Nizatidine, a small molecular compound, enhances killed H5N1 vaccine cell-mediated responses and protects mice from lethal viral challenge

Abstract

Nizatidine (NIZ), closely related to Cimetidine, is a histamine H2 receptor inverse agonist used primarily as an anti-acid drug. Recent studies showed that this class of compounds may also modulate immune responses. To evaluate adjuvant effects of NIZ on vaccine immune modulation, we formulated NIZ with a H5N1 killed viral antigen and tested in vitro and in vivo. NIZ activated DC maturation and stimulated Th1 and Th2 immune responses to H5N1 vaccine. As a result, it enhanced both antibody and T cell-mediated immune responses. We also observed that a single immunization into C57BL/6 mice blocked IL-10 upregulation and potentiated Th1/Th2 dual polarization. Importantly, the inoculation of H5N1 vaccine with NIZ significantly improved protection of animals from death after challenge and reduced virus loads in the lung tissues. Considering its water-soluble nature, compared with Cimetidine, Nizatidine may be a better choice to use as a vaccine adjuvant.

Keywords: Cimetidine; H5N1 vaccine; Nizatidine; adjuvant; killed viral antigen.

Figure 1. Effects of NIZ on APC activation in vitro. DC line DC2.4 and macrophage line RAW264.7 were treated with NIZ or CIM for 48 h. (A) The cells were immunostained for MHC-II, CD80, CD86 and CD40 and analyzed by FACS. (B) The percentage of cells bearing the markers in the DC2.4 cell line, summarized as the means of three independent experiments. (C) The percentage of cells bearing the markers in the RAW264.7 cell line summarized as means of three independent experiments. Cell fluorescence was gated for analysis and all data are presented as mean ± SD *P < 0.05, **P < 0.01 compared with medium treated cells.

Figure 2. Analysis of splenocytes of C57BL/6 mice after immunization with H5N1 with or without NIZ. Samples were collected on day 3 after immunization and stimulated by 1 ug/ml H5N1 killed antigen for 12 h in vitro. (A) The CD11c⁺ cells were gated and analyzed for CD80, CD86, CD40, and MHC-II in total CD11c⁺ cells. (B) All the splenocytes were gated and intracellular immuno-staining for IL-12, TNF-α, or IL-10 analyzed by FACS. A representative result from three independent experiments is shown. All data are presented as mean ± SD *P < 0.05, **P < 0.01 compared with immunization with antigen alone.

Figure 3. Effects of NIZ on IgG level. Serum samples were collected 14 d after a single immunization of C57BL/6 mice. Anti-H5N1 titer was determined by ELISA (A), or hemagglutination inhibition (B). Mean titers (n = 6) are expressed in log10 or log2. Representative results from three independent experiments are shown. All data are presented as mean ± SD *P < 0.05, **P < 0.01 compared with control mice vaccinated with killed viral antigen alone.

Figure 4. Effects of NIZ on T cell function. The splenocytes were separated 7 d after a single immunization and used to perform intracellular staining or for proliferation assay (n = 6). (A) The level of T-cell proliferation was assayed using MTT assay. The splenocytes were stimulated for 3 d in vitro using killed H5N1 antigen as a specific antigen, BSA as a non-specific antigen, or anti-CD3 as a positive control. Anti-CD28 was added as a co-stimulant in all reactions. Proliferative response was expressed as stimulation index (SI). Splenocytes were stimulated for 12 h in vitro using killed H5N1 antigen as a specific antigen and blocked with BFA for 8 h. The lymphocytes were first gated on CD4⁺ T cells then stained for intracellular IFN-γ (B), IL-4 (C), Foxp3 (D), IL-10 (E) and TGF-β (F), and analyzed by FACS. Representative results from three independent experiments are shown. All data are presented as mean ± SD *P < 0.05, **P < 0.01 compared with mice vaccinated with killed viral antigen alone.

Figure 5. Effects of NIZ on the protective responses. Seven mice in each group were immunized once with the killed H5N1 antigen, with or without NIZ, 14 d before challenge with 10 LD₅₀ per animal. (A) Survival curves after H5N1 challenge were recorded. (B) Body weight change of challenged mice was measured every day and (C) viral loads in the lungs of each animal were measured 7 d after the challenge. All data are presented as mean ± SD *P < 0.05, **P < 0.01 compared with mice immunized with the killed H5N1 antigen alone.