A major roadblock in the development of novel vaccines is the formulation and delivery of the antigen. Liposomes composed of a dimethyldioctadecylammonium (DDA) backbone and the adjuvant trehalose-6-6-dibehenate (TDB, termed “cationic adjuvant formulation (CAF01)”, promote immunogenicity and protective efficacy of vaccines, most notably against infection with Mycobacterium tuberculosis. Specifically, the multicomponent antigen H56 delivered by CAF01 protects against tuberculosis in mice. Here we investigated whether the inclusion of immune-modulatory adjuvants into CAF01 modulates the immunogenicity of H56/CAF01 in vitro and in vivo. Based on our recent findings we selected the active sequence of the mycobacterial 19 kDa lipoprotein, Pam₃Cys, which interacts with Toll like receptor 2 to induce an antimicrobial pathway. H56/CAF01-Pam₃Cys liposomes were characterized for Pam₃Cys incorporation, size, toxicity and activation of primary human macrophages. Macrophages efficiently take up H56/CAF01-Pam₃Cys and trigger the release of significantly higher levels of TNF, IL-12 and IL-10 than H56/CAF01 alone. To evaluate the immunogenicity in vivo, we immunized mice with H56/CAF01-Pam₃Cys and measured the release of IFN-γ and IL-17A by lymph node cells and spleen cells. While the antigen-specific production of IFN-γ was reduced by inclusion of Pam₃Cys into H56/CAF01, the levels of IL-17A remained unchanged. In agreement with this finding, the concentration of the IFN-γ-associated IgG2a antibodies in the serum was lower than in H56/CAF01 immunized animals. These results provide proof of concept that Toll like-receptor agonist can be included into liposomes to modulate immune responses. The discordant results between the in vitro studies with human macrophages and in vivo studies in mice highlight the relevance and complexity of comparing immune responses in different species

中文翻译：

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Toll样受体激动剂Pam3Cys调节含有结核疫苗候选物H56的脂质体的免疫原性。

开发新型疫苗的主要障碍是抗原的配制和传递。由二甲基二十八烷基铵（DDA）骨架和辅助海藻糖-6-6-二山hen酸酯（TDB，称为“阳离子辅助剂（CAF01）”）组成的脂质体可提高疫苗的免疫原性和保护功效，尤其是针对结核分枝杆菌的感染。 CAF01传递的多组分抗原H56可以预防小鼠的结核病，在这里我们研究了CAF01中是否包含免疫调节佐剂是否在体内外调节H56 / CAF01的免疫原性。分枝杆菌19 kDa脂蛋白，Pam ₃Cys，与Toll样受体2相互作用以诱导抗菌途径。H56 / CAF01-Pam ₃ Cys脂质体的特征在于主要人巨噬细胞的Pam ₃ Cys掺入，大小，毒性和活化。巨噬细胞有效地吸收了H56 / CAF01-Pam ₃ Cys，并触发了比单独的H56 / CAF01显着更高水平的TNF，IL-12和IL-10释放。为了评估体内免疫原性，我们用H56 / CAF01-Pam ₃ Cys免疫小鼠，并测量淋巴结细胞和脾细胞释放的IFN-γ和IL-17A。通过加入Pam _3可以减少IFN-γ的抗原特异性产生半胱氨酸进入H56 / CAF01后，IL-17A水平保持不变。与该发现一致，血清中与IFN-γ相关的IgG2a抗体的浓度低于经H56 / CAF01免疫的动物。这些结果提供了可以在脂质体中包含Toll样受体激动剂以调节免疫应答的概念证明。人类巨噬细胞的体外研究与小鼠体内研究之间的不一致结果突显了比较不同物种免疫应答的相关性和复杂性