Isolation And Screening Of Neutralizing Monoclonal Antibodies Against Human Adenovirus

Human adenoviruses(HAdVs)are unenveloped linear double stranded DNA viruses belonging to the mammalian adenovirus genus of adenoviridae.HAdVs cause wide range of illness involving the upper or lower respiratory tract,gastrointestinal tract or conjunctiva.Severe clinical outcomes such as pneumonia,brochitis,nephritis,diarrhea,and hemorrhagic conjuncivitis can occur but rarely in people at young age or immunecompromised individuals,resulting in a serious health threat in the world.Outbreaks of adenovirus infection have occurred in many countries or regions around the world.In recent years,there have been outbreaks of adenoviruses in densely populated places such as in schools,childcare facilities and boot camps in China.There are many different of serologically distinct subtypes of adnovirus.Different subtypes of adnovisuses are endemic in different countries or regions in the world.Adenovirus type 3(HAdV3),adenovirus type 7(HAdV7)and adenovirus type 55(HAdV55)are among the most common subtypes of adenovirus infections in China.Currently,there is no specific drug treatment or effective vaccines for prevention of adenovirus infections.Specific immunoglobulin and specific monoclonal neutralizing antibody as therapeutics can be used just like natural humoral immunity for prevention of microbacterium and virus infection in high-risk population.Therefore,potent neutralizing monoclonal antibodies are urgently needed for development of therapeutics for prophylatic treatment of adenovirus infection.Especially those with broadly neutralizing activity against different subtypes of adnoviruses are more desirable because different subtypes of adnoviruses may be involved in different adnovirus infections.However,at present,no monoclonal neutralizing antibody drugs are available for the prevention and treatment of adenovirus infection in the world.Capsid of adenovirus is composed of three major proteins:penton,hexon and Fiber.The top of the Fiber is composed of three subunit domains,which form the trimer Knob(FK).Adenovirus FK protein is involved in not only the first step of binding and adsorption of adenovirus to cell surface receptor,but also the cause of serotype differences of adenoviruses together with hexon protein.Because sequences of the adenovirus FK fragment are more conserved than the hexon fragment among different subtypes of adenoviruses,antibodies targeting on the conserved antigenic epitope of the FK protein should more likely have broder neutralization spectrium against different subtypes of adenoviruses.Thus,purpose of this study is to isolate and characterize antibodies targeting on the FK protein with goal of obtaining neutralizing antibodies as tools to explore the neutralizing antigenic epitope for better design and development of adenovirus vaccine,and as potential candidates for development of antibody therapeutics against adenovirus infections.In this study,blood samples from healthy volunteers were assayed against both HAdV3and HAdV7 in neutralization assays for identifying the best candidate samples for use in antibody isolation.Recombinant FK proteins derived from adenoviruses including Ad3、Ad7、Ad11、Ad14 and Ad55 were produced and used as antigens for characterization of the isolated antibodies.Peripheral blood mononuclear cells(PBMC)corresponding to the blood samples with adenovirus neutralizing activity in neutralization assays were used for sorting antigen-specific single memory B cells by flow cytometry using FK proteins as probes labeled with fluorescence dyes.Immunoglobulin(Ig)variable regions of the light chain and heavy chain gene segments(V_LJ_L and V_HDJ_H)were amplified by RT/nested PCR from the sorted single B cells and used to produce recombinant m Abs.Total of 4recombinant antibodies were identified from the sorted single B cells to bind adenovirus FK proteins.One(TMAb03)of 4 isolated antibodies bound to all 5 FK proteins(Ad3、Ad7、Ad11、Ad14 and Ad55)with high affinity to bind FK proteins at KDs of 6.54E-10,1.00E-08,3.38E-07,4.53E-09 and 5.30E-08,respectively,in surface plasmon resonance(SPR)assays.Neutralization assays showed that m Ab TMAb03 neutralized HAdV7 and HAdV14 with neutralization IC50 titers of 0.2412μg/m L and 0.02617μg/m L,respectively.In summary,4 monoclonal antibodies specific to adenovirus FK protein were isolated from healthy volunteers.Among them,m Ab TMAb03 has broad-spectrum binding activity to adenovirus FK proteins of multiple subtypes(AD3,Ad7,AD11,Ad14 and ad55),has high affinity to bind adenovirus FK proteins and neutralizes adenovirus subtypes 7 and 14.Antibodies isolated from this study can be as tools to further explore important antigenic epitope for vaccine development and can be a candidate component for cocktail-based antibody therapeutics for prevention and treatment of adenovirus infection.