Isolating Of Neutralizing Antibodies Against Rift Valley Fever Virus And The Preliminary Study Of Their Protective Mechanism

The Rift Valley fever(RVF)is an arthropod-borne zoonotic infectious disease.The agent of the disease was isolated in the Rift valley in Kenya and named Rift Valley Fever Virus(RVFV),which can cause a self-limiting febrile illness,headache in humans,severe even to neurological disorders,vision loss,hemorrhagic fever and death.From 1950 to 1998,Rift Valley fever epidemic mainly occurred on the African continent and caused thousands of deaths.The Rift Valley Fever epidemic that broke out in Saudi Arabia in 2000 was the first outbreak outside the African continent,which caused hundreds of deaths.In 2016,the first imported case also appeared in China.This indicates that Rift Valley Fever has been spreading across regions.However,no vaccines or drugs approved for the prevention or treatment of Rift Valley Fever virus.Therefore,the development of Rift Valley Fever virus vaccine and antiviral drugs is of great significance for the prevention and treatment of Rift Valley Fever virus.Based on this situation,this study constructed a RVF vaccine candidate through recombinant human adenovirus serotype 4 vector,vaccinated a rhesus monkey and screened Rift Valley Fever virus neutralizing antibodies from this rhesus monkey to provide a new way for the treatment of Rift Valley Fever patients and identify the epitopes of neutralizing antibodies to provide theoretical basis for the development of Rift Valley Fever vaccines.The first is the preparation of immunogens.Recombinant human Adenovirus serotype 4 expressing RVFV Gn and Gc protein was constructed using human Adenovirus serotype 4 as a vector and named HAdV4-GnGcopt.The result showed that HAdV4-GnGcopt can express RVFV Gn and Gc protein in HEK293 cells.Meanwhile,the truncated Gn protein(154-469aa)and Gc protein(691-1119aa)were expressed using suspension mammalian cells Expi293F,and the results showed that both Gn and Gc proteins were expressed successfully.Finally,the truncated Gn and Gc proteins were purified using StrepTrap affinity chromatography columns.After the rhesus monkey primary immunized with HAdV4-GnGcopt twice,the rhesus monkey was boosted with Gn and Gc proteins.Then the levels of antibodies to Gn and Gc proteins were analyzed by ELISA.The results indicated that HAdV4-GnGcopt,Gn and Gc proteins successfully induced the generation of antibodies against Gn and Gc proteins in rhesus monkey.Consequently,the Gn or Gc protein-specific memory B cells in peripheral blood in rhesus monkey was screened via Single-cell sorting technology,and a total of 1,253 memory B cells were obtained,accounting for about 0.2%of the total cells.Then,188 memory B cells were used to amplify the antibody genes through single-cell PCR technology,and a total of 104 pairs of antibodies were screened out,of which 57 were kappa light chains and 47 light chain lambda light chains.Finally,11 antibodies to Gn and 24 antibodies to Gc were screened by ELISA.In order to measure the neutralizing antibody level in rhesus monkey and the neutralizing activity of antibodies against Gn and Gc proteins,the Rift Valley Fever virus MP-12 strains expressing eGFP and Renilla luciferase were rescued using reverse genetic technology,named RVFV-SeGFP and RVFV-SRluc,respectively.Then,the susceptible cell line of Rift Valley Fever Virus was identified and the results demonstrated that Huh7(human liver cancer cell line)is a susceptible cell line to Rift Valley Fever virus.RVFV-SeGFP was used to determine the neutralizing antibody titer in rhesus monkey on day 210 after the first immunization.The results showed that HAdV4-GnGcopt,Gn and Gc proteins successfully stimulated the rhesus monkey to generate neutralizing antibodies against Rift Valley Fever Virus.Then,RVFV-SRluc was used to measure the neutralizing activity of 11 antibodies to Gn and 24 antibodies to Gc.The results showed that only 2 antibodies to Gn(1332F11 and 1331E4)owned good neutralizing activities,and the IC50 of the two antibodies are 0.1678 and 0.3388 ?g/mL,respectively.The method of truncating Gn was used to identify the binding peptides of 1332F11 and 1331E4.Although 1332F11 and 1331E4 are two different antibodies,the results showed that both of them bind to the peptide 409GSKKCTGDAAFCSAY423.Alanine-Scanning Mutagenesis was used to identify the critical residues of these two antibodies.The results indicated that the critical residues of 1332F11 and 1331E4 are very similar.According to the obtained critical residues of 1332F11 and 1331E4,software Discovery Studio 4.5 was used for antigen-antibody molecular docking and screening the optimal conformation of the antigen-antibody complex.The results showed that 1332F11 and 1331E4 are both close to the fusion loop in the Gc,although they bind to Gn through different orientations.Finally,according to the optimal conformation of antigen-antibody complex,we speculated that 1332F11 and 1331E4 exert neutralizing effects in the same way.It is speculated that 1332F11 and 1331E4 likely preclude the sterical rearrangement RVFV glycoprotein,hindering the exposure of fusion loop in Gc to the endosomal membranes.In summary,we successfully constructed a recombinant human adenovirus serotype 4 expressing Rift Valley Fever GnGc protein,and the recombinant adenovirus can elicit higher binding antibody levels in rhesus monkey.Its feasibility as a vaccine candidate for Rift Valley Fever Virus can be further explored.Meanwhile,we constructed an infectious clone of the Rift Valley Fever Virus MP-12 strain,which provides important method for the future development of vaccines and antiviral drugs.Finally,we isolated two neutralizing antibodies against Rift Valley Fever Virus from rhesus monkey,explained the possible mechanism of their neutralizing effect and provided theoretical basis for the treatment of patients and the development of vaccines.