Journal: Nature Communications

Article Title: Potent neutralization by antibodies targeting the MPXV A28 protein

doi: 10.1038/s41467-025-66344-0

Figure Lengend Snippet: a Neutralization kinetics of VACV IHDJ by anti-MPXV mAbs at IC 50 concentrations with 1.5% NHS (3% during pre-incubation). NHS and mAbs were added at 10-min intervals from 2 h before to 2 h after infection. b Neutralization in the presence of 1.5% NHS (3% during pre-incubation) and 6 consecutive 4-fold dilutions of C5-IN-1 starting from 10 μM. c Same as ( b ) but with 8 consecutive 5-fold dilutions of a C3 inhibitor AMY-101 starting from 10 μM. d Neutralization in the presence of 1.5% NHS (3% during pre-incubation) or C1q- or C3-depleted human serum. e C1q deposition on VACV IHDJ virions ( ~ 5 × 10⁶ PFU) incubated with 1.5% NHS and 10 μg/mL mAbs. Left panel: flow cytometry plots, pre-gated for virions using GFP followed by staining for C1q-AF647. Right panel: median fluorescence intensity. f-h TEM visualization of mAb and complement factors C1q, and C3 deposition on virions. Purified MV particles were incubated with 10 μg/mL of either mAb 7M1162 anti-H3 ( f ), 8M2110 anti-A28 ( g ) or MGO53 isotype control ( h ) and 1.5% NHS. IgG, C1q, and C3 deposition were visualized using anti-human IgG gold nanoparticles (10 nm) or secondary mouse anti-C1q and mouse anti-C3, followed by labeling with anti-mouse gold nanoparticles (20 nm). Anti-H3 and anti-A28 mAbs are shown in magenta and green, respectively; anti-M1 (7D11) in black; MGO53 in gray. IC 50 concentrations for panels ( a – d ) were: 15 μg/mL (7M1162), 0.125 μg/mL (8M2110), 1.25 μg/mL (10M2146), and 62.5 μg/mL (10M2154). Spearman correlation was used in ( a ) to determine the relationship between pre-incubation times and neutralization. Standard curves were determined by fitting values using simple linear regression for ( a ) and Inhibitor vs. normalized response (Variable slopes) nonlinear regression for ( b - c ). Standard deviation of the mean is shown for all values in ( a – d ). The percentages of complement sources indicated represent the final concentrations in the infected wells. Data are representative of two independent experiments with similar results using technical replicates: n = 3 for ( a ), n = 5 for ( b ), n = 6 for ( c ), and n = 3 for ( d ). Source data are provided as a Source Data file.

Article Snippet: For experiments involving selective complement inhibitors, mAbs and NHS mixtures were incubated with either 6 consecutive 4-fold dilutions, starting from 10 μM of a selective complement C5 inhibitor, C5-IN-1 (Compound 7- MedChemExpress), or 8 consecutive 5-fold dilutions, starting from 10 μM of a selective complement C3 inhibitor, CP40 (AMY-101- MedChemExpress).

Techniques: Neutralization, Incubation, Infection, Flow Cytometry, Staining, Fluorescence, Purification, Control, Labeling, Standard Deviation