Rapamycin alters the cytokine profile in the cerebrospinal fluid after gene delivery.

<p>Rapamycin alters the cytokine profile in the cerebrospinal fluid after gene delivery.</p

GFP-specific T cell responses as spot forming cells (SFC)/10⁶ PBMC after rapamycin treatment.

<p>T cell responses in PBMC were measured by ELISPOT at 14, 28, and 84 days after AAV9/GFP delivery in (A) AAV9/GFP only (controls); (B) AAV9/GFP + rapamycin. Arrow indicates undetectable (< 10 SFC/10⁶ PBMC) GFP-specific responses at necropsy (84 days).</p

IHC was carried out to visualize GFP expression in the lumbar spinal cord.

<p>Shown are representative 40 micron lumbar spinal cord sections of all study macaques, stained for GFP. Magnified insets show examples of GFP-positive motor neurons. Macaque ID numbers are provided in each panel, along with a qualitative scoring of ventral horn expression. The (-) indicates no GFP-positive staining above that seen in uninjected NHPs; (+) indicates some positive cells observed, above that seen in uninjected macaques; (++) indicates relatively strong expression in a large number of neurons.</p

GFP expression in lumbar spinal cords of NHPs monitored by IHC.

<p>GFP expression in lumbar spinal cords of NHPs monitored by IHC.</p

Cytotoxic GFP-specific T cells decreased after rapamycin treatment.

<p>Multiparameter flow cytometry was used to identify CD8+ CD107+ T cells in PBMC after stimulation with GFP peptide pools. CD8+CD107+ expression in PBMC was monitored 28 and 84 days after transgene delivery in (A) AAV9/GFP (controls); (B) AAV9/GFP and rapamycin.</p

Magnitude and breadth of GFP-specific T cell responses reduced after treatment with rapamycin.

<p>Multiparameter flow cytometry to assess T cell responses was performed using peptide (GFP or AAV9 peptide pools)-stimulated PBMC. PBMC were stained with antibodies to detect the following markers: CD4, CD8, IFN-γ, IL2, Ki67, and TNFα. Panels A and B depict GFP- specific T cell responses in two treatment groups: AAV9/GFP (controls) and AAV9/GFP + rapamycin. Panels C and D depict AAV9-specific T cell responses.</p

DNA immunization induces influenza-specific T cell responses.

<p>A) IFN-<b>γ</b> analysis was performed on peripheral blood mononuclear cells. Vaccine-induced responses to specific influenza antigens 2 weeks post third vaccination (week 14). Mean response for each animal is plotted (n = 8/group). Error bars represent SEM. B) Responses of vaccinated animals to influenza peptide pools corresponding to subsets of HA and NP as well as M2e at 2 weeks post third vaccination (week 14). C) Total IFN-<b>γ</b> responses against HA, NP, and M2e in vaccinated and control animals at 2 weeks post third vaccination (week 14). Mean of duplicate samples is plotted; error bars represent SEM. Responses after third immunization are significantly elevated over baseline; p = 0.0008 as calculated by Mann-Whitney U test.</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