Characterization Of Ultra-potent Human Neutralizing Antibody Repertoires Against MERS-CoV From A Recovered Patient

Middle East respiratory syndrome coronavirus(MERS-CoV)was the second highly pathogenic coronavirus in six human coronaviruses since Sever Acute respiratory syndrome(SARS).As of March 2018,data from the World Health Organization(WHO)showed that more than 27 countries reported MERS cases,including 2189 laboratory-confirmed cases,782 deaths,and up to 35%mortality.To date,no effective preventive vaccines and antiviral applications have been reported.The use of passive immunotherapy and human neutralizing monoclonal antibodies in the emergency treatment of new infectious diseases has been reported.Several studies have suggested that the use of MERS-CoV specific human neutralizing antibodies has been considered as an effective treatment option.At present,although there has been some progress on the study of human specific neutralizing antibodies against MERS-CoV,most human antibodies do not have strong neutralizing activity.Therefore,more and more MERS-CoV-specific human-neutralizing antibodies still need to be developed,and a more comprehensive and in-depth analysis of the epitopes and molecular mechanisms are needed to explore,which provides the basis for vaccine development and clinical application of neutralizing antibodies.In this study,we isolated and expanded B cells from the whole blood of a convalescent patient who was the first imported MERS-CoV infected patient in China in 2015.First,ELISA was used to screen the cloned wells that could specifically bind to the MERS-CoV spine protein,and then the neutralizing activity of positive wells was analyzed and grouped according to the MERS-CoV-S pseudovirus neutralization assay.The RT-PCR technique was used to clone the variable region of the potent-positive[Inhibition Rate(IR)? 90%]B cells into the expression vectors and immunogenetics(IMGT/N-QUEST)analysis was perform after sequencing.Pairs of VH and VL/VK clones(in the same or different wells)were transiently co-transfected into HEK-293T cells for expression and purification to obtain the human monoclonal antibody library that neutralizing targeted MERS-CoV-S protein.Next,we performed functional analysis of the human monoclonal antibody library by S protein binding ELISA and pseudovirus neutralization experiments in vitro.Two human monoclonal antibodies(MERS-GD27 and MERS-GD33)with the highest neutralizing activity in vitro were analysised by competition ELISA,Biolayer Interometry technology(BLI),neutralization test of pseudovirus mutants,and Plaque Reduction Neutralization Test(PRNT)of live MERS-CoV.Further,MERS-GD27 was performed for functional and structural studies in vitro and in vivo by antibody-dependent cell-mediated cytotoxicity(ADCC),antibody-dependent enhancement(ADE),hu-DPP4 transgenic mice,and the crystal structure analysis of monoclonal antibody and RBD complex.Our main findings are as follows:1)2880-well B cells were isolated from PBMCs of the patient recovering from MERS-CoV infection by B-cell isolation technique,and 82 B-cell culture supernatants were tested against MERS-CoV S protein by enzyme-linked immunosorbent assay(ELISA)showing a positive reaction(2.85%of the total).Eleven wells showed the strongest neutralizing activity against the MERS-CoV S pseudovirus(IR ?90%,accounting for 13.41%of the total),and 68 wells showed moderate pseudoviral neutralization activity(50%<IR<90%,accounting for 82.93%of the total,3 wells showed weak pseudoviral neutralization activity(30%?IR<50%,accounting for 3.66%of the total).2)We obtained 110 heavy chain(VH)plasmids and 220 light chain(VL)plasmids from 11-well strong neutralizing active B cells,which were amplified by PCR and cloned.Antibody gene repertoires with potent neutralizing activity revealed that VH genes originated from 16 different germline genes,VL lambda and kappa genes originated from 16 and 9 different germline genes.Among the 22 strong neutralizing mAbs,distinct VH genes of the neutralizing antibody were originated from germline genes VH1-69,and distinct VL genes of the neutralizing antibody were originated from six different germline genes.VH segments showed very low levels of somatic hypermutation(SHM),ranging from zero to only three amino acid substitutions.The lengths of the VH complementarity-determining region 3(CDR3)for this VH vary from 16 to 18 amino acids.However,the VL genes were more diverse.SHM ranged from 0 to 11 amino acid substitutions.3)We produced 22 anti-MERS-CoV human neutralizing monoclonal antibodies by expression and purification.All of them had strong binding activity against S protein,of which 13 were homologous well-derived mAbs and 9 were different well-derived mAbs.Pseudovirus neutralization assays showed that 15 human mAbs showed superior neutralizing activity(IC50 value ? 0.005 ?g/ml).All of these antibody heavy chain germline genes were IGHV1-69.VH germline genes from the same well could show different neutralization activities when paired with different VLs.4)Two homologous well-derived mAbs MERS-GD27 and MERS-GD33 showed the strongest neutralizing activity in neutralization experiments in vitro(consistent with the reported neutralizing monoclonal antibody m336).The IC50 values were 0.0010 ?g/ml and 0.0013 ?g/ml,respectively.The PRNT results were similar to the results of the pseudovirus neutralization test.Antibody concentrations of 0.001 ?g/well had a significant inhibition of live MERS-CoV infection.5)MERS-GD27 and MERS-GD33 showed subnanomolar affinity for the MERS-CoV S protein[equilibrium dissociation constant(Kd)equivalent to 0.775 nM and 0.575 nM,respectively].13 mAbs could be divided into different groups based on their binding activities(high,medium,and low)of competing ability with MERS-GD27 or MERS-GD33.It was found that MERS-GD27 and MERS-GD33 did not bind to the same epitopes of S protein and therefore had a low level of competing activity(<20%).The other 11 mAbs showed discrepant competitive binding activity.6)MERS-GD27 and MERS-GD33 showed a broad spectrum of potent neutralizing activity in neutralizing experiments with 15 mutant MERS-CoV S glycoproteins.Pseudotyped viruses bearing 10 of 15 mutant MERS-CoV S glycoproteins could be neutralized by either MERS-GD27 or MERS-GD33.MERS-GD27 showed effective neutralizing activity against MERS-CoV,as indicated by the robust inhibition on infection by all but five pseudotyped viruses bearing the mutant S glycoprotein,and five mutations(L506F,D509G,V534A,E536K,and A556V)resulted in substantial increases in resistance against neutralization of MERS-GD27.MERS-GD33 differed from MERS-GD27 based on the neutralizing activity against the MERS-CoV pseudoviruses.MERS-GD33 could effectively neutralize the virus with the E536K mutation but could not neutralize the virus with the R511P mutation.7)The combination antibodies(MERS-GD27 and MERS-GD33)show strong synergistic effect in pseudovirus neutralization experiments in vitro.8)MERS-GD27 did not activate NK cells,therefore it could not induce ADCC and ADE effect in vitro.9)It showed significant prophylactic and therapeutic protective effects when transgenic mice infected with lethal virus(3LD50)were injected with 5 mg/kg of MERS-GD27.10)The crystal structure analysis of MERS-GD27 Fab and MERS-CoV RBD protein complexes showed that MERS-GD27 binding sites almost completely overlapped with the receptor binding sites,suggesting that it had a partially conserved neutralization mechanism with m336.In summary,we describe the isolation and characterization of 22 human monoclonal antibodies from the B cells of a naturally infected patient.Two of the most potent mAbs(MERS-GD27 and MERS-GD33)were selected to be further determined on the in vitro biological activities and and potential as candidate therapeutics in aninimal model.Furthermore,the structural basis for neutralization of MERS-GD27 against MERS-CoV was also explored.Our data pave the way for development and application of human mAbs against MERS-CoV infections.