Cross-neutralization ability of anti-MERS-CoV monoclonal antibodies against a variety of merbecoviruses

Abstract

In the 21st century, three severe human coronavirus infections have occurred. One of them is the Middle East respiratory syndrome coronavirus (MERS-CoV), a merbecovirus belonging to the family Coronaviridae, is a human pathogenic coronavirus first detected in 2012. Several monoclonal antibodies (mAbs) have been developed for both therapeutics and prevention of MERS-CoV infection. However, the extent to which these anti-MERS-CoV antibodies neutralize other merbecoviruses remains unclear. Here, we evaluated the cross-neutralization ability of ten anti-MERS-CoV mAbs against the pseudoviruses with the spike proteins of five merbecoviruses known to bind to dipeptidyl peptidase 4 (DPP4): three clades of MERS-CoV, a bat-derived merbecovirus (BtCoV-422) and a pangolin-derived merbecovirus (MjHKU4r-CoV). We show that all eight mAbs targeting the receptor-binding domain (RBD) potently neutralize all MERS-CoV clades, but not BtCoV-422 and MjHKU4r-CoV. Of these, the neutralization potency of one mAb, m336, against the MERS-CoV clade B declined due to the V530L substitution detected in certain isolates during the 2015 outbreak in South Korea. On the other hand, although BtCoV-422 was neutralized by the two non-RBD mAbs, 7D10 (targeting the N-terminal domain) and G4 (targeting the S2 subunit), MjHKU4r-CoV found to be resistant. Our findings suggest that combining multiple mAbs targeting different epitopes could be a promising strategy for prevention of future outbreaks caused by novel pathogenic merbecoviruses.

Keywords: MERS-CoV; merbecovirus; neutralizing antibody; outbreak; pandemic; spillover.

Figure 1

Phylogenetic analysis and infectivity of merbecoviruses having high usage of human DPP4. (A) Maximum likelihood-based tree of the merbecovirus complete genome sequences. The highlighted name with animal silhouettes represents the DPP4-using viruses used for experiment. Four representative betacoronaviruses were used as the outgroup. (B) Infectivity assay. HOS-human DPP4/TMPRSS2 cells were infected with pseudoviruses bearing each S. The amount of input virus was adjusted to match the amount of HIV-1 p24 capsid protein. Assays were performed in quadruplicate. The presented data are expressed as the average ± SD of relative light unit (RLU). Each dot indicates the result of an individual replicate.

Figure 2

Binding modes and cross-reactivity of MERS-CoV S RBD-targeting mAbs. (A) Types of MERS-CoV neutralizing antibodies targeting RBD. Antibodies are classified into three groups and shown as cartoon representation. The A/EMC/2012 RBD is colored in green and antibodies in gray. Group 1 antibodies include D12 (PDB ID: 4ZPT) and JC57-14 (PDB ID: 6C6Y). Group 2 antibodies include m336 (PDB ID: 4XAK), CDC2-C2 (PDB ID: 6C6Z) and MCA1 (PDB ID: 5GMQ), Group 3 antibody includes MERS-4 V2 (PDB ID: 5YY5), Group 4 antibody includes KNIH90-F1 (PDB ID: 4ZPT). (B) Neutralization breadth of RBD-targeting mAbs. The heatmap displays the fold change in 50% inhibitory concentration (IC₅₀) values, divided by an IC₅₀ against A/EMC/2012. The color scheme shows the IC₅₀ fold change against 3 clades of MERS-CoVs in addition to BtCoV-422 and MjHKU4r-CoV as the intensity of the blue color in the heatmap. The darkest blue indicates either the fold change more than 6 or the mAb failed to reach IC₅₀ at the highest concentration tested. The red indicates no neutralization (no NT).

Figure 3

V530L substitution of MERS-CoV B/KNIH002 escaping from neutralization by m336. (A) Epitopes of the RBD-targeting mAbs in the alignment of the RBD amino acids sequences of three MERS-CoV strains and two merbecoviruses. The amino acids 525–545 sequences is truncated to show the epitopes of mAbs marked in different colors. The yellow, blue, green and pink represent the epitopes of mAbs from Group 1, Group 2, Group 3 and Group 4, respectively. The amino acid at position 530, highlighted with a red color, differs among MERS-CoV strains: L is present in B/KNIH002, while V is present in A/EMC/2012 and C/HKU270. Binding sites of DPP4 are indicated by red asterisks. (B) Structural insights of the mutation in B/KNIH002 RBD bound by m336. The interaction regions in the complex structure of m336 and the A/EMC/2012 RBD (PDB: 4XAK) (left) and the region in the complex structure of m336 and the B/KNIH002 RBD estimated by AlphaFold3 (right) are shown. In the structures, gray represents the m336 heavy chain, green represents A/EMC/2012 RBD, and pink represents B/KNIH002 RBD. Representative interactions between m336 and A/EMC/2012 RBD are shown as lines with yellow dots. (C) Pseudovirus infectivity. HOS-human DPP4/TMPRSS2 cells were infected with pseudoviruses bearing each S. The amount of input virus was adjusted to match the amount of HIV-1 p24 capsid protein. Assays were performed in quadruplicate. The presented data are expressed as the average ± SD of relative light unit (RLU). Each dot indicates the result of an individual replicate. Statistically significant differences (**p < 0.01; ***p < 0.001) versus parental S were determined by two-sided Student’s t-test. (D) Neutralization assay with m336 and MERS-CoV point mutants at position 530. A neutralization assay was performed using pseudoviruses with A/EMC/2012, B/KNIH002, A/EMC/2012 V530L and B/KNIH002 L530V S proteins, and the mAb, m336. Statistically significant differences (**p < 0.01; *p < 0.05) versus each parental S protein were determined by two-sided Wilcoxon signed-rank tests.

Figure 4

Neutralization spectrum and epitopes of the anti NTD mAb and the anti-S2 mAb. (A) Binding modes of MERS-CoV neutralizing mAbs, 7D10 (PDB: 6 J11) and G4 (PDB: 5WQM), targeting NTD and S2 subunit of A/EMC/2012 RBD. The RBD is colored in green and antibodies in gray. (B) Neutralization breadth of the non-RBD targeting mAbs. The heatmap displays the fold change in 50% inhibitory concentration (IC₅₀) values, divided by an IC₅₀ against A/EMC/2012. The color scheme shows the IC₅₀ fold change against 3 clades of MERS-CoVs in addition to BtCoV-422 and MjHKU4r-CoV as the intensity of the blue color in the heatmap. The darkest blue indicates either the fold change more than 6 or the mAb failed to reach IC₅₀ at the highest concentration tested. The red indicates no neutralization (no NT). (C) The epitopes of 7D10 and G4 in the alignment of the NTD and S2 amino acids sequences of three MERS-CoV strains together with BtCoV-422 and MjHKU4r-CoV. The NTD ranging amino acid 18 to amino acid 35 is shown the upper table with the epitope recognized by 7D10 marked in brown. The amino acid at position 26 of C/HKU270, highlighted with a red color, differs from A/EMC/2012 and B/KNIH002. The S2 subunit ranging amino acid 1,170 to amino acid 1,217 is shown the lower table with the epitope recognized by G4 marked in violet. The amino acid at position 1,176, 1,183–4 and 1,215 of MjHKU4r-CoV differ from MERS-CoVs and BtCoV-422.