N-Glycosylation of Seasonal Influenza Vaccine Hemagglutinins: Implication for Potency Testing and Immune Processing

Abstract

Prior to each annual flu season, health authorities recommend three or four virus strains for inclusion in the annual influenza vaccine: a type A:H1N1 virus, a type A:H3N2 virus, and one or two type B viruses. Antigenic differences between strains are found in the glycosylation patterns of the major influenza virus antigen, hemagglutinin (HA). Here we examine the glycosylation patterns of seven reference antigens containing HA used in influenza vaccine potency testing. These reagents are supplied by the Center for Biologics Evaluation and Research (CBER) or the National Institute for Biological Standards and Control (NIBSC) for use in vaccine testing. Those produced in hen egg, Madin-Darby canine kidney (MDCK), and insect (Sf9) expression systems were examined. They are closely related or identical to antigens used in commercial vaccines. The reference antigens studied were used in the 2014-2015 influenza season and included A/California/07/2009 H1N1, A/Texas/50/2012 H3N2, and B/Massachusetts/02/2012. Released glycan and HA-specific glycopeptide glycosylation patterns were examined. We also examined the sensitivity of the single radial immunodiffusion (SRID) potency test to differences in HA antigen glycosylation. Based on deglycosylation studies applied using standard assay procedures, the SRID assay was not sensitive to any HA antigen glycosylation status from any cell system. Mapping of glycosites with their occupying glycan to functional regions, including antigenic sites, lectin interaction regions, and fusion domains, was performed and has implications for immune processing, immune responses, and antigenic shielding. Differences in glycosylation patterns, as dictated by the cell system used for expression, may impact these functions.IMPORTANCE In the present study, the glycosylation patterns of the 2014-2015 influenza vaccine season standard antigens A/California/07/2009 H1N1, A/Texas/50/2012 H3N2, and B/Massachusetts/02/2012 were revealed, and the sensitivity of the single radial immunodiffusion (SRID) potency test to glycosylation was tested. Differences in hemagglutinin glycosylation site composition and heterogeneity seen in antigens produced in different cell substrates suggest differences in processing and downstream immune responses. The SRID potency test used for vaccine release is not sensitive to differences in glycosylation under standard use conditions. This work reveals important differences in vaccine antigens and may point out areas where improvements may be made concerning vaccine antigen preparation, immune processing, and testing.

Keywords: glycan masking; glycopeptide; mass spectrometry; vaccine.

FIG 1

N-glycan subtype examples. N-glycan compositions were divided into the following five groups in this study: high-mannose, complex, hybrid, intermediate, and paucimannose types. Representative glycan cartoons are presented in Fig. 2. High-mannose glycans have the composition Man5-9GlcNAc2 (Fig. 2, m/z 1,579.90, 1,783.99, 1,988.08, and 2,192.14). Complex glycans consist of a trimannosyl core with additional residues replaced by one or more monosaccharides other than Man and typically extended by the addition of GlcNAc, GalNAc, Gal, and Fuc (Fig. 2, m/z 2,244.16, 2,418.17, and 2,693.14). Hybrid N-glycans here also consist of a trimannosyl core; one branch retains some or all of its Man residues, and only one arm is elongated, typically with GlcNAc, GalNAc, Gal, and Fuc (Fig. 2, m/z 2,029.11), in the same way as that for complex N-glycans. Intermediate N-glycans are similar to hybrid glycans, since only one arm is elongated, but it is limited to the addition of a GlcNAc. Here we define intermediate N-glycans as Man3-5GlcNAc3 structures with or without a core Fuc residue (Fig. 2, m/z 1,417.09 and 1,591.21). The distinction between intermediate and hybrid glycans is important for comparisons made between cell platforms. The latter are characteristic of insect cell glycosylation, being more abundant in insect cells than in eggs or MDCK cells. The paucimannose N-glycans consist of a trimannosyl core with no substituents on either terminal mannose residue. The core GlcNAc residue can be replaced by Fuc (Fig. 2, m/z 1,171.91 and 1,346.02) in some insect cell lines (10, 13), and an additional Fuc core substitution is possible, although this is usually not seen in proteins derived from the Sf9 cell lines used here. Green circles are Man, blue squares are GlcNAc, red triangles are Fuc, and yellow circles are Gal.

FIG 2

N-glycan profiles of H1N1 reference antigens from eggs and MDCK and Sf9 cells. Glycans were released from proteins, permethylated, and analyzed by MALDI-TOF MS. Highly abundant peaks are annotated with possible compositions.

FIG 3

(A) Relative abundances of permethylated N-glycans from H1N1 reference antigens. Measurement was performed in triplicate by MALDI-TOF MS. The composition of each glycan is displayed beneath the histogram. The relative abundance of each glycan is displayed as a percentage of the total abundance, with standard deviations displayed as error bars. (B) Individual subgroup glycans were summed, and the percentage of each subgroup is displayed in a pie diagram. Abbreviations: H, hexose; N, N-acetylglucosamine; dHex, deoxyhexose; NeuAc, sialic acid. High-mannose glycans are noted by the number of mannoses they have, i.e., Man9.

FIG 4

MS/MS spectra of glycopeptide NAGSIIISDTPVHDCN₂₇₆TTCQTPK of H1N1 HAs. The colors in the figure indicate the following: red, y ions; blue, b ions; green, y/b ions after neutral loss; pink, glycopeptides after neutral loss assigned by BiopharmaLynx; and gray, ions unassigned by BiopharmaLynx (some were assigned manually, as indicated). Monosaccharide symbols are as follows: blue squares, GlcNAc; green circles, Man; red triangles, Fuc; and yellow circles, Gal. Peptide fragments with neutral glycosyl loss are indicated. Oxonium ions at m/z 204.09, 366.14, 528.19, and 690.25 are indicated.

FIG 5

(A) Relative abundances of permethylated N-glycans from H3N2 reference antigens. Measurement was performed in triplicate by MALDI-TOF MS. The composition of each glycan is displayed underneath the histogram, abbreviated as described in the legend to Fig. 3. The relative abundance of each glycan is displayed as a percentage of the total abundance, with standard deviations displayed as error bars. (B) Individual subgroup glycans were summed, and the percentage of each subgroup is displayed in a pie diagram.

FIG 6

(A) Relative abundances of permethylated N-glycans from influenza B virus reference antigens. Measurement was performed in triplicate by MALDI-TOF MS. The composition of each glycan is displayed underneath the histogram, abbreviated as described in the legend to Fig. 3. The relative abundance of each glycan is displayed as a percentage of the total abundance, with standard deviations displayed as error bars. (B) Individual subgroup glycans were summed, and the percentage of each subgroup is displayed in a pie diagram.

FIG 7

Glycosylation and antigenic site mapping for hemagglutinins. (A) Most abundant glycans at each site in A/California/07/2009 H1N1 HA produced in eggs and MDCK and Sf9 cells. Shown are surface area representations of two sides of an HA monomer from PDB structure 3UBQ, which is 98% identical to California H1N1 HA. The RBS is indicated with a light blue circle. N-glycosylation sites are colored cyan. Antigenic sites are colored as follows: Sa, red; Sb, orange; H1C, yellow; Ca1, green; Ca2, pink; and Cb, blue. Numbers under glycoforms from the egg and MDCK cell lines are ratios of forms with 2 HexNAcs (high mannose) to those with 3 HexNAcs (complex and hybrid). Under the Sf9 cell line glycoforms, the ratios are percentages of sugars with 2 HexNAcs and 5 or more hexoses (high mannose), 2HexNAcs and fewer than 5 hexoses (paucimannose), and 3 or more HexNAcs (intermediate). (B) Most abundant glycans at each site in A/Texas/50/2012 H3N2 HA produced in eggs or MDCK cells. Shown are surface area representations of two sides of an HA monomer from PDB structure 4WE8, which is 98% identical to Texas H3N2 HA. The RBS is indicated with a purple circle. N-glycosylation sites are colored cyan. Antigenic sites are colored as follows: A, red; B, orange; C, yellow; D, green; and E, blue. Numbers under glycoforms are ratios of forms with 2 HexNAcs to those with 3 or more HexNAcs. (C) Most abundant glycans at each site in B/Massachusetts/02/2012 HA produced in eggs or MDCK cells. Shown are surface area representations of two sides of an HA monomer from PDB structure 4M40, which is 96% identical to B/Massachusetts/02/2012 HA. The RBS is indicated with a light blue circle. N-glycosylation sites are colored cyan. Antigenic sites are colored as follows: BA, red; BB1, orange; BB2, pink; BC, yellow; BD, green; and BE, blue. Numbers under glycoforms are ratios of forms with 2 HexNAcs to those with 3 or more HexNAcs. Figures were made with Pymol. Relevant antigenic site mapping reports used in this work are cited in the text.

FIG 8

SRID analysis of reference antigens. Reference antigens were treated with and without an Endo F1, F2, and F3 endoglycosidase cocktail. Resultant antigens were compared to untreated control antigen. Blue, control antigen; orange, incubation without enzyme; gray, incubation with enzyme cocktail. The reference antigen is shown at the top of each graph. Dilutions are shown on the x axis. Immunoprecipitation ring diameters are shown on the y axis.