antigens derived from different Borrelia garinii strains. The antigens may be the same or different.

[0183]

[0160] In a particular embodiment, the saRNA encodes at least three, at least four, at least five or at least six or more bacterial antigens derived from different Borrelia afzeli strains. The antigens may be the same or different.

[0184]

[0161] In certain embodiments, the saRNA encodes one or more chimeric antigens comprising subunits of any antigen described herein.

[0185]

[0162] In one embodiment, the saRNA encodes comprise the proximal portion from one OspA serotype, together with the distal portion from another OspA serotype. Attorney Docket No.: KEYL-001WO

[0186]

[0163] In amother embodiment, the saRNA encodes comprise the proximal portion from one OspC serotype, together with the distal portion from another OspC serotype.

[0187]

[0164] In one embodiment, the sequence encoding the at least one antigen is codon optimized, for example to ensure proper folding; bias GC content to increase RNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove / add post translation modification sites in encoded protein (e.g, glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and NA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or to reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art ■ non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and / or proprietary methods. Additional algorithms include LinearDesign (Lang et al.. 2023, PMID: 37130545) to optimize the RNA structure and codon selection simultaneously, In some embodiments, the open reading frame (ORF) sequence is optimized using optimization algorithms.

[0188]

[0165] Like mRNA, saRNA is a linear, single-stranded RNA molecule. The structure generally includes; (a) a 5' cap, which protects the RNA from degradation and serves as the initiation site for translation; (b) a 5' untranslated region (UTR), important for the regulation of translation and stability of the RNA; (b) a self-amplifying region (replicase) containing non-structural proteins that allow the RNA to replicate itself inside of the host cell, permitting high levels of protein expression from small amount of RNA (compared to non- replicating RNA); (c) a subgenomic promoter (SGP); (d) at least one antigen; (e) a 3’ UTR that, similar to the 5’ UTR, is important for regulation and stability of the RNA; and (f) a polyA tail that protects the RNA, aiding in stability, as well as translation.

[0189]

[0166] In a particular embodiment the saRNA comprises: (a) a 5’ untranslated region (UTR), important for the regulation of translation and stability of the RNA (b) an IRES to initiate translation; (b) a self-amplifying region (replicase) containing non-structural proteins that allow the RNA to replicate itself inside of the host cell, permitting high levels of protein expression from small amount of RNA (compared to non-replicating RNA); (c) a subgenomic promoter (SGP); (d) at least one antigen; (e) a 3' UTR that, similar to the 5’ UTR, is important for regulation and stability of the RNA; and (f) a polyA tail that protects the RNA, aiding in stability, as well as translation. Attorney Docket No.: KEYL-001WO

[0190]

[0167] In a particular embodiment the saRNA comprises: (a) a 5’ cap. which protects the RNA from degradation and serves as the initiation site for translation: (b) a 5’ untranslated region (UTR), important for the regulation of translation and stability of the RNA: (b) a self¬ amplifying region (replicase) containing non- structural proteins that allow the RNA to replicate itself inside of the host cell, permitting high levels of protein expression from small amount of RNA (compared to non-replicating RNA); (c) an IRES sequence; (d) at least one antigen; (e) a 3' UTR that, similar to the 5’ UTR. is important for regulation and stability of the RNA; and (f) a poly A tail that protects the RNA, aiding in stability, as well as translation.

[0191]

[0168] In a particular embodiment, the saRNA encodes at least one antigen derived from R burgdorferi sensu stricto, B. afzelii, B. garinii, B. bavariensis, B. spielmanii, and B. mayonii or a combination thereof. In one embodiment, the saRNA encodes at least six antigens derived one each from B. burgdorferi sensu stricto, B. afzelii, B. garinii, B. bavariensis, B. spielmanii, and B. mayonii to provide global, species-level coverage. The antigen may be the same or different,

[0192]

[0169] In a particular embodiment, the saRNA encodes at least one OspA or a fragment thereof from serotype 1. serotype 2, serotype 3, serotype 4, serotype 5. serotype 6, serotype 7 or a combination thereof to provides serotype diversity.

[0193]

[0170] In a particular embodiment, the saRNA encodes at least one OspC or fragment thereof from genotype A, B. C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S. T, U or combinations thereof.

[0194]

[0171] The UTRs, the replicase and / or the SGP may be derived from a virus. In one embodiment, the virus is a positive-sense single-stranded RNA [(+)ssRNA] viruses, for example, alphaviruses, flaviviruses, lentiviruses, measles viruses, or rhabdo viruses.

[0195]

[0172] In a particular embodiment, the saRNA encodes one or more conserved antigens or fragments thereof, for example, epitopes from VlsE (e.g., IR6 / C6), DbpA / B, FLaB or BBK32) to ensure cross-strain immunity and long-term durability.

[0196]

[0173] In certain embodiments, the saRNA encodes OspA + BBK32 + DbpB.

[0197]

[0174] In certain embodiments, the saRNA encodes OspC + VlsE IR6 + FlaB.

[0198]

[0175] In certain embodiments, the saRNA encodes DbpA + BBK32 + Glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

[0199]

[0176] In certain embodiments, the saRNA encodes OspA (serotypes 1-7) + OspC (A-U genotypes) + Borrelia enolase protein.

[0200]

[0177] In certain embodiments, the saRNA encodes VlsE IR6 + DbpB + BBK32 + Boreelia Complement Regulator- Acquiring Surface Proteins (CRASP) proteins. Attorney Docket No.: KEYL-001WO

[0201]

[0178] In certain embodiments, the saRNA encodes at least one antigen, adjuvant or combination thereof that is membrane-tethered upon expression. In certain embodiments, membrane-tethering enhances antigen presentation, stability, immune recognition, or a combination thereof compared to a soluble form of the same antigen. The degree of enhancement may vary. In certain embodiment, the enhancement is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least 2-fold, at least 3-fold, at least 4-fold or at least 5-fold or more.

[0202]

[0179] The increase in antigen presentation may be measured by any suitable method, for example, utilizing flow cytometry for peptide-MHC complexes, co-culture assays measuring T cell cytokine release, proliferation assays of antigen-specific T cells, microscopy or imaging of antigen localization, in vivo immunization studies comparing IgG titers and protection.

[0203]

[0180] The increase in antigen stability may be measured by any suitable method, for example, utilizing ELISA binding assays, flow cytometry with peptide-MHC complexes, T cell activation assays, in vivo immunization studies.

[0204]

[0181] The increase in immune recognition may be measured by any suitable method, for example, utilizing flow cytometry for T cell activation markers, ELI Spot or intracellular cytokine staining for cytokine responses, CFSE or CTV proliferation assays, peptide-MHC complex quantification, tetramer staining, antibody binding assays such as ELISA or SPR / BLI, uptake and trafficking imaging, in vivo immunization endpoints.

[0205]

[0182] Non-limiting examples of Venezuelan Equine Encephalitis Virus (VEEV), Semliki Forest Virus (SFV), Sindbis Virus (SIN), Chikungunya Virus (CHIKV), Eastern Equine Encephalitis Virus (EEEV), Mayaro Virus (MAYV), Getah Virus (GETV), Ross River Virus (RRV), Una Virus (UNAV), Middleburg Virus (MIDV), O'nyong nyong virus (ONNV), Barmah Forest Virus (BFV), Mucambo Virus (MUCV), Tonate Virus (TONV), Everglades Virus (EVEV), Rio Negro Virus (RNV), Highlands J Virus (HJV), Western Equine Encephalitis Virus (WEEV), and Aura Virus (AURAV).

[0206]

[0183] In a particular embodiment, the UTRs, the replicase and / or the SGP are derived from Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), Sindbis virus (SINV), or Semliki Forest virus (SFV) or combinations thereof.

[0207]

[0184] The 5' cap is a modified nucleotide structure located at the 5' terminus of the saRNA that protects the RNA from degradation and enables recruitment of translation initiation factors. In certain embodiments, the 5' cap is linked to the RNA through a 5'-5' triphosphate bridge. The 5' cap may be configured as Cap 0 (m7GpppN), Cap 1 (m7GpppNm), or Cap 2 Attorney Docket No.: KEYL-001WO

[0208] (m7GpppNmNm), wherein N is a ribonucleotide and subscript m denotes 2'-O-methylation of the ribose. In certain embodiments, the 5' cap comprises a co-transcriptionally incorporated cap analog that enforces correct forward orientation during RNA synthesis. In certain embodiments, the cap analog is a trinucleotide Cap 1 -directing structure of the general form: m7G(5')-ppp-(5')-Ni-p-N2, wherein Ni and N2 are independently selected from natural or chemically modified nucleotides, and Ni may be 2'-O-methylated to impose the Cap 1 structure and reduce innate immune sensing. In certain preferred embodiments, the 5' cap comprises a Cap 1 -directing trinucleotide selected from: m7G(5')-ppp-(5')-Am-pU, or m7G(5')-ppp-(5')-Am-pG, wherein Amdenotes 2'-O-methyladenosine and U or G are uridine or guanosine residues, optionally 2'-O-methylated. Such Cap 1 -imposing structures increase translation efficiency, improve RNA stability, reduce interferon pathway activation, and are preferred for saRNA constructs intended for durable antigen expression. Non-limiting categories of cap structures suitable for use herein include Cap 0 structures, Cap 1 structures, Cap 2 structures, co-transcriptionally incorporated trinucleotide or tetranucleotide cap analogs phosphorothioate-modified cap analogs, inverted cap analogs, acyclic cap analogs, carbocyclic or L-ribose cap analogs, and combinations thereof. In certain embodiments, the 5' cap is introduced post-transcriptionally by enzymatic capping. In some embodiments, an uncapped or partially capped RNA is treated with an RNA guanylyltransferase and a 5'-triphosphatase to generate a Cap 0 structure (m⁷GpppN). In some embodiments, the Cap 0 structure is further converted to a Cap 1 structure (m7GpppNm) through 2'-O-methylation of the ribose at the first transcribed nucleotide (N) by an RNA 2'-O-methyltransferase. In certain embodiments, the Cap 1 structure is further converted to a Cap 2 structure (m7GpppNmNm) through 2'-O-methylation of the ribose at the second transcribed nucleotide (N+i) by a second RNA 2'-O-methyltransferase. In certain embodiments, the enzymatic capping reaction is performed on in vitro transcribed RNA following purification of the RNA or directly in crude transcription reaction mixtures. In some embodiments, S-adenosylmethionine (SAM) is included as a methyl donor for the formation of Cap 1 or Cap 2 structures. In some embodiments, the enzymatic capping procedure results in at least about 80%, at least about 85%, at least about 90%, at least about 95%, or greater than 98% conversion of uncapped RNA to the desired capped RNA species. In certain embodiments, the use of post-transcriptional capping enables the same RNA sequence to be formulated with different cap structures (e.g., Cap 0, Cap 1, or Cap 2) to tune innate immune sensing, translation efficiency, or antigen expression kinetics depending on the intended clinical application. Attorney Docket No.: KEYL-001WO

[0209]

[0185] The “5'-UTR” or “5'-UTR element” is located 5' (i.e., upstream) of a coding sequence and is transcribed but not translated. The 5' UTR plays a crucial role in the regulation of gene expression, including the initiation of translation and mRNA stability. A 5'-UTR may comprise elements for controlling gene expression, also called regulatory elements, such as ribosomal binding sites, miRNA binding sites, and the like. In certain embodiments, the 5' UTR is the genomic 5' UTR associated with the replicase open reading frame. In other embodiments, the 5' UTR is the subgenomic 5' UTR positioned upstream of an antigen coding sequence. Either or both UTRs may be varied independently to tune antigen expression levels. In certain embodiments, the 5' UTR is a heterologous UTR, i.e., a UTR found in nature associated with a different ORF. In other embodiments, the 5' UTR is a synthetic or de novo-engineered UTR that does not occur in nature. In certain embodiments, the 5' UTR is derived from a positivesense single-stranded RNA virus, including but not limited to alphaviruses (e.g., Venezuelan equine encephalitis virus (VEEV), Semliki Forest vims (SFV), Sindbis vims (SINV)), flaviviruses, or other (+)ssRNA viral genomes. In other embodiments, the 5' UTR is a synthetic sequence optimized to reduce secondary stmcture and enhance ribosome accessibility, thereby improving translation efficiency. In certain embodiments, the 5' UTR comprises a Kozak consensus motif or other translation-enhancing sequence. In certain embodiments, the 5' UTR is engineered to reduce activation of innate immune receptors, including but not limited to IFIT, RIG-I and MDA5, while preserving or enhancing translational initiation efficiency. The 3' untranslated region (“3' UTR”) is located downstream of a coding sequence and upstream of the poly(A) tail. The 3' UTR contributes to RNA stability, translation efficiency, intracellular localization, and interactions with host RNA-binding or regulatory proteins. In certain embodiments, the 3' UTR is configured in the context of a self-amplifying RNA (saRNA) genome, wherein the saRNA exists in vivo in at least two forms: (i) the full-length genomic RNA encoding the replicase, and (ii) one or more subgenomic RNAs encoding one or more antigens. In certain embodiments, the 3' UTR is present on both the genomic RNA and the subgenomic RNA(s). In other embodiments, the 3' UTR sequences of the genomic and subgenomic RNAs differ from one another to modulate differential stability or translation efficiency. In certain embodiments, the 3' UTR is derived from a positive-sense single-stranded RNA (+ssRNA) virus, such as an alphavirus (e.g., Venezuelan equine encephalitis virus (VEEV), Semliki Forest virus (SFV), or Sindbis virus (SINV)). In such embodiments, the 3' UTR may comprise one or more conserved sequence elements (CSEs) required for recognition by the replicase complex and efficient replication of the genomic and / or subgenomic RNAs. In certain embodiments, the 3' UTR comprises two or more tandem 3' UTR elements. The Attorney Docket No.: KEYL-001WO

[0210] elements may be homologous or heterologous and may be selected to promote increased RNA stability, enhanced translation efficiency, reduced activation of innate immune sensors (e.g., RIG-I, MDA5, PKR, OAS, or TLR7 / 8), and / or optimal cytoplasmic localization. In certain embodiments, the 3' UTR is synthetic or engineered de novo to (i) minimize destabilizing motifs, (ii) reduce inhibitory secondary structure, and / or (iii) tune antigen expression kinetics independently from replicase expression. In particular embodiments, distinct 3' UTR designs are used for the genomic RNA and the subgenomic RNA(s) to achieve controlled, staged, or sustained antigen production. In certain embodiments, a double, triple or quadruple UTR such as a 5’ UTR or 3’ UTR may be used in the saRNA constructed disclosed herein.

[0211]

[0186] The replicase comprises one or more nonstructural proteins (nsPs) necessary for transcription and replication of the self-amplifying RNA (saRNA). In certain embodiments, the replicase is encoded within genomic RNA and is translated directly from the genomic RNA following cellular uptake and delivery to the cytoplasm. The replicase proteins assemble into an RNA-dependent RNA polymerase (RdRp) complex that supports (i) replication of the full-length genomic RNA and (ii) transcription of one or more subgenomic RNAs that direct antigen expression. In certain embodiments, the replicase region comprises four nonstructural proteins (nsPl-nsP4), which are initially translated as a single polyprotein precursor (P1234). Following translation, Pl 234 undergoes regulated proteolytic processing to generate individual nsPs, which together form the active replicase complex. nsPl is involved in RNA capping and membrane association, nsP2 exhibits protease and helicase activities, nsP3 contributes to host interaction and RNA amplification scaffolding, and nsP4 provides the core RNA polymerase function.

[0212]

[0187] In certain embodiments, the replicase and / or nsP processing sites are derived from a positive-sense single-stranded RNA virus, such as an alphavirus (e.g., Venezuelan equine encephalitis virus (VEEV), Semliki Forest virus (SFV), or Sindbis virus (SINV)). In other embodiments, the replicase is a chimeric, codon-optimized, fidelity-engineered, attenuated, or otherwise modified replicase exhibiting altered replication kinetics, reduced cytopathic effect, enhanced antigen expression duration, or reduced innate immune activation.

[0213]

[0188] In some embodiments, the sequence, codon usage, or secondary structure of the replicase region is engineered to tune antigen expression kinetics, including adjusting replication rate, subgenomic transcription efficiency, or the temporal relationship between replicase expression and antigen expression. In certain embodiments, the replicase is derived from a Venezuelan equine encephalitis virus (VEEV) TC-83 and / or TRD lineages. Attorney Docket No.: KEYL-001WO

[0214]

[0189] In some embodiments, the replicase comprises one or more substitutions in nsP2, including but not limited to P773S and D584N. These substitutions are associated with modulation of replicase activity and cellular host response and may be used to adjust replication kinetics, cytopathic effect, antigen expression duration, and / or innate immune activation. In certain embodiments, the nsP2 substitutions are present individually or in combination. In some embodiments, the replicase is otherwise identical to that of a wild-type or vaccine lineage alphavirus except for such defined substitutions. In one embodiment, the self-amplifying RNA comprises a single subgenomic promoter configured to direct transcription of one antigenencoding subgenomic RNA. In one embodiment, the self-amplifying RNA comprises at least two subgenomic promoters, each independently controlling transcription of a corresponding antigen-encoding subgenomic RNA. In one embodiment, the self-amplifying RNA comprises three or more subgenomic promoters, wherein each subgenomic promoter directs transcription of a distinct antigen-encoding subgenomic RNA, thereby enabling multivalent antigen expression from a single saRNA molecule. In one embodiment, the two or more subgenomic promoters are identical in sequence. In one embodiment, the two or more subgenomic promoters are heterologous, synthetic, or sequence-divergent from one another to tune transcriptional efficiency and relative antigen expression levels. In one embodiment, the subgenomic promoter is derived from an alphavirus 26S promoter, including but not limited to VEEV, SFV, or SINV. In one embodiment, the subgenomic promoter is synthetic, consensus-optimized, or structure-optimized to enhance transcription efficiency, reduce RNA secondary structure, or increase replicase recognition. In one embodiment, the relative expression levels of encoded antigens are adjusted by altering the sequence, positioning, or promoter strength of one or more subgenomic promoters. The 3' poly(A) tail is an untranslated sequence of adenosine nucleotides located at the 3' end of the RNA that contributes to RNA stability, translation efficiency, and protection from exonucleolytic degradation. In certain embodiments, the poly(A) tail has a length of from about 25 to about 400 adenosine residues, including but not limited to about 50-400, about 50-300, about 50-250, or about 60-250 residues. In one embodiment, the poly(A) tail is about 100 adenosine nucleotides in length. In certain embodiments, the poly(A) tail is structured, comprising a first poly(A) segment, followed by a single non-adenosine nucleotide, followed by a second poly(A) segment, e.g., Ax-N-Ay, wherein N is a nucleotide other than adenosine (e.g., guanosine or cytidine), and x and y are independently selected lengths (e.g., 10-200 nucleotides each). Such structured poly(A) tails may reduce plasmid recombination, increase transcriptional stability, and / or enhance ribosomal loading. In one embodiment, the poly(A) tail comprises a Aw-G-Aw(SEQ Attorney Docket No.: KEYL-001WO

[0215] ID NO: 16) or A60-G-A60(SEQ ID NO: 17) architecture, wherein the single non- adenosine residue interrupts homopolymeric stretch length to increase construct stability during DNA template propagation and to maintain efficient translation following transcription. In certain embodiments, the poly(A) tail is encoded in the DNA template, whereas in other embodiments, the poly(A) tail is added post-transcriptionally, such as by a poly(A) polymerase reaction, allowing control over tail length and uniformity.

[0216]

[0190] In certain embodiments, the compositions disclosed herein comprise the saRNA formulated in a pharmaceutically acceptable carrier. In one embodiment, the saRNA is formulated in a lipid nanoparticle (LNP) comprising an ionizable lipid (or cationic lipid), cholesterol, a phospholipid, and a polyethylene glycol (PEG)-lipid, or functional equivalents thereof. The LNP may fully encapsulate the saRNA or may present the saRNA electrostatically complexed to the LNP surface. In other embodiments, the saRNA is formulated in a liposome, polymeric nanoparticle, peptide-based delivery system, virus-like particle (VLP), exosome, or other non- viral or viral delivery vehicle configured to facilitate cellular uptake and cytoplasmic release.

[0217]

[0191] In certain embodiments, the composition further comprises one or more pharmaceutically acceptable excipients, including buffers, salts, sugars, tonicity agents, bulking agents, cryoprotectants, stabilizers, and / or antioxidants. Non- limiting examples include citrate buffer, phosphate-buffered saline (PBS), sucrose, trehalose, mannitol, or combinations thereof, to some embodiments, the composition is provided in a lyophilized form and reconstituted prior to administration.

[0218]

[0192] In certain embodiments, the composition further comprises an adjuvant, which may be encoded within the saRNA itself (e.g., an immune-stimulatory cytokine such as IL- 12) or co-formulated or co-delivered (e.g., a TLR agonist, STING agonist, saponin-based adjuvant, or alum). In embodiments wherein the adjuvant is encoded within the saRNA, the adjuvant may be secreted, cytosolic, or membrane-tethered to localize immune stimulation to the site of antigen expression.

[0219]

[0193] In one embodiment, the adjuvant is IL- 12. IL- 12 plays a central role in lymphocyte proliferation and activation. It consists of a heterodimeric protein comprised of two subunits p35 (IL- 12 A) and p40 (IL-12B).

[0220]

[0194] The IL-12 may be expressed as a single-chain or heterodimeric form.

[0221]

[0195] In certain embodiments, the IL-12 is engineered to increase stability, secretion or localization. Attorney Docket No.: KEYL-001WO

[0222]

[0196] In one embodiment, the IL-12 comprises one or more mutations (e.g,, one, two, three, four, five or more mutations) to increase stability, secretion, localization or a combination thereof.

[0223]

[0197] In certain embodiments, the IL-2 is engineered to increase half-life. In one embodiment, the IL- 12 is a fusion protein engineered to increase half-life. The fusion domain may vary. In one embodiment, the fusion domain is an Fc domain, an albumin domain, an albumin binding domain, a transferrin domain or the like, In one embodiment, the fusion domain is another cytokine, such as IL-15.

[0224]

[0198] In certain embodiments, the IL-12 is engineered to permit targeting to a specific tissue or cell. In one embodiment, an antibody, antibody fragment or localizing domain (e.g., transmembrane domain) is fused to the IL-12,

[0225]

[0199] In certain embodiments, the self-amplifying RNA disclosed herein is a combination vaccine, i.e., encoding at least one antigen from Borrelia and at least one antigen from another virus, bacteria or fungi.

[0226]

[0200] In one embodiment, the self-amplifying RNA encodes at least one antigen from Borrelia and at least one antigen from the group of bacteria selected from Anaplasma phagocytophilum, Ehrlichia species, Borrelia miyamotoi, Borrelia mayonii, Rickettsia rickettsii (Rocky Mountain spotted fever), Francisella tularensis (tularemia), and Bartonella species.

[0227]

[0201] In one embodiment, the self-amplifying RNA encodes at least one antigen from Borrelia and at least one antigen from the group of viruses selected from Powassan virus and its subtype Deer Tick Virus.

[0228]

[0202] In one embodiment, the self-amplifying RNA encodes at least one antigen from Borrelia and at least one antigen from a parasite, e.g., Babesia microti.

[0229]

[0203] In certain embodiments, the composition is administered intramuscularly, subcutaneously, intradermally, or intranasally. In certain embodiments, the composition is administered as a single dose. In other embodiments, the composition is administered in two or more doses, wherein one or more subsequent doses boost the immune response.

[0230] Methods of Use

[0231]

[0204] Also disclosed are methods of using the compositions disclosed herein.

[0232]

[0205] In one embodiment a method is provided for producing an immune response comprising administering any composition disclosed herein to a subject, thereby producing an immune response. Attorney Docket No.: KEYL-001WO

[0233]

[0206] The immune response may be a cellular, humoral, innate, or adaptive reaction of the immune system to the one or more antigens. An immune response may include antibody production, B cell activation, T cell activation (including CD4+ and / or CD8+ responses), innate immune signaling, and / or cytokine or chemokine expression. In certain embodiments, the immune response comprises both humoral and cellular components.

[0234]

[0207] In certain embodiments, the immune response is a localized immune response, i.e., at the site(s) of infection. In one embodiment, the localized immune response permits increased efficacy but limits side effects (e.g., fever, fatigue, widespread inflammation and / or tissue damage). In certain embodiments, the immune response is localized to the skin, joints (e.g., knees), nervous system (e.g., peripheral nerves), heart or a combination thereof.

[0235]

[0208] The immune response may be measured by any suitable method. Representative, non¬ limiting measures include cytokine release assays (e.g., IFN-y ELISA) antibody detection (e.g., IgG, IgE binding assays), cell activation markers flow cytometry for T-cell or B-cell activity) and / or predictive modeling (e.g,, using biological and demographic data).

[0236]

[0209] In one embodiment, the method results in the production of production of antibodies that bind to a Borrelia species.

[0237]

[0210] In a particular embodiment, the method results in an IgG response that is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7 -fold. 8-fold, 9-fold or 10-fold higher or more than baseline, i.e., prior to administration of the saRNA construct disclosed herein.

[0238]

[0211] In a particular embodiment, the method results in an IgG responses that is relative to IgG response in a subject that is not vaccinated with the saRNA construct disclosed herein, or relative to an alternative vaccine against Lyme disease.

[0239]

[0212] In certain embodiments, the IgG response lasts for about 3, about 6, about 9, or about 12 months or more and provides protection against bacterial challenge. In one embodiment, the IgG response lasts for about one, about two. about three, about four or about five years or more and provide protection against bacterial challenge. In one embodiment, the method comprises administering any of the compositions disclosed herein to a subject in an amount effective to prevent Lyme disease or a Borrelia infection. In certain embodiments, the method comprising administered a booster dose to prevent Lyme disease or Borrelia infection, e.g., after about 6, about 9, about 12, about 16 or about 18 months or more.

[0240]

[0213] In one embodiment, the method results in an immune response that is enhanced relative to an immune response to an mRNA expressing the same antigen(s). The degree of increase may vary. In one embodiment, the immune response is enhanced about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%,, about 2-fold, 2- Attorney Docket No.: KEYL-001WO

[0241] fold, about 3-fold, about 5-fold, about 8-fold, about 10-fold, about 15-fold or longer compared to a mRNA encoding the same antigen(s).

[0242]

[0214] In certain embodiment, the method results in an IgG response that is sustained (maintained overtime) relative to an IgG response from a mRNA vaccine comprising the same antigenfs) as the self-amplifying RNA disclosed herein. In certain embodiments, the IgG response is maintained about 25%, about 50% about 100%, about 2-fold, about 3-fold, about 5-fold, about 8-fold, about 10-fold. about 15-fold or longer compared to a mRNA encoding the same antigen(s).

[0243]

[0215] In a particular embodiment, the method disclosed herein results in an increase of about 25 to about 50%. more particularly, about 30 to about 40% in IgG relative (in comparison to) to the same method using an mRNA encoding the same antigen(s) and a protective level of IgG is maintained for at least 12 months, more particularly, about 12 to about 14, about 12 to about 16 or about 12 to about 18 months. Optionally, a booster is administered at about 6, about 12, months.

[0244]

[0216] Lyme disease, also known as Lyme borreliosis occurs in stages characterized by different clinical manifestations, with remissions and exacerbations. Stage I, early infection, consists of a localized infection of the skin. Stage 2 follows days or weeks later and is characterized by disseminated infection. Stage 3 occurs months or years later and represents persistent infection. The methods disclosed herein may be used to prevent infection or to treat infection at any of stages 1-3. The subject may be at particular risk for Lyme disease or a Borrelia infection. For example, the subject may live in or be traveling to a geographic region where ticks are highly prevalent.

[0245]

[0217] The result of prevention may be an immune response that protects a subject against Lyme disease or another Borrelia infection. Prevention involves administering to a subject the antigen(s) in a manner to induce an immune reaction against the antigenfs). Thereafter, the subject may be exposed to or challenged with Borrelia. If the response is protective, Borrelia will not be detectable in the subject or will be detected at a lower levels than without vaccination. Detection can be accomplished by any means known in the art, including, for example PCR, ELISA or Western Blot.

[0246]

[0218] In embodiments where the method involves treatment, the subject may have been previously diagnosed with Lyme disease according to any suitable method. The result of treatment may be, for example, a reduction or amelioration in one or more symptoms of Borrelia infection. Attorney Docket No.: KEYL-001WO

[0247]

[0219] Representative, non-limiting examples of diagnostic methods include a two-tier serologic test for antibodies to B. burgdorferi, comprising a first ELISA assay followed by a second Western blot, where both are positive.

[0248]

[0220] Non-limiting examples of symptoms of Lyme disease or Borrelia infection that may be impacted by the methods described herein include rash (erythema migrans), fever, headache, tiredness, stiffness. facial paralysis, cardiac symptoms (e.g., carditis, valve disease, conduction system disease), neurological symptoms, arthritis or a combination thereof. Depending on the Borrelia species involved, clinical symptoms may vary. Borrelia burgdorferi infection in the United States is generally associated with more diverse symptoms than infection with other Borrelia species.

[0249]

[0221] The reduction in symptoms may be compared to symptoms prior to treatment or in a population of subjects, untreated vs. treated patients (e.g., control in a clinical trial). The reduction may vary. In one embodiment, the reduction is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100%.

[0250]

[0222] The result of treatment may also or alternatively be clearance of persistent infection.

[0251]

[0223] In a particular embodiment, the result of treatment is resolution of Erythema migrans (EM), normalization of cardiac conduction or a combination thereof.

[0252]

[0224] In a particular embodiment, the result of treatment is patient-reported reduction in fatigue, pain, and / or cognitive symptoms.

[0253]

[0225] In a particular embodiment, the result of treatment is return to normal daily activities, improved quality of life scores or a combination thereof.

[0254]

[0226] The result of treatment can be measured at any suitable time. In one embodiment, the result is measured at 5, 7, 10, 12 or 14 days after treatment.

[0255]

[0227] In a particular embodiment, the result of treatment is no recurrence of EM or systemic symptoms during follow-up, for example, at 3, 4, 5 or 6 months after treatment.

[0256]

[0228] In one embodiment, the results of treatment may be determined differently based on the stage of the subject at time of administration.

[0257]

[0229] In a particular embodiment, the subject is stage 1 and the result of treatment is (i) resolution of EM rash; (ii) absence of new lesions during follow up; and / or (iit) relief of systemic symptoms.

[0258]

[0230] In another particular embodiment, the subject is stage 2 and the result of treatment is (i) neurologic improvement (resolution of facial palsy, meningitis symptoms), cardiac recovery (normalization of AV block or conduction abnormalities), and / or (iii) absence of relapse during follow-up, e.g., 3, 4, 5 or 6 months. Attorney Docket No.: KEYL-001WO

[0259]

[0231] In a further particular embodiment, the subject is stage 3 and the result of treatment is (i) reduction in joint swelling and pain, improvement in neurologic deficits or neuropathy, improvement in quality-of-life scores (e.g.. SF-36. fatigue scales), and / or prevention of PTLDS (Post-Treatment Lyme Disease Syndrome).

[0260]

[0232] PTLDS is defined as the constellation of subjective symptoms including fatigue, cognitive dysfunction, or myalgia that result in disability and persist for at least 6 months after adequate treatment for a patient who has met diagnostic criteria for Lyme disease. (Wormser GP, et al (2006) Clin Infect Dis 43: 1089— 1134), Between 0.5 and 13.1% of patients with Lyme disease have reported nonspecific symptoms 6 months or later following treatment.

[0261]

[0233] In certain embodiments, the result of treatment is improved relatively to an mRNA comprising the same antigen(s). The degree of improvement may vary. In certain embodiments, the improvement is statistically significant. The duration of improvement may also vary, e.g., differ by a matter of days, weeks, months or years.

[0262]

[0234] The dose administered according to the methods outlined herein may vary. In certain embodiments, the dose administered to a subject is sufficient to prevent disease (delay its onset, or slow or stop its progression). One skilled in the art will recognize that dosage will depend upon a variety of factors including the strength of the composition employed, as well as the age, species, condition, and body weight of the subject. The size of the dose will also be determined by the route, timing, and frequency of administration as well as the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular composition and the desired physiological effect.

[0263]

[0235] In one embodiment, the dose is less than about 50 micrograms, less than about 45 micrograms, less than about 40 micrograms, less than about 35 micrograms, less than about 30 micrograms, less than about 25 micrograms, less than about 20 micrograms, less than about 15 micrograms or less than about 10 micrograms, but in each case greater than about 0.1 micrograms.

[0264]

[0236] In one embodiment, the dose is between about 1 microgram and 10 micrograms in humans, more particularly, between about 1 and about 9 micrograms, about 1 and about 8 micrograms, about 1 and about 7 micrograms, about 1 and about 6 micrograms, about 1 and about 5 micrograms, about 1 and about 4 micrograms, about 1 and about 3 micrograms or about 1 and about 2 micrograms.

[0265]

[0237] In one embodiment, the dose is between about 1 and about 3 micrograms, about 3 and about 5 micrograms, about 5 and about 7 micrograms or about 7 and about 10 micrograms. Attorney Docket No.: KEYL-001WO

[0266]

[0238] In certain embodiments, the dose is about 1, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5 about 7, about 7.5, about 8 and about 8.5, about 9, about 9.5 or about 10 micrograms or more.

[0267]

[0239] In certain embodiments, the dose is less than about 1 mg, more particularly, less than about.900 mg, less than about.800 mg. less than about.700 mg, less than about.600 mg, or less than about.500 micrograms.

[0268]

[0240] In one embodiment, the administration is a sole administration, i.e., a single dose. In certain embodiments, the sole administration is sufficient to provide prevention or treatment of infection or Lyme disease. Protection may be extended, for example, for months into years.

[0269]

[0241] In one embodiment, protection lasts for about one, about two, about three, about four, about five, about six. about seven, about eight, about nine, about 10, about 11 or about 12. months or more.

[0270]

[0242] In another embodiment, protection lasts tor about 18, about 24, about 36 or about 48 months or more,

[0271]

[0243] In certain embodiments, protection lasts for about 2, about 3, about 4, about 5. about 6, about 7 or about 8 years or more.

[0272]

[0244] In one embodiment, the administration is a sole administration, i.e., a single dose. In certain embodiments, the sole administration is sufficient to provide prevention or treatment of infection oi Lyme disease. Pi tection may be extended, for example, for months into years.

[0273]

[0245] In another embodiment, there are multiple administrations. Multiple administrations can include any number of two or more administrations, including two, three, four, five or six or more administrations. One skilled in the art can readily determine the number of administrations to perform or the desirability of performing one or more additional administrations according to methods known in the art for monitoring therapeutic methods and other monitoring methods provided herein. Accordingly, the methods provided herein include methods of providing to the subject one or more administrations of a pharmaceutical composition, where the number of administrations can be determined by monitoring the subject, and based on the results of the monitoring, determining whether or not to provide one or more additional administrations. Deciding to provide more than one administration (e.g., one, two or three or more) may optionally be based on one or more results of monitoring.

[0274]

[0246] The period between administrations can be any of a variety of time periods. The period can be determined by a variety of factors, including monitoring steps, as described in relation to the number of administrations. In one embodiment, the period can be a function of the time required for a subject to mount an immune response or greater the period required to mount an Attorney Docket No.: KEYL-001WO

[0275] immune response. In certain embodiments, the period can be about one week, about two weeks, about three weeks, about four weeks, about two months, about three months or about four months or more.

[0276]

[0247] The compositions disclosed herein (saRNA, pharmaceutical compositions) can optionally be administered in combination with one or more additional therapeutic agents. The therapeutic agent may be any suitable therapeutic agent, including an antibiotic or immune- modulators, e.g., drugs, monoclonal antibodies or cytokines, for example, such as interleukin-12 (IL- 12) or interleukin -15 (IL-15). The additional therapeutic agent may be co-adrninistered or may be expressed from the same saRNA molecule if genetically encodable.

[0277] EXAMPLES

[0278] Example 1. Synthesis of a self-amplifying RNA encoding multiple antigens associated with Lyme disease.

[0279]

[0248] This example describes the construction and synthesis of a self-amplifying RNA encoding multiple antigens associated with the causative agent of Lyme disease. A selfamplifying RNA vector was constructed with sequences derived from VEEV. The coding sequence for OspA derived from the B31 strain of Borreliella burgdorferi was preceded by a Kozak sequence and inserted downstream of the subgenomic promoter. The region with significant homology to the human LFA1 peptide was altered by substituting the amino acids YV at positions 165 and 166 with FT, and the amino acid T at position 170 with K, as previously detailed by Willett et al. (Willett et al., 2004, PMID: 14742868). After two stop codons, a short spacer sequence was inserted before an IRES derived from coxsackievirus B3 (CVB3) virus. After the IRES sequence, the coding sequence for OspC derived from the B31 strain of Borrealia burgdorferi was inserted. BspQI cut sites were removed from both coding sequences of OspA and OspC by introducing silent mutations as needed. The coding sequence of OspC was followed by the VEEV 3’ UTR, a 100 nucleotide polyA tract and a BspQI linearization site. The resulting saRNA vector was cloned into a backbone containing a pUC ori and kanamycin resistance gene. The map for the reference plasmid is depicted in Figure 1.

[0280] The plasmid sequences were assembled by Gibson assembly. The resulting assembled plasmid was transformed into NEB stable cells followed by outgrowth at 30C for 3 hours. The cells are plated on pre-warmed kanamycin containing plates and are grown overnight at 30C. Single colonies were isolated and grown in kanamycin containing LB media. Plasmid was isolated with the ZymoPure MidiPrep kit. The sequence was verified by whole plasmid sequencing. The resulting plasmid DNA was linearized with BspQI as recommended by New England Attorney Docket No.: KEYL-001WO

[0281] Biolabs. Following linearization, the DNA was purified with the Zymo DNA Clean & Concentrator kit. To produce the RNA, in vitro transcription was performed as follows:

[0282] 10X IVT Buffer 1 uL

[0283] CleanCap AU (100 mM) 0.4 uL

[0284] ATP (100 mM) 0.5 uL

[0285] 5 -methylcytidine (100 mM) 0.5 uL

[0286] GTP (100 mM) 0.5 uL

[0287] UTP (100 mM) 0.5 uL

[0288] Enzyme (NEB T7 Polymerase) 0.5 uL

[0289] YIPP (NEB) 0.5 uL

[0290] Superase Inhibitor (Invitrogen) 1 uL

[0291] DNA Template 1 ug

[0292] Water to 10 uL

[0293]

[0294]

[0249] The IVT reactions were conducted at 37C for 3 hours. The DNA template was degraded by the addition of 0.5 uL of TURBO DNase for 10 minutes at 37C. The RNA preparations were purified with the NEB Monarch purification kit prior and were eluted in RNA Storage Solution (Invitrogen). The RNA integrity and size were validated by denaturing gel electrophoresis. A total of 100 ng of saRNA was transfected into HEK293 cells cultured in a 96-well plate using MessengerMax reagent (ThermoFisher Scientific). Control transfections were performed with GFP-encoding saRNA or mock conditions. After 24 hours, cells were lysed on ice using 1% NP-40 lysis buffer supplemented with a complete Mini protease inhibitor tablet. Lysates were kept on ice for 30 minutes, followed by centrifugation to clear cell debris, and supernatants are transferred to fresh tubes. Protein concentration was quantified using a BCA assay (Pierce Rapid Gold BCA reagent), and samples are normalized accordingly. Normalized protein samples were combined with 4X NuPAGE LDS sample buffer and 10X NuPAGE reducing agent, then heated at 70°C for 10 minutes. The samples were electrophoresed on 4-12% Bis-Tris Mini gels at 90V for 45 minutes in IX MES running buffer. Each sample was analyzed multiple times for detection of different protein targets. Proteins were transferred to nitrocellulose membranes using the iBlot2 system. Membranes were blocked with 2.5% milk in PBS-T for 1 hour at room temperature. OspA expression was detected by incubating the membrane with a 1:1000 dilution of OspA rabbit antibody (Rockland, 200-401-C13) in 2.5% milk PBS-T for 3 hours at room temperature. For OspC Attorney Docket No.: KEYL-001WO

[0295] expression, a 1:1000 dilution of OspC rabbit antibody (Rockland, 200-401-C11) was used. Beta-actin was detected as a loading control using a 1:1000 dilution of beta- actin mouse antibody (Cell Signaling Technologies, 8H10D10). Following primary antibody incubation, membranes were washed three times with IX PBS-T for 5 minutes per wash. OspA and OspC were detected with anti-rabbit HRP conjugate (Cell Signaling Technologies, 7074), and betaactin was detected with anti-mouse DyLight 680 (Invitrogen, 35518). Secondary antibody incubations were performed for 30 minutes at room temperature, followed by three 5-minute washes with IX PBS-T. HRP-stained membranes were developed using SuperSignal West Pico substrate and imaged on an iBright imager. The DyLight 680-stained membranes were directly imaged using the same system. The expression of OspA was detected only in the lysate derived from the cells transfected with the saRNA encoding both OspA and OspC. The expression of OspC was detected only in the lysate derived from the cells transfected with the saRNA encoding both OspA and OspC. The expression of both Lyme disease antigens were observed after transfection with the multivalent self-amplifying RNA vector (FIG.2).

[0296] Example 2. Administration of a self-amplifying encoding Lyme disease antigens.

[0297]

[0250] This example details the preparation, administration, and evaluation of a selfamplifying RNA (saRNA) encoding OspA and / or OspC antigens, encapsulated within lipid nanoparticles (LNPs) for delivery. C57BL / 6 and BALB / c mice are immunized to assess antibody titers, T cell responses, and protection following challenge with Borrelia burgdorferi. The purified saRNA is formulated into LNPs comprised of ALC-0315, DSPC, ALC-0159, and Cholesterol. Groups of C3H, C57BL / 6 and BALB / c mice (n = 5 per strain) receive 50 pL of the LNP-formulated saRNA via intramuscular injection on Day 0, with a subset receiving a booster dose on Day 21. Control groups are administered saline. Serum samples are collected weekly to monitor immune response. Anti-OspA and / or anti-OspC IgG titers are measured using ELISA, with endpoint titrations to calculate geometric mean titers. At the end of the study, spleens and lymph nodes are harvested for T cell response analysis. Single-cell suspensions are prepared and stimulated with OspA and / or OspC peptide pools, followed by intracellular cytokine staining and flow cytometry to assess CD4+ and CD8+ T cell responses, including production of IFN-y, TNF-a, and IL-2. In separate challenge study, immunized and control C3H mice are challenged with Borrelia burgdorferi (B31 strain) intradermally or by tick challenge to assess vaccine efficacy. Mice are monitored over 2-4 weeks for infection markers, including erythema, joint swelling, and weight loss. At the conclusion of the observation period, tissues (ear, ankle joint, heart) are collected for quantitative PCR analysis Attorney Docket No.: KEYL-001WO

[0298] of Borrelia burgdorferi DNA load. Histological analysis is also conducted to evaluate inflammation and tissue pathology related to Lyme disease.

[0299] Example 3. Screening for self-amplifying RNA constructs conferring optimal immunogenicity against Lyme disease associated proteins.

[0300]

[0251] This example describes the development and evaluation of self-amplifying RNA (saRNA) encoding OspA and / or OspC proteins. Multiple constructs are evaluated and compared. Differences between the constructs include the presence of absence of different transmembrane domains appended onto the OspA and / or OspC coding sequences with or without a flexible glycine-serine linker sequence in between. Additionally, other candidate sequences include the addition of a signal peptide derived from human or mouse origin. The constructs are evaluated in vitro for expression levels on the cell surface, secreted into the cell culture supernatant, and cytosolic expression. The constructs are evaluated in vivo to determine the immunological properties of each candidate sequence. An ideal sequence will raise a neutralizing antibody response and / or a T cell response against all of the encoded Lyme disease antigens.

[0301] Example 4. A self-amplifying RNA encoding Lyme disease associated antigens elicits a strong and durable antibody response in mice.

[0302]

[0252] A self-amplifying RNA encoding OspA and OspC was constructed as described in Example 1. The RNA was prepared by in vitro transcription with 100% substitution of cytidine with 5-methylcytidine. The resulting RNA was encapsulated in LNPs comprised of 46.3 mol% ALC-0315, 42.7 mol% Cholesterol, 9.4 mol% DSPC, and 1.6 mol% ALC-0159. Female BALB / C mice approximately 20 weeks of age (n = 5 / group) were intramuscularly administered the LNPs containing 1000 ng or 10 ng of saRNA. As a control, an additional group of mice received PBS. Blood was collected by submandibular bleed on days 14, 28, and 112. The blood samples were allowed to clot for 15 minutes at room temperature before centrifugation at 3000g for 10 minutes. The resulting serum was frozen at -80C until analysis by ELISA. ELISA plates were coated overnight at 4C with 1 pg / mL of OspA protein from Native Antigen Company (REC32017) or OspC protein (REC32019). The serum samples were serially diluted and incubated for 2 hours after blocking. To detect the resulting IgG antibodies binding the coated antigens, a 1:10,000 dilution of anti-mouse IgG HRP secondary antibody was added for a 15-minute incubation. Between steps 5 washes were performed with a plate washer. To each well, 100 pL of TMB substrate was added and the plates were incubated in the dark for 15 minutes. To stop the reaction, 50 LIL of 2N sulfuric acid was added to each well. Both the 1000 ng and 10 ng doses elicited strong antibody responses against OspA (FIG.3 & FIG.4). The antibody Attorney Docket No.: KEYL-001WO

[0303] response was maintained for at least 114 days. Furthermore, antibody responses were observed against the encoded OspC antigen (FIG.5 & FIG.6). The mice that received PBS did not have an antibody response against either antigen, demonstrating that the saRNA vaccine elicited an antibody response against Lyme disease associated antigens. Unexpectedly, the titers were comparable between the single administration of 10 ng and 1000 ng. In a Borrelia burgdorferi neutralization experiment, the serum from day 112 from mice that received either 10 ng or 1000 ng significantly inhibited growth. Without wishing to be bound by theory, these results suggest the dose sparing advantage and replicating durable nature of self-amplifying RNA.

[0304] Example 5. Synthesis of a self-amplifying RNA encoding a single antigen associated with Lyme disease.

[0305]

[0253] This example describes the construction and synthesis of a self-amplifying RNA encoding a single antigen associated with the causative agent of Lyme disease. A selfamplifying RNA vector is constructed with sequences derived from VEEV. The coding sequence for OspA derived from a strain of Borreliella is preceded by a Kozak sequence and inserted downstream of the subgenomic promoter. In the case of Borrelia burgdorferi, a region with significant homology to the human LFA1 peptide is altered by substituting the amino acids YV at positions 165 and 166 with FT, and the amino acid T at position 170 with K, as previously detailed by Willett et al. (Willett et al., 2004, PMID: 14742868). BspQI cut sites are removed from the antigen coding sequence by introducing silent mutations. The coding sequence of OspA is followed by the VEEV 3’ UTR, a polyA tract and a BspQI linearization site. The resulting saRNA vector is cloned into a backbone containing a pUC ori and kanamycin resistance gene. The plasmid sequences are assembled by Gibson assembly. The resulting assembled plasmid is transformed into NEB stable cells followed by outgrowth at 30C for 3 hours. The cells are plated on pre-warmed kanamycin containing plates and are grown overnight at 30C. Single colonies are isolated and grown in kanamycin containing LB media. Plasmid is isolated with the ZymoPure MidiPrep kit. The sequence is verified by whole plasmid sequencing. The resulting plasmid DNA is linearized with BspQI. Following linearization, the DNA is purified and used as a template for in vitro transcription. To produce the RNA, in vitro transcription is performed with co-transcriptional capping. The resulting RNA is purified and encapsulated into lipid nanoparticles. The lipid nanoparticles are diluted in a suitable buffer to a desired concentration. The administration of the lipid nanoparticles containing the Lyme disease antigen encoding saRNA to a host results in an immune response. The immune response prevents the transmission of the relevant Borrelia strain upon exposure via tick bite. Attorney Docket No.: KEYL-001WO

[0306] Example 6. Self-Amplifying RNA Encoding OspA Induces Sustained Antibody Responses in Humans.

[0307]

[0254] In this example, a self-amplifying RNA encoding OspA is administered to a human subject. Following administration, serum is collected at multiple time points. The vaccine elicits antibodies specific for OspA that persist for at least 12 weeks, and in certain subjects at least 20 weeks. Antibody titers remain above baseline even in some subjects that did not receive a booster dose. Without being bound by theory, the replicase-mediated intracellular RNA amplification promotes prolonged antigen expression, thereby maintaining germinal center activity and supporting long-lived plasma cell development.

[0308] Example 7. Self-Amplifying RNA Vaccine Reduces Infection Following Tick Challenge.

[0309]

[0255] In this example, subjects vaccinated with the disclosed self-amplifying RNA encoding OspA are exposed to tick vectors harboring Borrelia burgdorferi. Vaccinated subjects exhibit reduced bacterial burden in blood and tissues relative to unvaccinated controls. Reduction in pathogen load is observed in both acute and late timepoints. In certain instances, the infection is prevented altogether. Without being bound by theory, sustained antigen presentation maintains antibody responses that provide ongoing protection through the full duration of the seasonal exposure period.

[0310] Example 8. Durability of Immune Responses Across Multiple Geographic Strains of Borrelia.

[0311]

[0256] In this example, multiple self-amplifying RNA constructs encoding OspA variants derived from multiple Borrelia strains are administered to a subject. Exemplary sequences are described by SEQ IDs 2, 3, 4, 5, 6, 7, 8, 9, 10, 11. The antigen-specific immune response exhibits reactivity to multiple geographically distinct variants. Antibody and / or T cell responses persist for extended durations. The durability of the immune response remains consistent even when the encoded antigens are derived from different strains. Without being bound by theory, persistent antigen expression and multivalent antigen selection broaden protective coverage across circulating strains.

[0312] Example 9. Design of a computationally optimized broadly protective OspA antigen to be expressed from self-amplifying RNA.

[0313]

[0257] This example describes the computational workflow used to generate a broadly reactive (COBRA) OspA consensus antigen. OspA protein sequences from pathogenic Borrelia genospecies were collected from public repositories and strain genomes, yielding 169 accessions. Sequences were screened to remove low-quality, truncated, or frame-shifted entries, and exact or near-duplicate records are collapsed, resulting in 91 non-redundant Attorney Docket No.: KEYL-001WO

[0314] sequences. A global multiple-sequence alignment was generated, and unsupervised clustering was performed on the aligned sequences; cluster validation identifies an optimal partition into eight clusters. For each cluster, a position-wise majority-rale consensus sequence is computed. A final COBRA consensus sequence is then derived by weighted voting across the eight cluster-level consensuses, with weights proportional to cluster sizes. Summary outputs include the final consensus FASTA, an identity matrix and heatmap comparing cluster consensuses, and a table mapping input sequences to cluster assignments.

[0315] Example 10. Broad protective immunity against diverse Borrelia strains following immunization with COBRA OspA saRNA.

[0316]

[0258] This example describes an evaluation of the breadth of protection conferred by a selfamplifying RNA encoding a computationally optimized broadly reactive (COBRA) OspA antigen. C3H mice are immunized by intramuscular administration of LNP-formulated saRNA encoding COBRA OspA (SEQ ID NO: 8) at doses of 10 ng or 1000 ng in a 50 pL injection volume. Control groups receive (i) PBS, (ii) LNP-formulated non-replicating mRNA encoding COBRA OspA, or (iii) LNP-formulated saRNA encoding a strain-specific OspA derived from B. burgdorferi B31 (SEQ ID NO: 2). Seram is collected at Weeks 2, 4, 8, and 12 postimmunization to assess antibody binding to a panel of recombinant OspA proteins derived from six phylogenetically distinct Borrelia species (B. burgdorferi, B. afzelii, B. garinii, B. mayonii, B. spielmanii, and B. bavariensis) using ELISA. Functional antibody activity is evaluated using complement-mediated killing assays and in vitro spirochete growth inhibition assays performed separately for each strain. At Day 28 post-immunization, animals are challenged with Ixodes ticks infected with one of three distinct Borrelia strains representing North American, Western European, and Eastern European phylogenetic clusters. The challenge strains include B. burgdorferi, B. afzelii, and B. garinii. Animals are monitored for signs of infection, including erythema, joint swelling, and weight change. At Day 35 post-challenge, tissues including ear, heart, and ankle joint are collected for Borrelia DNA quantification by qPCR. Histological evaluation of joint and heart tissue is performed to assess inflammatory infiltration and synovial pathology. Mice immunized with the COBRA OspA saRNA exhibit antibody binding to all tested OspA variants, whereas animals immunized with strain-specific OspA primarily bind antigens from the homologous strain. Following tick challenge, animals immunized with COBRA OspA saRNA display reduced or undetectable Borrelia DNA levels across all three challenge strains, while animals receiving strain-specific OspA saRNA show reduced pathogen load only in B31 challenge groups. Minimal inflammatory pathology is observed in mice immunized with COBRA OspA saRNA, while control and strain-restricted Attorney Docket No.: KEYL-001WO

[0317] vaccine groups exhibit joint and cardiac inflammation. These results indicate that the COBRA OspA saRNA construct elicits broad protective immunity across antigenically diverse Borrelia strains.

[0318] Example 11. A self-amplifying RNA vaccine encoding Lyme disease associated antigens is protective in mice.

[0319]

[0259] Mice (n =4 / group) are intramuscularly vaccinated with a 100 ng dose of saRNA (SEQ ID 1) encapsulated in LNPs comprised of SMI 02, DSPC, Cholesterol, and DMG-PEG2K. As a negative control, an additional cohort of mice receive PBS. After 3 weeks, the mice are boosted by an additional 100 ng dose of saRNA or PBS. On day 41, serum is collected to confirm antibody titers against OspA and OspC by ELISA. Between days 42 and 49, the mice are challenged with ticks harboring Borrelia burgdorferi. On day 70, the mice are euthanized, and qPCR is performed to assess Borrelia burgdorferi genome levels within the heart and joint tissues. The 100 ng saRNA vaccinations result in protective effects and no copies of genomic DNA are detected in the heart and joint. In contrast, the mice that received PBS have detectable genomic in the heart and joint. Cultures of heart and bladder tissue are performed to detect viable Borrelia. In the PBS control treated mice, the cultures are all positive for both the heart and bladder. The mice that received the saRNA vaccine does not have any positive cultures, indicating that the vaccine is effective at preventing transmission of the Borrelia bacteria from the tick challenge.

[0320] Example 12. Development of a multivalent saRNA vaccine against Lyme disease that covers geographic variants.

[0321]

[0260] In this example, multiple bivalent self-amplifying RNA constructs encoding OspA variants. The two trivalent saRNA constructs each encode three distinct OspA serotypes selected from Table 1 below. They may encode the serotypes in any order and combinations, resulting in two constructs covering all six serotypes. Exemplary sequences are described by SEQ IDs 9, 10. The two trivalent saRNA constructs encoding three distinct OspA variants each (6 total) are administered to a subject. The antigen-specific immune response exhibits crossreactivity to multiple geographically distinct variants. Antibody and / or T cell responses persist for extended durations. The durability of the immune response remains consistent even when the encoded antigens are derived from different strains. Without being bound by theory, persistent antigen expression and multivalent antigen selection broaden protective coverage across circulating strains. Attorney Docket No.: KEYL-001WO

[0322] Table 1.) Representative OspA serotypes, genospecies, and strains. The strains in the table below are exemplary and not exhaustive, different strains and genospecies may exist for the different serotypes included in a multivalent vaccine.

[0323] OspA serotype Genospecies Representative strain

[0324] ST 1 B. burgdorferi sensu stricto B31

[0325] ST2 B. afzelii K78

[0326] ST3 B. garinii PBr

[0327] ST4 B. bavariensis PBi

[0328] ST5 B. garinii PHei

[0329] ST6 B. garinii DK29

[0330] Example 13. Development of a multivalent saRNA vaccine against Lyme disease that covers geographic variants.

[0331]

[0261] In this example, a single self-amplifying RNA construct encodes six OspA variants derived from distinct serotypes. The construct contains three OspA sequences (STI, ST2, ST3) downstream of the first subgenomic promoter, linked together by (GGGS)3 linkers (SEQ ID NO: 18). At the end of the ST3 antigen is a stop codon followed by a spacer and an IRES sequence. After the IRES, three additional OspA sequences (ST4, ST5, ST6) are present, linked together by (GGGS)3 linkers (SEQ ID NO: 18). An exemplary sequence is described by SEQ ID 11. The multivalent saRNA construct encoding the six distinct OspA variants each is administered to a subject. The antigen-specific immune response induces protection to multiple geographically distinct variants. Antibody and T cell responses persist for extended durations. The durability of the immune response remains consistent even when the encoded antigens are derived from different strains. Without being bound by theory, persistent antigen expression and multivalent antigen selection broaden protective coverage across circulating strains.

Claims

Attorney Docket No.: KEYL-001WOWHAT IS CLAIMED IS:

1. A self-amplifying RNA construct comprising:a. a 5’ cap structure,b. a nucleic acid sequence comprising, 5’ to 3 ’:(i) a nucleotide sequence encoding nonstructural proteins nsp 1 -nsp4 or functional variants thereof;(ii) at least one subgenomic promoter (SGP);(iii) a nucleotide sequence encoding one or more antigens derived from a Borrelia species operably linked to the subgenomic promoter, wherein optionally, the one or more antigens are engineered to comprise a transmembrane domain;(iv) a poly(A) tail.

2. The self-amplifying RNA construct of claim 1, wherein the one or more antigens are selected from the group consisting of outer surface protein A (OspA), outer surface protein B (OspB), outer surface protein C (OspC), outer surface protein D (OspD), outer surface protein E (OspE), outer surface protein F (OspF), decorin binding protein A (DbpA), decorin binding protein B (DbpB), Erp proteins, VlsE, Ag45, P66 protein, BmpA (P39), CspA, BB0405, BptA, P13, RevA, RevB, Lmpl, BBK07, BBK12, or combinations thereof.

3. The self-amplifying RNA construct of claims 1-2, wherein the Borrelia species is selected from the group consisting of Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii, Borrelia mayonii, Borrelia spielmanii, Borrelia bavariensis, Borrelia lusitaniae or combinations thereof.

4. The self- amplifying RNA constructs of claims 1-3, wherein the Borrelia species is Borrelia burgdorferi.

5. The self-amplifying RNA construct of claims 1-4, wherein the self-replicating RNA virus is a positive-sense single-stranded RNA virus.

6. The self-amplifying RNA construct of claims 1-5, wherein the self- replicating RNA virus is an alphavirus.

7. The self-amplifying RNA of claims 1-4, wherein the self-replicating RNA virus is selected from the group consisting of: Venezuelan Equine Encephalitis Virus (VEEV), Semliki Forest Virus (SFV), Sindbis Virus (SIN), Chikungunya Virus (CHIKV), Eastern Equine Encephalitis Virus (EEEV), Mayaro Virus (MAYV), Getah VirusAttorney Docket No.: KEYL-001WO(GETV), Ross River Virus (RRV), Una Virus (UNAV), Middleburg Virus (MIDV), O'nyong nyong virus (ONNV), Barmah Forest Virus (BFV), Mucambo Virus (MUCV), Tonate Virus (TONV), Everglades Virus (EVEV), Rio Negro Virus (RNV), Highlands J Virus (HJV), Western Equine Encephalitis Virus (WEEV), and Aura Virus (AURAV), or engineered variants thereof.

8. The self-amplifying RNA construct of claims 1-7, wherein the nucleic acid sequence is selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NOTO, SEQ ID NO: 11 or a variant thereof.

9. The self-amplifying RNA construct of claims 1-8, wherein the nucleic acid sequence encodes two or more Borrelia antigens.

10. The self-amplifying RNA construct of claim 9, wherein the two or more Borrelia antigens are selected from the group consisting of outer surface protein A (OspA), outer surface protein B (OspB), outer surface protein C (OspC), outer surface protein D (OspD), outer surface protein E (OspE), outer surface protein F (OspF), decorin binding protein A (DbpA), decorin binding protein B (DbpB), Erp proteins, VlsE, Ag45, P66 protein, BmpA (P39), CspA, BB0405, BptA, Pl 3, RevA, RevB, Lmpl, BBK07, BBK12, or any combinations thereof.

11. The self-amplifying RNA construct of claim 10, wherein the two or more Borrelia antigens are selected from the group consisting of OspA, OspC, DbpA, DbpB, VlsE, or any combinations thereof.

12. The self-amplifying RNA construct of claim 9-11, wherein the two or more borrelia antigens are OspA, OspC or fragments thereof.

13. The self-amplifying RNA construct of claim 12, wherein the OspA and OspC or fragments thereof are derived from Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii, Borrelia mayonii, Borrelia spielmanii, Borrelia bavariensis, Borrelia lusitaniae or combinations thereof.

14. The self-ampliying RNA construct of claim 12-13, wherein the OspA and OspC are derived from Borrelia burgdorferi or variants thereof.

15. The self-amplifying RNA construct of claims 9-14, wherein the nucleic acid sequence encoding the first antigen is operably linked to the nucleotide encoding the second antigen by a nucleotide sequence selected from the group consisting of a 2A sequence, an IRES sequence or a sequence encoding a second subgenomic promoter.Attorney Docket No.: KEYL-001WO16. The self-amplifying RNA construct of claims 1-8, wherein the nucleic acid sequence encodes three or more Borrelia antigens.

17. The self-amplifying RNA construct of claims 16, wherein the three or more Borrelia antigens are selected from the group consisting of outer surface protein A (OspA), outer surface protein B (OspB), outer surface protein C (OspC), outer surface protein D (OspD), outer surface protein E (OspE), outer surface protein F (OspF), decorin binding protein A (DbpA), decorin binding protein B (DbpB), Erp proteins, VlsE, Ag45, P66 protein, BmpA (P39), CspA, BB0405, BptA, Pl 3, RevA, RevB, Lmpl, BBK07, BBK12, or any combinations thereof.

18. The self-amplifying RNA construct of claims 16-17, wherein a nucleotide sequence selected from the group consisting of a 2A sequence, an IRES sequence or a sequence encoding a second or third subgenomic promoter operably links (i) the nucleotide sequence encoding the first and second antigen and (ii) the nucleotide sequence encoding the second and third antigen.

19. The self- amplifying RNA construct of claims 1-14, wherein the nucleic acid sequence encoding at least one antigen is operably linked to a nucleic acid sequence encoding a transmembrane domain.

20. The self- amplifying RNA construct of claims 9-19, wherein the nucleic acid sequence encoding a first antigen is operably linked to a nucleic acid sequence encoding a first transmembrane domain and the nucleic acid sequence encoding a second antigen is operably linked to a nucleic acid sequence encoding a second transmembrane domain.

21. The self- amplifying RNA construct of claims 1-20, wherein the transmembrane domain is selected from the group consisting of domains derived from Hemagglutinin (HA), SARS-CoV-2 Spike, ICAM-1, CD8 alpha, CD4, CD28, PDGFR, T-cell receptor alpha, MHC class I, B7-1 / CD80, GP64, VSV-G, erythropoietin receptor, IL-2 receptor alpha, ICOS, 4-1BB (CD137), 0X40 (CD134), Fas, HVEM (TNFRSF14), CD3 zeta, CTLA-4, CD27, SLAMF7, CD2, CD19, CD22, CD33, CD44, CD74, CD99, CD 150, CD200, CCR5, CXCR4, CD40, CD83, Notch, LFA-1 (CD 11 a), integrin beta-1 (ITGB1), integrin alpha-L (ITGAL), integrin alpha-V (ITGAV), transferrin receptor (CD71), E-cadherin, N-cadherin, CD7, CD10, CD45, CD123, CCR7, CX3CR1, CD47, SIRP-alpha, TROP2, TNFR1, TNFR2, RANK (TNFRSF11A), CD137L (4-1BBL), LIGHT (TNFSF14), TRAILR1 (DR4). TRAILR2 (DR5), TLR2, TLR4, TLR9, CR2 (CD21), CD79a, CD79b, CD86, CD23, Fc receptor gamma (FcRy), or CD 18.Attorney Docket No.: KEYL-001WO22. The self-amplifying RNA construct of claims 1-21, wherein the nucleic acid sequence further comprises at least one 5’ untranslated region (UTR) derived from a 5’ UTR of a self-replicating RNA virus, positioned 5’ to the nucleotide sequence encoding nonstructural proteins nspl-nsp4 or functional variants thereof.

23. The self-amplifying RNA construct of claims 1-22, wherein the nucleic acid sequence further comprises a 3’ untranslated region (UTR), derived from a 3’ UTR of a selfreplicating RNA virus, positioned between the nucleotide sequence encoding one or more antigens derived from a Borrelia species and the poly(A) tail.

24. A plasmid comprising a nucleotide sequence corresponding to the nucleic acid sequence of the self-amplifying RNA construct of claims 1-23.

25. A pharmaceutical composition comprising the self-amplifying RNA construct of claims 1-23 or the plasmid of claim 24 and a pharmaceutically acceptable carrier.

26. The pharmaceutical composition of claim 25, further comprising a second selfamplifying RNA construct, wherein the sequence of the first and second selfamplifying constructs are different.

27. The pharmaceutical composition of claim 26, wherein the two or more constructs encode at least one different antigen.

28. A method of eliciting an immune response in a subject in need thereof, comprising administering a self-amplifying RNA construct according to claims 1-23, the plasmid of claim 24 or the pharmaceutical composition of claims 25-27 to the subject, thereby eliciting an immune response.

29. A method of preventing Lyme disease, comprising administering a self-amplifying RNA construct according to claims 1-23, the plasmid of claim 24 or the pharmaceutical composition of claims 25-27 to a subject in need thereof, thereby preventing Lyme disease.

30. A method of treating Lyme disease, comprising administering a self-amplifying RNA construct according to claims 1-23, the plasmid of claim 24 or the pharmaceutical composition of claims 25-27 to a subject in need thereof, thereby treating Lyme disease.

31. The method of claim 30, wherein the subject has been diagnosed with Stage 1, 2 or 3 Lyme disease.

32. The method of claims 30-31, wherein treating comprises a reduction or elimination of one or more symptoms of Lyme disease.Attorney Docket No.: KEYL-001WO33. The method of claims 30-31, wherein treating comprises an eradication of persistent infection.

34. The method of claims 29-33, wherein the pharmaceutical composition comprises a lipid nanoparticle (LNP) formulation.

35. The method of claims 29-34, wherein the self-amplifying RNA construct or pharmaceutical composition is disposed within or on a microneedle patch.

36. A method of inducing an immune response in a subject in need thereof, comprising administering two or more self-amplifying RNA constructs of claims 1-23 or plasmids of claim 24 to the subject, wherein the nucleotide sequences of the two or more constructs are different and the administration is sequential.

37. A method of preventing Lyme Disease in a subject in need thereof, comprising administering two or more self-amplifying RNA constructs of claims 1-23 or plasmids of claim 24 to the subject, wherein the nucleotide sequences of the two or more constructs are different and wherein the administration is sequential.

38. A method of treating Lyme Disease in a subject in need thereof, comprising administering two or more self-amplifying RNA constructs of claims 1-23 or plasmids of claim 24 to the subject, wherein the nucleotide sequences of the two or more constructs are different and wherein the administration is sequential.

39. The method of claim 38, the subject has been diagnosed with Stage 1, 2 or 3 Lyme disease.

40. The method of claim 38-39, wherein the treating comprises a reduction or elimination of one or more symptoms of Lyme disease.

41. The method of claims 38-39, wherein the treating comprises an eradication of persistent infection.

42. A method of producing the self-amplifying RNA construct of any of claims 1-23 comprising:a. synthesizing the RNA using in vitro transcription (IVT) from a linearized DNA template;b. capping the RNA co-transcriptionally or post-transcriptionally with a cap analog; andc. purifying the RNA using chromatography techniques to remove contaminants.

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