Sustained IFN signaling is associated with delayed development of SARS-CoV-2-specific immunity (preprint)

Plasma RNAemia, delayed antibody responses and inflammation predict COVID-19 outcomes, but the mechanisms underlying these immunovirological patterns are poorly understood. We profile 782 longitudinal plasma samples from 318 hospitalized COVID-19 patients. Integrated analysis using k-means reveal four patient clusters in a discovery cohort: mechanically ventilated critically-ill cases are subdivided into good prognosis and high-fatality clusters (reproduced in a validation cohort), while non-critical survivors are delineated by high and low antibody responses. Only the high-fatality cluster is enriched for transcriptomic signatures associated with COVID-19 severity, and each cluster has distinct RBD-specific antibody elicitation kinetics. Both critical and non-critical clusters with delayed antibody responses exhibit sustained IFN signatures, which negatively correlate with contemporaneous RBD-specific IgG levels and absolute SARS-CoV-2-specific B and CD4+ T cell frequencies. These data suggest that the Interferon paradox previously described in murine LCMV models is operative in COVID-19, with excessive IFN signaling delaying development of adaptive virus-specific immunity.

Colocalization of expression transcripts with COVID-19 outcomes is rare across cell states, cell types and organs. (preprint)

Identifying causal genes at GWAS loci can help pinpoint targets for therapeutic interventions. Expression studies can disentangle such loci but signals from expression quantitative trait loci (eQTLs) often fail to colocalize-which means that the genetic control of measured expression is not shared with the genetic control of disease risk. This may be because gene expression is measured in the wrong cell type, physiological state, or organ. We tested whether Mendelian randomization (MR) could identify genes at loci influencing COVID-19 outcomes and whether the colocalization of genetic control of expression and COVID-19 outcomes was influenced by cell type, cell stimulation, and organ. We conducted MR of cis-eQTLs from single cell (scRNA-seq) and bulk RNA sequencing. We then tested variables that could influence colocalization, including cell type, cell stimulation, RNA sequencing modality, organ, symptoms of COVID-19, and SARS-CoV-2 status among individuals with symptoms of COVID-19. The outcomes used to test colocalization were COVID-19 severity and susceptibility as assessed in the Host Genetics Initiative release 7. Most transcripts identified using MR did not colocalize when tested across cell types, cell state and in different organs. Most that did colocalize likely represented false positives due to linkage disequilibrium. In general, colocalization was highly variable and at times inconsistent for the same transcript across cell type, cell stimulation and organ. While we identified factors that influenced colocalization for select transcripts, identifying 33 that mediate COVID-19 outcomes, our study suggests that colocalization of expression with COVID-19 outcomes is partially due to noisy signals even after following quality control and sensitivity testing. These findings illustrate the present difficulty of linking expression transcripts to disease outcomes and the need for skepticism when observing eQTL MR results, even accounting for cell types, stimulation state and different organs.

Proteomic Characterization of Acute Kidney Injury in Patients Hospitalized with SARS-CoV2 Infection (preprint)

Background Acute kidney injury (AKI) is a known complication of COVID-19 and is associated with an increased risk of in-hospital mortality. Unbiased proteomics using biological specimens can lead to improved risk stratification and discover pathophysiological mechanisms. Methods Using measurements of ~4000 plasma proteins in two cohorts of patients hospitalized with COVID-19, we discovered and validated markers of COVID-associated AKI (stage 2 or 3) and long-term kidney dysfunction. In the discovery cohort (N= 437), we identified 413 higher plasma abundances of protein targets and 40 lower plasma abundances of protein targets associated with COVID-AKI (adjusted p <0.05). Of these, 62 proteins were validated in an external cohort (p <0.05, N =261). Results We demonstrate that COVID-AKI is associated with increased markers of tubular injury (NGAL) and myocardial injury. Using estimated glomerular filtration (eGFR) measurements taken after discharge, we also find that 25 of the 62 AKI-associated proteins are significantly associated with decreased post-discharge eGFR (adjusted p <0.05). Proteins most strongly associated with decreased post-discharge eGFR included desmocollin-2, trefoil factor 3, transmembrane emp24 domain-containing protein 10, and cystatin-Cindicating tubular dysfunction and injury. Conclusions Using clinical and proteomic data, our results suggest that while both acute and long-term COVID-associated kidney dysfunction are associated with markers of tubular dysfunction, AKI is driven by a largely multifactorial process involving hemodynamic instability and myocardial damage.

Alternative splicing in the lung influences COVID-19 severity and respiratory diseases. (preprint)

Hospital admission for COVID-19 remains common despite the successful development of vaccines and treatments. Thus, there is an ongoing need to identify targets for new COVID-19 therapies. Alternative splicing is an essential mechanism for generating functional diversity in protein isoforms and influences immune response to infection. However, the causal role of alternative splicing in COVID-19 severity and its potential therapeutic relevance is not fully understood. In this study, we evaluated the causal role of alternative splicing in COVID-19 severity and susceptibility using Mendelian randomization (MR). To do so, we performed two-sample MR to assess whether cis-sQTLs spanning 8,172 gene splicing in 5,295 genes were associated with COVID-19 outcomes in the COVID-19 Host Genetics Initiative, including up to 158,840 COVID-19 cases and 2,782,977 population controls. We identified that alternative splicing in lungs, rather than total RNA expression of OAS1, ATP11A, DPP9 and NPNT, was associated with COVID-19 severity. MUC1 splicing was associated with COVID-19 susceptibility. Further colocalization analyses supported a shared genetic mechanism between COVID-19 severity with idiopathic pulmonary fibrosis at ATP11A and DPP9 loci, and with chronic obstructive lung diseases at NPNT. We lastly showed that ATP11A, DPP9, NPNT, and MUC1 were highly expressed in lung alveolar epithelial cells, both in COVID-19 uninfected and infected samples. Taken together, these findings clarify the importance of alternative splicing of proteins in the lung for COVID-19 and other respiratory diseases, providing isoform-based targets for drug discovery.

Proteome-wide Mendelian randomization implicates nephronectin as an actionable mediator of the effect of obesity on COVID-19 severity (preprint)

Obesity is a major risk factor for COVID-19 severity; however, the underlying mechanism is not fully understood. Considering that obesity influences the human plasma proteome, we sought to identify circulating proteins mediating the effects of obesity on COVID-19 severity. We first screened 4,907 plasma proteins to identify proteins influenced by body mass index (BMI) using Mendelian randomization (MR). This yielded 1,216 proteins, whose effects on COVID-19 severity were assessed, again using MR. This two-step approach identified nephronectin (NPNT), for which a one standard deviation increase was associated with severe COVID-19 (odds ratio = 1.71, 95% CI: 1.45-2.02, P = 1.63 x 10-10). Colocalization analyses indicated that an NPNT splice isoform drove this effect. Overall, NPNT mediates 3.7% of the total effect of BMI on severe COVID-19. Finally, we found that decreasing body fat mass and increasing fat-free mass can lower NPNT levels and thus may improve COVID-19 outcomes. These findings provide actionable insights into how obesity influences COVID-19 severity.

Proteomic Characterization of Acute Kidney Injury in Patients Hospitalized with SARS-CoV2 Infection (preprint)

Acute kidney injury (AKI) is a known complication of COVID-19 and is associated with an increased risk of in-hospital mortality. Unbiased proteomics using longitudinally collected biological specimens can lead to improved risk stratification and discover pathophysiological mechanisms. Using longitudinal measurements of ~4000 plasma proteins in two cohorts of patients hospitalized with COVID-19, we discovered and validated markers of COVID-associated AKI (stage 2 or 3) and long-term kidney dysfunction. In the discovery cohort (N= 437), we identified 413 upregulated and 40 downregulated proteins associated with COVID-AKI (adjusted p <0.05). Of these, 62 proteins were validated in an external cohort (p <0.05, N =261). We demonstrate that COVID-AKI is associated with increased markers of tubular injury (NGAL) and myocardial injury. Using estimated glomerular filtration (eGFR) measurements taken after discharge, we also find that 25 of the 62 AKI-associated proteins are significantly associated with decreased post-discharge eGFR (adjusted p <0.05). Proteins most strongly associated with decreased post-discharge eGFR included desmocollin-2, trefoil factor 3, transmembrane emp24 domain-containing protein 10, and cystatin-C indicating tubular dysfunction and injury. Using longitudinal clinical and proteomic data, our results suggest that while both acute and long-term COVID-associated kidney dysfunction are associated with markers of tubular dysfunction, AKI is driven by a largely multifactorial process involving hemodynamic instability and myocardial damage.

Circulating proteins to predict adverse COVID-19 outcomes (preprint)

Predicting COVID-19 severity is difficult, and the biological pathways involved are not fully understood. To approach this problem, we measured 4,701 circulating human protein abundances in two independent cohorts totaling 986 individuals. We then trained prediction models including protein abundances and clinical risk factors to predict adverse COVID-19 outcomes in 417 subjects and tested these models in a separate cohort of 569 individuals. For severe COVID-19, a baseline model including age and sex provided an area under the receiver operator curve (AUC) of 65% in the test cohort. Selecting 92 proteins from the 4,701 unique protein abundances improved the AUC to 88% in the training cohort, which remained relatively stable in the testing cohort at 86%, suggesting good generalizability. Proteins selected from different adverse COVID-19 outcomes were enriched for cytokine and cytokine receptors, but more than half of the enriched pathways were not immune-related. Taken together, these findings suggest that circulating proteins measured at early stages of disease progression are reasonably accurate predictors of adverse COVID-19 outcomes. Further research is needed to understand how to incorporate protein measurement into clinical care.

Integrated immunovirological profiling validates plasma SARS-CoV-2 RNA as an early predictor of COVID-19 mortality (preprint)

Despite advances in COVID-19 management, it is unclear how to recognize patients who evolve towards death. This would allow for better risk stratification and targeting for early interventions. However, the explosive increase in correlates of COVID-19 severity complicates biomarker prioritisation. To identify early biological predictors of mortality, we performed an immunovirological assessment (SARS-CoV-2 viral RNA, cytokines and tissue injury markers, antibody responses) on plasma samples collected from 144 hospitalised COVID-19 patients 11 days after symptom onset and used to test models predicting mortality within 60 days of symptom onset. In the discovery cohort (n=61, 13 fatalities), high SARS-CoV-2 vRNA, low RBD-specific IgG levels, low SARS-CoV-2-specific antibody-dependent cellular cytotoxicity, and elevated levels of several cytokines and lung injury markers were strongly associated with increased mortality in the entire cohort and the subgroup on mechanical ventilation. Model selection revealed that a three-variable model of vRNA, age and sex was very robust at identifying patients who will succumb to COVID-19 (AUC=0.86, adjusted HR for log-transformed vRNA=3.5; 95% CI: 2.0-6.0). This model remained robust in an independent validation cohort (n=83, AUC=0.85). Quantification of plasma SARS-CoV-2 RNA can help understand the heterogeneity of disease trajectories and identify patients who may benefit from new therapies.

Failure to replicate the association of rare loss-of-function variants in type I IFN immunity genes with severe COVID-19 (preprint)

A recent report found that rare predicted loss-of-function (pLOF) variants across 13 candidate genes in TLR3- and IRF7-dependent type I IFN pathways explain up to 3.5% of severe COVID-19 cases. We performed whole-exome or whole-genome sequencing of 1,934 COVID-19 cases (713 with severe and 1,221 with mild disease) and 15,251 ancestry-matched population controls across four independent COVID-19 biobanks. We then tested if rare pLOF variants in these 13 genes were associated with severe COVID-19. We identified only one rare pLOF mutation across these genes amongst 713 cases with severe COVID-19 and observed no enrichment of pLOFs in severe cases compared to population controls or mild COVID-19 cases. We find no evidence of association of rare loss-of-function variants in the proposed 13 candidate genes with severe COVID-19 outcomes.

Circulating Proteins Influencing COVID-19 Susceptibility and Severity: a Mendelian Randomization Study (preprint)

Proteins detectable in peripheral blood may influence COVID-19 susceptibility or severity. However, understanding which circulating proteins are etiologically involved is difficult because their levels may be influenced by COVID-19 itself and also subject to confounding factors. To identify circulating proteins influencing COVID-19 susceptibility and severity we undertook a large-scale two-sample Mendelian randomization (MR) study, since this study design can rapidly scan hundreds of circulating proteins and reduces bias due to confounding and reverse causation. We began by identifying the genetic determinants of 955 circulating proteins in up to 10,708 SARS-CoV-2 uninfected individuals, retaining only single nucleotide polymorphisms near the gene encoded by the circulating protein. We then undertook an MR study to estimate the effect of these proteins on COVID-19 susceptibility and severity using the Host Genetics Initiative. We found that a standard deviation increase in OAS1 levels was associated with reduced COVID-19 death or ventilation (N = 2,972 cases / 284,472 controls; OR = 0.48, P = 7x10-8), COVID-19 hospitalization (N = 6,492 / 1,012,809; OR = 0.60, P = 2x10-7) and COVID-19 susceptibility (N = 17,607 / 1,345,334; OR = 0.81, P = 6x10-5). Results were consistent despite multiple sensitivity analyses probing MR assumptions. OAS1 is an interferon-stimulated gene that promotes viral RNA degradation. Other potentially implicated proteins included IL10RB. Available medicines, such as interferon-beta-1b, increase OAS1 and could be explored for their effect on COVID-19 susceptibility and severity.

Vitamin D and Covid-19 Susceptibility and Severity: a Mendelian Randomization Study (preprint)

Introduction Increased vitamin D levels, as reflected by 25OHD measurements, has been proposed to protect against Covid-19 disease based on in-vitro, observational, and ecological studies. However, vitamin D levels are associated with many confounding variables and thus associations described to date may not be causal. Vitamin D MR studies have provided results that are concordant with large-scale vitamin D randomized trials. Here, we used two-sample MR to assess the effect of circulating 25OHD levels on Covid-19 susceptibility. Methods Genetic variants strongly associated with 25OHD levels in a 443,734-participant genome-wide association study (GWAS) were used as instrumental variables. GWASs of Covid-19 susceptibility and severity from the Covid-19 Host Genetics Initiative were used to test the effect of 25OHD levels on these outcomes. Cohorts from the Covid-19 Host Genetics Initiative GWAS included up to 966,395 individuals of European ancestry. Results Genetically increased 25OHD levels by one standard deviation on the logarithmic scale had no clear effect on susceptibility but tended to increase the odds ratio of hospitalization (OR = 2.34; 95% CI: 1.33, 4.11) and severe disease (OR = 2.21; 95% CI: 0.87, 5.55). Extensive sensitivity analyses probing the assumptions of MR provided consistent estimates. Conclusion These findings do not support a protective role of increased 25OHD levels on Covid-19 outcomes and may suggest harm. At present, individuals should not use vitamin D supplements to protect against Covid-19 outcomes, and on-going supplementation trials should closely monitor for signals of harm.