Preparation And Protective Analysis Of Recombinant Caninized Antibody Against H3N2 Subtype Canine Influenza Virus

Canine influenza is a viral disease caused by canine influenza virus(CIV).The clinical features include bronchitis,pneumonia,and even death.In naturally,influenza viruses from a variety of sources can infect canine and are prone to make the viral genes recombination.H3N2 is the main canine influenza virus subtype prevalent in Asia,and it can also infect cats.As a companion animal,canine may act as an intermediate host for human influenza(such as seasonal H1N1),which poses a great threat to human environment.Therefore,the prevention and control of canine influenza has an important public health significance.Currently,monoclonal antibodies,which have been studied a lot among therapeutic antibodies,have some limitations in clinical application due to their heterogenicity.So,the complete caninized antibodies are an ideal choice.1.Preparation of recombinant caninized antibodies against H3N2 subtype canine influenza virus from peripheral blood lymphocytesIn order to prepare recombinant caninized antibody scFv-Fc,total RNA was extracted from CIV-infected canine peripheral blood lymphocytes.IgG heavy chain(VH),light chain(VL)and Fc fragment were amplified by PCR.VH and VL were ligated by an elastic peptide chain linker using overlap extension PCR,and then transformed into Escherichia coli.Positive colonies were verified by PCR and gene sequencing.After screening,the target scFv was constructed with Fc fragment to recombinant caninized antibody scFv-Fc.Protein analysis indicated that a single-chain antibody(scFv)of about 50 kD and a recombinant antibody(scFv-Fc)of about 89 kD were obtained.The antibody activity assay showed that the scFv and scFv-Fc could specifically bind to CIV,and 1 mg/mL of scFv showed the ELISA titer of 1:210,the hemagglutination inhibition(HI)titer of 1:27,and the neutralizing antibody titer of 1:20.The HI titer of 1 mg/mL scFv-Fc was 1:26,the ELISA titer was 1:25,and the neutralization activity titer was 1:20.2.Preparation of recombinant caninized antibodies against H3N2 subtype canine influenza virus by phage display systemIn order to prepare a high neutralizing activity recombinant caninized antibody,pCANTAB 5E phage display system was used to obtain a phage antibody library.A phage antibody library with the capacity of 8.4×105 CFU/mL was obtained through four rounds biological scouring screens.The phage-ELISA detection result showed the positive rate of the antibody library was over 86%.19 phage antibodies were outstanding in the phageELISA values,and their P/N were all greater than 4.The 19 positive antibody strains were pre-expressed in the host bacterium HB2151,and then,6 strains with high detection value were screened by anti-E-tag ELISA,which were recombined with canine Fc fragment to construct the prokaryotic expression vector.Finally,4 strains of them were successfully constructed.By a series of confirming,all the four recombinant canine antibodies had a higher ELISA titer of 1:211,and two of them(166-Fc and 150-Fc)had the same neutralization activity titer of 1:160.3.Protective analysis of recombinant caninized antibody against H3N2 canine subtype influenza virusIn order to study whether the recombinant antibody has protection in vivo,the recombinant antibody no.166-Fc with 06 virus was evaluated in BALB/c mice and beagles.Mice experiment set the virus group,negative group(PBS),antibody prophylaxis group and antibody therapeutic group.Intraperitoneal injection of maximum protection dose of 20 mg/kg of antibody 24 h before(antibodies prophylaxis group)and after(antibody therapeutic group)challenge.Measuring different periods of relevant indications(weight,lung organ related cytokines and viral load,etc.).Virus group began to reduce weight 2 days after challenge,to heaven the lowest on the 4th day.Lungs and feces viral loads went up on the 2nd day after challenge,reaching the highest on the 6th day.Virus group present a significant difference(P<0.001)with antibody prophylaxis group and therapeutic group,but the viral loads between antibody prophylaxis group and therapeutic group had no difference.Cytokines(IL-6,KL-6,IFN-?,TNF-?)in mice lungs present the same trend and conclusion as the viral loads.Mice lung pathological section results showed that virus group and antibody groups suffer from lung tissue damage on the 6th day but antibody groups were lighter than the virus group.This effect on the 10th day was more obvious.Canine antibody prophylaxis experiment set virus group,negative group(PBS)and antibody group.The maximum antibody protection dose of 20 mg/kg was hypodermic injected 48 hours before challenge.Measuring different periods of relevant indications(temperature,weight,serum cytokines and viral loads,etc.).Antibody prophylaxis experiment of canine showed that virus group appeared clinical symptoms such as weight loss,fervescence.Clinical symptoms reach the highest on the 6th days,showing significant differences(P<0.001)with the antibody group and PBS group.The viral loads in serum,nasopharyngeal swabs and anal swabs of virus group on the 2nd,4th,6th,8th,10th day had significant differences(P<0.001)compared with antibody group.The results of serum cytokines in canine showed that the concentration of KL-6 in the virus group increased and then decreased with the course of disease,and reached the highest level on the 8th day,which was significantly different from the antibody group and the PBS group(P<0.05).Respectively,IFN-y and TNF-? tended to decrease first and then rise,which were significantly different from the PBS group(P<0.05).There was no significant difference in body weight,body temperature,clinical assignment and expression levels of cytokines between the antibody group and the PBS group.Therefore,recombinant caninized antibody can reduce weight loss,fever and other clinical symptoms caused by the virus in canine and mice,and can alleviate the damage to the lung to a certain extent.In summary,in this study,the complete caninized antibody against H3N2 subtype canine influenza virus with neutralizing activity were prepared,and the antibody showed good immune protection in both mice and canine infection models.