Study On The Protection Effect Of Single-chain Fragment Variable Antibodies Against Porcine Major Diarrhea Pathogens Infection

Piglet diarrhea accounts for more than 50%of the total mortality in newborn piglets.It is one of the most important diseases that cause economic losses to the world pig industry.Current major pathogens that causing diarrhea in piglets include porcine epidemic diarrhea virus(PEDV),transmissible gastroenteritis virus(TGEV)and enterotoxigenic Escherichia coli(ETEC).The sicked piglets show symptoms including diarrhea,dehydration,growth retardation and eventually death.The prevention methods against PEDV and TGEV are to immunize the sows by vaccination.The piglets passively obtain antibodies by sucking the sow's milk after birth.However,vaccines usually do not produce high levels of milk-derived antibodies in sows,and the duration time for each piglet suckling milk are different,it is difficult to ensure that each piglet receives enough antibodies to fight against viruses.The prevention and control of ETEC infections depend on antibiotics,however,excessive use of antibiotics will lead to the emergence of drug-resistant ETEC.From these aspects,it is extremely important to seek a new type of immune preparation that effectively fight against diarrhea pathogen infection.For gastrointestinal pathogen infection,oral administration of antibodies or antibody derivatives to animal is an effective method for controlling infectious disease.However,traditional immune preparations have some disadvantages,including poor product uniformity and the risk of inducing immune rejection to animals.With the development of biotechnology,genetically engineered antibodies have become a research a hot spot in the preparation of novel antibody reagents.The most representative of genetically engineered antibodies is single-chain fragment variable(scFv).Many studies have reported treatment of diseases using scFv antibodies in the field of human medicine,but there are few related studies in the veterinary field.In particular,research on porcine-derived scFv antibodies and the application of scFv antibodies in the prevention and treatment of porcine diarrhea pathogen infection are still a blank,and these research is of great significance for the development of new strategies for the prevention and treatment of porcine diarrhea pathogens.In this assay,we first constructed a porcine scFv phage display library,screened scFv antibodies against the porcine diarrhea pathogens(PEDV,TGEV ETEC),and studied the biological characteristics and protective effects of scFv antibodies.The research contents are as follows:1 Acquisition of scFv antibodies against porcine major diarrhea pathogensIn order obtain scFv antibodies against porcine major diarrhea pathogens,the porcine-derived scFv phage display library was constructed,the library capacity was calculated to be 5.5×10⁷cfu/m L(V_H-Linker-V_L?)and 7.2×10⁷cfu/m L(V_H-Linker-V_L?),respectively.Twelve clones were randomly selected and their sequences were derived from the porcine antibody sequences and have diversity.Purified PEDV,TGEV or ETEC k88ac fimbriae was served as antigens,and the constructed porcine derived scFv-phage display library was screened.By using phage-ELISA,three anti-PEDV scFv antibodies(PZZ 21,PZZ 24 and PZZ 35),four anti-TGEV scFv antibodies(TZZ 14,TZZ 19,TZZ 43 and TZZ 46)and two anti-ETEC k88ac scFv antibodies(EZZ 3 and EZZ 24)were obtained.The sequences of positive scFv antibodies were ligated into prokaryotic vector for protein expression.After purification,soluble scFv proteins were obtained.When these pig-derived scFv antibodies are applied to piglets,they avoid the immune rejection of piglets against antibodies due to difference species between antibodies and treated animal.2 Research on biological characteristics of scFv antibodies2.1 Biological characteristics of scFv antibodies against PEDV and TGEVTo develop novel immunological reagents against PEDV and TGEV infection,the biological properties of anti-PEDV scFv antibodies and anti-TGEV antibodies were investigated.The stability test showed that during 4?storage,the activities of scFv antibodies were prolonged after the addition of protease inhibitors.Affinity tests confirmed that scFv antibodies have an affinity of 10⁷-10⁸M^-1,indicating a good binding affinity.The viral proteins recognized by scFv antibodies were analyzed,it was found that PZZ 21,PZZ 24 and PZZ 35 both bind to the S1 subunit of PEDV S protein,TZZ 14,TZZ 19 and TZZ 46 specifically bind to the S protein of TGEV,while TZZ 43 binds to the N protein of TGEV.Virus neutralization assays confirmed that above mentioned anti-S protein scFv antibodies have antiviral effects,and the combination of PZZ 21,PZZ 24 and PZZ 35 had a better anti-viral effect than a single scFv.TZZ 14,TZZ 19 and TZZ 46 also showed synergic antiviral effects.We also analyzed how the scFv antibodies neutralize PEDV virus infection through invasion/adhesion test,results showed that PZZ 21,PZZ 24 and PZZ 35 exert antiviral effects during virus adhesion,but have no effect during virus invasion.2.2 Biological characteristics of scFv antibodies against ETEC K88ac fambriaeETEC K88 fambriae mediate bacterial adhesion and colonization on the surface of intestinal epithelial cells,which is a prerequisite for diarrhea.Cell adhesion inhibition experiment confirmed that EZZ 3 and EZZ 24 can inhibit ETEC adhesion to cells,and the inhibition rates was 51.9±2.7%and 46.7±2.1%,respectively.When the EZZ 3and EZZ 24 were used in combination,the inhibition rate reached 62.6±1.5%.Furthermore,a mouse ETEC-infected diarrhea model was established to analyze the protective effects of EZZ 3 and EZZ 24 on mice.Animal experiment showed that compared with ETEC challenge group,mice in the scFv-treated group do not have obvious clinical diarrhea,and the structure of jejunum was intact.In addition,scFv treatment does not cause damage to mouse tissues and affect the growth of mice,showing biosafety for application.3 Preparation of nanoparticle-based system for scFv antibodies oral deliveryThe above studies have proved that anti-PEDV and TGEV scFv antibodies neutralize viral infections in vitro,anti-ETEC scFv antibodies have protective effects on mouse diarrhea caused by ETEC.However,the digestive tract environment of piglets is complex,after oral administration of the scFv antibodies,their efficiency may be affected by the acidic environment of the stomach and protease.In order to improve the stability of scFv antibodies in the gastrointestinal tract,the scFv molecules were coated into carboxymethyl chitosan-based p H-responsive nanoparticle(CMCS/CS-scFv).By optimizing the input ratio and reaction conditions,the particle size of the CMCS/CS-scFv was 292±21 nm,and PDI was 0.24±0.12.The CMCS/CS-scFv nanoparticles exhibited a spherical shape under transmission electron microscopy,and the size was uniform.The release characteristic of scFv antibodies in the CMCS/CS-scFv nanoparticles were p H sensitive,the release rate of CMCS/CS-scFv nanoparticles in the gastric acid environment was low,and the release rate in the alkaline environment was fast.In addition,CMCS/CS-scFv nanoparticles showed no toxic to intestinal cells and piglets,indicating their application safety.This study provides a scientific basis for the development of novel scFv-based pharmaceutical preparations.4.Protective effects CMCS/CS-scFv nanoparticles on diarrhea induced by porcine pathogensIn order to verify the protective function of CMCS/CS-scFv on piglet diarrhea,this study first compared the protective effects of scFv and CMCS/CS-scFv on piglets infected with a single pathogen.The piglet protection experiment of PEDV found that oral administration with CMCS/CS-PZZ(PZZ including scFv PZZ 21,PZZ 24 and PZZ 35)and PZZ have a protective effect on piglets infected with PEDV,and the diarrhea index of piglets after CMCS/CS-PZZ treatment is significantly lower than oral administrated with PZZ(p<0.05),showing better protective effect.The protection experiment of TGEV also confirmed that the CMCS/CS-TZZ(TZZ including scFv TZZ 14,TZZ 19 and TZZ 46)treatment group have better protective effects on piglet diarrhea than the TZZ treatment group,and the fecal virus load was significantly lower than the TZZ treatment group(p<0.05).Animal ETEC infection experiment confirmed that CMCS/CS-EZZ treatment provides protection for piglets infected with ETEC,and the protection effect of CMCS/CS-EZZ after oral administration is better than EZZ oral administration.Considering the situation of diarrhea pathogen mixed infection,CMCS/CS-scFv(PZZ,TZZ,EZZ)was further used to study their protective effects on PEDV,TGEV and ETEC combined infection.Results showed that the number of virions and bacteria in the feces was significantly lower in CMCS/CS-scFv treated group than that of the challenge group(p<0.05),and the survival rate is significantly high in CMCS/CS-scFv treated group than that of the challenge group.This study demonstrates the protective effect of combined CMCS/CS-scFv on diarrhea pathogen mixed infection,which lays a foundation for large-scale clinical application and formulation optimization.5.Protective effects of Lactococcus lactis that secreting scFv on diarrhea induced by porcine pathogensWhen scFv antibodies are used to treat piglet diarrhea,the longer it stays in the intestine,the better the effect will get.In order to keep the scFv antibodies released in the intestine,inspired by the live carrier vaccine of lactic acid bacteria,this study combined anti-porcine diarrhea pathogen scFv antibodies with Lactococcus lactis(L.lactis)expression system to construct a recombinant L.lactis that secreting scFv antibodies.In vitro experiments confirmed that recombinant L.lactis is acid-base tolerance.In vivo experiments confirmed that recombinant L.lactis colonized in intestinal mucosa and continued secreting scFv antibodies after oral administration.Cell protection experiment confirmed that secreted scFv antibodies are active,anti-virus scFv antibodies showed an neutralization effect on PEDV and TGEV infected cells,anti-K88ac scFv antibodies showed an inhibitory effect on ETEC adhesion cells.Animal protection experiments confirmed that recombinant L.lactis protect piglets after oral administration of diarrhea pathogens,the symptoms of diarrhea caused by PEDV,TGEV or ETEC infection are alleivated.This study provides a scientific basis for the development of a novel scFv-based oral delivery system for the treatment of piglet diarrhea.In summary,a porcine scFv antibody phage display library was constructed,and active anti-PEDV,TGEV or ETEC k88ac fimbriae scFv antibodies were obtained.Viral protein recognized by anti-PEDV and TGEV scFv antibodies and the neutralizing effects of scFv antibodies were analyzed in vitro,the effect of anti-K88ac scFv antibodies against ETEC induced diarrhea were analyzed based on cell adhesion inhibition experiments and mouse diarrhea model.Furthermore,CMCS/CS-scFv nanoparticles capable of resisting the acidic environment and protease in the stomach were prepared,and CMCS/CS-scFv increased the oral stability of scFv antibodies.Piglet challenge experiments confirmed that both scFv and CMCS/CS-scFv have protective effects against diarrhea caused by PEDV,TGEV or ETEC.Moreover,oral administration with CMCS/CS-scFv alone or in combination has a better protective effect than scFv antibodies.Moreover,recombinant L.lactis that secreting scFv antibodies were prepared,and it was confirmed that the recombinant L.lactis-scFv has a good protective effect on diarrhea pathogens in piglets after oral administration.This study explores a new way for the prevention and treatment of piglet diarrhea pathogens,and also provides a useful reference for the development of antibody preparations to control other animal diseases.