Different Immunity Elicited by Recombinant H5N1 Hemagglutinin Proteins Containing Pauci-Mannose, High-Mannose, or Complex Type N-Glycans

Shih-Chang Lin,Suh-Chin Wu,Ben Dionne,Michael Butler,Ming-Hsi Huang,Chi-Huey Wong,Jia-Tsrong Jan,Chung-Yi Wu,Brian James Ward

doi:10.1371/journal.pone.0066719

Abstract

Highly pathogenic avian influenza H5N1 viruses can result in poultry and occasionally in human mortality. A safe and effective H5N1 vaccine is urgently needed to reduce the pandemic potential. Hemagglutinin (HA), a major envelope protein accounting for approximately 80% of spikes in influenza virus, is often used as a major antigen for subunit vaccine development. In this study, we conducted a systematic study of the immune response against influenza virus infection following immunization with recombinant HA proteins expressed in insect (Sf9) cells, insect cells that contain exogenous genes for elaborating N-linked glycans (Mimic) and mammalian cells (CHO). While the antibody titers are higher with the insect cell derived HA proteins, the neutralization and HA inhibition titers are much higher with the mammalian cell produced HA proteins. Recombinant HA proteins containing tri- or tetra-antennary complex, terminally sialylated and asialyated-galactose type N-glycans induced better protective immunity in mice to lethal challenge. The results are highly relevant to issues that should be considered in the production of fragment vaccines.

Highlights

Pathogenic avian influenza (HPAI) viruses such as H5N1, H7N7, and H9N2 can result in poultry and occasionally in human mortality [1]
According to our present results, recombinant HA glycoproteins containing complex terminally sialylated and asialyated-galactose type N-glycans induced higher levels of HI and neutralizing antibodies and better protective immunity in mice compared to recombinant HA glycoproteins containing pauci-mannose or highmannose type N-glycans
While the antibody titers are higher with the insect cell derived HA proteins, the neutralization titers are much higher with the mammalian cell produced HA proteins with complex type N-linked glycans and the antibodies protect mice to lethal challenge

Summary

Introduction

Pathogenic avian influenza (HPAI) viruses such as H5N1, H7N7, and H9N2 can result in poultry and occasionally in human mortality [1]. Recent reports indicate that the involvement of HA and PB2 amino acid substitutions leads to easier transmission in ferrets, suggesting that HPAI H5N1 viruses have the potential to evolve and be transmitted between mammals, posing the risk of a human pandemic [4,5]. Anti-H5N1 neutralizing antibodies have been elicited in mice, chickens, and ferrets using recombinant HA proteins expressed in mammalian and insect cells [6,7,8], plant cells [9,10], and E. coli cells [11,12,13,14,15]. At least two research teams have reported that single GlcNAc glycans of complex N-linked HA glycoproteins increase receptor binding in sialic acid and neutralizing antibody titers in mice [7,17]

Methods

Results

Conclusion