Cátedra de Microbiología, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina.
Immunology Unit & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hannover, Germany.
Front Immunol. 2019 Jun 26;10:1422. doi: 10.3389/fimmu.2019.01422. eCollection 2019.
The development of new subunit vaccines has promoted the rational design of adjuvants able to induce a strong T-cell activation by targeting specific immune receptors. The S-layer is a (glyco)-proteinaceous envelope constituted by subunits that self-assemble to form a two-dimensional lattice that covers the surface of different species of and . Due to their ability to self-assemble in solution, they are attractive tools to be used as antigen/hapten carriers or adjuvants. Recently, we have demonstrated that S-layer glycoprotein from CIDCA 8348 (SLP-8348) enhanced the LPS-induced response on macrophages in a Ca-dependent manner, but the receptors involved in these immunomodulatory properties remain unknown. Therefore, we aim to determine the C-type lectin receptors (CLRs) recognizing this bacterial surface glycoprotein as well as to investigate the role of glycans in both the immunogenicity and adjuvant capacity of SLP-8348. Here, using a mild periodate oxidation protocol, we showed that loss of SLP-8348 glycan integrity impairs the cell-mediated immune response against the protein. Moreover, our data indicate that the adjuvant capacity of SLP-8348 is also dependent of the biological activity of the SLP-8348 glycans. In order to evaluate the CLRs involved in the interaction with SLP-8348 an ELISA-based method using CLR-hFc fusion proteins showed that SLP-8348 interacts with different CLRs such as Mincle, SingR3, and hDC-SIGN. Using BMDCs derived from CLR-deficient mice, we show that SLP-8348 uptake is dependent of Mincle. Furthermore, we demonstrate that the SLP-8348-induced activation of BMDCs as well as its adjuvant capacity relies on the presence of Mincle and its signaling adaptor CARD9 on BMDCs, since SLP-8348-activated BMDCs from Mincle or CARD9 mice were not capable to enhance OVA-specific response in CD4 T cells purified from OT-II mice. These findings significantly contribute to the understanding of the role of glycans in the immunomodulation elicited by bacterial SLPs and generate a great opportunity in the search for new adjuvants derived from non-pathogenic microorganisms.
新型亚单位疫苗的发展促进了佐剂的合理设计,这些佐剂能够通过靶向特定免疫受体诱导强烈的 T 细胞活化。S-层是由亚基自组装形成二维晶格的(糖)蛋白包膜,覆盖不同种属的 和 的表面。由于它们能够在溶液中自组装,因此它们是用作抗原/半抗原载体或佐剂的有吸引力的工具。最近,我们已经证明,来自 CIDCA 8348(SLP-8348)的 S-层糖蛋白以 Ca 依赖性方式增强了 LPS 诱导的巨噬细胞反应,但参与这些免疫调节特性的受体仍不清楚。因此,我们旨在确定识别这种细菌表面糖蛋白的 C 型凝集素受体(CLRs),并研究 SLP-8348 的糖基在免疫原性和佐剂能力中的作用。在这里,我们使用温和的过碘酸钠氧化方案表明,SLP-8348 糖完整性的丧失会损害针对该蛋白的细胞介导免疫反应。此外,我们的数据表明,SLP-8348 的佐剂能力也依赖于 SLP-8348 糖基的生物学活性。为了评估与 SLP-8348 相互作用涉及的 CLRs,我们使用基于 ELISA 的方法使用 CLR-hFc 融合蛋白表明,SLP-8348 与不同的 CLRs 相互作用,如 Mincle、SingR3 和 hDC-SIGN。使用来自 CLR 缺陷型小鼠的 BMDCs,我们表明 SLP-8348 的摄取依赖于 Mincle。此外,我们证明,SLP-8348 诱导的 BMDC 活化及其佐剂能力依赖于 BMDC 上 Mincle 和其信号传导衔接子 CARD9 的存在,因为来自 Mincle 或 CARD9 小鼠的 SLP-8348 激活的 BMDCs 不能增强从 OT-II 小鼠纯化的 CD4 T 细胞对 OVA 的特异性反应。这些发现极大地促进了对糖基在细菌 SLP 诱导的免疫调节中的作用的理解,并为从非致病性微生物中寻找新的佐剂提供了很大的机会。