《Carbohydr Polym》|中国科学院杭州医药研究所：金耳多糖的结构表征及其对季节性流感 mRNA 疫苗的免疫增强活性

中国科学院杭州医药研究所余陈欢教授团队在《Carbohydrate Polymers》发表突破性研究，首次从金耳（Tremella aurantia）子实体中鉴定出一种具有显著免疫增强作用的新型岩藻糖基化葡萄糖醛酸木糖甘露聚糖（TAP2），为开发新一代mRNA疫苗佐剂提供了创新解决方案。研究团队通过超声辅助H₂O₂-Cu²⁺降解技术，将天然金耳多糖（TAP，分子量1178.77 kDa）可控降解获得三个组分，其中TAP2（16.96 kDa）在保留完整单糖组成（甘露糖、葡萄糖醛酸、木糖和岩藻糖）的同时，展现出最优的免疫刺激活性和最低细胞毒性。结构解析显示，该多糖具有1,3-α-D-甘露糖主链及葡萄糖醛酸/木糖侧链的独特构型。在季节性流感HA mRNA疫苗小鼠模型中，TAP2作为佐剂显著提升血凝抑制效价，增加流感特异性IgG抗体水平，并有效提高致死剂量流感病毒攻击后的存活率。机制研究表明，TAP2的最佳分子尺寸和硫酸化岩藻糖分支通过激活TLR4/MyD88/NF-κB信号通路，显著促进树突状细胞成熟。该研究不仅首次阐明了金耳多糖的结构-活性关系，更为开发基于天然多糖的mRNA疫苗佐剂奠定了理论基础。

流感病毒是一种高传染性病原体，对人类健康构成严重威胁。其特点是突变速度快、传播范围广，可引发季节性流行，甚至可能导致全球性大流行。截至目前，接种疫苗仍是防控流感最有效的策略。然而，由于流感病毒极易发生抗原漂移和抗原转换，通过既往感染或疫苗接种获得的免疫力可能很快失效。传统疫苗对老年人、儿童及免疫功能低下人群的保护作用有限，且病毒突变常导致疫苗毒株与流行毒株不匹配。大量研究表明，佐剂（如 MF59、AS03）能显著增强免疫应答、降低抗原用量、提高抗体水平并延长保护时间，进而扩大对变异毒株的交叉保护作用，提升疫苗效力。但部分佐剂可能激活黏膜免疫，减少频繁接种的需求，但其免疫原性效果仍需进一步临床验证。因此，筛选安全有效的佐剂不仅能提高流感疫苗的保护效力，还能为应对其他快速突变病原体提供宝贵的技术思路，具有重要的公共卫生战略价值。

天然多糖因其具有免疫调节特性、良好的生物相容性且毒性较低，展现出作为流感疫苗佐剂的巨大潜力，成为传统佐剂的理想替代品。越来越多的证据表明，包括黄芪多糖、香菇多糖和葡聚糖在内的多种多糖，能够激活树突状细胞和巨噬细胞，促进 Th1/Th2 免疫平衡，增强体液免疫和细胞免疫应答，同时可能对多种病毒感染产生交叉保护作用。金耳（Tremella aurantia），又称金耳，是一种独特的药食两用真菌，其子实体呈黄色、叶状且质地胶质。在临床应用中，它可作为免疫功能低下者和高血压患者的营养补充剂，还能减轻肿瘤放化疗带来的副作用。然而，目前对橙黄金耳多糖的结构特征与药理特性仍缺乏深入了解。

本研究首先从金耳子实体中分离出一种高分子量多糖（命名为 TAP），随后采用超声辅助过氧化氢 - 醋酸铜法对其进行水解，得到 3 种水解产物（分别命名为 TAP1～TAP3）。后续研究重点围绕这些水解产物的结构表征，以及它们对树突状细胞（DC）活化的免疫调节作用展开；同时，通过检测接种季节性流感血凝素（HA）mRNA 脂质纳米颗粒疫苗的小鼠体内血凝抑制（HA）效价、流感特异性抗体水平及细胞因子表达，进一步探究了 TAP2 的免疫调节效应。

Fig. 1. Isolation, degradation and preliminary structural analysis of polysaccharides from T. aurantia fruiting bodies. (A) The crude polysaccharide was eluted with NaCl gradient solution (0–2.0 M) on ion exchange column. The fraction (F2) was further eluted with 0.5 M NaCl solution on gel filtration column to collect the main fraction designated as TAP. (B) TAP was hydrolyzed by ultrasonic-assisted H2O2‑copper acetate method. After TAP was hydrolyzed for 1 h and 4 h, the products were further eluted with 0.5 M NaCl solution on gel filtration column to collect the main fraction designated as TAP1 and TAP2, respectively. 4 h*, TAP3 was obtained from the hydrolyzed products by supplementing 10 % H2O2 at 30-min intervals during the 4 h hydrolysis process. (C) The molecular weights of TAP and TAP1-TAP3 were detected by HPGPC and (D) their monosaccharide compositions were detected by HPLC.

Fig. 2. Immunoenhancing activities of TAP and its hydrolyzed products in vitro and in vivo. (A) The cytotoxicity of TAP and TAP1-TAP3 on the proliferation of mouse DC2.4 dendritic cells. (B) Effects of TAP and TAP1-TAP3 on the secretion of IL-2 and IL-12p70 in DC2.4 cells. (C) The schedule of immunization and serum biochemical analysis. Effects of TAP and TAP1-TAP3 on the production of influenza specific-IgG titers at 15, 30, 90, and 150 days after 2nd vaccination in mice. AL, aluminum hydroxide; VA, influenza HA mRNA-encoding lipid nanoparticle vaccine. Data were shown as Mean ± SD (n = 5). Different letters indicated significant differences (P < 0.05) by Tukey's test.

Fig. 3. Chemical structural characterization of TAP. (A) The UV, IR, 1H NMR, 13C NMR, HSQC and HMBC spectra of TAP2. (B) The presumed structures of TAP2

Fig. 4. Immunoenhancing activity of TAP2 in vitro. (A) Effects of TAP2 on proliferation and IFN-γ secretion of splenocytes in vitro. (B) Effects of TAP2 on the expressions of DC surface biomarkers CD86 and MHC-II in DC2.4 DCs. (C) Effects of TAP2 on the secretion of TNF-α, IL-1β and IL-6 in DC2.4 DCs. Data were shown as Mean ± SEM (n = 5). Different letters indicated significant differences (P < 0.05) by Tukey's test.

Fig. 5. Immunoenhancing activity of TAP2 in vivo. (A) Effects of TAP2 on HI titers and influenza-specific antibody levels in serum of immunized mice. (B) Effects of TAP2 on the populations of different immune cells in total CD45+ immune cells derived from the spleen of immunized mice. ND, not detected. Data were shown as Mean ± SD (n = 10). Different letters indicated significant differences (P < 0.05) by Tukey's test.

Fig. 6. Protective effects of TAP2 against influenza virus-induced death and pneumonia in immunized mice. (A) Effects of TAP2 on survival rates of pre-immunized mice challenged with lethal dose of influenza virus. Data were shown as Mean ± SD (n = 12). (Bsingle bondC) Effects of TAP2 on pulmonary pathological damage and viral loads in pre-immunized mice challenged with lethal dose of influenza virus. Data were shown as Mean ± SD (n = 5). ND, not detected. Different letters indicated significant differences (P < 0.05) by Tukey's test.

Fig. 7. Potential targets of TAP2 in DCs. (A) The expressions of CD86+ and MHC-II as well as the production of TNF-α in DCs stimulated by TAP2 in the absence or presence of anti-TLR2, anti-TLR4, anti-MR, anti-CR3, or anti-dectin-1 antibody. (B) The binding energies between TAP2 and its targets. Red dot line indicated the threshold of the interaction between ligand and receptor. (C) The interaction between TAP2 and TLR4. (D) Effects of TAP2 on the expressions of key proteins in TLR4 signaling. Data were shown as Mean ± SD (n = 5). Different letters indicated significant differences (P < 0.05) by Tukey's test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

本研究发现，TAP2（一种从橙黄金耳中分离得到、结构明确的新型多糖）是极具潜力的流感 mRNA 疫苗佐剂候选物。其效用源于独特的结构特征，尤其是硫酸化岩藻糖分支与适宜的分子量（Mw），这两种特征使其能够高效且特异性地激活 TLR4（ Toll 样受体 4）通路。该通路的激活可促进树突状细胞（DC）成熟，诱导产生强效且持久的抗体应答，以及强大的细胞毒性 T 淋巴细胞应答——这些作用共同为机体抵御致命性流感病毒感染提供了优异的保护效果。这些研究结果不仅填补了金耳属真菌多糖研究领域的重要知识空白，更为真菌多糖作为下一代疫苗佐剂的药物研发提供了坚实的作用机制基础。研究建议，未来应重点开展三方面研究：阐明 TAP2 精确的精细结构；在雪貂等更高级别的动物模型中评估其效用与安全性；探索其作为佐剂在其他疫苗平台中的应用潜力，以及诱导机体对不同流感毒株产生交叉保护性免疫的能力。

原文链接：

https://doi.org/10.1016/j.carbpol.2025.124660