Identification Of Neutral Epitope Of Fowl Adenovirus Serotype-4 Fiber1 And Construction Of Its Epitope-VLP Vaccine

Hepatitis-hydropericardium syndrome(HHS)caused by Fowl Adenovirus serotype-4(FAdV-4)brings huge economic losses to the poultry industry.Commercially available vaccines are inactivated vaccines and yolk antibody vaccines,but they have drawbacks in terms of effectiveness.Therefore,it is important to develop novel,safe and highly effective FAdV-4 vaccines.With the rapid development of new vaccines,epitope vaccines that stimulate neutralizing antibody production are of great interest.The preparation of epitope vaccines requires the resolution of the key antigens and neutralizing epitopes of the virus that stimulate the body to produce neutralizing antibodies.FAdV-4 Fiber1,Fiber2,Hexon and penton proteins can stimulate antibody production in birds,but the key antigen for FAdV-4 to induce high affinity neutralizing antibodies are not clear.In tHis study,a large number of monoclonal antibodies against FAdV-4 were screened using single B-cell recombinant antibody technology,from which monoclonal antibody with neutralizing and recognized linear epitope was obtained.Further,the target antigens of the antibodies and their binding epitopes were resolved,and virus-like particle(VLP)based epitope vaccines were prepared.The main research contents and results are as follows.(1)Twenty monoclonal antibodies were screened against the AH-FADV-4 strain.New Zealand rabbits were inoculated four times with attenuated AH-FADV-4 strain.The average neutralization potency of serum on day 7 after the 4th inoculation reached 1:287.Monoclonal antibody p10 was obtained in rabbit spleen cells using phage display technique.The variable region of p10 was constructed to the constant region of the mouse-derived antibody IgG to obtain a rabbit-mouse chimeric antibody.There are 192 antigen-specific single B cells screened by fluorescence-activated cell sorting using CD4,CD8,IgM,IgG,and chimeric antibodies labeled strain as markers.Furthermore,72 groups of paired VH and VLκ genes were obtained by nested PCR amplification,from which 20 monoclonal antibodies with stable and high expression were obtained.(2)Obtained of neutralizing monoclonal antibody 47a and resolution of its antigen and liner antigen-binding epitopeThe 17a,47a,31b and 52b of the 20 monoclonal antibodies inhibited more than 95%of FAdV-4 virus at 5 μg,and 17a and 47a still showed strong neutralization effect at 0.2 μg,where 47a recognized linear epitopes.Adsorption test showed that 47a inhibited FAdV-4 adsorption to LMH cells.Moreover,the target antigen of 47a was identified as FAdV-4 Fiberl protein using mass spectrometry,ELISA,Western blot,and SPR.The predicted binding epitope between 47a and FAdV-4 Fiberl protein was determined by molecular simulation and molecular docking.The predicted results combined with truncated expression,ELISA and western blot results showed that the precise amino acid binding epitope between 47a and FAdV-4 Fiberl protein was 124KQANGALMVK133.(3)Preparation of epitope VLP vaccines and evaluation of their efficacyFirst,animal regression tests determined a lethality rate of 93.33%for AH-FAdV-4 strain with 105 TCID50.Subsequently,epitope 124KQANGALMVK133 was genetically fused to AP205 VLP.The prokaryotic expression system expressed the fused gene and obtained the epitope VLP protein of approximately 18 kDa in size.Immunization at a dose of 50 μg/bird resulted in 100%protection of both the eukaryotic expressed Fiberl protein and the prokaryotic expressed epitope VLP vaccine after challenge.The birds in the negative and blank control groups all died within 9 d with typical HHS.In addition,dose optimization tests showed that doses below 50 μg/bird produced incomplete protection;therefore,50 μg/bird is the optimal dose for immunization.In addition,the chicks did not show any discomfort in the overdose safety test,indicating that tHis vaccine is safe.In summary,20 monoclonal antibodies against FAdV-4 were screened,from which four neutralizing monoclonal antibodies were identified.Among them,47a target Fiberl protein and the epitope of the recognized target antigen Fiberl is 124KQANGALMVK133.Finally,the antigenic epitope was fused with AP205 VLP to obtain a highly protective epitope VLP vaccine,which provides theoretical and technical support for novel vaccine development.