Lyme disease vaccines comprising an adjuvant and a borrelia outer surface protein a antigen

Abstract

The present disclosure relates to immunogenic compositions comprising a 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 stimulating an immune response against Borrelia in a subject. The present disclosure also relates to kits of, uses of, and methods of using the immunogenic compositions.

Description

LYME DISEASE VACCINES COMPRISING AN ADJUVANT AND A BORRELIA OUTER SURFACE PROTEIN A ANTIGENCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. provisional application No. 63 / 512,872 filed July 10, 2023, the contents of which is incorporated by reference in its entirety.SUBMISSION OF SEQUENCE LISTING AS XML FILE

[0002] The present application is being filed along with a Sequence Listing in electronic format. The contents of the electronic Sequence Listing (377882009240SEQLIST.xml; Size: 86,006 bytes; and Date of Creation: July 9, 2024) is herein incorporated by reference in its entirety.FIELD

[0003] The present disclosure relates to immunogenic compositions comprising 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 stimulating an immune response against Borrelia in a subject. The present disclosure also relates to articles of manufacturing comprising, uses of, and methods of using the immunogenic compositions.BACKGROUND

[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 from the genus Borrelia. A monovalent serotype 1-OspA-based vaccine (LYMErix™) was approved and marketed in the USA for the prevention of Lyme disease caused by B. burgdorferi sensu stricto (s.s. but the vaccine is no longer available. Currently, there is no commercially available preventative medicament for Lyme disease. Thus, there is an unmet need for a safe and effective Lyme disease vaccine.SUMMARY

[0005] Provided herein in some embodiments is an immunogenic composition, comprising (i) a Borrelia outer surface protein A (OspA) antigen or an RNA polynucleotide encoding the OspA antigen and (ii) an adjuvant selected from the group consisting of an aluminum saltadjuvant; a toll-like receptor 9 (TLR9) agonist; a liposome-based adjuvant comprising a lipopolysaccharide and a saponin; and an oil-in-water emulsion comprising squalene, a sorbitan ester, and a polyoxyethylene sorbitan ester.

[0006] Also provided herein in some embodiments is an immunogenic composition, comprising (i) a Borrelia outer surface protein A (OspA) antigen and (ii) an adjuvant selected from the group consisting of an aluminum salt adjuvant; a toll-like receptor 9 (TLR9) agonist; a liposome-based adjuvant comprising a lipopolysaccharide and a saponin; and an oil-in- water emulsion comprising squalene, a sorbitan ester, and a polyoxyethylene sorbitan ester.

[0007] In some of any embodiments, the immunogenic composition does not comprise an RNA polynucleotide encoding a non-OspA orre / zrz outer surface protein antigen.

[0008] In some of any embodiments, the immunogenic composition does not comprise a non-OspA Borrelia outer surface protein antigen.

[0009] In some of any embodiments, the adjuvant is an aluminum salt adjuvant. In some of any embodiments, the immunogenic composition further comprises a second adjuvant. In some of any embodiments, the second adjuvant is selected from a TLR9 agonist; a liposomebased adjuvant comprising a lipopolysaccharide and a saponin; and an oil-in-water emulsion comprising squalene, a sorbitan ester, and a polyoxyethylene sorbitan ester.

[0010] In some of any embodiments, the adjuvant is a TLR9 agonist. In some of any embodiments, the immunogenic composition further comprises a second adjuvant. In some of any embodiments, the second adjuvant is selected from an aluminum salt adjuvant; a liposome-based adjuvant comprising a lipopolysaccharide and a saponin; and an oil-in-water emulsion-based adjuvant comprising squalene, a sorbitan ester, and a polyoxyethylene sorbitan ester.

[0011] In some of any embodiments, the adjuvant is a liposome-based adjuvant comprising a lipopolysaccharide and a saponin. In some of any embodiments, the immunogenic composition further comprises a second adjuvant. In some of any embodiments, the second adjuvant is selected from an aluminum salt adjuvant; a TLR9 agonist; and an oil-in-water emulsion-based adjuvant comprising squalene, a sorbitan ester, and a polyoxyethylene sorbitan ester.

[0012] In some of any embodiments, the adjuvant is an oil-in-water emulsion-based adjuvant comprising squalene, a sorbitan ester, and a polyoxyethylene sorbitan ester. In some of any embodiments, the immunogenic composition further comprises a second adjuvant. Insome of any embodiments, the second adjuvant is selected from an aluminum salt adjuvant; a TLR9 agonist; and a liposome-based adjuvant comprising a lipopolysaccharide and a saponin.

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

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

[0015] Also provided herein in some embodiments is an immunogenic composition, comprising a Borrelia outer surface protein A (OspA) antigen and a toll-like receptor 9 (TLR9) agonist oligonucleotide of from 10 to 35 nucleotides in length comprising the sequence 5’-AACGTTCGAG-3’ (SEQ ID NO:2) and an unmethylated cytidine-phospho- guanosine (CpG) motif.

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

[0017] Also provided herein in some embodiments is an immunogenic composition, comprising a Borrelia outer surface protein A (OspA) antigen and the toll-like receptor 9 (TLR9) agonist oligonucleotide 5’-TGACTGTGAA CGTTCGAGAT GA-3’(SEQ ID NO:1), the oligonucleotide comprising an unmethylated cytidine-phospho-guanosine (CpG) motif.

[0018] In some of any embodiments, the immunogenic composition further comprises a second adjuvant. In some of any embodiments, the second adjuvant is selected from an aluminum salt adjuvant; a liposome-based adjuvant comprising a lipopolysaccharide and a saponin; and an oil-in-water emulsion-based adjuvant comprising squalene, a sorbitan ester, and a polyoxyethylene sorbitan ester.

[0019] In some of any embodiments, the second adjuvant is an aluminum salt adjuvant.

[0020] Also provided herein in some embodiments is an immunogenic composition, comprising: (i) a Borrelia outer surface protein A (OspA) antigen, (ii) the toll-like receptor 9 (TLR9) agonist oligonucleotide 5’-TGACTGTGAA CGTTCGAGAT GA-3’(SEQ ID NO:1), the oligonucleotide comprising an unmethylated cytidine-phospho-guanosine (CpG) motif, and (iii) an aluminum salt adjuvant.

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

[0022] In some of any embodiments, the immunogenic composition comprises from about 0.25 to about 1.25 mg Al3+, from about 0.25 mg to about 0.50 mg Al3+, or from about 0.30 mg to about 0.40 mg Al3+.

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

[0024] In some of any embodiments, the oligonucleotide comprises at least one phosphorothioate linkage. In some of any embodiments, the oligonucleotide comprises only phosphorothioate linkages. In some of any embodiments, the oligonucleotide comprises a combination of one or more phosphodiester linkages and one or more phosphorothioate linkages.

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

[0026] In some of any embodiments, the lipopolysaccharide is a lipid A or a derivative thereof. In some of any embodiments, the lipopolysaccharide is a monophosphoryl lipid A or a derivative thereof. In some of any embodiments, the lipopolysaccharide is 3-O-desacyl-4'- monophosphoryl lipid A. In some of any embodiments, the lipopolysaccharide is 3-deacyl- phosphorylated hexa-acyl disaccharide.

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

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

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

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

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

[0032] In some of any embodiments, the liposome-based adjuvant further comprises a neutral lipid. In some of any embodiments, the neutral lipid is a phosphatidylcholine. In some of any embodiments, the phosphatidylcholine is eggyolk phosphatidylcholine, dioleoyl phosphatidylcholine (DOPC), or dilauryl phosphatidylcholine. In some of any embodiments, the phosphatidylcholine is DOPC.

[0033] In some of any embodiments, the sorbitan ester is sorbitan ester 20. In some of any embodiments, the sorbitan ester is sorbitan ester 85.

[0034] In some of any embodiments, the polyoxyethylene sorbitan ester is polyoxyethylene sorbitan ester 20. In some of any embodiments, the polyoxyethylene sorbitan ester is polyoxyethylene sorbitan ester 80.

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

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

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

[0038] In some of any embodiments, the OspA antigen is two or more OspA antigens. In some of any embodiments, the two or more OspA antigens are a first OspA antigen and a second OspA antigen. In some of any embodiments, the two or more OspA antigens are a first OspA antigen, a second OspA antigen, and a third OspA antigen. In some of any embodiments, each of the two or more OspA antigens have non-identical amino acid sequences.

[0039] In some of any embodiments, the OspA antigen (e.g., the first, second, and / or third OspA antigen) is independently a 7>. burgdorferi, B. afzelii, B. bavariensis, B. garinii, B. mayonii, B. lusitaniae, B. bissettii, B. valasiana, or B. spielmanii OspA antigen. In some of any embodiments, the OspA antigen is aB. burgdorferi OspA antigen.

[0040] In some of any embodiments, the OspA antigen (e.g., the first, second, and / or third OspA antigen) independently comprises one or more epitopes from an OspA protein. In some of any embodiments, the one or more epitopes comprise at least 2, 3, 4, or 5 epitopes from across one or more OspA proteins. In some of any embodiments, the one or more epitopes comprise at least 2, 3, 4, or 5 epitopes from across at least 2, 3, 4, or 5 OspA serotypes.

[0041] In some of any embodiments, the one or more epitopes comprise an epitope selected from the sequences of amino acids set forth in SEQ ID NO:4-49. In some of any embodiments, the one or more epitopes are independently selected from the sequences of amino acids set forth in SEQ ID NO:4-49.

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

[0043] In some of any embodiments, the OspA antigen (e.g., the first, second, and / or 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 to the sequence of amino acids set forth in any of SEQ ID NO:50-62.

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

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

[0046] In some of any embodiments, the OspA antigen (e.g., the first, second, and / or third OspA antigen) independently is an OspA chimeric antigen comprising a plurality of epitopes from one or more OspA proteins. In some of any embodiments, the plurality of epitopes comprise at least 2, 3, 4, 5, or 6 epitopes from across at least 2, 3, 4, 5, or 6 OspA serotypes.

[0047] In some of any embodiments, the plurality of epitopes comprise an epitope selected from the sequences of amino acids set forth in SEQ ID NO:4-49. In some of any embodiments, the plurality of epitopes are independently selected from the sequences of amino acids set forth in SEQ ID NO: 4-49.

[0048] In some of any embodiments, the plurality of epitopes comprise an epitope selected from the sequences of amino acids set forth in SEQ ID NO:9, 10, 33, 34, 36, 47, and 49. In some of any embodiments, the plurality of epitopes comprise are independently selected from the sequences of amino acids set forth in SEQ ID NO:9, 10, 33, 34, 36, 47, and 49.

[0049] In some of any embodiments, the OspA antigen (e.g., the first, second, and / or third OspA antigen) independently comprises a sequence of amino acids that 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 to the sequence of amino acids set forth in any one of SEQ ID NO:63-82.

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

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

[0052] In some of any embodiments, the OspA antigen (e.g., the first, second, and / or third OspA antigen) independently comprises a sequence of amino acids that 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 to the sequence of amino acids set forth in any one of SEQ ID NO: 79-81.

[0053] In some of any embodiments, the immunogenic composition comprises a first OspA antigen comprising a sequence of amino acids that 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 to the sequence of amino acids set forth in SEQ ID NO:79 and a second OspA antigen comprising a sequence of amino acids that 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 to the sequence of amino acids set forth in SEQ ID NO: 80. In some of any embodiments, the immunogenic composition comprises a first OspA antigen comprising the sequence of amino acids set forth in SEQ ID NO:79 and a second OspA antigen comprising the sequence of amino acids set forth in SEQ ID NO:80.

[0054] In some of any embodiments, the immunogenic composition comprises a first OspA antigen comprising a sequence of amino acids that 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 to the sequence of amino acids set forth in SEQ ID NO:79 and a second OspA antigen comprising a sequence of amino acids that 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 to the sequence of amino acids set forth in SEQ ID NO:81. In some of any embodiments, the immunogenic composition comprises a first OspA antigen comprising the sequence of amino acids set forth in SEQ ID NO:79 and a second OspA antigen comprising the sequence of amino acids set forth in SEQ ID NO:81.

[0055] In some of any embodiments, the immunogenic composition comprises a first OspA antigen comprising a sequence of amino acids that 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 to the sequence of amino acids set forth in SEQ ID NO: 80 and a second OspA antigen comprising a sequence of amino acids that 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 to the sequence of amino acids set forth in SEQ ID NO:81. In some of any embodiments, the immunogenic composition comprises a first OspA antigen comprising the sequence of amino acids set forth in SEQ ID NO: 80 and a second OspA antigen comprising the sequence of amino acids set forth in SEQ ID NO:81.

[0056] In some of any embodiments, the immunogenic composition comprises a first OspA antigen comprising a sequence of amino acids that 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 to the sequence of amino acids set forth in SEQ ID NO:79, a second OspA antigen comprising a sequence of amino acids that 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 to the sequence of amino acids set forth in SEQ ID NO: 80, and a third OspA antigen comprising a sequence of amino acids that 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 to the sequence of amino acids set forth in SEQ ID NO:81. In some of any embodiments, the immunogeneic composition comprises a first OspA antigen comprising the sequence of amino acids set forth in a SEQ ID NO:79, a second OspA antigen comprising the sequence of amino acids set forth in SEQ ID NO:80, and a third OspA antigen comprising the sequence of amino acids set forth in SEQ ID NO:81.

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

[0058] In some of any embodiments, the OspA antigen (e.g., the first, second, and / or third OspA antigen) independently is lipidated.

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

[0060] In some of any embodiments, the immunogenic composition is for stimulating an immune response against Borrelia in a subject by administering to the subject an effectiveamount of the immunogenic composition. In some of any embodiments, the immunogenic composition is for protecting a subject from infection by Borrelia by administering to the subject an effective amount of the immunogenic composition. In some of any embodiments, the immunogenic composition is for preventing a subject from contracting Lyme disease by administering to the subject an effective amount of the immunogenic composition.

[0061] In some of any embodiments, the subject a human subject.

[0062] In some of any embodiments, the human subject is at least 18 years of age. In some of any embodiments, the human subject is from 18 years to 70 years of age.

[0063] In some of any embodiments, the human subject is under 18 years of age. In some of any embodiments, the human subject is from 2 years to 5 years of age. In some of any embodiments, the human subject is from 5 years to 18 years of age.

[0064] Also provided herein in some embodiments is a method for stimulating an immune response against Borrelia in a subject, comprising administering to a subject an effective amount of any of the provided immunogenic compositions.

[0065] Also provided herein in some embodiments is a method for protecting a subject from infection by Borrelia, comprising administering to a subject an effective amount of any of the provided immunogenic compositions.

[0066] Also provided herein in some embodiments is a method for preventing a subject from contracting Lyme disease, comprising administering to a subject an effective amount of any of the provided immunogenic compositions.

[0067] In some of any embodiments, the immunogenic composition is administered by intramuscular injection.

[0068] In some of any embodiments, a first dose and a second dose of the immunogenic composition is administered to the subject. In some of any embodiments, the second dose of the immunogenic composition is administered from about 2 weeks to about 10 weeks after the administration of the first dose of the immunogenic composition. In some of any embodiments, 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.

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

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

[0071] In some of any embodiments, a plurality of booster doses is administered to the subject. In some of any embodiments, the first booster dose is administered at least about 1 year or at least about 2 years after the administration of the first dose. In some of any embodiments, a booster dose is administered yearly.

[0072] In some of any embodiments, the subject a human subject.

[0073] In some of any embodiments, the human subject is at least 18 years of age. In some of any embodiments, the human subject is from 18 years to 70 years of age.

[0074] In some of any embodiments, the human subject is under 18 years of age. In some of any embodiments, the human subject is from 2 years to 5 years of age. In some of any embodiments, the human subject is from 5 years to 18 years of age.

[0075] Also provided herein in some embodiments is use of any of the provided immunogenic compositions for stimulating an immune response against Borrelia in a subject by administering to the subject an effective amount of the immunogenic composition.

[0076] Also provided herein in some embodiments is use of any of the provided immunogenic compositions for protecting a subject from infection by Borrelia by administering to the subject an effective amount of the immunogenic composition.

[0077] Also provided herein in some embodiments is use of any of the provided immunogenic compositions for preventing a subject from contracting Lyme disease by administering to the subject an effective amount of the immunogenic composition.

[0078] Also provided herein in some embodiments is use of any of the provided immunogenic compositions in the manufacture of a medicament for stimulating an immune response against Borrelia in a subject.

[0079] Also provided herein in some embodiments is use of any of the provided immunogenic compositions in the manufacture of a medicament for protecting a subject from infection by Borrelia.

[0080] Also provided herein in some embodiments is use of any of the provided immunogenic compositions in the manufacture of a medicament for preventing a subject from contracting Lyme disease.

[0081] In some of any embodiments, the immunogenic composition is administered by intramuscular injection.

[0082] In some of any embodiments, a first dose and a second dose of the immunogenic composition is administered to the subject. In some of any embodiments, the second dose of the immunogenic composition is administered from about 2 weeks to about 10 weeks after the administration of the first dose of the immunogenic composition. In some of any embodiments, 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.

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

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

[0085] In some of any embodiments, a plurality of booster doses is administered to the subject. In some of any embodiments, the first booster dose is administered at least about 1 year or at least about 2 years after the administration of the first dose. In some of any embodiments, a booster dose is administered yearly.

[0086] In some of any embodiments, the subject a human subject.

[0087] In some of any embodiments, the human subject is at least 18 years of age. In some of any embodiments, the human subject is from 18 years to 70 years of age.

[0088] In some of any embodiments, the human subject is under 18 years of age. In some of any embodiments, the human subject is from 2 years to 5 years of age. In some of any embodiments, the human subject is from 5 years to 18 years of age.

[0089] Also provided herein in some embodiments is a vial comprising any of the provided immunogenic compositions.

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

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

[0092] Also provided herein in some embodiments is a pre-filled syringe comprising any of the provided immunogenic compositions.

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

[0094] Also provided herein in some embodiments is a kit comprising: a) a first composition comprising an adjuvant selected from an aluminum salt adjuvant; a toll-like receptor 9 (TLR9) agonist; a liposome-based adjuvant comprising a lipopolysaccharide and a saponin; and an oil-in-water emulsion-based adjuvant comprising squalene, a sorbitan ester, and a polyoxyethylene sorbitan ester; b) a second composition comprising a Borrelia outer surface protein A (OspA) antigen; and c) instructions for combining the first composition and the second composition to prepare any of the provided immunogenic compositions.

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

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

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

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

[0099] In some of any embodiments, the kit further comprises: d) a further set of instructions for administration of the immunogenic composition to a subject to stimulate an immune response against Borrelia in the subject.

[0100] Also provided herein in some embodiments is a kit comprising: a) any of the provided immunogenic compositions; and b) instructions for administration of the immunogenic composition to a subject to stimulate an immune response against Borrelia in the subject.

[0101] In some of any embodiments, the kit further comprises a syringe for intramuscular injection of the immunogenic composition. In some of any embodiments, the syringe is prefilled with the immunogenic composition.

[0102] Also provided herein in some embodiments is a kit comprising: a) any of the provided vials; and b) instructions for administration of the immunogenic composition to a subject to stimulate an immune response against Borrelia in the subject.

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

[0104] Also provided herein in some embodiments is a kit comprising: a) any of the provided pre-filled syringes; and b) instructions for administration of the immunogenic composition to a subject to stimulate an immune response against Borrelia in the subject.

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

[0106] In some of any embodiments, the administration is according to any of the provided methods.

[0107] In some of any embodiments, the subject a human subject.

[0108] In some of any embodiments, the human subject is at least 18 years of age. In some of any embodiments, the human subject is from 18 years to 70 years of age.

[0109] In some of any embodiments, the human subject is under 18 years of age. In some of any embodiments, the human subject is from 2 years to 5 years of age. In some of any embodiments, the human subject is from 5 years to 18 years of age.BRIEF DESCRIPTION OF DRAWINGS

[0110] FIG. 1 shows a schematic of the three chimeric OspA antigens, each of which combines two different serotypes from OspA. On top is a schematic of the OspA antigen, which is divided into the C-term portion (top; solid color) and N-term portion (bottom; hatched pattern). On the bottom are the three chimeric OspA antigens: S 1 / 2 (left), S6 / 4 (middle), and S5 / 3 (right), which contain different N-term and C-term portions from the indicated serotypes.

[0111] FIGS. 2A-2G show antibody response using ELISA to different indicated OspA antigens following administration of one of three OspA antigens or mixture of OspA antigens (non-lipidated OspA chimeric antigens, non-lipidated OspA full-length serotype 1 (SI) antigen, or lipidated OspA chimeric antigens) without adjuvant (0) or with 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). FIG. 2A shows antibody response to OspA S5 / 3 following administration of the indicated non-lipidated OspA chimeric antigens with different adjuvant formulations. FIG. 2B shows antibody response to OspA S6 / 4 following administration of the indicated non-lipidated OspA chimeric antigens with different adjuvant formulations. FIG. 2C shows antibody response to OspA S 1 / 2 following administration of the indicated non-lipidated OspA antigens with different adjuvant formulations. FIG. 2D shows antibody response to full-length OspA SI antigen following administration of the indicated non- lipidated OspA antigens with different adjuvant formulations. FIG. 2E shows antibody response to OspA S 1 / 2 following administration of the indicated lipidated OspA chimeric antigens with different adjuvant formulations. FIG. 2F shows antibody response to OspA S5 / 3 following administration of the indicated lipidated OspA chimeric antigens with different adjuvant formulations. FIG. 2G shows antibody response to OspA S6 / 4 following administration of the indicated lipidated OspA antigens with different adjuvant formulations. Fold-change increases (FIs) in titer levels between certain immunogenic compositions arealso indicated at the top of the graphs. In addition, the median values of the antibody titers are also shown at the top of each conditions for FIGS. 2E-2G.

[0112] FIGS. 3A-3C show antibody response using ELISA to the OspA chimeric antigen S 1 / 2 (FIG. 3A), S5 / 3 (FIG. 3B), and S6 / 4 (FIG. 3C) post-lstinjection (marked as “1”) or post-2ndinjection (marked as “2”) of lipidated OspA chimeric antigens and the indicated adjuvant formulation. Fold-change increases (FIs) in titer levels between the 1stand 2ndinjections are also indicated at the top of each immunogenic composition.DETAILED DESCRIPTION

[0113] The present disclosure relates to immunogenic compositions comprising 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 stimulating an immune response against Borrelia in a subject. The present disclosure also relates to articles of manufacture comprising, uses of, and methods of using the immunogenic compositions.

[0114] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0115] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.General Techniques and Definitions

[0116] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art.

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

[0118] The phrase “comprising” as used herein is open-ended, indicating that such embodiments may include additional elements. In contrast, the phrase “consisting of’ is closed, indicating that such embodiments do not include additional elements (except for trace impurities). The phrase “consisting essentially of’ is partially closed, indicating that such embodiments may further comprise elements that do not materially change the basic characteristics of such embodiments.

[0119] The term “about” as used herein in reference to a value encompasses from 90% to 110% of that value (e.g., about 3000 pg of CpG adjuvant refers to 2700 pg to 3300 pg 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 oligonucleosides; or combinations thereof. The oligonucleotide can be linearly or circularly configured, or the oligonucleotide can include both linear and circular segments. Oligonucleotides are polymers of nucleosides joined, generally, through phosphodiester linkages, although alternate linkages, such as phosphor othioate esters, may also be used in oligonucleotides. A nucleoside consists of a purine (adenine (A) or guanine (G) or derivative thereof) or pyrimidine (thymine (T), cytosine (C), or uracil (U), or derivative thereof) base bonded to a sugar. The four nucleoside units (or bases) in DNA are called deoxyadenosine, deoxyguanosine, thymidine, and deoxycytidine. A nucleotide is a phosphate ester of a nucleoside.

[0121] The terms “CpG”, “CpG motif’, and “cytosine-phosphate-guanosine” as used herein refer to an unmethylated cytidine-phospho-guanosine dinucleotide, which when present in an oligonucleotide contributes to a measurable immune response in vitro, in vivo, and / or ex vivo. Examples of measurable immune responses include antigen-specific antibody production, secretion of cytokines, and activation or expansion of lymphocyte populations, such as NK cells, CD4+ T lymphocytes, CD8+ T lymphocytes, or B lymphocytes. Preferably, the CpG- containing oligonucleotide preferentially activates a Thl-type response.

[0122] The term “antigen” refers to a substance that is recognized and bound specifically by an antibody or by 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 the present disclosure, the term “antigen” typically refers to a polypeptide or protein antigen at least eight amino acid residues in length, which may comprise one or more post-translational modifications.

[0123] The terms “polypeptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and are not limited to a certain length unless otherwise specified. Polypeptides may include natural amino acid residues or a combination of natural and non-natural amino acid residues. The terms also include post-expression modifications of the polypeptide, for example glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, the polypeptides may include modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity (e.g., antigenicity).

[0124] The terms “isolated” and “purified” as used herein refers to a material that is removed from at least one component with which it is naturally associated (e.g., removed from its original environment). The term “isolated” when used in reference to a recombinant protein refers to a protein that has been removed from the culture medium of the host cell that produced the protein. In some embodiments, an isolated protein (e.g., Borrelia 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 a “sufficient amount” of a substance is an amount sufficient to effect beneficial or desired results, including clinical results, and as such, an “effective amount” depends upon the context in which it is being applied. In the context of administering an immunogenic composition, an effective amount includes sufficient antigen and adjuvant(s) to stimulate an immune response (preferably a seroprotective level of antibody to the antigen).

[0126] In the present disclosure, the terms “individual” and “subject” refer to a mammal. Mammals include humans, non-human primates (e.g., monkeys), farm animals, sport animals, rodents (e.g., mice and rats), and pets (e.g., dogs and cats).

[0127] The term “dose” as used herein in reference to an immunogenic composition refers to a measured portion of the immunogenic composition taken by (administered to or received by) a subject at any one time.

[0128] “ Stimulation” of a response or parameter includes eliciting and / or enhancing that response or parameter when compared to otherwise same conditions except for a parameter of interest, or alternatively, as compared to another condition. For example, “stimulation” of an immune response means an increase in the response. Depending upon the parameter measured, the increase may be from 2-fold to 200-fold or over, from 5-fold to 500-fold or over, from 10-fold to 1000-fold or over, or from 2, 5, 10, 50, or 100-fold to 200, 500, 1,000, 5,000, or 10,000-fold.

[0129] Conversely, “inhibition” of a response or parameter includes reducing and / or repressing that response or parameter when compared to otherwise same conditions except for a parameter of interest, or alternatively, as compared to another condition. For example, “inhibition” of an immune response means a decrease in the response. Depending upon the parameter measured, the decrease may be from 2-fold to 200-fold, from 5-fold to 500-fold or over, from 10-fold to 1000-fold or over, or from 2, 5, 10, 50, or 100-fold to 200, 500, 1,000, 2,000, 5,000, or 10,000-fold.

[0130] The relative terms “higher” and “lower” refer to a measurable increase or decrease, respectively, in a response or parameter when compared to otherwise same conditions except for a parameter of interest, or alternatively, as compared to another condition. For instance, a “higher antibody titer” refers to an antigen-reactive antibody titer as a consequence of administration of an immunogenic composition of the present disclosure that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold above an antigen-reactive antibody titer as a consequence of a control condition. Likewise, a “lower antibody titer” refers to an antigen-reactive antibody titer as a consequence of a control condition that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold below an antigen-reactive antibody titer as a consequence of administration of an immunogenic composition of the present disclosure.

[0131] As used herein, the term “immunization” refers to a process that increases a subject’s reaction to antigen and therefore improves its ability to resist or overcome infection and / or resist disease.

[0132] The term “vaccination” as used herein refers to the introduction of a vaccine into a body of a subject.

[0133] “Adjuvant” refers to a substance which, when added to a composition comprising an antigen, enhances or potentiates an immune response to the antigen in a subject upon exposure.

[0134] As used herein, “percent (%) amino acid sequence identity”, “percent identity”, and “sequence identity” when used with respect to an amino acid sequence (reference polypeptide sequence) is defined as the percentage of amino acid residues in a candidate sequence (e.g., the subject antigen) that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance 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 needed to achieve maximal alignment over the full length of the sequences being compared.

[0135] An amino acid substitution may include replacement of one amino acid in a polypeptide with another amino acid. Amino acid substitutions may be introduced into an antigen of interest and the products screened for a desired activity, e.g., increased stability and / or immunogenicity.

[0136] Amino acids generally can be grouped according to the following common sidechain properties:(1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;(3) acidic: Asp, Glu;(4) basic: His, Lys, Arg;(5) residues that influence chain orientation: Gly, Pro; and(6) aromatic: Trp, Tyr, Phe.

[0137] Conservative amino acid substitutions will involve exchanging a member of one of these classes with another member of the same class. Non-conservative amino acid substitutions will involve exchanging a member of one of these classes with a member of another class.I. Immunogenic Compositions

[0138] The present disclosure relates to immunogenic compositions containing a. Borrelia outer surface protein A (OspA) antigen or an RNA polynucleotide encoding the OspA antigen and an adjuvant. In some embodiments, the immunogenic composition contains a Borrelia OspA antigen and an adjuvant. In some embodiments, the adjuvant is any described herein, for instance any described in Section I-A. In some embodiments, the OspA antigen is any described herein, for instance any described in Section I-B. In some embodiments, the immunogenic composition contains an additional component, for instance any described in Section I-C.

[0139] In some aspects, the immunogenic compositions are for stimulating an immune response against Borrelia in a subject. In some embodiments, the antigen and adjuvant are present in the immunogenic composition in amounts effective to stimulate an immune response against Borrelia in a subject. In some embodiments, the stimulating is by administering to the subject an effective amount of the immunogenic composition.

[0140] In some embodiments, the immunogenic composition is for protecting a subject from infection by Borrelia. In some embodiments, the antigen and adjuvant are present in the immunogenic composition in amounts effective to stimulate an immune response against Borrelia in a subject. In some embodiments, the protecting is by administering to the subject an effective amount of the immunogenic composition.

[0141] In some embodiments, the immunogenic composition is for preventing a subject from contracting Lyme disease. In some embodiments, the antigen and adjuvant are present in the immunogenic composition in amounts effective to stimulate an immune response against Borrelia in a subject. In some embodiments, the preventing is by administering to the subject an effective amount of the immunogenic composition.

[0142] In some embodiments, the subject is a human subject. In some embodiments, the human subject is at least 18 years of age. In some embodiments, the human subject is about 18 to about 70 years of age. In some embodiments, the human subject is about 18 to about 55 years of age. In some embodiments, the human subject is under 18 years of age. In some embodiments, the human subject is about 2 to about 5 years of age. In some embodiments, the human subject is about 5 to about 18 years of age.

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

[0144] Adjuvants that can be used in the present immunogenic compositions include mineral salts, such as aluminum salts, e.g., aluminum hydroxide, aluminum hydroxide gels (e.g., Rehydragel®), aluminum phosphate, and calcium phosphate; surface-active agents and microparticles, e.g., nonionic block polymer surfactants, cholesterol, virosomes, 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)), proteosomes, immune stimulating complexes, cochleates, quarterinary amines (dimethyl diocatadecyl ammonium bromide (DDA)), pyridine, vitamin A, vitamin E, and poly(lactide-co-glycolide) microparticles (Shah et al., Methods Mol Biol, 1494: 1-14, 2017); bacterial products, such as the RIBI adjuvant system (Ribi Inc.), cell wall skeleton of Mycobacterum phlei (Detox®), muramyl dipeptides (MDP) and tripeptides (MTP), monophosphoryl lipid A, Bacillus Calmete-Guerin, heat labile E. coli enterotoxins, cholera toxin, trehalose dimycolate, and CpG oligodeoxnucleotides; cytokines and hormones, e.g., interleukins (IL-1, IL-2, IL-6, IL-12, IL-15, and IL-18), granulocyte-macrophage colony stimulating factor, dehydroepiandrosterone, and 1,25-dihydroxy vitamin D3; polyanions, e.g., dextran; polyacrylics (e.g., polymethylmethacrylate and Carbopol 934P); carriers, e.g., tetanus toxid, diptheria toxoid, cholera toxin B subnuit, mutant heat labile enterotoxin of enterotoxigenic E. coli (rmLT), and heat shock proteins; oil-in-water emulsions, e.g., AMPHIGEN® (Hydronics, USA); water-in-oil emulsions, e.g., Freund's complete and incomplete adjuvants; and liposome-based adjuvants. Further adjuvants that can be used in the present immunogenic compositions 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 the group consisting of an aluminum salt adjuvant; a toll-like receptor 9 (TLR9) agonist; a liposome-based adjuvant; and an oil-in-water emulsion-based adjuvant. In some embodiments, the adjuvant is selected from the group consisting of an aluminum salt adjuvant; a TLR9 agonist that is an oligonucleotide containing an unmethylated cytidine-phospho-guanosine (CpG) motif; a liposome-based adjuvant containing a lipopolysaccharide and a saponin; and an oil-in-water emulsion-based adjuvant containing squalene, a sorbitan ester, and a polyoxyethylene sorbitan ester.

[0146] In some embodiments, the adjuvant is an aluminum salt adjuvant, such as any described herein, for instance in Section I-A-l. In some embodiments, the adjuvant is a TLR9 agonist, e.g., a TLR9 agonist that is an oligonucleotide containing an unmethylated CpG motif, such as any described herein, for instance in Section I-A-2. In some embodiments, the adjuvant is a liposome-based adjuvant, e.g., a liposome-based adjuvant containing a lipopolysaccharide and a saponin, such as any described herein, for instance in Section I-A-3. In some embodiments, the adjuvant is an oil-in-water emulsion-based adjuvant, e.g., an oil- in-water emulsion-based adjuvant containing squalene, a sorbitan ester, and a polyoxyethylene sorbitan ester, such as any described herein, for instance in Section I-A-4.

[0147] In some embodiments, the immunogenic composition contains a second adjuvant that is different from the first adjuvant. In some embodiments, the first and second adjuvants are individually any of the adjuvants described herein. In some embodiments, the first and second adjuvants are selected from the group consisting of an aluminum salt adjuvant; a TLR9 agonist; a liposome-based adjuvant; and an oil-in-water emulsion-based adjuvant. In some embodiments, the first and second adjuvants are selected from the group consisting of an aluminum salt adjuvant; a TLR9 agonist that is an oligonucleotide containing an unmethylated CpG motif; a liposome-based adjuvant containing a lipopolysaccharide and a saponin; and an oil-in-water emulsion-based adjuvant containing squalene, a sorbitan ester, and a polyoxyethylene sorbitan ester.

[0148] In some embodiments, the second adjuvant is a polycationic carrier. In some embodiments, the first adjuvant is a TLR9 agonist, e.g., a TLR9 agonist that is an oligonucleotide containing an unmethylated CpG motif, and the second adjuvant is a polycationic carrier. In some embodiments, the first adjuvant, e.g., TLR9 agonist, forms cationic complexes with the polycationic carrier. In some embodiments, the development of the cationic complexes leads to a heightened immune response, relative to the immune response induced by the first adjuvant alone. In some embodiments, the polycationic carrier is any described in 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 polyethylenimine (PEI). In some embodiments, the polycationic carrier is an aluminum salt adjuvant, such as any described herein, for instance in Section I-A-l.

[0149] In some embodiments, the second adjuvant is an aluminum salt adjuvant, which can be any described herein and in any of the amounts described herein. In some embodiments, the first adjuvant is a TLR9 agonist, e.g., a TLR9 agonist that is an oligonucleotidecontaining an unmethylated CpG motif, and the second adjuvant is an aluminum salt adjuvant. In some embodiments, the first adjuvant is a liposome-based adjuvant, e.g., a liposome-based adjuvant containing a lipopolysaccharide and a saponin, and the second adjuvant is an aluminum salt adjuvant. In some embodiments, the first adjuvant is an oil-in- water emulsion-based adjuvant, e.g., an oil-in-water emulsion-based adjuvant containing squalene, a sorbitan ester, and a 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 embodiments, the aluminum salt adjuvant is potassium aluminum sulfate.

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

[0151] In some embodiments, the first adjuvant is a lipopolysaccharide, and the second adjuvant is an aluminum salt adjuvant. In some embodiments, the lipopolysaccharide is adsorbed on the aluminum salt adjuvant. In some embodiments, the lipopolysaccharide is a lipid A or a derivative thereof. In some embodiments, the lipopolysaccharide is a monophosphoryl lipid A or a derivative thereof. In some embodiments, the lipopolysaccharide is 3-O-desacyl-4'-monophosphoryl lipid A. In some embodiments, the lipopolysaccharide is 3-deacyl-phosphorylated hexa-acyl disaccharide. 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-desacyl-4'-monophosphoryl lipid A, and the second adjuvant is aluminum hydroxide. In some embodiments, the first and second adjuvants together are the adjuvant AS04. In some embodiments, 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):eabbl025, 2020 Jun 19). In some embodiments, 3M-052 is formulated in poly(lactic-co-gly colic) 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 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 is composed of nanoparticles containing a saponin. In some embodiments, the saponin is extracted from Quillaja saponaria (soapbark) trees. In some embodiments, the nanoparticles contain a saponin, cholesterol, and a phospholipid. 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 to 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 from about 0.25 to about 1.25 mg Al3+. In some embodiments, the immunogenic composition contains from about 0.5 to about 1 mg Al3+. In some embodiments, the immunogenic composition contains from about 0.25 mg to about 0.50 mg Al3+. In some embodiments, the immunogenic composition contains from about 0.30 mg to about 0.40 mg Al3+. 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 Al3+.2. Oligonucleotide Toll-Like Receptor 9 (TLR9) Agonists

[0156] Toll-like receptors (TLRs) are expressed in and on dendritic cells and other innate immune cells and are among the most important receptors for stimulating a response to the presence of invading pathogens. Humans have multiple types of TLRs that are similar in structure 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 harnessed to enhance adaptive responses.

[0157] In some embodiments, the adjuvant in the present immunogenic compositions is an oligonucleotide TLR9 agonist. In some embodiments, the oligonucleotide contains an unmethylated cytidine-phospho-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 to stimulate T helper 1 (Thl) cell responses (Coffman et al., Immunity, 33:492-503, 2010). Based on structure and biological function, CpG-ODNs have been divided into three general classes: CpG-A, CpG-B, and CpG-C (Campbell, Methods Mol Biol, 1494:15-27, 2017). The degree of B cell activation varies between the classes, with CpG-A ODNs being weak, CpG-C ODNs being good, and CpG-B ODNs being strong B cell activators. Oligonucleotide TLR9 agonists in the present immunogenic compositions are preferably good B cell activators (CpG-C ODN) or more preferably strong (CpG-B ODN) B cell activators.

[0158] Oligonucleotide TLR9 agonists often contain a palindromic sequence following the general formula of 5’-purine-purine-CG-pyrimidine-pyrimidine-3’ or 5’-purine-purine-CG- pyrimidine-pyrimidine-CG-3’ (U.S. Patent No. 6,589,940). TLR9 agonism is also observed with certain non-palindromic CpG-enriched phosphorothioate oligonucleotides, but may be affected by changes in the nucleotide sequence. Additionally, TLR9 agonism can be abolished by methylation of the cytosine within the CpG dinucleotide. In some embodiments, the TLR9 agonist in the present immunogenic compositions is an oligonucleotide of from 8 to 35 nucleotides in length. In some embodiments, the TLR9 agonist in the present immunogenic compositions is an oligonucleotide of from 8 to 35 nucleotides in length comprising the sequence 5’-AACGTTCG-3’. In some embodiments, the oligonucleotide is greater than 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, and theoligonucleotide is less than 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, or 24 nucleotides in length. In some embodiments, the TLR9 agonist is an oligonucleotide of from 10 to 35 nucleotides in length. In some embodiments, the TLR9 agonist is an oligonucleotide of from 10 to 35 nucleotides in length comprising the sequence 5’-AACGTTCGAG-3’ (SEQ ID NO:2). In some embodiments, the oligonucleotide is greater than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, and the oligonucleotide is less than 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, or 24 nucleotides in length. In some embodiments, the oligonucleotide does not comprise the sequence 5’-TCGTCGTTTT GTCGTTTTGT CGTT-3’ (ODN 2006 or ODN 7909, set forth in SEQ ID NO:3).

[0159] Researchers at Dynavax Technologies Corporation (Emeryville, CA) have identified a 22-mer phosphorothioate linked oligodeoxynucleotide, CpG 1018® adjuvant, which contains specific sequences that can substantially enhance the immune response to coadministered antigens across species (Campbell, Methods Mol Biol, 1494: 15-27, 2017). CpG 1018® adjuvant(5’-TGACTGTGAA CGTTCGAGAT GA-3’, set forth in SEQ ID NO: 1) was chosen after screening a broad panel of oligonucleotides for immunostimulatory activity in vitro and in vivo. CpG 1018® adjuvant is a CpG-B ODN that is active in mice, rabbits, dogs, baboons, cynomolgus monkeys, and humans. In some embodiments, the oligonucleotide is from 22 to 35 nucleotides in length and comprises the sequence 5’-TGACTGTGAA CGTTCGAGAT GA-3’ (SEQ ID NO: 1). In some embodiments, the oligonucleotide is greater than 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides in length, and the oligonucleotide is less than 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, or 24 nucleotides in length, provided the maximum is below the minimum length.

[0160] Although the exemplary oligonucleotide CpG 1018® adjuvant is a CpG-ODN, oligonucleotides that are not fully DNA molecules can also be used in the present immunogenic compositions. In some embodiments, the oligonucleotide is a DNA / RNA chimeric molecule in which the CpG(s) and the palindromic sequence are deoxyribonucleic acids, and one or more nucleic acids outside of these regions are ribonucleic acids. In some embodiments, the oligonucleotide is linear. In other embodiments, the oligonucleotide is circular or includes hairpin loop(s). 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 can be modifications of the 3 ’OH or 5 ’OH group, modifications of thenucleotide base, modifications of the sugar component, and modifications of the phosphate group. Modified bases may be included in the palindromic sequence of the oligonucleotide as long as the modified base(s) maintains the same specificity for its natural complement through Watson-Crick base pairing (e.g., the palindromic portion is still self-complementary). In some embodiments, the oligonucleotide comprises a non-canonical base. In some embodiments, the oligonucleotide comprises a modified nucleoside. In some embodiments, the modified nucleoside is selected from the group consisting of 2’-deoxy-7-deazaguanosine, 2’-deoxy-6-thioguanosine, arabinoguanosine, 2’-deoxy-2’substituted-arabinoguanosine, and 2’-O-substituted-arabinoguanosine.

[0162] The oligonucleotide may contain a modification of the phosphate group. For example, in addition to phosphodiester linkages, phosphate modifications include methyl phosphonate, phosphorothioate, phosphoramidate (bridging or non-bridging), phosphotriester, and phosphorodi thioate and may be used in any combination. Other nonphosphate linkages may also be used. In some embodiments, the oligonucleotide contains only phosphorothioate backbones. In some embodiments, the oligonucleotide contains only phosphodiester backbones. In some embodiments, the oligonucleotide contains a combination of phosphate linkages in the phosphate backbone, such as a combination of phosphodiester and phosphorothioate linkages. Oligonucleotides with phosphorothioate backbones can be more immunogenic than those with phosphodiester backbones and can 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 includes at least one, two, or three internucleotide phosphorothioate ester linkages. In some embodiments, when a plurality of oligonucleotide molecules is present in a pharmaceutical composition containing at least one excipient, both stereoisomers of the phosphorothioate ester linkage are present in the plurality of oligonucleotide molecules. In some embodiments, all of the intemucleotide linkages of the oligonucleotide are phosphorothioate linkages, or said another way, the oligonucleotide has a phosphorothioate backbone.

[0163] In some embodiments, the immunogenic composition contains from about 375 pg to about 6000 pg of the oligonucleotide. In some embodiments, the immunogenic composition contains from about 750 pg to about 6000 pg of the oligonucleotide. In some embodiments, the immunogenic composition contains from about 750 pg to about 3000 pg of the oligonucleotide. In some embodiments, the immunogenic composition contains from about750 pg to about 1500 pg of the oligonucleotide. In some embodiments, the immunogenic composition contains from about 1500 pg to about 3000 pg of the oligonucleotide. In some embodiments, the immunogenic composition contains from about 3000 pg to about 6000 pg of the oligonucleotide. In some embodiments, the immunogenic composition contains greater than or equal to about 750, 1000, 1250, or 1500 pg of the oligonucleotide, and less than or equal to about 6000, 5000, 4000, or 3000 pg of the oligonucleotide. In some embodiments, the immunogenic composition contains about 375, 750, 1500, 3000, or 6000 pg of the oligonucleotide. In some embodiments, the immunogenic composition contains about 375 pg of the oligonucleotide. In some embodiments, the immunogenic composition contains about 750 pg of the oligonucleotide. In some embodiments, the immunogenic composition contains about 1000 pg of the oligonucleotide. In some embodiments, the immunogenic composition contains about 1500 pg of the oligonucleotide. In some embodiments, the immunogenic composition contains about 3000 pg of the oligonucleotide. In some embodiments, the immunogenic composition contains about 6000 pg of the oligonucleotide.

[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 with organic bases (for example, organic amines) such as N-Me-D-glucamine, N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, choline, tromethamine, dicyclohexylamines, and t-butyl amines, and salts with amino acids such as arginine, lysine, and the like. In some embodiment, the oligonucleotide is in the ammonium, sodium, lithium, or potassium salt form. In some embodiments, the oligonucleotide is in the sodium salt form.3. Liposome-Based Adjuvants

[0165] In some embodiments, the adjuvant in the present immunogenic compositions is a liposome-based adjuvant, such as any described in Tretiakova et al., Biochem (Mose) Suppl Ser A Membr Cell 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 includes a lipopolysaccharide. In some embodiments, the liposome-based adjuvant includes a saponin. In some embodiments, the liposome-based adjuvant includes a lipopolysaccharide and a saponin.

[0166] In some embodiments, the lipopolysaccharide is a lipid A or a derivative thereof. In some embodiments, the lipopolysaccharide is a monophosphoryl lipid A or a derivative thereof. In some embodiments, the lipopolysaccharide is naturally occurring. In someembodiments, the lipopolysaccharide is synthetic. In some embodiments, the lipopolysaccharide is 3-O-desacyl-4'-monophosphoryl lipid A. In some embodiments, the lipopolysaccharide is 3-deacyl-phosphorylated hexa-acyl disaccharide.

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

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

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

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

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

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

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

[0175] In some embodiments, the ratio of saponin to sterol is from 1 : 1 to 1 : 100 w / w. In some embodiments, the ratio of saponin to sterol is from 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 a phosphatidylcholine. In some embodiments, thephosphatidylcholine is eggyolk phosphatidylcholine, dioleoyl phosphatidylcholine (DOPC), or dilauryl phosphatidylcholine. In some embodiments, the phosphatidylcholine is DOPC.

[0177] In some embodiments, the liposome-based adjuvant contains 3-O-desacyl-4'- monophosphoryl lipid A and QS-21. In some embodiments, the liposome-based adjuvant contains 3-O-desacyl-4'-monophosphoryl lipid A, QS-21, and cholesterol. In some embodiments, the liposome-based adjuvant contains 3-O-desacyl-4'-monophosphoryl lipid A, QS-21, cholesterol, and DOPC. In some embodiments, the liposome-based adjuvant is AS01, AS01B, or AS01E, for instance 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-deacyl- phosphorylated hexa-acyl disaccharide and QS-21. In some embodiments, the liposomebased adjuvant is ALFQ.4. Oil-in-Water Emulsion-Based Adjuvants

[0179] In some embodiments, the adjuvant in the present immunogenic compositions is an oil-in-water emulsion-based adjuvant, such as any 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 a sorbitan ester (also known as a Span). In some embodiments, the oil-in-water emulsion-based adjuvant contains a polyoxyethylene sorbitan ester (also known as a Tween). In some embodiments, the oil-in-water emulsion-based adjuvant contains squalene, a sorbitan ester, and a polyoxyethylene sorbitan ester.

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

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

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

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

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

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

[0186] In some embodiments, the oil-in-water emulsion-based adjuvant contains an additional immune-enhancing component. In some embodiments, the oil-in-water emulsionbased adjuvant contains a vitamin. In some embodiments, the oil-in-water emulsion-based adjuvant contains vitamin E.

[0187] In some embodiments, the oil-in-water emulsion-based adjuvant contains squalene, vitamin E, and polyoxyethylene sorbitan ester 80. In some embodiments, the oil-in-water emulsion-based adjuvant is AS03. In some embodiments, the immunogenic composition contains about 11 mg squalene, about 12 mg vitamin E, and about 5 mg polyoxyethylene sorbitan ester 80.B. Borrelia Outer Surface Protein A (OspA) Antigens and RNA Polynucleotides

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

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

[0190] The RNA polynucleotide can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) permit sustained or delayed release; (4) alter the biodistribution to specific tissues or cell types; (5) increase the translation of encoded protein in vivo; and / or (6) alter the release profile of encoded protein in vivo. Excipients can include solvents, dispersion media, diluents, other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, hyaluronidase, nanoparticle mimics, and combinations thereof. Exemplary formulations for the RNA polynucleotide are described in US 10,898,574, US 10,703,789, and US 10,577,403.

[0191] In some embodiments, the RNA polynucleotide is formulated in lipid nanoparticles. Examples of lipid nanoparticle compositions and methods of making same are described, for example, in 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 a cationic lipid. In some embodiments, the lipid nanoparticles contain a sterol. In some embodiments, the lipid nanoparticles contain a PEG-modified lipid. In some embodiments, the lipid nanoparticles contain a neutral lipid. In some embodiments, the lipid nanoparticles contain a cationic lipid, a sterol, and a PEG-modified lipid. In some embodiments, the lipid nanoparticles contain a cationic lipid, a neutral lipid, a sterol, and a PEG-modified lipid.

[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 andamino alcohol lipids. In another aspect, the lipid may be a cationic lipid such as DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA and amino alcohol lipids. The amino alcohol cationic lipid may be the lipids described in and / or made by the methods described in U.S. Patent Publication No. US20130150625. For example, the cationic lipid may be 2-amino-3-[(9Z, 12Z)-octadeca-9,12-dien-l-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien- l-yloxy]methyl}propan-l-ol (Compound 1 in US20130150625); 2-amino-3-[(9Z)-octadec-9- en-l-yloxy]-2-{[(9Z)-octadec-9-en-l-yloxy]methyl}propan-l-ol (Compound 2 in US20130150625); 2-amino-3 -[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]-2- [(octyloxy)methyl]propan-l-ol (Compound 3 in US20130150625); and 2-(dimethylamino)-3- [(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]-2-{ [(9Z, 12Z)-octadeca-9, 12-dien- 1 - yloxy]methyl}propan-l-ol (Compound 4 in US20130150625); or any pharmaceutically acceptable salt or stereoisomer thereof.

[0193] In some embodiments, the lipid nanoparticles contain an ionizable cationic lipid, for example, 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), or di((Z)-non-2-en-l-yl) 9- ((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), and further contain a neutral lipid, a sterol and a molecule capable of reducing particle aggregation, for example a PEG or PEG-modified lipid.

[0194] In some embodiments, the lipid nanoparticle formulation consists essentially of (i) at least one lipid selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl- [l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3- DMA), and di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); (ii) a neutral lipid selected from DSPC, DPPC, POPC, DOPE and SM; (iii) a sterol, e.g., cholesterol; and (iv) a PEG-lipid, e.g., PEG-DMG or PEG-cDMA, in a molar ratio of about 20-60% cationic lipid: 5-25% neutral lipid: 25-55% sterol; 0.5-15% PEG-lipid. In some embodiments, the lipid nanoparticles contain 50% ionizable cationic lipid, 10% neutral lipid, 38.5% sterol, and 1.5% PEG-modified lipid. In some embodiments, the neutral lipid is 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), the sterol is cholesterol, and the PEG- modified lipid is l,2-dimyristoyl-racalycero-3-methoxypolyethylene glycol-2000 (PEG- DMG) or PEG-cDMA.

[0195] Seven principal OspA serotypes have been recognized among European isolates (designated serotypes 1 to 7, see Wilske et al., J. Clin. Microbiol. 31 :340-50, 1993). In some aspects, OspA serotypes tend to correlate with species, for instance with serotype 1corresponding to B. burgdorferi s.s., serotype 2 corresponding to B. afzelii, and serotypes 3 to 7 corresponding to B. garinii. Serotype 4, in some aspects, is alternatively considered to correspond to B. bavariensis (Margos et al., Appl. Environ. Microbiol. 75: 5410-6, 2009).

[0196] In some embodiments, the OspA antigen is any of the OspA antigens described in WO2021207615, W02021205022, WO2010132758, 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 7,582,304, US 7,605,248, and US20090324638.

[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 is understood that reference to the OspA antigen in the disclosure herein independently describes embodiments of any one of the OspA antigens 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 includes two or more OspA antigens. For example, each of the OspA antigens have non-identical amino acid sequences that can be overlapping or entirely non-overlapping sequences.

[0199] In some embodiments, the OspA antigen is aB. burgdorferi OspA antigen. In some embodiments, the OspA antigen is aB. afzelii OspA antigen. In some embodiments, the OspA antigen is aB. bavariensis OspA antigen. In some embodiments, the OspA antigen is a B. garinii OspA antigen. In some embodiments, the OspA antigen is a B. mayonii OspA antigen. In some embodiments, the OspA antigen is aB. lusitaniae OspA antigen. In some embodiments, the OspA antigen is aB. bissettii OspA antigen. In some embodiments, the OspA antigen is aB. spielmanii OspA antigen. In some embodiments, the OspA antigen is a B. valasiana OspA antigen.

[0200] In some embodiments, each of the different OspA antigens is an OspA antigen from a different Borrelia species. In some embodiments, the OspA antigen includes a first OspA antigen and a second OspA antigen. In some embodiments, the first OspA antigen and the second OspA antigen have non-identical amino acid sequences. In some embodiments, the two or more OspA antigens further includes a third OspA antigen. In some embodiments, the first OspA antigen, the second OspA antigen, and the third OspA antigen have non-identical 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 2, 3, 4, 5, or 6 epitopes from one or more OspA proteins. In some embodiments, the one or more epitopes are from the same OspA protein. In some embodiments, the one or more epitopes are from the same OspA serotype. In some embodiments, the one or more epitopes are from multiple OspA proteins. In some embodiments, the one or more epitopes contain at least 2, 3, 4, 5, or 6 epitopes from at least 2, 3, 4, 5, or 6 OspA proteins. In some embodiments, the one or more epitopes are from multiple OspA serotypes. In some embodiments, the one or more epitopes contain at least 2, 3, 4, 5, or 6 epitopes from at least 2, 3, 4, 5, or 6 OspA serotypes.

[0204] In some embodiments, the one or more epitopes include an OspA epitope contained in any of the sequences of amino acids set forth in SEQ ID NO:50-82. In some embodiments, the one or more epitopes are independently selected from OspA epitopes contained in any of the sequences of amino acids set forth in SEQ ID NO:50-82. In some embodiments, the one or more epitopes include any of the linear OspA epitopes contained in the sequences of amino acids set forth in SEQ ID NO: 50-82. In some embodiments, the one or more epitopes include any of the conformational OspA epitopes contained in the sequences of amino acids set forth in SEQ ID NO:50-82. In some embodiments, the one or more epitopes include linear and conformational OspA epitopes contained in any of the sequences of amino acids set forth in SEQ ID NO:50-82.

[0205] In some embodiments, the one or more epitopes include an OspA epitope contained in any of the sequences of amino acids set forth in SEQ ID NO:50-62. In some embodiments, the one or more epitopes are independently selected from OspA epitopes contained in any of the sequences of amino acids set forth in SEQ ID NO:50-62. In some embodiments, the one or more epitopes include any of the linear OspA epitopes contained in the sequences of amino acids set forth in SEQ ID NO: 50-62. In some embodiments, the one or more epitopes include any of the conformational OspA epitopes contained in the sequences of amino acidsset forth in SEQ ID NO:50-62. In some embodiments, the one or more epitopes include linear and conformational OspA epitopes contained in any of the sequences of amino acids set forth in SEQ ID NO:50-62.

[0206] In some embodiments, the one or more epitopes include an OspA epitope contained in any of the sequences of amino acids set forth in SEQ ID NO:63-82. In some embodiments, the one or more epitopes are independently selected from OspA epitopes contained in any of the sequences of amino acids set forth in SEQ ID NO:63-82. In some embodiments, the one or more epitopes include any of the linear OspA epitopes contained in the sequences of amino acids set forth in SEQ ID NO:63-82. In some embodiments, the one or more epitopes include any of the conformational OspA epitopes contained in the sequences of amino acids set forth in SEQ ID NO:63-82. In some embodiments, the one or more epitopes include linear and conformational OspA epitopes contained in any of the sequences of amino acids set forth in SEQ ID NO:63-82.

[0207] In some embodiments, the one or more epitopes are independently selected from the sequences of amino acids set forth in SEQ ID NO:4-49. In some embodiments, the one or more epitopes are independently selected from any of the linear epitopes whose sequences of amino acids are set forth in 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 of the sequences of amino acids set forth in SEQ ID NO: 50-82 that contains 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 thereto. In some embodiments, the OspA antigen has any of the sequences of amino acids set forth in SEQ ID NO:50-82 that contains 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 contiguous amino acids thereof. In some embodiments, the OspA antigen has any of the sequences of amino acids set forth in SEQ ID NO:50-82 that contains 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 of the sequences of amino acids set forth in SEQ ID NO: 50-62 that contains 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 thereto. In someembodiments, the OspA antigen has any of the sequences of amino acids set forth in SEQ ID NO:50-62 that contains 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 contiguous amino acids thereof. In some embodiments, the OspA antigen has any of the sequences of amino acids set forth in SEQ ID NO:50-62 that contains 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 to the sequence of amino acids set forth in any 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 contiguous amino acids of the sequence of amino acids set forth in any of SEQ ID NO:50-62.

[0212] In some embodiments, the OspA antigen comprises the sequence of amino acids set forth in any 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:50. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:51. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of aminoacids set forth in SEQ ID NO:52. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:53. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:54. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:55. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:56. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:57. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:58. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:59. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:60. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of aminoacids set forth in SEQ ID NO:61. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:62. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 a plurality of 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 are from the same OspA protein. In some embodiments, the plurality of epitopes are from the same OspA serotype. In some embodiments, the plurality of epitopes are from multiple OspA proteins. In some embodiments, the plurality of epitopes contain at least 2, 3, 4, or 5 epitopes from at least 2, 3, 4, 5, or 6 OspA proteins. In some embodiments, the plurality of epitopes are from multiple OspA serotypes. In some embodiments, the plurality of epitopes contain at least 2, 3, 4, 5, or 6 epitopes from at least 2, 3, 4, 5, or 6 OspA serotypes.

[0228] In some embodiments, the plurality of epitopes include an OspA epitope contained in any of the sequences of amino acids set forth in SEQ ID NO:50-82. In some embodiments, the plurality of epitopes are independently selected from OspA epitopes contained in any of the sequences of amino acids set forth in SEQ ID NO:50-82. In some embodiments, the plurality of epitopes include any of the linear OspA epitopes contained in the sequences of amino acids set forth in SEQ ID NO: 50-82. In some embodiments, the plurality of epitopes include any of the conformational OspA epitopes contained in the sequences of amino acids set forth in SEQ ID NO:50-82. In some embodiments, the plurality of epitopes include linear and conformational OspA epitopes contained in any of the sequences of amino acids set forth in SEQ ID NO:50-82.

[0229] In some embodiments, the plurality of epitopes include an OspA epitope contained in any of the sequences of amino acids set forth in SEQ ID NO:50-62. In some embodiments, the plurality of epitopes are independently selected from OspA epitopes contained in any ofthe sequences of amino acids set forth in SEQ ID NO:50-62. In some embodiments, the plurality of epitopes include any of the linear OspA epitopes contained in the sequences of amino acids set forth in SEQ ID NO: 50-62. In some embodiments, the plurality of epitopes include any of the conformational OspA epitopes contained in the sequences of amino acids set forth in SEQ ID NO:50-62. In some embodiments, the plurality of epitopes include linear and conformational OspA epitopes contained in any of the sequences of amino acids set forth in SEQ ID NO:50-62.

[0230] In some embodiments, the plurality of epitopes include an OspA epitope contained in any of the sequences of amino acids set forth in SEQ ID NO:63-82. In some embodiments, the plurality of epitopes are independently selected from OspA epitopes contained in any of the sequences of amino acids set forth in SEQ ID NO:63-82. In some embodiments, the plurality of epitopes include any of the linear OspA epitopes contained in the sequences of amino acids set forth in SEQ ID NO:63-82. In some embodiments, the plurality of epitopes include any of the conformational OspA epitopes contained in the sequences of amino acids set forth in SEQ ID NO:63-82. In some embodiments, the plurality of epitopes include linear and conformational OspA epitopes contained in any of the sequences of amino acids set forth in SEQ ID NO:63-82.

[0231] In some embodiments, the plurality of epitopes are independently selected from the sequences of amino acids set forth in SEQ ID NO:4-49. In some embodiments, the plurality of epitopes are independently selected from any of the linear epitopes whose sequences of amino acids are set forth in 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 of the sequences of amino acids set forth in SEQ ID NO:50-82 that contains 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 thereto. In some embodiments, the OspA antigen has any of the sequences of amino acids set forth in SEQ ID NO:50-82 that contains 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 contiguous amino acids thereof. In some embodiments, the OspA antigen has any of the sequences of amino acids set forth in SEQ ID NO: 50-82 that contains 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 of the sequences of amino acids set forth in SEQ ID NO: 63 -82 that contains 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 thereto. In some embodiments, the OspA antigen has any of the sequences of amino acids set forth in SEQ ID NO: 63 -82 that contains 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 contiguous amino acids thereof. In some embodiments, the OspA antigen has any of the sequences of amino acids set forth in SEQ ID NO: 63 -82 that contains one or more linear epitopes.

[0234] In some embodiments, the OspA antigen is an OspA chimeric antigen containing a plurality of epitopes from one or more OspA proteins. In some embodiments, the plurality of epitopes are linear epitopes. In some embodiments, the OspA antigen is an OspA chimeritope. In some embodiments, the OspA antigen has any of the sequences of amino acids set forth in SEQ ID NO:50-82 that is an OspA chimeric antigen containing a plurality of 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 thereto. In some embodiments, the OspA antigen has any of the sequences of amino acids set forth in SEQ ID NO:50-82 that is an OspA chimeric antigen containing a plurality of linear epitopes or has at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 contiguous amino acids thereof. In some embodiments, the OspA antigen has any of the sequences of amino acids set forth in SEQ ID NO: 50-82 that is an OspA chimeric antigen containing a plurality of linear epitopes.

[0235] In some embodiments, the OspA antigen is an OspA chimeric antigen containing a plurality of epitopes from one or more OspA proteins. In some embodiments, the plurality of epitopes are linear epitopes. In some embodiments, the OspA antigen is an OspA chimeritope. In some embodiments, the OspA antigen has any of the sequences of amino acids set forth in SEQ ID NO:63-82 that is an OspA chimeric antigen containing a plurality of 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 thereto. In some embodiments, the OspA antigen has any of the sequences of amino acids set forth in SEQ ID NO:63-82 that is an OspA chimeric antigen containing a plurality of linear epitopes or has at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or250 contiguous amino acids thereof. In some embodiments, the OspA antigen has any of the sequences of amino acids set forth in SEQ ID NO:63-82 that is an OspA chimeric antigen containing a plurality of 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 to the sequence of amino acids set forth in any 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 contiguous amino acids of the sequence of amino acids set forth in any of SEQ ID NO:63-82.

[0238] In some embodiments, the OspA antigen comprises the sequence of amino acids set forth in any 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:63. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:64. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:65. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:66. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:67. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:68. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:69. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:70. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:71. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:72. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:73. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:74. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:75. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:76. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:77. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:78. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:79. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:80. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:81. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:82. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth 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 to the sequence of amino acids set forth in any 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 contiguous amino acids of the sequence of amino acids set forth in any of SEQ ID NO:79-81. In some embodiments, the OspA antigen comprises the sequence of amino acids set forth in any of SEQ ID NO:79-81.

[0260] In some embodiments, the immunogenic composition comprises a first OspA antigen that 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 to the sequence of amino acids set forth in SEQ ID NO:79 and a second OspA antigen that 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:79. In some embodiments, the first OspA antigen comprises the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:80. In some embodiments, the second OspA antigen comprises the sequence of amino acids set forth in SEQ ID NO:80.

[0261] In some embodiments, the immunogenic composition comprises a first OspA antigen that 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 to the sequence of amino acids set forth in SEQ ID NO:79 and a second OspA antigen that 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:79. In some embodiments, the first OspA antigen comprises the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:81. In some embodiments, the second OspA antigen comprises the sequence of amino acids set forth in SEQ ID NO:81.

[0262] In some embodiments, the immunogenic composition comprises a first OspA antigen that 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 to the sequence of amino acids set forth in SEQ ID NO:80 and a second OspA antigen that 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:80. In some embodiments, the first OspA antigen comprises the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:81. In some embodiments, the second OspA antigen comprises the sequence of amino acids set forth in SEQ ID NO:81.

[0263] In some embodiments, the immunogenic composition comprises a first OspA antigen that 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 to the sequence of amino acids set forth in SEQ ID NO:79, a second OspA antigen that 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 to the sequence of amino acids set forth in SEQ ID NO: 80, and a third OspA antigen that 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 to the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:79. In some embodiments, the first OspA antigen comprises the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO: 80. In some embodiments, the second OspA antigen comprises the sequence of amino acids set forth 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 contiguous amino acids of the sequence of amino acids set forth in SEQ ID NO:81. In some embodiments, the third OspA antigen comprises the sequence of amino acids set forth in SEQ ID NO:81.

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

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

[0266] Lipidation may confer adjuvant properties or stabilize the OspA antigen. Lipidation of a protein may occur using a number of different known methods (see, e.g., 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, lipidation occurs through a chemical lipidation process. In some embodiments, lipidation occurs using an E. coli expression system. In some embodiments, lipidation is assisted through the presence of a lipidation signal peptide sequence, such as those present on an 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 the enzymes diacylglyceryl transferase, signal peptidase II and transacylase. In some embodiments, the first step is the transfer of a diacylglyceride to the cysteine sulphydryl group of the unmodified prolipoprotein, followed by the cleavage of the signal peptide by signal peptidase II and, finally, the acylation of the a- amino group of the N-terminal cysteine of the apolipoprotein. The result is the placement of one lipid and a glycerol group substituted with two further lipids on the N-terminal cysteine residue of the polypeptide. The lipidation signal sequence, which is cleaved off during lipidation, is not present in the final polypeptide sequence.

[0267] In some embodiments, the lipidated OspA antigen is purified. In some embodiments, purification occurs using detergent extraction and / or ion-exchange chromatography. In some embodiments, purification occurs using ion-exchange chromatography and Triton XI 00 extraction. In some embodiments, the purification process decreases endotoxin levels. In some embodiments, the purification decreases the endotoxin level below 10 EU / mg.

[0268] In some embodiments, the OspA antigen is lipidated through the addition or presence of a lipid moiety to the OspA antigen. In some embodiments, lipid moieties, such as fatty acids, are 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 / orN-terminus. In some embodiments, lipid moieties, such as fatty acids, are added to the OspA antigen at the N-terminus. In some embodiments, lipid molecules, such as fatty acids, and glycerol are added to the OspA antigen at the N-terminus. In some embodiments, the lipidated OspA antigen has a 3 fatty acid group and a glycerol added to the N-terminus of the lipidated OspA antigen.

[0269] In some embodiments, a lipid moiety comprises one to three lipids, such as C14-20 alkyl and / or C14-20 alkenyl. In some embodiments, a lipid moiety comprises saturated fatty acids (ranging from Ci4-Cis), unsaturated fatty acids (Cie / Cis), and / or palmitate (C16:0) as major components. In some embodiments, the lipid moiety is attached to a glycerol. In some embodiments, the lipid moiety is attached to an N-terminal cysteine of the polypeptide of the OspA antigen. In some embodiments, the N-terminal cysteine is present on a signal peptide sequence. In some embodiments, the lipid moiety is attached at the amino terminal cysteine residue of the signal peptide sequence. In some embodiments, the lipid moiety is attached at the sulfhydryl group of the cysteine residue of the signal peptide sequence. In some embodiments, the N-terminal cysteine can be lipidated on both the amino group (-HN2) and sulfhydryl group (-SH). In some embodiments, the N-terminal cysteine can be lipidated to form around 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 lipidation signal peptide sequence. Lipidation signal peptide sequences are known in the art (see, e.g., US 8,986,704 and US 11,305,000). Exemplary lipidation signal peptide sequences include, but are not limited to, the lipidation signal peptide sequences set forth in SEQ ID NOs: 83-85. In some embodiments, the OspA antigen further comprises a lipidation signal peptide sequence as set forth in SEQ ID NO: 83. In some embodiments, the OspA antigen further comprises a lipidation signal peptide sequence as set forth in SEQ ID NO: 84. In some embodiments, the OspA antigen further comprises a lipidation signal peptide sequence as set forth 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 set forth in SEQ ID NO: 83.

[0271] Lipidation signal peptide sequences are cleaved off during processing, thus the lipidated OspA antigens may not contain the lipidation signal peptide sequence in the mature lipidated protein.C. Additional Components

[0272] The immunogenic compositions of the present disclosure may comprise 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 for the present immunogenic compositions include solvents, bulking agents, buffering agents, tonicity adjusting agents, preservatives, surfactants, and emulsifying agents (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, a bulking agent, a buffering agent, and a tonicity adjusting agent (e.g., sodium chloride in saline may serve as both an aqueous vehicle and a tonicity adjusting agent). 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 compositions contains an aqueous vehicle as a solvent. Suitable vehicles 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 buffering agent. Buffering agents control 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 certain other embodiments, the buffer is a Tris buffer. Other suitable buffers include amino acids such as arginine, glycine, histidine, and lysine. The buffering agent may further comprise hydrochloric acid or sodium hydroxide. In some embodiments, the buffering agent maintains the pH of the immunogenic composition within a 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 from about 6 to 9 in which 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 tonicity adjusting agent. Suitable tonicity adjusting agents include dextrose, glycerol, sodium chloride, glycerin, and mannitol.

[0277] In some embodiments, the immunogenic composition contains a bulking agent. Bulking agents are particularly useful when the immunogenic composition is to be lyophilized before administration. In some embodiments, the bulking agent is a protectant that aids in the stabilization and prevention of degradation of the active agents during freeze or spray drying and / or during storage. Suitable bulking agents include sugars (e.g., mono-, di- , and polysaccharides) such as sucrose, lactose, trehalose, mannitol, sorbital, 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 the monosodium salt of glutamic acid, 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 is in a single use container, and thus does not necessitate inclusion of a preservative.II. Articles of Manufacture

[0280] The present disclosure also provides vials, syringes (e.g., pre-filled syringes), and kits containing any of the immunogenic compositions described herein, such 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 vial, syringe, or kit contains a single dose of the immunogenic composition, such as a 0.3 mL to 1.5 mL dose.

[0281] The present disclosure also provides kits containing a first composition and a second composition. In some embodiments, the first composition contains an adjuvant, which can be any adjuvant described herein, such as in Section I-A. In some embodiments, the second composition contains a Borrelia outer surface protein A (OspA) antigen, which can be any described herein, such as in Section I-B. 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 contains instructions for combining the first composition and the second composition to prepare any of the immunogenic compositions described herein, such 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 0.3 mL to 1.5 mL dose.

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

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

[0285] In some embodiments, the kit further contains instructions for administration of the immunogenic composition to a subject to stimulate an immune response against Borrelia in the subject. In some embodiments, the administration is according to any of the methods described herein, for instance any described in Section III. In some embodiments, the subject is a human subject. In some embodiments, the human subject is at least 18 years of age. In some embodiments, the human subject is about 18 to about 70 years of age. In some embodiments, the human subject is about 18 to about 55 years of age. In some embodiments, the human subject is under 18 years of age. In some embodiments, the human subject is about 2 to about 5 years of age. In some embodiments, the human subject is about 5 to about 18 years of age.

[0286] In some embodiments, the kit contains an immunogenic composition packaged appropriately. In some embodiments, the kit contains i) a first composition and ii) a second composition that are packaged appropriately, such that the first composition and the second composition may be combined to form an immunogenic composition. For example, if the immunogenic composition is a freeze-dried power, a vial with a resilient stopper can be used so that the powder may be easily resuspended by injecting fluid (e.g., sterile water, saline, etc.) through the resilient stopper. In some embodiments, the kit contains a device for administration (e.g., syringe). In some embodiments, the syringe is pre-filled with the immunogenic composition. In some embodiments, the administration is by intramuscular injection. The instructions relating to the use of the immunogenic composition generally include information as to dosage, schedule, and route of administration for the intended methods of use.III. Methods Of Use

[0287] The present disclosure also relates to methods for stimulating an immune response against Borrelia in a subject, involving administering to the subject an effective amount of any of the immunogenic compositions described herein, for instance in Section I. In some embodiments, the antigen and adjuvant are present in the immunogenic composition in amounts effective to stimulate an immune response against Borrelia in a subject.

[0288] The present disclosure also relates to methods for protecting a subject from infection by Borrelia, involving administering to the subject an effective amount of any of the immunogenic compositions described herein, for instance in Section I. In some embodiments, the antigen and adjuvant are present in the immunogenic composition in amounts effective to stimulate an immune response against Borrelia in a subject.

[0289] The present disclosure also relates to methods for preventing a subject from contracting Lyme disease, involving administering to the subject an effective amount of any of the immunogenic compositions described herein, for instance in Section I. In some embodiments, the antigen and adjuvant are present in the immunogenic composition in amounts effective to stimulate an immune response against Borrelia in a subject.

[0290] In some embodiments, the subject is a human subject. In some embodiments, the human subject is at least 18 years of age. In some embodiments, the human subject is about 18 to about 70 years of age. In some embodiments, the human subject is about 18 to about 55 years of age. In some embodiments, the human subject is under 18 years of age. In some embodiments, the human subject is about 2 to about 5 years of age. In some embodiments, the human subject is about 5 to about 18 years of age.

[0291] In some embodiments, the methods of the present disclosure are suitable for increasing the immune response against Borrelia in the subject relative to a baseline immune response against Borrelia in the subject prior to administration of the immunogenic composition. Stimulating an immune response, which means increasing the immune response, can arise from eliciting a de novo immune response (e.g., as a consequence of an initial vaccination regimen) or enhancing an existing immune response (e.g., as a consequence of a booster vaccination regimen). In some embodiments, stimulating an immune response includes one or more of stimulating cytokine production; stimulating B lymphocyte proliferation; stimulating antibody production; stimulating interferon pathway- associated gene expression; stimulating chemoattractant-associated gene expression; andstimulating plasmacytoid dendritic cell maturation. In some embodiments, the immune response prevents the subject from infection with Borrelia. In some embodiments, the immune response prevents the subject from contracting Lyme disease.

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

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

[0294] In some embodiments, the second dose is administered from about 6 weeks to about 10 weeks after the administration of the first dose. In some embodiments, the second dose is administered from about 7 weeks to about 9 weeks after the administration of the first dose. In some embodiments, the second dose is administered about 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 from about 4 months to about 8 months after the first dose. In some embodiments, the third dose is administered from about 5 months to about 7 months after the first dose. In some embodiments, the third dose is administered from about 22 weeks to about 26 weeks after the first dose. In some embodiments, the third dose is administered about 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, the booster dose is administered at least 1year after the first dose. In some embodiments, the booster dose is administered at least 2 years after the first dose.

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

[0298] In some embodiments, the booster dose is administered from about 10 months to about 14 months after the first dose. In some embodiments, the booster dose is administered about 1 year after the first dose.

[0299] In some embodiments, the booster dose is administered from about 20 months to about 28 months after the first dose. In some embodiments, the booster dose is administered about 2 years after the first dose.

[0300] In some embodiments, a plurality of booster doses is administered to the subject. In some embodiments, a plurality of yearly booster doses is administered to the subject. In some embodiments, the first booster dose is administered at least about 1 year or at least about 2 years after the administration of the first dose. In some of any embodiments, a booster dose is administered yearly.

[0301] In some embodiments, a first and second dose of the immunogenic composition is administered to the subject, wherein the second dose is administered about 1 month after the first dose.

[0302] In some embodiments, a first, second, and booster dose of the immunogenic composition is administered to the subject, wherein the second dose is administered about 1 month after the first dose, and the booster dose is administered at least 2 years after the first dose. In some embodiments, a first, second, and booster dose of the immunogenic composition is administered to the subject, wherein the second dose is administered about 1 month after the first dose, and the booster dose is administered about 2 years after the first dose.

[0303] In some embodiments, a first and second dose of the immunogenic composition is administered to the subject, wherein the second dose is administered about 2 months after the first dose.

[0304] In some embodiments, a first, second, and booster dose of the immunogenic composition is administered to the subject, wherein the second dose is administered about 2 months after the first dose, and the booster dose is administered at least 1 year after the first dose. In some embodiments, a first, second, and booster dose of the immunogenic composition is administered to the subject, wherein the second dose is administered about 2 months after the first dose, and the booster dose is administered about 1 year after the first dose.

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

[0306] In some embodiments, a first, second, and third dose of the immunogenic composition is administered to the subject, wherein the second dose is administered about 2 months after the first dose, and the third dose is administered about 6 months after the first dose.

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

[0308] In some embodiments, a first, second, third, and plurality of booster doses of the immunogenic composition is administered to the subject, wherein the second dose is administered about 2 months after the first dose, the third dose is administered about 6months after the first dose, the first booster dose is administered about 1 year after the first dose, and a booster dose is administered every year.

[0309] In some embodiments, the immunogenic composition is administered by intramuscular injection. In some embodiments, the injection site is in an arm of the subject. In some embodiments, the injection site is in a deltoid muscle of the subject.EXEMPLARY EMBODIMENTS

[0310] Among the provided embodiments are:1. An immunogenic composition, comprising (i) a Borrelia outer surface protein A (OspA) antigen or an RNA polynucleotide encoding the OspA antigen and (ii) an adjuvant selected from the group consisting of an aluminum salt adjuvant; a toll-like receptor 9 (TLR9) agonist; a liposome-based adjuvant comprising a lipopolysaccharide and a saponin; and an oil-in-water emulsion comprising squalene, a sorbitan ester, and a polyoxyethylene sorbitan ester.2. An immunogenic composition, comprising (i) a Borrelia outer surface protein A (OspA) antigen and (ii) an adjuvant selected from the group consisting of an aluminum salt adjuvant; a toll-like receptor 9 (TLR9) agonist; a liposome-based adjuvant comprising a lipopolysaccharide and a saponin; and an oil-in-water emulsion comprising squalene, a sorbitan ester, and a polyoxyethylene sorbitan ester.3. The immunogenic composition of embodiment 1 or embodiment 2, wherein the immunogenic composition does not comprise an RNA polynucleotide encoding a non- OspA Borrelia outer surface protein antigen.4. The immunogenic composition of any one of embodiments 1-3, wherein the immunogenic composition does not comprise a non-OspA Borrelia outer surface protein antigen.5. The immunogenic composition of any one of embodiments 1-4, wherein the adjuvant is an aluminum salt adjuvant.6. The immunogenic composition of embodiment 5, further comprising a second adjuvant.7. The immunogenic composition of embodiment 6, wherein the second adjuvant is selected from a TLR9 agonist; a liposome-based adjuvant comprising a lipopolysaccharide and a saponin; and an oil-in-water emulsion comprising squalene, a sorbitan ester, and a polyoxyethylene sorbitan ester.8. The immunogenic composition of any one of embodiments 1-4, wherein the adjuvant is a TLR9 agonist.9. The immunogenic composition of embodiment 8, further comprising a second adjuvant.10. The immunogenic composition of embodiment 9, wherein the second adjuvant is selected from an aluminum salt adjuvant; a liposome-based adjuvant comprising a lipopolysaccharide and a saponin; and an oil-in-water emulsion-based adjuvant comprising squalene, a sorbitan ester, and a polyoxyethylene sorbitan ester.11. The immunogenic composition of any one of embodiments 1-4, wherein the adjuvant is a liposome-based adjuvant comprising a lipopolysaccharide and a saponin.12. The immunogenic composition of embodiment 11, further comprising a second adjuvant.13. The immunogenic composition of embodiment 12, wherein the second adjuvant is selected from an aluminum salt adjuvant; a TLR9 agonist; and an oil-in-water emulsion-based adjuvant comprising squalene, a sorbitan ester, and a polyoxyethylene sorbitan ester.14. The immunogenic composition of any one of embodiments 1-4, wherein the adjuvant is an oil-in-water emulsion-based adjuvant comprising squalene, a sorbitan ester, and a polyoxyethylene sorbitan ester.15. The immunogenic composition of embodiment 14, further comprising a second adjuvant.16. The immunogenic composition of embodiment 15, wherein the second adjuvant is selected from an aluminum salt adjuvant; a TLR9 agonist; and a liposome-based adjuvant comprising a lipopolysaccharide and a saponin.17. The immunogenic composition of any one of embodiments 1-4, 7-10, 13, and 16, wherein the TLR9 agonist is an oligonucleotide comprising an unmethylated cytidinephospho-guanosine (CpG) motif.18. The immunogenic composition of embodiment 17, wherein the oligonucleotide is from 10 to 35 nucleotides in length.19. The immunogenic composition of embodiment 17 or embodiment 18, wherein the oligonucleotide comprises the sequence 5’-AACGTTCGAG-3’ (SEQ ID NO:2).20. The immunogenic composition of any one of embodiments 17-19, wherein the oligonucleotide comprises the sequence of 5’-TGACTGTGAA CGTTCGAGAT GA-3’(SEQ ID NO: 1).21. An immunogenic composition, comprising a Borrelia outer surface protein A(OspA) antigen and a toll-like receptor 9 (TLR9) agonist oligonucleotide of from 10 to 35 nucleotides in length comprising the sequence 5’-AACGTTCGAG-3’ (SEQ ID NO:2) and an unmethylated cytidine-phospho-guanosine (CpG) motif.22. The immunogenic composition of embodiment 21, wherein the oligonucleotide comprises the sequence of 5’-TGACTGTGAA CGTTCGAGAT GA-3’(SEQ ID NO: 1).23. An immunogenic composition, comprising a Borrelia outer surface protein A (OspA) antigen and the toll-like receptor 9 (TLR9) agonist oligonucleotide 5’- TGACTGTGAA CGTTCGAGAT GA-3’(SEQ ID NO: 1), the oligonucleotide comprising an unmethylated cytidine-phospho-guanosine (CpG) motif.24. The immunogenic composition of any one of embodiments 21-23, further comprising a second adjuvant.25. The immunogenic composition of embodiment 24, wherein the second adjuvant is selected from an aluminum salt adjuvant; a liposome-based adjuvant comprising a lipopolysaccharide and a saponin; and an oil-in-water emulsion-based adjuvant comprising squalene, a sorbitan ester, and a polyoxyethylene sorbitan ester.26. The immunogenic composition of 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) Borrelia outer surface protein A (OspA) antigen, (ii) the toll-like receptor 9 (TLR9) agonist oligonucleotide 5’- TGACTGTGAA CGTTCGAGAT GA-3’(SEQ ID NO: 1), the oligonucleotide comprising an unmethylated cytidine-phospho-guanosine (CpG) motif, and (iii) an aluminum salt adjuvant.28. The immunogenic composition of 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 of 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 of any one of embodiments 1-29, wherein the immunogenic composition comprises from about 0.25 to about 1.25 mg Al3+, from about 0.25 mg to about 0.50 mg Al3+, or from about 0.30 mg to about 0.40 mg Al3+.31. The immunogenic composition of any one of embodiments 17-30, wherein the oligonucleotide is a single-stranded oligodeoxynucleotide.32. The immunogenic composition of any one of embodiments 17-30, wherein the oligonucleotide is fully RNA.33. The immunogenic composition of any one of embodiments 17-30, wherein the oligonucleotide is an RNA / DNA chimera.34. The immunogenic composition of any one of embodiments 17-33, wherein the oligonucleotide comprises at least one phosphorothioate linkage.35. The immunogenic composition of any one of embodiments 17-34, wherein the oligonucleotide comprises only phosphorothioate linkages.36. The immunogenic composition of any one of embodiments 17-34, wherein the oligonucleotide comprises a combination of one or more phosphodiester linkages and one or more phosphorothioate linkages.37. The immunogenic composition of any one of embodiments 1-4, 7-10, 13, and 16-36, wherein the immunogenic composition comprises from about 375 pg to about 6000 pg of the TLR9 agonist or from about 750 pg to about 3000 pg of the TLR9 agonist.38. The immunogenic composition of any one of embodiments 1-4, 7-10, 13, and 16-36, wherein the immunogenic composition comprises about 375 pg, about 750 pg, about 1000 pg, about 1500 pg, about 3000 pg, or about 6000 pg of the TLR9 agonist.39. The immunogenic composition of any one of embodiments 1-4, 7, 10-13, 16- 20, 25, 26, and 28-38, wherein the lipopolysaccharide is a lipid A or a derivative thereof.40. The immunogenic composition of any one of embodiments 1-4, 7, 10-13, 16- 20, 25, 26, and 28-39, wherein the lipopolysaccharide is a monophosphoryl lipid A or a derivative thereof.41. The immunogenic composition of any one of embodiments 1-4, 7, 10-13, 16- 20, 25. 26, and 28-40, wherein the lipopolysaccharide is 3-O-desacyl-4'-monophosphoryl lipid A.42. The immunogenic composition of any one of embodiments 1-4, 7, 10-13, 16- 20, 25, 26, and 28-40, wherein the lipopolysaccharide is 3-deacyl-phosphorylated hexa-acyl di saccharide.43. The immunogenic composition of any one of embodiments 1-4, 7, 10-13, 16- 20, 25, 26, and 28-42, wherein the immunogenic composition comprises from about 1 pg to about 100 pg, from about 10 pg to about 50 pg, from about 20 pg to about 30 pg, from about 21 pg to about 29 pg, from about 22 pg to about 28 pg, from about 23 pg to about 27 pg, or from about 24 pg to about 26 pg of the lipopolysaccharide.44. The immunogenic composition of any one of embodiments 1-4, 7, 10-13, 16- 20, 25, 26, and 28-42, wherein the immunogenic composition comprises from about 1 pg to about 30 pg, from about 5 pg to about 15 pg, from about 6 pg to about 14 pg, from about 7 pg to about 13 pg, from about 8 pg to about 12 pg, or from about 9 pg to about 11 pg of the lipopolysaccharide.45. The immunogenic composition of any one of embodiments 1-4, 7, 10-13, 16- 20, 25, 26, and 28-42, wherein the immunogenic composition comprises from about 1 pg to about 9 pg, from about 2 pg to about 8 pg, from about 3 pg to about 7 pg, or from about 4 pg to about 6 pg of the lipopolysaccharide.46. The immunogenic composition of 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 Quillaja saponaria Molina.47. The immunogenic composition of 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 of 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 of 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 of any one of embodiments 1-4, 7, 10-13, 16- 20, 25, 26, and 28-49, wherein the immunogenic composition comprises from about 1 pg to about 100 pg, from about 10 pg to about 50 pg, from about 20 pg to about 30 pg, from about 21 pg to about 29 pg, from about 22 pg to about 28 pg, from about 23 pg to about 27 pg, or from about 24 pg to about 26 pg of the saponin.51. The immunogenic composition of any one of embodiments 1-4, 7, 10-13, 16- 20, 25, 26, and 28-49, wherein the immunogenic composition comprises from about 1 pg to about 30 pg, from about 5 pg to about 15 pg, from about 6 pg to about 14 pg, from about 7 pg to about 13 pg, from about 8 pg to about 12 pg, or from about 9 pg to about 11 pg of the saponin.52. The immunogenic composition of any one of embodiments 1-4, 7, 10-13, 16- 20, 25, 26, and 28-49, wherein the immunogenic composition comprises from about 1 pg to about 9 pg, from about 2 pg to about 8 pg, from about 3 pg to about 7 pg, or from about 4 pg to about 6 pg of the saponin.53. The immunogenic composition of 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 of embodiment 53, wherein the sterol is P- sitosterol, stigmasterol, ergosterol, ergocalciferol, or cholesterol.55. The immunogenic composition of embodiment 53 or embodiment 54, wherein the sterol is cholesterol.56. The immunogenic composition of any one of embodiments 53-55, wherein the ratio of saponin to sterol is from 1 : 1 to 1 : 100 w / w or from 1 : 1 to 1 :5 w / w, optionally about1 : 1 w / w.57. The immunogenic composition of any one of embodiments 1-4, 7, 10-13, 16- 20, 25, 26, and 28-56, wherein the liposome-based adjuvant further comprises a neutral lipid.58. The immunogenic composition of embodiment 57, wherein the neutral lipid is a phosphatidylcholine.59. The immunogenic composition of embodiment 58, wherein the phosphatidylcholine is eggyolk phosphatidylcholine, dioleoyl phosphatidylcholine (DOPC), or dilauryl phosphatidylcholine.60. The immunogenic composition of embodiment 58 or embodiment 59, wherein the phosphatidylcholine is DOPC.61. The immunogenic composition of any one of embodiments 1-4, 7, 10, 13-20, 25, 26, and 28-60, wherein the sorbitan ester is sorbitan ester 20.62. The immunogenic composition of any one of embodiments 1-4, 7, 10, 13-20, 25, 26, and 28-60, wherein the sorbitan ester is sorbitan ester 85.63. The immunogenic composition of any one of embodiments 1-4, 7, 10, 13-20, 25, 26, and 28-62, wherein the polyoxyethylene sorbitan ester is polyoxyethylene sorbitan ester 20.64. The immunogenic composition of any one of embodiments 1-4, 7, 10, 13-20, 25, 26, and 28-62, wherein the polyoxyethylene sorbitan ester is polyoxyethylene sorbitan ester 80.65. The immunogenic composition of any one of embodiments 1-4, 7, 10, 13-20, 25, 26, and 28-64, wherein the immunogenic composition comprises from about 2% w / v to about 7% w / v, from about 3% w / v to about 6% w / v, or from about 4% w / v to about 5% w / v of the squalene.66. The immunogenic composition of any one of embodiments 1-4, 7, 10, 13-20, 25, 26, and 28-65, wherein the immunogenic composition comprises from about 0.2% w / v to about 0.8% w / v, from about 0.3% w / v to about 0.7% w / v, or from about 0.4% w / v to about 0.6% w / v of the sorbitan ester.67. The immunogenic composition of any one of embodiments 1-4, 7, 10, 13-20, 25, 26, and 28-66, wherein the immunogenic composition comprises from about 0.2% w / v to about 0.8% w / v, from about 0.3% w / v to about 0.7% w / v, or from about 0.4% w / v to about 0.6% w / v of the polyoxyethylene sorbitan ester.68. The immunogenic composition of any one of embodiments 1-67, wherein the OspA antigen is two or more OspA antigens.69. The immunogenic composition of embodiment 68, wherein the two or more OspA antigens are a first OspA antigen and a second OspA antigen.70. The immunogenic composition of embodiments 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 of any one of embodiments 68-70, wherein each of the two or more OspA antigens have non-identical amino acid sequences.72. The immunogenic composition of any one of embodiments 1-71, wherein the OspA antigen is independently a . burgdorferi, B. afzelii, B. bavariensis, B. garinii, B. mayonii, B. lusitaniae, B. bissettii, B. valasiana, or B. spielmanii OspA antigen.73. The immunogenic composition of any one of embodiments 1-72, wherein the OspA antigen is independently a . burgdorferi OspA antigen.74. The immunogenic composition of any one of embodiments 1-73, wherein the OspA antigen independently comprises one or more epitopes from an OspA protein.75. The immunogenic composition of embodiment 74, wherein the one or more epitopes comprise at least 2, 3, 4, 5, or 6 epitopes from across one or more OspA proteins.76. The immunogenic composition of embodiment 74 or embodiment 75, wherein the one or more epitopes comprise at least 2, 3, 4, 5, or 6 epitopes from across at least 2, 3, 4, 5, or 6 OspA serotypes.77. The immunogenic composition of any one of embodiments 74-76, wherein the one or more epitopes comprise an epitope selected from the sequences of amino acids set forth in SEQ ID NO:4-49.78. The immunogenic composition of any one of embodiments 74-77, wherein the one or more epitopes are independently selected from the sequences of amino acids set forth in SEQ ID NO:4-49.79. The immunogenic composition of any one of embodiments 74-78, wherein the one of more epitopes comprise an epitope selected from the sequences of amino acids set forth in SEQ ID NO:9, 10, 33, 34, 36, 47, and 49.80. The immunogenic composition of 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 to the sequence of amino acids set forth in any of SEQ ID NO:50-62.81. The immunogenic composition of 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 contiguous amino acids of the sequence of amino acids set forth in any of SEQ ID NO:50-62.82. The immunogenic composition of any one of embodiments 1-78, 80, and 81, wherein the OspA antigen independently comprises the sequence of amino acids set forth in any of SEQ ID NO:50-62.83. The immunogenic composition of any one of embodiments 1-78, wherein the OspA antigen independently is an OspA chimeric antigen comprising a plurality of epitopes from one or more OspA proteins.84. The immunogenic composition of embodiment 83, wherein the plurality of epitopes comprise at least 2, 3, 4, 5, or 6 epitopes from across at least 2, 3, 4, 5, or 6 OspA serotypes.85. The immunogenic composition of embodiment 83 or embodiment 84, wherein the plurality of epitopes comprise an epitope selected from the sequences of amino acids set forth in SEQ ID NO:4-49.86. The immunogenic composition of any one of embodiments 83-85, wherein the plurality of epitopes are independently selected from the sequences of amino acids set forth in SEQ ID NO: 4-49.87. The immunogenic composition of any one of embodiments 83-86, wherein the plurality of epitopes comprise an epitope selected from the sequences of amino acids set forth in SEQ ID NO:9, 10, 33, 34, 36, 47, and 49.88. The immunogenic composition of any one of embodiments 83-87, wherein the plurality of epitopes comprise are independently selected from the sequences of amino acids set forth in SEQ ID NO:9, 10, 33, 34, 36, 47, and 49.89. The immunogenic composition of any one of embodiments 1-78 and 83-86, wherein the OspA antigen independently comprises a sequence of amino acids that 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 to the sequence of amino acids set forth in any one of SEQ ID NO:63-82.90. The immunogenic composition of 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 contiguous amino acids of the sequence of amino acids set forth in any one of SEQ ID NO:63-82.91. The immunogenic composition of any one of embodiments 1-78 and 83-90, wherein the OspA antigen independently comprises the sequence of amino acids set forth in any one of SEQ ID NO:63-82.92. The immunogenic composition of any one of embodiments 1-78 and 83-89, wherein the OspA antigen independently comprises a sequence of amino acids that 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 to the sequence of amino acids set forth in any one of SEQ ID NO:79-81.93. The immunogenic composition of any one of embodiments 1-78, 83-89, and 92, wherein the immunogenic composition comprises a first OspA antigen comprising a sequence of amino acids that 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 to the sequence of amino acids set forth in SEQ ID NO:79 and a second OspA antigen comprising a sequence of amino acids that 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 to the sequence of amino acids set forth in SEQ ID NO:80.94. The immunogenic composition of any one of embodiments 1-78, 83-89 and 91-93, wherein the immunogenic composition comprises a first OspA antigen comprising the sequence of amino acids set forth in SEQ ID NO:79 and a second OspA antigen comprising the sequence of amino acids set forth in SEQ ID NO:80.95. The immunogenic composition of any one of embodiments 1-78, 83-89, and 92, wherein the immunogenic composition comprises a first OspA antigen comprising a sequence of amino acids that 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 to the sequence of amino acids set forth in SEQ ID NO:79 and a second OspA antigen comprising a sequence of amino acids that 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 to the sequence of amino acids set forth in SEQ ID NO:81.96. The immunogenic composition of any one of embodiments 1-78, 83-89, 91, 92, and 95, wherein the immunogenic composition comprises a first OspA antigencomprising the sequence of amino acids set forth in SEQ ID NO:79 and a second OspA antigen comprising the sequence of amino acids set forth in SEQ ID NO:81.97. The immunogenic composition of any one of embodiments 1-78, 83-89, and 92, wherein the immunogenic composition comprises a first OspA antigen comprising a sequence of amino acids that 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 to the sequence of amino acids set forth in SEQ ID NO:80 and a second OspA antigen comprising a sequence of amino acids that 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 to the sequence of amino acids set forth in SEQ ID NO:81.98. The immunogenic composition of any one of embodiments 1-78, 83-89, 91, 92, and 97, wherein the immunogenic composition comprises a first OspA antigen comprising the sequence of amino acids set forth in SEQ ID NO:80 and a second OspA antigen comprising the sequence of amino acids set forth in SEQ ID NO:81.99. The immunogenic composition of any one of embodiments 1-78, 83-89, 92, and 93, wherein the immunogenic composition comprises a first OspA antigen comprising a sequence of amino acids that 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 to the sequence of amino acids set forth in SEQ ID NO:79, a second OspA antigen comprising a sequence of amino acids that 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 to the sequence of amino acids set forth in SEQ ID NO:80, and a third OspA antigen comprising a sequence of amino acids that 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 to the sequence of amino acids set forth in SEQ ID NO:81.100. The immunogenic composition of any one of embodiments 1-78, 83-89, and 91-94, wherein the immunogeneic composition comprises a first OspA antigen comprising the sequence of amino acids set forth in SEQ ID NO:79, a second OspA antigen comprising the sequence of amino acids set forth in SEQ ID NO:80, and a third OspA antigen comprising the sequence of amino acids set forth in SEQ ID NO:81.101. The immunogenic composition of any one of embodiments 1-100, wherein the OspA antigen independently is non-lipidated.102. The immunogenic composition of any one of embodiments 1-101, wherein the OspA antigen independently is lipidated.103. The immunogenic composition of any one of embodiments 1-102, wherein the immunogenic composition is a 0.3 mL to 1.5 mL dose.104. The immunogenic composition of any one of embodiments 1-103, wherein the immunogenic composition is for stimulating an immune response against Borrelia in a subject by administering to the subject an effective amount of the immunogenic composition.105. The immunogenic composition of any one of embodiments 1-104, wherein the immunogenic composition is for protecting a subject from infection by Borrelia by administering to the subject an effective amount of the immunogenic composition.106. The immunogenic composition of any one of embodiments 1-104, wherein the immunogenic composition is for preventing a subject from contracting Lyme disease by administering to the subject an effective amount of the immunogenic composition.107. The immunogenic composition of any one of embodiments 1-104, wherein the subject a human subject.108. The immunogenic composition of embodiment 107, wherein the human subject is at least 18 years of age.109. The immunogenic composition of embodiment 107 or embodiment 108, wherein the human subject is from 18 years to 70 years of age.110. The immunogenic composition of embodiment 107, wherein the human subject is under 18 years of age.111. The immunogenic composition of embodiment 107 or embodiment 110, wherein the human subject is from 2 years to 5 years of age.112. The immunogenic composition of embodiment 107 or embodiment 110, wherein the human subject is from 5 years to 18 years of age.113. A method for stimulating an immune response against Borrelia in a subject, comprising administering to a subject an effective amount of the immunogenic composition of any one of embodiments 1-112.114. A method for protecting a subject from infection by Borrelia, comprising administering to a subject an effective amount of the immunogenic composition of any one of embodiments 1-112.115. A method for preventing a subject from contracting Lyme disease, comprising administering to a subject an effective amount of the immunogenic composition of any one of embodiments 1-112.116. The method of any one of embodiments 113-115, wherein the immunogenic composition is administered by intramuscular injection.117. The method of any one of embodiments 113-116, wherein a first dose and a second dose of the immunogenic composition is administered to the subject.118. The method of embodiment 117, wherein the second dose of the immunogenic composition is administered from about 2 weeks to about 10 weeks after the administration of the first dose of the immunogenic composition.119. The method of 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 of any one of embodiments 117-119, wherein a third dose of the immunogenic composition is administered to the subject.121. The method of embodiment 120, wherein the third dose of the immunogenic composition is administered from about 4 months to about 8 months after the administration of the first dose of the immunogenic composition.122. The method of embodiment 120 or embodiment 121, wherein the third dose of the immunogenic composition is administered about 6 months after the administration of the first dose of the immunogenic composition.123. The method of any one of embodiments 113-122, wherein a booster dose of the immunogenic composition is administered to the subject.124. The method of embodiment 123, wherein the booster dose is administered at least about 1 year or at least about 2 years after the administration of the first dose.125. The method of any one of embodiments 113-124, wherein a plurality of booster doses is administered to the subject.126. The method of 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 of embodiment 125 or embodiment 126, wherein a booster dose is administered yearly.128. The method of any one of embodiments 113-127, wherein the subject is a human subject.129. The method of embodiment 128, wherein the human subject is at least 18 years of age.130. The method of embodiment 128 or embodiment 129, wherein the human subject is from 18 years to 70 years of age.131. The method of embodiment 128, wherein the human subject is under 18 years of age.132. The method of embodiment 128 or embodiment 131, wherein the human subject is from 2 years to 5 years of age.133. The method of embodiment 128 or embodiment 131, wherein the human subject is from 5 years to 18 years of age.134. Use of the immunogenic composition of any one of embodiments 1-112 for stimulating an immune response against Borrelia in a subject by administering to the subject an effective amount of the immunogenic composition.135. Use of the immunogenic composition of any one of embodiments 1-112 for protecting a subject from infection by Borrelia by administering to the subject an effective amount of the immunogenic composition.136. Use of the immunogenic composition of any one of embodiments 1-112 for preventing a subject from contracting Lyme disease by administering to the subject an effective amount of the immunogenic composition.137. Use of the immunogenic composition of any one of embodiments 1-112 in the manufacture of a medicament for stimulating an immune response against Borrelia in a subject.138. Use of the immunogenic composition of any one of embodiments 1-112 in the manufacture of a medicament for protecting a subject from infection by Borrelia.139. Use of the immunogenic composition of any one of embodiments 1-112 in the manufacture of a medicament for preventing a subject from contracting Lyme disease.140. The use of any one of embodiments 134-139, wherein the immunogenic composition is administered by intramuscular injection.141. The use of any one of embodiments 134-140, wherein a first dose and a second dose of the immunogenic composition is administered to the subject.142. The use of embodiment 141, wherein the second dose of the immunogenic composition is administered from about 2 weeks to about 10 weeks after the administration of the first dose of the immunogenic composition.143. The use of 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 of any one of embodiments 141-143, wherein a third dose of the immunogenic composition is administered to the subject.145. The use of embodiment 144, wherein the third dose of the immunogenic composition is administered from about 4 months to about 8 months after the administration of the first dose of the immunogenic composition.146. The use of embodiment 144 or embodiment 145, wherein the third dose of the immunogenic composition is administered about 6 months after the administration of the first dose of the immunogenic composition.147. The use of any one of embodiments 134-146, wherein a booster dose of the immunogenic composition is administered to the subject.148. The use of embodiment 147, wherein the booster dose is administered at least about 1 year or at least about 2 years after the administration of the first dose.149. The use of any one of embodiments 134-148, wherein a plurality of booster doses is administered to the subject.150. The use of 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 of embodiment 149 or embodiment 150, wherein a booster dose is administered yearly.152. The use of any one of embodiments 133-151, wherein the subject is a human subject.153. The use of embodiment 152, wherein the human subject is at least 18 years of age.154. The use of embodiment 152 or embodiment 153, wherein the human subject is from 18 years to 70 years of age.155. The use of embodiment 152, wherein the human subject is under 18 years of age.156. The use of embodiment 152 or embodiment 155, wherein the human subject is from 2 years to 5 years of age.157. The use of embodiment 152 or embodiment 155, wherein the human subject is from 5 years to 18 years of age.158. A vial comprising the immunogenic composition of any one of embodiments 1-112.159. The vial of embodiment 158, wherein the immunogenic composition is in liquid form.160. The vial of embodiment 158, wherein the immunogenic composition is in lyophilized form.161. The vial of any one of embodiments 158-160, wherein the vial comprises a single dose of the immunogenic composition.162. The vial of embodiment 161, wherein the single dose is a 0.3 mL to 1.5 mL dose.163. A pre-filled syringe comprising the immunogenic composition of any one of embodiments 1-112.164. The pre-filled syringe of embodiment 163, wherein the syringe comprises a single dose of the immunogenic composition.165. The pre-filled syringe of embodiment 164, wherein the single dose is a 0.3 mL to 1.5 mL dose.166. A kit compri sing : a) a first composition comprising an adjuvant selected from an aluminum salt adjuvant; a toll-like receptor 9 (TLR9) agonist; a liposome-based adjuvant comprising a lipopolysaccharide and a saponin; and an oil-in-water emulsion-based adjuvant comprising squalene, a sorbitan ester, and a polyoxyethylene sorbitan ester; b) a second composition comprising a Borrelia outer surface protein A (OspA) antigen; and c) instructions for combining the first composition and the second composition to prepare the immunogenic composition of any one of embodiments 1-112.167. The kit of embodiment 166, wherein the second composition is in liquid form.168. The kit of embodiment 166, wherein the second composition is in lyophilized form.169. The kit of any one of embodiments 166-168, wherein the instructions are for preparing a single dose of the immunogenic composition.170. The kit of any one of embodiments 166-169, wherein the kit is for a single dose of the immunogenic composition.171. The kit of embodiment 169 or embodiment 170, wherein the single dose is a 0.3 mL to 1.5 mL dose.172. The kit of any one of embodiments 166-171, wherein the first and / or second composition comprises a second adjuvant.173. The kit of embodiment 172, wherein the second adjuvant is an aluminum salt adjuvant.174. The kit of 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 of embodiment 173 or embodiment 174, wherein the aluminum salt adjuvant comprises aluminum hydroxide.176. The kit of any one of embodiments 166-175, further comprising: d) a further set of instructions for administration of the immunogenic composition to a subject to stimulate an immune response against Borrelia in the subject.177. A kit compri sing : a) the immunogenic composition of any one of embodiments 1-112; and b) instructions for administration of the immunogenic composition to a subject to stimulate an immune response against Borrelia in the subject.178. The kit of embodiment 176 or embodiment 177, further comprising a syringe for intramuscular injection of the immunogenic composition.179. The kit of embodiment 178, wherein the syringe is pre-filled with the immunogenic composition.180. A kit comprising: a) the vial of any one of embodiments 158-162; and b) instructions for administration of the immunogenic composition to a subject to stimulate an immune response against Borrelia in the subject.181. The kit of embodiment 180, further comprising a syringe for intramuscular injection of the immunogenic composition.182. A kit compri sing : a) the pre-filled syringe of any one of embodiments 163-165; and b) instructions for administration of the immunogenic composition to a subject to stimulate an immune response against Borrelia in the subject.183. The kit of embodiment 182, wherein the syringe is for intramuscular injection of the immunogenic composition.184. The kit of any one of embodiments 176-183, wherein the administration is according to the method of any one of embodiments 124-144.185. The kit of any one of embodiments 176-184, wherein the subject is a human subject.186. The kit of embodiment 185, wherein the human subject is at least 18 years of age.187. The kit of embodiment 185 or embodiment 186, wherein the human subject is from 18 years to 70 years of age.188. The kit of embodiment 185, wherein the human subject is under 18 years of age.189. The kit of embodiment 185 or embodiment 188, wherein the human subject is from 2 years to 5 years of age.190. The kit of embodiment 185 or embodiment 188, wherein the human subject is from 5 years to 18 years of age.EXAMPLESEXAMPLE 1Effects of Adjuvant on Immune Response to Lyme Disease Immunogenic Composition

[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 afzelii. OspA serotype 3, 5, and 6 are derived from Borrelia garinii. OspA serotype 4 is derived from Borrelia bavariensis.

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

[0314] Three different antigens or mixture of antigens were tested in the immunogenic compositions: (a) a non-lipidated full-length version of the OspA Serotype 1 antigen (Borrelia Burgdorferi B31 strain) (OspA SI) as set forth in SEQ ID NO: 50, (b) a mixture of non-lipidated OspA chimeric antigens containing three chimeric OspA antigens covering 6 serotypes (OspA chimeric antigens), and (c) lipidated OspA chimeric antigens that are the same as the three chimeric OspA chimeric antigens described in (b) but are further lipidated. The OspA chimeric antigens in the mixture of (b) above are schematized in the bottom of FIG. 1, and contain three different chimeric OspA antigens, each of which combines two different serotypes from OspA. The first chimeric antigen (Sl / 2), which covers part of the C- terminal portion of OspA serotype 2 and part of the N-terminal portion of OspA serotype 1, is- n -set forth in SEQ ID NO: 79. The second chimeric antigen (S6 / 4), which covers part of the C- terminal portion of OspA serotype 4 and part of the N-terminal portion of OspA serotype 6, is set forth in SEQ ID NO: 80. The third chimeric antigen (S5 / 3), which covers part of the C- terminal portion of OspA serotype 3 and part of the N-terminal portion of OspA serotype 5, is set forth in SEQ ID NO: 81. For the lipidated versions of the chimeric antigens of (c) above, the three aforementioned chimeric antigens included additional fatty acids and glycerol appended to the N-terminus of each of the OspA chimeric antigens. In addition, a His tag as set forth in SEQ ID NO: 87 was appended to the C-terminus of each antigen. For example, the OspA SI antigen with the appended His tag is set forth in SEQ ID NO: 86.

[0315] Female C3H / HeN mice were divided across twelve treatment groups containing eight animals each, each of which received one of twelve immunogenic compositions with a given OspA antigen or mixture of OspA antigens and adjuvant formulation as set forth in Table El below. Each type of antigen or mixture of antigens contained either no adjuvant, alum alone, CpG alone, or both CpG and alum. The immunogenic compositions adjuvanted with alum alone contained antigens adsorbed to 50 micrograms of aluminum hydroxide (Alhydrogel 2%, Al(0H)3). The immunogenic compositions adjuvanted with CpG alone contained antigens mixed with 10 micrograms of CpG. The immunogenic compositions adjuvanted with alum and CpG contained antigens adsorbed to 50 microgram of alum and subsequently mixed with 10 micrograms of CpG. All antigens and adjuvants were diluted in sterile saline.Table El: Study Design

[0316] Female mice were injected intramuscularly at Day 0 and on Day 21. Blood was drawn on Day 21 and Day 35 for measurement of serum antibody level by ELISA. Responses were measured for each mouse. The antibody endpoint titer was defined as the reciprocal of the highest dilution from neat serum needed to achieve 0.5 OD. Positive controls for each ELISA plate were included to control inter-plate variation. Positive controls included defined antibody levels of mouse serum samples from a previous study. Negative controls included serum from unvaccinated mice.

[0317] Antibody levels in Day 35 serum, i.e., post-2ndinjection, from mice administered immunogenic compositions containing non-lipidated (non-lip) OspA chimeric antigens were measured against OspA S5 / 3 (FIG. 2A), OspA S6 / 4 (FIG. 2B), OspA Sl / 2 (FIG. 2C; left side), or full-length OspA SI (FIG. 2D; left side). Antibody levels in Day 35 serum from mice administered immunogenic compositions containing non-lip OspA SI were measured against either OspA Sl / 2 (FIG. 2C; right side) or full-length OspA SI (FIG. 2D; right side). Finally, antibody levels in Day 35 serum from mice administered immunogenic compositions containing lipidated OspA chimeric antigens were measured against OspA Sl / 2 (FIG. 2E), OspA S5 / 3 (FIG. 2F), or OspA S6 / 4 (FIG. 2G). For each Figure, fold-change increases (FI) in antibody levels between the two indicated conditions, as measured by comparing the median antibody levels from one indicated condition versus the other indicated condition, are also shown. In addition, for FIGS. 2E-2G, median antibody levels are also shown above each condition.

[0318] For mice administered immunogenic compositions containing non-lipidated OspA antigen(s) in FIGS. 2A-2D, the use of any adjuvant, including alum, CpG, and CpG + alum, induced higher OspA antibody titers as compared to use without adjuvants for each OspA antigen tested against. Further, the use of a combination of CpG + alum formulated with any non-lipidated OspA antigen(s) led to the highest OspA titer for each OspA antigen tested against, with a FI between 2.8-4.1 greater as compared to use of alum alone. In addition, as shown in FIG. 2D, the use of either the OspA chimeric antigens or full-length OspA SI led to similar antibody responses against full-length OspA SI, indicating that both the OspA chimeric antigens and SI are similarly immunogenic to full-length OspA SI and thatantibodies against OspA Sl / 2 can recognize the main SI epitopes present on the full length OspA SI. In other words, the OspA Sl / 2 chimeric antigen retains the immunogenicity of the full-length OspA SI antigen.

[0319] In contrast, as shown in FIGS. 2E-2G, for mice administered an immunogenic composition containing lipidated OspA chimeric antigens, use of alum resulted in only a limited increase of OspA antibody titers as compared to use without adjuvants. However, use of either CpG or CpG + alum significantly enhanced OspA antibody titers as compared to no adjuvant or alum alone.

[0320] Taken together, these results indicate that both the non -lipidated and lipidated OspA chimeric antigens are immunogenic to OspA. Further, the use of an adjuvant formulation containing CpG, including with or without alum, enhanced the OspA antibody response against several OspA antigens when combined with several different OspA antigens or mixture of OspA antigens.

[0321] In addition to assessing antibody titer following the second injection, antibody response kinetics between the 1st and 2nd injections in serum from mice treated with lipidated OspA chimeric antigens were also assessed. FIGS. 3A-3C show antibody titers against OspA Sl / 2, OspA S5 / 3, and OspA S6 / 4, respectively, for each lipidated OspA formulation tested, in serum from Day 21 after the first injection (1) and from Day 35 after the second injection (2). Fold-change increases (FI) in antibody values, calculated by comparing the median antibody levels induced by a given composition at Day 35 versus the median antibody levels induced by the same composition at Day 21, are also shown above each condition. The median antibody titer values are also shown in Table E2.Table E2: Antibody Titer Values

[0322] The results indicate that the first dose of immunogenic composition, when formulated with both CpG and alum, resulted in the greatest antibody titer as compared to when formulated with no adjuvant, alum alone, or CpG alone. Thus, the combination of OspA antigens, CpG, and alum has a synergistic effect leading to a greater magnitude in immune response (i.e., following a first dose prior to receiving a second dose). Further, the second dose of immunogenic composition, when formulated with both CpG and alum, increases OspA antibody titers from between 5.6-8.8 fold depending on serotype tested.

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

[0324] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced. Therefore, the examples should not be construed as limiting the scope of the disclosure, which is delineated by the appended claims.SEQUENCES

Claims

CLAIMS1. An immunogenic composition, comprising (i) a Borrelia outer surface protein A (OspA) antigen or an RNA polynucleotide encoding the OspA antigen and (ii) an adjuvant selected from the group consisting of an aluminum salt adjuvant; a toll-like receptor 9 (TLR9) agonist; a liposome-based adjuvant comprising a lipopolysaccharide and a saponin; and an oil-in-water emulsion comprising squalene, a sorbitan ester, and a polyoxyethylene sorbitan ester.

2. An immunogenic composition, comprising (i) a Borrelia outer surface protein A (OspA) antigen and (ii) an adjuvant selected from the group consisting of an aluminum salt adjuvant; a toll-like receptor 9 (TLR9) agonist; a liposome-based adjuvant comprising a lipopolysaccharide and a saponin; and an oil-in-water emulsion comprising squalene, a sorbitan ester, and a polyoxyethylene sorbitan ester.

3. The immunogenic composition of claim 1 or claim 2, wherein the immunogenic composition does not comprise an RNA polynucleotide encoding a non-OspA Borrelia outer surface protein antigen.

4. The immunogenic composition of any one of claims 1-3, wherein the immunogenic composition does not comprise a non-OspA Borrelia outer surface protein antigen.

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

6. The immunogenic composition of claim 5, further comprising a second adjuvant.

7. The immunogenic composition of claim 6, wherein the second adjuvant is selected from a TLR9 agonist; a liposome-based adjuvant comprising a lipopolysaccharide and a saponin; and an oil-in-water emulsion comprising squalene, a sorbitan ester, and a polyoxyethylene sorbitan ester.

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

9. The immunogenic composition of claim 8, further comprising a second adjuvant.

10. The immunogenic composition of claim 9, wherein the second adjuvant is selected from an aluminum salt adjuvant; a liposome-based adjuvant comprising a lipopolysaccharide and a saponin; and an oil-in-water emulsion-based adjuvant comprising squalene, a sorbitan ester, and a polyoxyethylene sorbitan ester.

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

12. The immunogenic composition of claim 11, further comprising a second adjuvant.

13. The immunogenic composition of claim 12, wherein the second adjuvant is selected from an aluminum salt adjuvant; a TLR9 agonist; and an oil-in-water emulsionbased adjuvant comprising squalene, a sorbitan ester, and a polyoxyethylene sorbitan ester.

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

15. The immunogenic composition of claim 14, further comprising a second adjuvant.

16. The immunogenic composition of claim 15, wherein the second adjuvant is selected from an aluminum salt adjuvant; a TLR9 agonist; and a liposome-based adjuvant comprising a lipopolysaccharide and a saponin.

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

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

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

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

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

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

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

24. The immunogenic composition of any one of claims 21-23, further comprising a second adjuvant.

25. The immunogenic composition of claim 24, wherein the second adjuvant is selected from an aluminum salt adjuvant; a liposome-based adjuvant comprising a lipopolysaccharide and a saponin; and an oil-in-water emulsion-based adjuvant comprising squalene, a sorbitan ester, and a polyoxyethylene sorbitan ester.

26. The immunogenic composition of 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) Borrelia outer surface protein A (OspA) antigen, (ii) the toll-like receptor 9 (TLR9) agonist oligonucleotide 5’- TGACTGTGAA CGTTCGAGAT GA-3’(SEQ ID NO: 1), the oligonucleotide comprising an unmethylated cytidine-phospho-guanosine (CpG) motif, and (iii) an aluminum salt adjuvant.

28. The immunogenic composition of 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 of any one of claims 1-7, 10, 13, 16-20, and25-28, wherein the aluminum salt adjuvant comprises aluminum hydroxide.

30. The immunogenic composition of any one of claims 1-29, wherein the immunogenic composition comprises from about 0.25 to about 1.25 mg Al3+, from about 0.25 mg to about 0.50 mg Al3+, or from about 0.30 mg to about 0.40 mg Al3+.

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

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

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

34. The immunogenic composition of any one of claims 17-33, wherein the oligonucleotide comprises at least one phosphorothioate linkage.

35. The immunogenic composition of any one of claims 17-34, wherein the oligonucleotide comprises only phosphorothioate linkages.

36. The immunogenic composition of any one of claims 17-34, wherein the oligonucleotide comprises a combination of one or more phosphodiester linkages and one or more phosphorothioate linkages.

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

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

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

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

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

42. The immunogenic composition of any one of claims 1-4, 7, 10-13, 16-20, 25, 26, and 28-40, wherein the lipopolysaccharide is 3-deacyl-phosphorylated hexa-acyl di saccharide.

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

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

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

46. The immunogenic composition of 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 Quillaja saponaria Molina.

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

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

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

53. The immunogenic composition of 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 of claim 53, wherein the sterol is P-sitosterol, stigmasterol, ergosterol, ergocalciferol, or cholesterol.

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

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

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

58. The immunogenic composition of claim 57, wherein the neutral lipid is a phosphatidylcholine.

59. The immunogenic composition of claim 58, wherein the phosphatidylcholine is eggyolk phosphatidylcholine, dioleoyl phosphatidylcholine (DOPC), or dilauryl phosphatidylcholine.

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

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

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

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

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

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

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

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

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

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

70. The immunogenic composition of 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 of any one of claims 68-70, wherein each of the two or more OspA antigens have non-identical amino acid sequences.

72. The immunogenic composition of any one of claims 1-71, wherein the OspA antigen is independently a . burgdorferi, B. afzelii, B. bavariensis, B. garinii, B. mayonii, B. lusitaniae, B. bissettii, B. valasiana, or B. spielmanii OspA antigen.

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

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

75. The immunogenic composition of claim 74, wherein the one or more epitopes comprise at least 2, 3, 4, 5, or 6 epitopes from across one or more OspA proteins.

76. The immunogenic composition of claim 74 or claim 75, wherein the one or more epitopes comprise at least 2, 3, 4, 5, or 6 epitopes from across at least 2, 3, 4, 5, or 6 OspA serotypes.

77. The immunogenic composition of any one of claims 74-76, wherein the one or more epitopes comprise an epitope selected from the sequences of amino acids set forth in SEQ ID NO:4-49.

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

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

80. The immunogenic composition of 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 to the sequence of amino acids set forth in any of SEQ ID NO:50-62.

81. The immunogenic composition of 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 contiguous amino acids of the sequence of amino acids set forth in any of SEQ ID NO:50-62.

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

83. The immunogenic composition of any one of claims 1-78, wherein the OspA antigen independently is 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 comprise at least 2, 3, 4, 5, or 6 epitopes from across at least 2, 3, 4, 5, or 6 OspA serotypes.

85. The immunogenic composition of claim 83 or claim 84, wherein the plurality of epitopes comprise an epitope selected from the sequences of amino acids set forth in SEQ ID NO:4-49.

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

87. The immunogenic composition of any one of claims 83-86, wherein the plurality of epitopes comprise an epitope selected from the sequences of amino acids set forth in SEQ ID NO:9, 10, 33, 34, 36, 47, and 49.

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

89. The immunogenic composition of any one of claims 1-78 and 83-86, wherein the OspA antigen independently comprises a sequence of amino acids that 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 to the sequence of amino acids set forth in any one of SEQ ID NO:63-82.

90. The immunogenic composition of 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 contiguous amino acids of the sequence of amino acids set forth in any one of SEQ ID NO:63-82.

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

92. The immunogenic composition of any one of claims 1-78 and 83-89, wherein the OspA antigen independently comprises a sequence of amino acids that 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 to the sequence of amino acids set forth in any one of SEQ ID NO: 79-81.

93. The immunogenic composition of any one of claims 1-78, 83-89, and 92, wherein the immunogenic composition comprises a first OspA antigen comprising a sequence of amino acids that 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 to the sequence of amino acids set forth in SEQ ID NO:79 and a second OspA antigen comprising a sequence of amino acids that 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 to the sequence of amino acids set forth in SEQ ID NO:80.

94. The immunogenic composition of any one of claims 1-78, 83-89 and 91-93, wherein the immunogenic composition comprises a first OspA antigen comprising the sequence of amino acids set forth in SEQ ID NO:79 and a second OspA antigen comprising the sequence of amino acids set forth in SEQ ID NO:80.

95. The immunogenic composition of any one of claims 1-78, 83-89, and 92, wherein the immunogenic composition comprises a first OspA antigen comprising a sequence of amino acids that 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 to the sequence of amino acids set forth in SEQ ID NO:79 and a second OspA antigen comprising a sequence of amino acids that 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 to the sequence of amino acids set forth in SEQ ID NO:81.

96. The immunogenic composition of any one of claims 1-78, 83-89, 91, 92, and 95, wherein the immunogenic composition comprises a first OspA antigen comprising the sequence of amino acids set forth in SEQ ID NO:79 and a second OspA antigen comprising the sequence of amino acids set forth in SEQ ID NO:81.

97. The immunogenic composition of any one of claims 1-78, 83-89, and 92, wherein the immunogenic composition comprises a first OspA antigen comprising a sequence of amino acids that 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 to the sequence of amino acids set forth in SEQ ID NO:80 and a second OspA antigen comprising a sequence of amino acids that 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 to the sequence of amino acids set forth in SEQ ID NO:81.

98. The immunogenic composition of any one of claims 1-78, 83-89, 91, 92, and 97, wherein the immunogenic composition comprises a first OspA antigen comprising the sequence of amino acids set forth in SEQ ID NO:80 and a second OspA antigen comprising the sequence of amino acids set forth in SEQ ID NO:81.

99. The immunogenic composition of any one of claims 1-78, 83-89, 92, and 93, wherein the immunogenic composition comprises a first OspA antigen comprising a sequence of amino acids that 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 to the sequence of amino acids set forth in SEQ ID NO:79, a second OspA antigen comprising a sequence of amino acids that 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 to thesequence of amino acids set forth in SEQ ID NO:80, and a third OspA antigen comprising a sequence of amino acids that 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 to the sequence of amino acids set forth in SEQ ID NO:81.

100. The immunogenic composition of any one of claims 1-78, 83-89, and 91-94, wherein the immunogeneic composition comprises a first OspA antigen comprising the sequence of amino acids set forth in SEQ ID NO:79, a second OspA antigen comprising the sequence of amino acids set forth in SEQ ID NO:80, and a third OspA antigen comprising the sequence of amino acids set forth in SEQ ID NO:81.

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

102. The immunogenic composition of any one of claims 1-101, wherein the OspA antigen independently is lipidated.

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

104. The immunogenic composition of any one of claims 1-103, wherein the immunogenic composition is for stimulating an immune response against Borrelia in a subject by administering to the subject an effective amount of the immunogenic composition.

105. The immunogenic composition of any one of claims 1-104, wherein the immunogenic composition is for protecting a subject from infection by Borrelia by administering to the subject an effective amount of the immunogenic composition.

106. The immunogenic composition of any one of claims 1-104, wherein the immunogenic composition is for preventing a subject from contracting Lyme disease by administering to the subject an effective amount of the immunogenic composition.

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

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

109. The immunogenic composition of claim 107 or claim 108, wherein the human subject is from 18 years to 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 of claim 107 or claim 110, wherein the human subject is from 2 years to 5 years of age.

112. The immunogenic composition of claim 107 or claim 110, wherein the human subject is from 5 years to 18 years of age.

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

114. A method for protecting a subject from infection by Borrelia, comprising administering to a subject an effective amount of the immunogenic composition of any one of claims 1-112.

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

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

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

118. The method of claim 117, wherein the second dose of the immunogenic composition is administered from about 2 weeks to about 10 weeks after the 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 1 month or about 2 months after the administration of the first dose of the immunogenic composition.

120. The method of 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 from about 4 months to about 8 months after the 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 about 6 months after the administration of the first dose of the immunogenic composition.

123. The method of 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 booster dose is administered at least about 1 year or at least about 2 years after the administration of the first dose.

125. The method of any one of claims 113-124, wherein a plurality of booster doses is 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 yearly.

128. The method of 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 of age.

130. The method of claim 128 or claim 129, wherein the human subject is from 18 years to 70 years of age.

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 from 2 years to 5 years of age.

133. The method of claim 128 or claim 131, wherein the human subject is from 5 years to 18 years of age.

134. Use of the immunogenic composition of any one of claims 1-112 for stimulating an immune response against Borrelia in a subject by administering to the subject an effective amount of the immunogenic composition.

135. Use of the immunogenic composition of any one of claims 1-112 for protecting a subject from infection by Borrelia by administering to the subject an effective amount of the immunogenic composition.

136. Use of the immunogenic composition of any one of claims 1-112 for preventing a subject from contracting Lyme disease by administering to the subject an effective amount of the immunogenic composition.

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

138. Use of the immunogenic composition of any one of claims 1-112 in the manufacture of a medicament for protecting a subject from infection by Borrelia.

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

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

141. The use of any one of claims 134-140, wherein a first dose and a second dose of the immunogenic composition is administered to the subject.

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

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

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

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

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

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

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

150. The use of 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 of claim 149 or claim 150, wherein a booster dose is administered yearly.

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

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

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

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

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

157. The use of claim 152 or claim 155, wherein the human subject is from 5 years to 18 years of age.

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

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

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

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

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

163. A pre-filled syringe comprising the immunogenic composition of 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 0.3 mL to 1.5 mL dose.

166. A kit compri sing : a) a first composition comprising an adjuvant selected from an aluminum salt adjuvant; a toll-like receptor 9 (TLR9) agonist; a liposome-based adjuvant comprising a lipopolysaccharide and a saponin; and an oil-in-water emulsion-based adjuvant comprising squalene, a sorbitan ester, and a polyoxyethylene sorbitan ester; b) a second composition comprising a Borrelia outer surface protein A (OspA) antigen; and c) instructions for combining the first composition and the second composition to prepare the immunogenic composition of any one of claims 1-112.

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

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

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

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

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

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

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

174. The kit of 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 of claim 173 or claim 174, wherein the aluminum salt adjuvant comprises aluminum hydroxide.

176. The kit of any one of claims 166-175, further comprising: d) a further set of instructions for administration of the immunogenic composition to a subject to stimulate an immune response against Borrelia in the subject.

177. A kit compri sing : a) the immunogenic composition of any one of claims 1-112; and b) instructions for administration of the immunogenic composition to a subject to stimulate an immune response against Borrelia in the subject.

178. The kit of claim 176 or claim 177, further comprising 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 of any one of claims 158-162; and b) instructions for administration of the immunogenic composition to a subject to stimulate an immune response against Borrelia in the subject.

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

182. A kit compri sing : a) the pre-filled syringe of any one of claims 163-165; and b) instructions for administration of the immunogenic composition to a subject to stimulate an immune response against Borrelia in the subject.

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

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

185. The kit of 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 of age.

187. The kit of claim 185 or claim 186, wherein the human subject is from 18 years to 70 years of age.

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

189. The kit of claim 185 or claim 188, wherein the human subject is from 2 years to 5 years of age.

190. The kit of claim 185 or claim 188, wherein the human subject is from 5 years to 18 years of age.

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