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Fortin, A.

Publications and source records attributed to Fortin, A..

8 recordsLinked to original sources
bioRxiv2025

No Evidence of Direct Activation of Human Neutrophil Responses by Multivalent Prefusion Trimeric SARS-CoV-2 Spike Protein ex vivo

The SARS-CoV-2 Spike (S) protein is essential for viral entry and serves as the primary immunogen in most COVID-19 vaccines. While its role in adaptive immunity is well defined, its potential to contribute directly to innate immune activation and remains incompletely understood. Neutrophils, in particular, are prominent effectors in COVID-19 severity, yet how they respond directly to the S protein presented in a multivalent format is unclear. Here, we investigated whether the S protein can directly activate human neutrophils ex vivo using two biologically relevant models: nanoparticles displaying multivalent stabilized prefusion trimeric S glycoprotein, and purified {beta}-propiolactone-inactivated SARS-CoV-2 virions. Neutrophils were exposed to nanoparticles or inactivated virus, either alone or pre-coated with monoclonal or polyclonal anti-S antibodies. Nanoparticles displaying Respiratory Syncytial Virus (RSV) Fusion (F) protein and purified {beta}-propiolactone-inactivated RSV served as comparators. Across all models and conditions tested, the S protein did not induce significant neutrophil responses. No consistent effects were observed on cell viability, surface marker expression, reactive oxygen species production, neutrophil extracellular trap formation, cytokine release, or inflammatory gene expression--even in the presence of anti-S antibodies mimicking immune complexes. Results with F-nanoparticles and inactivated RSV were similarly modest. These findings indicate that the trimeric prefusion S protein, whether displayed multivalently on nanoparticles or in the context of inactivated viral particles, is insufficient to trigger robust neutrophil activation. This work provides insight into the innate immune profile of the S protein and suggests that its use in vaccine platforms is unlikely to directly provoke neutrophil-mediated inflammatory responses.

immunology↗
bioRxiv2025

Validation and multi-site deployment of a lyophilized qRT-PCR reagent for the molecular diagnosis of avian influenza and rabies in Sub-Saharan African regions

Molecular methods are widely accepted as gold standard techniques for the laboratory diagnosis of most human and animal pathogens. However, most molecular protocols rely on reagents that need to be transported and stored at a freezing temperature, a requirement that might affect their reliability in areas where the cold chain cannot be guaranteed. Over the years, several lyophilized molecular products have been marketed to circumvent this issue. We therefore evaluated the feasibility of replacing liquid reagents with freeze-dried formulations for the molecular diagnosis of avian influenza (AIV) and rabies (RABV) viruses, two priority zoonotic pathogens widely spread in Sub-Saharan Africa. Among the available kits, we selected the Qscript lyo 1-step kit (Quantabio) due to its easy-to-use features, single-reaction format, and preliminary performance assessment. Through a more in-depth evaluation, we determined its analytical and diagnostic performance and formulation stability, and obtained results comparable to those of standard liquid master mixes. Notably, for the detection of divergent lyssaviruses, the lyophilized reagents sensitivity was affected by suboptimal complementarity between the oligonucleotides and the target sequences. Finally, a multi-site evaluation in four veterinary diagnostic laboratories located in Sub-Saharan Africa demonstrated the successful deployment of AIV and RABV assays utilizing freeze-dried reagents, which can interchangeably replace liquid master mixes. Altogether our results indicate that the Qscript lyo 1-step kit (Quantabio) represents a valid alternative to wet reagents for the molecular diagnosis of avian influenza and rabies, and has the potential for broader applications to other relevant infectious diseases upon proper validation. Author summaryMolecular diagnostic protocols rely on reagents that need to be transported and stored at freezing temperatures. Meeting this requirement can be challenging in areas where the maintenance of the cold chain is not guaranteed, such as in sub-Saharan Africa. Our study aimed to assess the feasibility of using lyophilized reagents as a replacement for liquid reagents in the molecular diagnosis of two widespread zoonotic pathogens in Sub-Saharan Africa, namely avian influenza and rabies. To accomplish this, we selected a commercially available lyophilized reagent based on its format and performance characteristics. We conducted a laboratory validation to assess the use of the lyophilized reagent throughout the entire diagnostic process. We also conducted a reproducibility test involving African laboratories as potential end-users. Our findings confirm that the lyophilized reagent can replace traditional liquid reagents to diagnose rabies and avian influenza and suggest its possible use for a wider range of infectious diseases after undergoing appropriate validation.

microbiology↗
bioRxiv2024

Broadly Reactive Anti-VHH Antibodies for Characterizing, Blocking, or Activating Nanobody-Based CAR-T Cells

Production of chimeric antigen receptor T cell (CAR-T) therapies is dependent on the use of antibody reagents to label, isolate, and/or expand T cell products. We sought to create antibody-based tools that directly target the variable domain of heavy-chain only antibodies (VHH or nanobody) used in some CAR molecules. Two murine antibodies were identified which bind to distinct epitopes in the conserved framework regions of llama-derived VHHs, and not to human VH domains. We produced a high-quality dual-clonal anti-VHH antibody product which reacts with over 98% of VHH proteins, regardless of their antigenic specificity. Anti-VHH binding did not disrupt VHH/antigen interaction, and thus could be used for secondary labeling to assess cellular or tissue reactivity of VHH molecules. Despite not interfering with antigen binding, anti-VHH antibodies potently inhibited VHH-CAR function, blocking CAR-T activation and cytolytic killing of target cells. When immobilized, anti-VHH antibodies could also be applied for activation and expansion of VHH CAR-T cells, inducing 730-fold mean expansion, >94% CAR purity, with retained CD8/CD4 heterogeneity. Functionally, anti-VHH antibody-expanded CAR-T cells maintained strong antigen specific activity without functional exhaustion. Overall, these data identify a useful new tool for understanding and manipulating VHH-based CAR-T cells. Funding SourceThis work was funded by the National Research Council Canada Disruptive Technology Solutions Cell and Gene Therapy challenge program, and BioCanRx Declaration of interestsThe anti-VHH antibodies reported here are the subject of a provisional patent application by the National Research Council of Canada

immunology↗
bioRxiv2020

Pinpointing of cysteine oxidation sites in vivo by high-resolution proteomics reveals mechanism of redox-dependent inhibition of STING

Protein function is regulated by post-translational modifications, among which reversible oxidation of Cys (Cys ox-PTM) emerged as a key regulatory mechanism of cellular responses. The redox regulation of virus-host interactions is well documented, but in most cases, proteins subjected to Cys ox-PTM remain unknown. The identification of Cys ox-PTM sites in vivo is essential to underpin our understanding of the mechanisms of the redox regulation. In this study, we present a proteome-wide identification of reversible Cys ox-PTM sites in vivo during stimulation by oxidants using a maleimide-based bioswitch method coupled to mass spectrometry. We identified 2720 unique Cys ox-PTM sites encompassing 1473 proteins with distinct abundance, location and functions. Label-free quantification (LFQ)-based analysis revealed the enrichment of ox-PTM in numerous pathways, many relevant to virus-host interaction. Here, we focused on the oxidation of STING, the central adaptor of the innate immune type I interferon pathway induced upon detection of cytosolic DNA. We provide the first in vivo demonstration of reversible oxidation of Cys148 and Cys206 of STING. Molecular analyses led us to establish a new model in which Cys148 oxidation is constitutive, while Cys206 oxidation is inducible by oxidative stress or by the natural ligand 23-cGAMP. We show that oxidation of Cys206 has an inhibitory function to prevent STING hyperactivation through the constraint of a conformational change associated with the formation of inactive polymers containing intermolecular disulfide bonds. This provides new ground for the design of therapies targeting STING relevant to autoinflammatory disorders, immunotherapies and vaccines. Brief summary of the main resultsThe function of proteins is regulated by post-translational modifications, among which reversible oxidation of Cys recently emerged as a key component. Comprehension of redox regulation of cellular responses requires identification of specific oxidation sites in vivo. Using a bioswitch method to specifically label Cys subjected to reversible oxidation coupled to mass spectrometry, we identified thousands of novel oxidation sites. Many are relevant to virus-host interaction pathways. Here, we focused on the oxidation of STING, an adaptor critical for activating the innate immune type I interferon pathway engaged upon cytosolic DNA sensing. Molecular studies led us to establish a new model in which STING Cys148 is oxidized at basal levels, while Cys206 oxidation is induced by oxidative stress and ligand binding. We show that oxidation of Cys206 has an inhibitory function to prevent STING hyperactivation. This study provides ground for novel research avenues aimed at designing therapeutics that target this pathway.

immunology↗
bioRxiv2019

Odor Modulates the Temporal Dynamics of Fear Memory Consolidation

Systems consolidation (SC) theory proposes that recent, contextually rich memories are stored in the hippocampus (HPC). As these memories become remote, they are believed to rely more heavily on cortical structures within the prefrontal cortex (PFC), where they lose much of their contextual detail and become schematized. Odor is a particularly evocative cue for intense remote memory recall and despite these memories being remote, they are highly contextual. In instances such as post-traumatic stress disorder (PTSD), intense remote memory recall can occur years after trauma, which seemingly contradicts SC. We hypothesized that odor may shift the organization of salient or fearful memories such that when paired with an odor at the time of encoding, they are delayed in the de-contextualization process that occurs across time, and retrieval may still rely on the HPC, where memories are imbued with contextually rich information, even at remote time points. We investigated this by tagging odor- and non-odor-associated fear memories in male c57BL/6 mice and assessed recall and c-Fos expression in the dorsal CA1 (dCA1) and prelimbic cortex (PL) 1 d or 21 d later. In support of SC, our data showed that recent memories were more dCA1-dependent whereas remote memories were more PL-dependent. However, we also found that odor influenced this temporal dynamic biasing the memory system from the PL to the dCA1 when odor cues were present. Behaviorally, inhibiting the dCA1 with activity-dependent DREADDs had no effect on recall at 1 d and unexpectedly caused an increase in freezing at 21 d. Together, these findings demonstrate that odor can shift the organization of fear memories at the systems level.

neuroscience↗
bioRxiv2018

RNASeq analysis identifies non-canonical role of STAT2 and IRF9 in the regulation of a STAT1-independent antiviral and immunoregulatory transcriptional program induced by IFNβ and TNFα

IFN{beta} typically induces an antiviral and immunoregulatory transcriptional program through the activation of ISGF3 (STAT1, STAT2 and IRF9) transcriptional complexes. The response to IFN{beta} is context-dependent and is prone to crosstalk with other cytokines, such as TNF IFN{beta} and TNF synergize to drive a specific delayed transcriptional program. Previous observation led to the hypothesis that an alternative STAT1-independent pathway involving STAT2 and IRF9 might be involved in gene induction by the combination of IFN{beta} and TNF. Using genome wide transcriptional profiling by RNASeq, we found that the costimulation with IFN{beta} and TNF induces a broad antiviral and immunoregulatory transcriptional program independently of STAT1. Additionally, STAT2 and IRF9 are involved in the regulation of only a subset of these STAT1-independent genes. Consistent with the growing literature, STAT2 and IRF9 act in concert to regulate a subgroup of these genes. Unexpectedly, STAT2 and IRF9 were also engaged in specific independent pathways to regulate distinct sets of IFN{beta} and TNF-induced genes. Altogether these observations highlight the existence of distinct previously unrecognized non-canonical STAT1-independent, but STAT2 and/or IRF9-dependent pathways in the establishment of a delayed antiviral and immunoregulatory transcriptional program in conditions where elevated levels of both IFN{beta} and TNF are present.

immunology↗
bioRxiv2017

DUSP1 promotes apoptosis and suppresses cell migration, while leaving the JIP1-protected cytokine production intact, during paramyxovirus infection.

The host antiviral response involves the induction of interferons and proinflammatory cytokines, but also the activation of cell death pathways, including apoptosis, to limit viral replication and spreading. This host defense is strictly regulated to eliminate the infection while limiting tissue damage that is associated with virus pathogenesis. Post-translational modifications, most notably phosphorylation, are key regulators of the antiviral defense implying an important role of protein phosphatases. Here, we investigated the role of the dual-specificity phosphatase 1 (DUSP1) in the host defense against human respiratory syncytial virus (RSV), a pathogenic virus of the Pneumoviridae family, and Sendai virus (SeV), a model virus being developed as a vector for anti-RSV vaccine. We found that DUSP1 is upregulated before being subjected to proteasomal degradation. DUSP1 does not inhibit the antiviral response, but negatively regulates virus-induced JNK/p38 MAPK phosphorylation. Interaction with the JNK-interacting protein 1 scaffold protein prevents dephosphorylation of JNK by DUSP1, likely explaining that AP-1 activation and downstream cytokine production are protected from DUSP1 inhibition. Importantly, DUSP1 promotes SeV-induced apoptosis and suppresses cell migration in RSV-infected cells. Collectively, our data unveil a previously unrecognized selective role of DUSP1 in the regulation of tissue damage and repair during infections by RSV and SeV.

microbiology↗