SARS-CoV-2 targets neurons of 3D human brain organoids.

COVID-19 pandemic caused by SARS-CoV-2 infection is a public health emergency. COVID-19 typically exhibits respiratory illness. Unexpectedly, emerging clinical reports indicate that neurological symptoms continue to rise, suggesting detrimental effects of SARS-CoV-2 on the central nervous system (CNS). Here, we show that a Düsseldorf isolate of SARS-CoV-2 enters 3D human brain organoids within 2 days of exposure. We identified that SARS-CoV-2 preferably targets neurons of brain organoids. Imaging neurons of organoids reveal that SARS-CoV-2 exposure is associated with altered distribution of Tau from axons to soma, hyperphosphorylation, and apparent neuronal death. Our studies, therefore, provide initial insights into the potential neurotoxic effect of SARS-CoV-2 and emphasize that brain organoids could model CNS pathologies of COVID-19.

Immunoadsorption as a method of antibody donation during the COVID-19 pandemic.

BACKGROUND AND OBJECTIVES: Initial therapeutic efforts to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) included the use of plasma from convalescent donors containing anti-SARS-CoV-2 antibodies. High-neutralizing antibody titres are required for therapeutic efficacy. This study aims to show that immunoadsorption followed by tangential flow filtration can be used to obtain antibody concentrates with high-neutralizing capacities. MATERIALS AND METHODS: Eligible donors (n = 10, five males and three females) underwent immunoadsorption using adsorber columns specific for human antibodies. Glycine-washed out eluates of 1.5 L volume were further concentrated by tangential flow filtration using 30 kDa ultrafiltration membranes. The same membranes were applied for diafiltrations to exchange residual glycine for 0.9% normal saline. RESULTS: Antibody concentrates were obtained within 8 h from the start of donation and had 4.58 ± 1.95, 3.28 ± 1.28 and 2.02 ± 0.92 times higher total IgG, IgA and IgM concentrations, 3.29 ± 1.62 and 3.74 ± 0.6 times higher SARS-CoV-2 N and S antibody concentrations and 3.85 ± 1.71 times higher SARS-CoV-2 S-specific IgG concentrations compared to the donors' peripheral blood. The specific SARS-CoV-2 virus neutralization capacities increased in all but one concentrate. All antibody concentrates (50-70 mL final volume) passed microbiological tests, were free of hazardous glycine levels and could be stored at -80°C and 4°C for 1 year with 20 ± 3% antibody loss. CONCLUSION: Immunoadsorption followed by tangential flow filtration is a feasible procedure to collect IgG, IgA and IgM as well as SARS-CoV-2 N- and S-specific antibody concentrates of low volume, free of albumin and coagulation factors. Whether these concentrates can be used as passive immunisation in infected patients remains to be elucidated.

Human brain organoids to explore SARS-CoV-2-induced effects on the central nervous system.

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.

The impact of BNT162b2 mRNA vaccine on adaptive and innate immune responses.

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.

Induction of trained immunity by influenza vaccination - impact on COVID-19.

Non-specific protective effects of certain vaccines have been reported, and long-term boosting of innate immunity, termed trained immunity, has been proposed as one of the mechanisms mediating these effects. Several epidemiological studies suggested cross-protection between influenza vaccination and COVID-19. In a large academic Dutch hospital, we found that SARS-CoV-2 infection was less common among employees who had received a previous influenza vaccination: relative risk reductions of 37% and 49% were observed following influenza vaccination during the first and second COVID-19 waves, respectively. The quadrivalent inactivated influenza vaccine induced a trained immunity program that boosted innate immune responses against various viral stimuli and fine-tuned the anti-SARS-CoV-2 response, which may result in better protection against COVID-19. Influenza vaccination led to transcriptional reprogramming of monocytes and reduced systemic inflammation. These epidemiological and immunological data argue for potential benefits of influenza vaccination against COVID-19, and future randomized trials are warranted to test this possibility.

Efficacy of BCG Vaccination Against Respiratory Tract Infections in Older Adults During the Coronavirus Disease 2019 Pandemic.

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.

Let It Go: HIV-1 cis-Acting Repressive Sequences.

After human immunodeficiency virus type 1 (HIV-1) was identified in the early 1980s, intensive work began to understand the molecular basis of HIV-1 gene expression. Subgenomic HIV-1 RNA regions, spread throughout the viral genome, were described to have a negative impact on the nuclear export of some viral transcripts. Those studies revealed an intrinsic RNA code as a new form of nuclear export regulation. Since such regulatory regions were later also identified in other viruses, as well as in cellular genes, it can be assumed that, during evolution, viruses took advantage of them to achieve more sophisticated replication mechanisms. Here, we review HIV-1 cis-acting repressive sequences that have been identified, and we discuss their possible underlying mechanisms and importance. Additionally, we show how current bioinformatic tools might allow more predictive approaches to identify and investigate them.

Humoral response to SARS-CoV-2 and seasonal coronaviruses in COVID-19 patients.

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.

Age-dependent Immune Response to the Biontech/Pfizer BNT162b2 Coronavirus Disease 2019 Vaccination.

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.

Sensitivity of anti-SARS-CoV-2 serological assays in a high-prevalence setting.

Evaluation and power of seroprevalence studies depend on the performed serological assays. The aim of this study was to assess four commercial serological tests from EUROIMMUN, DiaSorin, Abbott, and Roche as well as an in-house immunofluorescence and neutralization test for their capability to identify SARS-CoV-2 seropositive individuals in a high-prevalence setting. Therefore, 42 social and working contacts of a German super-spreader were tested. Consistent with a high-prevalence setting, 26 of 42 were SARS-CoV-2 seropositive by neutralization test (NT), and immunofluorescence test (IFT) confirmed 23 of these 26 positive test results (NT 61.9% and IFT 54.8% seroprevalence). Four commercial assays detected anti-SARS-CoV-2 antibodies in 33.3-40.5% individuals. Besides an overall discrepancy between the NT and the commercial assays regarding their sensitivity, this study revealed that commercial SARS-CoV-2 spike-based assays are better to predict the neutralization titer than nucleoprotein-based assays are.

COVID-19 antibody donation using immunoadsorption: Report of two cases.

For more than a year the whole world is suffering from the COVID-19 pandemic with no treatment option in sight. Administration of plasma from convalescent donors containing anti-SARS-CoV-2 antibodies, though promising according to case reports, failed to show a clear benefit in a greater number of trials. One reason could be varying and low antibody contents in a majority of plasma units hampering standardization and clinical efficacy. Besides, other plasma components unnecessarily transfused like coagulation factors might promote hypercoagulation seen in severe COVID-19 etiopathology. We therefore hypothesized that instead of collecting whole plasma units, convalescent donors could donate solely immunoglobulins by undergoing immunoadsorption, a mode of therapy regularly applied in autoimmune diseases. Here, we report the results of the first two antibody donations performed at the University Hospital Düsseldorf. In both cases, immunoadsorptions were very well tolerated with no side effects. Collected and neutralized eluates were concentrated using tangential flow filtration increasing the concentration of immunoglobulins 10fold as compared to peripheral blood and leading to probably eight times more neutralizing antibodies than in one plasma unit. Therefore, immunoadsorption can be used as a method of antibody donation. Whether these donated antibodies can be used as passive immunization in acutely infected patients remains to be elucidated.

Altered HIV-1 mRNA Splicing Due to Drug-Resistance-Associated Mutations in Exon 2/2b.

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.

Gymnotic Delivery of LNA Mixmers Targeting Viral SREs Induces HIV-1 mRNA Degradation.

Transcription of the HIV-1 provirus generates a viral pre-mRNA, which is alternatively spliced into more than 50 HIV-1 mRNAs encoding all viral proteins. Regulation of viral alternative splice site usage includes the presence of splicing regulatory elements (SREs) which can dramatically impact RNA expression and HIV-1 replication when mutated. Recently, we were able to show that two viral SREs, GI3-2 and ESEtat, are important players in the generation of viral vif, vpr and tat mRNAs. Furthermore, we demonstrated that masking these SREs by transfected locked nucleic acid (LNA) mixmers affect the viral splicing pattern and viral particle production. With regard to the development of future therapeutic LNA mixmer-based antiretroviral approaches, we delivered the GI3-2 and the ESEtat LNA mixmers "nakedly", without the use of transfection reagents (gymnosis) into HIV-1 infected cells. Surprisingly, we observed that gymnotically-delivered LNA mixmers accumulated in the cytoplasm, and seemed to co-localize with GW bodies and induced degradation of mRNAs containing their LNA target sequence. The GI3-2 and the ESEtat LNA-mediated RNA degradation resulted in abrogation of viral replication in HIV-1 infected Jurkat and PM1 cells as well as in PBMCs.

Cellular rejuvenation to combat HIV-1-related neurocognitive impairment.

HIV-1-related neurocognitive impairment affects a significant proportion of people living with HIV, and accelerated brain aging has been implicated in its pathogenesis. This forum explores the application of cellular rejuvenation strategies to target molecular mechanisms of brain aging, promote neuronal health, and combat cognitive decline.

MMR vaccination induces trained immunity via functional and metabolic reprogramming of γδ T cells.

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.

A pan-respiratory antiviral chemotype targeting a transient host multi-protein complex.

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.

Asperphenalenones Isolated from the Biocontrol Agent Clonostachys rosea and Their Antimicrobial Activities.

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.

RT-PCR Testing of Organ Culture Medium for Corneal Storage Fails to Detect SARS-CoV-2 Infection Due to Lack of Viral Replication.

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.

Design of D-Amino Acids SARS-CoV-2 Main Protease Inhibitors Using the Cationic Peptide from Rattlesnake Venom as a Scaffold.

The C30 endopeptidase (3C-like protease; 3CLpro) 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 3CLpro.

Multi-Omics Integration Reveals Only Minor Long-Term Molecular and Functional Sequelae in Immune Cells of Individuals Recovered From COVID-19.

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.