Preparation And Application Of Monoclonal Antibodies Against Circulating Clades Of H5 Subtype Avian Influenza Virus

The H5N1 subtype highly pathogenic avian influenza virus(AIV)was first isolated in China in 1996 in Guangdong Province and named A/Goose/Guangdong/1/96(GD/96).Since then,the GD/96-like virus has been prevalent in many regions of China and evolved numerous genotypes,and has rapidly spread to many countries across Asia,Europe and Africa.The virus has caused infection and death of large numbers of birds,resulting in significant economic losses to the poultry industry around the world.Not only that,the virus could also jump the species barrier to transmit directly from avian to human,posing an enormous threat to public health security.As the H5N1 virus mutates rapidly,it has diverged into several HA clades with genetic and antigenic differences,and reassorted with multiple subtypes of NA to generate the H5NX viruses,making the prevention and control situation more and more challenging.Therefore,in terms of early warning and effective control of the outbreak,it is particularly important to strengthen the epidemiological surveillance of H5 viruses and analyze the evolutionary variation patterns,plus develop rapid diagnostic agents and vaccine products.In this study,based on the long-term predominant prevalence of clade 2.3.4.4 and clade 2.3.2.1 H5 subtype AIV in China,we prepared monoclonal antibodies against the HA and NP proteins,and preliminarily studied their applications in the analysis of viral antigenic variation and broad-spectrum detection.1.Preparation and identification of monoclonal antibodies against clade 2.3.4.4 and clade 2.3.2.1 H5 subtype avian influenza virusDifferent H5 viruses of circulating clade 2.3.4.4(strain QD1),clade 2.3.4.4d(strain 0205),clade 2.3.2.1d(strain 1033)and clade 2.3.2.1e(strain HF9)were selected to prepare whole-virus inactivated vaccines after inactivation and ultracentrifugation.The vaccines were then subcutaneously administered to the abdominal region of 6-week-old BALB/c mice by multi-point injection,respectively.After 3 times of such routine immunization,the mice were further boosted in the manner of peritoneal injection.Another 3 days later,fusion between spleen cells and myeloma cells were conducted to prepare hybridoma cells.Subsequently,hemagglutination inhibition(HI)and enzyme linked immunosorbent assay(ELISA)were applied to detect the positive fusion wells.After 3 successive subcloning,a total of 17 positive hybridoma cell lines targeting the HA protein were screened,including 2B2,2E10,4F3,6F4,6C11,5F2,4E3,7E9 against the immunogen of strain 0205,and 1G10,2G5,3D9 against strain 1033,plus 3A6,6F6,6A5,6H2,5H4,7G3 against strain HF9.In addition,3 positive hybridoma cell lines of 3D4,4B9 and 5F10 targeting the NP protein against the immunogen of strain QD1 were also obtained.The HI titers of each HA monoclonal antibody(mAb)ascites were between 11?14 log2,and the neutralization titers ranged from 1:508 to 1:28963.While the ELISA titers of the NP mAb ascites were all above 1:320000.Further characterization of those 20 mAb showed that they all belonged to the IgG antibody class except for 2G5 and 6H2 being of IgA,while all mAb possessed the sensitive reaction activity in indirect immunofluorescence assay(IFA).Particularly,the HA mAb of 3D9,1G10,6A5,3A6,7G3,5H4 and the NP mAb of 3D4 and 5F10 were all able to react specifically with the corresponding immunogenic HA and NP protein at 75 kDa and 55 kDa in the Western blot(WB)assay.2.Application of H5 subtype avian influenza virus HA protein monoclonal antibodies in antigenic variationTo explore the antigenic variation inter-clade and intra-clade of clade 2.3.4.4 and clade 2.3.2.1 H5 subtype AIV plus the related molecular basis,33 H5 strains distributing in different circulating clades were selected to conduct the cross HI assay with the 17 HA mAb that had been prepared in Chapter 1.The results showed that the the 17 HA mAb had varied reactivity with the tested H5 viruses,and the 33 strains could be classified into 8 antigenic types.Furthermore,the molecular biology software Mega 7.0 was used for HA gene sequence alignment of those H5 viruses with different antigenic patterns.As revealed by the reactivity of H5 viruses with mAb 3A6,6F6,2G5 and the 8 mAb against the immunogen of clade 2.3.4.4 virus,site 205(H5 numbering)was speculated to be the key amino acid that causing the antigenic alteration.In addition,the response spectrum of mAb 6A5 with the H5 viruses screened two sets of multi-site amino acid mutations containing positions 82,102,104,172,179,190,204 and positions 88,157,199,respectively.Those two sets containing positions might have a combined effect in influencing antigenic variation.Moreover,mAb 7G3 and 5H4 additionally screened positions 156 and 200.And the different reactivity of mAb 3D9 indicated that sites 102 and 199 might have a combined effect in influencing antigenic variation.3.Application of H5 subtype avian influenza virus NP protein monoclonal antibodies in broad-spectrum detectionGiven that NP protein is a highly conserved protein of influenza A viruses,the potential application for broad-spectrum detection of the 3 NP mAb prepared in Chapter 1 was further evaluated.AIV of different HA subtypes from HI to H11,as well as influenza viruses of swine and human origin,and influenza B viruses were selected to infect MDCK cells for IFA determination.The results showed that the 3 NP mAb of 3D4,4B9 and 5F10 all had good broad-spectrum activity against influenza A viruses of different HA subtypes and different host sources but did not react with influenza B viruses,indicating the specificity to just influenza A viruses.Subsequently,the mAb ascites was purified by Protein G affinity chromatography column and labeled with Horseradish Peroxidase(HRP).And a double-antibody sandwich ELISA method was established by using purified mAb 3D4 as the capture antibody and HRP-4B9 as the detection antibody through pairwise tests.After optimization,the best encapsulation concentration of capture antibody was finally determined as 1.25 ?g/mL,and the best dilution of detection antibody was 1:8000.The optimal reaction conditions were as follows:the capture antibody was overnight coated with 100 ?L/well at 4?,1%gelatin was selected for closure in 200 ?L/well at 37? for 2 h,the antigen to be detected was in 100?L/well for reaction at 37? for 2 h,the capture antibody was incubated in 100 ?L/well at 37?for 1 h,and the TMB reaction was terminated after 15 min and the OD450nm value was measured.The evaluation of this double-antibody sandwich ELISA assay showed that the method had strong specificity and good broad-spectrum property in detection of influenza A virus.The minimum detection level was 0.3710~2 TCID50/mL for H1N1 and 0.9810~2 TCID50/mL for H9N2,with the sensitivity significantly higher than that of the Hemagglutination(HA)test.Additionally,no error detection or leak detection occurred in the detection test of clinical samples,suggesting that the method would have good practical application prospects.