Potent neutralization of Rift Valley fever virus by human monoclonal antibodies through fusion inhibition

Abstract

Rift Valley fever virus (RVFV), an emerging arboviral and zoonotic bunyavirus, causes severe disease in livestock and humans. Here, we report the isolation of a panel of monoclonal antibodies (mAbs) from the B cells of immune individuals following natural infection in Kenya or immunization with MP-12 vaccine. The B cell responses of individuals who were vaccinated or naturally infected recognized similar epitopes on both Gc and Gn proteins. The Gn-specific mAbs and two mAbs that do not recognize either monomeric Gc or Gn alone but recognized the hetero-oligomer glycoprotein complex (Gc+Gn) when Gc and Gn were coexpressed exhibited potent neutralizing activities in vitro, while Gc-specific mAbs exhibited relatively lower neutralizing capacity. The two Gc+Gn-specific mAbs and the Gn domain A-specific mAbs inhibited RVFV fusion to cells, suggesting that mAbs can inhibit the exposure of the fusion loop in Gc, a class II fusion protein, and thus prevent fusion by an indirect mechanism without direct fusion loop contact. Competition-binding analysis with coexpressed Gc/Gn and mutagenesis library screening indicated that these mAbs recognize four major antigenic sites, with two sites of vulnerability for neutralization on Gn. In experimental models of infection in mice, representative mAbs recognizing three of the antigenic sites reduced morbidity and mortality when used at a low dose in both prophylactic and therapeutic settings. This study identifies multiple candidate mAbs that may be suitable for use in humans against RVFV infection and highlights fusion inhibition against bunyaviruses as a potential contributor to potent antibody-mediated neutralization.

Keywords: Phlebovirus; Rift Valley fever virus; adaptive immunity; antibodies; monoclonal; virus internalization.

Fig. 1.

Competition binding of RVFV-specific mAbs on Gc-Gn coexpressing cells. We tested 18 mAbs in competition-binding assays. The mAbs are displayed in four groups (designated A [with A1, A2, or A3 subgroups], B, C, or D) based on their ability to compete for binding with each other. The values shown are the percentage of binding that occurred during competition compared to noncompeted binding of the mAb and derived from the mean fluorescence intensity (MFI). MFI values were normalized against a mock transfected control. The values are also indicated by the box fill color; darker colors toward black indicate higher competition, and lighter colors toward white indicate less competition, on a gradient scale. Values shown are the average of three technical replicates from three independent experiments.

Fig. 2.

Mutation library screening for loss-of-binding to identify critical residues. Expression clones containing key mutations in (A) Gn (for groups A2 and A3) and (B) Gc (for groups B, C, and D) from loss-of-binding screening are shown. The color of the mAb clone names corresponds to the competition group assigned in Fig. 1. The reactivity of each mAb tested for mutants is shown as a percentage of the reactivity of that mAb to wt Gn or Gc. A larger library of mutants was tested, but here, results are shown only for residues that reduced binding of at least one mAb. Amino acids were considered critical if the level of binding was <30% of wt binding. Data shown are the average of technical duplicates from two independent experiments. The location of residues at which amino acid substitution disrupts mAb binding are mapped onto the crystal structure of Gn (A) or Gc (B) and are designated by color-coded spheres. Color coding is as follows: gray (RVFV-142), shades of blue (residues that influence the binding of multiple competition group A2 mAbs), magenta (residues that influence the binding of mAbs in both A2 and A3 competition groups), light orange (RVFV-429), dark orange (RVFV-226), purple (group B mAbs), light green (group C mAb, RVFV-128), and yellow (group D mAb, RVFV-249). A side-view of Gn (A) is shown and is also shown rotated 180°. Gn (domain A, B, or C in cyan, green, or magenta) or Gc (domain I in red, domain II in yellow, or domain III in blue) are shown as cartoon. Gc models has been adapted from PDB (Protein Data Bank):4HJ1, and the Gn model has been adapted from PDB:5Y0W as their respective templates.

Fig. 3.

Fusion inhibition is mediated by RVFV mAbs against Gn domain A or Gc+Gn epitopes but not Gn domain B or Gc mAbs. (A) Representative mAbs from each domain of Gn, one Gc binding mAb, and Gc+Gn–specific mAbs were titrated in a pre- versus postattachment assay to determine relative contribution of neutralization before or after attachment in Vero cells. (B–D) A FFWO assay was used to assess antibody inhibition of viral fusion with Vero cell membranes under low-pH conditions at the cell surface to mimic acidified endosomes. Virus was adsorbed on a Vero cell monolayer at 4 °C for 45 mins. Virus then was removed, and RVFV-specific mAbs were added for 30 min. Cells were exposed to a prewarmed pH 5.5 medium or a control neutral pH medium for 2 min at 37 °C. The medium was removed, and the cells were incubated in supplemented DMEM for 16 h before fixing on plate, permeabilizing, and staining for viral antigen. Quantification of intracellular viral antigens was done by counting infected cells relative to a virus-only control on a BioSpot CTL plate reader. Three independent experiments were performed in triplicate for each antibody for all assays. (A) Pre- versus postattachment of representative mAbs. (B) Full dilution curves for 10 IgG mAbs in the FFWO assay. (C) Relative infection ratio at 30 µg/mL for 20 IgG mAbs in the FFWO assay. (D) Relative infection ratio at 15 or 30 µg/mL for 3 mAbs tested as Fab fragments in the FFWO assay.

Fig. 4.

RVFV-140, RVFV-268, and RVFV-226 increase survival when used as prophylaxis or therapy against lethal RVFV infection in mice. (A) Prophylaxis study in a C57BL/6 mouse model of RVFV infection. MAb (200 or 10 µg) was administered once by the IP route to mice (n = 14) 2 h prior to SC inoculation of 300 PFU of RVFV (ZH501 strain). (B) Therapeutic study in a BALB/c mouse model of RVFV infection. MAb (200 µg) was administered once by the IP route to mice (n = 14) either 2 or 4 d after SC inoculation of 100 PFU of RVFV (ZH501 strain). Kaplan–Meier survival curves were assessed by a Mantel–Cox logrank test. Weight data are represented as the group mean and SE (SEM) of the percent change in weight of surviving animals relative to starting weight. Error bars represent the SEM. Viral titer data were assayed using an infectious cell culture assay in technical triplicate. In both studies, human mAb DENV 2D22 (specific to an unrelated target, dengue virus) was used as the negative control, and mice were monitored daily from day 0 to 21 d.p.i. for survival and body weight. **P = 0.0021; ***P = 0.0002; ****P < 0.0001.