Kurt Vermeire

Alcohol-induced dose dumping is a significant concern for modified-release oral dosage forms, as it can compromise the intended release behaviour. For enteric-coated formulations, alcohol exposure may lead to loss of enteric functionality and unintended premature drug release in the stomach. Although previous studies have mainly assessed the loss of enteric performance in delayed-release dosage forms, the mechanisms underlying alcohol-induced changes remain poorly understood. In this study, the behaviour of isolated hydroxypropyl methylcellulose phthalate (HPMC-P) films was investigated in aqueous ethanolic media containing 0-40% v/v ethanol. Film permeability was evaluated using paracetamol, nicotinamide and tritiated water as model permeants. No significant change in permeability was observed at ethanol concentrations up to 20% v/v. However, exposure to 40% v/v ethanol significantly increased permeability. Under these conditions, permeability differences between paracetamol (4.4±0.6 E-05 cm/s) and nicotinamide (2.4±0.3 E-05 cm/s) were observed and were initially attributed to differences in drug solubility in hydroalcoholic medium. However, studies with tritiated water eliminated solubility-related effects and provided direct evidence of ethanol-induced changes in film permeability. Microscopic imaging revealed ethanol concentration dependent pore formation within the films. Mass loss study showed negligible film erosion even at 40% v/v ethanol, despite the substantial increase in permeability. These findings demonstrate that increased film permeability originates primarily from ethanol-induced microstructural changes rather than polymer erosion. Overall, this work provides mechanistic insights into the effects of ethanol exposure on HPMC-P films and improves understanding of alcohol-induced alterations in enteric-coated formulations.

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 Taï 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 Taï 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.

The structural instability of influenza hemagglutinin (HA) is related to its function in low pH-mediated membrane fusion, which requires prior cleavage of the premature HA0 by a host protease. Mapping the precise determinants of HA cleavability and stability has implications for risk assessment of zoonotic influenza A viruses, viral transmissibility, and vaccine production. Here, we conducted random mutagenesis on the early 2009 pandemic H1 HA, followed by selection of acid-stable viruses, and detailed profiling of the mutant HAs. We identified mutation D346N, which is located in the cleavage loop and renders H1 HA0 12-fold resistant to trypsin activation. Its cleavage by transmembrane serine protease 2 (TMPRSS2) was proved not to be affected. Likewise, we found that the poor cleavage of H16 HA0, which is unusual in carrying an N346 residue, is evident with exogenous trypsin and when non-activated particles encounter TMPRSS2 only during entry. Since H16 HA also exhibits a very low fusion pH, we propose that the gull H16N3 virus may carry a much more stable HA than other avian influenza A viruses. In addition, our random mutagenesis study on H1 HA yielded four acid-stabilizing mutations, which decrease the pH and efficiency of membrane fusion, and subtly impair the viral entry and replication in human airway-derived epithelial cells. These mutations are situated in the globular head, vestigial esterase, and membrane-proximal part of H1 HA, in regions involved in the refolding of HA at low pH. Collectively, our mutagenesis approach revealed determinants of HA cleavability and stability, with relevance for viral surveillance and vaccine production. IMPORTANCE: The presence of influenza A viruses (IAV) throughout the animal world, particularly avian species, represents a constant threat for zoonotic infections or a new influenza pandemic. To be transmissible among humans, a zoonotic IAV requires a hemagglutinin (HA) that is activated by host proteases and exhibits appropriate stability. Mapping the underlying determinants also matters for producing HA-based vaccines with high shelf stability. Through random mutagenesis and selection of acid-stable viruses, we discovered a mutation that renders HA resistant to exogenous trypsin activation. The natural occurrence of this residue in H16 HA, combined with the prominent acid stability of this HA subtype, suggests that the gull H16N3 virus may differ from other avian IAVs in carrying an environmentally stable HA. Besides, we identified four stabilizing mutations located in different parts of HA. Hence, our study delivers insight into factors that modulate HA cleavability and acid stability, with relevance for viral surveillance and vaccine production.

The aim of this study was to gain detailed insights into how different plasticizers influence the permeability of isolated hydroxypropyl methyl cellulose phthalate (HPMC-P) films. Since the migration of plasticizers from film coatings may critically affect their functionality, it is essential to monitor the impact of their leaching on the associated film properties. In this work, the leaching behavior of plasticizers from isolated HPMC-P films was estimated using their thermal properties by modulated temperature differential scanning calorimetry (MT-DSC) at varying plasticizer concentrations. The films plasticized with acetyl triethyl citrate (ATEC) and dibutyl sebacate (DBS) exhibited lower permeability compared to those plasticized with polyethylene glycol 400 (PEG 400). This reduced permeability can be attributed to the slower leaching rates of ATEC and DBS and to the persistent hydrophobic character that they impart to the films, even after prolonged aqueous exposure. In contrast, films plasticized with PEG 400 demonstrated the highest permeability because of the hydrophilicity induced in the polymeric films. However, a subsequent decline in their diffusion rate was observed once PEG 400 had leached out of the films. Furthermore, the correlation between leaching behavior and flux profiles provided an understanding of the influence of plasticizer on film permeability based on their polarity. Moreover, saturation in plasticization efficiency was noted with DBS-based films at higher plasticizer concentrations (greater than 10% w/w). Overall, our study highlights the importance of selecting appropriate plasticizers for the development of polymer films and provides insights into tailoring film permeability and drug release kinetics.

Sudan virus (SUDV; species Orthoebolavirus sudanense) remains a major public health threat, yet research is limited by restricted access to biosafety level 4 (BSL-4) facilities. To address this, we developed a biologically contained SUDV lacking the essential VP30 gene, restricting replication to VP30-expressing cells. We demonstrate efficient virus rescue, strict functional and genetic containment, and replication kinetics comparable to wild-type SUDV in permissive cells. Using heterologous VP30-expressing cell lines, we observe strong cross-functionality among orthoebolaviruses, whereas Marburg virus VP30 shows minimal activity, highlighting genus-specific constraints. The system supports high-throughput antiviral screening and confirms robust activity of remdesivir. In addition, resistance profiling identified substitutions at residue F549 of viral polymerase L as key determinants for remdesivir resistance, with additional mutations at M675. Together, this biologically contained SUDV system enables safe study of viral replication, antiviral discovery, and resistance evolution under lower biosafety conditions.

Nipah virus is a highly lethal, zoonotic paramyxovirus that has caused recurring outbreaks in several South and Southeast Asian countries since its discovery in Malaysia in 1998. Symptoms of infection include severe respiratory and neurological disease, often resulting in death. As no approved vaccines or antivirals are currently available to reduce the burden of this virus, it is classified as a biosafety level 4 pathogen. There is an urgent need for systems that enable research in a lower biocontainment setting, especially since the World Health Organization declared Nipah virus a priority pathogen for pandemic concern. In the past, several minigenome systems have already been developed as safe alternatives to working with infectious virus; however, these systems remain relatively inefficient and lack robustness and reliability for further applications. Therefore, we developed novel optimized RNA polymerase II-driven minigenomes with nanoluciferase or enhanced green fluorescent protein reporter genes. Both systems outperform previously designed Nipah virus minigenomes, are easily operable, and can be implemented for antiviral compound screenings.