Biogenesis of Influenza A Virus Hemagglutinin Cross-Protective Stem Epitopes

<div><p>Antigenic variation in the globular domain of influenza A virus (IAV) hemagglutinin (HA) precludes effective immunity to this major human pathogen. Although the HA stem is highly conserved between influenza virus strains, HA stem-reactive antibodies (StRAbs) were long considered biologically inert. It is now clear, however, that StRAbs reduce viral replication in animal models and protect against pathogenicity and death, supporting the potential of HA stem-based immunogens as drift-resistant vaccines. Optimally designing StRAb-inducing immunogens and understanding StRAb effector functions require thorough comprehension of HA stem structure and antigenicity. Here, we study the biogenesis of HA stem epitopes recognized in cells infected with various drifted IAV H1N1 strains using mouse and human StRAbs. Using a novel immunofluorescence (IF)-based assay, we find that human StRAbs bind monomeric HA in the endoplasmic reticulum (ER) and trimerized HA in the Golgi complex (GC) with similar high avidity, potentially good news for producing effective monomeric HA stem immunogens. Though HA stem epitopes are nestled among several <i>N</i>-linked oligosaccharides, glycosylation is not required for full antigenicity. Rather, as <i>N</i>-linked glycans increase in size during intracellular transport of HA through the GC, StRAb binding becomes temperature-sensitive, binding poorly to HA at 4°C and well at 37°C. A <i>de novo</i> designed, 65-residue protein binds the mature HA stem independently of temperature, consistent with a lack of <i>N</i>-linked oligosaccharide steric hindrance due to its small size. Likewise, StRAbs bind recombinant HA carrying simple <i>N</i>-linked glycans in a temperature-independent manner. Chemical cross-linking experiments show that <i>N</i>-linked oligosaccharides likely influence StRAb binding by direct local effects rather than by globally modifying the conformational flexibility of HA. Our findings indicate that StRAb binding to HA is precarious, raising the possibility that sufficient immune pressure on the HA stem region could select for viral escape mutants with increased steric hindrance from <i>N</i>-linked glycans.</p></div

Structural and Antigenic Variation among Diverse Clade 2 H5N1 Viruses

<div><p>Antigenic variation among circulating H5N1 highly pathogenic avian influenza A viruses mandates the continuous production of strain-specific pre-pandemic vaccine candidates and represents a significant challenge for pandemic preparedness. Here we assessed the structural, antigenic and receptor-binding properties of three H5N1 HPAI virus hemagglutinins, which were recently selected by the WHO as vaccine candidates [A/Egypt/N03072/2010 (Egypt10, clade 2.2.1), A/Hubei/1/2010 (Hubei10, clade 2.3.2.1) and A/Anhui/1/2005 (Anhui05, clade 2.3.4)]. These analyses revealed that antigenic diversity among these three isolates was restricted to changes in the size and charge of amino acid side chains at a handful of positions, spatially equivalent to the antigenic sites identified in H1 subtype viruses circulating among humans. All three of the H5N1 viruses analyzed in this study were responsible for fatal human infections, with the most recently-isolated strains, Hubei10 and Egypt10, containing multiple residues in the receptor-binding site of the HA, which were suspected to enhance mammalian transmission. However, glycan-binding analyses demonstrated a lack of binding to human α2-6-linked sialic acid receptor analogs for all three HAs, reinforcing the notion that receptor-binding specificity contributes only partially to transmissibility and pathogenesis of HPAI viruses and suggesting that changes in host specificity must be interpreted in the context of the host and environmental factors, as well as the virus as a whole. Together, our data reveal structural linkages with phylogenetic and antigenic analyses of recently emerged H5N1 virus clades and should assist in interpreting the significance of future changes in antigenic and receptor-binding properties.</p></div

Development of a high-throughput assay to detect antibody inhibition of low pH induced conformational changes of influenza virus hemagglutinin

<div><p>Many broadly neutralizing antibodies (bnAbs) bind to conserved areas of the hemagglutinin (HA) stalk region and can inhibit the low pH induced HA conformational changes necessary for viral membrane fusion activity. We developed and evaluated a high-throughput virus-free and cell-free ELISA based low pH induced HA Conformational Change Inhibition Antibody Detection Assay (HCCIA) and a complementary proteinase susceptibility assay. Human serum samples (n = 150) were tested by HCCIA using H3 recombinant HA. Optical density (OD) ratios of mAb HC31 at pH 4.8 to pH 7.0 ranged from 0.87 to 0.09. Our results demonstrated that low pH induced HA conformational change inhibition antibodies (CCI) neutralized multiple H3 strains after removal of head-binding antibodies. The results suggest that HCCIA can be utilized to detect and characterize CCI in sera, that are potentially broadly neutralizing, and serves as a useful tool for evaluating universal vaccine candidates targeting the HA stalk.</p></div

Identification of HA species recognized by StRAbs.

<p>(A) IAV PR8-infected MDCK cells were labeled with [³⁵S]-Met and chased at 37°C. Detergent cell lysates were treated with an irrelevant mAb (10G-4 to the VSV N protein; no depletion) or depleted of HA monomers (mHA depleted) or HA trimers (tHA depleted) using the anti-HA head mAbs Y8-10C2 or H17-L2, respectively, at 4°C. Cell extracts were then incubated with the StRAbs C179 or 1F02 also at 4°C in a second round of immunoprecipitation (IP). Collected HA species were analyzed by non-reducing SDS-PAGE and fluorography. (B–M) MDCK cells were infected with IAV PR8 in the absence (no treatment) or presence of 10 µM monensin. Cells were fixed, permeabilized, and incubated with the human StRAbs 1F02 (B–G) and 2G02 (H–M) (green channel) and rabbit pAbs to NA (red channel). DNA was labeled using DAPI (blue channel). Stained cells were examined by fluorescence confocal microscopy. Bars: 10 µm. Arrowheads point NA co-localizing with HA monomers in the nuclear envelope (ER). (N) MDCK cells were infected with IAV PR8 in the presence of 10 µM monensin and processed for IF confocal microscopy using 2-fold serial dilutions of the purified anti-HA head mAb H28-E23 (control) or the StRAbs 1F02 and 2G02. Fluorescence intensities of the ER (HA monomers) and GC (HA trimers) are expressed as arbitrary units (a.u.). Data are represented as mean ± SEM from ∼100 cells/Ab dilution.</p

Steric hindrance due to <i>N</i>-linked glycan processing shields HA from StRAb binding.

<p>(A–C) PyMOL images of the crystal structures of mouse Fab C179 in complex with the IAV A/Japan/305/57 (H2N2) HA monomer [RSCB protein database entry: 4HLZ) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004204#ppat.1004204-Dreyfus1" target="_blank">[32]</a> (A)]; the IAV PR8 HA monomer used in this study [shown for comparison only; RSCB protein database entry: 1RVX) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004204#ppat.1004204-Gamblin1" target="_blank">[35]</a> (B)]; and HB80.4 in complex with the IAV A/Brevig Mission/1/18 (H1N1) HA monomer [RSCB protein database entry: 4EEF) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004204#ppat.1004204-Whitehead1" target="_blank">[36]</a> (C)], showing glycosylation-prone Asn residues within or around of the stem region of HA (red; H3 numbering scheme). The HA1 and HA2 peptides are displayed in purple and pink, respectively. (D) Detergent extracts from [³⁵S]-Met-labeled and chased IAV PR8-infected MDCK cells at 37°C were treated with an irrelevant mAb (10G-4 to the VSV N protein; no depletion) or depleted of HA monomers (mHA depleted) or HA trimers (tHA depleted) using the anti-HA head mAbs Y8-10C2 or H17-L2, respectively, at 4°C as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004204#ppat-1004204-g001" target="_blank">Fig. 1A</a>. Detergent cell lysates were then incubated with 2.4 µg/ml FLAG-tagged HB80.4 also at 4°C. HA species in complex with HB80.4 were immunocollected with the anti-FLAG mAb M2 at 4°C and analyzed by non-reducing SDS-PAGE and fluorography. pHA: processed, glycosylated HA.</p

Processing of HA glycans in the distal Golgi complex shields HA from StRAb binding.

<p>(A and B) IAV PR8-infected MDCK cells were pulse-labeled with [³⁵S]-Met and chased at 37°C. At the end of each chase time point cells were detergent-lysed and then subjected to IP with the anti-HA head mAbs H17-L2 and H28-E23 (A) or the StRAbs C179 and 1F02 (B) at 4°C. Immunocollected HA species were analyzed by non-reducing SDS-PAGE and fluorography. pHA: processed, glycosylated HA. (C) Detergent extracts from [³⁵S]-Met-labeled and chased MDCK cells infected with IAV PR8 were treated with an irrelevant mAb (10G-4 to the VSV N protein; no depletion) or HA-depleted with the mAbs H28-E23 (control), C179, and 1F02 at 4°C as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004204#ppat-1004204-g001" target="_blank">Fig. 1A</a> before being incubated with H17-L2 also at 4°C in a second round of IP. Precipitated HA species were visualized by SDS-PAGE under non-reducing conditions and fluorography.</p

Inhibition of <i>N</i>-linked glycosylation or <i>N</i>-linked oligosaccharide processing restores proper StRAb binding to HA.

<p>(A–C) MDCK cells infected with IAV PR8 were treated with tunicamycin (A) or a mixture of DMN and SWN (B and C) for 30 min before being labeled with [³⁵S]-Met and chased in continuous presence of the inhibitors at 37°C. (A and B) Detergent cell extracts were subjected to IP using the anti-HA head mAbs H17-L2 and H28-E23 or the StRAbs C179 and 1F02 at 4°C. Immunocollected proteins were resolved by non-reducing SDS-PAGE and visualized by fluorography. n-gHA: non-glycosylated HA; n-pHA: non-processed, glycosylated HA. (C) Detergent cell lysates were incubated with an irrelevant mAb (10G-4 to the VSV N protein; no depletion) or HA-depleted with the mAbs H28-E23 (control), C179, and 1F02 at 4°C before being treated with H17-L2 also at 4°C in a second round of IP as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004204#ppat-1004204-g002" target="_blank">Fig. 2C</a>. Precipitated HA species were analyzed by non-reducing SDS-PAGE and fluorography.</p

Structural variation among the different H5N1 clades.

<p>(A) Surface representation of the Hubei10 trimeric HA indicating the positions of surface exposed residue substitutions among Clade 1 (Viet04), clade 2.3.4 (Anhui05), clade 2.2.1 (Egypt10) and clade 2.3.2.1 (Hubei10). Positions containing single substitutions are colored cyan and positions containing multiple substitutions are colored magenta. (B) Amino acid consensus sequences of H5N1 HA clades at positions equivalent to the HA antigenic sites, Ca, Cb, Sa and Sb, of human H1N1 viruses <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075209#pone.0075209-Caton1" target="_blank">[39]</a>, are shown. Clade 1 (Viet04), clade 2.3.4 (Anhui05), clade 2.2.1 (Egypt10) and clade 2.3.2.1 (Hubei10) are highlighted in red. Structural positions of these equivalent sites are highlighted on the Hubei10 trimeric structure (Ca; pale yellow, Cb; wheat, Sa; pale green, Sb; pale blue). Asparagine residues that are potentially N-glycosylated are colored orange.</p

Structural comparison between H5 hemagglutinins.

<p>(A) Structural alignment of Anhui05 (green), Egypt10 (blue) and Hubei10 (purple) onto Viet04 (yellow) reveals how structurally related these clades are. (B) Alignment of the receptor-binding site (RBS) reveals conserved structural features and residues. (C) Compared to Viet04, a total of eleven residue differences in and around the RBS are present. Amino acid residues in each structure are numbered consecutively according to the ectodomain fragment of the mature HA1 protein. *Deletion of Leu129 in Egypt10 produces a shift in the numbering of residues 129–324 in Egypt10 relative to structurally equivalent residues in Anhui05 and Hubei10.</p

Principle of HCCIA.

<p><b>A.</b> The linear schematic of the rHA as described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199683#pone.0199683.ref041" target="_blank">41</a>] with some modifications, not to scale. N-terminal baculovirus GP67 signal peptide, HA ectodomain, mutated thrombin cleavage site from LVPRGS to LVPAGS, foldon, and 6 histidine-Tag. <b>B.</b> The structure shown in the schematic was modified from a published HA structure (PDB No. 1HTM and 4WE4) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199683#pone.0199683.ref041" target="_blank">41</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199683#pone.0199683.ref043" target="_blank">43</a>]. Though trimeric rHA was used in the HCCIA, the H3 rHA is shown as a monomer in Fig 1. In the absence of CCI, H3 rHA would undergo conformational changes at the pH of fusion and HC31 (HC67 for H3 rHA, or 1/87 mAb for H2 rHA) would lose the ability to bind to the rHA, while HC3 (shown in black) would bind to the H3 rHA when in its fusogenic conformation. <b>C.</b> In the presence of CCI (shown in green), H3 rHA would remain “locked” in its metastable pre-fusion conformation and maintain recognition by both HC3 (shown in black) and HC31 (shown in red), as well as HC67 or 1/87 mAb for H2 rHA.</p