Characterization of mAb6-9-1 monoclonal antibody against hemagglutinin of avian influenza virus H5N1 and its engineered derivative, single-chain variable fragment antibody

Abstract

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 peptide libraries. The comparative structural model of the HA variant used for antibody generation was developed to further facilitate epitope mapping. Based on the sequences of the affinity-selected polypeptides and the structural model of HA the epitope was 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 inhibiting activity and its antigenic specificity. Additionally, total RNA isolated from the hybridoma cell line 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 differences in specificity were discussed.

The Natural Oligoribonucleotides Functionalized by D-Mannitol Affected Interactions of Hemagglutinin with Glycan Receptor Indicating Anti-Influenza Activity

“…Based on the structural data, conserved residues involved in the interactions with glycan receptors in the hemagglutinin revealed the interface for binding sialic acid located at the membrane-distal tips of the RBD domain. The same epitopes are recognized specifically by IgG-type [27] or monoclonal mAb6-9-1 anti-bodies [28]. For the HA3 strain the conserved motif comprised α-helix (residues 190 EQTNLYVQA 199 ) together two loops (residues 130 VTQNGGSNACK 140 , and 220 RPWVGQSGRI 230 ) on the two sides of central helix (numbering according to the sequence of HA3) (Figure 1) [8].…”

Irradiation by a Combination of Different Peak-Wavelength Ultraviolet-Light Emitting Diodes Enhances the Inactivation of Influenza A Viruses

“…Therefore, IAV infections are a globally important issue not only in humans but also in their livestock. Avian influenza (AI) viruses are classified by their pathogenic strength based on the feasibility of infection in chickens [5]. The critical genetic difference determining the low pathogenic (LPAI) or highly pathogenic (HPAI) phenotype depends on the HA cleavage site [6].…”