Generation And Application Of The Monoclonal Antibodies Against Human Adenoviruses(HAdVs)

Human adenoviruses(HAdVs)belong to the genus Mastadenovirus in the Adenoviridae family.The viruses,as one of the most common pathogens associated with community acquired pneumonia(CAP),usually result in outbreaks or epidemics in human population.It has a broad spectrum of tissue-tropism and infects the epithelial cells of the respiratory tract,conjunctiva,gastrointestinal tract and genitourinary tract.The viruses are classified into Species(or Subgroup)A to G.Recently,mounting novel adenovirus genome types via gene recombination within subgroup viruses have been identified and there are at least 103 types so far.The double-stranded DNA genome of HAdVs encodes around 40 proteins,among which,there are three important structural proteins,namely Penton base,Hexon and Fiber.Hexon,as the main antigenic target for immune response,has the antigenic region that is common to all mammalian adenoviruses.Moreover,ε of Hexon loop region and y of fiber knob trigger type-specific neutralizing antibodies.Fiber and penton base are involved in receptor binding and viral entry into host cells.HAdVs infection is usually self-limited in the immunocompetent.However,the severe or even fatal infection is usually found in the younger less than 5-year-olds,the aged,the ones with a compromised or deficient immunity as well as the recipients after solid organ or hematopoietic stem cell transplantation.Until now,the national surveillance network on HAdVs and HAdVs infection has not been setup.The need of early detection or alarming of HAdVs infection is required.Furthermore,the therapeutic antiviral agents targeting HAdVs specifically are not available in clinical practice.The available methods for the diagnosis of HAdVs infection include virus isolation,immunofluorescent test for viral antigen and nuclear acid testing(NAT).However,these approaches require well-equipped laboratories,well-trained personals and are time-consuming and difficult for use in the remote and undeveloped areas.Therefore,it is of great significance to search for a diagnosis method with lower cost and simple operation.Additionally,the recovery of the severe treated by intravenous globulin(IVIG)via neutralizing inflammatory factors or blocking viral infection confirmed the protective effects of Abs.High baseline of anti-HAdV-E4(>1:32)could block viral infection.The monoclonal antibodies(mAbs)targeting αv integrin inhibit the internalization of viral particles.Thus,the mAbs with neutralizing activity is expected for potential clinical use.In this study,we described the isolation and characterization of the mouse mAbs designed to direct HAdVs types in clinic as much as possible or has neutralizing activity.By immunizing mice with HAdV-B3,we isolated 39 mAbs using hybridoma technology and the functional characteristics of the mAbs were investigated in ChapterⅠ.The immunochromatographic assay(ICA)using mAbs 7E6 and 3C11 with a cross-reactivity to a serial of tested HAdVs subgroups and high-affinity to Hexon is established and is evaluated in Chapter Ⅱ.Ⅰ.Generation and characterization of mAbs against HAdVs.1.Through subcutaneous multi-point immunization of mice with concentrated human adenovirus particles,splenocyte-myeloma fusion and virus particle binding ELISA screening methods,39 human adenovirus-specific monoclonal antibodies were obtained.All positive clones specifically bind to HAdV-B3 virion.Among them,24 mAbs bind to HAdV-C2,B3,C6 and B7 virion,one cross-reacts with HAdV-B3,C6 and B7 virion,and 10 cross-reacts with HAdV-B3 and B7 virion,4 mAbs only specifically bind with HAdV-B3 virion.2.The Antigen binding activity of 39 mAbs to the Hexon and Fiber protein was tested.19 mAbs specifically bind with Hexon protein,and the other 20 strains of mAbs are non-Hexon binding.3.Using Plaque reduction assay,the neutralizing activity of the 39 mAbs against live virus HAdV-B3 and HAdV-C1 virus was evaluated.Four mAbs,2H4,5B12,3A3 and 1H10 specifically neutralized HAdV-B3,with an IC50 of 1.252,2.619,11.16 and 14.06μg/ml,respectively.2H4 is a Hexon-specific mAbs,5B12,3A3and 1H10 is a non-Hexon-specific neutralizing mAbs.4.Two high-affinity antibodies to Hexon protein,7E6 and 3C11 were obtained and their EC50 value for Hexon-binding are 4.60x10-3 and 9.87x10-2μg/ml,respectively.Ⅱ.Establishment and evaluation of an ICA for rapid detecting of HAdVs.1.Two high-affinity monoclonal antibodies 7E6 and 3C11,with a Hexon-binding EC50 of 5pg/ml and 0.3pg/ml,respectively,are cross-reactive with HAdV-B3,C5 and B7.The pair was processed into ICA test.2.The specificity of the ICA was assayed by testing a serial of respiratory viruses and enteroviruses including human adenovirus,bovine adenovirus,influenza A virus,influenza B virus,murine norovirus,rotavirus,Reovirus and Enterovirus 71.The ICA can specifically bind to mammalian adenovirus,and no visible band was detected in other 6 types of viruses at the titers of 104-106TCID50/ml.3.The sensitivity of ICA to different types of HAdVs is evaluated.The ICA can specifically detect HAdV-C1,C2,B3,E4,C5,C6,B7,D10 and gastrointestinal HAdV-G41,with a limit range of 0.16-103TCID50/ml,respectively.4.The ICA was also tested by original throat swabs of 10 HAdVs patients confirmed by nuclear acid testing(NAT).Seven clinic samples with successful virus isolation tested by this ICA showed positive result.Conclusions1.In this study,a total of 39 mAbs specific for HAdVs was obtained.Ten monoclonal antibodies bind specifically to Hexon.Among them,7E6 and 3C11 have ultra-high affinity.214、5B12、3A3 and 1H10 with HAdV-3 neutralizing activity.The mAbs with neutralizing activity provides a potential application for clinical therapy and prevention.2.An ICA for rapid detecting of HAdVs using mAb 7E6 and 3C11 was successfully established.This ICA show good performance on different subgroups or types of HAdVs as well as throat swab specimen and is an ideal choice for POCT.