Research On Influenza Virus Neutralizing Antibody Cloning And New Method For Modification

BackgroupInfluenza is a major social and public health problem which endangering human health.Even seasonal influenza kills 25-50 thousands people worldwide every year.In recent years,the emergence of H1N1,H5N1,H7N9 epedemic challenged the epedemic prevention and control.Since the influenza vaccine provides limited protection,anti-influenza virus medicine is subjected to serious drug resistant mutation,especially there is lack of effective treatment measures for severe influenza patients,anti-viral neutralization antibody is considered to be a more potential medicine.The strategies are important to develop neutralizing antibodies with better protective effects,including antibody modification and optimization of antibody isolation and cloning technology.Currently,Luciferase viral neutralization assay and bispecific antibody modification are important methods for antibody research and development.Objective and MethodsIn this study,combined with single cell culture of plasma cell and single cell PCR technique,the microneutralization antibody screening assay(BMN)with influenza Luciferase virus(IAV-Luc)was optimized to isolate and clone anti influenza neutralization antibody.At the same time,we developed double specific antibody against two subtypes of influenza virus with DVD-Ig and Cross-Mab techniques.1.The plasma cells were isolated from the peripheral blood of the volunteers immunized with seasonal influenza vaccine,and the single cells were culture in a medium with IL-6.The antiviral activity of culture supernatant was detected by the microneutralization assay(BMN)with a low titer virus.The antibody genes in the well with neutralization activity were cloned by single cell PCR and inserted into a expression vector.The cloned antibodies were characterized by HAI,virus neutralization,antigen binding assay.2.By using variable region of heavychain and light chain of antibody A206(neutralizing H1/California/7/2009)and 4E6(neutralizing H5/Hong Kong/482/1997),two bispecific antibodies were constructed with techniques of DVD-Ig or Cross-Mabs.The activity of bispecific antibodies were compared to A206 and 4E6 with the viral neutralization and antigen binding assay.Res?LtsA BMN assay was successfully established to determine the neutralization activity in the c?Lture supernatant of single plasma cell.We cloned 8 neutralization antibodies against H1/CA09(Ab-2C,4E,3A,5A,B1,D5,4A,D7)from the single plasma cell with viral neutralization activity.All the 8 antibodies could bind HA protein of H1/CA09 and neutralize H1/CA09 virus.The antibodies Ab-2C,4E,3A,4A and 5A had hemagglutination inhibitory activity,which indicating that they recognized receptor binding site of HA sphere.Using DVD-Ig or Cross-Mab technique,we successfully constructed two bispecific antibodies against two subtypes of influenza viruses H1/CA09 and H5/HK97.Compared to maternal antibodies,the virus neutralization activity of two bispecific antibodies decreased,and which decreased more significantly of the bispecific antibody based on Cross-Mab technique.ConclusionsThe direct use of single cell PCR to clone neutralization antibody genes from a single B lymphocyte was heavy workload and low efficient.After screening neutralization activity of a single B lymphocyte culture supernatant by the microneutralization assay,it reduced the workload and improved efficient to clone antibody gene from the B cells with neutralization activity.It provided an efficient tool for the subsequent research and development of neutralizing antibodies against influenza viruses.The successful construction of anti-H1/CA09 and H5/HK97 bispecific neutralizing antibodies with DVD-Ig and Cross-Mab techniques laid a technical basis,which would be helpful to develop broad-spectrum neutralizing antibodies against different subtype of influenza viruses.