SARS-CoV-2 Infection Biomarkers Reveal an Extended RSAD2 Dependant Metabolic Pathway (preprint)

We present compelling evidence for the existence of an evolutionary adaptive response to viral agents such as SARS-CoV-2, that results in the human in vivo biosynthesis of a family of compounds with potential antiviral activity. Using nuclear magnetic resonance (NMR) spectroscopy, we detected a characteristic spin-system motif indicative of the presence of an extended panel of urinary and serum metabolites during the acute viral phase. The structure of eight of nucleoside analogues was elucidated (six of which have not previously been reported in human urine), and subsequently confirmed by total-synthesis and matrix spiking. The molecular structures of the nucleoside analogues and their correlation with an array of serum cytokines, including IFN-2, IFN-{gamma} and IL-10, suggest an association with the viperin enzyme contributing to an endogenous innate immune defense mechanism against viral infection.

Stratification of hypertensive COVID-19 patients by quantitative NMR spectroscopy of serum metabolites, lipoproteins and inflammation markers (preprint)

Background: The exact pathophysiology of humans suffering from the multifaceted SARS-CoV-2 infection is not yet conclusively understood and risk stratification is needed. Novel diagnostic approaches like the nuclear magnetic resonance spectroscopy (NMR) based quantification of metabolites, lipoproteins, and inflammation markers has helped to identify typical alterations in the blood serum of COVID-19 patients. However, important confounders such as age, sex, and comorbidities, which strongly influence the metabolome, were often not considered. Therefore, the aim of this NMR study was to consider gender, as well as arterial hypertension (AHT) which affects more than 1.2 billion people worldwide, when investigating COVID-19-positive serum samples in a large age-matched cohort. As AHT is a risk factor for severe COVID-19 disease, this study focuses on comparing metabolomic characteristics of COVID-19 patients with and without AHT. Methods and Findings: NMR serum data from 329 COVID-19 patients were compared with 305 individuals from a healthy age and sex-matched control cohort. 134 of the 329 COVID-19 patients were affected by AHT. These were analyzed together with NMR data from 58 hypertensives without COVID-19. In addition to metabolite, lipoprotein, and glycoprotein data from NMR, common laboratory parameters were considered. Statistical comparison of the COVID-19 cohort with the control cohort reproduced results of previous studies. However, several differences emerged when AHT was considered. Especially, the previously described triglyceride-rich lipoprotein profile was no longer observed in COVID-19 patients, nor was an increase in ketone bodies. Typical metabolic changes that were apparent in COVID-19 patients in both sexes and with AHT were an increase in C- reactive protein (CRP) and the ratio of total glycoprotein (Glyc) to supramolecular phospholipids composite (SPC) which is an inflammatory NMR parameter. Further alterations were a decrease in glutamine, leucine, isoleucine, and lysine, citric acid, HDL-4 particles, and total cholesterol. Typical metabolic cardiovascular risk markers could be detected in hypertensive COVID-19 patients, as well as higher inflammatory NMR parameters than in normotensive COVID-19 patients. Conclusion: We could show that a more precise picture of COVID-19 blood serum parameters emerge when AHT is considered which accordingly should be included in future studies and would help for a refined patient stratification.

Anti-SARS-CoV-2 antibody containing plasma improves outcome in patients with hematologic or solid cancer and severe COVID-19 via increased neutralizing antibody activity. A randomized clinical trial (preprint)

Cancer patients are at high risk of severe COVID-19 with high morbidity and mortality. Further, impaired humoral response renders SARS-CoV-2 vaccines less effective and treatment options are scarce. Randomized trials using convalescent plasma are missing for high-risk patients. Here, we performed a multicenter trial (https://www.clinicaltrialsregister.eu/ctr-search/trial/2020-001632-10/DE) in hospitalized patients with severe COVID-19 within four risk groups (1, cancer; 2, immunosuppression; 3, lab-based risk factors; 4, advanced age) randomized to standard of care (CONTROL) or standard of care plus convalescent/vaccinated anti-SARS-CoV-2 plasma (PLASMA). For the four groups combined, PLASMA did not improve clinically compared to CONTROL (HR 1.29; p=0.205). However, cancer patients experienced shortened median time to improvement (HR 2.50, p=0.003) and superior survival in PLASMA vs. CONTROL (HR 0.28; p=0.042). Neutralizing antibody activity increased in PLASMA but not in CONTROL cancer patients (p=0.001). Taken together, convalescent/vaccinated plasma may improve COVID-19 outcome in cancer patients unable to intrinsically generate an adequate immune response.

Scalable RT-LAMP-based SARS-CoV-2 testing for infection surveillance with applications in pandemic control (preprint)

Throughout the current SARS-CoV-2 pandemic, limited diagnostic testing capacity prevented sentinel testing of the population, demonstrating the need for novel testing strategies and infrastructures. Here, we describe the set-up of an alternative testing platform, which allows scalable surveillance testing as an acute pandemic response tool and for pandemic preparedness purposes, exemplified by SARS-CoV-2 diagnostics in an academic environment. The testing strategy involves self-sampling based on gargling saline, pseudonymized sample handling, automated 96-well plate-based RNA extraction, and viral RNA detection using a semi-quantitative multiplexed colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay with an analytical sensitivity comparable to RT-quantitative polymerase chain reaction (RT-qPCR). We provide standard operating procedures and an integrated software solution for all workflows, including sample logistics, LAMP assay analysis by colorimetry or by sequencing (LAMP-seq), and communication of results to participants and the health authorities. Using large sample sets including longitudinal sample series we evaluated factors affecting the viral load and the stability of gargling samples as well as the diagnostic sensitivity of the RT-LAMP assay. We performed >35,000 tests during the pandemic, with an average turnover time of fewer than 6 hours from sample arrival at the test station to result announcement. Altogether, our work provides a blueprint for fast, sensitive, scalable, cost- and labor-efficient RT-LAMP diagnostics. As RT-LAMP-based testing requires advanced, but non-specialized laboratory equipment, it is independent of potentially limiting clinical diagnostics supply chains.

Prevalence, determinants, and impact on general health and working capacity of post-acute sequelae of COVID-19 six to 12 months after infection: a population-based retrospective cohort study from southern Germany (preprint)

Background: Post-acute sequelae of SARS-CoV-2 infection have commonly been described after COVID-19, but few population-based studies have examined symptoms six to 12 months after acute SARS-CoV-2 infection and their associations with general health recovery and working capacity. Methods: This population-based retrospective cohort study in four geographically defined regions in southern Germany included persons aged 18-65 years with PCR confirmed SARS-CoV-2 infection between October 2020 and March 2021. Symptom frequencies (six to 12 months after versus before acute infection, expressed as prevalence differences [PD] and ratios [PR]), symptom severity and clustering, risk factors and associations with general health recovery, and working capacity were analysed. Findings: Among a total of 11,710 subjects (mean age 44.1 years, 59.8% females, 3.5% previously admitted with COVID-19, mean follow-up time 8.5 months) the most prevalent symptoms with PDs >20% and PRs >5% were rapid physical exhaustion, shortness of breath, concentration difficulties, chronic fatigue, memory disturbance, and altered sense of smell. Female sex and severity of the initial infection were the main risk factors. Prevalence rates, however, appeared substantial among both men and women who had a mild course of acute infection, and PCS considerably affected also younger subjects. Fatigue (PD 37.2%) and neurocognitive impairment (PD 31.3%) as symptom clusters contributed most to reduced health recovery and working capacity, but chest symptoms, anxiety/depression, headache/dizziness and pain syndromes were also prevalent and relevant for working capacity, with some differences according to sex and age. When considering new symptoms with at least moderate impairment of daily life and [≤]80% recovered general health or working capacity, the overall estimate for post-COVID syndrome was 28.5% (age- and sex-standardised rate 26.5%). Interpretation The burden of self-reported post-acute symptoms and possible sequelae, notably fatigue and neurocognitive impairment, remains considerable six to 12 months after acute infection even among young and middle-aged adults after mild acute SARS-CoV-2 infection, and impacts general health and working capacity.

Early and rapid identification of COVID-19 patients with neutralizing type I-interferon auto-antibodies by an easily implementable algorithm (preprint)

Purpose Six-19% of critically ill COVID-19 patients display circulating auto-antibodies against type I interferons (IFN-AABs). Here, we establish a clinically applicable strategy for early identification of IFN-AAB-positive patients for potential subsequent clinical interventions. Methods We analysed sera of 430 COVID-19 patients with severe and critical disease from four hospitals for presence of IFN-AABs by ELISA. Binding specificity and neutralizing activity were evaluated via competition assay and virus-infection-based neutralization assay. We defined clinical parameters associated with IFN-AAB positivity. In a subgroup of critically ill patients, we analyzed effects of therapeutic plasma exchange (TPE) on the levels of IFN-AABs, SARS-CoV-2 antibodies and clinical outcome. Results The prevalence of neutralizing AABs to IFN- and IFN-{omega} in COVID-19 patients was 4.2% (18/430), while being undetectable in an uninfected control cohort. Neutralizing IFN-AABs were detectable exclusively in critically affected, predominantly male (83%) patients (7.6% IFN- and 4.6% IFN-{omega} in 207 patients with critical COVID-19). IFN-AABs were present early post-symptom onset and at the peak of disease. Fever and oxygen requirement at hospital admission co-presented with neutralizing IFN-AAB positivity. IFN-AABs were associated with higher mortality (92.3% versus 19.1 % in patients without IFN-AABs). TPE reduced levels of IFN-AABs in three of five patients and may increase survival of IFN-AAB-positive patients compared to those not undergoing TPE. Conclusion IFN-AABs may serve as early biomarker for development of severe COVID-19. We propose to implement routine screening of hospitalized COVID-19 patients according to our algorithm for rapid identification of patients with IFN-AABs who most likely benefit from specific therapies.

Can we predict antibody responses in SARS-CoV-2? A cohort analysis. (preprint)

BackgroundAfter infection with severe acute respiratory syndrome coronavirus (SARS-CoV-2), Immunoglobulin G (IgG) antibodies and virus-specific neutralizing antibodies (nAbs) develop. This study describes antibody responses in a cohort of recovered COVID-19 patients to identify predictors. MethodsWe recruited patients with confirmed SARS-CoV-2 infection from Heidelberg, Germany. Blood samples were collected three weeks after COVID-19 symptoms ended. Participants with high antibody titers were invited for follow-up visits. IgG titers were measured by the Euroimmun Assay, and nAbs titers in a SARS-CoV-2 infection-based assay. Results281 participants were enrolled between April and August 2020 with IgG testing, 145 (51.6%) had nAbs, and 35 (12.5%) had follow-up. The median IgG optical density (OD) ratio was 3.1 (Interquartile range (IQR) 1.6-5.1), and 24.1% (35/145) had a nAb titer>1:80. Higher IgG titers were associated with increased age and more severe disease, and higher nAbs were associated with male gender and CT-value of 25-30 on RT-PCR at diagnosis. The median IgG OD ratio on follow-up was 3.7 (IQR 2.9-5.9), a median increase of 0.5 (IQR -0.3-1.7). Six participants with follow-up nAbs all had titers [≤] 1:80. ConclusionsWhile age and disease severity were correlated with IgG responses, predictive factors for nAbs in convalescent patients remain unclear.

COVID-19 severity and complications associated with low diversity, dysbiosis and predictive metagenome features of the oropharyngeal microbiome. (preprint)

Coronavirus 2 (SARS-CoV-2) infection and the resulting COVID-19 illness vary from asymptomatic disease, mild upper respiratory tract infection, pneumonia1, to a life-threatening multi-organ failure with case fatality rates ranging from 0.27–13.4%2,3. Despite increasing knowledge of the clinical and immunological features underlying COVID-191,4−6, biological variables explaining the course of infection and its severity remain elusive. At the entry site of SARS-CoV2, the oropharyngeal microbiome represents a hub integrating viral and immune signals at the start of the infection7–10. To evaluate the role of the oropharyngeal microbiome in COVID-19, we performed a multi-center, cross-sectional clinical study analyzing the oropharyngeal microbial metagenomes in healthy adults, patients with non-SARS-CoV-2 infections, or with mild, moderate and severe COVID-19 encompassing a total of 345 participants. Significantly reduced microbiome diversity and high dysbiosis were observed in hospitalized patients with severe COVID-19, which was further associated with a loss of microbial genes and metabolic pathways. In this cohort, diversity measures were also associated with need for intensive care treatments as major clinical parameters in COVID-19. We further applied random forest machine learning to unravel microbial features for segregating clinical outcomes in hospitalized cases, and observed oropharyngeal microbiome abundances of Haemophilus or Streptococcus species as most important features. These findings provide insights into the role of the oropharyngeal microbiome in SARS-CoV-2 infection, and may suggest new biomarkers for COVID-19 severity.

Lack of antibodies against seasonal coronavirus OC43 nucleocapsid protein identifies patients at risk of critical COVID-19 (preprint)

Most COVID-19 patients experience a mild disease; a minority suffers from critical disease. We report about a biomarker validation study regarding 296 patients with confirmed SARS-CoV-2 infections from four tertiary care referral centers in Germany and France. Patients with critical disease had significantly less anti-HCoV OC43 nucleocapsid protein antibodies compared to other COVID-19 patients (p=0.007). In multivariate analysis, OC43 negative inpatients had an increased risk of critical disease, higher than the risk by increased age or BMI, and lower than the risk by male sex. A risk stratification based on sex and OC43 serostatus was derived from this analysis. Our results indicate that prior infections with seasonal human coronaviruses can protect against a severe course of COVID-19. Anti-OC43 antibodies should be measured for COVID-19 inpatients and considered as part of the risk assessment. We expect individuals tested negative for anti-OC43 antibodies to particularly benefit from vaccination, especially with other risk factors prevailing.

Serological response and disease-specific neutralizing antibodies in kidney transplant recipients with SARS-CoV-2 infection – a case series (preprint)

Background Since SARS-CoV-2 is a highly contagious virus without an available disease-specific medication, the hope is focused on a sustained immunity after SARS-CoV-2 infection and a near-term successful vaccination therapy. A sufficient anti-SARS-CoV-2 antibody production with neutralizing antibodies is crucial to prevent further viral spreading and for protection against prospective reinfection. Kidney transplant recipients may have a potentially aggravated risk for COVID-19 complications as well as a reduced vaccine response due to the allograft protecting immunosuppressive therapy. However, little is known about the strength and duration of their immunological response upon SARS-CoV-2 infection.Case presentation Here we report on 4 kidney transplant recipients proven to have SARS-CoV-2 infection by positive PCR testing, focusing on their immunological response with the production of disease-specific neutralizing antibodies. All kidney transplant recipients developed a sufficient antibody response including specific neutralizing antibodies against SARS-CoV-2 within 2 to 3 weeks after the first onset of symptoms that sustained during the follow-up of 15 weeks. After 6 weeks, the virus was eliminated in all patients. Most important, the serological response and viral shedding were achieved and sustained in the presence of immunosuppression. Acute kidney graft deterioration was common but reconstituted in all transplant recipients during follow-up. Conclusions Immunocompromised kidney transplant recipients showed a functional serological response with disease-specific neutralizing antibodies upon SARS-CoV-2 infection, a basic prerequisite for a prospective successful vaccination response. 

From multiplex serology to serolomics: A novel approach to the antibody response against the SARS-CoV-2 proteome (preprint)

Background: The emerging SARS-CoV-2 pandemic entails an urgent need for specific and sensitive high-throughput serological assays to assess SARS-CoV-2 epidemiology. We therefore aimed at developing a fluorescent-bead based SARS-CoV-2 multiplex serology assay for detection of antibody responses to the SARS-CoV-2 proteome. Methods: Proteins of the SARS-CoV-2 proteome and protein N of SARS-CoV-1 and common cold Coronaviruses (ccCoVs) were recombinantly expressed in E. coli or HEK293 cells. Assay performance was assessed in a Covid-19 case cohort (n=48 hospitalized patients from Heidelberg) as well as n=85 age- and sex-matched pre-pandemic controls from the ESTHER study. Assay validation included comparison with home-made immunofluorescence and commercial Enzyme-linked immunosorbent (ELISA) assays. Results: A sensitivity of 100% (95% CI: 86%-100%) was achieved in Covid-19 patients 14 days post symptom onset with dual sero-positivity to SARS-CoV-2 N and the receptor-binding domain of the spike protein. The specificity obtained with this algorithm was 100% (95% CI: 96%-100%). Antibody responses to ccCoVs N were abundantly high and did not correlate with those to SARS-CoV-2 N. Inclusion of additional SARS-CoV-2 proteins as well as separate assessment of immunoglobulin (Ig) classes M, A, and G allowed for explorative analyses regarding disease progression and course of antibody response. Conclusion: This newly developed SARS-CoV-2 multiplex serology assay achieved high sensitivity and specificity to determine SARS-CoV-2 sero-positivity. Its high throughput ability allows epidemiologic SARS-CoV-2 research in large population-based studies. Inclusion of additional pathogens into the panel as well as separate assessment of Ig isotypes will furthermore allow addressing research questions beyond SARS-CoV-2 sero-prevalence.

SARS-CoV-2 seroprevalence and clinical features of COVID-19 in a German liver transplant recipient cohort: a prospective serosurvey study. (preprint)

In liver transplant (LT) recipients with severe COVID-19 fatal outcome has been reported in a substantial subset of patients. Whether LT recipients are at increased risk for severe COVID-19 compared to the general population is controversial. Here we report the first results of a SARS-CoV-2 serosurvey in a large LT recipient cohort. Taking into account known risk factors, LT recipients a priori represented a high-risk cohort for severe COVID-19 with 101/219 (46.1 %) presenting with more than 2 risk factors for severe COVID-19. Out of 219 LT recipients 8 (3.7%) were either tested positive for nasopharyngeal SARS-CoV-2 RNA or anti-SARS-CoV-2 serum IgG. 5/8 (62.5 %) did not show any clinical signs of infection, 3/8 (37.5%) had self-limited disease, none required hospitalization for COVID-19. 5/8 (67.5%) SARS-CoV-2 positive patients showed high utilization of the healthcare system. 2/8 (25 %) had known exposure to infected health care personal. A majority of 65.4 % often or always avoided outside family social contacts. Face masks were commonly worn by all patients. In summary, LT recipients showed a SARS-CoV-2 seroconversion rate similar to the general population with a substantial percentage of unrecognized infections. The health care system can be the assumed source of infection in most of these cases.

SARS-CoV-2 infection induces a pro-inflammatory cytokine response through cGAS-STING and NF-κB (preprint)

SARS-CoV-2 is a novel virus that has rapidly spread, causing a global pandemic. In the majority of infected patients, SARS-CoV-2 leads to mild disease; however, in a significant proportion of infections, individuals develop severe symptoms that can lead to permanent lung damage or death. These severe cases are often associated with high levels of pro-inflammatory cytokines and low antiviral responses which can lead to systemic complications. We have evaluated transcriptional and cytokine secretion profiles from infected cell cultures and detected a distinct upregulation of inflammatory cytokines that parallels samples taken from infected patients. Building on these observations, we found a specific activation of NF-{kappa}B and a block of IRF3 nuclear translocation in SARS-CoV-2 infected cells. This NF-{kappa}B response is mediated by cGAS-STING activation and could be attenuated through STING targeting drugs. Our results show that SARS-CoV-2 curates a cGAS-STING mediated NF-{kappa}B driven inflammatory immune response in epithelial cells that likely contributes to inflammatory responses seen in patients and might be a target to suppress severe disease symptoms.

Hypoferremia predicts hospitalization and oxygen demand in COVID-19 patients (preprint)

BackgroundIron metabolism might play a crucial role in cytokine release syndrome in COVID-19 patients. Therefore we assessed iron metabolism markers in COVID-19 patients for their ability to predict disease severity. MethodsCOVID-19 patients referred to the Heidelberg University Hospital were retrospectively analyzed. Patients were divided into outpatients (cohort A, n=204), inpatients (cohort B, n=81), and outpatients later admitted to hospital because of health deterioration (cohort C, n=23). ResultsIron metabolism parameters were severely altered in patients of cohort B and C compared to cohort A. In multivariate regression analysis including age, gender, CRP and iron-related parameters only serum iron and ferritin were significantly associated with hospitalization. ROC analysis revealed an AUC for serum iron of 0.894 and an iron concentration <6{micro}mol/l as the best cutoff-point predicting hospitalization with a sensitivity of 94.7% and a specificity of 67.9%. When stratifying inpatients in a low- and high oxygen demand group serum iron levels differed significantly between these two groups and showed a high negative correlation with the inflammatory parameters IL-6, procalcitonin, and CRP. Unexpectedly, serum iron levels poorly correlate with hepcidin. ConclusionWe conclude that measurement of serum iron can help predicting the severity of COVID-19. The differences in serum iron availability observed between the low and high oxygen demand group suggest that disturbed iron metabolism likely plays a causal role in the pathophysiology leading to lung injury. KEY POINTSO_LIIron metabolism parameters are severely altered in COVID-19 patients. C_LIO_LIMeasurement of serum iron can help predicting the severity of COVID-19. C_LI

Microscopy-based assay for semi-quantitative detection of SARS-CoV-2 specific antibodies in human sera (preprint)

Emergence of the novel pathogenic coronavirus Sars-CoV-2 and its rapid pandemic spread presents numerous questions and challenges that demand immediate attention. Among these is the urgent need for a better understanding of humoral immune response against the virus and assessment of seroprevalence levels in the population, both of which form the basis for developing public health strategies to control viral spread. For this, sensitive, specific and quantitative serological assays are required. Here we describe the development of a semi-quantitative high-content microscopy-based assay for detection of three major classes (IgG, IgA and IgM) of SARS-CoV-2 specific antibodies in human samples. The possibility to detect antibodies against the entire viral proteome together with a robust semi-automated image analysis workflow resulted in improvement of sensitivity and specificity compared to an approved ELISA-based diagnostic test. Combining both methods resulted in maximum specificity in a negative control cohort, while maintaining high sensitivity. The procedure described here is compatible with high-throughput microscopy approaches and may be applied for serological analysis of other virus infections.