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