As one of the pathogen-associated molecular patterns (PAMPs), flagellin is recently utilized as a potent adjuvant for many subunit vaccines. In this study, a truncated flagellin (tFL) with deletion of the hypervariable regions was adopted as a carrier–adjuvant by chemical conjugation with a chimeric malaria antigen M.RCAg-1 (M312) via a heterobifunctional polyethylene glycol (PEG) linker. After booster immunization in mice without any extra adjuvants, the M312–PEG–tFL conjugates elicited M312-specific antibody titers 100–1000 times higher than M312 and 10–100 times higher than the physical mixture of M312 and tFL. The elicited specific antibodies could recognize the native parasites, and the immunofluorescence assay (IFA) titer was 2100 for M312–P5k–tFL, which was about 7 times higher than M312. Furthermore, the IFA titers of the conjugates were comparable to the positive control of complete Freund’s adjuvant (CFA). Compared to M312, the M312–PEG–tFL conjugates enhanced the proliferation index, lymphocyte activation, and memory T-cell generation. IgG subclasses of sera and cytokines analysis of splenocytes showed that conjugation with tFL could slightly trigger the Th1 polarization, while the antigen alone predominantly induced a Th2-biased immune response. Furthermore, a more-efficient innate immune response was provoked by the M312–PEG–tFL conjugates, as determined by the detection of antigen-specific TNF-α secretion by splenocytes. Our results indicated that tFL mainly retained the function as an agonist of TLR5. Conjugation of antigen to tFL could induce strong humoral and moderate cellular immune responses. Thus, conjugation of antigen to tFL as a potent carrier–adjuvant is an effective strategy for developing a promising protein-based vaccine.

中文翻译：

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带有嵌合鞭毛蛋白的嵌合嵌合疟疾抗原促成的迅速而稳定的体液免疫。

作为一种与病原体相关的分子模式（PAMP），鞭毛蛋白最近被用作许多亚基疫苗的有效佐剂。在这项研究中，通过杂合双功能聚乙二醇（PEG）接头与嵌合疟疾抗原M.RCAg-1（M312）化学偶联，将截短的鞭毛蛋白（tFL）与高可变区缺失作为载体佐剂。在没有任何额外佐剂的小鼠中加强免疫后，M312–PEG–tFL偶联物产生的M312特异性抗体效价比M312高100–1000倍，比M312和tFL的物理混合物高10–100倍。诱发的特异性抗体可以识别天然寄生虫，M312–P5k–tFL的免疫荧光测定（IFA）效价为2100，约为M312的7倍。此外，结合物的IFA效价与完全弗氏佐剂（CFA）的阳性对照相当。与M312相比，M312–PEG–tFL共轭物可增强增殖指数，淋巴细胞活化和记忆T细胞生成。血清的IgG亚类和脾细胞的细胞因子分析表明，与tFL结合可以轻微触发Th1极化，而单独的抗原则主要诱导Th2偏向的免疫反应。此外，M312–PEG–tFL偶联物激发了更有效的先天免疫反应，这通过检测脾细胞分泌的抗原特异性TNF-α来确定。我们的结果表明tFL主要保留了作为TLR5激动剂的功能。抗原与tFL的结合可以诱导强烈的体液和中度细胞免疫应答。因此，