Characterization of mAb6-9-1 monoclonal antibody against hemagglutinin of avian influenza virus H5N1 and its engineered derivative, single-chain variable fragment antibody
Hemagglutinin (HA), as a major surface antigen of influenza virus, is widely used as a target for production of neutralizing antibodies. Monoclonal antibody, mAb6-9-1, directed against HA of highly pathogenic avian influenza virus A/swan/Poland/305-135V08/2006(H5N1) was purified from mouse hybridoma cells culture and characterized. The antigenic specificity of mAb6-9-1 was verified by testing its cross-reactivity with several variants of HA. The mimotopes recognized by mAb6-9-1 were selected from two types of phage display libraries. The comparative structural model of the HA variant used for antibody generation was developed to further facilitated epitope mapping. Based on the sequences of the affinity-selected polypeptides and the structural model of HA the epitope has been located to the region near the receptor binding site (RBS). Such localization of the epitope recognized by mAb6-9-1 is in concordance with its moderate hemagglutination inhibition activity and its antigenic specificity. Additionally, total RNA from hybridoma cells secreting mAb6-9-1 was used for obtaining two variants of cDNA encoding recombinant single-chain variable fragment (scFv) antibody. To ensure high production level and solubility in bacterial expression system, the scFv fragments were produced as chimeric proteins in fusion with thioredoxin or displayed on a phage surface after cloning into the phagemid vector. Specificity and affinity of the recombinant soluble and phage-bound scFv were assayed by suitable variants of ELISA test. The observed slight differences in specificity are discussed.
Al-Majhdi F (2007) Structure of the sialic acid binding site in influenza A virus: hemagglutinin. Journal of Biological Sciences 7: 113-122.
Ascione A, Capecchi B, Campitelli L, Imperiale V, Flego M, Zamboni S, Gellini M, Alberini I, Pittiglio E, Donatelli I, Temperton N J, Cianfriglia M (2009) Human monoclonal antibodies in single chain fragment variable format with potent neutralization activity against influenza virus H5N1. Antiviral Research 83: 238-244. doi: 10.1016/j.antiviral.2009.05.005.
Bublil E M, Freund NT., Mayrose I, Penn O, Roitburd-Berman A, Rubinstein ND, Pupko T, Gershoni JM (2007) Stepwise prediction of conformational discontinuous B-Cell Epitopes using the mapitope algorithm. Proteins-Structure Function and Bioinformatics 68: 294-304. doi: 10.1002/prot.21387.
Cao Y, Marks JD, Huang Q, Rudnick, SI, Xiong C, Hittelman WN, Wen X, Marks JW, Cheung LH, Boland K, Li C, Adams GP, Rosenblum MG (2012a) Single-Chain Antibody-Based Immunotoxins Targeting Her2/neu: Design Optimization and Impact of Affinity on Antitumor Efficacy and Off-Target Toxicity. Molecular Cancer Therapeutics 11: 143-153. doi: 10.1158/1535-7163.MCT-11-0519.
Cao Z, Meng J, Li X, Wu R, Huang Y, He Y (2012b) The Epitope and Neutralization Mechanism of AVFluIgG01, a Broad-Reactive Human Monoclonal Antibody against H5N1 Influenza Virus. Plos One 7. doi: 10.1371/journal.pone.0038126.
Choo AB, Dunn RD, Broady KW, Raison RL (2002) Soluble expression of a functional recombinant cytolytic immunotoxin in insect cells. Protein Expr Purif 24: 338-347. doi: 10.1006/prep.2001.1589.
Dobhal S, Chaudhary VK, Singh A, Pandey D, Kumar A, Agrawal S (2013) Expression of recombinant antibody (single chain antibody fragment) in transgenic plant Nicotiana tabacum cv. Xanthi. Mol Biol Rep 40: 7027-7037. doi: 10.1007/s11033-013-2822-x.
Dorsam H, Braunagel M, Kleist C, Moynet D, Welschof M (2002) Screening of phage displayed antibody libraries. The protein protocols handbook: 1073-1082.
Du L, Jin L, Zhao G, Sun S, Li J, Yu H, Li Y, Zheng B-J, Liddington RC, Zhou Y, Jiang S (2013) Identification and Structural Characterization of a Broadly Neutralizing Antibody Targeting a Novel Conserved Epitope on the Influenza Virus H5N1 Hemagglutinin. Journal of Virology 87: 2215-2225.
Ho M, Nagata S, Pastan I (2006) Isolation of anti-CD22 Fv with high affinity by Fv display on human cells. Proc Natl Acad Sci U S A 103: 9637-9642.
Jarocka U, Sawicka R, Gora-Sochacka A, Sirko A, Dehaen W, Radecki J, Radecka H (2016) An electrochemical immunosensor based on a4,4-thiobisbenzenethiol self-assembled monolayer for the detectionof hemagglutinin from avian influenza virus H5N1 Sensors and Acttuators B: Chemical 228: 25-30.
Jarocka U, Sawicka R, Góra-Sochacka A, Sirko A, Zagórski-Ostoja W, Radecki J, Radecka H (2014) An immunosensor based on antibody binding fragments attached to gold nanoparticles for the detection of peptides derived from avian influenza hemagglutinin H5. Sensors (Basel) 14, 15714-15728. doi: 10.3390/s140915714.
Jurado P, de Lorenzo V, Fernandez LA (2006) Thioredoxin fusions increase folding of single chain Fv antibodies in the cytoplasm of Escherichia coli: Evidence that chaperone activity is the prime effect of thioredoxin. Journal of Molecular Biology 357: 49-61.doi: 10.1016/j.jmb.2005.12.058.
Kaku Y, Noguchi A, Okutani A, Inoue S, Tanabayashi K, Yamamoto Y, Hotta A, Suzuki M, Sugiura N, Yamada A (2012) Altered specificity of single-chain antibody fragments bound to pandemic H1N1-2009 influenza virus after conversion of the phage-bound to the soluble form. BMC Res Notes 5: 483. doi: 10.1186/1756-0500-5-483.
Kalenik B, Sawicka R, Góra-Sochacka A, Sirko A (2014) Influenza prevention and treatment by passive immunization. Acta Biochim Pol 61: 573-587.
Kyte J, Doolittle RF (1982) A SIMPLE METHOD FOR DISPLAYING THE HYDROPATHIC CHARACTER OF A PROTEIN. Journal of Molecular Biology 157: 105-132. doi: 10.1016/0022-2836(82)90515-0.
Ladiges W, Osman GE (2001) Molecular characterization of immunoglobulin genes. In Basic methods in antibody production and characterization. Howard G, Bethell D eds, pp 169-192. CRC Press LLC.
Li Y, Zhang X, Xu Q, Fu Q, Zhu Y, Chen S, Peng D, Liu X (2013) Characterisation and haemagglutinin gene epitope mapping of a variant strain of H5N1 subtype avian influenza virus. Veterinary Microbiology 162: 614-622.
Mayrose I, Penn O, Erez E, Rubinstein ND, Shlomi T, Freund NT, Bublil EM, Ruppin E, Sharan R, Gershoni JM, Martz E, Pupko T (2007a) Pepitope: epitope mapping from affinity-selected peptides. Bioinformatics 23: 3244-3246. doi: 10.1093/bioinformatics/btm493.
Mayrose I, Shlomi T, Rubinstein ND, Gershoni JM, Ruppin E, Sharan R, Pupko T (2007b) Epitope mapping using combinatorial phage-display libraries: a graph-based algorithm. Nucleic Acids Research 35: 69-78. doi: 10.1093/nar/gkl975.
Meng EC, Pettersen EF, Couch GS, Huang CC, Ferrin TE (2006) Tools for integrated sequence-structure analysis with UCSF Chimera. Bmc Bioinformatics 7. doi: 10.1186/1471-2105-7-339.
Oriuchi N, Higuchi T, Hanaoka H, Iida Y, Endo K (2005) Current status of cancer therapy with radiolabeled monoclonal antibody. Annals of Nuclear Medicine 19: 355-365.
Peng Y, Zou Y, Li H, Li K, Jiang T (2014) Inferring the antigenic epitopes for highly pathogenic avian influenza H5N1 viruses. Vaccine 32: 671-676. doi: 10.1016/j.vaccine.2013.12.005.
Rouet R, Lowe D, Dudgeon K, Roome B, Schofield P, Langley D, Andrews J, Whitfeld P, Jermutus L, Christ D (2012) Expression of high-affinity human antibody fragments in bacteria. Nature Protocols 7: 364-373. doi: 10.1038/nprot.2011.448.
Sali A, Blundell TL (1993) COMPARATIVE PROTEIN MODELING BY SATISFACTION OF SPATIAL RESTRAINTS. Journal of Molecular Biology 234: 779-815. doi: 10.1006/jmbi.1993.1626.
Shepelyakovskaya AO, Laman AG, Lomonosova AV, Fursova KK, Savinov GV, Vertiev YV, Brovko FA, Grishin EV (2011) Effect of the format of antibodies on their specificity. Mol Immunol 49: 433-440. doi: 10.1016/j.molimm.2011.09.017.
Smith GJD, Donis RO, WHO/OIE/FAO Working group (2012) Continued evolution of highly pathogenic avian influenza A (H5N1): updated nomenclature. Influenza and Other Respiratory Viruses 6: 1-5. doi: 10.1111/j.1750-2659.2011.00298.x.
Smith GJD, Donis RO, World Health Organization, W. (2015) Nomenclature updates resulting from the evolution of avian influenza A(H5) virus clades 184.108.40.206a, 2.2.1, and 2.3.4 during 2013-2014. Influenza and Other Respiratory Viruses 9: 271-276. doi: 10.1111/irv.12324.
Sonoda H, Kumada Y, Katsuda T, Yamaji H (2010) Functional expression of single-chain Fv antibody in the cytoplasm of Escherichia coli by thioredoxin fusion and co-expression of molecular chaperones. Protein Expression and Purification 70: 248-253. doi: 10.1016/j.pep.2009.11.003.
Szewczyk B, Bienkowska-Szewczyk K, Krol E (2014) Introduction to molecular biology of influenza A viruses. Acta Biochimica Polonica 61: 397-401.
Wu R, Li X, Leung H-C, Cao Z, Qiu Z, Zhou Y, Zheng B-J, He Y (2014) A novel neutralizing antibody against diverse clades of H5N1 influenza virus and its mutants capable of airborne transmission. Antiviral Research 106: 13-23. http://dx.doi.org/10.1016/j.antiviral.2014.03.005.
Zhang X, Qi X, Zhang Q, Zeng X, Shi Z, Jin Q, Zhan F, Xu Y, Liu Z, Feng Z, Jiao Y (2013) Human 4F5 single-chain Fv antibody recognizing a conserved HA1 epitope has broad neutralizing potency against H5N1 influenza A viruses of different clades. Antiviral Research 99: 91-99. http://dx.doi.org/10.1016/j.antiviral.2013.05.001.
Acta Biochimica Polonica is an open access quarterly and publishes four issues a year. All contents are distributed under the Creative Commons Attribution-ShareAlike 4.0 International (CC BY-SA 4.0) license. Everybody may use the content following terms: Attribution — You must give appropriate credit, provide a link to the license, and indicate if changes were made, ShareAlike — If you remix, transform, or build upon the material, you must distribute your contributions under the same license as the original. There are no additional restrictions — You may not apply legal terms or technological measures that legally restrict others from doing anything the license permits.
Copyright for all published papers © stays with the authors.
Copyright for the journal: © Polish Biochemical Society.