Lyme disease vaccine and method of use thereof

Abstract

Provided is an immunogenic composition for use in raising immunity against the agent of Lyme disease. The composition is based on variants of CspZ and comprises a polypeptide of SEQ ID NO: 1, except that (a) the amino acid whose position corresponds to amino acid 188 of SEQ ID NO: 1 is any amino acid other than tyrosine; and the amino acid whose position corresponds to amino acid 192 of SEQ ID NO: 1 is any amino acid other than tyrosine; and (b) one or both of the amino acids acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 may be an amino acid other than inosine and an amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 may be an amino acid other than cysteine. Also provided is a polynucleotide encoding any of the foregoing polypeptides and a method of vaccinating against Lyme disease comprising administering the polypeptide or RNA encoding the polypeptide to a subject.

Description

LYME DISEASE VACCINE AND METHOD OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a which claims benefit of priority from U.S. Provisional Patent Application No.63 / 709.040, 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 0332098AWO_Sequence_Listing_XML.xml; Size: 21,887 bytes; and Date of Creation: October 17, 2025, is herein incorporated by reference in its entirety. BACKGROUND

[0004] Active immunization aims to trigger host immunity in eliminating pathogens during current and subsequent infection. Some surface antigens from infectious agents are less immunogenic, resulting in inefficient pathogen-elimination for the benefit of survival. Lyme disease is the most common vector-borne disease in the Northern hemisphere, with the number of human cases continuously rising (approximately 476,000 cases in the U.S. reported on 2022), while no effective prevention is commercially available).

[0005] As causative agents, multiple species of the spirochete bacteria, Borrelia burgdorferi sensu lato (also known as Borrelia burgdorferi or Lyme borreliae) are carried by infected Ixodes ticks and migrate to vertebrate hosts through tick bites. Amongst those Lyme borreliae species, B. burgdorferi sensu stricto (hereafter B. burgdorferi) is the most prevalent human infectious Lyme borreliae species in North America whereas other human infectious species (e.g., B. afzelii, B. garinii, and B. bavariensis) are prevalent in Eurasia. Upon invasion, Lyme borreliae establish colonization in the tick bite sites of skin and disseminatethrough the bloodstream to distal organs, causing arthritis, carditis and / or neurological symptoms (i.e., neuroborreliosis).

[0006] A human Lyme disease vaccine (LYMERIXTM) was commercialized 20 years ago but then withdrawn from market because of various concerns. A second-generation vaccine is under clinical trial. However, these vaccines target a Lyme borreliae protein, OspA, which is solely produced when bacteria are in the ticks but not after bacteria invade humans. That results in no memory, anti-OspA immune responses after pathogen invasion. Therefore, maintaining long-lasting, effective titers of pathogen-killing antibodies is required to allow any OspA-targeted vaccines efficacious. Such a requirement has reflected the need of constant boosters for OspA-targeted vaccines to maintain protective levels of antibodies, a significant challenge of Lyme disease vaccine development

[0018] .

[0007] Lyme disease bacteria produce an outer surface protein, CspZ (also known as BbCRASP-2). CspZ promotes bacterial dissemination to distal tissues by evading complement, the first line innate immune defense from vertebrate animals in the blood, through binding and recruiting a host complement inhibitor, factor H (FH). Although CspZ is not found in every Lyme borreliae strains

[0025] , serologically confirmed and / or symptomatic human Lyme disease patients in North America and Eurasia all develop elevated levels of antibodies that recognize CspZ [26, 27]. For example, 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). These findings suggest the production of CspZ in most human infectious Lyme borreliae strains or species. Additionally, CspZ is only produced after Lyme borreliae invade vertebrate hosts, likely by triggering enhanced memory immune responses, underscoring the potential of targeting this protein as an alternative Lyme disease vaccine candidate [28, 29]. However, vaccination with CspZ did not protect mice from Lyme borreliae colonization and Lyme disease-associated manifestations [26, 30-32], indicating the need to engineer this antigen in improving the protective immune responses.

[0008] The present disclosure is directed to overcoming these and other deficiencies in the art. SUMMARY

[0009] Provided is an immunogenic composition, including a polypeptide of SEQ ID NO: 1, except that: an amino acid whose position corresponds to amino acid 188 of SEQ ID NO: 1 may include any amino acid other than tyrosine and an amino acid whose positioncorresponds to amino acid 192 of SEQ ID NO: 1 may include any amino acid other than tyrosine; and one or both of (a) an amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 may include an amino acid other than inosine and (b) an amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 may include an amino acid other than cysteine. The amino acid whose position corresponds to amino acid 188 of SEQ ID NO: 1 may include alanine. The amino acid whose position corresponds to amino acid 192 of SEQ ID NO: 1 may include alanine. The amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 may include tyrosine. The amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 may include serine.

[0010] The amino acid whose position corresponds to amino acid 188 of SEQ ID NO: 1 may include alanine and the amino acid whose position corresponds to amino acid 192 of SEQ ID NO: 1 may include alanine. The amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 may include an amino acid other than inosine and the amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 may include an amino acid other than cysteine. The amino acid whose position corresponds to amino acid 164 or SEQ ID NO: 1 may include tyrosine and the amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 may include serine.

[0011] The amino acid whose position corresponds to amino acid 188 of SEQ ID NO: 1 may include alanine, the amino acid whose position corresponds to amino acid 192 of SEQ ID NO: 1 may include alanine, the amino acid whose position corresponds to amino acid 164 or SEQ ID NO: 1 may include tyrosine, and the amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 may include serine.

[0012] The amino acid sequence may be selected from that of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, or as encoded by a polynucleotide sequence including that of SEQ ID NO: 3 or SEQ ID NO: 14.

[0013] The immunogenic composition may further including one or more adjuvant. The one or more adjuvant may be covalently attached to the polypeptide. One or more of the one or more adjuvant may not be covalently attached to the polypeptide. One or more of the one or more adjuvant may be selected from aluminum salt, AS04, AS03, monophosphoryl lipid A, poly(I:C), a CpG DNA adjuvant, MF59, an emulsion adjuvant including squalene and water, a combination adjuvant including block copolymer CRL-8300, squalene, a sorbitan monooleateor, N-[1-(2,3-Dioleoyloxy)propyl] -N,N,N-trimethylammonium salt (DOTAP), 3 β-[N-(N',N'-dimethylaminoethane) -carbamoyl] cholesterol (DC-chol liposome), an aluminum hydroxide wet gel suspension, α-galactosylceramide, and a virosomal adjuvant.

[0014] One or more of: an amino acid whose position corresponds to amino acid 34 of SEQ ID NO: 1 may include any amino acid other than cysteine; an amino acid whose position corresponds to amino acid 48 of SEQ ID NO: 1 may include any amino acid other than threonine; an amino acid whose position corresponds to amino acid 61 of SEQ ID NO: 1 may include any amino acid other than inosine; an amino acid whose position corresponds to amino acid 86 of SEQ ID NO: 1 may include any amino acid other than phenylalanine; an amino acid whose position corresponds to amino acid 96 of SEQ ID NO: 1 may include any amino acid other than inosine; an amino acid whose position corresponds to amino acid 117 of SEQ ID NO: 1 may include any amino acid other than lysine; an amino acid whose position corresponds to amino acid 123 of SEQ ID NO: 1 may include any amino acid other than valine, and an amino acid whose position corresponds to amino acid 174 of SEQ ID NO: 1 may include any amino acid other than glycine.

[0015] One or more of: the amino acid whose position corresponds to amino acid 34 of SEQ ID NO: 1 may include serine; the amino acid whose position corresponds to amino acid 48 of SEQ ID NO: 1 may include proline; the amino acid whose position corresponds to amino acid 61 of SEQ ID NO: 1 may include threonine; the amino acid whose position corresponds to amino acid 86 of SEQ ID NO: 1 may include proline; the amino acid whose position corresponds to amino acid 96 of SEQ ID NO: 1 may include threonine; the amino acid whose position corresponds to amino acid 117 of SEQ ID NO: 1 may include glutamic acid; the amino acid whose position corresponds to amino acid 123 of SEQ ID NO: 1 may include methionine; and the amino acid whose position corresponds to amino acid 174 of SEQ ID NO: 1 may include methionine.

[0016] From one to thirty six, from one to twenty four, from one to sixteen, from one to eight, or from one to four amino acids whose position corresponds to an amino acid of from 1-163 in SEQ ID NO: 1 may not be the same as the corresponding amino acid in SEQ ID NO: 1. From one to four, from one to three, of from one to two amino acids corresponding to amino acids whose position corresponds to amino acid of from 193-207 in SEQ ID NO: 1 may not be the same as the corresponding amino acid in SEQ ID NO: 1.

[0017] Provided is a method of vaccinating a subject, including administering to the subject the immunogenic composition. The method may include treating a subject having Lyme disease or suspected of having been infected with or exposed to Borrelia burgdorferi, including administering to the subject a composition including the immunogenic composition. Administering may include administering the immunogenic composition more than once. The method may include administering the composition at least two times.Administration of the immunogenic composition fewer than three times may prevent the subject from contracting Lyme disease. The subject may be a rodent, a cat, a dog, a cattle, or a human.

[0018] Provided is a composition, including a means for immunizing a subject against a CspZ-expressing species of Borrelia, and one or more adjuvant, wherein administering the immunogenic composition to a subject fewer than three times prevents exposure of the subject to the CspZ-expressing species of Borrelia from causing Lyme disease in the subject.

[0019] One or more of the one or more adjuvant may be covalently attached to the polypeptide. One or more of the one or more adjuvant may not be not covalently attached to the polypeptide. One or more of the one or more adjuvant may be selected from aluminum salt, AS04, AS03, monophosphoryl lipid A, poly(I:C), a CpG DNA adjuvant, MF59, an emulsion adjuvant including squalene and water, a combination adjuvant including block copolymer CRL-8300, squalene, a sorbitan monooleateor, N-[1-(2,3-Dioleoyloxy)propyl] - N,N,N-trimethylammonium salt (DOTAP), 3 β-[N-(N',N'-dimethylaminoethane) -carbamoyl] cholesterol (DC-chol liposome), an aluminum hydroxide wet gel suspension, α- galactosylceramide, and a virosomal adjuvant.

[0020] Provided is a polynucleotide encoding any foregoing polypeptide. The polynucleotide sequence may include that of SEQ ID NO: 3. The polynucleotide may further include a signal peptide. The signal peptide may direct the polypeptide for secretion by a cell when the polypeptide is expressed in the cell. The signal peptide may be selected from a signal peptide of immunoglobulin kappa, a signal peptide of tissue plasminogen activator, a signal peptide of serum albumin, a signal peptide of growth factor, and a signal peptide of interleukin 2. The signal peptide may be a serum albumin signal peptide. The signal peptide may be human serum albumin signal peptide. The signal peptide sequence may include that of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:12, or SEQ ID NO: 13.

[0021] The polynucleotide sequence may include that of SEQ ID NO: 3. The polynucleotide sequence may include that of SEQ ID NO: 14. The polynucleotide may include RNA. Provided is a cell including the polynucleotide. Provided is a vector including the polynucleotide. The vector may include a lipid nanoparticle, a viral vector, or a plasmid.

[0022] Provided is a method of vaccinating a subject, including administering to the subject a composition including the polynucleotide. Provided is a method of treating a subject having Lyme disease or suspected of having been infected with or exposed to Borrelia burgdorferi, including administering to the subject a composition including thepolynucleotide. Administering may include administering the immunogenic composition more than once. The method may include administering the composition at least two times. Administration of the composition fewer than three times may the subject from contracting Lyme disease. The subject may be a rodent, a cat, a dog, a cattle, or a human.

[0023] Provided is an immunogenic composition, including a polypeptide of SEQ ID NO: 1, except that: an amino acid whose position corresponds to amino acid 188 of SEQ ID NO: 1 may include any amino acid other than tyrosine and an amino acid whose position corresponds to amino acid 192 of SEQ ID NO: 1 may include any amino acid other than tyrosine; and one or both of (a) an amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 may include an amino acid other than inosine and (b) an amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 may include an amino acid other than cysteine; and an aluminum salt adjuvant and a CpG adjuvant. The aluminum salt adjuvant may include aluminum hydroxide. The aluminum salt adjuvant may include an aluminum hydroxide wet gel suspension or AS04.

[0024] The amino acid whose position corresponds to amino acid 188 of SEQ ID NO: 1 may include alanine. The amino acid whose position corresponds to amino acid 192 of SEQ ID NO: 1 may include alanine. The amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 may include tyrosine. The amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 may include serine. The amino acid whose position corresponds to amino acid 188 of SEQ ID NO: 1 may include alanine and the amino acid whose position corresponds to amino acid 192 of SEQ ID NO: 1 may include alanine.

[0025] The amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 may include an amino acid other than inosine and the amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 may include an amino acid other than cysteine. The amino acid whose position corresponds to amino acid 164 or SEQ ID NO: 1 may include tyrosine and the amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 may include serine. The amino acid whose position corresponds to amino acid 188 of SEQ ID NO: 1 may include alanine, the amino acid whose position corresponds to amino acid 192 of SEQ ID NO: 1 may include alanine, the amino acid whose position corresponds to amino acid 164 or SEQ ID NO: 1 may include tyrosine, and the amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 may include serine.

[0026] The immunogenic composition may have an amino acid sequence selected from that of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.

[0027] The immunogenic composition may further include one or more additional adjuvant. The one or more of the one or more additional adjuvant may covalently attached to the polypeptide. The one or more additional adjuvant may not be covalently attached to the polypeptide. The one or more additional adjuvant may be selected from AS03, monophosphoryl lipid A, poly(I:C), MF59, an emulsion adjuvant including squalene and water, a combination adjuvant including block copolymer CRL-8300, squalene, a sorbitan monooleateor, N-[1-(2,3-Dioleoyloxy)propyl] -N,N,N-trimethylammonium salt (DOTAP), 3 β-[N-(N',N'-dimethylaminoethane) -carbamoyl] cholesterol (DC-chol liposome), an aluminum hydroxide wet gel suspension, α-galactosylceramide, and a virosomal adjuvant.

[0028] One or more of an amino acid whose position corresponds to amino acid 34 of SEQ ID NO: 1 may include any amino acid other than cysteine, an amino acid whose position corresponds to amino acid 48 of SEQ ID NO: 1 may include any amino acid other than threonine, an amino acid whose position corresponds to amino acid 61 of SEQ ID NO: 1 may include any amino acid other than inosine, an amino acid whose position corresponds to amino acid 86 of SEQ ID NO: 1 may include any amino acid other than phenylalanine, an amino acid whose position corresponds to amino acid 96 of SEQ ID NO: 1 may include any amino acid other than inosine, an amino acid whose position corresponds to amino acid 117 of SEQ ID NO: 1 may include any amino acid other than lysine, an amino acid whose position corresponds to amino acid 123 of SEQ ID NO: 1 may include any amino acid other than valine, and an amino acid whose position corresponds to amino acid 174 of SEQ ID NO: 1 may include any amino acid other than glycine.

[0029] One or more of the amino acid whose position corresponds to amino acid 34 of SEQ ID NO: 1 may include serine, the amino acid whose position corresponds to amino acid 48 of SEQ ID NO: 1 may include proline, the amino acid whose position corresponds to amino acid 61 of SEQ ID NO: 1 may include threonine, the amino acid whose position corresponds to amino acid 86 of SEQ ID NO: 1 may include proline, the amino acid whose position corresponds to amino acid 96 of SEQ ID NO: 1 may include threonine, the amino acid whose position corresponds to amino acid 117 of SEQ ID NO: 1 may include glutamic acid, the amino acid whose position corresponds to amino acid 123 of SEQ ID NO: 1 may include methionine, and the amino acid whose position corresponds to amino acid 174 of SEQ ID NO: 1 may include methionine.

[0030] From one to thirty six, from one to twenty four, from one to sixteen, from one to eight, or from one to four amino acids whose position corresponds to an amino acid of from 1-163 in SEQ ID NO: 1 may not be the same as the corresponding amino acid in SEQID NO: 1. From one to four, from one to three, of from one to two amino acids corresponding to amino acids whose position corresponds to amino acid of from 193-207 in SEQ ID NO: 1 may not be the same as the corresponding amino acid in SEQ ID NO: 1.

[0031] Provided is a method of vaccinating a subject, including administering to the subject the immunogenic composition. The method may include treating a subject having Lyme disease or suspected of having been infected with or exposed to Borrelia burgdorferi, including administering to the subject a composition including the immunogenic composition. Administering may include administering the immunogenic composition more than once. The method may include administering the composition at least two times. Administration of the immunogenic composition fewer than three times may prevent the subject from contracting Lyme disease. The subject may be a rodent, a cat, a dog, a cattle, or a human.

[0032] Provided is a composition, including a means for immunizing a subject against a CspZ-expressing species of Borrelia, and an aluminum salt adjuvant and a CpG adjuvant, wherein administering the immunogenic composition to the subject fewer than three times may prevent exposure of the subject to the CspZ-expressing species of Borrelia from causing Lyme disease in the subject. The aluminum salt adjuvant may include aluminum hydroxide. The aluminum salt adjuvant may include an aluminum hydroxide wet gel suspension or AS04.

[0033] The immunogenic composition may further include one or more additional adjuvant, wherein one or more of the one or more adjuvant is selected from AS03, monophosphoryl lipid A, poly(I:C), MF59, an emulsion adjuvant including squalene and water, a combination adjuvant including block copolymer CRL-8300, squalene, a sorbitan monooleateor, N-[1-(2,3-Dioleoyloxy)propyl] -N,N,N-trimethylammonium salt (DOTAP), 3 β-[N-(N',N'-dimethylaminoethane) -carbamoyl] cholesterol (DC-chol liposome), an aluminum hydroxide wet gel suspension, α-galactosylceramide, and a virosomal adjuvant. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] 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:

[0035] Figure 1. The high-resolution structures of CspZ-YA and the mutagenesis of amino acids in CspZ-YA by structure-based vaccine design. (A) Crystal structure of CspZ- YA (gray; PDB ID 9F1V) is superimposed with B. burgdorferi B31 CspZ (blue) from CspZand SCR6-7 (gold) complex (PDB ID 9F7I; rmsd 0.9 Å) and B. burgdorferi B31 CspZ (green) from CspZ and SCR7 (red) complex (PDB ID 6ATG; rmsd 0.74 Å). The inlet figures showed the loop region between helices H and I helices, indicating the residues Tyr207 and Tyr211 in CspZ from B. burgdorferi strain B31 and the mutated residues Ala207 and Ala211 in CspZ-YA. Residues Lys212 and Lys213 found in the loop region in CspZ and in the extended helix I in CspZ-YA are indicated. The interaction between residues Arg206 and Glu186 in CspZ is indicated. The structure is presented from top and side views. (B) Design landscape of CspZ-YA (PDB ID 9F1V) shown as a ribbon diagram with the side chains of mutations shown as spheres. Insets highlight the position of selected stabilizing mutations. Side chains in each inset are shown as dark red sticks with sulfur atoms in yellow, nitrogen atoms in blue and oxygen atoms in red.

[0036] Figure 2. Table showing that mice repeatedly immunized with CspZ- YAC187S or CspZ-YAI183Y had sera with more robust levels of borreliacidal activity than that from CspZ-YA-vaccinated mice.

[0037] Figure 3. CD spectra demonstrate no impacts of secondary structures by mutating indicating amino acids of CspZ-YA. Far-UV CD analysis of (A) untagged CspZ- YA (CspZ-YAUT) and CspZ-YAC187S (CspZ-YAC187SUT), and (B) histidine tagged CspZ- YA and the mutant proteins derived from this protein. The molar ellipticity, Φ, was measured from 190-250nm for 10μM of each protein in PBS.

[0038] Figure 4. Figure showing that mice repeatedly immunized with CspZ-YAC187Sor CspZ-YAI183Y had sera with more robust levels of borreliacidal activity than that from CspZ-YA-vaccinated mice. (A) C3H / HeN mice received inoculation of PBS (control) or immunization of CspZ-YA or the mutant proteins derived from this protein at 0 day for the group of mice that were immunized once. The second group of mice that were immunized twice received the abovementioned proteins or PBS at 14 days initial immunization (dpii). The third group of mice that were immunized three times received the abovementioned proteins or PBS at 14 and 28 dpii. At 14 days post last immunization (14dpli), sera from these mice were collected for analyses of the titers of CspZ IgG and bactericidal activities. At 21 dpli, nymphal ticks carrying B. burgdorferi B31-A3 were placed on those mice and allowed to feed until repletion. Mice were sacrificed at 42 dpli for seropositivity, histopathology, and bacterial burden quantification. Mice inoculated with PBS that are not fed on by nymphs were included as an uninfected control group. (B to G) Sera were collected at 14dpli from C3H / HeN mice immunized (B and C) once, (D and E) twice, or (F and G) three times. These mice were immunized with PBS (control) or untagged CspZ-YA (CspZ-YAUT) or its derivedmutant proteins, or histidine tagged CspZ-YA (CspZ-YA), or its derived mutant proteins (Six mice for CspZ-YAUT- or CspZ-YAC187SUT-immunized mice whereas five mice for the rest of immunization groups of mice). These sera were serially diluted as indicated, and mixed with guinea pig complement and B. burgdorferi B31-A3 (5 × 105 cells ml-1). After being 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. (A, C, and E) The survival percentage was derived from the proportion of serum-treated to untreated spirochetes. Data shown are the mean ± SEM of the survival percentage from three replicates in one representative experiment. (B, D, and F) The 50% borreliacidal dilution of each serum sample, representing the dilution rate that effectively killed 50% of spirochetes, was obtained from curve-fitting and extrapolation of Panel A, C, and E. Data shown are the geometric mean ± geometric standard deviation of the borreliacidal titers from three experiments. The exact values are shown in Figure 2. PBS- inoculated mouse sera displayed no bactericidal activity (“NK”, no killing). N.d. indicates “not determined.” Statistical significance (p < 0.05, Kruskal Wallis test with the two-stage step-up method of Benjamini, Krieger, and Yekutieli) of differences in borreliacidal titers between groups are indicated (“#”).

[0039] Figure 5. Immunization twice with CspZ-YAC187S or CspZ-YAI183Y but not CspZ-YA protected mice from seroconversion, borrelial tissue colonization, and Lyme disease-associated arthritis. Five PBS- or lipidated OspA (OspA)-, or histidine tagged CspZ- YA- , or CspZ-YAI183Y (I183Y)-, or CspZ-YAI183Y / C187S (I183Y / C187S)- or six untagged CspZ-YA (CspZ-YAUT)- or CspZ-YAC187S(C187S)-immunized C3H / HeN mice that were immunized twice in the fashion described in Fig.1 by indicated proteins. At 21days post last immunization, these mice were then fed on by nymphs carrying B. burgdorferi B31-A3. Mice inoculated with PBS that are not fed on by nymphs were included as an uninfected control group (uninfect.). (A) Seropositivity was determined by measuring the levels of IgG against C6 peptides in the sera of those mice at 42 days post last immunization using ELISA. The mouse was considered as seropositive if that mouse had IgG levels against C6 peptides greater than the threshold, the mean plus 1.5-fold standard deviation of the IgG levels against C6 peptides from the PBS-inoculated, uninfected mice (dotted line). The number of mice in each group with the anti-C6 IgG levels greater than the threshold (seropositive) is shown. Data shown are the geometric mean ± geometric 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 are presented. N.d. indicates “not determined.” (B to F) B. burgdorferi (Bb) burdens at (B) nymphs after when feeding to repletion or (C) the tick feeding site (“Inoc. Site”), (D) bladder, (E) heart, and (F) knees, were quantitatively measured at 42 days post last immunization, shown as the number of Bb per 100ng total DNA. Data shown are the geometric mean ± geometric standard deviation of the spirochete burdens from each group of mice. Asterisks indicate the statistical significance (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. (G) Tibiotarsus joints at 42 days post last immunization were collected to assess inflammation by staining these tissues using hematoxylin and eosin. Representative images from one mouse per group are shown. Top panels are lower-resolution images (joint, ×10 [bar, 160 µm]); bottom panels are higher-resolution images (joint, 2×20 [bar, 80 µm]) of selected areas (highlighted in top panels). Arrows indicate infiltration of immune cells. (Inlet figure) To quantitate inflammation of joint tissues, at least ten random sections of tibiotarsus joints from each mouse were scored on a scale of 0-3 for the severity of arthritis. Data shown are the mean inflammation score ± standard deviation of the arthritis scores from each group of mice. Asterisks indicate the statistical significance (p < 0.05, Kruskal Wallis test with the two-stage step-up method of Benjamini, Krieger, and Yekutieli) of differences in inflammation relative to uninfected mice.

[0040] Figure 6. Immunization once or three times with CspZ-YA, CspZ-YAC187S, or CspZ-YAI183Yshowed indistinguishable protectivity for seroconversion and borrelial tissue colonization. Five PBS- or lipidated OspA (OspA)-, or histidine tagged CspZ-YA- or CspZ- YAI183Y(I183Y)-, or six untagged CspZ-YA (CspZ-YAUT)- or CspZ-YAC187S(C187S)- immunized C3H / HeN mice that were immunized (A to F) once or (G to L) three times by indicated proteins in the fashion as described in Fig.1. At 21 days post last immunization, these mice were then fed on by nymphs carrying B. burgdorferi B31-A3. Mice inoculated with PBS that are not fed on by nymphs were included as an uninfected control group (uninfect.). (A and G) Seropositivity was determined by measuring the levels of IgG against C6 peptides in the sera of those mice at 42 days post last immunization using ELISA. The mouse was considered as seropositive if that mouse had IgG levels against C6 peptides greater than the threshold, the mean plus 1.5-fold standard deviation of the IgG levels against C6 peptides from the PBS-inoculated, uninfected mice (dotted line). The number of mice in each group with the anti-C6 IgG levels greater than the threshold (seropositive) is shown. Data shown are the geometric mean ± geometric standard deviation of the titers of anti-C6 IgG. Statistical significances (p < 0.05, Kruskal-Wallis test with the two-stage step-upmethod of Benjamini, Krieger, and Yekutieli) of differences in IgG titers relative to (*) uninfected mice are presented. (B to F, H to L) B. burgdorferi (Bb) burdens at (B and H) nymphs after when feeding to repletion or (C and I) the tick feeding site (“Inoc. Site”), (D and J) bladder, (E and K) heart, and (F and L) knees, were quantitatively measured at 42 days post last immunization, shown as the number of Bb per 100ng total DNA. Data shown are the geometric mean ± geometric standard deviation of the spirochete burdens from each group of mice. Asterisks indicate the statistical significance (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.

[0041] Figure 7. More than 90% of Lyme disease human patients develop CspZ antibodies. Sera from 38 patients with seropositive for Lyme disease infection (Two tier pos.; Positive in Two tier test) were determined for the titers of antibodies that recognize CspZ using ELISA, as described in the section “ELISA” of the Materials and Methods. Ten serum samples from humans residing in non-endemic area of Lyme disease were included as negative control (Neg. ctrl.) and to set up the threshold value of titers that can be used to determine CspZ antibody positivity. That threshold value was mean 1.5-folds of standard deviation extrapolated from the values of negative control human sera. Thirty six out of 38 serum samples (94.7%) yield greater anti-CspZ IgG titers than the threshold values and was thus considered positive for CspZ antibodies. Shown is the geometric mean ± geometric standard deviation of the titers. Statistical significance (p < 0.05, Kruskal Wallis test with the two-stage step-up method of Benjamini, Krieger, and Yekutieli) of differences in CspZ-IgG titers between groups are indicated (“#”).

[0042] Figure 8. CspZ antibodies originated from humans or mice recognized CspZ- YAC187Sor CspZ-YAI183Yat indistinguishable levels from CspZ-YA. (A to D) Sera from 36 patients with both seropositive for Lyme disease (“Two tier positive”; Positive in two tier test) in Fig.7 were included. (A) These sera were applied to determined their levels of recognition to histidine tagged CspZ-YA, CspZ-YAC187S or CspZI183Y using ELISA as described in the section “ELISA” in Materials and Methods. Ten serum samples from humans residing in non-endemic area of Lyme disease were included as negative control. Data shown are the geometric mean ± geometric standard deviation of levels of recognition in each group of serum samples. Statistical significance (p < 0.05, Kruskal Wallis test with the two-stage step-up method of Benjamini, Krieger, and Yekutieli) of differences in levels of recognition by groups are indicated (“#”).(E to H) Sera from five histidine tagged CspZ-YA- or CspZ-YAI183Y (I183Y)-, or six untagged CspZ-YA (CspZ-YAUT)- or CspZ-YAC187S(C187S)-immunized C3H / HeN mice that were immunized twice in the fashion described in Fig.1 were collected at 14dpli. PBS-inoculated mice were included as control. For each serum sample, the levels of its recognition by histidine tagged CspZ-YA, CspZ-YAC187S or CspZ-YAI183Y were measured using ELISA. Data shown are the geometric mean ± geometric standard deviation of levels of recognition. Statistical significance (p < 0.05, Kruskal Wallis test with the two-stage step-up method of Benjamini, Krieger, and Yekutieli) of differences in levels of recognition by groups are indicated (“#”). For each serum sample originated from (B to D) humans or (F to H) mice, the values the levels ofrecognition by (B and F) CspZ-YA vs. CspZ-YAC187S, (C CspZ-YA vs. CspZ- YAI183Y, or (D and H) CspZ-YAC187Svs. CspZ-YAI183Ywere plotted. The correlation of these values derived from recognition by each of indicated CspZ-YA proteins was quantitatively determined using Spearman analysis and shown as R values. P values are also shown to demonstrate the statistical significance (p < 0.05, Spearman analysis) of the correlation between indicated values in X- and Y-axis in panel B to D and F to H. (I) Superimposed crystal structures of CspZ-YA (gray; PDB ID 9F1V), CspZ-YAC187S (brown; PDB ID 9F21) and the predicted structure of CspZ-YAI183Y (green). Side chains as thin bonds in all three proteins are illustrated. (inlet figure) Shown is the region in CspZ-YA, CspZ-YAC187S and CspZ-YAI183Ywhere mutations (Ala207, Ala211, Cys187, Ser187, Ile183 and Tyr183) were introduced. Residues associated with mutations are illustrated as thick bonds, but all other residues in all three proteins are represented as thin bonds. All the interactions observed between the amino acid side chains in CspZ-YA are indicated as dotted lines.

[0043] Figure 9. The comparison of CspZ, CspZ-YA, CspZ-YAC187S, and CspZ- YAI183Y structures suggest the protein stability impacted by the C187S and I183Y mutagenesis. (A) Shown is the top view of the superimposed structures for CspZ (blue; PDB ID 9F7I), CspZ-YAUT (gray; PDB ID 9F1V), CspZ-YAC187SUT (brown; PDB ID 9F21), and AlphaFold predicted structure of CspZ-YAI183Y(green). (B to C) Shown is the region around buried Cys187 in (B) CspZ-YA and (C) CspZ-YAC187S. Distances in angstroms to the closest atoms in the neighboring side chains are indicated. (D to F) The crystal structures of (D) CspZ from B. burgdorferi B31, (E) CspZ-YA, and (F) the predicted structure of CspZ- YAI183Y show the hydrophobic core accounting for helices G, H and I and the residues Tyr207, Tyr211, Ile183, and Cys187 in CspZ and the equivalent residues in CspZ-YA and CspZ-YAI183Y.

[0044] Figure 10. CspZ-YAC187S and CspZ-YAI183Y maintained the recognition by protective CspZ IgGs at higher temperature for longer period of time. (A and B) UntaggedCspZ-YA (CspZ-YAUT) or CspZ-YAC187S(CspZ-YAC187SUT) or histidine tagged CspZ-YA or CspZ-YAI183Y (10µM) in PBS buffer were subjected to the thermoshift assays described in the materials and methods. (A) Shown is the fluorescence intensities of each of the CspZ-YA proteins under the temperatures ranging from 25 to 99oC from one representative experiment. (B) The melting temperature (Tm) was extrapolated from the maximal positive derivative values of the fluoresces intensity (d(RFU) / dT) as bars. Data shown are the mean ± standard deviation of the Tm values for each of the CspZ-YA proteins from eight experiments. Statistical significance (p < 0.05, Kruskal Wallis test with the two-stage step-up method of Benjamini, Krieger, and Yekutieli) of differences in percent binding between groups are indicated (“#”). (C and D) One µg CspZ-YAUT, CspZ-YAC187SUT, CspZ-YA, or CspZ-YAI183Ywas incubated at 4 or 37oC for 6- or 24-h prior to being coated on microtiter plate wells. The microtiter plate wells immobilized with each of these proteins before incubation (0-h) were included as unincubated control. The ability of the CspZ monoclonal IgG, (C) 1139c or (D) 1193c, to recognize each of these CspZ-YA proteins were determined using ELISA in the section “Accelerated stability study” in Materials and Methods. The work was performed on four independent experiments (one replicate per experiment). Data are expressed as the percent binding, derived by normalizing the levels of bound 1139c or 1193c from the wells coated with each of the CspZ-YA proteins in different incubating conditions normalized to that in the unincubated control. Data shown are the mean ± standard deviation of the percent binding of 1139c or 1193c from four experiment. Statistical significance (p < 0.05, Kruskal Wallis test with the two-stage step-up method of Benjamini, Krieger, and Yekutieli) of differences in percent binding between groups are indicated (“#”).

[0045] Figure 11. The antibodies 1139c and 1193c efficiently recognize CspZ-YA, prevent human FH-binding, and promote lysis and opsonophagocytosis of B. burgdorferi. (A and B) The 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 areexpressed 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 IC50values 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 representative experiment, 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 BA50values are shown as the mean ± SD of from three experiments.

[0046] Figure 12. The 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.

[0047] Figure 13. Immunization of CspZ-YA and its mutant proteins triggered indistinguishable levels of antibodies against CspZ. Sera were collected at 14dpli from C3H / HeN mice immunized (A) once, (B) twice, or (C) three times in the fashion as describedin Fig.1. These mice were immunized with PBS (control) or untagged CspZ-YA (CspZ- YAUT) or its derived mutant protein, or histidine tagged CspZ-YA (CspZ-YA), or its derived mutant proteins (Six mice for CspZ-YAUT- or CspZ-YAC187SUT-immunized mice whereas five mice for the rest of immunization groups of mice). The levels of total IgG against CspZ were determined using quantitative ELISA. Data shown are the geometric mean ± geometric standard deviation of the titers of anti-CspZ antibodies from five mice per group. Asterisks indicate the statistical significances (p < 0.05, Kruskal Wallis test with the two-stage step-up method of Benjamini, Krieger, and Yekutieli) of differences in antibody titers relative to the sera from PBS-inoculated mice.

[0048] Figure 14 depicts results illustrating Lyme disease bacterial colonization mediated by CspZ-YAC187S vaccination with different adjuvants.

[0049] Figure 15. The formulation of Alum-CpG or Alum-αGal allowed CspZ- YAC187S to induced antibodies with greatest titers and bactericidal activities in mice. (A) The schematic diagram shows pre-adolescent C3H / HeN receiving two inoculations at 0- and 14- days post initial immunization (dpii) with TBS (control) or CspZ-YAC187S formulated with indicated adjuvants, followed by the infection via feeding by I. scapularis nymph carrying B. burgdorferi strain B31-A3 at 35dpii and the euthanasia at 21 days post nymph feeding (dpf) (or 56dpii) (B-G) Sera were collected at (B, D, E) 14dpii for immunization Once (Immun.1) and (C, F, G) 28dpii for immunization twice (Immun.2). (B-C) The levels of total IgG against CspZ-YAC187Sin the sera were determined using quantitative ELISA. Data shown are the geometric mean ± geometric standard deviation of the titers of anti-CspZ-YAC187S antibodies from five mice per group. (D-G) The sera were diluted as indicated, and mixed with guinea pig complement and B. burgdorferi B31-A3 for 24 hours. Surviving spirochetes were quantified from three fields of view microscopically in three independent experiments. (D, F) The survival percentage was derived from the proportion of serum-treated to untreated spirochetes. Data shown are the mean ± SEM of the survival percentage from three replicates in one representative experiment. (E, G) The BA50 value, representing the dilution rate that effectively killed 50% of spirochetes, was obtained from curve-fitting and extrapolation of Panel C and F. Data shown are the geometric mean ± geometric standard deviation of the borreliacidal titers from five mice per group in three experiments per mouse sample and shown in Table 2. (“NK”), no killing. Statistical significance (p < 0.05, Kruskal Wallis test with the two-stage step-up method of Benjamini, Krieger, and Yekutieli) of IgG titers or borreliacidal titers from an indicated group of mice was compared to the titers from TBS- inoculated mice (“*”) or between indicated groups were shown as “#”.

[0050] Figure 16. CspZ-YAC187Sformulated with Alum-CpG or Alum-αGal protected mice from seroconversion, borrelial colonization and Lyme disease-associated joint inflammation. Five pre-adolescent C3H / HeN mice were inoculated with TBS or CspZ- YAC187S formulated with indicated adjuvants, followed by infection using (A) nymphs carrying B. burgdorferi B31-A3 in the fashion described in Fig.1A. Mice inoculated with TBS that are not fed on by nymphs were included as an uninfected control group (uninfect.). (B) Seropositivity was determined by measuring the levels of IgG against C6 peptides in the sera of those mice at 21 dpf using ELISA. The mouse was considered as seropositive if that mouse had IgG levels against C6 peptides greater than the threshold, the mean plus 1.5-fold standard deviation of the IgG levels against C6 peptides from the TBS-inoculated, uninfected mice (dotted line). The number of mice in each group with the anti-C6 IgG levels greater than the threshold (seropositive) is shown. Data presented here are the geometric mean ± geometric 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 are presented. (A, C-F) B. burgdorferi (Bb) burdens at (A) nymphs after when feeding to repletion or (C) the tick feeding site (“Bite Site”), (D) bladder, (E) heart, and (F) knees, were quantitatively measured at 21dpf, shown as the number of (A) Bb per tick or (C-F) per 100ng total DNA. Data shown are the geometric mean ± geometric standard deviation of the spirochete burdens from each group of mice. Asterisks indicate the statistical significance (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. (G-H) Tibiotarsus joints at 42 dpli were collected to assess inflammation by staining these tissues using hematoxylin and eosin. (G) Representative images from one mouse per group are shown. Top panels are lower-resolution images (joint, ×10 [bar, 160 µm]); bottom panels are higher-resolution images (joint, 2×20 [bar, 80 µm]) of selected areas (highlighted in top panels). Arrows indicate infiltration of immune cells. (H) To quantitate inflammation of joint tissues, at least ten random sections of tibiotarsus joints from each mouse were scored on a scale of 0-3 for the levels of inflammation. Data shown are the mean inflammation score ± standard deviation of the inflammatory scores from each group of mice. Asterisks indicate the statistical significance (p < 0.05, Kruskal Wallis test with the two-stage step-up method of Benjamini, Krieger, and Yekutieli) of differences in inflammation relative to uninfected mice.

[0051] Figure 17. The titers and bactericidal activities of antibodies triggered by Alum-CpG formulated CspZ-YAC187S were detectable for up to eight months. (A) Theschematic diagram shows pre-adolescent C3H / HeN receiving at 0 (Imm.1), and both 0 and 14 (Imm.2) dpii with CspZ-YAC187S formulated with Alum and CpG. Mice inoculated with TBS at 0 and 14 dpii, or TMG-formulated OspA at 0, 14, and 28dpii were included as control. Sera were collected at 14, 42, 70, 98, 126, 154, 182, 210, 238, and 273 days post last immunization (dpli). (B-C) The levels of total IgG against (B) OspA and (C) CspZ-YAC187Sin the sera were determined using quantitative ELISA. Data shown are the geometric mean ± geometric standard deviation of the titers of (B) anti-OspA and (C) CspZ-YAC187S IgGs from five mice per group. (D) The sera were diluted as indicated, and mixed with guinea pig complement and B. burgdorferi B31-A3 for 24 hours. Surviving spirochetes were quantified from three fields of view microscopically in three independent experiments. The survival percentage was derived from the proportion of serum-treated to untreated spirochetes. Data shown are the mean ± SEM of the survival percentage from three replicates in one representative experiment. The BA50 value, representing the dilution rate that effectively killed 50% of spirochetes, was obtained from curve-fitting and shown in Table 3. Asterisks represent that no killing was detected in indicated groups of mice and time points.

[0052] Figure 18. Natural infection triggered CspZ-targeting bactericidal antibodies linked to prevention of seroconversion, borrelial colonization and joint inflammation in Alum-CpG-CspZ-YAC187Svaccinated mice. (A-K) As shown in Fig.4A, five pre-adolescent C3H / HeN mice were inoculated with TBS or CspZ-YAC187S formulated with Alum and CpG, followed by infection at 273dpli At 273 dpli using I. scapularis nymphs carrying B. burgdorferi strain B31-A3. The sera were collected at 4, 7, 10, 14, and 21 dpf. These mice were euthanized at 21dpf. Mice inoculated with TBS that are not fed on by nymphs were included as an uninfected control group (uninfect.). (A-B) The levels of total IgG against (A) OspA and (B) CspZ-YAC187Sin the sera were determined using quantitative ELISA. Data shown are the geometric mean ± geometric standard deviation of the titers of (A) anti-OspA and (B) CspZ-YAC187SIgGs from five mice per group. (C) The sera were diluted as indicated, and mixed with guinea pig complement and B. burgdorferi B31-A3 for 24 hours. Surviving spirochetes were quantified from three fields of view microscopically in three independent experiments. The survival percentage was derived from the proportion of serum-treated to untreated spirochetes. Data shown are the mean ± SEM of the survival percentage from three replicates in one representative experiment. The BA50 value, representing the dilution rate that effectively killed 50% of spirochetes, was obtained from curve-fitting and shown in Table S8. Asterisks represent that no killing was detected in indicated groups of mice and time points. (E) Seropositivity was determined by measuring the levels of IgG against C6peptides in the sera of those mice at 21 dpf using ELISA. The mouse was considered as seropositive if that mouse had IgG levels against C6 peptides greater than the threshold, the mean plus 1.5-fold standard deviation of the IgG levels against C6 peptides from the TBS- inoculated, uninfected mice (dotted line). The number of mice in each group with the anti-C6 IgG levels greater than the threshold (seropositive) is shown. Data shown are the geometric mean ± geometric standard deviation of the titers of anti-C6 IgG. (D, F-I) B. burgdorferi (Bb) burdens at (D) replete nymphs or (F) the tick feeding site (“Bite Site”), (I) bladder, (J) heart, and (K) knees, were quantitatively measured at 21dpf, shown as the number of (D) Bb per tick or (F-I) per 100ng total DNA. Data shown are the geometric mean ± geometric standard deviation of the spirochete burdens from each group of mice. (J-K) Tibiotarsus joints at 21dpf were collected to assess inflammation by staining these tissues using hematoxylin and eosin. (J) Representative images from one mouse per group are shown. Top panels are lower- resolution images (joint, ×10 [bar, 160 µm]); bottom panels are higher-resolution images (joint, 2×20 [bar, 80 µm]) of selected areas (highlighted in top panels). Arrows indicate infiltration of immune cells. (K) To quantitate inflammation of joint tissues, at least ten random sections of tibiotarsus joints from each mouse were scored on a scale of 0-3 for the levels of inflammation. Data shown are the mean inflammation score ± standard deviation of the inflammatory scores from each group of mice. Asterisks indicate the statistical significance (p < 0.05, Kruskal Wallis test with the two-stage step-up method of Benjamini, Krieger, and Yekutieli) of differences in (D, F-I) bacterial burdens, (D) C6 IgG titers, (K) inflammatory scores relative to uninfected mice.

[0053] Figure 19. The differential gene expressions in the mice inoculated with Alum, Alum-CpG, or Alum-αGal formulated CspZ-YAC187S vs. TBS. Five pre-adolescent C3H / HeN mice per group were inoculated with TBS or CspZ-YAC187Sformulated with Alum, Alum and CpG, and Alum and αGal at 0 and 14dpii in the fashion described in Fig.1A. At 28dpii, the expression levels of genes in the spleen from each group of the mice were determined. Differentially expressed genes (DEGs) are defined by an adjusted P values less than 0.05 and absolute (log2fold change) ≥0.58 or ≤0.58. The DEGs in the mice inoculated with TBS vs. CspZ-YAC187S formulated with (A) Alum, (B) Alum and CpG, or (C) Alum and αGal were plotted as a Volcano plots. The up- and down-regulated DEGs were shown in red and blue, respectively. The details of the immune-related DEGs were shown in Table S2 to 4.

[0054] Figure 20. The levels of anti-OspA IgG titers determined in the mice immunized with OspA or CspZ-YAC187S formulated with different adjuvants from 14 to 273 dpli. Five pre-adolescent C3H / HeN receiving at 0 (Imm.1) or both 0 and 14 (Imm.2) dpiiwith CspZ-YAC187Sformulated with Alum and CpG. Mice inoculated with TBS at 0 and 14 dpii, or TMG-formulated OspA at 0, 14, and 28dpii were included as control. Sera were collected at (A) 14, (B) 42, (C) 70, (D) 98, (E) 126, (F) 154, (G) 182, (H) 210, (I) 238, and (J) 273 dpli. The levels of total IgG against OspA in the sera were determined using quantitative ELISA. Data shown are the geometric mean ± geometric standard deviation of the titers of anti-OspA IgG from five mice per group. Statistical significance (p < 0.05, Kruskal Wallis test with the two-stage step-up method of Benjamini, Krieger, and Yekutieli) of greater anti- OspA IgG titers in the indicated group were shown to be compared with the titers from TBS- inoculated mice (“*”).

[0055] Figure 21. The levels of anti-CspZ-YAC187SIgG titers determined in the mice immunized with OspA or CspZ-YAC187S formulated with different adjuvants from 14 to 273 dpli. Five pre-adolescent C3H / HeN receiving at 0 (Imm.1) or both 0 and 14 (Imm.2) dpii with CspZ-YAC187S formulated with Alum and CpG. Mice inoculated with TBS at 0 and 14 dpii, or TMG-formulated OspA at 0, 14, and 28dpii were included as control. Sera were collected at (A) 14, (B) 42, (C) 70, (D) 98, (E) 126, (F) 154, (G) 182, (H) 210, (I) 238, and (J) 273 dpli. The levels of total IgG against CspZ-YAC187Sin the sera were determined using quantitative ELISA. Data shown are the geometric mean ± geometric standard deviation of the titers of anti-CspZ-YAC187Santibodies from five mice per group. Statistical significance (p < 0.05, Kruskal Wallis test with the two-stage step-up method of Benjamini, Krieger, and Yekutieli) of greater anti-CspZ-YAC187SIgG in the indicated group were shown to be compared with the titers from TBS-inoculated mice (“*”) or between indicated groups (“#”).

[0056] Figure 22. The levels of bactericidal antibodies determined in the mice immunized with OspA or CspZ-YAC187S formulated with different adjuvants from 14 to 273 dpli. Five pre-adolescent C3H / HeN receiving at 0 (Imm.1), or both 0 and 14 (Imm.2) dpii with CspZ-YAC187S formulated with Alum and CpG. Mice inoculated with TBS at 0 and 14 dpii, or TMG-formulated OspA at 0, 14, and 28dpii were included as control. Sera were collected at (A-B) 14, (C-D) 42, (E-F) 70, (G-H) 98, (I-J) 126, (K-L) 154, (M-N) 182, (O-P) 210, (Q-R) 238, and (S-T) 273 dpli. The sera were diluted as indicated, and mixed with guinea pig complement and B. burgdorferi B31-A3 for 24 hours. Surviving spirochetes were quantified from three fields of view microscopically in three independent experiments. (A, C, E, G, I, K, M, O, Q, and S) The survival percentage was derived from the proportion of serum-treated to untreated spirochetes. Data shown are the mean ± SEM of the survival percentage from three replicates in one representative experiment. (B, D, F, H, J, L, N, P, R, and T) The BA50 value, representing the dilution rate that effectively killed 50% ofspirochetes, was obtained from curve-fitting and extrapolation of Panel A, C, E, G, I, K, M, O, Q, and S for indicated time points. Data shown are the geometric mean ± geometric standard deviation of the borreliacidal titers from five mice per group in three experiments per mouse sample and shown in Table 3. Statistical significance (p < 0.05, Kruskal Wallis test with the two-stage step-up method of Benjamini, Krieger, and Yekutieli) of greater BA50was shown between indicated groups (“#”).

[0057] Figure 23. The levels of anti-OspA IgG titers determined in the miceimmunized with OspA or CspZ- formulated with different adjuvants from 4 to 21 dpf. Five pre-adolescent C3H / HeN receiving at 0 (Imm.1), and both 0 and 14 (Imm.2) dpii with CspZ-YAC187Sformulated with Alum and CpG. Mice inoculated with TBS at 0 and 14 dpii, or TMG-formulated OspA at 0, 14, and 28dpii were included as control. The mice were infected using I. scapularis nymphs carrying B. burgdorferi strain B31-A3 at 273 dpli. Mice inoculated with TBS that are not fed on by nymphs were included as an uninfected control group (uninfect.). The sera were collected at (A) 4, (B) 7, (C) 10, (D) 14, and (E) 21 dpf. The levels of total IgG against OspA in the sera were determined using quantitative ELISA. Data shown are the geometric mean ± geometric standard deviation of the titers of anti-OspA IgG from five mice per group.

[0058] Figure 24. The levels of anti-CspZ-YAC187SIgG titers determined in the mice immunized with OspA or CspZ-YAC187S formulated with different adjuvants from 4 to 21 dpf. Five pre-adolescent C3H / HeN receiving at 0 (Imm.1), and both 0 and 14 (Imm.2) dpii with CspZ-YAC187S formulated with Alum and CpG. Mice inoculated with TBS at 0 and 14 dpii, or TMG-formulated OspA at 0, 14, and 28dpii were included as control. The mice were infected using I. scapularis nymphs carrying B. burgdorferi strain B31-A3 at 273 dpli. Mice inoculated with TBS that are not fed on by nymphs were included as an uninfected control group (uninfect.). The sera were collected at (A) 4, (B) 7, (C) 10, (D) 14, and (E) 21 dpf. The levels of total IgG against CspZ-YAC187Sin the sera were determined using quantitative ELISA. Data shown are the geometric mean ± geometric standard deviation of the titers of anti-CspZ-YAC187SIgG from five mice per group. Statistical significance (p < 0.05, Kruskal Wallis test with the two-stage step-up method of Benjamini, Krieger, and Yekutieli) of greater anti-CspZ-YAC187SIgG titers in the indicated group were shown to be compared with the titers from uninfected mice (“*”).

[0059] Figure 25. The levels of bactericidal antibodies determined in the mice immunized with OspA or CspZ-YAC187S formulated with different adjuvants from 4 to 21 dpf. Five pre-adolescent C3H / HeN receiving at 0 (Imm.1), and both 0 and 14 (Imm.2) dpiiwith CspZ-YAC187Sformulated with Alum and CpG. Mice inoculated with TBS at 0 and 14 dpii, or TMG-formulated OspA at 0, 14, and 28dpii were included as control. The mice were infected using I. scapularis nymphs carrying B. burgdorferi strain B31-A3 at 273 dpli. Mice inoculated with TBS that are not fed on by nymphs were included as an uninfected control group (uninfect.). The sera were collected at (A-B) 4, (C-D) 7, (E-F) 10, (G-H) 14, and (I-J) 21 dpf. The sera were diluted as indicated, and mixed with guinea pig complement and B. burgdorferi B31-A3 for 24 hours. Surviving spirochetes were quantified from three fields of view microscopically in three independent experiments. (A, C, E, G, and I) The survival percentage was derived from the proportion of serum-treated to untreated spirochetes. Data shown are the mean ± SEM of the survival percentage from three replicates in one representative experiment. (B, D, F, H, and J) The BA50 value, representing the dilution rate that effectively killed 50% of spirochetes, was obtained from curve-fitting and extrapolation of Panel A, C, E, G, and I for indicated time points. Data shown are the geometric mean ± geometric standard deviation of the borreliacidal titers from five mice per group in three experiments per mouse sample and shown in Table S8. Statistical significance (p < 0.05, Kruskal Wallis test with the two-stage step-up method of Benjamini, Krieger, and Yekutieli) of greater BA50 was shown between indicated groups (“#”). DETAILED DESCRIPTION

[0060] This disclosure relates to an immunogenic composition including a polypeptide having the amino acid sequence of SEQ ID NO: 1, or one or more variant thereof as disclosed herein, or any polynucleotide such as DNA or RNA encoding any of the foregoing. Variants of SEQ ID NO: 1 are also given as examples of SEQ ID NO: 2 and for all of the following disclosure and examples and for all purposes disclosed herein, a variant of SEQ ID NO: 1, wherever disclosed herein, may alternatively be referred to as SEQ ID NO: 2 or an according example thereof. The immunogenic composition may include one of more adjuvant. Administering the immunogenic composition to a subject may cause resistance or immunity to infection or development of disease or disease symptoms, such as Lyme disease or symptoms thereof, otherwise cause by exposure to a Lyme-causing Borreliae bacteria, such as Borrelia burgdorferi, or Borrelia afzelii or Borrelia garinii. Such symptoms of Lyme disease may include 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 / orinformation-processing capabilities, meningitis symptoms, pain in the neck, mid / lower back, or spine, and fatigue.

[0061] An immunogenic composition as disclosed herein may include a polypeptide having a sequence of CspZ, or partial sequence thereof, or a portion or fragment of any of the foregoing, further including an amino acid substitution at one or more amino acid relative to a corresponding amino acid of CspZ. A modification may include one or more amino acid substitution in a region corresponding to the FH binding region of CspZ, which modification may prevent, reduce, inhibit, or minimize binding of the polypeptide to FH. Examples of such amino acid substitutions within an FH-binding domain of CspZ are disclosed in US Patent Application Publication No.2019 / 0201516 A1, the disclosure of which is incorporated herein by reference in its entirety. Such examples include one or both of an amino acid substitution at amino acids 188 and 192 of SEQ ID NO: 1 (which correspond to tyrosine residues at amino acids 207 and 111 of Borrelia burgdorferi CspZ), such as alanine, or an amino acid other than tyrosine, independently at one or both of amino acids corresponding to amino acid 188 and 192 of SEQ ID NO: 1.

[0062] The polypeptide may further include one or more additional amino acid substitution, such as at a position corresponding to, independently, one or more of amino acid 34, 48, 61, 86, 96, 117, 123, 164, 168, and 174 of SEQ ID NO: 1 (which correspond to amino acids 53, 67, 80, 105, 115, 136, 142, 183, 187, and 193, respectively, of Borrelia burgdorferi CspZ). Said additional one or more amino acid substitution may result in an immunogenic composition that, when administered to a subject, may promote development of an immune response that protects against, prevents, minimizes symptoms or severity thereof, development, or contraction of disease caused by subsequent exposure to a Lyme-disease causing bacteria, including a Lyme-disease causing bacteria that expresses CspZ such as Borrelia burgdorferi. Said immune response may occur following fewer than three administrations of the immunogenic composition to a subject, such as following one or two administrations. Administration of any polynucleotide, such as DNA or RNA, of any sequence that encodes any foregoing protein immunogen may be administered to a subject to be taken up by cells of the subject, wherein transcriptional / translational cellular machinery lead to cellular production and release of the protein immunogen. Vaccination resulting from administration of an RNA molecule encoding a protein immunogen, to stimulate production of the immunogen by cells of the recipient, has been widely demonstrated as effective. See Leong et al., 2025, Revolutionizing immunization: a comprehensive review of mRNA vaccine technology and applications. Virol J 22, 71; and Zhang et al., 2024, Efficient signalsequence of mRNA vaccines enhances the antigen expression to expand the immune protection against viral infection. J Nanobiotechnol 22, 295, which references are hereby incorporated by reference in their entireties.

[0063] Said additional one or more amino acid substitution may result in an immunogenic composition that, when administered to a subject previously exposed to a Lyme-disease causing bacteria, including a Lyme-disease causing bacteria that expresses CspZ such as Borrelia burgdorferi, may promote development of an immune response that protects against, prevents, minimizes symptoms or severity thereof, development, or contraction of disease caused by subsequent exposure to a Lyme-disease causing bacteria, including a Lyme-disease causing bacteria that expresses CspZ such as Borrelia burgdorferi. Said immune response may occur following fewer than three administrations of the immunogenic composition to a subject, such as following one or two administrations.

[0064] Said additional one or more amino acid substitution may result in an immunogenic composition that, when administered to a subject diagnosed with or suspected of having Lyme disease, including Lyme disease caused by or suspected to have been caused by previous exposed to a Lyme-disease causing or potentially Lyme-disease causing bacteria, including a Lyme-disease causing bacteria that expresses or may express CspZ such as Borrelia burgdorferi, may promote development of an immune response that protects against, prevents, minimizes symptoms or severity thereof, development, or contraction or worsening of disease caused by exposure to the Lyme-disease causing bacteria, including a Lyme- disease causing bacteria that expresses CspZ such as Borrelia burgdorferi. Said immune response may occur following fewer than three administrations of the immunogenic composition to a subject, such as following one or two administrations.

[0065] CspZ having substitutions for tyrosine at positions 207 and 211 of CspZ of Borrelia burgdorferi (e.g. alanine substitutions for these tyrosine residues, such as at amino acids 188 and 192 of SEQ ID NO: 1) does not bind to FH to expose the epitopes around FH- binding site (CspZ-Y207A / Y211A, namely “CspZ-YA”) [31, 32]. 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 [32, 34].

[0066] Disclosed herein is an immunogen corresponding to CspZ-YA or a fragment thereof by mutagenizing amino acids to induce bacterial killing immune responses and preventing Lyme disease and symptoms thereof. Improved stability of CspZ-YA mutant proteins in mammalian-adapted conditions identified is also disclosed herein. For example, asdisclosed herein, an immunogen including one or more additional amino acid substitution, such as at a position corresponding to, independently, one or more of amino acid 34, 48, 61, 86, 96, 117, 123, 164, 168, and 174 of SEQ ID NO: 1 (which correspond to amino acids 53, 67, 80, 105, 115, 136, 142, 183, 187, and 193, respectively, of Borrelia burgdorferi CspZ) may exhibit greater stability under physiological conditions such as body temperature following administration, prolonging the ability of the immunogen to maintain an ability to stimulate development of an immune response recognizing CspZ, such as to an FH binding domain of CspZ. Such greater stability may promote development of immunity to Lyme- disease caused by Borrelia species that express CspZ, including but not limited to B. burgdorferi (e.g., Borrelia afzelii expresses CspZ and inoculation with an immunogen as disclosed herein may prevent or promote development or symptoms or treat or ameliorate symptoms of Lyme disease caused by exposure to any CspZ-expressing Borrelia species, or suspected exposure thereto, or Lyme disease caused by such exposure). For example, as a significant improvement over other immunogens, administration of fewer than three inoculations with an immunogen as disclosed herein may promote effective and lasing immunization, whereas more than two inoculations with other potential immunogens has previously been reported as being required for development of an effective prophylactic immune response. Administration of fewer than three inoculations with an immunogen as disclosed herein may promote effective and lasing treatment of Lyme disease and symptoms thereof. More than two inoculations with an immunogenic composition as disclosed herein may also be performed, for prevention of Lyme or treatment of Lyme disease or Lyme- related symptoms or suspected Lyme or suspected exposure to a Lyme-causing agent such as a species of Borrelia expressing CspZ such as B. burgdorferi or B. afzelii.

[0067] In an example, an immunogenic polypeptide may include one or more amino acid or sequence thereof in addition to a polypeptide immunogen having the amino acid sequence of SEQ ID NO: 1. For example, it may include one or more additional amino acid sequence, C-terminal thereto, N-terminal thereto, or both. It may include one or more amino acid substitution in of SEQ ID NO: 1, in addition to the one or more substitution at one or more of amino acid 34, 48, 61, 86, 96, 117, 123, 164, 168, and 174 of SEQ ID NO: 1 (which correspond to amino acids 53, 67, 80, 105, 115, 136, 142, 183, 187, and 193, respectively, of Borrelia burgdorferi CspZ). 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 acidthat 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).

[0068] 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.

[0069] An immunogenic composition may include one or more substitution, including a conservative amino acid substitution. Thus, it may include an amino acid sequence that has less than 100% sequence identity with SEQ ID NO: 1 (beyond one or more substitution at one or more of amino acid 34, 48, 61, 86, 96, 117, 123, 164, 168, and 174 of SEQ ID NO: 1). For example, it may include one amino acid substitution, such as a conservative amino acidsubstitution, compared to a sequence as set out in SEQ ID NO: 1 in addition to one or more substitution at one or more of amino acid 34, 48, 61, 86, 96, 117, 123, 164, 168, and 174 of SEQ ID NO: 1. For example, it may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 ,7, 18 ,19, 20, or more amino acid substitutions, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 ,7, 18 ,19, 20, or more conservative amino acid substitutions, provided the immunogen promotes an immune response against Lyme-disease causing species of Borrelia expressing CspZ such as B. burgdorferi or B. afzelii.

[0070] One or more amino acid in an immunogenic polypeptide as disclosed herein may be an R-amino acid or an L-amino acid. One or more amino acid in an immunogenic polypeptide as disclosed herein may be a standard amino acid (i.e., selected from Alanine, Arginine, Asparagine, Aspartic Acid, Cysteine, Glutamic acid, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, Valine, Selenocysteine, N-formylmethionine, and Pyrrolysine).

[0071] 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 groupor to the side chain, not naturally present on the amino acid and are included as examples where an amino acid is referred to herein.

[0072] Any one or more of the following amino acid substitutions may be made, in any combination, to a CspZ-YA polypeptide immunogen, such as to an immunogen having the amino acid sequence as set out in SEQ ID NO: 1. An amino acid whose position corresponds to amino acid 34 of SEQ ID NO: 1 may include any amino acid other than cysteine, an amino acid whose position corresponds to amino acid 48 of SEQ ID NO: 1 may include any amino acid other than threonine, an amino acid whose position corresponds to amino acid 61 of SEQ ID NO: 1 may include any amino acid other than inosine, an amino acid whose position corresponds to amino acid 86 of SEQ ID NO: 1 may include any amino acid other than phenylalanine, an amino acid whose position corresponds to amino acid 96 of SEQ ID NO: 1 may include any amino acid other than inosine, an amino acid whose position corresponds to amino acid 117 of SEQ ID NO: 1 may include any amino acid other than lysine, an amino acid whose position corresponds to amino acid 123 of SEQ ID NO: 1 may include any amino acid other than valine, an amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 may include any amino acid other than inosine, an amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 may include any amino acid other than cysteine, and an amino acid whose position corresponds to amino acid 174 of SEQ ID NO: 1 may include any amino acid other than glycine. The foregoing correspond to amino acids 53, 67, 80, 105, 115, 136, 142, 183, 187, and 193, respectively, of Borrelia burgdorferi CspZ.

[0073] Any one or more of the following, independently, may be included in a polypeptide immunogen as disclosed herein. The amino acid whose position corresponds to amino acid 34 of SEQ ID NO: 1 may include serine, the amino acid whose position corresponds to amino acid 48 of SEQ ID NO: 1 may include proline, the amino acid whose position corresponds to amino acid 61 of SEQ ID NO: 1 may include threonine, the amino acid whose position corresponds to amino acid 86 of SEQ ID NO: 1 may include proline, the amino acid whose position corresponds to amino acid 96 of SEQ ID NO: 1 may include threonine, the amino acid whose position corresponds to amino acid 117 of SEQ ID NO: 1 may include glutamic acid, the amino acid whose position corresponds to amino acid 123 of SEQ ID NO: 1 may include methionine, the amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 may include tyrosine, the amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 may include serine, and the amino acid whose position corresponds to amino acid 174 of SEQ ID NO: 1 may include methionine. The foregoingcorrespond to amino acids 53, 67, 80, 105, 115, 136, 142, 183, 187, and 193, respectively, of Borrelia burgdorferi CspZ.

[0074] An immunogen may have the amino acid sequence as set out in SEQ ID NO: 1: except that an amino acid whose position corresponds to amino acid 34 of SEQ ID NO: 1 may include any amino acid other than cysteine. An immunogen may have the amino acid sequence as set out in SEQ ID NO: 1: except that an amino acid whose position corresponds to amino acid 34 of SEQ ID NO: 1 may include serine.

[0075] An immunogen may have the amino acid sequence as set out in SEQ ID NO: 1: except that an amino acid whose position corresponds to amino acid 48 of SEQ ID NO: 1 may include any amino acid other than threonine. An immunogen may have the amino acid sequence as set out in SEQ ID NO: 1: except that an amino acid whose position corresponds to amino acid 48 of SEQ ID NO: 1 may include proline.

[0076] An immunogen may have the amino acid sequence as set out in SEQ ID NO: 1: except that an amino acid whose position corresponds to amino acid 61 of SEQ ID NO: 1 may include any amino acid other than inosine. An immunogen may have the amino acid sequence as set out in SEQ ID NO: 1: except that an amino acid whose position corresponds to amino acid 61 of SEQ ID NO: 1 may include threonine.

[0077] An immunogen may have the amino acid sequence as set out in SEQ ID NO: 1: except that an amino acid whose position corresponds to amino acid 86 of SEQ ID NO: 1 may include any amino acid other than phenylalanine. An immunogen may have the amino acid sequence as set out in SEQ ID NO: 1: except that an amino acid whose position corresponds to amino acid 86 of SEQ ID NO: 1 may include proline.

[0078] An immunogen may have the amino acid sequence as set out in SEQ ID NO: 1: except that an amino acid whose position corresponds to amino acid 96 of SEQ ID NO: 1 may include any amino acid other than inosine. An immunogen may have the amino acid sequence as set out in SEQ ID NO: 1: except that an amino acid whose position corresponds to amino acid 96 of SEQ ID NO: 1 may include threonine.

[0079] An immunogen may have the amino acid sequence as set out in SEQ ID NO: 1: except that an amino acid whose position corresponds to amino acid 117 of SEQ ID NO: 1 may include any amino acid other than lysine. An immunogen may have the amino acid sequence as set out in SEQ ID NO: 1: except that an amino acid whose position corresponds to amino acid 117 of SEQ ID NO: 1 may include glutamic acid.

[0080] An immunogen may have the amino acid sequence as set out in SEQ ID NO: 1: except that an amino acid whose position corresponds to amino acid 123 of SEQ ID NO: 1may include any amino acid other than valine. An immunogen may have the amino acid sequence as set out in SEQ ID NO: 1: except that an amino acid whose position corresponds to amino acid 123 of SEQ ID NO: 1 may include methionine.

[0081] An immunogen may have the amino acid sequence as set out in SEQ ID NO: 1: except that an amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 may include any amino acid other than inosine. An immunogen may have the amino acid sequence as set out in SEQ ID NO: 1: except that an amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 may include tyrosine.

[0082] An immunogen may have the amino acid sequence as set out in SEQ ID NO: 1: except that an amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 may include any amino acid other than cysteine. An immunogen may have the amino acid sequence as set out in SEQ ID NO: 1: except that an amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 may include serine.

[0083] An immunogen may have the amino acid sequence as set out in SEQ ID NO: 1: except that an amino acid whose position corresponds to amino acid 174 of SEQ ID NO: 1 may include any amino acid other than glycine. An immunogen may have the amino acid sequence as set out in SEQ ID NO: 1: except that an amino acid whose position corresponds to amino acid 174 of SEQ ID NO: 1 may include methionine.

[0084] An immunogen may have the amino acid sequence as set out in SEQ ID NO: 1: except that an amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 may include any amino acid other than inosine and an amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 may include any amino acid other than cysteine. An immunogen may have the amino acid sequence as set out in SEQ ID NO: 1: except that an amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 may include tyrosine and an amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 may include serine.

[0085] An immunogenic composition as disclosed herein may have any of the foregoing amino acid sequences, except that, in addition, from one to thirty six, from one to twenty four, from one to sixteen, from one to eight, or from one to four amino acids whose position corresponds to an amino acid of from 1-163 in SEQ ID NO: 1 are not the same as the corresponding amino acid in SEQ ID NO: 1. An immunogenic composition as disclosed herein may have any of the foregoing amino acid sequences, except that, in addition, from one to four, from one to three, of from one to two amino acids corresponding to amino acids whose position corresponds to amino acid of from 193-207 in SEQ ID NO: 1 are not thesame as the corresponding amino acid in SEQ ID NO: 1. From one to thirty six, from one to twenty four, from one to sixteen, from one to eight, or from one to four amino acids whose position corresponds to an amino acid of from 1-163 in SEQ ID NO: 1 and from one to four, from one to three, of from one to two amino acids corresponding to amino acids whose position corresponds to amino acid of from 193-207 in SEQ ID NO: 1may be an amino acid other than the corresponding amino acid(s) in SEQ ID NO: 1.

[0086] An amino acid substitution that is a conservative substitute for any one or more of the foregoing specifically identified substitutions may also be included. Every combination of any one or more of the foregoing, including without limitation a substitution at all ten of the foregoing amino acids, is explicitly included herein, as could be envisioned by a skilled person without the requirement of listing every such substitution or combination. Any of the foregoing examples that cause an immune response to a Lyme disease-causing species of Borrelia expressing CspZ such as B. burgdorferi or B. afzelii is each included in the present disclosure.

[0087] Also disclosed herewith is a polynucleotide encoding any immunogenic polypeptide disclosed herein. A person possessing ordinary skill in the art can envision a polynucleotide sequence encoding each and every immunogenic polypeptide disclosed herein, including all variations in sequences of each and every given single immunogenic polypeptide made possible because of codon degeneracy, whereby certain amino acids may be coded for by more than one triplet codon of nucleotides. Every polynucleotide encoding a polypeptide having the sequence set out in SEQ ID NO: 1 and all variants thereof having any one or more of the amino acid substitutions disclosed in the preceding paragraphs, in any combination, 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. Disclosed herein in a cell transfected with or expressing any of the foregoing polynucleotides, and a method of transfecting a cell with any of the foregoing polynucleotides, including by contacting the cell or an organism with any of the foregoing vectors or any of the foregoing polynucleotides.

[0088] 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, UUCAmino Acid Abbreviation Number of Codons (Degeneracy) Codons 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, AGCAlanine 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

[0089] A non-limiting implementation of a polynucleotide encoding an immunogen as disclosed herein is given in SEQ ID NO: 3. The polynucleotide sequence set out in SEQ ID NO: 3 encodes an immunogen as disclosed herein corresponding to SEQ ID NO: 1, except that, in the immunogen resulting from translation of the polynucleotide sequence set out in SEQ ID NO: 3, the amino acid corresponding to amino acid 168 of SEQ ID NO: 1 includes serine. The polynucleotide having a sequence as set out in SEQ ID NO: 3, or other polynucleotide sequence encoding an implementation of an immunogen as disclosed herein, may be DNA, or RNA, such as may be transcribed or transcribable therefrom.

[0090] A non-limiting implementation of a polynucleotide encoding an immunogen as disclosed herein is given in SEQ ID NO: 14. The polynucleotide sequence set out in SEQ ID NO: 14 encodes an immunogen as disclosed herein corresponding to SEQ ID NO: 1, except that, in the immunogen resulting from translation of the polynucleotide sequence set out in SEQ ID NO: 3, the amino acid corresponding to amino acid 168 of SEQ ID NO: 1includes serine. The polynucleotide sequence set out in SEQ ID NO: 14 also encodes an N- terminal signal sequence that is cleaved from the peptide during intracellular trafficking when the polynucleotide is expressed in a cell and processed during such trafficking so as to direct the immunogen, cleaved from the signal peptide, to be secreted from the cell. Examples of signal sequences that may be encoded for by polynucleotides such as RNA that also encode a peptide immunogen are known and described, such as in Zhang, et al. Efficient signal sequence of mRNA vaccines enhances the antigen expression to expand the immune protection against viral infection. J Nanobiotechnol 22, 295 (2024), which is incorporated by reference herein in its entirety. Such signal sequences or signal peptides may be included in any peptide immunogen as disclosed herein, as well as in any polynucleotide encoding any immunogen as disclosed herein without limitation, such as RNA or DNA. The polynucleotide having a sequence as set out in SEQ ID NO: 14, or other polynucleotide sequence encoding an implementation of an immunogen as disclosed herein, may be DNA, or RNA, such as may be transcribed or transcribable therefrom.

[0091] In an example, an immunogenic composition, such as a vaccine including an immunogenic polypeptide as disclosed herein, may include an adjuvant. An adjuvant is a composition included as part of an immunogenic composition, such as in a vaccine, for enhancing an immunogenic response in a subject to which it is administered. Many adjuvants are known to skilled artisans and may be administered to a subject with an immunogenic composition as disclosed herein for the purpose of stimulating or promoting an immune response against Borrelia burgdorferi or other Lyme-causing CspZ expressing Borrelia species. Non-limiting examples of adjuvants that could be included in a vaccine with an immunogenic polypeptide as disclosed herein include an aluminum salt, manganese, AS04, AS03, monophosphoryl lipid A (MPLA), poly(I:C), poly-ICLC, a CpG DNA adjuvant (for example but not limited to CpG 1018, CpG ODN 7909, or IC31), MF59 or other emulsion adjuvant, an emulsion adjuvant including squalene and water, a virosomal adjuvant, a cytokine, TITERMAX ® Gold adjuvant (a combination adjuvant including block copolymer CRL-8300, squalene, a sorbitan monooleateor), N-[1-(2,3-Dioleoyloxy)propyl] -N,N,N- trimethylammonium salt (DOTAP), 3 β-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-chol liposome), α-galactosylceramide, AS01, Qs-21, glucopyranosyl lipid A (GLA) (e.g., as an aqueous nanosuspension, stable emulsion, liposome, or aluminum hydroxide), an imidazoquinoline (e.g., without limitation, imiquimod (R837), resiquimod (R848), or 3M-052), a cyclic dinucleotide (such as but not limited to 2’,3’-cGAMP, 3’,3’- cGAMP, c-di-GMP, c-di-AMP, ADU-S100, MK-1454, BMS-986301, SB-11285, or IMSA-101), or any combination of any two or more of the foregoing. An immunogenic composition may include, without limitation, all possible combinations of any two or more of the foregoing adjuvants, all of which are explicitly included herein as if individually recited. Also included are immunogenic compositions including any one of the foregoing adjuvant. Other adjuvants that may be included in an immunogenic composition as disclosed herein, and explicitly included without limitation as optional component, alone or in any combination with the foregoing or following adjuvants, of any immunogenic composition disclosed herein, may include other toll-like receptor agonists, ISA51, Matrix M, or Advax. Examples of adjuvants for possible inclusion in an immunogenic composition as disclosed herein are disclosed in, for example, Iwasaki et al., 2020, Why and How Vaccines Work, Cell, Volume 183, Issue 2, 290 - 295; Zhao et al., 2023, Vaccine adjuvants: mechanisms and platforms. Sig Transduct Target Ther 8, 283; and Pulendran, et al, 2021, Emerging concepts in the science of vaccine adjuvants. Nat Rev Drug Discov 20, 454–475, all of which are hereby incorporated by reference herein in their entireties.

[0092] In an implementation disclosed herein, more than one adjuvant may be included. An adjuvant may include both an aluminum salt adjuvant and a CpG adjuvant, in combination. The aluminum salt adjuvant may include aluminum hydroxide, such as in, but not limited to, a wet gel suspension or AS04.

[0093] Including any one or more of the foregoing adjuvants or combinations thereof together with an immunogenic composition as disclosed herein may promote development of an immunological response. Any one or more of the foregoing could be combined with any immunogenic composition in accordance with the present disclosure for creation of a vaccine or stimulating immunogenicity to Borrelia burgdorferi or other CspZ-expressing Lyme disease-causing organism including for example B. afzelii. An immunogenic polypeptide as disclosed herein may be mixed with one or more adjuvant to form an immunogenic composition, wherein the one or more adjuvant is not covalently linked to the immunogenic polypeptide. An immunogenic polypeptide as disclosed herein may be covalently attached to one or more adjuvant to form an immunogenic composition, such as by modification of a side chain of an amino acid of, amino terminus of, or carboxyl terminus of the immunogenic polypeptide, permitting covalent attachment of the one or more adjuvant thereto.

[0094] 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 immunogenic composition as disclosed herein may be administered to prevent the development of Lyme disease orsymptoms thereof, or reduce severity of Lyme disease or symptoms thereof, following administration of the immunogenic composition to a subject. The subject may receive administration of the immunogenic composition 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 immunogenic composition 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 immunogenic composition 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. A therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder. The compositions may be administered to a patient 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.

[0095] Disclosed herein is a means for immunizing a subject against a CspZ- expressing species of Borrelia. Examples of such means include a polypeptide having an amino acid sequence as set out in SEQ ID NO: 1 except that any one or more of an amino acid whose position corresponds to amino acid 34 of SEQ ID NO: 1 may include any amino acid other than cysteine, an amino acid whose position corresponds to amino acid 48 of SEQ ID NO: 1 may include any amino acid other than threonine, an amino acid whose positioncorresponds to amino acid 61 of SEQ ID NO: 1 may include any amino acid other than inosine, an amino acid whose position corresponds to amino acid 86 of SEQ ID NO: 1 may include any amino acid other than phenylalanine, an amino acid whose position corresponds to amino acid 96 of SEQ ID NO: 1 may include any amino acid other than inosine, an amino acid whose position corresponds to amino acid 117 of SEQ ID NO: 1 may include any amino acid other than lysine, an amino acid whose position corresponds to amino acid 123 of SEQ ID NO: 1 may include any amino acid other than valine, an amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 may include any amino acid other than inosine, an amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 may include any amino acid other than cysteine, and an amino acid whose position corresponds to amino acid 174 of SEQ ID NO: 1 may include any amino acid other than glycine, an amino acid whose position corresponds to amino acid 34 of SEQ ID NO: 1 may include serine, an amino acid whose position corresponds to amino acid 48 of SEQ ID NO: 1 may include proline, an amino acid whose position corresponds to amino acid 61 of SEQ ID NO: 1 may include threonine, an amino acid whose position corresponds to amino acid 86 of SEQ ID NO: 1 may include proline, an amino acid whose position corresponds to amino acid 96 of SEQ ID NO: 1 may include threonine, an amino acid whose position corresponds to amino acid 117 of SEQ ID NO: 1 may include glutamic acid, an amino acid whose position corresponds to amino acid 123 of SEQ ID NO: 1 may include methionine, an amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 may include tyrosine, an amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 may include serine, an amino acid whose position corresponds to amino acid 174 of SEQ ID NO: 1 may include methionine, an amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 may include any amino acid other than inosine and an amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 may include any amino acid other than cysteine, and an amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 may include tyrosine and an amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 may include serine.

[0096] Disclosed herein is an immunogenic composition for immunizing a subject against a CspZ-expressing species of Borrelia, wherein the composition includes a polypeptide having an amino acid sequence as set out in SEQ ID NO: 1 except that any one or more of an amino acid whose position corresponds to amino acid 34 of SEQ ID NO: 1 may include any amino acid other than cysteine, an amino acid whose position corresponds to amino acid 48 of SEQ ID NO: 1 may include any amino acid other than threonine, an aminoacid whose position corresponds to amino acid 61 of SEQ ID NO: 1 may include any amino acid other than inosine, an amino acid whose position corresponds to amino acid 86 of SEQ ID NO: 1 may include any amino acid other than phenylalanine, an amino acid whose position corresponds to amino acid 96 of SEQ ID NO: 1 may include any amino acid other than inosine, an amino acid whose position corresponds to amino acid 117 of SEQ ID NO: 1 may include any amino acid other than lysine, an amino acid whose position corresponds to amino acid 123 of SEQ ID NO: 1 may include any amino acid other than valine, an amino acid whose position corresponds to amino acid 174 of SEQ ID NO: 1 may include any amino acid other than glycine, an amino acid whose position corresponds to amino acid 34 of SEQ ID NO: 1 may include serine, an amino acid whose position corresponds to amino acid 48 of SEQ ID NO: 1 may include proline, an amino acid whose position corresponds to amino acid 61 of SEQ ID NO: 1 may include threonine, an amino acid whose position corresponds to amino acid 86 of SEQ ID NO: 1 may include proline, an amino acid whose position corresponds to amino acid 96 of SEQ ID NO: 1 may include threonine, an amino acid whose position corresponds to amino acid 117 of SEQ ID NO: 1 may include glutamic acid, an amino acid whose position corresponds to amino acid 123 of SEQ ID NO: 1 may include methionine, and an amino acid whose position corresponds to amino acid 174 of SEQ ID NO: 1 may include methionine, and a means for raising the Tm of the immunogenic polypeptide to at least 60.5oC, or at least 61oC, or at least 61.5oC, or at least 62oC, or at least 62.5oC, wherein the means may include any one of more of an amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 substituted with any amino acid other than inosine, an amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 substituted with any amino acid other than cysteine, an amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 substituted with tyrosine, an amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 substituted with serine, an amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 substituted with any amino acid other than inosine and an amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 substituted with any amino acid other than cysteine, and an amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 substituted with tyrosine and an amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 substituted with serine.

[0097] Disclosed herein is an immunogenic composition for immunizing a subject against a CspZ-expressing species of Borrelia, wherein the composition includes a polypeptide having an amino acid sequence as set out in SEQ ID NO: 1 except that any oneor more of an amino acid whose position corresponds to amino acid 34 of SEQ ID NO: 1 may include any amino acid other than cysteine, an amino acid whose position corresponds to amino acid 48 of SEQ ID NO: 1 may include any amino acid other than threonine, an amino acid whose position corresponds to amino acid 61 of SEQ ID NO: 1 may include any amino acid other than inosine, an amino acid whose position corresponds to amino acid 86 of SEQ ID NO: 1 may include any amino acid other than phenylalanine, an amino acid whose position corresponds to amino acid 96 of SEQ ID NO: 1 may include any amino acid other than inosine, an amino acid whose position corresponds to amino acid 117 of SEQ ID NO: 1 may include any amino acid other than lysine, an amino acid whose position corresponds to amino acid 123 of SEQ ID NO: 1 may include any amino acid other than valine, an amino acid whose position corresponds to amino acid 174 of SEQ ID NO: 1 may include any amino acid other than glycine, an amino acid whose position corresponds to amino acid 34 of SEQ ID NO: 1 may include serine, an amino acid whose position corresponds to amino acid 48 of SEQ ID NO: 1 may include proline, an amino acid whose position corresponds to amino acid 61 of SEQ ID NO: 1 may include threonine, an amino acid whose position corresponds to amino acid 86 of SEQ ID NO: 1 may include proline, an amino acid whose position corresponds to amino acid 96 of SEQ ID NO: 1 may include threonine, an amino acid whose position corresponds to amino acid 117 of SEQ ID NO: 1 may include glutamic acid, an amino acid whose position corresponds to amino acid 123 of SEQ ID NO: 1 may include methionine, and an amino acid whose position corresponds to amino acid 174 of SEQ ID NO: 1 may include methionine, and a means for preventing Lyme disease in a subject administered the immunogenic composition fewer than three times, or one or two times, wherein the means may include any one or more of an amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 substituted with any amino acid other than inosine, an amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 substituted with any amino acid other than cysteine, an amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 substituted with tyrosine, an amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 substituted with serine, an amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 substituted with any amino acid other than inosine and an amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 substituted with any amino acid other than cysteine, and an amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 substituted with tyrosine and an amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 substituted with serine.

[0098] modRNA

[0099] A nucleoside is a molecule including a nitrogenous base (i.e., a nucleobase) linked to a pentose (e.g., deoxyribose or ribose) sugar. Nitrogenous bases which form nucleosides include adenine, guanine, cytosine, 5-methyl cytosine, uracil, and thymine. Suitable ribonucleosides (which include ribose as the pentose sugar) include, e.g., adenosine (A), guanosine (G), 5-methyluridine (m5U), uridine (U), and cytidine (C). Nucleotides are molecules including a nucleoside (e.g., a ribonucleoside) and a phosphate group. Ribonucleotides include, e.g., adenosine monophosphate, adenosine diphosphate, adenosine triphosphate, guanosine monophosphate, guanosine diphosphate, guanosine triphosphate, cytidine monophosphate, cytidine diphosphate, cytidine triphosphate, uridine monophosphate, uridine diphosphate, uridine triphosphate, and derivatives thereof.

[0100] Nucleoside-modified RNA, or modRNA, is a synthetic modified RNA that can be used for expression of a gene of interest. Chemical modifications to a ribonucleotide included in modRNA may stabilize an RNA molecule, blunt an immune response, or enhance transcription. Additionally, unlike delivery of protein agents directly to a cell, which can activate the immune system, the delivery of modRNA can be achieved without immune impact. For example, substitution of uridine and cytidine with pseudouridine or N1- methylpseudouridine and 5-methylcytidine, respectively, drastically reduces the immune response elicited from exogenous RNA without such substitutions. Stability and translational efficiency from an RNA molecule may also be increased by including a 3´-O-Me- m7G(5')ppp(5')G Anti Reverse Cap Analog (ARCA) at the 5′ end of the RNA molecule.

[0101] modRNA may encompass an RNA molecule with at least uridine substituted with pseudouridine. modRNA may encompass an RNA molecule with at least cytidine substituted with 5-methylcytidine. modRNA may encompass an RNA molecule including the modified nucleoside 5-methylcytidine (5mC). modRNA may encompass an RNA molecule including the modified nucleoside 2-Thiouridine-5′-Triphosphate (2-thio ψU). modRNA may encompass an RNA molecule with at least the modified nucleoside1-Methylpseudouridine-5′- Triphosphate (1-mψU). modRNA may encompass an RNA molecule with at least the modified nucleoside N1-methyl-pseudouridine (N1mΨ) substituted for uridine. modRNA may encompass an RNA molecule wherein at least 5′ triphosphates are removed. modRNA may encompass an RNA molecule wherein at least a 3′-O-Me-m7G(5′)ppp(5′)G Anti Reverse Cap Analog (ARCA) cap or C32H43N15O24P4 CleanCap Reagent AG is included in a 5′ untranslated regions of the RNA molecule.

[0102] modRNAs may be prepared by in vitro transcription. modRNA may be in vitro transcribed, e.g., from a linear DNA template using one or more reagents selected from a cap analog, guanosine triphosphate, adenosine triphosphate, cytidine triphosphate, uridine triphosphate, and derivatives thereof. A cap analog may be selected from Anti-Reverse Cap Analog (ARCA) 3′-O-Me-m7G(5′)ppp(5′)G, standard cap analog m7G(5′)ppp(5′)G, unmethylated cap analog G(5′)ppp(5′)G, methylated cap analog for A+1 sites m7G(5′)ppp(5′)A, and unmethylated cap analog for A+1 sites G(5′)ppp(5′)A. In certain examples, a cap analog is Anti-Reverse Cap Analog (ARCA) 3′-O-Me-m7G(5′)ppp(5′)G. According to some examples, modRNA may be in vitro transcribed from a plasmid template using one or more reagents selected from 3′-O-Me-m7G(5′)ppp(5′)G, guanosine triphosphate, adenosine triphosphate, cytidine triphosphate, N1-methylpseudouridine-5-triphosphate, and any one or more of the aforementioned examples of modRNA, or others, without limitation and in any combination.

[0103] An RNA vaccine of any nucleotide sequence encoding any protein immunogen disclosed herein may include a 3′ UTR (e.g., from 130 and 280 nucleotides in length) with a length and sequence to prevent reduced half-life of the RNA and to promote high levels of translation. Examples of such 3′ UTR for use in RNA vaccines are known. Some non-liming implementations of a 3′ UTR that may be included in any polynucleotide disclosed herein having a sequence encoding any immunogenic protein disclosed herein may include one or more (e.g., two) copies of a 3′ UTR sequence of the human alpha-globin or β- globin genes (such as two repeats of the human β-globin 3′ UTR) or the human PSMB3. See Leong et al., 2025, Revolutionizing immunization: a comprehensive review of mRNA vaccine technology and applications. Virol J 22, 71, which is hereby incorporated by reference in its entirety.

[0104] Additional suitable modifications to a modRNA or mRNA molecule are well known in the art (see, e.g., U.S. Patent No.8,278,036; U.S. Patent No.10,086,043; and U.S. Patent Application Publication No.2018 / 0353618; which are hereby incorporated by reference in their entireties for all purposes). In some embodiments, the nucleoside that is modified in the modRNA is a uridine (U), a cytidine (C), an adenine (A), or guanine (G). The modified nucleoside can be, for example, m5C (5-methylcytidine), m6A (N6- methyladenosine), s2U (2-thiouridien), ψ (pseudouridine), or Um (2-O-methyluridine). Some exemplary chemical modifications of nucleosides in the modRNA molecule may further include, for example and without limitation, pyridine-4-one ribonucleoside, 5-aza-uridine, 2- thio-5-aza uridine, 2-thiouridine, 4-thio pseudouridine, 2-thio pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl uridine, 1-carboxymethyl pseudouridine, 5-propynyl uridine, 1-propynyl pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl pseudouridine, 5-taurinomethyl-2-thio uridine, 1-taurinomethyl-4-thio uridine, 5-methyl uridine, 1-methyl pseudouridine, 4-thio-1-methyl pseudouridine, 2-thio-1-methyl pseudouridine, 1-methyl-1-deaza pseudouridine, 2-thio-1-methyl-1-deaza pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio dihydrouridine, 2-thio dihydropseudouridine, 2- methoxyuridine, 2-methoxy-4-thio uridine, 4-methoxy pseudouridine, 4-methoxy-2-thio pseudouridine, 5-aza cytidine, pseudoisocytidine, 3-methyl cytidine, N4-acetylcytidine, 5- formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio cytidine, 2-thio-5-methyl cytidine, 4-thio pseudoisocytidine, 4-thio-1-methyl pseudoisocytidine, 4-thio-1-methyl-1-deaza pseudoisocytidine, 1-methyl-1-deaza pseudoisocytidine, zebularine, 5-aza zebularine, 5- methyl zebularine, 5-aza-2-thio zebularine, 2-thio zebularine, 2-methoxy cytidine, 2- methoxy-5-methyl cytidine, 4-methoxy pseudoisocytidine, 4-methoxy-1-methyl pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza adenine, 7-deaza-8-aza adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6- isopentenyladenosine, N6-(cis-hydroxyisopentenyl) adenosine, 2-methylthio-N6-(cis- hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6- threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6- dimethyladenosine, 7-methyladenine, 2-methylthio adenine, 2-methoxy adenine, inosine, 1- methyl inosine, wyosine, wybutosine, 7-deaza guanosine, 7-deaza-8-aza guanosine, 6-thio guanosine, 6-thio-7-deaza guanosine, 6-thio-7-deaza-8-aza guanosine, 7-methyl guanosine, 6- thio-7-methyl guanosine, 7-methylinosine, 6-methoxy guanosine, 1-methylguanosine, N2- methylguanosine, N2,N2-dimethylguanosine, 8-oxo guanosine, 7-methyl-8-oxo guanosine, 1- methyl-6-thio guanosine, N2-methyl-6-thio guanosine, or N2,N2-dimethyl-6-thio guanosine.

[0105] In an example, modifications made to the modRNA are independently selected from 5-methylcytosine, pseudouridine, and 1-methylpseudouridine.

[0106] In some embodiments, the modRNA includes a modified uracil selected from the group consisting of pseudouridine (ψ), pyridine-4-one ribonucleoside, 5-aza uridine, 6- aza uridine, 2-thio-5-aza uridine, 2-thio uridine (s2U), 4-thio uridine (s4U), 4-thio pseudouridine, 2-thio pseudouridine, 5-hydroxy uridine (ho5U), 5-aminoallyl uridine, 5-halo uridine (e.g., 5-iodom uridine or 5-bromo uridine), 3-methyl uridine (m3U), 5-methoxy uridine (mo5U), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester(mcmo5U), 5-carboxymethyl uridine (cm5U), 1-carboxymethyl pseudouridine, 5- carboxyhydroxymethyl uridine (chm5U), 5-carboxyhydroxym ethyl uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio uridine (mcm5s2U), 5-aminomethyl-2-thio uridine (nm5s2U), 5-methylaminomethyl uridine (mnm5U), 5-methylaminomethyl-2-thio uridine (mnm5s2U), 5-methylaminomethyl-2-seleno uridine (mnm5se2U), 5-carbamoylmethyl uridine (ncm5U), 5-carboxymethylaminomethyl uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio uridine (cmnm5s2U), 5-propynyl uridine, 1-propynyl pseudouridine, 5-taurinomethyl uridine (τcm5U), 1-taurinomethyl pseudouridine, 5-taurinomethyl-2-thio uridine (™5s2U), 1-taurinomethyl-4-thio pseudouridine, 5-methyl uridine (m5U, e.g., having the nucleobase deoxythymine), 1-methyl pseudouridine (m1ψ), 5-methyl-2-thio uridine (m5s2U), 1-methyl-4-thio pseudouridine (m1s4ψ), 4-thio-1-methyl pseudouridine, 3-methyl pseudouridine (m3ψ), 2-thio-1-methyl pseudouridine, 1-methyl-1-deaza pseudouridine, 2-thio-1-methyl-1-deaza pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl dihydrouridine (m5D), 2-thio dihydrouridine, 2-thio dihydropseudouridine, 2-methoxy uridine, 2-methoxy-4- thio uridine, 4-methoxy pseudouridine, 4-methoxy-2-thio pseudouridine, N1-methyl pseudouridine, 3-(3-amino-3-carboxypropyl) uridine (acp3U), 1-methyl-3-(3-amino-3- carboxypropyl) pseudouridine (acp3ψ), 5-(isopentenylaminomethyl) uridine (inm5U), 5- (isopentenylaminomethyl)-2-thio uridine (inm5s2U), α-thio uridine, 2′-O-methyl uridine (Um), 5,2′-O-dimethyl uridine (m5Um), 2′-O-methyl pseudouridine (ψm), 2-thio-2′-O-methyl uridine (s2Um), 5-methoxycarbonylmethyl-2′-O-methyl uridine (mcm5Um), 5- carbamoylmethyl-2′-O-methyl uridine (ncm5Um), 5-carboxymethylaminomethyl-2′-O-methyl uridine (cmnm5Um), 3,2′-O-dimethyl uridine (m3Um), 5-(isopentenylaminomethyl)-2′-O- methyl uridine (inm5Um), 1-thio uridine, deoxythymidine, 2′-F-ara uridine, 2′-F uridine, 2′- OH-ara uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-3-(1-E-propenylamino) uridine.

[0107] In some embodiments, the modRNA includes a modified cytosine selected from the group consisting of 5-aza cytidine, 6-aza cytidine, pseudoisocytidine, 3-methyl cytidine (m3C), N4-acetyl cytidine (act), 5-formyl cytidine (f5C), N4-methyl cytidine (m4C), 5-methyl cytidine (m5C), 5-halo cytidine (e.g., 5-iodo cytidine), 5-hydroxymethyl cytidine (hm5C), 1-methyl pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio cytidine (s2C), 2-thio-5-methyl cytidine, 4-thio pseudoisocytidine, 4-thio-1-methyl pseudoisocytidine, 4-thio-1-methyl-1-deaza pseudoisocytidine, 1-methyl-1-deaza pseudoisocytidine, zebularine, 5-aza zebularine, 5-methyl zebularine, 5-aza-2-thio zebularine, 2-thio zebularine, 2-methoxy cytidine, 2-methoxy-5-methyl cytidine, 4-methoxypseudoisocytidine, 4-methoxy-1-methyl pseudoisocytidine, lysidine (k2C), alpha-thio cytidine, 2′-O-methyl cytidine (Cm), 5,2′-O-dimethyl cytidine (m5Cm), N4-acetyl-2′-O- methyl cytidine (ac4Cm), N4,2′-O-dimethyl cytidine (m4Cm), 5-formyl-2′-O-methyl cytidine (f5Cm), N4,N4,2′-O-trimethyl cytidine (m42Cm), 1-thio cytidine, 2′-F-ara cytidine, 2′-F cytidine, and 2′-OH-ara cytidine.

[0108] In some embodiments, the modRNA includes a modified adenine selected from the group consisting of 2-amino purine, 2,6-diamino purine, 2-amino-6-halo purine (e.g., 2-amino-6-chloro purine), 6-halo purine (e.g., 6-chloro purine), 2-amino-6-methyl purine, 8-azido adenosine, 7-deaza adenine, 7-deaza-8-aza adenine, 7-deaza-2-amino purine, 7-deaza-8-aza-2-amino purine, 7-deaza-2,6-diamino purine, 7-deaza-8-aza-2,6-diamino purine, 1-methyl adenosine (m1A), 2-methyl adenine (m2A), N6-methyl adenosine (m6A), 2- methylthio-N6-methyl adenosine (ms2m6A), N6-isopentenyl adenosine (i6A), 2-methylthio- N6-isopentenyl adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl) adenosine (io6A), 2- methylthio-N6-(cis-hydroxyisopentenyl) adenosine (ms2io6A), N6-glycinylcarbamoyl adenosine (g6A), N6-threonylcarbamoyl adenosine (t6A), N6-methyl-N6-threonylcarbamoyl adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl adenosine (ms2g6A), N6,N6-dimethyl adenosine (m62A), N6-hydroxynorvalyIcarbamoyl adenosine (hn6A), 2-methylthio-N6- hydroxynorvalylcarbamoyl adenosine (ms2hn6A), N6-acetyl adenosine (ac6A), 7-methyl adenine, 2-methylthio adenine, 2-methoxy adenine, alpha-thio adenosine, 2′-O-methyl adenosine (Am), N6,2′-O-dimethyl adenosine (m6Am) N6,N6,2′-O-trimethyl adenosine (m62Am), 1,2′-O-dimethyl adenosine (m1Am), 2′-O-ribosyl adenosine (phosphate) (Ar(p)), 2- amino-N6-methyl purine, 1-thio adenosine, 8-azido adenosine, 2′-F-ara adenosine, 2′-F adenosine, 2′-OH-ara adenosine, and N6-(19-amino-pentaoxanonadecyl) adenosine.

[0109] In some embodiments, the modRNA includes a modified guanine selected from the group consisting of inosine (I), 1-methyl inosine (m1I), wyosine (imG), methylwyosine (mimG), 4-demethyl wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW), hydroxywybutosine (OHyW), undermodified hydroxywybutosine (OHyWy), 7-deaza guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl queuosine (galQ), mannosyl queuosine (manQ), 7-cyano-7-deaza guanosine (preQ0), 7-aminomethyl-7-deaza guanosine (preQ1), archaeosine (G+), 7-deaza-8-aza guanosine, 6-thio guanosine, 6-thio-7-deaza guanosine, 6-thio-7-deaza-8-aza guanosine, 7- methyl guanosine (m7G), 6-thio-7-methyl guanosine, 7-methyl inosine, 6-methoxy guanosine, 1-methyl guanosine (m1G), N2-methyl-guanosine (m2G), N2,N2-dimethyl guanosine (m22G), N2,7-dimethyl guanosine (m2,7G), N2, N2,7-dimethyl guanosine (m2,2,7G), 8-oxo guanosine, 7-methyl-8-oxo guanosine, 1-methio guanosine, N2-methyl-6-thio guanosine, N2,N2-dimethyl- 6-thio guanosine, alpha-thio guanosine, 2′-O-methyl guanosine (Gm), N2-methyl-2′-O-methyl guanosine (m2Gm), N2,N2-dimethyl-2′-O-methyl guanosine (m22Gm), 1-methyl-2′-O-methyl guanosine (m1Gm), N2,7-dimethyl-2′-O-methyl guanosine (m2,7Gm), 2′-O-methyl inosine (1m), 1,2′-O-dimethyl inosine (m1Im), 2′-O-ribosyl guanosine (phosphate) (Gr(p)), 1-thio guanosine, O6-methyl guanosine, 2′-F-ara guanosine, and 2′-F guanosine.

[0110] modRNA may include, for example, a non-natural or modified nucleotide. The non-natural or modified nucleotide may include, for example, a backbone modification, sugar modification, or base modification. The non-natural or modified nucleotide may include, for example, a base modification. In some embodiments, the base modification is selected from the group consisting of 2-amino-6-chloropurine riboside 5′ triphosphate, 2-aminoadenosine 5′ triphosphate, 2-thiocytidine 5′ triphosphate, 2-thiouridine 5′ triphosphate, 4-thiouridine 5′ triphosphate, 5-aminoallylcytidine 5′ triphosphate, 5-aminoallyluridine 5′ triphosphate, 5- bromocytidine 5′ triphosphate, 5-bromouridine 5′ triphosphate, 5-iodocytidine 5′ triphosphate, 5-iodouridine 5′ triphosphate, 5-methylcytidine 5′ triphosphate, 5-methyluridine 5′ triphosphate, 6-azacytidine 5′ triphosphate, 6-azauridine 5′ triphosphate, 6-chloropurine riboside 5′-triphosphate, 7-deazaadenosine 5′ triphosphate, 7-deazaguanosine 5′ triphosphate, 8-azaadenosine 5′ triphosphate, 8-azidoadenosine 5′ triphosphate, benzimidazole riboside 5′ triphosphate, N1-methyladenosine 5′ triphosphate, N1-methylguanosine 5′ triphosphate, N6- methyladenosine 5′ triphosphate, O6-methylguanosine 5′ triphosphate, N1-methyl- pseudouridine 5′ triphosphate, puromycin 5′-triphosphate, and xanthosine 5′ triphosphate. Thus, according to some embodiments, modRNA includes N1-methyl-pseudouridine 5′ triphosphate.

[0111] Nanoparticles

[0112] A nanoparticle is a composition of matter having a nanoscale-dimension size, such as a diameter from about 1 nm to about 100 nm, though may refer to compositions having a larger diameter as well, such as up to 500 nm. A nanoparticle may provide enhanced cellular uptake and stability of a an RNA molecule as described herein. Packaging RNA in a nanoparticle may protect it from extracellular degradation processes that may otherwise occur following, for example, systemic or other administration of an RNA molecule, thereby increasing cellular uptake.

[0113] A nanoparticle may also improve cellular uptake by providing a mechanism for cellular entry, such as fusion of a nanoparticle’s membrane with a cellular membrane for delivery of the nanoparticle’s payload to an intracellular compartment. A variety of materialsare known to be suitable for nanoparticles for intracellular delivery of their payloads such as lipid or phospholipid micelles or liposomes, metal nanoparticles, such as gold, aluminum, iron nanoparticles, polyacrylamide, polyacrylate, or chitosan nanoparticles, a polymer-based nanoparticle such as a poly lactic-co-glycolic nanoparticle, may be used in accordance with the present disclosure, with an RNA molecule packaged in any type of nanoparticle suitable for an intended purpose, synthesized according to standard methods.

[0114] A nanoparticle including an RNA polynucleotide molecule encoding an immunogen as disclosed herein, or a DNA molecule encoding the foregoing RNA molecule, including all variations or implementations disclosed herein, may include a lipoplex or a lipid nanoparticle (LNP). LNPs may include four components: ionizable cationic lipids, neutral lipids such as phospholipids, a steroid such as cholesterol, and a polymer conjugated lipid such as polyethylene glycol (PEG)-lipids. LNPs may be prepared by mixing lipids dissolved in ethanol rapidly with nucleic acid in an aqueous buffer. The LNP may include any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated. An LNP may include one or more cationic lipids, and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and pegylated lipids. An LNP may include a cationic lipid, a neutral lipid, a steroid, a polymer conjugated lipid; and the RNA molecule or DNA molecule, encapsulated within or associated with the lipid nanoparticle.

[0115] An RNA polynucleotide molecule encoding an immunogen as disclosed herein, or a DNA molecule encoding the foregoing RNA molecule, may be included in a vector. A vector may include a lipid nanoparticles (LNP), charged polymers, coordinative polymers, boronic acid polymers, fluorinated polymers, gold nanoparticles, silica nanoparticles, virus-like particles (VLPs), exosomes, or an extracellular contractile injection system (eCIS) derived from the entomopathogenic bacterium P. asymbiotica virulence cassette (PVC). See Chan, and Tsourkas, 2024, Intracellular Protein Delivery: Approaches, Challenges, and Clinical Applications. BME Front., 5:0035, incorporated by reference herein in its entirety for all purposes.

[0116] A lipid nanoparticle as a vector may include an ionizable lipid, such as DOTMA, DODAP, DLin-DMA, DLin-KC2-DMA, DLin-MC3-DMA, or any combination of two or more of the foregoing, including together with other constituents conventionally included in LNPs, such as phospholipids, cholesterol, and PEGylated lipids. See Schlich et al., 2021, Cytosolic delivery of nucleic acids: The case of ionizable lipid nanoparticles. Bioeng Transl Med.;6(2):e10213.

[0117] Methods and compositions for forming thermostable polynucleotide- containing formulations capable of inducing an immune response are known (see, e.g., WO2025029323A1, which is incorporated by reference herein in its entirety). Such thermostable formulations may be made including any polynucleotide as disclosed herein without exception. Such a thermostable polynucleotide-containing formulation may, in a non- limiting implementation, include a nanoparticle. It may include one or more non-reducing disaccharide agents; such as trehalose, sucrose, lactose, or a combination thereof or combinations thereof. It may further include one or more aqueous solution; optionally wherein the aqueous solution includes histidine, H2O, or a combination thereof. It may further include hydroxypropyl-beta-cyclodextrin, lysine, leucine, silk fibroin, inulin, alternative sugars including one or more of dextrose, maltodextrin, sorbitol, mannitol, dextran, maltotriose, or a combination thereof.

[0118] The thermostable polynucleotide-containing formulation may include a nonionic starch derivative, other starch, polysaccharide, or substitutable agent thereof. The nonionic starch derivative or substitutable agent thereof may include one or more of hydroxy ethyl starch, succinylated gelatin, or other nonionic starch or derivative thereof.

[0119] The thermostable polynucleotide-containing formulation may include one or more polymers. The one or more polymers may include one or more of polyvinyl alcohol (PVA), polyacrylic acid (PAA), polyethylene glycol (PEG), polymethacrylate-based copolymers (e.g., Eudragit), povidone or other high molecular weight polymer or other low solubility polymer, or combination thereof. The polymer may be PVA.

[0120] The thermostable polynucleotide-containing formulation may include at least one cellulose agent. The at least one cellulose agent may include at least one of carboxy methyl cellulose (low, medium, and / or high molecular weight), chitosan, pectin, dextran, or combination thereof. The thermostable polynucleotide-containing formulation may include one or more additional volatilizing agents, including an alcohol or the like; optionally methanol or ethanol. The thermostable polynucleotide-containing formulation may include one or more volatile salts; optionally one or more of ammonium acetate, ammonium formate, ammonium carbamate, ammonium carbonate, ammonium bicarbonate, tri ethyl ammonium acetate, tri ethyl ammonium formate, triethylammonium carbonate, trimethylamine acetate trimethylamine formate, trimethylamine carbonate, pyridinal acetate and pyridinal formate, or combinations thereof.

[0121] The formulation may be essentially dry and include at least one partial or complete coating for encapsulating the essentially dry composition including apolynucleotide encoding an immunogen as disclosed herein. The complete coating or encapsulation may be an atomic layer deposition (ALD) applied coating or encapsulation of the essentially dry composition. Each layer of the one or more coating layers may include one or more coating layers of metallo-organic-, metal oxides-, metal alkoxides-, and / or aluminum-based material. Each layer of the one or more coating layers may include one or more of aluminum oxide, aluminum alkoxide, silicon dioxide, zinc dioxide, titanium dioxide, silicon nitride, or combinations thereof.

[0122] Such a formulation may be prepared by combining such of the foregoing components as intended for inclusion therein to create a liquid formulation; and at least one of: atomizing the liquid formulation in a gas stream to create particulates, and spray-drying the liquid formulation to create an essentially dry formulation of particulates and / or microparticles. At least 1.0% of the polynucleotide may be stable, wherein less than 70.0%, or less than 60%, or less than 50%, or less than 40%, of the polynucleotide is degraded. The composition may be stable for at least one month or more at elevated temperatures; optionally, wherein elevated temperatures are at least room temperature up to about 60° C, or up to about 70° C. The one or more nonreducing disaccharides may be present in a weight-to- volume concentration from about 1.0% to about 20.0% in the liquid formulation. The particulates and / or microparticles may further include at least one complete coating for encapsulating the particulates or the microparticles of the polynucleotide. The complete coating or encapsulation may include introducing the particulates or microparticles of the essentially dry formulation to a fluidized bed of an atomic layer deposition (ALD) reactor and applying one or more coating layers to the particulates or the essentially dry formulation.

[0123] Each layer of the one or more coating layers may include one or more coating layers of a metalloorganic-, metal oxide-, metal alkoxide-, and / or aluminum-based material. Each layer of the one or more coating layers includes one or more coating layers of aluminum oxide, aluminum alkoxide, silicon dioxide, Zinc dioxide. titanium dioxide. and silicon nitride or combinations thereof or compositions thereof.

[0124] Drying gas used for the spray-drying may have a relative humidity of less than 50%, or less than 40%, or less than 30%, or less than 20%, or less than 10% or less than 5%. Drying gas used for the spray-drying process may have an inlet gas or nozzle temperature of less than 200° C, less than 190° C, or less than 180° C, or less than 170° C, or less than 160° C, or less than 150° C, or less than 140° C, or less than 130° C, or less than 120° C, or less than 110° C, or less than 100° C, or less than 90° C, or less than 80° C, or less than 70° C, or less than 60° C, or 50 °C or less.

[0125] The liquid formulation may further include at least one alcohol agent and permitting a lower drying temperature including 200.0° C or less for spray-drying the particulates or liquid formulation. Drying gas flow rates for the spray-drying may include about 0.01 to about 5.0 m3 / min, or about 0.1 to about 2.5 m3 / min, or about 0.1 to about 1.5 m3 / min, or about 0.1-1.2 m3 / min. Atomizing gas flow rates may include about 0.01 to about 100 L / min, or about 0.05 to about 75 L / min, or about 0.01 to about 75 L / min, or about 0.1 to about 50 L / min. Feed flow rates for the spray-drying may include about 0.01 to about 50 g / min, about 0.05 to about 40 g / min. or about 0.1-35 g / min.

[0126] Preparation of such a formulation may further include using cyclone gas injected below the spray drying chamber for separating the particulates or the essentially dry formulation, and wherein cyclone gas flow rates include about 1.0 to about 1,000 L / min, about 1.0 to about 750.0 L / min, about 1.0 to about 500.0 L / min, or about 1.0-400.0 L / min. Drying gas for the spray-drying may include at least one of ambient air, dehumidified air, or dry nitrogen, or any combination thereof. A spray -dryer for spray-drying may include a two- fluid atomizing nozzle, an ultrasonic atomizing nozzle, or any nozzle determined to effectively atomize the liquid formulation. The liquid formulation containing the alcohol agent may be fed into a spray-dryer at a rate such that any drying and nozzle gas maintains the spray-dryer below the lower flammability limit for any evolved volatile components and reduces a chance of alcohol catching on fire.

[0127] Disclosed herein is a method for eliciting an immune response against Borrelia burgdorferi, including administering to a subject a pharmaceutical composition including a thermostable polynucleotide-containing formulation as disclosed herein, wherein the polynucleotide (DNA or RNA) encodes an immunogen as disclosed herein. Also disclosed herein is a DNA molecule encoding an RNA polynucleotide molecule encoding an immunogen as disclosed herein. The DNA molecule 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. The DNA molecule may be an oligonucleotide. Optionally, the DNA molecule may include one or more sequence promoting transcription of the RNA molecule as disclosed herein such that it may be produced by a cell that includes the DNA molecule. A vector may contain the necessary elements that permit transcribing an RNA molecule from the DNA molecule, and, optionally, translating the gene or interest into a polypeptide. The subject may have Lyme disease or be suspected of having been infected with or exposed to Borrelia burgdorferi, for example.

[0128] A vector may be an episomal vector (i.e., does not integrate into the genome of a host cell), or can be vectors that integrate into the host cell genome. The term vector may thus also be defined as a gene delivery vehicle that facilitates gene transfer into a target cell. This definition includes both non-viral and viral vectors. Non-viral vectors, including either a DNA or RNA payload, include but are not limited to cationic lipids, liposomes, nanoparticles, PEG, PEI, plasmid vectors (e.g. pUC vectors, bluescript vectors (pBS) and pBR322 or derivatives thereof that are devoid of bacterial sequences (minicircles)) transposons-based vectors (e.g. PiggyBac (PB) vectors or Sleeping Beauty (SB) vectors), etc. Viral vectors are derived from viruses and include but are not limited to retroviral, lentiviral, adeno-associated viral, adenoviral, herpes viral, hepatitis viral vectors or the like. Typically, but not necessarily, viral vectors are replication-deficient as they have lost the ability to propagate in a given cell since viral genes essential for replication have been eliminated from the viral vector. A vector may be an adeno-associated viral (AAV) vector. AAV vectors are preferably used as self-complementary, double-stranded AAV vectors (scAAV) in order to overcome one of the limiting steps in AAV transduction (i.e. single-stranded to double- stranded AAV conversion) (McCarty, 2001, 2003; Nathwani et al, 2002, 2006, 2011; Wu at al., 2008), although the use of single-stranded AAV vectors (ssAAV) are also encompassed herein.

[0129] While it may be possible for the compounds disclosed herein to be administered as the raw chemical, it is preferable to present them as a pharmaceutical composition. According to a further aspect, the present invention provides a pharmaceutical composition including a compound of formula I or a pharmaceutically acceptable salt thereof, together with one or more pharmaceutically carriers thereof and optionally one or more other therapeutic ingredients. The carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. In one embodiment, the pharmaceutically acceptable carrier is selected from the group consisting of a liquid filler, a solid filler, a diluent, an excipient, a solvent, and an encapsulating material.

[0130] Pharmaceutically acceptable carriers or excipients (e.g., additives such as diluents, immunostimulants, adjuvants, antioxidants, preservatives and solubilizing agents) are nontoxic to the cell or subject being exposed thereto at the dosages and concentrations employed. Examples of pharmaceutically acceptable carriers include water, e.g., buffered with phosphate, citrate and another organic acid. Representative examples of pharmaceutically acceptable excipients that may be useful in the present disclosure includeantioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; adjuvants (e.g., 3-glucan or granulocyte colony stimulating factor (GCSF)); hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

[0131] Disclosed herein is a cell transfected with or expressing any of the foregoing polynucleotides (DNA or RNA), and a method of transfecting or transducing a cell with any of the foregoing polynucleotides, including by contacting the cell, or an organism, with any of the foregoing vectors or any of the foregoing polynucleotides.

[0132] Included in the present disclosure is a method of administering to an organism such as a human an immunogen as disclosed herein (e.g., variants of SEQ ID NO: 1, such as represented by variations disclosed for SEQ ID NO: 2, all permutations of which are explicitly included herein, as are all polynucleotides encoding them, without limitation, to induce immunity or prevent contraction of Lyme disease, or administering any of the foregoing polynucleotides, be it DNA or RNA, to induce expression of the peptide immunogen by cells of the organism such as to activate an immune response. Any permutations of any of the foregoing examples disclosed throughout this disclosure, which combinations, permutations, and implementations are intentionally an explicitly included in examples of administering the expression regulatory system to a subject. Administering may include administering intravenously, administering intramuscularly, administering intraorally, administering intranasally, administering intraperitoneally, or any combination of the foregoing.

[0133] A any polynucleotide (RNA or DNA) having a sequence that encodes any of the immunogenic proteins disclosed herein may be administered to a subject to induce immunity to Lyme disease-causing borreliae, eliciting an immune response. In an implementation, an RNA vaccine may include an RNA molecule encoding an immunogen as disclosed herein as disclosed herein (such as encodes any variant of SEQ ID NO: 1 or of SEQ ID NO: 2, such as, without limitation, SEQ ID NO: 3 or SEQ ID NO: 14). Such an RNA molecule may be formulated with any suitable delivery system such as a lipid nanoparticle, liposome, polymeric nanoparticle, or other carrier suitable for in vivo delivery and cellular uptake. An RNA vaccine may be administered to a mammalian subject, including a human,through any route capable of achieving delivery to cells for translation of the encoded antigen. Exemplary routes of administration include intramuscular (IM), subcutaneous (SC), intradermal (ID), or intranasal administration. In certain embodiments, intramuscular administration may be performed by injection into a large muscle group (e.g., the deltoid or quadriceps).

[0134] Any such RNA vaccine may be delivered as a single dose or as part of a multi- dose regimen. In some embodiments, a priming dose may be followed by one or more booster doses administered at intervals sufficient to enhance or sustain the immune response, for example between 1 and 12 weeks after an initial dose. A dose may include between 1 µg and 500 µg of RNA per administration, although higher or lower doses may be used depending on the formulation, delivery system, and intended use. RNA may be provided in a sterile, buffered aqueous solution or suspension suitable for parenteral administration. The total injection volume may range from 0.1 mL to 2 mL per site.

[0135] Following administration, an RNA vaccine is taken up by host cells, which translate the encoded antigen and present it to the immune system, thereby inducing a humoral and / or cellular immune response specific to the target antigen. The response may be measured by standard immunological assays, including ELISA, neutralization assays, or T- cell activation assays. EXAMPLES

[0136] The following examples are intended to illustrate particular embodiments of the present disclosure, but are by no means intended to limit the scope thereof.

[0137] EXAMPLE 1:

[0138] An approach of structure-based vaccine design identified the targeted amino acids of CspZ-YA for mutagenesis. Availability of the high resolution of the candidate vaccinogen is the useful for structure-based vaccine design. We thus first crystallized and obtained the structure of recombinant untagged CspZ-YA protein (CspZ-YAUT ) (1.90 Å) (Fig.1A, Fig.2). We then superimposed CspZ-YAUTwith the previously determined crystal structures of CspZ from the CspZ-FH complex

[0035] . The superimposed structures revealed that the Y211A mutation led to the extension of the N-terminal helix I by three residues (Ala211, Lys212, Lys213) (inlet figures in Fig.1A). The extended helix I and the Y207A mutation resulted in an altered conformation of the loop between helix H and I (loop H / I, inlet figures in Fig.1A). Such an orientation of the loop in CspZ-YA thus prevented the Arg206 (located on the loop H / I) from interacting with Glu186 (inlet figures of Fig.1A).Overall, our newly-resolved, high-resolution CspZ-YA structure indicates the extended helix I and the altered loop H / I as the mechanisms underlying the lack of FH-binding ability in this mutant protein

[0036] .

[0139] We then set to design variants by substituting amino acids that are predicted to enhance the stability of CspZ-YA. The resulting substitutions include T67P, I80T, F105P, I115T, K136E, V142M, I183Y, and G193M on CspZ-YA, falling into one of four categories: (1) prolines at loop regions to favor folding entropy, (2) polar residues to reduce surface hydrophobicity, (3) bulky hydrophobic residues to fill internal cavities. and (4) creating charge repulsion to disrupt FH binding (Fig.1B). Additionally, the presence of cysteine residues could lead to the formation of unwanted intermolecular disulfide bonds, resulting in the risk of protein aggregations [37-40]. We thus designed additional substitutions targeting two cysteine residues of CspZ-YA (i.e., C53S and C187S in Fig.1B). Notably, C187S substitution might potentially form polar interaction with Glu214, and I183Y substitution is meant to fill the cavity newly created by Y207A / Y211A mutations on CspZ-YA backbone (inlet figures of Fig.1B). The recombinant version of these proteins was produced in E. coli with histidine tags (for the mutants of T67P, I80T, F105P, I115T, K136E, V142M, I183Y, and G193M) or as no tags (for the mutants of C53S and C187S). The histidine-tagged and untagged proteins of CspZ-YA were also produced as control. We found that CspZ-YAC53S was aggregated and insoluble (data not shown) while other CspZ-YA mutant proteins were soluble and did not show differences of global secondary structures, compared to CspZ-YA (Fig.3). Therefore, all variants except CspZ-YAC53S were moved forward to the following studies.

[0140] Mutagenizing I183Y and C187S of CspZ-YA permitted to trigger robust borreliocidal antibodies with reduced immunization frequency. To characterize the role of these mutagenized amino acids in impacting immunogenicity, we inoculated mice with each of these CspZ-YA mutant proteins, CspZ-YA, or CspZ-YAUTwith different frequency. The titers of CspZ IgG in the sera at fourteen days post last immunization (14dpli) were determined (Fig.4A). In any immunization frequency, the mice inoculated with any CspZ- YA proteins or mutant variants mounted significantly higher titers of CspZ IgG than those from PBS-inoculated, control mice (Fig.13). The titers of resulting CspZ IgG elevated as the immunization frequency increased (Fig.13). However, no significantly different titers of CspZ IgG were triggered among different CspZ-YA variants at any given immunization frequencies (Fig.13A to C). These results indicate the mutagenesis of these amino acids does not alter the overall IgG titers of CspZ after vaccination.

[0141] We then examined the ability of sera at 14dpli in killing Lyme borreliae using B. burgdorferi strain B31-A3 as a model, the genotype that is most prevalent in North America. We compared these sera’s BA50 values, the dilution rate of the sera to kill 50% of bacteria. The sera from the mice immunized with any CspZ-YA proteins or variants with any immunization frequency showed an immunization frequency-dependent bacterial killing (Fig. 4B, D, and F). The BA50 values from any CspZ-YA proteins or variants increased when the immunization is more frequent (Fig.4H). The CspZ-YA variants displayed no significantly different BA50 values from their parental CspZ-YA proteins during immunization once (Fig. 4C). However, CspZ-YAC183SUT and CspZ-YAI183Y but no other CspZ-YA variants triggered significantly greater levels of BA50 values than the parental CspZ-YA proteins when immunization was performed twice or three times (Fig.4E and G). These results highlighted the most robust bacterial killing efficacy by the antibodies induced during the immunization of CspZ-YAC187SUT and CspZ-YAI183Y.

[0142] The mutations of I183Y and C187S allowed CspZ-YA to protect mice from Lyme disease infection with reduced immunization frequency. We aimed to determine the ability of I183Y and C187S to reduce the protective immunization frequency of CspZ-YA against Lyme disease infection. We thus infected mice by permitting Ixodes scapularis nymphal ticks carrying B. burgdorferi B31-A3 feeding on the mice immunized with CspZ- YA proteins or variants under different immunization frequency (Fig.4A). We also included two controlled groups, PBS-inoculated mice and the mice immunized with lipidated OspA, the control vaccine antigen. At 42 dpli, we measured the bacterial burdens at different tissues and replete nymphs and detected the levels of IgG against C6 peptide, the commonly used Lyme disease serodiagnostic target (Fig.4A)

[0041] .

[0143] We found that the fed nymphs from the mice inoculated with PBS, OspA, or each of the CspZ-YA proteins or variants under any immunization frequency accounted for similar, detectable levels of bacterial burdens in fed nymphs (Fig. 5A, 6A and 6G). This is in agreement with the documented findings that vaccination of OspA or CspZ-YA does not eliminate B. burgdorferi in fed ticks [32, 42]. The mice immunized once with any tested antigens were all seropositive (Fig.6B) and had significantly greater bacterial loads in indicated tissues than uninfected mice (Fig.6C to F). In contrast, the mice immunized three times with any tested antigens but not PBS-inoculated, B. burgdorfei-infected mice were seronegative of C6 IgG (Fig.6H) and showed no significantly different levels of bacterial burdens in tissues than uninfected mice (Fig.6I to L). However, while the mice inoculated twice with PBS, OspA, CspZ-YAUT or CspZ-YA were seropositive of C6 IgG, all five CspZ-YAC187SUT-inoculated mice and four out of five CspZ-YAI183Y-inoculated mice were seronegative (Fig.5B). Consistently, unlike the mice inoculated twice with PBS, OspA, CspZ-YAUTor CspZ-YA having significant greater bacterial loads at tissues, CspZ- YAC187SUT- or CspZ-YAI183Y-inoculated mice showed no significantly different levels of bacterial burdens at tissues, compared to uninfected mice (Fig.5C to F). These results indicated that CspZ-YAC187SUT or CspZ-YAI183Y but not CspZ-YA and OspA with twice immunization allowed prevention from B. burgdorferi colonization and Lyme disease seropositivity.

[0144] We next determined the severity of Lyme disease-associated arthritis in the mice during immunization twice by histologically examining the mouse ankles. At 21dpli, we found elevated number of neutrophils and monocytes infiltrating into the tendon, connective tissues, and muscles from the OspA-, CspZ-YAUT- or CspZ-YA-immunized mice or PBS- inoculated mice (arrows in Fig.5G). However, no CspZ-YAI183Y- or CspZYAC187S-vaccinated mice developed the visual signs of arthritis (Fig. 5G). These data reflect to the scores of arthritic severity, demonstrating OspA-, CspZ-YAUT- or CspZ-YA-immunized mice but not CspZ-YAI183Y- or CspZYAC187S-vaccinated mice had significantly greater levels of arthritic phenotypes than uninfected mice (inlet figure of Fig.5G). Overall, the mutagenesis of I183Y and C187S allows CspZ-YA vaccination to prevent seropositivity, B. burgdorferi colonization, and Lyme disease-associated arthritis at reduced immunization frequency.

[0145] The mutations of I183Y and C187S did not alter the surface epitopes of CspZ- YA. One hypothesis to address the mechanisms underlying mutagenesis-mediated efficacy enhancement is that CspZ-YAI183Yand CspZ-YAC187SUTdeveloped distinct epitopes from CspZ-YA. Similar to the work described previously [26, 27], we found majority of the Lyme disease seropositive patients (36 out of 38), having elevated levels of CspZ IgGs (Fig.7). We thus tested this hypothesis by comparing the ability of CspZ IgGs in the sera from those 36 patients to recognize CspZ-YA, CspZ-YAI183Y, and CspZ-YAC187SUT. We found the levels of these sera recognized by CspZ-YA, CspZ-YAI183Y, and CspZ-YAC187SUT not significantly different from each other, but significantly greater than those from Lyme disease seronegative humans in non-endemic area (Fig.8A). A significantly positive correlation was detected for each patient serum sample to recognize CspZ-YA, CspZ-YAI183Y, and CspZ- YAC187SUT (Fig.8B to D). We also compared the CspZ-IgGs produced in the mice immunized twice with CspZ-YA, CspZ-YAUT, CspZ-YAI183Y, and CspZ-YAC187SUT to recognize each of these proteins in the same fashion. We found that similar levels of recognition by CspZ-YA, CspZ-YAI183Y, and CspZ-YAC187SUT for the sera from each immunization group of mice, andsuch levels of recognition are greater than those from PBS-inoculated control mice (Fig.8E). Additionally, when combining the values of recognition from different immunization groups of mice, we observed a significantly positive correlation for those sera to recognize CspZ- YA, CspZ-YAI183Y, and CspZ-YAC187SUT (Fig.8F to H). Such indistinguishable human or mouse CspZ IgGs recognition by CspZ-YA, CspZ-YAI183Y, and CspZ-YAC187SUT supports the possibility that mutagenesis of I183Y and C187S does not change the epitopes of CspZ-YA. We also crystallized and obtained the structure of the CspZ-YAC187SUT (2.00 Å) and did the AlphaFold prediction for CspZ-YAI183Y to further examine the impact of those mutations for the epitopes. Superimposed structures of CspZ-YAUT, CspZ-YAC187SUT, and CspZ-YAI183Y showed no significant difference on surface epitopes (Fig. 8I). This is consistent with the fact that Ile183 and Cys187 of CspZ-YA are buried between helices H and I (Fig.8J). Taken together, our structural and immunogenicity results demonstrated no alteration of surface epitopes after the mutation of I183 and C187 in CspZ-YA.

[0146] The mutations of I183Y and C187S promoted stability of the CspZ-YA structures recognized by CspZ-targeted, Lyme borrelia-killing monoclonal antibodies. Both Ile183 and Cys187 are located on and buried in helix H, raising a possibility that efficacy improvement selectively through the I183Y and C187S mutagenesis can be attributed to the stabilization and / or enhancement of the intramolecular interactions. We attempted to use protein crystal structures to investigate this possibility and did not find the formation of the additional non-covalent, intramolecular interactions in the high-resolution structure of CspZ- YAC187SUT, in comparison with that of CspZ-YAUT (Fig.9A to C). However, we observed the close proximity of Cys187 to other residues in helices helix G, H, and I, ranging from 3.7 to 4.0 Å to helix G, H, and I, respectively, (Fig.9B). Notably, a buried free-cysteine residue, like Cys187, has the tendency to undergo red-ox reactions on its thiol group, increasing the occupied space and lead to conformational abnormality and structural instability [37, 39, 43]. As serine does not have tendency to red-ox reactions, C187S mutagenesis would possibly stabilize the intramolecular interactions, specifically among helices G, H, and I. Further, the high-resolution structure of CspZ revealed a hydrophobic core formed among Tyr207, Phe210, and Tyr211 (Fig.9C). Unlike CspZ, a cavity was found in CspZ-YAUT because of the replacement of two bulky and non-polar residues, Tyr 207 and Tyr 211, by alanine, as well as the orientation of Phe210 away from the hydrophobic core (the red highlight in Fig. 9D). Moreover, the altered conformation in CspZ-YAUT prevented Arg206 from interacting with Glu186, exacerbating the cavity-mediated structural instability. We found that the AlphaFold predicted structure of CspZ-YAI183Y showed that the cavity is filled by a bulky,non-polar residue (i.e., Tyr-183), restoring the hydrophobic core (Fig.9E). This is consistent with the intention to achieve a more stabilized structure of CspZ-YAI183Y (Fig.1B).

[0147] We next examined if the CspZ-YAI183Yand CspZ-YAC187SUT have greater thermostability than that of CspZ-YA, as suggested by high resolution structures of these proteins. We compared CspZ-YAI183Yand CspZYAC187SUTwith their parental CspZ-YA proteins for their Tm values, the temperature in which 50% of the structures is unfolded. We found indistinguishable Tm values between CspZ-YAI183Y and CspZYAC187SUT (61.87 and 62.72oC for CspZ-YAI183Y and CspZYAC187SUT, respectively) (Fig.10A and B, Table 2). Table 2. The thermostability of CspZ-YA proteins. CspZ-YAUTCspZ-YA - C187S - I183Y nep p p .

[0148] In contrast, the Tm values of CspZ-YAI183Y and CspZYAC187SUT are significantly greater than those from CspZ-YA proteins (57.58 and 58.46oC for CspZ-YA and CspZYAUT, respectively), indicating the stability enhancement of CspZ-YA by I183Y and C187S mutagenesis (Fig.10A to C, Table 2). We next examined the impact of I183Y and C187S mutagenesis in modulating longevity of the protective epitopes in the CspZ-YA structures. We thus generated a CspZ-YA IgG that contains the Fc region of human IgG1 and F(ab’)2 from 1139 or 1193, our two monoclonal CspZ-YA IgG documented to eliminate Lyme borreliae

[0034] . The resulting IgGs, namely 1139c and 1193c, were verified for their ability to bind CspZ-YA (Fig.11A and B), block FH-binding ability of CspZ (Fig.11C), promote lysis of B. burgdorferi (Fig.11D), and confer opsonophagocytosis (Fig.11E). We then examined the ability of 1139c or 1193c to bind to CspZ-YAI183Y, CspZYAC187SUT, CspZ-YA, or CspZ-YAUT that were pre-incubated at 4 or 37 oC for different period of time. 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 (Fig. 10D and E). However, CspZ-YA and CspZ-YAUTbut not CspZ-YAI183Yand CspZYAC187SUT previously incubated at 37oC for 24-h had significantly lower levels of recognition, compared to those proteins prior to incubation (Fig.10D and E). These findings demonstrated longevity enhancement of CspZ-YA proteins in the mammalian adaptedconditions by I183Y and C187S mutagenesis, specifically on the structures that promote protective antibody induction.

[0149] DISCUSSION

[0150] Using native microbial surface antigens as vaccine targets face multiple challenges, one of which is that native antigens are often not immunogenic, lacking the ability to provide protective immunity [4]. While other native antigens are immunogenic when tested in vitro, conformational changes may occur after those antigens bind their host ligands in vivo to promote pathogen invasion. Such conformational alteration of antigens could result in the potential of the induced antibodies to not constantly recognize pathogens and / or inhibit the pathogen invasion, decreasing vaccine efficacy [8, 44-47]. These difficulties were initially found in some viral proteins but recently reported in native antigens from non-viral pathogens, such as Lyme disease bacteria [31, 32]. One strategy to overcome these hurdles is through amino acid mutagenesis to lock those antigens in certain structures that favor the induction of pathogen-killing antibodies (

[0034] , for review paper, see

[0048] ). For example, immunization with the native version of a Lyme borreliae FH-binding protein, CspZ, does not prevent Lyme disease infection [26, 30-32]. We previously generated CspZ- YA lacking FH-binding activity through the mutagenesis of Y207A and Y211A and found CspZ-YA to induce robust levels of borreliacidal antibodies that prevent the infection [31, 32]. In this study, our newly obtained high-resolution structure of CspZ-YA allowed the comparison with the structure of the parental CspZB31, showing the formation of an extended helix I by replacing Y211 to alanine, and a more flexible loop H / I from the replacement of Y207 to alanine (Fig.1A). These structural changes mechanistically support the inability of CspZ-YA to bind to FH, as both Y207 and Y211 are within or adjacent to the FH-binding interface based on our previously reported complexed structure of CspZB408 and the SCR6-7 domain of FH (ref ). Using CspZ as a model, our work here thus structurally demonstrated the mechanisms underlying the antigen engineering concept of unmasking the protective epitopes to promote vaccine efficacy.

[0151] The duration to maintain antibody’s effective titers determines the required immunization frequency of a vaccine to provide protectivity. However, how the antigens continue to maintain designated structures that can trigger long-lasting bactericidal antibodies remains to be an unresolved issue. Structure-based vaccine design is one of the recently developed strategies to promote the longevity of pathogen-killing antibodies after immunization. According to the existing high-resolution structures of an antigen, a series of amino acids are mutated with the goal to enhance the antigen stability by promoting suitableintramolecular interactions [6, 7]. In this study, the mutagenesis of Y207A and Y211A reduced the hydrophobic interactions and created a cavity at the C-terminal H / I loop and helix H of CspZ-YA, destabilizing protein’s folding conformation (Fig.9B). Introduction of the amino acids with great hydrophobicity on their side chains (i.e., isoleucine, leucine, valine, phenylalanine, and tyrosine) ideally would fill the cavity to maintain the protein stability [49-51]. Here we included the mutation of I183Y for cavity filling (Fig.9F), elevating the efficacy of CspZ-YA-triggered bactericidal antibodies and prevent bacterial colonization and disease manifestations at lower immunization frequency. The stability enhancement by I183Y mutagenesis and the structure comparison between CspZ-YAI183Y vs. CspZ-YA prove the concept of cavity filling at one of the first times in a bacterial vaccinogen.

[0152] We ascertained the impact of Cys187 on CspZ-YA in protein stability by replacing this residue with serine. We did not observe CspZ-YA and CspZ-YAC187S forming apparent aggregates. However, the stability of CspZ-YAC187Swas significantly enhanced, and this mutant CspZ-YA offered reduced immunization frequency for protection from B. burgdorferi colonization and Lyme disease-associated arthritis. In fact, the red-ox tendency of Cys187 has been thought as a factor to destabilize the protein because of the increasing size of cysteine’s side chain to pack this residue and the neighboring residues. This sparked off the concept by replacing cysteine to serine, which has less tendency of red-ox to enhance the protein stability

[0055] . Our newly resolved high-resolution structures of CspZ-YA and CspZ- YAC187S, showing significantly reduced distance between the neighboring residues and Ser187 on CspZ-YAC187S, compared to Cys187 on CspZ-YA. Our results thus provide clear evidence to support the concept of cysteine-based mutation strategy.

[0153] We found our CspZ-targeted and protective monoclonal antibodies to recognize CspZ-YAI183Y and CspZ-YAC187S, better than CspZ-YA at higher temperature for longer period of time (i.e., 37oC for 24-h). As mammalian body temperatures stay consistent in 37oC, both mutations would allow CspZ-YA to persist in the designated structures to promote the longevity of resulting protective antibodies, thus suitable as vaccines in human or other mammal uses. Additionally, I183 and C187 are both located on helix H. The structural comparison of CspZ-YAC187Sand CspZ-YAI183Y / C187Swith their parental CspZ-YA correlated I183Y- and C187S-mediated stability enhancement with the facilitation of suitable helix H-I interactions on CspZ-YA (Fig.9). Although I183 and C187 are not located on or surrounding the FH-binding interface of CspZ, the N-terminus of helix H and the loop H / I are within and immediately adjacent to the FH-binding sites

[0035] (Brangulis et al. unpublished).Therefore, it is possible that suitable helix H-I interactions of CspZ-YA vaccines prolongs the structures of the protective epitopes within or adjacent to the FH-binding interface of CspZ, triggering greater levels of protective antibodies. Testing this possibility would require the elucidation of the high-resolution complexed structure of CspZ-YA and those CspZ-targeted and protective antibodies (i.e., 1139c and 1193c), warranting further investigations.

[0154] The more stable structures suggested by structure-based vaccine design would also aid the process development in the downstream of vaccine production and ease the required conditions for transportation and storage, promoting the commercialization plan

[0056] . It is noteworthy that structure-based vaccine design should avoid the mutations of the amino acids close to the proposed or known protective epitopes [6, 7]. This is the rationale guided by the previously resolved high resolution structure of CspZ-FH

[0035] , and, indeed I183Y and C187S do not alter the surface epitopes demonstrated in this work (Fig.8I). In this study, we mutagenized CspZ-YA as a model to test the concept of structure-based vaccine design in decreasing the minimal immunization frequency that allows protectivity. The results elucidate the mechanisms underlying such a concept using a Lyme disease subunit vaccine as a model. Additionally, the fact that CspZ-YAI183Yor CspZ-YAC187Sproviding lower protective immunization frequency than OspA offers the opportunity using these mutated proteins as vaccines to overcome the needs of constant immunization for OspA- targeted vaccines. Finally, the breakthrough of vaccine design sparked off by recent pandemics underscores the importance for structure-guided approaches for efficacy optimization of vaccines. Using Lyme disease as a model, this concept-proof study provides mechanistic insights of structural-based vaccine design and illustrates the possibility to revisit the previously tested but inefficacious antigens. This work would hopefully facilitate the establishment of a pipeline for vaccine design that can be extended to combat other newly emerging infectious diseases.

[0155] Figure 14 illustrates prevention of Lyme disease bacterial colonization mediated by CspZ-YAC187S vaccination administered with different adjuvants. Five mice per group were inoculated with an immunogenic composition including Alhydrogel (Alum)-, Pam3CSK4 (Pam3)-, α-galactosylceramide (α-Gal)-, or TiterMax-Gold (TMG, control)- formulated CspZ-YAC187Stwice: at day 0 and 14. Five mice per group immunized with TMG- formulated lapidated OspA or inoculated with PBS in the same fashion were included as control. At day 35 post initial immunization, these mice were then fed on by nymphs carrying B. burgdorferi B31-A3. B. burgdorferi (Bb) burdens at (top left) nymphs after when feeding to repletion or the tick feeding site (“Inoc. Site”), heart, knees, and bladder werequantitatively measured at day 46 post last immunization, shown as the number of Bb per 100ng total DNA. Data shown are the geometric mean ± geometric standard deviation of the spirochete burdens from each group of mice. Asterisks indicate the statistical significance (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 (“PBS”).

[0156] MATERIALS AND METHODS

[0157] Ethics Statement. All mouse experiments were performed in strict accordancewith all provisions of the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the PHS Policy on Humane Care and Use of Laboratory Animals. The protocol (Docket Number 22-451) was approved by the Institutional Animal Care and Use Agency of Wadsworth Center, New York State Department of Health. All efforts were made to minimize animal suffering. This study also involves secondary use of deidentified archival patient sera collected in previous studies and was approved by the Institutional Review Board (IRB) of New York State Department of Health and Baylor College of Medicine underprotocol 565944-1 and H-46178, respectively. Analysis of deidentified patient data wascarried out under a waiver of consent.

[0158] Mouse, ticks, bacterial strains, hybridoma, and human serum samples. Four-week-old, female C3H / HeN mice were purchased from Charles River (Wilmington, MA, USA). Although 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 stringent criteria to define the protectivity. BALB / c C3-deficient mice were from in-house breeding colonies

[0057] and Ixodes scapularis tick larvae were obtained from BEI Resources (Manassas, VA). Escherichia coli strain BL21(DE3) andderivatives were grown at 37^ C or other appropriate temperatures in Luria-Bertani broth oragar, supplemented with kanamycin (50µg / mL). B. burgdorferi strain B31-A3 were grown at33^ C in BSK II complete medium

[0058] . Cultures of B. burgdorferi B31-A3 were tested withPCR to ensure a full plasmid profile before use [59, 60]. 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

[0061] . The negativecontrol human sera are the sera collected from 10 individuals residing in non-endemic area of Lyme disease.

[0159] Cloning, expression and purification of OspA, CspZ, CspZ-YA, and CspZ- YA-derived mutant proteins. The DNA encoding histidine tagged CspZ, CspZ-YA and CspZ- YA-derived mutant proteins (Table 3) was codon-optimized based on E. coli codon usage preference and synthesized by Synbiotech (Monmouth Junction, NJ), followed by subcloning into the pET28a using BamHI / SalI restriction sites. Table 3. Strains and plasmids used in this study. Strain or plasmid Genotype or characteristic B. burgdorferi . ri - - 53 67 80 th e- 36 42 e- 87 39 B.pET28a-CspZ-YA KanR; pET28a encoding protein residue 20 to 236 of CspZ with tyrosine- 207 and -211 simultaneously replaced by alanine residues e- th th th th th th th th th thCspZ-YAI183Yand CspZ-YAC187Sinclude amino acid substitutions to CspZ-YA at positions corresponding to amino acids 164 and 168, respectively, of SEQ ID NO: 1, specifically I164Y and C168S.

[0160] These plasmids were transformed into E. coli BL21 (DE3). The DNA encoding untagged CspZ and this protein-derived mutant proteins were codon-optimized based on E. coli codon usage preference, synthesized and subcloned into the pET41a using NdeI / XhoI sites by GenScript (Piscataway, NJ). 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. The generation of histidine-tagged CspZ and this protein-derived mutant proteins is described previously

[0062] . To purify the untagged CspZ-YA, and CspZ-YAC187S, we followed the procedure as described

[0062] . Because lipidation is required for recombinant OspA proteins to protect mice from Lyme disease infection [63, 64], the lipidated OspA was included as a control. To generate the lipidated OspA, a previous developed purification process was followed

[0064] .For structural studies, the encoding regions of CspZ-YA and CspZ-YAC187Swere cloned into the pETm-11 expression vector containing an N-terminal 6xHis tag, followed by a tobacco etch virus (TEV) protease cleavage site. Both proteins were expressed in E. coli BL21 (DE3) and purified by affinity chromatography as described previously for CspZ

[0033] .

[0161] Generation of CspZ-YA antibodies, #1139c and #1193c. Protective mouse monoclonal antibodies (mAbs) 1139 and 1193 against CspZ were developed previously

[0034] . These two mAbs were further modified 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 mAbs (1139c and 1193c) were then transiently produced in CHO cells, followed by purification with Protein A affinity chromatography.

[0162] Circular dichroism (CD) spectroscopy. CD analysis was performed on a Jasco 810 spectropolarimeter (Jasco Analytical Instrument, Easton, MD) under nitrogen. CD spectra were measured at room temperature (RT, 25°C) in a 1mm path length quartz cell. Spectra of each of the CspZ-YA proteins (10μM) were recorded in phosphate based saline buffer (PBS) at RT, and three far-UV CD spectra were recorded from 190-250nm in 1nm increments for far-UV CD. The background spectrum of PBS without proteins was subtracted from the protein spectra. CD spectra were initially analyzed by the software Spectra Manager Program (Jasco). Analysis of spectra to extrapolate secondary structures was performed using the K2D3 analysis programs

[0065] .

[0163] Mouse immunization and infection. C3H / HeN Mice were immunized as described, with slight modifications

[0032] . Fifty µl of PBS (control) or 25µg of untagged or histidine tagged CspZ-YA or its mutant proteins, or untagged, lipidated OspA in 50µl of PBS was thoroughly mixed with 50µl TiterMax Gold adjuvant (Norcross, GA, USA), resulting in total 100µl of the inoculum. This inoculum was introduced into C3H / HeN mice subcutaneously once at 0 days post initial immunization (dpii), twice at 0 and 14dpii, or three times at 0, 14, and 28dpii (Fig.1). At 14 days post last immunization (dpli), blood was collected via submandibular bleeding to isolate serum for the determination of ability in recognizing CspZ, CspZ-YA and the mutant proteins derived from CspZ-YA, as described in the sections of “ELISAs” and “Borreliacidal assays”, respectively (Fig.1). At 7 dpli, B. burgdorferi B31-A3-infected flat nymphs were placed in a chamber on the immunized or PBS-inoculated C3H / HeN mice as described (Fig.1)

[0030] . Five nymphs were allowed to feed to repletion on each mouse, and a subset of nymphs was collected pre- and post-feeding. At 21 dpli, tick bite sites of skin, bladder, knees, and heart were collected to determine thebacterial burdens, and ankles were also collected at 21dpli to determine the severity of arthritis described in the section “Quantification of spirochete burdens and histological analysis of arthritis. (Fig.1).” At this time point, blood was also collected via cardiac puncture bleeding to isolate serum for the determination of seropositivity described in the section “ELISAs” (Fig.1).

[0164] For the mice inoculated with IgGs, C3H / HeN mice were immunized as described, with slight modifications

[0034] . Basically, C3H mice were intraperitoneally inoculated with irrelevant human IgG (control), #1139c or #1193c (1 mg / kg) (Fig.12A). 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 [32, 57]. 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 in the section “Quantification of spirochete burdens and histological analysis of arthritis (Fig.12A).” Blood was also collected via cardiac puncture bleeding to isolate sera for the determination of seropositivity described in the section “ELISAs” (Fig.12A).

[0165] ELISAs. To measure the titers of anti-CspZ IgG in the serum samples (Fig. 13), one µg of histidine-tagged CspZ was coated on ELISA plate wells as described

[0032] . To determine the ability of anti-CspZ IgG in the sera to recognize CspZ-YA, and CspZ-YAI183Y, and CspZ-YAC187S(Fig.8A), each of these proteins with histidine tags (1 µg) was coated on ELISA plate wells as in the same fashion. The procedures following the protein coating are as described previously

[0032] . For each serum sample, the maximum slope of optical density / minute of all the dilutions of the serum samples was multiplied by the respective dilution factor, and the greatest value was used as arbitrary unit (A.U) to represent the antibody titers for the experiment to obtain anti-CspZ IgG (Fig.13) or the ability of the anti- CspZ IgG in the sera to recognize CspZ-YA and different CspZ-YA mutant proteins (Fig. 8A). The quality of the correlation for the ability of those CspZ IgG in recognizing CspZ-YA vs. CspZ-YAC187S, CspZ-YA vs. CspZ-YAI183Y, or CspZ-YAC187Svs. CspZ-YAI183Ywas determined by the R and P values of Spearman analysis, which was calculated using dose- response stimulation fitting in GraphPad Prism 9.3.1.

[0166] 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.5A, 6A, and 12A). This methodology has been commonly used for human Lyme disease diagnosis

[0066] and performed as described in our previous work

[0034] . 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.

[0167] We also determined the ability of #1139c or #1193c to prevent FH from binding to CspZ (Fig.11C), which was performed as described previously with modifications

[0034] . 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

[0034] . 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.11C), representing the IgG concentration that blocks 50% of FH binding, was calculated using dose-response stimulation fitting in GraphPad Prism 9.3.1.

[0168] Borreliacidal assays. The ability of serum samples (Fig.4B to G) or CspZ IgG (#1139c and #1193c, Fig.11D) to eradicate B. burgdorferi B31-A3 was determined as described with modifications [31, 32]. 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 33OC 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 Fig. 4B to G) or the concentration of IgGs (for #1139c and #1193c in Fig.11D) that kills 50% of spirochetes, was calculated using dose-response stimulation fitting in GraphPad Prism 9.3.1.

[0169] Quantification of spirochete burdens and histological analysis of arthritis. DNA was extracted from the indicated mouse tissues to determine the bacterial burdens (Fig. 5B to F, 6B to F and H to L, and 12C to G), using quantitative PCR analysis as described

[0032] . 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 100ng of total DNA. For the ankles that were applied to histological analysis of Lyme disease associated- arthritis (Fig.5G), the analysis was performed as described

[0032] . 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).

[0170] Crystallization and structure determination. Initial crystallization trials of CspZ-YA and CspZ-YAC187Swere performed in 96-well sitting drop crystallization plates (SWISSCI AG, Neuheim, Switzerland), using sparse-matrix screens JCSG+ and Structure Screen 1&2 from Molecular Dimensions (Newmarket, UK). Tecan Freedom EVO100 workstation (Tecan Group, Männedorf, Switzerland) was used to set up the plates by mixing 0.4 μl of protein with 0.4 μl of precipitant. After initial crystal hits, the corresponding crystallization conditions were optimized by varying the quantities of the components in the precipitant solution to obtain crystals suitable for harvesting. Diffraction data for CspZ- YAUT was collected from crystals grown in 0.2M ammonium acetate, 0.1 M sodium citrate (pH 6.5) and 30% PEG 4000, but for CspZ-YAC187SUTgrown in 2.2M ammonium citrate, 0.1 M HEPES (pH 7.5) and 2% PEG 400. Before harvesting and storing the crystals in liquid nitrogen, crystals for CspZ-YAUT were subjected to cryoprotectant made of the precipitant solution with additional 10% glycerol. Diffraction data for CspZ-YAUT were collected at the Diamond Light Source (Oxfordshire, UK) beamline I03 but the data for CspZ-YAC187SUTat the MX beamline instrument BL 14.1 at Helmholtz-Zentrum (Berlin, Germany)

[0067] . Reflections were indexed by XDS and scaled by AIMLESS from the CCP4 suite [68, 69]. Initial phases for CspZ-YAUT and CspZ-YAC187SUT were obtained by molecular replacement using Phaser

[0070] , with the crystal structure of B. burgdorferi CspZ as a search model (PDB ID 4CBE). After molecular replacement, the protein models were built automatically in BUCCANEER

[0071] . The crystal structures were improved by manual rebuilding in COOT

[0072] . Crystallographic refinement was performed using REFMAC5

[0073] . A summary of thedata collection, refinement and validation statistics for CspZ-YA and CspZ-YAC187Sare given in Table 1. Table 1. Statistics for Data and Structure Quality. Dataset CspZ-YAUTCspZ-YAUT-C187SSpace group P212121 P212121 Unit cell dimensions a (Å) 31.47 31.55 b (Å) 41.55 41.68 c (Å) 162.56 162.81 Wavelength (Å) 0.9762 0.9184 Resolution (Å) 162.56-1.90 41.68-2.00 Highest resolution bin (Å) 1.94-1.90 2.05-2.00 No. of reflections 231969 189839 No. of unique reflections 17550 15299 Completeness (%) 99.5 (100.0) 99.9 (100.0) Rmerge 0.09 (0.46) 0.09 (0.38) I / σ (I) 15.5 (4.8) 19.3 (6.2) Multiplicity 13.2 (13.5) 12.4 (13.5) Refinement Rwork 0.190 (0.245) 0.248 (0.204) Rfree 0.241 (0.308) 0.335 (0.337) Average B-factor (Å2) Overall 28.4 29.8 From Wilson plot 17.0 17.5 No. of atoms Protein 1743 1743 Water 199 170 RMS deviations from ideal Bond lengths (Å) 0.009 0.008 Bond angles (o) 1.530 1.457 Ramachandran outliers (%) Residues in most favored regions (%) 94.42 94.88 Residues in allowed regions (%) 4.65 4.19 Outliers (%) 0.93 0.93 Values in parentheses are for the highest resolution bin.

[0171] Protein 3D structure prediction using AlphaFold. AlphaFold v2.0

[0074] was used to predict the 3D structure for CspZ-YAI183Yas described previously for B. burgdorferi PFam12 family proteins

[0075] .

[0172] 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, 500nM) 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.

[0173] Phagocytosis assays The phagocytosis assays were performed as described previously with modifications

[0076] . 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 3.3uM 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 (107 bacteria) 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, 50rpm 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 a FACSCalibur 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.

[0174] Fluorescence-based thermoshift assays 10µM of indicated wild-type and mutant CspZ-YA proteins was applied to 7500 Fast Real-Time PCR System (Thermo Scientific) with a temperature range of 25–95 °C. All reactions were in 20µl of the final volume in 96-well plates using Protein Thermal Shift™ Dye Kit (ThermoFisher Scientific) at1:1,000 dilution in PBS buffer. Thermostability was identified by measuring the protein- unfolding concentration (melting temperature, Tm), extrapolated by obtaining the temperature with maximal positive derivative values of the fluorescence intensity using the 7500 Fast Real-Time PCR System software (Thermo Scientific).

[0175] 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

[0032] . 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

[0032] , #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.

[0176] Statistical analyses. Significant differences were determined with a Kruskal- Wallis test with the two-stage step-up method of Benjamini, Krieger, and Yekutieli

[0077] , two- tailed Fisher test (for seropositivity in Fig.5A, 6A, and 12B)

[0078] , or Spearman analysis (for correlation analysis in Fig.8B to D and F to H)

[0079] , using GraphPad Prism 9.3.1. A p-value < 0.05 was used to determine significance.

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[0178] EXAMPLE 2

[0179] The present example demonstrates the effectiveness of different adjuvants used in connection with an immunogen as disclosed herein. CspZ-YAC187Swas formulated with different adjuvants documented for human use and defined the resulting immunomodulation and the efficacy of Lyme disease prevention after vaccination in the murine model. We also demonstrate the durability and memory features of protective antibodies triggered by these vaccine regimens, aiming to identify underlying immunological basis and develop a prolonged memory immunity-driven Lyme disease vaccine.

[0180] CpG or αGal allowed the highest titers and bactericidal activities triggered by Alum-CspZ-YAC187S vaccination. To define the regimens that permit the highest levels of IgG titers and protective antibodies, we formulated CspZ-YAC187Swith different adjuvants, including Alum (Aluminum hydroxide / Alhydro gel) and PAM3 (PAM3CSK4) as they have been used in humans. The resulting anti-CspZ-YAC187SIgG titers were compared with those from formulation with titermax gold (TMG), an adjuvant with only documented preclinical use but effective in CspZ-YAC187Sformulation for prevention against Lyme disease infection under two immunizations. After the mice immunized at 0- and 14-days post initial immunization (dpii), the sera were collected at 14 and 28dpii for the mice immunized once (Immun. Once) and twice (Immun. Twice), respectively (Fig.15, A). We found indistinguishable levels of anti-CspZ-YAC187SIgG induced by the regimens containing each of these tested adjuvants under once or twice immunization (Fig.15, B-C). We then determined the ability of the sera to kill B. burgdorferi strain B31-A3 (OspC type A, the most common genotype from patients in North America) by providing the values of BA50, the dilution rate of the sera that can eradicate 50% of the bacteria (Fig.15, D-G, Table 2).

[0181] Table 2:

[0182] Sera from TMG but not Alum or PAM3 formulated CspZ-YAC187Sdisplayed detectable BA50 under once immunization (Fig.15, D-E, Table 2). Sera from TMG formulated CspZ-YAC187Ssignificantly higher values of BA50than those from Alum or PAM3 formulation under twice immunization (Fig.15, F-G, Table 2). These results showed the formulation of Alum or PAM3 alone to not provide better capability for CspZ-YAC187S to induce antibodies than TMG formulation.

[0183] Alum was considered as a “delivery system” for antigen formulation and has been commonly co-formulated with other adjuvants grouped as “immune potentiators” to manipulate the immune responses toward pathogen-killing for better efficacy. Two immune potentiators, CpG or αGal, thus were added into Alum-formulated CspZ-YAC187S prior to being introduced into mice (Fig.15, A). We found the titers of anti-CspZ-YAC187S IgG when the regimen contains Alum-CpG or Alum-αGal were significantly greater than those containing TMG under once immunization or TMG, Alum, or PAM3 under twice immunization (Fig.15, B-C). Additionally, the BA50values from Alum-CpG or Alum-αGal formulated CspZ-YAC187S were significantly higher than those from that antigen with TMG formulations under once immunization (Fig.15, D-E, Table 2) and TMG, PAM3, or Alum formulations under twice immunization (Fig.15, F-G, Table 2). These results indicate the ability of CpG and αGal as immune potentiators to boost the antibody titers and B. burgdorferi killing activities triggered by Alum formulated CspZ-YAC187S.

[0184] CpG- or αGal- formulations in Alum-CspZ-YAC187Spermitted the prevention against Lyme disease infection under two immunizations. We attempted to determine the impact of adjuvant formulations for CspZ-YAC187Svaccines into Lyme disease protectivity.CspZ-YAC187Swas thus formulated with TMG, Alum, or PAM3, or the combination of Alum and CpG or Alum and αGal (Fig.15, A). After two immunizations at 0- and 14dpii, the mice were allowed to be fed on by Ixodes scapularis nymphal ticks carrying B. burgdorferi strain B31-A3 at 35dpii (Fig.15, A). Uninfected mice or TBS-inoculated and B. burgdorferi- infected mice were included as control. We found indistinguishable bacterial burdens in the replete nymphs after those nymphs feeding on the mice immunized with tested regimens of CspZ-YAC187S, agreeing with the documented no impacts of B. burgdorferi survivability in fed nymphs associated with mice vaccinated with TMG-CspZ-YAC187S(Fig.16, A). We then determined the B. burgdorferi seropositivity by detecting the levels of IgG against C6 peptides and bacterial burdens in different tissues at 21-days post nymph feeding (dpf)(Fig. 15, A). As expected, all five TMG-CspZ-YAC187S-immunized mice were C6 seronegative and had indistinguishable bacterial burdens at the tick bite site of skin, bladder, heart, and knee joints than the uninfected mice (Fig.15, B-F). We found that all five mice inoculated with TBS (control) or Alum-formulated CspZ-YAC187Sand four out of five mice immunized with PAM3-formulated CspZ-YAC187S turned positive for anti-C6 IgG, with the titers significantly greater than the uninfected mice (Fig.15, B). This result is consistent with the finding of significantly higher B. burgdorferi burdens in the tested tissues of the mice inoculated with TBS or Alum or PAM3-formulated CspZ-YAC187S, compared to those from uninfected mice (Fig.15, C-F). However, all five mice inoculated with CspZ-YAC187S formulated with Alum- CpG or Alum-αGal turned C6 seronegative and had indistinguishable levels of bacterial burdens in the tested tissues from the uninfected mice (Fig.15, C-F). These results demonstrated the ability of either CpG or αGal to permit Alum-formulated CspZ-YAC187Sas efficacious vaccines to prevent B. burgdorferi tissue colonization and seropositivity preclinically.

[0185] We next determined in those mice for the severity of the inflammation at ankle joints triggered by Lyme disease causing bacteria histologically at 21 dpf (Fig.15, A). We observed increasing number of infiltrated mononuclear cells in the muscles, tendons, and connective tissues from all five mice inoculated with TBS or Alum- or four of five mice immunized with PAM3-formulated CspZ-YAC187S (arrows in Fig.16, G). The scores of inflammations from those mice were indistinguishable among each other but significantly higher than uninfected mice (Fig.16, H). However, all five mice immunized with CspZ- YAC187S formulated with Alum and CpG or Alum and αGal did not develop any signs of joint inflammation, similar to the uninfected mice or the mice inoculated with TMG-CspZ-YAC187S (Fig.16, G). The mice vaccinated with CspZ-YAC187S formulated with Alum and CpG orAlum and αGal also showed similar levels of inflammation scores at joints, compared to uninfected mice or TMG-CspZ-YAC187S-immunized mice (Fig.16, H). Overall, the formulation of CpG or αGal allows Alum-CspZ-YAC187Svaccination to prevent Lyme disease-associated joint inflammation.

[0186] Addition of CpG or αGal in Alum-CspZ-YAC187Sskewed distinct pathways for similar effector immune functions of pathogen killing. We next examined the impacts of adding CpG or αGal by comparing the transcriptomics of the mice immunized with Alum vs. Alum-CpG or Alum-αGal formulated CspZ-YAC187S.RNA extracted from the spleens of those mice immunized twice at 28dpii (14-days post last immunization) were applied for RNA sequencing (RNAseq) (Fig.15, A). Compared with the TBS-inoculated mice, mice inoculated with Alum-CpG formulated CspZ-YAC187S had less DEGs but Alum-αGal formulated CspZ-YAC187Shaving more DEGs, suggesting the modulation of CpG or αGal in transcriptomics of Alum-CspZ-YAC187S immunized mice (Fig.19). Comparing to the Alum- inoculated mice immunized with Alum-CpG or Alum-αGal formulated CspZ-YAC187Syielded 8 or 100 upregulated DEGs, respectively, but only three DEGs (2.9%) were in common. In contrast, no DEGs were in common among 32 or 28 downregulated DEGs by Alum-CpG or Alum-αGal formulated CspZ-YAC187S, respectively. When comparing the transcriptomics of Alum-CpG vs. Alum, we observed the upregulation of the genes involved in immunoglobulin production upregulated and anti-inflammatory cytokines. Identifying the DEGs under the regimens of Alum-αGal vs. Alum showed upregulation of the genes for immunoglobulin production, the recognition of antigen-presenting cells modulation of Th1 cell activation, and immune cell trafficking and downregulation of the genes for anti- inflammatory functions. These results suggest that CpG and αGal modulated in different fashion for the responses to Alum-CspZ-YAC187S-inoculation.

[0187] We next defined the differences of the immune pathways modulated by CpG or αGal to Alum-CpGC187S-vaccination by applying spleen transcriptomics from immunized mice into a Gene Set Enrichment Analysis (GSEA). In the comparison of Alum vs. Alum- CpG or Alum-αGal, three and five of top 10 enriched and upregulated pathways, respectively, but none of the top 10 enriched and downregulated pathways are considered immune-related. We thus delineated all upregulated immune-related pathways modulated by Alum-CpG or Alum-αGal using statistical cutoff (FDR q-value<0.1, NES >1.5). Consistent with CpG as the ligand of TLR9 to account for innate immune signaling , type 1 IFN pathways including IFNα / β and NFκB pathways were upregulated in the comparison of CspZ-YAC187S formulated with Alum vs. Alum-CpG. Such a signaling appears to promotenot only innate immune defenses but crosstalk with effective adaptive immunity. In the comparison of CspZ-YAC187S formulated with Alum vs. Alum-αGal, we also observed the activation of not only similar effective adaptive cells but NK cells involved in pathogen killing. However, this activation is likely facilitated uniquely through the signaling and activation of classical dendritic cells (cDCs) to present antigens to Th1 cells. These pathways were also highlighted in the comparison of mice immunized with Alum-CpG vs. Alum-αGal formulated CspZ-YAC187S. These results agree with the documented activation of cDC through the binding of αGal to iNKT cell. Overall, our findings support CpG to trigger innate immune signaling whereas αGal in inducing iNKT-cDC-Th1 activation but result in similar effector immune functions for pathogen killing.

[0188] Vaccination with Alum-CpG formulated CspZ-YAC187S triggered up to eight months of protective antibody durability. The abovementioned results identified Alum-CpG and Alum-αGal as the lead regimen to formulate CspZ-YAC187S. However, CpG but not αGal has been a FDA-approved adjuvants used in commercially available human vaccines. We thus prioritized CpG to formulate Alum-CspZ-YAC187S and sought to determine the durability of this lead regimens of adjuvant formulation in triggering protective antibodies triggered by CspZ-YAC187S vaccination. Mice were immunized once at 0- or twice at 0- and 14dpii, followed by the sera collection at different time points post last immunization (pli) to determine the levels of anti-OspA or -CspZ-YAC187S IgG titers or bactericidal antibodies (BA50) (Fig.17, A). The PBS-inoculated mice or the mice immunized three times with TMG- OspA were included as control (Fig.17, A). We detected OspA IgGs until 182dpli in TMG- OspA-inoculated mice but not in any mice inoculated with CspZ-YAC187Sor PBS (Fig.17, B and Fig.20). As expected, TMG-OspA or PBS-inoculated mice did not yield detectable CspZ-YAC187SIgGs (Fig.17, C and Fig.21). Immunization twice with CspZ-YAC187Sallowed longer duration of detectable CspZ-YAC187S IgGs (238dpli) than immunization once with that antigen (210dpli) (Fig.17, C and 20). Immunization twice with CspZ-YAC187Salso permitted greater levels of CspZ-YAC187S IgGs than immunization once with that antigen at any given time points (Fig.17, C and Fig.20). We then measured the BA50values of those mice and found while the sera from TBS-inoculated mice did not have any detectable bacterial killing activities time points, those from TMG-OspA immunized mice can be detectable until 182dpli (Fig.17, D and Fig.22). We observed that mice developed detectable bactericidal activities until 210dpli under immunization once with CspZ-YAC187S and 238dpli under immunization twice with that antigen (Fig. 17, D and Fig.22). These results showed the capability of CspZ-YAC187S to trigger protective antibodies depending on theimmunization frequency, and that capability can last for up to eight months after vaccination.

[0189] Immunization with Alum-CpG-CspZ-YAC187S recalled early bactericidal antibodies and protected mice from Lyme disease infection after vaccine-triggered antibodies waned. We next determined if natural infection could recall the CspZ-targeting immune responses after CspZ-YAC187S-triggered protective antibodies waned to undetectable levels. The abovementioned mice were not infected until the levels of their bactericidal antibodies and IgG titers against immunogens (OspA or CspZ-YAC187S) are undetectable, which is 273dpli (Fig.17, A). We then collected sera from indicated time points to determine levels of the recalled IgGs and bactericidal antibodies by natural infection of B. burgdorferi (Fig.17, A). We observe indistinguishable levels of anti-OspA IgGs in the mice immunized with TMG-OspA or Alum-CpG formulated CspZ-YAC187S from TBS-inoculated mice at any tested time points after nymph tick feeding (Fig.18, A, Fig.23). These results agree with downregulation of ospA expression after B. burgdorferi invades vertebrate hosts. The titers of anti-CspZ-YAC187SIgGs in TMG-OspA-inoculated mice were not significantly higher than those from TBS-treated mice until 10dpf (Fig.18, B, Fig.24), consistent with detectable CspZ-targeting antibodies reported at later stages during natural infection. In contrast, the titers of anti-CspZ-YAC187S IgGs from the mice vaccinated with Alum-CpG formulated CspZ-YAC187Swere significantly greater than those from TBS-treated mice as early as 4dpf and throughout all time points (Fig.18, B, Fig.24). Further, sera from 10dpf and beyond displayed detectable bactericidal activities in all mouse groups (BA50values), similar to other documented work , only the mice vaccinated with Alum-CpG formulated CspZ-YAC187S had detectable BA50values earlier than 10dpf (i.e., 4 and 7dpf) (Fig.18, C, Fig.25, Table 3).

[0190] Table 3:

[0191] These results suggest an early, recalled, and bacterial killing antibody responses triggered after natural infection of mice immunized with Alum-CpG-formulated CspZ- YAC187S.

[0192] We next tested if this CspZ-YAC187Svaccination-specific, natural infection- triggered bactericidal activities link to protectivity by determining anti-C6 seropositivity, B. burgdorferi burdens at tissues, and ankle joint inflammation histologically at 21dpf (Fig.17, A). We detected indistinguishable bacterial burdens in fed nymphs as expected. At 21dpf, all five TBS- or TMG-OspA inoculated mice were seropositive and had significantly higher B. burgdorferi burdens in all tested tissues than uninfected mice (Fig.18, D-I). Those mice also displayed elevated numbers of mononuclear cells infiltrating into connective tissues of ankle joints with the inflammation scores significantly higher than those from uninfected mice (Fig. 18, J-K). However, one and zero out of five mice turned seropositive in the mice immunizedwith Alum-CpG formulated CspZ-YAC187S, and the bacterial burdens in the tested tissues of those mice were indistinguishable from those from uninfected mice (Fig.18, D-I). Similarly, one and zero out of five mice vaccinated with Alum-CpG formulated CspZ-YAC187Sdeveloped the abovementioned signs of join inflammation, resulting in the scores of those mice to be similar to those from uninfected mice (Fig.18, J-K). Taken together, these results link the memory antibody responses triggered by Alum-CpG formulated CspZ-YAC187S vaccination to the prevention of seropositivity, bacterial colonization, and B. burgdorferi- associated joint inflammation in the murine Lyme disease model.

[0193] MATERIALS AND METHODS

[0194] Mouse, ticks, and bacterial strains. Four-week-old, female C3H / HeN mice were purchased from Charles River (Wilmington, MA, USA). Although 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 stringent criteria to define the protectivity. BALB / c C3-deficient mice were from in-house breeding colonies

[0056] 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 appropriatetemperatures in Luria-Bertani broth or agar, supplemented with kanamycin (50µg / mL). B. burgdorferi strain B31-A3 were grown at 33°C in BSK II complete medium

[0057] . Cultures of B. burgdorferi B31-A3 were tested with PCR to ensure a full plasmid profile [58, 59].

[0195] Purification of OspA and CspZ-YAC187S. The purification of untagged and lipidated OspA and untagged CspZ-YAC187Swere as described

[0061]

[0063] . The E. coli strain BL21(DE3) producing OspA and CspZ-YAC187S used in this study are listed (Table S4). The recombinant protein expression was induced with 1 mM Isopropyl-β-D-1- thiogalactopyranoside (IPTG). OspA was lipidaded as lipidation is required for recombinant OspA proteins to protect mice from Lyme disease infection [62, 63].

[0196] Mouse immunization and infection. C3H / HeN Mice were immunized as described, with slight modifications. Twenty-five µg of untagged tagged CspZ-YAC187Sor untagged, lipidated OspA (control) in 50µl of Tris-buffered saline (TBS) buffer was thoroughly mixed with 50µl TiterMax Gold adjuvant (Norcross, GA, USA), resulting in total 100µl of the inoculum. Twenty-five untagged tagged CspZ-YAC187S was also formulated with PAM3 (10μg, PAM3CSK4, InvivoGen, CA, USA), Alum (400μg, Alhydrogel, Corda, NJ, USA), Alum (400μg) mixed with CpG (20μg, CpG1826, InvivoGen, CA, USA), or Alum (400μg) mixed with αGal (1μg, α- galactosylceramide, Avanti, AL, USA) in 50µl of Tris- buffered saline buffer with 0.05% Pluronic F-127 and 7% sucrose (TBS). In addition, fifty µl of TBS only was included as control. The inoculum was introduced into C3H / HeN mice subcutaneously once at 0- or twice at 0- and 14-days post initial immunization (Fig.15, A and Fig.17, A). Blood was collected via submandibular bleeding at 14-days post last immunization (dpli) (Fig.115, A) or 14, 42, 70, 98, 126, 154, 182, 210, 238, and 273dpli (Fig.17, A). Sera obtained from the blood were used to determine the IgG titers of CspZ- YAC187Sor OspA, and the bactericidal activities, described in the section “ELISAs” and “Bactericidal assays”, respectively. At 21dpli (Fig. 15, A) or 273dpli (Fig.17, A), B. burgdorferi B31-A3-infected flat nymphs were placed in a chamber on the those C3H / HeN mice as described. Five nymphs were allowed to feed to repletion on each mouse, and a subset of nymphs was collected pre- and post-feeding. At 21 days post nymph feeding (dpf), tick bite sites of skin, bladder, knees, and heart were collected to determine the bacterial burdens, and ankles were also collected at 21 dpli to determine the severity of arthritis described in the section “Quantification of spirochete burdens and histological analysis of arthritis.” At this time point, blood was also collected via cardiac puncture bleeding to isolate sera for the determination of seropositivity described in the section “ELISAs.”

[0197] ELISAs. The determination of the titers of anti-OspA, -CspZ-YAC187SIgG, or -C6 IgG in the serum samples were as described with modifications. Specifically for anti-C6 IgG, this methodology has been commonly used for human Lyme disease diagnosis. One µg of untagged and lipidated OspA, untagged CspZ-YAC187S, or C6 peptides was coated on ELISA plate wells as described. The procedures following the protein coating are as described previously. For each serum sample, the maximum slope of optical density / minute of all the dilutions of the serum samples was multiplied by the respective dilution factor, and the greatest value was used as arbitrary unit (A.U) to represent the antibody titers for the experiment to obtain anti-OspA, -CspZ-YAC187S, or -C6 IgG. For anti-C6 IgG, 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.

[0198] Borreliacidal assays. The ability of serum samples to eradicate B. burgdorferi B31-A3 was determined as described with modifications [32, 33]. Briefly, the sera collected from mice immunized with lipidated OspA or CspZ-YAC187S under different adjuvant formulations at different immunization frequency were heat-treated to inactivate complement. The rest procedure was performed in the similar fashion as described [32, 33]. Surviving spirochetes at 24-h of incubation with Guinea pig sera and different dilution rate of serum samples 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 that kills 50% of spirochetes, was calculated using dose-response stimulation fitting in GraphPad Prism 9.3.1.

[0199] Quantification of spirochete burdens and histological analysis of arthritis. DNA was extracted from the indicated mouse tissues to determine the bacterial burdens using quantitative PCR analysis as described. Basically, the forward and reverse primers with the sequences as GTGGATCTATTGTATTAGATGAGGCTCTCG and GCCAAAGTTCTGCAACATTAACACCTAAAG, respectively, were used to amplify the recA gene of B. burgdorferi strain B31-A3. 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 from mouse tissues or presented as the number of bacteria per tick for B. burgdorferi in ticks. The histological analysis was applied to ankles for Lyme disease-associated arthritis. Basically, images were 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 withtwo or more foci of infiltration), or 3 (severe inflammation with focal and diffuse infiltration covering a large area).

[0200] mRNA Extractions for RNASeq analyses The spleens were placed immediately on dry ice and stored at -80C° and then submitted for RNA extraction and RNAseq analysis by GENEWIZ (Waltham, MA). Turbo Capture mRNA Kits (Qiagen) was used for total RNA extraction of 50 mg of tissues. A drill and polypropylene pestles were used to lyse TRIzol® suspended tissues in Eppendorf Tubes, followed by the treatment of DNAse I as described in the vendor’s manuals. Elutions were prepared in DNAse and RNAse free water, and the quality of RNA provided by Nanodrop Eight Spectrophotometer (ThermoFisher Scientific), 260 / 280 targeting 1.95-2.04. RINe (RNA Integrity Number) values greater than 6.5 with strong indications of robust 18S and 28S (28S / 18S) peaks, with minimal evidence of degraded small RNA at the lower threshold range, were submitted for further processing. The RNA samples were processed for mRNA enrichment using Dynabeads mRNA Purification Kit per vendor’s manual (ThermoFisher). The mRNA sequencing was then carried out by polyA selection using Illumina HiSeq, PE 2x150 (150 bp paired end).

[0201] RNASeq analysis After the sequencing, the reads were trimmed via Trimmomatic v.0.36, mapped sequence reads to the Mus musculus strain C3H / HeJ reference genome via ENSEMBL using the STAR aligner v.2.5.2b. The gene hit counts (calculation of reads / gene / sample) were determined using feature counts from the Subread package v.1.5.2. The results from Salmon were imported into R v.4.2.3 with tximport and a DESeq2 object was created with the DESeq dataset from tximport function. Differential expression analyses were performed by DESeq2 with minreplicates for replace set to 30.

[0202] RNASeq results from the spleens of the mice immunized with CspZ-YAC187Sformulated with Alum, Alum and CpG, or Alum and αGal were first be compared with those from the control (TBS-inoculated mice). Additionally, the results from the spleens of the mice vaccinated with Alum and CpG or Alum and αGal were compared with those with Alum. VolcaNoseR was used to plot and explore differentially expressed genes (DEGs) in volcano plot format (Goedhart and Luijsterburg, 2020). Adjusted p-values ≤ 0.05 and log2 fold changes ≥ 0.58 or ≤ -0.58 were used as the initial cutoffs defining differentially expressed genes (DEG). The number shared and unique DEGs among the mice immunized with CspZ-YAC187S formulated Alum, Alum and CpG, or Alum and αGal was visualized as Venn diagram with Venny 2.1(Oliveros, 2007-2015). Heat map analysis was performed. Up and downregulation gene lists were also analyzed using Gene set enrichment analysis(GSEA) software v3.0 available from the Broad Institute (http: / / software.broadinstitute.org / gsea / index.jsp). GSEA was performed with default algorithm as 1000 permutations, minimum term size of 15, and maximum term size of 500. Enriched gene sets were assigned based on FDR q-value < 0.1 and NES >1.5.

[0203] Flow Cytometry of splenocytes The preparation of splenocyte from the mice immunized with CspZ-YAC187S formulated with different adjuvants and the flow cytometry analysis of these splenocytes were performed in the same fashion as described previously. Basically, Spleens were rinsed in sterile phosphate based saline (PBS) buffer, then transferred to a gentleMACS C Tube containing 3mL of sterile PBS. The tissue was homogenized using a gentleMACS Dissociator (Miltenyi Biotech, Surrey, UK). Red blood cells in the spleen homogenates were subsequently lysed with ACK lysis buffer (Lonza, 10-548E). The lysis solution was diluted 5-fold with RPMI medium supplemented with 10% fetal bovine serum FBS, 1× penicillin-streptomycin (Pen-Strep), and l-glutamine (cRPMI medium). Then, splenocytes were pelleted by centrifugation for 5 min at 300 × g. Splenocytes were resuspended in 5 mL of cRPMI medium and passed through 40mm strainers (BD Biosciences, 352340). Cells were counted using acridine orange-propidium iodide (AOPI) live / dead dye and a Cellometer Auto 2000 automated cell counter. Then, 1 × 106live splenocytes were incubated for each sample in a 96-well non-tissue culture plate. For intracellular staining, splenocytes were incubated with 50 ng / mL phorbol 12-myristate 13- acetate (PMA)–500 ng / ml ionomycin, or medium only for 5 h at 37°C in 5% CO2. These cells were then analyzed by flow cytometry and Luminex.

[0204] To measure CD4- and CD8- responses, splenocytes were collected 5 hours post restimulation for flow cytometry labeling, washed with PBS, and stained with Live / Dead NIR fixable viability dye, anti-CD3e PE (phycoerythrin) / Dazzle 594, anti-CD4 Alexa Fluor 700, and anti-CD8a peridinin chlorophyll protein (PerCP)-Cy5.5. Tfh was evaluated with anti-CD44 fluorescein isothiocyanate (FITC), anti-CD185 (CXCR5) BV421 Brilliant Violet 421 (BV-421) and anti-CD279 (PD-1) PE (phycoerythrin)-Cy7 (PE-Cy7). To evaluate intracellular cytokine production, 4.1 mg / ml brefeldin A was added to splenocytes during the 5 h of restimulation. Splenocytes were stained for surface markers as described above, fixed with BD Cytofix / Cytoperm, and permeabilized according to the manufacturer’s instructions. Permeabilized splenocytes were stained with anti-IFNγ Alexa Fluor 647, anti-TNF Brilliant Violet 605 (BV-605), IL-4 Brilliant Violet 711 (BV-711) and IL-21 phycoerythrin (PE). Samples were acquired on an Attune instrument, and at least 100,000 total events in a live gate were analyzed using FlowJo software. Cells were gated on forward and side scatter forlymphocytes, exclusion of viability dye, and singlet populations. T cells, Tfh cells, CXCR5+CD8+ cells, B cells in the spleen were determined based on FMO. Data were plotted using GraphPad Prism software.

[0205] Statistical analyses. Significant differences were determined with a Kruskal- Wallis test with the two-stage step-up method of Benjamini, Krieger, and Yekutieli and two- tailed Fisher test (for seropositivity in Fig.16, B and Fig.18, B)

[0078] using GraphPad Prism 9.3.1. A p-value < 0.05 was used to determine significance.

[0206] EXAMPLE 3

[0207] An RNA vaccine including an RNA molecule having any nucleotide sequence that encodes any of the protein immunogens disclosed herein, with or without any of the signal peptide sequences disclosed herein, will be administered to a subject to induce an immune response. An RNA molecule may have the sequence set out in SEQ ID NO: 3, such as SEQ ID NO: 14. The RNA may e complexed as part of a lipid nanoparticle as disclosed herein. It may be in a composition with any variation of a thermostable polynucleotide- containing formulation as disclosed herein, or any lipid nanoparticle, liposome, or other suitable for RNA vaccine delivery for stimulating an immune response and protection from development of Lyme disease in a subject exposed or potentially exposed to a Lyme disease- causing Borrelia.

[0208] Following one or more administration of such an RNA vaccine, the subject would exhibit reduced susceptibility to development or contraction of Lyme disease even if bitten by an organism harboring a Lyme disease-causing Borrelia, which exposure would other wise have or would have been likely to have caused Lyme disease in the subject. In some subjects administered such an RNA vaccine, subsequent exposure to an organism harboring a Lyme disease-causing Borrelia may yet result in development or contracting Lyme disease, but its duration of symptoms (one or more of rash, fever, chills, fatigue, muscle and joint ache, headache, swollen lymph nodes, facial palsy (drooping on one or both sides of the face), neck stiffness, numbness, tingling, or shooting pains in the limbs, heart palpitations, dizziness, fainting, chest pain or shortness of breath, eye inflammation or vision changes, arthritis, , recurrent or persistent swelling and pain in large joints (such as the knees), nerve pain (neuropathy), memory, concentration, cognitive, or mood difficulties), severity of such symptoms, or both may be substantially reduced compared to a subject who had not received such an RNA vaccine. A subject may be administered such an RNA vaccine one, or more than once and, if more than once, multiple administrations may occur within weeks and / or a year or more of each other.

[0209] 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.

[0210] 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 matter disclosed 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.

[0211] SEQUENCES

[0212] SEQ ID NO: 1: Csp-YA immunogen DVSRLNQRNINELKIFVEKAKYYSIKLDAIYNECTGAYNDIMTYSEGTFSDQSKVNQ AISIFKKDNKIVNKFKELEKIIEEYKPMFLSKLIDDFAIELDQAVDNDVSNARHVADSY KKLRKSVVLAYIESFDVISSKFVDSKFVEASKKFVNKAKEFVEENDLIALECIVKTIGD MVNDREINSRSRANNFAKKEADFLGAAVELEGAYKAIKQTLL

[0213] SEQ ID NO: 2: Csp-YA immunogen DVSRLNQRNINELKIFVEKAKYYSIKLDAIYNEX34TGAYNDIMTYSEGX48FSDQSKV NQAISX61FKKDNKIVNKFKELEKIIEEYKPMX84LSKLIDDFAX96ELDQAVDNDVSNA RHVADSYX117KLRKSX123VLAYIESFDVISSKFVDSKFVEASKKFVNKAKEFVEENDL X164ALEX168IVKTIX174DMVNDREINSRSRX188NNFX192KKEADFLGAAVELEGAYKAI KQTLL, wherein (a)(i) X188is any amino acid other than Y or is A, X192is any amino acid other than Y or is A, X188 is any amino acid other than Y or is A and X192 is any amino acid other than Y or is A, X188is any amino acid other than Y and X192is A, X188is A and X192is any amino acid other than Y, or X188 is A and X192 is A, and (a)(ii) X164is any amino acid other than I or is Y, X168is any amino acid other than C or is S, X164 is any amino acid other than I or is Y and X168 is any amino acid other than C or is S, X164is any amino acid other than I and X168is S, X164is Y and X168is any amino acid other than C, or X164 is Y and X168 is S, and, optionally (b) independently, any one or more of X34 is C or is any amino acid other than C or is S, X48 is T or is any amino acid other than T or is P, X61 is I or is any amino acid other than I or is T, X86 is F or is any amino acid other than F or is P, X96 is I or is any amino acid otherthan I or is T, X117is K or is any amino acid other than K or is E, X123is V or is any amino acid other than V or is M, and X174 is G or is any amino acid other than G or is M.

[0214] SEQ ID NO: 3: Csp-YA immunogen nucleotide sequence GATGTGTCCCGTCTGAATCAGCGTAATATCAATGAGCTCAAGATATTCGTTGAGA AGGCGAAGTACTACAGCATCAAGCTGGACGCCATCTACAACGAGTGTACTGGAG CTTATAATGATATTATGACTTATTCAGAGGGGACATTCTCCGACCAGAGCAAGGT CAACCAGGCCATAAGCATCTTCAAGAAGGACAACAAGATCGTGAATAAGTTTAA GGAGCTGGAGAAGATCATCGAGGAGTACAAACCGATGTTCTTGTCCAAGCTCAT AGACGATTTCGCGATCGAGTTGGACCAGGCTGTCGACAACGATGTGAGCAACGC ACGACACGTGGCCGACAGCTACAAGAAGCTGCGGAAGAGTGTCGTGCTTGCTTA CATCGAGAGCTTCGACGTGATCTCGTCGAAGTTCGTCGACAGCAAGTTCGTGGAG GCCTCCAAGAAGTTCGTCAACAAGGCGAAGGAGTTCGTCGAGGAGAACGACTTG ATCGCCCTGGAGTCGATCGTGAAAACCATCGGTGACATGGTCAACGACAGGGAG ATCAACAGTCGTTCTCGGGCGAACAACTTCGCCAAGAAGGAGGCGGACTTCTTG GGGGCCGCCGTCGAGCTTGAAGGTGCTTATAAGGCCATCAAGCAGACCTTGCTCT AA

[0215] SEQ ID NO: 4: Csp-YA immunogen

[0216] DVSRLNQRNINELKIFVEKAKYYSIKLDAIYNECTGAYNDIMTYSEGTF SDQSKVNQAISIFKKDNKIVNKFKELEKIIEEYKPMFLSKLIDDFAIELDQAVDNDVSN ARHVADSYKKLRKSVVLAYIESFDVISSKFVDSKFVEASKKFVNKAKEFVEENDLIAL ESIVKTIGDMVNDREINSRSRANNFAKKEADFLGAAVELEGAYKAIKQTLL

[0217] SEQ ID NO: 5: Csp-YA immunogen

[0218] DVSRLNQRNINELKIFVEKAKYYSIKLDAIYNECTGAYNDIMTYSEGTF SDQSKVNQAISIFKKDNKIVNKFKELEKIIEEYKPMFLSKLIDDFAIELDQAVDNDVSN ARHVADSYKKLRKSVVLAYIESFDVISSKFVDSKFVEASKKFVNKAKEFVEENDLYA LESIVKTIGDMVNDREINSRSRANNFAKKEADFLGAAVELEGAYKAIKQTLL

[0219] SEQ ID NO: 6: Csp-YA immunogen DVSRLNQRNINELKIFVEKAKYYSIKLDAIYNECTGAYNDIMTYSEGTFSDQSKVNQ AISIFKKDNKIVNKFKELEKIIEEYKPMFLSKLIDDFAIELDQAVDNDVSNARHVADSY KKLRKSVVLAYIESFDVISSKFVDSKFVEASKKFVNKAKEFVEENDLIALESIVKTIGD MVNDREINSRSRXNNFXKKEADFLGAAVELEGAYKAIKQTLL

[0220] SEQ ID NO: 7: Csp-YA immunogen DVSRLNQRNINELKIFVEKAKYYSIKLDAIYNECTGAYNDIMTYSEGTFSDQSKVNQ AISIFKKDNKIVNKFKELEKIIEEYKPMFLSKLIDDFAIELDQAVDNDVSNARHVADSYKKLRKSVVLAYIESFDVISSKFVDSKFVEASKKFVNKAKEFVEENDLYALESIVKTIG DMVNDREINSRSRXNNFXKKEADFLGAAVELEGAYKAIKQTLL

[0221] SEQD NO: 8: Signal sequence MKWVTFISLLFLFSSAYS

[0222] SEQ ID NO: 9: Signal sequence METDTLLLWVLLLWVPGSTG

[0223] SEQ ID NO: 10: Signal sequence MKYLLLLLGAGLLLLLALVSASGRSTK

[0224] SEQ ID NO: 11: Signal sequence MYRMQLLSCIALSLALVTNS

[0225] SEQ ID NO: 12: Signal sequence MTPLQLLVLLWSGLVLLGVAPPGSAN

[0226] SEQ ID NO: 13: Signal sequence METPAWPRVPRPETAVARTLLLGWVFAQVA GAS

[0227] SEQ ID NO: 14: Csp-YA immunogen nucleotide sequence ATGAAATGGGTCACCTTTATCAGCCTGCTGTTCCTGTTCAGCAGCGCCTACAGCG ATGTGTCCCGTCTGAATCAGCGTAATATCAATGAGCTCAAGATATTCGTTGAGAA GGCGAAGTACTACAGCATCAAGCTGGACGCCATCTACAACGAGTGTACTGGAGC TTATAATGATATTATGACTTATTCAGAGGGGACATTCTCCGACCAGAGCAAGGTC AACCAGGCCATAAGCATCTTCAAGAAGGACAACAAGATCGTGAATAAGTTTAAG GAGCTGGAGAAGATCATCGAGGAGTACAAACCGATGTTCTTGTCCAAGCTCATA GACGATTTCGCGATCGAGTTGGACCAGGCTGTCGACAACGATGTGAGCAACGCA CGACACGTGGCCGACAGCTACAAGAAGCTGCGGAAGAGTGTCGTGCTTGCTTAC ATCGAGAGCTTCGACGTGATCTCGTCGAAGTTCGTCGACAGCAAGTTCGTGGAGG CCTCCAAGAAGTTCGTCAACAAGGCGAAGGAGTTCGTCGAGGAGAACGACTTGA TCGCCCTGGAGTCGATCGTGAAAACCATCGGTGACATGGTCAACGACAGGGAGA TCAACAGTCGTTCTCGGGCGAACAACTTCGCCAAGAAGGAGGCGGACTTCTTGG GGGCCGCCGTCGAGCTTGAAGGTGCTTATAAGGCCATCAAGCAGACCTTGCTCTA A

Claims

WHAT IS CLAIMED IS:

1. An immunogenic composition, comprising a polypeptide of SEQ ID NO: 1, except that an amino acid whose position corresponds to amino acid 188 of SEQ ID NO: 1 comprises any amino acid other than tyrosine and an amino acid whose position corresponds to amino acid 192 of SEQ ID NO: 1 comprises any amino acid other than tyrosine, and one or both of (a) an amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 comprises an amino acid other than inosine and (b) an amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 comprises an amino acid other than cysteine.

2. The immunogenic composition of claim 1, wherein the amino acid whose position corresponds to amino acid 188 of SEQ ID NO: 1 comprises alanine.

3. The immunogenic composition of claim 1 or claim 2, wherein the amino acid whose position corresponds to amino acid 192 of SEQ ID NO: 1 comprises alanine.

4. The immunogenic composition of any one of claims 1 through 3, wherein the amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 comprises tyrosine.

5. The immunogenic composition of any one of claims 1 through 4, wherein the amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 comprises serine.

6. The immunogenic composition of any one of claims 1 through 5, wherein the amino acid whose position corresponds to amino acid 188 of SEQ ID NO: 1 comprises alanine and the amino acid whose position corresponds to amino acid 192 of SEQ ID NO: 1 comprises alanine.

7. The immunogenic composition of any one of claims 1 through 6, wherein the amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 comprises an amino acid other than inosine and the amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 comprises an amino acid other than cysteine.

8. The immunogenic composition of any one of claims 1 through 7, wherein the amino acid whose position corresponds to amino acid 164 or SEQ ID NO: 1 comprises tyrosine and the amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 comprises serine.

9. The immunogenic composition of any one of claims 1 through 8, wherein the amino acid whose position corresponds to amino acid 188 of SEQ ID NO: 1 comprises alanine, the amino acid whose position corresponds to amino acid 192 of SEQ ID NO: 1 comprises alanine, the amino acid whose position corresponds to amino acid 164 or SEQ ID NO: 1 comprises tyrosine, and the amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 comprises serine.

10. The immunogenic composition of any one of claims 1 through 9, having an amino acid sequence selected from that of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, or as encoded by a polynucleotide sequence comprising that of SEQ ID NO: 3 or SEQ ID NO:

14.

11. The immunogenic composition of any one of claim 1 through 10, further comprising one or more adjuvant.

12. The immunogenic composition of any one of claim 1 through 10, further comprising one or more adjuvant, wherein one or more of the one or more adjuvant is covalently attached to the polypeptide.

13. The immunogenic composition of any one of claim 1 through 10, further comprising one or more adjuvant, wherein one or more of the one or more adjuvant is not covalently attached to the polypeptide.

14. The immunogenic composition of any one of claim 1 through 10, further comprising one or more adjuvant, wherein one or more of the one or more adjuvant is selected from aluminum salt, AS04, AS03, monophosphoryl lipid A, poly(I:C), a CpG DNA adjuvant, MF59, an emulsion adjuvant comprising squalene and water, a combination adjuvant comprising block copolymer CRL-8300, squalene, a sorbitan monooleateor, N-[1- (2,3-Dioleoyloxy)propyl] -N,N,N-trimethylammonium salt (DOTAP), 3 β-[N-(N',N'- dimethylaminoethane) -carbamoyl] cholesterol (DC-chol liposome), an aluminum hydroxide wet gel suspension, α-galactosylceramide, and a virosomal adjuvant.

15. The immunogenic composition of any one of claims 1 through 14, wherein one or more of an amino acid whose position corresponds to amino acid 34 of SEQ ID NO: 1 comprises any amino acid other than cysteine, an amino acid whose position corresponds to amino acid 48 of SEQ ID NO: 1 comprises any amino acid other than threonine, an amino acid whose position corresponds to amino acid 61 of SEQ ID NO: 1 comprises any amino acid other than inosine,an amino acid whose position corresponds to amino acid 86 of SEQ ID NO: 1 comprises any amino acid other than phenylalanine, an amino acid whose position corresponds to amino acid 96 of SEQ ID NO: 1 comprises any amino acid other than inosine, an amino acid whose position corresponds to amino acid 117 of SEQ ID NO: 1 comprises any amino acid other than lysine, an amino acid whose position corresponds to amino acid 123 of SEQ ID NO: 1 comprises any amino acid other than valine, and an amino acid whose position corresponds to amino acid 174 of SEQ ID NO: 1 comprises any amino acid other than glycine.

16. The immunogenic composition of any one of claims 1 through 15, wherein one or more of the amino acid whose position corresponds to amino acid 34 of SEQ ID NO: 1 comprises serine, the amino acid whose position corresponds to amino acid 48 of SEQ ID NO: 1 comprises proline, the amino acid whose position corresponds to amino acid 61 of SEQ ID NO: 1 comprises threonine, the amino acid whose position corresponds to amino acid 86 of SEQ ID NO: 1 comprises proline, the amino acid whose position corresponds to amino acid 96 of SEQ ID NO: 1 comprises threonine, the amino acid whose position corresponds to amino acid 117 of SEQ ID NO: 1 comprises glutamic acid, the amino acid whose position corresponds to amino acid 123 of SEQ ID NO: 1 comprises methionine, and the amino acid whose position corresponds to amino acid 174 of SEQ ID NO: 1 comprises methionine.

17. The immunogenic composition of any one of claims 1 through 16, wherein from one to thirty six, from one to twenty four, from one to sixteen, from one to eight, or from one to four amino acids whose position corresponds to an amino acid of from 1-163 in SEQ ID NO: 1 are not the same as the corresponding amino acid in SEQ ID NO:

1.

18. The immunogenic composition of any one of claims 1 through 17, wherein from one to four, from one to three, of from one to two amino acids corresponding to aminoacids whose position corresponds to amino acid of from 193-207 in SEQ ID NO: 1 are not the same as the corresponding amino acid in SEQ ID NO:

1.

19. A method of vaccinating a subject, comprising administering to the subject the immunogenic composition of any one of claims 1 through 18.

20. A method of treating a subject having Lyme disease or suspected of having been infected with or exposed to Borrelia burgdorferi, comprising administering to the subject a composition comprising the immunogenic composition of any one of claims 1 through 18.

21. The method of claim 19 or 20, wherein administering comprises administering the immunogenic composition more than once.

22. The method of claim 21, wherein the method comprises administering the composition at least two times.

23. The method of claim 22, wherein administration of the immunogenic composition fewer than three times prevents the subject from contracting Lyme disease.

24. The method of any one of claims 19 through 23 wherein in the subject is a rodent, a cat, a dog, a cattle, or a human.

25. A composition, comprising a means for immunizing a subject against a CspZ-expressing species of Borrelia, and one or more adjuvant, wherein administering the immunogenic composition to the subject fewer than three times prevents exposure of the subject to the CspZ-expressing species of Borrelia from causing Lyme disease in the subject.

26. The composition of claim 25, wherein one or more of the one or more adjuvant is covalently attached to the polypeptide.

27. The composition of claim 25, wherein one or more of the one or more adjuvant is not covalently attached to the polypeptide.

28. The immunogenic composition of any one of claims 25 through 27, wherein one or more of the one or more adjuvant is selected from aluminum salt, AS04, AS03, monophosphoryl lipid A, poly(I:C), a CpG DNA adjuvant, MF59, an emulsion adjuvant comprising squalene and water, a combination adjuvant comprising block copolymer CRL- 8300, squalene, a sorbitan monooleateor, N-[1-(2,3-Dioleoyloxy)propyl] -N,N,N- trimethylammonium salt (DOTAP), 3 β-[N-(N',N'-dimethylaminoethane) -carbamoyl]cholesterol (DC-chol liposome), an aluminum hydroxide wet gel suspension, α- galactosylceramide, and a virosomal adjuvant.

29. A polynucleotide encoding the polypeptide of any one of claims 1 through 18.

30. The polynucleotide of claim 29, wherein the polynucleotide sequence comprises that of SEQ ID NO:

3.

31. The polynucleotide of claim 29 or 30, wherein the polypeptide further comprises a signal peptide.

32. The polynucleotide of claim 31, wherein the signal peptide directs the polypeptide for secretion by a cell when the polypeptide is expressed in the cell.

33. The polynucleotide of claim 31 or 32, wherein the signal peptide is selected from a signal peptide of immunoglobulin kappa, a signal peptide of tissue plasminogen activator, a signal peptide of serum albumin, a signal peptide of growth factor, and a signal peptide of interleukin 2.

34. The polynucleotide of claim 33, wherein the signal peptide is a serum albumin signal peptide.

35. The polynucleotide of claim 33 or 34, wherein the signal peptide is human serum albumin signal peptide.

36. The polynucleotide of any one of claims 31 through 35, wherein the signal peptide sequence comprises that of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:12, or SEQ ID NO:

13.

37. The polynucleotide sequence of claim 36 wherein the polynucleotide sequence comprises that of SEQ ID NO:

3.

38. The polynucleotide sequence of claim 36 wherein the polynucleotide sequence comprises that of SEQ ID NO:

14.

39. The polynucleotide of any one of claims 29 through 38, wherein the polynucleotide comprises RNA.

40. A cell comprising the polynucleotide of any one of claim 29 through 39.

41. A vector comprising the polynucleotide of any one of 29 through 39.

42. The vector of claim 41, comprising a lipid nanoparticle, a viral vector, or a plasmid.

43. A method of vaccinating a subject, comprising administering to the subject a composition comprising the polynucleotide of any one of claims 29 through 39 or the vector of claim 41 or 42.

44. A method of treating a subject having Lyme disease or suspected of having been infected with or exposed to Borrelia burgdorferi, comprising administering to the subject a composition comprising the polynucleotide of any one of claims 28 through 38 or the vector of claim 41 or 42.

45. The method of claim 43 or 44, wherein administering comprises administering the immunogenic composition more than once.

46. The method of claim 45, wherein the method comprises administering the composition at least two times.

47. The method of claim 46, wherein administration of the composition fewer than three times prevents the subject from contracting Lyme disease.

48. The method of any one of claims 41 through 47 wherein in the subject is a rodent, a cat, a dog, a cattle, or a human.

49. An immunogenic composition, comprising a polypeptide of SEQ ID NO: 1, except that an amino acid whose position corresponds to amino acid 188 of SEQ ID NO: 1 comprises any amino acid other than tyrosine and an amino acid whose position corresponds to amino acid 192 of SEQ ID NO: 1 comprises any amino acid other than tyrosine, and one or both of (a) an amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 comprises an amino acid other than inosine and (b) an amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 comprises an amino acid other than cysteine; and an aluminum salt adjuvant and a CpG adjuvant.

50. The immunogenic composition of claim 49 wherein the aluminum salt adjuvant comprises aluminum hydroxide.

51. The immunogenic composition of claim 49 or 50 wherein the aluminum salt adjuvant comprises an aluminum hydroxide wet gel suspension or AS04.

52. The immunogenic composition of any one of claims 49 through 51, wherein the amino acid whose position corresponds to amino acid 188 of SEQ ID NO: 1 comprises alanine.

53. The immunogenic composition of any one of claims 49 through 52, wherein the amino acid whose position corresponds to amino acid 192 of SEQ ID NO: 1 comprises alanine.

54. The immunogenic composition of any one of any one of claims 49 through 53, wherein the amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 comprises tyrosine.

55. The immunogenic composition of any one of claims 49 through 54, wherein the amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 comprises serine.

56. The immunogenic composition of any one of claims 49 through 54, wherein the amino acid whose position corresponds to amino acid 188 of SEQ ID NO: 1 comprises alanine and the amino acid whose position corresponds to amino acid 192 of SEQ ID NO: 1 comprises alanine.

57. The immunogenic composition of any one of claims 49 through 56, wherein the amino acid whose position corresponds to amino acid 164 of SEQ ID NO: 1 comprises an amino acid other than inosine and the amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 comprises an amino acid other than cysteine.

58. The immunogenic composition of any one of claims 49 through 57, wherein the amino acid whose position corresponds to amino acid 164 or SEQ ID NO: 1 comprises tyrosine and the amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 comprises serine.

59. The immunogenic composition of any one of claims 49 through 58, wherein the amino acid whose position corresponds to amino acid 188 of SEQ ID NO: 1 comprises alanine, the amino acid whose position corresponds to amino acid 192 of SEQ ID NO: 1 comprises alanine, the amino acid whose position corresponds to amino acid 164 or SEQ ID NO: 1 comprises tyrosine, and the amino acid whose position corresponds to amino acid 168 of SEQ ID NO: 1 comprises serine.

60. The immunogenic composition of any one of claims 49 through 59, having an amino acid sequence selected from that of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, or as encoded by a polynucleotide sequence comprising that of SEQ ID NO:

14.

61. The immunogenic composition of any one of claim 49 through 60, further comprising one or more additional adjuvant.

62. The immunogenic composition of any one of claim 49 through 60, further comprising one or more additional adjuvant, wherein one or more of the one or more additional adjuvant is covalently attached to the polypeptide.

63. The immunogenic composition of any one of claim 49 through 60, further comprising one or more additional adjuvant, wherein one or more of the one or more additional adjuvant is not covalently attached to the polypeptide.

64. The immunogenic composition of any one of claim 49 through 60, further comprising one or more additional adjuvant, wherein one or more of the one or more additional adjuvant is selected from AS03, monophosphoryl lipid A, poly(I:C), MF59, an emulsion adjuvant comprising squalene and water, a combination adjuvant comprising block copolymer CRL-8300, squalene, a sorbitan monooleateor, N-[1-(2,3-Dioleoyloxy)propyl] - N,N,N-trimethylammonium salt (DOTAP), 3 β-[N-(N',N'-dimethylaminoethane) -carbamoyl] cholesterol (DC-chol liposome), an aluminum hydroxide wet gel suspension, α- galactosylceramide, and a virosomal adjuvant.

65. The immunogenic composition of any one of claims 49 through 64, wherein one or more of an amino acid whose position corresponds to amino acid 34 of SEQ ID NO: 1 comprises any amino acid other than cysteine, an amino acid whose position corresponds to amino acid 48 of SEQ ID NO: 1 comprises any amino acid other than threonine, an amino acid whose position corresponds to amino acid 61 of SEQ ID NO: 1 comprises any amino acid other than inosine, an amino acid whose position corresponds to amino acid 86 of SEQ ID NO: 1 comprises any amino acid other than phenylalanine, an amino acid whose position corresponds to amino acid 96 of SEQ ID NO: 1 comprises any amino acid other than inosine, an amino acid whose position corresponds to amino acid 117 of SEQ ID NO: 1 comprises any amino acid other than lysine, an amino acid whose position corresponds to amino acid 123 of SEQ ID NO: 1 comprises any amino acid other than valine, and an amino acid whose position corresponds to amino acid 174 of SEQ ID NO: 1 comprises any amino acid other than glycine.

66. The immunogenic composition of any one of claims 49 through 65, wherein one or more of the amino acid whose position corresponds to amino acid 34 of SEQ ID NO: 1 comprises serine,the amino acid whose position corresponds to amino acid 48 of SEQ ID NO: 1 comprises proline, the amino acid whose position corresponds to amino acid 61 of SEQ ID NO: 1 comprises threonine, the amino acid whose position corresponds to amino acid 86 of SEQ ID NO: 1 comprises proline, the amino acid whose position corresponds to amino acid 96 of SEQ ID NO: 1 comprises threonine, the amino acid whose position corresponds to amino acid 117 of SEQ ID NO: 1 comprises glutamic acid, the amino acid whose position corresponds to amino acid 123 of SEQ ID NO: 1 comprises methionine, and the amino acid whose position corresponds to amino acid 174 of SEQ ID NO: 1 comprises methionine.

67. The immunogenic composition of any one of claims 49 through 66, wherein from one to thirty six, from one to twenty four, from one to sixteen, from one to eight, or from one to four amino acids whose position corresponds to an amino acid of from 1-163 in SEQ ID NO: 1 are not the same as the corresponding amino acid in SEQ ID NO:

1.

68. The immunogenic composition of any one of claims 49 through 67, wherein from one to four, from one to three, of from one to two amino acids corresponding to amino acids whose position corresponds to amino acid of from 193-207 in SEQ ID NO: 1 are not the same as the corresponding amino acid in SEQ ID NO:

1.

69. A method of vaccinating a subject, comprising administering to the subject the immunogenic composition of any one of claims 49 through 68.

70. A method of treating a subject having Lyme disease or suspected of having been infected with or exposed to Borrelia burgdorferi, comprising administering to the subject a composition comprising the immunogenic composition of any one of claims 49 through 68.

71. The method of claim 69 or 70, wherein administering comprises administering the immunogenic composition more than once.

72. The method of claim 71, wherein the method comprises administering the composition at least two times.

73. The method of claim 72, wherein administration of the immunogenic composition fewer than three times prevents the subject from contracting Lyme disease.

74. The method of any one of claims 69 through 73 wherein in the subject is a rodent, a cat, a dog, a cattle, or a human.

75. A composition, comprising a means for immunizing a subject against a CspZ-expressing species of Borrelia, and an aluminum salt adjuvant and a CpG adjuvant, wherein administering the immunogenic composition to the subject fewer than three times prevents exposure of the subject to the CspZ-expressing species of Borrelia from causing Lyme disease in the subject.

76. The immunogenic composition of claim 75 wherein the aluminum salt adjuvant comprises aluminum hydroxide.

77. The immunogenic composition of claim 75 or 76 wherein the aluminum salt adjuvant comprises an aluminum hydroxide wet gel suspension or AS04.

78. The immunogenic composition of any one of claims 75 through 77, further comprising one or more additional adjuvant, wherein one or more of the one or more adjuvant is selected from AS03, monophosphoryl lipid A, poly(I:C), MF59, an emulsion adjuvant comprising squalene and water, a combination adjuvant comprising block copolymer CRL-8300, squalene, a sorbitan monooleateor, N-[1-(2,3-Dioleoyloxy)propyl] - N,N,N-trimethylammonium salt (DOTAP), 3 β-[N-(N',N'-dimethylaminoethane) -carbamoyl] cholesterol (DC-chol liposome), an aluminum hydroxide wet gel suspension, α- galactosylceramide, and a virosomal adjuvant.

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