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. 2013 May 29;5(187):187ra70.
doi: 10.1126/scitranslmed.3005996.

Glycosylations in the globular head of the hemagglutinin protein modulate the virulence and antigenic properties of the H1N1 influenza viruses

Rafael A Medina  1 , Silke Stertz, Balaji Manicassamy, Petra Zimmermann, Xiangjie Sun, Randy A Albrecht, Hanni Uusi-Kerttula, Osvaldo Zagordi, Robert B Belshe, Sharon E Frey, Terrence M Tumpey, Adolfo García-Sastre
Affiliations

Glycosylations in the globular head of the hemagglutinin protein modulate the virulence and antigenic properties of the H1N1 influenza viruses

Rafael A Medina et al. Sci Transl Med. .

Abstract

With the global spread of the 2009 pandemic H1N1 (pH1N1) influenza virus, there are increasing worries about evolution through antigenic drift. One way previous seasonal H1N1 and H3N2 influenza strains have evolved over time is by acquiring additional glycosylations in the globular head of their hemagglutinin (HA) proteins; these glycosylations have been believed to shield antigenically relevant regions from antibody immune responses. We added additional HA glycosylation sites to influenza A/Netherlands/602/2009 recombinant (rpH1N1) viruses, reflecting their temporal appearance in previous seasonal H1N1 viruses. Additional glycosylations resulted in substantially attenuated infection in mice and ferrets, whereas deleting HA glycosylation sites from a pre-pandemic virus resulted in increased pathogenicity in mice. We then more directly investigated the interactions of HA glycosylations and antibody responses through mutational analysis. We found that the polyclonal antibody response elicited by wild-type rpH1N1 HA was likely directed against an immunodominant region, which could be shielded by glycosylation at position 144. However, rpH1N1 HA glycosylated at position 144 elicited a broader polyclonal response able to cross-neutralize all wild-type and glycosylation mutant pH1N1 viruses. Moreover, mice infected with a recent seasonal virus in which glycosylation sites were removed elicited antibodies that protected against challenge with the antigenically distant pH1N1 virus. Thus, acquisition of glycosylation sites in the HA of H1N1 human influenza viruses affected not only their pathogenicity and ability to escape from polyclonal antibodies elicited by previous influenza virus strains but also their ability to induce cross-reactive antibodies against drifted antigenic variants.

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Conflict of interest statement

Competing Interest: The authors declare no competing interests. The findings and conclusions in this report are those of the authors and do not necessarily reflect the views of the funding agency.

Figures

Fig. 1
Fig. 1. Acquisition of glycosylation sites in HA of human H1N1 subtype over time prior to the emergence of the 2009 H1N1 virus
Amino acid alignment of antigenic sites in the HA1 of seasonal H1N1 strains circulating in humans since 1918 until just prior to the emergence of the 2009 H1N1 pandemic virus. Four representative 2009 H1N1 pandemic isolates are also included as reference. Alignment shows selected prototypical reference strains. Colored shading (purple, yellow and green) depicts known antigenic sites listed on top. Yellow boxes represent conserved glycosylations and red boxes represent glycosylations that appear and disappear over time. Arrows on the right show the year glycosylations appeared at the indicated residue positions.
Fig. 2
Fig. 2. Modeling of H1N1 glycosylations over time
(A) Time line illustrating the year of acquisition of glycosylations in the globular head of the HA protein. Numbers in red indicate the amino acid position of the glycosylation site that are conserved amongst human H1N1 isolates since their emergence in 1918, and numbers in black indicate the amino acid position of the glycosylation sites that appeared in the specific years shown at the bottom. Arrows denote the persistence of glycosylation sites through time, and circles represent their disappearance. Discontinuous lines indicate the time period from 157 to 177 when H1N1 viruses did not circulate in humans. (B) Representation of an HA monomer with the antigenic sites highlighted in red and the glycosylation sites in yellow. The stem region of HA is denoted in silver. Amino acid positions refer to the H1 nomenclature (sites 71, 142, 144, 172 and 177 correspond to H3 numbering 58, 128, 130, 158 and 163, respectively). Numbers in red indicate conserved glycosylation sites. (C) Structural modeling of the trimeric HA with glycosylations as they appeared over time from 1918 to the emergence of the 2009 pH1N1 virus. The glycan structures for the sites shown in yellow have been modeled onto the Cal/09 HA (PDB 3LZG) and are also depicted in yellow. All models were made with MacPyMol.
Fig. 3
Fig. 3. Phenotypic characterization of HA glycosylation mutant 2009 pH1N1 viruses
(A) Plaque morphology of rescued A/Netherlands/602/2009 HA glycosylation mutant viruses in MDCK cells. (B) Western blot analysis of whole cell lysates obtained from MDCK cells infected at an MOI of 5 for 12 h. Lysates were run under non-reducing conditions and blots were detected with rabbit polyclonal antiserum 3951 raised against a PR8 virus lacking H1, which had been removed by acid and DTT treatment. (C) Growth kinetics of rescued viruses in differentiated human tracheobrochial epithelial cells infected at an MOI of 0.001. Data are shown as the average of virus titrations conducted in triplicates for each time point shown by standard plaques assay in MDCK cells. The error bars represent the +/− SD at each time point.
Fig. 4
Fig. 4. 2009 pH1N1 viruses with additional glycosylations in the HA are attenuated in mice and ferrets
(A – E) Infection of 9-week-old female C57B/6 mice with Neth/09 glycosylation mutant viruses. Groups of 5 mice per recombinant virus were infected i.n. with the indicated doses. Body weight represents the average of each group and the error bars indicate the +/− SD at each time point. (F) Viral titer in the lungs of mice infected with 1×103 pfu of each mutant virus were obtained on days 2 (circle), 5 (square), and 7 (triangle) p.i. as shown. Black bars represent the average viral titer for 2 (arrows) or 3 mice per group at each time point as compared to the rNeth/09 WT virus. N.D. = Not detected. (G) Body weight changes in ferrets infected (n=3 per group) with the indicated viruses. Weights are shown as the average and the error bars represent the +/− SD. at each time point. Statistically significant difference (*) of the overall body weight of ferrets during the infection period was estimated with the Wilcoxon-matched pairs test. (H) Viral titers in nasal washes obtained every other day from ferrets shown in (G). (I) Viral load in tissues from ferrets (n=3) at day 3 p.i. with the indicated viruses. Values are represented as in (F).
Fig. 5
Fig. 5. HI activity of human sera after vaccination against pandemic 2009 H1N1
Pre- and post-vaccination sera samples obtained from 52 subjects enrolled in clinical trials to test the safety and immunogenicity of an inactivated 2009 H1N1 influenza vaccine, were tested for their HI activity against WT Neth/09 and the glycosylation mutant viruses. For each subject the difference in HI activity between post- and pre-vaccination (HI titer post-vaccination – HI titer pre-vaccination in number of wells) was determined to normalize for pre-existing HI activity. For each virus the distribution of increase in HI activity post vaccination is plotted. The median of the distribution is marked with a red line and statistically significant differences were determined with the Wilcoxon rank sum test.
Fig. 6
Fig. 6. Deletion of glycosylation sites in the HA of Tx/91 increases virulence in mice and cross-protects against the 2009 pH1N1 strain
Phenotypic characterization of recombinant influenza A viruses carrying either the wild type or glycosylation deletion mutant A/Texas/36/1991 HAs (N71K + S73N, T144D, N177K) and the remainder 7 genes from PR8 (viruses are 7:1 rPR8 expressing Tx/91 HA). (A) Western blot analysis of lysates obtained from MDCK cells infected at an MOI of 5 for 12 h with the respective glycosylation deletion mutant viruses. Lysates were run under reducing conditions and blots were detected with the polyclonal 3951 antibody. (B) 8-week-old female C57B/6 mice infected with 1×104 pfu of each virus shown. Average body weight of mice n=5 per group. Error bars denote the +/− SD for each time point. (C) Mice infected in panel B were allowed to seroconvert for 27 days at which time they were challenged with 100 LD50 of Neth/09. Percent survival is shown. (D) Mice infected with 1×104 pfu of each glycosylation deletion mutant, in which the same glycosylation sites were removed by an alternative set of amino acid substitutions to those used in B and C. At 29 days p.i. mice were challenged with 100 LD50 of Neth/09. Percent survival after challenge is shown. The student’s t-test was used to determine significance of body weight loss and the log-rank test was used to assess significance (* P<0.05) of survival outcome.
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