Enhanced Ability of Oligomeric Nanobodies Targeting MERS Coronavirus Receptor-Binding Domain

Abstract

Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV), an infectious coronavirus first reported in 2012, has a mortality rate greater than 35%. Therapeutic antibodies are key tools for preventing and treating MERS-CoV infection, but to date no such agents have been approved for treatment of this virus. Nanobodies (Nbs) are camelid heavy chain variable domains with properties distinct from those of conventional antibodies and antibody fragments. We generated two oligomeric Nbs by linking two or three monomeric Nbs (Mono-Nbs) targeting the MERS-CoV receptor-binding domain (RBD), and compared their RBD-binding affinity, RBD⁻receptor binding inhibition, stability, and neutralizing and cross-neutralizing activity against MERS-CoV. Relative to Mono-Nb, dimeric Nb (Di-Nb) and trimeric Nb (Tri-Nb) had significantly greater ability to bind MERS-CoV RBD proteins with or without mutations in the RBD, thereby potently blocking RBD⁻MERS-CoV receptor binding. The engineered oligomeric Nbs were very stable under extreme conditions, including low or high pH, protease (pepsin), chaotropic denaturant (urea), and high temperature. Importantly, Di-Nb and Tri-Nb exerted significantly elevated broad-spectrum neutralizing activity against at least 19 human and camel MERS-CoV strains isolated in different countries and years. Overall, the engineered Nbs could be developed into effective therapeutic agents for prevention and treatment of MERS-CoV infection.

Keywords: Coronavirus; MERS-CoV; cross-neutralization; nanobodies; receptor-binding domain; therapeutic antibodies.

Figure 1

Construction and characterization of dimeric and trimeric nanobodies (Nbs) targeting the Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV). (A) Heavy chain-only antibody (HcAb) consists of two constant heavy domains (CH2 and CH3) and heavy chain variable domains (VHHs). Monomeric Nb (Mono-Nb) was constructed previously by linking a MERS-CoV receptor-binding domain (RBD)-specific VHH and a C-terminal His₆, and dimeric Nb (Di-Nb) and trimeric Nb (Tri-Nb) were constructed by linking two or three Mono-Nbs with GGGGS linkers and a C-terminal His₆ tag for easy purification. (B) SDS-PAGE (left) and Western blot (right) analysis of MERS-CoV RBD-specific Nbs. The molecular weight marker (in kDa) is shown on the left. MERS-CoV RBD-targeting Mono-Nb was included as comparison, and MERS-CoV RBD-specific mAb (MERS mAb) and SARS-CoV RBD-specific mAb (SARS mAb) were used as controls. Anti-llama antibody was used for Western blot analysis.

Figure 2

Detection of binding between MERS-CoV RBD-specific Nbs and MERS-CoV RBD proteins. (A) ELISA for binding between Di-Nb or Tri-Nb and RBD wild-type (RBD-WT) protein of the EMC2012 strain and mutant proteins containing RBD mutations from strains isolated from human and camel in 2012, 2013, 2014, and 2015. MERS-CoV RBD-targeting Mono-Nb was used for comparison, and MERS-CoV RBD-specific mAb (MERS mAb) and SARS-CoV RBD-specific mAb (SARS mAb) were included as controls. Data are presented as mean A450 ± standard error (s.e.m.) (n = 2). Experiments were repeated twice, yielding similar results. Significant differences in median effective concentration (EC₅₀) ± s.e.m. were observed between Di-Nb and Mono-Nb, as well as between Tri-Nb and Mono-Nb, indicated by red and green asterisk (*, **, and ***), respectively. Concentration (in nM) was calculated based on predicted molecular weights of 16, 32, 48, and 150 kDa for Mono-Nb, Di-Nb, Tri-Nb, and mAb, respectively. (B) Surface Plasmon Resonance (SPR) analysis of binding between Di-Nb or Tri-Nb and RBD protein (i.e., RBD-WT). MERS-CoV RBD-targeting Mono-Nb was used for comparison. Binding parameters are shown in each figure.

Figure 3

Detection of the neutralizing activity of MERS-CoV RBD-specific Nbs and their inhibition of RBD–DPP4 binding. (A) Neutralizing activity of Di-Nb and Tri-Nb against the prototypic MERS-CoV (EMC2012 strain). The neutralizing activity of the Nbs is expressed as the Nb concentration (nM) that completely inhibited the cytopathic effect (CPE) of MERS-CoV in at least 50% of the wells (50% neutralization dose: ND₅₀). Data are expressed as mean ND₅₀ ± s.e.m. (n = 3). (B) Inhibition of binding between Di-Nb or Tri-Nb and hDPP4 protein, as determined by ELISA. Percentage inhibition is expressed as RBD–hDPP4 binding in the presence or absence of Nbs based on the formula (1 – [RBD–hDPP4-Nb]/[RBD–hDPP4]) × 100. Data are presented as mean percentage inhibition ± s.e.m. (n = 2). (C) Inhibition of binding between Di-Nb or Tri-Nb and hDPP4-expressing Huh-7 cells, as determined by flow cytometry analysis. Percentage inhibition is expressed as RBD–Huh-7 binding in the presence or absence of Nbs, which is calculated based on the formula (1–[RBD–Huh-7–Nb]/[RBD–Huh-7]) × 100. Data are presented as mean percentage inhibition ± s.e.m. (n = 2). For (A)–(C), MERS-CoV RBD-targeting Mono-Nb was used for comparison, and MERS mAb and SARS mAb were included as controls. Experiments were repeated twice, yielding similar results. Significant differences among groups were compared by ND₅₀ ± s.e.m. (A) or median inhibitory concentration (IC₅₀) ± s.e.m. (A,C). Significant differences between Di-Nb and Mono-Nb are shown as red asterisk, and those between Tri-Nb and Mono-Nb are shown as green asterisk (** and ***).