HK1207982B

HK1207982B - Mutant fragments of ospa and methods and uses relating thereto

Info

Publication number: HK1207982B
Application number: HK15108670.3A
Authority: HK
Inventors: 帕尔．科姆斯泰特; 乌尔班．伦德贝格; 安德烈亚斯．迈因克; 马库斯．汉纳; 沃尔夫冈．舒勒; 本亚明．维策尔; 克里斯托夫．赖尼施; 布里吉特．格罗曼; 罗伯特．施勒格尔
Original assignee: 瓦尔尼瓦奥地利有限责任公司
Priority date: 2012-07-06
Filing date: 2013-07-08
Publication date: 2018-07-13

Description

Mutant fragments of OspA and methods and uses related thereto

Technical Field

The present invention relates to compositions and methods for the prevention and treatment of Borrelia (Borrelia) infection. In particular, the present invention relates to a polypeptide comprising a mutant fragment of an outer surface protein a (ospa), a nucleic acid encoding said polypeptide, a pharmaceutical composition comprising said polypeptide and/or said nucleic acid (particularly suitable for use as a medicament in a method of treating or preventing borrelia infection), a method of treating or preventing borrelia infection and a method of immunizing a subject.

Background

Lyme borreliosis or lyme disease is the most commonly reported tick-borne disease in europe and north america. The disease is caused by insect-vector transmitted gram-negative spirochete borrelia burgdorferi (b.burgdorferi s.l.) and is an infection that may involve a variety of organs or tissues, leading to skin, heart, musculoskeletal and neurological disorders. In most countries lyme borreliosis is not an on-going disease and no accurate data on annual incidence is available. In the united states, the causative agent is borrelia burgdorferi (b. burgdorferi s.s) in the narrow sense and borrelia lyme disease is localized in the north eastern, middle atlantic and upper central and north states. In 2010, a total of about 30,000 cases of lyme borreliosis were reported by the united states centers for disease control and prevention (CDC). In europe, borrelia afzelii (b.afzelii) and borrelia garinii (b.garinii) are the main causative agents of lyme borreliosis, and borrelia burgdorferi and borrelia bavariensis (b.bavariensis) in the sense of geographical location contribute to lyme borreliosis to a lesser extent. The prevalence of lyme borreliosis varies considerably in different european countries and generally increases gradually from west to east. In most European countries, the number of reported cases of lyme borreliosis has increased gradually since the early 90 s of the 20 th century (e.g., Czech republic, Isania, Litotan; see Lymeborreliosis in Europe, WHO 2006), and the geographical distribution of cases has also expanded.

Borrelia belong to the family spirochaetaceae, which is subdivided into the medically important genera Treponema (Treponema), Leptospira (Leptospira) and borrelia. Borrelia burgdorferi, a spiral, motile gram-negative bacterium about 10-20 μm long and 0.2-0.5 μm wide, grows under microaerophilic conditions. The spirochete cell wall is composed of a cytoplasmic membrane surrounded by peptidoglycan and several flagella and then by a loosely associated outer membrane.

Lyme borreliosis usually occurs in stages, each stage being characterized by different clinical manifestations of remission and worsening. Stage 1, early infection, consisting of topical infection of the skin; days or weeks later, stage 2, disseminated infection; months to years later, stage 3, persistent infection. However, infection is variable; some patients only suffer from local infections of the skin, while others show only late-stage manifestations of the disease, such as arthritis. Different clinical symptoms of lyme borreliosis are also caused by infection with different generalized borrelix burgdorferi species. Borrelia burgdorferi in the sense causes more frequently articular manifestations (arthritis) and cardiac problems, borrelia afzelii mainly causes cutaneous symptoms (migratory erythema EM and chronic atrophic acrodermatitis ACA), while borrelia galschlii is implicated in the majority of lyme neuroborreliosis cases.

Topical infection-the most common symptom of stage 1 of infection is migratory erythema, which occurs in 70% -80% of the infected population. This skin damage is often followed by flu-like symptoms such as myalgia, joint pain, headache and fever. These non-specific symptoms occur in 50% of patients with migratory erythema.

Disseminated infection-during stage 2, bacteria move from the site of infection into the bloodstream, reaching distant tissues and organs. Neurological, cardiovascular and arthritic conditions that occur at this stage include meningitis, cranial neuropathy and intermittent inflammatory arthritis.

Persistent infection-phase 3 of the infection is chronic and occurs months to years after a tick bite. The most common symptom in north america is rheumatoid arthritis caused by borrelia burgdorferi infection in the narrow sense. Borrelia garinii-induced persistent infection of the central nervous system leads to more severe neurological symptoms during stage 3, and borrelia aryabhattai-induced persistent infection of the skin leads to chronic atrophic limb dermatitis.

In some risk groups such as farmers, forestry workers, hikers, runners or vacationers, as in children under the age of 15 and adults between the ages of 39 and 59, the seropositivity and incidence of disease are increased with no gender preference. This increased incidence of lyme borreliosis is associated with changes in forest habitats and social factors. Environmental changes such as fragmentation of forests have resulted in a dramatic reduction in rodent predators such as foxes and prey birds, which in turn has resulted in an increase in the number of rats and subsequently an increase in the number of ticks. Recently, unbalanced re-forestation has increased the number of deer and thus ticks. The expansion of suburban areas and the increased use of woodland areas for recreational uses such as camping and hiking have brought people into deeper contact with a greater number of borrelia tick carriers. All of these factors together lead to a broader distribution of borrelia and a higher incidence of lyme borreliosis.

Antimicrobial agents are the primary method of treating borrelia infections. The antibiotics used depend on the stage of the disease, symptoms and the patient's allergies to the drugs. The length of the course of antibiotic therapy also depends on the stage of the disease and the severity of the symptoms. Early stage lyme borrelia disease is commonly treated with oral tetracycline such as doxycycline and semi-synthetic penicillin such as amoxicillin or penicillin V. Arthritis and neurological conditions are treated with high doses of intravenous penicillin G or ceftriaxone. Up to 30% of patients with borrelia lyme do not exhibit the early symptoms characteristic of borrelia infection, which makes both diagnosis and treatment problematic. Antibiotic courses can be long (up to several months) and sometimes ineffective, and are therefore highly controversial in the field of borrelia, particularly during advanced disease. Even with effective treatment of borrelia, patients can suffer from debilitating fatigue, pain or neurological symptoms after years, which are referred to as post-treatment lyme disease symptoms. Often, the use of antibiotics can have undesirable consequences, such as the development of resistance by the target microorganism. Finally, antibiotic therapy can effectively cure lyme borreliosis, but does not provide protection against subsequent infection.

Monovalent serotype 1-OspA-based vaccines (LYMErix)^TM) Approved in the united states and marketed for the prevention of lyme disease caused by borrelia burgdorferi sensu stricta. However, the heterogeneity of OspA sequences in different serotypes in europe and elsewhere has prevented effective protection with OspA-based vaccines from only one serotype.

Chimeric OspA molecules comprising a proximal portion from one OspA serotype along with a distal portion from another OspA serotype can be used for the prevention and treatment of lyme disease or lyme borreliosis while retaining the antigenic features of the two parent polypeptides (WO2011/143617, WO 2011/143623).

Currently, there is no prophylactic agent on the market for lyme borreliosis, and there is therefore a need in the art to develop such an agent that can provide effective protection against borrelia present in the united states, europe and elsewhere, and in particular to develop an agent that can provide effective protection against several borrelia serotypes simultaneously.

Summary of The Invention

The present invention relates to a polypeptide comprising a mutant fragment of borrelia outer-surface protein a (ospa), a nucleic acid encoding the polypeptide, a vector comprising such nucleic acid molecule and a host cell comprising such vector. Furthermore, the present invention provides a method for producing such a polypeptide and a method for producing a cell expressing such a polypeptide. In addition, the invention provides antibodies that specifically bind such polypeptides; hybridoma cells that produce such antibodies; methods for producing such antibodies; pharmaceutical compositions comprising such polypeptides, nucleic acid molecules, vectors or antibodies; the use of such a polypeptide, nucleic acid molecule, vector or antibody for the preparation of a medicament or pharmaceutical composition, in particular for use as a vaccine or in a method for the treatment or prevention of borrelia infection; methods for diagnosing infections and for treating or preventing borrelia infections and methods of immunizing subjects.

Efforts to develop subunit vaccines for the prevention of lyme borreliosis have largely focused on the use of borrelia outer surface protein a (ospa) as an antigen. The OspA protein is expressed by borrelia only when in the tick vector intestine. Thus, OspA antibodies generated by vaccination do not fight infection in vivo, but rather enter the gut of ticks when they ingest blood. In the intestine, the antibodies neutralize spirochetes and block bacterial migration from the midgut of ticks to salivary glands, i.e., block the route by which borrelia enter the vertebrate host. Thus, OspA-specific antibodies prevent borrelia from being delivered from tick vectors into human hosts.

Lipidated forms of OspA from Borrelia burgdorferi strain ZS7 were commercially developed with aluminum hydroxide from SmithKlineBeecham (now GlaxoSmithKline (GSK)) as vaccines against Borrelia (LYMERix)^TM) For use in the U.S. market. Three doses of LYMERix are required over a one year period^TMFor optimal protection. The vaccine efficacy against lyme borreliosis was 49% after the first two doses and 76% after the third dose. However, in LYMERix^TMShortly after commercial availability, it exited the market in 2002. The reasons cited are problems with the practical use of vaccines, such as the need for booster injections every year or every other year, and the relatively high cost of this method of prevention compared to antibiotic treatment of early infections. In addition, a problem is found in LYMERix^TMAn autoimmune response can be elicited in a subset of the population due to sequence homology to human proteins, although this has not been demonstrated. Furthermore, this vaccine does not provide cross-protection against clinically important borrelia species.

Accordingly, in one embodiment, it is an object of the present invention to provide an improved vaccine for the prevention of lyme borreliosis. Preferably, the vaccine is easy to produce while being protective, safe and more effective than existing therapies and/or providing protection against more than one borrelia species.

The problem underlying the present invention is solved by a polypeptide comprising a mutant fragment of an outer surface protein a (OspA), wherein said mutant fragment consists of the C-terminal domain of a borrelia OspA protein and differs from the corresponding wild-type fragment at least by the introduction of at least one disulfide bond.

Surprisingly, it was found that the introduction of at least one disulfide bond in the mutant fragment increases the protective capacity of the polypeptide comprising the mutant OspA fragment relative to the polypeptide comprising the wild-type OspA fragment, as shown in the in vivo infection model. As shown in the examples, the introduction of at least one disulfide bond in the C-terminal fragment of OspA of Borrelia afzelii increases its protective capacity relative to the wild-type OspA fragment without a disulfide bond. The data provided in tables 2 and 3 indicate the protective ability of the mutant fragment with the introduced disulfide bond ("S2D 1-5") compared to the wild-type OspA fragment ("S2D 0"), since fewer animals were infected after immunization with the mutant OspA fragment compared to the wild-type OspA fragment. Some of the mutant OspA fragments tested provided protection comparable to that conveyed by the positive control antigen non-lipidated full-length OspA protein.

Detailed Description

Thus, in a first aspect, the present invention relates to a polypeptide comprising a mutant fragment of the outer surface protein a (OspA), wherein the mutant fragment consists of the C-terminal domain of borrelia OspA and differs from the corresponding wild-type fragment by at least the introduction of at least one disulfide bond.

The term Borrelia burgdorferi in general encompasses at least 13 Borrelia species (Table A-1). These species occur in different geographical areas and live in nature and in a wide range of animal hosts with endemic animal disease cycles involving ticks of the ricinus communis (Ixodes ricinus) complex, also known as the ixodersulatus complex. Four borrelia species are responsible for most human infections: borrelia burgdorferi, Borrelia afzelii, Borrelia bavaria and Borrelia garinii. Three other species borrelia ruhatitania (b.lusitaniae), borrelia bicistributa (b.bissetti) and borrelia starmannii (b.spielmanii) have occasionally been detected in humans, but their role in lyme borrelia disease is currently uncertain. New borrelia species are still being reported.

Table A-1.

As detailed above, borrelia outer surface protein a (ospa) is an abundant borrelia immunogenic lipoprotein of particular interest due to its potential as a vaccine candidate. OspA of borrelia burgdorferi generalizes a basic lipoprotein with a molecular weight of about 30kDa and encoded on a linear plasmid. An important aspect of OspA proteins is their N-terminal lipidation; that is, the N-terminal cysteine residue is substituted with a fatty acid having a chain length between C14 and C19 with or without a double bond (a feature that enhances the immunogenicity of the OspA protein). Synthetic peptides that have been shown to be poorly immunogenic induce a stronger antibody response upon lipidation; for example, in covalent attachment to Pam₃Cys (Bessler and Jung, Research Immunology (1992) 143: 548-. In addition, Pam is shown₃The Cys moiety enhances the immune response to OspA in mice by virtue in part of its interaction with TLR-2 (Yoder et al (2003) Infection and Immunity 71: 3894-3900). Thus, lipidation of the OspA C-terminal fragment would be expected to enhance the immunogenicity and protective capacity of the fragment.

Analysis of isolates of Borrelia burgdorferi generalised obtained in the northern United states and Europe has revealed that OspA has antigenic variation and several different groups can be defined based on serology. anti-OspA mabs have been reported that bind specific N-terminal and C-terminal epitopes. X-ray crystallography and NMR analysis have been used to identify immunologically important hypervariable domains in OspA and have mapped the LA-2 epitope at C-terminal amino acid 203-257 (Ding et al, mol.biol.302: 1153-64, 2000). Previous studies have shown that the production of antibodies against the C-terminal epitope LA-2 following vaccination with OspA is associated with protective immunity (Van Hoecke et al Vaccine (1996)14 (17-18): 1620-6 and Steere et al, N Engl JMed (1998) 339: 209-215). Antibodies to LA-2 have been shown to block borrelia transmission from ticks to the host (Golde et al, infection Immun (1997)65 (3): 882-. These studies indicate that the C-terminal portion of the OspA protein may be sufficient for inducing protective immunity. It should be noted that the sequence of the C-terminal part of OspA is highly conserved between Borrelia serotypes less than the N-terminal part (see FIG. 1).

Based on information from the studies listed above, along with other studies, truncated forms of OspA (also referred to herein as "OspA fragments" or "monomers") comprising the C-terminal portion are used in the present invention. These truncated forms of OspA have been shown to have lower protection than the full-length OspA protein. However, it was surprisingly found in the course of the present invention that the introduction of a disulfide bond in a truncated form (also referred to herein as a "mutant OspA fragment" or "mutant fragment") overcomes this disadvantage. While not limited to a particular mechanism, the improved protection is believed to be due to increased OspA fragment stability, as shown in assays measuring thermostability.

According to the invention, the mutant OspA fragment may be derived from any borrelia species; however, due to their relevance in the medical field (especially for humans), borrelia burgdorferi sensu stricto, borrelia afzelii, borrelia bavaria and borrelia garinii are preferred. In this regard, these four borrelia species can be further classified according to their OspA serotypes, which have been determined by analysis using monoclonal antibodies specific for the corresponding OspA proteins. Serotypes 1-7, which cause infection by most human borrelia, along with their prevalence, are shown in table a-2 below.

TABLE A-2 indications and prevalence of serotypes of Borrelia burgdorferi, Borrelia afzelii, Borrelia bavaria, and Borrelia garinii. Borrelia isolated from human cerebrospinal fluid or skin or from tick vectors were serotyped by probing whole cell lysates with mouse monoclonal antibodies, each specific for a particular epitope of OspA (as described by Wilske et al, j.of Clin Microbiol (1993)31 (2): 340-.

The structure of the OspA protein from Borrelia burgdorferi strain B31 in the narrow sense was determined by Li et al (Proc Natl Acad Sci (1997) 94: 3584-3589). It consists of an N-terminal (. beta. -strand 1 to 4) and a central. beta. -sheet (. beta. -strand 5 to 14N [ N-terminal part ]), a cylindrical sheet 1 (. beta. -strand 14C [ C-terminal part ] to 16), a cylindrical sheet 2 (. beta. -strand 17 to 21) and a C-terminal. alpha. -helix. The term "OspA C-terminal domain" or "wild-type fragment" or "C-terminal portion" in reference to OspA as used throughout the specification shall mean the C-terminal amino acid sequence of OspA, i.e. OspA lacks at least the N-terminal β -sheet (including β -strands 1 to 4). In OspA from Borrelia burgdorferi strain B31, the N-terminal fold consists of amino acids 17 to 70 (after post-translational cleavage of the 16aa long lipidated signal peptide).

The C-terminal OspA fragment of the invention may also include an N-terminal lipidation signal sequence, such as the lipidation signal sequence of amino acids 1 to 16 of OspA (SEQ ID NO: 14) or OspB (SEQ ID NO: 15) from Borrelia burgdorferi strain B31, from E.coli (referred to herein as the "lpp lipidation signal" (SEQ ID NO: 16)) or any other signal sequence, for example as defined below.

Lipidation of proteins with N-terminal lipidation signal sequences, such as those present on primary OspA polypeptides, occurs in e.coli expression vectors by the stepwise action of the enzymes diacylglycerol transferase, signal peptidase II and transacylase, respectively. The first step is to transfer the diacylglycerol to the cysteine thiol group of the unmodified pre-lipoprotein, then to cleave the signal peptide by signal peptidase II and finally to acylate the α -amino group of the N-terminal cysteine of the pre-lipoprotein. The result is the placement of one lipid and a glyceryl moiety substituted with two additional lipids on the polypeptide's N-terminal cysteine residue. The lipidation signal sequence cleaved during lipidation is not present in the final polypeptide sequence.

According to the invention, the mutant OspA fragment can be a lipidated protein,also known as lipoproteins, where the lipid moiety together with the glycerols is also known as "Lip". According to the invention, Lip comprises one to three lipids, such as C, linked to glycerol and an N-terminal cysteine of a polypeptide of the invention_14-20Alkyl and/or C_14-20Alkenyl, or preferably wherein Lip is a moiety of formula (I):

wherein R is₁、R₂Or R₃One of them is C₁₄-C₂₀Alkyl or alkenyl, and each of the others is independently C₁₄-C₂₀Alkyl or C₁₄-C₂₀Alkenyl, and X is an amino acid sequence linked to a cysteine residue represented by formula (I). More preferably, the Lip plus N-terminal cysteine of the polypeptide is N-palmitoyl-S- (2RS) -2, 3-bis- (palmitoyloxy) propylcysteine (referred to herein as "Pam₃Cys ") and is linked to the amino acid sequence of the invention via the carboxyl group C of the cysteine. In the above formula (I), R₁、R₂And R₃May be a palmitoyl moiety and X is an amino acid sequence linked to a cysteine residue.

According to the present invention, the C-terminal domain of OspA from a strain other than Borrelia burgdorferi B31 in the narrow sense is defined as follows: (i) lacks at least amino acids 17 to 70 and/or (ii) lacks at least an N-terminal domain homologous to amino acids 17 to 70 of OspA from Borrelia burgdorferi B31, narrow. In addition, the OspA C-terminal domain according to the invention may also lack an additional central folding portion, in particular an additional chain, as defined by Li and co-workers thereof (Li et al, supra), such as an amino acid portion from amino acids 17 to 82, 93, 105, 118 or 119, preferably 17 to 129, more preferably 1 to 125, 1 to 129 or 1 to 130 of any borrelia, in particular borrelia burgdorferi B31, or a homologous portion of the OspA protein from a borrelia species other than borrelia burgdorferi B31.

In the context of the present invention, the OspA C-terminal domain is also referred to as an "OspA fragment" or "fragment of OspA".

"mutant fragment" in the context of the polypeptides of the invention and as used throughout the specification shall mean an OspA C-terminal fragment which differs from the wild-type fragment at least in at least two introduced cysteines which may form disulfide bonds, as defined above. Without being bound by this theory, it is hypothesized that the disulfide bonds stabilize the fragments in a configuration that favors antibody binding induction. Folding of the wild-type C-terminal Fragment of OspA shows reduced temperature stability compared to the full-length protein (Koide et al, Structure-Based Design of a Second-generation distance vaccine Based on a C-terminal Fragment of Bo rrelia burgdorferi OspA, J.Mol.biol. (2005) 350: 290-. For the present invention, the sequence of the C-terminal domain of Borrelia burgdorferi B31OspA in the narrow sense has been analyzed in silico to determine the location of the introduced disulfide bridge for enhancing the stability of the folding of this C-terminal domain. The results of the analysis have been spot-tuned to homologous OspA fragments of other borrelia species, assuming that folding is conserved between species.

Typically, disulfide bonds can be introduced by introducing one or more cysteine residues, wherein a disulfide bond (S-S bridge) is formed between the thiol groups of two cysteine residues. If the disulfide bond is formed with a cysteine present in the wild-type fragment, only one cysteine residue needs to be introduced. One or preferably two cysteines may be introduced by amino acid addition or preferably substitution.

The OspA mutant fragment may further comprise an additional mutation relative to the wild type. As detailed above, the structural and surface domains of OspA are known in the art. Thus, the mutant fragment may comprise further mutations, in particular mutations which are at sites which are not on the surface of the protein and/or which are not involved in the immune response and thus do not affect the antigenic capacity. These mutations include one or more amino acid deletions (particularly small (e.g., up to 10 amino acids) deletions), one or more amino acid additions (particularly at the C-terminus or N-terminus), one or more amino acid substitutions (particularly one or more conservative amino acid substitutions). Examples of conservative amino acid substitutions include, but are not limited to, those listed below:

preferred mutations include alterations in selected fragment portions, for example, where the sequence having sequence similarity to the human leukocyte function-associated antigen (hLFA-l) present in borrelia burgdorferi sensu stricto is modified, for example, by replacement with a homologous sequence from an OspA protein from another borrelia species. The rationale for this modification is to reduce the risk of inducing immunological cross-reactions with human proteins. It is also possible to add signal sequences for lipidation, or to add marker proteins (e.g., for identification or purification) in the final or intermediate fragments.

In some embodiments, the amino acid sequence of the mutant OspA fragment has 60%, preferably at least 70%, more preferably at least 80%, more preferably 85%, more preferably 90%, even more preferably 95% sequence identity to the wild-type fragment. In another embodiment, the sequences are at most 10%, at most 9%, at most 8%, at most 7%, at most 6%, 5%, 4%, 3%, 2%, most preferably at most 1% different due to sequence additions, deletions or substitutions.

As known in the art and as used herein, identity is a relationship between two or more polypeptide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between such sequence strings. Identity can be easily calculated. Although there are a variety of methods for measuring between two polynucleotide or two polypeptide sequences, the term is well known to the skilled artisan (e.g., Sequence Analysis in Molecular Biology, von Heinje, g., Academic Press, 1987). Preferred methods of determining identity are designed to obtain the maximum match between the tested sequences. Methods of determining identity are encoded in computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to, the GCG program package (Devereux, J. et al, 1984), BLASTP, BLASTN, and FASTA (Altschul, S. et al, 1990).

In contrast to the mutant OspA fragment, the "wild-type fragment" in the context of the present invention relates to a fragment of naturally occurring borrelia OspA. The wild-type fragment is obtained by N-terminal deletion, but it does not contain internal deletions (other than the signal sequence as detailed herein) or mutations. Relative to the mutant OspA fragments, the wild-type fragments consist of the same OspA part (OspA of the same length and same chain, etc.) and differ only in the mutations detailed above, in particular in the introduction of at least one disulfide bond or the replacement of sequences with human homology such as hLFA-l (see above).

According to a preferred embodiment of the invention, the polypeptide of the invention does not comprise or consist of a full length OspA polypeptide having at least one introduced disulfide bond.

In one embodiment of the invention, the mutant OspA fragment differs from the corresponding wild-type fragment only by the introduction of at least one, preferably exactly one, disulfide bond.

Polypeptides are single linear polymers of amino acids linked by peptide bonds, and in some cases also disulfide bonds. According to the invention, the polypeptide also comprises one or more post-translational modifications; i.e. a linked biochemical functional group, such as a linked acetate, phosphate, lipid or carbohydrate, preferably one or more lipids, more preferably 1 to 3C, linked to the N-terminal cysteine together with glycerol₁₄-C₂₀An alkyl or alkenyl moiety, even more preferably 1 to 3 palmitoyl groups, most preferably three palmitoyl groups (Pam)₃)。

According to the invention, the polypeptide of the invention comprises a mutant OspA fragment as described above. According to the invention, it does not comprise (i) an N-terminal fold as defined above and (ii) optionally one or more additional central fold chains as defined above. However, the polypeptide may comprise one or more functional sequences, such as a signal sequence, e.g. a lipidated signal sequence or a post-translational modification such as lipidation.

In another embodiment of the invention, the polypeptide of the invention consists of: (i) one or more mutant OspA fragments optionally linked by a linker, e.g. as defined below; and (ii) optionally one or more amino acids heterologous to OspA, in particular a signal sequence; and (iii) optional post-translational modifications such as lipidation.

The polypeptide of the invention has protective ability. As detailed above, the introduction of disulfide bonds into mutant OspA fragments increases the protective capacity of the polypeptide relative to a polypeptide comprising the corresponding fragment without disulfide bonds. In some embodiments, the protective capacity is increased by at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90% relative to a polypeptide comprising a corresponding fragment without disulfide bonds.

The term protective ability describes the ability to protect a subject against borrelia infection. With respect to the polypeptides of the invention, protective ability relates to the ability of the polypeptides to induce an immune response that protects a subject against borrelia infection. Protective capacity can be tested by administering the polypeptide to a subject in a manner that induces an immune response against the polypeptide. Thereafter, the subject was challenged with borrelia. The subject's response to the infection is monitored. In particular, the presence of borrelia in a subject may be determined. For example, the polypeptide is protective if borrelia cannot be detected in the subject. The presence of borrelia can be determined by detecting borrelia-specific nucleic acids (e.g. by PCR) or borrelia-specific antibodies (e.g. by ELISA or western blot) or by detecting borrelia itself (e.g. culturing an organ or tissue in growth medium and verifying the presence of borrelia by microscopy). Specifically, the protective capacity reported as a percentage ("pc") for a particular dose is defined as follows:

pc (%) ═ total test subject number-borrelia infected subject number)/total test subject number x100

The difference in protective ability (. DELTA.pc) can be determined, for example, by comparing the protective ability of a mutant OspA fragment having a disulfide bond (pc [ bonded ]) with that of an OspA fragment having no disulfide bond (pc [ non-bonded ]). According to the invention, the polypeptides to be compared differ only by the introduction of at least one disulfide bond. The change in protective capacity (Δ pc) caused by the introduction of disulfide bonds was determined as follows:

Δ pc ═ pc [ sample ] -pc [ control ])

For example,. DELTA.pc ═ pc [ bonded ] -pc [ unbonded ])

If Apc is greater than zero (> 0), the protective capacity of the sample (e.g., a mutated OspA fragment with disulfide bonds) is higher than that of the control (e.g., an OspA fragment without disulfide bonds), assuming all other parameters (e.g., dose and assay) are the same. Conversely, if Δ pc is less than zero (< 0) and all other parameters (e.g., dose and assay) are assumed to be the same, then the protective capacity of the sample (e.g., a mutated OspA fragment with disulfide bonds) is less than that of the comparator (e.g., an OspA fragment without disulfide bonds).

Preferably, the protective capacity of the polypeptides of the invention is assessed by an in vivo challenge assay, wherein mice immunized with the polypeptides of the invention or placebo controls are challenged with borrelia introduced into the immunized subject by hypodermic needle (needle challenge method) or by introduction of a tick vector (tick challenge method).

Against the desired Borrelia strain (e.g., Borrelia burgdorferi, strain N40) by 20-fold and 50-fold Infectious Dose (ID)₅₀In between the dose borrelia is introduced subcutaneously into mice and the needle challenge method is carried out comparing the infection rate in challenged mice,the mouse is first immunized with the first polypeptide of the first aspect or with a suitable placebo (negative) control such as buffer or adjuvant alone. ID₅₀Defined as the dose at which 50% of challenged mice were infected. The dose of borrelia was measured in a variety of bacteria. Challenge doses can vary widely and are strain-related; therefore, the ID must first be determined by an attack experiment₅₀To assess the virulence of the strain. Four weeks after the needle challenge, blood and tissue were collected for use in a readout method to determine the infection status. These readout methods can be, for example, a VlsE ELISA (for serum) or qPCR (for harvested tissue) as described herein for identifying borrelia, or other methods.

By applying at least one tick nymph (e.g., scleroderma ricini) infected with borrelia (e.g., borrelia avermitilis, strain ISl) to mice immunized with said first polypeptide of the first aspect; and b) applying at least one infected tick nymph to a second mouse immunized with the second polypeptide of the first aspect; and c) comparing the infection rates of the two mice, typically six weeks after challenge, to carry out the tick challenge method. Preferably, each polypeptide to be tested is assayed or tested using a panel of mice. Suitable tests are also described and illustrated in the examples. Assessment of the infection status can be performed using a VlsE ELISA for serum or qPCR for collected tissues or using other suitable methods.

In a preferred embodiment of the invention, the product of the invention, such as for example a polypeptide of the invention comprising a mutated OspA fragment with a disulfide bond, is administered to a subject 3 times at a dose of 30 μ g, preferably 10 μ g, preferably 5.0 μ g, preferably 1.0 μ g, preferably 0.3 μ g or less, which has a protective capacity of 50% or more, preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, most preferably 99% or more. In one embodiment, the protective capacity is assessed in an in vivo challenge method, preferably a tick challenge method, more preferably a needle challenge method (e.g., as described in the examples). It was unexpectedly observed that immunization with OspA mutant fragments of one serotype could provide cross-protection against another serotype (example 4, table 4). Based on this finding, it is expected that the dose of the polypeptide of the present invention can be reduced even further.

In a preferred embodiment, the difference in protective capacity (Δ pc) between the polypeptide comprising a mutated OspA fragment having a disulfide bond of the invention and a placebo (negative) control is at least 50%, in particular at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably 90%, even more preferably 95%, most preferably at least 95%, when administered to a subject 3 times at a dose of 30 μ g, preferably 10 μ g, preferably 5.0 μ g, preferably 1.0 μ g, preferably 0.3 μ g or less.

In a preferred embodiment of the invention, the C-terminal domain is defined as a fragment consisting of at least the C-terminal 150 amino acids of the OspA protein. In one embodiment, the C-terminal domain is between 140 and 152 amino acids in length. In another embodiment, the C-terminal domain consists of no more than the last 152 amino acids, preferably the last 151 amino acids, more preferably the last 150 amino acids of the OspA protein. In an alternative embodiment, the C-terminal domain consists of not less than the last 140 amino acids, preferably the last 141 amino acids, preferably the last 142 amino acids, most preferably the last 143 amino acids of the OspA protein. The last amino acid of an OspA protein is defined herein as the C-most contiguous amino acid sequence of the OspA protein.

In another embodiment, the C-terminal domain of the borrelia OspA protein comprises, consists essentially of, or consists of: (i) an amino acid at position 126, 131 or 130 to position 273 of OspA from Borrelia afzelii strain K78 or (ii) a homologous domain of an amino acid of OspA from a Borrelia strain other than Borrelia afzelii strain K78.

The polypeptide of the invention may comprise, consist essentially of or consist of: (i) one or more of these mutated fragments optionally linked by, for example, a linker as defined below; and (ii) optionally one or more amino acids heterologous to OspA, in particular a signal sequence or site for post-translational modification such as lipidation; and (iii) optionally post-translational modifications, such as lipidation.

According to the invention, the polypeptide of the invention may comprise, consist essentially of or consist of: an element as defined herein, in particular one or more mutant OspA fragments; and optionally one or more additional elements such as heterologous domains, linker peptides, signal sequences or sites for lipidation. In this context "consisting essentially of …" means that the element may have some small amino acid changes, such as amino acid additions, substitutions or deletions, relative to the above sequence, preferably involving at most 10%, 5%, 4%, 3%, 2% or 1% of the amino acids of the element as defined herein.

According to the invention, at least one disulfide bond is introduced into the OspA fragment. This can preferably be achieved by introducing at least 1 or 2 cysteines, in particular 2 cysteines, into the fragment so as to allow the formation of at least one disulfide bond. If another cysteine is available for disulfide bonding in the fragment, only one cysteine may be introduced. However, it is preferred to introduce two cysteines. The cysteine is introduced by amino acid addition or substitution, preferably substitution. In the case of addition, a cysteine is inserted into the amino acid sequence between two amino acids, and in the case of substitution one amino acid is replaced with a cysteine.

In accordance with the present invention, OspA may be from any borrelia strain, in particular from those specified herein, such as borrelia burgdorferi, borrelia garinii, borrelia afzelii, borrelia andersoni (b.andersoni), borrelia bicolor, borrelia farinosa, borrelia ruhatensis, borrelia strobilani, borrelia stuartii, borrelia japan, borrelia tenyi, borrelia terrestris or borrelia cibaceri, borrelia bavaria, preferably from borrelia burgdorferi stricta, borrelia aldii, borrelia bavari or borrelia gariae. Preferably, OspA is from Borrelia afzelii, in particular strain K78, OspA serotype 2(SEQ ID NO: 19); borrelia burgdorferi, specifically strain B31, OspA serotype 1(SEQ ID NO: 20); borrelia garinii, specifically strain PBr, OspA serotype 3(SEQ ID NO: 21); borrelia bavaria, specifically strain PBi, OspA serotype 4(SEQ ID NO: 22); borrelia garinii, specifically strain PHei, OspA serotype 5(SEQ ID NO: 23); borrelia garinii was specifically strain DK29, OspA serotype 6(SEQ ID NO: 24) or Borrelia garinii was specifically strain T25, OspA serotype 7(SEQ ID NO: 25). The amino acid sequences (full length) of these OspA proteins are given below.

TABLE A-3 accession numbers for OspA sequences from selected Borrelia species strains. Abbreviations: borrelia afzelii, Bbu Borrelia burgdorferi, Bga Borrelia galbana, Bsp Borrelia sprengiensis, Bbi Borrelia besseiensis, Bva Borrelia farenhei, Btu Borrelia terenhei (Borrelia turicatae), Bdu Borrelia dahliae (Borrelia dittonii), blau Borrelia ruthensis, Bja Borrelia Japan, gb gene Bank, EMBL, tr unit prot/treble, sp unit prot/swisprot, prf Protein Research Foundation (Protein Research Foundation), dbj japanese DNA database (DNA database of pdpddb), bj database (ddj database), ddj database (database), and ddj database (database)

According to the present invention, disulfide bonds may be formed between cysteines introduced at any position of the OspA fragment to allow or support proper fragment folding. The positions may be selected based on the known structure of OspA as detailed above. In a preferred embodiment, the polypeptide of the invention comprises at least one disulfide bond between: between any of positions 182+/-3 and any of positions 269+/-3 (disulfide bond type 1); between any of positions 182+/-3 and any of positions 272+/-3 (disulfide bond type 2); between any of positions 244+/-3 and any of positions 259+/-3 (disulfide bond type 3); between any of positions 141+/-3 and any of positions 241+/-3 (disulfide bond type 4); between any of positions 165+/-3 and any of positions 265+/-3 (disulfide bond type 5); between any of positions 185+/-3 and any of positions 272+/-3 (disulfide bond type 6); between any of positions 199+/-3 and any of positions 223+/-3 (disulfide bond type 7); between any of positions 243+/-3 and any of positions 262+/-3 (disulfide bond type 8); between any of positions 184+/-3 and any of positions 204+/-3 (disulfide bond type 9); between any of positions 201+/-3 and any of positions 214+/-3 (disulfide bond type 10); between any of positions 246+/-3 and any of positions 259+/-3 (disulfide bond type 11); and/or between any of positions 167+/-3 and any of positions 178+/-3 (disulfide bond type 12), either at the position of OspA 2 of borrelia afzelii, in particular borrelia afzelii K78, or at the homologous amino acid position of OspA from a borrelia species other than borrelia afzelii, such as borrelia burgdorferi sensu stricta, in particular strain B31, serotype 1; borrelia garinii, in particular strain PBr, serotype 3; borrelia bavaria, in particular strain PBi, serotype 4; borrelia garinii, in particular strain PHei, serotype 5; borrelia garinii, specifically strain DK29, serotype 6; or borrelia garinii, in particular strain T25, serotype 7.

More specifically, the polypeptide of the invention comprises at least one disulfide bond between any of the following positions: between positions 182 and 269 (disulfide bond type 1); between positions 182 and 272 (disulfide bond type 2); between positions 244 and 259 (disulfide bond type 3); between positions 141 and 241 (disulfide bond type 4); between positions 165 and 265 (disulfide bond type 5); between positions 185 and 272 (disulfide bond type 6); between positions 199 and 223 (disulfide bond type 7); between positions 243 and 262 (disulfide bond type 8); between positions 184 and 204 (disulfide bond type 9); between positions 201 and 214 (disulfide bond type 10); between positions 246 and 259 (disulfide bond type 11); and/or between positions 167 and 178 (disulfide bond type 12), either at the position of borrelia afzelii, in particular borrelia afzelii K78 serotype 2OspA, or at the position of the homologous amino acids of OspA from borrelia other than borrelia afzelii, for example borrelia burgdorferi sensu stricto, in particular strain B31, serotype 1; borrelia garinii, in particular strain PBr, serotype 3; borrelia bavaria, in particular strain PBi, serotype 4; borrelia garinii, in particular strain PHei, serotype 5; borrelia garinii, specifically strain DK29, serotype 6; or borrelia garinii, in particular strain T25, serotype 7.

Table a-4. positions of cysteine substitutions in OspA proteins with the named disulfide bond types and serotype 2.

Even more preferred are disulfide bond types 1 to 5, especially disulfide bond types 1 to 4.

It should be noted that:

position 182+/-3 is an abbreviation for position 179, 180, 181, 182, 183, 184 or 185, preferably 182.

Position 269+/-3 is an abbreviation for position 266, 267, 268, 269, 270, 271 or 272, preferably 269.

Position 272+/-3 is an abbreviation for position 269, 270, 271, 272, 273, 274 or 275, preferably 272.

Position 244+/-3 is an abbreviation for position 241, 242, 243, 244, 245, 246 or 247, preferably 244.

The positions 259+/-3 are abbreviations for the positions 256, 257, 258, 259, 260, 261 or 262, preferably 259.

Position 141+/-3 is an abbreviation for position 138, 139, 140, 141, 142, 143 or 144, preferably 141.

Position 241+/-3 is an abbreviation for position 238, 239, 240, 241, 242, 243 or 244, preferably 241.

Position 165+/-3 is an abbreviation for position 162, 163, 164, 165, 166, 167 or 168, preferably 165.

Position 265+/-3 is an abbreviation for position 262, 263, 264, 265, 266, 267, or 268, preferably 265.

Position 185+/-3 is an abbreviation for position 182, 183, 184, 185, 186, 187 or 188, preferably 185.

Position 199+/-3 is an abbreviation for position 196, 197, 198, 199, 200, 201, or 202, preferably 199.

Position 223+/-3 is an abbreviation for position 220, 221, 222, 223, 224, 225 or 226, preferably 223.

Position 243+/-3 is an abbreviation for position 240, 241, 242, 243, 244, 245 or 246, preferably 143.

Position 262+/-3 is an abbreviation for position 259, 260, 261, 262, 263, 264 or 265, preferably 262.

Position 184+/-3 is an abbreviation for position 181, 182, 183, 184, 185, 186 or 187, preferably 184.

Position 204+/-3 is an abbreviation for position 201, 202, 203, 204, 205, 206 or 207, preferably 204.

Position 201+/-3 is an abbreviation for position 198, 199, 200, 201, 202, 203 or 204, preferably 201.

Position 214+/-3 is an abbreviation for position 211, 212, 213, 214, 215, 216 or 217, preferably 214.

Position 246+/-3 is an abbreviation for position 243, 244, 245, 246, 247, 248 or 249, preferably 246.

Position 167+/-3 is an abbreviation for position 164, 165, 166, 167, 168, 169 or 170, preferably 167.

Position 178+/-3 is an abbreviation for position 175, 176, 177, 178, 179, 180 or 181, preferably 178.

In a preferred embodiment, the mutant fragment is derived from amino acids at positions 126, 130 or 131 to 273 of the wild-type sequence of OspA of borrelia afzelii strain K78 serotype 2(SEQ id no: 18) and differs only in the introduction of at least one disulfide bond, in particular wherein the at least one disulfide bond is between positions 182 and 269 (disulfide bond type 1); between positions 182 and 272 (disulfide bond type 2); between positions 244 and 259 (disulfide bond type 3); between positions 141 and 241 (disulfide bond type 4); between positions 165 and 265 (disulfide bond type 5); between positions 185 and 272 (disulfide bond type 6); between positions 199 and 223 (disulfide bond type 7); between positions 243 and 262 (disulfide bond type 8); between positions 184 and 204 (disulfide bond type 9); between positions 201 and 214 (disulfide bond type 10); between positions 246 and 259 (disulfide bond type 11); and/or between positions 167 and 178 (disulfide bond type 12), or at homologous fragments and positions of OspA from borrelia species other than borrelia afzelii, such as borrelia burgdorferi sensu stricta, specifically strain B31, serotype 1; borrelia garinii, specifically strain PBr, serotype 3; borrelia bavaria, specifically strain PBi, serotype 4; borrelia garinii, specifically strain PHei, serotype 5; borrelia garinii, specifically strain DK29, serotype 6; or borrelia garinii, in particular strain T25, serotype 7.

In a more preferred embodiment, the mutant fragment has an amino acid sequence selected from the group consisting of: SEQ ID NO: 167. SEQ ID NO: 168. SEQ ID NO: 169. SEQ ID NO: 170. SEQ ID NO: 171. SEQ ID NO: 172. SEQ ID NO: 173. SEQ ID NO: 174. SEQ ID NO: 175. SEQ ID NO: 176. SEQ ID NO: 177. SEQ ID NO: 178 and a peptide having the sequence of SEQ ID NO: 2 to 13, wherein the cysteine has not been replaced, has 80%, more preferably 85%, more preferably 90%, even more preferably 95% sequence identity. Additional details regarding mutations and sequence identity are given above.

As detailed above, the polypeptides of the invention may comprise a signal sequence. Lipidation has been shown to confer adjuvant properties to OspA. Thus, a lipidated form of a polypeptide of the invention or a polypeptide comprising an lipidation signal is preferred. In a preferred embodiment, the polypeptide of the invention comprises a lipidation signal, preferably of the Borrelia outer-surface protein OspA or OspB (SEQ ID NO: 14 and 15, respectively) or more preferably of the E.coli lpp lipidation signal sequence (SEQ ID NO: 16). The OspA fragment of the invention comprising the lipidation signal is lipidated during processing and the lipidation signal peptide is cleaved off. Thus, the signal peptide is no longer present in the mature lipidated protein.

The lipidated protein according to the invention was labeled with "Lip" at the N-terminus to indicate the addition of 3 fatty acid groups and one glycerol to the polypeptide (see figure 4). Suitable lipidation signals as described above include MKKYLLGIGLILALIA (SEQ ID NO: 14), MRL LIGFALALALIG (SEQ ID NO: 15) and MKATKLVLGAVILGSTLLAG (SEQ ID NO: 16). Because the lipid moiety and glycerol are attached to a cysteine residue present at the N-terminus of the full-length wild-type OspA protein, the OspA C-terminal fragment for lipidation may additionally comprise a peptide comprising the cysteine residue followed by additional amino acids, referred to herein as a "lipidated peptide" or "LP" (see fig. 1 and 2). For example, a sequence such as CSS or CKQN (SEQ ID NO: 211) immediately C-terminal to the lipidated signal sequence provides an N-terminal cysteine residue for lipidation upon cleavage of the lipidated signal peptide. The lipidated cysteine-containing peptide is present in the final lipidated polypeptide of the invention.

It has been found that the OspA protein of borrelia burgdorferi in the narrow sense comprises a sequence (also referred to herein as "hLFA-1-like sequence") that has the ability to bind to a T-cell receptor and also has the ability to bind to the human leukocyte function-associated antigen (hLFA-1). The similarity of this OspA region to hLFA-1 can result in a cross-reactive immune response when administering borrelia burgdorferi OspA stricto to a human subject and can induce autoimmune diseases, particularly autoimmune arthritis, in susceptible individuals. Thus, in a preferred embodiment, the polypeptide of the invention does not comprise a sequence having the ability to bind to a T cell receptor and having the ability to bind to human leukocyte function-associated antigen (hLFA-1), and in particular does not comprise amino acid sequence GYVLEGTLTAE (SEQ ID NO: 17). To this end, the hLFA-1-like sequence, specifically the amino acid sequence GYVLEGTLTAE (SEQ ID NO: 17), may be replaced with a homologous sequence from an OspA protein of another Borrelia species, specifically NFTLEGKVAND (SEQ ID NO: 18).

In a preferred embodiment, the polypeptides of the invention comprising at least one disulfide bond establish substantially the same protective capacity against Borrelia infection as the polypeptides relative to at least one of the wild-type full-length OspA proteins derived from at least one Borrelia strain, in particular Borrelia afzelii K78, OspA serotype 2(SEQ ID NO: 19); borrelia burgdorferi, specifically strain B31, serotype 1(SEQ ID NO: 20); borrelia garinii, in particular strain PBr, serotype 3(SEQ ID NO: 21); borrelia bavaria, specifically strain PBi, serotype 4(SEQ ID NO: 22); borrelia garinii, in particular strain PHei, serotype 5(SEQ ID NO: 23); borrelia garinii, specifically strain DK29, serotype 6(SEQ ID NO: 24); or Borrelia garinii, in particular strain T25, serotype 7(SEQ ID NO: 25).

To provide cross-protection against different borrelia species or OspA serotypes, it is necessary to develop multivalent vaccines. Thus, in another preferred embodiment, the polypeptide of the first aspect comprises at least two mutant fragments from two different borrelia serotypes as defined above. In a preferred embodiment, the polypeptide of the first aspect comprises at least two mutant OspA fragments selected from the group consisting of:

-a fragment with disulfide bond type 1 and a fragment with disulfide bond type 2;

-a fragment with disulfide bond type 1 and a fragment with disulfide bond type 3;

-a fragment with disulfide bond type 1 and a fragment with disulfide bond type 4;

-a fragment with disulfide bond type 1 and a fragment with disulfide bond type 5;

-a fragment with disulfide bond type 1 and a fragment with disulfide bond type 6;

-a fragment with disulfide bond type 1 and a fragment with disulfide bond type 7;

-a fragment with disulfide bond type 1 and a fragment with disulfide bond type 8;

-a fragment with disulfide bond type 1 and a fragment with disulfide bond type 9;

-a fragment with disulfide bond type 1 and a fragment with disulfide bond type 10;

-a fragment with disulfide bond type 1 and a fragment with disulfide bond type 11;

-a fragment with disulfide bond type 1 and a fragment with disulfide bond type 12;

-a fragment with disulfide bond type 2 and a fragment with disulfide bond type 3;

-a fragment with disulfide bond type 2 and a fragment with disulfide bond type 4;

-a fragment with disulfide bond type 2 and a fragment with disulfide bond type 5;

-a fragment with disulfide bond type 2 and a fragment with disulfide bond type 6;

-a fragment with disulfide bond type 2 and a fragment with disulfide bond type 7;

-a fragment with disulfide bond type 2 and a fragment with disulfide bond type 8;

-a fragment with disulfide bond type 2 and a fragment with disulfide bond type 9;

-a fragment with disulfide bond type 2 and a fragment with disulfide bond type 10;

-a fragment with disulfide bond type 2 and a fragment with disulfide bond type 11;

-a fragment with disulfide bond type 2 and a fragment with disulfide bond type 12;

-a fragment with disulfide bond type 3 and a fragment with disulfide bond type 4;

-a fragment with disulfide bond type 3 and a fragment with disulfide bond type 5;

-a fragment with disulfide bond type 3 and a fragment with disulfide bond type 6;

-a fragment with disulfide bond type 3 and a fragment with disulfide bond type 7;

-a fragment with disulfide bond type 3 and a fragment with disulfide bond type 8;

-a fragment with disulfide bond type 3 and a fragment with disulfide bond type 9;

-a fragment with disulfide bond type 3 and a fragment with disulfide bond type 10;

-a fragment with disulfide bond type 3 and a fragment with disulfide bond type 11;

-a fragment with disulfide bond type 3 and a fragment with disulfide bond type 12;

-a fragment with disulfide bond type 4 and a fragment with disulfide bond type 5;

-a fragment with disulfide bond type 4 and a fragment with disulfide bond type 6;

-a fragment with disulfide bond type 4 and a fragment with disulfide bond type 7;

-a fragment with disulfide bond type 4 and a fragment with disulfide bond type 8;

-a fragment with disulfide bond type 4 and a fragment with disulfide bond type 9;

-a fragment with disulfide bond type 4 and a fragment with disulfide bond type 10;

-a fragment with disulfide bond type 4 and a fragment with disulfide bond type 11;

-a fragment with disulfide bond type 4 and a fragment with disulfide bond type 12;

-a fragment with disulfide bond type 5 and a fragment with disulfide bond type 6;

-a fragment with disulfide bond type 5 and a fragment with disulfide bond type 7;

-a fragment with disulfide bond type 5 and a fragment with disulfide bond type 8;

-a fragment with disulfide bond type 5 and a fragment with disulfide bond type 9;

-a fragment with disulfide bond type 5 and a fragment with disulfide bond type 10;

-a fragment with disulfide bond type 5 and a fragment with disulfide bond type 11;

-a fragment with disulfide bond type 5 and a fragment with disulfide bond type 12;

-a fragment with disulfide bond type 6 and a fragment with disulfide bond type 7;

-a fragment with disulfide bond type 6 and a fragment with disulfide bond type 8;

-a fragment with disulfide bond type 6 and a fragment with disulfide bond type 9;

-a fragment with disulfide bond type 6 and a fragment with disulfide bond type 10;

-a fragment with disulfide bond type 6 and a fragment with disulfide bond type 11;

-a fragment with disulfide bond type 6 and a fragment with disulfide bond type 12;

-a fragment with disulfide bond type 7 and a fragment with disulfide bond type 8;

-a fragment with disulfide bond type 7 and a fragment with disulfide bond type 9;

-a fragment with disulfide bond type 7 and a fragment with disulfide bond type 10;

-a fragment with disulfide bond type 7 and a fragment with disulfide bond type 11;

-a fragment with disulfide bond type 7 and a fragment with disulfide bond type 12;

-a fragment with disulfide bond type 8 and a fragment with disulfide bond type 9;

-a fragment with disulfide bond type 8 and a fragment with disulfide bond type 10;

-a fragment with disulfide bond type 8 and a fragment with disulfide bond type 11;

-a fragment with disulfide bond type 8 and a fragment with disulfide bond type 12;

-a fragment with disulfide bond type 9 and a fragment with disulfide bond type 10;

-a fragment with disulfide bond type 9 and a fragment with disulfide bond type 11;

-a fragment with disulfide bond type 9 and a fragment with disulfide bond type 12;

-a fragment with a disulfide bond type 10 and a fragment with a disulfide bond type 11;

-a fragment with disulfide bond type 10 and a fragment with disulfide bond type 12;

-a fragment with disulfide bond type 11 and a fragment with disulfide bond type 12;

-and

particularly wherein

-the fragment having disulfide bond type 1 has SEQ ID NO: 2 or an amino acid sequence identical to SEQ ID NO: 2, wherein the cysteine has not been replaced, has at least 80%, more preferably 85%, more preferably 90%, even more preferably 95% sequence identity;

-the fragment having disulfide bond type 2 has SEQ ID NO: 3 or an amino acid sequence identical to SEQ ID NO: 3, wherein the cysteine is not substituted, has at least 80%, more preferably 85%, more preferably 90%, even more preferably 95% sequence identity;

-the fragment with disulfide bond type 3 has the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence identical to SEQ ID NO: 4, wherein the cysteine has not been replaced, has at least 80%, more preferably 85%, more preferably 90%, even more preferably 95% sequence identity;

-the fragment having disulfide bond type 4 has SEQ ID NO: 5 or an amino acid sequence identical to SEQ ID NO: 5, wherein the cysteine has not been replaced, with at least 80%, more preferably 85%, more preferably 90%, even more preferably 95% sequence identity;

-the fragment with disulfide bond type 5 has the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence identical to SEQ ID NO: 6, wherein the cysteine has not been replaced, has at least 80%, more preferably 85%, more preferably 90%, even more preferably 95% sequence identity;

-the fragment with disulfide bond type 6 has the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence identical to SEQ ID NO: 7, wherein the cysteine has not been replaced, has at least 80%, more preferably 85%, more preferably 90%, even more preferably 95% sequence identity;

-the fragment with disulfide bond type 7 has the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence identical to SEQ ID NO: 8, wherein the cysteine has not been replaced, has at least 80%, more preferably 85%, more preferably 90%, even more preferably 95% sequence identity;

-the fragment with disulfide bond type 8 has the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence identical to SEQ ID NO: 9, wherein the cysteine has not been replaced, has at least 80%, more preferably 85%, more preferably 90%, even more preferably 95% sequence identity;

-the fragment with disulfide bond type 9 has the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence identical to SEQ ID NO: 10, wherein the cysteine has not been replaced, has at least 80%, more preferably 85%, more preferably 90%, even more preferably 95% sequence identity;

-the fragment with disulfide bond type 10 has the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence corresponding to SEQ ID NO: 11, wherein the cysteine has not been replaced, with at least 80%, more preferably 85%, more preferably 90%, even more preferably 95% sequence identity;

-the fragment having disulfide bond type 11 has SEQ ID NO: 12 or an amino acid sequence identical to SEQ ID NO: 12, wherein the cysteine has not been replaced, has at least 80%, more preferably 85%, more preferably 90%, even more preferably 95% sequence identity; and/or

-the fragment with disulfide bond type 12 has the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence identical to SEQ ID NO: 13, wherein the cysteine has not been replaced, with at least 80%, more preferably 85%, more preferably 90%, even more preferably 95% sequence identity.

Note that additional details regarding mutations and sequence identity are given above.

TABLE A-5 nomenclature of heterodimers of non-lipidated and lipidated mutant OspA fragments described in the present invention and SEQ ID NO.

Serotype (1-6) (see table a-2); d ═ disulfide bond type (see table a-4);

lip ═ lipidation: glycerol and the N-terminal addition of fatty acid residues.

In another preferred embodiment, the polypeptide according to the first aspect comprises at least two or three mutated fragments linked by one or more linkers. Linkers are rather short amino acid sequences used to join two fragments. It should be designed to avoid any negative impact on the fragments, their interaction in the subject to be treated or vaccinated, or their protective ability. Preference is given to short linkers of up to 21 amino acids, in particular up to 15 amino acids, especially up to 12 or 8 amino acids. More preferably, one or more linkers consist of small amino acids, such as glycine, serine and alanine, in order to reduce or minimize interaction with the fragment. Examples or preferred linkers include linkers comprising or consisting of poly G, e.g. (G)₈(SEQ ID NO：36)、(G)₁₂(SEQ ID NO：37)、GAGA(SEQ ID NO：38)、(GAGA)₂(SEQ ID NO：39)、(GAGA)₃(SEQ ID NO：40)、(GGGS)₂(SEQ ID NO: 41) or (GGGS)₃(SEQ ID NO: 42). A more preferred linker is a "LN 1" peptide linker ", which is a fusion consisting of two separate loop regions from the N-terminal half of OspA of Borrelia burgdorferi strain B31 in the narrow sense (aa65-74 and aa 42-53 with the amino acid exchange at position 53: D53S) having the following sequence: GTSDKNNGSGSKEKNKDGKYS (SEQ ID NO: 184).

In another preferred embodiment, the polypeptide according to the first aspect comprises: a polypeptide having a total size of at most 500 amino acids comprising two or three different mutated fragments as defined in the preferred embodiment of the first aspect; or a polypeptide consisting essentially of two or three mutated fragments, one or two linkers, and optionally an N-terminal cysteine; and/or a polypeptide consisting essentially of two or three different mutant fragments, an N-terminal extension of a fragment consisting of up to 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12 or 11 amino acids, preferably up to 10, 9, 8, 7 or 6 amino acids, still more preferably up to 5, 4, 3, 2 or 1 amino acids, and optionally an N-terminal cysteine, wherein the N-terminal extension is located directly N-terminal to the fragment of the corresponding Borrelia OspA. The N-terminal cysteine may optionally be followed by a short peptide linker of 1 to 10 amino acids in length and preferably takes the form of an N-terminal CSS peptide or CKQN peptide (SEQ ID NO: 211).

In a second aspect, the present invention relates to a nucleic acid encoding a polypeptide according to the first aspect.

The invention further provides a nucleic acid encoding a polypeptide of the invention. For the purposes of the present invention, the term "nucleic acid" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA including single-and double-stranded regions/forms.

The term "nucleic acid encoding a polypeptide" as used herein encompasses a polynucleotide comprising a sequence encoding a peptide or polypeptide of the invention. The term also encompasses polynucleotides comprising a single contiguous or discontinuous region encoding a peptide or polypeptide (e.g., a polynucleotide interrupted by integrated phage, integrated insert sequences, integrated vector sequences, integrated transposon sequences, or by RNA editing or genomic DNA recombination) along with additional regions (which may also comprise coding and/or non-coding sequences).

One of ordinary skill in the art will appreciate that due to the degeneracy of the genetic code, there are many nucleotide sequences that encode the polypeptides described herein. Some of these polynucleotides have little similarity to the nucleotide sequence of any native (i.e., naturally occurring) gene. Nevertheless, polynucleotides that vary due to differences in codon usage, such as polynucleotides optimized for codon usage in humans and/or primates and/or E.coli, are specifically contemplated by the present invention.

The sequence encoding the desired polypeptide may be synthesized in whole or in part using chemical methods well known in the art (see Caruthers, M.H., et al, Nucl. acids Res. Symp. Ser. pp. 215-223 (1980); Horn et al, Nucl. acids sRs. Symp. Ser. pp. 225-232 (1980)). Alternatively, the amino acid sequence of a polypeptide or portion thereof may be chemically synthesized in order to produce the protein itself. For example, peptide synthesis can be performed using various solid phase techniques (Roberge et al, Science 269: 202-204(1995)) and automated synthesis can be achieved, for example, using an ASI 431A peptide synthesizer (Perkin Elmer, Palo Alto, Calif.).

In addition, polynucleotide sequences of the present invention can be engineered using methods generally known in the art to alter polypeptide coding sequences for a variety of reasons, including, but not limited to, alterations that alter the cloning, processing, and/or expression of a gene product. For example, DNA shuffling by random fragmentation and PCR recombination of gene fragments and synthetic oligonucleotides can be used to engineer nucleotide sequences. In addition, site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, alter codon bias, create splice variants or introduce mutations, and the like.

In another aspect of the invention, the invention relates to a vector comprising a nucleic acid of the invention linked to an inducible promoter such that upon induction of the promoter the polypeptide encoded by the nucleic acid is expressed. In a preferred embodiment, the vector is pET28b (+).

Another aspect of the invention includes the vector wherein the inducible promoter is activated by adding preferably sufficient IPTG (isopropyl. beta. -D-l-thiogalactoside) to the growth medium. Optionally, the concentration of IPTG is between 0.1 and 10mM, 0.1 and 5mM, 0.1 and 2.5mM, 0.2 and 10mM, 0.2 and 5mM, 0.2 and 2.5mM, 0.4 and 10mM, 1 and 5mM, 2.5 and 10mM, 2.5 and 5mM, 5 and 10 mM. Alternatively, the promoter may be induced by changing temperature or pH.

Nucleic acid molecules as used herein generally refers to any ribonucleic acid molecule or deoxyribonucleic acid molecule, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for example, a nucleic acid molecule as used herein refers to at least single-and double-stranded DNA, a hybrid molecule comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or a mixture of single-and double-stranded regions. As used herein, the term nucleic acid molecule includes DNA or RNA as described above containing one or more modified bases. Thus, a DNA or RNA molecule having a backbone modified for stability or other reasons is a "nucleic acid molecule," as that term is intended herein. Furthermore, DNA or RNA species comprising unusual bases such as inosine or modified bases such as tritylated bases to name just two examples are also nucleic acid molecules as defined herein. It will be appreciated that a wide variety of modifications have been made to DNA and RNA molecules for many useful purposes known to those skilled in the art. The term nucleic acid molecule as used herein includes such chemically, enzymatically or metabolically modified forms of nucleic acid molecules as well as chemical forms of DNA and RNA which are characteristic of viruses and cells, including simple and complex cells, among others. The term nucleic acid molecule also encompasses short nucleic acid molecules commonly referred to as oligonucleotides. The terms "polynucleotide" and "nucleic acid" or "nucleic acid molecule" are used interchangeably herein.

The nucleic acids according to the invention may be chemically synthesized. Alternatively, the nucleic acid may be isolated from borrelia and modified by methods known to those skilled in the art. The same applies to the polypeptides according to the invention.

In addition, the nucleic acids of the invention can be functionally linked to any desired sequence, whether a borrelia regulatory sequence or a heterologous regulatory sequence, a heterologous leader sequence, a heterologous marker sequence or a heterologous coding sequence, using standard techniques such as cloning, in order to generate a fusion gene.

The nucleic acid molecules of the invention may be in the form of RNA, such as mRNA or cRNA, or in the form of DNA, including, for example, cDNA and genomic DNA, obtained by cloning or produced by chemical synthesis techniques or by a combination thereof. The DNA may be triplex, double stranded or single stranded. The single-stranded DNA may be the coding strand, also referred to as the sense strand, or it may be the non-coding strand, also referred to as the antisense strand.

The nucleic acids of the invention may be comprised in a vector or a cell. The vector may comprise the above-mentioned nucleic acids in a vector-replicable manner and may express the protein encoded by the nucleotide sequence in a host cell.

For recombinant production of a polypeptide of the invention, a host cell may be genetically engineered to incorporate an expression system or portion thereof or a nucleic acid of the invention. Introduction of nucleic acids into host cells can be accomplished by a number of standard laboratory manuals such as, for example, Davis et al, BASICETHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook et al, MOLECULAR CLONING: ALABORATORY MANUAL, 2 nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), such as calcium phosphate transfection, DEAE-dextran mediated transfection, translocation, microinjection, cationic lipid mediated transfection, electroporation, conjugation, transduction, scrape loading (scrape loading), ballistic introduction and infection.

Representative examples of suitable hosts include gram-negative bacterial cells such as cells of Escherichia coli, Acinetobacter, Actinobacillus, Bordetella (Bordetella), Brucella, Campylobacter, cyanobacteria, Enterobacter, Erwinia, Francisella (Francisella), helicobacter, Haemophilus, Klebsiella, Legionella, Moraxella (Moraxella), Neisseria (Neisseria), Pasteurella (Pasteurella), Proteus, Pseudomonas, Salmonella, Serratia, Shigella, Treponema, Vibrio, Yersinia (Yersinia). In one embodiment, the host cell is an E.coli cell. In a preferred embodiment, the host cell is an E.coli BL21(DE3) cell or E.coli BL21Star^TM(DE3) cells.

Alternatively, gram positive bacterial cells may also be used. A wide variety of expression systems can be used to produce the polypeptides of the invention. In one embodiment, the vector is derived from a bacterial plasmid. Generally, any system or vector suitable for maintaining, propagating or expressing a polynucleotide and/or expressing a polypeptide in a host may be used for expression in this regard. The appropriate DNA sequence may be inserted into the expression system by any of a variety of well-known and conventional techniques, such as those listed in Sambrook et al, Molecula clone, A LABORATORY MANUAL, (supra).

In one embodiment of the invention, the cells are grown under selective pressure, for example in the presence of an antibiotic, preferably kanamycin. In another embodiment, the cells are grown in the absence of an antibiotic.

A wide variety of expression vectors may be used to express the polypeptides according to the invention. In general, any vector suitable for maintaining, propagating or expressing a nucleic acid to express a polypeptide in a host may be used for expression in this regard. According to this aspect of the invention, the vector may be, for example, a plasmid vector, a single-or double-stranded phage vector, or a single-or double-stranded RNA or DNA viral vector. The starting plasmids disclosed herein are commercially available, publicly available, or can be constructed from available plasmids by conventional application of well-known published procedures. In certain aspects, preferred in the vector are those for expression of the nucleic acid molecule and the polypeptide according to the invention. The nucleic acid construct within the host cell may be used in a conventional manner to produce a gene product encoded by the recombinant sequence. Alternatively, the polypeptide according to the invention may be produced synthetically by conventional peptide synthesizers.

Furthermore, the invention relates to a host cell comprising such a vector. Representative examples of suitable host cells include bacteria such as streptococci, staphylococci, E.coli, Streptomycete, and Bacillus subtilis; fungi, such as yeast and aspergillus; insect cells, if fly S2 and noctuid Sf9 cells; mammalian cells, such as CHO, COS, HeLa, C127, 3T3, BHK, 293, or Bowes melanoma cells; and plant cells. Cell-free translation systems may also be used to produce such proteins using RNA derived from the DNA constructs of the invention.

In order to actually express a desired amino acid sequence by introducing the vector according to the present invention into a host cell, the vector may further include other sequences for controlling expression (e.g., a promoter sequence, a terminator sequence, and an enhancer sequence) and gene markers (e.g., a neomycin resistance gene and a kanamycin resistance gene) for selecting microorganisms, insect cells, animal culture cells, and the like, in addition to the nucleic acid sequence according to the present invention. Furthermore, the vector may comprise the nucleic acid sequence according to the invention in repetitive form (e.g. in tandem). The vector can be constructed based on the procedures and means conventionally used in the field of genetic engineering.

The host cell may be cultured in an appropriate medium, and the protein according to the present invention may be obtained from the culture product. The protein according to the invention can be recovered from the culture medium and purified in a conventional manner.

Furthermore, the problem underlying the present invention is further solved by a method for producing a polypeptide as defined above, said method being characterized by the steps of:

a) introducing a vector encoding the polypeptide into a host cell;

b) growing the host cell under conditions that allow expression of the polypeptide;

c) homogenizing the host cell; and

d) the host cells are homogenized and subjected to a purification step.

The invention further relates to a method for producing a polypeptide as defined above, characterized by the following steps:

a) introducing a nucleic acid encoding a polypeptide into a vector;

b) introducing the vector into a host cell;

c) growing the host cell under conditions that allow expression of the polypeptide;

d) homogenizing the host cell;

e) enriching the polypeptide in a liquid phase by phase separation; and

f) further purification was performed on a gel filtration column.

a) introducing a nucleic acid encoding a polypeptide into a vector;

b) introducing the vector into a host cell;

d) homogenizing the host cell;

e) enriching the polypeptide in a liquid phase by phase separation;

g) purifying on a gel filtration column; and

h) optionally, further processing on a buffer exchange column.

In another aspect, the underlying problem of the present invention is solved by an antibody or at least an effective part thereof specifically binding to at least a selected part of a polypeptide as defined above.

In a preferred embodiment, the antibody is a monoclonal antibody.

In another preferred embodiment, the effective portion comprises a Fab fragment, a F (ab) N fragment, a F (ab)₂Fragment or F_vAnd (3) fragment.

In yet another embodiment of the invention, the antibody is a chimeric antibody.

In yet another embodiment, the antibody is a humanized antibody.

In a preferred aspect, the antibodies of the invention specifically bind to the mutant OspA fragment polypeptides of the invention, but do not bind to the corresponding wild-type OspA fragment polypeptides. In a more preferred aspect, the antibody specifically binds to a disulfide bond of a mutant OspA fragment of the invention.

The term "specificity" refers to the number of different types of antigens or antigenic determinants that a particular antigen binding molecule or antigen binding protein (e.g., nanobody or polypeptide of the invention) can bind. The specificity of an antigen binding protein can be determined based on affinity and/or avidity. Equilibrium constant (K) from dissociation of antigen from antigen binding protein_D) The expressed affinity is a measure of the strength of binding between an antigenic determinant and an antigen binding site on an antigen binding protein. K_DThe smaller the value of (a), the stronger the binding strength between the antigenic determinant and the antigen-binding molecule (alternatively, the affinity may also be expressed as an affinity constant (K)_A) Which is 1/K_D)。

As will be clear to the skilled person (e.g. based on further disclosure herein), affinity may be determined in a manner known per se depending on the specific antigen concerned. Avidity is a measure of the strength of binding between an antigen binding molecule, such as an antibody of the invention or an effective portion thereof, and an associated antigen. Avidity relates to both the affinity between an antigenic determinant and an antigen binding site on an antigen binding molecule and the number of associated binding sites present on the antigen binding molecule. Typically, an antigen binding protein (e.g., an antibody of the invention or an effective portion thereof) will be present at 10^-5To 10^-12Mole/liter or less and preferably 10^-7To 10^-12Mole/liter or less and more preferably 10^-8To 10^-12Dissociation constant (K) in mol/l_D) (i.e., at 10)⁵To 10¹²Liter/mole or more and preferably 10⁷To 10¹²Liter/mole or more and more preferably 10⁸To 10¹²Association constant (K) in liters/mole_A) Bind its antigen. Greater than 10⁴Any K in mol/l_DValue (or less than 10)⁴M^-1Liter/moleErzhi K_AValue) is generally considered to indicate non-specific binding. Preferably, the monovalent immunoglobulin sequences of the invention will bind to the desired antigen with an affinity of less than 500nM, preferably less than 200nM, more preferably less than 10nM, such as less than 500 pM. Specific binding of an antigen binding protein to an antigen or antigenic determinant may be determined in any suitable manner known per se, including, for example, scatchard analysis and/or competitive binding assays, such as Radioimmunoassays (RIA), Enzyme Immunoassays (EIA) and sandwich competition assays, as well as different variations thereof known per se in the art, as well as other techniques mentioned herein.

As will be clear to the skilled person, the dissociation constant may be the actual dissociation constant or the apparent dissociation constant. Methods for determining the dissociation constant will be clear to the skilled person and include, for example, the techniques mentioned herein. In this respect, it will also be clear that it may not be possible to measure more than 10^-4Mol/l or 10^-3Mole/liter (e.g., 10)^-2Moles/liter). Optionally, the skilled person will also appreciate that it may be based on an (actual or apparent) association constant (K)_A) By the relation [ K_D＝1/K_A]The (actual or apparent) dissociation constant is calculated.

Affinity indicates the strength or stability of molecular interactions. Affinity is usually given as K_DOr dissociation constant in moles/liter (or M). Affinity can also be expressed as the association constant K_AWhich is equal to 1/K_DAnd has (mol/l)^-1(or M)^-1) The unit of (c). In the present specification, the stability of an interaction between two molecules (such as an amino acid sequence, nanobody or polypeptide of the invention and its intended target) will be mainly expressed as the K of its interaction_DValue, it will be clear to the skilled person in view of the relation K_A＝1/K_DThrough which K is_DThe value indicating the strength of the molecular interaction can also be used to calculate the corresponding K_AThe value is obtained. Said K_DThe value also characterizes the strength of the molecular interaction in a thermodynamic sense, since it passes through the well-known relationship DG ═ rt_D) (equivalents)Ln (K) in DG ═ rt_A) Is related to the binding free energy (DG), where R equals the gas constant, T equals absolute stability, and ln indicates the natural logarithm.

Biological interacting K considered interesting (e.g. specific)_DIn the range of usually 10^-10M (0.1nM) to 10^-5M (10000 nM). The stronger the interaction, the K_DThe lower.

In a preferred embodiment, the K of the antibody of the invention_DIs 10^-12M and 10^-5M, preferably less than 10^-6Preferably less than 10^-7Preferably less than 10^-8M, preferably less than 10^-9M, more preferably less than 10^-10M, even more preferably less than 10^-11M, most preferably less than 10^-12M。

Said K_DCan also be expressed as the complex dissociation rate constant (expressed as k)_off) With its association rate (denoted as k)_on) Ratio of (so that K)_D＝k_off/k_onAnd K_A＝k_on/k_off). Off rate k_offHaving the unit s^-1(where s is the SI unit symbol in seconds). Association rate k_onHaving a unit M^-1s^-1. The association rate can be in the range of 10²M^-1s^-1To about 10⁷M^-1s^-1Close to the diffusion-limited association rate constant of bimolecular interactions. The dissociation rate is represented by the relation t_1/2＝ln(2)/k_offBut rather the half-life of a given molecular interaction. The dissociation rate can be 10^-6s^-1(with t for several days_1/2Almost irreversible complex) to 1s^-1(t_1/20.69 s).

The affinity of the molecular interaction between two molecules can be measured by different techniques known per se, such as the well-known Surface Plasmon Resonance (SPR) biosensor technique (see, e.g., Ober et al, Intern. immunology, 13, 1551. sup. 1559, 2001), in which one molecule is immobilizedOn the biosensor chip and another molecule passes through the immobilized molecule under flow conditions, thereby generating k_on、k_offMeasured value and thus K_D(or K)_A) The value is obtained. This can be done, for example, using the well-known BIACORE instrument.

It will also be clear to the skilled person that if the measurement process affects the intrinsic binding affinity of the implied molecule to some extent, e.g. due to coating related artefacts on a biosensor of one molecule, the measured K_DCan be combined with the appearance K_DAnd correspondingly. Furthermore, if one molecule contains more than one recognition site for another molecule, the apparent K can be measured_D. In this case, the measured affinity may be influenced by the affinity of the interaction of the two molecules.

Another way in which affinity can be assessed is the 2-step ELISA (enzyme-linked immunosorbent assay) procedure of Friguet et al (J.Immunol. methods, 77, 305-19, 1985). This method establishes a solution phase binding equilibrium measurement and avoids possible artefacts related to the adsorption of one of the molecules on a support such as a plastic.

However, K_DThe absolute measurement of (a) can be quite labor intensive; thus, the apparent K is usually determined_DValues in order to assess the binding strength of the two molecules. It should be noted that the apparent K is the value of K as long as all measurements are made in a consistent manner (e.g., keeping assay conditions constant)_DThe measured value can be used as the true K_DAnd thus in this document, K_DAnd apparent K_DShould be treated with equal importance or relevance.

Finally, it should be noted that in many cases, an experienced scientist can judge whether it is convenient to determine binding affinity with respect to some reference molecule. For example, to assess the binding strength between molecules a and B, one may for example use biotin or other forms (fluorophores for fluorescence detection, for light absorption detection) known to bind to B and which are easily detectable with fluorophores or chromophores or other chemical moieties (e.g. in ELISA or flow cytometry)Chromophore, biotin for streptavidin-mediated ELISA detection)) a suitably labeled reference molecule C. Typically, the reference molecule C is maintained at a fixed concentration and the concentration of a is varied for a given concentration or amount of B. Thus, an Inhibitory Concentration (IC) was obtained₅₀A value corresponding to the concentration of a at which the signal measured for C in the absence of a is halved. Suppose, K_D _ref(K of reference molecule)_D) And the total concentration c of the reference molecule_refIt is known that the apparent K of the interaction A-B can be obtained from the following formula_D：K_D＝IC_5o/(1+c_ref/K_Dref). Note that if c_ref＜＜K_DrefThen K is_D≈IC₅₀. Assume in a consistent manner (e.g., hold c)_refImmobilization) IC for the compared binders_5oMeasurement can be made through IC₅₀The strength or stability of the molecular interaction is evaluated and this measurement is judged to be equal to K throughout this text_DOr equal to apparent K_D。

Another aspect of the invention relates to a hybridoma cell line producing an antibody as defined above.

Furthermore, the problem underlying the present invention is further solved by a method for the production of an antibody as defined above, said method being characterized by the steps of:

a) initiating an immune response in a non-human animal by administering to said animal a polypeptide as defined above;

b) removing antibody-containing body fluid from the animal; and

c) producing antibodies by subjecting the antibody-containing body fluid to additional purification steps.

The invention further relates to a method for producing an antibody as defined above, characterized by the following steps:

b) removing spleen or splenocytes from the animal;

c) producing hybridoma cells of said spleen or splenocytes;

d) selecting and cloning hybridoma cells specific for the polypeptide;

e) producing the antibody by culturing the cloned hybridoma cells; and

f) optionally, additional purification steps are performed.

Another aspect of the invention relates to a pharmaceutical composition comprising an antibody as specified above.

A further aspect relates to an antibody as defined above or a pharmaceutical composition comprising an antibody as defined above for use in the treatment or prevention of borrelia species (more preferably pathogenic borrelia species as disclosed herein, more preferably including borrelia burgdorferi sensu stricto, borrelia afzelii, borrelia bavariensis and borrelia garinii) infection.

In another aspect, the problem underlying the present invention is solved by the use of an antibody as defined above for the preparation of a pharmaceutical composition for the treatment or prevention of borrelia species (more preferably pathogenic borrelia species as disclosed herein, more preferably including borrelia burgdorferi sensu stricto, borrelia afzelii, borrelia bavaria and borrelia garinii) infection.

In a third aspect, the present invention relates to a pharmaceutical composition comprising a polypeptide according to the first aspect and/or a nucleic acid according to the second aspect. The pharmaceutical composition may optionally comprise any pharmaceutically acceptable carrier or excipient, such as a buffering substance, a stabilizer or another active ingredient, in particular an ingredient known in connection with pharmaceutical compositions and/or vaccine manufacture. Preferably, the pharmaceutical composition is for use as a medicament, in particular as a vaccine or for the prevention or treatment of infections caused by borrelia species, more preferably pathogenic borrelia species as disclosed herein, more preferably including borrelia burgdorferi sensu stricto, borrelia afzelii, borrelia bavariensis and borrelia garinii, and/or other pathogens against which antigens comprised in the vaccine are directed.

In one embodiment, the pharmaceutical composition further comprises an adjuvant. The selection of suitable adjuvants to be mixed with the bacterial toxins or conjugates prepared using the methods of the present invention is known to those skilled in the art. Suitable adjuvants include aluminium salts such as aluminium hydroxide or aluminium phosphate, but may also be other metal salts such as calcium, magnesium, iron or zinc salts, or may be an insoluble suspension of acylated tyrosine or acylated sugars, cationically or anionically derivatised saccharides or polyphosphazenes. In a preferred embodiment, the pharmaceutical composition is adjuvanted with aluminium hydroxide.

In another embodiment, the pharmaceutical composition further comprises an immunostimulatory substance, preferably selected from the group consisting of: a polycationic polymer, in particular a polycationic peptide; an immunostimulatory Oligodeoxynucleotide (ODN), in particular an oligo (ddC) ₁₃ (SEQ ID NO:32); a peptide containing at least two LysLeuLys motifs, in particular the peptide KLKLLLLLKLK (SEQ ID NO:33); a neuroactive compound, in particular human growth hormone; aluminium hydroxide, aluminium phosphate, Freund's complete or incomplete adjuvant or a combination thereof. Preferably, the immunostimulatory substance is a combination of a polycationic polymer and an immunostimulatory deoxynucleotide or a combination of a peptide containing at least two LysLeuLys motifs and an immunostimulatory deoxynucleotide, preferably a combination of KLKLLLLLKLK (SEQ ID NO:33) and oligo (ddC) ₁₃ (SEQ ID NO: 32). More preferably, said polycationic peptide is a polyarginine.

In another embodiment, the pharmaceutical composition comprises sodium phosphate, sodium chloride, L-methionine, sucrose, and Tween-20, at a pH of 6.7 +/-0.2. Preferably, the pharmaceutical composition also comprises aluminium hydroxide, preferably at a concentration of 0.15%.

In one embodiment, the formulation comprises between 5mM and 50mM sodium phosphate, between 100 and 200mM sodium chloride, between 5mM and 25mM L-methionine, between 2.5% and 10% sucrose, between 0.01% and 0.1% Tween20, and between 0.1% and 0.2% (w/v) aluminum hydroxide. More preferably, the formulation comprises 10mM sodium phosphate, 150mM sodium chloride, 10mM L-methionine, 5% sucrose, 0.05% Tween20, and 0.15% (w/v) aluminum hydroxide, pH 6.7. + -. 0.2. Even more preferably, the preparation comprises at least one, at least two, at least three mutant OspA heterodimers according to the invention.

In one embodiment, the pharmaceutical composition comprises 3 heterodimers, preferably Lip-S1D1-S2D1(SEQ ID NO: 186), Lip-S4D1-S3D1(SEQ ID NO: 194), and Lip-S5D1-S6D1(SEQ ID NO: 190). Preferably, the three heterodimers are mixed in a molar ratio of 1: 2: 1, 1: 3: 1, 1: 2, 1: 3, 1: 2, 1: 2: 3, 1: 3: 2, 1: 3, 2: 1, 2: 1: 2, 2: 1: 3, 2: 1, 2: 3: 2, 2: 3, 3: 1, 3: 1: 2, 3: 1: 3, 3: 2: 1, 3: 2, 3: 2: 3, 3: 1, 3: 2, most preferably 1: 1.

In one embodiment, the pharmaceutical composition comprises two heterodimers, preferably Lip-S1D1-S2D1(SEQ ID NO: 186) and Lip-S5D1-S6D1(SEQ ID NO: 190), Lip-S1D1-S2D1(SEQ ID NO: 186) and Lip-S4D1-S3D1(SEQ ID NO: 194) or Lip-S4D1-S3D1(SEQ ID NO: 194) and Lip-S5D1-S6D1(SEQ ID NO: 190) in a molar ratio of 1: 2, 1: 3, 2: 1, 3: 3, preferably 1: 1.

In one embodiment, the pharmaceutical composition or vaccine of the invention further comprises at least one additional antigen (generally referred to herein as a "combination vaccine"). In a preferred embodiment, the at least one further antigen is derived from a borrelia species causing lyme borreliosis. In various aspects, the at least one additional antigen is derived from another pathogen, preferably a tick-borne pathogen. In another aspect, the pathogen causes rocky mountain Spotted Fever, Human Granulocyte Ehrlichiosis (HGE), Sennett Fever (Sennetsu Fever), human monocyte ehrlichiosis (HM), borderline disease, conk rickettsiosis (Boutonneus Fever), rickettsiosis of Parkery (Rickettsia parkerissis), Southern Tick-Associated Rash disease (Southern Tick-Associated Rash Ill.) (STARI), Haititi Spotted Fever (Helvetica Spotted Fever), D rickettsiosis, 364 African Spotted Fever, relapsing fever (relasing heat), tularemia, Colorado tick fever (Colorado tick heat), tick-borne encephalitis (TBE, also known as FSME), crimean-congo hemorrhagic fever, type Q fever, eastern hemorrhagic fever (ompk hemorrhagic fever), kesanol forest disease (Kyasanur forest disease), powassan encephalitis (powassan encephalitis), hartland virus disease (Heartland virus disease), or Babesiosis (Babesiosis). In another aspect, the disease is japanese encephalitis.

In another embodiment, the at least one further antigen is derived from a carrier-transmitted, preferably tick-transmitted, pathogen selected from the group comprising: borrelia helminthosporium (Borrelia hermsii), Borrelia deltoides (Borrelia parkeri), Borrelia dorferi (Borrelia duttoni), Borrelia gondii (Borrelia miyamotoi), Borrelia terlii (Borrelia citricatae), Rickettsia rickettsii (Rickettsia occidentalis), Rickettsia australis (Rickettsia australis), Comorella rickettsii (Rickettsia occiori), Rickettsia helveticus (Rickettsia helvetica), Francisella tularensis (Francisella terrestris), anaplasma phagocytophilum (anaplama phagocytophilum), glandular fever Ehrlichia (Ehrlichia sennetsu), chahrlichia (Ehrlichia chaffeensis), bernet rickettsia (Coxiella burnetii), and Borrelia solitarica (Borrelia lonestrari), tick-borne encephalitis virus (TBEV akaFSME virus), Colorado Tick Fever Virus (CTFV), crimean-congo hemorrhagic fever virus (CCHFV), woodruff hemorrhagic fever virus (OHFV), Japanese Encephalitis Virus (JEV), and babesia species.

In another aspect, the combination vaccine of the invention comprises a combination of any of the vaccine compositions discussed herein and at least one second vaccine composition. In some aspects, the second vaccine composition protects against a vector-transmitted disease, preferably a tick-transmitted disease. In various aspects, the second vaccine composition has a seasonal immunization program (seasonal immunization schedule) that is compatible with immunization against borrelia infection or lyme borreliosis. In other aspects, the combination vaccine is useful in the prevention of a variety of diseases, for the geographic location of prevalence of such diseases.

In one aspect, the second vaccine composition is a vaccine selected from the group consisting of: tick-borne encephalitis vaccines, japanese encephalitis vaccines and rocky mountain spotted fever vaccines. In a preferred aspect, the vaccine composition is(Baxter)、(Novartis Vaccines)、(MicrogenPO) or TBE Moscow(Chumakov Institute of Poliomyolitis and Viral Encephalitides of Russian Academy of Medical Sciences). In another preferred aspect, the vaccine composition is(Valneva SE)、(Biological E, Ltd.) or(Sanofi Pasteur)。

Further provided is a vaccine comprising the pharmaceutical composition, which vaccine may further comprise a pharmaceutically acceptable excipient. In a preferred embodiment, the excipient is L-methionine.

The invention also includes immunogenic compositions. In some aspects, the immunogenic compositions of the invention comprise any of the compositions discussed herein and a pharmaceutically acceptable carrier. In various aspects, the immunogenic composition has the property of inducing the production of antibodies that specifically bind to an outer membrane protein a (ospa) protein. In certain aspects, the immunogenic composition has the property of inducing the production of antibodies that specifically bind borrelia. In particular aspects, the immunogenic composition has the property of inducing the production of antibodies that neutralize borrelia. In some aspects, the antibody is produced by an animal. In other aspects, the animal is a mammal. In even other aspects, the mammal is a human.

Vaccine formulations comprising the pharmaceutical compositions of the present invention may be used to protect mammals susceptible to borrelia infection or to treat mammals suffering from borrelia infection by administering the vaccine via a systemic or mucosal route. Such administration may include injection via intramuscular, intraperitoneal, intradermal, or subcutaneous routes; or via mucosal administration to the oral/digestive, respiratory or genitourinary tract. Although the vaccine of the present invention may be administered as a single dose, the components thereof may also be co-administered together at the same time or at different times.

In one aspect of the invention, there is provided a vaccine kit comprising a vial containing a pharmaceutical composition of the invention (optionally in lyophilized form) and further comprising a vial containing an adjuvant as described herein. In this aspect of the invention, it is envisaged that the adjuvant will be used to reconstitute the lyophilised immunogenic composition. In another aspect, the pharmaceutical composition of the present invention may be pre-mixed in a vial, preferably in a syringe.

Another aspect of the invention is a method of preventing or treating borrelia infection, the method comprising administering to a host an immunoprotective dose of a pharmaceutical composition or vaccine or kit of the invention. In one embodiment, there is provided a method of preventing or treating primary and/or recurrent episodes of borrelia infection, the method comprising administering to a host an immunoprotective dose of a pharmaceutical composition or vaccine or kit of the invention.

Another aspect of the invention is a pharmaceutical composition of the invention for use in the treatment or prevention of borrelia disease. In one embodiment, a pharmaceutical composition for treating or preventing borrelia infection is provided.

Another aspect of the invention is the use of a pharmaceutical composition or vaccine or kit of the invention in the manufacture of a medicament for the treatment or prevention of borrelia infection. In one embodiment, there is provided a pharmaceutical composition of the invention for use in the manufacture of a medicament for the treatment or prevention of borrelia infection.

The invention also includes methods for inducing an immune response in a subject. In various aspects, such methods comprise the step of administering to the subject any of the immunogenic compositions or vaccine compositions discussed herein in an amount effective to induce an immune response. In certain aspects, the immune response comprises production of anti-OspA antibodies.

The invention includes methods for preventing or treating borrelia infection or lyme borreliosis in a subject. In various aspects, such methods comprise the step of administering to the subject any of the vaccine compositions discussed herein or any of the combination vaccines discussed herein in an amount effective to prevent or treat borrelia infection or lyme borreliosis.

The invention includes the use of a polypeptide, nucleic acid, antibody, pharmaceutical composition or vaccine of the invention for the manufacture of a medicament. Other related aspects are also provided in the present invention.

The inventors expect that the terms "comprising", "comprises" and "comprising" herein may be optionally substituted with "consisting of," consisting of, and "consisting of," respectively, in each case. The term "comprising" means "including". Thus, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated compound or composition (e.g., nucleic acid, polypeptide, antibody) or step or group of compounds or steps but not the exclusion of any other compound, composition, step or group thereof. The abbreviation "e.g." is derived from latin languages such as (exempli gratia) and is used herein to indicate non-limiting examples. The abbreviation "e.g." is therefore synonymous with the term "e.g".

Embodiments herein relating to a "vaccine composition" of the invention may also be used in relation to embodiments of a "pharmaceutical composition" of the invention, and vice versa.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms in molecular biology can be found in Benjamin Lewis, Genes V, Oxford University Press, 1994(ISBN 0-19-854287-9); kendrew et al (ed), The Encyclopedia of Molecular Biology, Blackwell Science Ltd, published 1994(ISBN 0-632-02182-9); and Robert a.meyers (ed), Molecular Biology and biotiotechnology: a Comprehensive Desk Reference, VCH Publishers, Inc., 1995(ISBN 1-56081-.

The singular terms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "plurality" means two or more. It is further understood that all base sizes or amino acid sizes and all molecular weight or molecular mass values given for a nucleic acid or polypeptide are approximations and are provided for description. In addition, the numerical limits given with respect to the concentration or level of a substance, such as an antigen, may be approximate.

Preferred carriers or excipients for the polypeptides according to the invention in its different embodiments or the nucleic acid molecules according to the invention are immunostimulating compounds such as adjuvants for further stimulating an immune response to the polypeptides according to the invention or their encoding nucleic acid molecules.

Adjuvants useful in the compositions of the present invention include, but are not limited to:

A. mineral-containing composition

Mineral-containing compositions suitable for use as adjuvants in the present invention include mineral salts, such as aluminum and calcium salts. The present invention includes mineral salts such as hydroxides (e.g., oxyhydroxides), phosphates (e.g., hydroxyphosphates, orthophosphates), sulfates, etc., or mixtures of different mineral compounds, wherein the compounds are in any suitable form (e.g., gel form, crystalline form, amorphous form, etc.) and wherein adsorption is preferred. The mineral-containing composition can also be formulated as a metal salt particle.

One useful aluminum phosphate adjuvant is amorphous aluminum hydroxyphosphate, in which PO is₄The molar ratio Al is between 0.84 and 0.92. Another useful aluminum-based adjuvant is AS04, which is a combination of aluminum hydroxide + monophosphoryl lipid a (mpl).

B. Oil emulsions

Oil emulsion compositions suitable for use AS adjuvants in the present invention include squalene-in-water emulsions such AS MF59 (5% squalene, 0.5% Tween 80 and 0.5% Span 85, formulated AS submicron particles using microfluidizers), AS03 (squalene, DL- α -tocopherol and Tween 80) and AF03 (squalene, Tween),80 andb1 PH). Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA) may also be used.

Useful oil-in-water emulsions typically comprise at least one oil and at least one surfactant, wherein the one or more oils and the one or more surfactants are biodegradable (metabolizable) and biocompatible. The oil droplets in the emulsion are typically less than 1 μm in diameter, and these small sizes are achieved using a microfluidizer to provide a stable emulsion. Droplets less than 220nm in size are preferred because they can be subjected to filter sterilization.

The emulsion may comprise oils, such as those from animal (e.g. fish) or vegetable sources. Vegetable oil sources include nuts, seeds and grains. The nut oils are exemplified by the most commonly used peanut oil, soybean oil, coconut oil, and olive oil. For example, jojoba oil obtained from jojoba beans may be used. Seed oils include safflower oil, cottonseed oil, sunflower oil, sesame seed oil, and the like. Corn oil is the most readily available in the grain group, but other grain oils such as wheat, oats, rye, rice, moss bran (teff), triticale, and the like may also be used. The 6-10 carbon fatty acid esters of glycerol and 1, 2-propanediol, while not naturally present in seed oils, can be prepared by hydrolysis, isolation and esterification of suitable materials starting from nuts and seed oils. Fats and oils from mammalian milk are metabolizable and thus can be used to practice the invention. Procedures for isolation, purification, saponification, and other means required to obtain pure oils from animal sources are well known in the art. Most fish contain metabolizable oil that can be easily recovered. For example, cod liver oil, shark liver oil, and whale oil such as spermaceti exemplify several fish oils that may be used herein. Many branched oils are biosynthesized with 5-carbon isoprene units and are commonly referred to as terpenoids. Shark liver oil comprises the branched, unsaturated terpenoid 2, 6, 10, 15, 19, 23-hexamethyl-2, 6, 10, 14, 18, 22-tetracosane, called squalene, which is particularly preferred herein. The saturated analog of squalene, squalane, is also a preferred oil. Fish oils including squalene and squalane can be readily obtained from commercial sources or can be obtained by methods known in the art. Other preferred oils are tocopherols (see below). Mixtures of oils may be used.

Surfactants can be classified according to their 'HLB' (hydrophilic/lipophilic balance). Preferred surface-active substances according to the inventionThe agent has an HLB of at least 10, preferably at least 15 and more preferably at least 16. The present invention may be used with surfactants including, but not limited to: polyoxyethylene sorbitan ester surfactants (commonly known as Tween), in particular polysorbate 20 and polysorbate 80; copolymers of Ethylene Oxide (EO), Propylene Oxide (PO) and/or Butylene Oxide (BO) in DOWF AX^TMTrade names such as linear EO/PO block copolymers; octoxynol, the number of repeating ethoxy (oxy-1, 2-ethanediyl) groups of which may vary, of which octoxynol-9 (Triton X-100 or tert-octylphenoxypolyethoxyethanol) is of particular interest; (octylphenoxy) polyethoxyethanol (IGEPALCA-630/NP-40); phospholipids, such as phosphatidylcholine (lecithin); nonylphenol ethoxylates, e.g. Tergitol^TMNP series; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethylene glycol monolauryl ether (Brij 30); and sorbitan esters (commonly referred to as SPAN), such as sorbitan trioleate (SPAN 85) and sorbitan laurate. Nonionic surfactants are preferred. Preferred surfactants for inclusion in the emulsion are Tween 80 (polyoxyethylene sorbitan oleate), Span 85 (sorbitan trioleate), lecithin and Triton X-100.

Mixtures of surfactants may be used, for example, a Tween 80/Span 85 mixture. Combinations of polyoxyethylene sorbitan esters such as polyoxyethylene sorbitan monooleate (Tween 80) and octoxynol such as t-octylphenoxypolyethoxyethanol (Triton X-100) are also suitable. Another useful combination includes laureth 9 plus polyoxyethylene sorbitan esters and/or octoxynol.

Preferred amounts of surfactants (wt%) are: polyoxyethylene sorbitan esters (e.g. Tween 80): 0.01% to 1%, specifically about 0.1%; octyl or nonyl phenoxy polyoxyethanols (such as Triton X-100 or other detergents of the Triton series): 0.001% to 0.1%, specifically 0.005% to 0.02%; polyoxyethylene ethers (e.g. laureth 9): 0.1% to 20%, preferably 0.1% to 10% and in particular 0.1% to 1% or about 0.5%.

Preferably, substantially all (e.g., at least 90% by number) of the oil droplets have a diameter of less than 1 μm, e.g., < 750nm, < 500nm, < 400nm, < 300nm, < 250nm, < 220nm, < 200nm, or less. One particularly useful sub-microemulsion consists of squalene, Tween 80 and Span 85. The composition of the emulsion by volume may be about 5% squalene, about 0.5% polysorbate 80 and about 0.5% Span 85. These ratios were 4.3% squalene, 0.5% polysorbate 80 and 0.48% Span 85 by weight. The MF59 emulsion advantageously includes citrate ions, for example 10mM sodium citrate buffer.

C. Saponin preparation

Saponin formulations may also be used as adjuvants in the present invention. Saponins are a heterogeneous population of sterol glycosides and triterpene glycosides that can be found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponins from the bark of the quillaja Molina tree (quillaja apionaria Molina tree) have been extensively studied as adjuvants. Saponins are also commercially available from sarsapium palmatum (Smilax ornata) (sarsapsa), carnation (gymnophila paniculata) (bradlea veil) and soapwort (Saponaria officinalis) (soaproot). Saponin adjuvant formulations include purified formulations such as QS21 and lipid formulations such as ISCOMs. QS21 as Stimulon^TMAnd (5) selling.

The saponin composition has been purified using HPLC and RP-HPLC. Specific purified fractions obtained using these techniques have been identified, including QS7, QS 17, QS 18, QS21, QH-A, QH-B, and QH-C. Preferably, the saponin is QS 21. The saponin preparation may also include sterols, such as cholesterol.

The combination of saponin and cholesterol can be used to form unique particles known as Immune Stimulating Complexes (ISCOMs). ISCOMs also typically include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOMs. Preferably, the ISCOM includes one or more of QS7, QS 17, QS 18, QS21, QH-A, QH-B and QH-C. Optionally, the ISCOM may lack additional detergent.

D. Virosomes and virus-like particles

Virosomes and virus-like particles (VLPs) may also be used as adjuvants in the present invention. These structures typically comprise one or more proteins from the virus, optionally combined or formulated with phospholipids. They are generally non-pathogenic, non-replicative, and generally do not contain any native viral genome. Viral proteins may be recombinantly produced or isolated from whole viruses. These viral proteins suitable for use in virosomes or VLPs include proteins derived from: influenza virus (e.g., HA or NA), hepatitis B virus (e.g., core or capsid protein), hepatitis E virus, measles virus, Sindbis virus, rotavirus, foot and mouth disease virus, retrovirus, Norwalk virus, human papilloma virus, HIV, RNA-phage, Q β -phage (e.g., coat protein), GA-phage, fr-phage, AP 205-phage, and Ty (e.g., retrotransposon Ty protein pi).

E. Bacterial or microbial derivatives

Adjuvants suitable for use in the present invention include bacterial or microbial derivatives, such as non-toxic derivatives of enteric bacterial Lipopolysaccharide (LPS), lipid A derivatives, immunostimulatory oligonucleotides and ADP-ribosylating toxins and detoxified derivatives thereof.

Non-toxic derivatives of LPS include monophosphoryl lipid a (MPL) and 3-O-deacyl MPL (3 dMPL). 3dMPL is a 3 de-O-acylated monophosphoryl lipid A having 4, 5 or 6 acylated chains. Such "small particles" of 3dMPL are small enough to be sterile filtered through a 0.22 μm membrane. Other non-toxic LPS derivatives include monophosphoryl lipid a mimetics, such as aminoalkyl glucosaminide phosphate derivatives, e.g. RC-529, and synthetic phospholipid dimer E6020.

Lipid A derivatives include lipid A derivatives from E.coli such as OM-174. Immunostimulatory oligonucleotides suitable for use as adjuvants in the present invention include nucleotide sequences containing a CpG motif (a dinucleotide sequence comprising an unmethylated cytosine linked to a guanosine by a phosphate bond). Double stranded RNA and oligonucleotides comprising palindromic or poly (dG) sequences have also been shown to be immunostimulatory.

CpG may include nucleotide modifications/analogs, such as phosphorothioate modifications, and may be double-stranded or single-stranded. The CpG sequence may be directed against TLR9, such as the motifs GTCGTT or TTCGTT. The CpG sequence may be specific for inducing a Th1 immune response, such as a CpG-A ODN, or it may be more specific for inducing a B cell response, such as a CpG-B ODN. Preferably, the CpG is a CpG-A ODN.

Preferably, the CpG oligonucleotides are constructed such that the 5' end is accessible for receptor recognition. Optionally, two CpG oligonucleotide sequences may be linked at their 3' ends to form an "immunogenice". Particularly useful adjuvants based on immunostimulatory oligonucleotides are referred to asThus, adjuvants for use in the present invention may comprise a mixture of: (i) oligonucleotides (e.g., between 15-40 nucleotides) that include at least one (and preferably a plurality) of CpI motifs (i.e., cytosines are linked to inosine to form dinucleotides); and (ii) a polycationic polymer, such as an oligopeptide (e.g., between 5-20 amino acids) comprising at least one (and preferably a plurality) of Lys-Arg-Lys tripeptide sequences. The oligonucleotide may be a polynucleotide comprising the 26-mer sequence 5' - (dIdC)₁₃3' (SEQ ID NO: 32) deoxynucleotides. The polycationic polymer may be a peptide comprising the 11-mer amino acid sequence KLKLLLLLKLK (SEQ ID NO: 33).

Polycationic compounds derived from natural sources include HIV-REV or HIV-TAT (derivatized cationic peptides, antennapedia peptides, chitosan or other chitin derivatives) or other peptides obtained from these peptides or proteins by biochemical or recombinant production. Other preferred polycationic compounds are antimicrobial peptide precursors (cathelins) or related or derived substances from antimicrobial peptide precursors. For example, the mouse antimicrobial peptide precursor is a peptide having the amino acid sequence NH₂-RLAGLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPE-COOH (SEQ ID NO: 31). CorrelationOr the derivatized antimicrobial peptide precursor material comprises all or a portion of an antimicrobial peptide precursor sequence having at least 15-20 amino acid residues. Derivatization may include substitution or modification of the natural amino acid by amino acids that are not among the 20 standard amino acids. Furthermore, additional cationic residues may be introduced into such antimicrobial peptide precursor molecules. These antimicrobial peptide precursor molecules are preferably combined with the antigen. These antimicrobial peptide precursor molecules have surprisingly been shown to be effective as adjuvants for antigens as well, without the addition of further adjuvants. It is thus possible to use such antimicrobial peptide precursor molecules as effective adjuvants in vaccine formulations with or without additional immune activating substances.

Bacterial ADP-ribosylating toxins and detoxified derivatives thereof are useful as adjuvants in the present invention. Preferably, the protein is derived from Escherichia coli (E.coli heat labile enterotoxin "LT"), Vibrio cholerae (cholera toxin "CT"), or Bordetella pertussis (pertussis toxin "PT"). The use of detoxified ADP-ribosylated toxins as mucosal adjuvants and as parenteral adjuvants is known. The toxin or toxoid is preferably in the form of a holothurin, which comprises both a and B subunits. Preferably, the a subunit comprises a detoxifying mutation; preferably, the B subunit is not mutated. Preferably, the adjuvant is a detoxified LT mutant, such as LT-K63, LT-R72, LT-G192, or dmLT. A useful CT mutant is CT-E29H.

F. Human immunomodulator

Human immunomodulators suitable for use as adjuvants in the present invention include cytokines such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., interferon-gamma), macrophage colony stimulating factor, and tumor necrosis factor. A preferred immunomodulator is IL-12.

G. Bioadhesive and mucoadhesive agents

Bioadhesives and mucoadhesives may also be used as adjuvants in the present invention. Suitable bioadhesives include esterified hyaluronic acid microspheres or mucoadhesives such as polyacrylic acid, polyvinyl alcohol, polyvinyl pyrrolidone, polysaccharides and crosslinked derivatives of carboxymethylcellulose. Chitosan and its derivatives may also be used as adjuvants in the present invention.

H. Microparticles

Microparticles may also be used as adjuvants in the present invention. Microparticles (i.e., particles having a diameter of about 100nm to about 150 μm, more preferably about 200nm to about 30 μm, and most preferably about 500nm to about 10 μm) that are optionally treated to have a negatively charged surface (e.g., with SDS) or a positively charged surface (e.g., with a cationic detergent, such as CTAB) are formed from biodegradable and non-toxic materials (e.g., poly (alpha-hydroxy acids), polyhydroxybutyric acid, polyorthoesters, polyanhydrides, polycaprolactone, poly (lactide-co-glycolide), etc.).

I. Liposomes

Examples of liposomal formulations suitable for use as adjuvants are known.

J. Polyoxyethylene ethers and polyoxyethylene ester formulations

Adjuvants suitable for use in the present invention include polyoxyethylene ethers and esters. Such formulations also include a polyoxyethylene sorbitan ester surfactant in combination with an octoxynol and a polyoxyethylene alkyl ether or alkyl ester surfactant in combination with at least one additional non-ionic surfactant such as an octoxynol. Preferred polyoxyethylene ethers are selected from the group consisting of: polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-stearyl ether, polyoxyethylene-8-stearyl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether.

K. Muramyl peptides

Examples of muramyl peptides suitable for use as adjuvants in the present invention include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), and N-acetyl-L-alanyl-D-isoglutamyl-L-alanine-2- (1 '-2' -dipalmitoyl-5N-glycero-3-hydroxyphosphoryloxy) -ethylamine MTP-PE).

An imidazoquinolone compound.

Examples of imidazoquinolone compounds suitable for use as adjuvants in the present invention include imiquimod and its homologs (e.g., "resiquimod 3M").

The invention may also include combinations of aspects of one or more of the adjuvants identified above.

Preferably, the immunostimulatory compound in the pharmaceutical preparation according to the invention is selected from the group consisting of: a polycationic substance (in particular a polycationic peptide), an immunostimulatory nucleic acid molecule (preferably an immunostimulatory deoxynucleotide), an oil-in-water or water-in-oil emulsion, MF59, an aluminium salt, freund's complete adjuvant, freund's incomplete adjuvant, a neuroactive compound (in particular human growth hormone) or a combination thereof.

The use of aluminium hydroxide and/or aluminium phosphate adjuvants is particularly preferred, and antigens are typically adsorbed to these salts.

Furthermore, the pharmaceutical composition according to the present invention is a pharmaceutical composition comprising at least any one of the following compounds or a combination thereof: a nucleic acid molecule according to the invention, a polypeptide according to the invention in its various embodiments, a vector according to the invention, a cell according to the invention and an antibody according to the invention. In this regard, any of these compounds may be used in combination with one or more non-sterile or sterile carriers for a cell, tissue or organism, such as a pharmaceutical carrier suitable for administration to a subject. Such carriers can include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should meet the requirements of the mode of administration.

In one embodiment, the pharmaceutical composition comprises a stabilizer. The term "stabilizer" refers to a substance or vaccine excipient that protects the immunogenic composition of the vaccine from adverse conditions (such as those created during heating or freezing) and/or extends the stability or shelf life of the immunogenic composition under stable and immunogenic conditions or conditions. Examples of stabilizers include, but are not limited to, sugars such as sucrose, lactose, and mannose; sugar alcohols, such as mannitol; amino acids such as glycine or glutamic acid; and proteins such as human serum albumin or gelatin.

The pharmaceutical compositions of the present invention may be administered in any effective and convenient manner, including, for example, by topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, intratracheal, or intradermal routes, among others. In a preferred embodiment, the pharmaceutical composition is administered subcutaneously or intramuscularly, most preferably intramuscularly.

The active agent of the pharmaceutical composition of the invention may be administered to the individual in therapy or as a prophylactic agent, as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic.

Alternatively, the composition, preferably the pharmaceutical composition, may be formulated for topical application, for example in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouth washes, impregnated dressings and sutures and aerosols, and may contain suitable conventional additives including, for example, preservatives, solvents to promote drug penetration and emollients in ointments and creams. Such topical formulations may also contain compatible conventional carriers such as cream or ointment bases and ethanol or oleyl alcohol for lotions. Such carriers may constitute from about 1% to about 98% by weight of the formulation; typically they will constitute about 80% by weight of the formulation.

In addition to the therapies described above, the compositions of the present invention are generally useful as wound treatment agents to prevent bacterial adhesion to matrix proteins exposed to wound tissue and for prophylactic use in dental treatments as an alternative to or in combination with antibiotic prophylaxis.

In a preferred embodiment, the pharmaceutical composition is a vaccine composition. Preferably, such vaccine compositions are conveniently in injectable form. Conventional adjuvants may be used to enhance the immune response. Suitable unit doses for vaccination with protein antigens are between 0.02 and 3 μ g antigen per kg body weight for adults and between 0.2 and 10 μ g antigen per kg body weight for children, and such doses are preferably administered 1 to 3 times at intervals of 2 to 24 weeks.

Within the dosage range specified, no adverse toxic effects are expected with the compounds of the present invention, which would prevent administration of the compounds to the appropriate subject.

As another aspect, the invention includes kits comprising one or more pharmaceutical formulations for administration to a subject, the formulations being packaged in a manner that facilitates their use for administration to a subject. In a preferred embodiment, the kit comprises a final volume of 2mL of formulation, more preferably a final volume of 1 mL.

In a particular embodiment, the invention comprises a kit for producing a single dose administration unit. In various aspects, the kits each contain a first container having a dried protein and a second container having an aqueous formulation. Also included within the scope of the present invention are kits containing single and multi-chamber pre-filled syringes, e.g., liquid and powder syringes (lyosyringes).

In another embodiment, such a kit comprises a pharmaceutical formulation described herein (e.g., a composition comprising a therapeutic protein or peptide) packaged in a container, such as a sealed bottle or vessel, with a label describing the use of the compound or composition in practicing the method affixed to the container or included in the package. In one embodiment, the pharmaceutical formulation is packaged in the container such that the amount of headspace of the container (e.g., the amount of air between the liquid formulation and the top of the container) is sufficiently small. Preferably, the amount of headspace is negligible (i.e., nearly non-existent).

In one aspect, a kit comprises a first container having a therapeutic protein or peptide composition and a second container having a physiologically acceptable reconstituting solution for the composition. In one aspect, the pharmaceutical formulation is packaged in unit dosage form. The kit optionally further comprises equipment suitable for administering the pharmaceutical formulation according to a particular route of administration. In some aspects, the kit comprises a label describing the use of the pharmaceutical formulation.

The pharmaceutical composition may comprise a range of different antigens. Examples of antigens are completely inactivated or attenuated organisms, subfractions of these organisms, proteins or peptides in their simplest form. Antigens may also be recognized by the immune system in the form of glycosylated proteins or peptides and may also be or comprise polysaccharides or lipids. Since cytotoxic T-Cells (CTLs) recognize antigens in the form of short peptides (typically 8 to 11 amino acids in length) that bind to Major Histocompatibility Complex (MHC), short peptides can be used. B cells can recognize linear epitopes as short as 4 to 5 amino acids as well as three-dimensional structures (conformational epitopes).

In a preferred embodiment, the pharmaceutical composition of the third aspect further comprises a hyperimmune serum-reactive antigen or an active fragment or variant thereof against borrelia protein, such as for example the antigen, fragment and variant described in WO 2008/031133.

According to the present invention, the pharmaceutical composition according to the third aspect may be used as a medicament, in particular as a vaccine, in particular for use in combination with a particular disease or diseased condition caused by, associated with or associated with borrelia.

The pharmaceutical composition of the invention is useful as a medicament, in particular as a vaccine, in particular for use in combination with a disease or diseased condition related to or associated with borrelia caused by borrelia (more preferably any pathogenic borrelia species), and more preferably for use in a method for the treatment or prevention of borrelia infection, in particular borrelia burgdorferi sensu stricto, borrelia garinii, borrelia afzelii, borrelia andersoni, borrelia bavaria, borrelia besseyi, borrelia farinosa, borrelia rutinosa, borrelia strobila sporeai, borrelia japonica, borrelia tennici, borrelia terrestris or borrelia cibacii infection, preferably borrelia burgdorferi sensu stricto, borrelia aldii or borrelia gariae.

In this connection, it should be noted that different borrelia species (including borrelia burgdorferi in a broad sense) include several species and strains, including those disclosed herein. Diseases associated with, caused by or associated with said bacterial infection to be prevented and/or treated according to the invention include lyme borreliosis (lyme disease). Further aspects, symptoms, stages and subgroups of lyme borreliosis as also disclosed herein, as well as specific patient groups (included in the introductory part) suffering from such diseases, are incorporated herein by reference. More specifically, lyme borreliosis usually occurs in stages, with different clinical manifestations of remission and worsening at each stage. Early infection stage 1 consists of topical infection of the skin; days or weeks later, stage 2, disseminated infection; months to years later, stage 3, persistent infection. However, infection is variable; some patients only suffer from local skin infections, while others show only late-stage manifestations of the disease, such as arthritis.

In a fourth aspect, the present invention relates to a method of treating or preventing borrelia infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to the third aspect.

The term "subject" is used throughout the specification to describe an animal, preferably a mammal, more preferably a human, to which a treatment or method according to the invention is provided. For the treatment of those infections, conditions or disease states that are specific to a particular animal, such as a human patient, the term patient refers to that particular animal. Preferably, the subject is a human; however, medical uses of the composition may also include animals such as poultry (including chickens, turkeys, ducks, or geese), livestock (such as horses, cattle, or sheep), or companion animals (such as dogs or cats).

The term "effective amount" is used throughout the specification to describe the amount of a pharmaceutical composition of the invention that can be used to induce a desired result when used in the methods of the invention. In various aspects of the invention, the term effective amount is used in conjunction with treatment or prevention. In other aspects, the term effective amount is merely meant to include that amount of an agent which produces a result deemed beneficial or useful in a method according to the invention in which treatment or prevention of borrelia infection is sought.

The term effective amount with respect to the presently described compounds and compositions is used throughout the specification to describe the amount of a compound according to the present invention that is administered to a mammalian patient (including particularly human patients) suffering from borrelia-related disease to reduce or inhibit borrelia infection.

In a preferred embodiment, the method of immunizing a subject according to the fourth aspect comprises the step of administering to the subject a therapeutically effective amount of the pharmaceutical composition of the third aspect of the invention.

The method comprises inducing an immune response in an individual by gene therapy or otherwise by in vivo administration of a polypeptide or nucleic acid according to the invention, so as to stimulate the immune response to produce antibodies or cell-mediated T cell responses (cytokine-producing T cells or cytotoxic T cells) to protect the individual from a disease, whether or not the disease is already present in the individual.

The products of the invention (in particular polypeptides and nucleic acids) are preferably provided in isolated form and may be purified to homogeneity. The term "isolated" as used herein means "separated from its natural state by the hand of man", i.e., if it exists in nature, it has been altered or removed from its original environment, or both. For example, a naturally occurring nucleic acid molecule or polypeptide that is naturally present in a living organism in its natural state is not "isolated," but the same nucleic acid molecule or polypeptide that is isolated from the coexisting materials of its natural state is "isolated," as the term is used herein. As part of or after isolation, such nucleic acid molecules can be joined to other nucleic acid molecules such as DNA molecules for mutagenesis to form fusion genes, and for propagation or expression in, for example, a host. Isolated nucleic acid molecules, alone or joined to other nucleic acid molecules such as vectors, can be introduced into host cells in culture or whole organisms. Such DNA molecules may still be isolated, as the term is used herein, as they are not in their naturally occurring form or environment, when introduced into a host cell in culture or in a whole organism. Similarly, nucleic acid molecules and polypeptides may be present in compositions, such as media preparations, solutions for introducing nucleic acid molecules or polypeptides into, for example, cells, compositions or solutions for, for example, chemical or enzymatic reactions, which are not naturally occurring compositions and in which isolated nucleic acid molecules or polypeptides remain within the meaning of the term as used herein.

The invention is not limited to the particular methodology, protocols, and reagents described herein because they may vary. Furthermore, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention. As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the words "comprise", "comprising" and "includes" are to be construed as inclusive and not exclusive.

Unless defined otherwise, all technical and scientific terms and any abbreviations used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred methods and materials are described herein.

The invention is further illustrated by the following figures, tables, examples and sequence listing from which additional features, embodiments and advantages can be derived. Likewise, the particular modifications discussed should not be construed as limitations on the scope of the invention. It will be apparent to those skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the invention, and it is therefore to be understood that such equivalent embodiments are to be included herein.

Incorporating the invention

FIG. 1 shows an amino acid alignment of OspA serotypes 1-6 from Borrelia.

FIG. 2 schematically shows the generation of heterodimers of mutant OspA fragments according to the present invention.

FIG. 3 schematically represents the polypeptide components of one possible pharmaceutical composition "combination vaccine" of the invention comprising three different mutant OspA heterodimers.

FIG. 4 shows Pam₃Chemical Structure of Cys, Pam₃Cys is an example of a fatty acid-substituted cysteine, which would be present, for example, at the N-terminus of a lipidated polypeptide of the invention.

FIG. 5 shows the binding of antibodies from mice immunized with the mutant OspA fragment heterodimeric polypeptide of the present invention to the cell surface of Borrelia having OspA serotypes 1-6.

Table 1 shows the thermostability of the fold of mutant serotype 2OspA fragments having disulfide bond types D1 to D5 (see table a-4 for nomenclature) compared to wild type serotype 2OspA fragments without disulfide bonds (D0).

Table 2 shows protection of mice against borrelia afzelii (strain IS1) infection by the tick challenge method after immunization with mutant serotype 2OspA fragments having disulfide bond types D1 to D5 (for nomenclature, see table a-4), including a control group of mice immunized with PBS, full-length OspA, or wild-type serotype 2OspA fragment (S2D 0-His).

Table 3 shows protection of mice against borrelia adobergii (strain IS1) infection by the tick challenge method following immunization with lipidated mutant serotype 2OspA fragments (Lip-S2D1-His, Lip-S2D3-His and Lip-S2D4-His) having disulfide bond types D1, D3 and D4, including a control group of mice immunized with PBS and full-length OspA protein.

Table 4 shows the protective capacity of the mutant OspA heterodimers of the invention in an in vivo borrelia challenge model. Mice were immunized with Lip-S1D1-S2D1-His, Lip-S4D1-S3D1-His, Lip-S4D1-S3D1, or Lip-S5D1-S6D1-His and challenged with the indicated borrelia OspA serotype by either the tick challenge method or the needle challenge method as indicated. Control groups in each experiment were treated with Al (OH) alone₃And (4) adjuvant immunization.

Table 5 shows the protective capacity of the combination vaccine of the invention against in vivo challenge with OspA serotype 1 borrelia (strain N40, in needle challenge procedure) and OspA serotype 2 borrelia (strain IS1, in tick challenge procedure). Mice were immunized with the three antigens Lip-S1D1-S2D1, Lip-S4D1-S3D1, and Lip-S5D1-S6D1 together in a 1: 1 ratio (combination vaccine) or with the indicated control antigens, and were challenged with Borrelia by either the tick or needle challenge methods as indicated. Control groups in each experiment were treated with Al (OH) alone₃And (4) adjuvant immunization.

The figures and tables that may be referenced in this description are described in more detail below.

Figure 1 amino acid sequence alignment of OspA serotypes 1 to 6. The alignment shows that the membrane associated N-terminal portion of the protein has a more highly conserved amino acid sequence than the more exposed C-terminal portion.

FIG. 2 production of mutant OspA heterodimers of the invention comprising mutant OspA C-terminal fragments from two different OspA serotypes of Borrelia species. (A) Schematic representation of a nucleic acid encoding a lipidated mutant OspA heterodimer. The modules included 5 'to 3' the coding sequence for the lipidation signal sequence (Lip signal), a small cysteine-containing peptide for N-terminal lipidation (lipidation peptide ═ LP), a mutant C-terminal fragment of OspA with two non-native cysteines, a short linker peptide (LN1), followed by a second mutant OspA C-terminal fragment with two non-native cysteines. (B) The intermediate mutant OspA heterodimeric polypeptide comprises a new production directly after translation of the nucleic acid construct. From N-terminus to C-terminus, this polypeptide consists of a lipidation signal sequence (Lip signal), a cysteine-containing peptide (LP) for lipidation, a mutant OspA fragment with a non-natural disulfide bond, a short linker peptide (LN1), followed by a second mutant OspA fragment with a non-natural disulfide bond. (C) Final lipidation after post-translational modification of the mutant OspA heterodimeric polypeptide. From N-terminus to C-terminus, the heterodimer consists of a short cysteine-containing peptide with a lipidated N-terminal cysteine (indicated as "Lip"), a mutant OspA fragment stabilized by a disulfide bond, a linker peptide (LN1), and a second mutant OspA fragment stabilized by a disulfide bond. As shown, the lipidated signal sequence is cleaved off during the post-translational modification of the polypeptide.

Fig. 3 is an example of a preferred pharmaceutical composition according to the present invention. Three mutant OspA heterodimers, each comprising a mutant OspA fragment from two different borrelia OspA serotypes, are present in the composition, together providing OspA antigens from six different borrelia OspA serotypes. Such pharmaceutical compositions enable simultaneous immunization against six borrelia serotypes.

FIG. 4 Pam₃Chemical Structure schematic of Cys, Pam₃Cys is a fatty acid substitution of the N-terminal cysteine of the full-length wild-type OspA protein, as well as examples of lipidated mutant OspA fragment monomers and heterodimers of the invention. During the post-translational modification of full-length OspA proteins or polypeptides of the invention, the N-terminal lipidation signal sequence is cleaved off and the fatty acids (most commonly three palmitoyl moieties ("Pam"))₃")) is enzymatically covalently linked to the remaining residues of the N-terminal cysteine residue (the sulfur atom" S "is indicated by the arrow) polypeptide chain. Located in Pam₃The C-terminus of Cys residue is denoted by "Xn". (adapted from Bouchon et al (1997) Analytical Biochemistry 246: 52-61.)

FIG. 5 binding of antibodies from immunized mice to the cell surface of Borrelia spirochetes. Mice were immunized three times with 1 μ g each of the following indicated antigens at two week intervals, and sera were collected one week after the last dose: OspA serumLipidated and His-tagged full-length OspA proteins of forms 1 to 6; Lip-S1D1-S2D1, Lip-S4D1-S3D1, or Lip-S5D1-S6D1 alone; or Lip-S1D1-S2D1, Lip-S4D1-S3D1 and Lip-S5D1-S6D1 combined in a 1: 1 ratio ("combination vaccine"). Several serum dilutions were tested by cell staining and flow cytometry to learn binding to borrelia cell surfaces. Subtracting in the same manner as in the case of using Al (OH) alone₃Fluorescence intensity values observed when sera collected from adjuvant-immunized control mice were stained to account for non-specific binding. (borrelia used: borrelia burgdorferi, OspA serotype 1, strain N40; borrelia afzelii, OspA serotype 2, strain "C"; borrelia garinii, OspA serotype 3, strain "D"; borrelia bavaria, OspA serotype 4, strain Fin; borrelia garinii, OspA serotype 5, strain "E"; borrelia garinii, OspA serotype 6, strain "B")

TABLE 1 thermostability of non-lipidated His-tagged Borrelia aryabhattai K78 mutant serotype 2OspA fragments with differently arranged disulfide bonds. Mutant serotype 2OspA fragments with different cysteine bond types (see table a-4) were dissolved in 50mM tris-HCl, 150mM NaCl (pH 8.0) and tested for thermostability compared to wild type serotype 2OspA fragment (S2D 0). The presence of disulfide bonds results in an increase in melting temperature compared to the wild type serotype 2OspA fragment.

For nomenclature, see tables a-4 and a-5.

TABLE 2 protective capacity of reduced dose of non-lipidated His-tagged mutant serotype 2OspA fragments against Borrelia aryabhattai (serotype 2) infection by the tick challenge method. Five non-lipidated His-tagged mutant serotype 2OspA fragments were tested for protective capacity at two different doses (30 μ g and 5 μ g) and compared to wild type serotype 2OspA fragments. With Al (OH) alone₃Adjuvant immunisationGroups of mice or groups of mice immunized with non-lipidated full-length serotype 2OspA were used as negative and positive controls, respectively. All antigens were His-tagged and not lipidated. The data presented combine the results of several experiments performed under the same conditions.

TABLE 3 protective capacity of reduced dose of lipidated His-tagged mutant serotype 2OspA fragments against Borrelia aryabhattai infection by the tick challenge method. Three lipidated His-tagged mutant serotype 2OspA fragments with different disulfide bond types were tested for protective ability at three different doses (3.0. mu.g, 1.0. mu.g and 0.3. mu.g). With Al (OH) alone₃Groups of adjuvant-immunized mice or mice immunized with non-lipidated full-length serotype 2OspA were used as negative and positive controls, respectively. The data presented combine the results of several experiments performed under the same conditions.

Table 4. protective capacity of the mutant OspA heterodimers of the invention against borrelia challenge in vivo by either the needle challenge method or the tick challenge method. At two week intervals with indicated doses of OspA heterodimer or Al (OH) alone₃Groups of mice were immunized three times with adjuvant. Immunogens used were Lip-S1D1-S2D1-His (challenged with Borrelia OspA-ST1, experiments 1-3), Lip-S1D1-S2D1-His alone, Lip-S4D1-S3D1-His and Lip-S5D1-S6D1-His (challenged with Borrelia OspA-ST2, experiments 4-6), Lip-S4D1-S3D1 (challenged with Borrelia OspA-ST4, experiments 7 and 8) and Lip-S5D1-S6D1-His (challenged with Borrelia OspA-ST 4)5 challenge, experiments 9 and 10; challenge with borrelia OspA-ST6, experiments 11 and 12). Immunized mice were challenged two weeks after the last immunization with either the indicated tick challenge model or the needle challenge model.

A P-value; fisher exact test, two tails. Significance (< 0.05),. star high significance (< 0.01),. star very high significance (< 0.001)

TABLE 5 protective capacity of the mutant OspA heterodimer combination vaccines of the present invention against OspA serotype 1 and serotype 2 Borrelia challenge. Two week intervals with indicated doses of immunogen or Al (OH) alone₃Groups of mice were immunized three times with adjuvant. The immunogens used were a 1: 1 combination of mutant OspA heterodimers Lip-S1D1-S2D1, Lip-S4D1-S3D1 and Lip-S5D1-S6D1 (combination vaccine), Lip-S1D1-S2D1, Lip-OspA1-His and chimeric OspA ST1/ST 2. Immunized mice were challenged two weeks after the last immunization by either the tick challenge method (ST2, experiments 13 and 14) or the needle challenge method (ST1, experiments 15 and 16).

Examples

Example 1 evaluation of thermostability of mutant serotype 2OspA fragments

Experimental procedures

Thermal stability

The melting temperature (T.T.for the non-lipidated mutant serotype 2OspA fragment monomers) was determined by fluorescence-based thermal drift assay as described by Pantoliano et al (J.Biomol Screen 6: 429-440(2001))_m). Using fluorescent dyesOrange protein gel stain (supplied by Sigma company, usa as a 5000x concentrate in DMSO) was used to monitor protein unfolding. In each well, 7.5. mu.L of each pool was pooledOrange (diluted 1: 1000 from stock solution) and 17.5. mu.L of protein in buffer solution (1. mu.g or 2. mu.g). Protein samples were heated from 25 ℃ to 95 ℃ at a rate of 0.2 ℃/10s in a CFX96 real-time detection system (Bio-Rad, USA) and fluorescence changes were monitored. Fluorescence intensity was measured using excitation and emission wavelengths of 490nm and 575nm, respectively. Determination of T Using Bio-Rad CFX Manager 2.0 program_m. T of the non-lipidated His-tagged serotype 2OspA mutant fragment was measured in four different buffer systems_mThe value: 50mM Tris-HCl, 150mM NaCl (pH 9.0); 50mM Tris-HCl, 150mM NaCl (pH 8.0); PBS (pH 7.4); and 25mM HEPES, 150mM NaCl (pH 6.5), using the non-lipidated serotype 2OspA wild-type fragment (S2D0) as a control.

Results

In all cases, T of mutant serotype 2OspA fragment with introduced cysteine bond_mHigher than wild type serotype 2OspA fragment (S2D0) (see table 1). T tested in four different buffer systems_mWith similar results (dissolved in 50mM Tris-HCl, 150mM NaCl)Data for the protein in (pH 8.0) are shown in table 1), indicating that protein stability is similar over a wide pH range. This result demonstrates the hypothesis that the introduced disulfide bond stabilizes the OspA fragment.

Example 2 evaluation of protective Capacity of non-lipidated His-tagged mutant serotype 2OspA fragment monomers in the tick challenge procedure (ST2, Borrelia avermitilis)

Experimental procedures

Cloning and expression of recombinant proteins

GenScript, USA codon-optimizes wild-type serotype 2OspA fragments as well as mutant serotype 2OspA fragments with cysteine bond types 1-5 (SEQ ID NOS: 1, 2, 3, 4, 5 and 6, respectively) for E.coli expression. For purification purposes, a His-tag was added to the C-terminus of the non-lipidated mutant serotype 2OspA fragment. The gene fragment was cloned into the pET28b (+) vector (Novagen, USA), a vector containing the kanamycin resistance expression cassette and the T7 promoter. At 37 ℃ BL21Star by addition of IPTG^TM(DE3) expression of monomers in cells (Invitrogen, USA). Cells were collected by centrifugation after 4h and the pellet was stored at-70 ℃ for 12 months before further processing.

Purification of non-lipidated His-tagged wild-type OspA fragment monomeric proteins and mutant OspA fragment monomeric proteins

Mechanical disruption of cells by high pressure homogenization and application of soluble fraction containing His-tagged OspA fragments to Ni-Sepharose column (Ni Sepharose)^TM6Fast Flow; GE Healthcare, United Kingdom) and elute the His-tagged OspA fragment with an imidazole gradient (0-250 mM). The combined fractions were further purified on a gel filtration column (Superdex200, GEHealthcare) followed by a buffer exchange column (Sephadex G-25, GE Healthcare). His-tagged OspA fragment peaks were pooled based on analytical size exclusion column and reverse phase chromatography. After sterile filtration, the purified protein was stored at-20 deg.CAnd then until the formulation is formulated.

Immunization of mice

For all studies, female C3H/HeN (H-2) was used^k) Mice (Harlan, Italy). Five groups of 8-week-old mice were bled via the tail vein prior to each challenge, and preimmune sera were prepared and pooled. Five non-lipidated mutant serotype 2OspA fragment proteins (S2D 1-5, SEQ ID NOs: 2, 3, 4, 5 and 6, respectively) were tested in fifteen separate experiments. Three 100 μ L subcutaneous (s.c.) immunizations were administered at two week intervals. The doses used were 30 μ g and 5 μ g of the corresponding protein, respectively, tested in the 11 th experiment and 4 th experiment, respectively. All formulations contained a final concentration of 0.15% aluminum hydroxide (Al (OH)₃). One week after the third immunization, blood was collected and hyperimmune serum was prepared. In each experiment, the preparation method comprises the steps of preparing Al (OH)₃One group injected with PBS as a negative control and one group of mice was immunized with the wild-type C-terminal OspA fragment (SEQ ID NO: 1) S2D0 from Borrelia avermitilis strain K78. In each animal study, the full length non-lipidated wild-type OspA protein (SEQ ID NO: 209) from Borrelia avermitilis strain K78 was included (also formulated with 0.15% Al (OH))₃) The other group immunized served as a positive control. All animal experiments were performed according to austria regulations (bgb1nr.501/1989) and approved by "Magistratsabteilung 58".

Tick challenge and serum and tissue Collection on immunized mice (also referred to herein as the "tick challenge method")

Two weeks after the last immunization, the immunized mice were challenged with ticks. To challenge immunized mice with borrelia avermitilis, mice were challenged withCream (Reckitt Benckiser, uk) removed hair from the back of each mouse and a small vented container was adhered to the skin with super glue (Pattex, germany). Thereafter, one or two scleroderma ricini nymphs infected with the borrelia avermitilis strain IS1 were appliedEach mouse was allowed to attach and feed to exhaustion. The feeding status of each individual tick was monitored daily and only the mice from which at least one fully fed tick was collected were included in the final readout. There was no distinction between mice from which one or two fully fed ticks were collected.

Six weeks after tick application, blood was collected by orbital bleeding and final serum was prepared and used for VlsE ELISA analysis to determine the status of infection. Mice were then sacrificed by cervical dislocation and one ear was collected from each mouse, subjected to DNA extraction and subjected to nested PCR analysis to identify borrelia within the tissue.

Infection readout

Only mice where the applied ticks feed completely and available for collection were included in the final readout of the experiment. Mice were sacrificed 6 weeks after tick application and organs and final serum were collected. The final infection readout was based on two different assays (nested PCR targeting the 16S-23S intergenic region and VlsE (IR6) ELISA, as described in detail below).

Nested PCR targeting the 16S-23S intergenic region

DNA extraction and purification was performed using DNeasy blood and tissue kit (Qiagen, germany) according to the manufacturer's instructions on one ear from each mouse with the following modifications. Each ear was digested overnight at 60 ℃ in PCR-grade recombinant proteinase K (Roche, 14-22 mg/mL). The DNA was eluted in 50. mu.L of sterile deionized water and stored at-20 ℃ until further analysis. As a negative control, one blank purification column was included in each DNA extraction and purification, and the eluate was subjected to nested PCR. All DNA extracts were screened for the presence of borrelia DNA by a nested PCR procedure comprising 40 cycles of 94 ℃ for 30s, 56 ℃ for 30s and 72 ℃ for 60s using primers; forward primer 5'-GTATGTTTAGTGAGGGGGGTG-3' (SEQ ID NO: 26) and reverse primer 5'-GGATCATAGCTCAGGTGGTTAG-3' (SEQ ID NO: 27). From a reaction volume of 10. mu.L, 1. mu.L was used as template for the nested PCR reaction. The nested PCR step included 25 cycles of 94 ℃ for 30s, 60 ℃ for 30s, and 72 ℃ for 60s using primers; forward nested primer 5'-AGGGGGGTGAAGTCGTAACAAG-3' (SEQ ID NO: 28) and reverse nested primer 5'-GTCTGATAAACCTGAGGTCGGA-3' (SEQ ID NO: 29). In the final reaction volume, 5 μ L was separated on a 1% agarose gel containing ethidium bromide and bands were observed under UV-light.

In each PCR analysis, DNA purified from in vitro growth medium of borrelia avermitilis strain K78 was used as a positive control template. In addition, PBS was used as a negative control instead of the extracted DNA. 5 microliter of the final product was separated on a 1% agarose gel containing ethidium bromide and the bands were observed under UV-light.

ELISA Using constant region 6(IR6) of variable major protein-like sequence E protein (VlsE)

Biotinylated 25-mer peptide (MKKDDQIAAAMVLRGMAKDGQFALK) (SEQ ID NO: 30) derived from the sequence of Borrelia garinii strain IP90 was used for analysis (Liang FT et al (1999) JImmunol.163: 5566-73). Streptavidin pre-coated 96-well ELISA plates (Nunc, Denmark) were coated with 100. mu.L/well (1. mu.g/mL) of biotinylated peptide in PBS supplemented with 0.1% Tween20 (PBS/0.1T). The plates were incubated overnight at 4 ℃. After coating with the peptide, the plate was washed once with PBS/0.1T. The plates were then blocked with 100. mu.L/well PBS + 2% BSA for one hour at Room Temperature (RT) before being washed again with PBS/0.1T. Post challenge sera were tested for reactivity with the peptide at 1: 200, 1: 400, and 1: 800 dilutions in PBS + 1% BSA. Plates were incubated for 90min at RT, followed by three washes with PBS/0.1T. Then, each well received 50 μ L of polyclonal rabbit anti-mouse IgG 1.3 μ g/mL conjugated to HRP (Dako, Denmark) in PBS + 1% BSA. The plates were then incubated for 1h at RT. After three washes with PBS/0.1T, ABTS (50 μ L/well) was added as substrate (Sigma-Aldrich, USA) and the color was allowed to develop for 30 min. The absorbance was measured at 405 nm. All sera were tested twice; negative controls included PBS instead of serum, and plates were not coated with the peptide. Sera from mice that showed culture positive for borrelia aryabhattai infection were used as positive controls.

Results

Level of protection in tick challenge methods

High levels of protection were observed for all five stable OspA borrelia afzelii fragments at both doses tested (30 μ g and 5 μ g, see table 2). A high infection rate in the PBS control group indicates that these ticks are infected at a high frequency. In addition, the positive control, non-lipidated full-length OspA from borrelia afzelii strain K78, was very highly protective. Together, these control groups indicate high reliability of the experimental readout.

The protection results from the experiments testing 30 μ g doses (11 experiments in total) and 5 μ g doses (4 experiments in total) are summarized in table 2. The two methods used to verify infection, the VlsE ELISA and nested PCR, gave nearly identical results (data not shown), indicating that these read-out methods were used to assess the robustness of infection in the tick challenge method.

Example 3 evaluation of protective Capacity of lipidated His-tagged mutant serotype 2OspA fragment monomers against in vivo Borrelia challenge by the tick challenge method (ST2, Borrelia afzelii)

Experimental procedures

Cloning and expression of lipidated His-tagged mutant OspA fragment proteins

Serotype 2 mutant OspA fragments with cysteine bond types 1, 3 and 4(SEQ ID NOs 141, 143 and 144, respectively) were modified by adding a lipidation signal sequence (SEQ ID NO: 14) derived from OspA and directly followed by CKQN peptide (SEQ ID NO: 211) at the C-terminus to provide an N-terminal cysteine for lipidation. For purification purposes, all mutant OspA fragments were C-terminally tagged with histidine. Cloning of the Gene fragment into pET28b (+) vector (No)vagen), a vector containing a kanamycin resistance expression cassette and the T7 promoter. At BL21Star^TM(DE3) cells (Invitrogen) expressed lipidated monomers and after induction by IPTG, the growth temperature of the cells was reduced from 37 ℃ to 25 ℃ to promote efficient post-translational processing of the protein. Cells were collected by centrifugation after 4h and the pellet was stored at-70 ℃ for 12 months before further processing.

Purification of lipidated His-tagged wild-type OspA fragment monomeric proteins and mutant OspA fragment monomeric proteins

Cells were mechanically disrupted by high pressure homogenization and the lipidated His-tagged OspA fragment monomeric polypeptide was enriched in the liquid phase by phase separation using Triton X-114 as a detergent. The diluted detergent phase (20-fold to 30-fold) was then applied to a Ni-Sepharose column (Ni Sepharose)^TM6Fast Flow; GE Healthcare) and eluted the lipidated His-tagged OspA fragment by imidazole gradient (0-250 mM). The combined fractions were further purified on a gel filtration column (Superdex200, GEHealthcare) followed by a buffer exchange column (Sephadex G-25, GE Healthcare). Lipidated His-tagged OspA fragment peaks were pooled based on analytical size exclusion column and reverse phase chromatography. After sterile filtration, the purified protein was stored at-20 ℃ until formulation.

Immunization of mice

Three lipidated mutant OspA proteins (Lip-S2D1-His, Lip-S2D3-His and Lip-S2D4-His) were expressed and purified as described above. Use of Al (OH) alone as described in example 2₃Adjuvant and non-lipidated full-length serotype 2OspA were used as negative and positive controls for in vivo protection studies, respectively. All immunogens were formulated with 0.15% Al (OH)₃. Mice were injected subcutaneously three times at two week intervals with formulations containing 3.0 μ g, 1.0 μ g, or 0.3 μ g antigen and challenged with borrelia avermitilis-infected ticks (strain IS1) two weeks after the last immunization. Mice were sacrificed six weeks after tick challenge and infection was assessed.

Results

Level of protection in tick challenge methods

All three lipidated mutant OspA fragments conferred very high levels of protection against borrelia afzelii challenge even at the lowest tested dose (table 3). In the presence of Al (OH) alone₃High infection rate in adjuvant-immunized mice, indicating that these ticks are infected with high frequency. The positive control antigen, full-length non-lipidated OspA from borrelia afzelii strain K78, was also very highly protective. Taken together, these control groups indicate a high reliability of the infection method and thus give a high confidence of the results observed after immunization with the lipidated mutant OspA fragment.

Example 4 evaluation of the protective ability of the mutant OspA heterodimers of the present invention against in vivo Borrelia challenge by either needle challenge or tick challenge methods

Experimental procedures

Cloning and expression of lipidated His-tagged mutant OspA fragment heterodimers

GenScript, USA codon optimizes mutant OspA fragment monomers from Borrelia burgdorferi strain B31, Borrelia afzelii strain K78, Borrelia garinii strain PBr, Borrelia bavaria strain PBi, Borrelia garinii strain PHEi, and Borrelia garinii strain DK29 for E.coli expression. The hLFA-1-like epitope of OspA from Borrelia burgdorferi strain B31 (aa 164-174, SEQ ID NO: 17) was replaced by the non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18) from Borrelia avermitis strain K78. The lipidation signal sequence added to the heterodimer of the mutant OspA fragment is derived from the major outer membrane lipoprotein Lpp of e.coli and is directly followed by the CSS peptide at the C-terminus to provide the N-terminal cysteine for lipidation. The mutant OspA fragment heterodimer is generated by fusing different mutant OspA fragment monomers as described above via a 21 amino acid linker sequence toTwo separate loop regions derived from the N-terminal half of OspA from Borrelia burgdorferi strain B31 in the narrow sense ("LN 1"; aa65-74 and aa 42-53 with amino acid exchange D53S, SEQ ID NO: 184). For purification purposes, the heterodimer was constructed using a His-tag. The gene fragment was cloned into the pET28b (+) vector (Novagen), a vector containing the kanamycin resistance expression cassette and the T7 promoter. At BL21Star^TM(DE3) stable heterodimeric lipoproteins were expressed in cells (Invitrogen) and after induction by IPTG, the growth temperature of the cells was reduced from 37 ℃ to 25 ℃ to promote efficient post-translational processing of the proteins. Cells were collected by centrifugation after 4h and the pellet was stored at-70 ℃ for 12 months before further processing.

Purification of lipidated His-tagged mutant OspA fragment heterodimers

Cells were mechanically disrupted by high pressure homogenization and the lipidated His-tagged mutant OspA fragment heterodimer was enriched in the liquid phase by phase separation using Triton X-114 as detergent. The diluted detergent phase (20-fold to 30-fold) was then applied to a Ni-Sepharose column (Ni Sepharose)^TM6Fast Flow; GE Healthcare) and eluted lipidated His-tagged OspA heterodimers by imidazole gradient (0-250 mM). The combined fractions were further purified on a gel filtration column (Superdex200, GE Healthcare) followed by a buffer exchange column (Sephadex G-25, GE Healthcare). Lipidated His-tagged mutant OspA heterodimer peaks were pooled based on analytical size exclusion column and reverse phase chromatography. After sterile filtration, the purified heterodimer was stored at-20 ℃ until formulation.

Cloning and expression of lipidated His-tag-free mutant OspA fragment heterodimers

Constructs prepared as described above were used to generate constructs without His-tag by introducing a stop codon via PCR amplification. The gene fragment was cloned into the pET28b (+) vector (Merck Millipore), a vector containing the kanamycin resistance expression cassette and the T7 promoter. At BL21Star^TM(DE3) stable heterodimeric lipoproteins were expressed in cells (Invitrogen) and after induction by IPTG, the growth temperature of the cells was reduced from 37 ℃ to 25 ℃ to promote efficient post-translational processing of the proteins. Cells were collected by centrifugation after 4h and the pellet was stored at-70 ℃ for 12 months before further processing.

Purification of lipidated His-tag-free mutant OspA fragment heterodimers

Cells were mechanically disrupted by high pressure homogenization and the lipidated mutant OspA fragment heterodimer was enriched in the liquid phase by phase separation using Triton X-114 as a detergent. The diluted detergent phase is then subjected to anion exchange chromatography operating in unbound mode. The resulting stream was loaded onto a hydroxyapatite column (Bio-Rad) and the lipidated protein was eluted from the column by a linear salt gradient. The eluate was further purified on a DEAE-agarose column (GE Healthcare) in unbound mode, followed by a gel filtration column for buffer exchange (Superdex200, GE Healthcare). The lipidated mutant OspA heterodimer peaks were pooled based on analytical size exclusion column and SDS-PAGE. After sterile filtration, the purified heterodimer was stored at-20 ℃ until formulation.

Immunization of mice

For all studies, female C3H/HeN mice (Janvier, France) were used. Prior to each challenge, a group of ten 8-week-old mice was bled through the facial vein and pre-immune sera were prepared and pooled. Each 100 μ L subcutaneous (s.c.) immunization was administered three times at two week intervals. Each dose contained the amount of immunogen indicated in Table 4 (dose) and was formulated with a final concentration of 0.15% aluminum hydroxide (Al (OH)₃). One week after the third immunization, blood was collected from the facial vein and hyperimmune serum was prepared. In each experiment, the single Al (OH) was used₃The immunized group served as a negative control. All animal studies were performed according to austria regulations (bgb1nr.501/1989) and approved by "Magistratsabteilung 58".

To challenge immunized mice with borrelia avermitilis, mice were challenged withCream (Reckitt Benckiser) removed hair from the back of each mouse and a small vented container was adhered to the skin with super glue (Pattex). Thereafter, one or two scleroderma ricini nymphs infected with borrelia avermitilis strain IS1 were applied to each mouse, allowed to attach and feed until they were completely saturated and dropped. The feeding status of each individual tick was monitored daily and only the mice from which at least one fully fed tick was collected were included in the final readout.

Needle challenge of immunized mice with in vitro grown borrelia

Two weeks after the last immunization, mice were challenged with borrelia s.c. diluted in 100 μ L borrelia growth medium (BSK II). Challenge dose was strain-related, virulence of individual strains was used to determine ID₅₀The challenge experiment of (2) was evaluated. Dose range for needle challenge experiments is ID₅₀From 20 times to 50 times.

Sacrifice of mice and Collection of materials

Mice were sacrificed by cervical dislocation four weeks after needle challenge with the indicated borrelia species or six weeks after tick challenge with borrelia afzelii. Blood was collected by orbital bleeding and final serum was prepared and used in the VlsE ELISA to determine the status of infection. In addition, one ear was collected from each mouse, and DNA was extracted and subjected to quantitative pcr (qpcr) for identifying borrelia. The final infection readout was based on two different assays (VlsE ELISA and recA-targeted qPCR).

ELISA Using constant region 6 of VlsE (IR6)

Biotinylated 25-mer peptides (MKKDDQIAAAMVLRGMAKDGQFALK) derived from the sequence of Borrelia garinii strain IP90 were used for analysis (Liang FT, Alvarez AL, Gu Y, Nowling JM, Ramamotorthy R, Philipp MT. immunogenic conserved region with the variable domain of VlsE, the variable surface antigen of Borreliaburgdorferi. J Immunol.1999; 163: 5566-73). Streptavidin pre-coated 96-well ELISA plates (Nunc) were coated with 100. mu.L/well (1. mu.g/mL) of peptide in PBS supplemented with 0.1% Tween20 (PBS/0.1T). The plates were incubated overnight at 4 ℃. After coating with the peptide, the plate was washed once with PBS/0.1T. The plates were then blocked with 100. mu.L/well PBS + 2% BSA for one hour at Room Temperature (RT) before being washed again with PBS/0.1T. The reactivity of the post challenge sera with the peptides was tested with 1: 200, 1: 400 and 1: 800 dilutions in PBS + 1% BSA. Plates were incubated for 90min at RT, followed by three washes with PBS/0.1T. Then, each well received 50 μ L of polyclonal rabbit anti-mouse IgG at 1.3 μ g/mL conjugated to HRP (Dako) in PBS + 1% BSA. The plates were then incubated for 1h at RT. After three washes with PBS/0.1T, ABTS (50 μ L/well) was added as substrate (Sigma-Aldrich) and the color was allowed to develop for 30 min. The absorbance was measured at 405 nm. All sera were tested twice. Negative controls included PBS instead of serum, and the plates were not coated with the peptide. Sera from mice that showed culture positive for borrelia aryabhattai infection were used as positive controls.

recA-targeted qPCR

Oligonucleotide primers were designed against the recA gene in such a way that they could be used in qPCR for identifying all relevant Borrelia species causing Lyme borreliosis (forward primer: CATGCTCTTGATCCTGTTTA; reverse primer: CCCATTTCTCCATCTATCTC). The recA fragment was cloned from Borrelia burgdorferi strain N40 in the sense of pET28b (+) for use as a standard in each reaction. Chromosomal DNA extracted from mouse ears was diluted 1: 8 in water to reduce the matrix effect observed with undiluted DNA. Prepared from 10 μ L of SSoAdvanced for each experiment^TMGreen Supermix, 0.3. mu.L of each primer (10. mu.M) and 7.4. mu.L of water. 18 μ L of the host mixture was mixed with 2 μ L of diluted DNA extracted from bladder or ear in a microtiter plate and the DNA was amplified using the CFX96 real-time PCR detection system (Bio-Rad, USA). The DNA was denatured at 95 ℃ for 3 minutes, followed by 50 cycles of 95 ℃ for 15 seconds and 55 ℃ for 30 seconds. After amplification, DNA was prepared for melting curve analysis by denaturation at 95 ℃ for 30 seconds followed by 55 ℃ for 2 minutes. Melting curve analysis was performed by incubation at 55 ℃ for 5 seconds (with 0.5 ℃ increase per cycle) and at 95 ℃ for 5 seconds. On each plate, four sets of no-template controls (NTCs) were included, as well as two standard curves where the template copy number ranged from 10 to 10,000.

Results

The protective capacity of the lipidated mutant OspA fragment heterodimer was tested in 12 separate experiments. Mice were challenged in three experiments with each of the borrelia burgdorferi strain N40OspA serotype 1(ST1, needle challenge) or borrelia afzelii strain IS1OspA serotype 2(ST2, tick challenge), or in two experiments with each of the borrelia bavaria strain Scf OspA serotype 4(ST4, needle challenge), borrelia garinii strain "a" OspA serotype 5(ST5, needle challenge) or borrelia garinii strain "B" OspA serotype 6(ST6, needle challenge). In all experiments, Al (OH) alone was used₃The adjuvant-immunized mouse group was used as a negative control group. For the challenge with ticks, 1-2 ticks were applied to each mouse and only mice fed from at least one tick until fully saturated were included in the final readout. However, there was no difference between mice from which one or two fully fed ticks were collected. The protection data from twelve experiments are summarized in table 4.

Lipidated His-tagged OspA heterodimers (Lip-S1D1-S2D1-His) showed a highly statistically significant protection against OspA serotype 1 and OspA serotype 2 challenge in all six experiments compared to the negative control group (fisher exact test, two-tailed). Surprisingly, immunization with Lip-S4D1-S3D1-His and Lip-S5D1-S6D1-His also conferred high protection against OspA serotype 2 challenge (experiments 4-6), indicating that immunization with mutant OspA fragments of other serotypes may have cross-protective effects. Furthermore, immunization with Lip-S4D1-S3D1 conferred statistically significant protection against needle challenge with OspA serotype 4 Borrelia (experiments 7 and 8). Finally, immunization with Lip-S5D1-S6D1-His conferred protection against needle challenge with OspA serotype 5 (experiments 9 and 10) and against needle challenge with OspA serotype 6 (experiments 11 and 12). The infection status of each mouse was determined using a combination of VlsE ELISA and recA qPCR. When at least one method gives a positive result, the mouse is considered infected.

In summary, immunization with the mutant OspA fragment heterodimeric polypeptides of the invention confers protection against all borrelia serotypes tested and may also provide cross-protection in some cases.

Lipidated His-tagged OspA heterodimers (Lip-S1D1-S2D1-His) showed a highly statistically significant protection against OspA serotype 1 and OspA serotype 2 challenge in all six experiments compared to the negative control group (fisher exact test, two-tailed). Surprisingly, immunization with Lip-S4D1-S3D1-His and Lip-S5D1-S6D1-His also conferred high protective capacity against OspA serotype 2 challenge (experiments 4-6), indicating that immunization with mutant OspA fragments of other serotypes may have a cross-protective effect. Furthermore, immunization with Lip-S4D1-S3D1 conferred statistically significant protection against needle challenge with OspA serotype 4 Borrelia (experiments 7 and 8). Finally, immunization with Lip-S5D1-S6D1-His conferred protection against needle challenge with OspA serotype 5 (experiments 9 and 10) and OspA serotype 6 (experiments 11 and 12). The infection status of each mouse was determined using a combination of VlsE ELISA and recA qPCR. When at least one method gives a positive result, the mouse is considered infected.

Example 5 evaluation of the protective ability of the 1: 1 combination vaccine of the mutant OspA heterodimers of the present invention against OspA serotype 1 and serotype 2 Borrelia challenge in vivo by either the needle challenge method or the tick challenge method

Experimental procedures

Immunization of mice

For all studies, female C3H/HeN mice (Janvier, France) were used. Prior to each challenge, a group of ten 8-week-old mice was bled through the facial vein and pre-immune sera were prepared and pooled. Each 100 μ L s.c. immunization was administered three times at two week intervals. Groups of mice were immunized with a combination vaccine consisting of 1 μ g each of Lip-S1D1-S2D1, Lip-S4D1-S3D1, and Lip-S5D1-S6D 1. Three other OspA-based antigens were included in challenge experiments: Lip-OspA1-His (full-length serotype 1OspA, lipidated and His-tagged), lipidated chimeric OspA ST1/ST2, and Lip-S1D1-S2D1 alone. Negative (placebo) control was Al (OH) alone₃-an adjuvant. All antigens were expressed as aluminum hydroxide (Al (OH) with a final concentration of 0.15%₃) The PBS (9).

(chimeric OspA ST1/ST2(SEQ ID NO: 212) is an OspA chimera consisting of the first 10 amino acids of the N-terminal part of OspB (strain B31), amino acids 11-200 of serotype 1OspA fused to the last 201-255 amino acids of the C-terminal part of serotype 2OspA, and in which the hLFA-1-like sequence (146-170) of serotype 1OspA is replaced by a homologous sequence from serotype 2OspA the serotype 2OspA sequence is followed by two amino acids added due to a cloning site (XhoI) preceding the stop codon in the vector.)

One week after the third immunization, blood was collected from the facial vein and hyperimmune serum was prepared. All animal experiments were performed according to austria regulations (bgb1nr.501/1989) and approved by "magistratsabteiung 58".

Needle challenge of immunized mice with in vitro grown borrelia

Two weeks after the last immunization, mice were challenged with borrelia s.c. diluted in 100 μ L growth medium (BSKII). Challenge dose was strain-related, virulence of individual strains was used to determine ID₅₀The challenge experiment of (2) was evaluated. Dose range for needle challenge experiments is ID₅₀From 20 times to 50 times. Four weeks after the needle challenge, mice were sacrificed and blood and tissue were collected for use in a readout method to determine the infection status.

To challenge immunized mice with borrelia avermitilis, mice were challenged withCream (Reckitt Benckiser, uk) removed hair from the back of each mouse and a small vented container was adhered to the skin with super glue (Pattex, germany). Thereafter, one or two scleroderma ricini nymphs infected with borrelia avermitilis strain IS1 were applied to each mouse, allowed to attach and feed until they were completely saturated and dropped. The feeding status of each individual tick was monitored and only the mouse from which at least one fully fed tick was collected was included in the final readout.

Results

Lipidated mutant OspA fragment heterodimers without His-tag were combined in a 1: 1 ratio and tested for their protective ability against borrelia challenge. The immunization was challenged with Borrelia avermitilis (ST2, strain IS1, tick challenge) or with Borrelia burgdorferi sensu stricto (ST1, strain ZS7, needle challenge) in two experiments eachA mouse. Other OspA-based antigens included Lip-S1D1-S2D2 in all four experiments as well as Lip-OspA1-His and lipidated chimeric OspAST1/ST2 in experiments 15 and 16. With Al (OH) alone₃The adjuvant-immunized mouse group was used as a negative control group in each experiment. For the challenge with ticks, 1-2 ticks were applied to each mouse and only mice fed from at least one tick until fully saturated were included in the final readout. However, there was no difference between mice from which one or two fully fed ticks were collected. The protection data from four experiments are summarized in table 5.

The combination vaccine comprising three lipidated mutant OspA fragment heterodimers in a 1: 1 ratio conferred statistically significant protection in all four challenge experiments compared to the negative control group (fisher exact test, two-tailed). The infection status of each mouse was determined using a combination of vlsleelisa and recA qPCR. When at least one method gives a positive result, the mouse is considered infected.

Example 6 binding of antibodies from sera of mice immunized with mutant OspA fragment heterodimer to the cell surface of Borrelia

Experimental procedures

Immunization of mice

For all studies, female C3H/HeN mice were used. Prior to each challenge, a group of twelve 8 week old mice was bled via the facial vein and preimmune sera were prepared and pooled. Each 100 μ ls.c. immunization was administered three times at two week intervals. Each dose contained 1 μ g of each respective protein: Lip-S1D1-S2D1, Lip-S4D1-S3D1 and Lip-S5D1-S6D1 (combination vaccine), or 1 μ g of lipidated full-length OspA protein (ST1-ST6, as indicated) or 1 μ g of individual OspA heterodimer (Lip-S1D1-S2D1, Lip-S4D1-S3D1 or Lip-S5D1-S6D1, as indicated) with 0.15% final concentration of aluminum hydroxide as adjuvant. Negative (placebo) control was Al (OH) alone₃-an adjuvant. In the third immunizationOne week thereafter, blood was collected from the facial vein and hyperimmune serum was prepared. All animal experiments were performed according to austria regulations (bgb1nr.501/1989) and approved by "magistratsabteiung 58".

Flow cytometry to assess binding to borrelia

Mixing spiral body (1x 10)⁶) Mixed with an equal volume of 4% paraformaldehyde and incubated in 96-well plates (Nunclon96U, Nunc) for 2 hours at room temperature. The plate was centrifuged at 2,000g for 5 minutes and the supernatant discarded. Cells were washed with 150 μ L HBSS (HBSS-B) with 2% BSA, centrifuged as described above, and the supernatant discarded. They were heat inactivated by incubating the mouse sera at 56 ℃ for 35 minutes. The heat-inactivated serum was diluted in HBSS-B and sterile filtered by centrifugation at 4,000g for 3 minutes using a Costar spin-tube filter (0.22 μm, Corning, USA). Spirochetes were dissolved in 100 μ L serum and incubated for 45 min at room temperature. The plates were centrifuged at 2,000g for 15 minutes and the supernatant discarded. Cells were washed once with 150 μ L HBSS-B and then lysed in 100 μ L HBSS-B. One microliter of secondary antibody (PE-conjugated goat anti-mouse IgG, Beckman Coulter, USA) was added to the cells and incubated at room temperature for 45 minutes in the dark. The helices were washed once with 150 μ L HBSS-B and then dissolved in 200 μ L HBSS containing 2.5 μ M SYTO-17DNA dye and incubated for 10 min at room temperature in the dark. Stained spirochetes were pelleted by centrifugation at 2000g for 5 min and then dissolved in 200 μ L HBSS. Labeled spirochetes were measured with a FC500(Beckman Coulter) flow cytometer and SYTO-17 positive events were sorted. The values obtained with sera from placebo-immunized groups were subtracted from the values obtained with sera from heterodimer-immunized groups to account for non-specific binding.

Results

Binding of antibodies from the sera of hyperimmunized mice was observed in the case of different borrelia expressing all six OspA serotypes, indicating that antibodies generated in response to all antigens are functionally active and can bind native OspA in situ. The fluorescence intensity was linear over a large range of serum dilutions. For most OspA serotypes, the fluorescence intensity observed for sera produced with heterodimers is comparable to that observed for sera produced with lipidated full-length OspA.

EXAMPLE 7 formulation study

In order to optimize stability, studies on the formulation of the combination vaccine of the present invention were conducted. Different types of buffers and stabilizers were tested at different concentrations in combination with aluminum hydroxide and antigen. The optimal formulation of three heterodimers (120 μ g protein in total), 10mM sodium phosphate, 150mM sodium chloride, 10mM L-methionine, 5% sucrose, 0.05% Tween20 (polysorbate 20), and 0.15% (w/v) aluminum hydroxide, each at pH 6.7 ± 0.2, with 40 μ g/mL of each, was determined. #

Sequence of

SEQ ID NO：1

S2D 0-His: amino acids at positions 131-273 of the wild-type sequence of Borrelia afzelii strain K78OspA serotype 2, the C-terminal His-tag (GLEHHHHHH)

ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH

SEQ ID NO：2

S2D 1-His: the aa 131-273, C-terminal His-tag (GLEHHHHHH) of Borrelia afzelii strain K78OspA serotype 2 with disulfide bond type 1(aa 182 and 269)

ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALKGLEHHHHHH

SEQ ID NO：3

S2D 2-His: borrelia afzelii strain K78OspA serotype 2 with disulfide bond type 2(aa 182 and 272) aa 131-273, C-terminal His-tag (GLEHHHHHH)

ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNACKGLEHHHHHH

SEQ ID NO：4

S2D 3-His: the aa 131-273, C-terminal His-tag (GLEHHHHHH) of Borrelia afzelii strain K78OspA serotype 2 with disulfide bond types 3(aa 244 and 259)

ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTICVQKYDSAGTNLEGTCVEIKTLDELKNALKGLEHHHHHH

SEQ ID NO：5

S2D 4-His: borrelia aryabhattai strain K78OspA serotype 2 with disulfide bond type 4(aa 141 and 241) aa 131-273, C-terminal His-tag (GLEHHHHHH)

ELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH

SEQ ID NO：6

S2D 5-His: the aa 131-273, C-terminal His-tag (GLEHHHHHH) of Borrelia afzelii strain K78OspA serotype 2 with disulfide bond type 5(aa 165 and 265)

ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNCTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTCDELKNALKGLEHHHHHH

SEQ ID NO：7

S2D 6-His: aa 131-273, C-terminal His-tag (GLEHHHHHH) of Borrelia afzelii strain K78OspA serotype 2 with disulfide bond type 6(aa 185 and 272)

ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTCTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNACKGLEHHHHHH

SEQ ID NO：8

S2D 7-His: aa 131-273, C-terminal His-tag (GLEHHHHHH) of Borrelia afzelii strain K78OspA serotype 2 with disulfide bond types 7(aa 199 and 223)

ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTIEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTCALNDTNTTQATKKTGAWDSKTSTCTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH

SEQ ID NO：9

S2D 8-His: the aa 131-273, C-terminal His-tag (GLEHHHHHH) of Borrelia afzelii strain K78OspA serotype 2 with disulfide bond types 8(aa 243 and 262)

ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTCTVQKYDSAGTNLEGTAVECKTLDELKNALKGLEHHHHHH

SEQ ID NO：10

S2D 9-His: the aa 131-273, C-terminal His-tag (GLEHHHHHH) of Borrelia afzelii strain K78OspA serotype 2 with disulfide bond type 9(aa 184 and 204)

ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGCVTLSKEIAKSGEVTVALNDCNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH

SEQ ID NO：11

S2D 10-His: aa 131-273, C-terminal His-tag (GLEHHHHHH) of Borrelia afzelii strain K78OspA serotype 2 with disulfide bond type 10(aa 201 and 214)

ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVACNDTNTTQATKKTCAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH

SEQ ID NO：12

S2D 11-His: the aa 131-273, C-terminal His-tag (GLEHHHHHH) of Borrelia afzelii strain K78OspA serotype 2 with disulfide bond types 11(aa 246 and 259)

ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVCKYDSAGTNLEGTCVEIKTLDELKNALKGLEHHHHHH

SEQ ID NO：13

S2D 12-His: the aa 131-273, C-terminal His-tag (GLEHHHHHH) of Borrelia afzelii strain K78OspA serotype 2 with disulfide bond types 12(aa 167 and 178)

ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTCEGKVANDKVTCEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH

SEQ ID NO：14

Borrelia OspA lipidation signal

MKKYLLGIGLILALIA

SEQ ID NO：15

Borrelia OspB lipidation signal

MRLLIGFALALALIG

SEQ ID NO：16

Escherichia coli lpp lipidation signal

MKATKLVLGAVILGSTLLAG

SEQ ID NO：17

hLFA-1-like sequence from Borrelia burgdorferi strain B31

GYVLEGTLTAE

SEQ ID NO：18

non-hlFA-1-like sequence from Borrelia aryabhattai strain K78

NFTLEGKVAND

SEQ ID NO：19

Borrelia afzelii (strain K78; OspA serotype 2)

MKKYLLGIGLILALIACKQNVSSLDEKNSASVDLPGEMKVLVSKEKDKDGKYSLKATVDKIELKGTSDKDNGSGVLEGTKDDKSKAKLTIADDLSKTTFELFKEDGKTLVSRKVSSKDKTSTDEMFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEQTAVEIKTLDELKNALK

SEQ ID NO：20

Borrelia burgdorferi sensu stricto (strain B31, OspA serotype 1)

MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLELKGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKGYVLEGTLTAEKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIKNALK

SEQ ID NO：21

Borrelia garinii (strain PBr, OspA serotype 3)

MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGGMKVLVSKEKDKDGKYSLMATVEKLELKGTSDKSNGSGVLEGEKADKSKAKLTISQDLNQTTFEIFKEDGKTLVSRKVNSKDKSSTEEKFNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELKAALK

SEQ ID NO：22

Borrelia bavaria (strain PBi, OspA serotype 4)

MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGEMKVLVSKEKDKDGKYSLMATVDKLELKGTSDKSNGSGTLEGEKSDKSKAKLTISEDLSKTTFEIFKEDGKTLVSKKVNSKDKSSIEEKFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELKNALK

SEQ ID NO：23

Borrelia garinii (strain PHei, OspA serotype 5)

MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGGMKVLVSKEKDKDGKYSLMATVEKLELKGTSDKNNGSGTLEGEKTDKSKVKLTIAEDLSKTTFEIFKEDGKTLVSKKVTLKDKSSTEEKFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELKNALK

SEQ ID NO：24

Borrelia garinii (strain DK29, OspA serotype 6)

MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGGMTVLVSKEKDKDGKYSLEATVDKLELKGTSDKNNGSGTLEGEKTDKSKVKSTIADDLSQTKFEIFKEDGKTLVSKKVTLKDKSSTEEKFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELKNALK

SEQ ID NO：25

Borrelia garinii (strain T25, OspA serotype 7)

MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGEMKVLVSKEKDKDGKYSLEATVDKLELKGTSDKNNGSGVLEGVKAAKSKAKLTIADDLSQTKFEIFKEDGKTLVSKKVTLKDKSSTEEKFNDKGKLSEKVVTRANGTRLEYTEIQNDGSGKAKEVLKSLTLEGTLTADGETKLTVEAGTVTLSKNISESGEITVELKDTETTPADKKSGTWDSKTSTLTISKNSQKTKQLVFTKENTITVQKYNTAGTKLEGSPAEIKDLEALKAALK

SEQ ID NO：26

Forward primer

GTATGTTTAGTGAGGGGGGTG

SEQ ID NO：27

Reverse primer

GGATCATAGCTCAGGTGGTTAG

SEQ ID NO：28

Forward nested primers

AGGGGGGTGAAGTCGTAACAAG

SEQ ID NO：29

Reverse nested primers

GTCTGATAAACCTGAGGTCGGA

SEQ ID NO：30

25-mer peptides

MKKDDQIAAAMVLRGMAKDGQFALK

SEQ ID NO：31

Mouse antimicrobial peptide precursors

RLAGLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPE

SEQ ID NO：32

5’-(dIdC)₁₃-3’

dIdC dIdC dIdC dIdC dIdC dIdC dIdC dIdC dIdC dIdC dIdC dIdC dIdC

SEQ ID NO：33

KLK peptides

KLKLLLLLKLK

SEQ ID NO：34

Borrelia aryabhattai (strain K78, serotype 2), OspA aa 126-

FNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK

SEQ ID NO：35

Borrelia aryabhattai (strain K78, serotype 2), OspA aa 131-273

ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK

SEQ ID NO：36

Peptide linker

GGGGGGGG

SEQ ID NO：37

Peptide linker

GGGGGGGGGGGG

SEQ ID NO：38

Peptide linker

GAGA

SEQ ID NO：39

Peptide linker

GAGAGAGA

SEQ ID NO：40

Peptide linker

GAGAGAGAGAGA

SEQ ID NO：41

Peptide linker

GGGSGGGS

SEQ ID NO：42

Peptide linker

GGGSGGGSGGGS

SEQ ID NO：43

S1D4-S2D4_ aa: heterodimeric fusion proteins of OspA serotype 1 and OspA serotype 2, both with disulfide bond type 4, LN1 linker sequence, aa164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND

FNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALK

SEQ ID NO：44

Lip-S1D4-S2D4_ nt: both having the coding sequences of the fusion proteins of OspA serotype 1 and OspA serotype 2 of disulfide bond type 4,escherichia coli lpp lipidation signalLN1 linker sequence, aa164-174 of OspA serotype 1 substituted with non-hLFA-1-like sequence NFTLEGKVAND

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAGTCTCGGAAAAAATCATTACCCGTGCTTGCGGCACCCGTCTGGAATACACCGGCATTAAATCGGATGGCAGCGGCAAAGCGAAGGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAGACCACCCTGGTGGTGAAAGAAGGCACCGTTACGCTGA

GCAAAAACATTAGTAAGTCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAAAAGACGGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATTCCAAAAAGACCAAAGATCTGGTCTTCACGAAAGAATGCACCATCACGGTGCAGCAATATGACAGCAACGGTACCAAACTGGAAGGCTCTGCGGTGGAAATCACGAAACTGGATGAAATCAAAAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAATGCGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAAGAAGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAACAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAATGTACGATCACCGTGCAGAAATACGATAGTGCGGGTACCAACCTGGAAGGCACCGCTGTTGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAA

SEQ ID NO：45

Lip-S1D4-S2D4_ His _ aa: heterodimeric fusion proteins of OspA serotype 1 and OspA serotype 2, both with disulfide bond type 4, N-terminal CSS for lipid addition, N-terminal esterification, LN1 linker sequence, aa164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND, C-terminal His tag (GLEHHHHHH)

LioCSSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH

SEQ ID NO：46

Lip-S1D4-S2D4_ His _ nt: both having the coding sequences of heterodimeric fusion proteins of OspA serotype 1 and OspA serotype 2 of disulfide bond type 4,escherichia coli lpp lipidation signalLN1 linker sequence, aa164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND, C-terminal His tag (GLEHHHHHH)

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAGTCTCGGAAAAAATCATTACCCGTGCTTGCGGCACCCGTCTGGAATACACCGGCATTAAATCGGATGGCAGCGGCAAAGCGAAGGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAGACCACCCTGGTGGTGAAAGAAGGCACCGTTACGCTGAGCAAAAACATTAGTAAGTCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAAAAGACGGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATTCCAAAAAGACCAAAGATCTGGTCTTCACGAAAGAATGCACCATCACGGTGCAGCAATATGACAGCAACGGTACCAAACTGGAAGGCTCTGCGGTGGAAATCACGAAACTGGATGAAATCAAAAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAATGCGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAAGAAGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAACAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAATGTACGATCACCGTGCAGAAATACGATAGTGCGGGTACCAACCTGGAAGGCACCGCTGTTGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAAGGCCTCGAGCACCACCACCACCACCAC

SEQ ID NO：47

S1D1-S2D1_ aa: heterodimeric fusion proteins of OspA serotype 1 and OspA serotype 2 with disulfide bond type 1, LN1 linker sequence, aa164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND

FNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALK

SEQ ID NO：48

Lip-S1D1-S2D1_ nt: coding sequences for intermediate and final heterodimer fusion proteins of OspA serotype 1 and OspA serotype 2 having disulfide bond type 1,escherichia coli lpp lipidation signalLN1 linker sequence, aa164-174 of OspA serotype 1 substituted with non-hLFA-1-like sequence NFTLEGKVAND

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAGTCAGCGAAAAAATCATTACCCGCGCAGACGGCACCCGCCTGGAATACACCGGCATCAAATCGGACGGCAGCGGCAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAAACCACCCTGGTGGTGAAATGCGGCACCGTTACGCTGAGCAAAAACATTAGTAAATCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAGAAAACCGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATAGCAAGAAAACCAAAGATCTGGTCTTCACGAAAGAAAACACCATCACGGTGCAGCAATATGACAGCAATGGTACCAAACTGGAAGGCTCCGCTGTGGAAATCACGAAACTGGATGAAATCTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAAAACGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAATGCGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAATAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAAGATACGATCACCGTGCAGAAATACGACAGTGCGGGTACCAACCTGGAAGGCACGGCTGTTGAAATCAAAACCCTGGACGAACTGTGTAACGCCCTGAAA

SEQ ID NO：49

Lip-S1D1-S2D1_ His _ aa: heterodimeric fusion proteins of OspA serotype 1 and OspA serotype 2 with disulfide bond type 1, N-terminal CSS for lipid addition, N-terminal esterification, LN1 linker sequence, aa164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND, C-terminal His tag (GLEHHHHHH)

LipCSSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALKGLEHHHHHH

SEQ ID NO：50

Lip-S1D1-S2D1_ His _ nt: coding sequences of heterodimeric fusion proteins of OspA serotype 1 and OspA serotype 2 having disulfide bond type 1,escherichia coli lpp lipidation signalLN1 linker sequence, aa164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND, C-terminal His tag (GLEHHHHHH)

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAGTCAGCGAAAAAATCATTACCCGCGCAGACGGCACCCGCCTGGAATACACCGGCATCAAATCGGACGGCAGCGGCAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAAACCACCCTGGTGGTGAAATGCGGCACCGTTACGCTGAGCAAAAACATTAGTAAATCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAGAAAACCGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATAGCAAGAAAACCAAAGATCTGGTCTTCACGAAAGAAAACACCATCACGGTGCAGCAATATGACAGCAATGGTACCAAACTGGAAGGCTCCGCTGTGGAAATCACGAAACTGGATGAAATCTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAAAACGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAATGCGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAATAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAAGATACGATCACCGTGCAGAAATACGACAGTGCGGGTACCAACCTGGAAGGCACGGCTGTTGAAATCAAAACCCTGGACGAACTGTGTAACGCCCTGAAAGGCCTCGAGCACCACCACCACCACCAC

SEQ ID NO：51

S3D4-S4D4_ aa: heterodimeric fusion proteins of OspA serotype 3 and OspA serotype 4 with disulfide bond type 4, LN1 linker sequence

FNEKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPA

EIKDLAELKAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALK

SEQ ID NO：52

Lip-S3D4-S4D4_ nt: coding sequences for intermediate and final heterodimer fusion proteins of OspA serotype 3 and OspA serotype 4 with disulfide bond type 4,escherichia coli lpp lipidation signalLN1 linker sequence

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCAAACTGTCAGAAAAAGTGGTCACCCGCGCTTGTGGCACCCGCCTGGAATACACCGAAATCAAAAACGACGGCTCGGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGGAAGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAATGCACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCCCCGGCTGAAATCAAGGACCTGGCGGAACTGAAGGCGGCACTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGCTAAAGGTGAACTGTCGGAAAAAACCATCCTGCGCGCCTGTGGCACCCGCCTGGAATACACGGAAATCAAGTCGGACGGCACGGGCAAAGCAAAGGAACTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCGACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAATGCACGATCACCGTTCAAAAATATGATTCCGCAGGTACCAACCTGGAAGGCAACGCTGTGGAAATCAAAACCCTGGACGAACTGAAAAATGCTCTGAAG

SEQ ID NO：53

Lip-S3D4-S4D4_ His _ aa: heterodimeric fusion proteins of OspA serotype 3 and OspA serotype 4 with disulfide bond type 4, N-terminal CSS for lipid addition, N-terminal lipidation, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)

LipCSSFNEKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALKGLEHHHHHH

SEQ ID NO：54

Lip-S3D4-S4D4_ His _ nt: coding sequences of heterodimeric fusion proteins of OspA serotype 3 and OspA serotype 4 having disulfide bond type 4,escherichia coli lpp lipidation signalN-terminal CSS for lipid addition, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCAAACTGTCAGAAAAAGTGGTCACCCGCGCTTGTGGCACCCGCCTGGAATACACCGAAATCAAAAACGACGGCTCGGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGGAAGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAATGCACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCCCCGGCTGAAATCAAGGACCTGGCGGAACTGAAGGCGGCACTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGCTAAAGGTGAACTGTCGGAAAAAACCATCCTGCGCGCCTGTGGCACCCGCCTGGAATACACGGAAATCAAGTCGGACGGCACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCGACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAATGCACGATCACCGTTCAAAAATATGATTCCGCAGGTACCAACCTGGAAGGCAACGCTGTGGAAATCAAAACCCTGGACGAACTGAAAAATGCTCTGAAGGGTCTCGAGCAOCACCACCACCACCAC

SEQ ID NO：55

S3D1-S4D1_ aa: heterodimeric fusion proteins of OspA serotype 3 and OspA serotype 4, both with disulfide bond type 1, LN1 linker sequence

FNEKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALK

SEQ ID NO：56

Lip-S3D1-S4D1_ nt: both having the coding sequence of the intermediate and final heterodimer fusion proteins of OspA serotype 3 and OspA serotype 4 of disulfide bond type 1,escherichia coli lpp lipidation signalN-terminal CSS, LN1 linker sequences for lipid addition

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCAAACTGTCGGAAAAAGTGGTCACCCGCGCAAATGGCACCCGCCTGGAATACACGGAAATCAAAAACGATGGTAGCGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGTGCGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAAAACACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCACCGGCTGAAATCAAGGACCTGGCTGAACTGTGTGCGGCACTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGCTAAAGGTGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGGCACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAGATACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTCGAAATCAAAACCCTGGATGAACTGTGTAATGCTCTGAAG

SEQ ID NO：57

Lip-S3D1-S4D1_ His _ aa: heterodimeric fusion proteins of OspA serotype 3 and OspA serotype 4, both of which have disulfide bond type 1,escherichia coli lpp lipidation signalN-terminal CSS for lipid addition, N-terminal lipidation, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)

LipCSSFNEKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALKGLEHHHHHH

SEQ ID NO：58

Lip-S3D1-S4D1_ His _ t: both having the coding sequences of heterodimeric fusion proteins of OspA serotype 3 and OspA serotype 4 of disulfide bond type 1,escherichia coli lpp lipidation signalN-terminal CSS, LN1 linker sequence, C-terminal His tag (GLEHHHHHH) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCAAACTGTCGGAAAAAGTGGTCACCCGCGCAAATGGCACCCGCCTGGAATACACGGAAATCAAAAACGATGGTAGCGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGTGCGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTC for lipid additionTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAAAACACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCACCGGCTGAAATCAAGGACCTGGCTGAACTGTGTGCGGCACTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGCTAAAGGTGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGGCACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAGATACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTCGAAATCAAAACCCTGGATGAACTGTGTAATGCTCTGAAGGGTC

TCGAGCACCACCACCACCACCAC

SEQ ID NO：59

S5D4-S6D4_ aa: heterodimeric fusion proteins of OspA serotype 5 and OspA serotype 6, both with disulfide bond type 4, LN1 linker sequence

FNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALK

SEQ ID NO：60

Lip-S5D4-S6D4_ nt: both having the coding sequence of the intermediate and final heterodimer fusion proteins of OspA serotype 5 and OspA serotype 6 of disulfide bond type 4,escherichia coli lpp lipidation signalN-terminal CSS, LN1 linker sequences for lipid addition

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAATCAGTGAAAAAACCATTGTGCGTGCGTGTGGCACCCGTCTGGAATATACCGACATCAAGAGCGATAAAACGGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCGAAGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGACTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAATGCACGATCACCGTGCAAAACTATGATAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGGCAAAGGTGAAACGAGTGAAAAAACGATTGTTCGCGCCTGTGGCACCCGCCTGGAATGCACGGATATCAAGTCGGATGGTTCGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCCTGCTACGAAAAAGACCGGTAAATGGGACAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAATGCACGATCACCGTTCAACGCTATGATAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAA

SEQ ID NO：61

Lip-S5D4-S6D4_ His _ aa: heterodimeric fusion proteins of OspA serotype 5 and OspA serotype 6, both with disulfide bond type 4, N-terminal CSS for lipid addition, N-terminal lipidation, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)

LipCSSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALKGLEHHHHHH

SEQ ID NO：62

Lip-S5D4-S6D4_ His _ nt: both having the coding sequences of heterodimeric fusion proteins of OspA serotype 5 and OspA serotype 6 of disulfide bond type 4,escherichia coli lpp lipidation signalN-terminal CSS for lipid addition, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAATCAGTGAAAAAACCATTGTGCGTGCGTGTGGCACCCGTCTGGAATATACCGACATCAAGAGCGATAAAACGGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCGAAGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGACTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAATGCACGATCACCGTGCAAAACTATGATAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGGCAAAGGTGAAACGAGTGAAAAAACGATTGTTCGCGCCTGTGGCACCCGCCTGGAATACACGGATATCAAGTCGGATGGTTCGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGTCAA

AAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGACAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAATGCACGATCACCGTTCAACGCTATGATAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTCTCGAGCACCACCACCACCACCAC

SEQ ID NO：63

S5D1-S6D1_ aa: heterodimeric fusion protein of OspA serotype 6, LN1 linker sequence, both with disulfide bond type 1

FNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALK

SEQ ID NO：64

Lip-S5D1-S6D1_ nt: both having the coding sequence of the intermediate and final heterodimer fusion proteins of OspA serotype 6 of disulfide bond type 1,escherichia coli lpp lipidation signalN-terminal CSS, LN1 linker sequences for lipid addition

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAATCTCAGAAAAAACCATCGTCCGCGCTAACGGCACCCGCCTGGAATACACCGACATCAAATCAGACAAGACCGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCTGCGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGATTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAAGATACGATCACCGTGCAAAACTATGACAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGGCAAAGGTGAAACGAGCGAAAAGACCATCGTGCGTGCGAACGGTACCCGCCTGGAATATACGGACATTAAATCGGACGGCAGCGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGATAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAAGATACGATCACCGTTCAACGCTATGACAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAA

SEQ ID NO：65

Lip-S5D1-S6D1_ His _ aa: heterodimeric fusion protein of OspA serotype 6, both with disulfide bond type 1, N-terminal CSS for lipid addition, N-terminal lipidation, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)

LipCSSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALKGLEHHHHHH

SEQ ID NO：66

Lip-S5D1-S6D1_ His _ nt: both having the coding sequence of the heterodimeric fusion protein of OspA serotype 6 of disulfide bond type 1, Escherichia coli lpp lipidation signal, N-terminal CSS for lipid addition, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAATCTCAGAAAAAACCATCGTCCGCGCTAACGGCACCCGCCTGGAATACACCGACATCAAATCAGACAAGACCGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCTGCGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGATTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAAGATACGATCACCGTGCAAAACTATGACAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGGCAAAGGTGAAACGAGCGAAAAGACCATCGTGCGTGCGAACGGTACCCGCCTGGAATATACGGACATTAAATCGGACGGCAGCGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGATAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAAGATACGATCACCGTTCAACGCTATGACAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTCTCGAGCACCACCACCACCACCAC

SEQ ID NO：67

S2D4-S1D4_ aa: heterodimeric fusion proteins of OspA serotype 2 and OspA serotype 1, both with disulfide bond type 4, LN1 linker sequence, aa164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND

FNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALK

SEQ ID NO：68

Lip-S2D4-S1D4_ nt: both haveCoding sequences for intermediate and final heterodimer fusion proteins of OspA serotype 2 and OspA serotype 1 of disulfide bond type 4,escherichia coli lpp lipidation signalAa164-174 of OspA serotype 1 substituted by non-hLFA-1-like sequence NFTLEGKVAND, N-terminal CSS, LN1 linker sequence for lipid addition

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAATGCGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAAGAAGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAACAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAATGTACGATCACCGTGCAGAAATACCGATAGTGCGGGTACCAACCTGGAAGGCACCGCTGTTGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAGTCTCGGAAAAAATCATTACCCGTGCTTGCGGCACCCGTCTGGAATACACCGGCATTAAATCGGATGGCAGCGGCAAAGCGAAGGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAGACCACCCTGGTGGTGAAAGAAGGCACCGTTACGCTGAGCAAAAACATTAGTAAGTCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAAAAGACGGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATTCCAAAAAGACCAAAGATCTGGTCTTCACGAAAGAATGCACCATCACGGTGCAGCTTTATGACAGCAACGGTACCAAACTGGAAGGCTCTGCGGTGGAAATCACGAAACTGGATGAAATCAAAAATGCACTGAAA

SEQ ID NO：69

Lip-S2D4-S1D4_ His _ aa: heterodimeric fusion proteins of OspA serotype 2 and OspA serotype 1, both with disulfide bond type 4, N-terminal CSS for lipid addition, N-terminal esterification, LN1 linker sequence, aa 164-containing 174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND, C-terminal His tag (GLEHHHHHH)

LipCSSFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGLEHHHHHH

SEQ ID NO：70

Lip-S2D4-S1D4_ His _ nt: both having the coding sequences of heterodimeric fusion proteins of OspA serotype 2 and OspA serotype 1 of disulfide bond type 4,escherichia coli lpp lipidation signalN-terminal CSS, LN1 linker sequence for lipid addition, aa164-174, C-terminal His-tag of OspA serotype 1 substituted with non-hLFA-1-like sequence NFTLEGKVAND (GLEHHHHHH)

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAATGCGGCACCAAACT

GGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAAGAAGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAACAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAATGTACGATCACCGTGCAGAAATACGATAGTGCGGGTACCAACCTGGAAGGCACCGCTGTTGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAGTCTCGGAAAAAATCATTACCCGTGCTTGCGGCACCCGTCTGGAATACACCGGCATTAAATCGGATGGCAGCGGCAAAGCGAAGGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAGACCACCCTGGTGGTGAAAGAAGGCACCGTTACGCTGAGCAAAAACATTAGTAAGTCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAAAAGACGGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATTCCAAAAAGACCAAAGATCTGGTCTTCACGAAAGAATGCACCATCACGGTGCAGCAATATGACAGCAACGGTACCAAACTGGAAGGCTCTGCGGTGGAAATCACGAAACTGGATGAAATCAAAAATGCACTGAAAGGTCTCGAGCACCACCACCACCACCAC

SEQ ID NO：71

S2D1-S1D1_ aa: heterodimeric fusion proteins of OspA serotype 2 and OspA serotype 1, both with disulfide bond type 1, N-terminal CSS for lipid addition, LN1 linker sequence, aa164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND

FNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALK

SEQ ID NO：72

Lip-S2D1-S1D1_ nt: both having the coding sequence of the intermediate and final heterodimer fusion proteins of OspA serotype 2 and OspA serotype 1 of disulfide bond type 1, the Escherichia coli lpp lipidation signal, the N-terminal CSS for lipid addition, the LN1 linker sequence, aa164-174 of OspA serotype 1 replaced by the non-hLFA-1-like sequence NFTLEGKVAND

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAAAACGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAATGCGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAATAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAAGATACGATCACCGTGCAGAAATACGACAGTGCGGGTACCAACCTGGAAGGCACGGCTGTTGAAATCAAAACCCTGGACGAACTGTGTAACGCCCTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAGTCAGCGAAAAAATCATTACCCGCGCAGACGGCACCCGCCTGGAATACACCGGCATCAAATCGGACGGCAGCGGCAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAAACCACCCTGGTGGTGAAATGCGGCACCGTTACGCTGAGCAAAAACATTAGTAAATCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAGAAAACCGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATAGCAAGAAAACCAAAGATCTGGTCTTCACGAAAGAAAACACCATCACGGTGCAGCAATATGACAGCAATGGTACCAAACTGGAAGGCTCCGCTGTGGAAATCACGAAACTGGATGAAATCTGTAATGCACTGAAA

SEQ ID NO：73

Lip-S2D1-S1D1_ His _ aa: heterodimeric fusion proteins of OspA serotype 2 and OspA serotype 1, both with disulfide bond type 1, N-terminal CSS for lipid addition, LN1 linker sequence, aa164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND, N-terminal lipidation, C-terminal His tag (GLEHHHHHH)

LipCSSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALKGLEHHHHHH

SEQ ID NO：74

Lip-S2D1-S1D1_ His _ nt: both having the coding sequence of the heterodimeric fusion proteins OspA serotype 2 and OspA serotype 1 of disulfide bond type 1, Escherichia coli lpp lipidation signal, N-terminal CSS for lipid addition, LN1 linker sequence, aa64-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND, C-terminal His tag (GLEHHHHHH)

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAAAACGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAATGCGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAATAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAAGATACGATCACCGTGCAGAAATACGACAGTGCGGGTACCAACCTGGAAGGCACGGCTGTTGAAATCAAAACCCTGGACGAACTGTGTAACGCCCTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAGTCAGCGAAAAAATCATTACCCGCGCAGACGGCACCCGCCTGGAATACACCGGCATCAAATCGGACGGCAGCGGCAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAAACCACCCTGGTGGTGAAATGCGGCACCGTTACGCTGAGCAAAAACATTAGTAAATCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAGAAAACCGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATAGCAAGAAAACCAAAGATCTGGTCTTCACGAAAGAAAACACCATCACGGTGCAGCAATATGACAGCAATGGTACCAAACTGGAAGGCTCCGCTGTGGAAATCACGAAACTGGATGAAATCTGTAATGCACTGAAAGGTCTCGAGCACCACCACCACCACCAC

SEQ ID NO：75

S4D4-S3D4_ aa: heterodimeric fusion proteins of OspA serotype 4 and OspA serotype 3, both with disulfide bond type 4, N-terminal CSS for lipid addition, LN1 linker sequence

FNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALK

SEQ ID NO：76

Lip-S4D4-S3D4_ nt: both having the coding sequence of the intermediate and final heterodimer fusion proteins of OspA serotype 4 and OspA serotype 3 of disulfide bond type 4, the Escherichia coli lpp lipidation signal, the N-terminal CSS for lipid addition, the LN1 linker sequence

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGCTAAAGGTGAACTGTCGGAAAAAACCATCCTGCGCGCCTGTGGCACCCGCCTGGAATACACGGAAATCAAGTCGGACGGCACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCGACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAATGCACGATCACCGTTCAAAAATATGATTCCGCAGGTACCAACCTGGAAGGCAACGCTGTGGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAGGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTTAACGATAAGGGCAAACTGTCAGAAAAAGTGGTCACCCGCGCTTGTGGCACCCGCCTGGAATACACCGAAATCAAAAACGACGGCTCGGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGGAAGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAATGCACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCCCCGGCTGAAATCAAGGACCTGGCGGAACTGAAGGCGGCACTGAAA

SEQ ID NO：77

Lip-S4D4-S3D4_ His _ aa: heterodimeric fusion proteins of OspA serotype 4 and OspA serotype 3, both having disulfide bond type 4, N-terminal CSS for lipid addition, LN1 linker sequence, N-terminal lipidation, C-terminal His tag (GLEHHHHHH)

LipCSSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALKGLEHHHHHH

SEQ ID NO：78

Lip-S4D4-S3D4_ His _ nt: coding sequences of heterodimeric fusion proteins of OspA serotype 4 and OspA serotype 3, both having disulfide bond type 4, Escherichia coli lpp lipidation signal, N-terminal CSS for lipid addition, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGCTAAAGGTGAACTGTCGGAAAAAACCATCCTGCGCGCCTGTGGCACCCGCCTGGAATACACGGAAATCAAGTCGGACGGCACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCGACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAATGCACGATCACCGTTCAAAAATATGATTCCGCAGGTACCAACCTGGAAGGCAACGCTGTGGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAGGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTTAACGATAAGGGCAAACTGTCAGAAAAAGTGGTCACCCGCGCTTGTGGCACCCGCCTGGAATACACCGAAATCAAAAACGACGGCTCGGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGGAAGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAATGCACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCCCCGGCTGAAATCAAGGACCTGGCGGAACTGAAGGCGGCACTGAAAGGTCTCGAGCACCACCACCACCACCAC

SEQ ID NO：79

S4D1-S3D1_ aa: heterodimeric fusion proteins of OspA serotype 4 and OspA serotype 3, both with disulfide bond type 1, LN1 linker sequence

FNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALK

SEQ ID NO：80

Lip-S4D1-S3D1_ nt: both having the coding sequence of the intermediate and final heterodimer fusion proteins of OspA serotype 4 and OspA serotype 3 of disulfide bond type 1, the Escherichia coli lpp lipidation signal, the N-terminal CSS for lipid addition, the LN1 linker sequence

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGGAGGTTGCTCAAGCTTCAATGCTAAGGGCGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGGCACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAGATACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTCGAAATCAAAACCCTGGATGAACTGTGTAACGCCCTGAAGGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTTAACGATAAGGGCAAACTGTCGGAAAAAGTGGTCACCCGCGCAAATGGCACCCGCCTGGAATACACGGAAATCAAAAACGATGGTAGCGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGTGCGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAAAACACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCACCGGCTGAAATCAAGGACCTGGCTGAACTGTGTGCGGCACTGAAA

SEQ ID NO：81

Lip-S4D1-S3D1_ His _ aa: heterodimeric fusion proteins of OspA serotype 4 and OspA serotype 3, both having disulfide bond type 1, N-terminal CSS for lipid addition, LN1 linker sequence, N-terminal lipidation, C-terminal His tag (GLEHHHHHH)

LipCSSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGIEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALKGLEHHHHHH

SEQ ID NO：82

Lip-S4D1-S3D1_ His _ nt: both having the coding sequence of heterodimeric fusion proteins of OspA serotype 4 and OspA serotype 3 of disulfide bond type 1, Escherichia coli lpp lipidation signal, N-terminal CSS for lipid addition, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAATGCTAAGGGCGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGGCACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAAGATACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTCGAAATCAAAACCCTGGATGAACTGTGTAACGCCCTGAAGGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTTAACGATAAGGGCAAACTGTCGGAAAAAGTGGTCACCCGCGCAAATGGCACCCGCCTGGAATACACGGAAATCAAAAACGATGGTAGCGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGTGCGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAAAACA CGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCACCGGCTGAAATCAAGGACCTGGCTGAACTGTGTGCGGCACTGAAAGGTCTCGAGCACCACCACCACCACCAC

SEQ ID NO：83

S6D4-S5D4_ aa: heterodimeric fusion proteins of OspA serotype 6 and OspA serotype 5, both with disulfide bond type 4, LN1 linker sequence

FNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALK

SEQ ID NO：84

Lip-S6D4-S5D4_ nt: both having the coding sequence of the intermediate and final heterodimer fusion proteins of OspA serotype 6 and OspA serotype 5 of disulfide bond type 4, the Escherichia coli lpp lipidation signal, the N-terminal CSS for lipid addition, the LN1 linker sequence

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGGCAAAGGTGAAACGAGTGAAAAAACGATTGTTCGCGCCTGTGGCACCCGCCTGGAATACACGGATATCAAGTCGGATGGTTCGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGACAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAATGCACGATCACCGTTCAACGCTATGATAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAATCAGTGAAAAAACCATTGTGCGTGCGTGTGGCACCCGTCTGGAATATACCGACATCAAGAGCGATAAAACGGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCGAAGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGACTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAA

ACCAAGCAGCTGGTCTTCACGAAAGAATGCACGATCACCGTGCAAAACTATGATAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAA

SEQ ID NO：85

Lip-S6D4-S5D4_ His _ aa: heterodimeric fusion proteins of OspA serotype 6 and OspA serotype 5, both with disulfide bond type 4, N-terminal CSS for lipid addition, LN1 linker sequence, N-terminal lipidation, C-terminal His tag (GLEHHHHHH)

LipCSSFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALKGLFHHHHHH

SEQ ID NO：86

Lip-S6D4-S5D4_ His _ nt: coding sequences of heterodimeric fusion proteins of OspA serotype 6 and OspA serotype 5, both having disulfide bond type 4, Escherichia coli lpp lipidation signal, N-terminal CSS for lipid addition, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGGCAAAGGTGAAACGAGTGAAAAAACGATTGTTCGCGCCTGTGGCACCCGCCTGGAATACACGGATATCAAGTCGGATGGTTCGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGACAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAATGCACGATCACCGTTCAACGCTATGATAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAATCAGTGAAAAAACCATTGTGCGTGCGTGTGGCACCCGTCTGGAATATACCGACATCAAGAGCGATAAAACGGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCGAAGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGACTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAATGCACGATCACCGTGCAAAACTATGATAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTCTCGAGCACCACCACCACCACCAC

SEQ ID NO：87

S6D1-S5D1_ aa: heterodimeric fusion proteins of OspA serotype 6 and OspA serotype 5, both with disulfide bond type 1, LN1 linker sequence

FNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALK

SEQ ID NO：88

Lip-S6D1-S5D1_ nt: both having the coding sequence of the intermediate and final heterodimer fusion proteins of OspA serotype 6 and OspA serotype 5 of disulfide bond type 1, the Escherichia coli lpp lipidation signal, the N-terminal CSS for lipid addition, the LN1 linker sequence

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGGCAAAGGTGAAACGAGCGAAAAGACCATCGTGCGTGCGAACGGTACCCGCCTGGAATATACGGACATTAAATCGGACGGCAGCGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGATAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAAGATACGATCACCGTTCAACGCTATGACAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAATCTCAGAAAAAACCATCGTCCGCGCTAACGGCACCCGCCTGGAATACACCGACATCAAATCAGACAAGACCGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTG

GAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCTGCGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGATTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAAGATACGATCACCGTGCAAAACTATGACAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAA

SEQ ID NO：89

Lip-S6D1-S5D1_ His _ aa: heterodimer fusion proteins of OspA serotype 6 and OspA serotype 5, both having disulfide bond type 1, LN1 linker sequence, N-terminal lipidation, C-terminal His tag (GLEHHHHHH)

LipCSSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALKGLEHHHHHH

SEQ ID NO：90

Lip-S6D1-S5D1_ His _ nt: both having the coding sequence of heterodimeric fusion proteins of OspA serotype 6 and OspA serotype 5 of disulfide bond type 1, Escherichia coli lpp lipidation signal, N-terminal CSS for lipid addition, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGGCAAAGGTGAAACGAGCGAAAAGACCATCGTGCGTGCGAACGGTACCCGCCTGGAATATACGGACATTAAATCGGACGGCAGCGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGATAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAAGATACGATCACCGTTCAACGCTATGACAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAATCTCAGAAAAAACCATCGTCCGCGCTAACGGCACCCGCCTGGAATACACCGACATCAAATCAGACAAGACCGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCTGCGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGATTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAAGATACGATCACCGTGCAAAACTATGACAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTCTCGAGCACCACCACCACCACCAC

SEQ ID NO：91

S1D4-S2D1_ aa: heterodimeric fusion proteins of OspA serotype 1 with disulfide bond type 4 and OspA serotype 2 with disulfide bond type 1, LN1 linker sequence, aa164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND

FNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALK

SEQ ID NO：92

Lip-S1D4-S2D1_ nt: coding sequences for intermediate and final heterodimer fusion proteins of OspA serotype 1 with disulfide bond type 4 and OspA serotype 2 with disulfide bond type 1, escherichia coli lpp lipidation signal, N-terminal CSS for lipid addition, LN1 linker sequence, aa164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAGTCTCGGAAAAAATCATTACCCGTGCTTGCGGCACCCGTCTGGAATACACCGGCATTAAATCGGATGGCAGCGGCAAAGCGAAGGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAGACCACCCTGGTGGTGAAAGAAGGCACCGTTACGCTGAGCAAAAACATTAGTAAGTCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAAAAGACGGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATTCCAAAAAGACCAAAGATCTGGTCTTCACGAAAGAATGCACCATCACGGTGCAGCAATATGACAGCAACGGTACCAAACTGGAAGGCTCTGCGGTGGAAATCACGAAACTGGATGAAATCAAAAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAAAACGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAATGCGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAATAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAAGATACGATCACCGTGCAGAAATACGACAGTGCGGGTACCAACCTGGAAGGCACGGCTGTTGAAATCAAAACCCTGGACGAACTGTGTAACGCCCTGAAA

SEQ ID NO：93

Lip-S1D4-S2D1_ His _ aa: heterodimeric fusion proteins of OspA serotype 1 with disulfide bond type 4 and OspA serotype 2 with disulfide bond type 1, N-terminal CSS for lipid addition, LN1 linker sequence, aa 164-one 174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND, N-terminal lipidation, C-terminal His tag (GLEHHHHHH)

LipCSSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALKGLEHHHHHH

SEQ ID NO：94

Lip-S1D4-S2D1_ His _ nt: coding sequences of heterodimeric fusion proteins of OspA serotype 1 with disulfide bond type 4 and OspA serotype 2 with disulfide bond type 1, Escherichia coli lpp lipidation signal, N-terminal CSS for lipid addition, LN1 linker sequence, aa 164-rich 174 of OspA serotype 1 substituted by non-hLFA-1-like sequence NFTLEGKVAND, C-terminal His tag (GLEHHHHHH)

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAGTCTCGGAAAAAATCATTACCCGTGCTTGCGGCACCCGTCTGGAATACACCGGCATTAAATCGGATGGCAGCGGCAAAGCGAAGGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAGACCACCCTGGTGGTGAAAGAAGGCACCGTTACGCTGAGCAAAAACATTAGTAAGTCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAAAAGACGGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATTCCAAAAAGACCAAAGATCTGGTCTTCACGAAAGAATGCACCATCACGGTGCAGCAATATGACAGCAACGGTACCAAACTGGAAGGCTCTGCGGTGGAAATCACGAAACTGGATGAAATCAAAAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAAAACGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAATGCGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAATAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAAGATACGATCACCGTGCAGAAATACGACAGTGCGGGTACCAACCTGGAAGGCACGGCTGTTGAAATCAAAACCCTGGACGAACTGTGTAACGCCCTGAAAGGCCTCGAGCACCACCACCACCACCAC

SEQ ID NO：95

S1D1-S2D4_ aa: heterodimeric fusion proteins of OspA serotype 1 with disulfide bond type 1 and OspA serotype 2 with disulfide bond type 4, LN1 linker sequence, aa164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND

FNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVFITKLDEICNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALK

SEQ ID NO：96

Lip-S1D1-S2D4_ nt: coding sequences for intermediate and final heterodimer fusion proteins of OspA serotype 1 with disulfide bond type 1 and OspA serotype 2 with disulfide bond type 4, escherichia coli lpp lipidation signal, N-terminal CSS for lipid addition, LN1 linker sequence, aa164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAGTCAGCGAAAAAATCATTACCCGCGCAGACGGCACCCGCCTGGAATACACCGGCATCAAATCGGACGGCAGCGGCAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAAACCACCCTGGTGGTGAAATGCGGCACCGTTACGCTGAGCAAAAACATTAGTAAATCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAGAAAACCGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATAGCAAGAAAACCAAAGATCTGGTCTTCACGAAAGAAAACACCATCACGGTGCAGCAATATGACAGCAATGGTACCAAACTGGAAGGCTCCGCTGTGGAAATCACGAAACTGGATGAAATCTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAATGCGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAAGAAGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAACAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAATGTACGATCACCGTGCAGAAATACGATAGTGCGGGTACCAACCTGGAAGGCACCGCTGTTGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAA

SEQ ID NO：97

Lip-S1D1-S2D4_ His _ aa: heterodimeric fusion proteins of OspA serotype 1 with disulfide bond type 1 and OspA serotype 2 with disulfide bond type 4, N-terminal CSS for lipid addition, LN1 linker sequence, aa 164-one 174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND, N-terminal lipidation, C-terminal His tag (GLEHHHHHH)

LipCSSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH

SEQ ID NO：98

Lip-S1D1-S2D4_ His _ nt: coding sequences for heterodimeric fusion proteins of OspA serotype 1 with disulfide bond type 1 and OspA serotype 2 with disulfide bond type 4, Escherichia coli lpp lipidation signal, N-terminal CSS for lipid addition, LN1 linker sequence, aa 164-rich 174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND, C-terminal His tag (GLEHHHHHH)

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAGTCAGCGAAAAAATCATTACCCGCGCAGACGGCACCCGCCTGGAATACACCGGCATCAAATCGGACGGCAGCGGCAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAAACCACCCTGGTGGTGAAATGCGGCACCGTTACGCTGAGCAAAAACATTAGTAAATCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAGAAAACCGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATAGCAAGAAAACCAAAGATCTGGTCTTCACGAAAGAAAACACCATCACGGTGCAGCAATATGACAGCAATGGTACCAAACTGGAAGGCTCCGCTGTGGAAATCACGAAACTGGATGAAATCTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAATGCGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAAGAAGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAACAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAATGTACGATCACCGTGCAGAAATACGATAGTGCGGGTACCAACCTGGAAGGCACCGCTGTTGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAAGGCCTCGAGCACCACCACCACCACCAC

SEQ ID NO：99

S3D4-S4D1_ aa: heterodimeric fusion proteins of OspA serotype 3 with disulfide bond type 4 and OspA serotype 4 with disulfide bond type 1, LN1 linker sequence

FNEKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALK

SEQ ID NO：100

Lip-S3D4-S4D1_ nt: coding sequences for intermediate and final heterodimer fusion proteins of OspA serotype 3 with disulfide bond type 4 and OspA serotype 4 with disulfide bond type 1, escherichia coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 linker sequence

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCAAACTGTCAGAAAAAGTGGTCACCCGCGTTGTGGCACCCGCCTGGAATACACCGAAATCAAAAACGACGGCTCGGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGGAAGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAATGCACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCCCCGGCTGAAATCAAGGACCTGGCGGAACTGAAGGCGGCACTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGCTAAAGGTGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGGCACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAGATACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTCGAAATCAAAACCCTGGATGAACTGTGTAATGCTCTGAAG

SEQ ID NO：101

Lip-S3D4-S4D1_ His _ aa: heterodimeric fusion proteins of OspA serotype 3 with disulfide bond type 4 and OspA serotype 4 with disulfide bond type 1, N-terminal CSS for lipid addition, LN1 linker sequence, N-terminal lipidation, C-terminal His tag (GLEHHHHHH)

LipCSSFNEKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALKGLEHHHHHH

SEQ ID NO：102

Lip-S3D4-S4D1_ His _ nt: coding sequences of heterodimeric fusion proteins of OspA serotype 3 having disulfide bond type 4 and OspA serotype 4 having disulfide bond type 1, Escherichia coli lpp lipidation signal, N-terminal CSS for addition of lipid, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCAAACTGTCAGAAAAAGTGGTCACCCGCGCTTGTGGCACCCGCCTGGAATACACCGAAATCAAAAACGACGGCTCGGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGGAAGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAATGCACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCCCCGGCTGAAATCAAGGACCTGGCGGAACTGAAGGCGGCACTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGCTAAAGGTGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGGCACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAGATACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTCGAAATCAAAACCCTGGATGAACTGTGTAATGCTCTGAAGGGTCTCGAGCACCACCACCACCACCAC

SEQ ID NO：103

S3D1-S4D4_ aa: heterodimeric fusion proteins of OspA serotype 3 with disulfide bond type 1 and OspA serotype 4 with disulfide bond type 1, LN1 linker sequence

FNEKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPA

EIKDLAELCAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALK

SEQ ID NO：104

Lip-S3D1-S4D4_ nt: coding sequences for intermediate and final heterodimer fusion proteins of OspA serotype 3 with disulfide bond type 1 and OspA serotype 4 with disulfide bond type 1, Escherichia coli lpp lipidation signal, N-terminal CSS for lipid addition, LN1 linker sequence

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCAAACTGTCGGAAAAAGTGGTCACCCGCGCAAATGGCACCCGCCTGGAATACACGGAAATCAAAAACGATGGTAGCGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGTGCGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAAAACACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCACCGGCTGAAATCAAGGACCTGGCTGAACTGTGTGCGGCACTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGCTAAAGGTGAACTGTCGGAAAAAACCATCCTGCGCGCCTGTGGCACCCGCCTGGAATACACGGAAATCAAGTCGGACGGCACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCGACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAATGCACGATCACCGTTCAAAAATATGATTCCGCAGGTACCAACCTGGAAGGCAACGCTGTGGAAATCAAAACCCTGGACGAACTGAAAAATGCTCTGAAG

SEQ ID NO：105

Lip-S3D1-S4D4_ His _ aa: heterodimeric fusion proteins of OspA serotype 3 with disulfide bond type 1 and OspA serotype 4 with disulfide bond type 1, N-terminal CSS for lipid addition, LN1 linker sequence, N-terminal lipidation, C-terminal His tag (GLEHHHHHH)

LipCSSFNEKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALKGLEHHHHHH

SEQ ID NO：106

Lip-S3D1-S4D4_ His _ nt: coding sequences of heterodimeric fusion proteins of OspA serotype 3 having disulfide bond type 1 and OspA serotype 4 having disulfide bond type 1, Escherichia coli lpp lipidation signal, N-terminal CSS for addition of lipid, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCAAACTGTCGGAAAAAGTGGTCACCCGCGCAAATGGCACCCGCCTGGAATACACGGAAATCAAAAACGATGGTAGCGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGTGCGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAAAACACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCACCGGCTGAAATCAAGGACCTGGCTGAACTGTGTGCGGCACTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGCTAAAGGTGAACTGTCGGAAAAAACCATCCTGCGCGCCTGTGGCACCCGCCTGGAATACACGGAAATCAAGTCGGACGGCACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCGACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAATGCACGATCACCGTTCAAAAATATGATTCCGCAGGTACCAACCTGGAAGGCAACGCTGTGGAAATCAAAACCCTGGACGAACTGAAAAATGCTCTGAAGGGTCTCGAGCACCACCACCACCACCAC

SEQ ID NO：107

S5D4-S6D1_ aa: heterodimeric fusion proteins of OspA serotype 5 with disulfide bond type 4 and OspA serotype 6 with disulfide bond type 1, LN1 linker sequence

FNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALK

SEQ ID NO：108

Lip-S5D4-S6D1_ nt: coding sequences for intermediate and final heterodimer fusion proteins of OspA serotype 5 with disulfide bond type 4 and OspA serotype 6 with disulfide bond type 1, escherichia coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 linker sequence

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAATCAGTGAAAAAACCATTGTGCGTGCGTGTGGCACCCGTCTGGAATATACCGACATCAAGAGCGATAAAACGGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCGAAGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGACTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAATGCACGATCACCGTGCAAAACTATGATAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGGCAAAGGTGAAACGAGCGAAAAGACCATCGTGCGTGCGAACGGTACCCGCCTGGAATATACGGACATTAAATCGGACGGCAGCGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGATAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAAGATACGATCACCGTTCAACGCTATGACAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAA

SEQ ID NO：109

Lip-S5D4-S6D1_ His _ aa: heterodimeric fusion proteins of OspA serotype 5 with disulfide bond type 4 and OspA serotype 6 with disulfide bond type 1, N-terminal CSS for lipid addition, LN1 linker sequence, N-terminal lipidation, C-terminal His tag (GLEHHHHHH)

LipCSSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALKGLEHHHHHH

SEQ ID NO：110

Lip-S5D4-S6D1_ His _ nt: coding sequences of heterodimeric fusion proteins of OspA serotype 5 having disulfide bond type 4 and OspA serotype 6 having disulfide bond type 1, Escherichia coli lpp lipidation signal, N-terminal CSS for addition of lipid, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAATCAGTGAAAAAACCATTGTGCGTGCGTGTGGCACCCGTCTGGAATATACCGACATCAAGAGCGATAAAACGGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCGAAGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGACTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAATGCACGATCACCGTGCAAAACTATGATAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGGCAAAGGTGAAACGAGCGAAAAGACCATCGTGCGTGCGAACGGTACCCGCCTGGAATATACGGACATTAAATCGGACGGCAGCGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGATAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAAGATACGATCACCGTTCAACGCTATGACAGTGCGGGC

ACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTCTCGAGCACCACCACCACCACCAC

SEQ ID NO：111

S5D1-S6D4_ aa: heterodimeric fusion proteins of OspA serotype 5 with disulfide bond type 1 and OspA serotype 6 with disulfide bond type 4, LN1 linker sequence

FNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLFGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALK

SEQ ID NO：112

Lip-S5D1-S6D4_ nt: coding sequences for intermediate and final heterodimer fusion proteins of OspA serotype 5 with disulfide bond type 1 and OspA serotype 6 with disulfide bond type 4, escherichia coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 linker sequence

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAATCTCAGAAAAAACCATCGTCCGCGCTAACGGCACCCGCCTGGAATACACCGACATCAAATCAGACAAGACCGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCTGCGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGATTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAAGATACGATCACCGTGCAAAACTATGACAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGGCAAAGGTGAAACGAGTGAAAAAACGATTGTTCGCGCCTGTGGCACCCGCCTGGAATACACGGATATCAAGTCGGATGGTTCGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGACAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAATGCACGATCACCGTTCAACGCTATGATAGTGCGGGCACCAACCTGGAAGGCAAAGGCGTTGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAA

SEQ ID NO：113

Lip-S5D1-S6D4_ His _ aa: heterodimeric fusion proteins of OspA serotype 5 with disulfide bond type 1 and OspA serotype 6 with disulfide bond type 4, N-terminal CSS for lipid addition, LN1 linker sequence, N-terminal lipidation, C-terminal His tag (GLEHHHHHH)

LipCSSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRACGTRLEYTIKSDGDGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLIISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALKGLEHHHHHH

SEQ ID NO：114

Lip-S5D1-S6D4_ His _ nt: coding sequences of heterodimeric fusion proteins of OspA serotype 5 having disulfide bond type 1 and OspA serotype 6 having disulfide bond type 4, Escherichia coli lpp lipidation signal, N-terminal CSS for addition of lipid, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAATCTCAGAAAAAACCATCGTCCGCGCTAACGGCACCCGCCTGGAATACACCGACATCAAATCAGACAAGACCGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCTGCGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGATTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAAGATACGATCACCGTGCAAAACTATGACAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGGCAAAGGTGAAACGAGTGAAAAAACGATTGTTCGCGCCTGTGGCACCCGCCTGGAATACACGGATATCAAGTCGGATGGTTCGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGTCAA

SEQ ID NO：115

S2D4-S1D1_ aa: heterodimeric fusion proteins of OspA serotype 2 with disulfide bond type 4 and OspA serotype 1 with disulfide bond type 1, LN1 linker sequence, aa164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND

FNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALK

SEQ ID NO：116

Lip-S2D4-S1D1_ nt: coding sequences for intermediate and final heterodimer fusion proteins of OspA serotype 2 with disulfide bond type 4 and OspA serotype 1 with disulfide bond type 1, escherichia coli lpp lipidation signal, N-terminal CSS for lipid addition, LN1 linker sequence, aa164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAATGCGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAAGAAGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAACAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAATGTACGATCACCGTGCAGAAATACGATAGTGCGGGTACCAACCTGGAAGGCACCGCTGTTGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAGTCAGCGAAAAAATCATTACCCGCGCAGACGGCACCCGCCTGGAATACACCGGCATCAAATCGGACGGCAGCGGCAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAAACCACCCTGGTGGTGAAATGCGGCACCGTTACGCTGAGCAAAAACATTAGTAAATCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAGAAAACCGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATAGCAAGAAAACCAAAGATCTGGTCTTCACGAAAGAAAACACCATCACGGTGCAGCAATATGACAGCAATGGTACCAAACTGGAAGGCTCCGCTGTGGAAATCACGAAACTGGATGAAATCTGTAATGCACTGAAA

SEQ ID NO：117

Lip-S2D4-S1D1_ His _ aa: heterodimeric fusion proteins of OspA serotype 2 with disulfide bond type 4 and OspA serotype 1 with disulfide bond type 1, N-terminal CSS for lipid addition, LN1 linker sequence, aa 164-one 174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND, N-terminal lipidation, C-terminal His tag (GLEHHHHHH)

LipCSSFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALKGLEHHHHHH

SEQ ID NO：118

Lip-S2D4-S1D1_ His _ nt: coding sequences for heterodimeric fusion proteins of OspA serotype 2 with disulfide bond type 4 and OspA serotype 1 with disulfide bond type 1, Escherichia coli lpp lipidation signal, N-terminal CSS for lipid addition, LN1 linker sequence, aa 164-rich 174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND, C-terminal His tag (GLEHHHHHH)

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAATGCGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAAGAAGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAACAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAATGTACGATCACCGTGCAGAAATACGATAGTGCGGGTACCAACCTGGAAGGCACCGCTGTTGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAGTCAGCGAAAAAATCATTACCCGCGCAGACGGCACCCGCCTGGAATACACCGGCATCAAATCGGACGGCAGCGGCAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAAACCACCCTGGTGGTGAAATGCGGCACCGTTACGCTGAGCAAAAACATTAGTAAATCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAGAAAACCGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATAGCAAGAAAACCAAAGATCTGGTCTTCACGAAAGAAAACACCATCACGGTGCAGCAATATGACAGCAATGGTACCAAACTGGAAGGCTCCGCTGTGGAAATCACGAAACTGGATGAAATCTGTAATGCACTGAAAGGTCTCGAGCACCACCACCACCACCAC

SEQ ID NO：119

S2D1-S1D4_ aa: heterodimeric fusion proteins of OspA serotype 2 with disulfide bond type 1 and OspA serotype 1 with disulfide bond type 4, LN1 linker sequence, aa164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND

FNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALK

SEQ ID NO：120

Lip-S2D1-S1D4_ nt: coding sequences for intermediate and final heterodimer fusion proteins of OspA serotype 2 with disulfide bond type 1 and OspA serotype 1 with disulfide bond type 4, escherichia coli lpp lipidation signal, N-terminal CSS for lipid addition, LN1 linker sequence, aa164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAAAACGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAATGCGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAATAGCAAGAAAACCACGCAGCTGGTCTTCACCAAAACAAGATACGATCACCGTGCAGAAATACGACAGTGCGGGTACCAACCTGGAAGGCACGGCTGTTGAAATCAAAACCCTGGACGAACTGTGTAACGCCCTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAGTCTCGGAAAAAATCATTACCCGTGCTTGCGGCACCCGTCTGGAATACACCGGCATTAAATCGGATGGCAGCGGCAAAGCGAAGGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAGACCACCCTGGTGGTGAAAGAAGGCACCGTTACGCTGAGCAAAAACATTAGTAAGTCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAAAAGACGGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATTCCAAAAAGACCAAAGATCTGGTCTTCACGAAAGAATGCACCATCACGGTGCAGCAATATGACAGCAACGGTACCAAACTGGAAGGCTCTGCGGTGGAAATCACGAAACTGGATGAAATCAAAAATGCACTGAAA

SEQ ID NO：121

Lip-S2D1-S1D4_ His _ aa: heterodimeric fusion proteins of OspA serotype 2 with disulfide bond type 1 and OspA serotype 1 with disulfide bond type 4, N-terminal CSS for lipid addition, LN1 linker sequence, aa 164-one 174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND, N-terminal lipidation, C-terminal His tag (GLEHHHHHH)

LipCSSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGLEHHHHHH

SEQ ID NO：122

Lip-S2D1-S1D4_ His _ nt: coding sequences for heterodimeric fusion proteins of OspA serotype 2 with disulfide bond type 1 and OspA serotype 1 with disulfide bond type 4, Escherichia coli lpp lipidation signal, N-terminal CSS for lipid addition, LN1 linker sequence, aa 164-rich 174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND, C-terminal His tag (GLEHHHHHH)

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAAAACGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAATGCGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAATAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAAGATACGATCACCGTGCAGAAATACGACAGTGCGGGTACCAACCTGGAAGGCACGGCTGTTGAAATCAAAACCCTGGACGAACTGTGTAACGCCCTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAGTCTCGGAAAAAATCATTACCCGTGCTTGCGGCACCCGTCTGGAATACACCGGCATTAAATCGGATGGCAGCGGCAAAGCGAAGGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAGACCACCCTGGTGGTGAAAGAAGGCACCGTTACGCTGAGCAAAAACATTAGTAAGTCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAAAAGACGGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATTCCAAAAAGACCAAAGATCTGATCTTCACGAAAGAATGCACCATCACGGTGCAGCAATATGACAGCAACGGTACCAAACTGGAAGGCTCTGCGGTGGAAATCACGAAACTGGATGAAATCAAAAATGCACTGAAAGGTCTCGAGCACCACCACCACCACCAC

SEQ ID NO：123

S4D4-S3D1_ aa: heterodimeric fusion proteins of OspA serotype 4 with disulfide bond type 4 and OspA serotype 3 with disulfide bond type 1, LN1 linker sequence

FNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQQYDSAGTNLEGNAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEKDLAELCAALK

SEQ ID NO：124

Lip-S4D4-S3D1_ nt: coding sequences for intermediate and final heterodimer fusion proteins of OspA serotype 4 with disulfide bond type 4 and OspA serotype 3 with disulfide bond type 1, escherichia coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 linker sequence

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGCTAAAGGTGAACTGTCGGAAAAAACCATCCTGCGCGCGTGTGGGACCCGCCTGGAATACACGGAAATCAAGTCGGACGGCACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGGAAAGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCGACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAATGCACGATCACCGTTCAAAAATATGATTCCGCAGGTACCAACCTGGAAGGCAACGCTGTGGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAGGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTTAACGATAAGGGCAAACTGTCGGAAAAAGTGGTCACCCGCGCAAATGGCACCCGCCTGGAATACACGGAAATCAAAAACGATGGTAGCGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGTGCGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAAAACACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCACCGGCTGAAATCAAGGACCTGGCTGAACTGTGTGCGGCACTGAAA

SEQ ID NO：125

Lip-S4D4-S3D1_ His _ aa: heterodimeric fusion proteins of OspA serotype 4 with disulfide bond type 4 and OspA serotype 3 with disulfide bond type 1, N-terminal CSS for lipid addition, LN1 linker sequence, N-terminal CSS for lipid addition, N-terminal lipidation, C-terminal His tag (GLEHHHHHH)

LipCSSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRANGTRLEYTEIKNDGSG

KAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALKGLEHHHHHH

SEQ ID NO：126

Lip-S4D4-S3D1_ His _ nt: coding sequences of heterodimeric fusion proteins of OspA serotype 4 having disulfide bond type 4 and OspA serotype 3 having disulfide bond type 1, Escherichia coli lpp lipidation signal, N-terminal CSS for addition of lipid, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGCTAAAGGTGAACTGTCGGAAAAAACCATCCTGCGCGCCTGTGGCACCCGCCTGGAATACACGGAAATCAAGTCGGACGGCACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGAGAAAAGCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCGACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAATGCACGATCACCGTTCAAAAATATGATTCCGCAGGTACCAACCTGGAAGGCAACGCTGTGGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAGGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTTAACGATAAGGGCAAACTGTCGGAAAAAGTGGTCACCCGCGCAAATGGCACCCGCCTGGAATACACGGAAATCAAAAACGATGGTAGCGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGTGCGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAAAACACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCACCGGCTGAAATCAAGGACCTGGCTGAACTGTGTGCGGCACTGAAAGGTCTCGAGCACCACCACCACCACCAC

SEQ ID NO：127

S4D1-S3D4_ aa: heterodimeric fusion proteins of OspA serotype 4 with disulfide bond type 1 and OspA serotype 3 with disulfide bond type 4, LN1 linker sequence

FNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALK

SEQ ID NO：128

Lip-S4D1-S3D4_ nt: heterodimeric fusion proteins of OspA serotype 4 with disulfide bond type 1 and OspA serotype 3 with disulfide bond type 4, Escherichia coli lpp lipidation signal, N-terminal CSS for lipid addition, LN1 linker sequence

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAATGCTAAGGGCGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGGCACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAGATACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTCGAAATCAAAACCCTGGATGAACTGTGTAACGCCCTGAAGGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTTAACGATAAGGGCAAACTGTCAGAAAAAGTGGTCACCCGCGCTTGTGGCACCCGCCTGGAATACACCGAAATCAAAAACGACGGCTCGGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGGAAGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAATGCACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCCCCGGCTGAAATCAAGGACCTGGCGGAACTGAAGGCGGCACTGAAA

SEQ ID NO：129

Lip-S4D1-S3D4_ His _ aa: heterodimeric fusion proteins of OspA serotype 4 with disulfide bond type 1 and OspA serotype 3 with disulfide bond type 4, N-terminal CSS for lipid addition, LN1 linker sequence, N-terminal lipidation, C-terminal His tag (GLEHHHHHH)

LipCSSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALKGLEHHHHHH

SEQ ID NO：130

Lip-S4D1-S3D4_ His _ nt: coding sequences of heterodimeric fusion proteins of OspA serotype 4 having disulfide bond type 1 and OspA serotype 3 having disulfide bond type 4, Escherichia coli lpp lipidation signal, N-terminal CSS for addition of lipid, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAATGCTAAGGGCGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGGCACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAGATACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTCGAAATCAAAACCCTGGATGAACTGTGTAACGCCCTGAAGGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTTAACGATAAGGGCAAACTGTCAGAAAAAGTGGTCACCCGCGCTTGTGGCACCCGCCTGGAATACACCGAAATCAAAAACGACGGCTCGGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGGAAGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAATGCACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCCCCGGCTGAAATCAAGGACCTGGCGGAACTGAAGGCGGCACTGAAAGGTCTCGAGCACCACCACCACCACCAC

SEQ ID NO：131

S6D4-S5D1_ aa: heterodimeric fusion proteins of OspA serotype 6 with disulfide bond type 4 and OspA serotype 5 with disulfide bond type 1, LN1 linker sequence

FNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALK

SEQ ID NO：132

Lip-S6D4-S5D1_ nt: coding sequences for intermediate and final heterodimer fusion proteins of OspA serotype 6 with disulfide bond type 4 and OspA serotype 5 with disulfide bond type 1, escherichia coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 linker sequence

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGGCAAAGGTGAAACGAGTGAAAAAACGATTGTTCGCGCCTGTGGCACCCGCCTGGAATACACGGATATCAAGTCGGATGGTTCGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTAGGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGACAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAATGCACGATCACCGTTCAACGCTATGATAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAATCTCAGAAAAAACCATCGTCCGCGCTAACGGCACCCGCCTGGAATACACCGACATCAAATCAGACAAGACCGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCTGCGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGATTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAAGATACGATCACCGTGCAAAACTATGACAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAA

SEQ ID NO：133

Lip-S6D4-S5D1_ His _ aa: heterodimeric fusion proteins of OspA serotype 6 with disulfide bond type 4 and OspA serotype 5 with disulfide bond type 1, N-terminal CSS for lipid addition, LN1 linker sequence, N-terminal lipidation, C-terminal His tag (GLEHHHHHH)

LipCSSFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALKGLEHHHHHH

SEQ ID NO：134

Lip-S6D4-S5D1_ His _ nt: coding sequences of heterodimeric fusion proteins of OspA serotype 6 having disulfide bond type 4 and OspA serotype 5 having disulfide bond type 1, Escherichia coli lpp lipidation signal, N-terminal CSS for addition of lipid, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGGCAAAGGTGAAACGAGTGAAAAAACGATTGTTCGCGCCTGTGGCACCCGCCTGGAATACACGGATATCAAGTCGGATGGTTCGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGACAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAATGCACGATCACCGTTCAACGCTATGATAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAATCTCAGAAAAAACCATCGTCCGCGCTAACGGCACCCGCCTGGAATACACCGACATCAAATCAGACAAGACCGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCTGCGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGATTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAAGATACGATCACCGTGCAAAACTATGACAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTCTCGAGCACCACCACCACCACCAC

SEQ ID NO：135

S6D1-S5D4_ aa: heterodimeric fusion proteins of OspA serotype 6 with disulfide bond type 1 and OspA serotype 5 with disulfide bond type 4, LN1 linker sequence

FNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALK

SEQ ID NO：136

Lip-S6D1-S5D4_ nt: coding sequences for intermediate and final heterodimer fusion proteins of OspA serotype 6 with disulfide bond type 1 and OspA serotype 5 with disulfide bond type 4, escherichia coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 linker sequence

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGGCAAAGGTGAAACGAGCGAAAAGACCATCGTGCGTGCGAACGGTACCCGCCTGGAATATACGGACATTAAATCGGACGGCAGCGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGATAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAAGATACGATCACCGTTCAACGCTATGACAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAATCAGTGAAAAAACCATTGTGCGTGCGTGTGGCACCCGTCTGGAATATACCGACATCAAGAGCGATAAAACGGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCGAAGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGACTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAATGCACGATCACCGTGCAAAACTATGATAGCGCAGGT

ACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAA

SEQ ID NO：137

Lip-S6D1-S5D4_ His _ aa: heterodimeric fusion proteins of OspA serotype 6 with disulfide bond type 1 and OspA serotype 5 with disulfide bond type 4, N-terminal CSS for lipid addition, LN1 linker sequence, N-terminal lipidation, C-terminal His tag (GLEHHHHHH)

LipCSSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALKGLEHHHHHH

SEQ ID NO：138

Lip-S6D1-S5D4_ His _ nt: heterodimers of OspA serotype 6 having disulfide bond type 1 and OspA serotype 5 having disulfide bond type 4The coding sequence of the fusion protein is disclosed,escherichia coli lpp lipidation signalN-terminal CSS for lipid addition, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)

ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGGCAAAGGTGAAACGAGCGAAAAGACCATCGTGCGTGCGAACGGTACCCGCCTGGAATATACGGACATTAAATCGGACGGCAGCGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGATAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAAGATACGATCACCGTTCAACGCTATGACAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAATCAGTGAAAAAACCATTGTGCGTGCGTGTGGCACCCGTCTGGAATATACCGACATCAAGAGCGATAAAACGGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCGAAGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGACTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAATGCACGATCACCGTGCAAAACTATGATAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTCTCGAGCACCACCACCACCACCAC

SEQ ID NO：140

Lip-S2D 0-His: amino acids at positions 131-273 of the wild-type sequence of Borrelia afzelii strain K78OspA serotype 2, N-terminal CKQN for addition of lipids, C-terminal His-tag (GLEHHHHHH)

LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH

SEQ ID NO：141

Lip-S2D 1-His: aa131-273 of Borrelia afzelii strain K78OspA serotype 2 with disulfide bond type 1(aa 182 and 269), N-terminal CKQN for lipid addition, C-terminal His-tag (GLEHHHHHH)

LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALKGLEHHHHHH

SEQ ID NO：142

Lip-S2D 2-His: aa131-273 of Borrelia afzelii strain K78OspA serotype 2 with disulfide bond type 2(aa 182 and 272), N-terminal CKQN for lipid addition, C-terminal His tag (GLEHHHHHH)

LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNACKGLEHHHHHH

SEQ ID NO：143

Lip-S2D 3-His: aa131-273 of Borrelia aryabhattai strain K78OspA serotype 2 with disulfide bond type 3(aa 244 and 259), N-terminal CKQN for lipid addition, C-terminal His tag (GLEHHHHHH)

LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTICVQKYDSAGTNLEGTCVEIKTLDELKNALKGLEHHHHHH

SEQ ID NO：144

Lip-S2D 4-His: aa131-273 of Borrelia afzelii strain K78OspA serotype 2 with disulfide bond type 4(aa 141 and 241), N-terminal CKQN for lipid addition, C-terminal His-tag (GLEHHHHHH)

LipCKQNELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH

SEQ ID NO：145

Lip-S2D 5-His: aa131-273 of Borrelia aryabhattai strain K78OspA serotype 2 with disulfide bond type 5(aa 165 and 265), N-terminal CKQN for lipid addition, C-terminal His tag (GLEHHHHHH)

LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNCTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTCDELKNALKGLEHHHHHH

SEQ ID NO：146

Lip-S2D 6-His: aa131-273 of Borrelia aryabhattai strain K78OspA serotype 2 with disulfide bond type 6(aa 185 and 272), N-terminal CKQN for lipid addition, C-terminal His tag (GLEHHHHHH)

LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTCTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNACKGLEHHHHHH

SEQ ID NO：147

Lip-S2D 7-His: aa131-273 of Borrelia aryabhattai strain K78OspA serotype 2 with disulfide bond type 7(aa 199 and 223), N-terminal CKQN for lipid addition, C-terminal His-tag (GLEHHHHHH)

LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTCALNDTNTTQATKKTGAWDSKTSTCTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH

SEQ ID NO：148

Lip-S2D 8-His: aa131-273 of Borrelia afzelii strain K78OspA serotype 2 with disulfide bond type 8(aa 243 and 262), N-terminal CKQN for lipid addition, C-terminal His tag (GLEHHHHHH)

LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTCTVQKYDSAGTNLEGTAVECKTLDELKNALKGLEHHHHHH

SEQ ID NO：149

Lip-S2D 9-His: aa131-273 of Borrelia aryabhattai strain K78OspA serotype 2 with disulfide bond type 9(aa 184 and 204), N-terminal CKQN for lipid addition, C-terminal His tag (GLEHHHHHH)

LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGCVTLSKEIAKSGEVTVALNDCNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH

SEQ ID NO：150

Lip-S2D 10-His: aa131-273 of Borrelia aryabhattai strain K78OspA serotype 2 with disulfide bond type 10(aa 201 and 214), N-terminal CKQN for lipid addition, C-terminal His tag (GLEHHHHHH)

LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVACNDTNTTQATKKTCAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH

SEQ ID NO：151

Lip-S2D 11-His: aa131-273 of Borrelia aryabhattai strain K78OspA serotype 2 with disulfide bond type 11(aa 246 and 259), N-terminal CKQN for lipid addition, C-terminal His tag (GLEHHHHHH)

LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVCKYDSAGTNLEGTCVEIKTLDELKNALKGLEHHHHHH

SEQ ID NO：152

Lip-S2D 12-His: aa131-273 of Borrelia aryabhattai strain K78OspA serotype 2 with disulfide bond type 12(aa 167 and 178), N-terminal CKQN for lipid addition, C-terminal His tag (GLEHHHHHH)

LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTCEGKVANDKVTCEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH

SEQ ID NO：153

Lip-S2D 0: amino acids at positions 131-273 of the wild-type sequence of Borrelia afzelii strain K78OspA serotype 2, N-terminal CKQN for lipid addition

LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK

SEQ ID NO：154

Lip-S2D 1: aa131-273 of Borrelia aryabhattai strain K78OspA serotype 2 with disulfide bond type 1(aa 182 and 269), N-terminal CKQN for lipid addition

LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALK

SEQ ID NO：155

Lip-S2D 2: aa131-273 of Borrelia aryabhattai strain K78OspA serotype 2 with disulfide bond type 2(aa 182 and 272), N-terminal CKQN for lipid addition

LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNACK

SEQ ID NO：156

Lip-S2D 3: aa131-273 of serotype 2 of Borrelia aryabhattai strain K78OspA with disulfide bond types 3(aa 244 and 259), N-terminal CKQN for lipid addition

LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTICVQKYDSAGTNLEGTCVEIKTLDELKNALK

SEQ ID NO：157

Lip-S2D 4: aa131-273 of Borrelia aryabhattai strain K78OspA serotype 2 with disulfide bond types 4(aa 141 and 241), N-terminal CKQN for lipid addition

LipCKQNELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALK

SEQ ID NO：158

Lip-S2D 5: aa131-273 of Borrelia aryabhattai strain K78OspA serotype 2 with disulfide bond type 5(aa 165 and 265), N-terminal CKQN for lipid addition

LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNCTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTCDELKNALK

SEQ ID NO：159

Lip-S2D 6: aa131-273 of Borrelia aryabhattai strain K78OspA serotype 2 with disulfide bond type 6(aa 185 and 272), N-terminal CKQN for lipid addition

LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTCTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNACK

SEQ ID NO：160

Lip-S2D 7: aa131-273 of Borrelia aryabhattai strain K78OspA serotype 2 with disulfide bond type 7(aa 199 and 223), N-terminal CKQN for lipid addition

LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTCALNDTNTTQATKKTGAWDSKTSTCTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK

SEQ ID NO：161

Lip-S2D 8: aa131-273 of Borrelia aryabhattai strain K78OspA serotype 2 with disulfide bond type 8(aa 243 and 262), N-terminal CKQN for lipid addition

LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLWVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTCTVQKYDSAGTNLEGTAVECKTLDELKNALK

SEQ ID NO：162

Lip-S2D 9: aa131-273 of Borrelia aryabhattai strain K78OspA serotype 2 with disulfide bond type 9(aa 184 and 204), N-terminal CKQN for lipid addition

LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGCVTLSKEIAKSGEVTVALNDCNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK

SEQ ID NO：163

Lip-S2D 10: aa131-273 of Borrelia aryabhattai strain K78OspA serotype 2 with disulfide bond type 10(aa 201 and 214), N-terminal CKQN for lipid addition

LipCKQNELSAKTMTRENGTKLEYTWMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVACNDTNTTQATKKTCAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK

SEQ ID NO：164

Lip-S2D 11: aa131-273 of Borrelia aryabhattai strain K78OspA serotype 2 with disulfide bond type 11(aa 246 and 259), N-terminal CKQN for lipid addition

LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVCKYDSAGTNLEGTCVEIKTLDELKNALK

SEQ ID NO：165

Lip-S2D 12: aa131-273 of Borrelia aryabhattai strain K78OspA serotype 2 with disulfide bond type 12(aa 167 and 178), N-terminal CKQN for lipid addition

LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTCEGKVANDKVTCEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK

SEQ ID NO：166

S2D 0: amino acids at positions 131-273 of the wild-type sequence of the Borrelia afzelii strain K78OspA serotype 2

SEQ ID NO：167

S2D 1: aa131-273 of Borrelia aryabhattai strain K78OspA serotype 2 with disulfide bond type 1(aa 182 and 269)

ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALK

SEQ ID NO：168

S2D 2: aa131-273 of Borrelia aryabhattai strain K78OspA serotype 2 with disulfide bond type 2(aa 182 and 272)

ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNACK

SEQ ID NO：169

S2D 3: aa131-273 of Borrelia aryabhattai strain K78OspA serotype 2 with disulfide bond type 3(aa 244 and 259)

ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTICVQKYDSAGTNLEGTCVEIKTLDELKNALK

SEQ ID NO：170

S2D 4: aa131-273 of Borrelia aryabhattai strain K78OspA serotype 2 with disulfide bond types 4(aa 141 and 241)

ELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALK

SEQ ID NO：171

S2D 5: aa131-273 of Borrelia aryabhattai strain K78OspA serotype 2 with disulfide bond type 5(aa 165 and 265)

ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNCTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTCDELKNALK

SEQ ID NO：172

S2D 6: aa131-273 of Borrelia aryabhattai strain K78OspA serotype 2 with disulfide bond type 6(aa 185 and 272)

ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTCTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNACK

SEQ ID NO：173

S2D 7: aa131-273 of Borrelia aryabhattai strain K78OspA serotype 2 with disulfide bond type 7(aa 199 and 223)

ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTCALNDTNTTQATKKTGAWDSKTSTCTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK

SEQ ID NO：174

S2D 8: aa131-273 of Borrelia aryabhattai strain K78OspA serotype 2 with disulfide bond type 8(aa 243 and 262)

ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTCTVQKYDSAGTNLEGTAVECKTLDELKNALK

SEQ ID NO：175

S2D 9: aa131-273 of Borrelia aryabhattai strain K78OspA serotype 2 with disulfide bond type 9(aa 184 and 204)

ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGCVTLSKEIAKSGEVTVALNDCNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK

SEQ ID NO：176

S2D 10: aa131-273 of serotype 2 of the Borrelia aryabhattai strain K78OspA with disulfide bond types 10(aa 201 and 214)

ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVACNDTNTTQATKKTCAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK

SEQ ID NO：177

S2D 11: aa131-273 of serotype 2 of the Borrelia aryabhattai strain K78OspA with disulfide bond types 11(aa 246 and 259)

ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVCKYDSAGTNLEGTCVEIKTLDELKNALK

SEQ ID NO：178

S2D 12: aa131-273 of serotype 2 of the Borrelia aryabhattai strain K78OspA with disulfide bond types 12(aa 167 and 178)

ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTCEGKVANDKVTCEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK

SEQ ID NO：179

Borrelia burgdorferi sensu stricta (strain B31, serotype 1), OspA _ aa 126-

FNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIKNALK

SEQ ID NO：180

Borrelia garinii (strain PBr, serotype 3), OspA _ aa 126-

FNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELKAALK

SEQ ID NO：181

Borrelia bavaria (strain PBi, serotype 4), OspA _ aa 126-

FNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELKNALK

SEQ ID NO：182

Borrelia garinii (strain PHei, serotype 5), OspA _ aa 126-

FNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLIKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELKNALK

SEQ ID NO：183

Borrelia garinii (strain DK29, serotype 6), OspA _ aa 126-

FNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELKNALK

SEQ ID NO：184

LN1 peptide linker constructed from two separate loop regions from the OspAN-terminal half of Borrelia burgdorferi strain B31 (aa 65-74 and aa 42-53, amino acid exchange at position 53: D53S)

GTSDKNNGSGSKEKNKDGKYS

SEQ ID NO：185

Lip-S1D4-S2D4_ aa: heterodimeric fusion proteins of OspA serotype 1 and OspA serotype 2, both with disulfide bond type 4, N-terminal CSS for lipid addition, LN1 linker sequence, aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND

LipCSSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRECGTKLEYTEMKSDGT

GKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALK

SEQ ID NO：186

Lip-S1D1-S2D1_ aa: heterodimeric fusion proteins of OspA serotype 1 and OspA serotype 2 with disulfide bond type 1, N-terminal CSS for lipid addition, LN1 linker sequence, aa 164-like 174 of OspA serotype 1 substituted with non-hLFA-1-like sequence NFTLEGKVAND, N-terminal lipidation

LipCSSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALK

SEQ ID NO：187

Lip-S3D4-S4D4_ aa: heterodimeric fusion proteins of OspA serotype 3 and OspA serotype 4 with disulfide bond type 4, N-terminal CSS for lipid addition, LN1 linker sequence, N-terminal lipidation

LipCSSFNEKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALK

SEQ ID NO：188

Lip-S3D1-S4D1_ aa: heterodimeric fusion proteins of OspA serotype 3 and OspA serotype 4, both having disulfide bond type 1, N-terminal CSS for lipid addition, LN1 linker sequence, N-terminal lipidation

LipCSSFNEKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALK

SEQ ID NO：189

Lip-S5D4-S6D4_ aa: heterodimeric fusion proteins of OspA serotype 5 and OspA serotype 6, both with disulfide bond type 4, N-terminal CSS for lipid addition, LN1 linker sequence, N-terminal lipidation

LipCSSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALK

SEQ ID NO：190

Lip-S5D1-S6D1_ aa: heterodimeric fusion protein of OspA serotype 6, both with disulfide bond type 1, N-terminal CSS for lipid addition, LN1 linker sequence, N-terminal lipidation

LipCSSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALK

SEQ ID NO：191

Lip-S2D4-S1D4_ aa: heterodimeric fusion proteins of OspA serotype 2 and OspA serotype 1, both with disulfide bond type 4, N-terminal CSS for lipid addition, LN1 linker sequence, aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND, N-terminal lipidation

LipCSSFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALK

SEQ ID NO：192

Lip-S2D1-S1D1_ aa: heterodimeric fusion proteins of OspA serotype 2 and OspA serotype 1, both with disulfide bond type 1, N-terminal CSS for lipid addition, LN1 linker sequence, aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND, N-terminal lipidation

LipCSSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALK

SEQ ID NO：193

Lip-S4D4-S3D4_ aa: heterodimeric fusion proteins of OspA serotype 4 and OspA serotype 3, both having disulfide bond type 4, N-terminal CSS for lipid addition, LN1 linker sequence, N-terminal lipidation

LipCSSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALK

SEQ ID NO：194

Lip-S4D1-S3D1_ aa: heterodimeric fusion proteins of OspA serotype 4 and OspA serotype 3, both having disulfide bond type 1, N-terminal CSS for lipid addition, LN1 linker sequence, N-terminal lipidation

LipCSSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALK

SEQ ID NO：195

Lip-S6D4-S5D4_ aa: heterodimeric fusion proteins of OspA serotype 6 and OspA serotype 5, both with disulfide bond type 4, N-terminal CSS for lipid addition, LN1 linker sequence, N-terminal lipidation

LipCSSFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALK

SEQ ID NO：196

Lip-S6D1-S5D1_ aa: heterodimeric fusion proteins of OspA serotype 6 and OspA serotype 5, both having disulfide bond type 1, N-terminal CSS for lipid addition, LN1 linker sequence, N-terminal lipidation

LipCSSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALK

SEQ ID NO：197

Lip-S1D4-S2D1_ aa: heterodimeric fusion proteins of OspA serotype 1 with disulfide bond type 4 and OspA serotype 2 with disulfide bond type 1, N-terminal CSS for lipid addition, LN1 linker sequence, aa 164-one 174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND, N-terminal lipidation

LipCSSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALK

SEQ ID NO：198

Lip-S1D1-S2D 4_ aa: heterodimeric fusion proteins of OspA serotype 1 with disulfide bond type 1 and OspA serotype 2 with disulfide bond type 4, N-terminal CSS for lipid addition, LN1 linker sequence, aa 164-one 174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND, N-terminal lipidation

LipCSSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALK

SEQ ID NO：199

Lip-S3D4-S4D1_ aa: heterodimeric fusion proteins of OspA serotype 3 with disulfide bond type 4 and OspA serotype 4 with disulfide bond type 1, N-terminal CSS for lipid addition, LN1 linker sequence, N-terminal lipidation

LipCSSFNEKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALK

SEQ ID NO：200

Lip-S3D1-S4D4_ aa: heterodimeric fusion proteins of OspA serotype 3 with disulfide bond type 1 and OspA serotype 4 with disulfide bond type 1, N-terminal CSS for lipid addition, LN1 linker sequence, N-terminal lipidation

LipCSSFNEKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKVWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALK

SEQ ID NO：201

Lip-S5D4-S6D1_ aa: heterodimeric fusion proteins of OspA serotype 5 with disulfide bond type 4 and OspA serotype 6 with disulfide bond type 1, N-terminal CSS for lipid addition, LN1 linker sequence, N-terminal lipidation

LipCSSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALK

SEQ ID NO：202

Lip-S5D1-S6D4_ aa: heterodimeric fusion proteins of OspA serotype 5 with disulfide bond type 1 and OspA serotype 6 with disulfide bond type 4, N-terminal CSS for lipid addition, LN1 linker sequence, N-terminal lipidation

LipCSSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLS

KNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALK

SEQ ID NO：203

Lip-S2D4-S1D1_ aa: heterodimeric fusion proteins of OspA serotype 2 with disulfide bond type 4 and OspA serotype 1 with disulfide bond type 1, N-terminal CSS for lipid addition, LN1 linker sequence, aa 164-one 174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND, N-terminal lipidation

LipCSSFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAVVDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRADGTRLEYTGIKSDGS

GKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALK

SEQ ID NO：204

Lip-S2D1-S1D4_ aa: heterodimeric fusion proteins of OspA serotype 2 with disulfide bond type 1 and OspA serotype 1 with disulfide bond type 4, N-terminal CSS for lipid addition, LN1 linker sequence, aa 164-one 174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND, N-terminal lipidation

LipCSSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALK

SEQ ID NO：205

Lip-S4D4-S3D1_ aa: heterodimeric fusion proteins of OspA serotype 4 with disulfide bond type 4 and OspA serotype 3 with disulfide bond type 1, N-terminal CSS for lipid addition, LN1 linker sequence, N-terminal lipidation

LipCSSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALK

SEQ ID NO：206

Lip-S4D1-S3D4_ aa: coding sequences of intermediate and final heterodimer fusion proteins of OspA serotype 4 with disulfide bond type 1 and OspA serotype 3 with disulfide bond type 4, N-terminal CSS for lipid addition, LN1 linker sequence, N-terminal lipidation

LipCSSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALK

SEQ ID NO：207

Lip-S6D4-S5D1_ aa: heterodimeric fusion proteins of OspA serotype 6 with disulfide bond type 4 and OspA serotype 5 with disulfide bond type 1, N-terminal CSS for lipid addition, LN1 linker sequence, N-terminal lipidation

LipCSSFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALK

SEQ ID NO：208

Lip-S6D1-S5D4_ aa: heterodimeric fusion proteins of OspA serotype 6 with disulfide bond type 1 and OspA serotype 5 with disulfide bond type 4, N-terminal CSS for lipid addition, LN1 linker sequence, N-terminal lipidation

LipCSSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALK

SEQ ID NO：209

Borrelia afzelii (strain K78; OspA serotype 2) aa 17-273, deplated lipidation signal sequence (aa 1-16: MKKYLLGIGLILALIA), C-terminal His tag (GLEHHHHHH)

CKQNVSSLDEKNSASVDLPGEMKVLVSKEKDKDGKYSLKATVDKIELKGTSDKDNGSGVLEGTKDDKSKAKLTIADDLSKTTFELFKEDGKTLVSRKVSSKDKTSTDEMFNEKGELSAKTMTRENGTKLEYTEMK

SDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH

SEQ ID NO：210

Borrelia burgdorferi (OspA serotype 1, strain ZS7) aa 17-273, deplated lipidated signal sequence (aa 1-16: MKKYLLGIGLILALIA), C-terminal His tag (LEHHHHHHHH)

CSSFKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLELKGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKGYVLEGTLTAEKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIKNALKLEHHHHHH

SEQ ID NO：211

Cysteine-containing peptides from OspA

CKQN

SEQ ID NO：212

Chimeric OspA serotype 1/serotype 2, N-terminal lipidation

LipCAQKGAESIGSVSVDLPGEMKVLVSKEKDKNGKYDLIATVDKLELKGTSDKNNGSGVLEGVKTNKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITMADGTRLEYTGIKSDGTGKAKYVLKNFTLEGKVANDKTTLEVKEGTVTLSMNISKSGEVSVELNDTDSSAATKKTAVWNSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKLE

SEQ ID NO：213

Amino acids 126-274 of Borrelia galcherii strain T25 OspA serotype 7

FNDKGKLSEKVVTRANGTRLEYTEIQNDGSGKAKEVLKSLTLEGTLTADGETKLTVEAGTVTLSKNISESGEITVELKDTETTPADKKSGTWDSKTSTLTISKNSQKTKQLVFTKENTITVQKYNTAGTKLEGSPAEIKDLEALKAALK

SEQ ID NO：214

Forward oligonucleotide primer for RecA gene of borrelia

CATGCTCTTGATCCTGTTTA

SEQ ID NO：215

Histidine tag

GLEHHHHHH

SEQ ID NO：216

Reverse oligonucleotide primer for RecA gene of borrelia

CCCATTTCTCCATCTATCTC

The entire contents of all documents cited throughout this application, including references, issued patents, published patent applications, and co-pending patent applications, are hereby expressly incorporated by reference.

Claims

1. Polypeptide consisting of the lipidated amino acid sequence SEQ ID NO: 186.

2. A nucleic acid encoding the polypeptide of claim 1.

3. A vector comprising the nucleic acid molecule of claim 2.

4. A host cell comprising the nucleic acid of claim 2 or the vector of claim 3.

5. The host cell of claim 4, wherein the host cell is E.

6. A method for producing a cell expressing a polypeptide of claim 1, comprising transforming or transfecting a suitable host cell with the vector of claim 3.

7. A method for producing the polypeptide of claim 1, comprising expressing the nucleic acid of claim 2.

8. A method for producing the polypeptide according to claim 1, characterized by the steps of:

a) introducing a vector encoding the polypeptide into a host cell;

c) homogenizing the host cell; and

d) subjecting the host cell homogenate to a purification step.

9. A pharmaceutical composition comprising a polypeptide according to claim 1 and/or a nucleic acid according to claim 2.

10. The pharmaceutical composition of claim 9, further comprising a polypeptide having the amino acid sequence of SEQ ID NO 190.

11. The pharmaceutical composition of claim 9 or 10, further comprising a pharmaceutically acceptable excipient.

12. The pharmaceutical composition of claim 11, wherein the pharmaceutically acceptable excipient comprises L-methionine.

13. The pharmaceutical composition according to claim 9, further comprising at least one additional antigen derived from a borrelia species responsible for lyme borreliosis.

14. The pharmaceutical composition according to claim 13, wherein the borrelia species is borrelia bavariensis (b.bavariensis) or borrelia garinii (b.garrinii).

15. The pharmaceutical composition of claim 9, further comprising at least one additional antigen, wherein the at least one additional antigen is from a tick-borne pathogen.

16. The pharmaceutical composition of claim 15, wherein the tick-borne pathogen is selected from the group comprising: borrelia helminthosporium, borrelia deltoides, borrelia darwinii, borrelia gondii, borrelia tirpresent, rickettsia rickettii, rickettsia australis, conorelbirk, rickettsia helveticus, rickettsia parwazekii, francisella tularensis, anaplastic, ehrlichia, chefierlike, burnather rickettsia and borrelia lonthidae, tick-borne encephalitis virus (TBEV), corolapothyrifos heat transfer virus (CTFV), crimean-congo hemorrhagic fever virus (CCHFV), cougher luer forest virus (kf), powassan virus, hartlan virus, Oldv Hemorrhagic Fever Virus (OHFV) and some species of babesia.

17. Pharmaceutical composition according to claim 9, characterized in that it further comprises an immunostimulating substance.

18. The pharmaceutical composition of claim 17, wherein the immunostimulatory substance is selected from the group consisting of: polycationic polymers, immunostimulatory Oligodeoxynucleotides (ODNs), peptides containing at least two LysLeuLys motifs, neuroactive compounds, aluminium hydroxide or aluminium phosphate, freund's complete or incomplete adjuvant or combinations thereof.

19. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition is a vaccine.

20. Use of a polypeptide according to claim 1 in the manufacture of a medicament for the treatment or prevention of borrelia infection.

21. The use of claim 20, wherein the medicament is a vaccine.

22. The use of claim 20, wherein the borrelia infection is a borrelia burgdorferi sensu stricta, or a borrelia avermitis infection.