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OBJECTIVE Diabetes mellitus (DM) is a major risk factor for severe coronavirus disease 2019 (COVID-19) for reasons that are unclear. RESEARCH DESIGN AND METHODS We leveraged the International Study of Inflammation in COVID-19 (ISIC), a multicenter observational study of 2,044 patients hospitalized with COVID-19, to characterize the impact of DM on in-hospital outcomes and assess the contribution of inflammation and hyperglycemia to the risk attributed to DM. We measured biomarkers of inflammation collected at hospital admission and collected glucose levels and insulin data throughout hospitalization. The primary outcome was the composite of in-hospital death, need for mechanical ventilation, and need for renal replacement therapy. RESULTS Among participants (mean age 60 years, 58.2% males), those with DM (n = 686, 33.5%) had a significantly higher cumulative incidence of the primary outcome (37.8% vs. 28.6%) and higher levels of inflammatory biomarkers than those without DM. Among biomarkers, DM was only associated with higher soluble urokinase plasminogen activator receptor (suPAR) levels in multivariable analysis. Adjusting for suPAR levels abrogated the association between DM and the primary outcome (adjusted odds ratio 1.23 [95% CI 0.78, 1.37]). In mediation analysis, we estimated the proportion of the effect of DM on the primary outcome mediated by suPAR at 84.2%. Hyperglycemia and higher insulin doses were independent predictors of the primary outcome, with effect sizes unaffected by adjusting for suPAR levels. CONCLUSIONS Our findings suggest that the association between DM and outcomes in COVID-19 is largely mediated by hyperinflammation as assessed by suPAR levels, while the impact of hyperglycemia is independent of inflammation.

Background Heterologous vaccine regimens have been widely discussed as a way to mitigate intermittent supply shortages and to improve immunogenicity and safety of COVID-19 vaccines. We aimed to assess the reactogenicity and immunogenicity of heterologous immunisations with ChAdOx1 nCov-19 (AstraZeneca, Cambridge, UK) and BNT162b2 (Pfizer-BioNtech, Mainz, Germany) compared with homologous BNT162b2 and ChAdOx1 nCov-19 immunisation. Methods This is an interim analysis of a prospective observational cohort study enrolling health-care workers in Berlin (Germany) who received either homologous ChAdOx1 nCov-19 or heterologous ChAdOx1 nCov-19–BNT162b2 vaccination with a 10–12-week vaccine interval or homologous BNT162b2 vaccination with a 3-week vaccine interval. We assessed reactogenicity after the first and second vaccination by use of electronic questionnaires on days 1, 3, 5, and 7. Immunogenicity was measured by the presence of SARS-CoV-2-specific antibodies (full spike-IgG, S1-IgG, and RBD-IgG), by an RBD–ACE2 binding inhibition assay (surrogate SARS-CoV-2 virus neutralisation test), a pseudovirus neutralisation assay against two variants of concerns (alpha [B.1.1.7] and beta [B.1.351]), and anti-S1-IgG avidity. T-cell reactivity was measured by IFN-γ release assay. Findings Between Dec 27, 2020, and June 14, 2021, 380 participants were enrolled in the study, with 174 receiving homologous BNT162b2 vaccination, 38 receiving homologous ChAdOx1 nCov-19 vaccination, and 104 receiving ChAdOx1 nCov-19–BNT162b2 vaccination. Systemic symptoms were reported by 103 (65%, 95% CI 57·1–71·8) of 159 recipients of homologous BNT162b2, 14 (39%, 24·8–55·1) of 36 recipients of homologous ChAdOx1 nCov-19, and 51 (49%, 39·6–58·5) of 104 recipients of ChAdOx1 nCov-19–BNT162b2 after the booster immunisation. Median anti-RBD IgG levels 3 weeks after boost immunisation were 5·4 signal to cutoff ratio (S/co; IQR 4·8–5·9) in recipients of homologous BNT162b2, 4·9 S/co (4·3–5·6) in recipients of homologous ChAdOx1 nCov-19, and 5·6 S/co (5·1–6·1) in recipients of ChAdOx1 nCov-19– BNT162b2. Geometric mean of 50% inhibitory dose against alpha and beta variants were highest in recipients of ChAdOx1 nCov-19–BNT162b2 (956·6, 95% CI 835·6–1095, against alpha and 417·1, 349·3–498·2, against beta) compared with those in recipients of homologous ChAdOx1 nCov-19 (212·5, 131·2–344·4, against alpha and 48·5, 28·4–82·8, against beta; both p

Background Artemisinin-based combination therapy (ACT) for the treatment of malaria is highly effective, well tolerated and safe. Episodes of delayed haemolysis occur in up to 57.9% of patients with severe malaria treated with intravenous artesunate, mainly caused by ‘pitting’ of infected red blood cells in the spleen and the delayed loss of these once-infected RBCs (oiRBCs). Several reports indicate that post-treatment haemolysis (PTH) also occurs in uncomplicated malaria treated with oral ACT, calling for systematic investigation. Methods A prospective observational study to identify the incidence of PTH after oral ACT, defined as increased lactate dehydrogenase activity and low haptoglobin level on Day 14 after treatment. Patients were enrolled at two study centres in Germany and Italy. Study visits took place on Days 1, 3, 7, 14 and 28. Laboratory investigations included extended clinical routine laboratory tests, quantitative PfHRP2, anti-RBC antibodies and oiRBCs. The state of semi-immunity to malaria was assessed from childhood and ongoing exposure to Plasmodium spp. as per patient history. Results A total of 134 patients with uncomplicated malaria and 3-day ACT treatment were recruited. Thirty-seven (37.4%) of 99 evaluable patients with Pf and none of 9 patients with non-Pf malaria exhibited PTH on d14. Patients with PTH had higher initial parasitaemia, higher oiRBC counts on d3 and a 10-fold decrease in oiRBCs between d7 and d14 compared with patients without PTH. In patients with PTH, loss of haemoglobin was 4-fold greater in non-Africans than in Africans (−1.3 vs −0.3 g/dl). Semi-immune African patients with PTH showed markedly increased erythropoiesis on d14 compared with not semi-immune African and non-African patients with PTH. Conclusions PTH is common in patients with uncomplicated malaria and oral ACT. While the observed loss of haemoglobin will often not be clinically relevant, it could aggravate pre-existing anaemia, warranting follow-up examinations in populations at risk.

BackgroundNirmatrelvir/ritonavir is an effective therapy against SARS-CoV-2. Patients with end-stage renal disease (ESRD) are at high risk for severe COVID-19 and show impaired vaccine responses underlining the importance of antiviral therapy. However, use of nirmatrelvir/ritonavir is not recommended in these patients due to lack of clinical and pharmacokinetic data.ObjectiveTo investigate pharmacokinetics and hepatic tolerance of nirmatrelvir/ritonavir in patients with ESRD and haemodialysis (HD).Patients and methodsFour patients diagnosed with SARS-CoV-2 infection received nirmatrelvir/ritonavir 150/100mg twice daily as recommended for renal impairment; HD ran in two- to three-day intervals. Plasma and serum samples were drawn before and after each HD during the 5-day treatment and for ensuing 3-5 days.ResultsMedian peak levels of nirmatrelvir obtained two hours after medication pre-HD in three patients were 7745ng/mL on day 3 and 6653ng/mL on day 5; median post-HD levels (C6h) declined to 5765ng/mL (74%) and 5521ng/mL (83%), on days 3 and 5 of treatment, respectively. Three days after end of treatment, median levels were 365ng/mL pre-HD and 30ng/mL post-HD. Measurements of the fourth patient, six hours after drug intake pre-HD showed nirmatrelvir-levels of 3704ng/mL on treatment day 3 which fell to 2308ng/mL post-HD, at one hour before intake of the next dose (Cmin).ConclusionUse of nirmatrelvir/ritonavir in patients with ESRD results in high nirmatrelvir blood concentrations, which are still within the range known from patients without renal failure. No accumulation of nirmatrelvir took place and levels declined to zero within few days after end of treatment.

Background Venous thromboembolism (VTE) contributes significantly to COVID‐19 morbidity and mortality. The urokinase receptor system is involved in the regulation of coagulation. Levels of soluble urokinase plasminogen activator receptor (suPAR) reflect hyperinflammation and are strongly predictive of outcomes in COVID‐19. Whether suPAR levels identify patients with COVID‐19 at risk for VTE is unclear. Methods and Results We leveraged a multinational observational study of patients hospitalized for COVID‐19 with suPAR and D‐dimer levels measured on admission. In 1960 patients (mean age, 58 years; 57% men; 20% Black race), we assessed the association between suPAR and incident VTE (defined as pulmonary embolism or deep vein thrombosis) using logistic regression and Fine‐Gray modeling, accounting for the competing risk of death. VTE occurred in 163 (8%) patients and was associated with higher suPAR and D‐dimer levels. There was a positive association between suPAR and D‐dimer (β=7.34; P =0.002). Adjusted for clinical covariables, including D‐dimer, the odds of VTE were 168% higher comparing the third with first suPAR tertiles (adjusted odds ratio, 2.68 [95% CI, 1.51–4.75]; P Conclusions Higher suPAR was associated with incident VTE independently of D‐dimer in patients hospitalized for COVID‐19. Combining suPAR and D‐dimer identified patients at low VTE risk. Registration URL: https://www.clinicaltrials.gov ; Unique identifier: NCT04818866.

Monkeypox (MPX) is caused by the homonymous orthopoxvirus (MPXV) known since the 1970s to occur at low frequency in West and Central Africa. Recently, the disease has been spreading quickly in Europe and the US. The rapid rise of MPX cases outside previously endemic areas and the different clinical presentation prompt for a better understanding of the disease, including the development of clinical tests for rapid diagnosis and monitoring. Here, using Zeno SWATH MS - a latest-generation proteomic technology - we studied the plasma proteome of a group of MPX patients with a similar infection history and clinical severity typical for the current outbreak. Moreover, we compared their proteomes to those of healthy volunteers and COVID-19 patients. We report that MPX is associated with a strong and characteristic plasma proteomic response and describe MPXV infection biomarkers among nutritional and acute phase response proteins. Moreover, we report a correlation between plasma protein markers and disease severity, approximated by the degree of skin manifestation. Contrasting the MPX host response with that of COVID-19, we find a range of similarities, but also important differences. For instance, Complement factor H-related protein 1 (CFHR1) is induced in COVID-19, but suppressed in MPX, reflecting the different role of the complement system in the two infectious diseases. However, the partial overlap between MPX and COVID-19 host response proteins allowed us to explore the repurposing of a clinically applicable COVID-19 biomarker panel assay, resulting in the successful classification of MPX patients. Hence, our results provide a first proteomic characterization of the MPX human host response based on a case series. The results obtained highlight that proteomics is a promising technology for the timely identification of disease biomarkers in studies with moderate cohorts, and we reveal a thus far untapped potential for accelerating the response to disease outbreaks through the repurposing of multiplex biomarker assays.

The SARS-CoV-2 pandemic prompted a global vaccination effort and the development of numerous COVID-19 vaccines at an unprecedented scale and pace. As a result, current COVID- 19 vaccination regimens comprise diverse vaccine modalities, immunogen combinations and dosing intervals. Here, we compare vaccine-specific antibody and memory B cell responses following two-dose mRNA, single-dose Ad26.COV2.S and two-dose ChAdOx1 or combination ChAdOx1/mRNA vaccination. Plasma neutralizing activity as well as the magnitude, clonal composition and antibody maturation of the RBD-specific memory B cell compartment showed substantial differences between the vaccination regimens. While individual monoclonal antibodies derived from memory B cells exhibited similar binding affinities and neutralizing potency against Wuhan-Hu-1 SARS-CoV-2, there were significant differences in epitope specificity and neutralizing breadth against viral variants of concern. Although the ChAdOx1 vaccine was inferior to mRNA and Ad26.COV2.S in several respects, biochemical and structural analyses revealed enrichment in a subgroup of memory B cell neutralizing antibodies with distinct RBD-binding properties resulting in remarkable potency and breadth.

The recently emerged BA.2.75 Omicron sublineage of SARS-CoV-2 identified in numerous countries is rapidly increasing in prevalence in regions of India. Compared with BA.2, the spike protein of BA.2.75 differs in nine amino acid residues. To determine the impact of the spike mutations on polyclonal and monoclonal antibody activity, we investigated the neutralization sensitivity of BA.2.75 in comparison with B.1, BA.2, BA.2.12.1, and BA.4/5. Analysis of post-boost samples from 30 vaccinated individuals revealed significantly lower serum neutralizing activity against BA.2.75 than against BA.2. However, BA.2.75 was more sensitive to serum neutralization than the widely circulating BA.4/5 sublineages. Moreover, evaluation of 17 clinical-stage monoclonal antibodies demonstrated individual differences in Omicron sublineage activity. Notably, some authorized antibodies with low activity against other Omicron sublineages demonstrated high BA.2.75 neutralizing potency. Our results indicate a less pronounced degree of antibody evasion of BA.2.75 compared with BA.4/5 and suggest that factors beyond immune evasion may be required for an expansion of BA.2.75 over BA.4/5.

Berlin is amongst the cities most affected by the current monkeypox outbreak. Here, we report clinical characteristics of the first patients with confirmed monkeypox admitted to our center. We analyzed anamnestic, clinical, and laboratory data. Within a period of 2 weeks, six patients were hospitalized in our unit. All were MSM and had practiced condomless receptive anal intercourse in the weeks preceding admission. The chief complaint in all patients but one was severe anal pain unprecedented in severity. Investigations revealed proctitis, as well as anal and rectal ulcers with detection of monkeypox virus. Our findings support the hypothesis that sexual transmission plays a role in the current outbreak.

ObjectiveTo determine the immediate need for a fourth COVID-19 vaccination based on the neutralizing capacity in patients on methotrexate (MTX) therapy after mRNA booster immunization.MethodsIn this observational cohort study, neutralizing serum activity against SARS-CoV-2 wildtype (Wu01) and variant of concern (VOC) Omicron BA.1 and BA.2 were assessed by pseudovirus neutralization assay before, 4 and 12 weeks after mRNA booster immunization in 50 rheumatic patients on MTX, 26 of whom paused the medication. 44 non-immunosuppressed persons (NIP) served as control group.ResultsWhile the neutralizing serum activity against SARS-CoV-2 Wu01 and Omicron variants increased 67-to 73-fold in the NIP after booster vaccination, the serum activity in patients receiving MTX increased only 20-to 23-fold. As a result, significantly lower neutralizing capacities were measured in patients on MTX compared to the NIP at week 4. Patients who continued MTX treatment during vaccination had significantly lower neutralizing serum titres against all three virus strains at week 4 and 12 compared to patients who paused MTX and the control group, except for BA.2 at week 12. Patients who paused MTX reached comparably high neutralization titres as the NIP, except for Wu01 at week 12. Neutralization of omicron variants was significantly lower in comparison to wildtype in both groups.ConclusionPatients pausing MTX showed a similar vaccine response to NIP. Patients who continued MTX demonstrated an impaired booster response indicating a potential benefit of a second booster vaccination.

SARS-CoV-2 neutralizing antibodies play a critical role in prevention and treatment of COVID-19 but are challenged by viral evolution and antibody evasion, exemplified by the highly resistant Omicron BA.1 sublineage.1–12 Importantly, the recently identified Omicron sublineages BA.2.12.1 and BA.4/5 with differing spike mutations are rapidly emerging in various countries. By determining polyclonal serum activity of 50 convalescent or vaccinated individuals against BA.1, BA.1.1, BA.2, BA.2.12.1, and BA.4/5, we reveal a further reduction of BA.4/5 susceptibility to vaccinee sera. Most notably, delineation of the sensitivity to an extended panel of 163 antibodies demonstrates pronounced antigenic differences of individual sublineages with distinct escape patterns and increased antibody resistance of BA.4/5 compared to the most prevalent BA.2 sublineage. These results suggest that the antigenic distance from BA.1 and the increased resistance compared to BA.2 may favor immune escape-mediated expansion of BA.4/5 after the first Omicron wave. Finally, while most monoclonal antibodies in clinical stages are inactive against all Omicron sublineages, we identify promising novel antibodies with high pan-Omicron neutralizing potency. Our study provides a detailed understanding of the antibody escape from the most recently emerging Omicron sublineages that can inform on effective strategies to prevent and treat COVID-19.

SARS-CoV-2 neutralizing antibodies play a critical role in COVID-19 prevention and treatment but are challenged by viral evolution and the emergence of novel escape variants. Importantly, the recently identified Omicron sublineages BA.2.12.1 and BA.4/5 are rapidly becoming predominant in various countries. By determining polyclonal serum activity of 50 convalescent or vaccinated individuals against BA.1, BA.1.1, BA.2, BA.2.12.1, and BA.4/5, we reveal a further reduction in BA.4/5 susceptibility to vaccinee sera. Most notably, delineation of sensitivity to an extended 163-antibody panel demonstrates pronounced antigenic differences with distinct escape patterns among Omicron sublineages. Antigenic distance and/or higher resistance may therefore favor immune-escape-mediated BA.4/5 expansion after the first Omicron wave. Finally, while most clinical-stage monoclonal antibodies are inactive against Omicron sublineages, we identify promising antibodies with high pan-SARS-CoV-2 neutralizing potency. Our study provides a detailed understanding of Omicron-sublineage antibody escape that can inform on effective strategies against COVID-19.

To determine neutralizing activity against the severe acute respiratory syndrome coronavirus 2 ancestral strain and 4 variants of concern, we tested serum from 30 persons with breakthrough infection after 2-dose vaccination. Cross-variant neutralizing activity was comparable to that after 3-dose vaccination. Shorter intervals between vaccination and breakthrough infection correlated with lower neutralizing titers.

Global healthcare systems continue to be challenged by the COVID-19 pandemic, and there is a need for clinical assays that can help optimise resource allocation, support treatment decisions, and accelerate the development and evaluation of new therapies.We developed a multiplexed proteomics assay for determining disease severity and prognosis in COVID-19. The assay quantifies up to 50 peptides, derived from 30 known and newly introduced COVID-19-related protein markers, in a single measurement using routine-lab compatible analytical flow rate liquid chromatography and multiple reaction monitoring (LC-MRM). We conducted two observational studies in patients with COVID-19 hospitalised at Charité - Universitätsmedizin Berlin, Germany before (from March 1 to 26, 2020, n=30) and after (from April 4 to November 19, 2020, n=164) dexamethasone became standard of care. The study is registered in the German and the WHO International Clinical Trials Registry (DRKS00021688).The assay produces reproducible (median inter-batch CV of 10.9%) absolute quantification of 47 peptides with high sensitivity (median LLOQ of 143 ng/ml) and accuracy (median 96.8%). In both studies, the assay reproducibly captured hallmarks of COVID-19 infection and severity, as it distinguished healthy individuals, mild, moderate, and severe COVID-19. In the post-dexamethasone cohort, the assay predicted survival with an accuracy of 0.83 (108/130), and death with an accuracy of 0.76 (26/34) in the median 2.5 weeks before the outcome, thereby outperforming compound clinical risk assessments such as SOFA, APACHE II, and ABCS scores.Disease severity and clinical outcomes of patients with COVID-19 can be stratified and predicted by the routine-applicable panel assay that combines known and novel COVID-19 biomarkers. The prognostic value of this assay should be prospectively assessed in larger patient cohorts for future support of clinical decisions, including evaluation of sample flow in routine setting. The possibility to objectively classify COVID-19 severity can be helpful for monitoring of novel therapies, especially in early clinical trials.This research was funded in part by the European Research Council (ERC) under grant agreement ERC-SyG-2020 951475 (to M.R) and by the Wellcome Trust (IA 200829/Z/16/Z to M.R.). The work was further supported by the Ministry of Education and Research (BMBF) as part of the National Research Node 'Mass Spectrometry in Systems Medicine (MSCoresys)', under grant agreements 031L0220 and 161L0221. J.H. was supported by a Swiss National Science Foundation (SNSF) Postdoc Mobility fellowship (project number 191052). This study was further supported by the BMBF grant NaFoUniMedCOVID-19 - NUM-NAPKON, FKZ: 01KX2021. The study was co-funded by the UK's innovation agency, Innovate UK, under project numbers 75594 and 56328.

Elderly individuals are at high risk for severe COVID-19. Due to modest vaccine responses compared to younger individuals and the time elapsed since prioritized vaccinations, the emerging immune-evasive Omicron variant of SARS-CoV-2 is a particular concern for the elderly. Here we longitudinally determined SARS-CoV-2-neutralizing serum activity against different variants in a cohort of 37 individuals with a median age of 82 years. Participants were followed for 10 months after an initial two-dose BNT162b2 vaccination and up to 4.5 months after a BNT162b2 booster. Detectable Omicron-neutralizing activity was nearly absent after two vaccinations but elicited in 89% of individuals by the booster immunization. Neutralizing titers against the Wu01, Delta, and Omicron variants showed similar post-boost declines and 81% of individuals maintained detectable activity against Omicron. Our study demonstrates the mRNA booster effectiveness in inducing Omicron neutralizing activity and provides critical information on vaccine response durability in the highly vulnerable elderly population.

Purpose To investigate antimicrobial use and primary and nosocomial infections in hospitalized COVID-19 patients to provide data for guidance of antimicrobial therapy. Methods Prospective observational cohort study conducted at Charité–Universitätsmedizin Berlin, including patients hospitalized with SARS-CoV-2-infection between March and November 2020. Results 309 patients were included, 231 directly admitted and 78 transferred from other centres. Antimicrobial therapy was initiated in 62/231 (26.8%) of directly admitted and in 44/78 (56.4%) of transferred patients. The rate of microbiologically confirmed primary co-infections was 4.8% (11/231). Although elevated in most COVID-19 patients, C-reactive protein and procalcitonin levels were higher in patients with primary co-infections than in those without (median CRP 110 mg/l, IQR 51–222 vs. 36, IQR 11–101, respectively; p Conclusions Primary co-infections are rare, yet antimicrobial use was frequent, mostly based on clinical worsening and elevated inflammation markers without clear evidence for co-infection. More reliable diagnostic prospects may help to reduce overtreatment. Rates of nosocomial infections are substantial in severely ill patients on organ support and associated with worse patient outcome.

Protein glycosylation is a complex and heterogeneous post-translational modification. Specifically, the human plasma proteome is rich in glycoproteins, and as protein glycosylation is frequently dysregulated in disease, glycoproteomics is considered an underexplored resource for biomarker discovery. Here, we present OxoScan-MS, a data-independent mass spectrometric acquisition technology and data analysis software that facilitates sensitive, fast, and cost-effective glycoproteome profiling of plasma and serum samples in large cohort studies. OxoScan-MS quantifies glycosylated peptide features by exploiting a scanning quadrupole to assign precursors to oxonium ions, glycopeptide-specific fragments. OxoScan-MS reaches a high level of sensitivity and selectivity in untargeted glycopeptide profiling, such that it can be efficiently used with fast microflow chromatography without a need for experimental enrichment of glycopeptides from neat plasma. We apply OxoScan-MS to profile the plasma glycoproteomic in an inpatient cohort hospitalised due to severe COVID-19, and obtain precise quantities for 1,002 glycopeptide features. We reveal that severe COVID-19 induces differential glycosylation in disease-relevant plasma glycoproteins, including IgA, fibrinogen and alpha-1-antitrypsin. Thus, with OxoScan-MS we present a strategy for quantitatively mapping glycoproteomes that scales to hundreds and thousands of samples, and report glycoproteomic changes in severe COVID-19.

ObjectiveThe level of neutralising capacity against Omicron BA.1 and BA.2 after third COVID-19 vaccination in patients on paused or continuous methotrexate (MTX) therapy is unclear.MethodsIn this observational cohort study, neutralising serum activity against SARS-CoV-2 wild-type (Wu01) and variant of concern Omicron BA.1 and BA.2 were assessed by pseudovirus neutralisation assay before, 4 and 12 weeks after mRNA booster immunisation in 50 rheumatic patients on MTX, 26 of whom paused the medication. 44 non-immunosuppressed persons (NIP) served as control group.ResultsWhile the neutralising serum activity against SARS-CoV-2 Wu01 and Omicron variants increased 67–73 fold in the NIP after booster vaccination, the serum activity in patients receiving MTX increased only 20–23 fold. Patients who continued MTX treatment during vaccination had significantly lower neutralisation against all variants at weeks 4 and 12 compared with patients who paused MTX and the control group, except for BA.2 at week 12. Patients who paused MTX reached comparably high neutralising capacities as NIP, except for Wu01 at week 12. The duration of the MTX pause after—not before—was associated with a significantly higher neutralisation capacity against all three variants, with an optimal duration at 10 days after vaccination.ConclusionPatients pausing MTX after COVID-19 booster showed a similar vaccine response to NIP. Patients who continued MTX demonstrated an impaired response indicating a potentially beneficial second booster vaccination. Our data also suggest that a 1 week MTX break is sufficient if the last administration of MTX occurs 1–3 days before vaccination.

The Omicron variant of SARS-CoV-2 is causing a rapid increase in infections across the globe. This new variant of concern carries an unusually high number of mutations in key epitopes of neutralizing antibodies on the viral spike glycoprotein, suggesting potential immune evasion. Here we assessed serum neutralizing capacity in longitudinal cohorts of vaccinated and convalescent individuals, as well as monoclonal antibody activity against Omicron using pseudovirus neutralization assays. We report a near-complete lack of neutralizing activity against Omicron in polyclonal sera from individuals vaccinated with two doses of the BNT162b2 COVID-19 vaccine and from convalescent individuals, as well as resistance to different monoclonal antibodies in clinical use. However, mRNA booster immunizations in vaccinated and convalescent individuals resulted in a significant increase of serum neutralizing activity against Omicron. This study demonstrates that booster immunizations can critically improve the humoral immune response against the Omicron variant., Neutralization of the SARS-CoV-2 Omicron variant is markedly impaired in sera from recipients of two doses of the COVID-19 vaccine BNT162b2 or from convalescent individuals, but is robustly increased in both groups following a booster vaccine dose.

Global healthcare systems continue to be challenged by the COVID-19 pandemic, and there is a need for clinical assays that can both help to optimize resource allocation and accelerate the development and evaluation of new therapies. Here, we present a multiplex proteomic panel assay for the assessment of disease severity and outcome prediction in COVID-19. The assay quantifies 50 peptides derived from 30 COVID-19 severity markers in a single measurement using analytical flow rate liquid chromatography and multiple reaction monitoring (LC-MRM), on equipment that is broadly available in routine and regulated analytical laboratories. We demonstrate accurate classification of COVID-19 severity in patients from two cohorts. Furthermore, the assay outperforms established risk assessments such as SOFA and APACHE II in predicting survival in a longitudinal COVID-19 cohort. The prognostic value implies its use for support of clinical decisions in settings with overstrained healthcare resources e.g. to optimally allocate resources to severely ill individuals with high chance of survival. It can furthermore be helpful for monitoring of novel therapies in clinical trials.

COVID-19 mRNA vaccine BNT162b2 is highly immunogenic and effective, but recent studies have indicated waning anti-SARS-CoV-2 immune responses over time. Increasing infection rates has led authorities in several countries to initiate booster campaigns for vulnerable populations, including the elderly. However, the durability of vaccine-induced immunity in the elderly is currently unknown. Here, we describe interim results of a prospective cohort study comparing immune responses in a cohort of vaccinated elderly persons to those in healthcare workers (HCW), measured six months after first immunisation with BNT162b2. Anti-SARS-CoV-2 S1-, full Spike- and RBD-IgG seropositivity rates and IgG levels at six months were significantly lower in the elderly compared to HCW. Serum neutralization of Delta VOC measured by pseudovirus neutralisation test was detectable in 43/71 (60.6%, 95%CI: 48.9-71.1) in the elderly cohort compared to 79/83 in the HCW cohort (95.2%, 95%CI: 88.3-98.1) at six months post vaccination. Consistent with the overall lower antibody levels, SARS-CoV-2-S1 T cell reactivity was reduced in the elderly compared to HCW (261.6 mIU/ml, IQR:141.5-828.6 vs 1198.0 mIU/ml, IQR: 593.9-2533.6, pCollectively, these findings suggest that the established two-dose vaccination regimen elicits less durable immune responses in the elderly compared to young adults. Given the recent surge in hospitalisations, even in countries with high vaccination rates such as Israel, the current data may support booster vaccinations of the elderly. Further studies to determine long-term effectiveness of COVID-19 vaccines in high-risk populations and the safety and effectiveness of additional boosters are needed.

The secreted protein Cystatin C (CyC) is a cysteine protease inhibitor of incompletely characterized biomedical function, used clinically for estimation of glomerular filtration rate. Plasma CyC is elevated in many patients, especially when they receive glucocorticoid (GC) treatment. Here we empirically connect GCs with systemic regulation of CyC. First, we leveraged genome-wide association and structural equation modeling to determine the genetics of the latent trait CyC production in UK Biobank. Using multi-modal genomic, transcriptional, and experimental approaches, we demonstrated that CyC is a direct target of GC receptor, with GC-responsive CyC secretion exhibited by macrophages and cancer cells in vitro. Elevated serum CyC levels were positively correlated with GC levels in a murine model of cancer progression. Consistent with the coupling of CyC levels to GC signaling in a disease relevant manner, CyC predicted elevated all-cause and cancer-specific mortality in humans. These associations were orthogonally confirmed by a polygenic score (PGS) capturing germline predisposition to CyC production. This PGS predicted checkpoint immunotherapy failure in a combined clinical trial cohort of 685 metastatic cancer patients, with available germline exome sequencing. Taken together, our results demonstrate that CyC captures biomedically-relevant variations in endogenous GC activity, raising the possibility that CyC may be a direct effector of GC-induced immunosuppression and therefore a target for combination cancer immunotherapy.

COVID-19 has highly variable clinical courses. The search for prognostic host factors for COVID-19 outcome is a priority. We performed logistic regression for ICU admission against a polygenic score (PGS) for Cystatin C (CyC) production in patients with COVID-19. We analyzed the predictive value of longitudinal plasma CyC levels in an independent cohort of patients hospitalized with COVID-19. In four cohorts spanning European and African ancestry populations, we identified a significant association between CyC-production PGS and odds of critical illness (n cases=2,319), with the strongest association captured in the UKB cohort (OR 2.13, 95% CI 1.58-2.87, p=7.12e-7). Plasma proteomics from an independent cohort of hospitalized COVID-19 patients ( n cases = 131) demonstrated that CyC production was associated with COVID-specific mortality (p=0.0007). Our findings suggest that CyC may be useful for stratification of patients and it has functional role in the host response to COVID-19.

Cardiopulmonary Exercise Testing (CPET) provides a comprehensive assessment of pulmonary, cardiovascular and musculosceletal function. Reduced CPET performance could be an indicator for chronic morbidity after COVID-19.Patients ≥18 years with confirmed PCR positive SARS-CoV-2 infection were offered to participate in a prospective observational study of clinical course and outcomes of COVID-19. 54 patients completed CPET, questionnaires on respiratory quality of life and performed pulmonary function tests 12 months after SARS-CoV-2 infection.At 12 months after SARS-CoV-2 infection, 46.3% of participants had a peak performance and 33.3% a peak oxygen uptake of80% of the predicted values, respectively. Further impairments were observed in diffusion capacity and ventilatory efficiency. Functional limitations were particularly pronounced in patients after invasive mechanical ventilation and extracorporeal membrane oxygenation treatment. Ventilatory capacity was reduced80% of predicted values in 55.6% of participants, independent from initial clinical severity. Patient reported dyspnea and respiratory quality of life after COVID-19 correlated with CPET performance and parameters of gas exchange. Risk factors for reduced CPET performance 12 months after COVID-19 were prior intensive care treatment (OR 5.58, p = 0.004), SGRQ outcome25 points (OR 3.48, p = 0.03) and reduced DFunctional limitations causing chronic morbidity in COVID-19 survivors persist over 12 months after SARS-CoV-2 infection. These limitations were particularly seen in parameters of overall performance and gas exchange resulting from muscular deconditioning and lung parenchymal changes. Patient reported reduced respiratory quality of life was a risk factor for adverse CPET performance.

COVID-19 is highly variable in its clinical presentation, ranging from asymptomatic infection to severe organ damage and death. We characterized the time-dependent progression of the disease in 139 COVID-19 inpatients by measuring 86 accredited diagnostic parameters, such as blood cell counts and enzyme activities, as well as untargeted plasma proteomes at 687 sampling points. We report an initial spike in a systemic inflammatory response, which is gradually alleviated and followed by a protein signature indicative of tissue repair, metabolic reconstitution, and immunomodulation. We identify prognostic marker signatures for devising risk-adapted treatment strategies and use machine learning to classify therapeutic needs. We show that the machine learning models based on the proteome are transferable to an independent cohort. Our study presents a map linking routinely used clinical diagnostic parameters to plasma proteomes and their dynamics in an infectious disease., Graphical abstract, Demichev, Tober-Lau et al., present a time-resolved molecular map of the COVID-19, measuring plasma proteomes of patients with COVID-19 along with an extensive panel of clinical diagnostic parameters at 687-time points. They describe the specificity and dynamics, as well as the predictive and prognostic power of the molecular signatures in COVID-19.

Global healthcare systems are challenged by the COVID-19 pandemic. There is a need to optimize allocation of treatment and resources in intensive care, as clinically established risk assessments such as SOFA and APACHE II scores show only limited performance for predicting the survival of severely ill COVID-19 patients. Comprehensively capturing the host physiology, we speculated that proteomics in combination with new data-driven analysis strategies could produce a new generation of prognostic discriminators. We studied two independent cohorts of patients with severe COVID-19 who required intensive care and invasive mechanical ventilation. SOFA score, Charlson comorbidity index and APACHE II score were poor predictors of survival. Plasma proteomics instead identified 14 proteins that showed concentration trajectories different between survivors and non-survivors. A proteomic predictor trained on single samples obtained at the first time point at maximum treatment level (i.e. WHO grade 7) and weeks before the outcome, achieved accurate classification of survivors in an exploratory (AUROC 0.81) as well as in the independent validation cohort (AUROC of 1.0). The majority of proteins with high relevance in the prediction model belong to the coagulation system and complement cascade. Our study demonstrates that predictors derived from plasma protein levels have the potential to substantially outperform current prognostic markers in intensive care.Trial registrationGerman Clinical Trials Register DRKS00021688

SummarySevere COVID-19 is linked to both dysfunctional immune response and unrestrained immunopathogenesis, and it remains unclear if T cells also contribute to disease pathology. Here, we combined single-cell transcriptomics and proteomics with mechanistic studies to assess pathogenic T cell functions and inducing signals. We identified highly activated, CD16+ T cells with increased cytotoxic functions in severe COVID-19. CD16 expression enabled immune complex-mediated, T cell receptor-independent degranulation and cytotoxicity not found in other diseases. CD16+ T cells from COVID-19 patients promoted microvascular endothelial cell injury and release of neutrophil and monocyte chemoattractants. CD16+ T cell clones persisted beyond acute disease maintaining their cytotoxic phenotype. Age-dependent generation of C3a in severe COVID-19 induced activated CD16+ cytotoxic T cells. The proportion of activated CD16+ T cells and plasma levels of complement proteins upstream of C3a correlated with clinical outcome of COVID-19, supporting a pathological role of exacerbated cytotoxicity and complement activation in COVID-19.

Objectiveto assess reactogenicity and immunogenicity of heterologous prime-boost immunisations of ChAdOx1-nCoV19 (Vaxzevria, ChAdOx) followed by BNT162b2 (Comirnaty, BNT) compared to homologous BNT/BNT immunisation.Designprospective, observational cohort study.Settingunicenter study in a cohort of health care workers at a tertiary care center in Berlin, Germany.Participants340 health care workers immunised between 27 December 2020 and 21 May 2021 at Charité - Universitätsmedizin Berlin, GermanyMain outcome measuresthe main outcomes were reactogenicity assessed on days one, three, five and seven post prime and boost vaccination, and immunogenicity measured by serum SARS-CoV-2 full spike-, spike S1-, and spike RBD-IgG, virus neutralisation capacity, anti-S1-IgG avidity, and T cell reactivity measured by Interferon gamma release assay at 3-4 weeks post prime and boost immunisation.ResultsHeterologous ChAdOx/BNT booster vaccination was overall well-tolerated and reactogenicity was largely comparable to homologous BNT/BNT vaccination. Systemic reactions were most frequent after prime immunisation with ChAdOx (86%, 95CI: 79-91), and less frequent after homologous BNT/BNT (65%, 95CI: 56-72), or heterologous ChAdOx/BNT booster vaccination (48%, 95CI: 36-59). Serum antibody responses and T cell reactivity were strongly increased after both homologous and heterologous boost, and immunogenicity was overall robust, and comparable between both regimens in this cohort, with slightly increased S1-IgG avidity and T cell responses following heterologous booster immunisation.ConclusionsEvidence of rare thrombotic events associated with ChAdOx has led to recommendation of a heterologous booster with mRNA vaccines for certain age groups in several European countries, despite a lack of robust safety and immunogenicity data for this vaccine regimen. This interim analysis provides evidence that the currently recommended heterologous ChAdOx/BNT immunisation regimen with 10-12 week vaccine intervals is well tolerated and slightly more immunogenic compared to homologous BNT/BNT vaccination with three week vaccine intervals. Heterologous prime-boost immunisation for COVID-19 may be generally applicable to optimise logistics and improve immunogenicity and to mitigate potential intermittent supply shortages for individual vaccines.

ObjectivePatients with kidney failure have notoriously weak responses to common vaccines. Thus, immunogenicity of novel SARS-CoV-2 vaccines might be impaired in this group. To determine immunogenicity of SARS-CoV-2 vaccination in patients with chronic dialysis, we analyzed the humoral and T-cell response after two doses of mRNA vaccine Tozinameran (BNT162b2 BioNTech/Pfizer).Design, Settings, and ParticipantsThis observational study included 43 patients on dialysis before vaccination with two doses of Tozinameran 21 days apart. Overall, 36 patients completed the observation period. Serum samples were analyzed by SARS-CoV-2 specific antibodies ∼1 and ∼3-4 weeks after the second vaccination. In addition, SARS-CoV-2-specific T-cell responses were assessed at the later time point by an interferon-gamma release assay (IGRA). Outcomes at later timepoints were compared to a group of 44 elderly patients with no dialysis after immunization with Tozinameran.ExposuresBlood drawings during regular laboratory routine assessment right before start of dialysis therapy or at the time of vaccination and at follow-up study visits.Main Outcomes and MeasuresAssessment of immunogenicity after vaccination against SARS-CoV-2 in patients on and without dialysis.ResultsMedian age of patients on chronic dialysis was 74.0 years (IQR 66.0, 82.0). The proportion of males was higher (69.4%) than females. Only 20/36 patients (55.6%, 95%CI: 38.29-71.67) developed SARS-CoV-2-IgG antibodies at first sampling, whereas 32/36 patients (88.9%, 95%CI:73.00-96.38) demonstrated seropositivity at the second sampling. Seroconversion rates and antibody titers were significantly lower compared to a cohort of vaccinees with similar age but no chronic dialysis (>90% seropositivity). SARS-CoV-2-specific T-cell responses 3 weeks after second vaccination were detected in 21/31 vaccinated dialysis patients (67.7%, 95%CI: 48.53-82.68) compared to 42/44 (93.3%, 95%CI: 76.49-98.84) in controls of similar age.Conclusion and RelevancePatients on dialysis demonstrate a delayed, but robust immune response three weeks after the second dose, which indicates effective vaccination of this vulnerable group. However, the lower immunogenicity of Tozinameran in these patients needs further attention to develop potential countermeasures such as an additional booster vaccination.

Severe COVID-19 is linked to both dysfunctional immune response and unrestrained immunopathogenesis, and it remains unclear if T cells contribute to disease pathology. Here, we combined single-cell transcriptomics and proteomics with mechanistic studies assessing pathogenic T cell functions and inducing signals. We identified activated, CD16+ T cells with increased cytotoxic functions in severe COVID-19. CD16 expression enabled immune complex-mediated, T cell receptor-independent degranulation and cytotoxicity not found in other diseases. CD16+ T cells from COVID-19 patients promoted microvascular endothelial cell injury and release of neutrophil and monocyte chemoattractants. CD16+ T cell clones persisted beyond acute disease maintaining their cytotoxic phenotype. Age-dependent generation of C3a in severe COVID-19 induced activated CD16 + cytotoxic T cells. The proportion of activated CD16+ T cells and plasma levels of complement proteins upstream of C3a correlated with clinical outcome, supporting a pathological role of exacerbated cytotoxicity and complement activation in COVID-19. Funding: This work was supported by the German Research Foundation (DFG): SA1383/3-1 to B.S.; SFB-TR84 114933180 to L.E.S., S.B., P.G., S.H. and W.M.K. INST 37/1049-1, INST 216/981- 1, INST 257/605-1, INST 269/768-1, INST 217/988-1, INST 217/577-1, and EXC2151- 390873048 to J.L.S.; GRK 2168 – 272482170, ERA CVD (00160389 to J.L.S.; SFB 1454 – 432325352 to A.C.A. and J.L.S.; SFB TR57 and SPP1937 to J.N.; GRK2157 to A.-E.S.; and ME 3644/5-1 to H.E.M.; RTG2424 to N.B.; SFB-TRR219 322900939, BO3755/13-1 Project- ID 454024652 to P.B.; the Berlin University Alliance (BUA) (PreEP-Corona grant to L.E.S. and V.M.C.); the Berlin Institute of Health (BIH) (to L.E.S., V.M.C.,B.S. and W.M.K.); Helmholtz- Gemeinschaft Deutscher Forschungszentren, Germany (sparse2big to J.L.S.), EU projects SYSCID (733100 to J.L.S.); European Research Council Horizon 2020 (grant agreement No 101001791 to P.B.); the DZIF, Germany (TTU 04.816 and 04.817 to J.N.); the Hector Foundation (M89 to J.N.); the EU projects ONE STUDY (260687), BIO-DrIM (305147) and INsTRuCT (860003) to B.S.); German Registry of COVID-19 Autopsies through Federal Ministry of Health (ZMVI1-2520COR201 to P.B.); Federal Ministry of Education and Research (DEFEAT PANDEMICs, 01KX2021 and STOP-FSGS-01GM1901A to P.B.); the Berlin Senate to German Rheumatism Research Centre (DRFZ); the Berlin Brandenburg School for regenerative Therapies (BSRT) to C.B.; the German Federal Ministry of Education and Research (BMBF) projects RECAST (01KI20337) to B.S., V.M.C., L.E.S and M.R.; VARIPath (01KI2021) to V.M.C.; NUM COVIM (01KX2021) to L.E.S., V.M.C., F.K., J.L.S., J.N. and B.S.; RAPID to and S.H.,; SYMPATH to N.S. and W.M.K.; PROVID to S.H. and W.M.K.; ZissTrans (02NUK047E) to N.B; National Research Node ‘Mass spectrometry in Systems Medicine (MSCoresys) (031L0220A) to M.R. and N.B.; Diet–Body–Brain (DietBB) (01EA1809A) to J.L.S.; the UKRI/NIHR through the UK Coronavirus Immunology Consortium (UK-CIC), the Francis Crick Institute through the Cancer Research UK (FC001134), the UK Medical Research Council (FC001134), the Wellcome Trust (FC001134 and IA 200829/Z/16/Z) to M.R.; a Charite 3R project (to B.S., S.H., W.M.K.); and an intramural grant from the Department of Genomics & Immunoregulation at the LIMES Institute to A.C.A. We are grateful to the patients and donors volunteering to participate in this study making this research possible in the first place and wish for a speedy and full recovery. Conflict of Interest: V.M.C. is named together with Euroimmun GmbH on a patent application filed recently regarding SARS-CoV-2 diagnostics via antibody testing. A.R.S. and H.E.M. are listed as inventors on a patent application by the DRFZ Berlin in the field of mass cytometry. Ethical Approval: The study was approved by the Institutional Review board of Charite (EA2/066/20).

A serum proteome panel, which includes several type-3 cystatins and inter-α-trypsin inhibitors, predicts mortality in severe COVID-19 patients very well, in three independent cohorts., Here, we recorded serum proteome profiles of 33 severe COVID-19 patients admitted to respiratory and intensive care units because of respiratory failure. We received, for most patients, blood samples just after admission and at two more later time points. With the aim to predict treatment outcome, we focused on serum proteins different in abundance between the group of survivors and non-survivors. We observed that a small panel of about a dozen proteins were significantly different in abundance between these two groups. The four structurally and functionally related type-3 cystatins AHSG, FETUB, histidine-rich glycoprotein, and KNG1 were all more abundant in the survivors. The family of inter-α-trypsin inhibitors, ITIH1, ITIH2, ITIH3, and ITIH4, were all found to be differentially abundant in between survivors and non-survivors, whereby ITIH1 and ITIH2 were more abundant in the survivor group and ITIH3 and ITIH4 more abundant in the non-survivors. ITIH1/ITIH2 and ITIH3/ITIH4 also showed opposite trends in protein abundance during disease progression. We defined an optimal panel of nine proteins for mortality risk assessment. The prediction power of this mortality risk panel was evaluated against two recent COVID-19 serum proteomics studies on independent cohorts measured in other laboratories in different countries and observed to perform very well in predicting mortality also in these cohorts. This panel may not be unique for COVID-19 as some of the proteins in the panel have previously been annotated as mortality markers in aging and in other diseases caused by different pathogens, including bacteria.

BackgroundAdequate patient allocation is pivotal for optimal resource management in strained healthcare systems, and requires detailed knowledge of clinical and virological disease trajectories.MethodsA cohort of 168 hospitalized adult COVID-19 patients enrolled in a prospective observational study at a large European tertiary care center was analyzed.ResultsForty-four percent (71/161) of patients required invasive mechanical ventilation (IMV). Shorter duration of symptoms before admission (aOR 1.22 per day less, 95%CI 1.10-1.37, pConclusionOur results indicate a short duration of symptoms before admission as a risk factor for severe disease and different viral load kinetics in severely affected patients.

COVID-19 is highly variable in its clinical presentation, ranging from asymptomatic infection to severe organ damage and death. There is an urgent need for predictive markers that can guide clinical decision-making, inform about the effect of experimental therapies, and point to novel therapeutic targets. Here, we characterize the time-dependent progression of COVID-19 through different stages of the disease, by measuring 86 accredited diagnostic parameters and plasma proteomes at 687 sampling points, in a cohort of 139 patients during hospitalization. We report that the time-resolved patient molecular phenotypes reflect an initial spike in the systemic inflammatory response, which is gradually alleviated and followed by a protein signature indicative of tissue repair, metabolic reconstitution and immunomodulation. Further, we show that the early host response is predictive for the disease trajectory and gives rise to proteomic and diagnostic marker signatures that classify the need for supplemental oxygen therapy and mechanical ventilation, and that predict the time to recovery of mildly ill patients. In severely ill patients, the molecular phenotype of the early host response predicts survival, in two independent cohorts and weeks before outcome. We also identify age-specific molecular response to COVID-19, which involves increased inflammation and lipoprotein dysregulation in older patients. Our study provides a deep and time resolved molecular characterization of COVID-19 disease progression, and reports biomarkers for risk-adapted treatment strategies and molecular disease monitoring. Our study demonstrates accurate prognosis of COVID-19 outcome from proteomic signatures recorded weeks earlier.

Objectives Dexamethasone has become the standard of care for severe coronavirus disease 2019 (COVID-19), but its virological impact is poorly understood. The objectives of this work were to characterize the kinetics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) concentration in the upper respiratory tract (URT) and the antibody response in patients with (D+) and without (D–) dexamethasone treatment. Methods Data and biosamples from hospitalized patients with severe COVID-19, enrolled between 4th March and 11th December 2020 in a prospective observational study, were analysed. SARS-CoV-2 virus concentration in serial URT samples was measured using RT-PCR. SARS-CoV-2-specific immunoglobulins A and G (IgA and IgG) were measured in serum samples using S1-ELISA. Results We compared 101 immunocompetent patients who received dexamethasone (according to the inclusion criteria and dosage determined in the RECOVERY trial) to 93 immunocompetent patients with comparable disease severity from the first months of the pandemic, who had not been treated with dexamethasone or other glucocorticoids. We found no inter-group differences in virus concentration kinetics, duration of presence of viral loads >106 viral copies/mL (D+ median 17 days (IQR 13–24), D– 19 days (IQR 13–29)), or time from symptom onset until seroconversion (IgA: D+ median 11.5 days (IQR 11–12), D– 14 days (IQR 11.5–15.75); IgG: D+ 13 days (IQR 12–14.5), D– 12 days (IQR 11–15)). Conclusion Dexamethasone does not appear to lead to a change in virus clearance or a delay in antibody response in immunocompetent patients hospitalized with severe COVID-19.