Biology subjects

Neyts, J.

Publications and source records attributed to Neyts, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Antiviral sugar baits to reduce arbovirus transmission: a proof-of-concept study

Dengue virus (DENV) and chikungunya virus (CHIKV) remain major global health threats, yet approved antiviral therapies are lacking. Antiviral sugar baits (AVSBs) represent a novel transmission-blocking strategy that exploits the natural sugar-feeding behaviour of mosquitoes to deliver antiviral compounds directly to the mosquito vector. In this study, we evaluated the antiviral efficacy of AVSBs containing JNJ-A07, {beta}-D-N4-hydroxycytidine (NHC), molnupiravir (MPV), and 4'-fluorouridine (4'FlU) in Aedes aegypti mosquitoes. An ex vivo mosquito gut model was used to select antiviral concentrations and to assess the ability of 4'FlU to reach the mosquito midgut following sugar feeding. AVSBs were subsequently evaluated for their effects on mosquito attractiveness, longevity, fecundity, and fertility, as well as their ability to suppress DENV and CHIKV infection in vivo. 4'FlU potently inhibited CHIKV replication in ex vivo mosquito guts and retained antiviral activity following sugar-bait administration. None of the antiviral compounds affected mosquito attraction to the bait, while only modest and compound-specific effects on mosquito fitness were observed. AVSBs containing JNJ-A07 significantly reduced DENV infection and dissemination, whereas 4'FlU-containing AVSBs significantly reduced CHIKV infection and viral loads. In contrast, NHC- and MPV-containing AVSBs did not exhibit antiviral activity in vivo. Collectively, these findings provide proof-of-concept for AVSBs as a novel strategy to reduce arbovirus transmission by targeting viral replication within the mosquito vector, suggesting that AVSBs could complement existing arbovirus control measures.

microbiology↗

bioRxiv2026

Cross-protection against Bundibugyo by Ebola and Sudan vaccines

Ebola outbreaks continue to expand across Central Africa, yet available countermeasures remain limited and virus-specific. Whether available vaccines developed against the related Ebola virus (EBOV) and Sudan virus (SUDV) can confer cross-protection against Bundibugyo virus (BDBV) is incompletely understood. Here, we developed a BDBV surrogate challenge model and evaluated cross-protective immunity conferred by a set of monovalent vaccine candidates representing all three Orthoebolavirus species affecting humans. A yellow fever 17D-vectored BDBV vaccine (YF-BDB) expressing the BDBV glycoprotein (GP) as antigen conferred homologous protection in Ifnar-/- mice, preventing systemic viral dissemination and providing survival following surrogate BDBV challenge. Intriguingly, also vaccination with YF-EBO or YF-SUD protected mice from such BDBV surrogate challenge. Conversely, while YF-BDB and YF-EBO fully protected against respective heterologous EBOV or BDBV infection, they conferred only partial protection against lethal challenge in a comparable SUDV model. Serological analyses revealed comparable titers of cross-reactive antibodies across all vaccine groups. However, neutralization, especially cross-neutralization, was limited suggesting a major role for non-neutralizing mechanisms in protection. These findings demonstrate asymmetry in cross-protective immunity among Orthoebolavirus vaccines and indicate that SUDV GP-based antigens may provide broader protection than EBOV- or BDBV-targeted candidates. However, our data add to the preliminary yet limited evidence that EBOV GP-based vaccines might offer at least some degree of protection against BDBV, the virus driving current Public Health Emergency of International Concern (PHIEC) in the Central-East Africa region.

microbiology↗

bioRxiv2026

Rat hepatitis E virus infection has multiphasic viral replication kinetics in vivo

Background and AimsHepatitis E virus (HEV) infections are a growing threat to global public health. To obtain an in-depth understanding of HEV infections in untreated and ribavirin-treated rats, we characterized the early HEV viral kinetics using rat HEV (rHEV) as a surrogate model and using two routes of virus inoculation: intravenous (I.V.) or oral infection. Approach and ResultsWe frequently collected feces, serum, and tissue samples up to 60 days after infection in both infection models to characterize the rHEV viral kinetics. A ~2-week delay in quantifiable RNA levels in feces was observed in the oral versus the I.V. infection model. Early rHEV viral kinetics in feces were found to be multiphasic and showed good concordance with those in the various tissue compartments studied. Comparison of the viral kinetics in these samples also revealed that the liver may serve as the initial site of rHEV replication, followed by replication in the intestine and spleen. While a dosage of 60 mg/kg/day ribavirin was found optimal to maintain rHEV RNA levels at (nearly) undetectable in feces, levels were detectable in the liver and increased both in feces and liver after treatment discontinuation. ConclusionsWe found that the two rHEV infection models share similar multiphasic viral kinetics with the liver as the main site of viral replication. Additionally, the rHEV RNA load in feces could be used as a reliable proxy for that in the liver, spleen, and intestine. We also show that ribavirin at 60 mg/kg/day was partially effective in preventing viral rebound. These findings may aid in exploring the correlation between the infection phases and antiviral efficacy, ultimately guiding therapeutic decisions.

microbiology↗

bioRxiv2025

A high-throughput cell-based platform for Rhinovirus C research and antiviral drug discovery.

Rhinoviruses (RVs) are the most common cause of upper respiratory infections, yet RV-C is particularly associated with severe respiratory disease, including exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Despite its clinical significance, RV-C research and antiviral development have been hindered by the lack of scalable and biologically relevant models for high-throughput screening (HTS). Here, we present the development, optimization, and validation of a scalable high-content imaging-based platform for RV-C research. Our approach combines engineered HeLa cells expressing CDHR3C529Y, a genetically stable mGreenLantern-reporter RV-C15 virus, and a phenotypic selection strategy to identify monoclonal cell lines with maximal and sustained permissiveness. This approach enabled assay optimization in both 96- and 384-well formats, achieving robust performance (Z' > 0.7) and reproducibility across more than 100 runs with reference antiviral compounds. The platform was readily adapted to RV-A16, RV-B14, and additional RV-C types (C11 and C41), demonstrating broad applicability across rhinovirus species. A pilot screen of 10 240 compounds identified confirmed RV-C inhibitors, highlighting the readiness of the platform for integration into drug discovery pipelines. This platform provides a robust and scalable tool for systematic antiviral discovery and a foundation for mechanistic studies of RV-C replication. Significance statementRhinovirus C (RV-C) is strongly associated with severe respiratory disease such as asthma and COPD exacerbations, yet its study has been limited by the absence of scalable cell-based systems. We developed a high-content imaging-based high-throughput screening platform that supports efficient RV-C replication in engineered CDHR3-expressing cells and enables systematic antiviral discovery. The platform integrates a genetically stable fluorescent reporter virus with phenotypically selected permissive monoclonal cell lines, achieving high reproducibility and scalability in both 96- and 384-well formats. Its adaptability across multiple rhinovirus species establishes a unified framework for antiviral testing and mechanistic investigation. A pilot screen of 10K compounds confirmed the applicability of the platform to drug discovery campaigns.

microbiology↗

bioRxiv2025

Oropouche virus results in severe congenital disease in embryonic mice

The recent outbreak of Oropouche virus (OROV) in Latin-America with unprecedented reports of vertical transmission resulting in congenital malformations and fetal death, redefines the threat posed by this arbovirus. We here explore the teratogenic potential of an epidemic strain of the virus in a mouse model, whereby a low inoculum of OROV is injected into the placenta. The virus replicates efficiently in multiple embryonic organs, with high titers in the brain, and causes intrauterine growth restriction. Infected embryos display multi-organ pathology, including pneumonitis, myocarditis, steatohepatitis and severe neuropathology. Embryonic brains exhibit microcephaly, ventriculomegaly and extensive neural loss, mirroring findings in human fetal cases. A dramatic alteration of the neural cell population correlates with massive cell death, demonstrating the extreme cytotoxicity of OROV for the developing brain. The infection also triggers neuroinflammation characterized by cytokine upregulation, microglial activation and neutrophil infiltration. Altogether, our findings establish a link between congenital OROV infection and neurodevelopmental disease, highlighting its teratogenic potential.

microbiology↗

bioRxiv2025

Oral 4-fluorouridine completely suppresses epidemic Oropouche virus replication in mice and confers protection against virus-induced disease.

Oropouche virus (OROV) is an orthobunyavirus that causes increasingly frequent and severe outbreaks in Central and South America. We report that 4'-fluorouridine (4'-FlU) inhibits the in vitro replication of epidemic and pre-epidemic OROV strains in multiple cell lines. In vitro polymerase assays demonstrate that 4'-FlU (as its triphosphate) targets the Peribunyaviridae L protein, is incorporated during RNA synthesis and causes premature chain termination. Following 69 consecutive days of in vitro passages of OROV in the presence of suboptimal concentrations of 4'-FlU, no drug-resistant variants were identified in the viral polymerase. In stringent mouse (AG129) or Syrian hamster OROV-infection models, oral administration of 4'-FlU completely blocked viral replication and virus-induced disease, even when administration was delayed until 72 hours after infection. Our findings support exploring the potential of 4'-FlU for the management of OROV infections in humans.

microbiology↗

bioRxiv2025

Discovery of orally bioavailable Zika and West Nile Virus antiviral compounds targeting the NS2B-NS3 protease

Flaviviruses are a class of pathogenic viruses with pandemic potential that are typically transmitted via infected arthropods. In addition, Zika virus is sexually transmissible and causes congenital malformations if infection occurs during pregnancy. Although over 1.5 million people were infected during the 2015-2016 Zika outbreak, to date, there are no clinical-stage vaccines or antivirals. Herein, we report the discovery of potent inhibitors of the Zika virus NS2B-NS3 protease that also show activity against the West Nile virus NS2B-NS3 protease. Starting from a crystallographic fragment screen, we employed a pharmacophore approach coupled with high-throughput library chemistry to elaborate fragments in the active site. Potent, metabolically stable, non-covalent, non-peptidomimetic inhibitors were identified with antiviral activity in vitro. The lead compound was progressed to a three-day Zika virus challenge study in AG129 mice, where it displayed significant viral RNA load reduction in both mouse plasma and spleen.

microbiology↗

bioRxiv2025

Open-science discovery of DNDI-6510, a compound that addresses genotoxic and metabolic liabilities of the COVID Moonshot SARS-CoV-2 Mpro lead inhibitor

The 2020 SARS-CoV-2 coronavirus pandemic highlighted the urgent need for novel small molecule antiviral drugs. (S)-x38 DNDI-6510 is a non-covalent SARS-CoV-2 main protease inhibitor developed by the open science collaboration COVID Moonshot. Here, we report on the metabolic and toxicologic optimization of the lead series previously disclosed by the COVID Moonshot Initiative, leading up to the selection of (S)-x38 DNDI-6510 as the preclinical candidate. We describe the thorough profiling of the series, identifying key risks such as formation of genotoxic metabolites and high clearance, which were successfully addressed during lead optimization. In addition, we disclose the in vitro and in vivo evaluation of (S)-x38 DNDI-6510 in pharmacokinetic and pharmacodynamic models, exploring multiple approaches to ameliorate rodent-specific metabolic clearance, and show that both co-dosing of (S)-x38 DNDI-6510 with an ABT inhibitor and utilizing a metabolically humanized mouse model (8HUM) achieve significant improvements in exposure. Through comparisons of ABT co-dosing and humanized mouse models in efficacy experiments, we demonstrate that continuous exposure over cellular EC90 is required for SARS-CoV-2 antiviral efficacy in vivo in an antiviral model using a mouse-adapted SARS-CoV-2 strain. Finally, (S)-x38 DNDI-6510 was assessed in maximum tolerated dose experiments in two species, demonstrating significant in vivo PXR-linked auto-induction of metabolism, leading to the discontinuation of this compound. In summary, we report the successful effort to overcome series-specific AMES liabilities in a lead development program. Downstream optimization of existing series will require in-depth optimization of rodent-specific liabilities and metabolic induction profile.

pharmacology and toxicology↗

bioRxiv2025

Human Myelin Spheres for In Vitro Oligodendrocyte Maturation, Myelination and Neurological Disease Modeling

Demyelinating diseases, such as multiple sclerosis, damage the protective myelin sheaths of the central nervous system. The development of effective therapies has been hampered by the lack of models that accurately replicate human myelin biology. Here we present a novel method to generate human myelin spheres (MyS) by coculturing of hPSC-derived neuronal and oligodendrocyte precursor cells, to create myelinated neurons. Using multimodal analyses including confocal and (electron)microscopy, single-nuclei transcriptomics, lipidomics, and electrophysiology, we demonstrate myelination in MyS as early as six weeks into coculture. These myelinated structures mature over time into multilamellar and compacted myelin sheaths with lipid compositions and transcriptomic profiles mirror the temporal dynamics of in vivo human oligodendrocyte development and neuronal myelination, resembling those of late fetal/postnatal oligodendrocytes. By employing lysolecithin-induced demyelination and Rabies virus infection experiments, we demonstrate the potential of MyS as an innovative, physiologically relevant platform for studying myelin-related neurodegeneration and neuroinfection.

neuroscience↗

bioRxiv2025

The critical role of enterovirus 2A protease in viral translation, replication, and antagonism of host antiviral responses.

Enteroviruses dramatically remodel the cellular infrastructure for efficient replication and curtailing host antiviral responses. Proteases 2Apro and 3Cpro have been implicated in these processes based on in vitro studies, ectopic overexpression, and surrogate infection systems, but their relative contributions are unknown. Here, we replace the essential 2A cleavage site at the P1-P2 junction with an internal ribosome entry site (IRES), 3CD cleavage site, or T2A sequence, allowing us to catalytically inactivate 2Apro. Viruses with an inactive 2Apro are hampered in replication in cell lines and are severely attenuated in a Coxsackievirus B3 (CVB3) mouse pancreatitis infection model. We show that 2Apro, but not 3Cpro, is essential during infection for disturbing nucleocytoplasmic transport, shutting down host mRNA translation, suppressing stress granule formation, cleaving retinoic acid-inducible gene-1 (RIG-I)-like receptor (RLR) signaling pathway proteins and suppressing interferon-/{beta} (IFN-/{beta}) transcription, and overcoming the antiviral action of IFN-induced restriction factors. Moreover, using an advanced single-molecule live cell imaging approach, we reveal that 2Apro is important for the initial round of replication of the incoming viral RNA, which is a bottleneck for efficient infection. In conclusion, we establish that 2Apro plays a critical role in subverting antiviral responses and establishing a favorable host environment to expedite enterovirus replication.

microbiology↗

bioRxiv2025

Combinations of approved oral nucleoside analogues confer potent suppression of alphaviruses in vitro and in vivo

BackgroundAlphaviruses, including chikungunya virus (CHIKV), pose a significant global health threat, yet specific antiviral therapies remain unavailable. MethodsWe evaluated combinations of three oral directly acting antiviral drugs (sofosbuvir (SOF), molnupiravir (MPV), and favipiravir (FAV)), which are approved for other indications, against CHIKV, Semliki Forest virus (SFV), Sindbis virus (SINV), and Venezuelan Equine Encephalitis virus (VEEV) in vitro and in vivo. We assessed antiviral efficacy in human skin fibroblasts and liver cells, as well as in a mouse model of CHIKV-induced arthritis. FindingsIn human skin fibroblasts, synergistic antiviral effects were observed for combinations of MPV + SOF and FAV + SOF against CHIKV, and for FAV + SOF against SFV. In human liver cells, FAV + MPV conferred additive to synergistic activity against VEEV and SINV, while SOF synergized with FAV against SINV. In mice, MPV improved CHIKV-induced foot swelling and reduced systemic infectious virus titres. Combination treatment with MPV and SOF significantly reduced swelling and infectious virus titres compared to monotherapies of each drug. Sequencing of CHIKV RNA from joint tissue revealed that MPV caused dose- dependent increases in mutations in the CHIKV genome. Upon combination therapy of MPV with SOF, the number of mutations was significantly lower compared to monotherapy with several higher doses of MPV. InterpretationCombining these approved oral nucleoside analogues confers potent suppression of multiple alphaviruses in vitro and in vivo with enhanced control of viral genetic evolution in face of antiviral pressure. These drug combinations may ultimately lead to the development of potent combinations of pan-family alphavirus inhibitors. FundingThis work was supported by a PhD fellowship granted to S.V. by the Research Foundation - Flanders (FWO) (11D5923N). L.D.C. was also supported by Research Foundation - Flanders (FWO) PhD fellowship (11L1325N). Dr. Polyak and Schiffer are partially supported by R01AI121129. Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSAlphaviruses such as chikungunya virus (CHIKV), Sindbis virus (SINV), and Venezuelan Equine Encephalitis virus (VEEV) are a major threat for global health. Alphaviruses are responsible for debilitating diseases with major public health implications, yet no antiviral drugs are currently approved for treating these virus infections. Existing treatment options are limited to supportive care and are unlikely to provide protection against future outbreaks of other alphaviruses. Previous studies have shown that oral approved nucleoside analogues such as favipiravir (FAV), molnupiravir (MPV), and sofosbuvir (SOF) have antiviral activity against certain RNA viruses, including alphaviruses. However, systematic in vivo evaluations of these drugs and testing of drug combinations in both in vitro and in vivo settings are limited. Added value of this studyThis study provides a comprehensive evaluation of combinations of FAV, MPV and SOF against multiple alphaviruses in two human cell lines and a CHIKV mouse model. We demonstrate that certain combinations of these drugs confer synergistic antiviral activity, effectively suppressing CHIKV, SFV, SINV, and VEEV replication in vitro. Moreover, in vivo, we show for the first time that MPV treatment results in reduced CHIKV-induced foot swelling and systemic virus replication. Combining MPV with SOF enhances antiviral activity in mice as compared to monotherapy. By sequencing the viral genome, we show that MPV increases the number of mutations in a dose-dependent manner. Combination therapy of MPV and SOF reduces the number of mutations compared to higher doses of MPV. These findings highlight the potential of nucleoside analogue combinations as a promising therapeutic strategy against alphavirus infections. Implications of all the available evidenceThe results of this study suggest that combination therapy with approved nucleoside analogues could provide an effective treatment strategy for alphavirus infections. The observed increased efficacy of drug combinations supports the potential for dose optimization to enhance efficacy while reducing toxicity and development of resistance. Future research should focus on clinical evaluation of these drug combinations, pharmacokinetic studies, and further exploration of their impact on viral evolution. Given the expanding geographical distribution of alphaviruses and the lack of available treatments, these findings provide a foundation for developing pan-alphavirus antiviral therapies that could improve patient outcomes and global outbreak preparedness.

microbiology↗

bioRxiv2025

The combination of GS-441524 (remdesivir) and ribavirin results in a potent antiviral effect against human parainfluenza virus 3 infection in human airway epithelial cell cultures and in a mouse infection model

Human parainfluenza virus type 3 (HPIV-3) can cause severe respiratory diseases, particularly in young children, the elderly and immunocompromised. There are no approved antiviral drugs against this virus. We report that the combination of ribavirin with either remdesivir or its parent nucleoside GS-441524 results in a pronounced antiviral effect against HPIV-3 in LLC-MK2 cells and in human airway epithelial cells grown at the air-liquid interface. In AG129 mice intranasally inoculated with HPIV-3, the combined treatment of ribavirin and GS-441524 decreases infectious viral lung titers by >2.5 log10 to undetectable levels in 4 out of 11 mice and by 1.6 log10 in the remaining 7 mice as compared with vehicle. The lungs of all mice that received the combined treatment appear histologically normal or virtually normal, whereas 8 of 11 vehicle-treated mice present with bronchopneumonia. By contrast, ribavirin alone does not result in a reduction of infectious viral lung titers; GS-441524 alone reduces infectious viral lung titers by 1.2 log10. Moreover, several mice in the single-treatment groups exhibit severe lung pathology. These findings may warrant exploring this combination in patients with severe HPIV-3 infections and possibly also against infections with other viruses that are in vitro susceptible to these two drugs.

microbiology↗

bioRxiv2024

Antigenic imprinting dominates humoral responses to new variants of SARS-CoV-2 in a hamster model of COVID-19

The emergence of SARS-CoV-2 variants escaping immunity challenges the efficacy of current vaccines. Here, we investigated humoral recall responses and vaccine-mediated protection in Syrian hamsters immunized with the third-generation Comirnaty(R) Omicron XBB.1.5-adapted COVID-19 mRNA vaccine, followed by infection with either antigenically closely (EG.5.1) or distantly related (JN.1) Omicron subvariants. Vaccination with YF17D vector encoding a modified Gamma spike (YF-S0*) served as a control for pre-Omicron SARS-CoV-2 immunity. Our results show that both Comirnaty(R) XBB.1.5 and YF-S0* induce robust, however, poorly cross-reactive, neutralizing antibody (nAb) responses. In either case, total antibody and nAb levels increased following infection. Intriguingly, the specificity of these boosted nAbs did not match the respective challenge virus but was skewed towards the primary antigen used for immunization, suggesting a marked impact of antigenic imprinting; confirmed by antigenic cartography. Furthermore, limited cross-reactivity and rapid decline of nAbs induced by Comirnaty(R) XBB.1.5 with EG.5.1 and, more concerning, JN.1. raises doubts about sustained vaccine efficacy against recent circulating Omicron subvariants. Future vaccine design may have to address two major issues: (i) to overcome original antigenic sin that limits the breadth of a protective response towards emerging variants, and (ii) to achieve sustained immunity that lasts for at least one season.

immunology↗

bioRxiv2024

Thymidine kinase-expressing yellow fever 17D reporter virus facilitates prodrug activation and bioorthogonal labelling of infected cells

Tracking of viral replication and tissue tropism by in vivo imaging can help to unveil how live-attenuated vaccines such as the yellow fever 17D (17D) work, and likewise, to understand how adverse effects develop. Here we validate 17D-TK, a reporter virus derived from 17D that expresses herpes virus thymidine kinase (TK) that specifically converts nucleoside analogues such as Ganciclovir (GCV) to induce cell death, or difluoro-EdU (dF-EdU) for bioorthogonal labelling of infected cells by Click chemistry. 17D-TK induces a cytopathic effect in infected cell cultures, as well as mortality in intracranially inoculated mouse pups in a GCV dependent manner. Preferential phosphorylation of difluoro-EdU (dF-EdU) in 17D-TK infected cells allows to selectively stain cells that support 17D replication. Prospectively, 17D-TK can be used in combination with radiolabeled tracers for real-time detection and localization of sites of active viral replication in living animals using positron emission tomography (PET).

microbiology↗

bioRxiv2024

The triple combination of Remdesivir (GS-441524), Molnupiravir and Ribavirin is highly efficient in inhibiting coronavirus replication in human nasal airway epithelial cell cultures and in a hamster infection model

The use of fixed dose-combinations of antivirals with different mechanisms of action has proven a key in the successful treatment of infections with HIV and HCV. For the treatment of infections with SARS-CoV-2 and possible future epi-/pandemic coronaviruses, it will be important to explore the efficacy of combinations of different drugs, in particular to avoid resistance development, such as in patients with immunodeficiencies. As a first effort, we studied the antiviral potency of combinations of antivirals. To that end, we made use of primary human airway epithelial cell (HAEC) cultures grown at the air-liquid interface that were infected with the beta coronavirus OC43. We found that the triple combination of GS-441524 (parent nucleoside of remdesivir), molnupiravir, and ribavirin resulted in a more pronounced antiviral efficacy than what could be expected from a purely additive antiviral effect. The potency of this triple combination was next tested in SARS-CoV-2 infected hamsters. To that end, for each of the drugs, intentionally suboptimal or even ineffective doses were selected. Yet, in the lungs of all hamsters that received triple prophylactic therapy with suboptimal/inactive doses of GS-441524, molnupiravir, and ribavirin, no infectious virus was detectable. Our finding indicate that co-administration of approved drugs for the treatment of coronavirus infections should be further explored but also against other families of viruses with epidemic and pandemic potential for which no effective antiviral treatment is available.

microbiology↗

bioRxiv2024

Hepatitis E virus replication is facilitated by epithelial cell turnover and targets enteroendocrine cells in human intestinal organoids

Hepatitis E virus (HEV) is a leading pathogen causing acute viral hepatitis globally. While HEV is primarily spread fecal-orally, the role of the gut in HEV pathogenesis remains largely unexplored, including how HEV disseminates from gut to liver, and whether the gut is an HEV reservoir. We here aimed to illuminate HEV biology in the gut using human intestinal enteroids (HIEs). Three strategies were explored to establish an HEV-HIE model: three-dimensional (3D) HIEs, two-dimensional (2D) HIEs in transwell, and HEV RNA-electroporated HIEs. HEV particles produced by electroporated HIEs were characterized by western blot and gradient centrifugation. The intestinal tropism of HEV was investigated through confocal fluorescent microscopy and gene expression analysis. HEV infection in 3D-HIEs and 2D-HIEs showed limited replication, whereas HIEs electroporation led to a sustained increase in the release of non-enveloped infectious virions. These virions could re-infect new 3D-HIEs, yielding a [~]2 log10 increase in HEV RNA. In electroporated HIEs, high expression of the infectious ORF2 capsid form was observed in the supernatant. Importantly, 70% of all HEV-infected cells were identified as proliferative cells. ORF2 staining was also observed in absorptive enterocytes, goblet, and enteroendocrine cells. Overall, we established a robust HEV-HIE model that yields high titers of infectious non-enveloped virions. Proliferative cells and the fast intestinal epithelial cell turnover are important features that facilitate efficient HEV replication, and likely also its dissemination. This study suggests that the gut is an HEV reservoir, capable of producing some of the non-enveloped HEV shed in the feces. SignificanceHepatitis E virus (HEV) causes [~]20 million enterically transmitted cases of viral hepatitis globally. To better understand the largely unexplored role of the gut during an HEV infection, we used human intestinal enteroids (HIEs). We show that HEV-electroporated HIEs produce highly infectious non-envelopedHEV particles, resulting in a more efficient re-infection compared with traditional gold-standard methods. Importantly, we detected HEV in multiple human intestinal epithelial cell types within the human intestinal epithelium, with the majority of proliferative cells infected. This model offers valuable insights into the intestinal tropism of HEV and can serve to further investigate chronic HEV infection in the gut. Moreover, this underscores the importance of using physiologically relevant models to unravel details of HEV infection in the gut.

microbiology↗

bioRxiv2024

Yeast-based production platform for potent and stable heavy chain-only antibodies

Monoclonal antibodies are the leading drug of the biopharmaceutical market because of their high specificity and tolerability, but the current CHO-based manufacturing platform remains expensive and time-consuming leading to limited accessibility, especially in the case of diseases with high incidence and pandemics. Therefore, there is an urgent need for an alternative production system. In this study, we present a rapid and cost-effective microbial platform for heavy chain-only antibodies (VHH-Fc) in the methylotrophic yeast Komagataella phaffii (aka Pichia pastoris). We demonstrate the potential of this platform using a simplified single-gene VHH-Fc fusion construct instead of the conventional monoclonal antibody format, as this is more easily expressed in Pichia pastoris. We demonstrate that the Pichia-produced VHH-Fc fusion construct is stable and that a Pichia-produced VHH-Fc directed against the SARS-CoV-2 spike has potent SARS-CoV-2 neutralizing activity in vitro and in vivo. We expect that this platform will pave the way towards faster and cheaper development and production of broadly neutralizing single-chain antibodies in yeast.

microbiology↗

bioRxiv2023

ACE2 mimetic antibody potently neutralizes all SARS-CoV-2 variants and fully protects in XBB.1.5 challenged monkeys

The rapid evolution of SARS-CoV-2 to variants with improved transmission efficiency and reduced sensitivity to vaccine-induced humoral immunity has abolished the protective effect of licensed therapeutic human monoclonal antibodies (mAbs). To fill this unmet medical need and protect vulnerable patient populations, we isolated the P4J15 mAb from a previously infected, vaccinated donor, with <20 ng/ml neutralizing activity against all Omicron variants including the latest XBB.2.3 and EG.1 sub-lineages. Structural studies of P4J15 in complex with Omicron XBB.1 Spike show that the P4J15 epitope shares [~]93% of its buried surface area with the ACE2 contact region, consistent with an ACE2 mimetic antibody. Although SARS-CoV-2 mutants escaping neutralization by P4J15 were selected in vitro, these displayed lower infectivity, poor binding to ACE2, and the corresponding escape mutations are accordingly rare in public sequence databases. Using a SARS-CoV-2 XBB.1.5 monkey challenge model, we show that P4J15 confers complete prophylactic protection. We conclude that the P4J15 mAb has potential as a broad-spectrum anti-SARS-CoV-2 drug.

microbiology↗