Researchers from Mayo Clinic-Minnesota-USA along with experts from the University of California San Diego-California-USA have in a new study discovered that the SARS-CoV-2-ORF8 protein is the key factor that is causing COVID-19 disease severity.
The study findings identify the SARS-CoV-2 ORF8 protein as the pathogenic cause and mechanism of severe disease and suggest that NLRP3 inhibitors can be used to treat severe COVID-19.
The study findings were published on a preprint serve and are currently being peer reviewed. https://www.biorxiv.org/content/10.1101/2021.12.02.470978v1
Despite the enormous success in the development and implementation of effective vaccines to prevent SARS-CoV-2 infection, Covid-19 remains a major health, social, and economic threat to society. First, a significant population in the world remains unvaccinated and will continue to be impacted by the pandemic.
Second, immune response may wane with time requiring revaccination, or new emerging variants may invalidate the current vaccines. Third, many patients especially those undergoing cancer treatment, are poorly protected due to their compromised immunity.17,18
SARS-CoV-2 accessory protein ORF8, a 121 amino acid protein, is the least conserved protein in the betacoronavirus family.19,20 Here we report that ORF8 is a major secreted viral glycoprotein both in vitro and in patients with newly diagnosed Covid-19. Our data, summarized in Supplemental Figure 5, support the concept that the ORF8 protein in blood stimulates circulating CD14+/CD16+ monocytes to initiate immediate and systematic cytokine responses.
This is consistent with findings by others that monocytes are the single most affected subset in blood after SARS-CoV-2 infection.21 A low level of CD16+ monocytes in patients infected with SARS-CoV-2 is associated with severe disease,22 and is likely the result of inflammation-activated pyroptosis of monocytes mediated by activated Gasdermin D.
Our findings that secreted ORF8 is a key disease-causing viral factor and can be readily targeted with NLRP3 inhibitors offer a new treatment option to combat the current pandemic, especially those with persistently high ORF8 levels and severe disease.
The NLRP3 mediated inflammasome activation is a crucial part of innate immune response to pathogen-associated molecular patterns (PAMPs) or host danger associated patterns (DAMPs). When no threat signal is present, NLRP3 is in an inactive state where the middle ATPase activity-possessing NACHT domain (Fig 4D) is masked by its C-terminal LRR domain.
Upon infection, the inflammation pathway is initiated with the breakdown of pathogenic peptidoglycan by lysozyme (LYZ) 23 and cathepsin proteases (CTSB, CTSL, etc.) 24 followed by NLRP3 receptor mediated recognition of PAMPs/DAMPs leading to the production of pro-IL1b and pro-IL18.
Both cytokine precursors are subsequently cleaved by caspase 1 to release mature cytokines IL-1β/IL-18. Both NACHT and LRR domains are involved in NLRP3 activation through various post-translational modifications and protein recruitment.25 We use IL1b expression level as a sensitive readout for the NLRP3 pathway activation since IL-18 is expressed at much lower levels.
Our results suggest that ORF8 enters monocytes through a non-receptor mediated process followed by direct binding to the NACHT and/or LRR domains of inflammasome receptor protein NLRP3 to activate IL1b/IL18-mediated inflammatory responses.
It is not known if the binding interaction with both domains are mutually exclusive or inclusive, necessary and/or sufficient, for NLRP3 activation. Further studies are needed to determine the ORF8-NLRP3 binding mechanics.
Due to the lack of effective Covid-19 animal models, it is challenging to genetically test the pathogenic role and targetability of ORF8 protein in vivo. However, a SARS-CoV-2 variant (Δ382) found in Taiwan and Singapore with a complete loss of the ORF8 gene serves as a tailor-made natural genetic model for validating our findings.26,27
Young et al analyzed the clinical data of a cohort of 92 patients infected with the wildtype virus and 29 patients infected with Δ382 variant, and found that patients infected with the wildtype SARS-Cov2 exhibited much higher levels of pro-inflammatory cytokines than those infected with the Δ382 variant.
Furthermore, no patients with the Δ382 variant required supplemental oxygen whereas it was required in 26 (28%) patients infected with wildtype virus.
These observations provide yet another line of evidence supporting the link between ORF8 and Covid-19 disease severity, and an in vivo genetic proof of the potential role to target ORF8 to prevent severe Covid-19.
Additionally, various clinical studies and trials have shown that the IL1 receptor antagonist Anakinra significantly reduced the mortality risk in hospitalized patients with moderate to severe inflammation symptoms.28,29
We believe that the data from our present study suggests that targeting NLRP3 or ORF8 upstream of the IL1 receptor will provide more effective therapeutic benefit to patients with severe Covid-19 symptoms, and perhaps could also provide prophylactic benefit to patients at risk of developing severe Covid-19.