Analysis On Clinical Features Of H7N9 Patients And Preparation Of Humanized Monoclonal Antibodies Against Haemagglutinin

Objective Firstly,to analyze the clinical data of 37 cases of human infection with influenza A(H7N9)virus and to summarize the clinical features.Secondly,to construct phage display antibody library with peripheral blood lymphocytes c DNA from the survivors of H7N9 infected patients.Specifically,reverse transcribe PCR was used to amplify variable fragment of antibody genes and construct Fab antibody phage display library.The phage-display library can be used as a reserve to preparation of humanized high affinity Fab antibody.Avidin-magnetic beads and biotinated HA7 protein were used to pan the phage library for selection of HA7 binding phage clone,of which the plasmid were recovered and Fab antibody were expressed in prokaryotic expression system;The proteins were then purified and affinity against HA7 were measured by ELISA.The aim of this study is to obtain the humanized and specific H7N9 subtype antibody,and provide the basis for the prevention and control of human infection with H7N9 virus by passive immunization.Methods Collection of 37 H7N9 patients' clinical data in Guangdong region during December 2013 to March 2015 and serial blood sample in different disease progress periods(including within 2 weeks after the disease onset,2 to3 weeks,3weeks to discharge and follow up 12 weeks later).Descriptive analysis was used to analyze the clinical characteristics of 37 H7N9 patients' clinical data.On the other hand,plasma and PBMC were isolated from 37H7N9 patients' blood samples.To study the level of HA7 specific antibody in H7N9 patient plasma,ELISA was used to detect the affinity of 37 H7N9patients' plasma antibody to HA7 protein in each stage.PBMC whose corresponding plasma contained high titer of HA7 antibody were selected for total RNA extraction and then reverse transcription to prepare c DNA.Using a set of antibody specific primers,VH,VL gene fragment were amplified from the c DNA and Fab gene segment were assembled by overlap extension PCR,the 1.5kb Fab fragment were then digested and ligated with phagemid vector p Comb3 X.We used phage display technology to construct the phage antibody library of human Fab fragment and used magnetic bead separation technology to screen the specific Fab phage antibody.The positive Fab gene segments were expressed and Fab proteins were purified by Ni-NTA agarose.At last,we checked the protein size of the purified antibody by denatured and un-denatured SDS-PAGE and screening high binding monoclonal antibody by ELISA.Results(1)The level of plasma antibody targeting hemagglutinin in patients that infected with influenza A(H7N9)virus was significantly higher than that in healthy controls(P<0.001).2 to 3 weeks after disease onset,the level of the plasma antibody was significantly higher than that of within 2 weeks of disease onset(P<0.001)and 12 week follow-up(P<0.05).(2)The platform for phage display and Protein expression system is matured.The phage antibody library and prokaryotic expression vector were constructed successfully.The phage display library contains 200 million unique clones.(3)After 3 rounds of panning,we get two HA7 binding antibody Fab39 and Fab35.The two antibodies were expressed in prokaryotic expression system and purified by Ni-NTA agarose.The fully-human Fab antibodies have correct size by SDS-PAGE and high affinity against HA7 protein by ELISA.Conclusions(1)After human are infected with influenza A(H7N9)virus,the level of plasma antibody targeting hemagglutinin reach the peak at 2 to 3 weeks.(2)Human Fab phage antibody library construction and screening technology has been matured in our group.(3)The human monoclonal antibody with high affinity against type 7hemagglutinin has been prepared.It will lay the experimental foundation for the development of therapeutic monoclonal antibody against H7N9 virus in human.