Screening Of Neutralizing Nanobody Anti Hepatitis E Virus And Its Role In The Antiviral Effect

Hepatitis E virus（HEV）infection can cause Hepatitis E（HE）in humans and animals,which is usually a self-limited disease,but it can also cause fatality rate in pregnant women as high as 30%.HE is a zoonosis and there is no specific drug available at present.The genome of HEV contains three Open Reading frames（ORFs）,among which ORF2 encodes the capsid protein of the virus and contains major epitopes.ORF2 truncated protein p239（aa368-aa 606）can self-assemble into virus-like particles（VLP）,which can induce the immune response.Nanobody was from the variable domains of heavy chains of antibodies（VHHs）of camelid animals,with a natural absence of the light chain and the constant heavy-chain 1（CH1）domain of the heavy chain,which has been widely used in the diagnosis,detection and treatment of various diseases due to their advantages of small molecular weight,good solubility,stability and easy modification.Ferritin,is a kind of ferritin storage protein composed of 24 subunits,can self-assemble into a nanocage.After the fusion of nanobody and ferritin,the nanobody can be displayed on the surface of ferritin with the help of the characteristics of ferritin to enhance the affinity of nanoantibody and prolong the half-life.In the present study,a nanobody against the truncated HEV genotype 3 ORF2 protein g3-p239was expressed in prokaryotic system and fused with ferritin to express as fenobody for the study of their neutralizing activity and antiviral effect,in order to provide a new strategy for designing HEV therapeutic drugs.The results are listed as follows.1.The screening of nanobodies against g3-p239The mixture of 5 mg p239 including g3-p239（HEV-3,Kernow-C1 strain,Gen Bank JQ679013）,g1-p239（HEV-1,Sar55 strain,Gen Bank AF444002）,g3-rabbit p239（rabbit HEV-3,CHN-SX-r HEV strain,Gen Bank KX227751）and g4-p239（HEV-4,CHN-SD-s HEV strain,Gen Bank KF176351）and ap237（avian HEV,Ca HEV strain,Gen Bank JN997392）was emulsified with freund’s adjuvant before the immunization to the Bactrian camel via subcutaneous route for five times every two weeks.After the 4-year-old male Bactrian camel was immunized five times,serum samples were collected to isolate the peripheral blood mononuclear cell（PBMC）.Then,the c DNA was synthesized with the total RNA to amplify the VHH gene.Then,a phage library（6.3×10⁸ individual clones）was constructed and 12 nanobodies against g3-p239 were screened.2.The identification of neutralizing nanobodiesThe prokaryotic and eukaryotic expression vectors of 12 nanobodies were respectively constructed.Immunofluorescence assay（IFA）was used to identify the neutralizing activity of 12 nanobodies,the results showed that pufiried g3-p239-Nb1 and g3-p239-Nb55 could block the attachment of g3-p239 to Hep G2/C3A cells.Purified 12 HRP fused nanobodies were used as detecting antibodies,while g3-p239,g1-p239,g3-rabbit p239 and g4-p239 were used as coating antigens.ELISA showed that g3-p239-Nb55-HRP had the highest binding ability with g3-p239,and had the best cross-reaction with g1-p239,g3-rabbit p239 and g4-p239.In addition,HRP fused nanobodies were used for competitive ELISA when HEV Ig G positive swine serum was used as competitive antibody.The results showed that g3-p239-Nb55 had the strongest competitive rate against the HEV Ig G positive swine serum.According to the above results,g3-p239-Nb55 was selected for for the analysis of neutralizing activity.3.The preparation of fenobody-55The prokaryotic expression vector of ferritin fused g3-p239-Nb55（fenobody-55）was constructed was successfully expressed after IPTG induction.After purification by affinity chromatography,transmission electron microscope observation showed that fenobody-55self-assembled into a nanocaged structure.Addtionnaly,fenobody-55 also cross-reacted with different HEV p239 genotypes.The affinity of fenobody-55 for g3-p239 was about 20 times higher than that for g3-p239-Nb55 by Bio-Layer Interferometry（BLI）.The capture PCR assay showed that pretreated HEV could be captured by fenobody-55,indicating that fenobody-55 had a higher binding ability to native HEV than g3-p239-Nb55.At the same time,FITC-labeled g3-p239-Nb55 and fenobody-55 were injected into BALB/C mice through the tail vein,and serum was collected at different time points.The half-life of g3-p239-Nb55 and fenobody-55 was 31.8 min and 267.5 min,respectively.4.The identification of the neutralizing activity of g3-p239-Nb55 and fenobody-55The dose of g3-239 attaching to Hep G2/C3A cells was optimized,and 8μg and 16μg g3-239 were selected for IFA and Western blot analysis to identify g3-p239-Nb55 and fenobody-55 blocking the attachment of g3-p239 to Hep G2/C3A cells.The blocking effect of g3-p239-Nb55 and fenbody-55 to the attachment of g3-p239 to Hep G2/C3A cells was compared and analyzed.Comparing with NDV-Nb96 and NDV-fenobody-96,g3-p239-Nb55 and fenobody-55 could better block the adsorption of g3-239 to Hep G2/C3A cells in dose-depedent form.The blocking rates of g3-p239-Nb55 and fenobody-55 at 8μM were 79.7%and 97.7%,respectively,and the difference was statistically significant.Subsequently,the neutralization of g3-p239-Nb55 and fenobody-55 to the infection of HEV Kernow-C1/P6 in cells were analyzed.The adaptive Kernow-C1/P6 strain,which can stably multiply in Hep G2/C3A cells,wa detected by IFA and real-time fluorescence quantitative PCR.Compared with NDV-Nb96 and NDV-fenobody-96,g3-p239-Nb55 and fenobody-55 significantly inhibited the infection of Kernow-C1/P6 in Hep G2/C3A cells in a dose-dependent manner,with a blocking rate of 72.6%and 71.0%at 5μM,respectively.5.The identification of antiviral effect of g3-p239-Nb55 and fenobody-55 in vivoUsing SPF rabbits as animal models,r HEV was incubated overnight with g3-p239-Nb55 and fenobody-55,respectively,and challenged intravenously.Fecal and serum samples were collected weekly to detect fecal shedding and antibody levels.The results showed that after r HEV infection in rabbits,fecal sheding and increased antibody levels began at 3-week after infection（3 wpi）,with local lymphocytic phlebitis,peripheral phlebitis,and inflammatory cell infiltration in the liver.It is suggested that g3-p239-Nb55and fenobody-55 showed partial or complete protection against r HEV infection,respectively.6.The determination of epitope of g3-p239 recognized by g3-p239-Nb55In the present study,14 different truncated proteins of g3-p239 were designed and produced,including aa 393-aa 606,aa 380-aa 606,aa 373-aa 606,aa 415-aa 606,aa 440-aa606,aa 453-aa 606,aa 465-aa 606,aa 523-aa606,aa 368-aa 476,aa 393-aa 476,aa 393-aa534,aa393-aa558,aa393-aa 580 and aa 393-aa 593.ELISA results showed that 393-aa 606,aa 380-aa 606,aa 373-aa 606,aa 415-aa 606 and aa 440-aa 606 can react with g3-p239-Nb55-HRP,indicating that a conformational epitope composed of N-terminal and C-terminal was recogonized by g3-p239-Nb55.To determine the key aa participating in the interaction of g3-p239 and g3-p239-Nb55,the candidate epitope was predicted with the software Py MOL.The predicted aa residues of g3-p239 were discretely located in L370,L372,G392,G393,L395,Y397,Y443,W472,S474,V503,W548,E549,A550,G551 and T552.These key residues formed spatial structures which could be recognized by⁹⁸DMDDM¹⁰² of g3-p239-Nb55.To further verify the predicted epitope,the mutated g3-p239-Nb55-HRP,Nb55 ^{D98A-M99A-D100A-D101A-M102A}-HRP was successfully designed and produced.It was found that the binding ability of Nb55 ^{D98A-M99A-D100A-D101A-M102A}-HRP to g3-p239 was greatly decreased compared with that of g3-p239-Nb55-HRP.The predicted key amino acids of nanobody binding to antigen were proved to be correct,and indicated that the above amino acids of ORF2 protein may form a conformational epitope recognized by g3-p239-Nb55.Subsequently,the epitope recognized by the nanobody was determined as a novel conformational one,located on the surface of the viral particles and highly conserved among the different mammalian HEV isolates（genotype 1 to 8）.In summary,12 HEV g3-p239 specific nanoantibodies were screened by phage display technology and g3-p239-Nb55 was identifies with neutralizing ability to HEV.Subsequently,the polyvalent fenobody-55 was produced,whose neutralizing ability was proved to be higher than monovalent g3-p239-Nb55,and their antiviral effects were verified in rabbit model.Analysis of the binding sites of g3-p239-Nb55 and g3-p239 showed that they bound through conformational epitopes,and the key amino acid sites were highly conserved in genotype 1-8 HEV.The preparation of neutralizing nanoantibodies and fenobodies will provide new strategy and materials for the diagnosis and treatment of HEV,as well as the development of new drugs and vaccines.