Cooperativity mediated by rationally selected combinations of human monoclonal antibodies targeting the henipavirus receptor binding protein

Abstract

Hendra virus and Nipah virus (NiV), members of the Henipavirus (HNV) genus, are zoonotic paramyxoviruses known to cause severe disease across six mammalian orders, including humans. We isolated a panel of human monoclonal antibodies (mAbs) from the B cells of an individual with prior exposure to equine Hendra virus (HeV) vaccine, targeting distinct antigenic sites. The most potent class of cross-reactive antibodies achieves neutralization by blocking viral attachment to the host cell receptors ephrin-B2 and ephrin-B3, with a second class being enhanced by receptor binding. mAbs from both classes display synergistic activity in vitro. In a stringent hamster model of NiV Bangladesh (NiV_B) infection, antibodies from both classes reduce morbidity and mortality and achieve synergistic protection in combination. These candidate mAbs might be suitable for use in a cocktail therapeutic approach to achieve synergistic potency and reduce the risk of virus escape.

Keywords: B lymphocytes; Hendra virus; Nipah virus; antigen-antibody reactions; epitopes; henipavirus infections; monoclonal antibodies; therapy; viral antibodies.

Figure 1.. Identification of major antigenic sites for recognition of RBP by human mAbs

(A) Surface plasmon resonance competition binding of human antibodies against HeV-RBP. A first antibody was applied to a gold-coated sensor chip, and recombinant HeV-RBP head domain was associated to the coupled antibody. A second antibody was applied to the sensor chip to determine binding to RBP. Black boxes indicated a pairwise interaction in which the binding of the second antibody is blocked by the first. White indicates both antibodies could bind simultaneously. Gray indicates an intermediate competition phenotype. The matrix was assembled using the Carterra Epitope software. (B) Hydrogen-deuterium exchange mass spectrometry profiles for representative mAbs. A decrease (blue) or increase (red) in deuterium exchange on HeV-RBP in the presence of antibody is mapped onto the crystal structure of HeV-RBP (PDB: 6CMG). Structures are positioned in three orientations, with the top structure noting the ephrin-B2 binding pocket in yellow. HDX data mapped to the HeV-RBP amino acid sequence can be found at https://doi.org/10.17632/hzc4t8f4s2.1. (C) Half-maximal binding (blue) or neutralization (green) concentrations for antibodies against recombinant proteins or live HeV or NiV, respectively. (D) Neutralization curve plots for representative antibodies against HeV, NiV Malaysia, or NiV Bangladesh viruses. EC₅₀ values from a single experiment representative of three independent experiments performed in technical duplicate are shown. IC₅₀ values for neutralization are from a single independent experiment due to limitations of biosafety level 4 (BSL-4) resources. Data are represented as mean ± SD.

Figure 2.. Receptor blocking and structural studies

(A) Antibody binding to cell surface-displayed HeV-RBP when ephrin-B2 is bound. Cells transiently transfected with a cDNA encoding the full-length HeV-RBP were incubated with a saturating concentration of recombinantly expressed ephrin-B2. Without washing, cells were incubated with 2 μg/mL antibody, and binding was compared to binding of antibodies in the absence of ephrin-B2. The mAb m102.4 served as a control for receptor competition. Pooled data from three independent experiments are shown. Data are represented by mean ± SD. (B) Three-dimensional reconstruction from negative stain electron microscopy (nsEM) of dimeric HeV-RBP full ectodomain bound to HENV-103 Fab and HENV-117 Fab. The EM map is shown in gray, the Fabs are in purple and green, and the RBP head domain is colored by β-propeller. 2D classes are shown, with box size of 128 at Å per pixel (Å/pix) of 3.5.

Figure 3.. Therapeutic protection by human antibodies in hamster model of Nipah Bangladesh challenge

Survival curves (left) and weight maintenance (right) for hamsters treated with 10 mg/kg antibody (n = 5 per group) 24 h post-inoculation with 5 × 10⁶ PFU NiV Bangladesh by the intranasal route. An untreated control animal (n = 1) succumbed to infection 3 days post-inoculation. All weight maintenance charts include control animal in black. Two historical controls are plotted on survival curves and pooled with the experimental control to perform statistical analysis by the log rank Mantel-Cox test.

Figure 4.. Synergistic binding and neutralization

(A) Cooperative binding by HENV-103 and HENV-117 to cell surface-displayed HeV-RBP. Cells expressing HeV-RBP were incubated with unlabeled HENV-103 or HENV-117, followed by addition of a dilution series of Alexa Fluor 647 (AF647)-labeled HENV-103 or HENV-117. Cells were analyzed by flow cytometry and gated for AF647⁺ cells. Data were pooled from three independent experiments performed in technical duplicate. Data are represented by mean ± SD. (B) Dependence of HENV-117 effective concentration on HENV-103 binding enhancement. Cells were incubated with varying concentrations of unlabeled HENV-117, followed by incubation with AF647-labeled HENV-103 at 0.5 μg/mL, with enhancement calculated by comparing AF647⁺ cells to HENV-103 binding to HeV-RBP in the absence of HENV-117. Data from a single experiment performed in technical triplicate representative of three independent experiments are shown. Data are represented by mean ± SD. (C) Synergistic neutralization of rCedV-HeV by HENV-103 and HENV-117 combinations. Neutralization values at each matrix concentration (top) and calculated synergy scores (bottom) are shown. Serial dilutions of HENV-103 and HENV-117 were mixed with 4,000 PFU of rCedV-HeV-GFP for 2 h, followed by addition to Vero E6 cell monolayers in 96-well plates. Formalin-fixed cells were imaged using a CTL S6 analyzer to count GFP⁺ cells. Neutralization was calculated by comparing treatment to virus-only control wells, with GFP⁺ greater than control wells considered 0% neutralization. Values were imported into SynergyFinder using a zero interactions potency (ZIP) statistical model. Delta scores >10 indicate likely synergy. Two independent experiments were performed, with data from a single representative experiment shown.

Figure 5.. Antibody cocktail and corresponding bispecific antibody therapeutic activity in hamsters

(A) Neutralization of VSV-NiV_B by bispecific antibodies in comparison to equimolar antibody cocktail. Two independent experiments were performed in technical triplicate, with data from a single representative experiment shown. Data are represented by mean ± SD. (B) Syrian golden hamster challenge studies with HENV-103 and HENV-117 cocktail or corresponding bispecific antibodies. Challenge studies were performed as described above. p values represent statistical significance as determined by a Mantel-Cox log rank test. n = 5 animals were included in all groups, with control animals treated with PBS at 24 h post-inoculation.