Amiloride enhances adaptive immunity against HBV S2.

<p>Naïve C57 mice were immunized s.c. with pcD-S2 at various concentrations with or without amiloride in the hind footpad four times using the immunization scheme as shown (A). Seven days after the last immunization, animals (n = 3) were used to test anti-S2 IgG antibody titer (B) and delayed hypersensitivity (DTH) response after re-stimulation with 1 µg sAg <i>s.c.</i> in a hind footpad for 24 h (C). PBS was added as negative control. *, statistical significance among all groups. Another 3 animals were used to test HBV S208–215 specific lysis <i>in vitro</i> (D) and <i>in vivo</i> (E). Hepatocytes from HBV Alb1 trangenic mice were used as targets mixed with effectors from the immunized mice, or transferred into the immunized mice before the analysis of specific lysis <i>in vitro</i> (F) and <i>in vivo</i> (G). *, statistical significance between +/− amiloride.</p

Amiloride facilitates plasmid entry <i>in vitro</i>.

<p>Cy5-pEGFP entry into cell lines with or without 1 mM amiloride was analyzed after 2 h and is shown as the percentage of cells that were Cy5⁺. Expression of GFP was analyzed at day 3 and is shown as percentage of EGFP⁺Cy5⁺, on RAW264.7 (A, B, C), JAWSII (D, E), and DC2.4 (F, G). Data represent one of three independent experiments.</p

Amiloride increases the frequency of triple positive CD8 T cells.

<p>Splenocytes from mice immunized with pcD-S2 with or without amiloride (n = 3) were re-stimulated in vitro with 10 µg/ml S208–215 for 12 h (A-C), or 10 µg/ml HBsAg for 24 h (D), then cytokine secretion was blocked by monensin for 6 h. PMA or Ionomycin stimulating splenocyte of pcD-S2 immunized mice was added as positive controls. Cells stained with anti-CD3 and anti-CD8 were gated and then used for intracellular staining with single or multiple fluorescent-labeled antibodies. A shows the proportion of responsive cells in the total CD8⁺ T cells with or without amiloride treatment. These responsive cells were designated as either IFN-γ⁺, perforin⁺, or granzymeB⁺ cells. B shows the cytokine expression pattern in the responsive CD8 T cells with or without amiloride treatment. C shows the dose dependent effects of amiloride on the frequency of (IFN-γ⁺perforin⁺granzymeB⁺) triple positive cells. D shows the change in frequency of triple positive cells in response to HBsAg re-stimulation. The change in frequency of triple positive cells after co-culture with peritoneal macrophages followed by re-stimulation with S208–215 (E), or with spleno-DC (F) are also shown. Data represent three independent experiments with similar results.</p

Amiloride accelerates plasmid entry <i>in vivo</i>.

<p>Naïve C57 mice (n = 3) were immunized with Cy5-pEGFP <i>s.c.</i> in hind footpad with (right side) or without (left side) amiloride. Lymph node cells were collected after 4 h (Cy5⁺ cells) and after 24 h (GFP⁺ cells) and examined for proportion (B, D) and subtype (C, E) of stained cells from both draining and control lymph nodes. * in B and D, statistical significance between control and draining lymph nodes in all treated groups.</p

In vivo cytotoxic responses and antibody responses in immunized mice.

<p>C57BL/6 mice were immunized with 0.1 µg killed H5N1 vaccine in 100 ul of delivery vehicle, with vehicle alone, with vehicle containing PZQ (0.5 µg PZQ/0.1 µg antigen) or with vaccine in vehicle containing PZQ. (A) Analysis of in vivo cytotoxic lysis on day 7 after primary immunization. (B) The percentage of specific lysis summarized as the means of the three independent experiments. (C) Antibody levels in serum collected on day 14 after immunization and detected by ELISA. Data shown are representative from three independent experiments. *, p<0.05. ns, p>0.05. Killed V stands for the killed H5N1 vaccine and vehicle stands 7.5% ethanol in saline solution.</p

Analysis of antigen-specific cytokine productions in CD8⁺ T cells.

<p>Splenic cells were isolated from C57BL/6 mice on day 7 after immunization and stimulated with NP peptide for 6 h in culture. (A) Intracellular staining for IFN-γ, IL-17 and CD8⁺ was analyzed by FACS. (B) The percentages of positive-stained cells are summarized as the means from three independent experiments. (C) Co-staining for IFN-γ and IL-17 in CD8⁺ T cells. (D) The percentages were shown as the means of three independent experiments. **, p<0.01. ns, p>0.05.</p

Adoptive transfer of sensitized CD8⁺ T cells delays the development of virus infection.

<p>Each mouse received 5×10⁶ cells and was then challenged immediately with a lethal dose of H5N1 virus. (A) Viral loads in the lung of mice assayed on day 7 after infection. (B) Survival curves. Data are analyzed from three separate experiments and each had 5 animals. *, p<0.05. **, p<0.01. WT CD8 T cells stands for CD8⁺ T cells from wild type mice immunized with killed vaccine plus PZQ. IFN-γ KO CD8 T cells stand for CD8⁺ T cells from similarly immunized IFN-γ KO mice. IL-17KO CD8 T cells stands for CD8⁺ T cells from similarly immunized IL-17KO mice.</p

Effect of adding PZQ to high-dose (1 µg) killed H5N1 vaccine.

<p>Mice were immunized with high-dose killed H5N1 vaccine (0.5 µg PZQ/1 µg antigen). (A) In vivo cytotoxic lysis assay performed on day 7 after primary immunization. (B) The percentage of Tc cells at day 7. (C) Antibody levels in sera collected on day 14 after immunization and assayed by ELISA. (D) Weight change of surviving mice and (E) viral loads in the lungs. Mice were infected x days after immunization and viral load was measured 7 days later. (F) Survival curves. Data are analyzed from three separate experiments and each had 5 animals. *, p<0.05. ns, p>0.05. 1 µg V stands for 1 µg killed H5N1 vaccine.</p

Protection against a highly pathogenic avian influenza virus H5N1 infection.

<p>Mice were challenged one month after vaccination (0.5 µg PZQ/0.1 µg antigen). (A) Viral loads in the lungs on day 7 after virus infection. (B) Survival curves. Data are analyzed from three separate experiments and each had 5 animals. *, p<0.05.</p

In vivo cytotoxic responses in immunized wild-type, CD8KO, IFN-γKO and IL-17KO mice.

<p>Mice were tested at day 7 after immunization and the percentage of specific lysis is summarized. (A) In vivo cytotoxic lysis was tested in the wild-type and CD8KO mice. (B) In vivo cytotoxic lysis in IFN-γ KO mice. (C) In vivo cytotoxic lysis in IL-17KO mice. The same data from wild-type mice is shown in each panel for reference.*, p<0.05. **, p<0.01. ***, p<0.001. ns, p>0.05. KO stands for knockout.</p