Identification of a Highly Conserved Epitope on Avian Influenza Virus Non-Structural Protein 1 Using a Peptide Microarray

Abstract

Avian influenza virus (AIV) non-structural protein 1 (NS1) is a multifunctional protein. It is present at high levels in infected cells and can be used for AIV detection and diagnosis. In this study, we generated monoclonal antibody (MAb) D7 against AIV NS1 protein by immunization of BALB/c mice with purified recombinant NS1 protein expressed in Escherichia coli. Isotype determination revealed that the MAb was IgG1/κ-type subclass. To identify the epitope of the MAb D7, the NS1 protein was truncated into a total of 225 15-mer peptides with 14 amino acid overlaps, which were spotted for a peptide microarray. The results revealed that the MAb D7 recognized the consensus DAPF motif. Furthermore, the AIV NS1 protein with the DAPF motif deletion was transiently expressed in 293T cells and failed to react with MAb D7. Subsequently, the DAPF motif was synthesized with an elongated GSGS linker at both the C- and N-termini. The MAb D7 reacted with the synthesized peptide both in enzyme-linked immunosorbent assay (ELISA) and dot-blot assays. From these results, we concluded that DAPF motif is the epitope of MAb D7. To our knowledge, this is the first report of a 4-mer epitope on the NS1 protein of AIV that can be recognized by MAb using a peptide microarray, which is able to simplify epitope identification, and that could serve as the basis for immune responses against avian influenza.

A NS1-binding monoclonal antibody interacts with two residues that are highly conserved in seasonal as well as newly emerged influenza A virus

“…As mAbs targeting NS1 can be used for research and diagnostic assay development, multiple hybridomas have been generated to produce anti-NS1 antibodies. For those mAbs with epitope mapped, all of them have been shown to bind to motifs in either NS1(RBD) or NS1(ED) (Tan et al 2010;He et al 2013;Rahim et al 2013;Wen et al 2015;Sun et al 2016). In this study, we have generated a novel mAb 19H9 and found that it interacts with two residues, P85 and Y89, of NS1 that are highly conserved in seasonal as well as newly emerged highly pathogenic avian IAV strains.…”

Rapid Response to Pandemic Threats: Immunogenic Epitope Detection of Pandemic Pathogens for Diagnostics and Vaccine Development Using Peptide Microarrays

“… 101 , 102 The versatile application of peptide microarrays in unraveling epitope-specific antibody responses, drug and vaccine development approaches for influenza are highlighted by several studies. 50 − 55 …”

“…For example, a peptide microarray was used to map the epitope of a monoclonal antibody generated against the NS1 (nonstructural) protein of avian influenza virus. 55 As monoclonal antibodies are important in the field of diagnostics and treatment, characterizing the epitope (and eventually cross-reactivity) is essential for further applications. The authors reported a 4-mer epitope on the avian influenza virus NS1 protein that is recognized by a monoclonal antibody.…”

Generation and Characterization of the Monoclonal Antibody Panel Specific to the NS1 Protein of the Influenza A Virus

“…Monoclonal antibodies (MAbs) may serve as an efficient tool in such studies. MAbs are successfully used to map NS1 epitopes and to characterize the polymorphism of its structure, as well as to study the functions of this protein [7,[9][10][11].…”