Expression conditions and characterization of a novelly constructed lipoprotein intended as a vaccine to prevent human Haemophilus influenzae infections.
Expression conditions and characterization of a novelly constructed lipoprotein intended as a vaccine to prevent human Haemophilus influenzae infections.
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DOI：
10.1016/j.jbc.2023.105031
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发表时间：
2023-08
影响因子：
4.8
通讯作者：
Pichichero, Michael
中科院分区：
文献类型：
--
作者：
Kaur, Ravinder;Mangiafesto, Jill;Pryharski, Karin;Rasam, Sailee;Zagursky, Robert;Pichichero, Michael
细菌脂蛋白根据其脂质部分在结构上分为两组：二酰化（存在于革兰氏阳性细菌中）和三酰化（存在于一些革兰氏阳性细菌和大多数革兰氏阴性细菌中）。二酰化和三酰化脂质部分的不同之处在于单个酰胺连接的脂肪酸链。脂蛋白通过哺乳动物 Toll 样受体 2 (TLR2) 诱导宿主先天免疫反应。在本研究中，我们向重组 OMP26（流感嗜血杆菌的天然非脂化 (NL) 膜蛋白）中添加了脂质部分，并使用流式细胞术、LC/MS 和 MALDI-TOF 在不同表达条件下对其进行了广泛表征。我们还研究了 NL 和脂化 (L) OMP26 诱导 HEK Blue-hTLR2-TR1 和 hTLR-TLR6 细胞体外刺激的能力。我们的 L-OMP26 主要以二酰化形式表达，因此我们采用了富含载脂蛋白 N-乙酰转移酶 (Lnt) 的大肠杆菌菌株的额外基因副本，该菌株进一步酰化二酰基脂蛋白，以增强三酰化 L-OMP26 的产生。 L-OMP26 的二酰基和三酰基版本旨在作为人类疫苗，在小鼠模型中作为蛋白质疫苗成分进行了表征和评估。我们发现二酰基和三酰基L-OMP26蛋白制剂的免疫刺激活性显着不同，与三酰基化L-OMP26相比，二酰基化L-OMP26刺激更高的适应性免疫反应，并且与NL-OMP26相比，两者都刺激更高的适应性免疫反应。我们还构建并表征了 L-OMP26φNL-P6 融合蛋白，其中 NL-P6 蛋白（一种经常研究的流感嗜血杆菌疫苗候选物）与 L-OMP26 重组融合。我们在 L-OMP26φNL-P6 融合蛋白中观察到类似的脂化模式（主要是二酰化）。
Bacterial lipoproteins are structurally divided into two groups, based on their lipid moieties: diacylated (present in Gram-positive bacteria) and triacylated (present in some Gram-positive and most Gram-negative bacteria). Diacylated and triacylated lipid moieties differ by a single amide-linked fatty acid chain. Lipoproteins induce host innate immune responses by the mammalian Toll-like receptor 2 (TLR2). In this study, we added a lipid moiety to recombinant OMP26, a native nonlipidated (NL) membrane protein of Haemophilus influenzae, and characterized it extensively under different expression conditions using flow cytometry, LC/MS, and MALDI-TOF. We also investigated the ability of NL and lipidated (L) OMP26 to induce in vitro stimulation of HEK Blue-hTLR2-TR1 and hTLR-TLR6 cells. Our L-OMP26 was predominantly expressed in diacylated form, so we employed an additional gene copy of apolipoprotein N-acetyltransferase enzyme (Lnt)-rich Escherichia coli strain that further acylates the diacyl lipoproteins to enhance the production of triacylated L-OMP26. The diacyl and triacyl versions of L-OMP26, intended as a vaccine for use in humans, were characterized and evaluated as protein vaccine components in a mouse model. We found that the diacyl and triacyl L-OMP26 protein formulations differed markedly in their immune-stimulatory activity, with diacylated L-OMP26 stimulating higher adaptive immune responses compared with triacylated L-OMP26 and both stimulating higher adaptive immune response compared to NL-OMP26. We also constructed and characterized an L-OMP26φNL-P6 fusion protein, where NL-P6 protein (a commonly studied H. influenzae vaccine candidate) was recombinantly fused to L-OMP26. We observed a similar pattern of lipidation (predominantly diacylated) in the L-OMP26φNL-P6 fusion protein.
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影响因子：
3.1
作者：
Kaur, Ravinder;Pichichero, Michael
通讯作者：
Pichichero, Michael
影响因子：
3
作者：
Michel, Lea Vacca;Kaur, Ravinder;Pichichero, Michael E.
通讯作者：
Pichichero, Michael E.
影响因子：
3.2
作者：
Kurokawa, Kenji;Kim, Min-Su;Lee, Bok Luel
通讯作者：
Lee, Bok Luel
影响因子：
5.5
作者：
Farhat, Katja;Riekenberg, Sabine;Ulmer, Artur J.
通讯作者：
Ulmer, Artur J.
影响因子：
3.4
作者：
Kwok, Yan;Sung, Wang-Chou;Chong, Pele
通讯作者：
Chong, Pele