| ObjectiveTo construct a phage display library of specific nano-antibodies against the Middle East Respiratory Syndrome Coronavirus(MERS-COV)receptor binding domain(RBD)and to screen nano-antibodies with the phage display technology.To design and express different forms of specific nanobody molecules,and compare the biological activities and functional differences of these nanobody molecules.The study will set good foundations for the development of Nanobody drugs against MERS-CoV MethodsThe alpaca were immunized with MERS-CoV RBD recombinant protein,and PBMC were isolated from whole blood and total RNA was extracted after the last immunization.The VHH gene was amplified by PCR and used to construct recombinant phages.TG1 Escherichia coli was transformed by these recombinant phages.Phage display library of specific nano-antibodies against the Middle East Respiratory Syndrome Coronavirus were obtained.The nanobody was screened with phage display technology using RBD recombinant protein as antigen.Nano10 nanobody had been screened and expressed indifferent forms,including: 10-Fc which is a fused protein of Nano10 fragment and human IgG1 Fc fragment and is expected to form as a dimeric form;doublet or triplet antibody molecule-10-2、10-3 in which different copies of Nano10 molecules are ligated in tandem by a flexible linker.Then,the Pichia yeast was used to express different forms of recombinant nanobodies,and four kinds antibody molecules were purified with His-tags or Fc molecules:Nano10monomer(10-his),Nano10 and Fc fusion protein(10-Fc),Nano10 two-tandem molecule(10-2)and Nano10 three-tandem molecule(10-3).The reactivity and affinity of four forms of nanobody with MERS-CoV antigen was detected with indirect enzyme-linked immunosorbent assay and surface plasmon resonance(SPR)technology;Neutralizing activity of nanobodies of different modified forms was verified by neutralization test of pseudovirus and live virus;The blocking effects of nanobodies to block the binding of MERS-CoV RBDs to cell surface receptors was detected by flow cytometry technology.;The protective effect in vivo of candidate nanobody molecules was evaluated in the MERS-CoV sensitive mouse model(hDPP4 transgenic mice).The functional study of the screened nanoantibody was performed further.(1)The key neutralizing sites of the candidate antibody molecules was recognized by the method of site-directed mutagenesis of MERS-CoV RBD antigensto analyze the effect of antibody modification on recognition sites.(2)Antibody neutralization test by the binding of pseudoviruses with MERS-CoV receptor binding regions from different countries in different years was applied to identify the cross-neutralizing activity of candidate Nanobody molecules.(3)The heat stability of candidate nanobody molecules was evaluated by virus neutralization assay,flow cytometric receptor binding and blocking assay after placing nanobodies at different temperatures for different periods,.(4)themetabolic characteristics and antibody stability of the candidate Nanobody in vivo were analyzed by detecting the residue of the nanobody in the mouse serum at different time points after the injection of the nanobody..(5)The prevention and treatment function in vivo of candidate nanobodies was studied using the MERS-CoV infection lethal model ResultsThe MERS-CoV RBD specific nanobody phage library was constructed,and the Nanobody Nano10 with higher affinity was screened by RBD recombinant protein.Four different types of nanobody molecules have been designed(10-his、10-Fc、10-2、10-3),four kinds of nanobodies were successfully obtained by Pichia pastoris expression and purification.The results of the enzyme-linked immunosorbent assay showed that the four types of nanobody molecules and MERS-CoV recombinant antigen have good binding activity;Analysis of nanobody affinity by Surface Plasmon Resonance(SPR)techniques and results showed that modified nanobody molecules have better affinity.Four types of nanobody molecules have good virus neutralization activity in vitro;Flow cytometry experiments have shown that antibody molecules can effectively block the binding of viral RBD protein to cell surface receptors and exerts neutralizing activity by blocking the binding of viral RBD to the receptor.Although all the designed nanobodies have good neutralizing activity in vitro,in the experiment of in vivo,only nanobody 10-Fc can completely protect hDPP4 transgenic mice infected with lethal doses of MERS-CoV virus.Therefore,the dimeric antibody formed by fusing the Nanobody VHH fragment with the human IgG1 Fc fragment had the best antiviral activity in vivo.Further analysis of the neutralizing epitopes of the nanobodies revealed that Aspartate(Asp539)located at position 539 on the viralRBD is a key neutralizing site for candidate nanobody recognition.Furthermore,nanobody modification did not affect this recognition site.Sequence alignment showed that RBDAsp539 is highly conserved in the current MERS-CoV strain.Further neutralization experiments using RBD mutant pseudoviruses revealed that the candidate Nanobody 10-Fc has good neutralizing activity against MERS pseudovirus of different years in different regions,indicating that the candidate nanobody has good cross-neutralization activity against different MERS-CoV strains.In vitro experiments showed that the nanobody retained its good neutralizing activity after standing at 60°C for 7 days,And the in vivo metabolism experiment further found that the in vivo metabolic stability of the candidate nanobody 10-Fc is significantly higher than that of the monomeric antibody molecule 10-his.In vivo experiments showed that,all the hDPP4 transgenic mice,which were injected intraperitoneally with 10 mg/kg 10-Fc,lived with the treatment of nanoantibody at the-3 days and the +1 day and +3days respectively after lethal dose of MERS-CoV infection.The results indicated that the nanobody 10-Fc not only has good therapeutic effect but has preventive effects also.ConclusionIn this study,a high-affinity antibody molecule was screened using the established MERS-CoV RBD specific Nanobody phage display library.The screened antibody molecules were modified in different formats and expressed using the Pichia yeast expression system.By the comparative analysis,the antigen reactivity,affinity,virus neutralizing activity,binding and blocking effects to virus receptors,and in vivo protective effects of different types of candidate nanobodies were clarified.Furthermore,the modified nanobody 10-Fc was also confirmednot only retaining the epitope characteristics of theoriginal nanobody VHH monomer molecule,but also improving the stability and protective activity with more effective and protective immune effect.This study demonstrated an effective strategy for the modification and optimization of nanomolecule,which lays the foundation for further development of anti-MERS-CoV protective antibodies and vaccines. |
