Screening of random peptide library of hemagglutinin from pandemic 2009 A(H1N1) influenza virus reveals unexpected antigenically important regions.

Wanghui Xu,Lu Han,Zhanglin Lin,Lisa Ng Fong Poh

doi:10.1371/journal.pone.0018016

Abstract

The antigenic structure of the membrane protein hemagglutinin (HA) from the 2009 A(H1N1) influenza virus was dissected with a high-throughput screening method using complex antisera. The approach involves generating yeast cell libraries displaying a pool of random peptides of controllable lengths on the cell surface, followed by one round of fluorescence-activated cell sorting (FACS) against antisera from mouse, goat and human, respectively. The amino acid residue frequency appearing in the antigenic peptides at both the primary sequence and structural level was determined and used to identify “hot spots” or antigenically important regions. Unexpectedly, different antigenic structures were seen for different antisera. Moreover, five antigenic regions were identified, of which all but one are located in the conserved HA stem region that is responsible for membrane fusion. Our findings are corroborated by several recent studies on cross-neutralizing H1 subtype antibodies that recognize the HA stem region. The antigenic peptides identified may provide clues for creating peptide vaccines with better accessibility to memory B cells and better induction of cross-neutralizing antibodies than the whole HA protein. The scheme used in this study enables a direct mapping of the antigenic regions of viral proteins recognized by antisera, and may be useful for dissecting the antigenic structures of other viral proteins.

Highlights

The 2009 A(H1N1) influenza virus, which is referred to as the swine-origin influenza virus (S-OIV), caused the first global influenza pandemic in recent decades [1]
We report here the identification of antigenic peptides of the 2009 A(H1N1) influenza HA protein from a combinational library of viral protein fragments displayed on the surface of yeast cells and sorted by fluorescenceactivated cell sorting (FACS)
We used a novel statistical approach to identify antigenically important regions of the viral protein based on the frequency of each residue appearing in the antigenic peptides at both the primary sequence and structural level

Summary

Introduction

The 2009 A(H1N1) influenza virus, which is referred to as the swine-origin influenza virus (S-OIV), caused the first global influenza pandemic in recent decades [1]. Current treatment strategies for influenza-A viruses, such as vaccines and drugs, have not provided broad and lasting protection, partly due to the constantly evolving nature of the viral surface glycoprotein, hemagglutinin (HA) that allows it to avoid host immune attack. For many challenging diseases caused by viruses, the recognition of certain neutralizing epitopes by the immune system can provide broad and potent protection [4,5]. The antigenic structure of HA and the corresponding antibody response are not fully understood, complicating rational design of vaccines aimed at modulating antibody responses for targeting key epitopes

Methods

Results

Conclusion