Mapping immunodominant sites on the MERS-CoV spike glycoprotein targeted by infection-elicited antibodies in humans

Middle-East respiratory syndrome coronavirus (MERS-CoV) first emerged in 2012 and causes human infections in endemic regions. Most vaccines and therapeutics in development against MERS-CoV focus on the spike (S) glycoprotein to prevent viral entry into target cells. These efforts, however, are limited by a poor understanding of antibody responses elicited by infection along with their durability, fine specificity and contribution of distinct S antigenic sites to neutralization. To address this knowledge gap, we analyzed S-directed binding and neutralizing antibody titers in plasma collected from individuals infected with MERS-CoV in 2017–2019 (prior to the COVID-19 pandemic). We observed that binding and neutralizing antibodies peak 1 to 6 weeks after symptom onset/hospitalization, persist for at least 6 months, and broadly neutralize human and camel MERS-CoV strains. We show that the MERS-CoV S1 subunit is immunodominant and that antibodies targeting S1, particularly the RBD, account for most plasma neutralizing activity. Antigenic site mapping revealed that polyclonal plasma antibodies frequently target RBD epitopes, particularly a site exposed irrespective of the S trimer conformation, whereas targeting of S2 subunit epitopes is rare, similar to SARS-CoV-2. Our data reveal in unprecedented details the humoral immune responses elicited by MERS-CoV infection, which will guide vaccine and therapeutic design.


Figure S1
. Evaluation of MERS-CoV S-directed plasma IgG binding titers.Doseresponse curves of plasma IgG binding to prefusion-stabilized MERS-CoV EMC/2012 2P S for each of the 98 samples analyzed in this study by ELISA.Data are presented for one representative biological replicate.At least two biological replicates each using a unique batch of MERS-CoV S were conducted for each sample.
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It       Neutralization data are presented as mean ± standard error for the two technical replicates conducted.Data presented are from one representative biological replicate.Two independent biological replicates were performed using distinct batches of S1 protein for the antibody depletions as well as unique batches of S1 and S glycoproteins and pseudovirus for the ELISAs and neutralization assays, respectively.

Figure S8. IgG binding and neutralization curves following depletion with MERS-
CoV RBD and NTD.A) RBD IgG binding dose-response curves for each of the 28 samples included in the ELISA analysis after mock-depletion and depletion of RBDdirected antibodies.B) NTD IgG binding dose-response curves for each of the 28 samples included in the ELISA analysis after mock-depletion and depletion of NTDdirected antibodies.C) S IgG binding dose-response curves for each of the 28 samples included in the ELISA analysis after mock-depletion, depletion of RBD-directed or of NTDdirected antibodies.D) Dose-response curves for plasma neutralization of MERS-CoV S VSV after mock-depletion, depletion of RBD-directed or of NTD-directed antibodies.Neutralization data are presented as mean ± standard error for the two technical replicates conducted.Data presented are from one representative biological replicate.Two independent biological replicates were performed using distinct batches of RBD and NTD proteins for the antibody depletions as well as unique batches of RBD, NTD, and spike proteins and pseudovirus for the ELISAs and neutralization assays, respectively.

Figure S2 .
Figure S2.Evaluation of plasma neutralization potency against MERS-CoVEMC/2012.Dose-response curves of plasma neutralization of VSV pseudotyped with MERS-CoV EMC/2012 S for the 98 samples analyzed in this study.Data are presented as the mean ± standard error of two technical replicates from one representative biological replicate.At least two biological replicates with two technical replicates were completed for each sample using two distinct batches of pseudovirus.

Figure S3 .
Figure S3.Correlation analysis of S-directed IgG binding titers and neutralizing antibody titers.The sample with highest S IgG binding titer per individual was included in the analysis.The limits of detection (ED50 or ID50: ≤ 10) are indicated with dashed lines.

Figure S4 .
Figure S4.Evaluation of plasma neutralization potency against MERS-CoV variants and MjHKU4r-CoV-1.Dose-response curves for each of the 30 plasma samples included in the analysis using VSV pseudotyped with the indicated MERS-CoV variant or MjHKU4-CoV-1 S protein.Data are presented as mean ± standard error from one representative biological replicate.At least two biological replicates, each with two technical replicates, were conducted for each sample and each variant tested using unique batches of pseudovirus.

Figure S5 .
Figure S5.Evaluation of MERS-CoV S1-directed plasma IgG binding titers.Doseresponse curves of plasma IgG binding to MERS-CoV S1 analyzed by ELISA.The sample with the highest S IgG binding titer per individual was included in the analysis.Individual 22 was excluded due to sample volume limitations.Data obtained from one representative biological replicate are presented.Two biological replicates using distinct batches of S1 protein were completed for each sample.

Figure S6 .
Figure S6.Evaluation of plasma IgG binding and neutralizing activity following depletion of MERS-CoV S1-directed antibodies.A-B) Dose-response curves of S1-(A) and S-directed (B) IgG binding for each of the 27 samples included in the analysis upon mock-or S1-depletion analyzed by ELISA using MERS-CoV EMC/2012 S1 or prefusionstabilized 2P S. C) Dose-response curves of plasma neutralization of VSV pseudotyped

Figure S7 .
Figure S7.RBD and NTD IgG binding curves.A-B) Dose-response curves of RBD-(A) and NTD-directed (B) IgG binding for each of the 30 samples included in the analysis analyzed by ELISA using recombinantly expressed MERS-CoV EMC/2012 RBD or NTD.Data are presented from one representative biological replicate.At least two biological replicates were conducted using distinct batches of RBD or NTD.

Figure S9 .
Figure S9.Competition ELISA curves.A-B)Competition ELISA curves for the 30 plasma samples analyzed using the indicated S1-directed (A) and S2-directed (B) monoclonal antibodies and prefusion-stabilized MERS-CoV EMC/2012 2P S .Data are presented as mean ± standard error for two technical replicates from one representative biological replicate.Two independent biological replicates using unique batches of biotinylated spike protein as well as monoclonal antibodies were performed for each sample.