Lyme disease vaccine comprising adjuvant and borrelia outer surface protein antigen

Abstract

The present disclosure relates to immunogenic compositions comprising a Borrelia (Borrelia) outer surface protein A (OspA) antigen or an RNA polynucleotide encoding the OspA antigen, and an adjuvant. In some aspects, the immunogenic compositions are suitable for use in stimulating an immune response against Borrelia in a subject. The present disclosure also relates to kits of the immunogenic compositions, uses of the immunogenic compositions and methods of using the immunogenic compositions.

Description

Cross-reference to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 512,872, filed July 10, 2023, the contents of which are incorporated herein by reference in their entirety. Submitting sequence lists as XML files

[0002] This application is submitted together with the electronic sequence list. The contents of the electronic sequence list (377882009240SEQLIST.xml; size: 86,006 bytes; and creation date: July 9, 2024) are incorporated herein by reference in their entirety. Technical Field

[0003] This disclosure relates to immunogenic compositions comprising Borrelia outer surface protein A (OspA) antigen or RNA polynucleotide encoding said OspA antigen and an adjuvant. In some aspects, said immunogenic compositions are suitable for stimulating an immune response against Borrelia in a subject. This disclosure also relates to articles comprising said immunogenic compositions, uses of said immunogenic compositions, and methods of using said immunogenic compositions. Background Technology

[0004] Lyme borreliosis, or Lyme disease, is the most commonly reported tick-borne disease in Europe and North America. The disease is caused by infection with spirochetal bacteria belonging to the genus *Borrelia*. A vaccine based on a monovalent serotype 1-OspA (LYMErix™) was approved in the United States for the prevention of Lyme disease caused by *Borrelia burgdorferi sensu stricto(ss)*, but this vaccine is no longer available. Currently, there are no commercially available preventative drugs for Lyme disease. Therefore, there is an unmet need for a safe and effective Lyme disease vaccine. Summary of the Invention

[0005] In some embodiments, this document provides an immunogenic composition comprising (i) a spirochetal outer surface protein A (OspA) antigen or an RNA polynucleotide encoding an OspA antigen and (ii) an adjuvant selected from aluminum salt adjuvants; Toll-like receptor 9 (TLR9) agonists; liposome-based adjuvants comprising lipopolysaccharides and saponins; and an oil-in-water emulsion comprising squalene, sorbitol esters, and polyoxyethylene sorbitol esters.

[0006] In some embodiments, this document also provides an immunogenic composition comprising (i) a spirochetal outer surface protein A (OspA) antigen and (ii) an adjuvant selected from aluminum salt adjuvants; Toll-like receptor 9 (TLR9) agonists; liposome-based adjuvants comprising lipopolysaccharides and saponins; and an oil-in-water emulsion comprising squalene, sorbitol esters, and polyoxyethylene sorbitol esters.

[0007] In any of the embodiments, the immunogenic composition does not contain an RNA polynucleotide encoding a non-OspA spirochetal outer surface protein antigen.

[0008] In any of the embodiments, the immunogenic composition does not contain non-OspA spirochetal outer surface protein antigens.

[0009] In some embodiments, the adjuvant is an aluminum salt adjuvant. In some embodiments, the immunogenic composition further comprises a second adjuvant. In some embodiments, the second adjuvant is selected from TLR9 agonists; liposome-based adjuvants comprising lipopolysaccharides and saponins; and oil-in-water emulsions comprising squalene, sorbitol esters, and polyoxyethylene sorbitol esters.

[0010] In some embodiments, the adjuvant is a TLR9 agonist. In some embodiments, the immunogenic composition further comprises a second adjuvant. In some embodiments, the second adjuvant is selected from aluminum salt adjuvants; liposome-based adjuvants comprising lipopolysaccharides and saponins; and oil-in-water emulsion-based adjuvants comprising squalene, sorbitol esters, and polyoxyethylene sorbitol esters.

[0011] In some embodiments, the adjuvant is a liposome-based adjuvant comprising lipopolysaccharide and saponin. In some embodiments, the immunogenic composition further comprises a second adjuvant. In some embodiments, the second adjuvant is selected from aluminum salt adjuvants; TLR9 agonists; and oil-in-water emulsion-based adjuvants comprising squalene, sorbitol esters, and polyoxyethylene sorbitol esters.

[0012] In some embodiments, the adjuvant is an oil-in-water emulsion-based adjuvant comprising squalene, sorbitol ester, and polyoxyethylene sorbitol ester. In some embodiments, the immunogenic composition further comprises a second adjuvant. In some embodiments, the second adjuvant is selected from aluminum salt adjuvants; TLR9 agonists; and liposome-based adjuvants comprising lipopolysaccharides and saponins.

[0013] In some embodiments, the TLR9 agonist is an oligonucleotide comprising an unmethylated cytidine-phosphate-guanosine (CpG) motif. In some embodiments, the oligonucleotide is 10 to 35 nucleotides in length.

[0014] In some embodiments, the oligonucleotide comprises the sequence 5'-AACGTTCGAG-3' (SEQ ID NO:2). In some embodiments, the oligonucleotide comprises the sequence 5'-TGACTGTGAA CGTTCGAGAT GA-3' (SEQ ID NO:1).

[0015] In some embodiments, this document also provides an immunogenic composition comprising a spirochetal outer surface protein A (OspA) antigen and a Toll-like receptor 9 (TLR9) agonist oligonucleotide of 10 to 35 nucleotides in length, the oligonucleotide comprising the sequence 5'-AACGTTCGAG-3' (SEQ ID NO:2) and an unmethylated cytidine-phosphate-guanosine (CpG) motif.

[0016] In some embodiments, the oligonucleotide comprises the sequence 5'-TGACTGTGAA CGTTCGAGAT GA-3' (SEQ ID NO:1).

[0017] In some embodiments, this document also provides an immunogenic composition comprising a spirochetal outer surface protein A (OspA) antigen and a Toll-like receptor 9 (TLR9) agonist oligonucleotide 5'-TGACTGTGAA CGTTCGAGAT GA-3' (SEQ ID NO:1), said oligonucleotide comprising an unmethylated cytidine-phosphate-guanosine (CpG) motif.

[0018] In some embodiments, the immunogenic composition further comprises a second adjuvant. In some embodiments, the second adjuvant is selected from aluminum salt adjuvants; liposome-based adjuvants comprising lipopolysaccharides and saponins; and oil-in-water emulsion-based adjuvants comprising squalene, sorbitol esters, and polyoxyethylene sorbitol esters.

[0019] In some implementations, the second adjuvant is an aluminum salt adjuvant.

[0020] In some embodiments, this document also provides an immunogenic composition comprising: (i) a spirochetal outer surface protein A (OspA) antigen, (ii) a toll-like receptor 9 (TLR9) agonist oligonucleotide 5'-TGACTGTGAA CGTTCGAGAT GA-3' (SEQ ID NO:1), the oligonucleotide comprising an unmethylated cytidine-phosphate-guanosine (CpG) motif, and (iii) an aluminum salt adjuvant.

[0021] In some embodiments, the aluminum salt adjuvant comprises one or more of amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate. In some embodiments, the aluminum salt adjuvant comprises aluminum hydroxide.

[0022] In some embodiments, the immunogenic composition comprises about 0.25 mg to about 1.25 mg Al 3+ Approximately 0.25 mg to approximately 0.50 mg Al 3+ Or approximately 0.30 mg to approximately 0.40 mg Al 3+ .

[0023] In some embodiments, the oligonucleotide is a single-stranded oligodeoxynucleotide. In some embodiments, the oligonucleotide is entirely RNA. In some embodiments, the oligonucleotide is an RNA / DNA chimera.

[0024] In some embodiments, the oligonucleotide contains at least one phosphate thioester bond. In some embodiments, the oligonucleotide contains only phosphate thioester bonds. In some embodiments, the oligonucleotide contains a combination of one or more phosphodiester bonds and one or more phosphate thioester bonds.

[0025] In some embodiments, the immunogenic composition comprises about 375 µg to about 6000 µg of a TLR9 agonist or about 750 µg to about 3000 µg of a TLR9 agonist. In some embodiments, the immunogenic composition comprises about 375 µg, about 750 µg, about 1000 µg, about 1500 µg, about 3000 µg, or about 6000 µg of a TLR9 agonist.

[0026] In some embodiments, the lipopolysaccharide is lipid A or a derivative thereof. In some embodiments, the lipopolysaccharide is monophosphoryl lipid A or a derivative thereof. In some embodiments, the lipopolysaccharide is 3-O-deacylated-4'-monophosphoryl lipid A. In some embodiments, the lipopolysaccharide is 3-deacylated-phosphorylated hexaacyl disaccharide.

[0027] In some embodiments, the immunogenic composition comprises about 1 μg to about 100 μg, about 10 μg to about 50 μg, about 20 μg to about 30 μg, about 21 μg to about 29 μg, about 22 μg to about 28 μg, about 23 μg to about 27 μg, or about 24 μg to about 26 μg of lipopolysaccharide. In some embodiments, the immunogenic composition comprises about 1 μg to about 30 μg, about 5 μg to about 15 μg, about 6 μg to about 14 μg, about 7 μg to about 13 μg, about 8 μg to about 12 μg, or about 9 μg to about 11 μg of lipopolysaccharide. In some embodiments, the immunogenic composition comprises about 1 μg to about 9 μg, about 2 μg to about 8 μg, about 3 μg to about 7 μg, or about 4 μg to about 6 μg of lipopolysaccharide.

[0028] In some embodiments, the saponin is derived from the bark of the soapberry tree *Quillaja saponaria* Molina. In some embodiments, the saponin is Quil A or a derivative thereof. In some embodiments, the saponin is QS-17. In some embodiments, the saponin is QS-21.

[0029] In some embodiments, the immunogenic composition comprises about 1 μg to about 100 μg, about 10 μg to about 50 μg, about 20 μg to about 30 μg, about 21 μg to about 29 μg, about 22 μg to about 28 μg, about 23 μg to about 27 μg, or about 24 μg to about 26 μg of saponin. In some embodiments, the immunogenic composition comprises about 1 μg to about 30 μg, about 5 μg to about 15 μg, about 6 μg to about 14 μg, about 7 μg to about 13 μg, about 8 μg to about 12 μg, or about 9 μg to about 11 μg of saponin. In some embodiments, the immunogenic composition comprises about 1 μg to about 9 μg, about 2 μg to about 8 μg, about 3 μg to about 7 μg, or about 4 μg to about 6 μg of saponin.

[0030] In some embodiments, the liposome-based adjuvant further comprises a sterol. In some embodiments, the sterol is β-sitosterol, stigmasterol, ergosterol, ergocalciferol, or cholesterol. In some embodiments, the sterol is cholesterol.

[0031] In some embodiments, the ratio of saponin to sterol is 1:1 to 1:100 w / w or 1:1 to 1:5 w / w. In some embodiments, the ratio of saponin to sterol is about 1:1 w / w.

[0032] In some embodiments, the liposome-based adjuvant further comprises a neutral lipid. In some embodiments, the neutral lipid is phosphatidylcholine. In some embodiments, the phosphatidylcholine is egg yolk phosphatidylcholine, dioleoylphosphatidylcholine (DOPC), or dilauryl phosphatidylcholine. In some embodiments, the phosphatidylcholine is DOPC.

[0033] In some embodiments, the sorbitol ester is sorbitol ester 20. In some embodiments, the sorbitol ester is sorbitol ester 85.

[0034] In some embodiments, the polyoxyethylene sorbitol ester is polyoxyethylene sorbitol ester 20. In some embodiments, the polyoxyethylene sorbitol ester is polyoxyethylene sorbitol ester 80.

[0035] In some embodiments, the immunogenic composition comprises about 2% w / v to about 7% w / v, about 3% w / v to about 6% w / v, or about 4% w / v to about 5% w / v of squalene.

[0036] In some embodiments, the immunogenic composition comprises about 0.2% w / v to about 0.8% w / v, about 0.3% w / v to about 0.7% w / v, or about 0.4% w / v to about 0.6% w / v of sorbitol ester.

[0037] In some embodiments, the immunogenic composition comprises about 0.2% w / v to about 0.8% w / v, about 0.3% w / v to about 0.7% w / v, or about 0.4% w / v to about 0.6% w / v of polyoxyethylene sorbitol ester.

[0038] In some embodiments, the OspA antigen is two or more OspA antigens. In some embodiments, the two or more OspA antigens are a first OspA antigen and a second OspA antigen. In some embodiments, the two or more OspA antigens are a first OspA antigen, a second OspA antigen, and a third OspA antigen. In some embodiments, each of the two or more OspA antigens has a different amino acid sequence.

[0039] In any of the embodiments, the OspA antigen (e.g., the first OspA antigen, the second OspA antigen, and / or the third OspA antigen) is independently a Borrelia burgdorferi, Borrelia afzelii, Borrelia bavariensis, Borrelia garinii, Borrelia mayonii, Borrelia lusitaniae, Borrelia bissettii, Borrelia valasiana, or Borrelia spielmanii OspA antigen. In any of the embodiments, the OspA antigen is a Borrelia burgdorferi OspA antigen.

[0040] In some embodiments, the OspA antigen (e.g., a first OspA antigen, a second OspA antigen, and / or a third OspA antigen) independently comprises one or more epitopes from an OspA protein. In some embodiments, the one or more epitopes comprise at least two, three, four, or five epitopes from one or more OspA proteins. In some embodiments, the one or more epitopes comprise at least two, three, four, or five epitopes from at least two, three, four, or five OspA serotypes.

[0041] In some embodiments, the one or more epitopes comprise epitopes selected from the amino acid sequences shown in SEQ ID NO:4-49. In some embodiments, the one or more epitopes are independently selected from the amino acid sequences shown in SEQ ID NO:4-49.

[0042] In some embodiments, the one or more epitopes comprise an epitope selected from the amino acid sequences shown in SEQ ID NO:9, 10, 33, 34, 36, 47 and 49.

[0043] In any of some embodiments, the OspA antigen (e.g., the first OspA antigen, the second OspA antigen, and / or the third OspA antigen) independently has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any of SEQ ID NO:50-62.

[0044] In any of some embodiments, the OspA antigen (e.g., the first OspA antigen, the second OspA antigen, and / or the third OspA antigen) independently comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in any of SEQ ID NO:50-62.

[0045] In any of some embodiments, the OspA antigen (e.g., the first OspA antigen, the second OspA antigen, and / or the third OspA antigen) independently comprises the amino acid sequence shown in any one of SEQ ID NO:50-62.

[0046] In some embodiments, the OspA antigen (e.g., a first OspA antigen, a second OspA antigen, and / or a third OspA antigen) is independently an OspA chimeric antigen comprising multiple epitopes from one or more OspA proteins. In some embodiments, the multiple epitopes comprise at least two, three, four, five, or six epitopes from at least two, three, four, five, or six OspA serotypes.

[0047] In some embodiments, the plurality of epitopes comprises epitopes selected from the amino acid sequences shown in SEQ ID NO:4-49. In some embodiments, the plurality of epitopes are independently selected from the amino acid sequences shown in SEQ ID NO:4-49.

[0048] In some embodiments, the plurality of epitopes comprises epitopes selected from the amino acid sequences shown in SEQ ID NO: 9, 10, 33, 34, 36, 47, and 49. In some embodiments, the plurality of epitopes are independently selected from the amino acid sequences shown in SEQ ID NO: 9, 10, 33, 34, 36, 47, and 49.

[0049] In some embodiments, the OspA antigen (e.g., the first OspA antigen, the second OspA antigen, and / or the third OspA antigen) independently comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any of SEQ ID NO:63-82.

[0050] In any of some embodiments, the OspA antigen (e.g., the first OspA antigen, the second OspA antigen, and / or the third OspA antigen) independently comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in any of SEQ ID NO:63-82.

[0051] In any of some embodiments, the OspA antigen (e.g., the first OspA antigen, the second OspA antigen, and / or the third OspA antigen) independently comprises the amino acid sequence shown in any one of SEQ ID NO:63-82.

[0052] In some embodiments, the OspA antigen (e.g., the first OspA antigen, the second OspA antigen, and / or the third OspA antigen) independently comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any of SEQ ID NO:79-81.

[0053] In some embodiments, the immunogenic composition comprises a first OspA antigen and a second OspA antigen, the first OspA antigen comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 79, and the second OspA antigen comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 80. In some embodiments, the immunogenic composition comprises a first OspA antigen containing the amino acid sequence shown in SEQ ID NO: 79 and a second OspA antigen containing the amino acid sequence shown in SEQ ID NO: 80.

[0054] In some embodiments, the immunogenic composition comprises a first OspA antigen and a second OspA antigen, the first OspA antigen comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 79, and the second OspA antigen comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 81. In some embodiments, the immunogenic composition comprises a first OspA antigen containing the amino acid sequence shown in SEQ ID NO: 79 and a second OspA antigen containing the amino acid sequence shown in SEQ ID NO: 81.

[0055] In some embodiments, the immunogenic composition comprises a first OspA antigen and a second OspA antigen, the first OspA antigen comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 80, and the second OspA antigen comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 81. In some embodiments, the immunogenic composition comprises a first OspA antigen containing the amino acid sequence shown in SEQ ID NO: 80 and a second OspA antigen containing the amino acid sequence shown in SEQ ID NO: 81.

[0056] In some embodiments, the immunogenic composition comprises a first OspA antigen, a second OspA antigen, and a third OspA antigen, wherein the first OspA antigen comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:79; the second OspA antigen comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:80; and the third ... or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:80; and the third OspA antigen comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO The amino acid sequence shown in SEQ ID NO:81 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the immunogenic composition comprises a first OspA antigen containing the amino acid sequence shown in SEQ ID NO:79, a second OspA antigen containing the amino acid sequence shown in SEQ ID NO:80, and a third OspA antigen containing the amino acid sequence shown in SEQ ID NO:81.

[0057] In some implementations, the OspA antigen (e.g., the first OspA antigen, the second OspA antigen, and / or the third OspA antigen) is independently non-esterified.

[0058] In some implementations, the OspA antigen (e.g., the first OspA antigen, the second OspA antigen, and / or the third OspA antigen) is independently lipid-modified.

[0059] In some implementations, the immunogenic composition is in a dose of 0.3 mL to 1.5 mL.

[0060] In some embodiments, the immunogenic composition is used to stimulate an immune response against *Borrelia* spp. in a subject by administering an effective amount of the immunogenic composition to the subject. In some embodiments, the immunogenic composition is used to protect a subject from *Borrelia* spp. infection by administering an effective amount of the immunogenic composition to the subject. In some embodiments, the immunogenic composition is used to prevent a subject from developing Lyme disease by administering an effective amount of the immunogenic composition to the subject.

[0061] In some implementations, the subjects are human subjects.

[0062] In some embodiments, the human subject is at least 18 years old. In some embodiments, the human subject is between 18 and 70 years old.

[0063] In some embodiments, the human subjects are under 18 years of age. In some embodiments, the human subjects are between 2 and 5 years of age. In some embodiments, the human subjects are between 5 and 18 years of age.

[0064] In some embodiments, this document also provides a method for stimulating an immune response against the genus *Borrelia* in a subject, the method comprising administering to the subject an effective amount of any immunogenic composition provided.

[0065] In some embodiments, this document also provides a method for protecting a subject from infection with the genus *Treponema*, the method comprising administering to the subject an effective amount of any immunogenic composition provided.

[0066] In some embodiments, this document also provides a method for preventing a subject from developing Lyme disease, the method comprising administering to the subject an effective amount of any immunogenic composition provided.

[0067] In some implementations, the immunogenic composition is administered via intramuscular injection.

[0068] In some embodiments, a first dose and a second dose of the immunogenic composition are administered to the subject. In some embodiments, a second dose of the immunogenic composition is administered approximately 2 weeks to approximately 10 weeks after administration of the first dose. In some embodiments, a second dose of the immunogenic composition is administered approximately 1 month or approximately 2 months after administration of the first dose.

[0069] In some embodiments, a third dose of the immunogenic composition is administered to the subject. In some embodiments, a third dose of the immunogenic composition is administered approximately 4 to approximately 8 months after the administration of the first dose. In some embodiments, a third dose of the immunogenic composition is administered approximately 6 months after the administration of the first dose.

[0070] In some embodiments, a booster dose of the immunogenic composition is administered to the subject. In some embodiments, a booster dose is administered at least about 1 year or at least about 2 years after the first dose.

[0071] In some implementations, multiple booster doses are administered to the subject. In some implementations, the first booster dose is administered at least about 1 year or at least about 2 years after the first dose. In some implementations, a booster dose is administered annually.

[0072] In some implementations, the subjects are human subjects.

[0073] In some embodiments, the human subject is at least 18 years old. In some embodiments, the human subject is between 18 and 70 years old.

[0074] In some embodiments, the human subjects are under 18 years of age. In some embodiments, the human subjects are between 2 and 5 years of age. In some embodiments, the human subjects are between 5 and 18 years of age.

[0075] In some embodiments, this document also provides the use of any of the immunogenic compositions provided for stimulating an immune response against the genus *Treponema* in a subject by administering an effective amount of the immunogenic composition to the subject.

[0076] In some embodiments, this document also provides for the use of any immunogenic composition provided for protecting a subject from spirochetal infection by administering an effective amount of the immunogenic composition to the subject.

[0077] In some embodiments, this document also provides for the use of any immunogenic composition provided for preventing Lyme disease in a subject by administering an effective amount of the immunogenic composition to the subject.

[0078] In some embodiments, this document also provides for the use of any of the immunogenic compositions provided in the manufacture of a medicament for stimulating an immune response against the genus *Treponema* in a subject.

[0079] In some embodiments, this document also provides for the use of any of the immunogenic compositions provided in the manufacture of a medicament for protecting a subject from infection by the genus *Treponema*.

[0080] In some embodiments, this document also provides for the use of any of the immunogenic compositions provided in the manufacture of a medicament for preventing a subject from developing Lyme disease.

[0081] In some implementations, the immunogenic composition is administered via intramuscular injection.

[0082] In some embodiments, a first dose and a second dose of the immunogenic composition are administered to the subject. In some embodiments, a second dose of the immunogenic composition is administered approximately 2 weeks to approximately 10 weeks after administration of the first dose. In some embodiments, a second dose of the immunogenic composition is administered approximately 1 month or approximately 2 months after administration of the first dose.

[0083] In some embodiments, a third dose of the immunogenic composition is administered to the subject. In some embodiments, a third dose of the immunogenic composition is administered approximately 4 to approximately 8 months after the administration of the first dose. In some embodiments, a third dose of the immunogenic composition is administered approximately 6 months after the administration of the first dose.

[0084] In some embodiments, a booster dose of the immunogenic composition is administered to the subject. In some embodiments, a booster dose is administered at least about 1 year or at least about 2 years after the first dose.

[0085] In some implementations, multiple booster doses are administered to the subject. In some implementations, the first booster dose is administered at least about 1 year or at least about 2 years after the first dose. In some implementations, a booster dose is administered annually.

[0086] In some implementations, the subjects are human subjects.

[0087] In some embodiments, the human subject is at least 18 years old. In some embodiments, the human subject is between 18 and 70 years old.

[0088] In some embodiments, the human subjects are under 18 years of age. In some embodiments, the human subjects are between 2 and 5 years of age. In some embodiments, the human subjects are between 5 and 18 years of age.

[0089] In some embodiments, this document also provides vials containing any of the immunogenic compositions provided.

[0090] In some embodiments, the immunogenic composition is in liquid form. In some embodiments, the immunogenic composition is in lyophilized form.

[0091] In some embodiments, the vial contains a single dose of the immunogenic composition. The single dose is 0.3 mL to 1.5 mL.

[0092] In some embodiments, this document also provides pre-filled syringes comprising any of the immunogenic compositions provided.

[0093] In some embodiments, the syringe contains a single dose of the immunogenic composition. In some embodiments, the single dose is 0.3 mL to 1.5 mL.

[0094] In some embodiments, this document also provides a kit comprising: a) a first composition comprising an adjuvant selected from aluminum salt adjuvants; a Toll-like receptor 9 (TLR9) agonist; a liposome-based adjuvant comprising lipopolysaccharide and saponin; and an oil-in-water emulsion-based adjuvant comprising squalene, sorbitol ester, and polyoxyethylene sorbitol ester; b) a second composition comprising the outer surface protein A (OspA) antigen of *Treponema pallidum*; and c) instructions for combining the first and second compositions to prepare any immunogenic composition provided.

[0095] In some embodiments, the second composition is in liquid form. In some embodiments, the second composition is in lyophilized form.

[0096] In some embodiments, the instructions are for preparing a single dose of the immunogenic composition. In some embodiments, the kit is for a single dose of the immunogenic composition. In some embodiments, the single dose is a dose of 0.3 mL to 1.5 mL.

[0097] In some embodiments, the first composition and / or the second composition comprises a second adjuvant. In some embodiments, the first composition comprises a second adjuvant. In some embodiments, the second composition comprises a second adjuvant. In some embodiments, both the first composition and the second composition each comprise a second adjuvant.

[0098] In some embodiments, the second adjuvant is an aluminum salt adjuvant. In some embodiments, the aluminum salt adjuvant comprises one or more of amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate. In some embodiments, the aluminum salt adjuvant comprises aluminum hydroxide.

[0099] In some embodiments, the kit further comprises: d) an additional set of instructions for administering the immunogenic composition to a subject to stimulate an immune response against the genus *Treponema* in the subject.

[0100] In some embodiments, this document also provides a kit comprising: a) any immunogenic composition provided; and b) instructions for administering the immunogenic composition to a subject to stimulate an immune response against the genus *Borrelia* in the subject.

[0101] In some embodiments, the kit further includes a syringe for intramuscular injection of the immunogenic composition. In some embodiments, the syringe is pre-filled with the immunogenic composition.

[0102] In some embodiments, this document also provides a kit comprising: a) any vial provided; and b) instructions for administering the immunogenic composition to a subject to stimulate an immune response against the genus *Borrelia* in the subject.

[0103] In some embodiments, the kit further includes a syringe for intramuscular injection of the immunogenic composition.

[0104] In some embodiments, this document also provides a kit comprising: a) any pre-filled syringe provided; and b) instructions for administering the immunogenic composition to a subject to stimulate an immune response against the genus *Borrelia* in the subject.

[0105] In some implementations, the syringe is used for intramuscular injection of the immunogenic composition.

[0106] In some implementations, application is performed according to any method provided.

[0107] In some implementations, the subjects are human subjects.

[0108] In some embodiments, the human subject is at least 18 years old. In some embodiments, the human subject is between 18 and 70 years old.

[0109] In some embodiments, the human subjects are under 18 years of age. In some embodiments, the human subjects are between 2 and 5 years of age. In some embodiments, the human subjects are between 5 and 18 years of age. Attached Figure Description

[0110] Figure 1 This diagram illustrates three chimeric OspA antigens, each combining two different serotypes of OspA. The top image shows a schematic of the OspA antigens, divided into a C-terminal portion (top; solid color) and an N-terminal portion (bottom; shaded pattern). The bottom image shows the three chimeric OspA antigens: S1 / 2 (left), S6 / 4 (middle), and S5 / 3 (right), each containing different N-terminal and C-terminal portions from the indicated serotypes.

[0111] Figure 2A-2GThis demonstrates antibody responses to different OspA antigens, as detected by ELISA, after administration of one of three OspA antigens or a mixture of OspA antigens (non-lipidated OspA chimeric antigen, non-lipidated OspA full-length serotype 1 (S1) antigen, or lipidated OspA chimeric antigen), without the use of adjuvants (Ø) or with the use of one of the following adjuvant formulations: aluminum hydroxide (alum), CpG 1018. ® Adjuvant (Dynavax Technologies Corporation, Emeryville, CA; CpG) or a combination of CpG and alum (CpG + alum). Figure 2A The antibody response to OspA S5 / 3 after administration of the non-lipidated OspA chimeric antigen was shown when different adjuvant formulations were used. Figure 2B The antibody response to OspA S6 / 4 after administration of the non-lipidated OspA chimeric antigen was shown when different adjuvant formulations were used. Figure 2C The antibody response to OspA S1 / 2 after administration of the non-lipidated OspA antigen is shown when different adjuvant formulations are used. Figure 2D The antibody response to the full-length OspA S1 antigen was shown after administration of the non-esterified OspA antigen, with different adjuvant formulations. Figure 2E The antibody response to OspA S1 / 2 after administration of the lipotropic OspA chimeric antigen was shown when different adjuvant formulations were used. Figure 2F The antibody response to OspA S5 / 3 after administration of the esterified OspA chimeric antigen was shown with different adjuvant formulations. Figure 2G The antibody response to OspA S6 / 4 after administration of the lipotropic OspA antigen shown is illustrated using formulations with different adjuvants. The fold increase (FI) in titer levels between certain immunogenic compositions is also indicated at the top of the figure. Additionally, for… Figure 2E-2G The median antibody titer is also displayed at the top of each condition.

[0112] Figures 3A-3C The figures show the results of ELISA detection of OspA chimeric antigen S1 / 2 (labeled "1") after the first injection (labeled "1") or the second injection (labeled "2") of the lipotropic OspA chimeric antigen and the indicated adjuvant formulation. Figure 3A S5 / 3 Figure 3B ) and S6 / 4 ( Figure 3CThe antibody response is indicated. The fold increase in titer level (FI) between the first and second injections is also indicated on the top of each immunogenic composition. Detailed Implementation

[0113] This disclosure relates to immunogenic compositions comprising the outer surface protein A (OspA) antigen of *Borrelia* spp. or an RNA polynucleotide encoding said OspA antigen and an adjuvant. In some aspects, said immunogenic compositions are suitable for stimulating an immune response against *Borrelia* spp. in a subject. This disclosure also relates to articles comprising said immunogenic compositions, uses of said immunogenic compositions, and methods of using said immunogenic compositions.

[0114] All publications (including patent documents, scientific articles, and databases) mentioned in this application are incorporated herein by reference in their entirety for all purposes, to the extent that each individual publication is incorporated individually by reference. If any definition described herein contradicts or is otherwise inconsistent with the definitions described in patents, applications, published applications, and other publications incorporated herein by reference, the definitions described herein shall prevail over those incorporated herein by reference.

[0115] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the topics described. General technologies and definitions

[0116] Unless otherwise indicated, the practice of this disclosure will employ conventional techniques of molecular biology (including recombinant technologies), microbiology, cell biology, biochemistry, and immunology, which are within the scope of the art.

[0117] As used herein and in the appended claims, unless otherwise indicated, the singular forms “a”, “an”, and “the” include plural references. For example, “a” excipient includes one or more excipients.

[0118] As used herein, the phrase “comprising” is open-ended, indicating that such embodiments may include additional elements. In contrast, the phrase “consisting of” is closed-ended, indicating that such embodiments do not include additional elements (other than trace impurities). The phrase “substantially constitutes” is partially closed-ended, indicating that such embodiments may further include elements that do not substantially alter the essential characteristics of such embodiments.

[0119] As used herein, the term “about” refers to 90% to 110% of the value (e.g., about 3000 µg of CpG adjuvant means 2700 µg to 3300 µg of CpG adjuvant).

[0120] As used interchangeably herein, the terms “polynucleotide” and “oligonucleotide” include single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded RNA (ssRNA), and double-stranded RNA (dsRNA); modified oligonucleotides and oligonucleotides; or combinations thereof. Oligonucleotides can be linearly or cyclically configured, or may include both linear and cyclic segments. Oligonucleotides are nucleoside polymers typically linked by phosphodiester bonds, but alternative bonds, such as thiophosphates, can also be used in oligonucleotides. Nucleosides consist of purine (adenine (A) or guanine (G) or derivatives thereof) or pyrimidine (thymine (T), cytosine (C), or uracil (U) or derivatives thereof) bases bonded to sugars. The four nucleoside units (or bases) in DNA are called deoxyadenosine, deoxyguanosine, thymine, and deoxycytidine. Nucleotides are phosphate esters of nucleosides.

[0121] As used herein, the terms “CpG,” “CpG motif,” and “cytosine-phosphate-guanosine” refer to unmethylated cytosine-phosphate-guanosine dinucleotides that, when present in oligonucleotides, contribute to measurable immune responses in vitro, in vivo, and / or ex vivo. Examples of measurable immune responses include antigen-specific antibody production, cytokine secretion, and activation or expansion of lymphocyte populations such as NK cells, CD4+ T lymphocytes, CD8+ T lymphocytes, or B lymphocytes. Preferably, CpG-containing oligonucleotides preferentially activate Th1 responses.

[0122] The term "antigen" refers to a substance that is specifically recognized and bound by an antibody or a T-cell antigen receptor. Antigens can include peptides, polypeptides, proteins, glycoproteins, polysaccharides, complex carbohydrates, sugars, gangliosides, lipids, and phospholipids; portions thereof; and combinations thereof. In the context of this disclosure, the term "antigen" generally refers to a polypeptide or protein antigen of at least eight amino acid residues in length, which may contain one or more post-translational modifications.

[0123] Unless otherwise specified, the terms "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues and are not limited to a certain length. A peptide may include native amino acid residues or combinations of native and non-native amino acid residues. The term also includes post-expression modifications of the peptide, such as glycosylation, sialylation, acetylation, phosphorylation, etc. In some aspects, a peptide may include modifications relating to its natural or native sequence, provided that the protein maintains the desired activity (e.g., antigenicity).

[0124] As used herein, the terms "isolated" and "purified" refer to material that has been removed from at least one component naturally associated with it (e.g., removed from its original environment). When used with respect to recombinant proteins, the term "isolated" refers to a protein that has been removed from the culture medium of the host cells from which the protein was produced. In some embodiments, the isolated protein (e.g., a spirochetal antigen) is at least 75%, 90%, 95%, 96%, 97%, 98%, or 99% pure, as determined by high-performance liquid chromatography (HPLC).

[0125] An “effective amount” or “sufficient amount” of a substance is an amount sufficient to achieve beneficial or desired results, including clinical outcomes, and therefore, an “effective amount” depends on the context in which it is being applied. In the context of administering an immunogenic composition, an effective amount comprises sufficient antigen and one or more adjuvants to stimulate an immune response (preferably a serum-protective level of antibodies against said antigen).

[0126] In this publication, the terms “individual” and “subject” refer to mammals. “Mammalians” include humans, non-human primates (e.g., monkeys), livestock, locomotion animals, rodents (e.g., mice and rats), and pets (e.g., dogs and cats).

[0127] As used herein with respect to immunogenic compositions, the term “dosage” refers to a measured portion of the immunogenic composition taken (administered or received) by a subject at any given time.

[0128] A “stimulus” for a response or parameter includes the induction and / or enhancement of that response or parameter when compared to a condition that is otherwise identical except for the target parameter, or alternatively, when compared to another condition. For example, a “stimulus” for an immune response means an increase in the response. Depending on the parameter being measured, the increase can be from 2 to 200 times or more, from 5 to 500 times or more, from 10 to 1000 times or more, or from 2, 5, 10, 50, or 100 times to 200, 500, 1,000, 5,000, or 10,000 times.

[0129] Conversely, “suppression” of a response or parameter includes reducing and / or inhibiting the response or parameter when compared to conditions that are otherwise identical except for the target parameter, or alternatively, when compared to another condition. For example, “suppression” of an immune response means a reduction in the response. Depending on the parameter being measured, the reduction can be from 2 to 200 times, from 5 to 500 times or more, from 10 to 1000 times or more, or from 2, 5, 10, 50, or 100 times to 200, 500, 1,000, 2,000, 5,000, or 10,000 times.

[0130] The relative terms "higher" and "lower" refer to a measurable increase or decrease in response or parameter when compared to conditions that are otherwise identical except for the target parameter, or alternatively, when compared to another condition. For example, "higher antibody titer" means that the antigen-reactive antibody titer resulting from the administration of the immunogenic composition of this disclosure is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher than the antigen-reactive antibody titer resulting from the administration of the immunogenic composition of this disclosure. Similarly, "lower antibody titer" means that the antigen-reactive antibody titer resulting from the administration of the immunogenic composition of this disclosure is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times lower than the antigen-reactive antibody titer resulting from the administration of the immunogenic composition of this disclosure when the control condition is used.

[0131] As used herein, the term "immunization" refers to the process of increasing a subject's response to an antigen and thus improving their ability to resist or overcome infection and / or disease.

[0132] As used in this article, “vaccination” refers to the introduction of a vaccine into a subject’s body.

[0133] "Adjuvant" refers to a substance that, when added to a composition containing an antigen, enhances or strengthens the immune response of a subject to the antigen after exposure.

[0134] As used herein, when referring to amino acid sequences (reference polypeptide sequences), “amino acid sequence identity percentage (%)”, “identity percentage”, and “sequence identity” are defined as the percentage of amino acid residues in a candidate sequence (e.g., a subject antigen) that are identical to amino acid residues in a reference polypeptide sequence after alignment of the sequences and, where necessary, introduction of vacancies to achieve the maximum sequence identity percentage and without treating any conserved substitutions as part of the sequence identity. Alignments used to determine the amino acid sequence identity percentage can be performed in various ways within the scope of the art (e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0135] Amino acid substitution can involve replacing one amino acid in a polypeptide with another. Amino acid substitutions can be introduced into a target antigen, and the desired activity of the product (e.g., increased stability and / or immunogenicity) can be screened.

[0136] Amino acids can usually be grouped according to the following common side-chain characteristics: (1) Hydrophobicity: Leucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Alkaline: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; and (6) Aromatics: Trp, Tyr, Phe.

[0137] Conservative amino acid substitution involves the exchange of a member from one of these categories with another member from the same category. Non-conservative amino acid substitution involves the exchange of a member from one of these categories with a member from another category. I. Immunogenic Composition

[0138] This disclosure relates to immunogenic compositions containing a spirochetal outer surface protein A (OspA) antigen or an RNA polynucleotide encoding an OspA antigen and an adjuvant. In some embodiments, the immunogenic composition contains a spirochetal OspA antigen and an adjuvant. In some embodiments, the adjuvant is any adjuvant described herein, such as any adjuvant described in Section IA. In some embodiments, the OspA antigen is any OspA antigen described herein, such as any OspA antigen described in Section IB. In some embodiments, the immunogenic composition contains additional components, such as any components described in Section IC.

[0139] In some embodiments, the immunogenic composition is used to stimulate an immune response against *Borrelia* in a subject. In some embodiments, the antigen and adjuvant are present in the immunogenic composition in amounts that effectively stimulate an immune response against *Borrelia* in the subject. In some embodiments, stimulation is performed by administering an effective amount of the immunogenic composition to the subject.

[0140] In some embodiments, the immunogenic composition is used to protect a subject from infection with *Bretorius*. In some embodiments, the antigen and adjuvant are present in the immunogenic composition in amounts that effectively stimulate an immune response against *Bretorius* in the subject. In some embodiments, protection is achieved by administering an effective amount of the immunogenic composition to the subject.

[0141] In some embodiments, the immunogenic composition is used to prevent a subject from developing Lyme disease. In some embodiments, the antigen and adjuvant are present in the immunogenic composition in amounts that effectively stimulate an immune response against *Bretschneidera* in the subject. In some embodiments, prevention is carried out by administering an effective amount of the immunogenic composition to the subject.

[0142] In some embodiments, the subjects are human subjects. In some embodiments, the human subjects are at least 18 years old. In some embodiments, the human subjects are from about 18 years old to about 70 years old. In some embodiments, the human subjects are from about 18 years old to about 55 years old. In some embodiments, the human subjects are under 18 years old. In some embodiments, the human subjects are from about 2 years old to about 5 years old. In some embodiments, the human subjects are from about 5 years old to about 18 years old.

[0143] In some embodiments, the immunogenic composition is in a dose of 0.3 mL to 1.5 mL. In some embodiments, the immunogenic composition is in a dose of about 0.25 mL, 0.50 mL, 0.75 mL, or 1.0 mL. In some embodiments, the immunogenic composition is in a dose of about 0.50 mL. In some embodiments, the immunogenic composition is in a dose of about 0.75 mL. In some embodiments, the immunogenic composition is in a unit dose. A. Adjuvant

[0144] Adjuvants that can be used in the immunogenic compositions of this invention include mineral salts, such as aluminum salts (e.g., aluminum hydroxide, aluminum hydroxide gel (e.g., Rehydragel®), aluminum phosphate) and calcium phosphate; surfactants and microparticles, such as nonionic block polymer surfactants, cholesterol, virions, saponins (e.g., Quil A, QS-21, and GPI-0100, such as in Matrix-M™ (Lovgren et al., Biotechnol Appl Biochem, 10(2):161–172, 1988)), proteasomes, immunostimulatory complexes, spirochetes, quaternary ammonium (dimethyl dioctadecyl ammonium bromide (DDA)), pyridine, vitamin A, vitamin E, and poly(lactide-co-glycolic acid) microparticles (Shah et al., Methods Mol Biol, 1494:1-14, 2017); and bacterial products, such as the RIBI adjuvant system (Ribi). Inc.), the cell wall skeleton of Mycobacterium umphlei (Detox®), muramyl dipeptide (MDP) and muramyl tripeptide (MTP), monophospholipid A, Bacillus Calmete-Guerin, heat-labile Escherichia coli enterotoxin, cholera toxin, trehalose dimethicone and CpG oligodeoxynucleotides; cytokines and hormones such as interleukins (IL-1, IL-2, IL-6, IL-12, IL-15 and IL-18), granulocyte-macrophage colony-stimulating factor, dehydroepiandrosterone and 1,25-dihydroxyvitamin D3; polyanions such as dextran; polyacrylates (e.g., polymethyl methacrylate and Carbopol). 934P); carriers, such as tetanus toxin, diphtheria toxoid, cholera toxin B subunit, heat-labile enterotoxin mutant of enterotoxin-producing Escherichia coli (rmLT), and heat shock proteins; oil-in-water emulsions, such as AMPHIGEN® (Hydronics, USA); water-in-oil emulsions, such as Freund's complete and incomplete adjuvants; and liposome-based adjuvants. Further adjuvants that may be used in the immunogenic compositions of the present invention are described in Pulendran et al., Nat Rev Drug Discov 20:454–475, 2021; and Facciola et al., Vaccines (Basel) 10(5):819, 2022.

[0145] In some embodiments, the adjuvant is selected from aluminum salt adjuvants; Toll-like receptor 9 (TLR9) agonists; liposome-based adjuvants; and oil-in-water emulsion-based adjuvants. In some embodiments, the adjuvant is selected from aluminum salt adjuvants; TLR9 agonists, which are oligonucleotides containing an unmethylated cytidine-phosphate-guanosine (CpG) motif; liposome-based adjuvants containing lipopolysaccharides and saponins; and oil-in-water emulsion-based adjuvants containing squalene, sorbitol esters, and polyoxyethylene sorbitol esters.

[0146] In some embodiments, the adjuvant is an aluminum salt adjuvant, such as any of those described herein (e.g., in Section IA-1). In some embodiments, the adjuvant is a TLR9 agonist, for example, a TLR9 agonist of an oligonucleotide containing an unmethylated CpG motif, such as any of those described herein (e.g., in Section IA-2). In some embodiments, the adjuvant is a liposome-based adjuvant, for example, a liposome-based adjuvant containing lipopolysaccharide and saponin, such as any of those described herein (e.g., in Section IA-3). In some embodiments, the adjuvant is an oil-in-water emulsion-based adjuvant, for example, an oil-in-water emulsion-based adjuvant containing squalene, sorbitol ester, and polyoxyethylene sorbitol ester, such as any of those described herein (e.g., in Section IA-4).

[0147] In some embodiments, the immunogenic composition contains a second adjuvant different from the first adjuvant. In some embodiments, the first and second adjuvants are individually any adjuvant described herein. In some embodiments, the first and second adjuvants are selected from: aluminum salt adjuvants; TLR9 agonists; liposome-based adjuvants; and oil-in-water emulsion-based adjuvants. In some embodiments, the first and second adjuvants are selected from: aluminum salt adjuvants; TLR9 agonists, which are oligonucleotides containing unmethylated CpG motifs; liposome-based adjuvants containing lipopolysaccharides and saponins; and oil-in-water emulsion-based adjuvants containing squalene, sorbitol esters, and polyoxyethylene sorbitol esters.

[0148] In some embodiments, the second adjuvant is a polycationic carrier. In some embodiments, the first adjuvant is a TLR9 agonist, for example, a TLR9 agonist of an oligonucleotide containing an unmethylated CpG motif, and the second adjuvant is a polycationic carrier. In some embodiments, the first adjuvant (e.g., a TLR9 agonist) forms a cationic complex with the polycationic carrier. In some embodiments, the development of the cationic complex leads to an enhanced immune response relative to an immune response induced by the first adjuvant alone. In some embodiments, the polycationic carrier is any of those described in the following literature: Dong et al., ACS Appl. Mater. Interfaces 14(5):6331-6342, 2022; and Fu et al., European Polymer Journal 113:115-132, 2019. In some embodiments, the polycationic carrier is polycationic polyethyleneimine (PEI). In some embodiments, the polycationic carrier is an aluminum salt adjuvant, such as any of those described herein (e.g., in Section IA-1).

[0149] In some embodiments, the second adjuvant is an aluminum salt adjuvant, which may be any of those described herein and in any amount described herein. In some embodiments, the first adjuvant is a TLR9 agonist, for example, a TLR9 agonist containing an oligonucleotide with an unmethylated CpG motif, and the second adjuvant is an aluminum salt adjuvant. In some embodiments, the first adjuvant is a liposome-based adjuvant, for example, a liposome-based adjuvant containing lipopolysaccharide and saponin, and the second adjuvant is an aluminum salt adjuvant. In some embodiments, the first adjuvant is an oil-in-water emulsion-based adjuvant, for example, an oil-in-water emulsion-based adjuvant containing squalene, sorbitan ester, and polyoxyethylene sorbitan ester, and the second adjuvant is an aluminum salt adjuvant. In some embodiments, the aluminum salt adjuvant is one or more of amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate. In some embodiments, the aluminum salt adjuvant is one or both of aluminum hydroxide and aluminum phosphate. In some embodiments, the aluminum salt adjuvant is aluminum hydroxide. In some implementations, the aluminum salt adjuvant is potassium aluminum sulfate.

[0150] In some embodiments, the first adjuvant is a TLR9 agonist, for example, a TLR9 agonist containing an oligonucleotide with an unmethylated CpG motif, and the second adjuvant is potassium aluminum sulfate. In some embodiments, the first adjuvant is a liposome-based adjuvant, for example, a liposome-based adjuvant containing lipopolysaccharide and saponin, and the second adjuvant is potassium aluminum sulfate. In some embodiments, the first adjuvant is an oil-in-water emulsion-based adjuvant, for example, an oil-in-water emulsion-based adjuvant containing squalene, sorbitan ester, and polyoxyethylene sorbitan ester, and the second adjuvant is potassium aluminum sulfate.

[0151] In some embodiments, the first adjuvant is a lipopolysaccharide (LPS), and the second adjuvant is an aluminum salt adjuvant. In some embodiments, the LPS is adsorbed onto the aluminum salt adjuvant. In some embodiments, the LPS is lipid A or a derivative thereof. In some embodiments, the LPS is monophospholipid A or a derivative thereof. In some embodiments, the LPS is 3-O-deacylated-4'-monophospholipid A. In some embodiments, the LPS is 3-deacylated-phosphorylated hexaacylbiose. In some embodiments, the aluminum salt adjuvant is one or more of amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate. In some embodiments, the aluminum salt adjuvant is one or both of aluminum hydroxide and aluminum phosphate. In some embodiments, the aluminum salt adjuvant is aluminum hydroxide. In some embodiments, the aluminum salt adjuvant is potassium aluminum sulfate. In some embodiments, the first adjuvant is 3-O-deacylated-4'-monophospholipid A, and the second adjuvant is aluminum hydroxide. In some embodiments, the first and second adjuvants together are adjuvant ASO4. In some implementations, the adjuvant is AS04.

[0152] In some embodiments, the adjuvant is a synthetic TLR7 / 8 agonist. In some embodiments, the adjuvant is 3M-052 (Kasturi et al., Sci Immunol 5(48):eabb1025, June 19, 2020). In some embodiments, 3M-052 is formulated in poly(lactic acid-co-glycolic acid) or PLGA nanoparticles. In some embodiments, 3M-052 is formulated with an aluminum salt adjuvant. In some embodiments, 3M-052 is formulated with potassium aluminum sulfate. In some embodiments, the first adjuvant is 3M-052, and the second adjuvant is any adjuvant described herein. In some embodiments, the first adjuvant is 3M-052, and the second adjuvant is a TLR4 agonist. In some embodiments, the TLR4 agonist is GLA.

[0153] In some embodiments, the adjuvant is a saponin-based adjuvant. In some embodiments, the adjuvant consists of saponin-containing nanoparticles. In some embodiments, the saponin is extracted from the soapberry tree (Sapindus mukorossi). In some embodiments, the nanoparticles contain saponins, cholesterol, and phospholipids. In some embodiments, the adjuvant is Matrix-M™. 1. Aluminum salt adjuvants

[0154] In some embodiments, the adjuvant is an aluminum salt adjuvant. In some embodiments, the OspA antigen is adsorbed onto the aluminum salt adjuvant. In some embodiments, the aluminum salt adjuvant is one or more of amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate. In some embodiments, the aluminum salt adjuvant is one or both of aluminum hydroxide and aluminum phosphate. In some embodiments, the aluminum salt adjuvant is aluminum hydroxide. In some embodiments, the aluminum salt adjuvant is potassium aluminum sulfate.

[0155] In some embodiments, the immunogenic composition contains about 0.25 mg to about 1.25 mg of Al. 3+ In some embodiments, the immunogenic composition contains about 0.5 mg to about 1 mg of Al. 3+ In some embodiments, the immunogenic composition contains about 0.25 mg to about 0.50 mg of Al. 3+ In some embodiments, the immunogenic composition contains about 0.30 mg to about 0.40 mg of Al. 3+ In some embodiments, the immunogenic composition contains about 0.25 mg, about 0.50 mg, about 0.75 mg, about 1.00 mg, or about 1.25 mg of Al. 3+ . 2. Oligonucleotide Toll-like receptor 9 (TLR9) agonists

[0156] Toll-like receptors (TLRs) are expressed on or on dendritic cells and other innate immune cells, and are among the most important receptors stimulating responses to the presence of invading pathogens. Humans possess multiple types of TLRs that are structurally similar but recognize different parts of viruses or bacteria. By activating specific TLRs, it is possible to stimulate and control specific types of innate immune responses that can be used to enhance adaptive responses.

[0157] In some embodiments, the adjuvant in the immunogenic compositions of the present invention is an oligonucleotide TLR9 agonist. In some embodiments, the oligonucleotide contains an unmethylated cytidine-phosphate-guanosine (CpG) motif. TLR9 (CD289) recognizes unmethylated CpG motifs found in microbial DNA, which can be mimicked using synthetic CpG-containing oligodeoxynucleotides (CpG-ODNs). CpG-ODNs are known to enhance antibody production and stimulate T helper 1 (Th1) cell responses (Coffman et al., Immunity, 33:492-503, 2010). Based on structure and biological function, CpG-ODNs have been classified into three main classes: CpG-A, CpG-B, and CpG-C (Campbell, Methods Mol Biol, 1494:15-27, 2017). The degree of B cell activation varies among different categories, with CpG-A ODN being a weak B cell activator, CpG-C ODN a good B cell activator, and CpG-B ODN a strong B cell activator. The oligonucleotide TLR9 agonist in the immunogenic composition of the present invention is preferably a good B cell activator (CpG-C ODN) or more preferably a strong B cell activator (CpG-B ODN).

[0158] Oligonucleotide TLR9 agonists typically contain palindromic sequences of the following general formula: 5'-purine-purine-CG-pyrimidine-pyrimidine-3' or 5'-purine-purine-CG-pyrimidine-pyrimidine-CG-3' (US Patent No. 6,589,940). TLR9 agonism has also been observed in some non-palindromic CpG-rich phosphate thioester oligonucleotides, but this may be affected by variations in the nucleotide sequence. Furthermore, TLR9 agonism can be eliminated by methylation of cytosine within the CpG dinucleotide. In some embodiments, the TLR9 agonist in the immunogenic compositions of the present invention is an oligonucleotide of 8 to 35 nucleotides in length. In some embodiments, the TLR9 agonist in the immunogenic compositions of the present invention is an oligonucleotide of 8 to 35 nucleotides in length comprising the sequence 5'-AACGTTCG-3'. In some embodiments, the oligonucleotide is longer than 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides, and shorter than 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, or 24 nucleotides. In some embodiments, the TLR9 agonist is an oligonucleotide of 10 to 35 nucleotides in length. In some embodiments, the TLR9 agonist is an oligonucleotide of 10 to 35 nucleotides in length comprising the sequence 5'-AACGTTCGAG-3' (SEQ ID NO:2). In some embodiments, the oligonucleotide is longer than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides, and shorter than 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, or 24 nucleotides. In some embodiments, the oligonucleotide does not contain the sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (ODN 2006 or ODN 7909, as shown in SEQ ID NO:3).

[0159] Researchers at Dynavax Technologies Corporation (Emeryville, California) have identified phosphate-thioester-linked 22-meric oligodeoxynucleotide CpG 1018 ® Adjuvants containing specific sequences that can significantly enhance immune responses to co-administered antigens across species (Campbell, Methods Mol Biol, 1494:15-27, 2017). CpG 1018 ® adjuvant (5'-TGACTGTGAA CGTTCGAGAT GA-3', as shown in SEQ ID NO:1) was selected after screening a large group of oligonucleotides for in vitro and in vivo immunostimulatory activity. CpG 1018 ® The adjuvant is a CpG-B ODN active in mice, rabbits, dogs, baboons, cynomolgus monkeys, and humans. In some embodiments, the oligonucleotide is 22 to 35 nucleotides in length and contains the sequence 5'-TGACTGTGAA CGTTCGAGAT GA-3' (SEQ ID NO:1). In some embodiments, the oligonucleotide is longer than 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides, and shorter than 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, or 24 nucleotides, provided that the maximum length is lower than the minimum length.

[0160] Although the exemplary oligonucleotide CpG 1018 ® The adjuvant is CpG-ODN; oligonucleotides that are not entirely DNA molecules can also be used in the immunogenic compositions of the present invention. In some embodiments, the oligonucleotide is a DNA / RNA chimeric molecule, wherein one or more CpGs and palindromic sequences are deoxyribonucleic acid, and one or more nucleic acids outside these regions are ribonucleic acid. In some embodiments, the oligonucleotide is linear. In other embodiments, the oligonucleotide is circular or includes one or more hairpin loops. In some embodiments, the oligonucleotide is single-stranded. In some embodiments, the oligonucleotide is double-stranded.

[0161] In some embodiments, the oligonucleotide may contain modifications. Modifications may include modifications of 3'OH or 5'OH groups, modifications of nucleotide bases, modifications of sugar components, and modifications of phosphate groups. Modified bases may be included in the palindromic sequence of the oligonucleotide, provided that one or more modified bases maintain the same specificity to their natural complements via Watson-Crick base pairing (e.g., the palindromic portion remains self-complementary). In some embodiments, the oligonucleotide contains non-canonical bases. In some embodiments, the oligonucleotide contains modified nucleosides. In some embodiments, the modified nucleosides are selected from 2'-deoxy-7-deazonoguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxy-2'-substituted arabinoguanosine, and 2'-O-substituted arabinoguanosine.

[0162] Oligonucleotides can contain phosphate group modifications. For example, in addition to phosphodiester bonds, phosphate modifications include methylphosphonates, thiophosphates, aminophosphates (bridged or unbridged), triphosphates, and dithiophosphates, and can be used in any combination. Other non-phosphate bonds can also be used. In some embodiments, the oligonucleotide contains only a thiophosphate backbone. In some embodiments, the oligonucleotide contains only a phosphodiester backbone. In some embodiments, the oligonucleotide contains a combination of phosphate bonds in the phosphate backbone, such as a combination of phosphodiester bonds and thiophosphate bonds. Oligonucleotides with a thiophosphate backbone may have higher immunogenicity than those with a phosphodiester backbone and may be more resistant to degradation after injection into the host (Braun et al., J Immunol, 141:2084-2089, 1988; and Latimer et al., Mol Immunol, 32:1057-1064, 1995). In some embodiments, the oligonucleotide comprises at least one, two, or three internucleotide thiophosphate bonds. In some embodiments, when multiple oligonucleotide molecules are present in a pharmaceutical composition containing at least one excipient, both stereoisomers of the thiophosphate bonds are present in the multiple oligonucleotide molecules. In some embodiments, all internucleotide bonds in the oligonucleotide are thiophosphate bonds, or in other words, the oligonucleotide has a thiophosphate backbone.

[0163] In some embodiments, the immunogenic composition contains about 375 µg to about 6000 µg of oligonucleotides. In some embodiments, the immunogenic composition contains about 750 µg to about 6000 µg of oligonucleotides. In some embodiments, the immunogenic composition contains about 750 µg to about 3000 µg of oligonucleotides. In some embodiments, the immunogenic composition contains about 750 µg to about 1500 µg of oligonucleotides. In some embodiments, the immunogenic composition contains about 1500 µg to about 3000 µg of oligonucleotides. In some embodiments, the immunogenic composition contains about 3000 µg to about 6000 µg of oligonucleotides. In some embodiments, the immunogenic composition contains more than or equal to about 750 µg, 1000 µg, 1250 µg, or 1500 µg of oligonucleotides, and less than or equal to about 6000 µg, 5000 µg, 4000 µg, or 3000 µg of oligonucleotides. In some embodiments, the immunogenic composition contains about 375 µg, 750 µg, 1500 µg, 3000 µg, or 6000 µg of oligonucleotides. In some embodiments, the immunogenic composition contains about 375 µg of oligonucleotides. In some embodiments, the immunogenic composition contains about 750 µg of oligonucleotides. In some embodiments, the immunogenic composition contains about 1000 µg of oligonucleotides. In some embodiments, the immunogenic composition contains about 1500 µg of oligonucleotides. In some embodiments, the immunogenic composition contains about 3000 µg of oligonucleotides. In some embodiments, the immunogenic composition contains about 6000 µg of oligonucleotides.

[0164] In some embodiments, the oligonucleotide is in its pharmaceutically acceptable salt form. Exemplary basic salts include ammonium salts; alkali metal salts (such as sodium, lithium, and potassium salts); alkaline earth metal salts (such as calcium and magnesium salts); zinc salts; salts formed with organic bases (e.g., organic amines) (such as N-Me-D-glucosamine, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, choline, glycerol, dicyclohexylamine, and tert-butylamine); and salts formed with amino acids (such as arginine, lysine, etc.). In some embodiments, the oligonucleotide is in the form of an ammonium, sodium, lithium, or potassium salt. In some embodiments, the oligonucleotide is in the form of a sodium salt. 3. Liposome-based adjuvants

[0165] In some embodiments, the adjuvant in the immunogenic composition of the present invention is a liposome-based adjuvant, such as any of those described in the following literature: Tretiakova et al., Biochem (Mosc) Suppl Ser A MembrCell Biol, 16(1):1-20, 2022; or Alving et al., Expert Rev Vaccines 19(3):279-292, 2020. In some embodiments, the liposome-based adjuvant comprises lipopolysaccharide. In some embodiments, the liposome-based adjuvant comprises saponin. In some embodiments, the liposome-based adjuvant comprises both lipopolysaccharide and saponin.

[0166] In some embodiments, the lipopolysaccharide (LPS) is lipid A or a derivative thereof. In some embodiments, the LPS is monophosphoryl lipid A or a derivative thereof. In some embodiments, the LPS is naturally occurring. In some embodiments, the LPS is synthetic. In some embodiments, the LPS is 3-O-deacylated-4'-monophosphoryl lipid A. In some embodiments, the LPS is 3-deacylated-phosphorylated hexaacyl disaccharide.

[0167] In some embodiments, the immunogenic composition contains about 1 μg to about 100 μg of lipopolysaccharide. In some embodiments, the immunogenic composition contains about 10 μg to about 50 μg of lipopolysaccharide. In some embodiments, the immunogenic composition contains about 20 μg to about 30 μg of lipopolysaccharide. In some embodiments, the immunogenic composition contains about 21 μg to about 29 μg of lipopolysaccharide. In some embodiments, the immunogenic composition contains about 22 μg to about 28 μg of lipopolysaccharide. In some embodiments, the immunogenic composition contains about 23 μg to about 27 μg of lipopolysaccharide. In some embodiments, the immunogenic composition contains about 24 μg to about 26 μg of lipopolysaccharide.

[0168] In some embodiments, the immunogenic composition contains about 1 μg to about 30 μg of lipopolysaccharide. In some embodiments, the immunogenic composition contains about 5 μg to about 15 μg of lipopolysaccharide. In some embodiments, the immunogenic composition contains about 6 μg to about 14 μg of lipopolysaccharide. In some embodiments, the immunogenic composition contains about 7 μg to about 13 μg of lipopolysaccharide. In some embodiments, the immunogenic composition contains about 8 μg to about 12 μg of lipopolysaccharide. In some embodiments, the immunogenic composition contains about 9 μg to about 11 μg of lipopolysaccharide.

[0169] In some embodiments, the immunogenic composition contains about 1 μg to about 9 μg of lipopolysaccharide. In some embodiments, the immunogenic composition contains about 2 μg to about 8 μg of lipopolysaccharide. In some embodiments, the immunogenic composition contains about 3 μg to about 7 μg of lipopolysaccharide. In some embodiments, the immunogenic composition contains about 4 μg to about 6 μg of lipopolysaccharide.

[0170] In some embodiments, the saponin is naturally occurring. In some embodiments, the saponin is synthetic. In some embodiments, the saponin is derived from the bark of the Molina soapberry tree. In some embodiments, the saponin is Quil A or a derivative thereof. In some embodiments, the saponin is QS-17. In some embodiments, the saponin is QS-21.

[0171] In some embodiments, the immunogenic composition contains about 1 μg to about 100 μg of saponin. In some embodiments, the immunogenic composition contains about 10 μg to about 50 μg of saponin. In some embodiments, the immunogenic composition contains about 20 μg to about 30 μg of saponin. In some embodiments, the immunogenic composition contains about 21 μg to about 29 μg of saponin. In some embodiments, the immunogenic composition contains about 22 μg to about 28 μg of saponin. In some embodiments, the immunogenic composition contains about 23 μg to about 27 μg of saponin. In some embodiments, the immunogenic composition contains about 24 μg to about 26 μg of saponin.

[0172] In some embodiments, the immunogenic composition contains about 1 μg to about 30 μg of saponin. In some embodiments, the immunogenic composition contains about 5 μg to about 15 μg of saponin. In some embodiments, the immunogenic composition contains about 6 μg to about 14 μg of saponin. In some embodiments, the immunogenic composition contains about 7 μg to about 13 μg of saponin. In some embodiments, the immunogenic composition contains about 8 μg to about 12 μg of saponin. In some embodiments, the immunogenic composition contains about 9 μg to about 11 μg of saponin.

[0173] In some embodiments, the immunogenic composition contains about 1 μg to about 9 μg of saponin. In some embodiments, the immunogenic composition contains about 2 μg to about 8 μg of saponin. In some embodiments, the immunogenic composition contains about 3 μg to about 7 μg of saponin. In some embodiments, the immunogenic composition contains about 4 μg to about 6 μg of saponin.

[0174] In some embodiments, the liposome-based adjuvant further contains a sterol. In some embodiments, the sterol is β-sitosterol, stigmasterol, ergosterol, ergocalciferol, or cholesterol. In some embodiments, the sterol is cholesterol.

[0175] In some embodiments, the ratio of saponin to sterol is 1:1 to 1:100 w / w. In some embodiments, the ratio of saponin to sterol is 1:1 to 1:5 w / w.

[0176] In some embodiments, the liposome-based adjuvant further contains a neutral lipid. In some embodiments, the neutral lipid is phosphatidylcholine. In some embodiments, the phosphatidylcholine is egg yolk phosphatidylcholine, dioleoylphosphatidylcholine (DOPC), or dilauryl phosphatidylcholine. In some embodiments, the phosphatidylcholine is DOPC.

[0177] In some embodiments, the liposome-based adjuvant contains 3-O-deacylated-4'-monophosphoryllipid A and QS-21. In some embodiments, the liposome-based adjuvant contains 3-O-deacylated-4'-monophosphoryllipid A, QS-21, and cholesterol. In some embodiments, the liposome-based adjuvant contains 3-O-deacylated-4'-monophosphoryllipid A, QS-21, cholesterol, and DOPC. In some embodiments, the liposome-based adjuvant is AS01, AS01B, or AS01E, for example, as described in US 10,039,823. In some embodiments, the liposome-based adjuvant is AS01. In some embodiments, the liposome-based adjuvant is AS01B. In some embodiments, the liposome-based adjuvant is AS01E.

[0178] In some embodiments, the liposome-based adjuvant contains 3-deacylated hexaacyl disaccharide and QS-21. In some embodiments, the liposome-based adjuvant is ALFQ. 4. Adjuvants based on oil-in-water emulsions

[0179] In some embodiments, the adjuvant in the immunogenic compositions of the present invention is an oil-in-water emulsion-based adjuvant, such as any of those described in Zhang et al., BMC Vet Res 14:415, 2018. In some embodiments, the oil-in-water emulsion-based adjuvant contains squalene. In some embodiments, the oil-in-water emulsion-based adjuvant contains sorbitan ester (also known as Span). In some embodiments, the oil-in-water emulsion-based adjuvant contains polyoxyethylene sorbitan ester (also known as Tween). In some embodiments, the oil-in-water emulsion-based adjuvant contains squalene, sorbitan ester, and polyoxyethylene sorbitan ester.

[0180] In some embodiments, the sorbitol ester is sorbitol ester 20. In some embodiments, the sorbitol ester is sorbitol ester 85.

[0181] In some embodiments, the polyoxyethylene sorbitol ester is polyoxyethylene sorbitol ester 20. In some embodiments, the polyoxyethylene sorbitol ester is polyoxyethylene sorbitol ester 80.

[0182] In some embodiments, the immunogenic composition contains about 2% w / v to about 7% w / v of squalene. In some embodiments, the immunogenic composition contains about 3% w / v to about 6% w / v of squalene. In some embodiments, the immunogenic composition contains about 4% w / v to about 5% w / v of squalene.

[0183] In some embodiments, the immunogenic composition contains about 0.2% w / v to about 0.8% w / v of sorbitol ester. In some embodiments, the immunogenic composition contains about 0.3% w / v to about 0.7% w / v of sorbitol ester. In some embodiments, the immunogenic composition contains about 0.4% w / v to about 0.6% w / v of sorbitol ester.

[0184] In some embodiments, the immunogenic composition contains about 0.2% w / v to about 0.8% w / v of polyoxyethylene sorbitol ester. In some embodiments, the immunogenic composition contains about 0.3% w / v to about 0.7% w / v of polyoxyethylene sorbitol ester. In some embodiments, the immunogenic composition contains about 0.4% w / v to about 0.6% w / v of polyoxyethylene sorbitol ester.

[0185] In some embodiments, the adjuvant based on the oil-in-water emulsion contains squalene, sorbitol 85, and polyoxyethylene sorbitol 80. In some embodiments, the immunogenic composition contains about 2% w / v to about 7% w / v of squalene; about 0.2% w / v to about 0.8% w / v of sorbitol 85; and about 0.2% w / v to about 0.8% w / v of polyoxyethylene sorbitol 80. In some embodiments, the adjuvant based on the oil-in-water emulsion is MF59. In some embodiments, the adjuvant based on the oil-in-water emulsion is AddaVax™.

[0186] In some embodiments, the adjuvant based on the oil-in-water emulsion contains additional immune-enhancing components. In some embodiments, the adjuvant based on the oil-in-water emulsion contains vitamins. In some embodiments, the adjuvant based on the oil-in-water emulsion contains vitamin E.

[0187] In some embodiments, the adjuvant based on the oil-in-water emulsion contains squalene, vitamin E, and polyoxyethylene sorbitan ester 80. In some embodiments, the adjuvant based on the oil-in-water emulsion is AS03. In some embodiments, the immunogenic composition contains about 11 mg of squalene, about 12 mg of vitamin E, and about 5 mg of polyoxyethylene sorbitan ester 80. B. Borrelia contains outer surface protein A (OspA) antigen and RNA polynucleotide.

[0188] In some embodiments, the immunogenic composition contains the outer surface protein A (OspA) antigen of *Bretorius* or an RNA polynucleotide encoding the OspA antigen. In some embodiments, the immunogenic composition contains the *Bretorius* OspA antigen. In some embodiments, the immunogenic composition does not contain a non-OspA *Bretorius* outer surface protein antigen.

[0189] In some embodiments, the immunogenic composition contains an RNA polynucleotide encoding an OspA antigen. In some embodiments, the immunogenic composition does not contain an RNA polynucleotide encoding a non-OspA spirochetal outer surface protein antigen. In some embodiments, the RNA polynucleotide further comprises a 5' untranslated region (UTR) and a 3' UTR. In some embodiments, the RNA polynucleotide further comprises a poly(A) tail. In some embodiments, the RNA polynucleotide further comprises a 5' cap analog. In some embodiments, the RNA polynucleotide is a modified polynucleotide, as described in US 10,898,574, US 10,703,789, and US 10,577,403.

[0190] RNA polynucleotides can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) allow sustained or delayed release; (4) alter biodistribution to a specific tissue or cell type; (5) increase the translation of the encoded protein in vivo; and / or (6) alter the release characteristics of the encoded protein in vivo. Excipients may include solvents, dispersion media, diluents, other liquid media, dispersants or suspending agents, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, lipids, liposomes, lipid nanoparticles, polymers, lipid complexes, core-shell nanoparticles, peptides, proteins, hyaluronidase, nanoparticle mimics, and combinations thereof. Exemplary formulations of RNA polynucleotides are described in US 10,898,574, US 10,703,789, and US 10,577,403.

[0191] In some embodiments, RNA polynucleotides are formulated in lipid nanoparticles. Examples of lipid nanoparticle compositions and methods for preparing lipid nanoparticle compositions are described, for example, in the following literature: Semple et al. (2010) Nat. Biotechnol. 28:172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed., 51:8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578. In some embodiments, the lipid nanoparticles contain cationic lipids. In some embodiments, the lipid nanoparticles contain sterols. In some embodiments, the lipid nanoparticles contain PEG-modified lipids. In some embodiments, the lipid nanoparticles contain neutral lipids. In some embodiments, the lipid nanoparticles contain cationic lipids, sterols, and PEG-modified lipids. In some embodiments, the lipid nanoparticles contain cationic lipids, neutral lipids, sterols, and PEG-modified lipids.

[0192] Exemplary lipids include DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids, and amino alcohol lipids. In another aspect, the lipids can be cationic lipids, such as DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, and amino alcohol lipids. Amino alcohol cationic lipids can be the lipids described in U.S. Patent Publication No. US20130150625 and / or lipids prepared by the methods described in U.S. Patent Publication No. US20130150625. For example, cationic lipids can be 2-amino-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadec-9,12-dien-1-yloxy]methyl}prop-1-ol (compound 1 in US20130150625); 2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}prop-1-ol (compound 2 in US20130150625); 2-amino -3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]prop-1-ol (compound 3 in US20130150625); and 2-(dimethylamino)-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadec-9,12-dien-1-yloxy]methyl}prop-1-ol (compound 4 in US20130150625); or any pharmaceutically acceptable salt or stereoisomer thereof.

[0193] In some embodiments, the lipid nanoparticles contain ionizable cationic lipids, such as 2,2-dilinole-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinole-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), or di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butyryl)oxy)heptadecanoate (L319), and further contain neutral lipids, sterols, and molecules capable of reducing particle aggregation (e.g., PEG or PEG-modified lipids).

[0194] In some embodiments, the lipid nanoparticle formulation is substantially composed of: (i) at least one lipid selected from 2,2-dilinole-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinole-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butyryl)oxy)heptadecanoate (L319); (ii) neutral lipids selected from DSPC, DPPC, POPC, DOPE, and SM; (iii) sterols, such as cholesterol; and (iv) PEG-lipids, such as PEG-DMG or PEG-cDMA, in a molar ratio of about 20%-60% cationic lipids: 5%-25% neutral lipids: 25%-55% sterols: 0.5%-15% PEG-lipids. In some embodiments, the lipid nanoparticles contain 50% ionizable cationic lipids, 10% neutral lipids, 38.5% sterols, and 1.5% PEG-modified lipids. In some embodiments, the neutral lipids are 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), the sterols are cholesterol, and the PEG-modified lipids are 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000 (PEG-DMG) or PEG-cDMA.

[0195] Seven major OspA serotypes have been identified in European isolates (named serotypes 1 through 7, see Wilske et al., J. Clin. Microbiol. 31:340-50, 1993). In some respects, OspA serotypes tend to be species-related, for example, serotype 1 corresponds to Borrelia burgdorferi, serotype 2 to Borrelia auriculata, and serotypes 3 through 7 to Borrelia galbana. In some respects, serotype 4 is alternatively considered to correspond to Borrelia bavaria (Margos et al., Appl. Environ. Microbiol. 75: 5410-6, 2009).

[0196] In some implementations, the OspA antigen is any of the OspA antigens described in the following documents: WO2021207615, WO 2021205022, WO 2010132758, US 9,975,927, US 10,766,931, US 11,572,392, US 9,023,367, US 8,623,375, US 8,623,376, US 9,303,073, US 9,334,311, US 9,895,434, US 11,305,000, US 9,562,079, US 9,534,021, US 10,266,571, US 8,821,893, US 7,887,815, US US 7,582,304, US 7,605,248 and US 20090324638.

[0197] In some embodiments, the OspA antigen includes one or more OspA antigens, such as 1, 2, 3, 4, 5, or 6 OspA antigens. It should be understood that references to OspA antigens in this disclosure independently describe an embodiment of any one of the one or more OspA antigens.

[0198] In some embodiments, the OspA antigen is a single OspA antigen. In some embodiments, the OspA antigen comprises two or more OspA antigens. For example, each OspA antigen has a different amino acid sequence, which may be overlapping sequences or completely non-overlapping sequences.

[0199] In some embodiments, the OspA antigen is *Bretorius burgdorferi* OspA antigen. In some embodiments, the OspA antigen is *Bretorius auriculata* OspA antigen. In some embodiments, the OspA antigen is *Bretorius Bavaria* OspA antigen. In some embodiments, the OspA antigen is *Bretorius galbella* OspA antigen. In some embodiments, the OspA antigen is *Bretorius mesei* OspA antigen. In some embodiments, the OspA antigen is *Bretorius louvelii* OspA antigen. In some embodiments, the OspA antigen is *Bretorius bise* OspA antigen. In some embodiments, the OspA antigen is *Bretorius stearothermia* OspA antigen. In some embodiments, the OspA antigen is *Bretorius warwickii* OspA antigen.

[0200] In some embodiments, each of the different OspA antigens is an OspA antigen from a different species of the genus *Breospira*. In some embodiments, the OspA antigens include a first OspA antigen and a second OspA antigen. In some embodiments, the first OspA antigen and the second OspA antigen have different amino acid sequences. In some embodiments, the two or more OspA antigens further include a third OspA antigen. In some embodiments, the first OspA antigen, the second OspA antigen, and the third OspA antigen have different amino acid sequences.

[0201] In some embodiments, the OspA antigen is a naturally occurring OspA protein or a fragment thereof. In some embodiments, the OspA antigen is a recombinant OspA protein.

[0202] In some embodiments, the OspA antigen contains one or more epitopes from an OspA protein. In some embodiments, the one or more epitopes include at least one linear epitope. In some embodiments, the one or more epitopes are linear epitopes. In some embodiments, the one or more epitopes include at least one conformational epitope. In some embodiments, the one or more epitopes are conformational epitopes.

[0203] In some embodiments, the one or more epitopes contain at least two, three, four, five, or six epitopes from one or more OspA proteins. In some embodiments, the one or more epitopes originate from the same OspA protein. In some embodiments, the one or more epitopes originate from the same OspA serotype. In some embodiments, the one or more epitopes originate from multiple OspA proteins. In some embodiments, the one or more epitopes contain at least two, three, four, five, or six epitopes from at least two, three, four, five, or six OspA proteins. In some embodiments, the one or more epitopes originate from multiple OspA serotypes. In some embodiments, the one or more epitopes contain at least two, three, four, five, or six epitopes from at least two, three, four, five, or six OspA serotypes.

[0204] In some embodiments, the one or more epitopes include OspA epitopes contained in any amino acid sequence shown in SEQ ID NO:50-82. In some embodiments, the one or more epitopes are independently selected from OspA epitopes contained in any amino acid sequence shown in SEQ ID NO:50-82. In some embodiments, the one or more epitopes include any linear OspA epitope contained in the amino acid sequence shown in SEQ ID NO:50-82. In some embodiments, the one or more epitopes include any conformational OspA epitope contained in the amino acid sequence shown in SEQ ID NO:50-82. In some embodiments, the one or more epitopes include both linear and conformational OspA epitopes contained in any amino acid sequence shown in SEQ ID NO:50-82.

[0205] In some embodiments, the one or more epitopes include OspA epitopes contained in any amino acid sequence shown in SEQ ID NO:50-62. In some embodiments, the one or more epitopes are independently selected from OspA epitopes contained in any amino acid sequence shown in SEQ ID NO:50-62. In some embodiments, the one or more epitopes include any linear OspA epitope contained in the amino acid sequence shown in SEQ ID NO:50-62. In some embodiments, the one or more epitopes include any conformational OspA epitope contained in the amino acid sequence shown in SEQ ID NO:50-62. In some embodiments, the one or more epitopes include both linear and conformational OspA epitopes contained in any amino acid sequence shown in SEQ ID NO:50-62.

[0206] In some embodiments, the one or more epitopes include OspA epitopes contained in any amino acid sequence shown in SEQ ID NO:63-82. In some embodiments, the one or more epitopes are independently selected from OspA epitopes contained in any amino acid sequence shown in SEQ ID NO:63-82. In some embodiments, the one or more epitopes include any linear OspA epitope contained in the amino acid sequence shown in SEQ ID NO:63-82. In some embodiments, the one or more epitopes include any conformational OspA epitope contained in the amino acid sequence shown in SEQ ID NO:63-82. In some embodiments, the one or more epitopes include both linear and conformational OspA epitopes contained in any amino acid sequence shown in SEQ ID NO:63-82.

[0207] In some embodiments, the one or more epitopes are independently selected from the amino acid sequences shown in SEQ ID NO:4-49. In some embodiments, the one or more epitopes are independently selected from any linear epitopes shown in the amino acid sequences of SEQ ID NO:4-49.

[0208] In some embodiments, the OspA antigen is an OspA protein containing one or more conformational epitopes. In some embodiments, the OspA antigen has any amino acid sequence as shown in SEQ ID NO:50-82 containing one or more conformational epitopes, or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. In some embodiments, the OspA antigen has any amino acid sequence as shown in SEQ ID NO:50-82 containing one or more conformational epitopes, or has at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids. In some embodiments, the OspA antigen has any amino acid sequence as shown in SEQ ID NO:50-82 containing one or more conformational epitopes.

[0209] In some embodiments, the OspA antigen is an OspA protein containing one or more conformational epitopes. In some embodiments, the OspA antigen has any amino acid sequence as shown in SEQ ID NO:50-62 containing one or more conformational epitopes, or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. In some embodiments, the OspA antigen has any amino acid sequence as shown in SEQ ID NO:50-62 containing one or more conformational epitopes, or has at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids. In some embodiments, the OspA antigen has any amino acid sequence as shown in SEQ ID NO:50-62 containing one or more conformational epitopes.

[0210] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:50-62.

[0211] In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in any one of SEQ ID NO:50-62.

[0212] In some embodiments, the OspA antigen comprises an amino acid sequence shown in any one of SEQ ID NO:50-62.

[0213] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:50. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:50. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:50.

[0214] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:51. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:51. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:51.

[0215] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:52. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:52. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:52.

[0216] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:53. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:53. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:53.

[0217] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:54. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:54. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:54.

[0218] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:55. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:55. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:55.

[0219] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:56. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:56. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:56.

[0220] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:57. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:57. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:57.

[0221] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:58. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:58. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:58.

[0222] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:59. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:59. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:59.

[0223] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO: 60.

[0224] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:61. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:61. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:61.

[0225] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:62. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:62. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:62.

[0226] In some embodiments, the OspA antigen is an OspA chimeric antigen. In some embodiments, the OspA chimeric antigen contains multiple epitopes from one or more OspA proteins. In some embodiments, the OspA antigen is an OspA chimeritope.

[0227] In some embodiments, the plurality of epitopes originate from the same OspA protein. In some embodiments, the plurality of epitopes originate from the same OspA serotype. In some embodiments, the plurality of epitopes originate from multiple OspA proteins. In some embodiments, the plurality of epitopes contains at least two, three, four, five, or six epitopes from at least two, three, four, five, or six OspA proteins. In some embodiments, the plurality of epitopes originate from multiple OspA serotypes. In some embodiments, the plurality of epitopes contains at least two, three, four, five, or six epitopes from at least two, three, four, five, or six OspA serotypes.

[0228] In some embodiments, the plurality of epitopes includes OspA epitopes contained in any amino acid sequence shown in SEQ ID NO:50-82. In some embodiments, the plurality of epitopes are independently selected from OspA epitopes contained in any amino acid sequence shown in SEQ ID NO:50-82. In some embodiments, the plurality of epitopes includes any linear OspA epitope contained in the amino acid sequence shown in SEQ ID NO:50-82. In some embodiments, the plurality of epitopes includes any conformational OspA epitope contained in the amino acid sequence shown in SEQ ID NO:50-82. In some embodiments, the plurality of epitopes includes both linear and conformational OspA epitopes contained in any amino acid sequence shown in SEQ ID NO:50-82.

[0229] In some embodiments, the plurality of epitopes includes OspA epitopes contained in any amino acid sequence shown in SEQ ID NO:50-62. In some embodiments, the plurality of epitopes are independently selected from OspA epitopes contained in any amino acid sequence shown in SEQ ID NO:50-62. In some embodiments, the plurality of epitopes includes any linear OspA epitope contained in the amino acid sequence shown in SEQ ID NO:50-62. In some embodiments, the plurality of epitopes includes any conformational OspA epitope contained in the amino acid sequence shown in SEQ ID NO:50-62. In some embodiments, the plurality of epitopes includes both linear and conformational OspA epitopes contained in any amino acid sequence shown in SEQ ID NO:50-62.

[0230] In some embodiments, the plurality of epitopes includes OspA epitopes contained in any amino acid sequence shown in SEQ ID NO:63-82. In some embodiments, the plurality of epitopes are independently selected from OspA epitopes contained in any amino acid sequence shown in SEQ ID NO:63-82. In some embodiments, the plurality of epitopes includes any linear OspA epitope contained in the amino acid sequence shown in SEQ ID NO:63-82. In some embodiments, the plurality of epitopes includes any conformational OspA epitope contained in the amino acid sequence shown in SEQ ID NO:63-82. In some embodiments, the plurality of epitopes includes both linear and conformational OspA epitopes contained in any amino acid sequence shown in SEQ ID NO:63-82.

[0231] In some embodiments, the plurality of epitopes are independently selected from the amino acid sequences shown in SEQ ID NO:4-49. In some embodiments, the plurality of epitopes are independently selected from any linear epitope shown in the amino acid sequences of SEQ ID NO:4-49.

[0232] In some embodiments, the OspA antigen is an OspA protein containing one or more linear epitopes. In some embodiments, the OspA antigen has any amino acid sequence as shown in SEQ ID NO:50-82 containing one or more linear epitopes, or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. In some embodiments, the OspA antigen has any amino acid sequence as shown in SEQ ID NO:50-82 containing one or more linear epitopes, or has at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids. In some embodiments, the OspA antigen has any amino acid sequence as shown in SEQ ID NO:50-82 containing one or more linear epitopes.

[0233] In some embodiments, the OspA antigen is an OspA protein containing one or more linear epitopes. In some embodiments, the OspA antigen has any amino acid sequence as shown in SEQ ID NO:63-82 containing one or more linear epitopes, or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. In some embodiments, the OspA antigen has any amino acid sequence as shown in SEQ ID NO:63-82 containing one or more linear epitopes, or has at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids. In some embodiments, the OspA antigen has any amino acid sequence as shown in SEQ ID NO:63-82 containing one or more linear epitopes.

[0234] In some embodiments, the OspA antigen is an OspA chimeric antigen containing multiple epitopes from one or more OspA proteins. In some embodiments, the multiple epitopes are linear epitopes. In some embodiments, the OspA antigen is an OspA chimeric epitope. In some embodiments, the OspA antigen has any amino acid sequence as shown in SEQ ID NO:50-82 for an OspA chimeric antigen containing multiple linear epitopes, or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. In some embodiments, the OspA antigen has any amino acid sequence as shown in SEQ ID NO:50-82, which is an OspA chimeric antigen containing multiple linear epitopes, or has at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids. In some embodiments, the OspA antigen has any amino acid sequence as shown in SEQ ID NO:50-82, which is an OspA chimeric antigen containing multiple linear epitopes.

[0235] In some embodiments, the OspA antigen is an OspA chimeric antigen containing multiple epitopes from one or more OspA proteins. In some embodiments, the multiple epitopes are linear epitopes. In some embodiments, the OspA antigen is an OspA chimeric epitope. In some embodiments, the OspA antigen has any amino acid sequence as shown in SEQ ID NO:63-82 for an OspA chimeric antigen containing multiple linear epitopes, or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. In some embodiments, the OspA antigen has any amino acid sequence as shown in SEQ ID NO:63-82, which is an OspA chimeric antigen containing multiple linear epitopes, or has at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids. In some embodiments, the OspA antigen has any amino acid sequence as shown in SEQ ID NO:63-82, which is an OspA chimeric antigen containing multiple linear epitopes.

[0236] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:63-82.

[0237] In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in any one of SEQ ID NO:63-82.

[0238] In some embodiments, the OspA antigen comprises an amino acid sequence shown in any one of SEQ ID NO:63-82.

[0239] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:63. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:63. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:63.

[0240] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:64. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:64. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:64.

[0241] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:65. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:65. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:65.

[0242] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:66. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:66. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:66.

[0243] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:67. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:67. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:67.

[0244] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:68. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:68. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:68.

[0245] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:69. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:69. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:69.

[0246] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:70. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:70. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:70.

[0247] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:71. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:71. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:71.

[0248] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:72. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:72. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:72.

[0249] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:73. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:73. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:73.

[0250] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:74. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:74. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:74.

[0251] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:75. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:75. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:75.

[0252] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:76. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:76. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:76.

[0253] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:77. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:77. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:77.

[0254] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:78. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:78. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:78.

[0255] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:79. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:79. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:79.

[0256] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 80. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO: 80. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO: 80.

[0257] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:81. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:81. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:81.

[0258] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:82. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:82. In some embodiments, the OspA antigen comprises the amino acid sequence shown in SEQ ID NO:82.

[0259] In some embodiments, the OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:79-81. In some embodiments, the OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in any one of SEQ ID NO:79-81. In some embodiments, the OspA antigen comprises the amino acid sequence shown in any one of SEQ ID NO:79-81.

[0260] In some embodiments, the immunogenic composition comprises a first OspA antigen and a second OspA antigen, wherein the first OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 79, and the second OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 80. In some embodiments, the first OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:79. In some embodiments, the first OspA antigen comprises the amino acid sequence shown in SEQ ID NO:79. In some embodiments, the second OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:80. In some embodiments, the second OspA antigen comprises the amino acid sequence shown in SEQ ID NO:80.

[0261] In some embodiments, the immunogenic composition comprises a first OspA antigen and a second OspA antigen, wherein the first OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 81, and the second OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 81. In some embodiments, the first OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:79. In some embodiments, the first OspA antigen comprises the amino acid sequence shown in SEQ ID NO:79. In some embodiments, the second OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:81. In some embodiments, the second OspA antigen comprises the amino acid sequence shown in SEQ ID NO:81.

[0262] In some embodiments, the immunogenic composition comprises a first OspA antigen and a second OspA antigen, wherein the first OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 81, and the second OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 81. In some embodiments, the first OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:80. In some embodiments, the first OspA antigen comprises the amino acid sequence shown in SEQ ID NO:80. In some embodiments, the second OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:81. In some embodiments, the second OspA antigen comprises the amino acid sequence shown in SEQ ID NO:81.

[0263] In some embodiments, the immunogenic composition comprises a first OspA antigen, a second OspA antigen, and a third OspA antigen. The first OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:79. The second OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:80. The third OspA antigen has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:80. The amino acid sequence shown in NO:81 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the first OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:79. In some embodiments, the first OspA antigen comprises the amino acid sequence shown in SEQ ID NO:79. In some embodiments, the second OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:80. In some embodiments, the second OspA antigen comprises the amino acid sequence shown in SEQ ID NO:80. In some embodiments, the third OspA antigen comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:81. In some embodiments, the third OspA antigen comprises the amino acid sequence shown in SEQ ID NO:81.

[0264] In some embodiments, the OspA antigen is non-lipidized. In some embodiments, the first OspA antigen is non-lipidized. In some embodiments, the second OspA antigen is non-lipidized. In some embodiments, the third OspA antigen is non-lipidized.

[0265] In some embodiments, the OspA antigen is lipid-modified. In some embodiments, the first OspA antigen is lipid-modified. In some embodiments, the second OspA antigen is lipid-modified. In some embodiments, the third OspA antigen is lipid-modified. In some embodiments, the first OspA antigen and the second OspA antigen are lipid-modified. In some embodiments, the first OspA antigen and the third OspA antigen are lipid-modified. In some embodiments, the second OspA antigen and the third OspA antigen are lipid-modified. In some embodiments, the first OspA antigen, the second OspA antigen, and the third OspA antigen are lipid-modified.

[0266] Lipidification can confer adjuvant properties or stabilize the OspA antigen. Protein lipidification can be performed using many different known methods (see, for example, US 8,986,704, US 11,305,000, Bouchon et al., Anal Biochem, 1997, Hantke et al., Eur. J. Biochem, 1973, and Erdile et al., Infect Immun, 1993). In some embodiments, lipidification is performed via a chemical lipidification process. In some embodiments, lipidification is performed using an *E. coli* expression system. In some embodiments, lipidification is assisted by the presence of lipidation signal peptide sequences (such as those present on the OspA antigen). In some embodiments, the *E. coli* expression system is BL21 DE3. In some embodiments, the *E. coli* expression system uses any one or all of diacylglycerol transferase, signal peptidase II, and acyltransferase. In some embodiments, the first step involves transferring diacylglycerol to the cysteine ​​thiol group of the unmodified prolipoprotein, followed by cleavage of the signal peptide by signal peptidase II, and finally acylation of the α-amino group of the N-terminal cysteine ​​residue of the apolipoprotein. The result is the placement of a lipid and a glycerol group substituted by two other lipids on the N-terminal cysteine ​​residue of the peptide. The lipolysis signal sequence cleaved during lipolysis is not present in the final peptide sequence.

[0267] In some embodiments, the lipotropic OspA antigen is purified. In some embodiments, purification is performed using detergent extraction and / or ion-exchange chromatography. In some embodiments, purification is performed using ion-exchange chromatography and Triton X100 extraction. In some embodiments, the purification process reduces endotoxin levels. In some embodiments, purification reduces endotoxin levels to below 10 EU / mg.

[0268] In some embodiments, the OspA antigen is esterified by adding or presenting a lipid moiety to the OspA antigen. In some embodiments, a lipid moiety (such as a fatty acid) is added to the OspA antigen, such as at the C-terminus and / or N-terminus. In some embodiments, glycerol is added to the OspA antigen, such as at the C-terminus and / or N-terminus. In some embodiments, a lipid moiety (such as a fatty acid) is added to the OspA antigen at the N-terminus. In some embodiments, a lipid molecule (such as a fatty acid) and glycerol are added to the OspA antigen at the N-terminus. In some embodiments, the esterified OspA antigen has a 3-fatty acid group and a glycerol group added to the N-terminus of the esterified OspA antigen.

[0269] In some implementations, the lipid portion comprises one to three lipids, such as C 14-20 Alkyl and / or C 14-20 Alkenyl group. In some embodiments, the lipid moiety comprises saturated fatty acids (ranging from C... 14 -C 18 ), unsaturated fatty acids (C 16 / C 18 The lipid moiety is composed of glycerol and / or palmitate (C16:0) as the main component. In some embodiments, the lipid moiety is attached to glycerol. In some embodiments, the lipid moiety is attached to the N-terminal cysteine ​​residue of the OspA antigen polypeptide. In some embodiments, the N-terminal cysteine ​​is present on the signal peptide sequence. In some embodiments, the lipid moiety is attached to the N-terminal cysteine ​​residue of the signal peptide sequence. In some embodiments, the lipid moiety is attached to the thiol group of the cysteine ​​residue of the signal peptide sequence. In some embodiments, the N-terminal cysteine ​​may be esterified at both the amino group (-HN2) and the thiol group (-SH). In some embodiments, the N-terminal cysteine ​​may be esterified to form approximately 1-3 fatty acid modifications. In some embodiments, the lipid moiety attached to the N-terminal cysteine ​​is S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-N-palmitoylcysteine.

[0270] In some embodiments, the OspA antigen may further comprise a lipolysis signal peptide sequence. Lipolysis signal peptide sequences are known in the art (see, for example, US 8,986,704 and US 11,305,000). Exemplary lipolysis signal peptide sequences include, but are not limited to, the lipolysis signal peptide sequences shown in SEQ ID NO:83-85. In some embodiments, the OspA antigen further comprises a lipolysis signal peptide sequence as shown in SEQ ID NO:83. In some embodiments, the OspA antigen further comprises a lipolysis signal peptide sequence as shown in SEQ ID NO:84. In some embodiments, the OspA antigen further comprises a lipolysis signal peptide sequence as shown in SEQ ID NO:85. In some embodiments, the OspA antigen comprises an OspB leader sequence. In some embodiments, the OspB leader sequence comprises the sequence shown in SEQ ID NO:83.

[0271] The lipotropic signal peptide sequence is cleaved during processing, so the lipotropic OspA antigen may not contain the lipotropic signal peptide sequence found in mature lipotropic proteins. C. Other components

[0272] The immunogenic compositions disclosed herein may contain one or more additional components, such as one or more excipients, one or more stabilizers, one or more preservatives, and / or additional antigens.

[0273] Pharmaceutically acceptable excipients used in the immunogenic compositions of the present invention include solvents, fillers, buffers, tension modifiers, preservatives, surfactants, and emulsifiers (Pramanick et al., Pharma Times, 45:65-77, 2013). In some embodiments, the immunogenic composition contains an excipient that functions as one or more of a solvent, filler, buffer, and tension modifier (e.g., sodium chloride in saline can act as both an aqueous medium and a tension modifier). In some embodiments, the immunogenic composition contains an excipient that functions as one or both of a surfactant and an emulsifier (e.g., the nonionic surfactant polysorbate 80).

[0274] In some embodiments, the immunogenic composition contains an aqueous medium as a solvent. Suitable mediators include sterile water, saline solution, phosphate-buffered saline, and Ringer's solution. In some embodiments, the immunogenic composition is isotonic.

[0275] In some embodiments, the immunogenic composition contains a buffer. The buffer controls the pH to inhibit degradation of the active agent during processing, storage, and optionally reconstitution. Suitable buffers include salts comprising acetate, citrate, phosphate, sulfate, or Tris. In some embodiments, the buffer is not a phosphate-containing buffer. In some other embodiments, the buffer is a Tris buffer. Other suitable buffers include amino acids such as arginine, glycine, histidine, and lysine. The buffer may further comprise hydrochloric acid or sodium hydroxide. In some embodiments, the buffer maintains the pH of the immunogenic composition in the range of 6 to 9. In some embodiments, the pH is greater than (lower limit) 6, 7, or 8. In some embodiments, the pH is less than (upper limit) 9, 8, or 7. That is, the pH is in the range of about 6 to 9, where the lower limit is less than the upper limit. In some embodiments, the pH is about 6.5, about 7.0, or about 7.5.

[0276] In some embodiments, the immunogenic composition contains a tension modifier. Suitable tension modifiers include dextran, glycerol, sodium chloride, glycerin, and mannitol.

[0277] In some embodiments, the immunogenic composition contains a filler. The filler is particularly useful when the immunogenic composition is lyophilized prior to application. In some embodiments, the filler is a protective agent that helps stabilize the active agent and prevent its degradation during freeze-drying or spray drying and / or storage. Suitable fillers include sugars (e.g., monosaccharides, disaccharides, and polysaccharides), such as sucrose, lactose, trehalose, mannitol, sorbitol, glucose, and raffinose.

[0278] In some embodiments, the immunogenic composition contains a stabilizer. Suitable stabilizers include sugars, such as sucrose and lactose; amino acids, such as glycine or monosodium glutamate; and proteins, such as human serum albumin or gelatin.

[0279] In some embodiments, the immunogenic composition contains a preservative. Suitable preservatives include antioxidants and antimicrobial agents. In some embodiments, the immunogenic composition is prepared under sterile conditions and in a single-use container, therefore it does not need to contain a preservative. II. Products

[0280] This disclosure also provides vials, syringes (e.g., pre-filled syringes), and kits containing any of the immunogenic compositions described herein (as in Section I). In some embodiments, the immunogenic composition is in liquid form. In some embodiments, the immunogenic composition is in lyophilized form. In some embodiments, the provided vials, syringes, or kits contain a single dose of the immunogenic composition, such as a dose of 0.3 mL to 1.5 mL.

[0281] This disclosure also provides kits containing the first composition and the second composition. In some embodiments, the first composition contains an adjuvant, which can be any adjuvant described herein (as described in Section IA). In some embodiments, the second composition contains a spirochetal outer surface protein A (OspA) antigen, which can be any one described herein (as described in Section IB). In some embodiments, the second composition is in liquid form. In some embodiments, the second composition is in lyophilized form.

[0282] In some embodiments, the kit further includes instructions for combining the first and second compositions to prepare any immunogenic composition described herein (as in Section I). In some embodiments, the instructions are for preparing a single dose of the immunogenic composition. In some embodiments, the kit is for preparing a single dose of the immunogenic composition, such as a dose of 0.3 mL to 1.5 mL.

[0283] In some embodiments, combining the first composition with the second composition involves mixing the first composition and the second composition. In some embodiments, combining the first composition with the second composition involves mixing the first composition and the second composition in a single vial. In some embodiments, combining the first composition with the second composition involves mixing the first composition and the second composition in a single vial by gentle inversion. In some embodiments, mixing continues at room temperature for up to about one hour.

[0284] In some embodiments, the first composition contains a second adjuvant, which can be any adjuvant described herein (as described in Section 1A). In some embodiments, the second composition contains a second adjuvant, which can be any adjuvant described herein (as described in Section 1A).

[0285] In some embodiments, the kit further includes instructions for administering the immunogenic composition to a subject to stimulate an immune response against *Treponema pallidum*. In some embodiments, administration is performed according to any of the methods described herein (e.g., any of the methods described in Section III). In some embodiments, the subject is a human subject. In some embodiments, the human subject is at least 18 years old. In some embodiments, the human subject is from about 18 years to about 70 years old. In some embodiments, the human subject is from about 18 years to about 55 years old. In some embodiments, the human subject is under 18 years old. In some embodiments, the human subject is from about 2 years to about 5 years old. In some embodiments, the human subject is from about 5 years to about 18 years old.

[0286] In some embodiments, the kit contains a suitably packaged immunogenic composition. In some embodiments, the kit contains i) a first composition and ii) a second composition, both suitably packaged to allow combination to form an immunogenic composition. For example, if the immunogenic composition is a lyophilized powder, a vial with an elastic stopper can be used, allowing the powder to be easily resuspended by injecting a fluid (e.g., sterile water, saline, etc.) through the elastic stopper. In some embodiments, the kit contains a device for administration (e.g., a syringe). In some embodiments, the syringe is pre-filled with the immunogenic composition. In some embodiments, administration is performed by intramuscular injection. Instructions for use of the immunogenic composition typically include information on the dosage, schedule, and route of administration for the intended method of use. III. Usage Method

[0287] This disclosure also relates to a method for stimulating an immune response against the genus *Borrelia* in a subject, the method comprising administering to the subject an effective amount of any immunogenic composition described herein (e.g., in Section I). In some embodiments, the antigen and adjuvant are present in the immunogenic composition in amounts that effectively stimulate an immune response against the genus *Borrelia* in the subject.

[0288] This disclosure also relates to methods for protecting a subject from infection with *Borrelia*, the methods comprising administering to the subject an effective amount of any immunogenic composition described herein (e.g., in Section I). In some embodiments, the antigen and adjuvant are present in the immunogenic composition in amounts that effectively stimulate an immune response against *Borrelia* in the subject.

[0289] This disclosure also relates to a method for preventing a subject from developing Lyme disease, the method comprising administering to the subject an effective amount of any immunogenic composition described herein (e.g., in Section I). In some embodiments, the antigen and adjuvant are present in the immunogenic composition in amounts that effectively stimulate an immune response against the genus *Treponema* in the subject.

[0290] In some embodiments, the subjects are human subjects. In some embodiments, the human subjects are at least 18 years old. In some embodiments, the human subjects are from about 18 years old to about 70 years old. In some embodiments, the human subjects are from about 18 years old to about 55 years old. In some embodiments, the human subjects are under 18 years old. In some embodiments, the human subjects are from about 2 years old to about 5 years old. In some embodiments, the human subjects are from about 5 years old to about 18 years old.

[0291] In some embodiments, the methods of this disclosure are suitable for increasing the immune response against *Borrelia* in a subject relative to a baseline immune response against *Borrelia* prior to administration of the immunogenic composition. Stimulating an immune response means increasing an immune response, which can be achieved by inducing a new immune response (e.g., as a result of an initial vaccination regimen) or enhancing an existing immune response (e.g., as a result of a booster vaccination regimen). In some embodiments, stimulating an immune response includes one or more of the following: stimulating cytokine production; stimulating B lymphocyte proliferation; stimulating antibody production; stimulating the expression of genes related to the interferon pathway; stimulating the expression of genes related to chemical inducers; and stimulating the maturation of plasmacytoid dendritic cells. In some embodiments, the immune response prevents the subject from contracting *Borrelia* infection. In some embodiments, the immune response prevents the subject from developing Lyme disease.

[0292] In some embodiments, a first dose and a second dose of the immunogenic composition are administered to the subject. In some embodiments, a second dose of the immunogenic composition is administered approximately 2 weeks to approximately 12 weeks after administration of the first dose. In some embodiments, a second dose of the immunogenic composition is administered approximately 2 weeks to approximately 10 weeks after administration of the first dose. In some embodiments, a second dose is administered approximately 2 weeks to approximately 2 months after administration of the first dose. In some embodiments, a second dose is administered approximately 2 weeks to approximately 6 weeks after administration of the first dose. In some embodiments, a second dose is administered approximately 2 weeks to approximately 4 weeks after administration of the first dose. In some embodiments, a second dose is administered approximately 3 weeks after administration of the first dose.

[0293] In some embodiments, the second dose is administered approximately 2 to approximately 6 weeks after the first dose. In some embodiments, the second dose is administered approximately 3 to approximately 5 weeks after the first dose. In some embodiments, the second dose is administered approximately 1 month after the first dose.

[0294] In some embodiments, a second dose is administered approximately 6 to approximately 10 weeks after the first dose. In some embodiments, a second dose is administered approximately 7 to approximately 9 weeks after the first dose. In some embodiments, a second dose is administered approximately 2 months after the first dose.

[0295] In some embodiments, a third dose of the immunogenic composition is administered to the subject. In some embodiments, the third dose is administered approximately 4 to approximately 8 months after the first dose. In some embodiments, the third dose is administered approximately 5 to approximately 7 months after the first dose. In some embodiments, the third dose is administered approximately 22 to approximately 26 weeks after the first dose. In some embodiments, the third dose is administered approximately 6 months after the first dose.

[0296] In some embodiments, a booster dose of the immunogenic composition is administered to the subject. In some embodiments, a booster dose is administered at least one year after the first dose. In some embodiments, a booster dose is administered at least two years after the first dose.

[0297] In some embodiments, a booster dose is administered approximately 10 months to approximately 5 years after the first dose. In some embodiments, a booster dose is administered approximately 1 year to approximately 5 years after the first dose. In some embodiments, a booster dose is administered approximately 20 months to approximately 5 years after the first dose. In some embodiments, a booster dose is administered approximately 2 years to approximately 5 years after the first dose. In some embodiments, a booster dose is administered approximately 2 years to approximately 4 years after the first dose. In some embodiments, a booster dose is administered approximately 2 years to approximately 3 years after the first dose.

[0298] In some implementations, a booster dose is administered approximately 10 to 14 months after the first dose. In some implementations, a booster dose is administered approximately 1 year after the first dose.

[0299] In some implementations, a booster dose is administered approximately 20 to approximately 28 months after the first dose. In some implementations, a booster dose is administered approximately 2 years after the first dose.

[0300] In some implementations, multiple booster doses are administered to the subject. In some implementations, multiple annual booster doses are administered to the subject. In some implementations, the first booster dose is administered at least about one year or at least about two years after the first dose. In some implementations, a booster dose is administered annually.

[0301] In some implementations, a first dose and a second dose of the immunogenic composition are administered to the subject, wherein the second dose is administered approximately one month after the first dose.

[0302] In some embodiments, a subject is administered a first dose, a second dose, and a booster dose of the immunogenic composition, wherein the second dose is administered approximately one month after the first dose, and the booster dose is administered at least two years after the first dose.

[0303] In some implementations, a first dose and a second dose of the immunogenic composition are administered to the subject, wherein the second dose is administered approximately two months after the first dose.

[0304] In some embodiments, a subject is administered a first dose, a second dose, and a booster dose of the immunogenic composition, wherein the second dose is administered approximately two months after the first dose, and the booster dose is administered at least one year after the first dose.

[0305] In some embodiments, a subject is administered a first dose, a second dose, and a booster dose of the immunogenic composition, wherein the second dose is administered approximately 2 months after the first dose, and the booster dose is administered at least 2 years after the first dose.

[0306] In some implementations, a first, second, and third dose of the immunogenic composition is administered to the subject, wherein the second dose is administered approximately two months after the first dose and the third dose is administered approximately six months after the first dose.

[0307] In some embodiments, a subject is administered a first, second, third, and booster dose of the immunogenic composition, wherein the second dose is administered approximately 2 months after the first dose, the third dose is administered approximately 6 months after the first dose, and the booster dose is administered at least 2 years after the first dose.

[0308] In some implementations, the immunogenic composition is administered to the subject in a first dose, a second dose, a third dose, and multiple booster doses, wherein the second dose is administered approximately 2 months after the first dose, the third dose is administered approximately 6 months after the first dose, the first booster dose is administered approximately 1 year after the first dose, and booster doses are administered annually.

[0309] In some embodiments, the immunogenic composition is administered via intramuscular injection. In some embodiments, the injection site is in the subject's arm. In some embodiments, the injection site is in the subject's deltoid muscle. Exemplary Implementation

[0310] The provided implementation plan includes: 1. An immunogenic composition comprising (i) a spirochetal outer surface protein A (OspA) antigen or an RNA polynucleotide encoding said OspA antigen and (ii) an adjuvant selected from aluminum salt adjuvants; Toll-like receptor 9 (TLR9) agonists; liposome-based adjuvants comprising lipopolysaccharides and saponins; and an oil-in-water emulsion comprising squalene, sorbitol esters and polyoxyethylene sorbitol esters. 2. An immunogenic composition comprising (i) a spirochetal outer surface protein A (OspA) antigen and (ii) an adjuvant selected from aluminum salt adjuvants; Toll-like receptor 9 (TLR9) agonists; liposome-based adjuvants comprising lipopolysaccharides and saponins; and an oil-in-water emulsion comprising squalene, sorbitol esters and polyoxyethylene sorbitol esters. 3. The immunogenic composition according to embodiment 1 or embodiment 2, wherein the immunogenic composition does not contain an RNA polynucleotide encoding a non-OspA spirochetal outer surface protein antigen. 4. The immunogenic composition according to any one of embodiments 1-3, wherein the immunogenic composition does not contain non-OspA spirochetal outer surface protein antigens. 5. The immunogenic composition according to any one of embodiments 1-4, wherein the adjuvant is an aluminum salt adjuvant. 6. The immunogenic composition according to embodiment 5, wherein the immunogenic composition further comprises a second adjuvant. 7. The immunogenic composition according to embodiment 6, wherein the second adjuvant is selected from TLR9 agonists; liposome-based adjuvants comprising lipopolysaccharides and saponins; and oil-in-water emulsions comprising squalene, sorbitol esters and polyoxyethylene sorbitol esters. 8. The immunogenic composition according to any one of embodiments 1-4, wherein the adjuvant is a TLR9 agonist. 9. The immunogenic composition according to embodiment 8, wherein the immunogenic composition further comprises a second adjuvant. 10. The immunogenic composition according to embodiment 9, wherein the second adjuvant is selected from aluminum salt adjuvants; liposome-based adjuvants comprising lipopolysaccharides and saponins; and oil-in-water emulsion-based adjuvants comprising squalene, sorbitol esters and polyoxyethylene sorbitol esters. 11. The immunogenic composition according to any one of embodiments 1-4, wherein the adjuvant is a liposome-based adjuvant comprising lipopolysaccharide and saponin. 12. The immunogenic composition according to embodiment 11, wherein the immunogenic composition further comprises a second adjuvant. 13. The immunogenic composition according to embodiment 12, wherein the second adjuvant is selected from aluminum salt adjuvants; TLR9 agonists; and adjuvants based on oil-in-water emulsions comprising squalene, sorbitol esters and polyoxyethylene sorbitol esters. 14. The immunogenic composition according to any one of embodiments 1-4, wherein the adjuvant is an oil-in-water emulsion-based adjuvant comprising squalene, sorbitol ester and polyoxyethylene sorbitol ester. 15. The immunogenic composition according to embodiment 14, wherein the immunogenic composition further comprises a second adjuvant. 16. The immunogenic composition according to embodiment 15, wherein the second adjuvant is selected from aluminum salt adjuvants; TLR9 agonists; and liposome-based adjuvants comprising lipopolysaccharides and saponins. 17. The immunogenic composition according to any one of embodiments 1-4, 7-10, 13 and 16, wherein the TLR9 agonist is an oligonucleotide comprising an unmethylated cytidine-phosphate-guanosine (CpG) motif. 18. The immunogenic composition according to embodiment 17, wherein the oligonucleotide is 10 to 35 nucleotides in length. 19. The immunogenic composition according to embodiment 17 or embodiment 18, wherein the oligonucleotide comprises sequence 5'-AACGTTCGAG-3' (SEQ ID NO:2). 20. The immunogenic composition according to any one of embodiments 17-19, wherein the oligonucleotide comprises the sequence 5'-TGACTGTGAA CGTTCGAGAT GA-3' (SEQ ID NO:1). 21. An immunogenic composition comprising a spirochetal outer surface protein A (OspA) antigen and a Toll-like receptor 9 (TLR9) agonist oligonucleotide of 10 to 35 nucleotides in length, said oligonucleotide comprising the sequence 5'-AACGTTCGAG-3' (SEQ ID NO:2) and an unmethylated cytidine-phosphate-guanosine (CpG) motif. 22. The immunogenic composition according to embodiment 21, wherein the oligonucleotide comprises the sequence 5'-TGACTGTGAA CGTTCGAGAT GA-3' (SEQ ID NO:1). 23. An immunogenic composition comprising a spirochetal outer surface protein A (OspA) antigen and a Toll-like receptor 9 (TLR9) agonist oligonucleotide 5'-TGACTGTGAA CGTTCGAGAT GA-3' (SEQ ID NO:1), said oligonucleotide comprising an unmethylated cytidine-phosphate-guanosine (CpG) motif. 24. The immunogenic composition according to any one of embodiments 21-23, wherein the immunogenic composition further comprises a second adjuvant. 25. The immunogenic composition according to embodiment 24, wherein the second adjuvant is selected from aluminum salt adjuvants; liposome-based adjuvants comprising lipopolysaccharides and saponins; and oil-in-water emulsion-based adjuvants comprising squalene, sorbitol esters and polyoxyethylene sorbitol esters. 26. The immunogenic composition according to any one of embodiments 9, 10, 12, 13, 15-20, 24 and 25, wherein the second adjuvant is an aluminum salt adjuvant. 27. An immunogenic composition comprising: (i) a spirochetal outer surface protein A (OspA) antigen, (ii) a toll-like receptor 9 (TLR9) agonist oligonucleotide 5'-TGACTGTGAA CGTTCGAGAT GA-3' (SEQ ID NO:1), said oligonucleotide comprising an unmethylated cytidine-phosphate-guanosine (CpG) motif, and (iii) an aluminum salt adjuvant. 28. The immunogenic composition according to any one of embodiments 1-7, 10, 13, 16-20 and 25-27, wherein the aluminum salt adjuvant comprises one or more of amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate and potassium aluminum sulfate. 29. The immunogenic composition according to any one of embodiments 1-7, 10, 13, 16-20 and 25-28, wherein the aluminum salt adjuvant comprises aluminum hydroxide. 30. The immunogenic composition according to any one of embodiments 1-29, wherein the immunogenic composition comprises about 0.25 mg to about 1.25 mg A1 3+ Approximately 0.25 mg to approximately 0.50 mg Al 3+ Or approximately 0.30 mg to approximately 0.40 mg Al 3+ . 31. The immunogenic composition according to any one of embodiments 17-30, wherein the oligonucleotide is a single-stranded oligodeoxynucleotide. 32. The immunogenic composition according to any one of embodiments 17-30, wherein the oligonucleotide is entirely RNA. 33. The immunogenic composition according to any one of embodiments 17-30, wherein the oligonucleotide is an RNA / DNA chimera. 34. The immunogenic composition according to any one of embodiments 17-33, wherein the oligonucleotide comprises at least one phosphate thioester bond. 35. The immunogenic composition according to any one of embodiments 17-34, wherein the oligonucleotide contains only phosphate thioester bonds. 36. The immunogenic composition according to any one of embodiments 17-34, wherein the oligonucleotide comprises a combination of one or more phosphodiester bonds and one or more thiophosphate bonds. 37. The immunogenic composition according to any one of embodiments 1-4, 7-10, 13 and 16-36, wherein the immunogenic composition comprises about 375 µg to about 6000 µg of a TLR9 agonist or about 750 µg to about 3000 µg of the TLR9 agonist. 38. The immunogenic composition according to any one of embodiments 1-4, 7-10, 13 and 16-36, wherein the immunogenic composition comprises about 375 µg, about 750 µg, about 1000 µg, about 1500 µg, about 3000 µg or about 6000 µg of the TLR9 agonist. 39. The immunogenic composition according to any one of embodiments 1-4, 7, 10-13, 16-20, 25, 26 and 28-38, wherein the lipopolysaccharide is lipid A or a derivative thereof. 40. The immunogenic composition according to any one of embodiments 1-4, 7, 10-13, 16-20, 25, 26 and 28-39, wherein the lipopolysaccharide is monophosphoryl lipid A or a derivative thereof. 41. The immunogenic composition according to any one of embodiments 1-4, 7, 10-13, 16-20, 25, 26 and 28-40, wherein the lipopolysaccharide is 3-O-deacylated-4'-monophosphoryllipide A. 42. The immunogenic composition according to any one of embodiments 1-4, 7, 10-13, 16-20, 25, 26 and 28-40, wherein the lipopolysaccharide is a 3-deacylated hexaacyl disaccharide. 43. The immunogenic composition according to any one of embodiments 1-4, 7, 10-13, 16-20, 25, 26 and 28-42, wherein the immunogenic composition comprises about 1 μg to about 100 μg, about 10 μg to about 50 μg, about 20 μg to about 30 μg, about 21 μg to about 29 μg, about 22 μg to about 28 μg, about 23 μg to about 27 μg or about 24 μg to about 26 μg of the lipopolysaccharide. 44. The immunogenic composition according to any one of embodiments 1-4, 7, 10-13, 16-20, 25, 26 and 28-42, wherein the immunogenic composition comprises about 1 μg to about 30 μg, about 5 μg to about 15 μg, about 6 μg to about 14 μg, about 7 μg to about 13 μg, about 8 μg to about 12 μg or about 9 μg to about 11 μg of the lipopolysaccharide. 45. The immunogenic composition according to any one of embodiments 1-4, 7, 10-13, 16-20, 25, 26 and 28-42, wherein the immunogenic composition comprises about 1 μg to about 9 μg, about 2 μg to about 8 μg, about 3 μg to about 7 μg or about 4 μg to about 6 μg of the lipopolysaccharide. 46. ​​The immunogenic composition according to any one of embodiments 1-4, 7, 10-13, 16-20, 25, 26 and 28-45, wherein the saponin is derived from the bark of the Molina soapberry tree. 47. The immunogenic composition according to any one of embodiments 1-4, 7, 10-13, 16-20, 25, 26 and 28-46, wherein the saponin is Quil A or a derivative thereof. 48. The immunogenic composition according to any one of embodiments 1-4, 7, 10-13, 16-20, 25, 26 and 28-47, wherein the saponin is QS-17. 49. The immunogenic composition according to any one of embodiments 1-4, 7, 10-13, 16-20, 25, 26 and 28-47, wherein the saponin is QS-21. 50. The immunogenic composition according to any one of embodiments 1-4, 7, 10-13, 16-20, 25, 26 and 28-49, wherein the immunogenic composition comprises about 1 μg to about 100 μg, about 10 μg to about 50 μg, about 20 μg to about 30 μg, about 21 μg to about 29 μg, about 22 μg to about 28 μg, about 23 μg to about 27 μg or about 24 μg to about 26 μg of the saponin. 51. The immunogenic composition according to any one of embodiments 1-4, 7, 10-13, 16-20, 25, 26 and 28-49, wherein the immunogenic composition comprises about 1 μg to about 30 μg, about 5 μg to about 15 μg, about 6 μg to about 14 μg, about 7 μg to about 13 μg, about 8 μg to about 12 μg or about 9 μg to about 11 μg of the saponin. 52. The immunogenic composition according to any one of embodiments 1-4, 7, 10-13, 16-20, 25, 26 and 28-49, wherein the immunogenic composition comprises about 1 μg to about 9 μg, about 2 μg to about 8 μg, about 3 μg to about 7 μg or about 4 μg to about 6 μg of the saponin. 53. The immunogenic composition according to any one of embodiments 1-4, 7, 10-13, 16-20, 25, 26 and 28-52, wherein the liposome-based adjuvant further comprises a sterol. 54. The immunogenic composition according to embodiment 53, wherein the sterol is β-sitosterol, stigmasterol, ergosterol, ergocalciferol, or cholesterol. 55. The immunogenic composition according to embodiment 53 or embodiment 54, wherein the sterol is cholesterol. 56. The immunogenic composition according to any one of embodiments 53-55, wherein the ratio of saponin to sterol is 1:1 to 1:100 w / w or 1:1 to 1:5 w / w, optionally about 1:1 w / w. 57. The immunogenic composition according to any one of embodiments 1-4, 7, 10-13, 16-20, 25, 26 and 28-56, wherein the liposome-based adjuvant further comprises neutral lipids. 58. The immunogenic composition according to embodiment 57, wherein the neutral lipid is phosphatidylcholine. 59. The immunogenic composition according to embodiment 58, wherein the phosphatidylcholine is egg yolk phosphatidylcholine, dioleoyl phosphatidylcholine (DOPC) or dilauryl phosphatidylcholine. 60. The immunogenic composition according to embodiment 58 or embodiment 59, wherein the phosphatidylcholine is DOPC. 61. The immunogenic composition according to any one of embodiments 1-4, 7, 10, 13-20, 25, 26 and 28-60, wherein the sorbitol ester is sorbitol ester 20. 62. The immunogenic composition according to any one of embodiments 1-4, 7, 10, 13-20, 25, 26 and 28-60, wherein the sorbitol ester is sorbitol ester 85. 63. The immunogenic composition according to any one of embodiments 1-4, 7, 10, 13-20, 25, 26 and 28-62, wherein the polyoxyethylene sorbitol ester is polyoxyethylene sorbitol ester 20. 64. The immunogenic composition according to any one of embodiments 1-4, 7, 10, 13-20, 25, 26 and 28-62, wherein the polyoxyethylene sorbitol ester is polyoxyethylene sorbitol ester 80. 65. The immunogenic composition according to any one of embodiments 1-4, 7, 10, 13-20, 25, 26 and 28-64, wherein the immunogenic composition comprises about 2% w / v to about 7% w / v, about 3% w / v to about 6% w / v or about 4% w / v to about 5% w / v of the squalene. 66. The immunogenic composition according to any one of embodiments 1-4, 7, 10, 13-20, 25, 26 and 28-65, wherein the immunogenic composition comprises about 0.2% w / v to about 0.8% w / v, about 0.3% w / v to about 0.7% w / v or about 0.4% w / v to about 0.6% w / v of the dehydrated sorbitol ester. 67. The immunogenic composition according to any one of embodiments 1-4, 7, 10, 13-20, 25, 26 and 28-66, wherein the immunogenic composition comprises about 0.2% w / v to about 0.8% w / v, about 0.3% w / v to about 0.7% w / v or about 0.4% w / v to about 0.6% w / v of the polyoxyethylene sorbitol ester. 68. The immunogenic composition according to any one of embodiments 1-67, wherein the OspA antigen is two or more OspA antigens. 69. The immunogenic composition according to embodiment 68, wherein the two or more OspA antigens are a first OspA antigen and a second OspA antigen. 70. The immunogenic composition according to embodiment 68 or embodiment 69, wherein the two or more OspA antigens are a first OspA antigen, a second OspA antigen, and a third OspA antigen. 71. The immunogenic composition according to any one of embodiments 68-70, wherein each of the two or more OspA antigens has a different amino acid sequence. 72. The immunogenic composition according to any one of embodiments 1-71, wherein the OspA antigen is independently the OspA antigen of Borrelia burgdorferi, Borrelia aberrantii, Borrelia Bavaria, Borrelia galbella, Borrelia mellitus, Borrelia louvelii, Borrelia bisserii, Borrelia valgum, or Borrelia stearothermiae. 73. The immunogenic composition according to any one of embodiments 1-72, wherein the OspA antigen is independently a Borrelia burgdorferi OspA antigen. 74. The immunogenic composition according to any one of embodiments 1-73, wherein the OspA antigen independently comprises one or more epitopes from the OspA protein. 75. The immunogenic composition according to embodiment 74, wherein the one or more epitopes comprise at least two, three, four, five or six epitopes from one or more OspA proteins. 76. The immunogenic composition according to embodiment 74 or embodiment 75, wherein the one or more epitopes comprise at least two, three, four, five or six epitopes from at least two, three, four, five or six OspA serotypes. 77. The immunogenic composition according to any one of embodiments 74-76, wherein the one or more epitopes comprise epitopes selected from the amino acid sequences shown in SEQ ID NO:4-49. 78. The immunogenic composition according to any one of embodiments 74-77, wherein the one or more epitopes are independently selected from the amino acid sequences shown in SEQ ID NO:4-49. 79. The immunogenic composition according to any one of embodiments 74-78, wherein the one or more epitopes comprise an epitope selected from the amino acid sequences shown in SEQ ID NO: 9, 10, 33, 34, 36, 47 and 49. 80. The immunogenic composition according to any one of embodiments 1-78, wherein the OspA antigen independently has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 50-62. 81. The immunogenic composition according to any one of embodiments 1-78 and 80, wherein the OspA antigen independently comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 consecutive amino acids of the amino acid sequence shown in any one of SEQ ID NO:50-62. 82. The immunogenic composition according to any one of embodiments 1-78, 80 and 81, wherein the OspA antigen independently comprises the amino acid sequence shown in any one of SEQ ID NO:50-62. 83. The immunogenic composition according to any one of embodiments 1-78, wherein the OspA antigen is independently an OspA chimeric antigen comprising a plurality of epitopes from one or more OspA proteins. 84. The immunogenic composition according to embodiment 83, wherein the plurality of epitopes comprises at least two, three, four, five, or six epitopes from at least two, three, four, five, or six OspA serotypes. 85. The immunogenic composition according to embodiment 83 or embodiment 84, wherein the plurality of epitopes comprises epitopes selected from the amino acid sequences shown in SEQ ID NO:4-49. 86. The immunogenic composition according to any one of embodiments 83-85, wherein the plurality of epitopes are independently selected from the amino acid sequences shown in SEQ ID NO:4-49. 87. The immunogenic composition according to any one of embodiments 83-86, wherein the plurality of epitopes comprise epitopes selected from the amino acid sequences shown in SEQ ID NO: 9, 10, 33, 34, 36, 47 and 49. 88. The immunogenic composition according to any one of embodiments 83-87, wherein the plurality of epitopes are independently selected from the amino acid sequences shown in SEQ ID NO: 9, 10, 33, 34, 36, 47 and 49. 89. The immunogenic composition according to any one of embodiments 1-78 and 83-86, wherein the OspA antigen independently comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 63-82. 90. The immunogenic composition according to any one of embodiments 1-78 and 83-89, wherein the OspA antigen independently comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 consecutive amino acids of the amino acid sequence shown in any one of SEQ ID NO: 63-82. 91. The immunogenic composition according to any one of embodiments 1-78 and 83-90, wherein the OspA antigen independently comprises the amino acid sequence shown in any one of SEQ ID NO:63-82. 92. The immunogenic composition according to any one of embodiments 1-78 and 83-89, wherein the OspA antigen independently comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:79-81. 93. The immunogenic composition according to any one of embodiments 1-78, 83-89 and 92, wherein the immunogenic composition comprises a first OspA antigen and a second OspA antigen, the first OspA antigen comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:79, and the second OspA antigen comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:80. 94. The immunogenic composition according to any one of embodiments 1-78, 83-89 and 91-93, wherein the immunogenic composition comprises a first OspA antigen containing the amino acid sequence shown in SEQ ID NO:79 and a second OspA antigen containing the amino acid sequence shown in SEQ ID NO:80. 95. An immunogenic composition according to any one of embodiments 1-78, 83-89 and 92, wherein the immunogenic composition comprises a first OspA antigen and a second OspA antigen, the first OspA antigen comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 79, and the second OspA antigen comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 81. 96. The immunogenic composition according to any one of embodiments 1-78, 83-89, 91, 92 and 95, wherein the immunogenic composition comprises a first OspA antigen containing the amino acid sequence shown in SEQ ID NO:79 and a second OspA antigen containing the amino acid sequence shown in SEQ ID NO:81. 97. An immunogenic composition according to any one of embodiments 1-78, 83-89 and 92, wherein the immunogenic composition comprises a first OspA antigen and a second OspA antigen, the first OspA antigen comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 80, and the second OspA antigen comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 81. 98. The immunogenic composition according to any one of embodiments 1-78, 83-89, 91, 92 and 97, wherein the immunogenic composition comprises a first OspA antigen containing the amino acid sequence shown in SEQ ID NO:80 and a second OspA antigen containing the amino acid sequence shown in SEQ ID NO:81. 99. An immunogenic composition according to any one of embodiments 1-78, 83-89, 92, and 93, wherein the immunogenic composition comprises a first OspA antigen, a second OspA antigen, and a third OspA antigen, the first OspA antigen comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 79, the second OspA antigen comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 80, and the third ...3%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in NO:81 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. 100. The immunogenic composition according to any one of embodiments 1-78, 83-89 and 91-94, wherein the immunogenic composition comprises a first OspA antigen containing the amino acid sequence shown in SEQ ID NO:79, a second OspA antigen containing the amino acid sequence shown in SEQ ID NO:80 and a third OspA antigen containing the amino acid sequence shown in SEQ ID NO:81. 101. The immunogenic composition according to any one of embodiments 1-100, wherein the OspA antigen is independently non-lipotropic. 102. The immunogenic composition according to any one of embodiments 1-101, wherein the OspA antigen is independently lipid-modified. 103. The immunogenic composition according to any one of embodiments 1-102, wherein the immunogenic composition is in a dose of 0.3 mL to 1.5 mL. 104. The immunogenic composition according to any one of embodiments 1-103, wherein the immunogenic composition is used to stimulate an immune response against the genus *Treponema* in a subject by administering an effective amount of the immunogenic composition to the subject. 105. The immunogenic composition according to any one of embodiments 1-104, wherein the immunogenic composition is used to protect the subject from spirochetal infection by administering an effective amount of the immunogenic composition to the subject. 106. The immunogenic composition according to any one of embodiments 1-104, wherein the immunogenic composition is used to prevent the subject from developing Lyme disease by administering an effective amount of the immunogenic composition to the subject. 107. The immunogenic composition according to any one of embodiments 1-104, wherein the subject is a human subject. 108. The immunogenic composition according to embodiment 107, wherein the human subject is at least 18 years old. 109. The immunogenic composition according to embodiment 107 or embodiment 108, wherein the human subject is between 18 and 70 years old. 110. The immunogenic composition according to embodiment 107, wherein the human subject is under 18 years of age. 111. The immunogenic composition according to embodiment 107 or embodiment 110, wherein the human subject is 2 to 5 years old. 112. The immunogenic composition according to embodiment 107 or embodiment 110, wherein the human subject is aged 5 to 18 years. 113. A method for stimulating an immune response against the genus *Treponema* in a subject, the method comprising administering to the subject an effective amount of the immunogenic composition according to any one of embodiments 1-112. 114. A method for protecting a subject from infection with the genus *Treponema*, the method comprising administering to the subject an effective amount of the immunogenic composition according to any one of embodiments 1-112. 115. A method for preventing a subject from developing Lyme disease, the method comprising administering to the subject an effective amount of the immunogenic composition according to any one of embodiments 1-112. 116. The method according to any one of embodiments 113-115, wherein the immunogenic composition is administered by intramuscular injection. 117. The method according to any one of embodiments 113-116, wherein the subject is administered a first dose and a second dose of the immunogenic composition. 118. The method according to embodiment 117, wherein the second dose of the immunogenic composition is administered about 2 weeks to about 10 weeks after administration of the first dose of the immunogenic composition. 119. The method according to embodiment 117 or embodiment 118, wherein the second dose of the immunogenic composition is administered about 1 month or about 2 months after the administration of the first dose of the immunogenic composition. 120. The method according to any one of embodiments 117-119, wherein a third dose of the immunogenic composition is administered to the subject. 121. The method according to embodiment 120, wherein the third dose of the immunogenic composition is administered approximately 4 to approximately 8 months after administration of the first dose of the immunogenic composition. 122. The method according to embodiment 120 or embodiment 121, wherein the third dose of the immunogenic composition is administered approximately 6 months after the administration of the first dose of the immunogenic composition. 123. The method according to any one of embodiments 113-122, wherein a booster dose of the immunogenic composition is administered to the subject. 124. The method according to embodiment 123, wherein the enhanced dose is administered at least about 1 year or at least about 2 years after the administration of the first dose. 125. The method according to any one of embodiments 113-124, wherein multiple booster doses are administered to the subject. 126. The method according to embodiment 125, wherein the first booster dose is administered at least about 1 year or at least about 2 years after the administration of the first dose. 127. The method according to implementation scheme 125 or implementation scheme 126, wherein an enhanced dose is administered annually. 128. The method according to any one of embodiments 113-127, wherein the subject is a human subject. 129. The method according to embodiment 128, wherein the age of the human subject is at least 18 years. 130. The method according to embodiment 128 or embodiment 129, wherein the age of the human subject is between 18 and 70 years. 131. The method according to embodiment 128, wherein the human subject is under 18 years of age. 132. The method according to embodiment 128 or embodiment 131, wherein the age of the human subject is 2 to 5 years. 133. The method according to embodiment 128 or embodiment 131, wherein the human subject is between 5 and 18 years old. 134. The use of the immunogenic composition according to any one of embodiments 1-112 for stimulating an immune response against the genus *Treponema* in a subject by administering an effective amount of the immunogenic composition to the subject. 135. The use of the immunogenic composition according to any one of embodiments 1-112 for protecting a subject from infection with *Treponema pallidum* by administering an effective amount of the immunogenic composition to the subject. 136. The use of the immunogenic composition according to any one of embodiments 1-112 for preventing a subject from developing Lyme disease by administering an effective amount of the immunogenic composition to the subject. 137. Use of the immunogenic composition according to any one of embodiments 1-112 in the manufacture of a medicament for stimulating an immune response against the genus *Treponema* in a subject. 138. Use of the immunogenic composition according to any one of embodiments 1-112 in the manufacture of a medicament for protecting a subject from infection with the genus *Treponema*. 139. Use of the immunogenic composition according to any one of embodiments 1-112 in the manufacture of a medicament for preventing a subject from developing Lyme disease. 140. The use according to any one of embodiments 134-139, wherein the immunogenic composition is administered by intramuscular injection. 141. The use according to any one of embodiments 134-140, wherein the first and second doses of the immunogenic composition are administered to the subject. 142. The use according to embodiment 141, wherein the second dose of the immunogenic composition is administered about 2 weeks to about 10 weeks after the administration of the first dose of the immunogenic composition. 143. The use according to embodiment 141 or embodiment 142, wherein the second dose of the immunogenic composition is administered about 1 month or about 2 months after the administration of the first dose of the immunogenic composition. 144. The use according to any one of embodiments 141-143, wherein a third dose of the immunogenic composition is administered to the subject. 145. The use according to embodiment 144, wherein the third dose of the immunogenic composition is administered approximately 4 months to approximately 8 months after administration of the first dose of the immunogenic composition. 146. The use according to embodiment 144 or embodiment 145, wherein the third dose of the immunogenic composition is administered approximately 6 months after the administration of the first dose of the immunogenic composition. 147. The use according to any one of embodiments 134-146, wherein a booster dose of the immunogenic composition is administered to the subject. 148. The use according to embodiment 147, wherein the enhanced dose is administered at least about 1 year or at least about 2 years after the administration of the first dose. 149. The use according to any one of embodiments 134-148, wherein multiple booster doses are administered to the subject. 150. The use according to embodiment 149, wherein the first booster dose is administered at least about 1 year or at least about 2 years after the administration of the first dose. 151. The use as described in implementation scheme 149 or implementation scheme 150, wherein an enhanced dose is applied annually. 152. The use according to any one of embodiments 133-151, wherein the subject is a human subject. 153. The use according to embodiment 152, wherein the age of the human subject is at least 18 years. 154. The use according to embodiment 152 or embodiment 153, wherein the age of the human subject is 18 to 70 years. 155. The use according to embodiment 152, wherein the human subject is under 18 years of age. 156. The use according to embodiment 152 or embodiment 155, wherein the age of the human subject is 2 to 5 years. 157. The use according to embodiment 152 or embodiment 155, wherein the age of the human subject is 5 to 18 years. 158. A vial containing an immunogenic composition according to any one of embodiments 1-112. 159. The vial according to embodiment 158, wherein the immunogenic composition is in liquid form. 160. The vial according to embodiment 158, wherein the immunogenic composition is in lyophilized form. 161. The vial according to any one of embodiments 158-160, wherein the vial contains a single dose of the immunogenic composition. 162. The vial according to embodiment 161, wherein the single dose is a dose of 0.3 mL to 1.5 mL. 163. A pre-filled syringe comprising an immunogenic composition according to any one of embodiments 1-112. 164. The pre-filled syringe according to embodiment 163, wherein the syringe contains a single dose of the immunogenic composition. 165. The pre-filled syringe according to embodiment 164, wherein the single dose is a dose of 0.3 mL to 1.5 mL. 166. A kit comprising: a) A first composition comprising an adjuvant selected from aluminum salt adjuvants; Toll-like receptor 9 (TLR9) agonists; liposome-based adjuvants comprising lipopolysaccharides and saponins; and oil-in-water emulsion-based adjuvants comprising squalene, sorbitol esters and polyoxyethylene sorbitol esters. b) A second composition containing the outer surface protein A (OspA) antigen of *Breospira spp.*; and c) A specification for combining the first composition and the second composition to prepare an immunogenic composition according to any one of embodiments 1-112. 167. The kit according to embodiment 166, wherein the second composition is in liquid form. 168. The kit according to embodiment 166, wherein the second composition is in lyophilized form. 169. The kit according to any one of embodiments 166-168, wherein the instructions are for preparing a single dose of the immunogenic composition. 170. The kit according to any one of embodiments 166-169, wherein the kit is for a single dose of the immunogenic composition. 171. The kit according to embodiment 169 or embodiment 170, wherein the single dose is a dose of 0.3 mL to 1.5 mL. 172. The kit according to any one of embodiments 166-171, wherein the first composition and / or the second composition comprises a second adjuvant. 173. The kit according to embodiment 172, wherein the second adjuvant is an aluminum salt adjuvant. 174. The kit according to embodiment 173, wherein the aluminum salt adjuvant comprises one or more of amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate. 175. The kit according to embodiment 173 or embodiment 174, wherein the aluminum salt adjuvant comprises aluminum hydroxide. 176. The kit according to any one of embodiments 166-175, wherein the kit further comprises: d) Another set of instructions for administering the immunogenic composition to a subject to stimulate an immune response against the genus *Treponema* in the subject. 177. A kit comprising: a) The immunogenic composition according to any one of embodiments 1-112; and b) Instructions for use in administering the immunogenic composition to a subject to stimulate an immune response against the genus *Treponema* in the subject. 178. The kit according to embodiment 176 or embodiment 177, wherein the kit further comprises a syringe for intramuscular injection of the immunogenic composition. 179. The kit according to embodiment 178, wherein the syringe is pre-filled with the immunogenic composition. 180. A kit comprising: a) a vial according to any one of embodiments 158-162; and b) Instructions for use in administering the immunogenic composition to a subject to stimulate an immune response against the genus *Treponema* in the subject. 181. The kit according to embodiment 180, the kit further comprising a syringe for intramuscular injection of the immunogenic composition. 182. A kit comprising: a) A pre-filled syringe according to any one of embodiments 163-165; and b) Instructions for use in administering the immunogenic composition to a subject to stimulate an immune response against the genus *Treponema* in the subject. 183. The kit according to embodiment 182, wherein the syringe is used for intramuscular injection of the immunogenic composition. 184. The kit according to any one of embodiments 176-183, wherein the application is performed according to any one of embodiments 124-144. 185. The kit according to any one of embodiments 176-184, wherein the subject is a human subject. 186. The kit according to embodiment 185, wherein the human subject is at least 18 years old. 187. The kit according to embodiment 185 or embodiment 186, wherein the human subject is between 18 and 70 years old. 188. The kit according to embodiment 185, wherein the human subject is under 18 years of age. 189. The kit according to embodiment 185 or embodiment 188, wherein the human subject is 2 to 5 years old. 190. The kit according to embodiment 185 or embodiment 188, wherein the human subject is between 5 and 18 years old. Example Example 1 Effect of adjuvants on the immune response to Lyme disease immunogenic compositions

[0311] Abbreviations: OD (optical density); OspA (outer surface protein A); alum (aluminum hydroxide); ELISA (enzyme-linked immunosorbent assay); mcg (micrograms)

[0312] OspA serotypes: OspA serotype 1 is derived from Borrelia burgdorferi. OspA serotype 2 is derived from Borrelia auriculata. OspA serotypes 3, 5 and 6 are derived from Borrelia galbella. OspA serotype 4 is derived from Borrelia bavaria.

[0313] Experiments were conducted in mice to compare the effects of adjuvants administered with one of three different immunogenic compositions containing one or a mixture of three different OspA antigens, formulated with or without an adjuvant. The adjuvants tested included: 1) CpG (whose nucleotide sequence is shown in SEQ ID NO:1), 2) alum, or 3) a combination of CpG and alum.

[0314] Three different antigens or mixtures of antigens were tested in the immunogenic compositions: (a) the non-lipidized full-length form of OspA serotype 1 antigen (Breorhiza burgdorferi strain B31) (OspA S1), as shown in SEQ ID NO:50; (b) a mixture of non-lipidized OspA chimeric antigens containing three chimeric OspA antigens (OspA chimeric antigens) covering six serotypes; and (c) lipidized OspA chimeric antigens identical to the three chimeric OspA antigens described in (b) but further lipidized. The OspA chimeric antigens in the mixture described in (b) are schematically shown in [illustration missing]. Figure 1 The bottom of the compound contains three different chimeric OspA antigens, each combining two different serotypes of OspA. The first chimeric antigen (S1 / 2) is shown in SEQ ID NO:79, covering a portion of the C-terminal portion of OspA serotype 2 and a portion of the N-terminal portion of OspA serotype 1. The second chimeric antigen (S6 / 4) is shown in SEQ ID NO:80, covering a portion of the C-terminal portion of OspA serotype 4 and a portion of the N-terminal portion of OspA serotype 6. The third chimeric antigen (S5 / 3) is shown in SEQ ID NO:81, covering a portion of the C-terminal portion of OspA serotype 3 and a portion of the N-terminal portion of OspA serotype 5. For the esterified form of the chimeric antigens in (c) above, the three aforementioned chimeric antigens include additional fatty acids and glycerol attached to the N-terminus of each OspA chimeric antigen. Additionally, a His tag, as shown in SEQ ID NO:87, is attached to the C-terminus of each antigen. For example, the OspA S1 antigen with an additional His tag is shown in SEQ ID NO:86.

[0315] Female C3H / HeN mice were randomly assigned to twelve treatment groups, each containing eight animals. Each group received one of twelve immunogenic compositions having a given OspA antigen or mixture of OspA antigens and an adjuvant formulation, as shown in Table E1 below. Each type of antigen or antigen mixture contained no adjuvant, only alum, only CpG, or both CpG and alum. Immunogenic compositions using alum only as an adjuvant contained antigen adsorbed onto 50 μg of aluminum hydroxide (Alhydrogel 2%, Al(OH)3). Immunogenic compositions using CpG only as an adjuvant contained antigen mixed with 10 μg of CpG. Immunogenic compositions using alum and CpG as adjuvants contained antigen adsorbed onto 50 μg of alum and subsequently mixed with 10 μg of CpG. All antigens and adjuvants were diluted in sterile saline. Table E1: Research Design

[0316] Female mice were intramuscularly injected on days 0 and 21. Blood was drawn on days 21 and 35 for measuring serum antibody levels by ELISA. Response was measured for each mouse. The antibody endpoint titer was defined as the reciprocal of the highest dilution of pure serum required to reach 0.5 OD. Positive controls were included for each ELISA plate to control for inter-plate variability. Positive controls included definitive antibody levels from mouse serum samples from previous studies. Negative controls included serum from unvaccinated mice.

[0317] For OspA S5 / 3 ( Figure 2A OspA S6 / 4 ( Figure 2B ), OspA S1 / 2 ( Figure 2C (left side) or full length OspA S1 ( Figure 2D (Left) Antibody levels were measured in serum from mice administered an immunogenic composition containing a non-lipped (non-lipped) OspA chimeric antigen on day 35 (i.e., after the second injection). Antibody levels against OspA S1 / 2 ( Figure 2C (Right side) or full length OspA S1 ( Figure 2D (Right side) Antibody levels were measured in the serum of mice administered an immunogenic composition containing non-lipotropic OspA S1 on day 35. Finally, antibody levels against OspA S1 / 2 ( Figure 2E OspA S5 / 3 ( Figure 2F ) or OspA S6 / 4 ( Figure 2G Antibody levels were measured in the serum of mice administered an immunogenic composition containing lipotropic OspA chimeric antigen on day 35. For each graph, the fold increase (FI) in antibody levels between two indicator conditions is also shown, as measured by comparing the median antibody levels from one indicator condition to the other. Additionally, for Figure 2E-2G The median antibody level was also displayed for each condition.

[0318] for Figures 2A-2D Mice administered immunogenic compositions containing one or more non-lipotropic OspA antigens showed higher OspA antibody titers against each tested OspA antigen when any adjuvant (including alum, CpG, and CpG + alum) was used compared to mice without adjuvant. Furthermore, the combination of CpG + alum formulated with any one or more non-lipotropic OspA antigens resulted in the highest OspA titers against each tested OspA antigen, with FI significantly increased by 2.8–4.1 compared to using alum alone. Additionally, as... Figure 2DAs shown, using the OspA chimeric antigen or the full-length OspAS1 resulted in similar antibody responses against the full-length OspA S1, indicating that both the OspA chimeric antigen and S1 have similar immunogenicity against the full-length OspA S1, and that antibodies against OspA S1 / 2 can recognize the major S1 epitope present on the full-length OspA S1. In other words, the OspA S1 / 2 chimeric antigen retains the immunogenicity of the full-length OspA S1 antigen.

[0319] In comparison, such as Figure 2E-2G As shown, in mice administered an immunogenic composition containing an esterified OspA chimeric antigen, the use of alum resulted in only a limited increase in OspA antibody titers compared to the absence of adjuvant. However, the use of CpG or CpG + alum significantly enhanced OspA antibody titers compared to the absence of adjuvant or alum alone.

[0320] In summary, these results indicate that both non-esterified and esterified OspA chimeric antigens are immunogenic to OspA. Furthermore, when combined with several different OspA antigens or mixtures of OspA antigens, the use of adjuvant formulations containing CpG (with or without alum) enhanced OspA antibody responses against several OspA antigens.

[0321] In addition to assessing antibody titers after the second injection, antibody response kinetics between the first and second injections were evaluated in serum from mice treated with lipo-OspA chimeric antigen. Figures 3A-3C The antibody titers for each tested lipo-OspA formulation against OspA S1 / 2, OspA S5 / 3, and OspA S6 / 4 are shown in serum from day 21 (1) after the first injection and from day 35 (2) after the second injection. Above each condition, the fold increase (FI) of the antibody value calculated by comparing the median antibody level induced by the given composition on day 35 with the median antibody level induced by the same composition on day 21 is also shown. The median antibody titer values ​​are also shown in Table E2. Table E2: Antibody Titer Values

[0322] The results showed that the first dose of the immunogenic composition, when formulated with both CpG and alum, produced the highest antibody titer compared to formulations without adjuvant, with alum alone, or with CpG alone. Therefore, the combination of OspA antigen, CpG, and alum has a synergistic effect, leading to a greater immune response (i.e., after the first dose and before the second dose). Furthermore, when formulated with both CpG and alum, the second dose of the immunogenic composition increased the OspA antibody titer by 5.6–8.8-fold, depending on the serotype tested.

[0323] Overall, these results suggest that administration of CpG with an OspA-based Lyme disease immunogenic composition (with or without alum) can improve subsequent antibody responses to many different OspA serotypes, such as by providing a greater immune response.

[0324] Although the above disclosure has been described in detail by way of illustration and examples for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications may be made. Therefore, the embodiments should not be construed as limiting the scope of this disclosure, which is defined by the appended claims. sequence

Claims

1. An immunogenic composition comprising (i) a spirochetal outer surface protein A (OspA) antigen or an RNA polynucleotide encoding said OspA antigen and (ii) an adjuvant selected from aluminum salt adjuvants; Toll-like receptor 9 (TLR9) agonists; liposome-based adjuvants comprising lipopolysaccharides and saponins; and an oil-in-water emulsion comprising squalene, sorbitol esters and polyoxyethylene sorbitol esters.

2. An immunogenic composition comprising (i) a spirochetal outer surface protein A (OspA) antigen and (ii) an adjuvant selected from aluminum salt adjuvants; Toll-like receptor 9 (TLR9) agonists; liposome-based adjuvants comprising lipopolysaccharides and saponins; and an oil-in-water emulsion comprising squalene, sorbitol esters and polyoxyethylene sorbitol esters.

3. The immunogenic composition according to claim 1 or claim 2, wherein the immunogenic composition does not contain an RNA polynucleotide encoding a non-OspA spirochetal outer surface protein antigen.

4. The immunogenic composition according to any one of claims 1-3, wherein the immunogenic composition does not contain non-OspA spirochetal outer surface protein antigens.

5. The immunogenic composition according to any one of claims 1-4, wherein the adjuvant is an aluminum salt adjuvant.

6. The immunogenic composition according to claim 5, wherein the immunogenic composition further comprises a second adjuvant.

7. The immunogenic composition according to claim 6, wherein the second adjuvant is selected from TLR9 agonists; liposome-based adjuvants comprising lipopolysaccharides and saponins; and oil-in-water emulsions comprising squalene, sorbitol esters and polyoxyethylene sorbitol esters.

8. The immunogenic composition according to any one of claims 1-4, wherein the adjuvant is a TLR9 agonist.

9. The immunogenic composition according to claim 8, wherein the immunogenic composition further comprises a second adjuvant.

10. The immunogenic composition of claim 9, wherein the second adjuvant is selected from aluminum salt adjuvants; liposome-based adjuvants comprising lipopolysaccharides and saponins; and oil-in-water emulsion-based adjuvants comprising squalene, sorbitol esters and polyoxyethylene sorbitol esters.

11. The immunogenic composition according to any one of claims 1-4, wherein the adjuvant is a liposome-based adjuvant comprising lipopolysaccharide and saponin.

12. The immunogenic composition according to claim 11, wherein the immunogenic composition further comprises a second adjuvant.

13. The immunogenic composition of claim 12, wherein the second adjuvant is selected from aluminum salt adjuvants; TLR9 agonists; and adjuvants based on oil-in-water emulsions comprising squalene, sorbitol esters and polyoxyethylene sorbitol esters.

14. The immunogenic composition according to any one of claims 1-4, wherein the adjuvant is an oil-in-water emulsion-based adjuvant comprising squalene, sorbitol ester and polyoxyethylene sorbitol ester.

15. The immunogenic composition according to claim 14, wherein the immunogenic composition further comprises a second adjuvant.

16. The immunogenic composition of claim 15, wherein the second adjuvant is selected from aluminum salt adjuvants; TLR9 agonists; and liposome-based adjuvants comprising lipopolysaccharides and saponins.

17. The immunogenic composition according to any one of claims 1-4, 7-10, 13 and 16, wherein the TLR9 agonist is an oligonucleotide comprising an unmethylated cytidine-phosphate-guanosine (CpG) motif.

18. The immunogenic composition of claim 17, wherein the oligonucleotide is 10 to 35 nucleotides in length.

19. The immunogenic composition according to claim 17 or claim 18, wherein the oligonucleotide comprises sequence 5'-AACGTTCGAG-3' (SEQ ID NO:2).

20. The immunogenic composition according to any one of claims 17-19, wherein the oligonucleotide comprises the sequence 5'-TGACTGTGAA CGTTCGAGAT GA-3' (SEQ ID NO:1).

21. An immunogenic composition comprising a spirochetal outer surface protein A (OspA) antigen and a Toll-like receptor 9 (TLR9) agonist oligonucleotide of 10 to 35 nucleotides in length, said oligonucleotide comprising the sequence 5'-AACGTTCGAG-3' (SEQ ID NO:2) and an unmethylated cytidine-phosphate-guanosine (CpG) motif.

22. The immunogenic composition of claim 21, wherein the oligonucleotide comprises the sequence 5'-TGACTGTGAA CGTTCGAGAT GA-3' (SEQ ID NO:1).

23. An immunogenic composition comprising a spirochetal outer surface protein A (OspA) antigen and a Toll-like receptor 9 (TLR9) agonist oligonucleotide 5'-TGACTGTGAA CGTTCGAGAT GA-3' (SEQ ID NO:1), said oligonucleotide comprising an unmethylated cytidine-phosphate-guanosine (CpG) motif.

24. The immunogenic composition according to any one of claims 21-23, wherein the immunogenic composition further comprises a second adjuvant.

25. The immunogenic composition of claim 24, wherein the second adjuvant is selected from aluminum salt adjuvants; liposome-based adjuvants comprising lipopolysaccharides and saponins; and oil-in-water emulsion-based adjuvants comprising squalene, sorbitol esters and polyoxyethylene sorbitol esters.

26. The immunogenic composition according to any one of claims 9, 10, 12, 13, 15-20, 24 and 25, wherein the second adjuvant is an aluminum salt adjuvant.

27. An immunogenic composition comprising: (i) a spirochetal outer surface protein A (OspA) antigen, (ii) a toll-like receptor 9 (TLR9) agonist oligonucleotide 5'-TGACTGTGAA CGTTCGAGAT GA-3' (SEQ ID NO:1), said oligonucleotide comprising an unmethylated cytidine-phosphate-guanosine (CpG) motif, and (iii) an aluminum salt adjuvant.

28. The immunogenic composition according to any one of claims 1-7, 10, 13, 16-20 and 25-27, wherein the aluminum salt adjuvant comprises one or more of amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate and potassium aluminum sulfate.

29. The immunogenic composition according to any one of claims 1-7, 10, 13, 16-20 and 25-28, wherein the aluminum salt adjuvant comprises aluminum hydroxide.

30. The immunogenic composition according to any one of claims 1-29, wherein the immunogenic composition comprises about 0.25 mg to about 1.25 mg A1 3+ Approximately 0.25 mg to approximately 0.50 mg Al 3+ Or approximately 0.30 mg to approximately 0.40 mg Al 3 + .

31. The immunogenic composition according to any one of claims 17-30, wherein the oligonucleotide is a single-stranded oligodeoxynucleotide.

32. The immunogenic composition according to any one of claims 17-30, wherein the oligonucleotide is entirely RNA.

33. The immunogenic composition according to any one of claims 17-30, wherein the oligonucleotide is an RNA / DNA chimera.

34. The immunogenic composition according to any one of claims 17-33, wherein the oligonucleotide comprises at least one phosphate thioester bond.

35. The immunogenic composition according to any one of claims 17-34, wherein the oligonucleotide comprises only phosphate thioester bonds.

36. The immunogenic composition according to any one of claims 17-34, wherein the oligonucleotide comprises a combination of one or more phosphodiester bonds and one or more thiophosphate bonds.

37. The immunogenic composition according to any one of claims 1-4, 7-10, 13 and 16-36, wherein the immunogenic composition comprises about 375 µg to about 6000 µg of the TLR9 agonist or about 750 µg to about 3000 µg of the TLR9 agonist.

38. The immunogenic composition according to any one of claims 1-4, 7-10, 13 and 16-36, wherein the immunogenic composition comprises about 375 µg, about 750 µg, about 1000 µg, about 1500 µg, about 3000 µg or about 6000 µg of the TLR9 agonist.

39. The immunogenic composition according to any one of claims 1-4, 7, 10-13, 16-20, 25, 26 and 28-38, wherein the lipopolysaccharide is lipid A or a derivative thereof.

40. The immunogenic composition according to any one of claims 1-4, 7, 10-13, 16-20, 25, 26 and 28-39, wherein the lipopolysaccharide is monophosphoryl lipid A or a derivative thereof.

41. The immunogenic composition according to any one of claims 1-4, 7, 10-13, 16-20, 25, 26 and 28-40, wherein the lipopolysaccharide is 3-O-deacylated-4'-monophosphoryllipid A.

42. The immunogenic composition according to any one of claims 1-4, 7, 10-13, 16-20, 25, 26 and 28-40, wherein the lipopolysaccharide is a 3-deacylated hexaacyl disaccharide.

43. The immunogenic composition according to any one of claims 1-4, 7, 10-13, 16-20, 25, 26 and 28-42, wherein the immunogenic composition comprises about 1 μg to about 100 μg, about 10 μg to about 50 μg, about 20 μg to about 30 μg, about 21 μg to about 29 μg, about 22 μg to about 28 μg, about 23 μg to about 27 μg or about 24 μg to about 26 μg of the lipopolysaccharide.

44. The immunogenic composition according to any one of claims 1-4, 7, 10-13, 16-20, 25, 26 and 28-42, wherein the immunogenic composition comprises about 1 μg to about 30 μg, about 5 μg to about 15 μg, about 6 μg to about 14 μg, about 7 μg to about 13 μg, about 8 μg to about 12 μg or about 9 μg to about 11 μg of the lipopolysaccharide.

45. The immunogenic composition according to any one of claims 1-4, 7, 10-13, 16-20, 25, 26 and 28-42, wherein the immunogenic composition comprises about 1 μg to about 9 μg, about 2 μg to about 8 μg, about 3 μg to about 7 μg or about 4 μg to about 6 μg of the lipopolysaccharide.

46. ​​The immunogenic composition according to any one of claims 1-4, 7, 10-13, 16-20, 25, 26 and 28-45, wherein the saponin is derived from the bark of the Molina soapberry tree.

47. The immunogenic composition according to any one of claims 1-4, 7, 10-13, 16-20, 25, 26 and 28-46, wherein the saponin is Quil A or a derivative thereof.

48. The immunogenic composition according to any one of claims 1-4, 7, 10-13, 16-20, 25, 26 and 28-47, wherein the saponin is QS-17.

49. The immunogenic composition according to any one of claims 1-4, 7, 10-13, 16-20, 25, 26 and 28-47, wherein the saponin is QS-21.

50. The immunogenic composition according to any one of claims 1-4, 7, 10-13, 16-20, 25, 26 and 28-49, wherein the immunogenic composition comprises about 1 μg to about 100 μg, about 10 μg to about 50 μg, about 20 μg to about 30 μg, about 21 μg to about 29 μg, about 22 μg to about 28 μg, about 23 μg to about 27 μg or about 24 μg to about 26 μg of the saponin.

51. The immunogenic composition according to any one of claims 1-4, 7, 10-13, 16-20, 25, 26 and 28-49, wherein the immunogenic composition comprises about 1 μg to about 30 μg, about 5 μg to about 15 μg, about 6 μg to about 14 μg, about 7 μg to about 13 μg, about 8 μg to about 12 μg or about 9 μg to about 11 μg of the saponin.

52. The immunogenic composition according to any one of claims 1-4, 7, 10-13, 16-20, 25, 26 and 28-49, wherein the immunogenic composition comprises about 1 μg to about 9 μg, about 2 μg to about 8 μg, about 3 μg to about 7 μg or about 4 μg to about 6 μg of the saponin.

53. The immunogenic composition according to any one of claims 1-4, 7, 10-13, 16-20, 25, 26 and 28-52, wherein the liposome-based adjuvant further comprises a sterol.

54. The immunogenic composition according to claim 53, wherein the sterol is β-sitosterol, stigmasterol, ergosterol, ergocalciferol, or cholesterol.

55. The immunogenic composition according to claim 53 or claim 54, wherein the sterol is cholesterol.

56. The immunogenic composition according to any one of claims 53-55, wherein the ratio of saponin to sterol is 1:1 to 1:100 w / w or 1:1 to 1:5 w / w, optionally about 1:1 w / w.

57. The immunogenic composition according to any one of claims 1-4, 7, 10-13, 16-20, 25, 26 and 28-56, wherein the liposome-based adjuvant further comprises neutral lipids.

58. The immunogenic composition according to claim 57, wherein the neutral lipid is phosphatidylcholine.

59. The immunogenic composition according to claim 58, wherein the phosphatidylcholine is egg yolk phosphatidylcholine, dioleoyl phosphatidylcholine (DOPC), or dilauryl phosphatidylcholine.

60. The immunogenic composition according to claim 58 or claim 59, wherein the phosphatidylcholine is DOPC.

61. The immunogenic composition according to any one of claims 1-4, 7, 10, 13-20, 25, 26 and 28-60, wherein the sorbitol ester is sorbitol ester 20.

62. The immunogenic composition according to any one of claims 1-4, 7, 10, 13-20, 25, 26 and 28-60, wherein the sorbitol ester is sorbitol ester 85.

63. The immunogenic composition according to any one of claims 1-4, 7, 10, 13-20, 25, 26 and 28-62, wherein the polyoxyethylene sorbitol ester is polyoxyethylene sorbitol ester 20.

64. The immunogenic composition according to any one of claims 1-4, 7, 10, 13-20, 25, 26 and 28-62, wherein the polyoxyethylene sorbitol ester is polyoxyethylene sorbitol ester 80.

65. The immunogenic composition according to any one of claims 1-4, 7, 10, 13-20, 25, 26 and 28-64, wherein the immunogenic composition comprises about 2% w / v to about 7% w / v, about 3% w / v to about 6% w / v or about 4% w / v to about 5% w / v of the squalene.

66. The immunogenic composition according to any one of claims 1-4, 7, 10, 13-20, 25, 26 and 28-65, wherein the immunogenic composition comprises about 0.2% w / v to about 0.8% w / v, about 0.3% w / v to about 0.7% w / v or about 0.4% w / v to about 0.6% w / v of the dehydrated sorbitol ester.

67. The immunogenic composition according to any one of claims 1-4, 7, 10, 13-20, 25, 26 and 28-66, wherein the immunogenic composition comprises the polyoxyethylene dehydrated sorbitol ester at about 0.2% w / v to about 0.8% w / v, about 0.3% w / v to about 0.7% w / v or about 0.4% w / v to about 0.6% w / v.

68. The immunogenic composition according to any one of claims 1-67, wherein the OspA antigen is two or more OspA antigens.

69. The immunogenic composition according to claim 68, wherein the two or more OspA antigens are a first OspA antigen and a second OspA antigen.

70. The immunogenic composition according to claim 68 or claim 69, wherein the two or more OspA antigens are a first OspA antigen, a second OspA antigen, and a third OspA antigen.

71. The immunogenic composition according to any one of claims 68-70, wherein each of the two or more OspA antigens has a different amino acid sequence.

72. The immunogenic composition according to any one of claims 1-71, wherein the OspA antigen is independently the OspA antigen of Borrelia burgdorferi, Borrelia aberrantii, Borrelia Bavaria, Borrelia galbella, Borrelia mellitus, Borrelia louvelii, Borrelia bisserii, Borrelia valgum, or Borrelia stearothermiae.

73. The immunogenic composition according to any one of claims 1-72, wherein the OspA antigen is independently a Borrelia burgdorferi OspA antigen.

74. The immunogenic composition according to any one of claims 1-73, wherein the OspA antigen independently comprises one or more epitopes from the OspA protein.

75. The immunogenic composition of claim 74, wherein the one or more epitopes comprise at least two, three, four, five, or six epitopes from one or more OspA proteins.

76. The immunogenic composition according to claim 74 or claim 75, wherein the one or more epitopes comprise at least two, three, four, five or six epitopes from at least two, three, four, five or six OspA serotypes.

77. The immunogenic composition according to any one of claims 74-76, wherein the one or more epitopes comprise epitopes selected from the amino acid sequences shown in SEQ ID NO:4-49.

78. The immunogenic composition according to any one of claims 74-77, wherein the one or more epitopes are independently selected from the amino acid sequences shown in SEQ ID NO:4-49.

79. The immunogenic composition according to any one of claims 74-78, wherein the one or more epitopes comprise an epitope selected from the amino acid sequences shown in SEQ ID NO: 9, 10, 33, 34, 36, 47 and 49.

80. The immunogenic composition according to any one of claims 1-78, wherein the OspA antigen independently has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:50-62.

81. The immunogenic composition according to any one of claims 1-78 and 80, wherein the OspA antigen independently comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 consecutive amino acids of the amino acid sequence shown in any one of SEQ ID NO:50-62.

82. The immunogenic composition according to any one of claims 1-78, 80 and 81, wherein the OspA antigen independently comprises the amino acid sequence shown in any one of SEQ ID NO:50-62.

83. The immunogenic composition according to any one of claims 1-78, wherein the OspA antigen is independently an OspA chimeric antigen comprising a plurality of epitopes from one or more OspA proteins.

84. The immunogenic composition of claim 83, wherein the plurality of epitopes comprises at least two, three, four, five, or six epitopes from at least two, three, four, five, or six OspA serotypes.

85. The immunogenic composition according to claim 83 or claim 84, wherein the plurality of epitopes comprises epitopes selected from the amino acid sequences shown in SEQ ID NO:4-49.

86. The immunogenic composition according to any one of claims 83-85, wherein the plurality of epitopes are independently selected from the amino acid sequences shown in SEQ ID NO:4-49.

87. The immunogenic composition according to any one of claims 83-86, wherein the plurality of epitopes comprise epitopes selected from the amino acid sequences shown in SEQ ID NO: 9, 10, 33, 34, 36, 47 and 49.

88. The immunogenic composition according to any one of claims 83-87, wherein the plurality of epitopes are independently selected from the amino acid sequences shown in SEQ ID NO: 9, 10, 33, 34, 36, 47 and 49.

89. The immunogenic composition according to any one of claims 1-78 and 83-86, wherein the OspA antigen independently comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 63-82.

90. The immunogenic composition according to any one of claims 1-78 and 83-89, wherein the OspA antigen independently comprises at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 consecutive amino acids of the amino acid sequence shown in any one of SEQ ID NO: 63-82.

91. The immunogenic composition according to any one of claims 1-78 and 83-90, wherein the OspA antigen independently comprises the amino acid sequence shown in any one of SEQ ID NO:63-82.

92. The immunogenic composition according to any one of claims 1-78 and 83-89, wherein the OspA antigen independently comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:79-81.

93. The immunogenic composition according to any one of claims 1-78, 83-89, and 92, wherein the immunogenic composition comprises a first OspA antigen and a second OspA antigen, the first OspA antigen comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 79, and the second OspA antigen comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

80.

94. The immunogenic composition according to any one of claims 1-78, 83-89 and 91-93, wherein the immunogenic composition comprises a first OspA antigen containing the amino acid sequence shown in SEQ ID NO:79 and a second OspA antigen containing the amino acid sequence shown in SEQ ID NO:

80.

95. The immunogenic composition according to any one of claims 1-78, 83-89, and 92, wherein the immunogenic composition comprises a first OspA antigen and a second OspA antigen, the first OspA antigen comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 79, and the second OspA antigen comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

81.

96. The immunogenic composition according to any one of claims 1-78, 83-89, 91, 92 and 95, wherein the immunogenic composition comprises a first OspA antigen containing the amino acid sequence shown in SEQ ID NO:79 and a second OspA antigen containing the amino acid sequence shown in SEQ ID NO:

81.

97. The immunogenic composition according to any one of claims 1-78, 83-89, and 92, wherein the immunogenic composition comprises a first OspA antigen and a second OspA antigen, the first OspA antigen comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 80, and the second OspA antigen comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

81.

98. The immunogenic composition according to any one of claims 1-78, 83-89, 91, 92 and 97, wherein the immunogenic composition comprises a first OspA antigen containing the amino acid sequence shown in SEQ ID NO:80 and a second OspA antigen containing the amino acid sequence shown in SEQ ID NO:

81.

99. The immunogenic composition according to any one of claims 1-78, 83-89, 92, and 93, wherein the immunogenic composition comprises a first OspA antigen, a second OspA antigen, and a third OspA antigen, the first OspA antigen comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:79, the second OspA antigen comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:80, and the third ...98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:80, and the third OspA antigen comprising an amino acid sequence having at least 80%, 83%, 84%, 85%, 86%, 8 The amino acid sequence shown in NO:81 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

100. The immunogenic composition according to any one of claims 1-78, 83-89 and 91-94, wherein the immunogenic composition comprises a first OspA antigen containing the amino acid sequence shown in SEQ ID NO:79, a second OspA antigen containing the amino acid sequence shown in SEQ ID NO:80 and a third OspA antigen containing the amino acid sequence shown in SEQ ID NO:

81.

101. The immunogenic composition according to any one of claims 1-100, wherein the OspA antigen is independently non-lipotropic.

102. The immunogenic composition according to any one of claims 1-101, wherein the OspA antigen is independently lipid-modified.

103. The immunogenic composition according to any one of claims 1-102, wherein the immunogenic composition is in a dose of 0.3 mL to 1.5 mL.

104. The immunogenic composition according to any one of claims 1-103, wherein the immunogenic composition is used to stimulate an immune response against the genus *Treponema* in a subject by administering an effective amount of the immunogenic composition to the subject.

105. The immunogenic composition according to any one of claims 1-104, wherein the immunogenic composition is used to protect a subject from spirochetal infection by administering an effective amount of the immunogenic composition to the subject.

106. The immunogenic composition according to any one of claims 1-104, wherein the immunogenic composition is used to prevent the subject from developing Lyme disease by administering an effective amount of the immunogenic composition to the subject.

107. The immunogenic composition according to any one of claims 1-104, wherein the subject is a human subject.

108. The immunogenic composition of claim 107, wherein the human subject is at least 18 years old.

109. The immunogenic composition according to claim 107 or claim 108, wherein the human subject is between 18 and 70 years of age.

110. The immunogenic composition of claim 107, wherein the human subject is under 18 years of age.

111. The immunogenic composition according to claim 107 or claim 110, wherein the human subject is 2 to 5 years old.

112. The immunogenic composition according to claim 107 or claim 110, wherein the human subject is between 5 and 18 years old.

113. A method for stimulating an immune response against the genus *Borrelia* in a subject, the method comprising administering to the subject an effective amount of the immunogenic composition according to any one of claims 1-112.

114. A method for protecting a subject from infection with the genus *Treponema*, the method comprising administering to the subject an effective amount of the immunogenic composition according to any one of claims 1-112.

115. A method for preventing a subject from developing Lyme disease, the method comprising administering to the subject an effective amount of the immunogenic composition according to any one of claims 1-112.

116. The method according to any one of claims 113-115, wherein the immunogenic composition is administered by intramuscular injection.

117. The method according to any one of claims 113-116, wherein the first dose and the second dose of the immunogenic composition are administered to the subject.

118. The method of claim 117, wherein the second dose of the immunogenic composition is administered about 2 weeks to about 10 weeks after administration of the first dose of the immunogenic composition.

119. The method of claim 117 or claim 118, wherein the second dose of the immunogenic composition is administered about one month or about two months after the administration of the first dose of the immunogenic composition.

120. The method according to any one of claims 117-119, wherein a third dose of the immunogenic composition is administered to the subject.

121. The method of claim 120, wherein the third dose of the immunogenic composition is administered approximately 4 to approximately 8 months after administration of the first dose of the immunogenic composition.

122. The method of claim 120 or claim 121, wherein the third dose of the immunogenic composition is administered approximately 6 months after administration of the first dose of the immunogenic composition.

123. The method according to any one of claims 113-122, wherein a booster dose of the immunogenic composition is administered to the subject.

124. The method of claim 123, wherein the enhanced dose is administered at least about 1 year or at least about 2 years after the administration of the first dose.

125. The method according to any one of claims 113-124, wherein a plurality of booster doses are administered to the subject.

126. The method of claim 125, wherein the first booster dose is administered at least about 1 year or at least about 2 years after the administration of the first dose.

127. The method of claim 125 or claim 126, wherein a booster dose is administered annually.

128. The method according to any one of claims 113-127, wherein the subject is a human subject.

129. The method of claim 128, wherein the human subject is at least 18 years old.

130. The method of claim 128 or claim 129, wherein the human subject is between 18 and 70 years old.

131. The method of claim 128, wherein the human subject is under 18 years of age.

132. The method of claim 128 or claim 131, wherein the human subject is between 2 and 5 years old.

133. The method of claim 128 or claim 131, wherein the human subject is between 5 and 18 years old.

134. The use of the immunogenic composition according to any one of claims 1-112 for stimulating an immune response against the genus *Treponema* in a subject by administering an effective amount of the immunogenic composition to the subject.

135. The use of the immunogenic composition according to any one of claims 1-112 for protecting a subject from infection with *Treponema pallidum* by administering an effective amount of the immunogenic composition to the subject.

136. The use of the immunogenic composition according to any one of claims 1-112 for preventing Lyme disease in a subject by administering an effective amount of the immunogenic composition to the subject.

137. Use of the immunogenic composition according to any one of claims 1-112 in the manufacture of a medicament for stimulating an immune response against the genus *Treponema* in a subject.

138. Use of the immunogenic composition according to any one of claims 1-112 in the manufacture of a medicament for protecting a subject from infection with the genus *Treponema*.

139. Use of the immunogenic composition according to any one of claims 1-112 in the manufacture of a medicament for preventing a subject from developing Lyme disease.

140. The use according to any one of claims 134-139, wherein the immunogenic composition is administered by intramuscular injection.

141. The use according to any one of claims 134-140, wherein the first and second doses of the immunogenic composition are administered to the subject.

142. The use according to claim 141, wherein the second dose of the immunogenic composition is administered about 2 weeks to about 10 weeks after administration of the first dose of the immunogenic composition.

143. The use according to claim 141 or claim 142, wherein the second dose of the immunogenic composition is administered about one month or about two months after the administration of the first dose of the immunogenic composition.

144. The use according to any one of claims 141-143, wherein a third dose of the immunogenic composition is administered to the subject.

145. The use according to claim 144, wherein the third dose of the immunogenic composition is administered about 4 months to about 8 months after the administration of the first dose of the immunogenic composition.

146. The use according to claim 144 or claim 145, wherein the third dose of the immunogenic composition is administered approximately 6 months after the administration of the first dose of the immunogenic composition.

147. The use according to any one of claims 134-146, wherein a booster dose of the immunogenic composition is administered to the subject.

148. The use according to claim 147, wherein the enhanced dose is administered at least about 1 year or at least about 2 years after the administration of the first dose.

149. The use according to any one of claims 134-148, wherein a plurality of booster doses are administered to the subject.

150. The use according to claim 149, wherein the first booster dose is administered at least about 1 year or at least about 2 years after the administration of the first dose.

151. The use according to claim 149 or claim 150, wherein a booster dose is administered annually.

152. The use according to any one of claims 133-151, wherein the subject is a human subject.

153. The use according to claim 152, wherein the age of the human subject is at least 18 years.

154. The use according to claim 152 or claim 153, wherein the age of the human subject is between 18 and 70 years.

155. The use according to claim 152, wherein the human subject is under 18 years of age.

156. The use according to claim 152 or claim 155, wherein the age of the human subject is 2 to 5 years.

157. The use according to claim 152 or claim 155, wherein the human subject is between 5 and 18 years old.

158. A vial comprising the immunogenic composition according to any one of claims 1-112.

159. The vial according to claim 158, wherein the immunogenic composition is in liquid form.

160. The vial according to claim 158, wherein the immunogenic composition is in lyophilized form.

161. The vial according to any one of claims 158-160, wherein the vial contains a single dose of the immunogenic composition.

162. The vial of claim 161, wherein the single dose is a dose of 0.3 mL to 1.5 mL.

163. A pre-filled syringe comprising the immunogenic composition according to any one of claims 1-112.

164. The pre-filled syringe of claim 163, wherein the syringe comprises a single dose of the immunogenic composition.

165. The pre-filled syringe of claim 164, wherein the single dose is a dose of 0.3 mL to 1.5 mL.

166. A kit comprising: a) A first composition comprising an adjuvant selected from aluminum salt adjuvants; Toll-like receptor 9 (TLR9) agonists; liposome-based adjuvants comprising lipopolysaccharides and saponins; and oil-in-water emulsion-based adjuvants comprising squalene, sorbitol esters and polyoxyethylene sorbitol esters. b) A second composition containing the outer surface protein A (OspA) antigen of *Breospira spp.*; and c) A specification for combining the first composition and the second composition to prepare an immunogenic composition according to any one of claims 1-112.

167. The kit according to claim 166, wherein the second composition is in liquid form.

168. The kit according to claim 166, wherein the second composition is in lyophilized form.

169. The kit according to any one of claims 166-168, wherein the instructions are for preparing a single dose of the immunogenic composition.

170. The kit according to any one of claims 166-169, wherein the kit is for a single dose of the immunogenic composition.

171. The kit according to claim 169 or claim 170, wherein the single dose is a dose of 0.3 mL to 1.5 mL.

172. The kit according to any one of claims 166-171, wherein the first composition and / or the second composition comprises a second adjuvant.

173. The kit according to claim 172, wherein the second adjuvant is an aluminum salt adjuvant.

174. The kit according to claim 173, wherein the aluminum salt adjuvant comprises one or more of amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate.

175. The kit according to claim 173 or claim 174, wherein the aluminum salt adjuvant comprises aluminum hydroxide.

176. The kit according to any one of claims 166-175, wherein the kit further comprises: d) Another set of instructions for administering the immunogenic composition to a subject to stimulate an immune response against the genus *Treponema* in the subject.

177. A kit comprising: a) The immunogenic composition according to any one of claims 1-112; and b) Instructions for use in administering the immunogenic composition to a subject to stimulate an immune response against the genus *Treponema* in the subject.

178. The kit according to claim 176 or claim 177, wherein the kit further comprises a syringe for intramuscular injection of the immunogenic composition.

179. The kit of claim 178, wherein the syringe is pre-filled with the immunogenic composition.

180. A kit comprising: a) The vial according to any one of claims 158-162; and b) Instructions for use in administering the immunogenic composition to a subject to stimulate an immune response against the genus *Treponema* in the subject.

181. The kit of claim 180, further comprising a syringe for intramuscular injection of the immunogenic composition.

182. A kit comprising: a) A pre-filled syringe according to any one of claims 163-165; and b) Instructions for use in administering the immunogenic composition to a subject to stimulate an immune response against the genus *Treponema* in the subject.

183. The kit of claim 182, wherein the syringe is used for intramuscular injection of the immunogenic composition.

184. The kit according to any one of claims 176-183, wherein the application is performed by the method according to any one of claims 124-144.

185. The kit according to any one of claims 176-184, wherein the subject is a human subject.

186. The kit of claim 185, wherein the human subject is at least 18 years old.

187. The kit according to claim 185 or claim 186, wherein the human subject is between 18 and 70 years old.

188. The kit of claim 185, wherein the human subject is under 18 years of age.

189. The kit according to claim 185 or claim 188, wherein the human subject is 2 to 5 years old.

190. The kit according to claim 185 or claim 188, wherein the human subject is between 5 and 18 years old.

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