Generation And Application Of Monoclonal Antibodies Against The HA Of H6 Avian Influenza Virus

H6 subtype avian influenza virus(AIV)was first isolated from turkeys in the USA in 1965,and are enzootic and genetically diverse in both domestic poultry and wild birds.Currently,H6 AIVs are undergoing constant recombination with different subtypes of AIVs and may cause spillovers in mammals,including swine and humans.Altogether,this suggested to us that H6 IAVs pose a significant threat to public health.Hemagglutinin(HA)is the most abundant glycoprotein on the surface of AIV,which mediates binding to sialic acid on the host cells and facilitates fusion between the viral envelope and the host cell membrane.HA is the primary antigenic targets of neutralizing antibodies.Immunodominance patterns of several HA subtypes have been characterized,however,the antigenic domains of H6 were poor understood;at the same time,there is a lack of ELISA kits for detecting H6 viruses.1.Generation of monoclonal antibodies against the HA of H6 avian influenza virusIn this study,BALB/c mice were inflected intraperitoneally with(A/Eurasian teal/Jiangxi/2018WB0417(H6N2))(MA E-Teal/417)virus.Splenocytes from the immunized mouse were fused with mouse myeloma Sp2/0 cells.15 monoclonal antibodies(MAbs)against the HA of H6 virus were obtained by IFA,named as 1A5,1D4,2C5,3B9,3D7,4A7,4B4,4B8,4C2,4G2,5E9,5G2,6D2,6D11,and 6E3,respectively.To further confirm these HA-specificity and functional attributes,we tested these 15 MAbs in isotype detection,HI assays,western blot(WB)assays and ELISAs.MAb isotype detection showed that 15 MAbs were all IgG;1D4,3B9,4A7,4G2,5E9,6D11 were IgG1;2C5,3D7,6D2 were IgG2a;1 A5,4B4,4B8,4C2,5G2,6E3 were IgG2b.The HI assays results showed that 11 MAbs had HI titers(1A5,2C5,3B9,3D7,4B4,4B8,4C2,5G2,6D2,6D11,and 6E3),HI titers were 25-213.4 MAbs(1D4,4G2,4A7,and 5E9)had no HI titers.In WB assays,all these MAbs can efficiently bind HA protein in 293T cells transfected with pDP2002-HA,except for 6D11.In ELIS As,all MAbs showed stronger binding ability to MA E-Teal/417.To characterize the binding breadth of these MAbs against the HA of H6 virus,we tested the bind ability of these MAbs with other two H6 viruses which were different genotypes from E-Teal/417 with ELIS As and HI assays.The results showed that,all MAbs can bind to these two H6 viruses in the ELISA test,and 11 MAbs with HI characteristics can also bind to these two H6 viruses,however,the MAbs had different binding ability.These MAbs provides conditions for determining the antigen sites of H6.At the same time,four MAbs,1A5,4C2,5E9,and 6E3,which reacted well with multiple.strains of H6 virus ELISA,can be used to develop ELISA method for detecting H6 virus.2.Identification of critical amino acids mutations in epitopes in the HA of H6 influenza virus in the pressure of the antibodiesTo identify the antigenic domains of H6 targeting by these MAbs,escape mutants of MA E-Teal/417 were generated.The result showed that a total of 15 escape mutants were generated by these 11 MAbs.Through sequencing and analyzing the HA gene of escape mutants,9 critical amino acids in H6 antigenic domains were identified,located in the positions of 69,89,120,124,139,140,149,221,246.Through examined 2270 full length HA sequences of H6 viruses deposited in Influenza Research Database(http://www.fludb.org),we most found variations detected in the HA of these escape mutants have present in some of the natural H6 isolates.Since MAbs of 4C2 and 6E3 targeted amino acids in the HA positions of 89 and 140,which are comparatively conserved We used BALB/c mice as a model and tested the protective effect of these two MAbs against the virus.The results showed that these two MAbs had good preventive effects on mice and also had relatively good therapeutic effects.3.Establishment of a Double-MAb sandwich ELISA methodConsidering the current lack of ELISA kits for detecting H6 virus in the market,we established a Double-MAb sandwich ELISA method for detecting H6 antigen using four selected MAbs with good reactivity to all H6 viruses,namely 1A5,4C2,5E9,and 6E3 MAbs.We labeled these four MAbs with peroxidase and randomly combined them with unlabeled MAbs and labeled 4 MAbs with HRP for Double-MAb sandwich ELIS A testing;The confirmed results showed that the 5E9 MAb was used as a capture antibody,and HRP labeled MAb 6E3 was used as a detection antibody with good detection performance.We further optimized the ELISA method and determine the concentration of captured antibodies as 4 μg/mL,sealed with 2%BSA blocking solution at 37℃ for 60 minutes,and the detected antigen acted for 90 minutes at 37℃.Dilute HRP labeled 6E3 antibody to 0.76μg/mL using 1%skim milk,reaction at 37℃ for 60 minutes,and developed for 15 minutes using TMB.Simultaneously,the sensitivity,specificity,and stability of this ELISA method were tested.The results showed detectability of 2.32×103 TCID50/mL virus,which does not react with AIV-H1,AIV-H3,AIV-H4,AIV-H5,AIV-H7,AIV-H9,AIV-H10,AIV-H12,and FAdV-4,DAdV-3,IBV,NDV,GAstV,GPV,ALV,FAdV-8,IBDV viruses,shows good specificity;the coefficient of variation for intra batch repeated experiments is 1.55%6.26%,and the coefficient of variation for inter batch repeated experiments is 1.97%7.13%,indicating that this method has good stability.