Immunoprotection And Mechanism Of Anti-idiotypic Nanobody Of Porcine Circovirus Type 2 Cap Protein

Porcine circovirus type 2（PCV2）is the major pathogen causing Porcine Circovirus-Associated Disease（PCVAD）,which can result in serious economic loss in the pig industry.The capsid protein（Cap）of PCV2 is the only structural protein associated with PCV2 infection and immunity that contains antigenic epitopes of the virus.Vaccine immunization is the most important measure to prevent and control PCV2,but immunization failures occur from time to time as the genotypes of PCV2 epidemic strains continue to change.Anti-idiotypic antibody is a specific antibody against the idiotype on the variable region of the antibody.Immunologist Jerne proposed the immune network theory,which states that after a host comes into contact with an antigen,the body can produce antibody Ab1 against the antigen,and subsequent idiotypic variants of Ab1 can induce the production of anti-idiotypic antibodies.AId or Ab2).There are four types of Ab2:AB2α,ab2β,AB2γand ab2δ.Only Ab2βcan mimic the antigen,also known as the antigen"inner image",and can be used as an alternative antigen for the development of vaccines against idiotypes.However,the production of traditional anti-idiotypic antibody using monoclonal and polyclonal antibodies is complex and has a high failure rate.Nanobody（Nb）,or single-domain antibody,is derived from the heavy chain variable region（VHH）of antibodies in the family Camelidae,and is characterised by its small size,simple structure,high antigen-binding affinity,and remarkable stability under extreme conditions,which make it able to break through the constraints and challenges posed by the traditional monoclonal antibodies,and show great potential for development and application.Nanoantibodies have been of great fascination to numerous research areas for a long time,particularly in the area of diagnosing and treating illnesses.In this study,we used a monoclonal antibody specific for PCV2 Cap as Ab1 to immunize camelids,and phage display technology was used to screen for the anti-idiotypic nanobody and to express it in Picrosporum,which mimicked the neutralizing epitope of PCV2 Cap and thus could induce a protective immune response against PCV2b infection in mice and piglets.The main results are as follows:1.Identification of antiviral neutralization activity of PCV2 Cap-specific monoclonal antibodyThe mouse ascites containing the monoclonal antibody m Ab-1E7（Ab1）of PCV2 Cap was purified by saturated ammonium sulfate precipitation and Protein G affinity chromatography,and high purity m Ab-1E7 was obtained.IFA results showed that m Ab-1E7could specifically identify virus particles in PCV2b infected cells.Subsequently,PK-15 cells were used for virus neutralization test.The results showed that when the concentration of m Ab-1E7 was 12～24μg/m L,the inhibition rate reached 70%～90%,and the number of copies of PCV2 DNA in cells treated with m Ab-1E7 decreased significantly in a dose-dependent manner.The titer of daughter virus in cell supernatant decreased with the increase of m Ab-1E7 concentration.BABL/c mice were used as the animal model of PCV2b infection,and the neutralization effect of m Ab-1E7 in vivo was evaluated.The results showed that m Ab-1E7could block the infection of PCV2b,and no seroconversion was detected in infected mice.Viremia and viral load in lymph nodes were significantly reduced.These results indicate that m Ab-1E7 can neutralize PCV2b in vivo and in vitro.2.Evaluation of immunoprotection of anti-idiotypic nanobody of PCV2 Cap.In the early stage,m Ab-1E7 was used as Ab1 to immunize camels,and 12 anti-idiotypic nanobodies（Ab2）which can specifically bind m Ab-1E7 were screened by phage display technology,and named Nbs.Twelve strains of anti-idiotypic nanobodies were expressed by Pichia pastoris,purified and identified.The Ab2βanti-idiotypic nanobody（Nb61）was identified by blocking ELISA and detecting Ab3 in serum of mice immunized with anti-idiotypic nanobodies.The immune protection effect of Nb61 was evaluated in animal model BABL/c mice and susceptible piglets.After 14 days of immunization with Nb61,the specific antibody of PCV2 Cap was detected in the serum of mice.After 28 days of challenge with PCV2b,the viremia and viral load in lymph nodes of mice were significantly lower than those of non-immunized control group.The same result was observed in piglet experiment.In addition,the serum neutralization test of immunized piglets showed that the neutralization titer of immunized piglets reached 2^5.059 after 500μg Nb61 was immunized.The PCV2 antigen in the lymph nodes of piglets was detected by immunohistochemical test,and the results showed that the PCV2 antigen was significantly reduced.These results indicate that Nb61 has a good protective effect on mice and piglets infected with PCV2b.3.Immunoprotective mechanism of anti-idiotypic nanobody of PCV2 Cap.By comparing the amino acid sequences of Nb61 and PCV2 Cap,it was found that Nb61epitope ¹⁰¹NYNDFL¹⁰⁶ on CDR3 was consistent with 5 key amino acids on PCV2 Cap epitope⁷⁵NINDFL⁸⁰.The recognition epitopes of m Ab-1E7 were identified by the overlapping short peptides of synthetic Nb61 and PCV2 Cap.The results showed that m Ab-1E7 recognized the⁷⁵NINDFLPPG⁸³ region of PCV2 Cap and the ¹⁰¹NYNDFLG¹⁰⁷ region of Nb61,and there were five key amino acids in the two epitopes.This is consistent with previous sequence alignment results,confirming that Nb61 mimics the PCV2 Cap epitope ⁷⁵NINDFL⁸⁰.A monoclonal antibody,m Ab-3G4（Ab3）against Nb61,was produced and found to recognize the ⁷⁵NINDFL⁸⁰ epitopes of PCV2 Cap and neutralize PCV2 infection,similar to m Ab-1E7.In order to analyze the structural simulation mechanism,the antigen-antibody complex was obtained by molecular docking.After analysis,it was found that the binding spatial structure of m Ab-3G4 and m Ab-1E7 with PCV2 Cap was similar,and similar structures were also observed in the docking between m Ab-3G4 and m Ab-1E7 with Nb61.These results indicate that Nb61 mimics the epitope ⁷⁵NINDFL⁸⁰ of PCV2 Cap at the functional conformation and amino acid sequence level.In summary,the anti-idiotypic nanobodies were expressed by Pichia pastoris,and the Ab2βanti-idiotypic nanobody（Nb61）was identified by blocking ELISA and detecting Ab3 in serum of mice immunized with anti-idiotypic nanobodies.Nb61 mimicking the PCV2 Cap and can provide protection immune responses against PCV2b infection in the pigs and mice,which is the same as the PCV2 Cap.Mechanically,Nb61 mimics a neutralizing epitope⁷⁵NINDFL⁸⁰ of PCV Cap and elicits potent Ab3 in vivo to confer protection against PCV2infection.This study further confirmed the rationality of the"immune network theory"in nanobody technology,which provided a new idea for developing a new anti-idiotypic nanobody vaccine for PCV2,and also provided a reference for preventing and treating other severe infectious diseases of human beings and animals.