Compositions and methods for treating and / or preventing LYME disease

Abstract

Disclosed herein are compositions and methods for treating, and for the preventative treatment of Lyme disease. The compositions can include a conjugate vaccine polypeptide.

Description

COMPOSITIONS AND METHODS FOR TREATING AND / OR PREVENTING LYME DISEASECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 574,550, filed on April 4, 2024, the contents of which are herein incorporated by reference in their entireties.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under R21AI148982 awarded by the National Institute of Allergy and Infectious Disease and under W81XWH2210728 awarded by the Department of Defense. The government has certain rights in the invention.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The content of the electronic sequence listing (65005301 167. xml; Size: 51,257 bytes; and Date of Creation: March 14, 2025) is herein incorporated by reference in its entirety.FIELD

[0004] This invention relates to compositions and methods for treating and / or preventing Lyme disease. In particular, this invention relates to conjugate polypeptides comprising a T cell epitope derived from aBorrelia methyl-accepting chemotaxis protein (Mcp), compositions comprising the conjugate polypeptides, and methods of treatment using the conjugate polypeptides.BACKGROUND

[0005] Lyme disease is the most common vector-bome disease in the United States. Lyme disease is caused by the bacterium Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia bctvariensis, Borrelia mayonii and others. It is transmitted to humans through the bite of infected blacklegged ixodes ticks. Typical symptoms include fever, headache, fatigue, and a characteristic skin rash called erythema migrans. If left untreated, infection can spread to joints, the heart, and the nervous system. Lyme disease is diagnosed based on symptoms, physical findings (e.g.. rash), and the possibility of exposure to infected ticks.SUMMARY

[0006] In one aspect, a polypeptide is provided. The polypeptide can include a first portion comprising or consisting of at least one first amino acid sequence that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 1-5; and a second portion comprising or consisting of at least one second amino acid sequence that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 6-11.

[0007] In another aspect, a composition is provided. The composition can include one or more of the polypeptides disclosed herein.

[0008] In yet another aspect, a method for treating Lyme disease or for the preventative treatment of Lyme disease is provided. The method can include administering to a subject one or more of the polypeptides and / or compositions disclosed herein.

[0009] In another aspect, a carrier protein for a vaccine is provided. The carrier protein can comprise or consist of at least one first amino acid sequence that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 1-5.

[0010] In still another aspect, a polypeptide for a vaccine is provided. The polypeptide can comprise a carrier polypeptide portion comprising or consisting of at least one first amino acid sequence that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 1-5, covalently linked to another polypeptide portion.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIGS. 1A-1E. Identification and validation of Mcpl and Mcp4 Cd4+ T cell epitopes. (FIG. 1A) An immunopeptidomics approach was previously used to isolate and identify MHC class Il-bound peptides (CD4+ T cell epitopes) derived from the Lyme disease spirochete Borrelia burgdorferi (Bb) using liquid chromatography and tandem mass spectrometry (LC-MS / MS). (FIG. IB) Two peptides previously identified using this method were derived from Mcpl (amino acids 49-66) and Mcp4 (amino acids 442-462). Both were within the cytosolic methyl-accepting transducer domain (MATD). With the exception of Mcpl, other Mcp proteins from Borrelia burgdorferi contain a C-terminal MATD, two transmembrane alpha helices, and a periplasmic ligand-binding domain. (FIGS. 1C-1D) To validate the predicted peptide (FIG. 1C), synthesized peptides (FIG. ID) were previously analyzed by LC-MS / MS. Validated peptides are those whose fragments and retention times match those of the synthesized peptide. (FIG. IE) Mcp4 is immunogenic in mice infected with Bb for 4 weeks. Proliferation was measured by loss of cytosolic (proliferation) dye. MHC-II peptides derived from chicken ovalbumin (OVA) and a nonimmunogenic Bb protein LptD areused as comparison groups. Data are combined from two independent experiments using 3 mice per experiment. Immunogenicity was defined as peptides that elicit T cell proliferation greater than 3 standard deviations above the control (OVA) peptide (*P<0.05, ** P<0.01).

[0012] FIGS. 2A-2C. Structure of OspA / Mcp4-MATD and OspC / Mcp4-MATD conjugate vaccines. (FIG. 2A) Structure of Borrelia burgdorferi Mcp4 (BB0680) and the Mcp4382-670 Methyl-Accepting Transducer Domain (MATD). (FIG. 2B) Structure of the OspA / Mcp4- MATD conjugate vaccine containing the OspA N-terminal Pam3Cys lipid modification (top) or nonlipidated OspA (bottom). (FIG. 2C) Structure of OspC / Mcp4-MATD conjugate vaccine containing the OspC N-terminal Pam3Cys lipid modification (top) or nonlipidated OspC (bottom).

[0013] FIG. 3. Induction and affinity purification of conjugate protein and components. Size exclusion chromatography (SEC) of conjugate (61.86 kDa), OspA (30.57 kDa), and Mcp4 MATD (32.49 kDa) proteins was used to detect purified protein and was visualized by staining with Coomassie Blue.

[0014] FIGS. 4A-4C. Confirmation of proper folding of conjugate protein using circular dichroism spectra analysis and thermal unfolding. (FIG. 4A) Spectrum of conjugate protein and OspA component in phosphate and NaCl buffer (20 mM and 100 mM, respectively). Background from buffer salt content noted around 190 nm wavelength. (FIG. 4B) Thermal unfolding of conjugate protein and OspA from 20-90 °C at 1 °C / min increase. Inflection points indicate transition of protein from folded to unfolded state.

[0015] FIGS. 5A-5B. MATD carrier protein enhances CD4+ T cell responses to B. burgdorferi (Bb) challenge without altering antibody responses to OspA. Mice (3M / 3F per group) were immunized with conjugate protein, OspA alone, or adjuvant alone (15 pg / mouse), given a boost+adjuvant (15 pg / mouse) 21 days later, then infected with 2xl04B. burgdorferi or BSK uninfected control 21 days post-boost. (FIG. 5A) CD44+ CD6210CD4+ T effector cells in inguinal lymph nodes from mice as a percent of total CD4+ T cells were compared between BSK uninfected controls and Bb-challenged mice. (FIG. 5B) Enzy me-linked immunosorbent assay (ELISA) was used to assess relative levels of anti-OspA IgG in serum (1:800 dilution) of half of the mice per group. Two non-immunized, Bb-infected mice were used as a comparison group. Statistically significant differences between groups were determined by ANOVA (Sidak (5 A) or Turkey (4B) post-hoc, *p<0.05, ****p<0.0001).DETAILED DESCRIPTION

[0016] Lyme disease affects nearly 500.000 individuals in the U.S. annually and is caused by infection with the tick-bome spirochete Borrelia burgdorferi (Bb). Lack of a commercially - available human vaccine against the Lyme disease (LD) pathogen Bb is a major detriment to public health. Nonvaccine measures to limit pathogen transmission have had limited success, and while LD can usually be successfully treated with antibiotic therapy, at least 10% of patients with LD report lingering symptoms after antibiotics, particularly those not treated in a timely manner. Approximately half of untreated patients develop Lyme arthritis (LA), the most common late-stage disease manifestation, of whom -10-20% fail to fully resolve their symptoms even after 1-3 months of oral and / or IV antibiotic therapy, called post-infectious LA. In these patients, Bb peptidoglycan is detectible in inflamed joints for months to years after antibiotic therapy, and autoimmune T and B cell responses often accompany their disease. Both of these factors likely contribute to ongoing inflammation and tissue damage after spirochetal killing, making treatment of lingering symptoms immensely challenging. Diagnosis and treatment of post-infectious LA and other post-treatment LD syndromes continue to be significant public health concerns. The frequency and geographic distribution of LD in the U.S. and globally have increased dramatically, with current estimates at nearly half a million new cases per year in the U.S, and Bb seroprevalence globally at -14.5%. Thus, there is an urgent need to develop a safe and effective Lyme disease vaccine for use in humans.

[0017] Two vaccines based on the Bb outer surface lipoprotein OspA were developed in the 1990’s but are no longer available. LYMErix, one of these OspA-based Lyme disease vaccines, was safe and moderately effective. However, it required at least three doses to achieve -70% protection. This vaccine was pulled from the market shortly after FDA approval due to lack of demand, the need for frequent boosters, weak recommendations by the CDC, and pending lawsuits by Lyme disease activist who believed that the OspA vaccine caused significant adverse reactions. A commercially available Lyme disease vaccine for dogs that targets OspA and OspC subunits (VANGUARDDcrLyme, Zoetis, hereafter crLyme) has been developed for use in dogs. Current human Lyme disease vaccine candidate VLA15 from Pfizer and Valneva in Phase 3 trials use a protein subunit immunogen containing fragments of 6 OspA serotypes to generate a vaccine targeting North American and European Lyme disease spirochetes. Modema recently announced an mRNA LD vaccine development program targeting North American Bb strains (mRNA-1982) and European strains (mRNA-1975), although the specific immune targets are not yet publicly known (“Modema AnnouncesClinical and Program Updates at 4th Vaccines Day,” April 11, 2023, inv estors . modematx.com / news) .

[0018] Despite this recent progress, Lyme vaccines targeting OspA, and other surface lipoproteins suffer from several significant limitations. First, frequent boosters (every 6, 12, and / or 24 months) are required for OspA-based vaccines to maintain high antibody levels needed for protection. Second, due in part to the history of controversy surrounding LYMErix, there is lingering distrust of LD vaccines generally, and of OspA-based vaccines in particular. Third, OspA is rapidly downregulated during transmission and OspC is rapidly downregulated during the first few days or weeks following transmission, so vaccines targeting these antigens have only a small window of effectiveness. Fourth, genes encoding OspA, OspC, and other surface lipoproteins under consideration as vaccine targets (e.g., DbpA, CspZ. FtlA / B, and others) are often serologically diverse and are typically located on highly genetically variable plasmids. Together, these data indicate that vaccines targeting OspA, and other surface lipoproteins may be sub-optimal vaccine candidates.

[0019] Conjugate vaccines have been developed to combine a weak antigen of interest with a strong antigen as a carrier, thereby boosting an immune response to the desired antigen. Hemophilus influenzae type B (Hib) vaccines are examples of conjugate vaccines, in which the weak antigen of interest is a poorly immunogenic polysaccharide. To boost an immune response to this polysaccharide, a protein carrier, such as inactivated tetanus toxoid, is conjugated to the poorly immunogenic antigen to boost thymus-dependent (TD) antibody responses to the vaccine. A similar T-cell-directed vaccine component approach has recently been demonstrated to dramatically enhance and broaden SARS-CoV-2 immunization efficacy. Somewhat akin to limitations with OspA-based vaccines, COVID- 19 vaccines targeting the Spike protein, which is highly variable and is a relatively poor T cell immunogen, provide rapidly waning of protective immunity and immunized individuals are susceptible to reinfection by new variants. Recent findings have identified immunogenic T cell epitopes from non-spike proteins, and co-administration of T cell vaccine components directed against nonsurface antigens markedly enhanced viral clearance by the anti-Spike immune response, resulting in broad protection from infection and / or severe disease. The compositions and methods disclosed herein use a similar approach to conjugate a strong Borrelia burgdorferi antigen we have recently identified to OspA or OspC to enhance TD antibody responses to OspA / C. A major advantage of using Bb Mcps in this conjugate vaccine is that immune responses to the carrier proteins will also provide some degree of protection in cases of escape infections, whereas other carriers such as the tetanus toxoid, provides no such protection.

[0020] An immunopeptidomics approach was previously used to identify immunogenic MHC class II epitopes derived from Bb presented by human APCs in vitro and mouse APCs ex vivo (FIG. 1 A). One of the major objectives of that prior study was to identify T cell epitopes from Bb that could be used to improve LD vaccines. Numerous Bb T cell epitopes previously identified are in the process of being validated. One peptide previously identified from mice in the immunopeptidomics screen was derived from BB0680 (Mcp4 442-462), a methyl- accepting chemotaxis protein (FIG. IB; bottom). An epitope from Mcpl (Mcpl 49-66) presented on human APCs (FIG. IB; top) was also previously identified. Lyme disease spirochetes encode for five Mcps, all of which are chromosomally -encoded and highly conserv ed amongst both sensu lato and sensu stricto genospecies. The Mcp peptide previously identified in human and mouse APCs are from the methyl-accepting transducer domains of Mcpl and Mcp4, respectively. Mcp4 442-462 was validated using a synthesized peptide (FIG. ID) and is immunogenic in mice infected with B. burgdorferi (FIG. IE). Mcp4442-462 is one of several promiscuous epitopes derived from the conserved Mcp4 methyl-accepting transducer domain that are predicted to bind to MHC class II alleles in both mice and humans of various HL ADR types.

[0021] In various aspects, the present compositions and methods disclosed herein utilize a conjugate vaccine that incorporates both T-cell and B-cell immunogens to address the gaps in current LD vaccine technology-. In various aspects, as discussed further below, the conjugate vaccines disclosed herein incorporate a carrier protein that contains highly immunogenic T cell epitopes linked to outer surface lipoproteins.

[0022] Definitions and Terminology-

[0023] The disclosed compositions and methods may be further described using definitions and terminology as follows. The definitions and terminology- used herein are for the purpose of describing particular embodiments only, and are not intended to be limiting.

[0024] As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise.

[0025] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary- skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term w-hich are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.

[0026] As used herein, the terms "include" and “including’' have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0027] The phrase “such as” should be interpreted as “for example, including.” Moreover the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0028] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0029] All language such as “up to.” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3. 4, or 6 members, and so forth.

[0030] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in thesame. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”

[0031] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 50%, 55%, 60%, 65%, 70%, 75%. 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%. 99%, or more identity over a specified region, e.g.. of an entire nucleic acid or polypeptide sequence or individual portions or domains of a nucleic acid or polypeptide), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical.” This definition also refers to the complement of a test sequence, in the context of nucleic acids. By way of example, in embodiments, the identify exists over a region that is about or at least about 5, 10, 15. 20, 50, 100, or 1000, amino acids in length, to about, less than about, or at least about 220, 100 or 1000 amino acids or nucleotides in length. Optionally, the identity exists over a region that is at least about 5, 10, 15, 20, 21 amino acids in length to about 100, about 20 to about 75, about 30 to about 50 amino acids or nucleotides in length. Non-limiting examples of polypeptide sequences provided herein comprise sequences that are substantially identical to any one of SEQ ID NOs: 1-31. By way of example, polypeptides that are at least 80%. 81%. 82%. 83% 84%. 85%. 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any one of SEQ ID NOs: 1 -31 is provided herein. Polypeptides comprising a difference of 1, 2, 3, 4, 5, 6 or 7, 8, 9 or 10 amino acids as compared to any one of SEQ ID NOs: 1-31 are also contemplated herein.

[0032] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0033] An example of algorithms suitable for determining percent sequence identify and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. As will be appreciated by one of skill in the art, the software forperforming BLAST analyses is publicly available through the website of the National Center for Biotechnology Information (NCBI). In embodiments, BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins. In embodiments, a BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. In embodiments, T is referred to as the neighborhood word score threshold (Altschul et al., supra). In embodiments, these initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. In embodiments, the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. In embodiments, cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). In embodiments, for amino acid sequences, a scoring matrix is used to calculate the cumulative score. In embodiments, extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantify X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. In embodiments, the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. In embodiments, the NCBI BLASTN or BLASTP program is used to align sequences. In embodiments, the BLASTN or BLASTP program uses the defaults used by the NCBI. In embodiments, the BLASTN program (for nucleotide sequences) uses as defaults: a word size (W) of 28; an expectation threshold (E) of 10; max matches in a query range set to 0; match / mismatch scores of 1. 2: linear gap costs; the filter for low complexify regions used; and mask for lookup table only used. In embodiments, the BLASTP program (for amino acid sequences) uses as defaults: a word size (W) of 3; an expectation threshold (E) of 10; max matches in a query range set to 0; the BLOSUM62 matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992)); gap costs of existence: 11 and extension: 1; and conditional compositional score matrix adjustment.

[0034] The terms ‘’polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

[0035] The term “amino acid’' refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y- carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g.. homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0036] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0037] "Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG. which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual probe sequences.

[0038] As to amino acid sequences, one of skill will recognize that individual substitutions to a peptide, polypeptide, or protein sequence which alters a single amino acid is a“conservatively modified variant’' where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles.

[0039] The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0040] "Lyme disease" as used herein refers to an infection by aBorrelia bacterium, which is also known as Borreliosis. At least nine species of Borrelia are known to cause Lyme disease, including Borrelia afzelii, Borrelia bissettiae, Borrelia burgdorferi. Borrelia garinii, Borrelia lusitaniae, Borrelia mayonii. Borrelia spielmanii, and Borrelia valaisiana. Additional Borrelia strains include, without limitation, 'Q.burgdorferi-JDl. B.burgdorferi-N40, B.burgdorferi-B31 , B.burgdorferi-ZS7, and B.garinii-bavariensis . Lyme disease is commonly caused by the transfer of Borrelia bacteria to mammalian hosts by certain hard ticks of the Ixodidae family. In the U.S., Lyme disease is most commonly the result of the transfer of Borrelia burgdorferi bacteria by the blacklegged tick, also known as a deer tick (Ixodes scapularis or Ixodes pacificus'). Outside the U.S., Ixodes ricinus and Ixode persulcatus act as Borreliosis vectors. Without early detection and treatment, the bacteria travel through the bloodstream and affect various tissues in the host. The infection may develop into an inflammatory condition that affects multiple systems, starting with skin, joints, and nervous system and moving to organs. Symptoms can include one or more of fever, chills, headache, fatigue, muscle pain, joint pain, swollen lymph nodes, a bullseye rash, termed erythema migrans, facial palsy, and arthritis.

[0041] The term “subject’' may be used interchangeably with the terms “individual” and “patient” and includes human and non-human subjects. In some embodiments, subjects may be any animal that can be infected with a bacterium, such as Borrelia burgdorferi . In some embodiments, the subject is a mammal, such as a human, dog, cat, or livestock, such as cattle, pigs, or sheep. In some embodiments, a subject may include one or more reservoir host species, such as white-footed mice, chipmunks, other rodents, etc. As used herein, a “reservoir host species” is any species in which a pathogen, such as a bacterium, can survive and reproduce. Reservoir host species commonly serve as a source for the infection of other non-reservoir hostspecies. In some embodiments, a subject may include a wild, domesticated, or captive population of animals such as deer, elk, bison, etc. In some embodiments, a subject may include one or more wild or domesticated bird species, such as chickens, ducks, or turkeys.

[0042] As used herein, the terms “treat” or “treatment” encompass both “preventative” and “curative” treatment. “Preventative” treatment is meant to indicate a postponement of development of a disease, a symptom of a disease, or medical condition, suppressing symptoms that may appear, or reducing the risk of developing or recurrence of a disease or symptom. “Curative” treatment includes reducing the severity of or suppressing the worsening of an existing disease, symptom, or condition. Thus, treatment includes ameliorating or preventing the worsening of existing disease symptoms, preventing additional symptoms from occurring, ameliorating or preventing the underlying systemic causes of symptoms, inhibiting the disorder or disease, e.g., arresting the development of the disorder or disease, relieving the disorder or disease, causing regression of the disorder or disease, relieving a condition caused by the disease or disorder, or stopping the symptoms of the disease or disorder.

[0043] A non-limiting example of a treatment for a subject diagnosed with Lyme disease includes, but is not limited to. administration of a composition comprising one or more polypeptides that have at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to one or more of SEQ ID NOs: 1-31, or to one or more of SEQ ID NOs: 1-5 and 12-31. In some aspects, the composition comprises an adjuvant. In some embodiments, the composition comprises a vaccine.

[0044] A non-limiting example of a treatment for infection by Borrelia sp. including, but not limited to B. burgdorferi includes, but is not limited to, administration of a composition comprising one or more polypeptides that have at least 80%, 85%, 90%. 95%, 98%, 99%, or 100% identity to one or more of SEQ ID NOs: 1-31, or to one or more of SEQ ID NOs: 1-5 and 12-31. In some aspects, the composition comprises an adjuvant. In some embodiments, the composition comprises a vaccine.

[0045] A non-limiting example of a preventative treatment for a subject at risk of exposure to a tick causing Lyme disease includes, but is not limited to, administration of a composition comprising one or more polypeptides that have at least 80%. 85%. 90%. 95%, 98%, 99%, or 100% identity to one or more of SEQ ID NOs: 1-31, or to one or more of SEQ ID NOs: 1-5 and 12-31. In some aspects, the composition comprises an adjuvant. In some aspects, the composition comprises a vaccine.

[0046] A non-limiting example of a preventative treatment for a subj ect at risk for infection by Borrelia sp. including, but not limited to B. burgdorferi includes, but is not limited to,administration of a composition comprising one or more polypeptides that have at least 80%, 85%. 90%. 95%. 98%. 99%. or 100% identity to one or more of SEQ ID NOs: 1-31, or to one or more of SEQ ID NOs: 1-5 and 12-31. In some aspects, the composition comprises an adjuvant. In some aspects, the composition comprises a vaccine.

[0047] As used herein, the term "adjuvant" refers to ingredients used in some vaccines to create a stronger immune response in subjects receiving the vaccine. By way of example, but not by way of limitation, adjuvants include aluminum, monophosphoryl lipid A (MPL), MPL + aluminum salt, oil in water emulsion, e.g., composed of squalene, MPL and QS-21, cytosine phosphaguanine (CpG).

[0048] As used herein, the term "buffer" refers to a pH buffering agent that helps maintain the pH of an aqueous solution. Non-limiting examples of pH buffering agents include phosphate buffers (e.g. PBS), Tris, citric acid, histidine, glycine, HEPES, MOPS, and PIPES.

[0049] As used herein, the term "stabilizer" refers to an agent that stabilizes a component of a composition, such as a protein, antibody, and / or polypeptide, thereby helping to presen e its stability and integrity. Non-limiting examples of stabilizers include glycerol, polysorbates, polyethylene glycols (PEGs), glycine, histidine, arginine, sugars (e.g. trehalose), and polyols (e g., mannitol, sorbitol and glycerol).

[0050] As used herein, the term “macromolecular crowding agent” refers to an inert agent that excludes volume in solution from other macromolecules, such as proteins and antibodies. Non-limiting examples of macromolecular crowding agents include polyethylene glycols (PEGs), dextran, and high molecular weight, highly branched polysaccharides.

[0051] As used herein, the term "antioxidant" refers to an agent that inhibits oxidation reactions. Non-limiting examples of antioxidants include citrate, EDTA (ethylenediaminetetraacetic acid), methionine, ascorbic acid, and thiols.

[0052] As used herein, the term "preservative" refers to an agent that prevents or inhibits microbiological growth. Non-limiting examples of preservatives include CMIT (5-chloro-2- methylisothiazol-3(2H)-one), MIT (2-methylisothiazol-3(2H)-one), and sodium azide.

[0053] As used herein, the term "surfactant" refers to an agent that modulates the solubility of another molecule or agent in a solution. Non-limiting examples of surfactants include polysorbate 20, polysorbate 80, Triton X-100, and pluronic F68.

[0054] As used herein the term "standard two-tier test" (STT) refers to a decision tree that describes the steps required to currently test for Lyme disease. The first required test is the Enzyme Immunoassay (EIA) or Immunofluorescence Assay (IF A). If this test yields negative results, the provider should consider an alternative diagnosis; or in cases where the patient hashad symptoms for less than or equal to 30 days, the provider may treat the patient and follow up with a convalescent serum. If the first test yields positive or equivocal results, two options are available: 1) If the patient has had symptoms for less than or equal to 30 days, an IgM Western Blot is performed; 2) if the patient has had symptoms for more than 30 days, the IgG Western Blot is performed. The IgM should not be used if the patient has been ill for more than 30 days.

[0055] Compositions and Polypeptides

[0056] In various aspects, the compositions and / or polypeptides disclosed herein can include or be part of a conjugate vaccine for Lyme disease. In certain aspects, the conjugate vaccine can incorporate a carrier protein that contains highly immunogenic T cell epitopes linked to outer surface lipoproteins. In one or more aspects, this carrier protein is derived from the broadly conserved methyl-accepting transducer domains (MATD) from the Bb methyl- accepting chemotaxis proteins, or Mcps, or variant thereof. In certain aspects, the carrier protein can comprise or consist of at least one amino acid sequence that is at least 80%, 90%, 95%, 98%. 99%. or 100% identical to one or more of SEQ ID NOs: 1-5. In various aspects, more than one copy of the carrier protein may be present in the polypeptides and / or compositions disclosed herein. For instance, in one aspect, a first amino acid sequence that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 1-5, and a second amino acid sequence that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 1-5 can be present. In certain aspects, the carrier protein can be a T-cell immunogen.

[0057] In various aspects, the conjugate vaccine and / or polypeptides disclosed herein can also include an outer surface protein OspA or OspC or variant thereof, e.g., as the B-cell immunogen. For instance, in certain aspects, the conjugate vaccine and / or polypeptides can comprise or consist of at least one amino acid sequence that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 6-11. In various aspects, more than one copy of the B-cell immunogen may be present in the polypeptides and / or compositions disclosed herein. For instance, in one aspect, a first amino acid sequence that is at least 80%, 90%. 95%. 98%. 99%. or 100% identical to one or more of SEQ ID NOs: 6-11, and a second amino acid sequence that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 6-11 can be present.

[0058] As discussed above, the polypeptides and / or conjugate vaccines disclosed herein can include both a T-cell immunogen (e.g., one or more of the carrier proteins or variants disclosed above) and a B-cell immunogen (e.g., one or more of the outer surface protein OspAor OspC. or variant thereof). In various aspects a linker sequence can connect the T-cell immunogen to the B-cell immunogen. In certain aspects, the linker sequence can comprise a glycine-serine (GS) linker, an alpha helix-forming linker, a proline-rich linker, a cleavable linker, or a combination thereof. In certain aspects, the GS linker can comprise 2-22 amino acids. In the same or alternative aspects, in the GS linker, at least 50% of the amino acids are G and S, and optionally, the GS linker can comprise one or more of A. T, or a polar amino acid. In one or more aspects, the T and / or A may be added to this GS linker to maintain linker flexibility and the polar amino acids may be added to improve solubility. In one or more aspects, the alpha helix-forming linker, which may be used as a rigid linker, can comprise sequences of 1-5 repeating EAAAK motifs, and optionally, the EAAAK motifs can be flanked by A residues. In certain aspects, the proline linker, which may be used as a rigid linker, can comprise 6-24 amino acids consisting of XP repeats, where X is A, K, or E, or other nonproline amino acid. In one or more aspects, the cleavable linker can comprise or consist of a cyclopeptide containing a disulfide bond, or a protease-sensitive amino acid sequence that is an in vivo cleavable linker that is sensitive to proteases such as matrix metalloproteases, cathepsins, or furins.

[0059] In various aspects, the T-cell immunogen and the B-cell immunogen may be ordered in any manner in the polypeptides disclosed herein. For instance, in one aspect, the B- cell immunogen can be present at the N-terminal side and the carrier protein and / or T-cell immunogen can be present at the C-terminal side. In an alternative aspects, the T-cell immunogen can be present at the N-terminal side and the carrier protein and / or B-cell immunogen can be present at the C-terminal side.

[0060] In various aspects, example polypeptides, which may be used in conjugate vaccines and / or the compositions disclosed herein, include, but are not limited to an amino acid sequence that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 12-31.

[0061] The polypeptides, compositions, and / or conjugate vaccines disclosed herein will provide stronger B cell memory and higher-affinity antibodies, which will lengthen the duration of protection. Importantly, the highly conserved Mcp-derived carrier protein will broaden protection against disease from serologically distinct Bb genospecies in cases of escape infections.

[0062] In various aspects, to generate a conjugate vaccine, Borrelia burgdorferi (Bb) B31 outer surface lipoproteins OspA or OspC, with or without a lipid modification (an N-terminal tripalmitoyl-S-glyceryl-cysteine (ParmCys) lipid modification is shown as an example in FIG.2B), can be linked to the methyl -accepting transducer domains (MATD) of Bb methyl- accepting chemotaxis proteins Mcpl, Mcp2, Mcp3, Mcp4, or Mcp5. to generate a two- component Lyme disease conjugate vaccine. In one or more aspects, components can be joined with a linker protein sequence (Gly-Ser linker shown as an example in FIGS. 2B and 2C) between the C-terminal end of the OspA / OspC protein and the N-terminal end of the Mcp MATD (Mcp4 shown as an example). The lipidated conjugate vaccine can be administered without an adjuvant, whereas the nonlipidated conjugate vaccines will be administered with an exogenous adjuvant, in various aspects.

[0063] In various aspects, the polypeptides disclosed herein can be lipidated. In certain aspects, any lipidation can be used. In one aspect, the polypeptide can be lipidated with an N- terminal tripalmitoyl-S-glyceryl-cysteine (PamsCysj-motif

[0064] In various aspects, the compositions and / or conjugate vaccines can include one or more adjuvants. In certain aspects, an example adjuvant can comprise an alum component, e.g., an alum emulsion, and / or can comprise a natural or synthetic pattern recognition receptor ligand, and / or any other suitable adjuvant. In certain aspects, conjugate immunogens or vaccines containing the endogenous TLR2 ligand ParmCys lipid moiety can be administered without any additional adjuvant. Conjugate vaccines lacking this lipid moiety may be administered with an alum emulsion as an adjuvant, or with a natural or synthetic pattern recognition receptor ligand as an adjuvant, in various aspects.

[0065] In some embodiments, the compositions disclosed herein are provided in lyophilized form.

[0066] Methods

[0067] Disclosed herein are methods for treating or for the preventative treatment of Lyme disease and / or infection with Borrelia sp.

[0068] In some aspects, the method comprises treating the subject for Lyme disease and / or infection with Borrelia sp, or for the preventative treatment of Lyme disease and / or infection with Borrelia sp. In various aspects, the methods can include administering to a subject one or more of the polypeptides and / or compositions disclosed herein. For instance, in one aspect, the methods can include administering one or more of the polypeptides having an amino acid sequence that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 12-31. In the same or alternative aspects, the methods can include administering one or more polypeptides having: a first portion comprising or consisting of at least one first amino acid sequence that is at least 80%. 90%. 95%. 98%. 99%. or 100% identical to one or more of SEQ ID NOs: 1-5; and a second portion comprising or consisting of at least one second aminoacid sequence that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 6-11. In such aspects, the first portion and second portion are linked together with a linker sequence, such as any of the linker sequences discussed above.

[0069] In some aspects of any of the methods above, the subject previously tested positive for Lyme disease, for example, by standard two-tier (STT) testing.

[0070] In some aspects of any of the methods above, the subject exhibits one or more of the following symptoms: erythema migrans. facial palsy, and arthritis; and / or the subject already tested positive for Lyme disease in standard two-tier test (STT). In some aspects, the subject has exhibited the symptom for 3 months or less, 2 months or less, 1 month or less, 2 weeks or less, 1 week or less, 3 days or less, or 1 day or less.

[0071] In some aspects of any of the methods above, the subject has not previously been treated for Lyme disease.

[0072] In some aspects of any of the methods above, the subject is asymptomatic for Lyme disease, but may have come in contact with a blacklegged tick selected from Ixodes scapular is. Ixodes paci ficus. Ixodes ricinus, and Ixodes persulcatus . In some aspects, the possible contact between the subject and the tick was within 3 months, within 2 months, within 1 month, within 2 weeks, within 1 week, within 3 days, or within 1 day.

[0073] In some aspects of any of the methods above, the subject is asymptomatic for Lyme disease and has previously been treated for Lyme disease. In some aspects, the subject is asymptomatic for Lyme disease and has not previously been treated for Lyme disease.

[0074] In some aspects of any of the methods above, the subject has previously been treated for Lyme disease and exhibits one or more of the following symptoms: erythema migrans, facial palsy, and arthritis; and / or the subject previously tested positive for Lyme disease in standard two-tier test (STT). In some aspects, the subject has not received treatment for Lyme disease for at least 1 year, for at least 5 years, for at least 10 years, or for at least 15 years.

[0075] In some aspects of any of the methods above, the method comprises a step of treating the subject for Lyme disease.

[0076] In some aspects of any of the methods above, the subject is being treated for Lyme disease.

[0077] In various aspects of any of the methods above, the subject is being vaccinated for prevention of Lyme disease or for mitigation of any potential future symptoms of Lyme disease.

[0078] Informal Sequence Listing

[0079] Carrier Protein: Subunit of a methyl-accepting chemotaxis protein (Mcp) containing the methyl-accepting transducer domain (MATD): Entire sequence of Borrelia burgdorferi Mcpl (BB0578), or C-terminal cytosolic domains from B. burgdorferi Mcp2 (BB0596), Mcp3 (BB0597), Mcp4 (BB0680), or Mcp5 (BB0681), or homologs of Mcpl, Mcp2, Mcp3, Mcp4, or Mcp5 from other Borrelia burgdorferi sensu lato genospecies.

[0080] Mcpl (BB 0578. entire sequence) [SEQ ID NO: 1]

[0081] Note: Mcpl consists of a cytosolic MATD only and lacks transmembrane and periplasmic domains contained in other Bb Mcps.

[0082] MKKFSFFKKKKKAVDLNLLNVVGNDKKDLSTYEYKAPVKEMLKGFYHT KASISTLKVGFESLQRILFSEIEDFDQIFSTFVEMTNNARNQISVIFSDLGKNNKEKLNQ ISSVIVGIQGSLETISNFLGATNMISLNAKLEAARAKEYGKGFSVVADE1KRLSDQAK GVMNMISVKEIEEVSKDLISHNLKDLQIDIDKFFAEILEQLNYLESIFKRFSRSQEEFSS LIESLESIDANMAYYSRNCDSLIGSDTFMLSNDEFLKELEFIISDQFSWINNLRLLVEG QRTIFIQTDASKHGFGLFYKGLSPKNDAIRQLWEEVYIPYLNINKFAAEILIIFRVENRS DSGLRQAKDFLSQAESLSEEIVRKLEH1KKMVIELDNQG1SIFS

[0083] Mcp2 AA 381 -715 (BB 0596 MATD) [ SEQ ID NO : 2]

[0084] NSLQERVKLLKENGDHLFSEINKTHNTIKNSNQYIEKTQEEVEKQVEFISNTTNIIESL SKNIS SLDNSIETQ AAS VEQS S S AIEEMIGGIQ SITEITQKAAKSTEELKRFSD DGRKKQEEVITQIKEIFKNSTRLQEANSLISSIASQTNLLSMNAAIEASHAGEAGKGFA IVAEEIKDLAEQVTSQSESVASSINEIMDSITKTVNTSELTNKAFNQIFDSINLVVQVIE EINHTMQEQSIGSQEI LK ALNTMREITY EVKIGSNDMFRGNKEIISTIKLLGEINITVSNS MKGLKEEINTLVEAIERIKVLGTTNSSHISGISESINQFKTK

[0085] Mcp3 AA 381-735 (BB 0597 MATD) [SEQ ID NO: 3]

[0086] IISYISSKLDNLSSKSNESFEKIKFYSEDLNEYLEQIETAISNTESIDSSILVYE QLRDTFSRFEKSIVDILKGFES1ADPINDHNKYISEISSNFEESVSFFYSIDKNLEIFNKV ATINSTDIENIKSKVFDLNIVFENVNKNFADLLSQTNSLQSVNKLLVSISAQTNMLAM NAAIEAAKAGDAGKSFAVVAEEIRKLAINSGKYSKTIKDELKTVDSIIAVINSEIDTIY KNFIDIQDNVDNNFSRHEKVDLTLAKHFKEIGEFKERYLSHDTKIRDAKNMYKEIFN NHYFISGKFNNFSQDLKEFKVSKMNLDAVSSLQEYSSLVKSSKDKILKTKELIQKIND EIKDILF

[0087] Mcp4 AA 382-670 (BB_0680 MATD) [SEQ ID NO: 4]

[0088] NAIRVLVQDMVKGNLDKDYALDDDENTLDELGMLSLQVVKMKKAISVA ISSVLRNISYVNKASLEVASSSQNLSSSALQQASALEEMSANVEQIASGVNMSANNSY ETEQIALKTNENSQIGGRAVEESVIAMQDIVEKVSVIEEIARKTNLLALNAAIEAARAGDEGKGFAVVASEIRKLADLSKISALEIGELVEDNSKVATEAGVIFKEMLPEIEETANLVKKISEGSSKQSDQIAQFKMALDQVGEVVQSSASSSEQLSSMSDKMLEKSKELRKSVLFFKIKDSK

[0089] Mcp5 AA 381-633 (BB 0681 MATD) [SEQ ID NO: 5]

[0090] YEGLEQLRTNFSSVAKGVIENLDYLYENAIQIANASQNLSSGAVEQASTLEQMTANIEQISQGVSENTENAATTEKIAVNTNERTKEGHKSVVKAIEAMTVITEKIGIIDEITRQTNLLALNASIEAARVGEKGKGFEVVAAEVRKLADQSKESAREIID1ANRSLTVASRAGENFEQIVPGMEQTARLVKNISNESYKQSVQ1EQFKNAIEQVSQLVQTTASSSEELSAMSEKMLESVKDLKESVDYFKIEK

[0091] Lipoprotein conjugate vaccine component: Borrelia burgdorferi OspA or OspC or homolog sequences from other Borrelia burgdorferi sensu lato genospecies.

[0092] Borrelia burgdorferi OspA (BB A15) with Escherichia coli lipidation signal sequence (Pam3Cys residue underlined) [SEQ ID NO: 6]

[0093] MK / rKLVLGAVILGSTLLAGCSSKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLELKGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKGYVLEGTLTAEKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIKNALK

[0094] OspA (BB Al 5) with native lipidation signal sequence (Pam3Cys residue underlined) [SEQ ID NO: 7]

[0095] MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLELKGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKGYVLEGTLTAEKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIKNALK

[0096] Nonlipidated OspA (BB A15) containing a C17A mutation [SEQ ID NO: 8]

[0097] MKKYLLGIGLILALIAAKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLELKGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKGYVLEGTLTAEKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIKNALK

[0098] OspC (BB Bl 9) with native lipidation signal sequence (Pam3Cys residue underlined) [SEQ ID NO: 9]

[0099] MKKNTLSAILMTLFLFISCNNSGKDGNTSANSADESVKGPNLTEISKKITDSNAVLLAVKEVEALLSSIDEIAAKAIGKKIHQNNGLDTENNHNGSLLAGAYAISTLIKQKLDGLKNEGLKEKIDAAKKCSETFTNKLKEKHTDLGKEGVTDADAKEAILKTNGTKTKGAEELGKLFESVEVLSKAAKEMLANSVKELTSPVVAESPKKP

[0100] OspC (BB B19) with E. coli lipidation signal sequence (Pam3Cys residue underlined) [SEQ ID NO: 10]

[0101] MlGnKLVLGAVILGSILLAGCSSNNSGKDGNTSANSADESVKGPNLTEISKKITDSNAVLLAVKEVEALLSSIDEIAAKAIGKKIHQNNGLDTENNHNGSLLAGAYAISTLIKQKLDGLKNEGLKEKIDAAKKCSETFTNKLKEKHTDLGKEGVTDADAKEAILKTNGTKTKGAEELGKLFESVEVLSKAAKEMLANSVKELTSPVVAESPKKP

[0102] Nonlipidated OspC (BB B19) a C19A mutation [SEQ ID NO: 11]

[0103] MKKNTLSAILMTLFLFISANNSGKDGNTSANSADESVKGPNLTEISKKITDSNAVLLAVKEVEALLSSIDEIAAKAIGKKIHQNNGLDTENNHNGSLLAGAYAISTLIKQKLDGLKNEGLKEKIDAAKKCSETFTNKLKEKHTDLGKEGVTDADAKEAILKTNGTKTKGAEELGKLFESVEVLSKAAKEMLANSVKELTSPVVAESPKKP

[0104] Examples of conjugate proteins containing a flexible GS linker as an example (in bold).

[0105] OspA / Mcpl Conjugate protein with GS linker [SEQ ID NO: 12]

[0106] MKATKLVLGAVILGSTLLAGCSSKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLELKGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKGYVLEGTLTAEKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGSMKXFSFFKKKKKAVDLNLLNVVGNDKKDLSTYEYKAPVKEMLKGFYHTKASISTLKVGFESLQRILFSEIEDFDQIFSTFVEMTNNARNQISVIFSDLGKNNKEKLNQISSVIVGIQGSLETISNFLGATNMISLNAKLEAARAKEYGKGFSVVADEIKRLSDQAKGVMNMISVKEIEEVSKDLISHNLKDLQIDIDKFFAEILEQLNYLESIFKRFSRSQEEFSSLIESLESIDANMAYYSRNCDSLIGSDTFMLSNDEFLKELEFIISDQFSWINNLRLLVEGQRTIFIQTDASKHGFGLFYKGLSPKNDAIRQLWEEVYIPYLNINKFAAEILIIFRVENRSDSGLRQAKDFLSQAESLSEEIVRKLEHIKKMVIELDNQGISIFS

[0107] OspA / Mcp2 MATD Conjugate protein with GS linker [SEQ ID NO: 13]

[0108] MKATKLVLGAVILGSTLLAGCSSKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLELKGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKGYVLEGTLTAEKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGSNSLQERVKLLKENGDHLFSEINKTHNTIKNSNQYIEKTQEEVEKQVEFISNTTNIIESLSKNISSLDNSIETQAASVEQSSSAIEEMIGGIQSITEITQKAAKSTEELKRFSDDGRKKQEEVITQIKEIFKNSTRLQEANSLISSIASQTNLLSMNAAIEASHAGEAGKGFAIVAEE IKDLAEQVTSQSESVASSINEIMDSITKTVNTSELTNKAFNQIFDSINLVVQVIEEINHT MQEQSIGSQEILKALNTMREITYEVKIGSNDMFRGNKEIISTIKLLGEINITVSNSMKGLKEEINTLVEAIERIKVLGTTNSSHISGISESINQFKTK

[0109] OspA / Mcp3 MATD Conjugate protein with GS linker [SEQ ID NO: 14]

[0110] MKATKLVLGAVILGSTLLAGCSSKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLELKGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTL EVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKGYVLEGTLTAEKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGSIISYISSKLDNLSSKSNESFEKIKFYSEDLNEYLEQIETAISNTESIDSSILVYEQLRD TFSRFEKSIVDILKGFESIADPINDHNKYISEISSNFEESVSFFYSIDKNLEIFNKVATINS TDIENIKSKVFDLNIVFENVNKNFADLLSQTNSLQSVNKLLVSISAQTNMLAMNAAIEAAKAGDAGKSFAVVAEEIRKLAINSGKYSKTIKDELKTVDSIIAVINSEIDTIYKNFIDIQDNVDNNFSRHEKVDLTLAKHFKEIGEFKERYLSHDTKIRDAKNMYKEIFNNHYFISGKFNNFSQDLKEFKVSKMNLDAVSSLQEYSSLVKSSKDKILKTKELIQKINDEIKDILF

[0111] Osp A / Mcp4 MATD Conjugate protein with GS linker [SEQ ID NO: 15]

[0112] MKATKLVLGAVILGSTLLAGCSSKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLELKGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRADGTRLEYTGIKSDGSGK AKEVLKGYVLEGTLTAEKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTA AWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGSNAIRVLVQDMVKGNLDKDYALDDDENTLDELGMLSLQVVKMKKAISVAISSVLRNISYVNKASLEVASSSQNLSSSALQQASALEEMSANVEQIASGVNMSANNSYETEQIALKTNENSQIGGRAVEESVIAMQDIVEKVSVIEEIARKTNLLALNAAIEAARAGDE GKGFAVVASEIRKLADLSKISALEIGELVEDNSKVATEAGVIFKEMLPEIEETANLVKKISEGSSKQSDQIAQFKMALDQVGEVVQSSASSSEQLSSMSDKMLEKSKELRKSVLF FKIKDSK

[0113] OspA / Mcp5 MATD Conjugate protein with GS linker [SEQ ID NO: 16]

[0114] MKATKLVLGAVILGSTLLAGCSSKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLELKGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTL EVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRADGTRLEYTGIKSDGSGK AKEVLKGYVLEGTLTAEKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTA AWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIKNAL KGSYEGLEQLRTNFSSVAKGVIENLDYLYENAIQIANASQNLSSGAVEQASTLEQMT ANIEQISQGVSENTENAATTEKIAVNTNERTKEGHKSVVKAIEAMTVITEKIGIIDEITRQTNLLALNASIEAARVGEKGKGFEVVAAEVRKLADQSKESAREIIDIANRSLTVASR AGENFEQIVPGMEQTARLVKNISNESYKQSVQIEQFKNAIEQVSQLVQTTASSSEELS AMSEKMLESVKDLKESVDYFKIEK

[0115] OspA C17A / Mcpl conjugate protein with GS linker [SEQ ID NO: 17]

[0116] MKKYLLGIGLILALIAAKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLELKGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDG KTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKG YVLEGTLTAEKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGSMKKF SFFKKKKKAVDLNLLNVVGNDKKDLSTYEYKAPVKEMLKGFYHTKASISTLKVGFE SLQRILFSEIEDFDQIFSTFVEMTNNARNQISVIFSDLGKNNKEKLNQISSVIVGIQGSLE TISNFLGATNMISLNAKLEAARAKEYGKGFSVVADEIKRLSDQAKGVMNMISVKEIE EVSKDLISHNLKDLQIDIDKFFAEILEQLNYLESIFKRFSRSQEEFSSLIESLESIDANMA YYSRNCDSLIGSDTFMLSNDEFLKELEFIISDQFSWINNLRLLVEGQRTIFIQTDASKHGFGLFYKGLSPKNDAIRQLWEEVYIPYLNINKFAAEILIIFRVENRSDSGLRQAKDFLS QAESLSEEIVRKLEHIKKMVIELDNQGISIFS

[0117] OspA C17A / Mcp2 MATD conjugate protein with GS linker [SEQ ID NO: 18]

[0118] MKKYLLGIGLILALIAAKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDG KYDLIATVDKLELKGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKG YVLEGTLTAEKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTS TLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGSNSLQ ERVKLLKENGDHLFSEINKTHNTIKNSNQYIEKTQEEVEKQVEFISNTTNIIESLSKNIS SLDNSIETQAASVEQSSSAIEEMIGGIQSITEITQKAAKSTEELKRFSDDGRKKQEEVIT QIKEIFKNSTRLQEANSLISSIASQTNLLSMNAAIEASHAGEAGKGFAIVAEEIKDLAEQVTSQSESVASSINEIMDSITKTVNTSELTNKAFNQIFDSINLVVQVIEEINHTMQEQSIGSQEILKALNTMREITYEVKIGSNDMFRGNKEIISTIKLLGEINITVSNSMKGLKEEINT LVEAIERIKVLGTTNSSHISGISESINQFKTK

[0119] OspA C17A / Mcp3 MATD conjugate protein with GS linker [SEQ ID NO: 19]

[0120] MKKYLLGIGLILALIAAKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDG KYDLIATVDKLELKGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDG KTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKG YVLEGTLTAEKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTS TLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGSIISYIS SKLDNLSSKSNESFEKIKFYSEDLNEYLEQIETAISNTESIDSSILVYEQLRDTFSRFEKS IVDILKGFESIADPINDHNKYISEISSNFEESVSFFYSIDKNLEIFNKVATINSTDIENIKS KVFDLNIVFENVNKNFADLLSQTNSLQSVNKLLVSISAQTNMLAMNAAIEAAKAGD AGKSFAVVAEEIRKLAINSGKYSKTIKDELKTVDSIIAVINSEIDTIYKNFIDIQDNVDNNFSRHEKVDLTLAKHFKEIGEFKERYLSHDTKIRDAKNMYKEIFNNHYFISGKFNNFS QDLKEFKVSKMNLDAVSSLQEYSSLVKSSKDKILKTKELIQKINDEIKDILF

[0121] OspA C17A / Mcp4 MATD conjugate protein with GS linker [SEQ ID NO: 20]

[0122] MKKYLLGIGLILALIAAKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDG KYDLIATVDKLELKGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDG KTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKG YVLEGTLTAEKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTS TLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGSNAIR VLVQDMVKGNLDKDYALDDDENTLDELGMLSLQVVKMKKAISVAISSVLRNISYV NKASLEVASSSQNLSSSALQQASALEEMSANVEQIASGVNMSANNSYETEQIALKTN ENSQIGGRAVEESVIAMQDIVEKVSVIEEIARKTNLLALNAAIEAARAGDEGKGFAVV ASEIRKLADLSKISALEIGELVEDNSKVATEAGVIFKEMLPEIEETANLVKKISEGSSKQSDQIAQFKMALDQVGEVVQSSASSSEQLSSMSDKMLEKSKELRKSVLFFKIKDSK

[0123] OspA C17A / Mcp5 MATD conjugate protein with GS linker [SEQ ID NO: 21]

[0124] MKKYLLGIGLILALIAAKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDG KYDLIATVDKLELKGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDG KTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKG YVLEGTLTAEKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTS TLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGSYEGL EQLRTNFSSVAKGVIENLDYLYENAIQIANASQNLSSGAVEQASTLEQMTANIEQISQ GVSENTENAATTEKIAVNTNERTKEGHKSVVKAIEAMTVITEKIGIIDEITRQTNLLAL NASIEAARVGEKGKGFEVVAAEVRKLADQSKESAREIIDIANRSLTVASRAGENFEQIVPGMEQTARLVKNISNESYKQSVQIEQFKNAIEQVSQLVQTTASSSEELSAMSEKML ESVKDLKESVDYFKIEK

[0125] OspC / Mcpl Conjugate protein with GS linker [SEQ ID NO: 22]

[0126] MKATKLVLGAVILGSTLLAGCSSNNSGKDGNTSANSADESVKGPNLTEISKKITDSNAVLLAVKEVEALLSSIDEIAAKAIGKKIHQNNGLDTENNHNGSLLAGAYAI STLIKQKLDGLKNEGLKEKIDAAKKCSETFTNKLKEKHTDLGKEGVTDADAKEAILK TNGTKTKGAEELGKLFESVEVLSKAAKEMLANSVKELTSPVVAESPKKPGSMKKFS FFKKKKKAVDLNLLNVVGNDKKDLSTYEYKAPVKEMLKGFYHTKASISTLKVGFES LQRILFSEIEDFDQIFSTFVEMTNNARNQISVIFSDLGKNNKEKLNQISSVIVGIQGSLET ISNFLGATNMISLNAKLEAARAKEYGKGFSVVADEIKRLSDQAKGVMNMISVKEIEE VSKDLISHNLKDLQIDIDKFFAEILEQLNYLESIFKRFSRSQEEFSSLIESLESIDANMAY YSRNCDSLIGSDTFMLSNDEFLKELEFIISDQFSWINNLRLLVEGQRTIFIQTDASKHGF GLFYKGLSPKNDAIRQLWEEVYIPYLNINKFAAEILIIFRVENRSDSGLRQAKDFLSQAESLSEEIVRKLEHIKKMVIELDNQGISIFS

[0127] OspC / Mcp2 MATD Conjugate protein with GS linker [SEQ ID NO: 23]

[0128] MKATKLVLGAVILGSTLLAGCSSNNSGKDGNTSANSADESVKGPNLTEISKKITDSNAVLLAVKEVEALLSSIDEIAAKAIGKKIHQNNGLDTENNHNGSLLAGAYAI STLIKQKLDGLKNEGLKEKIDAAKKCSETFTNKLKEKHTDLGKEGVTDADAKEAILK TNGTKTKGAEELGKLFESVEVLSKAAKEMLANSVKELTSPVVAESPKKPGSNSLQER VKLLKENGDHLFSEINKTHNTIKNSNQYIEKTQEEVEKQVEFISNTTNIIESLSKNISSL DNSIETQAASVEQSSSAIEEMIGGIQSITEITQKAAKSTEELKRFSDDGRKKQEEVITQI KEIFKNSTRLQEANSLISSIASQTNLLSMNAAIEASHAGEAGKGFAIVAEEIKDLAEQV TSQSESVASSINEIMDSITKTVNTSELTNKAFNQIFDSINLVVQVIEEINHTMQEQSIGS QEILKALNTMREITYEVKIGSNDMFRGNKEIISTIKLLGEINITVSNSMKGLKEEINTLV EAIERIKVLGTTNSSHISGISESINQFKTK

[0129] OspC / Mcp3 MATD Conjugate protein with GS linker [SEQ ID NO: 24]

[0130] MKATKLVLGAVILGSTLLAGCSSNNSGKDGNTSANSADESVKGPNLTEISKKITDSNAVLLAVKEVEALLSSIDEIAAKAIGKKIHQNNGLDTENNHNGSLLAGAYAI STLIKQKLDGLKNEGLKEKIDAAKKCSETFTNKLKEKHTDLGKEGVTDADAKEAILK TNGTKTKGAEELGKLFESVEVLSKAAKEMLANSVKELTSPVVAESPKKPGSIISYISS KLDNLSSKSNESFEKIKFYSEDLNEYLEQIETAISNTESIDSSILVYEQLRDTFSRFEKSI VDILKGFESIADPINDHNKYISEISSNFEESVSFFYSIDKNLEIFNKVATINSTDIENIKSK VFDLNIVFENVNKNFADLLSQTNSLQSVNKLLVSISAQTNMLAMNAAIEAAKAGDA GKSFAVVAEEIRKLAINSGKYSKTIKDELKTVDSIIAVINSEIDTIYKNFIDIQDNVDNNFSRHEKVDLTLAKHFKEIGEFKERYLSHDTKIRDAKNMYKEIFNNHYFISGKFNNFSQ DLKEFKVSKMNLDAVSSLQEYSSLVKSSKDKILKTKELIQKINDEIKDILF

[0131] OspC / Mcp4 MATD Conjugate protein with GS linker [SEQ ID NO: 25]

[0132] MKATKLVLGAVILGSTLLAGCSSNNSGKDGNTSANSADESVKGPNLTEIS KKITDSNAVLLAVKEVEALLSSIDEIAAKAIGKKIHQNNGLDTENNHNGSLLAGAYAI STLIKQKLDGLKNEGLKEKIDAAKKCSETFTNKLKEKHTDLGKEGVTDADAKEAILK TNGTKTKGAEELGKLFESVEVLSKAAKEMLANSVKELTSPVVAESPKKPGSNAIRVL VQDMVKGNLDKDYALDDDENTLDELGMLSLQVVKMKKAISVAISSVLRNISYVNK ASLEVASSSQNLSSSALQQASALEEMSANVEQIASGVNMSANNSYETEQIALKTNEN SQIGGRAVEESVIAMQDIVEKVSVIEEIARKTNLLALNAAIEAARAGDEGKGFAVVAS EIRKLADLSKISALEIGELVEDNSKVATEAGVIFKEMLPEIEETANLVKKISEGSSKQS DQIAQFKMALDQVGEVVQSSASSSEQLSSMSDKMLEKSKELRKSVLFFKIKDSK

[0133] OspC / Mcp5 MATD Conjugate protein with GS linker [SEQ ID NO: 26]

[0134] MKATKLVLGAVILGSTLLAGCSSNNSGKDGNTSANSADESVKGPNLTEIS KKITDSNAVLLAVKEVEALLSSIDEIAAKAIGKKIHQNNGLDTENNHNGSLLAGAYAI STLIKQKLDGLKNEGLKEKIDAAKKCSETFTNKLKEKHTDLGKEGVTDADAKEAILK TNGTKTKGAEELGKLFESVEVLSKAAKEMLANSVKELTSPVVAESPKKPGSYEGLE QLRTNFSSVAKGVIENLDYLYENAIQIANASQNLSSGAVEQASTLEQMTANIEQISQG VSENTENAATTEKIAVNTNERTKEGHKSVVKAIEAMTVITEKIGIIDEITRQTNLLALN ASIEAARVGEKGKGFEVVAAEVRKLADQSKESAREIIDIANRSLTVASRAGENFEQIV PGMEQTARLVKNISNESYKQSVQIEQFKNAIEQVSQLVQTTASSSEELSAMSEKMLES VKDLKESVDYFKIEK

[0135] OspC C19A / Mcpl Conjugate protein with GS linker [SEQ ID NO: 27]

[0136] MKKNTLSAILMTLFLFISANNSGKDGNTSANSADESVKGPNLTEISKKITD SNAVLLAVKEVEALLSSIDEIAAKAIGKKIHQNNGLDTENNHNGSLLAGAYAISTLIK QKLDGLKNEGLKEKIDAAKKCSETFTNKLKEKHTDLGKEGVTDADAKEAILKTNGT KTKGAEELGKLFESVEVLSKAAKEMLANSVKELTSPVVAESPKKPGSMKKFSFFKK KKKAVDLNLLNVVGNDKKDLSTYEYKAPVKEMLKGFYHTKASISTLKVGFESLQRI LFSEIEDFDQIFSTFVEMTNNARNQISVIFSDLGKNNKEKLNQISSVIVGIQGSLETISNF LGATNMISLNAKLEAARAKEYGKGFSVVADEIKRLSDQAKGVMNMISVKEIEEVSK DLISHNLKDLQIDIDKFFAEILEQLNYLESIFKRFSRSQEEFSSLIESLESIDANMAYYSR NCDSLIGSDTFMLSNDEFLKELEFIISDQFSWINNLRLLVEGQRTIFIQTDASKHGFGLF YKGLSPKNDAIRQLWEEVYIPYLNINKFAAEILIIFRVENRSDSGLRQAKDFLSQAESL SEEIVRKLEHIKKMVIELDNQGISIFS

[0137] OspC C19A / Mcp2 MATD Conjugate protein with GS linker [SEQ ID NO: 28]

[0138] MKKNTLSAILMTLFLFISANNSGKDGNTSANSADESVKGPNLTEISKKITDSNAVLLAVKEVEALLSSIDEIAAKAIGKKIHQNNGLDTENNHNGSLLAGAYAISTLIK QKLDGLKNEGLKEKIDAAKKCSETFTNKLKEKHTDLGKEGVTDADAKEAILKTNGT KTKGAEELGKLFESVEVLSKAAKEMLANSVKELTSPVVAESPKKPGSNSLQERVKLL KENGDHLFSEINKTHNTIKNSNQYIEKTQEEVEKQVEFISNTTNIIESLSKNISSLDNSIE TQAASVEQSSSAIEEMIGGIQSITEITQKAAKSTEELKRFSDDGRKKQEEVITQIKEIFK NSTRLQEANSLISSIASQTNLLSMNAAIEASHAGEAGKGFAIVAEEIKDLAEQVTSQSE SVASSINEIMDSITKTVNTSELTNKAFNQIFDSINLVVQVIEEINHTMQEQSIGSQEILK ALNTMREITYEVKIGSNDMFRGNKEIISTIKLLGEINITVSNSMKGLKEEINTLVEAIER IKVLGTTNSSHISGISESINQFKTK

[0139] OspC C19A / Mcp3 MATD Conjugate protein with GS linker [SEQ ID NO: 29]

[0140] MKKNTLSAILMTLFLFISANNSGKDGNTSANSADESVKGPNLTEISKKITD SNAVLLAVKEVEALLSSIDEIAAKAIGKKIHQNNGLDTENNHNGSLLAGAYAISTLIKQKLDGLKNEGLKEKIDAAKKCSETFTNKLKEKHTDLGKEGVTDADAKEAILKTNGT KTKGAEELGKLFESVEVLSKAAKEMLANSVKELTSPVVAESPKKPGSIISYISSKLDNLSSKSNESFEKIKFYSEDLNEYLEQIETAISNTESIDSSILVYEQLRDTFSRFEKSIVDILK GFESIADPINDHNKYISEISSNFEESVSFFYSIDKNLEIFNKVATINSTDIENIKSKVFDLN IVFENVNKNFADLLSQTNSLQSVNKLLVSISAQTNMLAMNAAIEAAKAGDAGKSFA VVAEEIRKLAINSGKYSKTIKDELKTVDSIIAVINSEIDTIYKNFIDIQDNVDNNFSRHE KVDLTLAKHFKEIGEFKERYLSHDTKIRDAKNMYKEIFNNHYFISGKFNNFSQDLKEF KVSKMNLDAVSSLQEYSSLVKSSKDKILKTKELIQKINDEIKDILF

[0141] OspC C19A / Mcp4 MATD Conjugate protein with GS linker [SEQ ID NO: 30]

[0142] MKKNTLSAILMTLFLFISANNSGKDGNTSANSADESVKGPNLTEISKKITD SNAVLLAVKEVEALLSSIDEIAAKAIGKKIHQNNGLDTENNHNGSLLAGAYAISTLIK QKLDGLKNEGLKEKIDAAKKCSETFTNKLKEKHTDLGKEGVTDADAKEAILKTNGTKTKGAEELGKLFESVEVLSKAAKEMLANSVKELTSPVVAESPKKPGSNAIRVLVQD MVKGNLDKDYALDDDENTLDELGMLSLQVVKMKKAISVAISSVLRNISYVNKASLE VASSSQNLSSSALQQASALEEMSANVEQIASGVNMSANNSYETEQIALKTNENSQIG GRAVEESVIAMQDIVEKVSVIEEIARKTNLLALNAAIEAARAGDEGKGFAVVASEIRK LADLSKISALEIGELVEDNSKVATEAGVIFKEMLPEIEETANLVKKISEGSSKQSDQIA QFKMALDQVGEVVQSSASSSEQLSSMSDKMLEKSKELRKSVLFFKIKDSK

[0143] OspC C19A / Mcp5 MATD Conjugate protein with GS linker [SEQ ID NO: 31]

[0144] MKKNTLSAILMTLFLFISANNSGKDGNTSANSADESVKGPNLTEISKKITD SNAVLLAVKEVEALLSSIDEIAAKAIGKKIHQNNGLDTENNHNGSLLAGAYAISTLIK QKLDGLKNEGLKEKIDAAKKCSETFTNKLKEKHTDLGKEGVTDADAKEAILKTNGT KTKGAEELGKLFESVEVLSKAAKEMLANSVKELTSPVVAESPKKPGSYEGLEQLRTN FSSVAKGVIENLDYLYENAIQIANASQNLSSGAVEQASTLEQMTANIEQISQGVSENT ENAATTEKIAVNTNERTKEGHKSVVKAIEAMTVITEKIGIIDEITRQTNLLALNASIEA ARVGEKGKGFEVVAAEVRKLADQSKESAREIIDIANRSLTVASRAGENFEQIVPGME QTARLVKNISNESYKQSVQIEQFKNAIEQVSQLVQTTASSSEELSAMSEKMLESVKDL KESVDYFKIEK

[0145] In the foregoing description, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and / or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0146] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0147] Citations to a number of patent and non-patent references are made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.

[0148] The following examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.EXEMPLARY EMBODIMENTS

[0149] Embodiment !. A polypeptide comprising: a first portion comprising or consisting of at least one first amino acid sequence that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 1-5; and a second portion comprising or consisting of at least one second amino acid sequence that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 6-11.

[0150] Embodiment . The polypeptide of embodiment 1. further comprising a linker sequence connecting the first portion to the second portion.

[0151] Embodiment 3. The polypeptide of embodiment 2, wherein the linker sequence comprises a glycine-serine (GS) linker, an alpha helix-forming linker, a proline-rich linker, a cleavable linker, or a combination thereof.

[0152] Embodiment 4. The polypeptide of embodiment 3, wherein the GS linker comprises 2-22 amino acids, wherein at least 50% of the amino acids are G and S, and optionally, wherein the GS linker comprises one or more of A, T, or a polar amino acid.

[0153] Embodiment 5. The polypeptide of embodiment 3 or 4, wherein the alpha helixforming linker comprises sequences of 1-5 repeating EAAAK motifs, and optionally, wherein the EAAAK motifs are flanked by A residues.

[0154] Embodiment 6. The polypeptide of any one of embodiments 3-5, wherein the proline linker comprises 6-24 amino acids consisting of XP repeats, where X is A, K, or E, or other non-proline amino acid.

[0155] Embodiment 7. The polypeptide of any one of embodiments 3-6. wherein the cleavable linker comprises or consists of a cyclopeptide containing a disulfide bond, or a protease-sensitive amino acid sequence that is an in vivo cleavable linker that is sensitive to proteases such as matrix metalloproteases, cathepsins, or furins.

[0156] Embodiment 8. The polypeptide of any one of embodiments 1-7. wherein the polypeptide is arranged so that the second portion is at the N-terminal side and the first portion is at the C-terminal side.

[0157] Embodiment 9. The polypeptide of any one of embodiments 1-4 and 8, where the polypeptide comprises or consists of an amino acid sequence that is at least 80%, 90%, 95%. 98%. 99%. or 100% identical to one or more of SEQ ID NOs: 12-31.

[0158] Embodiment 10. The polypeptide of any one of embodiments 1-9, wherein the polypeptide is lipidated.

[0159] Embodiment 11. The polypeptide of embodiment 10, wherein the polypeptide is lipidated with an N-terminal tripalmitoyl-S-glyceryl-cysteine (PamsCys)-moti f.

[0160] Embodiment 12. The polypeptide of any one of embodiments 1-11, wherein the first portion comprises at least two first amino acid sequences that are each at least 80%, 90%, 95%. 98%. 99%. or 100% identical to one or more of SEQ ID NOs: 1-5.

[0161] Embodiment 13. The polypeptide of any one of embodiments 1-12, wherein the second portion comprises at least two second amino acid sequences that each are at least 80%, 90%, 95%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 6-11.

[0162] Embodiment 14. A composition comprising the polypeptide of any one of embodiments 1-13.

[0163] Embodiment 15. The composition of embodiment 14, further comprising an adjuvant.

[0164] Embodiment 16. The composition of embodiment 15, wherein the adjuvant comprises an alum component, e.g.. an alum emulsion, and / or wherein the adjuvant comprises a natural or synthetic pattern recognition receptor ligand.

[0165] Embodiment 17. The composition of embodiment 15 or 16, wherein the composition is in lyophilized form.

[0166] Embodiment 18. A method of treating Lyme disease, or a method for the preventative treatment of Lyme disease, the method comprising: administering to a subject the polypeptide of any one of embodiments 1-13, or the composition of any one of embodiments 14-17.

[0167] Embodiment 19. The method of embodiment 18, wherein the subject is asymptomatic for Lyme disease, but may have come in contact with a blacklegged tick selected from Ixodes scapular is or Ixodes pact ficus.

[0168] Embodiment 20. The method of embodiment 19, wherein the possible contact was within 3 months, 2 months, 1 month, 2 weeks. 1 week, 3 days, or 1 day.

[0169] Embodiment 21. The method of any one of embodiments 18-20. wherein the subject has not previously been treated for Lyme disease.

[0170] Embodiment 22. The method of any one of embodiments 18-20, wherein the subject is asymptomatic for Lyme disease and has previously been treated for Lyme disease.

[0171] Embodiment 23. The method of any one of embodiments 18-22, wherein the subject previously tested positive for Lyme disease by standard two-tier (STT) testing.

[0172] Embodiment 24. The method of embodiment 18, wherein the subject has previously been treated for Lyme disease and exhibits one or more of the following symptoms: erythema migrans, facial palsy, and arthritis; and / or the subject tests positive for Lyme disease in a standard two-tier test (STT).

[0173] Embodiment 25. The method of any one of embodiments 18-20 and 22-24, wherein the subject has not received treatment for Lyme disease for at least 1 year, at least 5 years, at least 10 years, or at least 15 years.

[0174] Embodiment 26. The method of any one of embodiments 18-25, wherein the Lyme disease is caused by a Borrelia sp. bacteria.

[0175] Embodiment 27. The method of embodiment 26, wherein the Lyme disease is caused by Borrelia burgdorferi.

[0176] Embodiment 28. A carrier protein for a vaccine, comprising or consisting of at least one first amino acid sequence that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 1-5.

[0177] Embodiment 29 A polypeptide for a vaccine, comprising a carrier polypeptide portion comprising or consisting of at least one first amino acid sequence that is at least 80%. 90%, 95%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 1-5, covalently linked to another polypeptide portion.EXAMPLE

[0178] A peptide within the Mcp4 cytosolic methyl-accepting transducer domain (MATD) amino acids 442-462 was identified using an immunopeptidomics approach to isolate and identify MHC class Il-bound peptides (CD4+ T cell epitopes) derived from the Lyme disease spirochete Borrelia burgdorferi (Bb). The Mcp4 peptide was synthesized and analyzed for immunogenicity in a mouse Lyme disease model. Black 6 mice were infected with Bb for 4 weeks. Following the 4 week infection, T cells were harvested from mouse lymph nodes and stimulated with the Mcp4 peptide, a peptide derived from chicken ovalbumin (OVA) that binds strongly to black mouse MHC-II molecules labeled as Ova, or a peptide derived from Bb protein LptD. After 5 days, T cell reactivity was assessed with a proliferation assay. As demonstrated in FIG. IE, the Mcp4 peptide exhibited improved T cell reactivity, as compared to the OVA and LptD peptides.

[0179] To demonstrate the efficacy of the conjugate vaccine approach for treating Lyme disease, a conjugate polypeptide (SEQ ID NO: 15) was synthesized, which included the Mcp4- MATD peptide (SEQ ID NO: 4; T cell immunogen) and an OspA peptide (SEQ ID NO: 6)B-cell immunogen), linked by a GS linker. The structure of the purified conjugate polypeptide and its constituent Mcp4-MATD and OspA peptides was confirmed by size exclusion chromatography (SEC) and Coomassie Blue staining (FIG. 3). Circular dichroism spectra analysis (FIG. 4A) and thermal unfolding analysis (FIGS. 4B-4C) confirmed the proper folding of the conjugate protein.

[0180] The immunogenic activity of the conjugate polypeptide was analyzed using an in vivo model of Lyme disease. Three groups of mice (treatment groups; 3M / 3F per group) were immunized with either (1) the conjugate protein (SEQ ID NO: 15), (2) the OspA peptide (SEQ ID NO: 6) alone, or (3) adjuvant alone (15 pg / mouse). Two non-immunized, Bb-infected mice were used as a control group. 21 days after the initial immunization, the treatment groups were given a booster immunization + adjuvant (15 pg / mouse), then infected with 2xl04B. burgdorferi or BSK medium for uninfected control 21 days post-boost.

[0181] To assess the ability of the conjugate polypeptide to elicit a T cell-associated immunogenic response, inguinal lymph node T cells were harvested from the BSK uninfected controls and Bb-challenged mice. The harvested T cells were analyzed to determine the proportion of CD44+ CD6210CD4+ T effector cells, as a percent of total CD4+ T cells. CD44+ CD6210CD4+ T effector cells are ‘'memory’’ T cells, which play a vital role in providing longterm immunity against pathogenic infection. As shown in FIG. 5A, the mice administered the conjugate polypeptide followed by the Bb challenge exhibited a significantly increased proportion of CD4+ T effector cells, as compared to mice administered the conjugate polypeptide without the Bb challenge. Notably, this Bb-induced elevation of CD4+ T effector cells was not present in the mice administered OspA alone or adjuvant alone.

[0182] To assess the effect of the conjugate polypeptide on the immune response to OspA, serum was collected from 3 mice per group and analyzed (1:800 dilution) by enzyme-linked immunosorbent assay (ELISA) to determine anti-OspA IgG levels. As demonstrated in FIG. 5C, there was no difference in serum anti-OspA IgG levels between mice administered the conjugate polypeptide and those administered OspA alone.

[0183] Collectively, these findings demonstrate that administration of a conjugate polypeptide comprising a T cell immunogen (e.g.. Mcp4-MATD) and a B cell immunogen (e.g., OspA) can enhance a protective immune response to immunization.

[0184] References

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[0186] 2. Paules CI, Marston HD, Bloom ME, Fauci AS. Tickbome Diseases -Confronting a Growing Threat. N Engl J Med. 2018;379(8): 701 -3.

[0187] 3. Wormser GP, McKenna D, Karmen CL, Shaffer KD, Silverman JH, Nowakowski J, et al. Prospective Evaluation of the Frequency and Severity of Symptoms in Lyme Disease Patients With Erythema Migrans Compared With Matched Controls at Baseline, 6 Months, and 12 Months. Clin Infect Dis. 2020.

[0188] 4. Lochhead RB, Strle K, Arvikar SL, Weis JJ. Steere AC. Lyme arthritis: linking infection, inflammation and autoimmunity7. Nat Rev Rheumatol. 2021;17(8):449-61.

[0189] 5. Steere AC, Schoen RT, Taylor E. The clinical evolution of Lyme arthritis. AnnIntern Med. 1987;107(5):725-31.

[0190] 6. Jutras BL, Lochhead RB. Kloos ZA, Biboy J, Strle K, Booth CJ, et al. Borrelia burgdorferi peptidoglycan is a persistent antigen in patients with Lyme arthritis. Proc Natl Acad Sci U S A. 2019;! 16(27): 13498-507.

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Claims

CLAIMS1. A polypeptide comprising: a first portion comprising or consisting of at least one first amino acid sequence that is at least 80%, 90%, 95%, 98%, 99%. or 100% identical to one or more of SEQ ID NOs:

4. 1-3, and 5; and a second portion comprising or consisting of at least one second amino acid sequence that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 6-11.

2. The polypeptide of claim 1, further comprising a linker sequence connecting the first portion to the second portion.

3. The polypeptide of claim 2, wherein the linker sequence comprises a glycine-serine (GS) linker, an alpha helix-forming linker, a proline-rich linker, a cleavable linker, or a combination thereof.

4. The polypeptide of claim 3, wherein the GS linker comprises 2-22 amino acids, wherein at least 50% of the amino acids are G and S, and optionally, wherein the GS linker comprises one or more of A, T, or a polar amino acid.

5. The polypeptide of claim 3, wherein the alpha helix-forming linker comprises sequences of 1-5 repeating EAAAK motifs, and optionally, wherein the EAAAK motifs are flanked by A residues.

6. The polypeptide of claim 3, wherein the proline linker comprises 6-24 amino acids consisting of XP repeats, where X is A, K, or E. or other non-proline amino acid.

7. The polypeptide of claim 3, wherein the cleavable linker comprises or consists of a cyclopeptide containing a disulfide bond, or a protease-sensitive amino acid sequence that is an in vivo cleavable linker that is sensitive to proteases such as matrix metalloproteases, cathepsins, or furins.

8. The polypeptide of claim 1, wherein the polypeptide is arranged so that the second portion is at the N-terminal side and the first portion is at the C-terminal side.

9. The polypeptide of claim 1 , where the polypeptide comprises or consists of an amino acid sequence that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 15, 12-14, and 16-31.

10. The polypeptide of claim 1, wherein the polypeptide is lipidated.

11. The polypeptide of claim 10, wherein the polypeptide is lipidated with an N-terminal tripalmitoyl-S-glyceryl-cysteine (ParmCysj-motif12. The polypeptide of claim 1, wherein the first portion comprises at least two first amino acid sequences that are each at least 80%, 90%, 95%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 4, 1-3, and 5.

13. The polypeptide of claim 1, wherein the second portion comprises at least two second amino acid sequences that each are at least 80%, 90%, 95%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 6-11.

14. A composition comprising the polypeptide of claim 1.

15. The composition of claim 14, further comprising an adjuvant.

16. The composition of claim 15, wherein the adjuvant comprises an alum component, e.g., an alum emulsion, and / or wherein the adjuvant comprises a natural or synthetic pattern recognition receptor ligand.

17. The composition of claim 15, wherein the composition is in lyophilized form.

18. A method of treating Lyme disease, or a method for the preventative treatment of Lyme disease, the method comprising: administering to a subject the polypeptide of claim 1, or the composition of claim 14.

19. The method of claim 18, wherein the subject is asymptomatic for Lyme disease, but may have come in contact with a blacklegged tick selected from Ixodes sccipularis or Ixodes paciflcus.

20. The method of claim 18, wherein the possible contact was within 3 months, 2 months, 1 month, 2 weeks, 1 week, 3 days, or 1 day.

21. The method of claim 18, wherein the subj ect has not previously been treated for Lyme disease.

22. The method of claim 18, wherein the subject is asymptomatic for Lyme disease and has previously been treated for Lyme disease.

23. The method of claim 18, wherein the subject previously tested positive for Lyme disease by standard two-tier (STT) testing.

24. The method of claim 18, wherein the subject has previously been treated for Lyme disease and exhibits one or more of the following symptoms: erythema migrans, facial palsy, and arthritis; and / or the subject tests positive for Lyme disease in a standard two-tier test (STT).

25. The method of claims 18, wherein the subject has not received treatment for Lyme disease for at least 1 year, at least 5 years, at least 10 years, or at least 15 years.

26. The method of claim 18, wherein the Lyme disease is caused by aBorrelia sp. bacteria.

27. The method of claim 26, wherein the Lyme disease is caused by Borrelia burgdorferi.

28. A carrier protein for a vaccine, comprising or consisting of at least one first amino acid sequence that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 4, 1-3, and 5.

29. A polypeptide for a vaccine, comprising a carrier polypeptide portion comprising or consisting of at least one first amino acid sequence that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 4, 1-3, and 5, covalently linked to another polypeptide portion.

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