SONG Jin-xing ,WANG Meng-xiang ,ZHANG Yi-xuan ,WAN Bo ,DU Yong-kun ,ZHUANG Guo-qing ,Ll Zi-bin,QlAO Song-lin,GENG RuiWU Ya-nan #,ZHANG Gai-ping ,

1 College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,P.R.China

2 International Joint Research Center of National Animal Immunology,Zhengzhou 450046,P.R.China

3 College of Animal Science and Veterinary Medicine,Tianjin Agricultural University,Tianjin 300384,P.R.China

4 Longhu Laboratory,Zhengzhou 450046,P.R.China

5 School of Advanced Agricultural Sciences,Peking University,Beijing 100871,P.R.China

6 Henan Provincial Key Laboratory of Animal Immunology,Henan Academy of Agricultural Sciences,Zhengzhou 450002,P.R.China

Abstract

African swine fever virus (ASFV) is a lethal pathogen that causes severe threats to the global swine industry and it has already had catastrophic socio-economic effects.To date,no licensed prophylactic vaccine exists.Limited knowledge exists about the major immunogens of ASFV and the epitope mapping of the key antigens.As such,there is a considerable requirement to understand the functional monoclonal antibodies (mAbs) and the epitope mapping may be of utmost importance in our understanding of immune responses and designing improved vaccines,therapeutics,and diagnostics.In this study,we generated an ASFV antibody phage-display library from ASFV convalescent swine PBMCs,further screened a specific ASFV major capsid protein (p72) single-chain antibody and fused with an IgG Fc fragment (scFv-83-Fc),which is a specific recognition antibody against ASFV Pig/HLJ/2018 strain.Using the scFv-83-Fc mAb,we selected a conserved epitope peptide (221MTGYKH226) of p72 retrieved from a phage-displayed random peptide library.Moreover,flow cytometry and cell uptake experiments demonstrated that the epitope peptide can significantly promote BMDCs maturation in vitro and could be effectively uptaken by DCs,which indicated its potential application in vaccine and diagnostic reagent development.Overall,this study provided a valuable platform for identifying targets for ASFV vaccine development,as well as to facilitate the optimization design of subunit vaccine and diagnostic reagents.

Keywords: ASFV,phage display antibody library,single chain antibody (scFv),p72,epitope

1.lntroduction

African swine fever (ASF) is a highly lethal hemorrhagic infectious disease that can be circulated in domestic swine and wild boars caused by ASFV infection,and with no effective vaccine or therapeutic strategy now (Revillaetal.2018; Dixonetal.2019).The disease has been endemic over the last years through eastern Europe and the Russian Federation,and has recently spread to the major pig production countries of China and Southeast Asia,posing a serious threat to global pork production and food security (Dixonetal.2020).African swine fever virus (ASFV),the causative agent of ASF,belongs to the group of nuclear cytoplasmic large DNA viruses (NCLDVs),which is the sole member of the family Asfarviridae and the only known DNA arbovirus transmitted by arthropod (Iyeretal.2001,2006).The genome sizes of different ASFV strains ranged from 170–194 kb,encoding 151–174 open reading frames (Liuetal.2019; Wangetal.2020).

The large genomic composition and complex virion structure of ASFV have led to the fact that the immune protection mechanisms against ASFV are still poorly understood.Studies have shown that passive transfer of ASFV hyperimmune antisera to naive swine resulted in protection against a lethal challenge infection with a virulent ASFV strain,clearly demonstrating that antibody-mediated humoral immunity plays a pivotal role in ASFV control and antibody is an important component of the protective immunity against ASFV (Onisketal.1994; Ariasetal.2017; Wangetal.2019).The scFv is small (25–27 kDa)monovalent antibody fragments comprising the Fv variable region heavy chain (VH) and the Fv variable region light chain (VL) domains connected by a short peptide linker (Lietal.2015).The genes of VHand VLdare joined together with short and flexible peptide linker or with disulfide bond (Glockshuberetal.1990).The scFv is the smallest unit of immunoglobulin molecule that holds a complete antigenbinding domain of an antibody,and are widely used for antigen targeting (Holliger and Hudson 2005; Weisser and Hall 2009; Ahamadi-Fesharakietal.2019).Due to the devoid of immunoglobulin Fc,scFv poses no risk of ADE induction and is relatively inexpensive because of their rapid mass production in non-mammalian or mammalian expression systems.

Currently,despite the development of antibody technology such as single B cell sorting,antibody screening using phage display technology still has unique advantages,including versatility,high efficiency,cost effectiveness and high throughput.Phage-display platforms have been widely used in the generation of recombinant antibodies,antibody engineering and many other applications (Ledsgaardetal.2022).Moreover,phage display allows the generation of antibodies against almost any target,including pathogens,toxins,or highly conserve antigens,and so on (Bradburyetal.2011).Although there have been studies reported the use of phage-display platforms to screen neutralizing antibody (Liuetal.2021; Panetal.2021; Stefanetal.2021),most were constructed from immunized or naive animals,and few reports were from ASFV convalescent swine for antibody screening.In addition,most of the reported ASFV linear epitopes have been screened from synthetic peptide arrays.To our knowledge,there have been no public reports of using phage display to obtain ASFV epitopes,and ASFV antibodies produced by natural infections are also poorly studied to date.

Analyzing the interaction profiles of ASFV key structural proteins and antibodies,screening ASFV protective antigens need to be explored.In this study,the phage antibody library from PBMCs of four ASFV convalescent swine was constructed to obtain a specific ASFV-scFv-83 targeting the key capsid protein p72 of ASFV.This study provides an effective screening method for ASFV antibodies and epitopes,which can also be extended to other viruses for quickly screening of potential antibodies and B cell epitope identification,and provides a very useful platform for discovering targets for ASFV vaccine development,laying the foundation for a safe and effective ASFV vaccine,and promoting the development of ASFV vaccines and diagnostic reagents.

2.Materials and methods

2.1.Sample collection

An immune-tolerant swine was collected from Puyang Pig Farm in Henan Province,China,negative for ASFV nucleic acid and positive for antibody.The sample performed was carried out in strict accordance with OIE ASFV standards.

2.2.Determination of anti-p72 antibody titer via ELlSA

The serum titer of p72-specific antibody for the ASFV convalescent swine was detected by enzyme-linked immunosorbent assay (ELISA).In brief,96-well plates were coated overnight at 4°C with 300 ng/well recombinant p72 in carbonate-bicarbonate buffer (CBS,pH 9.6),and then washed with PBST (with 0.5% (v/v) Tween 20,pH 7.4).After blocking with 200 μL 5% (w/v) skim milk (BD Difco,Sparks,MD) per well at 37°C for 1 h,and then mixed the four serially diluted serum samples.and incubated at 37°C for 1 h.Negative control was using the negative serum purchased from China Veterinary Culture Collection Center (Beijing,China).The plates were then washed with PBST and incubated with 1:5 000 diluted horseradish peroxidase (HRP)-mouse anti-pig IgG (Immunoway,Suzhou,China) antibodies at 37°C for 40 min.After washing thrice with PBST,enzymatic reaction was carried out using 100 μL of SureBlue Reserve TMB Microwell Peroxidase Substrate (Solarbio,Beijing,China) and the reaction stopped with a 50-μL volume of Stop Solution (2 mol L–1HCl).Finally,the optical density at 450 nm(OD450) was measured using Spark 10 mol L–1Plate Reader (TECAN,Austria,Switzerland).

2.3.Construction of the immune phage antibody library

For library construction,one ASFV convalescent swine was collected,which tested positive for serum while negative for nucleic acid.Further,using ELISA to detect the specific antibody titer of p72 recombinant protein in serum.Isolation and collection of PBMCs from peripheral blood and spleen of swine.Total RNA was extracted from PBMCs using the TRIzol reagent (TaKaRa,Beijing,China),and then cDNAs were synthesized using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme,Nanjing,China).The sequences encoding VHand the two types of VL(VLKand VLλ) were separately amplified with specific primers and subjected to form the scFv,and the fragments were joined by a flexible linker peptide (Gly4 Ser Gly4 Ser Gly3 Ala Ser).The primers used above were designed according to the swine antibody sequences published on IMGT (https://www.imgt.org/) and shown in Table 1.Subsequently,the scFv were subcloned into the phagemid vector pHEN2,which transformed by electroporation intoEscherichiacoliTG1 (Lucigen,USA) and subjected to culture on 2× Yeast Extract and tryptone agar plate with 100 μg mL–1ampicillin and 1% glucose (2× YT-AG) overnight at 37°C.The phage libraries were stored as glycerol stocks in liquid nitrogen until use.In addition,for the determination of the insertion rate,96 colonies were randomly picked,and colony-PCR with the F (5′-TGGAATTGTGAGCGGATAACAATT-3′) and R (5′-GTAAATGAATTTTCTGTATGAGG-3′) primers was performed (the target fragment of about 1 kb).Finally,yet importantly,for the determination of phage titer,phage libraries were amplifiedviahelper phage infection,purified using Poly (ethylene glycol) 8000 (Solarbio,Beijing,China) precipitation,and subjected to gradient dilution.

Table 1 Primers for PCR amplification of swine antibody variable regions

2.4.Biopanning of specific scFv library,and genetic analyses of phage antibodies

The 4 convalescent swine derived libraries were screened three rounds with the trimer p72 recombinant protein as the target substrate,which was stored in laboratory as previously described (Gengetal.2022).The concentrations of antigen in the first,second and third rounds of screening were 10,2.5,and 0.65 μg mL–1in PBS,respectively.The antigen was incubated at 4°C overnight,followed by blocking with 5% (w/v) skim milk in wells of 96-well plate (Immuno,Thermo Scientific,USA).The wells were washed with PBST,100 μL phage library (3.2×109pfu μL–1) was added and shaky incubated for 1 h at room temperature (RT).In order to reduce the nonspecific binding,the unbound phages were washed 10 times with PBST (PBS with 0.1%,0.3%,0.5% Tween-20 in three rounds of panning,respectively),and then eluted by 0.1 mol L–1glycine (pH 2.2) for 10 min at RT,followed neutralized by 2 mol L–1Tris (pH 9.6).ThenE.coliTG1 was infected with eluted phages to obtain the phage clones.

The specificity of the positive clones was further identified by phage ELISA.In brief,the colony was infected with phage and cultured in a 96-well plate.When OD value reaches 0.5,M13 helper phage was added.After 12 h incubation at 30°C,the phage supernatant was collected and identified by ELISA to show monoclonal anti-p72 scFv.A total of 100 μL phage supernatant was added to each well,and the scFv of phage was detected by horseradish peroxidase (HRP) conjugated anti-M13 antibody (1:5 000),while using BSA as the negative control.The reaction was carried out with 100 μL TMB,at 37°C for 15 min,away from light,and stoppedviathe addition of 50 μL/well H2SO4.The absorbance values were quantified at 450 nm.The positive clones were obtained and sent for sequencing.Analysis of complementarity determining regions (CDRs) 1,2,3 was used IMGT website (https://www.imgt.org/IMGT_vquest/analysis).

2.5.Cloning,expression and purification of fulllength antibodies

Following three rounds of panning,for larger scale production of scFv antibodies,the gene encoding scFv antibody clone was amplified by PCR and sub-cloned into modified IgG expression vector,and ensure that the VH–VLpairing of the selected single-chain antibody is appropriate.Expi293F cells were transiently transfected with recombinant plasmids for antibody expression and cultured at 37°C,5% CO2.The supernatants were then collected 5 d post transfection for sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS-PAGE) analysis and purified using Protein A Resin column (GenScript,Nanjing,China).

2.6.lndirect immunofluorescence assay (lFA) and Western blot

The specific binding of these scFv to the p72 protein and inactivated virus samples was further verified using IFA and Western blot,respectively.The inactivated virus samples isolated from porcine alveolar macrophages (PAM) cells infected with ASFV HLJ/18 strain,which was provided by Harbin Veterinary Research Institute,CAAS.In simple terms,HEK293T cells (2.5×105cells/well) were inoculated into 24-well plates 1 d before transfection,and then transfected with the recombinant full length p72 (GenBank: MK333180.1) fused with Flagtag at N-terminus when the cell density reached about 70%.At 24 h post-transfection,the coverslips containing cells were fixed with 4% paraformaldehyde for 30 min at RT.After three washes with PBS,the cells were permeabilized in PBS containing 0.1% Triton X-100 and blocked with 10% fetal calf serum (FBS).The primary scFv-83 were diluted with PBS containing 10% FBS and incubated with cells for 1 h at RT.After washing with PBS,cells were then stained with FITC-conjugated goat anti-pig IgG (Proteintech,Wuhan China) for 1 h at 37°C.The cells were finally washed in PBS and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI,Solarbio,Beijing,China).Cells were visualized using an LSM Confocal Microscope (ZEISS,Jena,Germany).

Inactivated ASFV samples were loaded on 12.5% SDS-PAGE and electrically transferred to polyvinylidene fluoride (PVDF) membrane (Merck Millipore,Germany).Following,the membrane was blocked with 5% BSA in 1× TBST (TRI-Buffered Saline,0.05% Tween-20),and incubated with the purified antibody (diluted at 200 μg mL–1) 4°C overnight.The PVDF membranes were probed with HRP-conjugated affinipure goat anti-pig IgG (H+L) antibody diluted at 1:5 000 (Proteintech,Wuhan,China),and revealed with the NcmECL Ultra (NCM,Suzhou,China).Chemiluminescence images were visualized with the Luminescent Image Analyzer (Amersham Imager 680,Japan,GE Health).

2.7.Epitope mapping

Furthermore,we used a commercial kit (Ph.D.TM-12 Phage Display Peptide Library Kit,New England Biolabs) to investigate the epitope recognized by the scFv-83.Biological screening,clonal selection,elution and phage amplification were performed according to the NEB Manual.

The Ph.D.TM-12 phage display peptide library was subjected to three rounds of panning using scFv-83 as the bait protein.For the first round of panning,a total 100 μL/well of bait protein (10 μg mL–1in 0.1 mol L–1NaHCO3,pH 8.6) was adsorbed onto 96-well ELISA plates overnight at 4°C and blocked with 200 μL/well blocking buffer (5% skim milk in PBST) at 37°C for 1 h.After rapid washing 5 times with TBST (TBS+0.1% (v/v) Tween-20),10 μL Ph.D.?-12 (≥1.0×1013pfu) were added into the 96-well plates containing in 100 μL CBS,the mixture was shaken well and incubated for 60 min at 37°C.After washing three times with TBST,100 μL/well elution buffer (0.2 mol L–1glycine-HCl,1 mg mL–1BSA,pH 2.2) was added to suspend and incubated for 10 min at 37°C,and then the eluate was collected in microcentrifuge tube,where 1 μL was used for titer determination and the remaining eluent was used for amplification.Subsequently,the eluent was added into 20 mL of the prepared ER2738 culture at early-log phase and incubated at 37°C for 4.5 h by shaking.The amplified phages were titrated on LB/IPTG/Xgal plates using blue/white method as output of the current round and input of the next round,respectively.The second and third rounds of panning were the same as described above.After three rounds of panning,20 blue clones were selected to extract single-strand DNA for sequencing verification and PCR amplification using universal primers,followed by purification of the PCR products using a commercial kit (Qiagen,Germany).The PCR products of 19 phage clones were send for Sanger sequencing with (–) 96gIII reverse primer.The 12-mer sequences were identified by analysis of the phage sequence characteristics.The linear homologous sequences were retrieved with blastp at NCBI (https://blast.ncbi.nlm.nih.gov/).

2.8.Epitope-binding using peptide-based ELlSA and dot-ELlSA

The C-terminal amidated synthetic model peptide was synthesized by GenScript Biochem Ltd.,China.The peptide dissolved in PBS to 1 mg mL–1with purity ≥95%.Labelling of peptide was performed by Sulfo-NHS-LCBiotin using the manufacturer’s protocol (Thermo Fisher,USA).Streptavidin-coated microtitre plates (BEAVER,Suzhou,China) were coated with biotin-labelled peptide (5 μg mL–1) in PBS overnight at 4°C.The wells were then washed three times with PBST,blocked with blocking buffer (5% skim milk in PBST) for 2 h at RT,and then washed twice with PBST.The purified scFv-83 was diluted to 200 μg mL–1was used as the primary antibody,after being incubated for 1 h,washed three times with PBST,and excess buffer was aspirated after the last wash.Goat anti-pig IgG conjugated HRP as secondary antibody (Proteintech,China) was performed at 37°C for 1 h.The plates were again washed six times with PBST.After excess buffer was aspirated,each well was added with 100 μL TMB substrate and incubated at 37°C for 15 min,keep in dark place.The reaction was stopped by H2SO4addition with 50 μL/well.The absorbance values were recorded at 450 nm using a microplate reader TECAN Spark 20M Multimode Microplate Reader (Tecan,Mannedorf,Switzerland).

Dropped the diluted peptide solution onto the nitrocellulose (NC) membrane.After drying,the membrane was blocked with 5% skim milk at 37°C for 1 h and incubated overnight with specific antibody scFv-83 at 4°C,then HRP-conjugated affinipure goat anti-pig IgG (H+L) was added and incubated for 1 h at RT.The immune reactions were detected with NcmECL Ultra (NCM,Suzhou,China).

2.9.Molecular docking

Homology modelling of the three-dimensional (3D) structure of scFv-83 was performed on the Phyre2 website (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) using the amino acid sequences.Crystal structure of p72 protein (PDB: 6KU9) was as the template and obtained from RCSB database (https://www.rcsb.org/).Molecular manipulation platform (MOE2019.01) was used for molecular docking,with the structures of antigen and antibody introduced into MOE.Structure preparation module was used to optimize the protein processing,including the removal of solvent molecules,water molecules,hydrogenation and energy minimization,etc.Finally,the processed target protein was defined as the docking receptor.Before docking,the force field of AMBER10:EHT and the implicit solvation model of the reaction field (R-field) were selected.The protein–protein docking module was selected for molecular docking.All molecular postures were sorted by London DG function,and then the positions of the top 100 postures were refined and scored by GBVI/WSA.Visualization and graphical generation of the post-docking complex were performed using PyMOL Software (www.pymol.org).

2.10.lnduction of BMDCs maturation in vitro

Bone marrow-derived dendritic cells (BMDCs) were prepared according to a previously reported method (Songetal.2023).Flow cytometry analysis was carried out after incubation with 30 μg mL–1of epitope peptide for 48 h.Cells were then stained with fluorescenceconjugated Monoclonal Mouse anti-CD11c-APC,CD80-FITC,and CD86-FITC antibodies (Biolegend,USA).LPS (500 ng mL–1) as positive control.Stained cells were analyzed by BD FACS Canto II (BD Biosiences) and data were analysed by FlowJo Software (Version X,TreeStar,Ashland,OR,USA).

2.11.Cell uptake studies

CLSM was employed to evaluate the intracellular uptake and biodistribution of peptide.Briefly,DC2.4 cells were plated on small sterilized glass shards in 24-well plates and incubated at 37°C (5% CO2) for at least 24 h.The synthetic peptide was coupled to FITC on the day before the experiment,The coupling method was performed as previously described (Songetal.2023).And the cells were treated with free FITC and FITC-peptide (0.05 mg mL–1) for 24 h,respectively.After incubation,cells were washed with PBS buffer.The cytoskeleton was stained with as described in the manufacturer instructions manual.Cells were then fixed with 4% paraformaldehyde for 15 min at room temperature,and permeabilization was performed using 0.1% Triton X-100 for 5 min at RT,after being washed twice with PBS.To show the cells,the iFluor594-phalloidin (MKBio,China) was used to stain the cytoskeleton (red) as described in the manufacturer’s instructions.Afterwards,nucleus was counterstained with DAPI.Fluorescence microscopy images were acquired using ZEN Software (Carl Zeiss LSM 880,Jena,Germany).Fluorescence CLSM images were measured at excitation/emission 485/580 nm (red) and then at excitation/emission 485/530 nm (green).

3.Results

3.1.Collection of blood samples from convalescent swine and determination of the anti-p72 titer

In order to construct the phage-display library,peripheral blood samples were collected from an ASFV convalescent swine,whose ASFV nucleic acid test results were negative.One of the samples was selected for the specific antibody titer test of the p72 recombinant protein,and the results showed that the titer is over 1:12 800 by ELISA (Fig.1).And PBMCs were isolated and pooled for the construction of immune phage antibody library.

Fig.1 Determination of specific antibody titer in convalescent swine serum by ELISA.The endpoint titer (1:12 800) was defined as the highest serum dilution that yielded a positive OD value.

3.2.Library construction,panning,and validation

The coding regions of VHand VLwere amplified from the cDNA of PBMCs of convalescent swine (Fig.2-A).After connecting with flexible spline,the full-length scFv gene was about 750 bp (Fig.2-B).The scFv expressed was a single polypeptide consisting of VHand VLjoined by a flexible peptide linker that allows the reconstitution of functional antigen-binding domains (Fig.2-C).The scFv gene was cloned into the pHEN2 phage vector and electroporation.A total of 24 colonies were randomly selected to identify the quality of the scFv library by colony PCR,and 19 out of the 24 colonies were correct (Fig.2-D),which indicated that the proportion of recombinant plasmids containing scFv was about 79%.Following phage amplification,the phage titer was calculatedviagradient dilution,and the hybrid phage display libraries sizes of scFv VHK-VHλwas 3.2×109pfu mL–1.

To isolate scFv specific for the ASFV p72 protein,we performed a biological screen and obtained 235 positive clones.The single colonies were randomly selected and further screened by monoclonal phage ELISA,and the results showed that eight of them could specifically bound to p72,that is,scFv-2,scFv-19,scFv-21,scFv-42,scFv-51,scFv-55,scFv-79,and scFv-83 (Fig.2-E).The output phage titer of each round and the output/input ratio were listed in Table 2.Obviously,after three rounds of enrichment,the harvest rate of phage antibodies was increased from 1.59×10–5to 6.5×10–2,which indicated that the specific phage antibodies were effectively enriched.The biological screening flowchart is shown in Fig.3.

Fig.3 Screening for p72-specific scFv from immue phage display library.Phage antibody screening is a basic method to sequential affinity screening for specific antibodies from a large excess of non-binding clones,which involves constructing the phage display library,immobilizing the target protein to a solid carrier and then exposing the phage library to allow specific binding.Multiple rounds of washing were performed to exclude non-specific binding,and the remaining binding phages were washed and re-amplified for subsequent further identification.

Table 2 Enrichment of the phage library during three rounds of panning

3.3.Production and identification of specific scFv

The eight specific scFv positive clones screened by monoclonal phage ELISA were sub-cloned into pFUSEhIgG-Fc2 expression vector for further specificity identification,and expressed in Expi293 System.Followed,the culture supernatant (scFv-2,scFv-19,scFv-21,scFv-42,scFv-51,scFv-55,scFv-79,and scFv-83) of the expressed cells purified by protein-A affinity chromatography,and subjected to SDS-PAGE (Fig.4-A).As described in Fig.4-B and C,scFv-83 could react not only with the p72 recombinant protein,but also with inactivated ASFV HLJ/2018 strain,as evidenced by IFA and Weston blot,severally.Moreover,the sequencing results showed that ASFV-scFv-83 sequence matched the genetic characteristics of the variable region (Fig.4-D),and it consists of a heavy chain encoded by the immunoglobulin heavy chain gene variable regionIGHV1-4＊01or theIGHV1-4＊02gene.

Fig.4 Expression and purification of ASFV-scFv-83-Fc.A,Coomassie blue staining of the purified scFv-83-Fc.Lane M,AB clonal Color Mixed Prestained Protein ladder; lanes 1 and 2,refolded purified specific scFv-83; lane 3,mock 293F cells.B,results of the IFA assay for the reactivity of scFv-83 and p72 recombinant protein in cells.Full-length p72 protein analogue transfected cells were incubated with scFv-83 and then added FITC-labeled goat anti-pig IgG (1:100 diluted).Cells were observed under fluorescence microscope (×200 magnification).Blue (DAPI) indicated the nucleus.C,reactivity of scFv-83 with the inactivated virus-infected samples.Lane 1,mock-infected PAM cells (mock); lane 2,ASFV HLJ/18 infected PAM cells.D,CDR analysis of scFv-83 by IMGT/VQUEST.

3.4.ldentification of the ASFV-specific scFv binding epitope on capsid protein p72

To understand the epitope of p72 for binding to scFv-83-Fc,phage-displayed random peptide libraries were screened using scFv-83-Fc as the target.Phages displaying ASFV specific scFv binding epitope were selected after three rounds of panning.The phage titer increased exponentially after three rounds.Of the 19 clones selected by ASFV scFv-83,PCR products of all clones were of the expected size.Moreover,the 19 clones were found to be identical by sequencing,indicating that the key sequences were effectively screened.According to BLASTp analysis,the linear epitope of p72 was221MTGYKH226,among which M221,T222,K225,and H226 were the key recognition sites.Fig.5-A showed the localization of these critical residues on the crystal structure of the ASFV p72 protein (PDB: 6KU9) in colored stick mode.Fig.5-B and C showed the results of peptide-based ELISA and dot-ELISA,respectively,indicating that ASFV scFv-83 recognizes the linear epitope on p72,specifically,the epitope on p72 including M221,T222,G223,Y224,K225,and H226 residues.It is remarkable that,none of the residues composing the epitope recognized by ASFV scFv-83 has been found mutated neither in genotype I nor in genotype II circulating in China currently (Fig.5-D).For visualization,a computer docking simulation was used to map the binding of ASFV scFv-83 and p72 (Fig.6-A and B).The binding sites of p72 mainly include D219,N569,R308,and T222,while those of ASFV scFv-83 mainly include Y160,T185,E128,and W240.The residues of the above binding sites can form a variety of interactions,including hydrogen bonds and hydrophobic interactions,which can effectively improve the stability of the antigen and ASFV scFv-83 complex.

Fig.5 The binding peptide mapping of p72.A,demonstration of the interaction of scFv with p72.The peptide showed rainbow color in stick mode,while scFv was represented green in cartoon.B,confirmation of p72-peptide with ELISA.C,confirmation of p72-peptide with dot-ELISA.D,protein sequence alignment of the identified epitope in different genotypes.14 of p72 sequences from different ASFV strains were aligned.The 221–226 aa epitope region identified by scFv-83 was circled in yellow.

Fig.6 The binding mode of p72/scFv-83 complex.A,structural analysis of p72/scFv-83 complex.Left,an overview of the backbone of p72/scFv-83 complex in cartoon; middle,structure of p72/scFv-83 complex in surface; right,the cartoon diagram visualization of the details of the binding between p72 and scFv-83.p72 was colored in green and scFv-83 was in red.The yellow dashes represented hydrogen bonds.B,the 2D binding mode of p72/scFv-83 complex.

3.5.Epitope peptide induces maturation of BMDCs and cellular uptake

The matured DCs can serve as APCs to present antigens to T cells,and cellular uptake are also crucial for antigen presentation to stimulate T cell immune responses.The expression of CD80+,CD86+ and MHCII+ on the surface of BMDCs was detected by flow cytometry after the concentration of 5×105cells/well plated on a 24-well plate and stimulated with the epitope peptide for 48 h.As shown in Fig.7,the percentages of CD80+,CD86+ and MHCII+ positive cells.Cells were stimulated with epitope peptide (30 μg mL–1) and LPS (500 ng mL–1),the expression of three co stimulatory molecules was significantly upregulated.In contrast,a much smaller increment was observed in the LPS-treated group.This indicates that the stimulation of epitope peptide on BMDCs activation is significantly enhanced.

Fig.7 Epitope peptide enhance BMDCs maturation and cellular uptake in vitro.Expression of CD80+,CD86+ and MHCII+ on BMDCs was measured following incubation with 48 h.Flow cytometry analysis of CD80+,CD86+ and MHCII+,were measured by flow cytometer.

The efficient cellular uptake is prerequisite for initiating immune response.The cellular uptake of peptide was evaluated after 24 h incubation with mouse dendritic cells (DC2.4).The fluorescence image in Fig.8 shows the uptake of FITC-labeled peptide in DC2.4.According to the CLSM images,an obvious intracellular uptake of FITCpeptide was observed FITC-peptide showed a strong fluorescence pattern in the cytoplasm.Compared with dissolved FITC,the green fluorescence-specific absorption peptide is obviously visible,and the dissolved FITC in the control group can only detect some free fluorescence signals.Therefore,the binding characteristics of peptide epitopes can be confirmed.The confocal laser scanning microscope showed the location of the peptide being uptake,further indicating that the epitope identified in our study can be ingested by antigen-presenting cells.To sum up,the CLSM data illustrate the specific intracellular absorption of the epitope.

Fig.8 Schematic showing the endocytosis of peptide by the mouse dendritic cell line DC2.4.Observation of the FITC-peptide distributed in cells,the red signals indicate the cytoskeleton,and the green signal indicate the FITC-labeled peptide.Nucleus was labeled by 4′,6-diamidino-2-phenylindole (blue).Scale bar is 10 μm.

4.Discussion

ASF has spread rapidly over the past decade,posing a serious risk of further expansion (Sánchez-Cordónetal.2018; Wenetal.2019).Despite some progress in vaccine research,there is still no safe and effective commercial vaccine available.In recent years,the epidemiological characteristics,pathogenicity and transmission of ASFV have been in-depth studied (Chapmanetal.2008; Jiaetal.2017; Sanchezetal.2017).But in fact,the molecular mechanism,pathogenesis and host immune response of ASFV infection still need to be further clarified.

Neutralizing antibody plays a significant role in the protective immune response to ASFV infection (Neilanetal.2004; Escribanoetal.2013).Noteworthily,p72,as the key capsid protein of viral particles,is not only the main antigen for ASFV serotyping,but also an effective target for inducing neutralizing antibodies (Gómez-Puertasetal.1996).So,p72 has been a functional antigen during the development of ASFV vaccine candidate.However,few studies have been reported on antibody production and epitopes of p72 after natural infection with ASFV.In this study,the identification method of ASFV p72 antibody involves the isolation of PBMC from four ASFV convalescent swine,construction of a high capacity (3.2×109pfu mL–1) phage immune library expressing the variable region pairs of IgG light and heavy chain,and screening specific antibodies,so as to explore the characteristics of antibody production in convalescent swine.Moreover,specific phage monoclone targeting p72 was isolated and ASFV-scFv-83 that recognized both the full-length p72 protein and inactivated virus samples was successfully identified.Unlike most direct screening methods for B-cell antibodies,phage display allows deep and high-throughput processing of antibody genes.It also allows enrichment of functional clones that may or may not have clonal expansion during the initial antibody response.In conclusion,the phage display antibody technique was successfully applied to the generation of recombinant antibodies.Here,a p72-specific antibody,ASFV-scFv-83,was identified from the phage library.Besides,ASFVscFv-83 is derived from the immune phage antibody library in the naturally infected state and can serve as the prototype for recombinant antibody modification,enhancing primitive affinity through mutations.Improvement of recombinant antibodies can be achieved by a variety of antibody engineering methods,such as optimization of isolation techniques or affinity maturation of antibody genes (Sheedyetal.2007).Furthermore,the generation of the swine monoclonal antibody,which can also be further engineered as a biosensor,and coupled with nanoparticles for ASFV detection and treatment,etc.Of course,further research is needed on the efficacy,safety and application potential of ASFV scFv-83.

Previous research had confirmed four epitopes identified by the monoclonal antibody: epitope aa 156–165,aa 265–280,aa 280–294,and aa 290–303 (Heimermanetal.2018).Here,a novel epitope was identified by Ph.D.12 technology,using ASFV-scFv-83 as the bait protein.Further,we evaluated the uptake of the epitope peptide by DCs,which clearly demonstrates that the binding peptide can be uptaken by DCs.As the most potent specific antigen-presenting cells,the antigen cross-presentation function of DCs plays a critical role in initiating an adaptive cytotoxic immune response against pathogens and is key to coordinating both innate and adaptive immune responses (Br?hleretal.2018; Belabedetal.2020).

Due to laboratory biosafety limitations,no neutralization experiment was performed,but importantly,scFv-83 could react with inactivated virus and recognize a linear B cell epitope on the p72 protein.In this regard,scFv-83 may have the potential to be a therapeutic antibody in future,and the epitope it recognizes may play an important role in ASF vaccine development.However,due to the limitations of the study,the reaction of this single-chain antibody with other ASFV strains has not been studied,and the epitope diversity and function of the obtained single-chain antibody need to be further explored.In order to screen single-chain antibodies with better performance,virus neutralization test and other screening tests are also needed.Moreover,identification of epitopes using swine mAbs also will provide insight into the immunogenicity of ASFV.In the future,the optimal design of engineered antibodies to evaluate the efficacy of scFv-83 and the role of Fc effector in the prevention of ASFV will be further studied.Our study provides theoretical support for the optimization of vaccine design and the development of diagnostic reagents and lays a foundation for studying the adaptive immunity.

5.Conclusion

In summary,we report the isolation of scFv-83 antibody specifically recognizing the ASFV/HLJ strain from ASFV convalescent swine antibody library,which could be an effective and quick way to generate fully swine antibodies against different antigens.Further,we screened a conserved epitope peptide targeted by the scFv-83 mAb using random peptide libraries.Flow cytometry analysis showed that the conservative epitope peptide can effectively promote the maturation of BMDCsinvitro.In addition,the evaluation of the phagocytosis of dendritic cells indicates that the DC2.4 cells can more efficiently absorb the epitope peptide,which might indicate their potential application in the development of vaccines and diagnostic reagents.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31941001 and 32002292),the Major Science and Technology Project of Henan Province,China (221100110600) and the Natural Science Foundation of Henan Province (202300410199).Sample of inactivated porcine alveolar macrophage (PAM) cells infected with ASFV Pig/HLJ/18 strain was kindly provided by Prof.Li Huang in Harbin Veterinary Research Institute,CAAS.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Ethical approval

All experimental procedures were approved and reviewed by the Institutional Animal Care and Use Committee of Henan Agricultural University,China.