Fig 1.
Evolution of N-glycosylation sites on H3 HA and broad neutralization of human H3N2 viruses from around 1990 by anti-glycan antibody 2G12.
(A) The number of non-conserved N-glycosylation sites on HA has evolved from one in 1968 to seven in 2019 on HA1 as shown in the stack chart. Ten glycan sites are cumulatively involved, where some are retained and others substituted from 1968 to 2019. The six completely conserved N-glycosylation sites in human H3N2 viruses (N8, N22, N38, N165, N285 in HA1, and N154 in HA2, which corresponds to N483 in HA0) are shown in white. (B) Neutralization activity of 2G12 IgG with different H3N2 strains was measured by a cell viability assay at four different antibody concentrations. The OD490 value is proportion to the cell density. Dashed line represents the OD490 for cell only control with no virus. (C) Neutralization activity of 2G12 IgG against two clinical isolates. The IC50 values of 2G12 IgG to A/TW/82486/2014 and A/TW/87302/2016 are 151 and 69 μg/ml, respectively. The error bars on each value are the standard deviation of three technical triplicates.
Fig 2.
Site-specific N-linked glycosylation of H3N2 HA glycoproteins and models of fully glycosylated HK68 and Vic11 HAs.
(A) Recombinant H3N2 HA glycoproteins were proteolytically digested and analyzed by LC-MS. Each graph summarizes the quantitative mass spectrometric analysis of the glycan populations at each individual N-linked glycosylation site, which are simplified into glycan categories. These categories are colored according to glycan compositions as per the key. The oligomannose-type glycoforms (M9 to M5; Man9GlcNAc2 to Man5GlcNAc2) are indicated in shades of green, hybrid-type glycans in dashed light pink, and complex-type glycans in pink. Glycosylation sites that are not present on individual HAs are shaded in grey, and sites where the glycosylation site is encoded, but glycan compositions could not be determined, are labelled as not determined (N.D.). (B) Experimentally observed glycans shown in Fig 2A are modelled onto the prefusion structure of trimeric HA from HK68 (PDB ID: 4FNK) [89] and Vic11 (PDB ID 4O5N) [15]. Glycans are colored according to the oligomannose-type glycan content as per the key, with the protein surface shown in grey.
Fig 3.
Evolution and spatial distribution of N-glycosylation sites on H3 HA and 2G12 neutralization.
(A) Front and top views of N-glycosylation sites on A/Brisbane/10/07 (H3N2) HA (PDB 6AOV) [91]. Each N-glycosylation site is shown only on protomer 1 in orange. Human receptor analogue, 6’-sialylated di-N-acetyllactosamine (6´-SLNLN), in the receptor binding sites of protomers 1 and 2 of the trimer are in green. (B) The distance from Cα of the Asn in each N-glycosylation site to the sialic acid of the sialoside receptor in the receptor binding domain (RBD) on the same protomer (protomer 1), and the corresponding distance to the RBD of the neighboring promoter (protomer 2) among conserved N-glycosylation sites (left panel) and non-conserved N-glycosylation sites (right panel). (C) In vitro neutralization of wild type and N165A HA mutant Pan99 and Bris07 viruses by 2G12, CR9114, and IgG311. The IC50 of 400 μg/ml indicated that the antibody has no neutralizing activity against H3N2 viruses.
Fig 4.
Virus escape mutants in a Pan99 library in the absence or presence of 2G12 and the fitness of N165/N246 mutations to Bris07 viruses.
(A) The relative fitness of each of the 256 variants in the mutant library is shown. The x-axis and y-axis represent the relative fitness without and with 2G12 selection, respectively. Each data point represents one variant. The relative fitness of wild type is set as 0. Variants with a gain in relative fitness in the presence of 2G12 selection (escape mutants) are in red. Variants with a high fitness without 2G12 selection, which did not escape 2G12 are in blue. These two groups were selected for sequence logo analyses [81]. (B) Variants at specific sites (HA1 144, 145, 160, 172, 192, 196, 226, 246). The upper panel indicates residue combinations of wild-type variants with superior fitness, whereas the bottom panel indicates sequence preferences of 2G12 escape mutants. (C) The fitness effect of mutations N165A, N246K, and N165A/N246K on Pan99 and Bris07 HA were measured by median tissue culture infectious dose (TCID50). Error bars represent standard deviation of quadruple measurements.
Fig 5.
Glycoforms at N165 and N246 and 2G12 binding mode to HA wildtype and glycan mutants.
(A) Site-specific glycosylation analysis of Bris07 HA glycan knockout mutants at N165 and N246 (left panel). Percentage differences in abundance of oligomannose-type glycans between glycan knockout mutants and WT Bris07 HA is also illustrated with decreased and increased abundance colored in red and blue (right panel), respectively. (B) Negative-stain electron microscopy (nsEM) images show the different modes of 2G12 recognition of Bris07 wild type (left panel) and N165A mutant (right panel) HA. Fab 2G12, as a domain-swapped dimer, and the HA trimer are indicated by arrows. 2G12 Fab involved in binding to two N246 glycans, one N246 glycan, and one N165 and possibly one N246 glycan are shown in green, purple, and yellow, respectively. Wild type and N165A HA are in white. (C) Front view of A/Brisbane/10/07 (H3N2) HA trimer (PDB 6AOV, in grey) [91]. Man9GlcNAc2 on N165 and N246 were modelled onto Bris07 wild type HA with Charmm-Gui [92]. 2G12 Fab dimer (PDB 6N2X, in cyan) [11] is shown bound to the lateral side (left panel) and apex of trimer in parallel (middle panel) or tilted (right panel) binding mode from the nsEM data in Fig 5B. Man9GlcNAc2 on N165 and N246 are in yellow and green, respectively. (D) Same as (C), but N165A is shown in magenta. N246 was modelled with Man8GlcNAc2. 2G12 is shown bound to the apex of Bris07 N165A trimer as in the nsEM reconstruction. The Man8GlcNAc2 at N246 is in green.
Fig 6.
Differential under-processing of glycan shields in different enveloped viruses.
(A) From left to right, HK68 HA, Severe acute respiratory syndrome coronavirus (SARS-CoV) spike, SARS-CoV-2 spike, MERS-CoV spike, Vic11 HA, LASV GPC, HIV-1 Env (PDB ID: 4FNK, 5X58, 6VSB, 5X59, 4O5N, 5VK2, 5ACO, respectively) [15,38,93–96]. Glycans are colored according to site-specific oligomannose-type glycan content [44,45], as per the key. (B) Glycans are colored according to Man8/9GlcNAc2 content (2G12 epitopes), as per the key.