Tukhvatulin Amir I, Dzharullaeva Alina S, Tukhvatulina Natalia M, Shcheblyakov Dmitry V, Shmarov Maxim M, Dolzhikova Inna V, Stanhope-Baker Patricia, Naroditsky Boris S, Gudkov Andrei V, Logunov Denis Y, Gintsburg Alexander L
N. F. Gamaleya Research Institute for Epidemiology and Microbiology, Gamaleya str.18, 123098 Moscow, Russia.
Cleveland BioLabs, Inc., Buffalo, New York, United States of America.
PLoS One. 2016 May 17;11(5):e0155650. doi: 10.1371/journal.pone.0155650. eCollection 2016.
Binding of pattern recognition receptors (PRRs) by pathogen-associated molecular patterns (PAMPs) activates innate immune responses and contributes to development of adaptive immunity. Simultaneous stimulation of different types of PRRs can have synergistic immunostimulatory effects resulting in enhanced production of molecules that mediate innate immunity such as inflammatory cytokines, antimicrobial peptides, etc. Here, we evaluated the impact of combined stimulation of PRRs from different families on adaptive immunity by generating alum-based vaccine formulations with ovalbumin as a model antigen and the Toll-like receptor 4 (TLR4) agonist MPLA and the Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) agonist MDP adsorbed individually or together on the alum-ovalbumin particles. Multiple in vitro and in vivo readouts of immune system activation all showed that while individual PRR agonists increased the immunogenicity of vaccines compared to alum alone, the combination of both PRR agonists was significantly more effective. Combined stimulation of TLR4 and NOD2 results in a stronger and broader transcriptional response in THP-1 cells compared to individual PRR stimulation. Immunostimulatory composition containing both PRR agonists (MPLA and MDP) in the context of the alum-based ovalbumin vaccine also enhanced uptake of vaccine particles by bone marrow derived dendritic cells (BMDCs) and promoted maturation (up-regulation of expression of CD80, CD86, MHCII) and activation (production of cytokines) of BMDCs. Finally, immunization of mice with vaccine particles containing both PRR agonists resulted in enhanced cellular immunity as indicated by increased proliferation and activation (IFN-γ production) of splenic CD4+ and CD8+ T cells following in vitro restimulation with ovalbumin and enhanced humoral immunity as indicated by higher titers of ovalbumin-specific IgG antibodies. These results indicate that combined stimulation of TLR4 and NOD2 receptors dramatically enhances activation of both the humoral and cellular branches of adaptive immunity and suggests that inclusion of agonists of these receptors in standard alum-based adjuvants could be used to improve the effectiveness of vaccination.
病原体相关分子模式（PAMPs）与模式识别受体（PRRs）的结合可激活先天免疫反应，并有助于适应性免疫的发展。同时刺激不同类型的PRRs可产生协同免疫刺激作用，导致介导先天免疫的分子如炎性细胞因子、抗菌肽等的产生增加。在此，我们通过制备以卵清蛋白为模型抗原、Toll样受体4（TLR4）激动剂MPLA和含核苷酸结合寡聚化结构域蛋白2（NOD2）激动剂MDP单独或共同吸附在铝佐剂 - 卵清蛋白颗粒上的铝佐剂疫苗制剂，评估了来自不同家族的PRRs联合刺激对适应性免疫的影响。免疫系统激活的多个体外和体内读数均显示，虽然与单独使用铝佐剂相比，单个PRR激动剂可提高疫苗的免疫原性，但两种PRR激动剂的组合效果显著更佳。与单个PRR刺激相比，TLR4和NOD2的联合刺激在THP - 1细胞中导致更强且更广泛的转录反应。在基于铝佐剂的卵清蛋白疫苗中同时含有两种PRR激动剂（MPLA和MDP）的免疫刺激组合物还增强了骨髓来源的树突状细胞（BMDCs）对疫苗颗粒的摄取，并促进了BMDCs的成熟（CD80、CD86、MHCII表达上调）和激活（细胞因子产生）。最后，用含有两种PRR激动剂的疫苗颗粒免疫小鼠，导致细胞免疫增强，表现为用卵清蛋白体外再刺激后脾CD4 +和CD8 + T细胞的增殖和激活（IFN - γ产生）增加，以及体液免疫增强，表现为卵清蛋白特异性IgG抗体滴度更高。这些结果表明，TLR4和NOD2受体的联合刺激显著增强了适应性免疫的体液和细胞分支的激活，并表明在标准铝佐剂中包含这些受体的激动剂可用于提高疫苗接种的有效性。
