Table 1.
Recombinant B. burgdorferi s.s. B31 proteins used in this study.
Fig 1.
Reactivity of LA-2 and LA-2 LALAPG with native OspA.
Representative flow cytometry assay of serially diluted LA-2 (dark blue) and LA-2 LALAPG (light blue overlay) reactivity with native OspA on the surface of B. burgdorferi s.s. B31. (Left) Fluorescence histogram overlays comparing binding properties. Percent and geometric mean fluorescence intensities of Alexa-647 fluorescently labeled events (under bracket) are indicated. (Right) Forward scatter (FSC) – side scatter (SSC) dot plot overlays comparing agglutination properties. Events increased in FSC and/or SSC (UL, UR, LR quadrants) demonstrate agglutination, and percent of events agglutinated is indicated.
Fig 2.
LA-2 LALAPG is deficient in complement deposition in vitro.
(A) Binding (MFI) of LA-2 IgG and LA-2 LALAPG to recombinant OspA. The asterisks indicate a significant difference between groups by Welch’s t-test (**P < 0.01). Quantification and comparison of human complement C3 deposition between LA-2 and LA-2 LALAPG in the context of OspA. The asterisk indicates a significant difference between groups by Welch’s t-test, where *P < 0.05. (C) Dose response of complement C3 deposition of LA-2 and LA-2 LALAPG in the context of OspA.
Fig 3.
Complement-dependent borreliacidal activity associated with LA-2 and LA-2 LALAPG.
Mid-log phase B. burgdorferi s.s. B31-5A4 carrying an IPTG-inducible mscarlet-I reporter (GGW979) were suspended (1.5 × 10⁶ cells per reaction) in BSKII medium supplemented with 20% guinea pig complement and 5 nM of the mAbs indicated on the x-axis (857−2, LA-2, LA-2 LALAPG, PB10), as detailed in the Materials and Methods. Following a 48-h incubation, the median fluorescence intensity (MFI; 569 nm excitation/611 nm emission) was determined. The bars are the mean of three independent experiments with each symbol being an independent experiment and the error bars indicating SD. The dashed red line represented 50% killing. The results demonstrate that 857−2 and LA-2 have potent borreliacidal activity as indicated by low normalized MFI, whereas LA-2 LALAPG and the isotype control were devoid of activity.
Table 2.
mAb passive protection in mouse model of tick-mediated B. burgdorferi s.s. B31 challenge.
Table 3.
mAb passive protection in mouse model of B. burgdorferi s.s. B31 ID challenge.
Fig 4.
LA-2 and LA-2 LALAPG prevent spirochete dissemination through skin.
Groups of 2−4 mice were administered LA-2, LA-2 LALAPG, or remained untreated, then intradermally challenged one day later with B. burgdorferi s.s. B31. On days 1, 3, and 7 post challenge, these groups of mice were euthanized, and skin biopsies were harvested at the injection site (IS), ~ 1 cm from the IS (S1), and ~3 cm from the IS (S2) and subsequently cultured. The pie chart subdivisions indicate the number of skin samples (mice) assayed per treatment and time point, with one sample collected from each skin site per mouse. Red subdivisions indicate culture positivity for motile spirochetes, while white subdivisions indicate no viable spirochetes detected in the culture. Shown are the combined results from two independent experiments. Created in BioRender. P, D. (2026) https://BioRender.com/74fgdnw.
Fig 5.
Cytometric bead array analysis of injection site skin biopsies of BALB/c mice treated with LA-2 and ID infected with B. burgdorferi s.s.
B31. On study day –1, mice were SC administered 30 µg/mL of LA-2 or were left untreated. Mice were ID injected with either B. burgdorferi s.s. B31 or PBS on day 0 and the injection site skin was harvested on day 5 for CBA analysis. Skin samples were diluted 1:2 in assay diluent for analysis. Statistics performed by one-way ANOVA, no matching or pairing, corrected for multiple comparisons using Tukey’s test. 95% confidence interval. *p < 0.05, **p < 0.01.