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

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

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

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

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

HB36.6 induces a transient cytokine response that is not required for protection.

<p>(a) Inflammatory cytokines were assayed by Bio-Plex using supernatants from lung homogenates obtained from BALB/c mice 2, 24 and 48 hours following administration with HB36.6 (6.0 mg/kg) or the scaffold protein (PDB ID 1u84) (6.0 mg/kg) (n = 10 mice per group). Fold change over naïve mice is shown. *P < 0.05. (b) Survival and weight change in SCID and MyD88-/- mice (n = 10 per group) that received 6.0 mg/kg of HB36.6, scaffold protein, or Protein Ctr (lysozyme) IN 2 hours before IN infection with 10 MLD₅₀ CA09 virus.</p

Intranasal delivery of HB36.6 affords prophylactic protection against lethal challenge by influenza virus.

<p>(a) Survival and weight change in BALB/c mice (n = 10 per group) that received 6.0 mg/kg of HB36.6 administered intranasally (IN) at 2, 24, or 48 hours before challenge with 10 MLD₅₀ CA09 virus. The Protein Control (Ctr) group received 6.0 mg/kg of lysozyme at 2 or 48 hours before challenge with 10 MLD₅₀ CA09 virus. (b) Survival and weight change in BALB/c mice (n = 5 per group) that received 0.01–1 mg/kg IN doses of HB36.6 2 hours before challenge with 10 MLD₅₀ of CA09 virus. (c) Survival and weight change in BALB/c mice (n = 10 per group) that received 3.0 mg/kg of HB36.6 IN 2 hours before IN infection with 10 MLD₅₀ of H1N1 CA09 virus, 6 MLD₅₀ H1N1 A/PR/8/34 (PR8), or 3 MLD₅₀ of H5N1 A/Duck/MN/1525/81 (MN81) virus. Mean and SEM are shown.</p

HB36.6 suppresses viral replication and inflammation in the lung.

<p>(a) Viral titers in nasal washes of untreated infected controls (Ctr) and mice that received 6.0 mg/kg HB36.6 either 1 day before (Prophylaxis, Pro) or 1 day after (Therapeutic, Ther) infection with 10 MLD₅₀ CA09 virus. Nasal washes collected on days 2, 4 and 6 post-infection were measured by determining the 50% tissue culture infectious dose (TCID₅₀) (bars indicate mean viral titer ±SD, n = 18 mice per group, three replicate experiments). (b) IHC staining of intracellular influenza NP (H1N1) of representative lung sections from uninfected (Naïve) and untreated infected controls (Control) and HB36.6-treated mice (Prophylactic and Therapeutic) at 4 days post-infection with 10 MLD₅₀ CA09 virus. Mouse lungs were not inflated with formalin and consequently resulted in lung collapse and a more hypercellular appearance in the uninfected control. Images selected show representative staining of influenza (NP) positive cells for each group. (c) Quantification of influenza positive cells in lung tissues was performed by measuring the area of positive staining compared to the total tissue on the slide (uniform random sampling of 50% lung tissue). (d) Inflammatory cytokines were assayed by Bio-Plex using supernatants from lung homogenates obtained from BALB/c mice on day 2 following infection with 10 MLD₅₀ CA09 virus (n = 8 mice per group). The fold change over naïve-uninfected mice is shown. For a, c and d, significant differences between the Pro and Ther groups to the Ctr group are shown: *P < 0.05, **P < 0.001.</p

Specificity-enhancing mutations and overall affinity-increasing substitutions using selection against different HA subtypes.

<p>(<b>a</b>) HB36.5, in complex with HA, colored by average residue enrichment in FACS sorts against 7 different HA subtypes. Final residue identity after mutagenesis and selection to obtain HB36.6 are labeled in black. Positions 54, 64, and 68 retained their HB36.5 identities. (<b>b</b>) Enrichment of substitutions at 12 key positions in HB36.5 in selections against each HA strain. Labels at the bottom indicate position in HB36.5; numbers at the top represent the different flu strains and subtypes (1: A/South Carolina/1/1918 (H1), 2: A/California/04/2009 (H1), 3: A/Vietnam/1203/2004 (H5), 4: A/Indonesia/05/2005 (H5), 5: A/Adachi/2/1957 (H2), 6: A/turkey/Wisconsin/1/1966 (H9), 7: A/duck/Alberta/60/1976 (H12)). At most positions, the enrichment profiles against the different HA strains are similar but, at several positions, they are quite distinct. At position 54, for example, arginine is highly conserved and substitutable for lysine in selections for binding against HAs 1–4, but is outcompeted by smaller charged/polar residues in selections against HAs 5–7 (red region at upper right of R54 panel). White cells indicate insufficient data (<15 sequences in the input library) and black boxes indicate the residue identities in HB36.6. (<b>c</b>) Origin of HA strain dependence of substitutions at HB36.5 position R54. Arg54 forms a hydrogen bond network with Asp and Arg residues in HAs 1–4. In HAs 5–7, the Asp is substituted by a Glu, disrupting the interface with Arg54 leading to a preference for smaller polar residues.</p