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Hardy, D.

Publications and source records attributed to Hardy, D..

At least 19 recordsLinked to original sources

Targeting Sec61 to Silence Pathogenic Light-Chain Production in AL Amyloidosis

Unstable immunoglobulin light chains (LCs) secreted by clonal plasma cells drive organ damage in AL amyloidosis, yet no therapy exist to directly suppress their biosynthesis. Here, we targeted the Sec61 translocon to block LC biogenesis. In patient-derived plasma cells, the Sec61 blocker mycolactone redirected nascent LCs to proteasomal degradation, inducing proteotoxic stress that synergized with bortezomib. In vivo, this combination reduced plasma-cell engraftment and LC secretion but caused toxicity, underscoring the need for Sec61 client selectivity. Proteomic profiling of a model patient-derived plasma cell line treated with the synthetic Sec61 inhibitors A317 and A347 revealed intermediate- and narrow-spectrum client selectivity, respectively, while retaining activity against the LC. However, unlike A317, the more selective A347 failed to inhibit diverse amyloidogenic LCs in a signal-peptide reporter assay. In primary plasma cells from an AL cohort, A317 consistently reduced pathogenic LC production while sparing matched non-malignant bone marrow cells. Together, these findings establish Sec61 as a therapeutic target in AL amyloidosis and identify the need to balance client selectivity with coverage of diverse amyloidogenic LC signal peptides.

cell biology↗

Single-cell spatially resolved transcriptomic characterization of the developing mouse cochlea

The cochlea, the sensory organ of hearing, functions as a frequency analyzer, analogous to a musical instrument. During development, while the medio-lateral axis supports differentiation of sensory cells and their surrounding supporting cells, the longitudinal axis underlies frequency-dependent properties of the cochlea. The combination of these two gene expression gradients defines unique physiological attributes of each cell intimately linked to its position within the cochlea. To determine which cochlear cell-types have a transcriptomic signature sensitive to these two gradients and identify the underlying gene regulatory networks, we took advantage of the advent of spatial single cell transcriptomics methodologies. We therefore generated a spatial transcriptomic atlas reaching single cell resolution based on the Visium HD technique, a sequencing-based technology that employ arrays of spatially barcoded probes to capture RNA molecules unbiasedly from histological tissue sections. Spatial transcriptional changes during embryonic stages, E14 and E16, as well as during postnatal development, P1 and P8, were investigated. Based on this dataset, not only cell-type assignment in single cell RNA-seq experiments could be validated, but the classification for some cell-types could be refined. Gradients of gene expression along the medio-lateral and longitudinal axes in multiple cell-types together with their temporal dynamics across development were also uncovered. Altogether, this atlas paves the way for deciphering gene regulatory networks controlling gene expression as a function of position in the cochlear cell types, providing a valuable resource for the design of efficient, robust and safe gene therapy strategies.

cell biology↗

Spatial organization of mAbs in complex with the virus glycoprotein determines the success of intravenous rabies therapy

Rabies is a complex disease that has defied efforts to develop an effective treatment. Although it can be successfully prevented through vector control or pre- or post-exposure prophylaxis, no therapeutic options exist for symptomatic rabies, and the disease is still responsible for about 59 000 human deaths each year. Following our successful attempts to use the human monoclonal antibodies RVC20 and RVC58 for intracerebroventricular treatment of rabies, we report here on the effects of these mAbs when administered intravenously and intramuscularly to symptomatic mice. Only a small proportion of mAbs was detected to cross the blood-brain barrier, yet this limited amount sufficiently cleared the viral infection and improved the survival rate of infected mice. Remarkably, these mAbs appeared more effective when used individually than when combined as a cocktail. RVC58 achieved an 80% survival rate, potentially due to its distinct binding topography, potent viral neutralization, and enhanced Fc{gamma}R engagement in the presence of virions. These findings suggest that monotherapy with a single mAb fully capable of engaging Fc{gamma}R may be possible, which could substantially lower the costs and improve treatment accessibility in under-resourced settings, potentially contributing to improved equity of global health. Taken together, this study supports mAb therapy as a promising preclinical approach for symptomatic rabies and provides a foundation for further development of an accessible treatment option.

microbiology↗

Fluke-borne viruses are a risk factor for diseases caused by carcinogenic trematodes

Three trematode parasites, Schistosoma haematobium, Opisthorchis viverrini, and Clonorchis sinensis are recognized group 1 carcinogens for their roles in squamous cell carcinoma of the urinary bladder and bile duct cancer (cholangiocarcinoma). We aimed to assess the presence of fluke viruses and their association with malignancy in fluke infections. We showed that all three carcinogenic trematodes harbored related viruses. Focusing on O. viverrini, we observed virus persistence at discrete life stages and viral tropism for the fluke tegument, and we provided molecular and serological evidence of parasitized hosts exposure to viral RNA and proteins. Serological analyses confirmed seroconversion of residents in endemic sites and revealed that antibody titers and avidity against fluke-borne viruses increase in individuals with hepatobiliary diseases. Random forest models discriminated efficiently between liver-fluke-infected participants without disease and those with periductal fibrosis or cholangiocarcinoma. Thus, we show that fluke-borne viruses are risk factors and diagnostic targets for fluke-associated cancer.

microbiology↗

A modified oncolytic measles virus exhibits strong immunotherapeutic potential through RIG-I activation by defective viral genomes

Oncolytic viruses can destroy tumors directly or by activating antitumor immunity, but balancing safety with potent immune stimulation remains challenging. Here, we show that deletion of the measles virus C protein, a key viral antagonist of innate immunity, reprograms the live-attenuated vaccine strain into a RIG-I-driven cancer immunotherapy. The resulting virus, MVdeltaC, accumulates defective viral genomes that activate RIG-I/MAVS signaling and trigger robust type I interferon and pro-inflammatory cytokine responses. MVdeltaC kills tumor cells more rapidly and efficiently than the parental virus and induces hallmarks of immunogenic cell death, including HMGB1 release and dendritic cell maturation. Intratumoral administration in immunocompetent mice bearing syngeneic neuroblastoma induced complete tumor regression in 90% of animals and established long-term antitumor memory. Antitumor responses were dependent on CD8 T and NK cells and were further enhanced by anti-CTLA-4 therapy or CD4 T-cell depletion. Prior measles immunization accelerated tumor clearance, indicating vaccine-boosted responses. MVdeltaC also controlled the growth of human mesothelioma, melanoma, and triple-negative breast cancer xenografts and patient-derived tumors in immunodeficient models. These findings establish MVdeltaC as a clinically ready, broad-spectrum immunotherapeutic that links RIG-I activation through defective viral genome generation to elicit potent and durable antitumor immunity. IMPACT StatementA modified measles virus lacking a viral innate immunity antagonist triggers potent antitumor responses via RIG-I sensing of defective viral genomes, revealing a new strategy for cancer immunotherapy.

immunology↗

Improved Immune Responses and Tuberculosis Protection by Aerosol Vaccination with recombinant BCG expressing ESX-1 from Mycobacterium marinum

The currently licensed anti-tuberculosis (TB) vaccine, Mycobacterium bovis BCG, provides limited protection against pulmonary TB in adolescents and adults, the main cause of TB transmission and mortality. To obtain an improved BCG-based vaccine candidate with increased immune signaling but still low virulence, we have previously generated a recombinant BCG strain named BCG::ESX-1Mmar, which is heterologously expressing ESX-1 functions of Mycobacterium marinum and thereby modulates the host innate immune responses via phagosomal rupture-associated induction of type I interferon production and enhanced inflammasome activity, leading to superior protection against TB disease in murine infection models. As protection may also vary with the route of vaccination, here, we have explored aerosol vaccination relative to subcutaneous vaccination, using BCG Pasteur and BCG::ESX-1Mmar. We found that mice vaccinated via the aerosol route with BCG Pasteur or BCG::ESX-1Mmar both yielded higher frequencies of CD4+ and CD8+ T effector memory (TEM) cells in the lungs compared to subcutaneously immunised mice, whereas comparable poly-functional Th1 (IL-2, TNF- and IFN-{gamma}) cytokine-producing subsets were observed in the spleens of all vaccinated mice. Significantly higher IL-17 responses without severe lung pathology were seen in the lungs of aerosol-vaccinated mice associated to local and transient inflammatory cytokine responses and immune cell infiltrations. Aerosol vaccination also elicited high amounts of humoral IgG and IgM responses in the bronchoalveolar lavage fluid and induced substantial lung CD69+ CD103+ T resident memory (TRM) cells, containing both CD4+ and CD8+ T subsets, in the airways of immunised mice, whereas this was not the case for subcutaneous vaccination. These effects led to significant improved protection against M. tuberculosis and reduced lung pathology in aerosol-vaccinated mice compared to subcutaneously vaccinated mice. Moreover, BCG::ESX-1Mmar induced enhanced T-cell immunity and superior protection compared to parental BCG Pasteur for both vaccination routes and thereby represents an interesting candidate for developing improved vaccination strategies against TB. Author summaryAnti-tuberculosis vaccine efficacy is influenced by multiple parameters, including the immunogenicity of the vaccine strain, the type of preclinical host model used, and the route of vaccination. Given recent advances in the field of mucosal vaccination, in the current study we were particularly interested to explore and compare aerosol-based vaccination with standard subcutaneous vaccination in a C57BL/6J mouse model using our recently developed recombinant BCG::ESX-1Mmar vaccine candidate in comparison with parental BCG Pasteur. Our results show that in this setting the protective efficacy of mucosal vaccination was superior to subcutaneous vaccination for both vaccine strains, whereby the use of BCG::ESX-1Mmar induced additional benefits in terms of bacterial load reduction compared to standard BCG Pasteur. Taken together, we propose that aerosol vaccination using BCG::ESX-1Mmar as live-attenuated vaccine candidate is a promising and powerful combination for obtaining improved protection against an M. tuberculosis challenge, a concept that can now be tested in other animal models in a perspective of a putative clinical trial.

microbiology↗

Hamsters with long Covid exhibits a neurodegenerative signature in the brainstem

After infection with SARS-CoV-2, patients may present with one or more symptoms that appear or persist over time, including fatigue, respiratory, cardiovascular and neurological disorders. Neurological symptoms include anxiety, depression and impaired short-term memory. However, the exact underlying mechanisms of long Covid are not yet decrypted. Using the golden hamster as a model, we provide further evidence that SARS-CoV-2 is neuroinvasive and can persist in the central nervous system, as we found viral RNA and replicative virus in the brainstem after 80 days of infection. Infected hamsters presented a neurodegenerative signature in the brainstem, with overexpression of innate immunity genes, impacted dopaminergic and glutamatergic synapses, altered energy metabolism. Finally, the infected hamsters manifested persistent signs of depression and impaired short-term memory, as well as late-onset signs of anxiety, as a valuable model to study long Covid. Conclusively, we provide evidence that virus-related and neurodegenerative and immunometabolic mechanisms coexist in the brainstem of infected hamsters and contribute to the manifestation of neuropsychiatric and cognitive symptoms. HighlightsO_LISARS-CoV-2 infects and persists in the brainstem of intranasally-inoculated hamsters C_LIO_LIPersistent neuropsychiatric and cognitive consequences are observed in SARS-CoV-2-infected hamsters C_LIO_LIThe brainstem present distinct transcriptome profiles in acute and in long Covid C_LIO_LIThe dopaminergic and glutamatergic systems are affected in long Covid C_LIO_LIThe SARS-CoV-2 infection affects the expression of genes related to neurodegenerative processes in acute and in long Covid C_LI

pathology↗

The SARS-CoV-2 envelope PDZ Binding Motif acts as a virulence factor disrupting host's epithelial cell-cell junctions

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the virus responsible for the COVID-19 pandemic, has significantly impacted global health, emphasizing the need to understand its pathogenicity and virulence mechanisms. SARS-CoV-2 disrupts the alveolar epithelial barrier and exacerbates airway inflammation, leading to acute respiratory failure, but the molecular details remain unclear. Additionally, SARS-CoV-2 infection causes neurological symptoms, potentially due to its weakly understood ability to cross the blood-brain barrier. The viral multifunctional Envelope (E) protein is crucial for its virulence, playing a key role in virus assembly, budding, and release. The E protein contains a PDZ-binding motif (PBM) that interacts with host PDZ domain-containing proteins, potentially affecting host signaling pathways and contributing to pathogenicity. This study focuses on the E protein PBM and its role in virulence, disrupting respiratory epithelial barriers and exacerbating airway inflammation. We generated recombinant mutant viruses lacking the PBM and conducted both in vitro and in vivo experiments to elucidate its impact on viral fitness, pathogenicity, and effects on the epithelial integrity. In vitro, the viral mutants showed delayed replication and reduced cytopathic effects. In vivo, experiments with hamsters revealed that PBM-deficient viruses caused less weight loss, lower viral loads, and reduced inflammation, indicating decreased pathogenicity. Histological analyses confirmed less airway damage in these hamsters compared to those infected with the wild-type virus. Additionally, PBM-deficient viruses had impaired interactions with tight junction proteins like ZO-1, a PDZ-containing protein, crucial for maintaining epithelial barrier integrity. Our findings also demonstrate that the PBM does not play a significant role in neuroinvasion during the acute phase of infection, as evidenced by comparable viral RNA loads across brain regions in infected hamsters, regardless of PBM presence. Histopathological and transcriptomic analyses further support this observation, suggesting that the PBM primarily affects specific epithelial barriers. Additionally, RNA-seq analysis on lung and brainstem from infected hamsters reveals that the PBM modulates inflammatory and immune responses, with a stronger impact in lung tissue than in the brainstem. PBM-deficient viruses induce lower levels of inflammation and cytokine expression, suggesting PBMs specific role in enhancing viral pathogenicity through the activation of pathways such as NF-{kappa}B and TNF. Thus, the E protein PBM plays a critical role in SARS-CoV-2s fitness, virulence, and pathogenicity, through the disruption of cell junctions and inflammation, underscoring its potential as a target for therapeutic interventions.

pathology↗

Myocarditis and neutrophil-mediated vascular leakage but not cytokine storm associated with fatal murine leptospirosis

Leptospirosis is a neglected re-emerging zoonosis caused by Leptospira spirochetes. Its pathophysiology remains mysterious, especially in the case of severe infection with L. interrogans. In the field of infectious diseases, the cause of death is rarely investigated in preclinical models. Here, for the first time, we identified unanticipated organ failures associated with death in a murine model of acute leptospirosis. Despite clinical similarities between bacterial sepsis and leptospirosis, striking differences were observed. Neither lung, liver, or kidney injury nor cytokine storm, or massive necroptosis could explain death. In contrast, severe leptospirosis was associated with high serum levels of the anti-inflammatory cytokine IL-10 and the chemokine RANTES, neutrophilia, pancreatitis and vascular damage. Unexpectedly, we demonstrated neutrophil-induced vascular permeability, making neutrophils a potential new therapeutic target. Strikingly, the main cause of death was myocarditis, an overlooked complication of human leptospirosis. These features are also found in patients, making this model a paradigm for better understanding human leptospirosis and designing novel therapeutic strategies.

immunology↗

A recombinant CHIKV-NLuc virus identifies chondrocytes as target of Chikungunya virus in a immunocompetent mouse model

First isolated in 1953 in Tanzania, the arthritogenic Chikungunya virus (CHIKV) re-emerged globally in 2005, leading to widespread outbreaks. Unlike other arboviruses, CHIKV predominantly induces symptomatic infections (72-96%), marked by fever, myalgia, polyarthralgia, and rash. Although rarely fatal, atypical forms such as encephalopathies can occur. Notably, 75.4% of patients experience persistent arthralgias for up to three years after the acute phase. Understanding CHIKVs pathophysiology in the joints is challenging due to the difficulties to obtain biological samples. The study employs a mouse model infected with a reporter virus expressing a Nano Luciferase to investigate the diseases transition to chronic arthritis. The murine model reveals viral replication in metatarsi joints, particularly in chondrocytes, confirmed in primary human chondrocytes undergoing viral-induced apoptosis. Ex vivo analysis confirmed viral replication in leg bones and articular cartilages, with histological evidence of focal erosive lesions and periarticular inflammation. The study further utilizes an in vivo imaging mouse model to monitor viral replication over time. Human chondrocytes prove susceptible to CHIKV infection, exhibiting active viral replication, bioluminescence activity, and increased viral production. CHIKV induced apoptosis, the upregulation of markers associated with cartilage remodeling and altered the cytokine production. This comprehensive study, utilizing advanced techniques and models, provides insights into CHIKVs ability to infect articular cartilages, shedding light on the mechanisms of chronic arthritis following infection.

microbiology↗

An oxadiazole-based compound potentiates anti- tuberculosis treatment by increasing host resistance via zinc poisoning

Anti-tuberculosis drugs, mostly developed over 60 years ago, combined with a poorly effective vaccine, have failed to eradicate tuberculosis. More worryingly, multi-resistant strains of Mycobacterium tuberculosis are constantly emerging. Innovative strategies are thus urgently needed to improve tuberculosis treatment. Recently, host-directed therapy has emerged as a promising strategy to be used in adjunct with existing or future antibiotics, by improving innate immunity or limiting immunopathology. Here, using high content imaging, we identified novel 1,2,4-oxadiazole-based compounds, that allow human macrophages to control MTB replication. Genome-wide gene expression analysis revealed that these molecules induced zinc remobilization inside cells, resulting in bacterial zinc intoxication. More importantly, we also demonstrated that, upon treatment with these novel compounds, M. tuberculosis became even more sensitive to anti-tuberculosis drugs, in vitro and in vivo, in a mouse model of tuberculosis. Manipulation of heavy metal homeostasis holds thus great promise to be exploited to develop host-directed therapeutic interventions.

microbiology↗

Distinct subtypes of grey matter heterotopia show subtype-specific morpho-electric neuronal properties and dynamics of epileptiform activity in mice

Grey matter heterotopia (GMH) are neurodevelopmental disorders associated with abnormal cortical function and epilepsy. Subcortical band heterotopia (SBH) and periventricular nodular heterotopia (PVNH) are two well-recognized GMH subtypes in which neurons are misplaced, either forming nodules lining the ventricles in PVNH, or forming bands in the white matter in SBH. Although both PVNH and SBH are commonly associated with epilepsy, it is unclear whether these two GMH subtypes differ in terms of pathological consequences or, on the contrary, share common altered mechanisms. Here, we studied two robust preclinical models of SBH and PVNH, and performed a systematic comparative assessment of the physiological and morphological diversity of heterotopia neurons, as well as the dynamics of epileptiform activity and input connectivity. We uncovered a complex set of altered properties, including both common and distinct physiological and morphological features across heterotopia subtypes, and associated with specific dynamics of epileptiform activity. Taken together, these results suggest that pro-epileptic circuits in GMH are, at least in part, composed of neurons with distinct, subtype-specific, physiological and morphological properties depending on the heterotopia subtype. Our work supports the notion that GMH represent a complex set of disorders, associating both shared and diverging pathological consequences, and contributing to forming epileptogenic networks with specific properties. A deeper understanding of these properties may help to refine current GMH classification schemes by identifying morpho-electric signatures of GMH subtypes, to potentially inform new treatment strategies.

neuroscience↗

Mice Humanized for Major Histocompatibility Complex and Angiotensin-Converting Enzyme 2 with High Permissiveness to SARS-CoV-2 Omicron Replication

Human Angiotensin-Converting Enzyme 2 (hACE2) is the major receptor enabling host cell invasion by SARS-CoV-2 via interaction with Spike glycoprotein. The murine ACE2 ortholog does not interact efficiently with SARS-CoV-2 Spike and therefore the conventional laboratory mouse strains are not permissive to SARS-CoV-2 replication. Here, we generated new hACE2 transgenic mice, which harbor the hACE2 gene under the human keratin 18 promoter, in C57BL/6 "HHD-DR1" background. HHD-DR1 mice are fully devoid of murine Major Histocompatibility Complex (MHC) molecules of class-I and -II and express only MHC molecules from Human Leukocyte Antigen (HLA) HLA 02.01, DRA01.01, DRB1.01.01 alleles, widely expressed in human populations. We selected three transgenic strains, with various hACE2 mRNA expression levels and distinctive profiles of lung and/or brain permissiveness to SARS-CoV-2 replication. Compared to the previously available B6.K18-ACE22Prlmn/JAX mice, which have limited permissiveness to SARS-CoV-2 Omicron replication, these three new hACE2 transgenic strains display higher levels of hACE2 mRNA expression, associated with high permissiveness to the replication of SARS-CoV-2 Omicron sub-variants. As a first application, one of these MHC- and ACE2-humanized strains was successfully used to show the efficacy of a lentiviral vector-based COVID-19 vaccine candidate.

microbiology↗

Early suppression of excitability in subcortical band heterotopia modifies epileptogenesis in rats

Malformations of cortical development represent a major cause of epilepsy in childhood. However, the pathological substrate and dynamic changes leading to the development and progression of epilepsy remain unclear. Here, we characterized an etiology-relevant rat model of subcortical band heterotopia (SBH), a diffuse type of cortical malformation associated with drug-resistant seizures in humans. We used longitudinal electrographic recordings to monitor the age-dependent evolution of epileptiform discharges during the course of epileptogenesis in this model. We found both quantitative and qualitative age-related changes in seizures properties and patterns, accompanying a gradual progression towards a fully developed seizure pattern seen in adulthood. We also dissected the relative contribution of the band heterotopia and the overlying cortex to the development and age-dependent progression of epilepsy using timed and spatially targeted manipulation of neuronal excitability. We found that an early suppression of neuronal excitability in SBH slows down epileptogenesis in juvenile rats, whereas epileptogenesis is paradoxically exacerbated when excitability is suppressed in the overlying cortex. However, in rats with active epilepsy, similar manipulations of excitability have no effect on chronic spontaneous seizures. Together, our data support the notion that complex developmental alterations occurring in both the SBH and the overlying cortex concur to creating pathogenic circuits prone to generate seizures. Our study also suggests that early and targeted interventions could potentially influence the course of these altered developmental trajectories, and favorably modify epileptogenesis in malformations of cortical development. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/506850v2_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@32b0deorg.highwire.dtl.DTLVardef@1f9c40dorg.highwire.dtl.DTLVardef@b6348borg.highwire.dtl.DTLVardef@33bb12_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

Neuroinvasion and anosmia are independent phenomena upon infection with SARS-CoV-2 and its variants

SUMMARYAnosmia was identified as a hallmark of COVID-19 early in the pandemic, however, with the emergence of variants of concern, the clinical profile induced by SARS-CoV-2 infection has changed, with anosmia being less frequent. Here, we assessed the clinical, olfactory and neuroinflammatory conditions of golden hamsters infected with the original Wuhan SARS-CoV-2 strain, its isogenic ORF7-deletion mutant and three variants: Gamma, Delta, and Omicron/BA.1. We show that infected animals developed a variant-dependent clinical disease including anosmia, and that the ORF7 of SARS-CoV-2 contributes to the induction of olfactory dysfunction. Conversely, all SARS- CoV-2 variants were found to be neuroinvasive, regardless of the clinical presentation they induce. Taken together, this confirms that neuroinvasion and anosmia are independent phenomena upon SARS-CoV-2 infection. Using newly generated nanoluciferase-expressing SARS-CoV-2, we validated the olfactory pathway as a major entry point into the brain in vivo and demonstrated in vitro that SARS-CoV-2 travels retrogradely and anterogradely along axons in microfluidic neuron-epithelial networks. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=151 HEIGHT=200 SRC="FIGDIR/small/505985v2_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@8225ceorg.highwire.dtl.DTLVardef@80342aorg.highwire.dtl.DTLVardef@e0dfd1org.highwire.dtl.DTLVardef@1861e13_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

Revealing invisible cell phenotypes with conditional generative modeling

Biological sciences, drug discovery and medicine rely heavily on cell phenotype perturbation and observation. Aside from dramatic events such as cell division or cell death, most cell phenotypic changes that keep cells alive are subtle and thus hidden from us by natural cell variability: two cells in the same condition already look different. While we show that deep learning models can leverage invisible features from microscopy images, to discriminate between close conditions, these features can yet hardly be observed and therefore interpreted. In this work, we show that conditional generative models can be used to transform an image of cells from any one condition to another, thus canceling cell variability. We visually and quantitatively validate that the principle of synthetic cell perturbation works on discernible cases such as high concentration drug treatments, nuclear translocation and golgi apparatus assays. We then illustrate its effectiveness in displaying otherwise invisible cell phenotypes triggered by blood cells under parasite infection, the presence of a disease-causing pathological mutation in differentiated neurons derived from iPSCs or low concentration drug treatments. The proposed approach, easy to use and robust, opens the door to the accessible discovery of biological and disease biomarkers.

bioinformatics↗

Splenic clearance of rigid erythrocytes as an inherited mechanism for splenomegaly and natural resistance to malaria

In malaria-endemic areas, subjects from specific groups like Fulani have a peculiar protection against malaria, with high levels of IgM but also frequent anemia and splenomegaly. The mechanisms underlying this phenotype remain elusive. In Benin, West Africa, we measured the deformability of circulating erythrocytes in genetically distinct groups (including Fulani) living in sympatry, using ektacytometry and microsphiltration, a mimic of how the spleen clears rigid erythrocytes. Compared to non-Fulani, Fulani displayed a higher deformability of circulating erythrocytes, pointing to an enhanced clearance of rigid erythrocytes by the spleen. This phenotype was observed in individuals displaying markers of Plasmodium falciparum infection. The heritability of this new trait was high, with a strong multigenic component. Five of the top 10 genes selected by a population structure-adjusted GWAS, expressed in the spleen, are potentially involved in splenic clearance of erythrocytes (CHERP, MB, PALLD, SPARC, PDE10A), through control of vascular tone, collagen synthesis and macrophage activity. In specific ethnic groups, genetically-controlled processes likely enhance the innate retention of infected and uninfected erythrocytes in the spleen, explaining splenomegaly, anemia, cryptic intrasplenic parasite loads, hyper-IgM, and partial protection against malaria. Beyond malaria-related phenotypes, inherited splenic hyper-filtration of erythrocytes may impact the pathogenesis of other hematologic diseases. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSThe genetic background of individuals influences their susceptibility to infectious diseases. Specific human groups, like the Fulani in Africa, react to malaria parasites (named Plasmodium) in a specific way. Upon infection, Fulani develop a grossly enlarged spleen, and high levels of anti-Plasmodium antibodies in their blood. They also carry smaller numbers of parasites in their blood, and thus are considered partially protected against malaria. The mechanisms underlying this natural protection, different from other natural protective mechanisms such as the sickle cell trait, are not well understood. Malaria impairs the deformability of red blood cells and the spleen is a key organ to controlling red blood cell quality. We have recently demonstrated that red blood cells containing live malaria parasites accumulate intensely in the spleen of subjects with long term exposure to these parasites. Enhanced retention of infected and uninfected red blood cells in the spleen would explain why the spleen is larger and why lower numbers of parasites are left in circulation. We thus explored whether the retention of infected and uninfected red blood cells could explain why Fulani are partially protected against malaria. Because it is unethical to perform spleen puncture or biopsies for research purposes, our explorations were indirect by carefully analyzing the properties of circulating red blood cells in a large number of subjects and by assessing whether observations could be explained by their genetic make-up. Added value of this studyIn more than 500 subjects, we confirmed the high frequency of large spleens in Fulani and, through 2 different methods, we demonstrated an enhanced deformability of their circulating red blood cells, that likely stems from the more efficient removal of the less deformable ones. This enhanced deformability was found to be inheritable based on carefully collected family links and refined analysis of genetic markers. Implications of all the available evidenceOur findings indicate that genes potentially driving the filtration of red blood cells by the spleen likely influence how subjects in specific groups in Africa and elsewhere react to malaria. While most previous hypotheses pointed to conventional immunological mechanisms as the trigger, we propose that a simple physiological mechanism that controls the quality of red blood cells may drive natural protection from malaria even before the intervention of immunological cells. A better understanding of these processes is of great importance in the context of malaria elimination efforts. These findings may also have an impact on the understanding of other red blood cell-related disorders, such as inherited red cell diseases, in which splenic filtration of abnormal red blood cells may precipitate splenic complications.

physiology↗

An intranasal lentiviral booster broadens immune recognition of SARS-CoV-2 variants and reinforces the waning mRNA vaccine-induced immunity that it targets to lung mucosa

As the COVID-19 pandemic continues and new SARS-CoV-2 variants of concern emerge, the adaptive immunity initially induced by the first-generation COVID-19 vaccines wains and needs to be strengthened and broadened in specificity. Vaccination by the nasal route induces mucosal humoral and cellular immunity at the entry point of SARS-CoV-2 into the host organism and has been shown to be the most effective for reducing viral transmission. The lentiviral vaccination vector (LV) is particularly suitable for this route of immunization because it is non-cytopathic, non-replicative and scarcely inflammatory. Here, to set up an optimized cross-protective intranasal booster against COVID-19, we generated an LV encoding stabilized Spike of SARS-CoV-2 Beta variant (LV::SBeta-2P). mRNA vaccine-primed and -boosted mice, with waning primary humoral immunity at 4 months post-vaccination, were boosted intranasally with LV::SBeta-2P. Strong boost effect was detected on cross-sero-neutralizing activity and systemic T-cell immunity. In addition, mucosal anti-Spike IgG and IgA, lung resident B cells, and effector memory and resident T cells were efficiently induced, correlating with complete pulmonary protection against the SARS-CoV-2 Delta variant, demonstrating the suitability of the LV::SBeta-2P vaccine candidate as an intranasal booster against COVID-19.

immunology↗