Cross-neutralization of 1918 and 2009 influenza viruses: role of glycans in viral evolution and vaccine design

Abstract

New strains of H1N1 influenza virus have emerged episodically over the last century to cause human pandemics, notably in 1918 and recently in 2009. Pandemic viruses typically evolve into seasonal forms that develop resistance to antibody neutralization, and cross-protection between strains separated by more than 3 years is uncommon. Here, we define the structural basis for cross-neutralization between two temporally distant pandemic influenza viruses--from 1918 and 2009. Vaccination of mice with the 1918 strain protected against subsequent lethal infection by 2009 virus. Both were resistant to antibodies directed against a seasonal influenza, A/New Caledonia/20/1999 (1999 NC), which was insensitive to antisera to the pandemic strains. Pandemic strain-neutralizing antibodies were directed against a subregion of the hemagglutinin (HA) receptor binding domain that is highly conserved between the 1918 and the 2009 viruses. In seasonal strains, this region undergoes amino acid diversification but is shielded from antibody neutralization by two highly conserved glycosylation sites absent in the pandemic strains. Pandemic HA trimers modified by glycosylation at these positions were resistant to neutralizing antibodies to wild-type HA. Yet, antisera generated against the glycosylated HA mutant neutralized it, suggesting that the focus of the immune response can be selectively changed with this modification. Collectively, these findings define critical determinants of H1N1 viral evolution and have implications for vaccine design. Immunization directed to conserved receptor binding domain subregions of pandemic viruses could potentially protect against similar future pandemic viruses, and vaccination with glycosylated 2009 pandemic virus may limit its further spread and transformation into a seasonal influenza.

Fig. 1

Cross-neutralization, HI reactivity, and specificity of antisera to 1918 SC and 2009 CA in contrast to a seasonal strain, 1999 NC. (A) Neutralization activity of antisera from mice immunized with the indicated HA plasmid expression vectors or no insert (control) plasmid was measured by luciferase assay with 1918 SC (left panel), 2009 CA (middle panel), or 1999 NC (right panel) HA-pseudotyped lentiviral vectors. (B) HI by antisera from mice immunized with control or the indicated HA expression vector was performed with 1918 SC HA-pseudotyped virus and 2009 CA and 1999 NC viruses. (C) Antisera from mice immunized with 1918 SC, 2009 CA, or 1999 NC HA plasmid were preabsorbed with HIV (control), 1918 SC, 2009 CA, or 1999 NC HA trimers, and the neutralization activities of the preabsorbed antisera were measured with 1918 SC, 2009 CA, and 1999 NC HA-pseudotyped lentiviral vectors. Percent reduction in neutralization was recorded at 1:800 serum dilution.

Fig. 2

Glycosylation patterns and RBD-A sequence conservation of human H1N1 HAs. (A) Ribbon diagrams (side and top views) of HA depicting N-linked glycosylation on the pandemic 1918 SC and 2009 CA strains (left panels) and the seasonal 1999 NC H1N1 strain (right panels). The asparagine side chains of glycosylation sites were rendered as blue Corey-Pauling-Koltun (CPK) models. The glycosylation sites 142 and 177 (1918 numbering) on the top of the RBD are circled by a dotted line. (B) Same as in (A), except that glycosylations were modeled as complex glycosylations (with the terminal sialic acid residues removed) with the GlyProt Server (29) and rendered as blue stick models. (C) The top panel illustrates the placement of these glycosylation sites on ribbon diagrams of 1918 SC HA using blue CPK models for glycosylated asparagine side chains. A red dotted circle on the leftmost ribbon diagram indicates the placement of the RBD-A region. The bottom panel depicts sequence identity between the 1918 pandemic strain and representative strains in the RBD-A region mapped onto a surface representation. Conserved residues are colored green and altered residues are colored red. The orientation is the same as in fig. S1 (middle two panels). PDB entry 1RUZ (1918 SC) was used for displaying the H1N1 pandemic strain HAs, and the seasonal H1N1 HAs were displayed using the structure of the A/PR/8/34 HA (PDB entry 1RU7). All structural panels were generated with the molecular graphics program UCSF Chimera (30).

Fig. 3

Addition of two glycosylation sites to 1918 SC or 2009 CA confers resistance to neutralization. (A) Inhibition of neutralizing antibodies to 1918 SC and 2009 CA measured on 1918 SC–pseudotyped and 2009 CA–pseudotyped lentiviral vectors or 1999 NC on 1999 NC reporter after absorption of sera with cells expressing the indicated HA or without absorption (Control). Percent reduction in neutralization was recorded at 1:400 serum dilution. (B and C) Comparable activity of 1918 SC and 2009 CA, and glycosylation mutants [1918 (2G) and 2009 (2G)] for viral entry using pseudotyped lentiviral vectors (left panels) and relative resistance of the 2G mutant pseudotyped versus wild-type reporters to neutralization by wild-type 1918 SC antisera (middle panel) or 2009 CA antisera (right panel) derived from DNA-vaccinated mice. (D) Neutralization of wild-type and glycosylation mutants of 1918 SC viruses by antisera from mice immunized twice with wild-type or glycosylation mutant 1918 SC HA DNA vaccines. Percent reduction in neutralization was measured at a 1:200 serum dilution. The sera raised to the 1918 2G were unable to neutralize the 1999 NC virus.