Type I interferons (IFN-I) are crucial components of the innate antiviral immune response. Previous research has linked impaired IFN-I responses to severe COVID-19, with genetic mutations affecting IFN-I pathways and neutralizing autoantibodies identified as risk factors for severe pneumonia. However, the efficacy of exogenous IFN-I treatment has been inconsistent, potentially due to late-stage administration and lack of patient stratification. This study aims to clarify the role of IFN-I in COVID-19 pathogenesis by examining IFN-I responses across different disease severities, considering various IFN-I subtypes, and employing sensitive and standardized assays. The two-step nature of IFN-I responses in COVID-19 is highlighted, where effective initial IFN-I activity is crucial for viral suppression. Failure in this initial step leads to viral dissemination, hyperinflammation, and ultimately, severe disease. The study seeks to understand this dichotomy and guide the development of more effective therapeutic strategies.

Existing literature demonstrates the critical role of IFN-I in early antiviral immunity against SARS-CoV-2. However, studies have reported conflicting findings regarding IFN-I levels and their association with disease severity, possibly due to variations in disease kinetics, patient populations, and assay methodologies. Studies have used interferon-stimulated genes (ISGs) as surrogates for IFN signaling, while challenges in directly measuring IFNα proteins have led to inconsistencies. The identification of genetic mutations in TLR3/7-dependent IFN-I pathways and neutralizing autoantibodies against IFNα/β/ω as risk factors for severe COVID-19 further underscores the importance of IFN-I in the immune response. Despite this, clinical trials using exogenous IFN-I have shown limited success, suggesting a complex interplay of factors influencing the effectiveness of IFN-I treatment.

The study employed multiple cohorts of COVID-19 patients with varying disease severities (moderate, severe, critical). Plasma samples were analyzed using different IFN assays, including a highly sensitive digital ELISA for IFNα subtypes and Luminex assays for IFNα2. These measurements were correlated with ISG scores and functional IFN activity (cytotoxicity assay). The study also investigated the induction of IFN-I proteins and downstream responses upon ex vivo whole blood stimulation with various immune agonists (Poly(I:C), R848, LPS). Intracellular proteomics analysis was conducted to examine changes in monocyte numbers and interferon signaling pathways (phospho-IRF3/7 and intracellular IFNα). Gene expression profiling using Nanostring technology was used to analyze differences in immunological and host responses in hospitalized versus non-hospitalized patients. The presence of neutralizing autoantibodies against IFNα and IFNβ was also assessed using a Gyros assay. Statistical analyses, including multinomial logistic regression, Kruskal-Wallis tests, Spearman correlation, and GSEA, were used to analyze the data.

The study found consistently reduced IFNα protein levels in severe and critical COVID-19 patients compared to moderate cases and healthy controls, when measured using sensitive digital ELISA. However, a commercial Luminex assay showed no difference between groups, emphasizing the assay's importance. IFNα2 levels measured by digital ELISA correlated well with ISG scores and functional activity, unlike the Luminex results. While IFNβ levels were detectable, there were no significant differences between groups. Analysis of another patient cohort confirmed lower IFNα2 levels in hospitalized patients, highlighting consistent findings across different populations. Autoantibodies against IFNα were present in a small percentage of patients (4/126), and they did not significantly impact the results. Functional studies of whole blood stimulation showed that hospitalized patients exhibited impaired IFN-I induction upon stimulation, while non-hospitalized patients showed a robust response. This impaired IFN response was not due to autoantibodies. Intracellular proteomics analysis revealed increased monocyte numbers in hospitalized patients but a decreased IFN-I response after stimulation. Gene expression analysis showed that hospitalized patients had pre-existing perturbations in immune responses, even before stimulation, and displayed a pronounced inflammatory response upon IFN-γ stimulation. These findings suggest that the impaired IFN-I response in hospitalized patients is a result of both cellular and intracellular perturbations.

The findings demonstrate a consistent association between impaired IFN-I responses and COVID-19 severity, even when correcting for confounding factors like age and sex. The use of highly sensitive assays was critical in revealing these differences. The study's findings offer a potential explanation for the inconsistent success of IFN-I-based therapies, as the impaired response may already be present by the time treatment is initiated. The pre-existing immune dysregulation in hospitalized patients underscores the need for early therapeutic intervention targeting the underlying cellular and signaling defects. The observed robust inflammatory response in hospitalized patients upon IFN-γ stimulation suggests potential therapeutic targets for mitigating the inflammatory cascade and improving outcomes. The study highlights the importance of using sensitive, standardized assays for studying IFN-I responses in COVID-19, as well as the need to focus on functional immune assays to better understand the complexities of the immune dysfunction in this disease.

This study provides strong evidence that impaired activation and regulation of type I interferon immunity contribute significantly to COVID-19 severity. The observed defects in IFN-I response are not solely attributable to neutralizing autoantibodies but involve cellular and intracellular perturbations. These findings have implications for therapeutic strategies, highlighting the need for early intervention targeting underlying immune dysregulation. Future research should focus on identifying specific therapeutic targets to restore IFN-I signaling and mitigate the inflammatory response in severe COVID-19.

The study is limited by the retrospective nature of some analyses and variations in patient characteristics across different cohorts. The relatively small sample size of some groups may affect the statistical power of some analyses. Further investigation is needed to fully elucidate the complex interplay of factors leading to impaired IFN-I responses and the optimal timing and target for therapeutic intervention.