Immune factors produced by PBMCs upon stimulation with Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1-derived exopolysaccharides inhibit HCoV-229E and SARS-CoV-2 replication

The COVID-19 pandemic entered a controllable phase due to vaccine and drug development. The development of defense strategies of enhancing host innate immunity remains imperative to counter the emergence of infectious SARS-CoV-2 variants. In this study, we examined the inhibitory effects of Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 (OLL1073R-1)-derived exopolysaccharides (R-1 EPS), a natural immunomodulator, against coronaviral infection. The inhibitory effect of R-1 EPS on HCoV-229E was evaluated first and subsequently extended to SARS-CoV-2. Cell culture supernatant was obtained by stimulating human peripheral blood mononuclear cells with R-1 EPS (R-1 sup), and the inhibitory effect of R-1 sup on coronaviruses was evaluated using the human lung cell line, MRC5. High levels of proinflammatory cytokines were detected in R-1 sup, and replication of both HCoV-229E and SARS-CoV-2 was significantly suppressed in MRC5 cells pretreated with R-1 sup. Transcriptomic analysis revealed that type I IFN signaling was upregulated in R-1 sup-treated MRC5 cells. Our results suggest that immune factors, including proinflammatory cytokines, in R-1 sup, enhance type I IFN response, resulting in a broad spectral inhibitory effect on coronaviruses. This study provides a scientific basis for the application of OLL1073R-1 and R-1 EPS as preventive strategies against SARS-CoV-2 and other coronaviruses. Overall design: This study investigates the changes in immune response of MRC5 upon treatment with PBMCs supernatant stimulated by R-1 EPS (R-1 sup). Three conditions were compared: untreated control (n=2), R-1 sup treatment (n=5), and untreated PBMCs supernatant (Control sup) treatment (n=5). Samples were collected 24 hours after treatment.

新冠疫情（COVID-19 pandemic）因疫苗与药物研发进入可控阶段。增强宿主先天免疫的防御策略开发，仍是应对传染性严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）变异株出现的必要举措。本研究针对一种天然免疫调节剂——德氏乳杆菌保加利亚亚种（Lactobacillus delbrueckii ssp. bulgaricus）OLL1073R-1衍生的胞外多糖（exopolysaccharides，R-1 EPS），探究其对冠状病毒感染的抑制作用。研究首先评估了R-1 EPS对人类冠状病毒229E（HCoV-229E）的抑制效果，随后将实验拓展至SARS-CoV-2。通过用R-1 EPS刺激人外周血单个核细胞（human peripheral blood mononuclear cells，PBMCs）获得细胞培养上清液（R-1 sup），并利用人肺细胞系MRC5评估了R-1 sup对冠状病毒的抑制作用。R-1 sup中检测到高水平的促炎细胞因子，且经R-1 sup预处理的MRC5细胞中，HCoV-229E与SARS-CoV-2的复制均受到显著抑制。转录组学分析显示，经R-1 sup处理的MRC5细胞中，I型干扰素（type I IFN）信号通路被上调。本研究结果表明，R-1 sup中的免疫因子（包括促炎细胞因子）可增强I型干扰素应答，从而对冠状病毒产生广谱抑制作用。本研究为OLL1073R-1与R-1 EPS作为预防SARS-CoV-2及其他冠状病毒的策略提供了科学依据。
实验整体设计：本研究探究了经R-1 EPS刺激的PBMCs上清液（R-1 sup）处理后，MRC5细胞的免疫应答变化。共设置3组对照进行比较：未处理对照组（n=2）、R-1 sup处理组（n=5）、未刺激PBMCs上清液（Control sup）处理组（n=5）。于处理后24小时收集样本。