{"paper_id":"519f969f-9291-4462-a611-d29948d1c113","body_text":"1\n1 An Fc-silent OspA monoclonal antibody passively protects mice from tick and \n2 intradermal Borrelia burgdorferi challenge\n3\n4\n5\n6 Daniel Palmer 1, Atieh Shemshadian2, Katherine Berman1, Graham G. Willsey1, Carol Lyn \n7 Piazza 1, Grace Freeman-Gallant1, Michael J Rudolph3, Jeff Bourgeois4, Linden Hu4, David J. \n8 Vance1,2, and Nicholas Mantis1,2,*\n9\n10\n11\n12 1Wadsworth Center, New York Department of Health, Division of Infectious Disease, Albany \n13 NY 12208; 2University of Albany, Department of Biomedical Sciences, Albany NY 12208; \n14 3New York Structural Biology Center, New York, NY 10027; 4Deparrment of Microbiology, \n15 Tufts University, Boston, MA 02111\n16\n17\n18\n19 *To whom correspondence should be addressed; nicholas.mantis@health.ny.gov\n20\n21\n22 Running title: Passive protection afforded by LA-2 LALAPG\n23\n24 Keywords: spirochete; antibody; complement; Fc receptor; skin; vector\n25\n26\n27\n28\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint \n\n2\n29\n30 ABSTRACT\n31 The monoclonal antibody, LA-2, has played a pivotal role in the development of Outer surface \n32 protein A (OspA)-based vaccines for Lyme disease, a multisystem illness caused by the tick-\n33 borne spirochete, Borrelia burgdorferi sensu lato. Of particular significance was the \n34 demonstration more than three decades ago that LA-2 equivalent antibody titers, defined by a \n35 competitive-inhibition ELISA, serve as a reliable correlate of vaccine-induced protection across \n36 different species, including humans. In vitro characterization of LA-2 has identified both \n37 complement-dependent and -independent activities, although which of these attributes contribute \n38 to protection against B. burgdorferi remains unresolved. To address this issue, we generated and \n39 characterized an “Fc-silent” version of LA-2 IgG1 carrying so-called LALAPG substitutions \n40 (L234A, L235A, P329G). We demonstrate that LA-2 LALAPG retained OspA binding activity \n41 but was severely attenuated in in vitro complement deposition and complement-dependent \n42 borreliacidal assays. Nonetheless, LA-2 LALAPG was as effective as LA-2 at passively \n43 protecting C3H mice against nymphal tick-mediated B. burgdorferi challenge. LA-2 LALAPG \n44 was also equivalent to LA-2 in passively protecting BALB/c mice against intradermal B. \n45 burgdorferi challenge. In the intradermal challenge model, viable spirochetes were not \n46 recoverable 24 h after injection from skin biopsies of mice treated with LA-2 or LA-2 LALAPG, \n47 and an influx of pro-inflammatory cytokines and chemokines to the injection site was abrogated. \n48 Collectively, these results suggest that LA-2’s primary mode of action involves direct physical \n49 interactions with the spirochete rather than complement-dependent killing. Elucidating these \n50 mechanisms may have implications for understanding the mechanistic correlates of OspA-based \n51 vaccine-induced immunity in humans. \n52\n53  \n54\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint \n\n3\n55 INTRODUCTION\n56 Over the past five decades, monoclonal antibodies (mAbs) have emerged as extraordinary \n57 tools in the identification of protective antigens and epitopes associated with pathogens of \n58 interest, leading to novel vaccines for viruses, bacteria and parasites 1-3. In the case of the Lyme \n59 disease spirochete, Borrelia burgdorferi sensu lato, multiple groups working in the 1990s \n60 generated large collections of mAbs that led to the identification Outer surface protein A (OspA) \n61 as a candidate Lyme disease vaccine antigen 4-15. OspA is a lipoprotein expressed at high levels \n62 by B. burgdorferi within the midgut of its arthropod vector, the black legged tick (Ixodes \n63 scapularis), where it is proposed to function as an adhesin 16. Structurally, OspA consists of 21 \n64 anti-parallel β -strands with a single C-terminal α-helix 17,18. The N-terminus is anchored in the \n65 spirochete outer membrane via a lipid moiety, while the C-terminus projects away (~80 Å) from \n66 the bacterial surface and is accessible to antibody attack 19,20. OspA is downregulated during or \n67 just after spirochete transmission to a mammalian host 21,22. As such, antibodies elicited by \n68 OspA-based vaccines are proposed to inhibit one or more steps in B. burgdorferi tick-mediated \n69 transmission, although the specific mechanisms by which this occurs remains to be fully \n70 elucidated. \n71 Among the many OspA mAbs characterized to date, LA-2 has played a particularly \n72 significant role in our understanding of OspA-mediated immunity. LA-2 was one of the first \n73 OspA-specific mAbs shown to passively protect mice from B. burgdorferi needle infection 6 and \n74 tick-mediated challenge 23. And, until just a few years ago, LA-2 was the only protective \n75 antibody whose epitope on OspA had been resolved at the structural level 18,24,25. In the context \n76 of Lyme disease vaccine development, LA-2 serological antibody “equivalence,” as defined by a \n77 competitive ELISA, proved to correlate with protection against tick-mediated B. burgdorferi \n78 infection in OspA-vaccinated mice and dogs 26. Remarkably, as part of a large randomized OspA \n79 vaccine trial, it was determined in a subset of individuals that LA-2 equivalent titers are also \n80 important biomarkers of Lyme disease susceptibility in humans, as individuals with confirmed \n81 Lyme disease had lower LA-2 equivalence than those who did not 27,28. LA-2 continues to be \n82 used as a benchmark in the development of next generation OspA vaccines 29 (M. Finn, personal \n83 communication).\n84 Despite LA-2’s central role in Lyme disease vaccine development, the exact mechanism \n85 by which LA-2 protects against B. burgdorferi infection remains incompletely defined. In fact, \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint \n\n4\n86 the basic question of whether complement is needed for LA-2’s protective activity has not been \n87 addressed. While LA-2 has potent complement-dependent borreliacidal activity in vitro 23,30,31, \n88 evidence indicates that complement (human or mouse) is not active in the tick midgut 32. Several \n89 complement-independent activities have been ascribed to LA-2, including effects on spirochete \n90 transmigration, that would be expected to impede B. burgdorferi from the tick midgut 33,34. \n91 Defining the contribution of complement in LA-2’s mechanism of action is important for \n92 understanding correlates of OspA-mediated immunity, especially as clinical trials of next \n93 generation OspA vaccines are ongoing 29,35,36. In this report, we generate an “Fc-silent” version \n94 of LA-2 IgG1 that is effectively devoid of in vitro complement-dependent borreliacidal activity \n95 and characterize its activity in mouse models of tick-mediated and intradermal B. burgdorferi \n96 challenge. \n97\n98 Materials and Methods\n99 Ethics statement. The mouse experiments described in this study were reviewed and approved \n100 by the Institutional Animal Care and Use Committees (IACUC) at the Wadsworth Center \n101 (protocol 23-459) and Tufts University-Tufts Medical Center (protocol B2024-50). The \n102 Wadsworth Center and Tufts University-Tufts Medical Center both comply with the Public \n103 Health Service Policy on Humane Care and Use of Laboratory Animals and were issued \n104 assurance numbers A3183-01 and A4059-01, respectively. Both facilities are fully accredited by \n105 the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). \n106 Obtaining this voluntary accreditation status reflects that these facilities’ Animal Care and Use \n107 Program meets all standards required by law and goes beyond the standards as it strives to \n108 achieve excellence in animal care and use. All animals were euthanized by carbon dioxide \n109 asphyxiation followed by cervical dislocation, as recommended by the Office of Laboratory \n110 Animal Welfare (OLAW), National Institutes of Health.\n111\n112 Recombinant B. burgdorferi B31 proteins. Recombinant OspA, DbpA and OspC type A \n113 derived from  B. burgdorferi strain B31 (Table 1) were expressed in E. coli as cited in Table 1.\n114\n  Table 1. Recombinant B. burgdorferi B31 proteins used in this study\nAntigen AA UniProt ID References\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint \n\n5\nOspA 18-273 P0CL66 24\nOspB 50–296 P17739 unpublished\nOspCA 38-201 Q07337 37\nDbpA 26-188 O50917 38\nDbpB 21–187 O50917 unpublished\nAbbreviations; AA, amino acid; UniProt (https://www.uniprot.org/)\n115\n116 LA-2 and LA-2 LALAPG IgG1 expression and purification. Codon optimized VH and VL \n117 DNA sequences of LA-2 (Antibody Registry RRID: AB_2619693) derived from PDB 1FJ1 18 \n118 were custom synthesized by Life Technologies (San Diego, CA) and cloned into TMV and PVX \n119 plant expression vectors containing codon-optimized human kappa and human IgG1 constant \n120 regions 39. The resulting plasmids were transformed into Agrobacterium tumefaciens. Four-\n121 week-old N. benthamiana plants were infiltrated with A. tumefaciens carrying plasmids for the \n122 expression of heavy and light chains of LA-2 LALAPG. Aerial plant parts were harvested after 7 \n123 days and extracted and clarified. The LA-2 LALAPG antibody was then purified with Protein A \n124 affinity and anion exchange chromatography 40. \n125\n126 Antibody affinity determinations by Biolayer interferometry (BLI). Affinity determinations \n127 were conducted using an Octet RED96e Biolayer Interferometer (Sartorius, Goettingen, \n128 Germany) with Data Acquisition 12.0 software. Biotinylated OspA (5 μg/mL) in PBS containing \n129 2% w/v BSA (“buffer”) was captured onto Octet SA (streptavidin) biosensors (Sartorius) for 5 \n130 min. After equilibration, sensors were immersed in two-fold serial dilutions of mAb starting at \n131 100 nM for 10 min. The sensors were then dipped into buffer for 30 min to allow for \n132 dissociation. The raw sensor data were loaded into the Data Analysis HT 12.0 software, grouped \n133 and fit using a 1:2 bivalent analyte model.\n134\n135 Flow cytometric analysis of B. burgdorferi surface labeling. B. burgdorferi strain B31 surface \n136 labeling with LA-2 and LA-2 LALAPG was performed essentially as described 31. LA-2 and \n137 LA-2 LALAPG were 2-fold serially diluted in PBS before incubation with viable B. burgdorferi \n138 B31. The ricin-specific mAb, PB10, was used as an IgG1 isotype control (10 µg/mL). Alexa \n139 Fluor 647-labeled goat anti-human IgG (H+L) (Invitrogen, Carlsbad, CA) was used as a \n140 secondary antibody. Samples were analyzed using a BD FACSCalibur (BD Biosciences, \n141 Franklin Lakes, NJ). Bacteria were gated on FSC and SSC to exclude debris, and 20,000 events \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint \n\n6\n142 were counted per condition. Agglutination was calculated as the percent of events in the UL + \n143 UR + LR quadrants. Data were analyzed using FlowJo v10.10.0 (BD Biosciences).\n144\n145 Complement-dependent borreliacidal assays. Complement-dependent borreliacidal assays \n146 were performed using a recombinant B. burgdorferi B31-5A4 strain carrying an IPTG-inducible \n147 mscarlet-I reporter (GGW979), as described 31. Briefly, GGW979 cultures were grown to mid-\n148 log phase in BSKII medium supplemented with gentamicin (50 µg/ml) at 32 °C under static \n149 conditions. Spirochetes were harvested by low-speed centrifugation and resuspended in phenol \n150 red–free BSKII containing gentamicin (50 µg/ml) to a final density of 3×10⁷ spirochetes/ml.\n151 Cell suspensions were then mixed 1:1 with phenol red–free BSKII supplemented with \n152 20% guinea pig complement (Sigma Aldrich, St. Louis, MO) and 20 nM of one of the following \n153 mAbs: LA-2, LA-2 LALAPG, 857-2, PB10. PB10, a ricin toxin-specific antibody, was used as \n154 an IgG1 isotype control. Reactions were set up in white 96-well assay plates (Co-Star). \n155 Following sample addition, reactions contained 1.5×10⁶ spirochetes, 5 nM of antibody and 10% \n156 guinea pig complement. Assay plates were then incubated overnight in a water jacketed \n157 incubator at 37°C with 5% CO 2. The following day, 1 mM IPTG was added to each well to \n158 induce mScarlet-I expression. Following a 48-h incubation at 37°C with 5% CO 2, the MFI was \n159 recorded at 569 nm (excitation)/611 nm (emission) using a Spectramax ID3 plate reader \n160 (Molecular Biosystems, San Jose, CA). Raw MFI data was then normalized as described 31. The \n161 data presented is the mean and SD of three independent experiments. \n162\n163 MIA. B. burgdorferi B31 antigens OspA, OspB, OspC, DbpA, and DbpB (Table 1) were \n164 coupled to Magplex-C microspheres (5 g antigen/ 1x10 6 microspheres) using a xMap Antibody \n165 Coupling Kit as recommended by the manufacturer (Luminex Corporation, Austin, TX). Beads \n166 were protected from light and stored at 2-8°C in xMAP AbC Wash Buffer (5x10 6 \n167 microspheres/mL) until use. Serum samples (1:100) and coupled microsphere stocks (1:50) were \n168 diluted in assay buffer (1 x PBS, 2% BSA, pH 7.4). The diluted sera (50 μL) and diluted \n169 microspheres (50 μL) were combined in black, clear-bottomed, non-binding, chimney 96-well \n170 plates (Greiner Bio-One, Monroe, North Carolina) and incubated at room temperature for 1 hr in \n171 a tabletop shaker (600 rpm). Plates were placed on a magnetic separator and washed three times \n172 using wash buffer (1 x PBS, 2% BSA, 0.02% TWEEN-20, 0.05% Sodium azide, pH 7.4). To \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint \n\n7\n173 detect seroconversion in the mice, goat anti-mouse IgG, Human-ads-PE (SouthernBiotech, \n174 Birmingham, Alabama) secondary antibody was diluted 1:500 in assay buffer, added (100 μL ) to \n175 each well, and allowed to incubate at room temperature for 30 min in a tabletop shaker (600 \n176 rpm). Alternatively, to detect remaining LA-2 or LA-2 LALAPG, PE labeled goat anti-Human \n177 IgG Fc, eBioscience (Invitrogen) secondary antibody was used. Plates were washed as \n178 previously stated. The microspheres were resuspended in 100 μL of wash buffer and placed back \n179 on the tabletop shaker (600 rpm) for 5 minutes prior to analysis using a FlexMap 3D (Luminex \n180 Corporation). To establish reactivity cutoffs for each antigen, the average median fluorescent \n181 intensity (MFI) of buffer-only wells was multiplied by six. MFIs for each mouse serum sample \n182 were divided by the antigen-specific reactivity cutoffs yielding an index value. An index value \n183 greater than 1 suggests reactivity above background for the given antigen.\n184\n185 Antibody-dependent complement deposition (ADCD) assay. We modified a flow cytometry-\n186 based HIV-1 antibody-dependent complement deposition (ADCD) for use with a Luminex \n187 instrument and OspA-coupled beads 41. Magplex-C microspheres coupled with recombinant B. \n188 burgdorferi antigens, OspA and OspCA, were diluted (1:50) and mixed 1: 1 (v/v) with primary \n189 antibodies, LA-2 and LA-2 LALAPG (10 g/mL), then seeded into a 96-well plates, covered in \n190 foil, and incubated for 1 h at room temperature (RT) with shaking. Plates were washed twice \n191 using a plate magnet and 190  L of wash buffer (PBS, 2% BSA, 0.02% Tween-20, 0.05% \n192 sodium azide, pH 7.4). Following the washes, 200 L of diluted human complement (1:50; Pel-\n193 Freez Biologicals, Rogers, AR) were added to each well and incubated for 20 min at RT with \n194 shaking. Plates were washed again then phycoerythrin-tagged mouse anti-C3/C3b/iC3b (1:100; \n195 BD) and phycoerythrin-tagged goat anti-human IgG Fc (1:500; Invitrogen) were added to their \n196 respected wells and incubated for 30 min. The plates were washed a final time before antibody-\n197 bead complexes were resuspended in 100 uL of wash buffer and incubated for 1 min while \n198 shaking. The plates were analyzed via a FlexMAP 3D instrument (Luminex Corporation) with \n199 results presented in median fluorescence intensity (MFI).\n200\n201 Mouse model of B. burgdorferi challenge by Ixodes scapularis nymphs. Animal studies were \n202 conducted with approval by the Institutional Animal Care and Use Committees (IACUC) at the \n203 Wadsworth Center and Tufts University-Tufts Medical Center. To generate B. burgdorferi B31 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint \n\n8\n204 infected Ixodes scapularis nymphs, C57BL/6J mice were injected subcutaneously with 10^5 \n205 cells/mL log growth phase of B. burgdorferi B31. Two to four weeks later, the mice were \n206 infested with naive Ixodes scapularis  larvae, which were allowed to feed to repletion. Replete \n207 larvae were harvested and allowed molt into mature infected nymphs. As controls, we used naive \n208 Ixodes scapularis nymphs procured from the Oklahoma State University tick rearing facility 42. \n209 For challenge studies, equal numbers of male and female C3H/HeN mice aged ~6 weeks \n210 (Charles River Laboratories, Kingston, NY) were acclimated in the Wadsworth Center’s \n211 vivarium for 1-2 weeks before the start of the experiment. On study day -1, mice were \n212 subcutaneously (SC) administered either LA-2, LA-2 LALAPG or an IgG1 isotype control (anti-\n213 Vibrio cholerae mAb ZAC-3) (120 g or 30 g per mouse) diluted in 200 µL of PBS. The \n214 following day, infected or naive nymphal ticks (5 per mouse) were placed on a shaved area of the \n215 mouse’s dorsum. Nymphs were collected from all mice 3-5 days post placement. Mice which \n216 had at least one tick that appeared to be at or near repletion at the time of collection were \n217 presumed to be successfully challenged. On study day 21, mice were euthanized, and blood was \n218 collected via cardiac puncture for serological analysis. Bladders were also collected for \n219 cultivation of spirochetes in 2 mL BSKII cultures treated with rifampicin (50 μg/mL), \n220 fosfomycin (20 μg/mL), and amphotericin B (2.5 μg/mL). Infection status was based on \n221 seroconversion using the MIA described above, as well as the presence or absence of live \n222 spirochetes in bladder cultures using dark-field microscopy, which were assessed weekly for one \n223 month.\n224\n225 Collection of engorged ticks, dissection, and determination of genome equivalents. One \n226 replete or near-replete nymph that had fed on each mouse was dissected to harvest the midgut \n227 tissues. Midgut tissues were extracted using the E.Z.N.A.® Mollusc & Insect DNA Kit (Omega \n228 Bio-tek, Inc., Norcross, GA) and real-time qPCR was performed to determine the Borrelia \n229 burden in midgut tissues. The single-copy B. burgdorferi flaB (flagellin) gene was amplified, and \n230 flaB copy number was standardized to total gDNA in each sample as measured using the Qubit 4 \n231 Fluorometer (Invitrogen) to determine normalized spirochete burdens in tick midguts.\n232\n233 Mouse model of intradermal B. burgdorferi challenge. Female BALB/c mice aged ~8 weeks \n234 (Taconic Biosciences, Germantown, NY) were acclimated in the Wadsworth Center’s vivarium \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint \n\n9\n235 for one week before the start of the experiment. On study day -1, mice were injected \n236 intraperitoneally (IP) with LA-2 or LA-2 LALAPG (0.1 - 120 g/mouse) in 200 µL PBS. The \n237 following day (study day 0), mice were challenged with mid-log phase B. burgdorferi strain \n238 B31-5A4 (1x10 5 cells) by intradermal (ID) injection. On day 21, the mice were euthanized, and \n239 blood was collected via cardiac puncture for serological analysis. Infection status was \n240 determined based on seroconversion using the MIA described above. \n241\n242 To assess the effects of LA-2 and LA-2 LALAPG on B. burgdorferi  skin dissemination, a mix of \n243 male and female BALB/c mice aged ~6 weeks were injected SC with 30 µg of LA-2 or LA-2 \n244 LALAPG in 200 µL PBS, or remained untreated. The following day (study day 0), mice were \n245 challenged with mid-log phase B. burgdorferi strain B31-5A4 (1x10 5 cells) by ID injection. \n246 Groups of mice were euthanized on days 1, 3 and 7, and ~5 mm skin biopsies were excised from \n247 the injection site (IS), ~1 cm away from the IS, and ~3 cm away from the IS. Biopsies were \n248 rinsed in PBS immediately after collection and placed in 2 mL BSKII medium supplemented \n249 with rifampicin (50 μg/mL), fosfomycin (20 μg/mL), and amphotericin B (2.5 μg/mL) for \n250 cultivation of spirochetes. The biopsy cultures were assessed weekly by dark-field microscopy \n251 over the course of four weeks for the presence of viable spirochetes.\n252\n253 Inflammatory cytokine and chemokine analysis in mouse skin biopsies. Groups of male and \n254 female BALB/c mice were SC administered 30 ug LA-2 per mouse on day -1 or left untreated. \n255 The following day (study day 0), mice were challenged with mid-log phase B. burgdorferi strain \n256 B31-5A4 (1x10 5 cells) by ID injection. 5 days-post infection, mice were euthanized, and a skin \n257 biopsy ~1 cm in diameter was collected from the injection site of each mouse in a cytokine \n258 extraction buffer containing 0.4M NaCl, 0.05% Tween 20, 0.5% Bovine Serum Albumin, 0.1 \n259 mM phenylmethylsulphonyl fluoride, and 20 Ki of aprotinin in 1X PBS. The solution containing \n260 the biopsy was homogenized at 5 m/s in a bead beater in 1-minute increments, with a 1-minute \n261 cool down between shakes. This was repeated five times, or until the biopsy was fully \n262 homogenized. This solution was centrifuged at 13,000 g for 10 minutes at 4°C, and the \n263 supernatant was collected and then prepared for cytometric bead array analysis. Skin \n264 homogenates were diluted 1:2 in assay diluent and processed using the BD Biosciences \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint \n\n10\n265 Cytometric Bead Array (CBA) Mouse Inflammation Kit following the manufacturer’s \n266 instructions. Samples were analyzed using a BD FACSCalibur (BD Biosciences).\n267\n268 Statistical analysis. Statistical procedures for experiments are described in the figure legends. \n269 All statistical analysis of data was performed in R, Graphpad Prism 9.0, and Microsoft Excel. \n270 In all experiments, p-values <0.05 are considered significant. \n271\n272 Results\n273 Recognition of recombinant and native OspA by LA-2 and LA-2 LALAPG. To generate an \n274 Fc-silent version of LA-2, codon optimized DNA sequences encoding the variable heavy chain \n275 (V H) was cloned in-frame into human IgG1 Fc and IgG1 LALAPG expression vectors. The IgG1 \n276 LALAPG derivative carries three-point mutations (L234A, L235A, P329G) relative to IgG1 that \n277 abolishes complement fixation activity and FcR recognition 43,44. The LA-2 VL coding sequence \n278 was inserted into a human kappa expression vector. The V H and VL plasmids were transformed \n279 into A. tumefaciens that was then used to infiltrate N. benthamiana . Aerial plant parts were \n280 harvested after 7 days and extracted and clarified antibodies were purified to homogeneity by \n281 Protein A affinity and anion exchange chromatography 40. By flow cytometry, LA-2 IgG1 and \n282 LA-2 LALAPG were equivalent in their ability to recognize native OspA on the surface of viable \n283 B. burgdorferi strain B31, as well as induce agglutination of those cells (Figure 1). Surface \n284 labeling was dose-dependent and resulted in a maximum of ~90% total cell labeling with a \n285 median fluorescence intensity (MFIs) exceeding 7500 for LA-2 LALAPG. LA-2 and LA-2 \n286 LALAPG also recognized recombinant OspA with similar apparent affinities as measured by \n287 BLI (Figure S1 ). \n288\n289 Figure 1. Reactivity of LA-2 and LA-2 LALAPG with native OspA. Representative flow \n290 cytometry assay of serially diluted LA-2 (dark blue) and LA-2 LALAPG (light blue overlay) \n291 reactivity with native OspA on the surface of B. burgdorferi B31. (Left) Fluorescence histogram \n292 overlays comparing binding properties. Percent and gMFIs of Alexa-647 fluorescently labeled \n293 events (under bracket) are indicated. (Right) Forward scatter (FSC) – side scatter (SSC) dot plot \n294 overlays comparing agglutination properties. Events increased in FSC and/or SSC (UL, UR, LR \n295 quadrants) demonstrate agglutination, and percent of events agglutinated is indicated.\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint \n\n11\n296\n297 LA-2 LALAPG IgG1 lacks complement fixation and borreliacidal activities. Introduction of \n298 the LALAPG (L234A, L235A, P329G) mutations into the Fc region of IgG1 is reported to \n299 essentially eliminate complement fixation activity 43,44. To examine this in the case of LA-2, we \n300 adopted an antibody-dependent complement deposition (ADCD) assay to OspA (Figure 2A) 41. \n301 Recombinant OspA was covalently coupled to fluorescent microspheres, then probed with LA-2 \n302 IgG1 and LA-2 LALAPG in the presence of human complement. Total mAb binding to the \n303 beads was determined using PE-labeled anti-human IgG, while complement deposition was \n304 measured using a PE-labeled anti-C3 antibody. The results confirmed that LA-2 and LA-2 \n305 LALAPG have equivalent capacities to bind OspA (Figure 2B). However, the two mAbs were \n306 starkly different in terms of complement fixation activity. LA-2 demonstrated a dose-dependent \n307 increase in C3 deposition that peaked at ~2  g/ml. LA-2 LALAPG, in contrast, was devoid of \n308 any activity even at 10 g/ml (Figure 2C). These results confirmed that LA-2 LALAPG is \n309 unable to fix complement via the classical pathway. \n310\n311 Figure 2. LA-2 LALAPG is deficient in complement deposition in vitro. (A) Binding (MFI) \n312 of LA-2 IgG and LA-2 LALAPG to recombinant OspA. The asterisks indicate a significant \n313 difference between groups by Welch’s t-test (**P<0.01). Quantification and comparison of \n314 human complement C3 deposition between LA-2 and LA-2 LALAPG in the context of OspA. \n315 The asterisk indicates a significant difference between groups by Welch’s t-test, where *P<0.05. \n316 (C) Dose response of complement C3 deposition of LA-2 and LA-2 LALAPG in the context of \n317 OspA. \n318\n319 To assess the capacity of LA-2 and LA-2 LALAPG to promote complement-dependent \n320 borreliacidal activity, we employed a recently developed fluorescence-based B. burgdorferi  \n321 reporter strain GGW979 31. GGW979 is a derivative of B. burgdorferi B31 that expresses the red \n322 fluorescent protein, mScarlet, under control of an IPTG-inducible promoter 45. In the assay, \n323 neither LA-2 nor LA-2 LALAPG had any measurable demonstrable borreliacidal activity in the \n324 absence of 5 nM complement (Figure 3). In the presence of exogenous complement, LA-2 IgG \n325 elicited dose-dependent borreliacidal activity at concentrations ranging from 20 to <1 g/ml \n326 (data not shown). LA-2 LALAPG, on the other hand, had no measurable complement-\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint \n\n12\n327 dependent borreliacidal activity, even at 20  g/ml. Collectively, these results demonstrate that \n328 the LA-2 LALAPG retains OspA binding activity but lacks complement-fixing activity. \n329\n330 Figure 3. Complement-dependent borreliacidal activity associated with LA-2 and LA-2 \n331 LALAPG. Mid-log phase B. burgdorferi strain B31-5A4 carrying an IPTG-inducible mscarlet-I \n332 reporter (GGW979) were suspended (1.5×10⁶ cells per reaction) in BSKII medium supplemented \n333 with 20% guinea pig complement and 5 nM of the mAbs indicated on the x-axis (857-2, LA-2, \n334 LA-2 LALAPG, PB10), as detailed in the Materials and Methods. Following a 48-h incubation, \n335 the median fluorescence intensity (MFI; 569 nm excitation/611 nm emission) was determined. \n336 The bars are the mean of three independent experiments with each symbol being an independent \n337 experiment and the error bars indicating SD. The dashed red line represented 50% killing. The \n338 results demonstrate that 857-2 and LA-2 have potent borreliacidal activity as indicated by low \n339 normalized MFI, whereas LA-2 LALAPG and the isotype control were devoid of activity. \n340\n341 LA-2 LALAPG protects mice from tick-mediated B. burgdorferi infection. Having \n342 established that LA-2 LALAPG is deficient in complement fixation, we next examined the \n343 mAb’s ability to protect mice from infection in a tick-mediated B. burgdorferi challenge. Groups \n344 of C3H/HeN mice were administered 120 or 30 g of LA-2 or LA-2 LALAPG by subcutaneous \n345 injection and challenged the following day with B. burgdorferi B31-infected Ixodes scapularis \n346 nymphs. An additional group of mice received an IgG1 isotype control (ZAC-3). On day 21, the \n347 mice were euthanized and assessed for B. burgdorferi infection by serology using a \n348 B.burgdorferi specific MIA and recovery of viable spirochetes from bladders. For statistical \n349 purposes, a mouse was scored as categorically infected if either readout (seroconversion, culture) \n350 was positive. By these metrics, LA-2 and LA-2 LALAPG were each protective at the 120 g \n351 dose (p<0.01), but only marginally effective at 30  g dose, relative to mice that received the \n352 isotype control (Table 2). Of particular importance, LA-2 and LA-2 LALAPG were statistically \n353 indistinguishable in terms of their protective efficacy (p>0.99).  These results demonstrate that \n354 LA-2 can protect mice from tick-mediated B. burgdorferi infection in the absence of complement \n355 fixation.\n356 B. burgdorferi numbers in the midgut of ticks that feed on OspA immunized mice were \n357 reported to decline or be eliminated entirely within days after engorgement suggesting that OspA \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint \n\n13\n358 antibodies exhibit borreliacidal activities within the tick gut 9,32,46. On the other hand, the “LA-2-\n359 like” mAb, C3.78, passively protected mice from tick-mediated B. burgdorferi infection without \n360 affecting spirochete numbers in the midgut, consistent with a mechanism of action not dependent \n361 on borreliacidal activity 47. To address this issue in the case of LA-2, we collected engorged ticks \n362 that had fed on ZAC-3-, LA-2- or LA-2 LALAPG-treated mice, dissected the midguts, then \n363 quantified spirochete burdens using qPCR. There was no significant reduction in spirochete \n364 burdens in the tick midgut in ticks that fed on LA-2 or LA-2 LALAPG-treated mice (Figure S2), \n365 as compared to the isotype control-treated group. Thus, LA-2 or LA-2 LALAPG do not appear to \n366 exhibit borreliacidal activity in the context of the tick midgut environment. \n367\nTable 2. mAb passive protection in mouse model of tick-mediated B. burgdorferi challenge\nreadout (# pos./# total) significance (p) b\nmAb Dose (µg) Tick serologya culture a vs. IC vs. LA-2\nLA-2 120 + 0/6 0/6 <0.01 -\nLALAPG 120 + 1/6 1/6 0.03 >0.99\nLA-2 30 + 2/5 1/5 0.18 -\nLALAPG 30 + 3/6 2/6 0.18 >0.99\nIC 30 + 5/5 5/5 - -\nIC 30 - 0/2 0/2 - -\na, number of positive mice/total mice per group. b, significance (Fisher’s exact test with Benjamani-Hochberg procedure \nfor the FDR) was determined using readout with highest infection status. p-values <0.05 are considered significant. IC = \nisotype control.  \n368\n369 LA-2 and LA-2 LALAPG limit skin dissemination of B. burgdorferi. While it is known that \n370 LA-2 and other OspA antibodies protect mice from B. burgdorferi dissemination even when \n371 spirochetes are delivered by injection, there are no reports examining whether this is dependent \n372 on complement 6,48,49. Considering the sensitivity of B. burgdorferi to the classical complement \n373 pathway 50, we reasoned that LA-2 would prevent disseminated infection following intradermal \n374 challenge, while LA-2 LALAPG would not. To test this, groups of mice were administered LA-2 \n375 or LA-2 LALAPG at 120 g per mouse then challenged the following day with viable B. \n376 burgdorferi B31 (105 cells) by intradermal injection. Three weeks later, mice were euthanized \n377 and assessed for seroconversion using the B. burgdorferi-specific MIA, described above. By this \n378 measure, all six mice in the LA-2 treated group and five of the six mice in the LA-2 LALAPG \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint \n\n14\n379 group were protected (Table 3). This demonstrates that neither Fc effector functions nor \n380 complement fixing activity are required for LA-2’s protective activity following intradermal \n381 challenge. \n382\nTable 3. mAb passive protection in mouse model of B. burgdorferi ID challenge\nDose \n(µg/mouse) LA-2 (p =)a LA-2 LALAPG (p =) a LA-2 vs. LA-2 \nLALAPGb\n120 0/6 (<0.01) 1/6 (0.02) >0.99\n1 1/6 (0.046) 2/6 (0.09) >0.99\n0.3 3/6 (0.54) 5/6 (>0.99) 0.82\n0.1 3/6 (0.27) 6/6 (>0.99) 0.27\n0 6/6 -- -\na, number of positive mice/total mice per group with significance compared to the infected control (0 µg/mouse \ndose) determined by Fisher’s exact test with Benjamani-Hochberg procedure for the FDR indicated in \nparentheses; b significance (p values), determined by Fisher’s exact test with Benjamani-Hochberg procedure \nfor the FDR, comparing numbers of infected mice between LA-2 and LA-2 LALALPG at each dose indicated.\n383\n384 To investigate how LA-2 and LA-2 LALAPG perform at limiting doses, we carried out a \n385 pilot study to establish the minimum amount of LA-2 required to protect BALB/c mice against \n386 B. burgdorferi B31 intradermal challenge. Those studies indicated that as little as 1 g of LA-2 \n387 IgG per mouse was sufficient to render B. burgdorferi B31 non-infectious (Table S1). We \n388 therefore compared LA-2 and LA-2 LALAPG side by side at doses of 1, 0.3 and 0.1 g mAb per \n389 animal in intradermal B. burgdorferi B31 challenge. At the 1  g dose, one of the six mice in the \n390 LA-2 treatment group was infected at day 21, while two of the six mice in the LA-2 LALAPG \n391 group were infected (Table 3). Although neither mAb conferred significant protection at the two \n392 lower doses tested (0.3 and 0.1  g per mouse), LA-2 treated mice fared slightly better than the \n393 LA-2 LALAPG treated mice in both cases. We conclude that LA-2 IgG protection in the \n394 intradermal challenge model is independent of Fc-mediated activities at high antibody \n395 concentrations but possibly important when antibody is limiting. \n396\n397 LA-2 and LA-2 LALAPG clear viable spirochetes from the B. burgdorferi skin. The fact that \n398 both LA-2 and LA-2 LALAPG treatments inhibited B. burgdorferi dissemination in the mouse \n399 model of intradermal challenge prompted us to examine spirochete burden in tissues at earlier \n400 time points. To do this, skin biopsies were collected on days 1, 3 and 7 at three locations: the \n401 injection site (IS) on the ventral side of the animal, ~1 cm from the IS, and ~3 cm from the IS on \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint \n\n15\n402 the dorsal side of the animal (Figure 4). Skin biopsies were cultured in BSKII medium for a \n403 month and scored regularly for appearance of viable spirochetes. In the control mice, viable \n404 spirochetes were recovered on days 1, 3 and 7, which coincides with the reported kinetics of B. \n405 burgdorferi dissemination 51,52. Moreover, three of the four mice also had positive knee and \n406 spleen cultures (data not shown). In contrast, skin biopsies from LA-2-treated mice were culture \n407 negative at all time points examined (Figure 4). The results were similar for LA-2 LALAPG \n408 treatment, with just one of three animals showing positive cultures on day 7 (Figure 4). These \n409 results indicate that, in the presence of LA-2 and LA-2 LALAPG, viable B. burgdorferi \n410 spirochetes are cleared rapidly at or very near the injection site, thereby arresting dissemination \n411 before it even gets started. \n412\n413 Figure 4. LA-2 and LA-2 LALAPG prevent spirochete dissemination through skin. Groups \n414 of mice were administered LA-2, LA-2 LALAPG or an isotype control by subcutaneous \n415 injection, then intradermally challenged one day later with B. burgdorferi. On days 1, 3, and 7 \n416 post challenge, mice were euthanized, and skin biopsies were harvested at the injection site (IS), \n417 ~ 1 cm from the IS (S1), and ~3 cm from the IS (S2). Biopsies were immersed in BSKII media to \n418 recover viable spirochetes. The pie charts indicate the number of skin samples assayed per \n419 treatment, with one sample collected from each skin site per mouse. Red subdivisions indicate \n420 positivity for motile spirochetes, while white subdivisions indicate no viable spirochetes \n421 detected. Shown are the combined results from two independent experiments.\n422\n423 The absence of viable spirochetes in the skin biopsies led us to hypothesize that local \n424 inflammation may contribute to LA-2-mediated spirochete clearance. To test this, we examined \n425 skin biopsy homogenates for the presence of mouse inflammatory chemokines and chemokines \n426 TNF-α, IFN-γ, MCP-1, IL-6, IL-10, and IL-12p70. At five days following injection, we observed \n427 elevated levels of TNF-α, IFN-γ, IL-6 and especially MCP-1 in untreated mice when compared \n428 to uninfected mice (Figure 5). However, in infected mice that were pretreated with LA-2, \n429 analytes showed cytokine concentrations similar to levels of uninfected mice (Figure 5). Thus, \n430 LA-2 treatment is not associated with residual inflammation in the skin and may clear the \n431 spirochetes before a cytokine response can be generated. Further analysis of mouse skin \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint \n\n16\n432 cytokines and chemokines, as well as immune cell infiltrates, collected at earlier infection time \n433 points is ongoing.  \n434\n435 Figure 5. BALB/c mice intradermal infected with B. burgdorferi and treated with LA-2, 5 \n436 days post-injection. On day –1, mice were given a subcutaneous injection of 30 µg/mL of LA-2 \n437 in 200 µL of 1X PBS, 30 µg/mL of ZAC-3 in 200 µL of 1X PBS, or 200 µL of 1X PBS. Mice \n438 were shaved on day 0 on their right and left flanks, then given an intradermal injection, on each \n439 flank, of 10^5 live B. burgdorferi cells in 50 µL of 1X PBS, or 50 µL of 1X PBS. Skin biopsies, \n440 approximately 1 cm in diameter, were collected in 1 mL of cytokine extraction buffer for \n441 cytometric bead array (CBA) analysis, 5 days post-injection. Skin samples were diluted 1:2 in \n442 assay diluent for analysis. Statistics performed by one-way ANOVA, no matching or pairing, \n443 corrected for multiple comparisons using Tukey’s test. 95% confidence interval. *p<0.05, \n444 **p<0.01.\n445\n446 Discussion\n447 In this report, we generated and characterized an “Fc-silent” derivative of LA-2 as a tool \n448 to investigate the role of complement in passive protection afforded by LA-2 in both tick- and \n449 needle-mediated B. burgdorferi challenge models. The Fc element of LA-2 was rendered silent \n450 by the addition of the so-called LALAPG substitutions (L234A, L235A, P329G), a modification \n451 that is gaining wide recognition for its research and clinical applications 44,53,54. We confirmed \n452 that LA-2 LALAPG retained OspA binding activity comparable to the parenteral LA-2 IgG1 but \n453 was markedly attenuated for in vitro complement fixation and complement-dependent \n454 borreliacidal activity. \n455 When tested in vivo, we found that LA-2 LALAPG was as effective as LA-2 IgG in \n456 passively protecting mice from tick-mediated B. burgdorferi  challenge, indicating that neither \n457 antibody-mediated complement fixation nor complement-dependent borreliacidal activity were \n458 necessary to inhibit spirochete infectivity. In this respect, our results agree with Gipson and de \n459 Silva who reported that the “LA-2-like” monoclonal antibody C3.78 blocks tick transmission of \n460 B. burgdorferi in the absence of host complement 8,47. Those studies were conducted using \n461 complement-deficient (C3) mice and C3.78 Fab fragments. de Silva and colleagues also \n462 demonstrated that C3-deficient mice actively immunized with OspA were also protected against \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint \n\n17\n463 B. burgdorferi infection, further reinforcing the notion that host complement is not required for \n464 transmission blocking activity of OspA vaccines 32. \n465 Furthermore, our results support a model in which LA-2 and LA-2 LALAPG inhibit B. \n466 burgdorferi transmission without affecting the number of spirochetes within the tick midgut. \n467 This observation is consistent with C3.78’s mode of action, in which low dose (~60  g/mouse) \n468 antibody protected mice from tick-mediated B. burgdorferi challenge without a concomitant \n469 reduction in spirochete numbers in tick tissues 47. In other words, antibody is proposed to block \n470 spirochete egress from the midgut by a non-borreliacidal mechanism. Gipson and de Silva and \n471 others have speculated that OspA antibodies like C3.78 influence the expression of spirochete \n472 genes and gene products required for transmission, including the requisite OspA to OspC \n473 transition 23,47. We favor a model in which OspA antibodies like LA-2 entrap spirochetes within \n474 the midgut by physically altering their transmigratory activity 34. Using a two compartment \n475 Transwell system, we reported recently that spirochete movement from the lower to upper \n476 chambers is reduced by >99% in the presence LA-2 or LA-2 LALAPG. Inhibition of \n477 transmigration coincided with LA-2’s ability to promote spirochete agglutination, alternations in \n478 membrane permeability, and even bleb formation 33,55. Exactly how LA-2 engagement with \n479 OspA results in changes in migratory activity remains obscure.  \n480 While LA-2 has the capacity to interfere with B. burgdorferi transmission within the \n481 context of the tick, it can also reduce infectivity of B. burgdorferi within the mouse. Indeed, LA-\n482 2 was originally identified as being capable of passively protecting scid mice from subcutaneous \n483 B. burgdorferi challenge 6. We confirmed and extended that original observation by \n484 demonstrating in both BALB/c and C3H mice that remarkably low doses of passively \n485 administered LA-2 were sufficient to not only confine but seemingly eliminate B. burgdorferi \n486 from the site of intradermal inoculation within hours. LA-2 LALAPG had similar properties, \n487 indicating that clearance of B. burgdorferi from the skin environment occurs without \n488 complement or Fc effector functions. These observations may be of clinical importance, as they \n489 suggest that if B. burgdorferi evades immunity within the context of the tick body, any \n490 spirochetes that still express OspA upon entry into the skin will encounter a second line of \n491 defense 21,56. While the underlying mechanism by which LA-2 promotes clearance of spirochetes \n492 from the skin environment without Fc effector functions is unknown, there are interesting \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint \n\n18\n493 parallels with antibody-mediated clearance of malaria parasites in this same environment that \n494 involve motility arrest and membrane shedding 57,58.\n495 In summary, we have demonstrated that LA-2, the well-characterized monoclonal \n496 antibody directed against the C-terminus of OspA, functions in both the tick and mammalian \n497 environments to limit B. burgdorferi infection without the need for Fc effector functions, such as \n498 complement fixation and FcR interactions. It is unclear whether LA-2 equivalence, as defined \n499 by a competitive ELISA, which correlates with immunity to Lyme disease in animal models and \n500 humans reflects functional activities in vivo or simply a proxy for other activities.  Nonetheless, \n501 our study makes a case for LA-2’s primary mode of action involving direct physical interactions \n502 with the spirochete rather than complement-dependent killing. Elucidating these mechanisms \n503 may have implications for our understanding of the mechanistic correlates of OspA-based \n504 vaccine-induced immunity in humans. \n505\n506 Acknowledgements\n507 We are grateful to Dr. Michael Pauly  and colleagues ZabBio for generating LA-2 LALAPG. We \n508 thank Drs. Renji Song and Jennifer Yates of the Wadsworth Center’s Immunology Core for \n509 assistance with flow cytometry and the Media and Cell Culture core for BSK II medium. We \n510 thank Ms. Elizabeth Cavosie (Wadsworth Center) for administrative assistance. BioRender was \n511 used for some figure generation. This work was supported by the National Institute of Allergy \n512 and Infectious Diseases (NIAID), National Institutes of Health, Department of Health and \n513 Human Services, Contract No. 75N93019C00040 (PI/PD Mantis). This content is solely the \n514 responsibility of the authors and does not necessarily represent the official views of the NIH.\n515\n516 References\n517 1. Lanzavecchia, A., Fruhwirth, A., Perez, L., and Corti, D. (2016). Antibody-guided \n518 vaccine design: identification of protective epitopes. Curr Opin Immunol 41, 62-67. \n519 10.1016/j.coi.2016.06.001.\n520 2. Principato, S., Pizza, M., and Rappuoli, R. (2020). Meningococcal factor H binding \n521 protein as immune evasion factor and vaccine antigen. FEBS Lett 594, 2657-2669. \n522 10.1002/1873-3468.13793.\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint \n\n19\n523 3. Yoo, R., Jore, M.M., and Julien, J.P. (2025). Targeting Bottlenecks in Malaria \n524 Transmission: Antibody-Epitope Descriptions Guide the Design of Next-Generation \n525 Biomedical Interventions. Immunol Rev 330, e70001. 10.1111/imr.70001.\n526 4. Fikrig, E., Barthold, S.W., Kantor, F.S., and Flavell, R.A. (1990). Protection of mice \n527 against the Lyme disease agent by immunizing with recombinant OspA. Science 250, \n528 553-556. 10.1126/science.2237407.\n529 5. Kramer, M.D., Schaible, U.E., Wallich, R., Moter, S.E., Petzoldt, D., and Simon, M.M. \n530 (1990). Characterization of Borrelia burgdorferi associated antigens by monoclonal \n531 antibodies. Immunobiology 181, 357-366. 10.1016/s0171-2985(11)80504-8.\n532 6. Schaible, U.E., Kramer, M.D., Eichmann, K., Modolell, M., Museteanu, C., and Simon, \n533 M.M. (1990). Monoclonal antibodies specific for the outer surface protein A (OspA) of \n534 Borrelia burgdorferi prevent Lyme borreliosis in severe combined immunodeficiency \n535 (scid) mice. Proc Natl Acad Sci U S A 87, 3768-3772. 10.1073/pnas.87.10.3768.\n536 7. Fikrig, E., Barthold, S.W., Marcantonio, N., Deponte, K., Kantor, F.S., and Flavell, R.A. \n537 (1992). Roles of OspA, OspB, and flagellin in protective immunity to Lyme borreliosis in \n538 laboratory mice. Infect Immun 60, 657-661. 10.1128/iai.60.2.657-661.1992.\n539 8. Sears, J.E., Fikrig, E., Nakagawa, T.Y., Deponte, K., Marcantonio, N., Kantor, F.S., and \n540 Flavell, R.A. (1991). Molecular mapping of Osp-A mediated immunity against Borrelia \n541 burgdorferi, the agent of Lyme disease. J Immunol 147, 1995-2000.\n542 9. de Silva, A.M., Telford, S.R., 3rd, Brunet, L.R., Barthold, S.W., and Fikrig, E. (1996). \n543 Borrelia burgdorferi OspA is an arthropod-specific transmission-blocking Lyme disease \n544 vaccine. J Exp Med 183, 271-275.\n545 10. Sădziene, A., Rosa, P.A., Thompson, P.A., Hogan, D.M., and Barbour, A.G. (1992). \n546 Antibody-resistant mutants of Borrelia burgdorferi: in vitro selection and \n547 characterization. J Exp Med 176, 799-809. 10.1084/jem.176.3.799.\n548 11. Sadziene, A., Thompson, P.A., and Barbour, A.G. (1993). In vitro inhibition of Borrelia \n549 burgdorferi growth by antibodies. J Infect Dis 167, 165-172. 10.1093/infdis/167.1.165.\n550 12. Sadziene, A., Jonsson, M., Bergström, S., Bright, R.K., Kennedy, R.C., and Barbour, \n551 A.G. (1994). A bactericidal antibody to Borrelia burgdorferi is directed against a variable \n552 region of the OspB protein. Infect Immun 62, 2037-2045. 10.1128/iai.62.5.2037-\n553 2045.1994.\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint \n\n20\n554 13. Wilske, B., Luft, B., Schubach, W.H., Zumstein, G., Jauris, S., Preac-Mursic, V., and \n555 Kramer, M.D. (1992). Molecular analysis of the outer surface protein A (OspA) of \n556 Borrelia burgdorferi for conserved and variable antibody binding domains. Med \n557 Microbiol Immunol 181, 191-207. 10.1007/bf00215765.\n558 14. Wilske, B., Preac-Mursic, V., Gobel, U.B., Graf, B., Jauris, S., Soutschek, E., Schwab, \n559 E., and Zumstein, G. (1993). An OspA serotyping system for Borrelia burgdorferi based \n560 on reactivity with monoclonal antibodies and OspA sequence analysis. J Clin Microbiol \n561 31, 340-350. 10.1128/jcm.31.2.340-350.1993.\n562 15. LaRocca, T.J., and Benach, J.L. (2008). The important and diverse roles of antibodies in \n563 the host response to Borrelia infections. Curr Top Microbiol Immunol 319, 63-103.\n564 16. Pal, U., de Silva, A.M., Montgomery, R.R., Fish, D., Anguita, J., Anderson, J.F., Lobet, \n565 Y., and Fikrig, E. (2000). Attachment of Borrelia burgdorferi within Ixodes scapularis \n566 mediated by outer surface protein A. J Clin Invest 106, 561-569. 10.1172/JCI9427.\n567 17. Li, H., and Lawson, C.L. (1995). Crystallization and preliminary X-ray analysis of \n568 Borrelia burgdorferi outer surface protein A (OspA) complexed with a murine \n569 monoclonal antibody Fab fragment. J Struct Biol 115, 335-337. 10.1006/jsbi.1995.1058.\n570 18. Ding, W., Huang, X., Yang, X., Dunn, J.J., Luft, B.J., Koide, S., and Lawson, C.L. \n571 (2000). Structural identification of a key protective B-cell epitope in Lyme disease \n572 antigen OspA. J Mol Biol 302, 1153-1164. 10.1006/jmbi.2000.4119.\n573 19. Li, H., Dunn, J.J., Luft, B.J., and Lawson, C.L. (1997). Crystal structure of Lyme disease \n574 antigen outer surface protein A complexed with an Fab. Proc Natl Acad Sci U S A 94, \n575 3584-3589.\n576 20. Schubach, W.H., Mudri, S., Dattwyler, R.J., and Luft, B.J. (1991). Mapping antibody-\n577 binding domains of the major outer surface membrane protein (OspA) of Borrelia \n578 burgdorferi. Infect Immun 59, 1911-1915. 10.1128/iai.59.6.1911-1915.1991.\n579 21. Caimano, M.J., Eggers, C.H., Gonzalez, C.A., and Radolf, J.D. (2005). Alternate sigma \n580 factor RpoS is required for the in vivo-specific repression of Borrelia burgdorferi plasmid \n581 lp54-borne ospA and lp6.6 genes. J Bacteriol 187, 7845-7852. 10.1128/JB.187.22.7845-\n582 7852.2005.\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint \n\n21\n583 22. Schwan, T.G., Piesman, J., Golde, W.T., Dolan, M.C., and Rosa, P.A. (1995). Induction \n584 of an outer surface protein on Borrelia burgdorferi during tick feeding. Proc Natl Acad \n585 Sci U S A 92, 2909-2913. 10.1073/pnas.92.7.2909.\n586 23. Wang, Y., Kern, A., Boatright, N.K., Schiller, Z.A., Sadowski, A., Ejemel, M., Souders, \n587 C.A., Reimann, K.A., Hu, L., Thomas, W.D., Jr., and Klempner, M.S. (2016). Pre-\n588 exposure Prophylaxis With OspA-Specific Human Monoclonal Antibodies Protects Mice \n589 Against Tick Transmission of Lyme Disease Spirochetes. J Infect Dis 214, 205-211. \n590 10.1093/infdis/jiw151.\n591 24. Schiller, Z.A., Rudolph, M.J., Toomey, J.R., Ejemel, M., LaRochelle, A., Davis, S.A., \n592 Lambert, H.S., Kern, A., Tardo, A.C., Souders, C.A., et al. (2021). Blocking Borrelia \n593 burgdorferi transmission from infected ticks to nonhuman primates with a human \n594 monoclonal antibody. J Clin Invest 131. 10.1172/JCI144843.\n595 25. Rudolph, M.J., Davis, S.A., Haque, H.M.E., Ejemel, M., Cavacini, L.A., Vance, D.J., \n596 Willsey, G.G., Piazza, C.L., Weis, D.D., Wang, Y., and Mantis, N.J. (2023). Structure of \n597 a transmission blocking antibody in complex with Outer surface protein A from the Lyme \n598 disease spirochete, Borreliella burgdorferi. Proteins. 10.1002/prot.26549.\n599 26. Golde, W.T., Piesman, J., Dolan, M.C., Kramer, M., Hauser, P., Lobet, Y., Capiau, C., \n600 Desmons, P., Voet, P., Dearwester, D., and Frantz, J.C. (1997). Reactivity with a specific \n601 epitope of outer surface protein A predicts protection from infection with the Lyme \n602 disease spirochete, Borrelia burgdorferi. Infect Immun 65, 882-889.\n603 27. Van Hoecke, C., Comberbach, M., De Grave, D., Desmons, P., Fu, D., Hauser, P., \n604 Lebacq, E., Lobet, Y., and Voet, P. (1996). Evaluation of the safety, reactogenicity and \n605 immunogenicity of three recombinant outer surface protein (OspA) lyme vaccines in \n606 healthy adults. Vaccine 14, 1620-1626. 10.1016/s0264-410x(96)00146-6.\n607 28. Steere, A.C., Sikand, V.K., Meurice, F., Parenti, D.L., Fikrig, E., Schoen, R.T., \n608 Nowakowski, J., Schmid, C.H., Laukamp, S., Buscarino, C., and Krause, D.S. (1998). \n609 Vaccination against Lyme disease with recombinant Borrelia burgdorferi outer-surface \n610 lipoprotein A with adjuvant. Lyme Disease Vaccine Study Group. N Engl J Med 339, \n611 209-215. 10.1056/NEJM199807233390401.\n612 29. Tahir, D., Geolier, V., Bruant, H., Le Flèche-Matéos, A., Mallet, A., Varloud, M., Civat, \n613 C., Girerd-Chambaz, Y., Montano, S., Pion, C., et al. (2025). A Lyme disease mRNA \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint \n\n22\n614 vaccine targeting Borrelia burgdorferi OspA induces strong immune responses and \n615 prevents transmission in mice. Mol Ther Nucleic Acids 36, 102514. \n616 10.1016/j.omtn.2025.102514.\n617 30. Haque, H.M.E., Ejemel, M., Vance, D.J., Willsey, G., Rudolph, M.J., Cavacini, L.A., \n618 Wang, Y., Mantis, N.J., and Weis, D.D. (2022). Human B Cell Epitope Map of the Lyme \n619 Disease Vaccine Antigen, OspA. ACS Infect Dis. 10.1021/acsinfecdis.2c00346.\n620 31. Vance, D.J., Basir, S., Piazza, C.L., Willsey, G.G., Haque, H.M.E., Tremblay, J.M., \n621 Rudolph, M.J., Muriuki, B., Cavacini, L., Weis, D.D., et al. (2024). Single-domain \n622 antibodies reveal unique borrelicidal epitopes on the Lyme disease vaccine antigen, outer \n623 surface protein A (OspA). Infect Immun 92, e0008424. 10.1128/iai.00084-24.\n624 32. Rathinavelu, S., Broadwater, A., and de Silva, A.M. (2003). Does host complement kill \n625 Borrelia burgdorferi within ticks? Infect Immun 71, 822-829. 10.1128/iai.71.2.822-\n626 829.2003.\n627 33. Frye, A.M., Ejemel, M., Cavacini, L., Wang, Y., Rudolph, M.J., Song, R., and Mantis, \n628 N.J. (2022). Agglutination of Borreliella burgdorferi by Transmission-Blocking OspA \n629 Monoclonal Antibodies and Monovalent Fab Fragments. Infect Immun, e0030622. \n630 10.1128/iai.00306-22.\n631 34. Bhattacharyya, A., and Mantis, N.J. (2025). OspA Antibodies Inhibit the In vitro \n632 Transmigration of <em>Borreliellia burgdorferi</em>. bioRxiv, \n633 2025.2011.2020.689610. 10.1101/2025.11.20.689610.\n634 35. Bézay, N., Wagner, L., Kadlecek, V., Obersriebnig, M., Wressnigg, N., Hochreiter, R., \n635 Schneider, M., Dubischar, K., Derhaschnig, U., Klingler, A., et al. (2024). Optimisation \n636 of dose level and vaccination schedule for the VLA15 Lyme borreliosis vaccine \n637 candidate among healthy adults: two randomised, observer-blind, placebo-controlled, \n638 multicentre, phase 2 studies. Lancet Infect Dis 24, 1045-1058. 10.1016/s1473-\n639 3099(24)00175-0.\n640 36. Lundberg, U., Hochreiter, R., Timofoyeva, Y., Kanevsky, I., Meinke, A., Anderson, A.S., \n641 and Simon, R. (2024). Preclinical Evidence for the Protective Capacity of Antibodies \n642 Induced by Lyme Vaccine Candidate VLA15 in People. Open Forum Infect Dis 11, \n643 ofae467. 10.1093/ofid/ofae467.\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint \n\n23\n644 37. Rudolph, M.J., Davis, S.A., Haque, H.M.E., Weis, D.D., Vance, D.J., Piazza, C.L., \n645 Ejemel, M., Cavacini, L., Wang, Y., Mbow, M.L., et al. (2023). Structural Elucidation of \n646 a Protective B Cell Epitope on Outer Surface Protein C (OspC) of the Lyme Disease \n647 Spirochete, Borreliella burgdorferi. mBio 14, e0298122. 10.1128/mbio.02981-22.\n648 38. Rudolph, M.J., Muriuki, B.M., Chen, Y., Vance, D.J., Vorauer, C., Piazza, C.L., \n649 Freeman-Gallant, G., Golonka, R.M., Mirabile, G., Guttman, M., et al. (2025). Germline \n650 encoded residues dominate the interaction of a human monoclonal antibody with decorin \n651 binding protein A of Borrelia burgdorferi. Front Immunol 16, 1611828. \n652 10.3389/fimmu.2025.1611828.\n653 39. Giritch, A., Marillonnet, S., Engler, C., van Eldik, G., Botterman, J., Klimyuk, V., and \n654 Gleba, Y. (2006). Rapid high-yield expression of full-size IgG antibodies in plants \n655 coinfected with noncompeting viral vectors. Proc Natl Acad Sci U S A 103, 14701-\n656 14706. 10.1073/pnas.0606631103.\n657 40. Swope, K., Morton, J., Pogue, G.P., Hume, S., Pauly, M.H., Shepherd, J., Simpson, C.A., \n658 Bratcher, B., Whaley, K.J., Zeitlin, L., et al. (2021). Manufacturing plant-made \n659 monoclonal antibodies for research or therapeutic applications. Methods Enzymol 660, \n660 239-263. 10.1016/bs.mie.2021.05.011.\n661 41. Fischinger, S., Fallon, J.K., Michell, A.R., Broge, T., Suscovich, T.J., Streeck, H., and \n662 Alter, G. (2019). A high-throughput, bead-based, antigen-specific assay to assess the \n663 ability of antibodies to induce complement activation. J Immunol Methods 473, 112630. \n664 10.1016/j.jim.2019.07.002.\n665 42. Kocan, K.M., de la Fuente, J., and Coburn, L.A. (2015). Insights into the development of \n666 Ixodes scapularis: a resource for research on a medically important tick species. Parasit \n667 Vectors 8, 592. 10.1186/s13071-015-1185-7.\n668 43. Schlothauer, T., Herter, S., Koller, C.F., Grau-Richards, S., Steinhart, V., Spick, C., \n669 Kubbies, M., Klein, C., Umaña, P., and Mössner, E. (2016). Novel human IgG1 and IgG4 \n670 Fc-engineered antibodies with completely abolished immune effector functions. Protein \n671 Eng Des Sel 29, 457-466. 10.1093/protein/gzw040.\n672 44. Wilkinson, I., Anderson, S., Fry, J., Julien, L.A., Neville, D., Qureshi, O., Watts, G., and \n673 Hale, G. (2021). Fc-engineered antibodies with immune effector functions completely \n674 abolished. PLoS One 16, e0260954. 10.1371/journal.pone.0260954.\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint \n\n24\n675 45. Bindels, D.S., Haarbosch, L., van Weeren, L., Postma, M., Wiese, K.E., Mastop, M., \n676 Aumonier, S., Gotthard, G., Royant, A., Hink, M.A., and Gadella, T.W., Jr. (2017). \n677 mScarlet: a bright monomeric red fluorescent protein for cellular imaging. Nat Methods \n678 14, 53-56. 10.1038/nmeth.4074.\n679 46. Fikrig, E., Telford, S.R., 3rd, Barthold, S.W., Kantor, F.S., Spielman, A., and Flavell, \n680 R.A. (1992). Elimination of Borrelia burgdorferi from vector ticks feeding on OspA-\n681 immunized mice. Proc Natl Acad Sci U S A 89, 5418-5421.\n682 47. Gipson, C.L., and de Silva, A.M. (2005). Interactions of OspA monoclonal antibody \n683 C3.78 with Borrelia burgdorferi within ticks. Infect Immun 73, 1644-1647. \n684 10.1128/IAI.73.3.1644-1647.2005.\n685 48. Simon, M.M., Schaible, U.E., Kramer, M.D., Eckerskorn, C., Museteanu, C., Müller-\n686 Hermelink, H.K., and Wallich, R. (1991). Recombinant outer surface protein a from \n687 Borrelia burgdorferi induces antibodies protective against spirochetal infection in mice. J \n688 Infect Dis 164, 123-132. 10.1093/infdis/164.1.123.\n689 49. Pine, M., Arora, G., Hart, T.M., Bettini, E., Gaudette, B.T., Muramatsu, H., Tombacz, I., \n690 Kambayashi, T., Tam, Y.K., Brisson, D., et al. (2023). Development of an mRNA-lipid \n691 nanoparticle vaccine against Lyme disease. Mol Ther 31, 2702-2714. \n692 10.1016/j.ymthe.2023.07.022.\n693 50. Kochi, S.K., and Johnson, R.C. (1988). Role of immunoglobulin G in killing of Borrelia \n694 burgdorferi by the classical complement pathway. Infect Immun 56, 314-321.\n695 51. Barthold, S.W., Persing, D.H., Armstrong, A.L., and Peeples, R.A. (1991). Kinetics of \n696 Borrelia burgdorferi dissemination and evolution of disease after intradermal inoculation \n697 of mice. Am J Pathol 139, 263-273.\n698 52. Shih, C.M., Pollack, R.J., Telford, S.R., 3rd, and Spielman, A. (1992). Delayed \n699 dissemination of Lyme disease spirochetes from the site of deposition in the skin of mice. \n700 J Infect Dis 166, 827-831. 10.1093/infdis/166.4.827.\n701 53. Damelang, T., Brinkhaus, M., van Osch, T.L.J., Schuurman, J., Labrijn, A.F., Rispens, \n702 T., and Vidarsson, G. (2023). Impact of structural modifications of IgG antibodies on \n703 effector functions. Front Immunol 14, 1304365. 10.3389/fimmu.2023.1304365.\n704 54. Johnson, N.V., Wall, S.C., Kramer, K.J., Holt, C.M., Periasamy, S., Richardson, S.I., \n705 Manamela, N.P., Suryadevara, N., Andreano, E., Paciello, I., et al. (2024). Discovery and \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint \n\n25\n706 characterization of a pan-betacoronavirus S2-binding antibody. Structure 32, 1893-\n707 1909.e1811. 10.1016/j.str.2024.08.022.\n708 55. Kudryashev, M., Cyrklaff, M., Baumeister, W., Simon, M.M., Wallich, R., and \n709 Frischknecht, F. (2009). Comparative cryo-electron tomography of pathogenic Lyme \n710 disease spirochetes. Mol Microbiol 71, 1415-1434. 10.1111/j.1365-2958.2009.06613.x.\n711 56. Caimano, M.J., Groshong, A.M., Belperron, A., Mao, J., Hawley, K.L., Luthra, A., \n712 Graham, D.E., Earnhart, C.G., Marconi, R.T., Bockenstedt, L.K., et al. (2019). The RpoS \n713 Gatekeeper in Borrelia burgdorferi: An Invariant Regulatory Scheme That Promotes \n714 Spirochete Persistence in Reservoir Hosts and Niche Diversity. Front Microbiol 10, 1923. \n715 10.3389/fmicb.2019.01923.\n716 57. Vanderberg, J.P., and Frevert, U. (2004). Intravital microscopy demonstrating antibody-\n717 mediated immobilisation of Plasmodium berghei sporozoites injected into skin by \n718 mosquitoes. Int J Parasitol 34, 991-996. 10.1016/j.ijpara.2004.05.005.\n719 58. Flores-Garcia, Y., Nasir, G., Hopp, C.S., Munoz, C., Balaban, A.E., Zavala, F., and \n720 Sinnis, P. (2018). Antibody-Mediated Protection against Plasmodium Sporozoites Begins \n721 at the Dermal Inoculation Site. mBio 9. 10.1128/mBio.02194-18.\n722\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}