The open reading frame 8 (ORF8) protein, encoded by the SARS-CoV-2 virus after infection, stimulates monocytes/macrophages to produce pro-inflammatory cytokines. We hypothesized that a positive ex vivo monocyte response to ORF8 protein pre-COVID-19 would be associated with subsequent severe COVID-19. We tested ORF8 ex vivo on peripheral blood mononuclear cells (PBMCs) from 26 anonymous healthy blood donors and measured intracellular cytokine/chemokine levels in monocytes by flow cytometry. The % monocytes staining positive in the sample and change in mean fluorescence intensity (ΔMFI) after ORF8 were used to calculate the adjusted MFI for each cytokine. We then tested pre-COVID-19 PBMC samples from 60 CLL patients who subsequently developed COVID-19 infection. Severe COVID-19 was defined as hospitalization due to COVID-19. In the 26 normal donor samples, the adjusted MFI for interleukin (IL)-1β, IL-6, IL-8, and CCL-2 were significantly different with ORF8 stimulation vs controls. We next analyzed monocytes from pre-COVID-19 PBMC samples from 60 CLL patients. The adjusted MFI to ORF8 stimulation of monocyte intracellular IL-1β was associated with severe COVID-19 and a reactive ORF8 monocyte response was defined as an IL- 1β adjusted MFI ≥ 0.18 (sensitivity 67%, specificity 75%). The median time to hospitalization after infection in CLL patients with a reactive ORF8 response was 12 days versus not reached for patients with a non-reactive ORF8 response with a hazard ratio of 7.7 (95% CI: 2.4-132, p=0.005). These results provide new insight on the monocyte inflammatory response to virus with implications in a broad range of disorders involving monocytes.

中文翻译：

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单核细胞对 SARS-CoV-2 蛋白 ORF8 的反应与慢性淋巴细胞白血病患者的严重 COVID-19 感染相关。

SARS-CoV-2 病毒感染后编码的开放阅读框 8 (ORF8) 蛋白会刺激单核细胞/巨噬细胞产生促炎细胞因子。我们假设，在 COVID-19 之前，单核细胞对 ORF8 蛋白的阳性离体反应可能与随后的严重 COVID-19 相关。我们在来自 26 名匿名健康献血者的外周血单核细胞 (PBMC) 上离体测试了 ORF8，并通过流式细胞术测量了单核细胞中的细胞内细胞因子/趋化因子水平。样品中染色阳性的单核细胞百分比和 ORF8 后平均荧光强度 (ΔMFI) 的变化用于计算每种细胞因子的调整后的 MFI。然后，我们测试了 60 名随后出现 COVID-19 感染的 CLL 患者的 COVID-19 前 PBMC 样本。重症 COVID-19 被定义为因 COVID-19 而住院。在 26 个正常供体样本中，ORF8 刺激后的白细胞介素 (IL)-1β、IL-6、IL-8 和 CCL-2 的调整后 MFI 与对照相比显着不同。接下来，我们分析了 60 名 CLL 患者的 COVID-19 前 PBMC 样本中的单核细胞。单核细胞胞内 IL-1β 的 ORF8 刺激调整后的 MFI 与严重的 COVID-19 相关，反应性 ORF8 单核细胞反应定义为 IL-1β 调整后的 MFI ≥ 0.18（敏感性 67%，特异性 75%）。具有反应性 ORF8 反应的 CLL 患者感染后到住院的中位时间为 12 天，而具有非反应性 ORF8 反应的患者则未达到住院时间，风险比为 7.7（95% CI：2.4-132，p=0.005）。这些结果为单核细胞对病毒的炎症反应提供了新的见解，对涉及单核细胞的广泛疾病具有影响。