Yung-Chieh Tseng, Chia-Yu Wu, Ming-Liang Liu, Ting-Hua Chen, Wan-Ling Chiang, Yueh-Hsiang Yu, Jia-Tsrong Jan, Kuo-I Lin, Chi-Huey Wong, and Che Ma

PNAS March 5, 2019 116 (10) 4200-4205

Significance

Glycosylation is important for the proper folding and function of glycoproteins. However, glycosylation on viral surface protein may mask the conserved epitopes and causes poor host immune responses and protection against viral infection. To expose more antigenic sites shielded by glycans, we developed a strategy to remove the glycans on the viral surface by modification of the traditional egg-based influenza vaccine production procedure. By means of immunization with the monoglycosylated split virus vaccine, mice were induced with broader immune responses, and thus better protection against cross-strain H1N1 virus infections. The results showed that the monoglycosylated split virus vaccine is a broadly protective vaccine and can be produced through simple modifications of the current vaccine manufacturing process.

Abstract

Each year influenza virus infections cause hundreds of thousands of deaths worldwide and a significant level of morbidity with major economic burden. At the present time, vaccination with inactivated virus vaccine produced from embryonated chicken eggs is the most prevalent method to prevent the infections. However, current influenza vaccines are only effective against closely matched circulating strains and must be updated and administered yearly. Therefore, generating a vaccine that can provide broad protection is greatly needed for influenza vaccine development. We have previously shown that vaccination of the major surface glycoprotein hemagglutinin (HA) of influenza virus with a single N-acetylglucosamine at each of the N-glycosylation sites [monoglycosylated HA (HA_mg)] can elicit better cross-protection compared with the fully glycosylated HA (HA_fg). In the current study, we produced monoglycosylated inactivated split H1N1 virus vaccine from chicken eggs by the N-glycosylation process inhibitor kifunensine and the endoglycosidase Endo H, and intramuscularly immunized mice to examine its efficacy. Compared with vaccination of the traditional influenza vaccine with complex glycosylations from eggs, the monoglycosylated split virus vaccine provided better cross-strain protection against a lethal dose of virus challenge in mice. The enhanced antibody responses induced by the monoglycosylated vaccine immunization include higher neutralization activity, higher hemagglutination inhibition, and more HA stem selectivity, as well as, interestingly, higher antibody-dependent cellular cytotoxicity. This study provides a simple and practical procedure to enhance the cross-strain protection of influenza vaccine by removing the outer part of glycans from the virus surface through modifications of the current egg-based process.

See: https://www.pnas.org/content/116/10/4200

Figure 1: Preparation of monoglycosylated virus in the embryonated chicken egg. (A) Schematic overview of monoglycosylated split virus vaccine production and viral HA with different glycan states. Viral HA_fg, HA with the typical complex type N-glycans; viral HA_hm, HA with high mannose type N-glycans; viral HA_mg, HA with GlcNAc at its N-glycosylation sites. Models were created with Protein Data Bank (PDB) ID code 3LZG by adding glycan using GlyProt (www.glycosciences.de/modeling/glyprot/php/main.php) and PDB files of lipid bilayer from Lipid Bilayer Membranes for RasMol (https://www.umass.edu/microbio/rasmol/bilayers.htm). The images are displayed using the PyMOL (https://pymol.org/2/) program. (B) Western blot analysis of viral HA in allantoic fluids that were inoculated with different concentrations of kifunensine. (C) Western blot analysis of viral HA in kifunensine-treated allantoic fluids with different concentration of Endo H digestion. In B and C, viral HA was detected by rabbit anti-HA polysera in Western blot analysis.