Rapid adaptation of established high-throughput molecular testing infrastructure for detection of monkeypoxvirus (preprint)

Background Since May 2022, a rising number of monkeypox-cases has been reported in non-endemic countries of the northern hemisphere. In contrast to previous clusters, infections seem predominantly driven by human-to-human transmission, rather than animal sources. In this study, we adapted two published qPCR assays (non-variola orthopoxvirus and monkeypoxvirus specific) for use as a lab-developed dual-target monkeypoxvirus-test on widely used automated high-throughput PCR-systems (cobas5800/6800/8800). Methods Selected assays were checked for in-silico inclusivity and exclusivity in current orthopoxvirus sequences, as well as for multiplex compatibility. Analytic performance was determined by serial dilution of monkeypoxvirus reference material, quantified by digital PCR. Cross reactivity was ruled out through a clinical exclusivity set containing various bloodborne and respiratory pathogens. Clinical performance was compared to a commercial manual RUO-kit using clinical remnant samples. Results Analytic lower limit of detection (LoD) was determined as 4.795 dcp/ml (CI95%: 3.598 - 8.633 dcp/ml) for both assays combined, with a dynamic range of at least 5 log-steps. The assay showed 100% positive and negative agreement with the manual RUO orthopoxvirus PCR test kit in clinical swab samples. Discussion While the full extend of the ongoing monkeypox outbreak remains to be established, the WHO and local health authorities are calling for increased awareness and efforts to limit further spread. For this, timely and scalable PCR tests are an important prerequisite. The assay presented here allows streamlined high-throughput molecular testing for monkeypoxvirus on existing hardware, broadly established previously for SARS-CoV-2 diagnostics.

Increased neutralization and IgG epitope identification after MVA-MERS-S booster vaccination against Middle East respiratory syndrome (preprint)

Vaccine development is essential for pandemic preparedness. We previously conducted a Phase 1 clinical trial of the vector vaccine candidate MVA-MERS-S against the Middle East respiratory syndrome coronavirus (MERS-CoV), expressing its full spike glycoprotein (MERS-CoV-S), as a homologous two-dose regimen (Days 0 and 28). Here, we evaluate a third vaccination with MVA-MERS-S in a subgroup of trial participants one year after primary immunization. A booster vaccination with MVA-MERS-S is safe and well-tolerated. Both binding and neutralizing anti-MERS-CoV antibody titers increase substantially in all participants and exceed maximum titers observed after primary immunization more than 10-fold. We identify four immunogenic IgG epitopes, located in the receptor-binding domain (RBD, n=1) and the S2 subunit (n=3) of MERS-CoV-S. The level of baseline anti-human coronavirus antibody titers does not impact the generation of anti-MERS-CoV antibody responses. Our data support the rationale of a booster vaccination with MVA-MERS-S and encourage further investigation in larger trials. One Sentence Summary A late booster vaccination with the vector vaccine MVA-MERS-S against MERS-CoV is safe and significantly increases humoral immunogenicity including responses to four IgG epitopes.

Respiratory Muscle Dysfunction in COVID-19 Patients with Persistent Symptoms (preprint)

In a cross-sectional analysis, we have identified a high prevalence of respiratory muscle dysfunction in persistently symptomatic patients after COVID-19 (‘Long COVID’). Respiratory muscle impairment in these patients was associated with exercise-induced deoxygenation, impaired exercise tolerance, activity and functional outcomes after COVID-19.

Digital PCR to quantify ChAdOx1 nCoV-19 copies in blood and tissues (preprint)

Vaccination with the adenoviral-vector based Astra Zeneca ChAdOx1 nCov-19 vaccine is efficient and safe. However, in rare cases vaccinated individuals developed life-threatening thrombotic complications, including thrombosis in cerebral sinus and splanchnic veins. Monitoring of the applied vector in vivo represents an important precondition to study the molecular mechanisms underlying vaccine-driven adverse effects now referred to as vaccine-induced immune thrombotic thrombocytopenia (VITT). We previously have shown that digital PCR is an excellent tool to quantify transgene copies in vivo . Here we present a highly sensitive digital PCR for in-situ quantification of ChAdOx1 nCoV-19 copies. Using this method, we quantified vector copies in human serum 24, 72 and 168 hours post vaccination, and in a variety of murine tissues in an experimental vaccination model 30 minutes post injection. We describe a method for high-sensitivity quantitative detection of ChAdOx1 nCoV-19 with possible implications to elucidate the mechanisms of severe ChAdOx1 nCov-19 vaccine complications.

Validation of a Prospective Urinalysis-Based Prediction Model for the Outcome of COVID-19 Disease: A Multicenter Cohort Study (preprint)

Background: Identifying preventive strategies in Covid-19 patients will help to improve resource-allocation and reduce mortality. In this Journal, we recently demonstrated in a post-mortem cohort that SARS-CoV-2 renal tropism was associated with kidney injury, disease severity and mortality. We also proposed an algorithm to predict the risk of adverse outcomes in Covid-19 patients harnessing urinalysis and protein/coagulation parameters on admission for signs of kidney injury. Here, we aimed to validate this hypothesis in a multicenter cohort.Methods: Patients hospitalized for Covid-19 at four tertiary centers were screened for an available urinalysis, serum albumin (SA) and antithrombin-III activity (AT-III) obtained prospectively within 48h upon admission. The respective presumed risk for an unfavorable course was categorized as “low”, “intermediate” or “high”, depending on a normal urinalysis, an abnormal urinalysis with SA ≥2 g/dl and AT-III ≥70%, or an abnormal urinalysis with at least one SA or AT-III abnormality. Time to ICU or death within ten days served as primary, in-hospital mortality and required organ support served as secondary endpoints.Findings: Among a total of N=223 screened patients, N=145 were eligible for enrollment, falling into the low (N=43), intermediate (N=84), and high risk (N=18) categories. The risk for ICU transfer or death was 100% in the high risk group and significantly elevated in the composite of high and intermediate risk as compared to the low risk group (63·7% vs. 27·9%; HR 2·6; 95%-CI 1·4 to 4·9; P=0·0020). Having an abnormal urinalysis was associated with mortality, need for mechanical ventilation, extra-corporeal membrane oxygenation (ECMO) or renal replacement therapy (RRT).Interpretation: Our data confirm that Covid-19-associated urine abnormalities on admission predict disease aggravation. This supports the conceptual relevance of Covid-19-associated kidney injury. By engaging a simple urine dipstick our algorithm allows for early preventive measures and appropriate patient stratification. Trial Registration: (ClinicalTrials.gov number NCT04347824)Funding Statement: This work was supported by the DFG (GR 1852/6-1 to OG; CRC1192 to JET, EH and TBH), (HU 1016/8-2, HU 1016/11-1, HU 1016/ 12-1 to TBH) and (GR 1852/6-1 to OG); by the BMBF (STOP-FSGS-01GM1518C and NephrESA-031L0191E to TBH), by the Else-Kröner Fresenius Foundation (Else Kröner-Promotionskolleg –iPRIME to TBH), and by the H2020-IMI2 consortium BEAt-DKD (115974 to TBH). In addition, the UMG Göttingen applied for Government funding (Covid-19 program) by The German Federal Ministry of Education and Research and the application currently is under consideration.Declaration of Interests: All authors report no conflict of interest in relation to this observational cohort-study. Ethics Approval Statement: According to the German Medicines Act, the study was approved by the leading institutional review board (IRB) of the UMG Göttingen (41/4/20), and all others.

Living Repository of Millions of T and B Cell Receptor Sequences from Patients with COVID-19 (preprint)

We profiled adaptive immunity in COVID-19 patients with active infection or after recovery and created a living repository of currently >14 million B and T cell receptor (BCR/TCR) sequences from blood of these patients. The B cell response showed converging IGHV3-driven BCR clusters closely associated with SARS-CoV-2 antibodies. Clonality and skewing of TCR repertoires was associated with interferon type I and III responses, early CD4+/CD8+ activation and counterregulation by BTLA, Tim-3, PD-1, TIGIT and CD73. Tfh, Th17-like and nonconventional (but not classical anti-viral) Th1 polarizations were induced. COVID-19 specific T cell responses were driven by TCR clusters shared between patients with a characteristic trajectory of clonotypes and traceability over the disease course. Our cohort – especially the subset with effective viral clearance – reveals fundamental insight into adaptive immunity to SARS-CoV-2 with the living repository providing a bottleneck resource for the scientific community urgently needed to inform therapeutic concepts and vaccine development.Conflict of Interest: The authors declare no competing interests.Ethical Approval: Blood collection was performed under institutional review board approvals number 2020-039 and 11/17.