Borrelia burgdorferi CSPZ-binding proteins and methods of use thereof

Abstract

Provided is an antibody or an antigen-binding fragment thereof capable of specifically binding to a Borrelia burgdorferi CspZ protein, and a method of administering said antibody or antigen-binding fragment thereof to a subject.

Description

BORRELIA BURGDORFERI CSPZ-BINDING PROTEINS AND METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority from U.S. Provisional Patent Application No.63 / 709,048, filed October 18, 2024, the entire contents of which are incorporated herein by reference. GOVERNMENT RIGHTS STATEMENT

[0002] This invention was made with Government support under grant number R21AI144891 awarded by the National Institutes of Health and grant number W81XWH-20- 1-0913 awarded by the Department of Defense. The Government has certain rights in the invention. SEQUENCE LISTING

[0003] The instant application contains an electronic sequence listing. The contents of the electronic sequence listing 0332099AWO_Sequence_Listing_XML.xml; Size: 35,009 bytes; and Date of Creation: October 17, 2025, is herein incorporated by reference in its entirety. BACKGROUND

[0004] Lyme disease (LD) is caused by Lyme borreliae spirochetes such as Borrelia burgdorferi sensu lato (also known as Borreliella burgdorferi sensu lato), transmitted by the bite of infected Ixodes ticks. LD is the most common vector-borne disease in the Northern hemisphere, with estimated 476,000 people diagnosed annually in the United States alone (1, 2). Among all spirochete species in the B. burgdorferi sensu lato species complex, B. burgdorferi sensu stricto (hereafter B. burgdorferi) and B. afzelii are the major causative species of LD in North America and Eurasia, respectively (2). Following transmission by an infected tick, Lyme borreliae spread from the bite site to various tissues, leading to severe systemic manifestations such as arthritis, carditis, and neuroborreliosis.

[0005] To survive in a host, Lyme borreliae need to evade multiple host immune responses, while establishing infection and throughout dissemination to distal tissues (8, 9). Part of the immune response in the bloodstream is complement system, which is composed of numerous serum proteins activated through three pathways that recombine to form differentprotein complexes to ultimately kill pathogens (10-12). Complement regulators are produced to inhibit this cascade to avoid damage to host cells (10, 13). For example, Factor H (FH) and FH-like protein 1 (FHL-1, a truncated form of FH) bind to the C3b in C3 and C5 convertases, leading to the degradation of C3b and inactivating all downstream processes (14). Lyme borreliae produce multiple outer surface proteins that recruit these complement regulators to its surface and promote the degradation of complement proteins upon binding, ultimately facilitating serum resistance and bloodstream survival of the spirochetes (15-21). These spirochete proteins include five distinct FH-binding proteins (collectively known as Complement Regulator Acquiring Surface Proteins (CRASPs)), including CspZ (CRASP-2) (22, 23). The cspZ gene is expressed only when spirochetes reside in vertebrate hosts but not in ticks (24), reflecting the induction of this gene in spirochetes under host environmental cues (e.g., blood and dialysis membrane chambers) (25-27). Additionally, when studied under blood treatment to overcome low expression during in vitro culturing, a cspZ-deficient B. burgdorferi mutant colonizes mouse tissues at reduced levels compared to the wild-type strain (27). These results suggest a role of CspZ to promote efficient spirochete dissemination.

[0006] Moreover, cspZ is highly conserved among Lyme borreliae strains (>80% sequence identity) and carried in all B. burgdorferi and B. afzelii strains isolated from human patients with systemic and more severe manifestations (e.g., arthritis and neuroborreliosis) (28-30). In addition, all LD patients in an anecdotal study develop antibodies that recognize CspZ (29). These observations raise the possibility of targeting CspZ as a human LD vaccine antigen. However, vaccination with wild-type CspZ does not protect mice from B. burgdorferi infection (29, 31-33), suggesting that anti-CspZ antibodies may be ineffective as treatments or prophylactics for LD, whereas such antibodies would provide useful for prevention and treatment of LD if available. CspZ having substitutions for tyrosine at positions 207 and 211 of CspZ of Borrelia burgdorferi (e.g., tyrosine-to-alanine substitutions at these positions of CspZ, referred to herein as CspZ-Y207A / Y211A or “CspZ-YA”) does not bind to FH, thereby exposing the epitopes in and around CspZ’s FH-binding site even in the presence of FH. CspZ-YA inoculation protects against the tickborne infection of multiple human-infectious Lyme borreliae strains and species and correlated such protectivity with the CspZ-induced antibodies that uniquely recognize the exposed epitopes surrounding the FH- binding site, as disclosed in US Patent Application Publication No.2019 / 0201516 A1, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Nevertheless, antibodies directed to the FH binding domain of CspZ are lacking.

[0007] The present disclosure is directed to overcoming these and other deficiencies in the art. SUMMARY

[0008] In one aspect, provided is an antibody or an antigen-binding fragment thereof capable of specifically binding to a Borrelia burgdorferi CspZ protein, including a heavy chain variable region including VHCDR1, VHCDR2, and VHCDR3, and a light chain variable region including VLCDR1, VLCDR2, and VLCDR3, wherein amino acid sequences of the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 include SEQ ID NOs: 1-6, respectively, or SEQ ID NOs: 7-12, respectively. Amino acid sequences of the VHCDR1, VHCDR2, and VHCDR3, VLCDR1, VLCDR2, and VLCDR3 may include SEQ ID NOs: 1-6, respectively. Amino acid sequences of the VHCDR1, VHCDR2, and VHCDR3, VLCDR1, VLCDR2, and VLCDR3 may include SEQ ID NOs: 7-12, respectively.

[0009] The antibody or antigen-binding fragment thereof may be selected from a Fab fragment, a (Fab′)2 fragment, a scFv fragment, and an IgG antibody. Binding of the antibody or antigen-binding fragment thereof to Borrelia burgdorferi or Borrelia afzelii CspZ protein may inhibit factor H binding to the Borrelia burgdorferi or Borrelia afzelii CspZ protein.

[0010] The antibody or antigen-binding fragment thereof may include an antibody. The antibody or antigen-binding fragment thereof may include heavy chain and light chain amino acid sequences independently having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 97% sequence identity, at least 99% sequence identity, or 100% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 14, respectively, or heavy chain and light amino acid sequences independently having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 97% sequence identity, at least 99% sequence identity, or 100% sequence identity with SEQ ID NO: 15 or SEQ ID NO: 16, respectively. The antibody or antigen-binding fragment thereof of may include heavy chain and light chain amino acid sequences independently having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 97% sequence identity, at least 99% sequence identity, or 100% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 14, respectively. The antibody or antigen-binding fragment thereof of may include heavy chain and light amino acid sequences independently having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 97% sequence identity, at least 99% sequence identity,or 100% sequence identity with SEQ ID NO: 15 or SEQ ID NO: 16, respectively. The antibody or antigen-binding fragment thereof may include monoclonal antibody 1193c or monoclonal antibody 1139c.

[0011] The antibody or antigen-binding fragment thereof may be bactericidal for Lyme-causing Borreliae bacteria. Administrating the antibody or antigen-binding fragment thereof to a subject infected with a Lyme-causing Borreliae bacteria may reduce seropositivity of the subject for the Lyme-causing Borreliae bacteria compared to a control subject infected with a Lyme-causing Borreliae bacteria but not administered the antibody or antigen-binding fragment thereof.

[0012] In an aspect, provided is a pharmaceutical composition, including any of the foregoing antibody or antigen-binding fragment thereof and a pharmaceutically acceptable excipient.

[0013] In an aspect, provided is a method of treatment, including administering any of the foregoing antibody or antigen-binding fragment thereof or pharmaceutical composition to a subject in need of such treatment. The subject may be a human.

[0014] Treatment may include administering the antibody or antigen-binding fragment thereof to the subject before the subject is or may be infected with a Lyme-causing Borreliae bacteria. Treatment may include administering the antibody or antigen-binding fragment thereof to the subject after the subject was or may have been infected with a Lyme- causing Borreliae bacteria. The Lyme-causing Borreliae bacteria may be Borrelia burgdorferi or Borrelia afzelii, The subject may have Lyme disease.

[0015] In an aspect, provided is a nucleic acid encoding any of the foregoing antibody or antigen-binding fragment thereof. Provided is a cell including the nucleic acid or a vector including the nucleic acid, or a cell transfected with the nucleic acid or vector.

[0016] In an aspect, provided is a cell expressing any of the foregoing antibody or antigen-binding fragment thereof of any one of claims 1 through 12.

[0017] In an aspect, provided is a hybridoma or engineered cell encoding any of the foregoing antibody or antigen-binding fragment thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] These and other features, aspects, and advantages of the present disclosure will become better understood when the following detailed description is read with reference to the accompanying drawings, wherein:

[0019] FIGs. lA-1E show IgGs that recognize both FH- and non-FH-binding sites of CspZ can be isolated separately from CspZ-YA-immunized rabbits and mice. (A) The integrity and purity assessment of CspZ-YA IgGs using SDS-PAGE. (B) A schematic diagram showing the purification process to retrieve CspZ-YA IgG (FH-binding sites) and CspZ-YA IgG (non-FH-binding sites). CspZ-conjugated resin was incubated with FH followed by loading CspZ-YA IgG (total). The bound and unbound fractions contain CspZ- YA IgG (non-FH-binding sites) and CspZ-YA IgG (FH-binding sites) IgG, respectively. After the bound fraction was eluted, the CspZ-YA IgG (non-FH-binding sites) was further purified using Protein A resin to remove contaminated FH. (C) A schematic diagram showing the experimental setup of panels D and E. (D and E) One microgram of CspZ was coated on ELISA plate wells, which were then incubated with human FH (500 nM), BSA (control), or PBS (control, data not shown), followed by the treatment of each of the CspZ-YA IgG samples. These IgGs include (D) CspZ-YA rabbit polyclonal IgGs that recognize the FH- binding site of CspZ (FH-binding sites) or non-FH-binding site (non-FH-binding sites) or (E) CspZ-YA mouse monoclonal IgGs (50 nM). The levels of bound CspZ-YA rabbit polyclonal and mouse monoclonal IgG were determined using HRP-conjugated goat antirabbit IgG (Sigma-Aldrich) and goat anti-mouse IgG (Sigma-Aldrich), respectively. Data were expressed as the percent IgG binding, derived by normalizing the levels of bound IgG from FH- or BSA-treated wells to that in PBS-treated wells. Data shown are the mean 6 standard error of the mean (SEM) of the percent IgG binding from three independent experiments. # indicates the statistical significance (P < 0.05, Mann-Whitney test) of different levels of percent IgG binding between indicated groups.

[0020] FIGs.2A-2C show mouse- and rabbit-derived IgGs that recognize the CspZ FH-binding site selectively block the human FH-binding activity of CspZ. (A) Experimental setup. (B and C) Each of the CspZ-YA IgGs was added into the CspZ-coated ELISA plate wells. These IgGs include (B) CspZ-YA rabbit polyclonal IgG (CspZ-YA IgG [total]), those IgGs that recognize the FH-binding site of CspZ (CspZ-YA IgG [FH-binding sites]), or non- FH-binding site (CspZ-YA IgG [non-FH-binding sites]), or (C) CspZ-YA mouse monoclonal IgGs at indicated concentrations (see Materials and Methods). The wells treated with irrelevant IgGs from rabbits (irr. rab. IgG) and mice (irr. ms. IgG) (50 nM) and PBS (data not shown) were included as a control in the same dose-dependent fashion. Each of those wells was then incubated with human FH, and the levels of bound FH were quantified using sheep anti-human FH and goat anti-sheep HRP IgG as primary and secondary antibodies, respectively. The work was performed on three independent experiments; within eachexperiment, samples were run in triplicate. Data are expressed as the percent human FH binding, derived by normalizing the levels of bound human FH from IgG-treated wells to those from PBS-treated wells. Data shown are the mean 6 SEM of the percent human FH binding from three replicates. Shown is one representative experiment. The concentrations of the IgG to inhibit 50% of human FH bound by CspZ (IC50) were obtained from curve-fitting and extrapolation of panels B and C and shown in Table 2.

[0021] FIGs.3A-3F show IgGs that recognize the CspZ FH-binding sites eliminate different Lyme borreliae species and strains more efficiently than those that bind to CspZ non-FH-binding sites. Each of the CspZ-YA IgG samples or irrelevant IgG samples from rabbits (irr. rab. IgG) or mice (irr. ms. IgG) or PBS (control, data not shown) was serially diluted as indicated and mixed with guinea pig complement and (A and B) B. burgdorferi strains B31-5A4 (Bb B31-5A4) or (C and D) 297 (Bb 297) or (E and F) B. afzelii strain VS461 (Ba VS461) (5 X 105cells mL-1). These IgGs include (A, C, E) CspZ-YA IgG (CspZ- YA IgG [total]), those IgGs that recognize the FH-binding site of CspZ (CspZ-YA IgG [FH- binding sites]), or those that recognize the non-FH-binding site (CspZ-YA IgG [non-FH- binding sites]), or (B, D, F) indicated CspZ-YA mouse monoclonal mouse IgGs at indicated concentrations. After incubated for 24 h, surviving spirochetes were quantified from three fields of view for each sample using dark-field microscopy. The work was performed on three independent experiments. The survival percentage was derived from the proportion of IgG-treated to PBS-treated spirochetes. Data shown are the mean 6 SEM of the survival percentage from three replicates. Shown is one representative experiment. The 50% borreliacidal activity of each IgG (BA50), representing the IgG concentrations that effectively killed 50% of spirochetes, was obtained and extrapolated from curve-fitting and shown in Table 3. Data shown are the mean 6 SEM of the borreliacidal titers from three experiments.

[0022] FIGs.4A-4E show CspZ-YA IgGs that recognize CspZ FH-binding sites selectively prevent B. burgdorferi B31-5A4 infection. (A) Timeframe of the IgG inoculation and B. burgdorferi infection. (B to E) C3H / HeN mice were inoculated with irr. IgG from rabbits (irr. rab. IgG) or mice (irr. ms. IgG) or CspZ-YA IgG samples (1 mg / kg, five mice per group). These CspZ-YA IgGs include total CspZ-YA IgG (total), those IgGs that recognize non-FH-binding site (non-FH-binding sites), or mouse monoclonal IgG no.1139 or 1193. At 24 h after IgG inoculation, these mice were fed on by I. scapularis nymphs carrying B. burgdorferi B31- 5A4 (Bb B31-5A4). An additional five mice inoculated with PBS but not fed on by ticks were included as the control (Uninfect.). The tissues were collected fromthose mice at 21dpf. Spirochete burdens at (B) the tick feeding site (bite site), (C) bladder, (D) ankles, and (E) heart were quantitatively measured at 21 dpf, shown as the number of spirochetes per 100 ng total DNA. Data shown are the geometric mean 6 geometric standard deviation of the spirochete burdens from five mice per group. Statistical significances (P < 0.05, Kruskal-Wallis test with the two-stage step-up method of Benjamini, Krieger, and Yekutieli) of differences in bacterial burdens relative to (*) uninfected mice or (#) between indicated groups of mice are presented.

[0023] FIG.5 shows passive inoculation of CspZ-YA IgGs does not eliminate B. burgdorferi B31-5A4 in ticks feeding on mice. C3H / HeN mice were inoculated with irrelevant IgG from rabbits (irr. rab. IgG) or mice (irr. ms. IgG), or CspZ-YA IgG samples (1mg / kg, five mice per group). These CspZ-YA IgGs include total CspZ-YA IgG (Total), those IgGs that recognize non-FH-binding site (non-FH-binding sites), or mouse monoclonal IgGs #1139 or 1193. At 24 hours after IgG inoculation, these mice were fed on by I. scapularis nymphs carrying B. burgdorferi B31-5A4 (Bb B31-5A4) and those nymphs feeding to repletion were collected. The nymphs prior to feeding were also included as control (Flat nymphs). Spirochete burdens at those nymphs were quantitatively measured and shown as the number of spirochetes per nymph (Bact. per tick). Data shown are the geometric mean ± geometric standard deviation of the bacterial burdens from eight flat nymphs or the nymphs feeding on mice inoculated with irrelevant rabbit IgG, total CspZ-YA IgG, or those IgG that recognize non-FH-binding site, or nine nymphs feeding on mice inoculated with irrelevant mouse IgG or the mouse monoclonal antibody #1139 or 1193.

[0024] FIG.6 shows CspZ-YA IgGs that recognize CspZ FH-binding sites selectively prevent seropositivity caused by B. burgdorferi B31-5A4 infection. C3H / HeN mice were inoculated with irr. IgG from rabbits (irr. rab. IgG) or mice (irr. ms. IgG), or CspZ-YA IgG samples (1 mg / kg, five mice per group). These CspZ-YA IgGs include total CspZ-YA IgG (Total), those IgGs that recognize non-FH-binding site (non-FH-binding sites), or mouse monoclonal IgG #1139 or 1193. At 24 hours after IgG inoculation, these mice were fed on by I. scapularis nymphs carrying B. burgdorferi B31-5A4 (Bb B31-5A4). An additional five mice inoculated with PBS but not fed on by ticks were included as the control (Uninfect.). The sera were collected from those mice at 21dpf. Seropositivity was determined by measuring the levels of IgG against C6 peptides in the sera of those mice were using ELISA. The mouse was considered as seropositive if that mouse had IgG levels against C6 peptides greater than the threshold, the mean plus three-fold standard deviation of the IgG levels against C6 peptides from the PBS-inoculated, uninfected mice (red dotted line). The numberof mice in each group with the anti-C6 IgG levels greater than the threshold (seropositive) is shown in Table 4. Data shown are the geometric mean ± geometric 8 standard deviation of the titers of anti-C6 IgG. Statistical significances (p < 0.05, Kruskal-Wallis test with the two- stage step-up method of Benjamini, Krieger, and Yekutieli) of differences in IgG titers relative to (*) uninfected mice or (#) between indicated groups of mice are presented.

[0025] FIGs.7A-7B show purity assessment for the purified his-tagged CspZ-YA and CspZ. Two to six micrograms of CspZ-YA or CspZ were loaded onto 14% or 4-20% tris- glycine SDS PAGE gels. The purity of each of these proteins was analyzed by densitometry and shown at the bottom panels.

[0026] FIGs.8A-8E show the chimeric monoclonal antibodies 1139c and 1193c efficiently recognize CspZ-YA, prevent human FH-binding, and promote lysis and opsonophagocytosis of B. burgdorferi. (A and B) The chimeric monoclonal antibody (A) #1139c or (B) #1193c was flowed over the chip surface, conjugated with indicated CspZ-YA. Binding was measured in response units (R.U.) by surface plasmon resonance. Shown is the mean ± standard deviation of the Kon, Koff, and KD values extrapolated from three experiments. One represented experiment is shown in this panel. (C) The monoclonal antibody #1139c or #1193c, or irrelevant human IgG (control, irr. hIgG) at indicated concentrations or PBS (control, data not shown) was added into the CspZ-coated ELISA plate wells. Each of those wells was then incubated with human FH, and the levels of bound FH were quantified using sheep anti-human FH and goat anti-sheep HRP IgG as primary and secondary antibodies, respectively. The work was performed on three independent experiments; within each experiment, samples were run in triplicate. Data are expressed as the percent human FH binding, derived by normalizing the levels of bound human FH from IgG-treated wells to that from PBS-treated wells. Data shown are the mean ± SEM of the percent human FH binding from three replicates. Shown is one representative experiment. The concentrations of the IgG to inhibit 50% of human FH bound by CspZ (IC50) was obtained from curve-fitting and shown in the inlet figure. The IC50 values are shown as the mean ± SD of from three experiments. (D) The monoclonal antibody #1139c or #1193c, or irrelevant human IgG (control, irr. hIgG) or PBS (control, data not shown) were serially diluted as indicated, and mixed with guinea pig complement and B. burgdorferi strains B31- A3 (5 × 105cells ml-1). After incubated for 24 hours, surviving spirochetes were quantified from three fields of view for each sample using dark-field microscopy. The work was performed on three independent experiments. The survival percentage was derived from the proportion of IgG-treated to PBS-treated spirochetes. Shown is one representativeexperiment, and in that experiment, the data points are the mean ± SEM of the survival percentage from three replicates. The 50% borreliacidal activity of each IgGs (BA50), representing the IgG concentrations that effectively killed 50% of spirochetes, was obtained and extrapolated from curve-fitting and shown in the inlet figure. The BA50 values are shown as the mean ± SD of from three experiments.

[0027] FIGs 9A-9G show the chimeric monoclonal antibodies 1139c and 1193c prevent seroconversion and tissue colonization caused by B. burgdorferi B31-A3 infection. (A) Timeframe of the IgG inoculation and B. burgdorferi infection. (B to G) Five C3H / HeN mice were inoculated with the monoclonal antibody #1139c or #1193c, or irrelevant human IgG (control, irr. hIgG) at the dose of 1 mg / kg. At 24 hours after IgG inoculation, these mice were fed on by I. scapularis nymphs carrying B. burgdorferi B31-A3 (Bb B31-A3). An additional five mice inoculated with PBS but not fed on by ticks were included as the control (Uninfect.). The tissues were collected from those mice at 4 days post nymph feeding. Spirochete burdens at (B) the tick feeding site (“Bite Site”), (C) bladder, (D) heart, and (E) knees were quantitatively measured at 21 dpf, shown as the number of spirochetes per 100ng total DNA. Data shown are the geometric mean ± geometric standard deviation of the spirochete burdens from five mice per group. Statistical significances (p < 0.05, Kruskal- Wallis test with the two-stage step-up method of Benjamini, Krieger, and Yekutieli) of differences in bacterial burdens relative to (*) uninfected mice are presented.

[0028] FIGs.10A-10C illustrate the structure and sequences of antibodies disclosed herein.10A shows structures of mouse, human, and chimeric mAbs.10B shows amino acid sequences of heavy and light chains of mAbs 1139c (“c” for “chimeric”) and 1193c (shown in the format of “Leader Sequence-VH / VL-hIgG1CH / hlgκCL,” where, optionally, a leader sequence may be present for cellular expression and cleaved during intracellular trafficking prior to formation of a mature antibody, which mature antibody may exclude the leader sequence, each CHsequence is a corresponding sequence from a human IgG1 CH, and each CL sequence is a corresponding sequence from a human Igκ CL.10C and 10D show amino acid sequences of regions of the heavy and light chains of mAbs 1139c and 1193c. Heavy and light chains of mouse mAbs 1139 and 1193 include the VRs and CDRs of chimeric mAbs 1139c and 1193c, respectively, but CHand CLregions having sequences corresponding to a mouse IgG1 CH and Igκ CL, respectively.

[0029] FIG.11 shows Table 4, seropositivity of mice passively immunized with anti- CspZ-YA IgGs followed by the infection of B. burgdorferi.DETAILED DESCRIPTION

[0030] This disclosure relates to an antibody antigen-binding fragment thereof that is capable of binding CspZ of an LD-causing spirochete. The antibody or antigen-binding fragment thereof may be an antibody, such as an IgG antibody or other antibody (e.g., an IgM, IgA, IgD, or IgE antibody), or fragment thereof such as a Fab fragment, a (Fab′)2 fragment, a scFv fragment, or other fragment. The antibody or antigen-binding fragment thereof has complementarity determining regions (CDR) of a variable region (VR), which include variable heavy chain CDR (VHCDR) and variable light chain CDR (VLCDR). The antibody or antigen-binding fragment thereof binds the FH-binding site of CspZ, such that it does not bind CspZ when FH is bound to CspZ’s FH-binding site, it competes with FH for binding to CspZ and inhibits FH binding to CspZ when the antibody or antigen-binding fragment thereof is bound to CspZ. Conversely, in the presence of an excess concentration of FH relative to the antibody or antigen-binding fragment thereof, which competes with the antibody or antigen-binding fragment thereof for binding to CspZ, Administering the antibody or antigen-binding fragment thereof to a subject may prevent development of LD or symptoms thereof in a subject exposed to a Csp-expressing LD-causing spirochete, and may reduce, ameliorate, eliminate, or otherwise treat such symptoms in a subject who was or is believed to have been exposed to a CspZ-expressing LD-causing spirochete. Compositions such as pharmaceutical compositions including any of the foregoing antibody or antigen- binding fragment thereof are also provide herein.

[0031] Administering an immunogen to subjects wherein the immunogen is a modified CspZ peptide with tyrosine-to-alanine substitutions in the FH-binding domain of the CspZ immunogen, preventing its binding to FH, prevents spirochete colonization and LD- associated manifestations (32, 33). See US Patent Application Publication Number 2019 / 0201516 A1, the entire content of which are hereby incorporated by reference. Correspondingly, disclosed herein are antibodies and antigen-binding fragments thereof comprising antibodies that eradicate colonization of CspZ-expressing LD-causing borreliae spirochetes and may be used to treat LD and symptoms thereof.

[0032] Antibodies and antigen-binding fragments thereof are structured so as to specifically bind to particular antigens. Regions of the peptides responsible for this antigen- binding ability are referred to as “complementarity determining regions” (CDR). CDR are a hypervariable region found both in the light chain and the heavy chain variable domains. Themore highly conserved portions of variable domains are called the framework regions (FRs). Amino acid positions of a hypervariable region of an antibody can vary, depending on the context and the various definitions. Variable domains of native heavy and light chains each comprise four framework regions connected by three CDRs. The CDRs in each chain are held together in close proximity by the FR regions in the order FR1-CDR1-FR2-CDR2-FR3- CDR3-FR4 and, with the CDRs from the other antibody chains, contribute to the formation of the target binding site of antibodies (see Kabat et al, Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md.1987; incorporated herein by reference). As used herein, numbering of immunoglobulin amino acid residues is done according to the immunoglobulin amino acid residue numbering system of Kabat et al, unless otherwise indicated. CDRs and FRs may be referred to herein alternatively in such forms as, for example, VHCDR1 or CDRV1(for variable chain CDR1), etc.

[0033] The term “antibody” (or abbreviated as Ab) refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive with, a particular antigen, and includes polyclonal, monoclonal, genetically engineered and otherwise modified forms of antibodies, including but not limited to chimeric antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bi- tri- and quad-specific antibodies, diabodies, triabodies, and tetrabodies), and antigen-binding fragments of antibodies, including e.g., Fab′, F(ab′)2, Fab, Fv, rIgG, and scFv fragments. Moreover, unless otherwise indicated, the term “monoclonal antibody” (mAb) is meant to include both intact molecules, as well as antibody fragments (such as, for example, Fab and F(ab′)2 fragments) that are capable of specifically binding to a target protein. Fab and F(ab′)2fragments lack the Fc fragment of an intact antibody, clear more rapidly from the circulation of the animal, and may have less non- specific tissue binding than an intact antibody (see Wahl et al., J. Nucl. Med.24:316, 1983; incorporated herein by reference).

[0034] The term “antigen-binding fragment,” as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to a target antigen. The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. The antibody fragments can be a Fab, F(ab′)2, scFv, SMIP, diabody, a triabody, an affibody, a nanobody, an aptamer, or a domain antibody. Examples of binding fragments encompassed of the term “antigen-binding fragment” of an antibody may include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1domains; (iv) a Fv fragment consisting of the VLand VHdomains of a single arm of an antibody, (v) a dAb including VH and VL domains; (vi) a dAb fragment (Ward et al., Nature 341:544-546, 1989), which consists of a VHdomain; (vii) a dAb which consists of a VHor a VL domain; (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a linker that enables them to be made as a single protein chain in which the VLand VHregions pair to form monovalent molecules (known as single-chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426, 1988, and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments can be screened for utility in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in some embodiments, by chemical peptide synthesis procedures known in the art.

[0035] As used herein, the term “scFv” refers to a single-chain Fv antibody in which the variable domains of the heavy chain and the light chain from an antibody have been joined to form one chain. scFv fragments contain a single polypeptide chain that includes the variable region of an antibody light chain (VL) (e.g., CDRL1, CDRL2, and / or CDRL3) and the variable region of an antibody heavy chain (VH) (e.g., CDRH1, CDRH2, and / or CDRH3) separated by a linker. The linker that joins the VL and VH regions of a scFv fragment can be a peptide linker composed of proteinogenic amino acids. Alternative linkers can be used to so as to increase the resistance of the scFv fragment to proteolytic degradation (e.g., linkers containing D-amino acids), in order to enhance the solubility of the scFv fragment (e.g., hydrophilic linkers such as polyethylene glycol-containing linkers or polypeptides containing repeating glycine and serine residues), to improve the biophysical stability of the molecule (e.g., a linker containing cysteine residues that form intramolecular or intermolecular disulfide bonds), or to attenuate the immunogenicity of the scFv fragment (e.g., linkers containing glycosylation sites). scFv molecules are known in the art and are described, e.g., in U.S. Pat. No.5,892,019, Flo et al., (Gene 77:51, 1989); Bird et al., (Science 242:423, 1988); Pantoliano et al., (Biochemistry 30:10117, 1991); Milenic et al., (Cancer Research 51:6363, 1991); and Takkinen et al., (Protein Engineering 4:837, 1991).

[0036] As skilled persons would appreciate, where an instance of antibody or antigen- binding fragment thereof is described herein as including each of a CDRL1 domain, a CDRL2domain, a CDRL3domain, a CDRH1domain, a CDRH2domain, and a CDRH3domain, any of the foregoing examples that do not possess all such domains are not included in such use of the term “antigen-binding fragment.”

[0037] As used herein, the term “chimeric” antibody refers to an antibody having variable domain sequences (e.g., CDR sequences) derived from an immunoglobulin of one source organism, such as rat or mouse, and constant regions derived from an immunoglobulin of a different organism (e.g., a human, another primate, pig, goat, rabbit, hamster, cat, dog, guinea pig, member of the bovidae family (such as cattle, bison, buffalo, elk, and yaks, among others), cow, sheep, horse, or bison, among others). Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229(4719): 1202-7; Oi et al, 1986, BioTechniques 4:214-221; Gillies et al, 1985, J. Immunol. Methods 125:191-202; U.S. Pat. Nos.5,807,715; 4,816,567; and 4,816,397; incorporated herein by reference.

[0038] Percent (%) sequence identity refers to the percentage of amino acid (or nucleic acid) residues of a sequence that are identical to the amino acid (or nucleic acid) residues of a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity (e.g., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment and non- homologous sequences can be disregarded for comparison purposes). Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software, such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, a reference sequence aligned for comparison with a candidate sequence may show that the candidate sequence exhibits from 50% to 100% sequence identity across the full length of the candidate sequence or a selected portion of contiguous amino acid (or nucleic acid) residues of the candidate sequence. The length of the candidate sequence aligned for comparison purposes may be, for example, at least 30%, (e.g., 30%, 40, 50%, 60%, 70%, 80%, 90%, or 100%) of the length of the reference sequence. When a position in the candidate sequence is occupied by the same amino acid residue as the corresponding position in the reference sequence, then the molecules are identical at that position.

[0039] As used herein, the phrase “specifically binds” refers to a binding reaction which is determinative of the presence of an antigen in a heterogeneous population of proteins and other biological molecules that is recognized, e.g., by an antibody or antigen-binding fragment thereof, with particularity. An antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of less than about 150 nM, such as less than about 150 nM, less than about 140 nM, less than about 135 nM, less than about 130 nM, less than about 120 nM, less than about 110 nM, or less than about 100 nM. For example, an antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of up to about 100 nM (e.g., between 1 pM and 100 nM), up to about 110 nM, up to about 120 nM, up to about 130 nM, up to about 135 nM, up to about 140 nM, or up to about 150 nM. An antibody or antigen-binding fragment thereof that does not exhibit specific binding to a particular antigen or epitope thereof will exhibit a KDof greater than about 150 nM (e.g., greater than 500 nm, 1 μM, 100 μM, 500 μM, or 1 mM) for that particular antigen or epitope thereof. A variety of Immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein or carbohydrate. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein or carbohydrate. See, Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual. Cold Spring Harbor Press, New York (1999), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

[0040] An antibody or antigen-binding fragment thereof as disclosed herein may reduce, inhibit, block, prevent, or otherwise decrease binding of FH to CspZ when the antibody of antigen-binding fragment thereof is bound to the CspZ protein. For example, the concentration of said antibody or antigen-binding fragment thereof to inhibit 50% of FH bound by CspZ (referred to as IC50) may be determined, such as by using an ELISA assay as disclosed herein. An antibody or antigen-binding fragment thereof may reduce, inhibit, block, prevent, or otherwise decrease binding of FH to CspZ when it has an IC50 value of less than about 5 nM, or less than about 10 nM, or less than about 20 nM, or less than about 30 nM, or less than about 40 nM, or less than about 50 nM, or less than about 75 nM, or less than about 100 nM, or less than about 200 nM, or less than about 300 nM, or less than about 400 nM, or less than about 500 nM, or less than about 750 nM, or less than about 1 µM.

[0041] Also disclosed herein is a polynucleotide encoding any antibody or antigen- binding fragment thereof disclosed herein. A person possessing ordinary skill in the art can envision a polynucleotide sequence encoding each and every antibody or antigen-binding fragment thereof disclosed herein, including all variations in sequences of each and every given single antibody or antigen-binding fragment thereof made possible because of codondegeneracy, whereby certain amino acids may be coded for by more than one triplet codon of nucleotides. Every polynucleotide encoding a polypeptide having the sequences of any antibody or antigen-binding fragment thereof (including without limitation any disclosed in FIGs 10A-10D or depicted or described therein or therewith) is explicitly included in the present disclosure. The polynucleotide may be included in a vector such as a plasmid or cosmid, transgenic organism such as a bacteria, an artificial chromosome, or a viral vector.

[0042] Codon degeneracy, meaning codons encoding particular amino acids and which can therefore be substituted one for the other for the encoding of an amino acid also encoded for by a different codon, is as follows: Amino Acid Abbreviation Number of Codons (Degeneracy) Codons Phenylalanine Phe (F) 2 UUU, UUC Leucine Leu (L) 6 UUA, UUG, CUU, CUC, CUA, CUG Isoleucine Ile (I) 3 AUU, AUC, AUA Methionine (Start) Met (M) 1 AUG Valine Val (V) 4 GUU, GUC, GUA, GUG Serine Ser (S) 6 UCU, UCC, UCA, UCG, AGU, AGC Proline Pro (P) 4 CCU, CCC, CCA, CCG Threonine Thr (T) 4 ACU, ACC, ACA, ACG Alanine Ala (A) 4 GCU, GCC, GCA, GCG Tyrosine Tyr (Y) 2 UAU, UAC Histidine His (H) 2 CAU, CAC Glutamine Gln (Q) 2 CAA, CAG Asparagine Asn (N) 2 AAU, AAC Lysine Lys (K) 2 AAA, AAG Aspartic acid Asp (D) 2 GAU, GAC Glutamic acid Glu (E) 2 GAA, GAG Cysteine Cys (C) 2 UGU, UGC Tryptophan Trp (W) 1 UGG Arginine Arg (R) 6 CGU, CGC, CGA, CGG, AGA, AGG Glycine Gly (G) 4 GGU, GGC, GGA, GGG Stop Codons — 3 UAA, UAG, UGA

[0043]

[0044] As used herein, the term “vector” includes a nucleic acid vector, e.g., a DNA vector, such as a plasmid, a RNA vector, virus or other suitable replicon (e.g., viral vector). Avariety of vectors have been developed for the delivery of polynucleotides encoding exogenous proteins into a prokaryotic or eukaryotic cell. Expression vectors of the invention contain a polynucleotide sequence as well as, e.g., additional sequence elements used for the expression of proteins and / or the integration of these polynucleotide sequences into the genome of a mammalian cell. Certain vectors that can be used for the expression of antibodies and antibody fragments of the invention include plasmids that contain regulatory sequences, such as promoter and enhancer regions, which direct gene transcription. Other useful vectors for expression of antibodies and antibody fragments contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements include, e.g., 5′ and 3′ untranslated regions, an internal ribosomal entry site (IRES), and polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector. The expression vectors of the invention may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker include genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.

[0045] As used herein, the term “percent (%) sequence identity” refers to the percentage of amino acid (or nucleic acid) residues of a candidate sequence that are identical to the amino acid (or nucleic acid) residues of a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity (e.g., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software, such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, a reference sequence aligned for comparison with a candidate sequence may show that the candidate sequence exhibits from 50% to 100% sequence identity across the full length of the candidate sequence or a selected portion of contiguous amino acid (or nucleic acid) residues of the candidate sequence. The length of the candidate sequence aligned for comparison purposes may be, for example, at least 30%, (e.g., 30%, 40, 50%, 60%, 70%, 80%, 90%, or 100%) of the length of the reference sequence. When a position in the candidate sequence is occupied by the same amino acid residue as thecorresponding position in the reference sequence, then the molecules are identical at that position.

[0046] The term “amino acid” or “any amino acid” as used here refers to any and all amino acids (i.e. organic molecules including an amino group and a carboxyl group, connected by a central carbon atom and including a side chain), including naturally occurring amino acids (e.g., α-amino acids, wherein the side chain is attached directly to the central carbon), unnatural amino acids, modified amino acids, and non-natural amino acids. It includes both D- and L-amino acids. Natural amino acids include those found in nature, such as, e.g., 23 aforementioned amino acids that combine into peptide chains to form the building-blocks of a vast array of proteins. These are primarily L stereoisomers, although a few D-amino acids occur in bacterial envelopes and some antibiotics. “Unnatural” or “non- natural” amino acids are non-proteinogenic amino acids (i.e., those not naturally encoded or found in the genetic code) that either occur naturally or are chemically synthesized. Over 140 unnatural amino acids are known and thousands of more combinations are possible. Examples of “unnatural” amino acids include β-amino acids (β3 and β2), homo-amino acids, proline and pyruvic acid derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, diamino acids, D-amino acids, alpha-methyl amino acids and N-methyl amino acids. Unnatural or non-natural amino acids also include modified amino acids. “Modified” amino acids include amino acids (e.g., natural amino acids) that have been chemically modified to include a group, groups, or chemical moiety, such as attached directly to the carboxyl or amino group or to the side chain, not naturally present on the amino acid and are included as examples where an amino acid is referred to herein.

[0047] In a polypeptide as disclosed herein, an amino acid of one type of class may be substituted by another amino acid in the same class, or having similar chemical or physical properties, as would be understood by skilled persons, in what is referred to as a conservative substitution. A conservative substitution is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. In general, a substitution of one amino acid within the following groups for another amino acid within the following groups represents a conservative substitution: (1) Aliphatic amino acids Glycine (Gly, G), Alanine (Ala, A), Valine (Val, V), Leucine (Leu, L), Isoleucine (Ile, I); (2) hydroxyl or sulfur / selenium-containing Serine (Ser, S), Cysteine (Cys, C), Selenocysteine (Sec, U), Threonine (Thr, T), Methionine (Met, M);Cyclic Proline (Pro, P); Aromatic Phenylalanine (Phe, F), Tyrosine (Tyr, Y), Tryptophan (Trp, W); Basic Histidine (His, H), Lysine (Lys, K), Arginine (Arg, R); Acidic and their amides Aspartate (Asp, D), Glutamate (Glu, E), Asparagine (Asn, N), Glutamine (Gln, Q).

[0048] As used herein, the terms “treatment” or “treating,” or “palliating” or “ameliorating” refer to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit and / or a prophylactic benefit. An antibody or antigen- binding fragment thereof as disclosed herein may be administered to prevent the development of Lyme disease or symptoms thereof, or reduce severity of Lyme disease or symptoms thereof, following administration of the antibody or antigen-binding fragment thereof to a subject. The subject may receive administration of the antibody or antigen-binding fragment thereof to prevent contracting Lyme disease, whether or not the subject is expected to or possibly expected to come into contact with a Lyme-disease causing organism or organism carrying a species of Borrelia such as Borrelia burgdorferi or Borrelia afzelii. The subject may receive administration of the antibody or antigen-binding fragment thereof to prevent contracting Lyme disease if the subject is expected to or possibly expected to come into contact with a Lyme-disease causing organism or organism carrying a species of Borrelia such as Borrelia burgdorferi or Borrelia afzelii. The subject may receive administration of the antibody or antigen-binding fragment thereof to prevent contracting Lyme disease, prevent the worsening of Lyme disease, or to otherwise treat Lyme disease, or any one or more symptoms thereof, such as if the subject is believed to or believed possibly to have come into contact with a Lyme-disease causing organism or organism carrying a species of Borrelia such as Borrelia burgdorferi or Borrelia afzelii, or if the subject has been diagnosed with Lyme disease or has confirmed symptoms of Lyme disease. Such symptoms may include any one or more of, without limitation, one or more of fever, chills, headache, muscle and joint aches, swollen lymph nodes, rash (e.g., erythema migrans rash), irregular heartbeat, heart palpitations, arthritis, facial palsy, pain, numbness, or tingling (e.g., in the hands or feet), neck stiffness, blurred or double vision, cognitive difficulties such as impaired thinking, memory, and / or information-processing capabilities, meningitis symptoms, pain in the neck, mid / lower back, or spine, and fatigue. Any of the aforementioned subject may be considered a subject in need of treatment.

[0049] A therapeutic benefit is achieved with the prevention, reduction, eradication or amelioration of one or more of the physiological symptoms associated with LD such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with LD or symptoms thereof, or improvement achieved prophylactically, notwithstandingthat the subject may still become afflicted with LD or symptoms thereof. The antibody or antigen-binding fragment thereof may be administered to a subject reporting one or more of the foregoing systems even though a diagnosis of Lyme disease may not have been made, such as if the subject was known or believed or suspected to have been or may have been exposed to a Lyme-disease causing organism or organism carrying a species of Borrelia such as Borrelia burgdorferi or Borrelia afzelii.

[0050] A pharmaceutical composition may include an antibody or antigen-binding fragment thereof as disclosed herein and a pharmaceutically acceptable excipient. A pharmaceutically acceptable excipient includes substances added to antibody formulations to improve protein stability and other properties such as bioavailability. They are considered inert and don't have a direct role in therapy, but may improve therapeutic or prophylactic effectiveness of the pharmaceutical composition. Some non-limiting examples of pharmaceutically acceptable excipients, as would be apprehended by skilled persons, may include any one or more of the following: tonicity-adjusting excipients, such as sodium chloride, an ionic excipient that adjusts tonicity, and sucrose, trehalose, mannitol, maltose, and sorbitol, non-ionic excipients that adjust osmolality; lyoprotectants, such as trehalose and sucrose, lyoprotectants for lyophilized powders; buffers or pH-modifying agents, such as histidine, citrate, succinate, acetate, phosphate, glutamate, adipic acid, aspartic acid, lactic acid, tromethamine, and 2-(N-morpholino)-ethanesulfonic acid; surfactants, such as polysorbate 20 or polysorbate 80, or poloxamer 188 or a polyethylene glycol such as PEG 3350; a viscosity-lowering excipient such as sodium chloride and the amino acids arginine, glycine, proline, and lysine. Pharmaceutical compositions may be prepared using, e.g., physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition. Osol. A. Ed. (1980); incorporated herein by reference), and in a desired form, e.g., in the form of lyophilized formulations or aqueous solutions. Other pharmaceutically acceptable excipients may also be included in a pharmaceutical composition as disclosed herein, appropriate the formulation of antibodies and antigen- binding fragments thereof, for administration to a subject for purposes of treatment and / or prophylaxis. EXAMPLES

[0051] The following examples are intended to illustrate particular embodiments of the present disclosure, but are by no means intended to limit the scope thereof.

[0052] Materials and Methods

[0053] Mouse, ticks, and bacterial strains. Three-week-old, female C3H / HeN mice were purchased from Charles River (Wilmington, MA, USA). Though such an age of the mice has not reached sexual maturity, the under development of immune system in this age of mice would allow such mice to be more susceptible to Lyme borreliae infection, increasing the signal to noise ratio of the readout. That will also provide more stringing criteria to define the protectivity. BALB / c C3-deficient mice were from in-house breeding colonies (48) and Ixodes scapularis tick larvae were obtained from BEI Resources (Manassas, VA). Escherichia coli strain BL21(DE3) and derivatives were grown at 37°C or other appropriate temperatures in Luria-Bertani broth or agar, supplemented with kanamycin (50µg / mL). Borrelia strains were grown at 33°C in BSK II complete medium (49), and these strains include B. burgdorferi strains B31-5A4 (50), 297 (31, 51), and VS461 (52) (Table 1). Cultures of B. burgdorferi strain B31-5A4 were tested with PCR to ensure a full plasmid profile before use (53, 54) whereas B. burgdorferi strain 297 and B. afzelii strain VS461 were maintained as fewer than 10 passages.

[0054] Table 1: Strains and plasmids used herein Strain or plasmid Genotype or characteristic Source B. burgdorferi B31-5A4 Clone 5A4 of B. burgdorferi B31 isolated (50) from I. scapularis ticks in US. 297 Clone A11 / B11 of B. burgdorferi 297 (31, 51) isolated from human Cerebrospinal fluid from US. B. afzelii VS461 Clone JL of B. afzelii VS461 isolated from (52) I. ricinus ticks in Switzerland. E. coli BL21(DE3) F−, ompT hsdSB (rB− mB−) gal dcm Novagene (DE3) BL21(DE3) / pET41a-CspZ BL21(DE3) producing residues 19 to 237 Present of CspZ followed by a TEV protease disclosure cleavage site and hexa-histidine BL21(DE3) / pET41a- BL21(DE3) producing residues 19 to 237 Present CspZ-YA of CspZ-YA followed by a TEV protease disclosure cleavage site and hexa-histidine Plasmids pET41a-CspZ KanRa; pET41a encoding protein residue Present 19 to 237 of CspZ followed by a TEV disclosure protease cleavage site and hexa-histidinepET41a-CspZ-YA KanR; pET41a encoding protein residue 19 Present to 237 of CspZ-YA followed by a TEV disclosure protease cleavage site and hexa-histidineaKanamycin resistant

[0055] Cloning, expression and purification of CspZ and CspZ-YA. The DNA encoding CspZ and CspZ-YA with a C-terminal TEV cleavage site (ENLYFQG) followed by a hexahistidine tag (his-tag) were codon-optimized based on E. coli codon usage preference and synthesized by GenScript (Piscataway, NJ, USA), followed by subcloning into the pET41a using NdeI / XhoI restriction sites. These plasmids were transformed into E. coli BL21 (DE3). The recombinant protein expression was induced with 1 mM Isopropyl-β-D-1- thiogalactopyranoside (IPTG). Once expression was confirmed, the clone with the highest expression for each construct was selected to create glycerol seed stocks.

[0056] To generate his-tagged CspZ and CspZ-YA, 9 L of Basal Salt Medium (BSM, 5 g / L of K2HPO4, 3.5 g / L of KH2PO4, 3.5 g / L of (NH4)2HPO4,15 g / L of glucose, 5 g / L of yeast extract, pH 7.2) was prepared and autoclaved. Before inoculation, 9 mL of K-12 Trace Salts Solution, 36 mL of 25% MgSO4-7H2O, 9 mL of 10% antifoam, 9 mL of 50 mg / mL kanamycin and 90 mL of 15 g / L CaCl2·2H2O were added aseptically.9 L of BSM was then inoculated with a CspZ or CspZ-YA seed culture to a starting OD600of 0.05. The culture was grown at 37°C until OD600 reached 0.5-1.0 and the induction phase was initiated. During the induction phase, the culture was induced with 0.2 mM IPTG at 22°C and pH 7.2. After 8 hours of induction, fed-batch feeding (50% v / v glucose) was optionally added at 2 mL / L / h. 30% dissolved oxygen (DO) was maintained throughout fermentation. The cell paste was harvested after 19-hour induction by centrifugation and stored at -80°C until purification. To further purify his-tagged CspZ or CspZ-YA, the cell paste was first thawed and resuspended in 50 mM phosphate buffer with 500 mM NaCl and 20 mM imidazole at pH 7.4 at a ratio of 20 mL buffer per gram and homogenized 3 times on ice at 15,000 psi with an EmulsiFlex-C3 high-pressure homogenizer. The lysed cells were centrifuged and only the supernatant with the soluble proteins was then further filtered with 0.45 µm filters and loaded onto two connecting 5 mL HisTrap IMAC FF columns (GE Healthcare) which were pre-equilibrated with 50mM phosphate buffer with 500 mM NaCl and 20 mM imidazole at pH 7.4. The columns were washed with 10 column volumes (CVs) of 50 mM phosphate buffer with 500 mM NaCl and 20 mM imidazole at pH 7.4 followed by eluted with a linear gradient of 20 mM to 500 mM imidazole over 20 CVs. The purified his-tagged CspZ or CspZ-YA wasdialyzed against PBS or TBS and verified with the integrity and purity greater than 90% using SDS-PAGE (Fig. S3) before storage at −80°C.

[0057] Generation of CspZ-YA mouse monoclonal and rabbit polyclonal antibodies. Purified his-tagged CspZ-YA was provided to Genemed Synthesis, Inc (San Antonio, TX) to generate CspZ-YA IgG (Total). The IgGs in the immunized rabbits containing CspZ-YA IgGs and naïve rabbit IgGs were purified using protein A chromatography and formulated in 1X PBS with 10% BSA, pH 7.4. To retrieve the CspZ-YA (Total), we first covalently conjugated CspZ onto AminoLink Plus Coupling Resin (ThermoFisher Scientific, Waltham, MA) based on the manufacturer’s instructions. The 5 mL IgGs from CspZ-YA immunized rabbits were subsequently mixed to 100 µL CspZ-conjugated resin slurry (resin to PBST buffer ratio as 1:1) for four hours to capture CspZ-YA IgG (Total). The resin was then washed twice with 1X PBST buffer (1X PBS with 0.05% Tween 20). Finally, the captured CspZ-YA IgG (Total) was eluted with 0.65ml of glycine buffer (0.1 M Glycine HCl at pH 2.5) and dialyzed against 1X PBST buffer.

[0058] To isolate the CspZ-YA IgG (FH-binding sites) and CspZ-YA IgG (non-FH binding sites) from CspZ-YA IgG (Total), 100 µL of CspZ-conjugated resin slurry was mixed with excess Factor H (Complement Technology, Tyler, TX, USA) for two hours, allowing the formation of CspZ-FH. This step is essential to block the FH binding site on CspZ. After washing off the excess FH using 1X PBST buffer that CspZ-FH resin was mixed with the CspZ-YA IgG (Total) for 90 minutes. After incubation and the centrifugation of the resin, the unbound fraction containing CspZ-YA IgG (FH-binding sites) was collected from the supernatant. The resulting resin was then washed with 1X PBST, and the bound fraction containing CspZ-YA IgG (non-FH-binding sites) was eluted using 0.1 M glycine HCl at pH 2.5. However, FH was found to be eluted with CspZ-YA IgG (non-FH-binding sites); thus, to remove that FH, the eluted proteins were further dialyzed against 1X PBST, followed by protein A affinity purification to capture the CspZ-YA IgG (non-FH-binding sites). The purified CspZ-YA IgG (non-FH-binding sites) was eventually eluted from protein A resin using the glycine buffer and dialyzed against 1X PBST.

[0059] The medium samples from each of the eight clones of mouse hybridoma cells producing CspZ-YA mouse monoclonal antibodies were generated by Protein and Monoclonal Antibody Production Core at Baylor College of Medicine. Each of these medium samples was then applied to NA Protein G Spin Columns to purify the CspZ-YA mouse monoclonal IgG (ThermoFisher Scientific) and then quantitated as described in the vendor’s manual.

[0060] ELISAs. To verify the ability of rabbit polyclonal and mouse monoclonal CspZ-YA IgGs in recognizing FH-binding sites of CspZ, we compared the levels of each of these IgGs in binding to FH-saturated CspZ with those in binding to CspZ proteins treated with BSA (control). One µg of histidine-tagged CspZ was coated on ELISA plate wells, followed by being blocked with 5% BSA in PBS buffer. Those wells were subsequently treated with human FH (500 nM, ComTech, Tayler, TX), BSA (control, 500 nM, Sigma- Aldrich, St. Louis, MO), or PBS (control), followed by incubation with each of the tested rabbit polyclonal and mouse monoclonal CspZ-YA IgGs (50 nM). HRP-conjugated goat anti- mouse IgG (Sigma-Aldrich) and goat anti-rabbit IgG (Sigma-Aldrich) were used to detect the binding of mouse monoclonal and rabbit polyclonal CspZ-YA IgGs, respectively. Tetramethylbenzidine solution (ThermoFisher) was added to each well and incubated for five minutes, then the reaction was stopped with hydrosulfuric acid. Plates were read at 405 nm using a Tecan Sunrise Microplate reader (Tecan, Morrisville, NC). The resulting absorption values were normalized to those from PBS-treated wells to obtain the percentage of CspZ-YA IgG binding.

[0061] To determine the ability of rabbit polyclonal and mouse monoclonal CspZ-YA IgGs in preventing FH binding to CspZ, the ELISA was performed as described with modifications (32). Each ELISA microtiter well was coated with one µg of histidine-tagged CspZ. After being blocked with 5% BSA in PBS buffer, the wells were incubated with PBS (control) or serially-diluted irrelevant mouse IgG (anti-green fluorescence protein of mouse IgG, ThermoFisher) or irrelevant rabbit IgG (anti-green fluorescence protein of rabbit IgG, ThermoFisher), or each of the mouse monoclonal or rabbit polyclonal CspZ-YA IgGs (0.4 nM, 0.8 nM, 1.6 nM, 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM) followed by being mixed with 500 nM of human FH. Sheep anti-human FH (1:200×, ThermoFisher) and then goat anti-sheep HRP (1:2000×, ThermoFisher) were added, and the levels of FH binding were detected by ELISA as mentioned above. Data were expressed as the proportion of FH binding from serum-treated to PBS-treated wells. The 50% inhibitory concentration (IC50) (Table 2), representing the IgG concentration that blocks 50% of FH binding, was calculated using dose-response stimulation fitting in GraphPad Prism 5.04.

[0062] Table 2. IC50 values of the CspZ-YA IgGs tested in this study for blocking FH binding to CspZ from B. burgdorferi B31-5A4 IC50 (nM) Rabbit IgGIrr. CspZ-YA IgG CspZ-YA IgG (Non-FH- CspZ-YA IgG (FH-binding rab. (Total) binding sites) sites) IgGani.b19.17±2.87 ni. 4.20±0.63 Mouse IgG Irr. 142 224 582 605 651 1009 1139 1193 ms. IgGcni. 82.02±10.12 ni. ni. 57.90±1.85 97.69±8.94 ni. 4.20±0.72 4.07±0.59aIrrelevant rabbit IgG, anti-green fluorescence protein of rabbit IgG.bNo FH binding inhibition was detected after incubation with 50nM of indicated IgG (the maximal IgG dose used in this study).cIrrelevant mouse IgG, anti-green fluorescence protein of mouse IgG.

[0063] The seropositivity of the mice after infection with B. burgdorferi was determined by detecting the presence or absence of the antibodies that recognize C6 peptides, which has been commonly used for human Lyme disease diagnosis (55).50 µl of serially diluted mouse serum (1:100×, 1:300×, 1:900×) from 21dpf was added to microtiter wells coated with C6 peptides ((55), Genemed Synthesis, Inc). Total IgG was detected using HRP- conjugated goat anti-mouse IgG (1:20,000×; Bethyl, Montgomery, TX, USA). After the incubation with antibodies for one hour, tetramethyl benzidine solution (ThermoFisher) was added, and the absorbance was detected at 620nm for 10 cycles of 60-second kinetic intervals with 10 seconds shaking duration using Tecan Sunrise Microplate reader as described above. For each serum sample, the maximum slope of optical density / minute of all the dilutions was multiplied by the respective dilution factor, and the greatest value was used as representative of antibody titers (arbitrary unit (A.U.)). The seropositive mice were defined as the mice with the serum samples yielding a value greater than the threshold, the mean plus three-fold standard deviation of the IgG values derived from the uninfected mice.

[0064] Borreliacidal assays. The ability of CspZ-YA mouse monoclonal and rabbit polyclonal IgGs to eradicate spirochetes was determined as described with modifications (32). Briefly, irrelevant mouse or rabbit IgG, or each of these CspZ-YA mouse monoclonal or rabbit polyclonal antibodies were serially diluted to the indicated concentrations (0.4 nM, 0.8 nM, 1.6 nM, 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM). The diluted IgGs were then mixed with complement-preserved guinea pig serum (Sigma-Aldrich; final concentration 5%). Note that this concentration of guinea pig sera serum has been verified to not killspirochetes in the absence of CspZ-YA antibodies (data not shown). The PBS treatment was included as a control. After incubated with the strains B31-5A4, 297, or VS461, the mixture was incubated at 33°C for 24 hours. Surviving spirochetes were quantified by directly counting the motile spirochetes using dark-field microscopy and expressed as the proportion of IgG-treated to PBS-treated Lyme borreliae. The 50% borreliacidal titer is shown in Table 3, representing the serum dilution rate that kills 50% of spirochetes, which was calculated using dose-response stimulation fitting in GraphPad Prism 5.04.

[0065] Table 3. BA50values of the CspZ-YA IgGs used in the present disclosure BA50(nM) Mixed Rabbit IgG withBb 297 nk. nd. nd. nd. nd. nd. nd. 2.48±0.12 1.27±0.02 Ba nk. nd. nd. nd. nd. nd. nd. 7.13±0.53 51.60±1.92 VS461 aIrrelevant rabbit IgG, anti-green fluorescence protein of rabbit IgG. bB. burgdorferi strain B31-5A4 cNo killing was detected after incubation with 50nM of indicated IgG (the maximal IgG dose used in this study). dB. burgdorferi strain 297 eNot determined fB. afzelii strain VS461 gIrrelevant mouse IgG, anti-green fluorescence protein of mouse IgG.

[0066] Generation of infected ticks. Generating infected I. scapularis ticks has been described previously (56). BALB / c C3-deficient mice were infected intradermally with 105of the strains B31-5A4 (27, 48). Ear tissues were collected via ear punch, and bacterial gDNA was purified for detection with qPCR to confirm infection (see section “IgG inoculation, B. burgdorferi infection, quantification of spirochete burdens”). Approximately 100 to 200 uninfected larvae were then allowed to feed to repletion on the infected mice as described previously (56). The engorged larvae were collected and allowed to molt into nymphs in a desiccator at room temperature with 95% relative humidity and light-dark control (light to dark, 16:8 hours).

[0067] IgG inoculation, B. burgdorferi infection, and quantification of spirochete burdens. Three-week-old, female C3H / HeN mice were subcutaneously inoculated with irrelevant mouse or rabbit IgG or each of the tested CspZ-YA mouse monoclonal or rabbit polyclonal IgGs (1 mg / kg). Five mice per group were used in this study. This number was justified by the power analysis. Using the means ± standard deviation from this study (10±10 for the uninfected control group, 55±10 for the infection group), a power analysis for attaining a statistically significant difference (p <0.05; a one-way ANOVA) between the negative control group and other groups with 95% probability requires at least 5 animals per group (57). This value is consistent with numbers that we and others have used in the past for similar studies (32, 58). At 24 hours after inoculation, five nymphs carrying B. burgdorferi strain B31-5A4 were allowed to feed to repletion on each mouse, and a subset of nymphs was collected pre- and post-feeding as described (32, 48). Mice were sacrificed at 21 dpf to collect the biting site of skin, knees, heart, and bladder. DNA was purified from these nymphs, and spirochetes were quantified as described with modifications (32). DNA was purified using EZ-10 Spin Column Animal Genomic DNA Mini-Prep Kit (Bio Basic, Inc., Markham, Ontario, CA). Spirochete burdens were quantified based on the amplification of recA from B. burgdorferi strain B31-5A4 using the primers ((BBRecAfp (5’- GTGGATCTATTGTATTAGATGAGGCTCTCG-3’) and BBRecArp (5’- GCCAAAGTTCTGCAACATTAACACCTAAAG-3’)) with qPCR using an Applied Biosystems 7500 Real-Time PCR system (ThermoFisher) in conjunction with PowerUp™ SYBR® Green Master Mix (ThermoFisher) as described (33, 59). The number of recA copies was calculated by establishing a threshold cycle (Cq) standard curve of a known number of recA genes extracted from strain B31-5A4, and burdens were reported as the number of recA copies per tick or normalized to 100ng of total DNA and reported as the number of recA copies per 100ng of total DNA.

[0068] Statistical analyses. Significant differences were determined with a Mann- Whitney test (between two groups)(60), Kruskal-Wallis test with the two-stage step-up method of Benjamini, Krieger, and Yekutieli (61) (more than two groups), or two-tailed Fisher test (for seropositivity in Table 1) (62) using GraphPad Prism 5.04. A p-value < 0.05 was used to determine significance.

[0069] Example 1: CspZ-YA immunization triggers IgGs that recognize both FH and non-FH-binding sites of CspZ.

[0070] To obtain antibodies that recognize CspZ-YA, sera generated from rabbit immunized with this recombinant protein was purified using a CspZ-immobilized resin tocapture all anti-CspZ-YA IgGs (CspZ-YA IgG (Total)) (lane 1 in Fig.1A). From this pool, we separated the IgGs that recognize the FH binding site (CspZ-YA IgG (FH-binding sites)) from those that recognize non-FH-binding sites of CspZ (CspZ-YA IgG (non-FH-binding sites)) (Fig.1B). After applying CspZ-YA IgGs (Total) to a resin functionalized with a CspZ- FH complex, we collected the unbound fraction containing CspZ-YA IgGs (FH-binding sites) (Lane 2 in Fig.1A, 1B). The bound proteins consisting mainly of CspZ-YA IgG (non-FH- binding sites) were then eluted (lane 3 in Fig.1A, 1B). This fraction also contained another protein with a molecular weight of approximately 120 kDa (lane 3 in Fig.1A), likely FH (lane 5 Fig.1A). The bound fraction was subsequently applied to a protein A column to remove the FH, resulting in purified CspZ-YA IgGs (non-FH-binding sites) (lane 4 in Fig.1A and 1B).

[0071] To verify the ability of these fractions to recognize (or not) FH-binding sites, we incubated CspZ-coated ELISA plate wells with FH or BSA (negative control), added CspZ-YA IgG (FH-binding sites) or CspZ-YA IgG (non-FH-binding sites) to those wells, and detected the levels of bound antibody (Fig.1C). We found that CspZ-YA IgG (non-FH- binding sites) bound to both FH- and BSA-treated wells indistinguishably, but the levels of bound CspZ-YA IgG (FH-binding sites) were significantly reduced in FH-treated wells, compared to those from BSA-treated wells (Fig.1D). These results demonstrate the ability of CspZ-YA IgG (FH-binding sites) to recognize FH-binding sites.

[0072] We further investigated whether CspZ-YA immunization triggered the production of IgGs that recognize both FH- and non-FH-binding sites in mice. To generate enough antibodies for this effort and for subsequent experiments, we obtained CspZ-YA mouse monoclonal IgGs from eight individual mouse hybridomas (#142, #224, #582, #605, #651, #1006, #1139, #1193). We then assessed if these antibodies recognize (or not) CspZ FH-binding sites. We found that the binding levels of mAbs #142, #224, 582, #605, #651, and #1006 to FH-treated wells did not significantly differ from those to BSA-treated wells (Fig.1E). However, antibodies #1139 and #1193 displayed significantly lower levels of binding to FH-treated wells than to BSA-treated wells (Fig.1E). These results grouped the CspZ-YA mouse monoclonal antibodies into those whose CspZ-binding activity is blocked by FH and those that are not. These findings demonstrate that IgGs that recognize either the FH- or non-FH-binding sites of CspZ can be isolated from CspZ-YA-immunized mice.

[0073] Example 2: The FH-binding activity of CspZ is selectively blocked by CspZ- YA IgGs that recognize FH-binding sites. Mouse sera after CspZ-YA immunization were documented to block FH-binding to CspZ (32). As the IgGs that recognize either FH- or non-FH-binding sites of CspZ can be isolated after such immunization, we sought to determine the ability of each of those IgGs to prevent FH-binding to CspZ. We thus incubated CspZ-YA IgG (FH-binding sites) or CspZ-YA IgG (non-FH-binding sites) with CspZ-coated ELISA plate wells, added FH to each of those wells, and determined the levels of bound FH (Fig. 2A). CspZ-YA IgGs before fractionation (CspZ-YA IgG (Total)) and irrelevant rabbit IgGs were included as a control. As expected, CspZ-YA IgGs (Total) but not irrelevant rabbit IgG antibodies inhibited FH binding to CspZ (Fig.2B, Table 2). We found that CspZ-YA IgGs (FH-binding sites) inhibit CspZ-FH binding in a dose-dependent manner, more efficiently than CspZ-YA IgGs (Total) (IC50 = 4.2nM). Conversely, CspZ-YA IgGs (non-FH-binding sites) did not inhibit the FH-binding activity of CspZ (Fig.2B, Table 2). We also determined the capability of each of the mouse monoclonal CspZ-YA IgGs to block FH binding to CspZ. We observed that those IgGs that do not bind to the FH-binding sites of CspZ (#142, #224, 582, #605, #651, #1006, #1139, #1193) did not reduce FH binding to CspZ while those that bound to the FH-binding sites of CspZ (#1139 and #1193) could (Fig.2C, see Table 2 for IC50). These results indicate that CspZ-YA IgGs that recognize FH-binding sites selectively prevent FH binding to CspZ.

[0074] Example 3: CspZ-YA IgGs that recognize CspZ FH-binding sites robustly killed Lyme borreliae in vitro.

[0075] We have shown that CspZ-YA vaccination triggers borreliacidal antibodies (32), raising the possibility of IgGs that recognize FH- and / or non-FH-binding sites of CspZ to kill Lyme borreliae. We examined this possibility and found that the irrelevant rabbit IgGs do not eliminate B. burgdorferi B31-5A4, but CspZ-YA IgGs (Total) killed those spirochetes in a dose-dependent manner (Fig.3A; the 50% borreliacidal activity of each IgG (BA50) was 7.4 nM, Table 3). We found that CspZ-YA IgGs (non-FH-binding sites) did not efficiently kill B. burgdorferi B31-5A4 since the results from this IgG did not allow accurate fitting to obtain a BA50value (Fig.3A, Table 3). However, CspZ-YA IgGs (FH-binding sites) robustly eradicated those spirochetes, with significantly lower BA50 values (BA50 = 2.56nM) than CspZ-YA IgGs (Total) (Fig.3A, Table 3). We also tested the bactericidal activity of each of the CspZ-YA mouse monoclonal IgGs in the same fashion and observed that irrelevant mouse IgG antibodies did not kill B. burgdorferi B31-5A4 (Fig.3B). We found that the IgGs that did not recognize the FH-binding sites of CspZ could be divided into two groups based on their bactericidal activities: Those IgGs that did not have any borreliacidal activities (#224, #582, #1009, Fig. 3B) and those that still killed bacteria but were not efficient enough to acquire accurate BA50 values (#142, #605, and #651, Fig.3B, Table 3). On the contrary,both IgGs that recognized the FH-binding sites of CspZ (#1139 and #1193, Fig.2C) efficiently killed spirochetes, with #1193 (BA50 = 1.23 nM) eradicating bacteria more efficiently than #1139 (BA50= 3.45 nM) (Fig.2C, Table 3). We further evaluated the ability of the IgGs that robustly killed B. burgdorferi B31-5A4 (CspZ-YA IgG (Total), CspZ-YA IgG (FH-binding sites), #1139, and #1193) to eradicate other strains and species of Lyme borreliae, including B. burgdorferi 297 and B. afzelii VS461. Similar to the results for B. burgdorferi B31-5A4, these IgGs eradicated B. burgdorferi 297 and B. afzelii VS461 in a dose-dependent manner (Fig.3C to 3E and Table 3). However, CspZ-YA IgGs (FH-binding sites) more robustly killed strain 297 than CspZ-YA IgGs (Total) but showed no significantly different efficiency in killing strain VS461, compared to CspZ-YA IgGs (Total) (Fig.3C and 3E and Table 3). In addition, #1139 displayed indistinguishable ability from #1193 to eliminate strain 297 but showed more robust killing of strain VS461 (Fig.3D and 3F and Table 3). These results not only showed the general ability of CspZ-YA IgGs that recognize the CspZ FH-binding sites to efficiently kill Lyme borreliae, but also exhibited that these antibodies varied in their efficiency against different bacterial strains and species.

[0076] Example 4: Passive immunization with CspZ-YA IgGs that recognize CspZ FH-binding sites reduced the seropositivity and levels of colonization by B. burgdorferi. Our previous efforts illustrate that passive inoculation of sera from CspZ-YA vaccination protect mice from Lyme borreliosis (32). This observation led to the hypothesis that the IgGs that recognized FH- and / or non-FH-binding sites of CspZ prevent B. burgdorferi infection. To test this hypothesis, we inoculated mice with CspZ-YA IgGs (non-FH-binding sites). Due to insufficient CspZ-YA IgGs (FH-binding sites) recovered after purification for this experiment, mAbs #1139 and 1193 were solely used to represent IgGs that recognize CspZ FH-binding sites. Those mice were then fed on by ticks carrying B. burgdorferi B31-5A4 at 1-day post-immunization (Fig.4A). In addition to uninfected mice, we also included the B31- 5A4-infected mice that were inoculated with CspZ-YA IgG (Total), irrelevant rabbit IgG, or irrelevant mouse IgG as a control (Fig.4A).

[0077] We found that after feeding on all groups of mice to full engorgement, ticks did not have significantly different levels of bacterial burdens (Fig. S1), consistent with the finding that CspZ is not produced in ticks (24). At 21-days post tick feeding (dpf), we determined the serology of all mice. The uninfected group was seronegative, whereas all five mice inoculated with irrelevant rabbit and mouse IgG were seropositive (Fig. S2, Table 4). Only one of five mice inoculated with CspZ-YA IgGs (Total) turned seropositive, a significantly lower number than the irrelevant control (Fig. S2, Table 4, shown in FIG.11).Conversely, all five mice injected with CspZ-YA IgGs (non-FH-binding sites) were seropositive, not significantly different from the rabbit IgG control (Fig. S2, Table 4). Notably, only one #1139-inoculated, and none of the #1193-inoculated, mice were seropositive (Fig. S2, Table 4). These results suggest that IgGs that recognize the FH-binding sites of CspZ reduced seroconversion of mice during infection.

[0078] We further determined the levels of spirochete colonization of mouse tissues at 21dpf. Mice inoculated with the irrelevant rabbit IgG control yielded significantly higher bacterial burdens at the bite site, in the bladder, at the ankles, and in the heart than uninfected mice (Fig.4B to E). Mice inoculated with CspZ-YA IgGs (Total) displayed significantly lower spirochete burdens for all tested tissues than the irrelevant rabbit IgG control (Fig.4B to E). Conversely, mice injected with CspZ-YA IgG (non-FH-binding sites) showed bacteria burdens indistinguishable from the irrelevant rabbit IgG control, and were significantly higher than uninfected mice (Fig.4B to E). Additionally, mice inoculated with irrelevant mouse IgG control developed significantly greater bacterial loads in all tissues than uninfected mice (Fig.4B to E). Except one non-protected mouse in the #1139-inoculated group, all #1139- and #1193-inoculated mice yielded indistinguishable bacterial burdens in all tissues from uninfected mice. Additionally, the #1193-inoculated mice had significantly lower spirochete loads in the distal tissues than the irrelevant mouse IgG-inoculated control mice. These results demonstrate the ability of IgG antibodies that recognize the FH-binding sites of CspZ to significantly reduce B. burgdorferi colonization.

[0079] DISCUSSION

[0080] The antibody repertoire induced by a specific antigen comprises different populations of antibodies that recognize distinct epitopes on that antigen. Some less abundant antibody fractions are considered “immunologically subdominant” (34). When the efficacious antibody population is immunologically subdominant, host adaptive immune responses may not efficiently eliminate pathogens and / or alleviate manifestations despite overall robust antibody titers (35). Likewise, if the protective epitopes induced by a vaccine antigen were immunologically subdominant that vaccine may be unable to prevent infection (36). Antigen engineering can enhance the abundance of the antibodies that recognize those immunologically subdominant protective epitopes, thereby improving vaccine efficacy (34). One of the strategies for antigen engineering is to remove the structures preventing exposure of the protective epitopes (e.g., surface polysaccharides that mask the protective epitopes of some viral antigens (37, 38)).

[0081] The antigen of interest, CspZ, is upregulated immediately after spirochetes infect hosts and binds to host FH (22, 24). Therefore, any protective epitopes in / around the FH-binding site may not be fully exposed to induce sufficient bactericidal antibodies (32, 33). In fact, high titers of antibodies against CspZ are in humans and mice after exposure to Lyme borreliae, but those spirochetes still persist during infection (28, 30, 32, 33). Additionally, we previously reported null protection in mice after immunization with CspZ, but full protection after vaccination with CspZ-YA, a CspZ mutant unable to bind its target ligand (28, 31-33). Thus, the protective epitopes may be immunologically subdominant, and engineering the antigen to remove the FH binding ability may have triggered the production of these protective antibodies. In the current study, we demonstrated the ability of the antibodies that specifically recognize the CspZ FH binding site to eradicate B. burgdorferi in vitro and in vivo, demonstrating that the protective epitopes were in / around the FH-binding site. Such an engineering of removing the ability to bind the target ligand in enhancing the levels of the protective antibodies has also been applied to other infectious agents, such as Fhbp, a Neisseria meningitidis antigen currently used as a human vaccine against meningococcal infection (39-41). However, unlike CspZ-YA, removing the FH-binding activity of Fhbp also magnified overall antibody responses (39, 42, 43). Taken together, our findings provide mechanistic insights into how such a precise antigen engineering would turn immunologically subdominant epitopes to be the major region that can induce efficacious antibodies against infectious agents.

[0082] However, an unanswered question is how those antibodies that recognize the mutant FH-binding sites promote spirochete clearance to protect mice from B. burgdorferi infection. We showed here that the protection from LD in vivo is selectively mediated by the CspZ-YA IgGs that recognize CspZ FH-binding sites. Together with another finding in the current study of antibody-mediated borreliacidal abilities in vitro by the IgGs that recognize CspZ FH-binding sites, it is possible that CspZ-YA IgGs-mediated killing is conferred by the Fc regions of IgGs. As that Fc region promotes classical pathway-mediated pathogen lysis and opsonophagocytosis directly or indirectly by the binding of antibodies to leukocytes (44), these mechanisms would play a role in the bacterial clearance caused by CspZ-YA IgGs. We also found that the CspZ-YA IgGs that recognize FH-binding sites blocks FH binding to CspZ, which leads to the possibility that these antibodies eliminate bacteria by preventing the ability of spirochetes to evade alternative pathway-mediated killing (e.g., opsonophagocytosis and pathogen lysis) (10). Delineating the role of each of these mechanisms in promoting the protective antibodies upon CspZ-YA vaccination warrants further investigations.

[0083] We found that the CspZ-YA IgGs that recognize FH-binding sites efficiently eliminate spirochetes across different strains and species of Lyme borreliae, but the extent of killing varied among the strains. This result is consistent with the fact that the sequences on the FH-binding interface of CspZ are highly similar among the variants of different strains within the same Lyme borreliae species (>98% identity) but moderately variable among the strains from different spirochete species (~80% identity) (29, 32). Moreover, CspZ-YA vaccination not only stops spirochete colonization at tissues but also prevents the development of LD-associated manifestations (i.e., arthritis) (30, 32). Though Lyme borreliae dissemination is a prerequisite for the onset of these manifestations, the severity of such disease symptoms has been shown to not necessarily be linked to the spirochete burdens in tissues (45). Therefore, the results from this study showing the absence of the spirochetes in the tissues from mice inoculated with CspZ-YA IgGs that recognize FH-binding sites may not fully address the mechanisms of manifestation prevention by CspZ-YA vaccines. Despite that, our finding does not preclude the possibility of the antibodies that recognize CspZ FH- binding sites killing spirochetes at the initial infection sites and blocking spirochetes from disseminating to distal tissues. In this study, the insufficient polyclonal CspZ-YA IgG (FH- binding sites) from CspZ-YA-immunized rabbits (~25µg isolated from six rabbits) justifies the use of #1139 and #1193 for the in vivo work. However, both #1139 and #1193 may account for only part of the antibody population in the CspZ-YA IgG (FH-binding sites). The fact of the protectivity provided by #1139 and #1193 does not rule out the possibility that the uncovered antibodies population in CspZ-YA IgG (FH-binding sites) have different phenotypes from #1139 and #1193, which is worth further investigation. Further, pre- exposure prophylaxis (PrEP) is a commonly studied strategy for LD prevention. In fact, in addition to vaccines, a yearly administrated monoclonal antibody against OspA, a protein that is required for tick-to-host transmission of spirochetes, is currently in clinical trials (46, 47). Our finding of the bactericidal effect by the antibodies recognizing the CspZ FH binding site illustrates that identifying monoclonal antibodies that bind these epitopes constitutes a promising option for a prophylactic agent against LD. Collectively, this study identified that the protective epitopes of CspZ-YA vaccines are proximal to the FH-binding site, which further elucidates the potential preventive mechanisms of this vaccine candidate. Such findings would eventually inform the strategy of precision antigen engineering in developing vaccines and monoclonal antibody-based prophylaxis against LD and other infectious diseases.

[0084] Example 5: Chimeric antibodies

[0085] In addition to the Y-to-A substitutions of CspZ-YA, further amino acid substitutions made to CspZ-YA (such as either of I183Y and C187S substitutions) promote the stability of the CspZ-YA epitopes recognized by CspZ-targeting, Lyme borrelia-killing monoclonal antibodies. The enhanced intramolecular interactions by I183Y and C187S mutagenesis raises a possibility of CspZ-YAI183Yand CspZ-YAC187Sto have increasing stability. We thus examined whether CspZ-YAI183Y and CspZ-YAC187S have greater thermostability than CspZ-YA. We found indistinguishable Tm values between CspZ- YAI183Yand CspZYAC187S(61.87 and 62.72oC, respectively). In contrast, the Tm-values of CspZ-YA were significantly lower, 57.58 and 58.46oC for histidine tagged and untagged CspZ-YA, respectively, indicating a stability enhancement through mutagenesis (Table 5).

[0086] Table 5. The thermostability of CspZ-YA proteins. CspZ-YA His-CspZ-YA - C187S - I183Y neep ca e pe e pe e . bHistidine-tagged CspZ-YA

[0087] We next examined the impact of I183Y and C187S mutagenesis on altering long-term stability of the protective epitopes in the CspZ-YA structures. We generated chimeric, monoclonal CspZ-YA IgGs that contain the Fc region of human IgG1 and F(ab’)2 from 1139 or 1193. The resulting chimeric IgGs, namely 1139c and 1193c, were first confirmed for their ability to bind to CspZ-YA (FIGs.8A and 8B), block the FH-binding ability of CspZ (FIG.8C), and promote lysis (FIG.8D) and opsonophagocytosis of B. burgdorferi (FIG.8E). We placed CspZ-YAI183Y, CspZYAC187S, or CspZ-YA (untagged and histidine tagged) at 4 or 37oC for different period of time and then examined the ability of 1139c or 1193c to bind to each of these CspZ-YA proteins. We found all CspZ-YA proteins or variants previously incubated at 4oC for 6- or 24-h or at 37oC for 6-h displayed similar levels of recognition to these proteins prior to incubation. However, CspZ-YA but not CspZ- YAI183Y and CspZ-YAC187S previously incubated at 37oC for 24-h had significantly lower levels of recognition, compared to those proteins prior to incubation, even though SE-HPLC did not indicate significant aggregation or degradation. These findings demonstrated long- term stability enhancement of CspZ-YA proteins in the physiological temperature by I183Y and C187S mutagenesis, specifically on the structures that promote protective antibody induction.

[0088] Anti-CspZ antibodies as disclosed herein recognize CspZ proteins, including CspZ-YA proteins, and recognize CspZ-YAI183Y and CspZ-YAC187S better than CspZ-YA at a higher temperature for a longer period of time (i.e., 37oC for 24-h). As mammalian body temperatures stay consistent at 37oC, both mutations would allow CspZ-YA to persist in the designated structures to promote the continuous production of resulting protective antibodies, thus suitable as vaccines for human or other mammal uses.

[0089] Materials and Methods

[0090] Hybridoma that produce the monoclonal antibodies #1139c or #1193c were cultivated in RPMI 1640 medium containing 10% FBS at 37oC with 5% of CO2. Thirty-eight deidentified two-tiered positive human serum samples were obtained from New York State Department of Health. These serum samples were previously collected from humans that were tested positive in two-tiered assays, which is the serological definition of Lyme disease infection. Mead et al., Updated CDC Recommendation for Serologic Diagnosis of Lyme Disease. MMWR Morb Mortal Wkly Rep.2019;68(32):703. The negative control human sera were collected from 10 individuals residing in a non-endemic area for Lyme disease.

[0091] Generation of humanized CspZ-YA antibodies, #1139c and #1193c. Protective mouse monoclonal antibodies (mAbs) 1139 and 1193 against CspZ were developed previously. Chen et al. CspZ FH-Binding Sites as Epitopes Promote Antibody-Mediated Lyme Borreliae Clearance. Infect Immun.2022;90(7):e0006222. These two mAbs were further humanized using the service provided by GenScript Probio (Piscataway, NJ). Briefly, DNA sequencing was performed using the hybridoma to identify the gene coding the variable domain of mAbs 1139 and 1193. Such genes were then grafted with the one coding for human IgG1. The two humanized chimeric mAbs (1139c and 1193c) were then transiently produced in CHO cells, followed by purification with Protein A affinity chromatography. Amino acid sequences for mAbs 1139c and 1193c were determined and are presented in FIGs 10A-10D.

[0092] For the mice inoculated with humanized monoclonal IgGs, C3H / HeN mice were immunized as described, with slight modifications. Chen et al. CspZ FH-Binding Sites as Epitopes Promote Antibody-Mediated Lyme Borreliae Clearance. Infect Immun. 2022;90(7):e0006222. C3H mice were intraperitoneally inoculated with irrelevant human IgG (control), #1139c or #1193c (1 mg / kg) (FIG.9). Five mice per group were used in this study. At 24 hours after inoculation, five nymphs carrying B. burgdorferi strain B31-A3 were allowed to feed to repletion on each mouse, and a subset of nymphs was collected pre- and post-feeding as described. Marcinkiewicz et al. The Factor H-Binding Site of CspZ as aProtective Target against Multistrain, Tick-Transmitted Lyme Disease. Infect Immun. 2020;88(5); Hart et al. Polymorphic factor H-binding activity of CspA protects Lyme borreliae from the host complement in feeding ticks to facilitate tick-to-host transmission. PLoS Pathog.2018;14(5):e1007106. Mice were sacrificed at 21 days post feeding (dpf) to collect the biting site of skin, bladder, knees, and heart to determine the bacterial burdens described below (FIG.9).” Blood was also collected via cardiac puncture bleeding to isolate sera for the determination of seropositivity described in the section “ELISAs” (FIG.9).

[0093] Additionally, the seropositivity of the mice after infection with B. burgdorferi was determined by detecting the presence or absence of the IgGs that recognize C6 peptides (FIG.9). This methodology has been commonly used for human Lyme disease diagnosis (Liang et al. C-terminal invariable domain of VlsE is immunodominant but its antigenicity is scarcely conserved among strains of Lyme disease spirochetes. Infect Immun. 2001;69(5):3224-31) and performed as described in our previous work. Chen et al. CspZ FH- Binding Sites as Epitopes Promote Antibody-Mediated Lyme Borreliae Clearance. Infect Immun.2022;90(7):e0006222. For each serum sample, the maximum slope of optical density / minute of all the dilutions was multiplied by the respective dilution factor, and the greatest value was used as representative of anti-C6 IgG titers (arbitrary unit (A.U.)). The seropositive mice were defined as the mice with the serum samples yielding a value greater than the threshold, the mean plus 1.5-fold standard deviation of the IgG values derived from the uninfected mice.

[0094] We also determined the ability of #1139c or #1193c to prevent FH from binding to CspZ (FIG.8C), which was performed as described previously with modifications. Chen et al. CspZ FH-Binding Sites as Epitopes Promote Antibody-Mediated Lyme Borreliae Clearance. Infect Immun.2022;90(7):e0006222. Basically, each ELISA microtiter well was coated with one µg of histidine-tagged CspZ. After being blocked with 5% BSA in PBS buffer, the wells were incubated with PBS (control) or serially-diluted irrelevant human IgG (Human IgG isotype control, Sigma-Aldrich, St. Louis, MO) #1139c or #1193c (0.4 nM, 0.8 nM, 1.6 nM, 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM) followed by being mixed with 500 nM of human FH. Sheep anti-human FH (1:200×, ThermoFisher; Waltham, MA) and then donkey anti-sheep HRP (1:2000×, ThermoFisher) were added, and the levels of FH binding were detected by ELISA as described previously. Chen et al. CspZ FH-Binding Sites as Epitopes Promote Antibody-Mediated Lyme Borreliae Clearance. Infect Immun. 2022;90(7):e0006222. Data were expressed as the proportion of FH binding from serum- treated to PBS-treated wells. The 50% inhibitory concentration (IC50) (the inlet figure of FIG.8C), representing the IgG concentration that blocks 50% of FH binding, was calculated using dose-response stimulation fitting in GraphPad Prism 9.3.1.

[0095] Borreliacidal assays. The ability of serum samples (FIGs.2B-2G) or monoclonal CspZ IgG (#1139c and #1193c, FIG.8D) to eradicate B. burgdorferi B31-A3 was determined as described with modifications. Marcinkiewicz et al. Eliminating Factor H- Binding Activity of Borrelia burgdorferi CspZ Combined with Virus-Like Particle Conjugation Enhances Its Efficacy as a Lyme Disease Vaccine. Front Immunol.2018;9:181; Marcinkiewicz et al. The Factor H-Binding Site of CspZ as a Protective Target against Multistrain, Tick-Transmitted Lyme Disease. Infect Immun.2020;88(5). Briefly, the sera collected from mice immunized with different CspZ-YA proteins at different immunization frequency were heat-treated to inactivate complement. Each of these serum samples or #1139c or #1193c was serially diluted, and mixed with complement-preserved guinea pig serum (Sigma-Aldrich) or heat-inactivated guinea pig serum (negative control). After adding the strain B. burgdorferi B31-A3, the mixture was incubated at 33°C for 24 hours. Surviving spirochetes were quantified by directly counting the motile spirochetes using dark-field microscopy and expressed as the proportion of serum-treated to untreated Lyme borreliae. The 50% borreliacidal activities (BA50), representing the serum dilution rate (for the serum samples in FIGs.2B-2G) or the concentration of IgGs (for #1139c and #1193c in Fig. S5D) that kills 50% of spirochetes, was calculated using dose-response stimulation fitting in GraphPad Prism 9.3.1.

[0096] Quantification of spirochete burdens and histological analysis of arthritis. DNA was extracted from the indicated mouse tissues to determine the bacterial burdens (FIGs.3B-3F, and 9C-9G), using quantitative PCR analysis as described. Marcinkiewicz et al. The Factor H-Binding Site of CspZ as a Protective Target against Multistrain, Tick- Transmitted Lyme Disease. Infect Immun.2020;88(5). Note that spirochete burdens were quantified based on the amplification of recA using the forward and reverse primers with the sequences as GTGGATCTATTGTATTAGATGAGGCTCTCG and GCCAAAGTTCTGCAACATTAACACCTAAAG, respectively. The number of recA copies was calculated by establishing a threshold cycle (Cq) standard curve of a known number of recA gene extracted from strain B31-A3, and burdens were normalized to 100 ng of total DNA. For the ankles that were applied to histological analysis of Lyme disease-associated arthritis (FIG.3G), the analysis was performed as described. Marcinkiewicz et al. The Factor H-Binding Site of CspZ as a Protective Target against Multistrain, Tick-Transmitted LymeDisease. Infect Immun.2020;88(5). The image was scored based on the severity of the inflammation as 0 (no inflammation), 1 (mild inflammation with less than two small foci of infiltration), 2 (moderate inflammation with two or more foci of infiltration), or 3 (severe inflammation with focal and diffuse infiltration covering a large area).

[0097] Surface Plasmon Resonance (SPR). Interactions of CspZ-YA with #1139c or #1193c were analyzed by SPR using a Biacore T200 (Cytiva, Marlborough, MA). Ten micrograms of #1139c or #1193c were conjugated to a Sensor Chip Protein G (Cytiva) by flowing each of these IgGs at the flow rate at 10μl / min, 25oC through that chip using PBS as the buffer. For quantitative SPR experiments, 10µL of increasing concentrations (0, 15, 31.25, 62.5, 125, 250, 500 nM) of CspZ-YA were injected into the control cell and the flow cell immobilized with #1139c or #1193c at 10μl / min, 25oC. To obtain the kinetic parameters of the interaction, sensogram data were fitted by means of BIAevaluation software version 3.0 (GE Healthcare), using the one step biomolecular association reaction model (1:1 Langmuir model), resulting in optimum mathematical fit with the lowest Chi-square values.

[0098] Phagocytosis assays. The phagocytosis assays were performed as described previously with modification. Schuijt et al. A tick mannose-binding lectin inhibitor interferes with the vertebrate complement cascade to enhance transmission of the Lyme disease agent. Cell Host Microbe.2011;10(2):136-46. B. burgdorferi B31-A3 were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE, Invitrogen) as described in vendor’s manual. Basically, the suspension of spirochetes (107) in BSK II media without rabbit sera, gelatin, and BSA was incubated with 3.3 mM of CFSE at room temperature for 10 minutes. To prepare the antibody-treated sera, normal or heat-inactivated human sera that are determined negative to anti-C6 IgGs were incubated with CFSE labeled spirochetes (107bacteria) in the presence of #1139c, #1193c, or irrelevant human IgG (Human IgG isotype control, Sigma-Aldrich) at room temperature for 10 minutes. Such spirochete suspension was then mixed with freshly isolated human neutrophils (PMNs) from a blood donor iQBioscience (Alameda, CA) at the ratio of 25 to 1 and shaking at 37oC, 50 rpm for 10 minutes. For each sample, the bacteria-PMNs mixture incubated on ice for 10 minutes immediately after mixing was included as control. Phagocytosis was stopped by transferring the bacteria-PMN mixtures to ice-cold Fluorescence-Activated Cell Sorting (FACS) buffer (PBS supplemented with 0.5% bovine serum albumin (BSA), 0.01% NaN3 and 0.35 mM EDTA) and stored at 4 °C. Samples continually kept on 4 °C were used as a control. PMNs were then washed suspended with ice-cold FACS-buffer prior to be applied to aFACSCalibur flow cytometer (Beckton Dickinson). The phagocytosis index of each sample was calculated as mean fluorescence intensity (MFI)×percentage (%) positive cells) at 37°C minus (MFI×% positive cells) at 4 °C. Each sample were performed in seven replicates in two different events.

[0099] Accelerated stability study. One µg of untagged CspZ-YA or CspZYAC183S, or histidine-tagged CspZ-YA or CspZYAI183Y was incubated at 4 or 37oC for 6- or 24-h prior to be coated on ELISA plate wells as described

[0033] . The ELISA plate wells immobilized with untagged or histidine-tagged CspZ-YA before incubation were included as control. After blocking those plate wells by PBS with tween 20 as described (Marcinkiewicz et al. The Factor H-Binding Site of CspZ as a Protective Target against Multistrain, Tick-Transmitted Lyme Disease. Infect Immun.2020;88(5)), #1139c or #1193c (1µM), was added to those wells, and the levels of binding between each of these antibodies with CspZ-YA proteins were determined by ELISA as described in the section “ELISAs.” Data were expressed as the proportion of #1139c- or #1193c-binding from the ELISA plate wells immobilized with the CspZ-YA proteins incubated at different conditions to those with the control wells.

[0100] Statistical analyses. Significant differences were determined with a Kruskal- Wallis test with the two-stage step-up method of Benjamini, Krieger, and Yekutieli (Benjamini YK, A. M. Yekutieli, D.. Adaptive linear step-up procedures that control the false discovery rate. Biometrika.2006;93:491-507), two-tailed Fisher test (for seropositivity in FIG.3A) (Fisher RA. Statistical methods for research workers.5th ed: Oliver&Boyd; 1934), or Spearman analysis (for correlation analysis in FIG.4B-4D and 4F-4H) (Spearman C. The proof and measurement of association between two things. Am J Psych.1904;15(1)), using GraphPad Prism 9.3.1. A p-value < 0.05 was used to determine significance.

[0101] As used herein, the term “about” refers to a value that is within a range of from 10% below to 10% above the value being described. For example, the term “about 5 nM” indicates a range of from 4.5 nM to 5.5 nM.

[0102] Although some non-limiting examples have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the present disclosure and these are therefore considered to be within the scope of the present disclosure as defined in the claims that follow.

[0103] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail herein (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matterdisclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein and may be used to achieve the benefits and advantages described herein.

[0104] References 1. Kugeler KJ, Schwartz AM, Delorey MJ, Mead PS, Hinckley AF. 2021. Estimating the Frequency of Lyme Disease Diagnoses, United States, 2010-2018. Emerg Infect Dis 27:616- 619. 2. Steere AC, Strle F, Wormser GP, Hu LT, Branda JA, Hovius JW, Li X, Mead PS.2016. Lyme borreliosis. Nat Rev Dis Primers 2:16090. 3. Gomes-Solecki M, Arnaboldi PM, Backenson PB, Benach JL, Cooper CL, Dattwyler RJ, Diuk-Wasser M, Fikrig E, Hovius JW, Laegreid W, Lundberg U, Marconi RT, Marques AR, Molloy P, Narasimhan S, Pal U, Pedra JHF, Plotkin S, Rock DL, Rosa P, Telford SR, Tsao J, Yang XF, Schutzer SE. 2019. Protective Immunity and New Vaccines for Lyme Disease. Clin Infect Dis doi:10.1093 / cid / ciz872. 4. Embers ME, Narasimhan S.2013. Vaccination against Lyme disease: past, present, and future. Front Cell Infect Microbiol 3:6. 5. Schuijt TJ, Hovius JW, van der Poll T, van Dam AP, Fikrig E. 2011. Lyme borreliosis vaccination: the facts, the challenge, the future. Trends Parasitol 27:40-7. 6. Shen AK, Mead PS, Beard CB. 2011. The Lyme disease vaccine--a public health perspective. Clin Infect Dis 52 Suppl 3:s247-52. 7. Steere AC, Drouin EE, Glickstein LJ.2011. Relationship between immunity to Borrelia burgdorferi outer-surface protein A (OspA) and Lyme arthritis. Clin Infect Dis 52 Suppl 3:s259-65. 8. Radolf JD, Caimano MJ, Stevenson B, Hu LT. 2012. Of ticks, mice and men: understanding the dual-host lifestyle of Lyme disease spirochaetes. Nat Rev Microbiol 10:87- 99. 9. Kurokawa C, Lynn GE, Pedra JHF, Pal U, Narasimhan S, Fikrig E. 2020. Interactions between Borrelia burgdorferi and ticks. Nat Rev Microbiol 18:587-600. 10. Reis ES, Mastellos DC, Hajishengallis G, Lambris JD. 2019. New insights into the immune functions of complement. Nat Rev Immunol 19:503-516. 11. Zipfel PF, Hallstrom T, Riesbeck K. 2013. Human complement control and complement evasion by pathogenic microbes--tipping the balance. Mol Immunol 56:152-60. 12. Blom AM.2017. The role of complement inhibitors beyond controlling inflammation. J Intern Med 282:116-128.13. Ricklin D, Reis ES, Lambris JD. 2016. Complement in disease: a defence system turning offensive. Nat Rev Nephrol 12:383-401. 14. Meri T, Amdahl H, Lehtinen MJ, Hyvarinen S, McDowell JV, Bhattacharjee A, Meri S, Marconi R, Goldman A, Jokiranta TS. 2013. Microbes bind complement inhibitor factor H via a common site. PLoS Pathog 9:e1003308. 15. Lin YP, Diuk-Wasser MA, Stevenson B, Kraiczy P. 2020. Complement Evasion Contributes to Lyme Borreliae-Host Associations. Trends Parasitol 36:634-645. 16. Skare JT, Garcia BL.2020. Complement Evasion by Lyme Disease Spirochetes. Trends Microbiol doi:10.1016 / j.tim.2020.05.004. 17. Dulipati V, Meri S, Panelius J. 2020. Complement evasion strategies of Borrelia burgdorferi sensu lato. FEBS Lett 594:2645-2656. 18. Blom AM, Hallstrom T, Riesbeck K. 2009. Complement evasion strategies of pathogens-acquisition of inhibitors and beyond. Mol Immunol 46:2808-17. 19. Miajlovic H, Smith SG. 2014. Bacterial self-defence: how Escherichia coli evades serum killing. FEMS Microbiol Lett 354:1-9. 20. Guerry P, Poly F, Riddle M, Maue AC, Chen YH, Monteiro MA.2012. Campylobacter polysaccharide capsules: virulence and vaccines. Front Cell Infect Microbiol 2:7. 21. Zipfel PF, Skerka C. 2009. Complement regulators and inhibitory proteins. Nat Rev Immunol 9:729-40. 22. Hartmann K, Corvey C, Skerka C, Kirschfink M, Karas M, Brade V, Miller JC, Stevenson B, Wallich R, Zipfel PF, Kraiczy P.2006. Functional characterization of BbCRASP- 2, a distinct outer membrane protein of Borrelia burgdorferi that binds host complement regulators factor H and FHL-1. Mol Microbiol 61:1220-36. 23. Lin YP, Frye AM, Nowak TA, Kraiczy P. 2020. New Insights Into CRASP-Mediated Complement Evasion in the Lyme Disease Enzootic Cycle. Front Cell Infect Microbiol 10:1. 24. Bykowski T, Woodman ME, Cooley AE, Brissette CA, Brade V, Wallich R, Kraiczy P, Stevenson B.2007. Coordinated expression of Borrelia burgdorferi complement regulator- acquiring surface proteins during the Lyme disease spirochete's mammal-tick infection cycle. Infect Immun 75:4227-36. 25. Tokarz R, Anderton JM, Katona LI, Benach JL. 2004. Combined effects of blood and temperature shift on Borrelia burgdorferi gene expression as determined by whole genome DNA array. Infect Immun 72:5419-32. 26. Brooks CS, Hefty PS, Jolliff SE, Akins DR. 2003. Global analysis of Borrelia burgdorferi genes regulated by mammalian host-specific signals. Infect Immun 71:3371-83.27. Marcinkiewicz AL, Dupuis AP, 2nd, Zamba-Campero M, Nowak N, Kraiczy P, Ram S, Kramer LD, Lin YP.2019. Blood treatment of Lyme borreliae demonstrates the mechanism of CspZ-mediated complement evasion to promote systemic infection in vertebrate hosts. Cell Microbiol 21:e12998. 28. Rogers EA, Marconi RT. 2007. Delineation of species-specific binding properties of the CspZ protein (BBH06) of Lyme disease spirochetes: evidence for new contributions to the pathogenesis of Borrelia spp. Infect Immun 75:5272-81. 29. Rogers EA, Abdunnur SV, McDowell JV, Marconi RT.2009. Comparative analysis of the properties and ligand binding characteristics of CspZ, a factor H binding protein, derived from Borrelia burgdorferi isolates of human origin. Infect Immun 77:4396-405. 30. Kraiczy P, Seling A, Brissette CA, Rossmann E, Hunfeld KP, Bykowski T, Burns LH, Troese MJ, Cooley AE, Miller JC, Brade V, Wallich R, Casjens S, Stevenson B.2008. Borrelia burgdorferi complement regulator-acquiring surface protein 2 (CspZ) as a serological marker of human Lyme disease. Clin Vaccine Immunol 15:484-91. 31. Coleman AS, Yang X, Kumar M, Zhang X, Promnares K, Shroder D, Kenedy MR, Anderson JF, Akins DR, Pal U. 2008. Borrelia burgdorferi complement regulator-acquiring surface protein 2 does not contribute to complement resistance or host infectivity. PLoS One 3:3010e. 32. Marcinkiewicz AL, Lieknina I, Yang X, Lederman PL, Hart TM, Yates J, Chen WH, Bottazzi ME, Mantis NJ, Kraiczy P, Pal U, Tars K, Lin YP. 2020. The Factor H-Binding Site of CspZ as a Protective Target against Multistrain, Tick-Transmitted Lyme Disease. Infect Immun 88. 33. Marcinkiewicz AL, Lieknina I, Kotelovica S, Yang X, Kraiczy P, Pal U, Lin YP, Tars K.2018. Eliminating Factor H-Binding Activity of Borrelia burgdorferi CspZ Combined with Virus-Like Particle Conjugation Enhances Its Efficacy as a Lyme Disease Vaccine. Front Immunol 9:181. 34. Caradonna TM, Schmidt AG. 2021. Protein engineering strategies for rational immunogen design. NPJ Vaccines 6:154. 35. Angeletti D, Gibbs JS, Angel M, Kosik I, Hickman HD, Frank GM, Das SR, Wheatley AK, Prabhakaran M, Leggat DJ, McDermott AB, Yewdell JW. 2017. Defining B cell immunodominance to viruses. Nat Immunol 18:456-463. 36. Altman MO, Angeletti D, Yewdell JW. 2018. Antibody Immunodominance: The Key to Understanding Influenza Virus Antigenic Drift. Viral Immunol 31:142-149.37. Dubrovskaya V, Tran K, Ozorowski G, Guenaga J, Wilson R, Bale S, Cottrell CA, Turner HL, Seabright G, O'Dell S, Torres JL, Yang L, Feng Y, Leaman DP, Vazquez Bernat N, Liban T, Louder M, McKee K, Bailer RT, Movsesyan A, Doria-Rose NA, Pancera M, Karlsson Hedestam GB, Zwick MB, Crispin M, Mascola JR, Ward AB, Wyatt RT. 2019. Vaccination with Glycan-Modified HIV NFL Envelope Trimer-Liposomes Elicits Broadly Neutralizing Antibodies to Multiple Sites of Vulnerability. Immunity 51:915-929 e7. 38. Liu WC, Jan JT, Huang YJ, Chen TH, Wu SC. 2016. Unmasking Stem-Specific Neutralizing Epitopes by Abolishing N-Linked Glycosylation Sites of Influenza Virus Hemagglutinin Proteins for Vaccine Design. J Virol 90:8496-508. 39. Beernink PT, Shaughnessy J, Braga EM, Liu Q, Rice PA, Ram S, Granoff DM. 2011. A meningococcal factor H binding protein mutant that eliminates factor H binding enhances protective antibody responses to vaccination. J Immunol 186:3606-14. 40. Granoff DM, Costa I, Konar M, Giuntini S, Van Rompay KK, Beernink PT. 2015. Binding of Complement Factor H (FH) Decreases Protective Anti-FH Binding Protein Antibody Responses of Infant Rhesus Macaques Immunized With a Meningococcal Serogroup B Vaccine. J Infect Dis 212:784-92. 41. Granoff DM, Giuntini S, Gowans FA, Lujan E, Sharkey K, Beernink PT. 2016. Enhanced protective antibody to a mutant meningococcal factor H-binding protein with low- factor H binding. JCI Insight 1:e88907. 42. Giuntini S, Beernink PT, Granoff DM. 2015. Effect of complement Factor H on anti- FHbp serum bactericidal antibody responses of infant rhesus macaques boosted with a licensed meningococcal serogroup B vaccine. Vaccine 33:7168-75. 43. Rossi R, Granoff DM, Beernink PT. 2013. Meningococcal factor H-binding protein vaccines with decreased binding to human complement factor H have enhanced immunogenicity in human factor H transgenic mice. Vaccine 31:5451-7. 44. Park HI, Yoon HW, Jung ST. 2016. The Highly Evolvable Antibody Fc Domain. Trends Biotechnol 34:895-908. 45. Li X, McHugh GA, Damle N, Sikand VK, Glickstein L, Steere AC. 2011. Burden and viability of Borrelia burgdorferi in skin and joints of patients with erythema migrans or lyme arthritis. Arthritis Rheum 63:2238-47. 46. Wang Y, Kern A, Boatright NK, Schiller ZA, Sadowski A, Ejemel M, Souders CA, Reimann KA, Hu L, Thomas WD, Jr., Klempner MS. 2016. Pre-exposure Prophylaxis With OspA-Specific Human Monoclonal Antibodies Protects Mice Against Tick Transmission of Lyme Disease Spirochetes. J Infect Dis 214:205-11.47. Anonymous. First Clinical Study of the Safety and Blood Levels of a Human Monoclonal Antibody (2217LS) Against Lyme Disease Bacteria in Healthy People. https: / / ClinicalTrials.gov / show / NCT04863287. 48. Hart T, Nguyen NTT, Nowak NA, Zhang F, Linhardt RJ, Diuk-Wasser M, Ram S, Kraiczy P, Lin YP. 2018. Polymorphic factor H-binding activity of CspA protects Lyme borreliae from the host complement in feeding ticks to facilitate tick-to-host transmission. PLoS Pathog 14:e1007106. 49. Barbour AG, Burgdorfer W, Grunwaldt E, Steere AC.1983. Antibodies of patients with Lyme disease to components of the Ixodes dammini spirochete. J Clin Invest 72:504-15. 50. Elias AF, Stewart PE, Grimm D, Caimano MJ, Eggers CH, Tilly K, Bono JL, Akins DR, Radolf JD, Schwan TG, Rosa P.2002. Clonal polymorphism of Borrelia burgdorferi strain B31 MI: implications for mutagenesis in an infectious strain background. Infect Immun 70:2139-50. 51. Steere AC, Grodzicki RL, Kornblatt AN, Craft JE, Barbour AG, Burgdorfer W, Schmid GP, Johnson E, Malawista SE.1983. The spirochetal etiology of Lyme disease. N Engl J Med 308:733-40. 52. Benoit VM, Fischer JR, Lin YP, Parveen N, Leong JM. 2011. Allelic variation of the Lyme disease spirochete adhesin DbpA influences spirochetal binding to decorin, dermatan sulfate, and mammalian cells. Infect Immun 79:3501-9. 53. Purser JE, Norris SJ. 2000. Correlation between plasmid content and infectivity in Borrelia burgdorferi. Proc Natl Acad Sci U S A 97:13865-70. 54. Bunikis I, Kutschan-Bunikis S, Bonde M, Bergstrom S.2011. Multiplex PCR as a tool for validating plasmid content of Borrelia burgdorferi. J Microbiol Methods 86:243-7. 55. Liang FT, Bowers LC, Philipp MT. 2001. C-terminal invariable domain of VlsE is immunodominant but its antigenicity is scarcely conserved among strains of Lyme disease spirochetes. Infect Immun 69:3224-31. 56. Kern A, Zhou CW, Jia F, Xu Q, Hu LT.2016. Live-vaccinia virus encapsulation in pH- sensitive polymer increases safety of a reservoir-targeted Lyme disease vaccine by targeting gastrointestinal release. Vaccine 34:4507-4513. 57. Faul F, Erdfelder E, Lang AG, Buchner A.2007. G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav Res Methods 39:175-91.58. Yang X, Coleman AS, Anguita J, Pal U. 2009. A chromosomally encoded virulence factor protects the Lyme disease pathogen against host-adaptive immunity. PLoS Pathog 5:e1000326. 59. Lin YP, Benoit V, Yang X, Martinez-Herranz R, Pal U, Leong JM. 2014. Strain- specific variation of the decorin-binding adhesin DbpA influences the tissue tropism of the lyme disease spirochete. PLoS Pathog 10:e1004238. 60. Mann HB, Whitney, D.R. 1947. On a test of whether one of two random variables is stochastically larger than the other. Ann Math Stat 18:50-60. 61. Benjamini YK, A. M. Yekutieli, D. . 2006. Adaptive linear step-up procedures that control the false discovery rate. Biometrika 93:491-507. 62. Fisher RA.1934. Statistical methods for research workers, 5th ed. Oliver&Boyd.

[0105] Sequences

[0106] Monoclonal antibody 1139c SEQ ID NO: 1 (VHCDR1): DTYMH SEQ ID NO: 2 (VHCDR2): RIDPANGNTKSDPKFQG SEQ ID NO: 3 (VHCDR3): FIYYGEGYAMDY SEQ ID NO: 4 (VLCDR1): RSSKSLLHSNGITYLY SEQ ID NO: 5 (VLCDR2): QMSNLAS SEQ ID NO: 6 (VLCDR3): AQNLEPPRT SEQ ID NO: 13 (VH): EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRIDPANG NTKSDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCSSFIYYGEGYAMDYWGQ GTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 14 (VL): DIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSN LASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLEPPRTFGGGTKLEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID NO: 17 (VHFR1): EVQLQQSGAELVKPGASVKLSCTASGFNIK SEQ ID NO: 18 (VHFR2): WVKQRPEQGLEWIG SEQ ID NO: 19 (VHFR3): KATITADTSSNTAYLQLSSLTSEDTAVYYCSS SEQ ID NO: 20 (VHFR4): WGQGTSVTVSS SEQ ID NO: 21 (CH): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 22 (VLFR1): DIVMTQAAFSNPVTLGTSASISC SEQ ID NO: 23 (VLFR2): WYLQKPGQSPQLLIY SEQ ID NO: 24 (VLFR3): GVPDRFSSSGSGTDFTLRISRVEAEDVGVYYC SEQ ID NO: 25 (VLFR4): FGGGTKLEIKRTV SEQ ID NO: 26 (CL): AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0107] Monoclonal antibody 1193c SEQ ID NO: 7 (VHCDR1): TSGMGVS SEQ ID NO: 8 (VHCDR2): HIYWDGDTRYNPSLKS SEQ ID NO: 9 (VHCDR3): RGITTATNWFAY SEQ ID NO: 10 (VLCDR1): SASSSVSYMY SEQ ID NO: 11 (VLCDR2): LTSNLAS SEQ ID NO: 12 (VLCDR3): QQWSSDPWT SEQ ID NO: 15 (VH): QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDGD TRYNPSLKSRLTISKDTSSNQIFLKITSVDTADTATYYCARRGITTATNWFAYWGQGT LVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 16 (VL): QIVLTQSPALMSASPGEKVTMTCSASSSVSYMYWYQQKPRSSPKPWIYLTSNLASGV PTRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSDPWTFGVGTKLEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 27 (VHFR1): QVTLKESGPGILQPSQTLSLTCSFSGFSLS SEQ ID NO: 28 (VHFR2): WIRQPSGKGLEWLA SEQ ID NO: 29 (VHFR3): RLTISKDTSSNQIFLKITSVDTADTATYYCAR SEQ ID NO: 30 (VHFR4): WGQGTLVTVSA SEQ ID NO: 31 (CH): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 32 (VLFR1): QIVLTQSPALMSASPGEKVTMTC SEQ ID NO: 33 (VLFR2): WYQQKPRSSPKPWIY SEQ ID NO: 34 (VLFR3): GVPTRFSGSGSGTSYSLTISSMEAEDAATYYC SEQ ID NO: 35 (VLFR4): FGVGTKLEIKRTV SEQ ID NO: 36 (CL): AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0108] Leader sequence SEQ ID NO: 37: MGWSCIILFLVATATGVHS

Claims

WHAT IS CLAIMED IS:

1. An antibody or an antigen-binding fragment thereof capable of specifically binding to a Borrelia burgdorferi CspZ protein, comprising a heavy chain variable region comprising VHCDR1, VHCDR2, and VHCDR3, and a light chain variable region comprising VLCDR1, VLCDR2, and VLCDR3, wherein amino acid sequences of the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 comprise SEQ ID NOs: 1-6, respectively, or SEQ ID NOs: 7-12, respectively.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein amino acid sequences of the VHCDR1, VHCDR2, and VHCDR3, VLCDR1, VLCDR2, and VLCDR3 comprise SEQ ID NOs: 1-6, respectively.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein amino acid sequences of the VHCDR1, VHCDR2, and VHCDR3, VLCDR1, VLCDR2, and VLCDR3 comprise SEQ ID NOs: 7-12, respectively.

4. The antibody or antigen-binding fragment thereof of any one of claims 1 through 3, selected from a Fab fragment, a (Fab′)2 fragment, a scFv fragment, and an IgG antibody.

5. The antibody or antigen-binding fragment thereof of any one of claims 1 through 4, wherein binding of the antibody or antigen-binding fragment thereof to Borrelia burgdorferi CspZ protein inhibits factor H binding to the Borrelia burgdorferi CspZ protein.

6. The antibody or antigen-binding fragment thereof of any one of claims 1 through 5, comprising an antibody.

7. The antibody or antigen-binding fragment thereof of any one of claims 1 through 6, whose heavy chain and light chain amino acid sequences independently have at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 97% sequence identity, at least 99% sequence identity, or 100% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 14, respectively, or whose heavy chain and light amino acid sequences independently have at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 97% sequence identity, at least 99% sequence identity, or 100% sequence identity with SEQ ID NO: 15 or SEQ ID NO: 16, respectively.

8. The antibody or antigen-binding fragment thereof of any one of claims 1 through 6, whose heavy chain and light chain amino acid sequences independently have at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, atleast 95% sequence identity, at least 97% sequence identity, at least 99% sequence identity, or 100% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 14, respectively.

9. The antibody or antigen-binding fragment thereof of any one of claims 1 through 6 whose heavy chain and light amino acid sequences independently have at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 97% sequence identity, at least 99% sequence identity, or 100% sequence identity with SEQ ID NO: 15 or SEQ ID NO: 16, respectively.

10. The antibody or antigen-binding fragment thereof of any one of claims 1 through 6, selected from monoclonal antibody 1193c and monoclonal antibody 1139c.

11. The antibody or antigen-binding fragment thereof of any one of claims 1 through 10, wherein he antibody is bactericidal for Lyme-causing Borreliae bacteria.

12. The antibody or antigen-binding fragment thereof of any one of claims 1 through 11, wherein administrating the antibody or antigen-binding fragment thereof to a subject infected with a Lyme-causing Borreliae bacteria reduces seropositivity of the subject for the Lyme-causing Borreliae bacteria compare to a control subject infected with a Lyme- causing Borreliae bacteria but not administered the antibody or antigen-binding fragment thereof.

13. A pharmaceutical composition, comprising the antibody or antigen-binding fragment thereof of any one of claims 1 through 12 and a pharmaceutically acceptable excipient.

14. A method of treatment, comprising administering an antibody or antigen- binding fragment thereof of any one of claims 1 through 12 or the pharmaceutical composition of claim 13 to a subject in need of such treatment.

15. The method of claim 14, wherein the subject is a human.

16. The method of claim 14 or 15, wherein the treatment comprises administering the antibody or antigen-binding fragment thereof to the subject before the subject is or may be infected with a Lyme-causing Borreliae bacteria.

17. The method of any one of claims 14 through 16, wherein the treatment comprises administering the antibody or antigen-binding fragment thereof to the subject after the subject was or may have been infected with a Lyme-causing Borreliae bacteria.

18. The method of claim 16 or 17, wherein the Lyme-causing Borreliae bacteria is Borrelia burgdorferi or Borrelia afzelii, 19. The method of any one of claims 14 through 18, wherein the subject has Lyme disease.

20. A nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1 through 12.

21. A cell comprising the nucleic acid of claim 20.

22. A vector comprising the nucleic acid of claim 20.

23. A cell expressing the antibody or antigen-binding fragment thereof of any one of claims 1 through 12.

24. A cell transfected with the vector of claim 23.

25. A hybridoma or engineered cell encoding the antibody or antigen-binding fragment thereof of any one of claims 1 through 12.

Images