SARS-CoV-2 Neutralizing Antibodies Generation And Adenovirus Neutralizing Antibodies Investigation

Background:Coronavirus(CoV)disease 2019(COVID-19),caused by the severe acute respiratory syndrome coronavirus(SARS)CoV-2,emerged worldwide at the end of the year 2019,causes serious harm to human life and health.So far,new variation strains continued to appear and enhanced the virus’ s transmission capacity and pathogenicity.The characteristics of evading neutralization by sera from vaccinated and convalescent individuals have caused great challenges to the epidemic prevention and control.SARS-CoV-2 neutralizing antibodies and vaccines are of crucial important for controlling the epidemic.This study first from the panning of broad spectrum and potent monoclonal neutralizing antibody in people infected with SARS-CoV-2,which can provide candidate drugs for treatment and can be used in the study of virus neutralization and escape.On the other hand,adenovirus vector vaccine is one of the most important technical platforms for SARS-CoV-2 vaccine in the world.As adenovirus vector vaccine can induce strong humoral and cellular immune response,however,the preexisting adenovirus neutralizing antibodies in the population may reduce the effect of adenovirus vector vaccine.Early studies in our laboratory found that the preexisting immunity of the most commonly used HAdV5 vector in Chinese population were as high as 77.34%.In order to determine whether the other subtypes of adenoviruses are more suitable as vaccine vectors,the second part of this study investigated the preexisting neutralizing antibodies against other subtypes of adenoviruses,including HAdV26 and HAdV35.Objective:1.Screening and cloning high-efficiency and broad-spectrum SARS-CoV-2neutralizing antibodies form peripheral blood of COVID-19 patients,and further analyzing the antibody binding antigen epitope and possible neutralization mechanism.2.To study the distribution of n Abs against HAdV26 and HAdV35 in Chinese population.Research method:1.Screening of neutralizing antibodies against novel SARS-CoV-2Ⅰ.Peripheral blood mononuclear cell(PBMC)from SARS-CoV-2 infected individuals were immortalized by Epstein-Barr virus.Enzyme-linked immunosorbent assay(ELISA)were used to screen antigen-specific B cells that can bind to SARS-CoV-2 spike(S)protein,then the heavy chain and light chain gene of antibody variable region were amplified by polymerase chain reaction(PCR)and constructed into full-length antibody expression vector.The cloned antibody genes were expressed in 293 cells and the antibodies were purified with protein A.Ⅱ.The binding activity of the antibodies to SARS-CoV-2 RBD protein and S protein was detected by ELISA,and the affinity of antibody with SARS-CoV-2 RBD protein and S protein was detected by bio layer interferometry(BLI).The antibodies with excellent binding activity and affinity were screened,and the neutralization activity of monoclonal antibody against SARS-CoV-2 wild type(WT)and other variants were detected by pseudovirus neutralization test and authentic virus neutralization test.Finally,the in vivo protective effect of monoclonal antibody was detected by animal experiment.Ⅲ.Through epitope competition experiment and bioinformatics methods,the possible bounding epitopes of antibodies were analyzed,and the possible mechanism of neutralization was speculated.2.Detection of neutralizing antibodies to HAdV26 and HAdV35 in general populationⅠ.A total of 1184 peripheral blood samples of healthy people were randomly collected from Guangdong Province and Shandong Province,including 942 from Guangdong Province and 260 from Shandong Province.Ⅱ.The replicative HAdV26 SEAP and HAdV35 SEAP recombinant viruses based on alkaline phosphatase(SEAP)reporter gene were constructed,and the micro-neutralization experimental methods were established.Ⅲ.The distribution of HAdV26 and HAdV35 neutralizing antibodies in general population was detected by micro-neutralization test.Research results:Our results showed that the monoclonal antibody N7D12 had excellent neutralizing activity against SARS-CoV-2 WT,with IC50 0.08μg/ml for pseudovirus neutralization test and 110.9ng/ml for authentic virus neutralization test.Then the neutralization activity of monoclonal antibody N7D12 against different variants of SARS-CoV-2 was detected by pseudovirus neutralization test and authentic virus neutralization test,we found that the monoclonal antibody N7D12 can easily neutralize SARS-CoV-2 WT and delta(b.1.617.2),but it had no neutralizing activity against beta(b.1.351)and Omicron(b.1.1.529).In the animal protection experiment,whether in the prevention group or the treatment group,the monoclonal antibody N7D12 can significantly reduce the viral load in the lung tissue and upper respiratory tract mucosa of Syrian hamsters.Further epitope competition experiment showed that the mutations of F490 and K417 significantly affected the binding activity of monoclonal antibody N7D12 to RBD protein.BLI analysis suggesting that antibody N7D12 can only bind the RBD in "up" state but not "down" state within the SARS-CoV-2 S protein.We found that the positive rate of neutralizing antibody for HAdV26 and HAdV35 were 47% and 15.8% respectively,HAdV26-seropositive individuals tended to have more moderate n Abs titers(201–1000),while low n Abs titers(72–200)were more frequently detected in HAdV35-seropositive individuals.Individuals younger than 20 showed very low seropositivity rates,the seropositivity rates of HAdV26 increased with age before 70 and decreased thereafter,while HAdV35 seropositivity rates did not show similar characteristics.Surprisingly,the seropositive rates and n Ab levels were higher in Guangdong province than those in Shandong province,but had no strong correlation with gender.Notably,the seroprevalence between HAdV26 and HAdV35 showed little correlation with each other,and no significant cross-neutralizing activity were detected either.Conclusion:1.We isolated the monoclonal antibody N7D12 from patients infected with SARS-CoV-2,which can effectively neutralize SARS-CoV-2 WT and delta(b.1.617.2),and has potential clinical application value for patients infected with corresponding variants,and preliminarily confirmed that K417 and F490 on RBD protein are the key functional sites of monoclonal antibody N7D12,which can provide reference for antibody drug development and vaccine design.2.This study also systematically detected the neutralizing antibodies of HAdV26 and HAdV35 in general population through a large cohort,and revealed the distribution characteristics of preexisting immunity against HAdV26 and HAdV35 in different regions and populations in China.In conclusion,the results of this study provide important basic data for the development of neutralizing antibodies and vaccines against SARS-CoV-2.