A Perspective on the Structural and Functional Constraints for Immune Evasion: Insights from Influenza Virus

Abstract

Influenza virus evolves rapidly to constantly escape from natural immunity. Most humoral immune responses to influenza virus target the hemagglutinin (HA) glycoprotein, which is the major antigen on the surface of the virus. The HA is composed of a globular head domain for receptor binding and a stem domain for membrane fusion. The major antigenic sites of HA are located in the globular head subdomain, which is highly tolerant of amino acid substitutions and continual addition of glycosylation sites. Nonetheless, the evolution of the receptor-binding site and the stem region on HA is severely constrained by their functional roles in engaging the host receptor and in mediating membrane fusion, respectively. Here, we review how broadly neutralizing antibodies (bnAbs) exploit these evolutionary constraints to protect against diverse influenza strains. We also discuss the emerging role of other epitopes that are conserved only in subsets of viruses. This rapidly increasing knowledge of the evolutionary biology, immunology, structural biology, and virology of influenza virus is invaluable for development and design of more universal influenza vaccines and novel therapeutics.

Keywords: antibody; evolution; hemagglutinin; influenza A virus; protein structure.

Figure 1. Major antigenic sites on HA

(a) The locations of the five major antigenic sites on H1 HA [40, 41] are shown on the HA trimer structure of A/California/04/2009 (PDB 3UBE) [84]. (b) The locations of the five major antigenic sites on H3 HA [–44] shown on the HA trimer structure of A/Hong Kong/1/1968 (PDB 2YPG) [85]. (a–b) The human receptor analog pentasaccharide lactoseries tetrasaccharide c (LSTc) is shown in sticks representation (yellow).

Figure 2. Natural substitutions and emerging N-glycosylation sites in the HA of human H3N2 viruses

(a) Stacked graph showing the cumulative average number of amino-acid substitutions in the HA in strains isolated from different years as compared to the ancestral strain, A/Hong Kong/1/1968. (b–c) HA residues that have different amino-acid identities between A/Hong Kong/1/1968 and the consensus sequence of the 2015 human H3N2 strains are shown as spheres on one protomer of A/Hong Kong/1/1968 HA trimer (PDB 4FNK) [69]. The other two protomers are shown in surface representation. Color scheme follows that of panel a. (c) Zoom-in view for the receptor binding site (RBS) of A/Hong Kong/1/1968 in complex with a human receptor analog pentasaccharide lactoseries tetrasaccharide c (LSTc) (PDB 2YPG) [85]. LSTc is shown as yellow sticks (carbons) with nitrogens in blue and oxygens in red. Residues within the RBS are labeled. Residues responsible for major antigenic changes are labeled with asterisks [48]. (d) Stacked graph showing the normalized percentage of strains that contain the indicated N-glycosylation sites. While the N-glycosylation site Asn81 disappeared in year 1977, nine other N-glycosylation sites have emerged in human H3N2 viruses since 1968. Five absolutely conserved N-glycosylation sites in human H3 strains (HA1 Asn22, Asn38, Asn165, Asn285; HA2 Asn154) are not included in the stacked graph. This analysis includes a total of 4625 sequences were downloaded from Influenza Research Database [190]. This plot is an updated version of the plot in Lee et al., 2014 [58]. (e) Oligomannoses (cyan) on A/Hong Kong/1/1968 (PDB 4FNK) [69] and A/Victoria/361/2011 (PDB 4O5N) [58] were modeled by Glyprot [191]. HA1 is colored in grey and HA2 is colored in black. The receptor-binding site (RBS) is colored in lime. The absolutely conserved N-glycosylation sites in human H3 strains (HA1 Asn22, Asn38, Asn165, Asn285; HA2 Asn154) are included in the display.

Figure 3. Binding footprints of RBS-targeted bnAbs

(a) Amino-acid sequence conservation is projected onto the HA structure of A/Hong Kong/1/1968 (PDB 4FNK) [69]. One protomer is shown in Cα ribbon representation and the other two protomers are shown in a surface representation. Human H1, H2, and H3 strains were used for computing the amino-acid sequence conservation. For each subtype, we randomly sampled at most 5 strains from each year for this analysis. (b) The sequence conservation of the HA RBS region of A/Hong Kong/1/1968 (PDB 4FNK) [69] is shown. Several highly conserved residues that interact with the sialic acid receptor are labeled. (c) The binding footprints of RBS-targeted bnAbs are shown on the structure of the HA RBS of A/Hong Kong/1/1968 (PDB 4FNK) [69]. Epitope usage represents the frequency of a specified residue being targeted by RBS-targeted bnAbs. Epitopes from nine RBS-targeted bnAbs, namely C05 [69], S139/1 [71], F045–092 [58], 2G1 [74], 8M2 [74], 5J8 [76], 1F1 [192], HC63 [79], and CH65 [75], were analyzed here. Locations of naturally occurring indels are indicated. (d–e) Same as panel b and c, except that the stem region is now shown. N38, which is a highly conserved N-glycosylation site in group 2 HAs, but not in group 1 HAs, is labeled. (e) Epitopes from nine stem-binding bnAbs, namely CR6261 [99], CR9114 [101], CR8043 [109], F10 [100], CR8020 [107], C179 [102], MEDI8852 [105], 39.29 [113], and FI6v3 [111], were analyzed here. Amino-acid substitutions associated with stem-binding bnAbs escape were annotated. HA1: T318K (C179 escape) [103]. HA2: D19N (CR8020 escape) [107], R25M (CR8043 escape) [109], G33E (CR8020 escape) [107], Q34R (CR8020 and CR8043 escape) [109], Q42K (39.29 escape) [118], D46Y/G (39.29 escape) [118], V52E (C179 escape) [103], and H111L (CR6261 escape) [108]. Of note, HA1 H111L is a buried behind the surface. “Site 1” indicates the epitope region of CR9114, CR6261, and other V_H1–69 encoded stem-binding bnAbs. “Site 2” indicates the epitope region of the two group 2-specific stem-binding bnAbs, namely CR8020 and CR8043. Site 2 is lower down the stem as compared to site 1. “Both” indicates the overlapping region of sites 1 and 2.

Figure 4. Binding of broadly neutralizing antibody (bnAb) to influenza hemagglutinin (HA)

(a) Interaction between RBS-targeted bnAbs and HA is shown. Binding of C05 [69] (cyan; PDB 4FP8), S139/1 [71] (pink; PDB 4GMS), F045–092 [58] (lime; PDB 4O58), 2G1 [74] (purple; PDB 4HG4), 8M2 [74] (orange; PDB 4HFU), 5J8 [76] (blue; PDB 4M5Z), 1F1 [192] (green; PDB 4GXU), HC63 [79] (gray; PDB 1KEN), and CH65 [75] (red; PDB 5UGY) Fabs to HA trimer (white). (b) Interaction between stem-binding bnAbs and HA is shown. Binding of CR6261 [99] (yellow; PDB 3GBN), CR9114 [101] (blue; PDB 4FQI), CR8043 [109] (orange; PDB 4NM8), CR8020 [107] (cyan; PDB 3SDY), C179 [102] (red; PDB 4HLZ), MEDI8852 [105] (pink; PDB 5JW4), 39.29 [113] (green; PDB 4KVN), and FI6v3 [111] (gray; PDB 3ZTJ) Fabs to HA trimer (white). The curved arrow indicates that the approach angle has to be from no more than perpendicular to at an upward disposition to avoid a steric clash with the membrane. CR6261 Fab is obscured by CR9114 Fab, as they both have very similar angles of approach to the HA and, therefore, is not visible in these views. (c) The approach angle of RBS-targeted bnAbs and stem-binding bnAbs was quantified by adapting the method described in [115]. Briefly, the trimer axis of HA is on the z-axis such that the x-y plane (horizontal plane) represents the viral membrane. Vertical plane represents the y-z plane. The long axis of the Fab is defined as the line connecting the averaged coordinate of Cα-atoms of the conserved cysteines in the constant domain to that in the variable domain. The angle between the long axis of the Fab and the horizontal plane or the vertical plane is shown. (d) Binding of H5M9 [128] (wheat; PDB 4MHJ) and CR8071 [101] (teal; PDB 4FQJ) Fabs to the same protomer of the HA trimer (white). To visually compare their epitope locations with other bnAb epitopes, the RBS and the epitope of a stem-binding bnAb CR6261 are colored in lime and pink, respectively.