Divisions of Viral Products, Veterinary Services, and Biostatistics, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA.
Vaccine. 2012 Jul 6;30(32):4859-65. doi: 10.1016/j.vaccine.2012.05.002. Epub 2012 May 15.
Subunit vaccines composed of recombinant or purified antigens have a good safety record but are poorly immunogenic and require adjuvants to activate innate immunity and facilitate antigen specific immune response. Of the many adjuvant formulations that are under development, very few are licensed mainly due to concerns about adverse side effects. The goal of our study was to develop in vitro assays that could predict toxicity of adjuvants in vivo. Pro-inflammatory cytokines IL-β, IL-6, TNF-α, and IL-8 were measured in human primary monocytes and the monocytoid cell line, MonoMac 6 (MM6), activated with a panel of TLR agonists or with adjuvants. A 0.5 EU/ml dose of Standard for endotoxin (previously shown to provide a margin between pyrogenic and non-pyrogenic substances in rabbits) was used as a comparator to establish a "safety threshold". FSL-1, Pam3CSK4, flagellin, and R848 TLR agonists but not Alum, MF59, Poly I:C, or MPL adjuvants induced cytokines in MM6 cells above the safety threshold. To confirm the predictive value of the in vitro assays, FSL-1 and flagellin were injected intramuscularly into New Zealand White (NZW) rabbits. Both TLR agonists induced fever within 6-8h post-injection followed 24-48 h later by increased C reactive protein (CRP). Importantly, an early peak in plasma prostaglandin E2 (PGE(2)) levels preceded rise in body temperature. In vitro production of PGE(2) in monocytes and MM6 cells was found following treatments with various TLR agonists but not with alum, MF59, MPL, or Poly I:C adjuvants. Together, our studies demonstrated a strong correlation between production of pro-inflammatory cytokines above a "safety threshold" and production of PGE(2)in vitro and an increase in body temperature in rabbits. The developed human cell based assays could provide an important tool for early screening of new molecular moieties and adjuvant formulations and may assist in selection of safer products.
亚单位疫苗由重组或纯化抗原组成，具有良好的安全性记录，但免疫原性差，需要佐剂来激活先天免疫并促进抗原特异性免疫反应。在许多正在开发的佐剂配方中，只有少数获得许可，主要是因为担心不良反应。我们的研究目标是开发能够预测体内佐剂毒性的体外检测方法。使用一组 TLR 激动剂或佐剂激活人原代单核细胞和单核细胞样细胞系 MonoMac 6（MM6）后，测量促炎细胞因子 IL-β、IL-6、TNF-α 和 IL-8。标准内毒素（先前已证明在兔体内提供热原性和非热原性物质之间的差距）的 0.5 EU/ml 剂量用作比较器以建立“安全阈值”。FSL-1、Pam3CSK4、鞭毛蛋白和 R848 TLR 激动剂，但不是 Alum、MF59、Poly I:C 或 MPL 佐剂，在 MM6 细胞中诱导的细胞因子超过安全阈值。为了确认体外检测的预测价值，将 FSL-1 和鞭毛蛋白肌肉内注射到新西兰白兔（NZW）中。两种 TLR 激动剂均在注射后 6-8 小时内引起发热，随后 24-48 小时后 C 反应蛋白（CRP）增加。重要的是，血浆前列腺素 E2（PGE2）水平的早期峰值先于体温升高。在使用各种 TLR 激动剂处理后，在单核细胞和 MM6 细胞中发现 PGE2 的体外产生，但在 Alum、MF59、MPL 或 Poly I:C 佐剂中没有发现。总之，我们的研究表明，在“安全阈值”以上产生促炎细胞因子与体内产生 PGE2 和兔子体温升高之间存在很强的相关性。开发的基于人类细胞的检测方法可以为新的分子部分和佐剂配方的早期筛选提供重要工具，并可能有助于选择更安全的产品。
