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Wastewater based epidemiology has become a widely used tool for monitoring trends of concentrations of different pathogens, most notably and widespread of SARS-CoV-2. Therefore, in 2022, also in Rhineland-Palatinate, the Ministry of Science and Health has included 16 wastewater treatment sites in a surveillance program providing biweekly samples. However, the mere viral load data is subject to strong fluctuations and has limited value for political deciders on its own. Therefore, the state of Rhineland-Palatinate has commissioned the University Medical Center at Johannes Gutenberg University Mainz to conduct a representative cohort study called SentiSurv, in which an increasing number of up to 12,000 participants have been using sensitive antigen self-tests once or twice a week to test themselves for SARS-CoV-2 and report their status. This puts the state of Rhineland-Palatinate in the fortunate position of having time series of both, the viral load in wastewater and the prevalence of SARS-CoV-2 in the population. Our main contribution is a calibration study based on the data from 2023-01-08 until 2023-10-01 where we identified a scaling factor ( 0.208 ± 0.031 ) and a delay ( 5.07 ± 2.30 days) between the virus load in wastewater, normalized by the pepper mild mottle virus (PMMoV), and the prevalence recorded in the SentiSurv study. The relation is established by fitting an epidemiological model to both time series. We show how that can be used to estimate the prevalence when the cohort data is no longer available and how to use it as a forecasting instrument several weeks ahead of time. We show that the calibration and forecasting quality and the resulting factors depend strongly on how wastewater samples are normalized., (© 2024. The Author(s).)

In Rhineland-Palatinate, Germany, a system of three data sources has been established to track the Covid-19 pandemic. These sources are the number of Covid-19-related hospitalizations, the Covid-19 genecopies in wastewater, and the prevalence derived from a cohort study. This paper presents an extensive comparison of these parameters. It is investigated whether wastewater data and a cohort study can be valid surrogate parameters for the number of hospitalizations and thus serve as predictors for coming Covid-19 waves. We observe that this is possible in general for the cohort study prevalence, while the wastewater data suffer from a too large variability to make quantitative predictions by a purely data-driven approach. However, the wastewater data and the cohort study prevalence are able to detect hospitalizations waves in a qualitative manner. Furthermore, a detailed comparison of different normalization techniques of wastewater data is provided., (© 2024. The Author(s).)

Long COVID (LC) occurs after at least 10% of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, yet its etiology remains poorly understood. We used 'omic" assays and serology to deeply characterize the global and SARS-CoV-2-specific immunity in the blood of individuals with clear LC and non-LC clinical trajectories, 8 months postinfection. We found that LC individuals exhibited systemic inflammation and immune dysregulation. This was evidenced by global differences in T cell subset distribution implying ongoing immune responses, as well as by sex-specific perturbations in cytolytic subsets. LC individuals displayed increased frequencies of CD4 + T cells poised to migrate to inflamed tissues and exhausted SARS-CoV-2-specific CD8 + T cells, higher levels of SARS-CoV-2 antibodies and a mis-coordination between their SARS-CoV-2-specific T and B cell responses. Our analysis suggested an improper crosstalk between the cellular and humoral adaptive immunity in LC, which can lead to immune dysregulation, inflammation and clinical symptoms associated with this debilitating condition., (© 2024. The Author(s).)

A multitude of alterations in the old immune system impair its functional integrity. Closely related, older individuals show, for example, a reduced responsiveness to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccines. However, systematic strategies to specifically improve the efficacy of vaccines in the old are missing or limited to simple approaches like increasing the antigen concentration or injection frequencies. We here asked whether the intrinsic, trimeric structure of the SARS-CoV-2 spike (S) antigen and/or a DNA- or protein-based antigen delivery platform affects priming of functional antibody responses particularly in old mice. The used S-antigens were primarily defined by the presence/absence of the membrane-anchoring TM domain and the closely interlinked formation/non-formation of a trimeric structure of the receptor binding domain (S-RBD). Among others, we generated vectors expressing prefusion-stabilized, cell-associated (TM + ) trimeric "S2-P" or secreted (TM - ) monomeric "S6-P ΔTM " antigens. These proteins were produced from vector-transfected HEK-293T cells under mild conditions by Strep-tag purification, revealing that cell-associated but not secreted S proteins tightly bound Hsp73 and Grp78 chaperones. We showed that both, TM-deficient S6-P ΔTM and full-length S2-P antigens elicited very similar S-RBD-specific antibody titers and pseudovirus neutralization activities in young (2-3 months) mice through homologous DNA-prime/DNA-boost or protein-prime/protein-boost vaccination. The trimeric S2-P antigen induced high S-RBD-specific antibody responses in old (23-24 months) mice through DNA-prime/DNA-boost vaccination. Unexpectedly, the monomeric S6-P ΔTM antigen induced very low S-RBD-specific antibody titers in old mice through homologous DNA-prime/DNA-boost or protein-prime/protein-boost vaccination. However, old mice efficiently elicited an S-RBD-specific antibody response after heterologous DNA-prime/protein-boost immunization with the S6-P ΔTM antigen, and antibody titers even reached similar levels and neutralizing activities as in young mice and also cross-reacted with different S-variants of concern. The old immune system thus distinguished between trimeric and monomeric S protein conformations: it remained antigen responsive to the trimeric S2-P antigen, and a simple change in the vaccine delivery regimen was sufficient to unleash its reactivity to the monomeric S6-P ΔTM antigen. This clearly shows that both the antigen structure and the delivery platform are crucial to efficiently prime humoral immune responses in old mice and might be relevant for designing "age-adapted" vaccine strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pflumm, Seidel, Klein, Groß, Krutzke, Kochanek, Kroschel, Münch, Stifter and Schirmbeck.)

SARS-CoV-2 triggered the most severe pandemic of recent times. To enter into a host cell, SARS-CoV-2 binds to the angiotensin-converting enzyme 2 (ACE2). However, subsequent studies indicated that other cell membrane receptors may act as virus-binding partners. Among these receptors, the epidermal growth factor receptor (EGFR) was hypothesized not only as a spike protein binder, but also to be activated in response to SARS-CoV-2. In our study, we aim at dissecting EGFR activation and its major downstream signaling pathway, the mitogen-activated signaling pathway (MAPK), in SARS-CoV-2 infection. Here, we demonstrate the activation of EGFR-MAPK signaling axis by the SARS-CoV-2 spike protein and we identify a yet unknown cross talk between ACE2 and EGFR that regulated ACE2 abundance and EGFR activation and subcellular localization, respectively. By inhibiting the EGFR-MAPK activation, we observe a reduced infection with either spike-pseudotyped particles or authentic SARS-CoV-2, thus indicating that EGFR serves as a cofactor and the activation of EGFR-MAPK contributes to SARS-CoV-2 infection., (© 2023 Engler et al.)

Antimicrobial peptides (AMPs) are major components of the innate immune defense. Accumulating evidence suggests that the antibacterial activity of many AMPs is dependent on the formation of amyloid-like fibrils. To identify novel fibril forming AMPs, we generated a spleen-derived peptide library and screened it for the presence of amyloidogenic peptides. This approach led to the identification of a C-terminal 32-mer fragment of alpha-hemoglobin, termed HBA(111-142). The non-fibrillar peptide has membranolytic activity against various bacterial species, while the HBA(111-142) fibrils aggregated bacteria to promote their phagocytotic clearance. Further, HBA(111-142) fibrils selectively inhibited measles and herpes viruses (HSV-1, HSV-2, HCMV), but not SARS-CoV-2, ZIKV and IAV. HBA(111-142) is released from its precursor by ubiquitous aspartic proteases under acidic conditions characteristic at sites of infection and inflammation. Thus, HBA(111-142) is an amyloidogenic AMP that may specifically be generated from a highly abundant precursor during bacterial or viral infection and may play an important role in innate antimicrobial immune responses., (© 2023. The Author(s).)

Background: Recent data on immune evasion of new SARS-CoV-2 variants raise concerns about the efficacy of antibody-based COVID-19 therapies. Therefore, in this study the in-vitro neutralization capacity against SARS-CoV-2 variant B.1 and the Omicron subvariants BA.1, BA.2 and BA.5 of sera from convalescent individuals with and without boost by vaccination was assessed., Methods and Findings: The study included 313 serum samples from 155 individuals with a history of SARS-CoV-2 infection, divided into subgroups without (n=25) and with SARS-CoV-2 vaccination (n=130). We measured anti-SARS-CoV-2 antibody concentrations by serological assays (anti-SARS-CoV-2-QuantiVac-ELISA (IgG) and Elecsys Anti-SARS-CoV-2 S) and neutralizing titers against B.1, BA.1, BA.2 and BA.5 in a pseudovirus neutralization assay. Sera of the majority of unvaccinated convalescents did not effectively neutralize Omicron sublineages BA.1, BA.2 and BA.5 (51.7%, 24.1% and 51.7%, resp.). In contrast, 99.3% of the sera of superimmunized individuals (vaccinated convalescents) neutralized the Omicron subvariants BA.1 and BA.5 and 99.6% neutralized BA.2. Neutralizing titers against B.1, BA.1, BA.2 and BA.5 were significantly higher in vaccinated compared to unvaccinated convalescents (p<0.0001) with 52.7-, 210.7-, 141.3- and 105.4-fold higher geometric mean of 50% neutralizing titers (NT50) in vaccinated compared to unvaccinated convalescents. 91.4% of the superimmunized individuals showed neutralization of BA.1, 97.2% of BA.2 and 91.5% of BA.5 with a titer ≥ 640. The increase in neutralizing titers was already achieved by one vaccination dose. Neutralizing titers were highest in the first 3 months after the last immunization event. Concentrations of anti-S antibodies in the anti-SARS-CoV-2-QuantiVac-ELISA (IgG) and Elecsys Anti-SARS-CoV-2 S assays predicted neutralization capacity against B.1 and Omicron subvariants BA.1, BA.2 and BA.5., Conclusions: These findings confirm substantial immune evasion of the Omicron sublineages, which can be overcome by vaccination of convalescents. This informs strategies for choosing of plasma donors in COVID-19 convalescent plasma programs that shall select specifically vaccinated convalescents with very high titers of anti-S antibodies., Competing Interests: SH, BJ, SK, CL, CV, RL, RM, PW, MS, HK, and HS are employees of the German Red Cross Blood Transfusion Service Baden-Württemberg-Hessen collecting plasma including convalescent plasma. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Seidel, Hoffmann, Jahrsdörfer, Körper, Ludwig, Vieweg, Albers, von Maltitz, Müller, Lotfi, Wuchter, Klüter, Kirchhoff, Schmidt, Münch and Schrezenmeier.)

Lysine-selective molecular tweezers (MTs) are supramolecular host molecules displaying a remarkably broad spectrum of biologic activities. MTs act as inhibitors of the self-assembly and toxicity of amyloidogenic proteins using a unique mechanism. They destroy viral membranes and inhibit infection by enveloped viruses, such as HIV-1 and SARS-CoV-2, by mechanisms unrelated to their action on protein self-assembly. They also disrupt biofilm of Gram-positive bacteria. The efficacy and safety of MTs have been demonstrated in vitro, in cell culture, and in vivo, suggesting that these versatile compounds are attractive therapeutic candidates for various diseases, infections, and injuries. A lead compound called CLR01 has been shown to inhibit the aggregation of various amyloidogenic proteins, facilitate their clearance in vivo, prevent infection by multiple viruses, display potent anti-biofilm activity, and have a high safety margin in animal models. The inhibitory effect of CLR01 against amyloidogenic proteins is highly specific to abnormal self-assembly of amyloidogenic proteins with no disruption of normal mammalian biologic processes at the doses needed for inhibition. Therapeutic effects of CLR01 have been demonstrated in animal models of proteinopathies, lysosomal-storage diseases, and spinal-cord injury. Here we review the activity and mechanisms of action of these intriguing compounds and discuss future research directions. SIGNIFICANCE STATEMENT: Molecular tweezers are supramolecular host molecules with broad biological applications, including inhibition of abnormal protein aggregation, facilitation of lysosomal clearance of toxic aggregates, disruption of viral membranes, and interference of biofilm formation by Gram-positive bacteria. This review discusses the molecular and cellular mechanisms of action of the molecular tweezers, including the discovery of distinct mechanisms acting in vitro and in vivo, and the application of these compounds in multiple preclinical disease models., (Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics.)

Host cell proteases such as TMPRSS2 are critical determinants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) tropism and pathogenesis. Here, we show that antithrombin (AT), an endogenous serine protease inhibitor regulating coagulation, is a broad-spectrum inhibitor of coronavirus infection. Molecular docking and enzyme activity assays demonstrate that AT binds and inhibits TMPRSS2, a serine protease that primes the Spike proteins of coronaviruses for subsequent fusion. Consequently, AT blocks entry driven by the Spikes of SARS-CoV, MERS-CoV, hCoV-229E, SARS-CoV-2 and its variants of concern including Omicron, and suppresses lung cell infection with genuine SARS-CoV-2. Thus, AT is an endogenous inhibitor of SARS-CoV-2 that may be involved in COVID-19 pathogenesis. We further demonstrate that activation of AT by anticoagulants, such as heparin or fondaparinux, increases the anti-TMPRSS2 and anti-SARS-CoV-2 activity of AT, suggesting that repurposing of native and activated AT for COVID-19 treatment should be explored., (© 2022 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

Certain viral pathogens can be shed into the human breast milk and cause infections in the infant upon breastfeeding. Thus, it is important to clarify whether viral RNA as well as infectious virus can be found in breast milk. The complexity of this body fluid poses several challenges for viral RNA isolation and detection of infectious virus. We here provide a protocol that allowed the identification of SARS-CoV-2 RNA in breast milk and the isolation of infectious virus after the virus has been artificially spiked into milk samples., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The COVID-19 course and immunity differ in children and adults. We analyzed immune response dynamics in 28 families up to 12 months after mild or asymptomatic infection. Unlike adults, the initial response is plasmablast-driven in children. Four months after infection, children show an enhanced specific antibody response and lower but detectable spike 1 protein (S1)-specific B and T cell responses than their parents. While specific antibodies decline, neutralizing antibody activity and breadth increase in both groups. The frequencies of S1-specific B and T cell responses remain stable. However, in children, one year after infection, an increase in the S1-specific IgA class switch and the expression of CD27 on S1-specific B cells and T cell maturation are observed. These results, together with the enhanced neutralizing potential and breadth of the specific antibodies, suggest a progressive maturation of the S1-specific immune response. Hence, the immune response in children persists over 12 months but dynamically changes in quality, with progressive neutralizing, breadth, and memory maturation. This implies a benefit for booster vaccination in children to consolidate memory formation., (© 2022. The Author(s).)

The current dogma of RNA-mediated innate immunity is that sensing of immunostimulatory RNA ligands is sufficient for the activation of intracellular sensors and induction of interferon (IFN) responses. Here, we report that actin cytoskeleton disturbance primes RIG-I-like receptor (RLR) activation. Actin cytoskeleton rearrangement induced by virus infection or commonly used reagents to intracellularly deliver RNA triggers the relocalization of PPP1R12C, a regulatory subunit of the protein phosphatase-1 (PP1), from filamentous actin to cytoplasmic RLRs. This allows dephosphorylation-mediated RLR priming and, together with the RNA agonist, induces effective RLR downstream signaling. Genetic ablation of PPP1R12C impairs antiviral responses and enhances susceptibility to infection with several RNA viruses including SARS-CoV-2, influenza virus, picornavirus, and vesicular stomatitis virus. Our work identifies actin cytoskeleton disturbance as a priming signal for RLR-mediated innate immunity, which may open avenues for antiviral or adjuvant design., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Most of the millions of people that are vaccinated against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), have previously been infected by related circulating human coronaviruses (hCoVs) causing common colds and will experience further encounters with these viruses in the future. Whether COVID-19 vaccinations impact neutralization of seasonal coronaviruses is largely unknown., Methods: We analyzed the capacity of sera derived from 24 individuals before and after heterologous ChAdOx1 nCoV-19 BNT162b2 prime-boost vaccination to neutralize genuine OC43, NL63, and 229E hCoVs, as well as viral pseudoparticles carrying the SARS-CoV-1, SARS-CoV-2, Middle East Respiratory Syndrome (MERS)-CoV, and hCoV-OC43, hCoV-NL63, and hCoV-229E spike proteins. Genuine hCoVs or spike containing pseudovirions were incubated with different concentrations of sera and neutralization efficiencies were determined by measuring viral RNA yields, intracellular viral nucleocapsid expression, or reporter gene expression in Huh-7 cells., Results: All individuals showed strong preexisting immunity against hCoV-OC43. Neutralization of hCoV-NL63 was more variable and all sera showed only modest inhibitory activity against genuine hCoV-229E. SARS-CoV-2 vaccination resulted in efficient cross-neutralization of SARS-CoV-1 but not of MERS-CoV. On average, vaccination significantly increased the neutralizing activity against genuine hCoV-OC43, hCoV-NL63, and hCoV-229E., Conclusions: Heterologous COVID-19 vaccination may confer some cross-protection against endemic seasonal coronaviruses., (© The Author(s) 2022. Published by Oxford University Press for the Infectious Diseases Society of America.)

In light of the decreasing immune protection against symptomatic SARS-CoV-2 infection after initial vaccinations and the now dominant immune-evasive Omicron variants, 'booster' vaccinations are regularly performed to restore immune responses. Many individuals have received a primary heterologous prime-boost vaccination with long intervals between vaccinations, but the resulting long-term immunity and the effects of a subsequent 'booster', particularly against Omicron BA.1, have not been defined. We followed a cohort of 23 young adults, who received a primary heterologous ChAdOx1 nCoV-19 BNT162b2 prime-boost vaccination, over a 7-month period and analysed how they responded to a BNT162b2 'booster'. We show that already after the primary heterologous vaccination, neutralization titers against Omicron BA.1 are recognizable but that humoral and cellular immunity wanes over the course of half a year. Residual responsive memory T cells recognized spike epitopes of the early SARS-CoV-2 B.1 strain as well as the Delta and BA.1 variants of concern (VOCs). However, the remaining antibody titers hardly neutralized these VOCs. The 'booster' vaccination was well tolerated and elicited both high antibody titers and increased memory T cell responses against SARS-CoV-2 including BA.1. Strikingly, in this young heterologously vaccinated cohort the neutralizing activity after the 'booster' was almost as potent against BA.1 as against the early B.1 strain. Our results suggest that a 'booster' after heterologous vaccination results in effective immune maturation and potent protection against the Omicron BA.1 variant in young adults., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Seidel, Zanoni, Groß, Krnavek, Erdemci-Evin, von Maltitz, Albers, Conzelmann, Liu, Weil, Mayer, Hoffmann, Pöhlmann, Beil, Kroschel, Kirchhoff, Münch and Müller.)

Besides pandemic SARS-CoV-2, also endemic seasonal human common cold coronaviruses (hCoVs) have a significant impact on human health and economy. Studies on hCoVs and the identification of antivirals are therefore crucial to improve human well-being. However, hCoVs have long been neglected and the methodology to study virus infection, replication and inhibition warrants being updated. We here evaluated the established plaque-based assays to determine viral titers and cell-to-cell spread and developed protocols for the immunodetection of the viral nucleocapsid protein by flow cytometry and in-cell ELISA to study infection rates at early time points. The developed protocols allow detection of hCoV-229E infection after 2, and hCoV-NL63 and -OC43 infection after 3 days at a single cell level or in a 96 well microtiter format, in large sample numbers without being laborious or expensive. Both assays can be applied to assess the susceptibility of cells to hCoV infection and replication, and to determine the efficacy of antiviral compounds as well as neutralizing antibodies in a sensitive and quantitative manner. Application revealed that clinically applied SARS-CoV-2 targeting monoclonal antibodies are inactive against hCoVs, but that the viral polymerase targeting antivirals remdesivir and molnupiravir are broadly active also against all three hCoVs. Further, the in-cell ELISA provided evidence that nirmatrelvir, previously shown to broadly inhibit coronavirus proteases, also prevents replication of authentic hCoVs. Importantly, the protocols described here can be easily adapted to other coronavirus strains and species as well as viruses of other families within a short time. This will facilitate future research on known and emerging (corona)viruses, support the identification of antivirals and increase the preparedness for future virus outbreaks., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Screening of a protein kinase inhibitor library identified SB431542, targeting activin receptor-like kinase 5 (ALK5), as a compound interfering with SARS-CoV-2 replication. Since ALK5 is implicated in transforming growth factor β (TGF-β) signaling and regulation of the cellular endoprotease furin, we pursued this research to clarify the role of this protein kinase for SARS-CoV-2 infection. We show that TGF-β1 induces the expression of furin in a broad spectrum of cells including Huh-7 and Calu-3 that are permissive for SARS-CoV-2. The inhibition of ALK5 by incubation with SB431542 revealed a dose-dependent downregulation of both basal and TGF-β1 induced furin expression. Furthermore, we demonstrate that the ALK5 inhibitors SB431542 and Vactosertib negatively affect the proteolytic processing of the SARS-CoV-2 Spike protein and significantly reduce spike-mediated cell-cell fusion. This correlated with an inhibitory effect of ALK5 inhibition on the production of infectious SARS-CoV-2. Altogether, our study shows that interference with ALK5 signaling attenuates SARS-CoV-2 infectivity and cell-cell spread via downregulation of furin which is most pronounced upon TGF-β stimulation. Since a TGF-β dominated cytokine storm is a hallmark of severe COVID-19, ALK5 inhibitors undergoing clinical trials might represent a potential therapy option for COVID-19.

Aims: Long-term sequelae may occur after SARS-CoV-2 infection. We comprehensively assessed organ-specific functions in individuals after mild to moderate SARS-CoV-2 infection compared with controls from the general population., Methods and Results: Four hundred and forty-three mainly non-hospitalized individuals were examined in median 9.6 months after the first positive SARS-CoV-2 test and matched for age, sex, and education with 1328 controls from a population-based German cohort. We assessed pulmonary, cardiac, vascular, renal, and neurological status, as well as patient-related outcomes. Bodyplethysmography documented mildly lower total lung volume (regression coefficient -3.24, adjusted P = 0.014) and higher specific airway resistance (regression coefficient 8.11, adjusted P = 0.001) after SARS-CoV-2 infection. Cardiac assessment revealed slightly lower measures of left (regression coefficient for left ventricular ejection fraction on transthoracic echocardiography -0.93, adjusted P = 0.015) and right ventricular function and higher concentrations of cardiac biomarkers (factor 1.14 for high-sensitivity troponin, 1.41 for N-terminal pro-B-type natriuretic peptide, adjusted P ≤ 0.01) in post-SARS-CoV-2 patients compared with matched controls, but no significant differences in cardiac magnetic resonance imaging findings. Sonographically non-compressible femoral veins, suggesting deep vein thrombosis, were substantially more frequent after SARS-CoV-2 infection (odds ratio 2.68, adjusted P < 0.001). Glomerular filtration rate (regression coefficient -2.35, adjusted P = 0.019) was lower in post-SARS-CoV-2 cases. Relative brain volume, prevalence of cerebral microbleeds, and infarct residuals were similar, while the mean cortical thickness was higher in post-SARS-CoV-2 cases. Cognitive function was not impaired. Similarly, patient-related outcomes did not differ., Conclusion: Subjects who apparently recovered from mild to moderate SARS-CoV-2 infection show signs of subclinical multi-organ affection related to pulmonary, cardiac, thrombotic, and renal function without signs of structural brain damage, neurocognitive, or quality-of-life impairment. Respective screening may guide further patient management., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Cardiology.)

Since diagnostic sampling material must be considered as infectious, we evaluated whether extraction buffers of SARS-CoV-2 rapid antigen test kits may inactivate SARS-CoV-2. Of concern, seven of nine tested buffers lacked potent virucidal activity. To reduce risk of infection during assay performance, virucidal antigen extraction buffers that efficiently inactivate virus should replace the extraction buffers in these commercially available point-of-care devices., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

The quality and persistence of children's humoral immune response following SARS-CoV-2 infection remains largely unknown but will be crucial to guide pediatric SARS-CoV-2 vaccination programs. Here, we examine 548 children and 717 adults within 328 households with at least one member with a previous laboratory-confirmed SARS-CoV-2 infection. We assess serological response at 3-4 months and 11-12 months after infection using a bead-based multiplex immunoassay for 23 human coronavirus antigens including SARS-CoV-2 and its Variants of Concern (VOC) and endemic human coronaviruses (HCoVs), and additionally by three commercial SARS-CoV-2 antibody assays. Neutralization against wild type SARS-CoV-2 and the Delta VOC are analysed in a pseudotyped virus assay. Children, compared to adults, are five times more likely to be asymptomatic, and have higher specific antibody levels which persist longer (96.2% versus 82.9% still seropositive 11-12 months post infection). Of note, symptomatic and asymptomatic infections induce similar humoral responses in all age groups. SARS-CoV-2 infection occurs independent of HCoV serostatus. Neutralization responses of children and adults are similar, although neutralization is reduced for both against the Delta VOC. Overall, the long-term humoral immune response to SARS-CoV-2 infection in children is of longer duration than in adults even after asymptomatic infection., (© 2022. The Author(s).)

The COVID-19 pandemic caused by SARS-CoV-2 has made the development of safe and effective vaccines a critical priority. To date, four vaccines have been approved by European and American authorities for preventing COVID-19, but the development of additional vaccine platforms with improved supply and logistics profiles remains a pressing need. Here we report the preclinical evaluation of a novel COVID-19 vaccine candidate based on the electroporation of engineered, synthetic cDNA encoding a viral antigen in the skeletal muscle. We constructed a set of prototype DNA vaccines expressing various forms of the SARS-CoV-2 spike (S) protein and assessed their immunogenicity in animal models. Among them, COVID-eVax-a DNA plasmid encoding a secreted monomeric form of SARS-CoV-2 S protein receptor-binding domain (RBD)-induced the most potent anti-SARS-CoV-2 neutralizing antibody responses (including against the current most common variants of concern) and a robust T cell response. Upon challenge with SARS-CoV-2, immunized K18-hACE2 transgenic mice showed reduced weight loss, improved pulmonary function, and lower viral replication in the lungs and brain. COVID-eVax conferred significant protection to ferrets upon SARS-CoV-2 challenge. In summary, this study identifies COVID-eVax as an ideal COVID-19 vaccine candidate suitable for clinical development. Accordingly, a combined phase I-II trial has recently started., Competing Interests: Declaration of interests A.C. and M.M. are Evvivax employees. E.M., F.P., G.R., A.M., L.L., L.L., E. Salvatori, M. Cappalletti, F.F.F., E.D., V.C., and L.A. are Takis employees. G. Caselli and L.R. are Rottapharm Biotech employees. Takis and Rottapharm Biotech are jointly developing COVID-eVax. M.I. participates in advisory boards/consultancies for or receives funding from Gilead Sciences, Roche, Third Rock Ventures, Amgen, Allovir, Asher Bio. L.G.G is a member of the board of directors at Genenta Science and Epsilon Bio and participates in advisory boards/consultancies for Gilead Sciences, Roche, and Arbutus Biopharma., (Copyright © 2021 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Heterologous COVID-19 vaccination regimens combining vector- and mRNA-based vaccines are already administered, but data on solicited adverse reactions, immunological responses and elicited protection are limited., Methods: To evaluate the reactogenicity and humoral as well as cellular immune responses towards most prevalent SARS-CoV-2 variants after a heterologous ChAdOx1 nCoV-19 BNT162b2 prime-boost vaccination, we analysed a cohort of 26 clinic employees aged 25-46 (median 30.5) years who received a ChAdOx1 nCoV-19 prime followed by a BNT162b2 boost after an 8-week interval. Serological data were compared to a cohort which received homologous BNT162b2 vaccination with a 3-week interval (14 individuals aged 25-65, median 42)., Findings: Self-reported solicited symptoms after ChAdOx1 nCoV-19 prime were in line with previous reports and more severe than after the BNT162b2 boost. Antibody titres increased significantly over time resulting in strong neutralization titres two weeks after the BNT162b2 boost and subsequently slightly decreased over the course of 17 weeks. At the latest time point measured, all analysed sera retained neutralizing activity against the currently dominant Delta (B.1.617.2) variant. Two weeks post boost, neutralizing activity against the Alpha (B.1.1.7) and immune-evading Beta (B.1.351) variant was ∼4-fold higher than in individuals receiving homologous BNT162b2 vaccination. No difference was observed in neutralization of Kappa (B.1.617.1). In addition, heterologous vaccination induced CD4 + and CD8 + T cells reactive to SARS-CoV-2 spike peptides of all analysed variants; Wuhan-Hu-1, Alpha, Beta, Gamma (P.1), and Delta., Interpretation: In conclusion, heterologous ChAdOx1 nCoV-19 / BNT162b2 prime-boost vaccination is not associated with serious adverse events and induces potent humoral and cellular immune responses. The Alpha, Beta, Delta, and Kappa variants of spike are potently neutralized by sera from all participants and reactive T cells recognize spike peptides of all tested variants. These results suggest that this heterologous vaccination regimen is at least as immunogenic and protective as homologous vaccinations and also offers protection against current variants of concern., Funding: This project has received funding from the European Union's Horizon 2020 research and innovation programme, the German Research Foundation, the BMBF, the Robert Koch Institute (RKI), the Baden-Württemberg Stiftung, the county of Lower Saxony, the Ministry for Science, Research and the Arts of Baden-Württemberg, Germany, and the National Institutes of Health., Competing Interests: Declaration of interest The authors have nothing to disclose., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

The COVID-19 pandemic continues to spread around the world and remains a major public health threat. Vaccine inefficiency, vaccination breakthroughs and lack of supply, especially in developing countries, as well as the fact that a non-negligible part of the population either refuse vaccination or cannot be vaccinated due to age, pre-existing illness or non-response to existing vaccines intensify this issue. This might also contribute to the emergence of new variants, being more efficiently transmitted, more virulent and more capable of escaping naturally acquired and vaccine-induced immunity. Hence, the need of effective and viable prevention options to reduce viral transmission is of outmost importance. In this study, we investigated the antiviral effect of iota-, lambda- and kappa-carrageenan, sulfated polysaccharides extracted from red seaweed, on SARS-CoV-2 Wuhan type and the spreading variants of concern (VOCs) Alpha, Beta, Gamma and Delta. Carrageenans as part of broadly used nasal and mouth sprays as well as lozenges have the potential of first line defense to inhibit the infection and transmission of SARS-CoV-2. Here, we demonstrate by using a SARS-CoV-2 spike pseudotyped lentivirus particles (SSPL) system and patient-isolated SARS-CoV-2 VOCs to infect transgenic A549ACE2/TMPRSS2 and Calu-3 human lung cells that all three carrageenan types exert antiviral activity. Iota-carrageenan exhibits antiviral activity with comparable IC 50 values against the SARS-CoV-2 Wuhan type and the VOCs. Altogether, these results indicate that iota-carrageenan might be effective for prophylaxis and treatment of SARS-CoV-2 infections independent of the present and potentially future variants.

SARS-CoV-2 is a novel human pathogenic coronavirus whose predilection for the respiratory tract has given rise to a rapid pandemic spread via airborne particles. Organ-specific susceptibility is substantially determined by the density of cell surface expression of ACE2, which is exploited by viral spike protein as a receptor molecule to mediate adhesion and, thus, to permit internalization of the viral genome into the host cell. Based on an ample data set derived from clinical studies and case reports, evidence suggests that distinct cell populations of the digestive and olfactory-gustatory system are equally equipped with membrane-bound ACE2, rendering them "vulnerable" to SARS-CoV-2. Numerous reports on concomitant gastrointestinal complaints and laboratory abnormalities are thought to reflect a relevant degree of organ dysfunction and underscore the tropism of SARS-CoV-2 for the digestive tract. Organoids are three-dimensional in vitro replicas of organ tissue which, owing to their organotypic complex cellular composition and functional resemblance to primary cells, are particularly appreciated for basic research in the field of infectious diseases. This review specifically addresses the involvement of digestive organs by SARS-CoV-2 and outlines the significant contribution of organoid- and primary-cell culture-based models to gaining a deeper understanding of the underlying pathophysiological processes., Competing Interests: Die Autorinnen/Autoren geben an, dass kein Interessenkonflikt besteht., (Thieme. All rights reserved.)

The COVID-19 pandemic has resulted in significant morbidity and mortality worldwide. To prevent severe infection, mass COVID-19 vaccination campaigns with several vaccine types are currently underway. We report pathological and immunological findings in 8 patients who developed vaccine-induced immune thrombotic thrombocytopenia (VITT) after administration of SARS-CoV-2 vaccine ChAdOx1 nCoV-19. We analyzed patient material using enzyme immune assays, flow cytometry and heparin-induced platelet aggregation assay and performed autopsies on two fatal cases. Eight patients (5 female, 3 male) with a median age of 41.5 years (range, 24 to 53) were referred to us with suspected thrombotic complications 6 to 20 days after ChAdOx1 nCoV-19 vaccination. All patients had thrombocytopenia at admission. Patients had a median platelet count of 46.5 x109/L (range, 8 to 92). Three had a fatal outcome and 5 were successfully treated. Autopsies showed arterial and venous thromboses in various organs and the occlusion of glomerular capillaries by hyaline thrombi. Sera from VITT patients contain high titer antibodies against platelet factor 4 (PF4) (OD 2.59±0.64). PF4 antibodies in VITT patients induced significant increase in procoagulant markers (P-selectin and phosphatidylserine externalization) compared to healthy volunteers and healthy vaccinated volunteers. The generation of procoagulant platelets was PF4 and heparin dependent. We demonstrate the contribution of antibody-mediated platelet activation in the pathogenesis of VITT.

The current SARS-CoV-2 pandemic has triggered the development of various SARS-CoV-2 neutralization tests. A wild-type virus (using African green monkey VeroE6 cells), a pseudovirus (using human Caco-2 cells), and a surrogate neutralization test platform were applied to characterize the SARS-CoV-2 neutralization potential of a cohort of 111 convalescent plasma donors over a period of seven months after diagnosis. This allowed an in-depth validation and assay performance analysis of these platforms. More importantly, we found that SARS-CoV-2 neutralization titers were stable or even increased within the observation period, which contradicts earlier studies reporting a rapid waning of Ab titers after three to four months. Moreover, we observed a positive correlation of neutralization titers with increasing age, number of symptoms reported, and the presence of the Rhesus Ag RhD. Validation of the platforms revealed that highest assay performances were obtained with the wild-type virus and the surrogate neutralization platforms. However, our data also suggested that selection of cutoff titers had a strong impact on the evaluation of neutralization potency. When taking strong neutralization potency, as demonstrated by the wild-type virus platform as the gold standard, up to 55% of plasma products had low neutralization titers. However, a significant portion of these products were overrated in their potency when using the surrogate assay with the recommended cutoff titer. In summary, our study demonstrates that SARS-CoV-2 neutralization titers are stable for at least seven months after diagnosis and offers a testing strategy for rapid selection of high-titer convalescent plasma products in a biosafety level 1 environment., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evades most innate immune responses but may still be vulnerable to some. Here, we systematically analyze the impact of SARS-CoV-2 proteins on interferon (IFN) responses and autophagy. We show that SARS-CoV-2 proteins synergize to counteract anti-viral immune responses. For example, Nsp14 targets the type I IFN receptor for lysosomal degradation, ORF3a prevents fusion of autophagosomes and lysosomes, and ORF7a interferes with autophagosome acidification. Most activities are evolutionarily conserved. However, SARS-CoV-2 Nsp15 antagonizes IFN signaling less efficiently than the orthologs of closely related RaTG13-CoV and SARS-CoV-1. Overall, SARS-CoV-2 proteins counteract autophagy and type I IFN more efficiently than type II or III IFN signaling, and infection experiments confirm potent inhibition by IFN-γ and -λ1. Our results define the repertoire and selected mechanisms of SARS-CoV-2 innate immune antagonists but also reveal vulnerability to type II and III IFN that may help to develop safe and effective anti-viral approaches., Competing Interests: Declaration of interests F.I.S. is a co-founder of DiosCURE Therapeutics SE and a consultant to IFM Therapeutics., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Pharmaceutical interventions are urgently needed to prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and transmission. As SARS-CoV-2 infects and spreads via the nasopharyngeal airways, we analyzed the antiviral effect of selected nasal and oral sprays on virus infection in vitro. Two nose sprays showed virucidal activity but were cytotoxic precluding further analysis in cell culture. One nasal and one mouth spray suppressed SARS-CoV-2 infection of TMPRSS2-expressing Vero E6 cells and primary differentiated human airway epithelial cultures. The antiviral activity in both sprays could be attributed to polyanionic ι- and κ-carrageenans. Thus, application of carrageenan-containing nasal and mouth sprays may reduce the risk of acquiring SARS-CoV-2 infection and may limit viral spread, warranting further clinical evaluation.

Background and Aims: The COVID-19 pandemic has spread worldwide and poses a severe health risk. While most patients present mild symptoms, descending pneumonia can lead to severe respiratory insufficiency. Up to 50% of patients show gastrointestinal symptoms like diarrhea or nausea, intriguingly associating with prolonged symptoms and increased severity. Thus, models to understand and validate drug efficiency in the gut of COVID-19 patients are of urgent need., Methods: Human intestinal organoids derived from pluripotent stem cells (PSC-HIOs) have led, due to their complexity in mimicking human intestinal architecture, to an unprecedented number of successful disease models including gastrointestinal infections. Here, we employed PSC-HIOs to dissect SARS-CoV-2 pathogenesis and its inhibition by remdesivir, one of the leading drugs investigated for treatment of COVID-19., Results: Immunostaining for viral entry receptor ACE2 and SARS-CoV-2 spike protein priming protease TMPRSS2 showed broad expression in the gastrointestinal tract with highest levels in the intestine, the latter faithfully recapitulated by PSC-HIOs. Organoids could be readily infected with SARS-CoV-2 followed by viral spread across entire PSC-HIOs, subsequently leading to organoid deterioration. However, SARS-CoV-2 spared goblet cells lacking ACE2 expression. Importantly, we challenged PSC-HIOs for drug testing capacity. Specifically, remdesivir effectively inhibited SARS-CoV-2 infection dose-dependently at low micromolar concentration and rescued PSC-HIO morphology., Conclusions: Thus, PSC-HIOs are a valuable tool to study SARS-CoV-2 infection and to identify and validate drugs especially with potential action in the gut., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Competing Interests: POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose.

To date, no effective vaccines or therapies are available against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative pandemic agent of the coronavirus disease 2019 (COVID-19). Due to their safety, efficacy and specificity, peptide inhibitors hold great promise for the treatment of newly emerging viral pathogens. Based on the known structures of viral proteins and their cellular targets, antiviral peptides can be rationally designed and optimized. The resulting peptides may be highly specific for their respective targets and particular viral pathogens or exert broad antiviral activity. Here, we summarize the current status of peptides inhibiting SARS-CoV-2 entry and outline the strategies used to design peptides targeting the ACE2 receptor or the viral spike protein and its activating proteases furin, transmembrane serine protease 2 (TMPRSS2), or cathepsin L. In addition, we present approaches used against related viruses such as SARS-CoV-1 that might be implemented for inhibition of SARS-CoV-2 infection., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)