Mechanistic Studies On Protective Immunity Of MERS-CoV Receptor-Binding Domain Against MERS-CoV Infection

Middle East respiratory syndrome(MERS)coronavirus(MERS-CoV)was first identified in June 2012.It has since spread to at least 27 countries and continues to threaten human health worldwide.As of March 08,2017,1,917 MERS cases,including 677 deaths(case fatality rate: >35%),have been reported to the WHO.No vaccines against MERS-CoV are currently available for human use.Nevertheless,progress has been made since the emergence of MERS-CoV in 2012,and a number of MERS vaccines have been developed and tested in preclinical stages.Receptor-binding domain(RBD)of MERS-CoV spike(S)protein contains a critical neutralizing domain,and has been extensively investigated as an important target for developing subunit candidate vaccines.In previous studies,different RBD fragments,immunization schedules and optimization,and protection of MERS-CoV RBD as a vaccine target had been explored deeply.However,there are still unanswered questions regarding the mechanisms of protective immunity of MERS-CoV RBD as a subunit vaccine.For example,there are various mutant MERS-CoV strains isolated from different regions and at different times throughout the course of the MERS outbreak from 2012 to 2015.Is it appropriate for the RBD with those mutant residues to serve as a vaccine target if those mutants can lead to significant decrease of the potency of protective immunity? Is it possible to improve the protective efficacy of the RBD vaccine by mimicking the native conformation of the RBD of MERS-CoV S protein? How to evaluate the contribution of a specific epitope of RBD in inducing protective immune responses? Is it possible to rationally design MERS-CoV RBD-based subunit vaccines to completely protect against lethal challenge of MERS-CoV by blocking immunodominant non-neutralizing epitopes? To answer those questions,here we designed the following experiments to conduct extensive and comprehensive research and further elucidate the mechanism of protective immunity of MERS-CoV RBD protein against MERS-CoV infection.1.Characterization of broad-spectrum protective immunity of recombinant MERS-CoV RBD(rRBD)protein against MERS-CoV infectionBased on the representative human MERS-CoV strains isolated from 2012 to2015 and camel MERS-CoV strains,we first constructed 5 MERS-CoV rRBD proteins,designated 2012-RBD,2013-RBD,2014-RBD,2015-RBD and Camel-RBD,respectively,using the RBD of prototypic MERS-CoV strain(EMC-RBD)as the template and site-directed mutagenesis method.We have also constructed eukaryotic expression vectors for package of pseudoviruses expressing S proteins of 17MERS-CoV strains identified during the 2012-2015 outbreaks,and 5 monoclonal antibody(mAb)escape MERS-CoV mutants.All rRBD proteins and pseudoviruses were expressed or packaged successfully in eukaryotic 293 T cell expression system.Co-immunoprecipitation(Co-IP),ELISA and flow cytometry assays were carried out to identify and quantify the binding between rRBD protein and dipeptidyl peptidase-4(DPP4),the receptor of MERS-CoV.Results revealed strong binding of these rRBD proteins with soluble human DPP4(hDPP4)and camel DPP4(cDPP4),as well as cell-associated hDPP4,indicating that all these rRBDs maintained good functionality.Antigenicity analysis by ELISA demonstrated no significant difference between above-mentioned rRBD proteins of divergent human and camel MERS-CoV strains and four neutralizing mAbs(mouse mAb Mersmab1 and human m Abs m336,m337,and m338),indicating that all rRBD proteins maintained good antigenicity.The purified rRBD proteins were then evaluated for their immunogenicity to induce cross-reactive antibody responses in immunized mice,and similarly high titers of MERS-CoV S1-specific cross-reactive antibodies were elicited.Like EMC-RBD,all rRBD proteins elicited similarly high titers of cross-neutralizing antibodies against 22 MERS pseudoviruses mentioned above and 2 live MERS-CoV strains.To elucidate the effect of critical mutations in the receptor-binding motif(RBM)of RBD to the functionality,antigenicity,and immunogenicity,we constructed and expressed 2additional RBD mutant proteins,RBD-FGG and RBD-FGGAA,with simultaneous mutations of multiple key residues in the RBM.Comprehensive evaluation of these two rRBD proteins using aforementioned methods exhibited significantly reduced functionality,antigenicity,and neutralizing immunogenicity as compared to EMC-RBD.However,these RBD mutants with diminished DPP4 binding also led to virus attenuation,suggesting that immunoevasion after RBD immunization is only accompanied by loss of viral fitness and that the outbreak of MERS-CoV strain with those mutants could not be founded in natural environment.Overall,this study provides impetus for further development of a safe,highly effective,and broad-spectrum RBD-based subunit vaccine to prevent MERS-CoV infection.2.Functional study of protective immunity of a trimeric RBD protein by mimicking the native conformation of MERS-CoV RBDThe results from the first section have confirmed that MERS-CoV RBD is an ideal target for development of effective and broad-spectrum subunit vaccine against MERS-CoV.Since the native S protein of MERS-CoV is presented as a trimer on the virus surface,here we further designed a RBD vaccine by mimicking the conformational trimeric structure of MERS-CoV S protein.In order to evaluate the mechanism of native conformation in subunit vaccine design,we constructed and expressed a RBD(e.g.,RBD-Fd)in trimeric conformation by fusing it with foldontrimerization motif in a hope to induce higher protective efficacy.Results have showed that the expressed RBD-Fd protein indeed formed a native trimeric structure,which was confirmed by SDS-PAGE and SEC-MALS.Co-IP assay and ELISA revealed that RBD-Fd bound strongly to DPP4 receptor,and potently inhibited the infection of MERS-CoV to its susceptible cells.Results from animal immunization and serum analysis demonstrated that RBD-Fd elicited robust RBD-specific neutralizing antibodies in mice,and maintained long-term neutralizing activity,being able to protecting 83% of the RBD-Fd-immunized mice from lethal MERS-CoV infection.This study emphasizes the importance of maintaining native conformational structure of envelope glycoprotein in developing subunit vaccines,verified the feasibility of designing MERS-CoV subunit vaccines based on the native conformational antigen,and elucidated the potential of developing MERS-CoV RBD as an important preventive and therapeutic drug.3.Effects and mechanistic studies of glycosylation of MERS-CoV RBD key epitopes on its protective immunity against MERS-CoV infectionThis section was designed to identify non-neutralizing and immunodominant epitopes and illuminate the function of specific neutralizing epitopes of MERS-CoV RBD in inducing neutralizing antibodies and protective immune responses,further revealing the mechanisms of protective immunity in the rational design of MERS-CoV RBD vaccines.Four distinct epitopes,including Arg511,Ala562,Val403,and Thr579,were selected on the MERS-CoV RBD based on its crystal structure and functional characteristics.Glycan probe was introduced to each of the four epitopes by site-directed mutations,and confirmed by SDS gel electrophoresis and mass spectrometry.AlphaScreen assay and fluorescence-activated cell sorting(FACS)were used to analyze the interaction between recombinant RBDs and hDPP4.The results demonstrated that the glycan probe located at residue 562 reduced the binding of the RBD to DPP4,the glycan probe located at residue 511 reduced the binding of the RBD to DPP4 even more,and the ones located at residues 403 and 579 had no impact on DPP4 binding.Functional analysis of epitope's interaction with RBD-specific neutralizing mAbs(hMS-1,m336-Fab,m337-Fab and m338-Fab)revealed that the glycan probe located at residue 511 abolished the binding of the RBD to hMS-1,reduced the binding of the RBD to m336-Fab and m337-Fab,and had no significant impact on the binding of the RBD to m338-Fab.In contrast,the glycan probes located at the other three residues,403,562 and 579,did not interfere with the binding of the RBD to any of these mAbs.Evaluation of the neutralizing immunogenicity of these RBD epitopes demonstrated that the RBDs containing a glycan probe at residues 579 and 511 induced significantly higher and lower neutralizing antibody titers,respectively,as compared with the wildtype RBD vaccine.Nevertheless,the RBDswith a glycan probe at residues 403 and 562 induced insignificant changes in neutralizing antibody titers in mouse sera.In order to evaluate an epitope's neutralizing immunogenicity,we introduced a novel concept “neutralizing immunogenicity index”(NII),and defined it as the contribution of an epitope to the vaccine's overall neutralizing immunogenicity.The NII was determined by masking the epitope with a glycan probe and then measuring the relative change of the vaccine's overall capacity to elicit neutralizing antibody titers.To prove this concept,we investigated the efficacy of an engineered MERS-CoV RBD vaccine with the epitope(e.g.,Thr579)masked by a negative NII score,and demonstrated significantly enhanced efficacy in protecting MERS-CoV susceptible hDPP4-transgenice mice from lethal MERS-CoV challenge.Therefore,this novel approach can be used as an effective tool to quantitatively evaluate the neutralizing immunogenicity of any epitope on the MERS-CoV RBD for designing vaccines.In this study,we have first confirmed that MERS-CoV RBD is an important vaccine target able to induce highly potent and broad-spectrum cross-neutralizing antibodies against infection of divergent circulating human and camel MERS-CoV strains.We have then improved the protective efficacy of RBD vaccine by facilitating it to form native conformational structure as well as masking the non-neutralizing epitopes with glycan probes and exposing the neutralizing epitopes on the RBD.We have further demonstrated the underlying mechanisms of protective immunity of MERS-CoV RBD vaccine against MERS-CoV infection.Overall,this study fills in a critical gap in subunit vaccine design,and can guide the rational design of subunit vaccines against MERS-CoV and other life-threatening viruses with surface glycoproteins.