Biology subjects

Neyts, J.

Publications and source records attributed to Neyts, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Ultrapotent SARS coronavirus-neutralizing single-domain antibodies that bind a conserved membrane proximal epitope of the spike

Currently circulating SARS-CoV-2 variants have gained complete or significant resistance to all SARS-CoV-2-neutralizing antibodies that have been used in the clinic. Such antibodies can prevent severe disease in SARS-CoV-2 exposed patients for whom vaccines may not provide optimal protection. Here, we describe single-domain antibodies (VHHs), also known as nanobodies, that can broadly neutralize SARS-CoV-2 with unusually high potency. Structural analysis revealed their binding to a unique, highly conserved, membrane proximal, quaternary epitope in the S2 subunit of the spike. Furthermore, a VHH-human IgG1 Fc fusion, efficiently expressed in Chinese hamster ovary cells as a stable antibody construct, protected hamsters against SARS-CoV-2 replication in a therapeutic setting when administered systemically at low dose. This VHH-based antibody represents a new candidate anti-COVID-19 biologic that targets the Achilles heel of the viral spike.

microbiology↗

bioRxiv2023

Genetic consequences of effective and suboptimal dosing with mutagenic drugs in a hamster model of SARS-CoV-2 infection

Mutagenic antiviral drugs have shown promising results against multiple viruses, yet concerns have been raised about whether their use might promote the emergence of new and harmful viral variants. Here, we examine the genetic consequences of effective and suboptimal dosing of favipiravir and molnupiravir in the treatment of SARS-CoV-2 infection in a hamster model. We identify a dose-dependent effect upon the mutational load in a viral population, with molnupiravir having a greater potency than favipiravir per mg/kg of treatment. The emergence of de novo variants was largely driven by stochastic processes, with evidence of compensatory adaptation but not of the emergence of drug resistance or novel immune phenotypes. Effective doses for favipiravir and molunpiravir correspond to similar levels of mutational load. Combining both drugs had an increased impact on both efficacy and mutational load. Our results suggest the potential for mutational load to provide a marker for clinical efficacy.

evolutionary biology↗

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↗

bioRxiv2022

Combination of the parent analogue of Remdesivir (GS-441524) and Molnupiravir results in a markedly potent antiviral effect in SARS-CoV-2 infected Syrian hamsters

Remdesivir was the first drug to be approved for the treatment of severe COVID-19; followed by molnupiravir (another prodrug of a nucleoside analogue) and the protease inhibitor nirmatrelvir. Combination of antiviral drugs may result in improved potency and help to avoid or delay the development of resistant variants. We set out to explore the combined antiviral potency of GS-441524 (the parent nucleoside of remdesivir) and molnupiravir against SARS-CoV-2. In SARS-CoV-2 (BA.5) infected A549-Dual hACE2-TMPRSS2 cells, the combination resulted in an overall additive antiviral effect with a synergism at certain concentrations. Next, the combined effect was explored in Syrian hamsters infected with SARS-CoV-2 (Beta, B.1.351); treatment was started at the time of infection and continued twice daily for four consecutive days. At 4 day 4 post-infection, GS-441524 (50 mg/kg, oral BID) and molnupiravir (150 mg/kg, oral BID) as monotherapy reduced infectious viral loads by 0.5 and 1.6 log10, respectively, compared to the vehicle control. When GS-441524 (50 mg/kg, BID) and molnupiravir (150 mg/kg, BID) were combined, infectious virus was no longer detectable in the lungs of 7 out of 10 of the treated hamsters (4.0 log10 reduction) and titers in the other animals were reduced by ~2 log10. The combined antiviral activity of molnupiravir which acts by inducing lethal mutagenesis and GS-441524, which acts as a chain termination appears to be highly effective in reducing SARS-CoV-2 replication/infectivity. The unexpected potent antiviral effect of the combination warrants further exploration as a potential treatment for COVID-19.

microbiology↗

bioRxiv2022

Nirmatrelvir-resistant SARS-CoV-2 is efficiently transmitted in Syrian hamsters

The SARS-CoV-2 main protease (3CLpro) is one of the promising therapeutic target for the treatment of COVID-19. Nirmatrelvir is the only the 3CLpro inhibitor authorized for treatment of COVID-19 patients at high risk of hospitalization; other 3Lpro inhibitors are in development. We recently repored on the in vitro selection of a SARS-CoV2 3CLpro (L50F-E166A-L167F; short 3CLprores) virus that is cross-resistant with nirmatrelvir and yet other 3CLpro inhibitors. Here, we demonstrate that the resistant virus replicates efficiently in the lungs of intranassaly infected hamsters and that it causes a lung pathology that is comparable to that caused by the WT virus. Moreover, 3CLprores infected hamsters transmit the virus efficiently to co-housed non-infected contact hamsters. Fortunately, resistance to Nirmatrelvir does not readily develop (in the clinical setting) since the drug has a relatively high barrier to resistance. Yet, as we demonstrate, in case resistant viruses emerge, they may easily spread and impact therapeutic options for others. Therefore, the use of SARS-CoV-2 3CLpro protease inhibitors in combinations with drugs that have a different mechanism of action, may be considered to avoid the development of drug-resistant viruses in the future.

microbiology↗

bioRxiv2022

The dissemination of Rift Valley fever virus to the eye and sensory neurons of zebrafish larvae is stat1 dependent.

The Rift Valley fever virus (RVFV) is listed by the WHO as priority disease and causes haemorrhagic fever, encephalitis, and permanent blindness. To study RVFV pathogenesis and identify small-molecule antivirals, we established a novel in vivo model using zebrafish larvae. Pericardial injection of RVFV resulted in ~4 log10 viral RNA copies/larva, which was inhibited by antiviral 2'-fluoro-2'-deoxycytidine. The optical transparency of the larvae allowed detection of RVFVeGFP in the liver and sensory nervous system, including the optic tectum and retina, but not the brain or spinal cord. Thus, RVFV-induced blindness likely occurs due to direct damage to the eye and peripheral neurons, rather than the brain. Treatment with JAK-inhibitor ruxolitinib, as well as knockout of stat1a but not stat1b, enhanced RVFV replication to ~6 log10 viral RNA copies/larva and ultra-bright livers, although without dissemination to sensory neurons or the eye, hereby confirming the critical role of stat1 in RVFV pathogenesis.

microbiology↗

bioRxiv2022

The substitutions L50F, E166A and L167F in SARS-CoV-2 3CLpro are selected by a protease inhibitor in vitro and confer resistance to nirmatrelvir

The SARS-CoV-2 main protease (3CLpro) has an indispensable role in the viral life cycle and is a therapeutic target for the treatment of COVID-19. The potential of 3CLpro-inhibitors to select for drug-resistant variants needs to be established. Therefore, SARS-CoV-2 was passaged in vitro in the presence of increasing concentrations of ALG-097161, a probe compound designed in the context of a 3CLpro drug discovery program. We identified a combination of amino acid substitutions in 3CLpro (L50F E166A L167F) that is associated with > 20x increase in EC50 values for ALG-097161, nirmatrelvir (PF-07321332) and PF-00835231. While two of the single substitutions (E166A and L167F) provide low-level resistance to the inhibitors in a biochemical assay, the triple mutant results in the highest levels of resistance (6x to 72x). All substitutions are associated with a significant loss of enzymatic 3CLpro activity, suggesting a reduction in viral fitness. Structural biology analysis indicates that the different substitutions reduce the number of inhibitor/enzyme interactions while the binding of the substrate is maintained. These observations will be important for the interpretation of resistance development to 3CLpro inhibitors in the clinical setting. Abstract ImportancePaxlovid is the first oral antiviral approved for treatment of SARS-CoV-2 infection. Antiviral treatments are often associated with the development of drug resistant viruses. In order to guide the use of novel antivirals it is essential to understand the risk of resistance development and to characterize the associated changes in the viral genes and proteins. In this work, we describe for the first time a pathway that allows SARS-CoV-2 to develop resistance against Paxlovid in vitro. The characteristics of in vitro antiviral resistance development may be predictive for the clinical situation. Therefore, our work will be important for the management of COVID-19 with Paxlovid and next generation SARS-CoV-2 3CLpro inhibitors.

microbiology↗

bioRxiv2022

A SCID mouse model to evaluate the efficacy of antivirals against SARS-CoV-2 infection

Ancestral SARS-CoV-2 lacks the intrinsic ability to bind to the mouse ACE2 receptor and therefore establishment of SARS-CoV-2 mouse models has been limited to the use of mouse-adapted viruses or genetically modified mice. Interestingly, some of the variants of concern, such as the beta B.1.351 variant, show an improved binding to the mouse receptor and hence better replication in different Wild type (WT) mice species. Here, we desribe the establishment of SARS-CoV-2 beta B.1.351 variant infection model in male SCID mice as a tool to assess the antiviral efficacy of potential SARS-CoV-2 small molecule inhibitors. Intranasal infection of male SCID mice with 105 TCID50 of the beta B.1.351 variant resulted in high viral loads in the lungs and moderate signs of lung pathology on day 3 post-infection (pi). Treatment of infected mice with the antiviral drugs Molnupiravir (200 mg/kg, BID) or Nirmatrelvir (300 mg/kg, BID) for 3 consecutive days significantly reduced the infectious virus titers in the lungs by 1.9 and 3.8 log10 TCID50/mg tissue, respectively and significantly improved lung pathology. Together, these data demonstrate the validity of this SCID mice/beta B.1.351 variant infection model as a convenient preclinical model for assessment of potential activity of antivirals against SARS-CoV-2. ImportanceUnlike the ancestral SARS-CoV-2 strain, the beta (B.1.351) VoC has been reported to replicate to some extent in WT mice (species C57BL/6 and BALB/c). We here demonstrate that infection of SCID mice with SARS-CoV-2 beta variant results in high viral loads in the lungs on day 3 post-infection (pi). Treatment of infected mice with the antiviral drugs Molnupiravir or Nirmatrelvir for 3 consecutive days markedly reduced the infectious virus titers in the lungs and improved lung pathology. The advantages of using this mouse model over the standard hamster infection models to assess the in vivo efficacy of small molecule antiviral drugs are (i) the use of a clinical isolate without the need to use mouse-adapted strains or genetically modified animals (ii) lower amount of the test drug is needed and (ii) more convenient housing conditions compared to bigger rodents such as hamsters.

microbiology↗

bioRxiv2022

Imprinted antibody responses against SARS-CoV-2 Omicron sublineages

SARS-CoV-2 Omicron sublineages carry distinct spike mutations and represent an antigenic shift resulting in escape from antibodies induced by previous infection or vaccination. We show that hybrid immunity or vaccine boosters result in potent plasma neutralizing activity against Omicron BA.1 and BA.2 and that breakthrough infections, but not vaccination-only, induce neutralizing activity in the nasal mucosa. Consistent with immunological imprinting, most antibodies derived from memory B cells or plasma cells of Omicron breakthrough cases cross-react with the Wuhan-Hu-1, BA.1 and BA.2 receptor-binding domains whereas Omicron primary infections elicit B cells of narrow specificity. While most clinical antibodies have reduced neutralization of Omicron, we identified an ultrapotent pan-variant antibody, that is unaffected by any Omicron lineage spike mutations and is a strong candidate for clinical development.

immunology↗

bioRxiv2022

ACE2 engagement exposes the fusion peptide to pan-coronavirus neutralizing antibodies

Coronaviruses use diverse Spike (S) glycoproteins to attach to host receptors and fuse with target cells. Using a broad screening approach, we isolated from SARS-CoV-2 immune donors seven monoclonal antibodies (mAbs) that bind to all human alpha and beta coronavirus S proteins. These mAbs recognize the fusion peptide and acquire high affinity and breadth through somatic mutations. Despite targeting a conserved motif, only some mAbs show broad neutralizing activity in vitro against alpha and beta coronaviruses, including Omicron BA.1 variant and bat WIV-1, and reduce viral titers and pathology in vivo. Structural and functional analyses show that the fusion peptide-specific mAbs bind with different modalities to a cryptic epitope which is concealed by prefusion-stabilizing 2P mutations and becomes exposed upon binding of ACE2 or ACE2-mimicking mAbs. This study identifies a new class of pan-coronavirus neutralizing mAbs and reveals a receptor-induced conformational change in the S protein that exposes the fusion peptide region.

immunology↗

bioRxiv2022

SARS-CoV-2 Omicron potently neutralized by a novel antibody with unique Spike binding properties

The SARS-CoV-2 Omicron variant exhibits very high levels of transmission, pronounced resistance to authorized therapeutic human monoclonal antibodies and reduced sensitivity to vaccine-induced immunity. Here we describe P2G3, a human monoclonal antibody (mAb) isolated from a previously infected and vaccinated donor, which displays picomolar-range neutralizing activity against Omicron BA.1, BA.1.1, BA.2 and all other current variants, and is thus markedly more potent than all authorized or clinically advanced anti-SARS-CoV-2 mAbs. Structural characterization of P2G3 Fab in complex with the Omicron Spike demonstrates unique binding properties to both down and up spike trimer conformations at an epitope that partially overlaps with the receptor-binding domain (RBD), yet is distinct from those bound by all other characterized mAbs. This distinct epitope and angle of attack allows P2G3 to overcome all the Omicron mutations abolishing or impairing neutralization by other anti-SARS-COV-2 mAbs, and P2G3 accordingly confers complete prophylactic protection in the SARS-CoV-2 Omicron monkey challenge model. Finally, although we could isolate in vitro SARS-CoV2 mutants escaping neutralization by P2G3 or by P5C3, a previously described broadly active Class 1 mAb, we found these viruses to be lowly infectious and their key mutations extremely rare in the wild, and we could demonstrate that P2G3/P5C3 efficiently cross-neutralized one anothers escapees. We conclude that this combination of mAbs has great prospects in both the prophylactic and therapeutic settings to protect from Omicron and other VOCs.

immunology↗

bioRxiv2022

Ivermectin does not protect against SARS-CoV-2 infection in the Syrian hamster model

Ivermectin, an FDA-approved antiparasitic drug, has been reported to have in vitro activity against SARS-CoV-2. An increasing off-label use of Ivermectin for COVID-19 has been reported. We here assessed the effect of Ivermectin in Syrian hamsters infected with the SARS-CoV-2 Beta (B.1.351) variant. Infected animals received a clinically relevant dose of Ivermectin (0.4 mg/kg subcutaneously dosed) once daily for four consecutive days after which the effect was quantified. Ivermectin monotherapy did not reduce lung viral load and even significantly worsened the SARS-CoV-2-induced lung pathology. Additionally, it did not potentiate the activity of Molnupiravir (Lagevrio) when combined with this drug. This study contributes to the growing body of evidence that Ivermectin does not result in a beneficial effect in the treatment of COVID-19. These findings are important given the increasing, dangerous off-label use of Ivermectin for the treatment of COVID-19.

microbiology↗

bioRxiv2022

Development and optimisation of a high-throughput screening assay for in vitro anti-SARS-CoV-2 activity: evaluation of 5676 phase 1 passed structures

Although vaccines are currently used to control the coronavirus disease 2019 (COVID-19) pandemic, treatment options are urgently needed for those who cannot be vaccinated and for future outbreaks involving new severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2) strains or coronaviruses not covered by current vaccines. Thus far, few existing antivirals are known to be effective against SARS-CoV-2 and clinically successful against COVID-19. As part of an immediate response to the COVID-19 pandemic, a high-throughput, high content imaging-based SARS-CoV-2 infection assay was developed in VeroE6-eGFP cells and was used to screen a library of 5676 compounds that passed phase 1 clinical trials. Eight candidates (nelfinavir, RG-12915, itraconazole, chloroquine, hydroxychloroquine, sematilide, remdesivir, and doxorubicin) with in vitro anti-SARS-CoV-2 activity in VeroE6-eGFP and/or Caco-2 cell lines were identified. However, apart from remdesivir, toxicity and pharmacokinetic data did not support further clinical development of these compounds for COVID-19 treatment.

immunology↗

bioRxiv2022

Biodistribution and Environmental Safety of a Live-attenuated YF17D-vectored SARS-CoV-2 Vaccine Candidate

New platforms are urgently needed for the design of novel prophylactic vaccines and advanced immune therapies. Live-attenuated yellow fever vaccine YF17D serves as vector for several licensed vaccines and platform for novel vaccine candidates. Based on YF17D, we developed YF-S0 as exceptionally potent COVID-19 vaccine candidate. However, use of such live RNA virus vaccines raises safety concerns, i.e., adverse events linked to original YF17D (yellow fever vaccine-associated neurotropic; YEL-AND, and viscerotropic disease; YEL-AVD). In this study, we investigated the biodistribution and shedding of YF-S0 in hamsters. Likewise, we introduced hamsters deficient in STAT2 signaling as new preclinical model of YEL-AND/AVD. Compared to parental YF17D, YF-S0 showed an improved safety with limited dissemination to brain and visceral tissues, absent or low viremia, and no shedding of infectious virus. Considering yellow fever virus is transmitted by Aedes mosquitoes, any inadvertent exposure to the live recombinant vector via mosquito bites is to be excluded. The transmission risk of YF-S0 was hence evaluated in comparison to readily transmitting YFV-Asibi strain and non-transmitting YF17D vaccine, with no evidence for productive infection of vector mosquitoes. The overall favorable safety profile of YF-S0 is expected to translate to other novel vaccines that are based on the same YF17D platform.

microbiology↗

bioRxiv2021

Remdesivir, Molnupiravir and Nirmatrelvir remain active against SARS-CoV-2 Omicron and other variants of concern.

We assessed the in vitro antiviral activity of remdesivir and its parent nucleoside GS-441524, molnupiravir and its parent nucleoside EIDD-1931 and the viral protease inhibitor nirmatrelvir against the ancestral SARS-CoV2 strain and the five variants of concern including Omicron. VeroE6-GFP cells were pre-treated overnight with serial dilutions of the compounds before infection. The GFP signal was determined by high-content imaging on day 4 post-infection. All molecules have equipotent antiviral activity against the ancestral virus and the VOCs Alpha, Beta, Gamma, Delta and Omicron. These findings are in line with the observation that the target proteins of these antivirals (respectively the viral RNA dependent RNA polymerase and the viral main protease Mpro) are highly conserved.

microbiology↗

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

Anti-SARS-CoV-2 human antibodies retaining neutralizing activity against SARS-CoV-2 B.1.1.529 (omicron)

SARS-CoV-2 B.1.1.529, designated omicron, was recently identified as a new variant of concern by WHO and is rapidly replacing SARS-CoV-2 delta as the most dominant variant in many countries. Unfortunately, because of the high number of mutations present in the spike of SARS-CoV-2 omicron, most monoclonal antibodies (mAbs) currently approved for treatment of COVID-19 lose their in vitro neutralizing activity against this variant. We recently described a panel of human anti-SARS-CoV-2 mAbs that potently neutralize SARS-CoV-2 Wuhan, D614G and variants alpha, beta, gamma and delta. In this work, we evaluated our mAb panel for potential in vitro activity against SARS-CoV-2 delta and omicron. Three mAbs from our panel retain neutralizing activity against both delta and omicron, with mAb 3B8 still resulting in complete neutralization at a concentration as low as 0.02 g/ml for both variants. Overall, our data indicate that mAb 3B8 may have the potential to become a game-changer in the fight against the continuously evolving SARS-CoV-2.

immunology↗

bioRxiv2021

A high-throughput neutralization assay for yellow fever serodiagnostics

Quick and accurate detection of neutralizing antibodies (nAbs) against yellow fever is essential in serodiagnosis during outbreaks, for surveillance and to evaluate vaccine efficacy in population-wide studies. All this requires serological assays that can process a large number of samples in a highly standardized format. Albeit being laborious, time-consuming and limited in throughput, classical plaque reduction neutralization test (PRNT) is still considered gold standard for the detection and quantification of nAbs due to its sensitivity and specificity. Here we report the development of an alternative fluorescence-based serological assay (SNTFLUO) with an equally high sensitivity and specificity that is fit for high-throughput testing with the potential for automation. Finally, our novel SNTFLUO was cross-validated in several reference laboratories and against international WHO standards showing its potential to be implemented in clinical use. SNTFLUO assays with similar performance are available for the Japanese encephalitis, Zika and dengue viruses amenable for differential diagnostics. IMPORTANCEFast and accurate detection of neutralizing antibodies (nAbs) against yellow fever virus (YFV) is key in yellow fever serodiagnosis, outbreak surveillance and monitoring of vaccine efficacy. Although classical PRNT still remains gold standard for measuring YFV nAbs, this methodology suffers from inherent limitations such as a low throughput and an overall high labor intensity. We present a novel fluorescence-based serum neutralization test (SNTFLUO) with equally high sensitivity and specificity that is fit for processing large number of samples in a highly standardized manner and has the potential to be implemented in clinical use. In addition, we present SNTFLUO assays with similar performance for Japanese encephalitis, Zika and dengue viruses opening new avenues for differential diagnostics.

microbiology↗