Gregg Kelsey A, Harberts Erin, Gardner Francesca M, Pelletier Mark R, Cayatte Corinne, Yu Li, McCarthy Michael P, Marshall Jason D, Ernst Robert K
Department of Microbial Pathogenesis, University of Maryland School of Dentistry, Baltimore, Maryland, USA.
Vaccine Platform Group, MedImmune, Gaithersburg, Maryland, USA.
mBio. 2017 May 9;8(3):e00492-17. doi: 10.1128/mBio.00492-17.
Adjuvant properties of bacterial cell wall components like MPLA (monophosphoryl lipid A) are well described and have gained FDA approval for use in vaccines such as Cervarix. MPLA is the product of chemically modified lipooligosaccharide (LOS), altered to diminish toxic proinflammatory effects while retaining adequate immunogenicity. Despite the virtually unlimited number of potential sources among bacterial strains, the number of useable compounds within this promising class of adjuvants are few. We have developed bacterial enzymatic combinatorial chemistry (BECC) as a method to generate rationally designed, functionally diverse lipid A. BECC removes endogenous or introduces exogenous lipid A-modifying enzymes to bacteria, effectively reprogramming the lipid A biosynthetic pathway. In this study, BECC is applied within an avirulent strain of to develop structurally distinct LOS molecules that elicit differential Toll-like receptor 4 (TLR4) activation. Using reporter cell lines that measure NF-κB activation, BECC-derived molecules were screened for the ability to induce a lower proinflammatory response than LOS. Their structures exhibit varied, dose-dependent, TLR4-driven NF-κB activation with both human and mouse TLR4 complexes. Additional cytokine secretion screening identified molecules that induce levels of tumor necrosis factor alpha (TNF-α) and interleukin-8 (IL-8) comparable to the levels induced by phosphorylated hexa-acyl disaccharide (PHAD). The lead candidates demonstrated potent immunostimulation in mouse splenocytes, human primary blood mononuclear cells (PBMCs), and human monocyte-derived dendritic cells (DCs). This newly described system allows directed programming of lipid A synthesis and has the potential to generate a diverse array of TLR4 agonist candidates. There is an urgent need to develop effective vaccines against infectious diseases that continue to be major causes of morbidity and mortality worldwide. Making effective vaccines requires selecting an adjuvant to strengthen an appropriate and protective immune response. This work describes a practical method, bacterial enzymatic combinatorial chemistry (BECC), for generating functionally diverse molecules for adjuvant use. These molecules were analyzed in cell culture for their ability to initiate immune stimulatory activity. Several of the assays described herein show promising cytokine production and costimulatory molecule expression results, suggesting that the BECC molecules may be useful in future vaccine preparations.
细菌细胞壁成分如单磷酰脂质A（MPLA）的佐剂特性已得到充分描述，并已获得美国食品药品监督管理局（FDA）批准用于如希瑞适（Cervarix）等疫苗。MPLA是化学修饰的脂寡糖（LOS）的产物，经过改造以减少毒性促炎作用，同时保留足够的免疫原性。尽管细菌菌株中潜在来源几乎无限，但这类有前景的佐剂中可用化合物的数量却很少。我们开发了细菌酶促组合化学（BECC）作为一种生成合理设计、功能多样的脂质A的方法。BECC将内源性或引入外源性脂质A修饰酶导入细菌，有效地重新编程脂质A生物合成途径。在本研究中，BECC应用于无毒菌株以开发结构不同的LOS分子，这些分子可引发不同的Toll样受体4（TLR4）激活。使用测量NF-κB激活的报告细胞系，筛选BECC衍生分子诱导比LOS更低促炎反应的能力。它们的结构在人和小鼠TLR4复合物中表现出不同的、剂量依赖性的、由TLR4驱动的NF-κB激活。额外的细胞因子分泌筛选鉴定出诱导肿瘤坏死因子α（TNF-α）和白细胞介素-8（IL-8）水平与磷酸化六酰二糖（PHAD）诱导水平相当的分子。主要候选物在小鼠脾细胞、人原代血液单核细胞（PBMC）和人单核细胞衍生的树突状细胞（DC）中表现出强大的免疫刺激作用。这个新描述的系统允许对脂质A合成进行定向编程，并有可能产生各种各样的TLR4激动剂候选物。迫切需要开发针对传染病的有效疫苗，这些传染病仍然是全球发病和死亡的主要原因。制备有效的疫苗需要选择一种佐剂来增强适当的保护性免疫反应。这项工作描述了一种实用的方法，即细菌酶促组合化学（BECC），用于生成功能多样的佐剂分子。在细胞培养中分析了这些分子启动免疫刺激活性的能力。本文所述的几种测定显示出有希望的细胞因子产生和共刺激分子表达结果，表明BECC分子可能在未来的疫苗制备中有用。
