A potent Gc neutralizing antibody reveals architecture-dependent bispecific protection against SFTSV

Figures

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  Figure 1.. Isolation and Characterization of mAbs from SFTS-Convalescent Donors. (A) Schematic workflow for screening SFTSV-specific antibodies using an scFv phage display antibody library. (B) Anti-Gn and anti-Gc endpoint titres in plasma from six convalescent SFTS donors measured by ELISA. Data are presented as mean ± SD (n = 6), with each symbol representing an individual. Statistical significance was determined by one-way ANOVA. Paired Student’s t-tests were used to compare antibody titres against Gn and Gc within the same individual. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. (C-D) Binding of isolated mAbs against recombinant SFTSV Gn (C) or Gc (D) in ELISA assays. (E) Neutralization of authentic SFTSV by individual Gc-mAbs in Vero cells. (F) Competitive binding of CAV-Gc-2H1, CAV-Gc-1B12, CAV-Gc-1F5 and SF83 to the Gc. Competitive ELISA assays were performed to assess epitope overlap among the four antibodies. (G) AlphaFold3-derived models showing relative positions of mapped epitopes on Gc (illustrative). The structural domains of Gc are colour-coded as follows: Domain I (red), Domain II (yellow), and Domain III (blue), fusion peptide (orange). The mAbs…

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  Figure 2.. Design, Expression, Binding and Neutralization of SFTSV bsAbs. (A) Schematic representation of SFTSV bsAbs. bsSCFV1-4 are in scFv-Ig format; bsDVD1-4 are in DVD-Ig format; bsCODV1-4 are in CODV-Ig format. Parental antibodies are colour-coded as follows: 40C10 in shades of green, CAV-Gc-2H1 in shades of blue, and JK8 in shades of yellow. Darker colours indicate VH domains, while lighter colours indicate VL domains. (B) Denaturing and reducing SDS-PAGE of the bsAbs constructed from the parental antibodies JK8 and CAV-Gc-2H1. The heavy and light chains of scFv-Ig format are ∼80 and 25 kDa, respectively. The heavy and light chains of DVD-Ig and CODV-Ig format are ∼65 kDa and 40 kDa, respectively. (C) Binding affinities (KD) of the three parental antibodies and all bsAb formats. Binding affinity measurements were performed using biolayer interferometry (BLI) on an Octet R8 instrument. Anti-human IgG Fc capture (AHC) biosensors were used to immobilize antibodies, followed by separate exposure to serial dilutions of monomeric Gn or Gc. Sensorgrams were fitted using a 1:1 Langmuir binding model to determine kinetic parameters. Responses with a maximum signal < 0.05 nm were defined as no…

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  Figure 3.. Neutralizing Mechanisms of bsAbs. (A) Membrane fusion mediated by SFTSV glycoproteins Gn and Gc, and its inhibition by bsAbs at varying concentrations were assessed using a split-GFP complementation reporter system. PBS-treated cells served as the control, and relative membrane fusion levels were normalized to this group. (B) Representative fluorescence images of cells treated with each bsAb at 10 μg/mL are shown. Nuclei are stained with DAPI (blue), and GFP-expressing proteins are shown in green. Syncytia edges are outlined in white. (C) Viral binding to cells was quantified by RT-qPCR in the presence of antibodies at concentrations of 1, 0.1 and 0.01 μg/mL. PBS-treated cells served as the control, and relative viral RNA levels were normalized to this group. Data are presented as mean ± SD from three independent experiments. Statistical significance was determined by comparing each group with the control group using two-way ANOVA followed by Dunnett’s multiple comparisons test. The corresponding P value thresholds are indicated as P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****).

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  Figure 4.. Evaluation of the prophylactic and therapeutic effects of mAb and bsAb in a mouse model of SFTSV infection. (A) Prophylactic effects of mAb and bsAb were evaluated in SFTSV-infected C57BL/6J-IFNAR-/- mice. Mouse survival and body weight were monitored daily. CAV-Gc-2H1 and bsSCFV3 were administered at 30 mg/kg one day prior to viral challenge. (B) Therapeutic strategy effects of mAb and bsAb were evaluated in SFTSV-infected C57BL/6J-IFNAR-/- mice. Mouse survival and body weight were monitored daily, with CAV-Gc-2H1 and bsSCFV3 administered at 30 or 10 mg/kg one day post-infection. (C) Hematoxylin and eosin (H&E) staining was used to evaluate tissue damage in infected mice treated with or without antibodies. Representative sections show widespread coagulative necrosis (white asterisks) and diffuse inflammatory infiltration (black arrows). Focal inflammatory infiltration within the liver parenchyma is indicated by yellow arrows. (D) Viral RNA levels in mouse serum and liver were quantified by RT-qPCR at the experimental endpoint following prophylactic and therapeutic antibody administration. Mice were euthanized either upon reaching predefined humane endpoints or at the scheduled…