Construction Of Anti-feline Parvovirus Therapeutic Chimeric Antibodies And Resolution Of Their Antigen-binding Epitope

Feline panleukopenia is caused by feline parvovirus(FPV)and the main symptoms are severe diarrhea,enteritis and vomiting,which are lethal to kittens.Currently,there is no specific drug for the treatment of feline panleukopenia,and rapid access to antibodies against FPV is benefit to treatment of cats.Therefore,monoclonal antibodies are potential drugs for the treatment of feline panleukopenia.Highly antiserum or murine-derived monoclonal antibodies are used for feline panleucopenia treatment,but poor specificity or species differences lead to diminished therapeutic efficacy and even allergy.Hence,the development of monoclonal antibodies may serve as a potential therapy for feline panleukopenia.To this end,this study isolated the FPV strain,obtained a therapeutic antibody pair with the feline backbone against this strain,and investigated the antiviral mechanism of the antibody pair.The main studies and results are as follows:1.Highly pathogenic FPV strain Hertz-FPV was isolatedDiseased material infected with FPV was inoculated with crandell reese feline kidney(CRFK)cells.There were typical cytopathic lesions in CRFK cells after 3 generations of blind transmission,and significant fluorescence was observed by indirect immunofluorescence.Subsequently,transmission electron microscopy observed virus particles of approximately 25 nm.The virus titer was 1 ×105 TCID50/mL.This strain belongs to genotype G1.When it was compared with the VP2 sequence of 134 FPV strains and 1 CPV strain,and the nucleotide and amino acid homology were higher than 98%,and the relationship was similar to strains in North China.This indicated that FPV strain was successfully isolated and obtained,which named Hertz-FPV.The cellular infection model showed that the infection rate of CRFK cells was approximately 85%at 16 h after inoculation with this strain.In addition,the hemagglutination potency was 1:512.Animal infection tests showed that this strain infected 8～10-week-old kittens,and the virus was detected in anal swabs on day 2～3,followed by clinical signs such as fever,severe diarrhea,vomiting,and depression.2.The rabbit-feline chimeric antibodies with different epitopes against Hertz-FPV strain were obtained.Sera from New Zealand rabbits immuno-inactivated Hertz-FPV strain had an average neutralizing antibody titer of 1:786 and a hemagglutination inhibition titers of 1:256.A total of 192 specific single B cells were sorted from the spleen of the New Zealand rabbit,and 63 paired antibody genes were amplified by nested PCR.After expression and validation,19 monoclonal antibodies were successfully obtained.Among them,six antibodies were FPV neutralizing,with A40,A50 and B13 showing the best results.The variable regions of these 3 neutralizing antibodies were spliced with the constant region of the feline-derived antibody IgG to obtain the feline-derived chimeric antibodies f-A40,f-A50 and f-B13.Finally,the chimeric antibodies f-A40 and f-A50 with non-competitive epitopes and high neutralization were screened by competitive pairing.3.Therapeutic effects and mechanism of rabbit-feline chimeric antibody pair.The chimeric antibodies were administered for 2 days to kittens artificially infected with FPV for 1 day.The kittens treated with chimeric antibodies paired(f-A40+f-50)did not die,and no organ lesions or infections occurred.Among the kittens treated with a single chimeric antibody(f-A40),one kitten was healthy,two kitten improved after the onset of symptoms,and one kitten died with organ lesions and infection.All of felines in the blank group died with severe organ lesions.Further,the antibody antiviral mechanism was investigated.The results showed that both f-A40 and f-A50 inhibited virus adsorption and the target antigen was FPV VP2 protein.Their affinities were both higher than 10-10 mol/L.In addition,f-A40 recognized VP2 protein through a conformational epitope and f-A50 recognized VP2 protein linear epitope 51NNQTEFKFLENG62.In summary,this study isolated a Hertz-FPV strain and established a cellular infection model,obtained rabbit-feline chimeric antibody pair against this strain,validated the therapeutic effects of the chimeric antibody pair,and provided insight into the target antigens and their neutralizing epitopes.This suggests a novel strategy for the screening of neutralizing antibodies and the treatment of feline panleucopenia,as well as a new target for the development of epitope vaccines.