Preparation And Epitope Analysis Of Monoclonal Neutralization Antibody Against Human Adenovirus Type 55

Human adenovirus type 55?HAdV55?is a new type B adenovirus found in recent years by recombination of HAdV11 and HAdV14.Because the pre-existing immunity of HAdV55 in the population is generally low,it is easy to break out in densely populated places such as hospitals,military,schools,etc.,which can cause severe respiratory diseases and even death.In order to reduce the harm caused by the outbreak of HAdV55,an effectively theropy method is urgently needed to prevent its spread in the population.Antibodies are a kind of protective protein produced by antibody-secreting cells after antigen stimulation.Monoclonal antibodies are highly specific antibodies produced by a single B-cell clone and only targeting a specific epitope.The antibody can block the infection by interaction or competive epitope with virus.It is widely used in clinical diagnosis,prevention and treatment of the disease.However,there are few reports on the preparation of HAdV55 antibody.Based on the existing HAdV55 candidate vaccine and HAdV14-55 hypervariable region chimeric virus in this laboratory,the subject intends to clone HAdV55-specific monoclonal antibodies from immunized macaques using single-cell PCR technology,and perform activity analysis epitope analysis.The main research work of this paper is summarized as follows:Research purposeA series of HAdV55-specific monoclonal antibodies were obtained.The binding activity and neutralization activity of monoclonal antibody were identified and its IC₅₀0 was determined.Antibody binding epitopes were identified by Ad14-55 HVR chimeric virus.MethodsWe immunized Chinese macaques with Ad55 that we had in our laboratory.Blood samples were collected and PBMC was isolated from macaques after immunization.B cells with specific anti-HAdV55 antibody were selected by flow cytometry.Monoclonal antibody gene sequence was obtained by single cell PCR.Western-blot was used to detect the expression of antibodies;binding effection was detected by ELISA;neutralization effection was detected based on the micro-neutralization experiment of alkaline phosphatase.The neutralization epitopes were analyzed by Ad14-55 HVR chimeric virus.ResultsA total of 276 memory B cells were selected from three 96-well plates using flow cytometry.Specific 47 pairs of antibody genes were cloned using single-cell PCR technology and 21 of them was verified first.Binding active antibodies,13 had neutralizing activity antibodies.Seven of these antibodies were expressed in large quantities,purified and concentrated.After titer determination and epitope analysis,one antibody interacted with the Fiber epitope of HAdV55 and six antibodies interacted with the Hexon epitope of HAdV55.These antibodies neutralized titer results show that the four monoclonal antibodies 28A1,28A5,28F3,and 29A10 have higher titers,and their IC50 are 0.2697ng/ml,0.1981ng/ml,0.4025ng/ml,0.5575ng/ml,respectively,can effectively neutralizes HAdV55 virus.Studies on neutralizing epitopes showed that neutralizing epitopes could be HVR1?HVR2 and HVR7,but further verification is needed.ConclusionThe HAdV55-specific antibody gene can be quickly cloned by single-cell PCR technology;the antibody cloned and expressed by single-cell PCR method has good binding activity and neutralizing activity;the neutralizing epitope of the antibody is identified by HAdV14-55HVR,and HAdV55 is specifed The neutralizing epitopes of the monoclonal antibodies may be HVR1,HVR2,HVR7,but more sample is needed to support this.