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The cellular nonsense-mediated decay (NMD) pathway recognizes and degrades mRNAs with unusual structural features, such as long 3' UTRs or overlapping reading frames, and therefore serves as a transcript quality control mechanism. A broad spectrum of today's knowledge about the nonsense-mediated mRNA decay pathway has been discovered using NMD reporter systems, mostly consisting of multiple exons, with a wild-type and a premature termination codon-containing variant. In a preliminary NMD study, we used the seven-exon triose phosphate isomerase (TPI) reporter and observed that in this well-known NMD reporter, surprisingly, not all splice sites are used constitutively, but additional cryptic splice sites are used. As this is more frequently observed in the construction of minigenes, especially when unknown splicing regulatory elements (SREs) are removed, for example, by shortening introns, this may affect the reliability of such reporters. To demonstrate how such minigenes can be improved in general with respect to constitutive splice site recognition, we restored an intron length in the TPI reporter or made bioinformatic adjustments to SREs or intrinsic strength of the splice sites themselves. As a result, this NMD reporter could be made more robust and specific for the evaluation of NMD sensitivity within a single transcript. The modifications of the TPI reporter shown here as examples can generally be used for the transfer of cellular multiexon transcripts to minigenes., (© 2025 Peter et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

We present a novel small molecule antiviral chemotype that was identified by an unconventional cell-free protein synthesis and assembly-based phenotypic screen for modulation of viral capsid assembly. Activity of PAV-431, a representative compound from the series, has been validated against infectious viruses in multiple cell culture models for all six families of viruses causing most respiratory diseases in humans. In animals, this chemotype has been demonstrated efficacious for porcine epidemic diarrhoea virus (a coronavirus) and respiratory syncytial virus (a paramyxovirus). PAV-431 is shown to bind to the protein 14-3-3, a known allosteric modulator. However, it only appears to target the small subset of 14-3-3 which is present in a dynamic multi-protein complex whose components include proteins implicated in viral life cycles and in innate immunity. The composition of this target multi-protein complex appears to be modified upon viral infection and largely restored by PAV-431 treatment. An advanced analog, PAV-104, is shown to be selective for the virally modified target, thereby avoiding host toxicity. Our findings suggest a new paradigm for understanding, and drugging, the host-virus interface, which leads to a new clinical therapeutic strategy for treatment of respiratory viral disease.

The measles, mumps, and rubella (MMR) vaccine protects against all-cause mortality in children, but the immunological mechanisms mediating these effects are poorly known. We systematically investigated whether MMR can induce long-term functional changes in innate immune cells, a process termed trained immunity, that could at least partially mediate this heterologous protection. In a randomized, placebo-controlled trial, 39 healthy adults received either the MMR vaccine or a placebo. Using single-cell RNA-Seq, we found that MMR caused transcriptomic changes in CD14+ monocytes and NK cells, but most profoundly in γδ T cells. Monocyte function was not altered by MMR vaccination. In contrast, the function of γδ T cells was markedly enhanced by MMR vaccination, with higher production of TNF and IFN-γ, as well as upregulation of cellular metabolic pathways. In conclusion, we describe a trained immunity program characterized by modulation of γδ T cell function induced by MMR vaccination.

Rbm3 (RNA-binding motif protein 3) is a stress responsive gene, which maintains cellular homeostasis and promotes survival upon various harmful cellular stimuli. Rbm3 protein shows conserved structural and molecular similarities to heterogeneous nuclear ribonucleoproteins (hnRNPs), which regulate all steps of the mRNA metabolism. Growing evidence is pointing towards a broader role of Rbm3 in various steps of gene expression. Here, we demonstrate that Rbm3 deficiency is linked to transcriptome-wide pre-mRNA splicing alterations, which can be reversed through Rbm3 co-expression from a cDNA. Using an MS2 tethering assay, we show that Rbm3 regulates splice site selection similar to other hnRNP proteins when recruited between two competing 5   ' splice sites. Furthermore, we show that the N-terminal part of Rbm3 encompassing the RNA recognition motif (RRM), is sufficient to elicit changes in splice site selection. On the basis of these findings, we propose a novel, undescribed function of Rbm3 in RNA splicing that contributes to the preservation of transcriptome integrity.

The seat of higher-order cognitive abilities in mammals, the neocortex, is a complex structure, organized in several layers. The different subtypes of principal neurons are distributed in precise ratios and at specific positions in these layers and are generated by the same neural progenitor cells (NPCs), steered by a spatially and temporally specified combination of molecular cues that are incompletely understood. Recently, we discovered that an alternatively spliced isoform of the TrkC receptor lacking the kinase domain, TrkC-T1, is a determinant of the corticofugal projection neuron (CFuPN) fate. Here, we show that the finely tuned balance between TrkC-T1 and the better known, kinase domain-containing isoform, TrkC-TK+, is cell type-specific in the developing cortex and established through the antagonistic actions of two RNA-binding proteins, Srsf1 and Elavl1. Moreover, our data show that Srsf1 promotes the CFuPN fate and Elavl1 promotes the callosal projection neuron (CPN) fate in vivo via regulating the distinct ratios of TrkC-T1 to TrkC-TK+. Taken together, we connect spatio-temporal expression of Srsf1 and Elavl1 in the developing neocortex with the regulation of TrkC alternative splicing and transcript stability and neuronal fate choice, thus adding to the mechanistic and functional understanding of alternative splicing in vivo., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The mRNA-based BNT162b2 protects against severe disease and mortality caused by SARS-CoV-2 via induction of specific antibody and T-cell responses. Much less is known about its broad effects on immune responses against other pathogens. Here, we investigated the adaptive immune responses induced by BNT162b2 vaccination against various SARS-CoV-2 variants and its effects on the responsiveness of immune cells upon stimulation with heterologous stimuli. BNT162b2 vaccination induced effective humoral and cellular immunity against SARS-CoV-2 that started to wane after six months. We also observed long-term transcriptional changes in immune cells after vaccination. Additionally, vaccination with BNT162b2 modulated innate immune responses as measured by inflammatory cytokine production after stimulation - higher IL-1/IL-6 release and decreased IFN-α production. Altogether, these data expand our knowledge regarding the overall immunological effects of this new class of vaccines and underline the need for additional studies to elucidate their effects on both innate and adaptive immune responses., Competing Interests: Declaration of Competing Interest M.G.N and L.A.B.J are scientific founders of TTxD and Lemba., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Clonostachys rosea is a fungus widely distributed on Earth and has a high capacity to adapt to complex environments in soil, plants, or sea. It is an endophyte that can be used as a potential biocontrol agent to protect plants from pathogenic fungi, nematodes, and insects. However, the spectrum of secondary metabolites produced by C. rosea has only scarcely been studied. In the present study, eight new phenalenones, asperphenalenones F-M ( 1 - 8 ), together with two known derivatives, asperphenalenones E and B ( 9 and 10 ), were isolated from the axenic rice culture of this fungus. The structures of the new compounds were elucidated by nuclear magnetic resonance, high-resolution electrospray ionization mass spectrometry, electronic circular dichroism, and gas chromatography-mass spectrometry analyses. Asperphenalenones J-M ( 5 - 8 ) are unusual phenalenone adducts that are conjugated to diterpenoid glycosides. Asperphenalenones F and H showed moderate antibacterial activity against methicillin-resistant Staphylococcus aureus , with minimal inhibitory concentrations of 12.5 and 25 μM, respectively. Asperphenalenone B exhibited low antiviral activity against the human immunodeficiency virus replication. Furthermore, asperphenalenones F and H exhibited low cytotoxicity against Jurkat cells, while all other compounds were devoid of cytotoxicity.

WT1 is a true chameleon, both acting as an oncogene and tumor suppressor. As its exact role in leukemogenesis is still ambiguous, research with model systems representing natural conditions surrounding the genetic alterations in WT1 is necessary. In a cohort of 59 leukemia/lymphoma cell lines, we showed aberrant expression for WT1 mRNA, which does not always translate into protein levels. We also analyzed the expression pattern of the four major WT1 protein isoforms in the cell lines and primary AML blasts with/without WT1 mutations and demonstrated that the presence of mutations does not influence these patterns. By introduction of key intronic and exonic sequences of WT1 into a lentiviral expression vector, we developed a unique tool that can stably overexpress the four WT1 isoforms at their naturally occurring tissue-dependent ratio. To develop better cellular model systems for WT1, we sequenced large parts of its gene locus and also other important myeloid risk factor genes and revealed previously unknown alterations. Functionally, inhibition of the nonsense-mediated mRNA decay machinery revealed that under natural conditions, the mutated WT1 alleles go through a robust degradation. These results offer new insights and model systems regarding the characteristics of WT1 in leukemia and lymphoma.

Biallelic germline mutations in BRCA2 occur in the Fanconi anemia (FA)-D1 subtype of the rare pediatric disorder, FA, characterized clinically by severe congenital abnormalities and a very high propensity to develop malignancies early in life. Clinical and genetic data from 96 FA-D1 patients with biallelic BRCA2 mutations were collected and used to develop a new cancer risk prediction score system based on the specific mutations in BRCA2. This score takes into account the location of frameshift/stop and missense mutations relative to exon 11 of BRCA2, which encodes the major sites for interaction with the RAD51 recombinase, and uses the MaxEnt and HBond splicing scores to analyze potential splice site perturbations. Among 75 FA-D1 patients with ascertained BRCA2 mutations, 66 patients developed 102 malignancies, ranging from one to three independent tumors per individual. The median age at the clinical presentation of peripheral embryonal tumors was 1.0, at the onset of hematologic malignancies 1.8 and at the manifestation of CNS tumors 2.7 years, respectively. Patients who received treatment lived longer than those without. Using our novel scoring system, we could distinguish three distinct cancer risk groups among FA-D1 patients: in the first, patients developed their initial malignancy at a median age of 1.3 years (n = 36, 95% CI = 0.9-1.8), in the second group at 2.3 years (n = 17, 95% CI = 1.4-4.4) and in the third group at 23.0 years (n = 22, 95% CI = 4.3-n/a). Therefore, this scoring system allows, for the first time, to predict the cancer manifestation of FA-D1 patients simply based on the type and position of the mutations in BRCA2., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The COVID-19 pandemic has greatly impacted the global economy and health care systems, illustrating the urgent need for timely and inexpensive responses to pandemic threats in the form of vaccines and antigen tests. Currently, antigen testing is mostly conducted by qualitative flow chromatography or via quantitative ELISA-type assays. The latter mostly utilize materials like protein-adhesive polymers and gold or latex particles. Here we present an alternative ELISA approach using inexpensive, biogenic materials and permitting quick detection based on components produced in the microbial model Ustilago maydis. In this fungus, heterologous proteins like biopharmaceuticals can be exported by fusion to unconventionally secreted chitinase Cts1. As a unique feature, the carrier chitinase binds to chitin allowing its additional use as a purification or immobilization tag. Recent work has demonstrated that nanobodies are suitable target proteins. These proteins represent a very versatile alternative antibody format and can quickly be adapted to detect novel antigens by camelidae immunization or synthetic libraries. In this study, we exemplarily produced different mono- and bivalent SARS-CoV-2 nanobodies directed against the spike protein receptor binding domain (RBD) as Cts1 fusions and screened their antigen binding affinity in vitro and in vivo. Functional nanobody-Cts1 fusions were immobilized on chitin forming an RBD tethering surface. This provides a solid base for future development of inexpensive antigen tests utilizing unconventionally secreted nanobodies as antigen trap and a matching ubiquitous and biogenic surface for immobilization., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The spliceosome, responsible for all mature protein-coding transcripts of eukaryotic intron-containing genes, consists of small uridine-rich nuclear ribonucleoproteins (UsnRNPs). The assembly of UsnRNPs depends, on one hand, on the arginine methylation of Sm proteins catalyzed by the PRMT5 complex. On the other hand, it depends on the phosphorylation of the PRMT5 subunit pICln by the Uncoordinated Like Kinase 1 (ULK1). In consequence, phosphorylation of pICln affects the stability of the UsnRNP assembly intermediate, the so-called 6 S complex. The detailed mechanisms of phosphorylation-dependent integrity and subsequent UsnRNP assembly of the 6 S complex in vivo have not yet been analyzed. By using a phospho-specific antibody against ULK1-dependent phosphorylation sites of pICln, we visualize the intracellular distribution of phosphorylated pICln. Furthermore, we detect the colocaliphosphor-pICln1 with phospho-pICln by size-exclusion chromatography and immunofluorescence techniques. We also show that phosphorylated pICln is predominantly present in the 6 S complex. The addition of ULK1 to in vitro produced 6 S complex, as well as the reconstitution of ULK1 in ULK1-deficient cells, increases the efficiency of snRNP biogenesis. Accordingly, inhibition of ULK1 and the associated decreased pICln phosphorylation lead to accumulation of the 6 S complex and reduction in the spliceosomal activity of the cell., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.)

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). In less than three years, an estimated 600 million infections with SARS-CoV-2 occurred worldwide, resulting in a pandemic with tremendous impact especially on economic and health sectors. Initially considered a respiratory disease, COVID-19, along with its long-term sequelae (long-COVID) rather is a systemic disease. Neurological symptoms like dementia or encephalopathy were reported early during the pandemic as concomitants of the acute phase and as characteristics of long-COVID. An excessive inflammatory immune response is hypothesized to play a major role in this context. However, direct infection of neural cells may also contribute to the neurological aspects of (long)-COVID-19. To mainly explore such direct effects of SARS-CoV-2 on the central nervous system, human brain organoids provide a useful platform. Infecting these three-dimensional tissue cultures allows the study of viral neurotropism as well as of virus-induced effects on single cells or even the complex cellular network within the organoid. In this review, we summarize the experimental studies that used SARS-CoV-2-infected human brain organoids to unravel the complex nature of (long)-COVID-19-related neurological manifestations., (© 2023 The Authors. Reviews in Medical Virology published by John Wiley & Sons Ltd.)

Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important prophylactic measure in kidney transplant recipients (KTRs), but the immune response is often impaired. Here, we examined the T-cell immune response against SARS-CoV-2 in 148 KTRs after 3 or 4 vaccine doses, including 35 KTRs with subsequent SARS-CoV-2 infection. The frequency of spike-specific T cells was lower in KTRs than in immunocompetent controls and was correlated with the level of spike-specific antibodies. Positive predictors for detection of vaccine-induced T cells were detection of spike-specific antibodies, heterologous immunization with messenger RNA and a vector vaccine, and longer time after transplantation. In vaccinated KTRs with subsequent SARS-CoV-2 infection, the T-cell response was greatly enhanced and was significantly higher than in vaccinated KTRs without SARS-CoV-2 infection. Overall, the data show a correlation between impaired humoral and T-cell immunity to SARS-CoV-2 vaccination and provide evidence for greater robustness of hybrid immunity in KTRs., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Gelsolin (GSN) is a structural actin-binding protein that is known to affect actin dynamics in the cell. Using mass spectrometry, we identified GSN as a novel Vpr-interacting protein. Endogenous GSN protein was expressed at detectable levels in monocyte-derived macrophages (MDM) and in THP-1 cells, but it was undetectable at the protein level in other cell lines tested. The HIV-1 infection of MDM was associated with a reduction in GSN steady-state levels, presumably due to the Vpr-induced degradation of GSN. Indeed, the coexpression of GSN and Viral protein R (Vpr) in transiently transfected HEK293T cells resulted in the Vpr-dependent proteasomal degradation of GSN. This effect was observed for Vprs from multiple virus isolates. The overexpression of GSN in HEK293T cells had no effect on Gag expression or particle release, but it reduced the expression and packaging of the HIV-1 envelope (Env) glycoprotein and reduced viral infectivity. An analysis of the HIV-1 splicing patterns did not reveal any GSN-dependent differences, suggesting that the effect of GSN on Env expression was regulated at a posttranscriptional level. Indeed, the treatment of transfected cells with lysosomal inhibitors reversed the effect of GSN on Env stability, suggesting that GSN reduced Env expression via enhanced lysosomal degradation. Our data identify GSN as a macrophage-specific host antiviral factor that reduces the expression of HIV-1 Env. IMPORTANCE Despite dramatic progress in drug therapies, HIV-1 infection remains an incurable disease that affects millions of people worldwide. The virus establishes long-lasting reservoirs that are resistant to currently available drug treatments and allow the virus to rebound whenever drug therapy is interrupted. Macrophages are long-lived cells that are relatively insensitive to HIV-1-induced cytopathicity and thus could contribute to the viral reservoir. Here, we identified a novel host factor, gelsolin, that is expressed at high levels in macrophages and inhibits viral infectivity by modulating the expression of the HIV-1 Env glycoprotein, which is critical in the spread of an HIV-1 infection. Importantly, the viral protein Vpr induces the degradation of gelsolin and thus counteracts its antiviral activity. Our study provides significant and novel insights into HIV-1 virus-host interactions and furthers our understanding of the importance of Vpr in HIV-1 infection and pathogenesis.

Modification of vaccination strategies is necessary to improve the immune response to SARS-CoV-2 vaccination in kidney transplant recipients (KTRs). This multicenter observational study analyzed the effects of the third SARS-CoV-2 vaccination in previously seronegative KTRs with the focus on temporary mycophenolate mofetil (MMF) dose reduction within propensity matched KTRs. 56 out of 174 (32%) previously seronegative KTRs became seropositive after the third vaccination with only three KTRs developing neutralizing antibodies against the omicron variant. Multivariate logistic regression revealed that initial antibody levels, graft function, time after transplantation and MMF trough levels had an influence on seroconversion (P < .05). After controlling for confounders, the effect of MMF dose reduction before the third vaccination was calculated using propensity score matching. KTRs with a dose reduction of ≥33% showed a significant decrease in MMF trough levels to 1.8 (1.2-2.5) μg/ml and were more likely to seroconvert than matched controls (P = .02). Therefore, a MMF dose reduction of 33% or more before vaccination is a promising approach to improve success of SARS-CoV-2 vaccination in KTRs., (© 2022 The Authors. Clinical Transplantation published by John Wiley & Sons Ltd.)

We provide follow-up data on the humoral immune response after COVID-19 vaccinations of two distinct cohorts aged below 60 and over 80 years to screen for age-related differences in the longevity and magnitude of the induction of the antibody responses post booster-vaccinations. While anti-SARS-CoV-2 spike-specific IgG and neutralization capacity waned rapidly after the initial vaccination schedule, additional boosters highly benefitted the humoral immune responses especially in the elderly cohort, including the neutralization of Omikron variants. Thus, adjusted COVID-19 booster vaccination schedules are an appropriate tool to overcome limitations in the success of vaccinations., 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 Müller, Andrée, Moskorz, Drexler, Hauka, Ptok, Walotka, Grothmann, Hillebrandt, Ritchie, Peter, Walker, Timm, Adams and Schaal.)

Determination of neutralizing antibody titers is still considered the gold standard for infection protection. A full virus neutralization test (VNT) with replication-competent, infectious SARS-CoV-2, is labor-intensive and requires Biosafety Level 3 certified laboratories. Therefore, several commercial SARS-CoV-2 surrogate virus neutralization tests (sVNTs) have been developed that aim to detect neutralizing antibodies targeting the receptor binding domain (RBD) of the viral spike glycoprotein (S). Neutralizing antibodies to the RBD block its interaction with the angiotensin-converting enzyme 2 (ACE2) receptor protein. Here, we compared a full virus neutralization test (VNT) with two SARS-CoV-2 surrogate virus neutralization tests (sVNT) and validated them in two cohorts of i) convalescent SARS-CoV-2-infected individuals and ii) COVID vaccinated individuals. The sVNTs showed highly different results both, compared to the VNT-titers and also between the two cohorts. This indicates that currently, sVNT provide a qualitative instead of a quantitative measurement of neutralizing antibodies. The findings in this work show that the cutoff levels for sVNTs might need to be readjusted for convalescent and vaccinated individuals., (Copyright © 2022. Published by Elsevier B.V.)

Correct pre-mRNA processing in higher eukaryotes vastly depends on splice site recognition. Beyond conserved 5'ss and 3'ss motifs, splicing regulatory elements (SREs) play a pivotal role in this recognition process. Here, we present in silico designed sequences with arbitrary a priori prescribed splicing regulatory HEXplorer properties that can be concatenated to arbitrary length without changing their regulatory properties. We experimentally validated in silico predictions in a massively parallel splicing reporter assay on more than 3000 sequences and exemplarily identified some SRE binding proteins. Aiming at a unified 'functional splice site strength' encompassing both U1 snRNA complementarity and impact from neighboring SREs, we developed a novel RNA-seq based 5'ss usage landscape, mapping the competition of pairs of high confidence 5'ss and neighboring exonic GT sites along HBond and HEXplorer score coordinate axes on human fibroblast and endothelium transcriptome datasets. These RNA-seq data served as basis for a logistic 5'ss usage prediction model, which greatly improved discrimination between strong but unused exonic GT sites and annotated highly used 5'ss. Our 5'ss usage landscape offers a unified view on 5'ss and SRE neighborhood impact on splice site recognition, and may contribute to improved mutation assessment in human genetics., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Older age is associated with increased severity and death from respiratory infections, including coronavirus disease 2019 (COVID-19). The tuberculosis BCG vaccine may provide heterologous protection against nontuberculous infections and has been proposed as a potential preventive strategy against COVID-19., Methods: In this multicenter, placebo-controlled trial, we randomly assigned older adults (aged ≥60 years; n = 2014) to intracutaneous vaccination with BCG vaccine (n = 1008) or placebo (n = 1006). The primary end point was the cumulative incidence of respiratory tract infections (RTIs) that required medical intervention, during 12 months of follow-up. Secondary end points included the incidence of COVID-19, and the effect of BCG vaccination on the cellular and humoral immune responses., Results: The cumulative incidence of RTIs requiring medical intervention was 0.029 in the BCG-vaccinated group and 0.024 in the control group (subdistribution hazard ratio, 1.26 [98.2% confidence interval, .65-2.44]). In the BCG vaccine and placebo groups, 51 and 48 individuals, respectively tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with polymerase chain reaction (subdistribution hazard ratio, 1.053 [95% confidence interval, .71-1.56]). No difference was observed in the frequency of adverse events. BCG vaccination was associated with enhanced cytokine responses after influenza, and also partially associated after SARS-CoV-2 stimulation. In patients diagnosed with COVID-19, antibody responses after infection were significantly stronger if the volunteers had previously received BCG vaccine., Conclusions: BCG vaccination had no effect on the incidence of RTIs, including SARS-CoV-2 infection, in older adult volunteers. However, it improved cytokine responses stimulated by influenza and SARS-CoV-2 and induced stronger antibody titers after COVID-19 infection., Clinical Trials Registration: EU Clinical Trials Register 2020-001591-15 ClinicalTrials.gov NCT04417335., (© The Author(s) 2022. Published by Oxford University Press for the Infectious Diseases Society of America.)

The C30 endopeptidase (3C-like protease; 3CL pro ) is essential for the life cycle of SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2) since it plays a pivotal role in viral replication and transcription and, hence, is a promising drug target. Molecules isolated from animals, insects, plants, or microorganisms can serve as a scaffold for the design of novel biopharmaceutical products. Crotamine, a small cationic peptide from the venom of the rattlesnake Crotalus durissus terrificus , has been the focus of many studies since it exhibits activities such as analgesic, in vitro antibacterial, and hemolytic activities. The crotamine derivative L-peptides (L-CDP) that inhibit the 3CL protease in the low µM range were examined since they are susceptible to proteolytic degradation; we explored the utility of their D-enantiomers form. Comparative uptake inhibition analysis showed D-CDP as a promising prototype for a D-peptide-based drug. We also found that the D-peptides can impair SARS-CoV-2 replication in vivo, probably targeting the viral protease 3CL pro .

The majority of COVID-19 patients experience mild to moderate disease course and recover within a few weeks. An increasing number of studies characterized the long-term changes in the specific anti-SARS-CoV-2 immune responses, but how COVID-19 shapes the innate and heterologous adaptive immune system after recovery is less well known. To comprehensively investigate the post-SARS-CoV-2 infection sequelae on the immune system, we performed a multi-omics study by integrating single-cell RNA-sequencing, single-cell ATAC-sequencing, genome-wide DNA methylation profiling, and functional validation experiments in 14 convalescent COVID-19 and 15 healthy individuals. We showed that immune responses generally recover without major sequelae after COVID-19. However, subtle differences persist at the transcriptomic level in monocytes, with downregulation of the interferon pathway, while DNA methylation also displays minor changes in convalescent COVID-19 individuals. However, these differences did not affect the cytokine production capacity of PBMCs upon different bacterial, viral, and fungal stimuli, although baseline release of IL-1Ra and IFN-γ was higher in convalescent individuals. In conclusion, we propose that despite minor differences in epigenetic and transcriptional programs, the immune system of convalescent COVID-19 patients largely recovers to the homeostatic level of healthy individuals., 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 Liu, Kilic, Li, Bulut, Gupta, Zhang, Qi, Peng, Tsay, Soon, Mekonnen, Ferreira, van der Made, van Cranenbroek, Koenen, Simonetti, Diavatopoulos, de Jonge, Müller, Schaal, Ostermann, Cornberg, Eiz-Vesper, van de Veerdonk, van Crevel, Joosten, Domínguez-Andrés, Xu, Netea and Li.)

We used enzyme-linked immunoassay methods to measure the prevalence and the levels of antibody responses to the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and four seasonal human coronaviruses (HCoV-OC43, HCoV-HKU1, HCoV 229E, and HCoV-NL63) in a cohort of 115 convalescent plasma donors infected with SARS-CoV-2 (1-61 days after symptom onset) compared to antibody levels in 114 individuals with no evidence of a recent infection with SARS-CoV-2. In the humoral response to the four seasonal coronaviruses, only HCoV-HKU1- and HCoV-229E-assays showed slightly elevated antibody levels in the COVID group compared to the control group. While in the COVID-group the levels of SARS-CoV-2 antibodies correlated significantly with disease severity, no association was found in the levels of antibodies against the seasonal coronaviruses. The most striking result in both groups was that the levels of antibodies against all tested coronaviruses, including the new SARS-CoV-2 showed a highly significant correlation with each other. There seems to be an individual predisposition to a weaker or stronger humoral immune response against all known seasonal human coronaviruses including the new SARS-CoV-2, which could lead to a definition of low and high responders against human coronaviruses with potential impact on the assessment of postinfection antibody levels and protection., (© 2021 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

Kidney transplant recipients (KTRs) are extremely vulnerable to SARS-CoV-2 infection and show an impaired immune response to SARS-CoV-2 vaccination. We analyzed factors related to vaccination efficiency in KTRs. In a multicenter prospective observational study (NCT04743947), IgG antibodies levels against SARS-CoV-2 spike S1 subunit and their neutralization capacity after SARS-CoV-2 vaccination were analyzed in 225 KTRs and compared to 176 controls. After the vaccination, 56 (24.9%) KTRs became seropositive of whom 68% had neutralizing antibodies. This immune response was significantly lower compared to controls (239 [78-519] BAU/ml versus 1826 [560-3180] BAU/ml for KTRs and controls, p < .0001). The strongest predictor for an impaired response was mycophenolate mofetil (MMF) treatment. Multivariate regression analysis revealed that MMF-free regimen was highly associated with seroconversion (OR 13.25, 95% CI 3.22-54.6; p < .001). In contrast, other immunosuppressive drugs had no significant influence. 187 out of 225 KTRs were treated with MMF of whom 26 (13.9%) developed antibodies. 23 of these seropositive KTRs had a daily MMF dose ≤1 g. Furthermore, higher trough MMF concentrations correlated with lower antibody titers (R -0.354, p < .001) supporting a dose-dependent unfavorable effect of MMF. Our data indicate that MMF dose modification could lead to an improved immune response., (© 2021 The Authors. American Journal of Transplantation published by Wiley Periodicals LLC on behalf of The American Society of Transplantation and the American Society of Transplant Surgeons.)

Concerns of possible transmission of SARS-CoV-2 from donors to patients by corneal transplantation have caused a decline in corneal transplantations. Graft culture media are routinely tested for infectious risks, but it is unclear whether this constitutes a viable means to avoid transmitting SARS-CoV-2 via keratoplasty. We found that SARS-CoV-2 RNA was not present in the medium after seven days of organ culture of corneas from donors (n = 4), who were SARS-CoV-2-positive upon tissue procurement. These medium samples showed no presence of viral RNA. To pursue this question under controlled conditions and further exclude the possibility of productive infection in corneal grafts, we inoculated corneoscleral discs from healthy donors (n = 8) with infectious SARS-CoV-2 and performed PCR testing of the culture medium at various time points. After seven days of culture, we also tested for SARS-CoV-2 RNA within the inoculated corneal tissue. The medium from tissue samples inoculated with SARS-CoV-2 showed no increase in viral RNA, which may indicate lack of viral replication in these corneal grafts. SARS-CoV-2-RNA was, however, detected on or in corneal tissue seven days after inoculation. Our data suggest that corneal grafts may not be permissive for replication of SARS-CoV-2 and demonstrates that PCR testing of culture media cannot safely exclude that tissue has been exposed to SARS-CoV-2. It also demonstrates the difficulty to differentiate between virus adherence and virus replication by PCR testing in SARS-CoV-2 exposed tissue.

The underlying molecular mechanism and their general effect on the replication capacity of HIV 1 drug-resistance-associated mutations is often poorly understood. To elucidate the effect of two such mutations located in a region with a high density of spicing regulatory elements on the HIV-1-splicing outcome, bioinformatic predictions were combined with transfection and infection experiments. Results show that the previously described R263K drug-resistance-associated integrase mutation has additionally a severe effect on the ESE2b splicing regulatory element (SRE) in exon 2b, which causes loss of SD2b recognition. This was confirmed by an R263R silent mutation with a similar predicted effect on the exon 2b SRE. In contrast, a V260I mutation and its silent counterpart with a lower effect on ESS2b did not exhibit any differences in the splicing pattern. Since HIV-1 highly relies on a balanced splicing reaction, changes in the splicing outcome can contribute to changes in viral replication and might add to the effect of escape mutations toward antiviral drugs. Thus, a classification of mutations purely addressing proteins is insufficient.

Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has led to the development of various vaccines. Real-life data on immune responses elicited in the most vulnerable group of vaccinees older than age 80 years old are still underrepresented despite the prioritization of the elderly in vaccination campaigns., Methods: We conducted a cohort study with 2 age groups, young vaccinees below the age of 60 years and elderly vaccinees over the age of 80 years, to compare their antibody responses to the first and second dose of the BNT162b2 coronavirus disease 2019 vaccination., Results: Although the majority of participants in both groups produced specific immunoglobulin G antibody titers against SARS-CoV-2 spike protein, titers were significantly lower in elderly participants. Although the increment of antibody levels after the second immunization was higher in elderly participants, the absolute mean titer of this group remained lower than the <60 years of age group. After the second vaccination, 31.3% of the elderly had no detectable neutralizing antibodies in contrast to the younger group, in which only 2.2% had no detectable neutralizing antibodies., Conclusions: Our data showed differences between the antibody responses raised after the first and second BNT162b2 vaccination, in particular lower frequencies of neutralizing antibodies in the elderly group. This suggests that this population needs to be closely monitored and may require earlier revaccination and/or an increased vaccine dose to ensure stronger long-lasting immunity and protection against infection., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)