Construction of a chimeric secretory IgA and its neutralization activity against avian influenza virus H5N1.

Cun Li,Xiaona Wang,Bo Zhang,Xiaoping An,Yigang Tong,Yong Huang,Baozhong Zhang,Zhiyi Zhang,Zhiqiang Mi,Azeem Mehmood Butt,Wenhui Zhang

doi:10.1155/2014/394127

Abstract

Secretory immunoglobulin A (SIgA) acts as the first line of defense against respiratory pathogens. In this assay, the variable regions of heavy chain (VH) and Light chain (VL) genes from a mouse monoclonal antibody against H5N1 were cloned and fused with human IgA constant regions. The full-length chimeric light and heavy chains were inserted into a eukaryotic expressing vector and then transfected into CHO/dhfr-cells. The chimeric monomeric IgA antibody expression was confirmed by using ELISA, SDS-PAGE, and Western blot. In order to obtain a dimeric secretory IgA, another two expressing plasmids, namely, pcDNA4/His A-IgJ and pcDNA4/His A-SC, were cotransfected into the CHO/dhfr-cells. The expression of dimeric SIgA was confirmed by using ELISA assay and native gel electrophoresis. In microneutralization assay on 96-well immunoplate, the chimeric SIgA showed neutralization activity against H5N1 virus on MDCK cells and the titer was determined to be 1 : 64. On preadministrating intranasally, the chimeric SIgA could prevent mice from lethal attack by using A/Vietnam/1194/04 H5N1 with a survival rate of 80%. So we concluded that the constructed recombinant chimeric SIgA has a neutralization capability targeting avian influenza virus H5N1 infection in vitro and in vivo.

Highlights

Endemic highly pathogenic avian influenza virus (AIV) H5N1 in poultry has been present since the first occurrence in 1997 in Hong Kong
Our results revealed that the recombinant Secretory immunoglobulin A (SIgA) could act as a preventative agent against H5N1 infection
The variable regions of the heavy (VH) and light (VL) chain genes of the anti-H5N1 HA neutralizing monoclonal antibody were cloned by RT-PCR [37]

Summary

Introduction

Endemic highly pathogenic avian influenza virus (AIV) H5N1 in poultry has been present since the first occurrence in 1997 in Hong Kong. AIV H5N1 circulates in waterfowl and domesticated avian species and has evolved into multiple phylogenetically distinct genotypes and clades [1,2,3], with geographically distinct groups in each country. H5N1 viruses occasionally infect humans, with high case-fatality rates. These viruses have repeatedly crossed the species barrier and caused highly lethal human infections. The wide distribution of highly pathogenic AIV H5N1 is a global threat to human health [4,5,6,7]. According to the latest WHO report [8], there have been 633 laboratory-confirmed highly pathogenic H5N1 AI cases worldwide from 2003 to 2013, with a mortality of 59.6%

Methods

Results

Conclusion