Characterization Of MAbs That Specific To Classical Swine Fever Virus Or Specially Differentiate Field Isolates From Vaccine Strain

Classical swine fever(CSF)is a highly contagious swine disease caused by CSF virus(CSFV),which significantly impairs the pig industry worldwide.And CSF is designated as a class A animal infectious disease in China.Hog cholera lapinized virus(HCLV),also called Chinese vaccine strain,(C strain),developed in China in the 1950s,is still recognized as the safest and most effective CSF vaccine in the world.Pandemic outbreaks of CSF in China was successfully controlled due to the widespread application of HCLV vaccine and replaced by sporadic outbreaks at present.Infected pigs without classical symptoms and immune tolerant gilts are still the main source of infection.Therefore,the serological diagnostic methods that can distinguish wild virus infection from vaccine immunity is very important to promote CSF eradication in China.Although there are commercial CSFV E2 subunit vaccines that can distinguish vaccine immunity from wild virus infection,but they can not block vertical transmission effectively.HCLV vaccine will still play an important role in CSF eradication in China since its incomparable efficacy and safety.However,the antigenicity difference of main antigenic proteins E2 or E^rns between HCLV vaccine and field CSFV strains is not clear.Screening and identifying monoclonal antibodies that can identify vaccine strains from field CSFV strains is critical to the development of serological diagnostic techniques.With the development of monoclonal antibody technology,a large number of antibodies have been applied to life science research and clinical diagnosis.Antibodies against CSFV E2 and E^rns protein have also been greatly developed and applied.However,there are few monoclonal antibodies identify the differential epitopes between HCLV vaccine and field CSFV strains.We characterized three m Abs against CSFV E2 protein(N0.1:3H3G6,No.3:6B211 and No.4:9A4H4)and one m Ab against HCLV-E^rns protein(N0.2:1104)in the previous study.In brief,the anti-SM-E2 m Ab3H3G6 reacted specifically with SM strain and most field isolates(80/94)within genotypes 1 and 2,but not with HCLV strain and field isolates within subgenotypes2.3(0/12).The anti-SM-E2 m Ab 9A4H4 reacted broadly with SM,HCLV and about half of the field isolates(57/106),while anti-HCLV-E2 m Ab 6B211 reacted well with HCLV and a limited number of field isolates(10/106)within sub-genotype 2.2,but weakly with SM strain and field CSFV strains within sub-genotype1.1.However,the anti-HCLV-E^rns m Ab 1104 only reacted with HCLV and Riems,except the virulent SM strain or any other field isolates.Therefore,m Ab 3H3G6,6B211 and 1104 can be used to distinguish CSFV field strains from HCLV vaccine.Those four m Abs recognized conformational epitopes and only 6B211 can neutralize CSFV.In the present study,9 m Ab hybridoma clones against SM-E2 were obtained through serials of selection and cloning processes.The hypotype of the m Ab HCL-002is Ig A,and the rest are Ig G1.According to the characterization,m Ab HCL-001,HCL-002 and HCL-010 can recognize a broad spectrum of CSFV strains used in this study.And m Ab HCL-002 reacted with SM,HCLV and strains within genotypes 1(4/4),most strains within sub-genotypes 2.1(66/68)and 2.2(24/24),but not react with strains within subgenotypes 2.3(0/12)and 3.1(0/1).And the reaction of HCL-007 and HCL-008 with CSFV was irregular.The m Ab HCL-009 can react with SM,2 field strains within sub-genotype 1.1 and 3 vaccine strains(HCLV-India,GPE-and Thiverval)but not with HCLV.Similarly,the m Ab HCL-018 can react with SM and 2 field strains within sub-genotype 1.1.However,HCL-014 can react with most of CSFV strains(104/108).According to the identification of 4 anti-HCLV-E^rns m Abs of Ig G1,the m Ab 1204 can react with all CSFV strains except HCLV-India and LPC,however the m Ab 1504,1904and 2004 only reacted with HCLV and Riems,similar to m Ab 1104.The results of virus neutralization test suggested that only HCL-008 can not neutralize SM strain,the HCL-007 and HCL-014 have weak neutralization ability against SM strain,and the rest of anti-SM-E2 m Abs can neutralize SM strain efficiently.However 4 anti-HCLV-E^rnsm Abs can not neutralize HCLV strain.The results of epitope type identification showed that the m Abs HCL-001,HCL-005 and HCL-010 recognized linear epitopes,and the other m Abs recognized conformational epitopes.In order to analyze the antigen epitopes recognized by the antibody,the regions of the epitopes were preliminarily identified by truncated expression,recombination.The epitopes recognized by monoclonal antibodies were successfully identified with amino acid sequence alignment analysis and site-directed mutagenesis.Anti-E2 m Abs 3H3G6,6B211 and 9A4H4 recognized regions containing critical motif ²¹³EPD²¹⁵,³⁷LXLNDG⁴² and ²⁷¹RXGP²⁷⁴.And the epitope recognized by HCL-001,HCL-005 and HCL-010 was ¹⁴³SPT¹⁴⁵,L¹⁴⁷ on E2.The epitope reacted with HCL-002 was⁹⁵GDD⁹⁷,¹⁵⁷REKPFPH(Y/N)R¹⁶⁴.The HCLV-E^rns-specific epitope recognized by 1104,1504,1904 and 2004 was ¹⁰⁰D,V¹⁰⁷,and CSFV E^rns-conservative epitopes recognized by 1204 was ³⁸CKGVP⁴²,W⁸¹.In order to explore the potential clinical application of the m Abs identified in this study,three broad-spectrum m Abs(HCL-001,HCL-002 and 1204)and two differential m Abs(3H3G6 and 1104)were selected for b ELISA based on the results of characterization.The results showed that the serum of negative pigs and CSFV-infected pigs can not block the 5 m Abs.However,the serum of HCLV-vaccinated pigs can block the m Abs HCL-001,HCL-002,1104 and 1204 except 3H3G6,and the serum of HCLV-vaccinated pig after challenge can block the 5 m Abs efficiently.According to the results of ELISA,the m Ab 3H3G6 was suitable for identifying post immune infection,and the m Ab HCL-002 and 1104 were suitable for evaluating the immunity of HCLV vaccine.This study analyzed the differential epitopes and conserved epitopes in E2 and E^rnsproteins of HCLV vaccine and CSFV field strains,providing new insights into the antigenic structure of CSFV glycoproteins and the molecular mechanism of immune escape of field strains.This work will provide new ideas for the optimization of CSF vaccine and the establishment of differential diagnosis methods in the future,which will contribute to the control and eradication of CSF in China.