Roles Of Anti-human Tim-1 Antibody In Preventing Ebola And Marburg Pseudovirus Infection And Antibody Optimization

BackgroundBoth Ebola virus?EBOV?and Marburg virus?MARV?are single-stranded RNA viruses,belonging to the Filoviridae family,which could infect human,non-human primates and other mammals.Ebola hemorrhagic fever and Marburg hemorrhagic fever were caused by these viruses are serious lethal infectious disease,which can cause severe immunosuppression and pathological damage,then eventually leading to multiple organ failure,Shock,and the onset of this syndrome is very fast.Ebola and Marburg viruses have been mainly distributed in Africa since the outbreak.However,with the development of economic globalization,Ebola virus and Marburg virus had broken through the geographical limitations,bringing risks and threats to the spread of diseases around the world.T-cell immunoglobulin and mucin-domain containing?Tim-1?is a key member of the Tim protein family,mainly expressed in CD4⁺T cells,CD8⁺T cells,Natural Killer cells?NKs?,macrophages,dendritic cells?DCs?,B cells,and mast cells,Binding their ligands,like phosphatidylserine?PS?and their family membersTim-4 can mediate clearance of apoptotic cells,activation of T cells,proliferation,and secretion of cytokines,which is also associated with a variety of immune dysfunctions.Recent studies have found that Tim-1 molecule can act as a surface receptor for a variety of potent retroviruses,and promotes viral infection by mediating the adhesion of the virus to host cells through interaction with phosphatidylserine?PS?on the viral membrane,such as hepatitis C virus?HCV?,dengue virus?DENV?,Ebola virus?EBOV?,Marburg virus?MARV?,and Zika virus?ZIKV?.The advent of antibody library technology had brought significant breakthroughs to the biopharmaceutical industry,and has broad application prospects of the field of clinical medicine.The basic principle is researching by genetic engineering techniques such as PCR,site-directed mutagenesis,DNA sequence analysis,transformation and transfection.The genotype of the antibody is conjugated to the phenotype.With certain screening procedures,the antigen-specific antibody gene is screened from the antibody library to enrich the antibody with high affinity and high potency.At present,the mammalian cell display platform is the most widely used technology in the eukaryotic expression antibody library display platform.The protein post-translation processing system has higher applicability,then expressed antibody is more similar to the natural antibody,which is often used for screening the full-length antibody library.The antibodies obtained by the screening are similar in structure to the natural antibodies produced by the body,and also have the characteristics of natural antibodies,which could play an important role in antibody research and development.Acute viruses such as Ebola and Marburg are serious threats to people's lives.Therefore,it is urgent to explore new intervention drugs and corresponding drug evaluation methods for the mechanism of viral infection.In the previous study,our laboratory successfully constructed a plasmid against human Tim-1,and then purified the anti-human Tim-1 antibody by eukaryotic cell expression system,and used the Ebola and Marburg pseudo virus technology to test the antibody.Functional evaluation,using bioinformatics technology,and molecular biology techniques to construct a human Tim-1 antibody library through a mammalian cell display system,it is expected to screen for high affinity and biological efficacy of anti-human Tim-1.The antibody lays a foundation for the development of anti-human Tim-1 antibody.ObjectiveUsing pseudo virus technology to evaluate the prevention and treatment result of anti-human Tim-1 antibody in Ebola and Marburg pseudo virus infection,and construct human Tim-1 antibody library by bioinformatics,proteomics and molecular biology techniques.The high-throughput sequencing technology is used to detect the quality of the antibody library,and the human Tim-1 antibody library is efficiently screened by flow cytometry to obtain higher affinity and function than the previous anti-human Tim-1 antibody.We has Purified anti-human Tim-1 antibody to provide a new experimental basis for the development and application of anti-human Tim-1 antibody.Methods?1?Ebola and Marburg pseudo viruses were prepared by artificial liposome method and transfected with EBOV-GP plasmid and MARV-GP plasmid,respectively.?2?The TCID₅₀ method was used to detect the pseudo virus titer of Ebola and Marburg,and the Ebola and Marburg pseudo virus titer values were calculated by the Reed-Muench method.?3?HEK-293T cells with high expression of Tim-1 were transfected with human-Tim-1 plasmid by artificial liposome method.FACS,Real-time quantitative PCR and Western blotting was used to detected the transfection efficiency.?4?HEK-293T cells with high expression of human-Tim-1 were infected by pseudo virus infection technique,and the fluorescence value of target cells was detected by dual luciferase reporter assay.?5?Eukaryotic expression of anti-human Tim-1 antibody,the purified anti-human Tim-1 antibody was identified by Coomassie blue staining assay,and its binding specificity to Tim-1 antigen was confirmed by ELISA and FACS experiments.?6?Pseudo virus infection technique and dual luciferase reporter assay were used to test the anti-infective activity of anti-human Tim-1 antibody against Ebola and Marburg pseudo virus.?7?The pseudo virus infection technique and the dual luciferase reporter assay were used to detect the synergistic effect of anti-human Tim-1 antibody with Ebola and Marburg pseudo virus specific antibodies.?8?Construction of a human Tim-1 mammalian cell membrane displayed antibody library and a human Tim-1 maternal antibody plasmid by molecular biology techniques.?9?High-efficiency screening of human Tim-1 antibody library by flow cytometry.?10?Extract the total DNA of the human Tim-1 antibody library.?11?Expression of a human Tim-1 antibody by a eukaryotic cell expression system.?12?FACS detects the affinity of human Tim-1 antibody to antigen.Results?1?Ebola and Marburg pseudo virus infection activities were detected by dual luciferase reporter assay,and the results showed that pseudo viruses such as Ebola and Marburg were successfully obtained.?2?Through the Reed-Muench method,the TCID₅₀ of the Ebola pseudo virus is 1×10^-1.71/0.1 mL,and the TCID50 of the Marburg pseudo virus is 1×10^-4.35/0.1 mL.?3?The transfection efficiency of human-Tim-1 plasmid was detected by FACS,Q-PCR and Western blotting.The results showed that the transfection efficiency of h?man-Tim-1 plasmid was positively correlated with the transfection quality.Human HEK-293T cells with high expression of Tim-1were obtained.?4?HEK-293T cells were transfected with human-Tim-1 plasmid and Vector plasmid,and then infected with pseudo viruses such as Ebola and Marburg viruses.The results showed that HEK-293T cells with high expression of human-Tim-1 significantly increased the susceptibility to pseudo viruses such as Ebola and Marburg viruses.?5?After eukaryotic expression of anti-human Tim-1 antibody,it was identified by Coomassie blue staining,and two bands of 55kDa and 26kDa were obtained by reduction electrophoresis,and one band was obtained by non-reduction electrophoresis above 180kDa,which was consistent with the position of IgG control antibody.The results showed that the laboratory successfully expressed anti-human Tim-1antibody.Specific binding of antigenic antibodies was confirmed by ELISA assay and FACS assay.?6?Neutralization of Ebola and Marburg pseudo viruses with different concentrations of anti-human Tim-1 antibodies.The results showed that anti-human Tim-1 antibody had good effect of neutralizing Ebola and Marburg pseudo virus infection compared with ricin toxin antibody.?7?Different concentrations of anti-human Tim-1 antibody were used in combination with Ebola and Marburg pseudo virus specific antibodies to detect the infection effects of Ebola and Marburg pseudo viruses.The results showed that anti-human Tim-1 antibody combined with Ebola and Marburg pseudo virus-specific antibodies significantly inhibited Ebola and Marburg pseudo virus infection of HEK-293T cells compared to ricin control antibody,indicating that the anti-human Tim-1 antibody has a good synergistic effect with Ebola and Marburg pseudo virus specific antibodies.?8?The human Tim-1 mammalian cell membrane display antibody library was successfully constructed by molecular biology technology,and its library capacity was 2.8×10⁸.The human Tim-1maternal antibody plasmid was also successfully constructed.?9?Four-stream sorting of human Tim-1 antibody library by flow cytometry was performed to obtain CHO cells containing human Tim-1 antibody library with similar affinity to human Tim-1 female antibody.?10?Total DNA cell extraction was performed on CHO cell lines after 4 rounds of flow sorting,to obtain a human Tim-1 antibody library plasmid which had similar affinity to human Tim-1 maternal antibody.?11?The selected human Tim-1 antibody plasmid was ligated to the eukaryotic expression vector,a brand-new human Tim-1 antibody was successfully expressed.?12?FACS experiments successfully screened human Tim-1 antibodies with similar affinity to the Tim-1 maternal antibody.ConclusionIn this study,anti-human Tim-1 antibody was prepared by eukaryotic expression and protein purification technology,and anti-human Tim-1 was detected by pseudo virus infection technology to have a good effect of neutralizing Ebola and Marburg pseudo virus infection.However,the anti-human Tim-1antibody we prepared was less effective in suppressing Ebola and Marburg virus than Ebola and Marburg specific antibodies.Furthermore,we successfully constructed a human Tim-1 antibody library using computer aided molecular design and molecular biology techniques,and detected antibody libraries by high-throughput sequencing technology.Quality,high-efficiency screening of the human Tim-1 antibody library by flow cytometry,and a human Tim-1 antibody library with similar affinity to the Tim-1 parental antibody was obtained,which was used for the development of human Tim-1 antibody,and provide a new basis of Ebola and Marburg viruses research.