Comparative Glycomics Analysis of Influenza Hemagglutinin (H5N1) Produced in Vaccine Relevant Cell Platforms

Abstract

Hemagglutinin (HA) is the major antigen in influenza vaccines and glycosylation is known to influence its antigenicity. Embryonated hen eggs are traditionally used for influenza vaccine production, but vaccines produced in mammalian and insect cells were recently licensed. This raises the concern that vaccines produced with different cell systems might not be equivalent due to differences in their glycosylation patterns. Thus, we developed an analytical method to monitor vaccine glycosylation through a combination of nanoLC/MSE and quantitative MALDI-TOF MS permethylation profiling. We then used this method to examine glycosylation of HA’s from two different influenza H5N1 strains produced in five different platforms, including hen eggs, three different insect cell lines (High Five™, expresSF+® and glycoengineered expresSF+), and a human cell line (HEK293). Our results demonstrated that (1) sequon utilization is not necessarily equivalent in different cell types, (2) there are quantitative and qualitative differences in the overall N-glycosylation patterns and structures produced by different cell types, (3) ~20% of the N-glycans on the HAs produced by High Five™ cells are core α1,3-fucosylated structures, which may be allergenic in humans, and (4) our method can be used to monitor differences in glycosylation during the cellular glycoengineering stages of vaccine development.

Avian and Human Seasonal Influenza Hemagglutinin Proteins Elicit CD4 T Cell Responses That Are Comparable in Epitope Abundance and Diversity

“…Proteins generated by this system form trimers and generate structures containing heterogeneous mixtures of N-linked glycans, consisting mainly of oligomannose and paucimannose but also containing high-mannose, intermediate-mannose, and hybrid species (30)(31)(32)(33). Mice were vaccinated subcutaneously and analyzed 10 to 11 days postvaccination.…”

“…It is known that HA from viruses that are adapted to circulate in humans contains more glycosylation sites in the globular head domain compared to avian HA (44). However, because unique features in alternative vaccine platforms (egg, mammalian, and insect cells) lead to distinct patterns of glycosylation, the role of glycosylation in protein vaccine immunogenicity is still an open question that will require further detailed study (31). In this regard, however, it is interesting to note that three epitopes restricted by HLA-DR1 that are shared within avian and human HA proteins elicit comparable CD4 T cell responses in the DR1 transgenic mice (Table 1; pH1, aa 437; H5, aa 433; H7, aa 431).…”

“…The recombinant HA proteins used were produced and purified from a baculovirusinsect cell (expresSF ϩ ) expression system and included A/California/04/09 (NR-13691), A/Brisbane/10/07 (NR-19238), A/Indonesia/05/05 (NR-10511), and A/Netherland/219/03 (NR-42022), obtained through BEI Resources (30,31). HLA-DR transgenic mice were immunized subcutaneously in the rear footpad with 1.0 g recombinant HA in the presence of alum, at a 1:1 ratio by volume.…”

Structural characterisation of glycosylation of paramyxovirus attachment and fusion proteins by mass spectrometry

“…Glycosylation is specific to various organisms, tissues and cell lines (22,45,46), as such, production of viral proteins in different expression systems can result in substantially different glycosylation profiles (47)(48)(49)(50). Defining glycosylation of viral surface proteins produced in vivo or in vitro is therefore important for the elucidation of host-virus interactions and for the design of viral therapeutics.…”

“…Interestingly, an analysis of released glycans from HN and F derived from Sendai virions propagated in eggs revealed acidic glycans that were not removed after sialidase digestion (298). Sulfated glycans have also been observed on the HA glycoprotein derived from influenza virions propagated in eggs (48,288,289). Given the observation of sulfation on other viral proteins produced in embryonic eggs and the presence of sulfated oxonium in the analysis of V4-VAR it is likely that glycans that were assigned ambiguously as "sulfated or phosphorylated" are in fact sulfated.…”