| BackgroundHuman infection of the H7N9 influenza virus was first isolated in China in2013 and again appeared in the southeast coastal area of China in October 2016,forming the fifth epidemic wave.According to data from the World Health Organization(WHO),as of December 2017,the number of epidemics reached 767,292 deaths were reported,and the mortality rate was approximately 38.1%.In the face of this sudden new subtype influenza virus,there has been a lack of effective protective vaccines.Although the first-line clinical antiviral drugs can inhibit the virus replication in the early stage,the efficacy of the infection is not obvious at the later stage,and it has been reported that a drug-resistant strain has been produced in clinical practice.Recent studies have shown that a insert mutations occur in the position of the viral hemagglutinin linker peptide,resulting in the mutation of H7N9 from a low pathogenic avian influenza virus(LPAIV)to a highly pathogenic avian influenza virus(HPAIV).As the virus continues to mutate,there is a potential for a new influenza pandemic.New preventive and therapeutic strategies are imminent.In the face of new infectious diseases,antiviral treatment relies on the selection of effective therapeutic drugs from existing drugs or the development of broad-spectrum antiviral drugs.The highly antiviral effect of monoclonal neutralizing antibodies is an important emergency response.At present,the commonly used technologies of antibody screening include cell hybridoma technology,phage display technology,yeast surface display technology,B cellimmortalization technology and single cell PCR technology.In recent years,with the maturation of single B cell technology,its full human source,high efficiency,high specificity,natural antibody pairing and other advantages make it widely used in the field of antibody preparation,become an important means to quickly respond to new and emerging infectious diseases.Part 1 The specificity and cross-reactivity of HA-antibodies in host sera induced by H7N9 influenza virus ObjectiveTo reveal the production and cross-reactivity of the anti-HA antibody in host sera elicited by the H7N9 influenza virus,and provide basis for the screen of broadly neutralizing antibody from the H7N9 infected individual and vaccine development.MethodsThe coding sequences for the ectodomains of H1,H3,H5 and H7 HA glycoproteins were synthesis,optimized and cloned into the pCDNA3.1-based vector.The recombinant HA proteins of H1,H3,H5 and H7 with C-tag were produced in the mammalian cell expression system.The capture ELISA was established by using one anti-tag antibody to capture the HA antigen in the supernatant of the transfected293 T cells.The binding of sera to H1,H3,H5,H7(2013)and H7(2017)from 22 cases of H7N9 infected patients were tested by the capture ELISA With H1N1-infected group and healthy donors as controls.Results1.The expression plasmids of recombinant HA proteins,H1,H3,H5,H7(2013)and H7(2017)were successfully constructed and verified by sequence analysis.The sizes of these proteins were 72 to 100 KD by western blot analysis.2.The obvious binding to both of H7N9 virus strains isolated in 2013 and 2017 years was observed in the sera from the 22 cases of H7N9 infected patients(P<0.01 by compared with healthy controls or H1N1-infected group).The sera antibodies induced by H7N9 virus were cross-reactive to H1,H5 from group 1(P<0.01 by compared with healthy controls),and H3 from group 2 virus(P<0.05 by compared with healthy controls).Conclusions1.The recombinant HA protein of influenza virus was successfully expressed using a mammalian cell expression system,and a capture ELISA for serum antibodydetection was established.2.H7N9 influenza virus can induce specific antibody responses in the body,and there is a binding reaction between the 2013 LPAI and the 2017 HPAI H7N9 avian influenza virus HA protein.3.H7N9 virus can induce antibody response to heterologous subtype HA(H1,H3,H5),which will support the screen of broadly neutralizing antibody from the H7N9 infected individual and vaccine development.Part 2 Isolation and characterization of human monoclonal antibodies against H7N9 virus ObjectiveTo obtain high-affinity neutralizing antibodies targeting the HA protein from the H7N9 infected individuals and provides an important target for the development of influenza vaccines,and a possible means for clinical treatment.MethodsIsolation of Peripheral Blood Mononuclear Cells from Patients with H7N9 Infection,Flow cytometry sorting of memory B cells labeled with IgD-IgM-CD27+CD38low fluorescent antibody,B cells are cultured in 96-well plates,The antibody-secreting positive wells of the B-cell culture supernatants were screened by the capture ELISA method,and the B-cell deposits corresponding to the positive wells were collected.The single-cell PCR technique was used to clone the heavy and light chain variable region genes in vitro,and ligated to the corresponding heavy and light chain vectors to construct antibody expression plasmids,and expressed in vitro.Finally,the affinity of antibodies was detected by Biolayer Interferometry(BLI);The binding of antibodies to H7(2013)、H7(2017)were tested by the capture ELISA With H1,H3,H5 as controls;Detection of antibodies binding to HA truncated fragments(HA,HA1,HA2,Headless)by ELISA,and antibody competition inhibition experiments were used to explore the antigen binding site of the antibodies;Virus microneutralization assay in vitro to detect antibody neutralizing virus ability.Results1.Isolation of antibodies and Affinity determination After selecting the memory B cells sorted by the flow cytometric sorting system,12 positive wells were screened using the H7 antigen capture ELISA.Collect B-cell deposits corresponding to positive wells,extracting RNA,reverse-transcriptionally synthesizing antibody cDNA,and amplifying the heavy and light chain variable region genes by 2 rounds of PCR,Eight paired pairs of light and heavy chains were obtained by electrophoresis and the size was about 350 bp.The products were recovered by gel and were respectively connected to the corresponding heavy and light chain carriers.Finally,5 antibody expression plasmids were successfully obtained and named: 6C3,1-2,19B2,23B7,24D6.5 pairs of antibodies have strong binding activity,the affinity(KD)between 1nM ~ 12 nM.2.Specificity and cross-reactivity of antibodies The 5 pairs of antibodies had obvious binding to the 2013 LPAI and the 2017 HPAI H7N9 avian influenza virus.Antibodies 6C3,19B2,23B7,24D6 have no cross reaction with proteins H1,H3,H5;Antibody 1-2 cross-reacts with H3 from Group 2,but does not cross-react with H1,H5 from Group 1.3.Analysis of HA binding sites of antibodies Antibodies 6C3,19B2,23B7,24D6 are clearly associated with HA and HA1,but not with HA2 and Headless;Antibody 1-2 is clearly associated with HA,HA2,and Headless,but not with HA1.Antibody competition inhibition test result display,Antibody 1-2 does not compete with antibodies 6C3,19B2,23B7,24D6;Antibody6C3 does not compete with 1-2 and competes with antibodies 19B2,23B7,24D6;Antibody 19B2 does not compete with 1-2,23B7,24D6 and competes with antibody6C3;Antibody 23B7 does not compete with 1-2,19B2 and competes with antibodies6C3,24D6;Antibody 24D6 has no competition with 1-2,19B2 and competes with antibody 6C3,23B7.4.Neutralizing activity of antibodies In vitro virus microneutralization experiment results show,Antibodies 6C3,19B2,23B7,24D6 have obvious neutralizing activity against LPAI H7N9 influenza virus and HPAI H7N9 influenza virus(The IC50 of the LPAI strain was 0.05742,4.832,0.7157,and 2.051μg/ml,respectively;the IC50 of the HPAI strain was 0.2718,1.006,0.9995,and 3.442μg/ml,respectively);And the neutralization activity of the virus is concentration-dependent.With the increase of the antibody concentration,the virus infection rate is significantly reduced.Conclusions1.Using B cell culture technology,5 HA-specific high-affinity monoclonal antibodies were successfully isolated from H7N9 infected individuals.2.The five antibodies showed obvious binding reaction to HA protein of LPAI strain and HPAI strain H7N9 avian influenza virus.3.Antibody 1-2 cross-reacts with other subtype HA proteins,but antibodies 6C3,19B2,23B7,24D6 do not;It is likely that the binding site of antibody 1-2 is located on the stem of HA and that antibodies 6C3,19B2,23B7,and 24D6 are located on the head of HA.In addition,the binding sites of antibodies 23B7 and 24D6 are the same,and the 6C3 binding site of antibodies is the most widely used.4.Antibodies 6C3,19B2,23B7,24D6 had obvious neutralizing activity against both LPAI strain and HPAI strain H7N9 avian influenza virus,and antibody 6C3 had the strongest neutralizing activity and had the potential as a therapeutic antibody. |
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