Screening And Identification Of Neutralizing Antibodies Against Marburg Virus

Marburg Virus(MARV)is a virulent pathogen that can cause severe hemorrhagic fever in humans and non-human primates(NHPs).The Marburg virus was first discovered and identified in 1967 in Marburg,Germany and was named after this city.Since its discovery,MARV has occasionally burst out several times in Africa,resulting in more than 400 deaths with a devastating mortality rate of 88%.Marburg virus spreads via direct contact with the blood,secretions,organs or other bodily fluids of infected people or animals through broken skin or mucous membranes.Due to its highly infectious and fatal nature,together with currently no clinically approved vaccines and therapeutic drugs,Marburg virus is considered to be a potential bioterrorism warfare agent,making relevant research of great military and social significance in prevention and counteraction of terrorism.Ebola virus,which belongs to the same filovirus family as Marburg virus,caused an outbreak in Western Africa in 2014 with unprecedented magnitude,raising worldwide concern of filovirus.Monoclonal antibodies are considered to be promising therapeutic drugs due to their specificity and their ability to exert therapeutic effects after exposure.In recent years,humanized and fully human antibody technology has developed vigorously,overcoming the shortcomings of traditional murine antibodies such as short half-life and obvious side effects,which greatly promotes the application of monoclonal antibodies in clinical treatment.At present,methods for preparing monoclonal antibodies mainly include hybridoma technology,phage antibody library technology,ribosome display technology,and single cell PCR technology.Among these methods,single cell PCR technology can directly amplify antibody genes from a single B lymphoid cell,with the advantages of rapid and high throughput as well as maintaining the natural pairing of light and heavy chains,making it one of the newest and most efficient antibody screening methods.Our laboratory has established a rapid screening platform for fully human monoclonal antibodies based on flow cytometry technology and single-cell PCR technology,which amplifies naturally-paired antibody genes from immune cells from vaccinated volunteers or patients undergoing rehabilitation.Effective antibodies can be screened for within one week through this platform.Based on this platform,our study was aimed at establishing a method for screening neutralizing antibodies from memory B cells of Rhesus macaques.When faced with a new infectious disease without proved vaccine or immune cells from reconavaiescents for antibody screening,we can rapidly obtain candidate neutralizing antibodies for clinical research using this method,which complements and improves the antibody screening platform,and provide technical reserves against other pathogens.In this study,one male and one female rhesus macaques were immunized four times with recombinant adenovirus type-5 vector-based MARV vaccine(rAd5-MARVopt)followed by one boost vaccination with recombinant MARV glycoprotein.Blood samples were harvested at day 0,7,14 and 28 after every vaccination.MARV GP specific and MARV neutralizing antibody serum titers at different time points were determined by ELISA and pseudovirion neutralization assay.The results indicated that serum antibody titers increased obviously at two or three weeks after vaccination and showed robust neutralizing activity against MARV pseudovirion,suggesting that an effective humoral immunological response has been elicited.In order to amplify the antibody genes of rhesus monkeys,we tried different cell sorting strategies and RT-PCR systems,and finally decided the strategy of sorting antibody secreting cells(ASCs)or antigen specific memory B cells,using SuperScript III Reverse Transcription Kit and multiplex primer combinations.Peripheral blood mononuclear cells(PBMCs)were isolated from blood sample at 14 days after the fifth vaccination using Ficoll-hypaque method.768 antibody secreting cells and 1344 antigen specific memory B cells were sorted through flow cytometry.116 and 336 pairs of antibody genes were amplified using single cell PCR respectively.Thus the positive rate is 15.1% for ASCs and 25% for memory B cells.Sequencing analysis of antibody genes revealed that the antibody genes are of good diversity and have no repetitive cloning.MARV GP?TM and MARV GP?muc proteins were expressed in mammalian cell expression system and insect cell expression system,providing experimental materials for antibody screening and function analysing.Through overlapping PCR,a linear expression cassette including a promoter-leader sequence,an antibody variable region gene,an antibody constant region gene,and a poly-A tail fragment was constructed for each antibody light or heavy chain gene.By transfecting HEK293 T cells,rapid and high-throughput expression of antibodies can be achieved.Then 79 antigen specific antibodies were screened by ELISA assay.We constructed expression vectors for binding antibodies,and performed antibody expression and purification,and further tested their neutralizing activities.It was found that two antibodies 2H5 and 3H5 can convey 73% and 79% neutralizing effectiveness respectively when used at the concentration of 100 ?g/m L.In summary,our study established a method for screening rhesus-derived mAbs based on flow cytometry and single cell PCR technology,providing technical reserves for the study of emerging infectious diseases.Several MARV specific mAbs as well as two neutralizing mAbs were obtained,which can be further investigated as candidate antibodies for the “cocktail therapy” against Marburg virus.