Importance of Neutralizing Monoclonal Antibodies Targeting Multiple Antigenic Sites on the Middle East Respiratory Syndrome Coronavirus Spike Glycoprotein To Avoid Neutralization Escape

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) causes a highly lethal pulmonary infection with ∼35% mortality. The potential for a future pandemic originating from animal reservoirs or health care-associated events is a major public health concern. There are no vaccines or therapeutic agents currently available for MERS-CoV. Using a probe-based single B cell cloning strategy, we have identified and characterized multiple neutralizing monoclonal antibodies (MAbs) specifically binding to the receptor-binding domain (RBD) or S1 (non-RBD) regions from a convalescent MERS-CoV-infected patient and from immunized rhesus macaques. RBD-specific MAbs tended to have greater neutralizing potency than non-RBD S1-specific MAbs. Six RBD-specific and five S1-specific MAbs could be sorted into four RBD and three non-RBD distinct binding patterns, based on competition assays, mapping neutralization escape variants, and structural analysis. We determined cocrystal structures for two MAbs targeting the RBD from different angles and show they can bind the RBD only in the "out" position. We then showed that selected RBD-specific, non-RBD S1-specific, and S2-specific MAbs given prophylactically prevented MERS-CoV replication in lungs and protected mice from lethal challenge. Importantly, combining RBD- and non-RBD MAbs delayed the emergence of escape mutations in a cell-based virus escape assay. These studies identify MAbs targeting different antigenic sites on S that will be useful for defining mechanisms of MERS-CoV neutralization and for developing more effective interventions to prevent or treat MERS-CoV infections.IMPORTANCE MERS-CoV causes a highly lethal respiratory infection for which no vaccines or antiviral therapeutic options are currently available. Based on continuing exposure from established reservoirs in dromedary camels and bats, transmission of MERS-CoV into humans and future outbreaks are expected. Using structurally defined probes for the MERS-CoV spike glycoprotein (S), the target for neutralizing antibodies, single B cells were sorted from a convalescent human and immunized nonhuman primates (NHPs). MAbs produced from paired immunoglobulin gene sequences were mapped to multiple epitopes within and outside the receptor-binding domain (RBD) and protected against lethal MERS infection in a murine model following passive immunization. Importantly, combining MAbs targeting distinct epitopes prevented viral neutralization escape from RBD-directed MAbs. These data suggest that antibody responses to multiple domains on CoV spike protein may improve immunity and will guide future vaccine and therapeutic development efforts.

Keywords: MERS-CoV; RBD; S1; escape mutation; monoclonal antibody; protection.

FIG 1

MAb binding specificity and neutralization potency. (A) Binding specificity. MAbs isolated from immunized NHPs and a MERS survivor were assayed by ELISA for binding to soluble receptor-binding domain (RBD) or S1 protein. RBD-specific MAbs (in red) bound to both RBD and S1 proteins, while S1-specific MAbs (in blue) bound only to S1. (B) Neutralization potency. Neutralization activity was measured using a MERS-CoV EMC S-pseudotyped lentivirus neutralization assay. NHP MAbs are shown in the left graph and human MAbs in the right graph (two NHP RBD-specific MAbs in black were included for comparison). Percent neutralization at the different MAb concentrations is shown. Data points represent the means of triplicate replicates with standard errors. (C) IC₅₀, IC₈₀, and IC₉₀ neutralization titers. ^aIC values represent average results from two technical replicates of neutralization testing using a pseudovirion entry assay or plaque reduction neutralization testing (PRNT). ^bThe PRNT IC₅₀ for MAb JC57-13 was not available (N/A), as >50% neutralization was obtained at the lowest concentration of MAb tested, 0.0032 μg ml⁻¹.

FIG 2

MAb neutralization breadth. RBD-specific MAbs (in red) (A) and S1-specific MAbs (in blue) (B) were measured for neutralization activity against 8 to 11 strains of MERS-CoV S-pseudotyped viruses as indicated, including strains identified from 2012 to 2015. CDC2-C2, CDC2-C5, CDC2-A2, and CDC2-A10 were isolated from a human donor, JC57-11, JC57-14, JC57-13, and FIB-H1 from immunized rhesus macaques, and G2 from an immunized mouse (20). Percent neutralization at the different MAb concentrations is shown. Data points represent the means of triplicate replicates with standard errors. (C) IC₅₀, IC₈₀, and IC₉₀ neutralization titers for panels A and B.

FIG 3

Structural characterization of macaque and human MERS virus-neutralizing antibodies. (A) Crystal structure of JC57-14 antibody bound to MERS England1 RBD. (B) Crystal structure of CDC-C2 bound to MERS England1 RBD. (C) JC57-14 and CDC-C2 are modeled using the MERS spike trimer structure (PDB code 5W9H) bound to a single RBD in the open conformation. (D) Antibody epitopes of JC57-14 and CDC-C2 are displayed on the surface of the RBD structure. Residues highlighted and labeled differing in natural viral variants were tested to characterize antibody epitopes.

FIG 4

Neutralization epitopes of RBD-specific MAbs. RBD-specific MAbs neutralize MERS-CoV by interacting with the different residues in the receptor-binding domain. (A) Neutralization curves. Six RBD-specific MAbs (CDC2-C2 and CDC2-C5 from a human, JC57-11 and JC57-14 from NHPs, and F11 and D12 from mice) were tested for neutralizing activity against pseudoviruses displaying MERS-CoV EMC S with engineered mutations in the RBD. EMC is shown in red, mutant constructs fully resistant to neutralization are shown in boldface blue type, mutant constructs partially resistant to neutralization are shown in lightface blue type, and mutant constructs with enhanced neutralization are shown in dark red. Data points represent the means of triplicate replicates with standard errors. The experiment was repeated once to ensure reproducible results; results from one of the two experiments are shown. (B) Fold changes in IC₅₀ neutralization titers corresponding to mutant forms of EMC S. Mutant S IC₅₀ neutralization titers (Fig. S5) are presented relative to native S (set equal to 1). Six MAbs display a total of four unique neutralization patterns, which are color-coded in blue, red, black, and purple.

FIG 5

Competition map of MAb binding by ELISA. RBD-specific MAbs (in red) and S1-specific MAbs (in blue) were biotinylated and used for competition binding to MERS-CoV S1 with MAbs listed in the leftmost column. Percent inhibition of analyte (biotinylated)-MAb binding by competitor (unlabeled) MAbs is indicated by color as shown in the color key. Competition binding curves are shown in Fig. S7A and S7B.

FIG 6

Passive transfer of RBD-, S1-, and S2-specific neutralizing MAbs protect against MERS-CoV infection. (A) Groups of 10 human DPP4-transgenic (hDPP4) mice were administered MAb CDC2-C2 (RBD specific), G2 (S1 specific), or G4 (S2 specific) (20) via the intraperitoneal route 1 day prior to challenge with MERS-CoV EMC. hDPP4 mice were challenged intranasally with 10⁶ TCID₅₀ of MERS-CoV (strain EMC/2012). At 3 days postinfection (dpi), four animals were sacrificed and lungs were collected for analyses. The remaining six animals were observed for 28 days for survival. (B) Survival curves of the different groups (6 hDPP4 mice per group). After challenge, hDPP4 mice were euthanized due to the severity of disease signs or at 28 dpi. (C and D) MERS-CoV viral titers in the lower respiratory tract (C) and in the brain (D) of hDPP4 mice at 3 dpi. Mean values ± SD were calculated. The dashed line indicates the cutoff limit of the TCID₅₀ assay.

FIG 7

S mutations associated with viral escape from neutralizing MAbs. EMC was serially passaged in the presence of the indicated human and NHP neutralizing anti-S MAbs, singly or combined, and S mutations associated with antibody escape in the 10th (P10) or 20th (P20) passage were determined by S gene sequence analysis of 11 to 15 plaque isolates (P10 single MAb and P20 double MAb) or three independent population virus cultures (P10 double MAb). Percentages of viral isolates or cultures containing the indicated mutation are shown. MAb combinations that prevented the emergence of RBD mutations are shown in red.