Biology subjects

Lemmens, V.

Publications and source records attributed to Lemmens, V..

7 recordsLinked to original sources

bioRxiv2026

A panel of biologically contained orthoebolaviruses for the screening of broad-spectrum antivirals

Filoviruses, particularly those of the Orthoebolavirus genus, pose ongoing public health threats due to their increasing frequency and geographic spread. However, research has been impeded by the biosafety level 4 classification of filoviruses. We previously reported the generation of biologically contained Ebola, Marburg and Sudan virus as lower biosafety level-compatible filovirus systems. In the present study, we expanded this repertoire to include biologically contained Tai Forest, Bundibugyo and Reston virus, thereby creating a near-complete toolkit for currently recognized human-relevant orthoebolaviruses. VP30-deficient viruses were generated with matching VP30 expressing cell lines. More specifically, we developed and optimized a dual-reporter system in VeroE6, Huh-7 and A549 cells, combining a virus-encoded enhanced green fluorescent protein reporter as readout for viral replication with a stably cellular-expressed nuclear mCherry marker for cytotoxicity assessment. Next, we screened two repurposing-oriented compound libraries comprising 640 small molecules against Tai Forest, Bundibugyo and Sudan virus, and subsequently cross-validated against Reston and Ebola virus. This approach identified multiple candidates with broad-spectrum activity across orthoebolaviruses, while also revealing virus-specific antivirals, with robust activity observed in both primate- and human-derived cell lines. Together, this work establishes a versatile and experimentally tractable lower biosafety level-compatible platform for the study of human-relevant orthoebolaviruses and the systematic discovery of broad-spectrum antiviral countermeasures against filoviruses.

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↗

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↗

bioRxiv2023

Single-dose YF17D-vectored Ebola vaccine candidate protects mice against both lethal surrogate Ebola and yellow fever virus challenge

Ebola virus (EBOV) and related filoviruses such as Sudan virus (SUDV) threaten global public health. Effective filovirus vaccines are available only for EBOV, yet restricted to emergency use considering a high reactogenicity and demanding logistics. Here we present YF-EBO, a live YF17D-vectored dual-target vaccine candidate expressing EBOV-glycoprotein (GP) as protective antigen. Safety of YF-EBO in mice was further improved over that of parental YF17D vaccine. A single dose of YF-EBO was sufficient to induce high levels of EBOV-GP specific antibodies and cellular immune responses, that protected against lethal infection using EBOV-GP-pseudotyped recombinant vesicular stomatitis virus (rVSV-EBOV) in interferon-deficient (Ifnar-/-) mice as surrogate challenge model. Concomitantly induced yellow fever virus (YFV)-specific immunity protected Ifnar-/- mice against intracranial YFV challenge. YF-EBO could thus help to simultaneously combat both EBOV and YFV epidemics. Finally, we demonstrate how to target other highly pathogenic filoviruses such as SUDV at the root of a current outbreak in Uganda.

immunology↗

bioRxiv2021

The omicron (B.1.1.529) SARS-CoV-2 variant of concern does not readily infect Syrian hamsters

The emergence of SARS-CoV-2 variants of concern (VoCs) has exacerbated the COVID-19 pandemic. End of November 2021, a new SARS-CoV-2 variant namely the omicron (B.1.1.529) emerged. Since this omicron variant is heavily mutated in the spike protein, WHO classified this variant as the 5th variant of concern (VoC). We previously demonstrated that the other SARS-CoV-2 VoCs replicate efficiently in Syrian hamsters, alike also the ancestral strains. We here wanted to explore the infectivity of the omicron variant in comparison to the ancestral D614G strain. Strikingly, in hamsters that had been infected with the omicron variant, a 3 log10 lower viral RNA load was detected in the lungs as compared to animals infected with D614G and no infectious virus was detectable in this organ. Moreover, histopathological examination of the lungs from omicron-infecetd hamsters revealed no signs of peri-bronchial inflammation or bronchopneumonia. Further experiments are needed to determine whether the omicron VoC replicates possibly more efficiently in the upper respiratory tract of hamsters than in their lungs.

microbiology↗

bioRxiv2021

Full protection against all four SARS-CoV-2 variants of concern (VOC) in hamsters requires revision of spike antigen used for vaccination.

Current first-generation COVID-19 vaccines are based on prototypic spike sequences from ancestral 2019 SARS-CoV-2 strains. However, the ongoing pandemic is fueled by variants of concern (VOC) that threaten to escape vaccine-mediated protection. Here we show in a stringent hamster model that immunization using prototypic spike expressed from a potent YF17D viral vector (1) provides vigorous protection against infection with ancestral virus (B lineage) and VOC Alpha (B.1.1.7), however, is insufficient to provide maximum protection against the Beta (B.1.351) variant. To improve vaccine efficacy, we created a revised vaccine candidate that carries an evolved spike antigen. Vaccination of hamsters with this updated vaccine candidate provides full protection against intranasal challenge with all four VOCs Alpha, Beta, Gamma (P.1) and Delta (B.1.617.2) resulting in complete elimination of infectious virus from the lungs and a marked improvement in lung pathology. Vaccinated hamsters did also no longer transmit the Delta variant to non-vaccinated sentinels. Hamsters immunized with our modified vaccine candidate also mounted marked neutralizing antibody responses against the recently emerged Omicron (B.1.1.529) variant, whereas the old vaccine employing prototypic spike failed to induce immunity to this antigenically distant virus. Overall, our data indicate that current first-generation COVID-19 vaccines need to be urgently updated to cover newly emerging VOCs to maintain vaccine efficacy and to impede virus spread at the community level. Significance StatementSARS-CoV-2 keeps mutating rapidly, and the ongoing COVID-19 pandemic is fueled by new variants escaping immunity induced by current first-generation vaccines. There is hence an urgent need for universal vaccines that cover variants of concern (VOC). In this paper we show that an adapted version of our vaccine candidate YF-S0* provides full protection from infection, virus transmission and disease by VOCs Alpha, Beta, Gamma and Delta, and also results in markedly increased levels of neutralizing antibodies against recently emerged Omicron VOC in a stringent hamster model. Our findings underline the necessity to update COVID-19 vaccines to curb the pandemic, providing experimental proof on how to maintain vaccine efficacy in view of an evolving SARS-CoV-2 diversity.

microbiology↗

bioRxiv2021

Glycine receptor α3K governs mobility and conductance of L/K splice variant heteropentamers

Glycine receptors (GlyRs) are ligand-gated pentameric chloride channels in the central nervous system. GlyR-3 is a possible target for chronic pain treatment and temporal lobe epilepsy. Alternative splicing into K or L variants determines the subcellular fate and function of GlyR-3, yet it remains to be shown whether its different splice variants can functionally co-assemble, and what the properties of such heteropentamers would be. Here, we subjected GlyR-3 to a combined fluorescence microscopy and electrophysiology analysis. We employ masked Pearsons and dual-color spatiotemporal correlation analysis to prove that GlyR-3 splice variants heteropentamerize, adopting the mobility of the K variant. Fluorescence-based single-subunit counting experiments revealed a variable and concentration ratio dependent hetero-stoichiometry. Via single-channel on-cell patch clamp we show heteropentameric conductances resemble those of the 3K splice variant. Our data are compatible with a model where 3 heteropentamerization fine-tunes mobility and activity of GlyR 3 channels, which is important to understand and tackle 3 related diseases. SummaryThe glycine receptor 3 is key to the central nervous systems physiology and involved in chronic pain and epilepsy. In this paper, Lemmens et al. reveal and functionally characterize 3 splice variant heteropentamerization via advanced single-molecule fluorescence image analysis. DeclarationsO_ST_ABSFundingC_ST_ABSWe acknowledge the UHasselt Advanced Optical Microscopy Centre (AOMC). Prof. Em. Marcel Ameloot, the Research Foundation Flanders (FWO, project G0H3716N) and the province of Limburg (Belgium) (tUL Impuls II) are acknowledged for funding the microscopy hardware. V. Lemmens is grateful for a doctoral scholarship from the UHasselt (17DOC11BOF) and KU Leuven (C14/16/053) Special Research Funds (BOF). Conflicts of interest / competing interestsNo conflicts of interest apply. Ethics approvalNot applicable Availability of data and materialAll data and material are available upon request. Code availabilityFluctuation imaging and co-localization analyses were performed in the software package PAM [71]. The software is available as source code, requiring MATLAB to run, or as pre-compiled standalone distributions for Windows or MacOS at http://www.cup.uni-muenchen.de/pc/lamb/software/pam.html or hosted in Git repositories under http://www.gitlab.com/PAM-PIE/PAM and http://www.gitlab.com/PAM-PIE/PAMcompiled. A detailed user manual is available at http://pam.readthedocs.io. Author contributionsConceptualization Meier J.C., Brone B. and Hendrix J.; Investigation and formal analysis Lemmens V. and Thevelein B.; Software development Hendrix J.; Writing the original draft Lemmens V., Thevelein B and Hendrix, J.; Review and editing by all authors.

biochemistry↗