The Construction And Neutralization Mechanism Of Nanobodies Against Highly Pathogenic Coronaviruses

Coronaviruses continuously and severely threaten people's health due to their fast transmission and bad results after infection.Up to date,there are three outbreaks of coronaviruses,which are severe acute respiratory syndrome caused by severe acute respiratory syndrome coronavirus(SARS-CoV)in 2003,Middle East respiratory syndrome caused by Middle East respiratory syndrome coronavirus(MERS-CoV)in2012 and coronavirus disease 2019(COVID-19)by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)in 2019.Unfortunately,only vaccines against SARS-CoV-2 are available now.There is neither special drug nor vaccine against SARS-CoV and MERS-CoV.Application of neutralizing antibody is an effective strategy of prevention and control of infectious diseases.The receptor-binding domain,RBD,in the spike protein of coronavirus,can strongly elicit protective antibody in vivo,and thus is an important target for monoclonal antibody or even vaccine research.Currently,there are many researches of monoclonal antibody against SARS-CoV and MERS-CoV,including human antibody,mouse antibody and humanized antibody.Nanobody is the smallest known antibody that can bind to antigen at present.Retaining high affinity and specifity against antigen,nanobody has the advantages of small molecular weight,easy to prepare,low immunogenicity and strong tissue permeability.It can be used for treatment of many diseases and thus has an extensive application prospect.There is little research about nanobody against MERS-CoV and none against SARS-CoV.Therefore,we performed a study of neutralizing nanobodies against MERS-CoV and SARS-CoV targeting their RBD.1.Study of neutralizing nanobody against MERS-CoV.In order to screen neutralizing nanobody against MERS-CoV,four rounds of bio-panning from MERS-CoV-RBD-specific nanobody library were performed using MERS-CoV S1 and S protein.After that,three nanobodies with high binding activity were identified.Using ELISA and bio-layer interferometry,a nanobody that recognized different epitope from Nb MS10(Nb MS10 was a nanobody against MERS-CoV identified previously),designated M34,was identified and expressed and prepared using protokaryon and eukaryon expression system.The binding affinity between M34 and MERS-CoV S1was measured using bio-layer interferometry(K_d=222 p M).MERS-CoV pseudovirus neutralization assay was performed to evaluate the neutralizing activity of M34(IC₅₀=15.2 ng/m L).Competitive ELSIA verified that M34 bound to MERS-CoV RBD in competition with DPP4.Further study showed that M34 blocked the binding between MERS-CoV RBD and DPP4 by flow cytometry and cell-cell fusion.Finally,molecular modeling and docking analysis was performed to predict the interface structure of M34binding to MERS-CoV RBD,which suggested that a hydrogen bond might form between the residue G54 in M34 and residue Y523 in RBD to realize the interreaction.2.Study of biparatope neutralizing antibody against MERS-CoV.To reduce the possibility of virus mutation when single antibody was used,the MERS-CoV nanobody M34 was linked with another nanobody Nb MS10 identified previously by a short peptide.A biparatope antibody was constructed and expressed by eukaryon expression system.The binding activity between the biparatope antibody and MERS-CoV S1 was detected by ELISA.The binding affinity was measured using bio-layer interferometry(K_d=36 p M).The neutralizing activity was evaluated using pseudovirus neutralization assay(IC₅₀=11.1 ng/m L).The results showed that the biological function such as affinity and neutralizing activity of the biparatope antibody was not infected.In stead,it gained neutralizing ability against D539A or D539N mutant MERS-CoV pseudovirus.3.Study of neutralizing nanobody against SARS-CoV.For the screening of neutralizing nanobody against SARS-CoV,alpacas was immunized with SARS-CoV RBD.After immunization,the peripheral blood mononuclear cells were isolated for the construction of phage-displayed nanobody library.Four rounds of bio-panning from the library were performed using SARS-CoV RBD and S1.Four nanobodies were identified using ELISA and the higest binding activity nanobody,designated S14,was selected for further study.The binding affinity was measured using bio-layer interferometry(K_d=143 p M).The neutralizing activity was evaluated using pseudovirus neutralization assay(IC₅₀=10.7 ng/m L).Competitive ELISA verified that S14 bind to SARS-CoV RBD in competition with ACE2.Flow cytometry revealed that S14 took effect by blocking the binding between SARS-CoV RBD and ACE2.Finally,molecular modeling and docking analysis was performed to predict the interface structure of S14binding to SARS-CoV RBD,which suggested that hydrogen bonds might form between the residue S33 and P99 in S14 and residue R449 in RBD to realize the interreaction.4.Study of bispecific antibody against MERS-CoV and SARS-CoV.For the multiple functions of antibody,by linking S14 with Nb MS10 using a short peptide,a bispecific antibody against MERS-CoV and SARS-CoV was constructed and expressed using eukaryon expression system.ELISA was performed to determin the binding activity between the bispecific antibody and MERS-CoV S1 or SARS-CoV S1.The binging affinity was measured using bio-layer interferometry(the K_d is 116 p M and 155p M,respectively).The neutralizing activity was evaluated using pseudovirus neutralization assay(the IC₅₀ is 7.00 ng/m L and 13.7 ng/m L,respectively).The results showed that the biological function such as affinity and neutralizing activity of the bispecific antibody was not infected.It gained the ability of neutralizing both MERS-CoV and SARS-CoV pseudovirus.This study aimed at exploring the specific nanobody against MERS-CoV and SARS-CoV targeting their RBD via phage play technology.After four rounds of bio-panning,nanobodies against MERS-CoV or SARS-CoV were identified respectively.Their biological function was evaluated using ELISA,bio-layer interferometry and pseudovirus neutralization assay.Their neutralization mechanism was investigated using competitive ELISA,flow cytometry and cell-cell fusion.Via gene engineering,a biparatope antibody against MERS-CoV and a bispecific antibody against MERS-CoV and SARS-CoV were constructed by linking nanobodies of different epitope against MERS-CoV and by linking nanobody against MERS-CoV and nanobody against SARS-CoV,respectively.And their biological function was evaluated using the same methods,including ELISA,bio-layer interferometry and pseudovirus neutralization assay.The results showed that the reconstructed antibodies kept the biological function before they were reconstructed.It proved that the antibody reconstruction was successful,which laid the foundation of developing therapy against MERS and SARS and provided a strategy of development of multi-function antibody.