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

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.

Rift Valley fever virus (RVFV) is an emerging arbovirus and zoonotic threat to human and animal health with pandemic potential because of the global presence of its vectors and hosts (1). First identified in 1931 (2), RVFV causes ongoing infections in Africa, with occasional rises in incidence related to the propagation of mosquitos due to alternating weather patterns (3). These outbreak episodes are characterized by mass livestock die-off events, particularly in young animals and abortions in pregnant females. Spillover events to humans occur by infected mosquito exposures or contact with the blood and/or organs of infected animals (4). Human-to-human transmission of RVFV has not been documented, but concern is growing that RVFV can be transmitted from mother to fetus in utero (5, 6). In humans, disease presentation ranges from a mild influenza-like illness to a potentially lethal hemorrhagic fever syndrome. The World Health Organization (W.H.O.) has reported more than 4,600 cases and 957 deaths from 2000 to 2016 due to RVFV infections, with a case fatality rate of more than 20% (7). RVFV outbreaks typically occur in sub-Saharan and North Africa, but in 2000, the first reported cases outside the African continent occurred in the Arabian Peninsula (8). Given the increasing observed mortality rate, spread to new regions, and potential use as a bioterrorist agent (9), the W.H.O. and the US National Institute of Allergy and Infectious Diseases have designated RVFV as a priority pathogen for urgent research and therapeutic development (10, 11).RVFV, a member of the Phlebovirus genus in the Phenuiviridae family of the Bunyavirales order (12) has a tripartite RNA genome containing large (L), medium (M), and small (S) gene segments. The sequences in the M segment encode for the viral envelope glycoproteins (Gc and Gn). These two glycoproteins allow for viral attachment, entry, fusion, and assembly when oriented as a pentamer of heterodimers (13–16). Data from animal and human vaccine trials indicate that high-serum neutralizing titers and protection (in animals) are associated with responses to Gn, Gc, or entire virion particles (17–23). Protection against experimental RVFV challenge is observed in vaccinated animals with high-serum titers of neutralizing antibodies (22, 23).On the viral surface, Gn and Gc organize into a pentamer or hexamers of Gc-Gn heterodimers with icosahedral symmetry (13, 14). Receptor engagement of DC-SIGN or possibly other receptors like heparan sulfate (24) by Gn allows for cellular attachment and initiates caveolae-mediated endocytosis (25, 26). Premature fusion and extension of the fusion loop is prevented by Gn, which provides a shield for the fusion loop in Gc (27). Upon exposure to acidic conditions in the late endosome, Gn repositions, and the class II fusion protein, Gc extends so that its fusion loop interacts with the host membrane (27–29). The extended Gc conformation subsequently rearranges so the membranes organize in close proximity to fuse (29). Given the broad host, tissue, and cell tropism of RVFV (24), inhibiting RVFV by blocking DC-SIGN engagement may not fully explain the neutralizing and protective capacity of potent mAbs. The fusion process, a common mechanistic feature of RVFV entry facilitating broad tropism, may be a desirable target to enable neutralization. These observations may explain why neutralization is observed in cells that do not express DC-SIGN.Here, we report the isolation of 20 human monoclonal antibodies (mAbs) from the circulating B cells of RVFV MP-12 vaccines or survivors of natural RVFV infection using neutralization as the primary screening method. This panel of antibodies targets diverse antigenic sites on Gc, Gn, and an undefined epitope that may present only in the glycoprotein complex (Gc+Gn) of properly hetero-oligomerized Gc-Gn. Here, we refer to mAbs that bind to coexpressed full-length Gc-Gn proteins but not to monomeric Gc or Gn as “Gc+Gn–specific mAbs.” Four competition groups for binding to viral antigen on the RVFV surface were recognized by neutralizing antibodies, and all groups contained antibodies that cross-neutralized diverse strains of RVFV. Antibodies from both vaccinated and naturally infected individuals primarily neutralized virus by targeting domain A on the Gn protein. Previously, mAbs derived from a single patient in China inhibited receptor engagement by binding to Gn domain A (30). We sought to explore the role of fusion inhibition as a second major mechanism of neutralization, by which mAbs could contribute to the overall potent neutralizing activity observed and prevent infection even in cells and tissues that do not readily express DC-SIGN. Fusion-inhibiting activity of anti-RVFV–specific mAbs has not been reported to date but has been observed for murine mAbs (31). We observed two classes of potently neutralizing antibodies, those that target domain A and cause partial inhibition of fusion and Gc+Gn–specific mAbs that cause complete inhibition of fusion. Studies of a representative mAb from each group indicate the Gn domain A-specific mAb loses fusion inhibition activity when used as Fab fragments, whereas the Gc+Gn–specific mAb retains fusion inhibition activity as a Fab, suggesting different mechanisms of action for these two classes of mAbs. Antibodies that recognize domain A, domain B, and Gc+Gn–specific epitopes neutralized RVFV with varying potencies, and all provided protection against lethal ZH501 wild-type (wt) virus challenge in prophylactic or therapeutic mouse models of experimental infection when used as a monotherapy. These results suggest these mAbs can be investigated as therapies against RVFV infection and that multiple antigenic sites of vulnerability for neutralization by inhibition of viral fusion exist on the virion surface.