| Zaire ebolavirus(EBOV)can cause acute severe hemorrhagic fever in human and nonhuman primates,with strong infectivity and lethality.The outbreak of the Ebola epidemic in West Africa in 2014 has led to the rapid development of related vaccines and antiviral drugs,but up to now there is no approved drug for the prevention or treatment of Ebola virus.The glycoprotein(GP)on the membrane plays a key role in the invasion of the virus,and is an important target for vaccine and antibody research.A novel recombinant adenovirus type-5 vector Ebola vaccine developed independently by our lab has passed Phase II clinical trials,showing good safety in the human body and can stimulate the immune response effectively.To achieve the protection effect,the vaccines need to be used before infection,so it is necessary to carry out the study of post-exposure treatment.The monoclonal antibody(mAb)has good specificity and pharmacokinetic characteristics,and is the current research focus of immunotherapy.Based on the above reasons,the aim of this study is to screen mAbs from the blood samples collected in clinical trials,and to study their neutralizing activities,binding epitopes and protective mechanisms,to provide an effective therapeutic method for the infection of Ebola virus.Firstly,GP-binding antibodies was selected from the vaccine samples by using the antibody screening platform based on flow sorting and single cell PCR technique.The neutralizing activity of different sera was evaluated by recombinant HIV-EBOV GP-Luciferase pseudovirus in HEK293 T cell in vitro.The neutralizing activity of sera was reflected by the expression level of luciferase reporter gene.The sample with relatively good neutral activity was chosen for the following fluorescent cell sorting.According to the selected surface molecular marker strategy,1920 antibody-secreting cells(ASCs)were isolated from peripheral blood mononuclear cells(PBMC).A total of 462 pairs of VH and VL genes were amplified from single ASCs by reverse transcription-PCR(RT-PCR)and nested PCR,the positive rate was 24%.To rapidly express the genes of antibodies,the paired heavy-and light-chain were constructed into a linear expression cassette respectively and then co-transfected into HEK293 T cells.The content of IgG and the binding activity to GP in supernatant were detected in ELISA respectively,and 32 strains of GP-positive antibody were eventually screened,the yield was 6.93%.Sequence homology analysis showed that these mAbs were different,and their subclass distribution was consistent with that in the human body under normal conditions.Secondly,four different forms of GP were produced to analyze the binding specificity of purified mAbs.The gene of heavy-and light-chains were cloned into pcDNA3.4 plasmid and then expressed in Expi293 cells,and mAbs were successfully purified by using Protein-A Sepharose.Four forms of truncated GP—GPecto,GPdmucin,sGP and GP1 were designed and constructed for further analysis of monoclonal antibodies.In order to determine the optimal expression system and to obtain purified GPs,GPdmucin,with moderate molecular weight and the most priority in the project,was chosen to express in prokaryotic,insect and mammalian cells,respectively.The GPdmucin expressed in prokaryotic cells was unstable,and had poor biological activity.In the insect expression system,insoluble GPdmucin was expressed intracellularly.Soluble GPdmucin was produced in Expi293 mammalian transient protein expression system,and the purity of which was over 90% after purification by Ni affinity chromatography.The biological activity of GPdmucin was confirmed with specific antibodies in ELISA.Then,the other three truncated GPs were expressed and purified in same way.The truncated GPs was used to further analyze the specificity and the binding regions of 17 specific antibodies.16 antibodies could bind to four truncated GPs,indicating that these antibodies bind to the sGP region which is the common region of all truncated GPs.mAb 17F3 only binds to GPecto and GPdmucin and cannot bind to sGP or GP1,suggesting that it binds to the GP2 subunit.Thirdly,the neutralizing activities of mAbs were evaluated in vitro and in vivo.In order to evaluate the protection activity of the selected mAbs,a neutralization experiment was carried out on HEK293 and Vero cells using HIV-EBOV GP and VSV-EBOV GP pesudoviruses,respectively.An antibody 1B3 exhibited a potent neutralizing ability for both pseudoviruses.A mouse model of EBOV infection was used in the National Microbiology Laboratory of Public Health Agency of Canada to determine the protective efficacy of 1B3 in vivo.Antibody 1B3 treatment significantly increased survival in infected mice,and the survival rates were 90% and 60%,respectively,for the animal groups treated with 1B3 at either 1 or 2 days post infection.The neutralizing activity of 1B3 against EBOV suggesting that 1B3 is a potential therapeutic antibody for EBOV infection.There is a conservative sequence homology and structural similarity between GPs of filovirus,especially between the RBD.Previous analysis showed that 1B3 may react with RBD,so we analyzed the broad-spectrum neutralizing activity of 1B3 to filovirus.The results showed that 1B3 could bind to BDBV GP,but could not neutralize BDBV pseudovirus in vitro,indicating that the binding pattern of 1B3 on BDBV was different from that on EBOV.Lastly,we further studied the binding epitopes and neutralization mechanism of mAb 1B3.The key amino acid sites of the combination of 1B3 and GP were predicted and analyzed with the help of the software Discovery Studio.The alanine mutants of GP was cloned into pDisplay vector and expressed on the cell surface.Then the changes in binding activities was verified through Western blot and flow fluorescence analysis.The key amino acids on GP were 117,118,171 and 172,the two pairs of adjacent amino acids at both ends of RBD.When these sites mutated,the binding activity of GP to 1B3 was lost.These four amino acids are conserved in RBD of the filovirus,and we further excluded the possibility of other non-conserved amino acids involved in the binding of 1B3 by constructing mutants.However,1B3,which binds to sGP,cannot bind to GPs of filovirus other than BDBV and EBOV,suggesting that some of the sites in GC may be involved in the binding of 1B3.We analyzed the epitopes of 1B3 by competitive binding assay using mAbs MIL77-1/-2/-3 of which the binding epitopes were known.MIL77-3 bound to GC was competed with 1B3 to bind to the GP,indicating that both binding epitopes were spatially close or overlapped.To study the protective mechanism of 1B3,GPdmucin was digested with thermolysin to excise the region of GC,forming the primed GP(GPcl).Additionally,we produced NPC1 Domain C(NPC1-C)in prokaryotic and mammalian system respectively.However,1B3 did not inhibit the binding of GPcl and NPC1-C,because the binding activity of 1B3 to GPcl was lost when the GC was excised.There are some critical sites for 1B3 binding on RBD,and 1B3 cannot bind to GP after cleavage of GC,indicating that 1B3 plays a protective role through the interaction with both domains.In digestion experiments,the GPdmucin post 1B3 attachment,could still be digested by thermolysin,suggesting that 1B3 does not exert protective activity through blocking the cleavage.In summary,we obtained several GP specific antibodies from the blood samples of vaccine clinical trial subjects,and most of the mAbs bound to the sGP region.The different forms of GP were successfully expressed in mammalian expression systems,and they could be used in analyzing of GP antibodies,evaluation of vaccine efficacy and research on viral pathogenesis.1B3 showed well neutralizing activity in both cells and mice,and was a promising therapeutic antibody for EBOV infection.Studies on binding epitopes and neutralization mechanism suggested that the protective activity of 1B3 might be achieved by interaction with both RBD and GC.This mechanism is different from the reported mAbs,which provides reference and help for the study of GP invasion and the design of the cocktail treatment strategies. |
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