Self-adjuvanting vaccines, consisting of recombinant protein antigens and covalently attached Toll-like receptor (TLR) agonists, have the ability to simultaneously and efficiently deliver antigen and TLR adjuvant to antigen presenting cells (APCs). Here, an enzyme-mediated ligation approach was used to overcome difficulties in producing homogeneous, molecularly defined self-adjuvanting vaccine products under native conditions. This process was optimized to allow the incorporation of the lipopeptide TLR2 agonist fibroblast-stimulating lipopeptide (FSL)-1 onto the N- or C-termini of recombinant protein antigens, employing the enzyme Staphylococcus aureus sortase A (SrtAsa) penta mutant. In addition, because SrtAsa-mediated ligations are reversible, a tryptophan zipper derived sequence was introduced into both reactants, which was demonstrated to improve ligation efficiency through the formation of a β-hairpin structure that hinders the reverse reaction. Finally, it was demonstrated that N- or C-terminal conjugation, and the incorporation of the β-hairpin structure, did not affect the TLR2-agonist activities of protein antigen TLR agonist conjugates. Overall, this SrtAsa-mediated ligation platform enabled production of antigen TLR2 agonist conjugates with enhanced ligation efficiency, with the conjugates demonstrating potent TLR2 signaling activation (EC50 <1nM).

中文翻译：

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一个自佐剂疫苗平台：特定位点分选酶的优化介导类似Toll受体2配体在重组蛋白抗原的羧基或氨基末端上的缀合。

由重组蛋白抗原和共价连接的Toll样受体（TLR）激动剂组成的自佐剂疫苗具有将抗原和TLR佐剂同时有效地递送至抗原呈递细胞（APC）的能力。在这里，使用酶介导的连接方法来克服在天然条件下生产均质的，分子定义的自佐剂疫苗产品方面的困难。优化此过程，以使用金黄色葡萄球菌分选酶A（SrtAsa）五突变体，将脂肽TLR2激动剂成纤维细胞脂肽（FSL）-1掺入重组蛋白抗原的N或C末端。此外，由于SrtAsa介导的连接是可逆的，因此将色氨酸拉链衍生的序列引入了两种反应物中，通过形成阻碍逆反应的β-发夹结构，可以提高连接效率。最后，证明了N-或C-末端的缀合以及β-发夹结构的掺入不影响蛋白抗原TLR激动剂缀合物的TLR2-激动剂活性。总体而言，该SrtAsa介导的连接平台能够产生具有增强的连接效率的抗原TLR2激动剂结合物，该结合物表现出有效的TLR2信号激活（EC50 <1nM）。不会影响蛋白质抗原TLR激动剂偶联物的TLR2激动剂活性。总体而言，该SrtAsa介导的连接平台能够产生具有增强的连接效率的抗原TLR2激动剂结合物，该结合物表现出有效的TLR2信号激活（EC50 <1nM）。不会影响蛋白质抗原TLR激动剂偶联物的TLR2激动剂活性。总体而言，该SrtAsa介导的连接平台能够产生具有增强的连接效率的抗原TLR2激动剂结合物，该结合物表现出有效的TLR2信号激活（EC50 <1nM）。