Characterization of novel monoclonal antibodies against the MERS-coronavirus spike protein and their application in species-independent antibody detection by competitive ELISA

Abstract

Since discovering the Middle East respiratory syndrome coronavirus (MERS-CoV) as a causative agent of severe respiratory illness in the Middle East in 2012, serological testing has been conducted to assess antibody responses in patients and to investigate the zoonotic reservoir of the virus. Although the virus neutralization test is the gold standard assay for MERS diagnosis and for investigating the zoonotic reservoir, it uses live virus and so must be performed in high containment laboratories. Competitive ELISA (cELISA), in which a labeled monoclonal antibody (MAb) competes with test serum antibodies for target epitopes, may be a suitable alternative because it detects antibodies in a species-independent manner. In this study, novel MAbs against the spike protein of MERS-CoV were produced and characterized. One of these MAbs was used to develop a cELISA. The cELISA detected MERS-CoV-specific antibodies in sera from MERS-CoV-infected rats and rabbits immunized with the spike protein of MERS-CoV. The MAb-based cELISA was validated using sera from Ethiopian dromedary camels. Relative to the neutralization test, the cELISA detected MERS-CoV-specific antibodies in 66 Ethiopian dromedary camels with a sensitivity and specificity of 98% and 100%, respectively. The cELISA and neutralization test results correlated well (Pearson's correlation coefficients=0.71-0.76, depending on the cELISA serum dilution). This cELISA may be useful for MERS epidemiological investigations on MERS-CoV infection.

Keywords: Competitive ELISA; Epidemiology; MERS coronavirus; Neutralizing antibody.

Fig. 1

Monoclonal antibodies recognize the receptor-binding domain (RBD) of MERS-CoV but not the RBDs of SARS-CoV, Bt-CoV-HKU4, or Bt-CoV-HKU5. (A) ELISA: the baculovirus expression system was used to generate the RBDs of MERS-CoV (amino acids 358–588 of the MERS-CoV S protein) and SARS-CoV (amino acids 318–510 of the SARS-CoV S protein). The RBDs served as antigens in an IgG-ELISA. The ability of the supernatants of the indicated hybridoma clones to recognize the RBDs was examined (left histogram). Rabbit anti-SARS-CoV serum was used to confirm the antigenicity of the SARS-CoV RBD (right histogram). The results were expressed as optical density (O.D.) (405–490 nm). (B) Immunofluorescence assay: the indicated monoclonal antibodies were labeled with FITC and incubated with HeLa 229 cells that expressed the Fc-tagged RBDs of MERS-CoV or the bat CoVs Bt-CoV-HKU4 and Bt-CoV-HKU5. The fluorescent signals were detected under a fluorescence microscope. An Alexa Fluor 488-labeled anti-mouse IgG served as a positive control.

Fig. 2

Neutralizing activity of MAbs 45E11, 45C2, and 43A9 against MERS-CoV. Approximately 100 pfu of MERS-CoV was mixed with serially diluted purified MAbs. The mixtures were then used to inoculate Vero cells in 12-well culture plates. After 4 days, the cells were fixed with 10% formalin and stained with crystal violet. The viral titers were expressed as% pfu relative to the viral titer in the absence of antibody. An unrelated MAb (MAb 9D3) served as a negative control.

Fig. 3

Characterization of mutant viruses that escaped from neutralization by MAbs. (A) Schematic depiction of the MERS-CoV receptor-binding domain (amino acids 367–607, black and hatched sections) and its receptor-binding motif (amino acids 484–567, hatched section). The amino acid substitutions detected in the S protein of the MERS-CoV mutants that escaped the 45E11, 45C2, or 43A9 MAbs are indicated. (B) Ability of the escape mutants to grow in the presence of the three MAbs, including the MAb from which they escaped originally. The MERS-CoV mutants that escaped 45E11 (MERS-K543N and MERS-Q544H), 45C2 (MERS-R542T and MERS- S546Y), and 43A9 (MERS-G462D) were mixed with 10 μg/mL of each MAb and incubated at 37 °C for 1 h. Vero cells were then inoculated with each mixture. After 2 days, the viral titer was determined by using the plaque forming assay. The viral titers are expressed as pfu/mL.

Fig. 4

Ability of the competitive ELISA based on the 45C2 monoclonal antibody (MAb) to detect MERS-CoV S protein-specific antibodies from infected rats and immunized rabbits. The 45C2 MAb was mixed with serially diluted sera from a rat that had been infected with MERS-CoV, two rabbits that had been immunized with the S protein of MERS-CoV, a rabbit that had been immunized with the N protein of MERS-CoV, a rabbit that had been immunized with the S protein of SARS-CoV, a rabbit that had been immunized with UV-inactivated SARS-CoV particles, and a rat that had been mock infected with MERS-CoV. The mixtures were then subjected to cELISA using whole UV-inactivated MERS-CoV antigen. The percent inhibitions relative to the ‘no antibody’ control that were obtained from three independent experiments and are expressed as means and standard deviations are shown. Serum samples that contain neutralization antibodies to live MERS-CoV are shown in the gray boxes.

Fig. 5

Correlation between percent inhibition in the 45C2-based competitive ELISA and the neutralization titers of 63 camel sera, as determined by using live MERS-CoV. Sixty-three sera from dromedary camels from Ethiopia were tested at various dilutions in the 45C2-based competitive ELISA and the neutralization test. The percent inhibition of each sample at the 256-fold dilution in the competitive ELISA is plotted on the y-axis. The neutralization titers (Log₁₀) are plotted on the x-axis. The Pearson’s correlation coefficient (r) was 0.74.