An Fc-silent OspA monoclonal antibody passively protects mice from tick and intradermal Borrelia burgdorferi challenge

1 1 An Fc-silent OspA monoclonal antibody passively protects mice from tick and 2 intradermal Borrelia burgdorferi challenge 3 4 5 6 Daniel Palmer 1, Atieh Shemshadian2, Katherine Berman1, Graham G. Willsey1, Carol Lyn 7 Piazza 1, Grace Freeman-Gallant1, Michael J Rudolph3, Jeff Bourgeois4, Linden Hu4, David J. 8 Vance1,2, and Nicholas Mantis1,2,* 9 10 11 12 1Wadsworth Center, New York Department of Health, Division of Infectious Disease, Albany 13 NY 12208; 2University of Albany, Department of Biomedical Sciences, Albany NY 12208; 14 3New York Structural Biology Center, New York, NY 10027; 4Deparrment of Microbiology, 15 Tufts University, Boston, MA 02111 16 17 18 19 *To whom correspondence should be addressed; [email protected] 20 21 22 Running title: Passive protection afforded by LA-2 LALAPG 23 24 Keywords: spirochete; antibody; complement; Fc receptor; skin; vector 25 26 27 28 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 The copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint 2 29 30 ABSTRACT 31 The monoclonal antibody, LA-2, has played a pivotal role in the development of Outer surface 32 protein A (OspA)-based vaccines for Lyme disease, a multisystem illness caused by the tick- 33 borne spirochete, Borrelia burgdorferi sensu lato. Of particular significance was the 34 demonstration more than three decades ago that LA-2 equivalent antibody titers, defined by a 35 competitive-inhibition ELISA, serve as a reliable correlate of vaccine-induced protection across 36 different species, including humans. In vitro characterization of LA-2 has identified both 37 complement-dependent and -independent activities, although which of these attributes contribute 38 to protection against B. burgdorferi remains unresolved. To address this issue, we generated and 39 characterized an “Fc-silent” version of LA-2 IgG1 carrying so-called LALAPG substitutions 40 (L234A, L235A, P329G). We demonstrate that LA-2 LALAPG retained OspA binding activity 41 but was severely attenuated in in vitro complement deposition and complement-dependent 42 borreliacidal assays. Nonetheless, LA-2 LALAPG was as effective as LA-2 at passively 43 protecting C3H mice against nymphal tick-mediated B. burgdorferi challenge. LA-2 LALAPG 44 was also equivalent to LA-2 in passively protecting BALB/c mice against intradermal B. 45 burgdorferi challenge. In the intradermal challenge model, viable spirochetes were not 46 recoverable 24 h after injection from skin biopsies of mice treated with LA-2 or LA-2 LALAPG, 47 and an influx of pro-inflammatory cytokines and chemokines to the injection site was abrogated. 48 Collectively, these results suggest that LA-2’s primary mode of action involves direct physical 49 interactions with the spirochete rather than complement-dependent killing. Elucidating these 50 mechanisms may have implications for understanding the mechanistic correlates of OspA-based 51 vaccine-induced immunity in humans. 52 53 54 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint 3 55 INTRODUCTION 56 Over the past five decades, monoclonal antibodies (mAbs) have emerged as extraordinary 57 tools in the identification of protective antigens and epitopes associated with pathogens of 58 interest, leading to novel vaccines for viruses, bacteria and parasites 1-3. In the case of the Lyme 59 disease spirochete, Borrelia burgdorferi sensu lato, multiple groups working in the 1990s 60 generated large collections of mAbs that led to the identification Outer surface protein A (OspA) 61 as a candidate Lyme disease vaccine antigen 4-15. OspA is a lipoprotein expressed at high levels 62 by B. burgdorferi within the midgut of its arthropod vector, the black legged tick (Ixodes 63 scapularis), where it is proposed to function as an adhesin 16. Structurally, OspA consists of 21 64 anti-parallel β -strands with a single C-terminal α-helix 17,18. The N-terminus is anchored in the 65 spirochete outer membrane via a lipid moiety, while the C-terminus projects away (~80 Å) from 66 the bacterial surface and is accessible to antibody attack 19,20. OspA is downregulated during or 67 just after spirochete transmission to a mammalian host 21,22. As such, antibodies elicited by 68 OspA-based vaccines are proposed to inhibit one or more steps in B. burgdorferi tick-mediated 69 transmission, although the specific mechanisms by which this occurs remains to be fully 70 elucidated. 71 Among the many OspA mAbs characterized to date, LA-2 has played a particularly 72 significant role in our understanding of OspA-mediated immunity. LA-2 was one of the first 73 OspA-specific mAbs shown to passively protect mice from B. burgdorferi needle infection 6 and 74 tick-mediated challenge 23. And, until just a few years ago, LA-2 was the only protective 75 antibody whose epitope on OspA had been resolved at the structural level 18,24,25. In the context 76 of Lyme disease vaccine development, LA-2 serological antibody “equivalence,” as defined by a 77 competitive ELISA, proved to correlate with protection against tick-mediated B. burgdorferi 78 infection in OspA-vaccinated mice and dogs 26. Remarkably, as part of a large randomized OspA 79 vaccine trial, it was determined in a subset of individuals that LA-2 equivalent titers are also 80 important biomarkers of Lyme disease susceptibility in humans, as individuals with confirmed 81 Lyme disease had lower LA-2 equivalence than those who did not 27,28. LA-2 continues to be 82 used as a benchmark in the development of next generation OspA vaccines 29 (M. Finn, personal 83 communication). 84 Despite LA-2’s central role in Lyme disease vaccine development, the exact mechanism 85 by which LA-2 protects against B. burgdorferi infection remains incompletely defined. In fact, .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint 4 86 the basic question of whether complement is needed for LA-2’s protective activity has not been 87 addressed. While LA-2 has potent complement-dependent borreliacidal activity in vitro 23,30,31, 88 evidence indicates that complement (human or mouse) is not active in the tick midgut 32. Several 89 complement-independent activities have been ascribed to LA-2, including effects on spirochete 90 transmigration, that would be expected to impede B. burgdorferi from the tick midgut 33,34. 91 Defining the contribution of complement in LA-2’s mechanism of action is important for 92 understanding correlates of OspA-mediated immunity, especially as clinical trials of next 93 generation OspA vaccines are ongoing 29,35,36. In this report, we generate an “Fc-silent” version 94 of LA-2 IgG1 that is effectively devoid of in vitro complement-dependent borreliacidal activity 95 and characterize its activity in mouse models of tick-mediated and intradermal B. burgdorferi 96 challenge. 97 98 Materials and Methods 99 Ethics statement. The mouse experiments described in this study were reviewed and approved 100 by the Institutional Animal Care and Use Committees (IACUC) at the Wadsworth Center 101 (protocol 23-459) and Tufts University-Tufts Medical Center (protocol B2024-50). The 102 Wadsworth Center and Tufts University-Tufts Medical Center both comply with the Public 103 Health Service Policy on Humane Care and Use of Laboratory Animals and were issued 104 assurance numbers A3183-01 and A4059-01, respectively. Both facilities are fully accredited by 105 the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). 106 Obtaining this voluntary accreditation status reflects that these facilities’ Animal Care and Use 107 Program meets all standards required by law and goes beyond the standards as it strives to 108 achieve excellence in animal care and use. All animals were euthanized by carbon dioxide 109 asphyxiation followed by cervical dislocation, as recommended by the Office of Laboratory 110 Animal Welfare (OLAW), National Institutes of Health. 111 112 Recombinant B. burgdorferi B31 proteins. Recombinant OspA, DbpA and OspC type A 113 derived from B. burgdorferi strain B31 (Table 1) were expressed in E. coli as cited in Table 1. 114 Table 1. Recombinant B. burgdorferi B31 proteins used in this study Antigen AA UniProt ID References .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint 5 OspA 18-273 P0CL66 24 OspB 50–296 P17739 unpublished OspCA 38-201 Q07337 37 DbpA 26-188 O50917 38 DbpB 21–187 O50917 unpublished Abbreviations; AA, amino acid; UniProt (https://www.uniprot.org/) 115 116 LA-2 and LA-2 LALAPG IgG1 expression and purification. Codon optimized VH and VL 117 DNA sequences of LA-2 (Antibody Registry RRID: AB_2619693) derived from PDB 1FJ1 18 118 were custom synthesized by Life Technologies (San Diego, CA) and cloned into TMV and PVX 119 plant expression vectors containing codon-optimized human kappa and human IgG1 constant 120 regions 39. The resulting plasmids were transformed into Agrobacterium tumefaciens. Four- 121 week-old N. benthamiana plants were infiltrated with A. tumefaciens carrying plasmids for the 122 expression of heavy and light chains of LA-2 LALAPG. Aerial plant parts were harvested after 7 123 days and extracted and clarified. The LA-2 LALAPG antibody was then purified with Protein A 124 affinity and anion exchange chromatography 40. 125 126 Antibody affinity determinations by Biolayer interferometry (BLI). Affinity determinations 127 were conducted using an Octet RED96e Biolayer Interferometer (Sartorius, Goettingen, 128 Germany) with Data Acquisition 12.0 software. Biotinylated OspA (5 μg/mL) in PBS containing 129 2% w/v BSA (“buffer”) was captured onto Octet SA (streptavidin) biosensors (Sartorius) for 5 130 min. After equilibration, sensors were immersed in two-fold serial dilutions of mAb starting at 131 100 nM for 10 min. The sensors were then dipped into buffer for 30 min to allow for 132 dissociation. The raw sensor data were loaded into the Data Analysis HT 12.0 software, grouped 133 and fit using a 1:2 bivalent analyte model. 134 135 Flow cytometric analysis of B. burgdorferi surface labeling. B. burgdorferi strain B31 surface 136 labeling with LA-2 and LA-2 LALAPG was performed essentially as described 31. LA-2 and 137 LA-2 LALAPG were 2-fold serially diluted in PBS before incubation with viable B. burgdorferi 138 B31. The ricin-specific mAb, PB10, was used as an IgG1 isotype control (10 µg/mL). Alexa 139 Fluor 647-labeled goat anti-human IgG (H+L) (Invitrogen, Carlsbad, CA) was used as a 140 secondary antibody. Samples were analyzed using a BD FACSCalibur (BD Biosciences, 141 Franklin Lakes, NJ). Bacteria were gated on FSC and SSC to exclude debris, and 20,000 events .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint 6 142 were counted per condition. Agglutination was calculated as the percent of events in the UL + 143 UR + LR quadrants. Data were analyzed using FlowJo v10.10.0 (BD Biosciences). 144 145 Complement-dependent borreliacidal assays. Complement-dependent borreliacidal assays 146 were performed using a recombinant B. burgdorferi B31-5A4 strain carrying an IPTG-inducible 147 mscarlet-I reporter (GGW979), as described 31. Briefly, GGW979 cultures were grown to mid- 148 log phase in BSKII medium supplemented with gentamicin (50 µg/ml) at 32 °C under static 149 conditions. Spirochetes were harvested by low-speed centrifugation and resuspended in phenol 150 red–free BSKII containing gentamicin (50 µg/ml) to a final density of 3×10⁷ spirochetes/ml. 151 Cell suspensions were then mixed 1:1 with phenol red–free BSKII supplemented with 152 20% guinea pig complement (Sigma Aldrich, St. Louis, MO) and 20 nM of one of the following 153 mAbs: LA-2, LA-2 LALAPG, 857-2, PB10. PB10, a ricin toxin-specific antibody, was used as 154 an IgG1 isotype control. Reactions were set up in white 96-well assay plates (Co-Star). 155 Following sample addition, reactions contained 1.5×10⁶ spirochetes, 5 nM of antibody and 10% 156 guinea pig complement. Assay plates were then incubated overnight in a water jacketed 157 incubator at 37°C with 5% CO 2. The following day, 1 mM IPTG was added to each well to 158 induce mScarlet-I expression. Following a 48-h incubation at 37°C with 5% CO 2, the MFI was 159 recorded at 569 nm (excitation)/611 nm (emission) using a Spectramax ID3 plate reader 160 (Molecular Biosystems, San Jose, CA). Raw MFI data was then normalized as described 31. The 161 data presented is the mean and SD of three independent experiments. 162 163 MIA. B. burgdorferi B31 antigens OspA, OspB, OspC, DbpA, and DbpB (Table 1) were 164 coupled to Magplex-C microspheres (5 g antigen/ 1x10 6 microspheres) using a xMap Antibody 165 Coupling Kit as recommended by the manufacturer (Luminex Corporation, Austin, TX). Beads 166 were protected from light and stored at 2-8°C in xMAP AbC Wash Buffer (5x10 6 167 microspheres/mL) until use. Serum samples (1:100) and coupled microsphere stocks (1:50) were 168 diluted in assay buffer (1 x PBS, 2% BSA, pH 7.4). The diluted sera (50 μL) and diluted 169 microspheres (50 μL) were combined in black, clear-bottomed, non-binding, chimney 96-well 170 plates (Greiner Bio-One, Monroe, North Carolina) and incubated at room temperature for 1 hr in 171 a tabletop shaker (600 rpm). Plates were placed on a magnetic separator and washed three times 172 using wash buffer (1 x PBS, 2% BSA, 0.02% TWEEN-20, 0.05% Sodium azide, pH 7.4). To .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint 7 173 detect seroconversion in the mice, goat anti-mouse IgG, Human-ads-PE (SouthernBiotech, 174 Birmingham, Alabama) secondary antibody was diluted 1:500 in assay buffer, added (100 μL ) to 175 each well, and allowed to incubate at room temperature for 30 min in a tabletop shaker (600 176 rpm). Alternatively, to detect remaining LA-2 or LA-2 LALAPG, PE labeled goat anti-Human 177 IgG Fc, eBioscience (Invitrogen) secondary antibody was used. Plates were washed as 178 previously stated. The microspheres were resuspended in 100 μL of wash buffer and placed back 179 on the tabletop shaker (600 rpm) for 5 minutes prior to analysis using a FlexMap 3D (Luminex 180 Corporation). To establish reactivity cutoffs for each antigen, the average median fluorescent 181 intensity (MFI) of buffer-only wells was multiplied by six. MFIs for each mouse serum sample 182 were divided by the antigen-specific reactivity cutoffs yielding an index value. An index value 183 greater than 1 suggests reactivity above background for the given antigen. 184 185 Antibody-dependent complement deposition (ADCD) assay. We modified a flow cytometry- 186 based HIV-1 antibody-dependent complement deposition (ADCD) for use with a Luminex 187 instrument and OspA-coupled beads 41. Magplex-C microspheres coupled with recombinant B. 188 burgdorferi antigens, OspA and OspCA, were diluted (1:50) and mixed 1: 1 (v/v) with primary 189 antibodies, LA-2 and LA-2 LALAPG (10 g/mL), then seeded into a 96-well plates, covered in 190 foil, and incubated for 1 h at room temperature (RT) with shaking. Plates were washed twice 191 using a plate magnet and 190  L of wash buffer (PBS, 2% BSA, 0.02% Tween-20, 0.05% 192 sodium azide, pH 7.4). Following the washes, 200 L of diluted human complement (1:50; Pel- 193 Freez Biologicals, Rogers, AR) were added to each well and incubated for 20 min at RT with 194 shaking. Plates were washed again then phycoerythrin-tagged mouse anti-C3/C3b/iC3b (1:100; 195 BD) and phycoerythrin-tagged goat anti-human IgG Fc (1:500; Invitrogen) were added to their 196 respected wells and incubated for 30 min. The plates were washed a final time before antibody- 197 bead complexes were resuspended in 100 uL of wash buffer and incubated for 1 min while 198 shaking. The plates were analyzed via a FlexMAP 3D instrument (Luminex Corporation) with 199 results presented in median fluorescence intensity (MFI). 200 201 Mouse model of B. burgdorferi challenge by Ixodes scapularis nymphs. Animal studies were 202 conducted with approval by the Institutional Animal Care and Use Committees (IACUC) at the 203 Wadsworth Center and Tufts University-Tufts Medical Center. To generate B. burgdorferi B31 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint 8 204 infected Ixodes scapularis nymphs, C57BL/6J mice were injected subcutaneously with 10^5 205 cells/mL log growth phase of B. burgdorferi B31. Two to four weeks later, the mice were 206 infested with naive Ixodes scapularis larvae, which were allowed to feed to repletion. Replete 207 larvae were harvested and allowed molt into mature infected nymphs. As controls, we used naive 208 Ixodes scapularis nymphs procured from the Oklahoma State University tick rearing facility 42. 209 For challenge studies, equal numbers of male and female C3H/HeN mice aged ~6 weeks 210 (Charles River Laboratories, Kingston, NY) were acclimated in the Wadsworth Center’s 211 vivarium for 1-2 weeks before the start of the experiment. On study day -1, mice were 212 subcutaneously (SC) administered either LA-2, LA-2 LALAPG or an IgG1 isotype control (anti- 213 Vibrio cholerae mAb ZAC-3) (120 g or 30 g per mouse) diluted in 200 µL of PBS. The 214 following day, infected or naive nymphal ticks (5 per mouse) were placed on a shaved area of the 215 mouse’s dorsum. Nymphs were collected from all mice 3-5 days post placement. Mice which 216 had at least one tick that appeared to be at or near repletion at the time of collection were 217 presumed to be successfully challenged. On study day 21, mice were euthanized, and blood was 218 collected via cardiac puncture for serological analysis. Bladders were also collected for 219 cultivation of spirochetes in 2 mL BSKII cultures treated with rifampicin (50 μg/mL), 220 fosfomycin (20 μg/mL), and amphotericin B (2.5 μg/mL). Infection status was based on 221 seroconversion using the MIA described above, as well as the presence or absence of live 222 spirochetes in bladder cultures using dark-field microscopy, which were assessed weekly for one 223 month. 224 225 Collection of engorged ticks, dissection, and determination of genome equivalents. One 226 replete or near-replete nymph that had fed on each mouse was dissected to harvest the midgut 227 tissues. Midgut tissues were extracted using the E.Z.N.A.® Mollusc & Insect DNA Kit (Omega 228 Bio-tek, Inc., Norcross, GA) and real-time qPCR was performed to determine the Borrelia 229 burden in midgut tissues. The single-copy B. burgdorferi flaB (flagellin) gene was amplified, and 230 flaB copy number was standardized to total gDNA in each sample as measured using the Qubit 4 231 Fluorometer (Invitrogen) to determine normalized spirochete burdens in tick midguts. 232 233 Mouse model of intradermal B. burgdorferi challenge. Female BALB/c mice aged ~8 weeks 234 (Taconic Biosciences, Germantown, NY) were acclimated in the Wadsworth Center’s vivarium .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint 9 235 for one week before the start of the experiment. On study day -1, mice were injected 236 intraperitoneally (IP) with LA-2 or LA-2 LALAPG (0.1 - 120 g/mouse) in 200 µL PBS. The 237 following day (study day 0), mice were challenged with mid-log phase B. burgdorferi strain 238 B31-5A4 (1x10 5 cells) by intradermal (ID) injection. On day 21, the mice were euthanized, and 239 blood was collected via cardiac puncture for serological analysis. Infection status was 240 determined based on seroconversion using the MIA described above. 241 242 To assess the effects of LA-2 and LA-2 LALAPG on B. burgdorferi skin dissemination, a mix of 243 male and female BALB/c mice aged ~6 weeks were injected SC with 30 µg of LA-2 or LA-2 244 LALAPG in 200 µL PBS, or remained untreated. The following day (study day 0), mice were 245 challenged with mid-log phase B. burgdorferi strain B31-5A4 (1x10 5 cells) by ID injection. 246 Groups of mice were euthanized on days 1, 3 and 7, and ~5 mm skin biopsies were excised from 247 the injection site (IS), ~1 cm away from the IS, and ~3 cm away from the IS. Biopsies were 248 rinsed in PBS immediately after collection and placed in 2 mL BSKII medium supplemented 249 with rifampicin (50 μg/mL), fosfomycin (20 μg/mL), and amphotericin B (2.5 μg/mL) for 250 cultivation of spirochetes. The biopsy cultures were assessed weekly by dark-field microscopy 251 over the course of four weeks for the presence of viable spirochetes. 252 253 Inflammatory cytokine and chemokine analysis in mouse skin biopsies. Groups of male and 254 female BALB/c mice were SC administered 30 ug LA-2 per mouse on day -1 or left untreated. 255 The following day (study day 0), mice were challenged with mid-log phase B. burgdorferi strain 256 B31-5A4 (1x10 5 cells) by ID injection. 5 days-post infection, mice were euthanized, and a skin 257 biopsy ~1 cm in diameter was collected from the injection site of each mouse in a cytokine 258 extraction buffer containing 0.4M NaCl, 0.05% Tween 20, 0.5% Bovine Serum Albumin, 0.1 259 mM phenylmethylsulphonyl fluoride, and 20 Ki of aprotinin in 1X PBS. The solution containing 260 the biopsy was homogenized at 5 m/s in a bead beater in 1-minute increments, with a 1-minute 261 cool down between shakes. This was repeated five times, or until the biopsy was fully 262 homogenized. This solution was centrifuged at 13,000 g for 10 minutes at 4°C, and the 263 supernatant was collected and then prepared for cytometric bead array analysis. Skin 264 homogenates were diluted 1:2 in assay diluent and processed using the BD Biosciences .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint 10 265 Cytometric Bead Array (CBA) Mouse Inflammation Kit following the manufacturer’s 266 instructions. Samples were analyzed using a BD FACSCalibur (BD Biosciences). 267 268 Statistical analysis. Statistical procedures for experiments are described in the figure legends. 269 All statistical analysis of data was performed in R, Graphpad Prism 9.0, and Microsoft Excel. 270 In all experiments, p-values <0.05 are considered significant. 271 272 Results 273 Recognition of recombinant and native OspA by LA-2 and LA-2 LALAPG. To generate an 274 Fc-silent version of LA-2, codon optimized DNA sequences encoding the variable heavy chain 275 (V H) was cloned in-frame into human IgG1 Fc and IgG1 LALAPG expression vectors. The IgG1 276 LALAPG derivative carries three-point mutations (L234A, L235A, P329G) relative to IgG1 that 277 abolishes complement fixation activity and FcR recognition 43,44. The LA-2 VL coding sequence 278 was inserted into a human kappa expression vector. The V H and VL plasmids were transformed 279 into A. tumefaciens that was then used to infiltrate N. benthamiana . Aerial plant parts were 280 harvested after 7 days and extracted and clarified antibodies were purified to homogeneity by 281 Protein A affinity and anion exchange chromatography 40. By flow cytometry, LA-2 IgG1 and 282 LA-2 LALAPG were equivalent in their ability to recognize native OspA on the surface of viable 283 B. burgdorferi strain B31, as well as induce agglutination of those cells (Figure 1). Surface 284 labeling was dose-dependent and resulted in a maximum of ~90% total cell labeling with a 285 median fluorescence intensity (MFIs) exceeding 7500 for LA-2 LALAPG. LA-2 and LA-2 286 LALAPG also recognized recombinant OspA with similar apparent affinities as measured by 287 BLI (Figure S1 ). 288 289 Figure 1. Reactivity of LA-2 and LA-2 LALAPG with native OspA. Representative flow 290 cytometry assay of serially diluted LA-2 (dark blue) and LA-2 LALAPG (light blue overlay) 291 reactivity with native OspA on the surface of B. burgdorferi B31. (Left) Fluorescence histogram 292 overlays comparing binding properties. Percent and gMFIs of Alexa-647 fluorescently labeled 293 events (under bracket) are indicated. (Right) Forward scatter (FSC) – side scatter (SSC) dot plot 294 overlays comparing agglutination properties. Events increased in FSC and/or SSC (UL, UR, LR 295 quadrants) demonstrate agglutination, and percent of events agglutinated is indicated. .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint 11 296 297 LA-2 LALAPG IgG1 lacks complement fixation and borreliacidal activities. Introduction of 298 the LALAPG (L234A, L235A, P329G) mutations into the Fc region of IgG1 is reported to 299 essentially eliminate complement fixation activity 43,44. To examine this in the case of LA-2, we 300 adopted an antibody-dependent complement deposition (ADCD) assay to OspA (Figure 2A) 41. 301 Recombinant OspA was covalently coupled to fluorescent microspheres, then probed with LA-2 302 IgG1 and LA-2 LALAPG in the presence of human complement. Total mAb binding to the 303 beads was determined using PE-labeled anti-human IgG, while complement deposition was 304 measured using a PE-labeled anti-C3 antibody. The results confirmed that LA-2 and LA-2 305 LALAPG have equivalent capacities to bind OspA (Figure 2B). However, the two mAbs were 306 starkly different in terms of complement fixation activity. LA-2 demonstrated a dose-dependent 307 increase in C3 deposition that peaked at ~2  g/ml. LA-2 LALAPG, in contrast, was devoid of 308 any activity even at 10 g/ml (Figure 2C). These results confirmed that LA-2 LALAPG is 309 unable to fix complement via the classical pathway. 310 311 Figure 2. LA-2 LALAPG is deficient in complement deposition in vitro. (A) Binding (MFI) 312 of LA-2 IgG and LA-2 LALAPG to recombinant OspA. The asterisks indicate a significant 313 difference between groups by Welch’s t-test (**P<0.01). Quantification and comparison of 314 human complement C3 deposition between LA-2 and LA-2 LALAPG in the context of OspA. 315 The asterisk indicates a significant difference between groups by Welch’s t-test, where *P<0.05. 316 (C) Dose response of complement C3 deposition of LA-2 and LA-2 LALAPG in the context of 317 OspA. 318 319 To assess the capacity of LA-2 and LA-2 LALAPG to promote complement-dependent 320 borreliacidal activity, we employed a recently developed fluorescence-based B. burgdorferi 321 reporter strain GGW979 31. GGW979 is a derivative of B. burgdorferi B31 that expresses the red 322 fluorescent protein, mScarlet, under control of an IPTG-inducible promoter 45. In the assay, 323 neither LA-2 nor LA-2 LALAPG had any measurable demonstrable borreliacidal activity in the 324 absence of 5 nM complement (Figure 3). In the presence of exogenous complement, LA-2 IgG 325 elicited dose-dependent borreliacidal activity at concentrations ranging from 20 to <1 g/ml 326 (data not shown). LA-2 LALAPG, on the other hand, had no measurable complement- .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint 12 327 dependent borreliacidal activity, even at 20  g/ml. Collectively, these results demonstrate that 328 the LA-2 LALAPG retains OspA binding activity but lacks complement-fixing activity. 329 330 Figure 3. Complement-dependent borreliacidal activity associated with LA-2 and LA-2 331 LALAPG. Mid-log phase B. burgdorferi strain B31-5A4 carrying an IPTG-inducible mscarlet-I 332 reporter (GGW979) were suspended (1.5×10⁶ cells per reaction) in BSKII medium supplemented 333 with 20% guinea pig complement and 5 nM of the mAbs indicated on the x-axis (857-2, LA-2, 334 LA-2 LALAPG, PB10), as detailed in the Materials and Methods. Following a 48-h incubation, 335 the median fluorescence intensity (MFI; 569 nm excitation/611 nm emission) was determined. 336 The bars are the mean of three independent experiments with each symbol being an independent 337 experiment and the error bars indicating SD. The dashed red line represented 50% killing. The 338 results demonstrate that 857-2 and LA-2 have potent borreliacidal activity as indicated by low 339 normalized MFI, whereas LA-2 LALAPG and the isotype control were devoid of activity. 340 341 LA-2 LALAPG protects mice from tick-mediated B. burgdorferi infection. Having 342 established that LA-2 LALAPG is deficient in complement fixation, we next examined the 343 mAb’s ability to protect mice from infection in a tick-mediated B. burgdorferi challenge. Groups 344 of C3H/HeN mice were administered 120 or 30 g of LA-2 or LA-2 LALAPG by subcutaneous 345 injection and challenged the following day with B. burgdorferi B31-infected Ixodes scapularis 346 nymphs. An additional group of mice received an IgG1 isotype control (ZAC-3). On day 21, the 347 mice were euthanized and assessed for B. burgdorferi infection by serology using a 348 B.burgdorferi specific MIA and recovery of viable spirochetes from bladders. For statistical 349 purposes, a mouse was scored as categorically infected if either readout (seroconversion, culture) 350 was positive. By these metrics, LA-2 and LA-2 LALAPG were each protective at the 120 g 351 dose (p<0.01), but only marginally effective at 30  g dose, relative to mice that received the 352 isotype control (Table 2). Of particular importance, LA-2 and LA-2 LALAPG were statistically 353 indistinguishable in terms of their protective efficacy (p>0.99). These results demonstrate that 354 LA-2 can protect mice from tick-mediated B. burgdorferi infection in the absence of complement 355 fixation. 356 B. burgdorferi numbers in the midgut of ticks that feed on OspA immunized mice were 357 reported to decline or be eliminated entirely within days after engorgement suggesting that OspA .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint 13 358 antibodies exhibit borreliacidal activities within the tick gut 9,32,46. On the other hand, the “LA-2- 359 like” mAb, C3.78, passively protected mice from tick-mediated B. burgdorferi infection without 360 affecting spirochete numbers in the midgut, consistent with a mechanism of action not dependent 361 on borreliacidal activity 47. To address this issue in the case of LA-2, we collected engorged ticks 362 that had fed on ZAC-3-, LA-2- or LA-2 LALAPG-treated mice, dissected the midguts, then 363 quantified spirochete burdens using qPCR. There was no significant reduction in spirochete 364 burdens in the tick midgut in ticks that fed on LA-2 or LA-2 LALAPG-treated mice (Figure S2), 365 as compared to the isotype control-treated group. Thus, LA-2 or LA-2 LALAPG do not appear to 366 exhibit borreliacidal activity in the context of the tick midgut environment. 367 Table 2. mAb passive protection in mouse model of tick-mediated B. burgdorferi challenge readout (# pos./# total) significance (p) b mAb Dose (µg) Tick serologya culture a vs. IC vs. LA-2 LA-2 120 + 0/6 0/6 0.99 LA-2 30 + 2/5 1/5 0.18 - LALAPG 30 + 3/6 2/6 0.18 >0.99 IC 30 + 5/5 5/5 - - IC 30 - 0/2 0/2 - - a, number of positive mice/total mice per group. b, significance (Fisher’s exact test with Benjamani-Hochberg procedure for the FDR) was determined using readout with highest infection status. p-values <0.05 are considered significant. IC = isotype control. 368 369 LA-2 and LA-2 LALAPG limit skin dissemination of B. burgdorferi. While it is known that 370 LA-2 and other OspA antibodies protect mice from B. burgdorferi dissemination even when 371 spirochetes are delivered by injection, there are no reports examining whether this is dependent 372 on complement 6,48,49. Considering the sensitivity of B. burgdorferi to the classical complement 373 pathway 50, we reasoned that LA-2 would prevent disseminated infection following intradermal 374 challenge, while LA-2 LALAPG would not. To test this, groups of mice were administered LA-2 375 or LA-2 LALAPG at 120 g per mouse then challenged the following day with viable B. 376 burgdorferi B31 (105 cells) by intradermal injection. Three weeks later, mice were euthanized 377 and assessed for seroconversion using the B. burgdorferi-specific MIA, described above. By this 378 measure, all six mice in the LA-2 treated group and five of the six mice in the LA-2 LALAPG .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint 14 379 group were protected (Table 3). This demonstrates that neither Fc effector functions nor 380 complement fixing activity are required for LA-2’s protective activity following intradermal 381 challenge. 382 Table 3. mAb passive protection in mouse model of B. burgdorferi ID challenge Dose (µg/mouse) LA-2 (p =)a LA-2 LALAPG (p =) a LA-2 vs. LA-2 LALAPGb 120 0/6 (0.99 1 1/6 (0.046) 2/6 (0.09) >0.99 0.3 3/6 (0.54) 5/6 (>0.99) 0.82 0.1 3/6 (0.27) 6/6 (>0.99) 0.27 0 6/6 -- - a, number of positive mice/total mice per group with significance compared to the infected control (0 µg/mouse dose) determined by Fisher’s exact test with Benjamani-Hochberg procedure for the FDR indicated in parentheses; b significance (p values), determined by Fisher’s exact test with Benjamani-Hochberg procedure for the FDR, comparing numbers of infected mice between LA-2 and LA-2 LALALPG at each dose indicated. 383 384 To investigate how LA-2 and LA-2 LALAPG perform at limiting doses, we carried out a 385 pilot study to establish the minimum amount of LA-2 required to protect BALB/c mice against 386 B. burgdorferi B31 intradermal challenge. Those studies indicated that as little as 1 g of LA-2 387 IgG per mouse was sufficient to render B. burgdorferi B31 non-infectious (Table S1). We 388 therefore compared LA-2 and LA-2 LALAPG side by side at doses of 1, 0.3 and 0.1 g mAb per 389 animal in intradermal B. burgdorferi B31 challenge. At the 1  g dose, one of the six mice in the 390 LA-2 treatment group was infected at day 21, while two of the six mice in the LA-2 LALAPG 391 group were infected (Table 3). Although neither mAb conferred significant protection at the two 392 lower doses tested (0.3 and 0.1  g per mouse), LA-2 treated mice fared slightly better than the 393 LA-2 LALAPG treated mice in both cases. We conclude that LA-2 IgG protection in the 394 intradermal challenge model is independent of Fc-mediated activities at high antibody 395 concentrations but possibly important when antibody is limiting. 396 397 LA-2 and LA-2 LALAPG clear viable spirochetes from the B. burgdorferi skin. The fact that 398 both LA-2 and LA-2 LALAPG treatments inhibited B. burgdorferi dissemination in the mouse 399 model of intradermal challenge prompted us to examine spirochete burden in tissues at earlier 400 time points. To do this, skin biopsies were collected on days 1, 3 and 7 at three locations: the 401 injection site (IS) on the ventral side of the animal, ~1 cm from the IS, and ~3 cm from the IS on .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint 15 402 the dorsal side of the animal (Figure 4). Skin biopsies were cultured in BSKII medium for a 403 month and scored regularly for appearance of viable spirochetes. In the control mice, viable 404 spirochetes were recovered on days 1, 3 and 7, which coincides with the reported kinetics of B. 405 burgdorferi dissemination 51,52. Moreover, three of the four mice also had positive knee and 406 spleen cultures (data not shown). In contrast, skin biopsies from LA-2-treated mice were culture 407 negative at all time points examined (Figure 4). The results were similar for LA-2 LALAPG 408 treatment, with just one of three animals showing positive cultures on day 7 (Figure 4). These 409 results indicate that, in the presence of LA-2 and LA-2 LALAPG, viable B. burgdorferi 410 spirochetes are cleared rapidly at or very near the injection site, thereby arresting dissemination 411 before it even gets started. 412 413 Figure 4. LA-2 and LA-2 LALAPG prevent spirochete dissemination through skin. Groups 414 of mice were administered LA-2, LA-2 LALAPG or an isotype control by subcutaneous 415 injection, then intradermally challenged one day later with B. burgdorferi. On days 1, 3, and 7 416 post challenge, mice were euthanized, and skin biopsies were harvested at the injection site (IS), 417 ~ 1 cm from the IS (S1), and ~3 cm from the IS (S2). Biopsies were immersed in BSKII media to 418 recover viable spirochetes. The pie charts indicate the number of skin samples assayed per 419 treatment, with one sample collected from each skin site per mouse. Red subdivisions indicate 420 positivity for motile spirochetes, while white subdivisions indicate no viable spirochetes 421 detected. Shown are the combined results from two independent experiments. 422 423 The absence of viable spirochetes in the skin biopsies led us to hypothesize that local 424 inflammation may contribute to LA-2-mediated spirochete clearance. To test this, we examined 425 skin biopsy homogenates for the presence of mouse inflammatory chemokines and chemokines 426 TNF-α, IFN-γ, MCP-1, IL-6, IL-10, and IL-12p70. At five days following injection, we observed 427 elevated levels of TNF-α, IFN-γ, IL-6 and especially MCP-1 in untreated mice when compared 428 to uninfected mice (Figure 5). However, in infected mice that were pretreated with LA-2, 429 analytes showed cytokine concentrations similar to levels of uninfected mice (Figure 5). Thus, 430 LA-2 treatment is not associated with residual inflammation in the skin and may clear the 431 spirochetes before a cytokine response can be generated. Further analysis of mouse skin .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint 16 432 cytokines and chemokines, as well as immune cell infiltrates, collected at earlier infection time 433 points is ongoing. 434 435 Figure 5. BALB/c mice intradermal infected with B. burgdorferi and treated with LA-2, 5 436 days post-injection. On day –1, mice were given a subcutaneous injection of 30 µg/mL of LA-2 437 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 438 were shaved on day 0 on their right and left flanks, then given an intradermal injection, on each 439 flank, of 10^5 live B. burgdorferi cells in 50 µL of 1X PBS, or 50 µL of 1X PBS. Skin biopsies, 440 approximately 1 cm in diameter, were collected in 1 mL of cytokine extraction buffer for 441 cytometric bead array (CBA) analysis, 5 days post-injection. Skin samples were diluted 1:2 in 442 assay diluent for analysis. Statistics performed by one-way ANOVA, no matching or pairing, 443 corrected for multiple comparisons using Tukey’s test. 95% confidence interval. *p<0.05, 444 **p<0.01. 445 446 Discussion 447 In this report, we generated and characterized an “Fc-silent” derivative of LA-2 as a tool 448 to investigate the role of complement in passive protection afforded by LA-2 in both tick- and 449 needle-mediated B. burgdorferi challenge models. The Fc element of LA-2 was rendered silent 450 by the addition of the so-called LALAPG substitutions (L234A, L235A, P329G), a modification 451 that is gaining wide recognition for its research and clinical applications 44,53,54. We confirmed 452 that LA-2 LALAPG retained OspA binding activity comparable to the parenteral LA-2 IgG1 but 453 was markedly attenuated for in vitro complement fixation and complement-dependent 454 borreliacidal activity. 455 When tested in vivo, we found that LA-2 LALAPG was as effective as LA-2 IgG in 456 passively protecting mice from tick-mediated B. burgdorferi challenge, indicating that neither 457 antibody-mediated complement fixation nor complement-dependent borreliacidal activity were 458 necessary to inhibit spirochete infectivity. In this respect, our results agree with Gipson and de 459 Silva who reported that the “LA-2-like” monoclonal antibody C3.78 blocks tick transmission of 460 B. burgdorferi in the absence of host complement 8,47. Those studies were conducted using 461 complement-deficient (C3) mice and C3.78 Fab fragments. de Silva and colleagues also 462 demonstrated that C3-deficient mice actively immunized with OspA were also protected against .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint 17 463 B. burgdorferi infection, further reinforcing the notion that host complement is not required for 464 transmission blocking activity of OspA vaccines 32. 465 Furthermore, our results support a model in which LA-2 and LA-2 LALAPG inhibit B. 466 burgdorferi transmission without affecting the number of spirochetes within the tick midgut. 467 This observation is consistent with C3.78’s mode of action, in which low dose (~60  g/mouse) 468 antibody protected mice from tick-mediated B. burgdorferi challenge without a concomitant 469 reduction in spirochete numbers in tick tissues 47. In other words, antibody is proposed to block 470 spirochete egress from the midgut by a non-borreliacidal mechanism. Gipson and de Silva and 471 others have speculated that OspA antibodies like C3.78 influence the expression of spirochete 472 genes and gene products required for transmission, including the requisite OspA to OspC 473 transition 23,47. We favor a model in which OspA antibodies like LA-2 entrap spirochetes within 474 the midgut by physically altering their transmigratory activity 34. Using a two compartment 475 Transwell system, we reported recently that spirochete movement from the lower to upper 476 chambers is reduced by >99% in the presence LA-2 or LA-2 LALAPG. Inhibition of 477 transmigration coincided with LA-2’s ability to promote spirochete agglutination, alternations in 478 membrane permeability, and even bleb formation 33,55. Exactly how LA-2 engagement with 479 OspA results in changes in migratory activity remains obscure. 480 While LA-2 has the capacity to interfere with B. burgdorferi transmission within the 481 context of the tick, it can also reduce infectivity of B. burgdorferi within the mouse. Indeed, LA- 482 2 was originally identified as being capable of passively protecting scid mice from subcutaneous 483 B. burgdorferi challenge 6. We confirmed and extended that original observation by 484 demonstrating in both BALB/c and C3H mice that remarkably low doses of passively 485 administered LA-2 were sufficient to not only confine but seemingly eliminate B. burgdorferi 486 from the site of intradermal inoculation within hours. LA-2 LALAPG had similar properties, 487 indicating that clearance of B. burgdorferi from the skin environment occurs without 488 complement or Fc effector functions. These observations may be of clinical importance, as they 489 suggest that if B. burgdorferi evades immunity within the context of the tick body, any 490 spirochetes that still express OspA upon entry into the skin will encounter a second line of 491 defense 21,56. While the underlying mechanism by which LA-2 promotes clearance of spirochetes 492 from the skin environment without Fc effector functions is unknown, there are interesting .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint 18 493 parallels with antibody-mediated clearance of malaria parasites in this same environment that 494 involve motility arrest and membrane shedding 57,58. 495 In summary, we have demonstrated that LA-2, the well-characterized monoclonal 496 antibody directed against the C-terminus of OspA, functions in both the tick and mammalian 497 environments to limit B. burgdorferi infection without the need for Fc effector functions, such as 498 complement fixation and FcR interactions. It is unclear whether LA-2 equivalence, as defined 499 by a competitive ELISA, which correlates with immunity to Lyme disease in animal models and 500 humans reflects functional activities in vivo or simply a proxy for other activities. Nonetheless, 501 our study makes a case for LA-2’s primary mode of action involving direct physical interactions 502 with the spirochete rather than complement-dependent killing. Elucidating these mechanisms 503 may have implications for our understanding of the mechanistic correlates of OspA-based 504 vaccine-induced immunity in humans. 505 506 Acknowledgements 507 We are grateful to Dr. Michael Pauly and colleagues ZabBio for generating LA-2 LALAPG. We 508 thank Drs. Renji Song and Jennifer Yates of the Wadsworth Center’s Immunology Core for 509 assistance with flow cytometry and the Media and Cell Culture core for BSK II medium. We 510 thank Ms. Elizabeth Cavosie (Wadsworth Center) for administrative assistance. BioRender was 511 used for some figure generation. This work was supported by the National Institute of Allergy 512 and Infectious Diseases (NIAID), National Institutes of Health, Department of Health and 513 Human Services, Contract No. 75N93019C00040 (PI/PD Mantis). 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It is made The copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted December 16, 2025. ; https://doi.org/10.64898/2025.12.14.694235doi: bioRxiv preprint