Research And Development Of Potent Neutralizing Antibody Against Human Cytomegalovirus And Its Epitope Identification

Human cytomegalovirus(HCMV)belongs to cytomegalovirus family which is included in Betaherpesvirinae.As a ds DNA virus,HCMV is the largest virus in human herpes virus group with 200 nm diameter and 230-240 kbp genome size.Human is the only host of HCMV which often leads to the increase of cell volume.Based on the time of infection,HCMV infection can be divided into congenital infection and acquired infection.Primary HCMV infection is normally identified as covert infection because of lack of clinical symptoms.Those hypo immunity patients,such as transplant patients and HIV patients could carry the virus for whole life and HCMV could be released from mucous membranes periodically.HCMV infection may cause several vital diseases,such as retinitis,pneumonitis and encephalitis.For pregnant women,HCMV infection could develop intrauterine infection and perinatal infection for fetus,which will give rise to birth defects,mental deficiency and developmental retardation.Most of HCMV infected patients are in latent infection state.Even if HCMV is replicated in vivo,it is mostly asymptomatic.At present,there are no effective and safe drugs for HCMV treatment.Therefore,the treatment of HCMV infection is still limited to symptomatic treatment.For now,drugs that inhibit viral replication(such as ganciclovir and propanoside)have been widely administered for HCMV prevention and treatment,but are always limited by side effects such as myelosuppression,nephrotoxicity,specific CTL reactions and drug resistance.Hence there is great importance and demand to develop potent neutralizing antibody and HCMV vaccine.Human cytomegalovirus is encoded more than 200 proteins,including pp65,pp150,IE1,glycoprotein B,g H,gc II and so on.At present,a number of antibodies which target g B,pp65,1E-1 and pp150 have been developed.However,most of them show poor neutralization activity and unsuitability for protection from HCMV infection.Recent studies have revealed that HCMV can infect fibroblasts,epithelial cells,endothelial cells and macrophages in two ways.Glycoprotein H(g H)complex g B,g H /g L /g O,g M/ g N are necessary for fibrocytes infection while g H,g L,UL128,UL130,and UL131 proteins are detrimental for epicytes,endotheliocytes and macrophages infection.Because the pentameric g H complex is missing in common laboratory strains,its importance in viral tropism,viral pathogenesis,and vaccine design was not fully appreciated.It is not surprising that Towne virus and AD169 virus induced poor neutralizing titers against viral infection of epithelial cells because of missing pentameric g H complex.Thus,the pentameric g H complex is likely a key antigen of HCMV neutralizing antibody.In this study,neutralizing antibodies against HCMV were developed by molecular cloning and antibody engineering techniques.The antibodies were tested at animal level and individual patient levels.The pentameric g H complex could be used as a target antigen for neutralizing antibody against HCMV.There are three parts of this present study:1.Development and characterization of rabbit neutralizing monoclonal antibody against HCMV.The analyses of the types of antibodies against HCMV and the characterization of neutralization activity and binding activity were performed in vivo.Rabbits were immunized with AD169 attenuated virus vaccine containing pentameric g H(g H,g L,p UL128,p UL130,and p UL131).The rabbit Hybridoma cell was obtained through cell fusion and specificity verification.Neutralizing activity and binding activity of antibodies were examined by virus neutralization test and ELISA.The variable region sequence of antibody was obtained by genetic engineering techniques.Based on the results of antibody specificity test,antibody variable region genome was analyzed by cluster online.45 rabbit m Ab against HCMV were successfully prepared,including 25 neutralizing antibodies.The m Abs with≥10-fold neutralizing capacity than HCMVHIG were identified as elite neutralizers(11/25).Among them 8 elite neutralizing antibodies could recognize pentameric g H complex.The majority of binding antibodes barely showed neutralization role and only recognize g B protein instead of pentameric g H complex.Genomic analyses for variable region suggested that 45 antibodies were originated from 26 B cell groups,in which the antigen binding activity and neutralization activity were consistent.The length of amino acid of HCDR3(15.9AA,P=0.009)and LCDR3(12.3AA,P=0.009)of neutralizing antibodies was significantly longer than that of binding antibodies.All information above implied that the antibodes which target to pentameric g H complex have good neutralization activity.2.Development and characterization of human-rabbit chimeric anti-HCMV antibody and humanized anti-HCMV antibodyThe purpose of this study is to develop neutralizing antibodies against HCMV by antibody engineering technology.Genetic engineering and antibody engineering were used to reconstruct human-rabbit chimeric antibody expression vector.Chimeric antibody and humanized antibody were expressed by 293 free style expression system and purified by protein A.Neutralizing activity and binding activity of antibodies were tested by virus neutralization test and ELISA.There were 5 human-rabbit chimeric antibodies and 3 humanized antibodies were successfully developed.The recombinant antibody is closer to human,and maintains neutralization and binding activity.It also proved the significance of CDR3 sequence in antibody construction.The sequence of VDJ region gene has a direct impact on the space epitope as well as the antibody expression.These results show that using antibody engineering technology to develop and optimizing anti-HCMV neutralizing antibodies could be achievable,which lays a theoretical foundation for the research and development of therapeutic antibodies.3.Screening,development and characterization of full human monoclonal antibodies against human HCMVThe purpose of this section is to test that pentameric g H complex is the key target antigen of neutralizing anti-HCMV antibodies produced by HCMV infected individuals.We collected peripheral blood from HCMV infected people with high titer neutralized antibody,and obtained memory B cells by immunomagnetic beads multi step screening.By utilizing the improved PCR amplification technology and primer design,the variable region gene sequence of the antibody was successfully obtained from most of single B cells.Genetic engineering and antibody engineering technology were used for reconstruction and expression of full human anti-HCMV antibody.Neutralizing activity and binding activity of antibodies were tested by virus neutralization test and ELISA.In this part,192 full human anti-HCMV antibodies were successfully obtained.21 antibody clones possessing neutralization performance were detected,and the target antigens of 15 antibodies were pentameric g H complex,accounting for 71.4% of the total neutralization antibody,which was consistent with our expectations.In particular,the light chains of 12 antibodies(12/21)were lambda chain,which accounts for 57.2% of the total neutralized antibody.The results showed that most of the neutralizing antibodies in HCMV infected individuals targeted pentameric g H complex.It is feasible to obtain full human elicit neutralizing antibodies from HCMV infected individuals with high titer.To sum up,whether to use an improved attenuated virus vaccine to immunize rabbits or to test memory B cells in patients with high titer HCMV infection,We have prove that pentameric g H complex is an substantial target antigen for neutralized antibody.Pentameric g H complex can provide new directions and technical guidelines for HCMV vaccine research and development.