Biology subjects

Goffinet, C.

Publications and source records attributed to Goffinet, C..

17 recordsLinked to original sources

bioRxiv2025

Synergistic latency reversal by the SMAC mimetic AZD5582 and iBET JQ1, combined with Nef ablation, facilitates immune-mediated elimination of latently HIV-1-infected T-cells

The shock-and-kill strategy can reactivate latent HIV-1 in vivo but has failed to meaningfully reduce the reactivatable reservoir, suggesting insufficient immunological visibility of infected cells. To investigate latency reversal agent (LRA)-specific shortcomings, we developed a novel HIV-1 latency model - Jurkat E6.1 subclonal T-cell lines, each harbouring a single, full-length, NL4.3-based provirus with a GFPOPT reporter in the Env V5 loop - and quantified HIV-1 reactivation, Env surface presentation across LRAs and synergistically acting combinations. Bryostatin-1 induced GBP5-mediated restriction of surface Env and resistance to apoptosis. Bryostatin-1/JQ1 synergised for HIV-1 reactivation and boosted levels of cell-surface Env, but failed to reduce resistance to apoptosis. AZD5582/JQ1 boosted cell-surface Env levels, without co-induction of GBP5, T-cell activation or apoptotic resistance. HIV-1 Nef antagonised cytotoxic killing of HIV-1-reactivating T-cells - its knockout, combined with AZD5582/JQ1, achieved the highest immune-mediated elimination of latently infected T-cells, representing a promising HIV cure approach.

microbiology↗

bioRxiv2024

MPXV Infects Human PBMCs in a Type I Interferon-Sensitive Manner

MPOX virus (MPXV), formerly known as monkeypox virus, led to a rapidly evolving pandemic starting May 2022, with over 90,000 cases reported beyond the African continent. This pandemic outbreak was driven by the MPXV variant Clade IIb. In addition, Clade I viruses circulating in the Democratic Republic of Congo (DRC) are drawing increased attention as cases constantly rise and Clade Ib, first identified in 2023, is now co-circulating with Clade Ia and seems to exhibit enhanced human-to-human transmissibility. While most infected individuals display a self-limiting disease with singular pox-like lesions, some endure systemic viral spread leading to whole-body rash with risks for necrosis, organ loss, and death. Intra-host dissemination and cellular tropism of MPXV are largely unexplored in humans. To establish a potential susceptibility of circulating immune cells to MPXV, we exposed human PBMCs from healthy donors ex vivo to a currently circulating MPXV clade IIb virus isolate in absence and presence of IFN-2a. qPCR of DNA extracted from cell lysates, but less from supernatants, revealed increasing MPXV DNA quantities that peaked at five to six days post-exposure, suggesting susceptibility of PBMCs to infection. IFN-2a pretreatment markedly reduced the quantity of MPXV DNA, suggesting that infection is sensitive to type I IFNs. Plaque assays from supernatants showed that infection gave rise to de novo production of infectious MPXV. In virus-inclusive scRNA-sequencing, monocytes, cycling NK cells and regulatory CD4+ T-cells scored positive for viral RNA, suggesting that these are the MPXV-susceptible cell types within the human PBMC population. Analysis of differentially expressed genes displayed a pronounced downregulation of expression pathways driving innate immunity in MPXV-infected cells, a well-established feature of poxviral infection. Pretreatment of PBMCs with current antivirals Cidofovir and Tecovirimat resulted in reduced amounts of viral antigen production and of released infectivity, suggesting suitability of the human PBMC infection model as a platform for evaluation of current and future antivirals and justifying trials to investigate Cidofovir and Tecovirimat as drugs reducing intra-patient viral spread. Together, our data suggest that human PBMCs are productively infected by MPXV which is accompanied by significant modulation of the cellular milieu. Our results have the potential to illuminate aspects of intra-host propagation of MPXV that may involve a lymphohematogenous route for replication and/or intra-host dissemination.

immunology↗

bioRxiv2024

Neural Network-Assisted Humanization of COVID-19 Hamster scRNAseq Data Reveals Matching Severity States in Human Disease

Translating findings from animal models to human disease is essential for dissecting disease mechanisms, developing and testing precise therapeutic strategies. The coronavirus disease 2019 (COVID-19) pandemic has highlighted this need, particularly for models showing disease severity-dependent immune responses. Single-cell transcriptomics (scRNAseq) is well poised to reveal similarities and differences between species at the molecular and cellular level with unprecedented resolution. However, computational methods enabling detailed matching are still scarce. Here, we provide a structured scRNAseq-based approach that we applied to scRNAseq from blood leukocytes originating from humans and hamsters affected with moderate or severe COVID-19. Integration of COVID-19 patient data with two hamster models that develop moderate (Syrian hamster, Mesocricetus auratus) or severe (Roborovski hamster, Phodopus roborovskii) disease revealed that most cellular states are shared across species. A neural network-based analysis using variational autoencoders quantified the overall transcriptomic similarity across species and severity levels, showing highest similarity between neutrophils of Roborovski hamsters and severe COVID-19 patients, while Syrian hamsters better matched patients with moderate disease, particularly in classical monocytes. We further used transcriptome-wide differential expression analysis to identify which disease stages and cell types display strongest transcriptional changes. Consistently, hamsters response to COVID-19 was most similar to humans in monocytes and neutrophils. Disease-linked pathways found in all species specifically related to interferon response or inhibition of viral replication. Analysis of candidate genes and signatures supported the results. Our structured neural network-supported workflow could be applied to other diseases, allowing better identification of suitable animal models with similar pathomechanisms across species. Key PointsO_LINeural networks can successfully match disease states between animal models and humans using single-cell data as shown for COVID-19 C_LIO_LIModerately diseased patients best matched Syrian hamster cells; severely diseased patients best matched Roborovski hamster neutrophils C_LI

immunology↗

bioRxiv2023

Single-cell-resolved interspecies comparison identifies a shared inflammatory axis and a dominant neutrophil-endothelial program in severe COVID-19

Key issues for research of COVID-19 pathogenesis are the lack of biopsies from patients and of samples at the onset of infection. To overcome these hurdles, hamsters were shown to be useful models for studying this disease. Here, we further leveraged the model to molecularly survey the disease progression from time-resolved single-cell RNA-sequencing data collected from healthy and SARS-CoV-2-infected Syrian and Roborovski hamster lungs. We compared our data to human COVID-19 studies, including BALF, nasal swab, and post-mortem lung tissue, and identified a shared axis of inflammation dominated by macrophages, neutrophils, and endothelial cells, which we show to be transient in Syrian and terminal in Roborovski hamsters. Our data suggest that, following SARS-CoV-2 infection, commitment to a type 1 or type 3-biased immunity determines moderate versus severe COVID-19 outcomes, respectively. One-Sentence SummaryActivation of different immunological programs upon SARS-CoV-2 infection determines COVID-19 severity.

immunology↗

bioRxiv2022

HIV-1 infection causes depletion of monocytic cells through a non-canonical cell death pathway

Programmed cell death is a regulatory mechanism to eliminate infected or damaged cells. Several programmed cell death pathways exist, including apoptosis, necroptosis and pyroptosis, which can be distinguished by the cellular molecules involved. Here we show that infection of monocytic cells with HIV-1 causes cell death, which is dose- and cell type dependent and occurs independently of nucleic acid sensing or interferon (IFN) signaling. Death is observed in the case of near full-length viruses that produce viral proteins upon infection, but not in case of a minimal lentiviral vector that does not express viral gene products, demonstrating the necessity of viral gene products or a near-full length RNA genome to trigger death. Inhibition of reverse transcription or integration rescues cells, indicating that a step after integration is responsible. Using mutant viruses, we further narrow down the step in the retroviral replication cycle that triggers death. Inhibition of diverse cell death pathways individually cannot rescue cells from death following infection, consistent with PANoptosis, a cellular death process that cannot be accounted for by any single programmed cell death pathway alone. Our results elucidate the viral and cellular determinants of cell death caused by HIV-1 infection and outline cellular responses that result in the depletion of specific cell populations.

microbiology↗

bioRxiv2022

Pharmacological inhibition of bromodomain and extra-terminal proteins induces NRF-2-mediated inhibition of SARS-CoV-2 replication and is subject to viral antagonism

Inhibitors of bromodomain and extra-terminal proteins (iBETs), including JQ-1, have been suggested as potential therapeutics against SARS-CoV-2 infection. However, molecular mechanisms underlying JQ-1-induced antiviral activity and its susceptibility to viral antagonism remain incompletely understood. iBET treatment transiently inhibited infection by SARS-CoV-2 variants and SARS-CoV, but not MERS-CoV. Our functional assays confirmed JQ-1-mediated downregulation of ACE2 expression and multi-omics analysis uncovered induction of an antiviral NRF-2-mediated cytoprotective response as an additional antiviral component of JQ-1 treatment. Serial passaging of SARS-CoV-2 in the presence of JQ-1 resulted in predominance of ORF6-deficient variants. JQ-1 antiviral activity was transient in human bronchial airway epithelial cells (hBAECs) treated prior to infection and absent when administered therapeutically. We propose that JQ-1 exerts pleiotropic effects that collectively induce a transient antiviral state that is ultimately nullified by an established SARS-CoV-2 infection, raising questions on their clinical suitability in the context of COVID-19.

microbiology↗

bioRxiv2022

SARS-CoV-2 entry route impacts a range of downstream viral and cellular processes

SARS-CoV-2 entry is promoted by both cell-surface TMPRSS2 and endolysosomal cathepsins. To investigate the impact of differentially routed virions on host and viral processes, lung epithelial cells expressing distinct combinations of entry factors were infected with authentic viruses. Entry route determined early rates of viral replication and transcription, egress and inhibitor sensitivity, with differences observed between virus strains. Transcriptional profiling revealed that induction of innate immunity was correlated to viral genome and transcript abundance in infected cells. Surface entry triggered early activation of antiviral responses, reducing cumulative virion production, while endolysosomal entry delayed antiviral responses and prolonged virus shedding due to extended cell viability. The likely molecular footprints of escape from antiviral effector targeting were also recorded in viral genomes and correlated with entry route-dependent immune status of cells. TMPRSS2 orthologues from diverse mammals, but not zebra fish, facilitated infection enhancement, which was more pronounced for ancestral strains. Leveraging RNA-seq and scRNA-seq datasets from SARS-CoV-2 infected hamsters, we validate aspects of our model in vivo. In summary, we demonstrate that distinct cellular and viral processes are linked to viral entry route, collectively modulating virus shedding, cell-death rates and viral genome evolution.

microbiology↗

bioRxiv2022

A live attenuated vaccine confers superior mucosal and systemic immunity to SARS-CoV-2 variants

Vaccines are a cornerstone in COVID-19 pandemic management. Here, we compare immune responses to and preclinical efficacy of the mRNA vaccine BNT162b2, an adenovirus-vectored spike vaccine, and the live-attenuated-virus vaccine candidate sCPD9 after single and double vaccination in Syrian hamsters. All regimens containing sCPD9 showed superior efficacy. The robust immunity elicited by sCPD9 was evident in a wide range of immune parameters after challenge with heterologous SARS-CoV-2 including rapid viral clearance, reduced tissue damage, fast differentiation of pre-plasmablasts, strong systemic and mucosal humoral responses, and rapid recall of memory T cells from lung tissue. Our results demonstrate that use of live-attenuated vaccines may offer advantages over available COVID-19 vaccines, specifically when applied as booster, and may provide a solution for containment of the COVID-19 pandemic.

immunology↗

bioRxiv2022

Non-productive exposure of PBMCs to SARS-CoV-2 induces cell-intrinsic innate immunity responses

Cell-intrinsic responses mounted in vivo in PBMCs during mild and severe COVID-19 differ quantitatively and qualitatively. Whether they are triggered by signals emitted by productively infected cells of the respiratory tract or are, at least partially, resulting from physical interaction with virus particles, remains unclear. Here, we analyzed susceptibility and expression profiles of PBMCs from healthy donors upon ex vivo exposure to SARS-CoV and SARS-CoV-2. In line with the absence of detectable ACE2 receptor expression, human PBMCs were refractory to productive infection. Bulk and single cell RNA-sequencing revealed JAK/STAT-dependent induction of interferon-stimulated genes, but not pro-inflammatory cytokines. This SARS-CoV-2-specific response was most pronounced in monocytes. SARS-CoV-2-RNA-positive monocytes displayed a lower ISG signature as compared to bystander cells of the identical culture. This suggests a preferential invasion of cells with a low ISG base-line profile or delivery of a SARS-CoV-2-specific sensing antagonist upon efficient particle internalization. Together, non-productive physical interaction of PBMCs with SARS-CoV-2-but not SARS-CoV particles stimulates JAK/STAT-dependent, monocyte-accentuated innate immune responses that resemble those detected in vivo in patients with mild COVID-19.

immunology↗

bioRxiv2021

Insights into standards of care: dexamethasone and antibodies against COVID-19 in hamster models

RationaleIn face of the ongoing SARS-CoV-2 pandemic, effective and well-understood treatment options are still scarce. While vaccines have proven instrumental in fighting SARS-CoV-2, their efficacy is challenged by vaccine hesitancy, novel variants and short-lasting immunity. Therefore, understanding and optimization of therapeutic options remains essential. ObjectivesWe aimed at generating a deeper understanding on how currently used drugs, specifically dexamethasone and anti-SARS-CoV-2 antibodies, affect SARS-CoV-2 infection and host responses. Possible synergistic effects of both substances are investigated to evaluate combinatorial treatments. MethodsBy using two COVID-19 hamster models, pulmonary immune responses were analyzed to characterize effects of treatment with either dexamethasone, anti-SARS-CoV-2 spike monoclonal antibody or a combination of both. scRNA sequencing was employed to reveal transcriptional response to treatment on a single cell level. Measurements and main resultsDexamethasone treatment resulted in similar or increased viral loads compared to controls. Anti-SARS-CoV-2 antibody treatment alone or combined with dexamethasone successfully reduced pulmonary viral burden. Dexamethasone exhibited strong anti-inflammatory effects and prevented fulminant disease in a severe COVID-19-like disease model. Combination therapy showed additive benefits with both anti-viral and anti-inflammatory potency. Bulk and single-cell transcriptomic analyses confirmed dampened inflammatory cell recruitment into lungs upon dexamethasone treatment and identified a candidate subpopulation of neutrophils specifically responsive to dexamethasone. ConclusionsOur analyses i) confirm the anti-inflammatory properties and indicate possible modes of action for dexamethasone, ii) validate anti-viral effects of anti-SARS-CoV-2 antibody treatment, and iii) reveal synergistic effects of a combination therapy and can thus inform more effective COVID-19 therapies.

molecular biology↗

bioRxiv2021

Post-entry, spike-dependent replication advantage of B.1.1.7 and B.1.617.2 over B.1 SARS-CoV-2 in an ACE2-deficient human lung cell line

Epidemiological data demonstrate that SARS-CoV-2 variants of concern (VOC) B.1.1.7 and B.1.617.2 are more transmissible and infections are associated with a higher mortality than non-VOC virus infections. Phenotypic properties underlying their enhanced spread in the human population remain unknown. B.1.1.7 virus isolates displayed inferior or equivalent spread in most cell lines and primary cells compared to an ancestral B.1 SARS-CoV-2, and were outcompeted by the latter. Lower infectivity and delayed entry kinetics of B.1.1.7 viruses were accompanied by inefficient proteolytic processing of spike. B.1.1.7 viruses failed to escape from neutralizing antibodies, but slightly dampened induction of innate immunity. The bronchial cell line NCI-H1299 supported 24- and 595-fold increased growth of B.1.1.7 and B.1.617.2 viruses, respectively, in the absence of detectable ACE2 expression and in a spike-determined fashion. Superior spread in NCI-H1299 cells suggests that VOCs employ a distinct set of cellular cofactors that may be unavailable in standard cell lines.

microbiology↗

bioRxiv2021

A realistic touch-transfer method reveals low risk of transmission for SARS-CoV-2 by contaminated coins and bank notes

The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created a significant threat to global health. While respiratory aerosols or droplets are considered as the main route of human-to-human transmission, secretions expelled by infected individuals can also contaminate surfaces and objects, potentially creating the risk of fomite-based transmission. Consequently, frequently touched objects such as paper currency and coins have been suspected as a potential transmission vehicle. To assess the risk of SARS-CoV-2 transmission by banknotes and coins, we examined the stability of SARS-CoV-2 and bovine coronavirus (BCoV), as surrogate with lower biosafety restrictions, on these different means of payment and developed a touch transfer method to examine transfer efficiency from contaminated surfaces to skin. Although we observed prolonged virus stability, our results, including a novel touch transfer method, indicate that the transmission of SARS-CoV-2 via contaminated coins and banknotes is unlikely and requires high viral loads and a timely order of specific events.

microbiology↗

bioRxiv2020

Longitudinal omics in Syrian hamsters integrated with human data unravel complexity of moderate immune responses to SARS-CoV-2

In COVID-19, the immune response largely determines disease severity and is key to therapeutic strategies. Cellular mechanisms contributing to inflammatory lung injury and tissue repair in SARS-CoV-2 infection, particularly endothelial cell involvement, remain ill-defined. We performed detailed spatiotemporal analyses of cellular and molecular processes in SARS-CoV-2 infected Syrian hamsters. Comparison of hamster single-cell sequencing and proteomics with data sets from COVID-19 patients demonstrated inter-species concordance of cellular and molecular host-pathogen interactions. In depth vascular and pulmonary compartment analyses (i) supported the hypothesis that monocyte-derived macrophages dominate inflammation, (ii) revealed endothelial inflammation status and T-cell attraction, and (iii) showed that CD4+ and CD8+ cytotoxic T-cell responses precede viral elimination. Using the Syrian hamster model of self-limited moderate COVID-19, we defined the specific roles of endothelial and epithelial cells, among other myeloid and non-myeloid lung cell subtypes, for determining the disease course.

immunology↗

bioRxiv2020

Human IFITM3 restricts Chikungunya virus and Mayaro virus infection and is susceptible to virus-mediated counteraction

Interferon-induced transmembrane (IFITM) proteins restrict infection by enveloped viruses through interfering with membrane fusion and virion internalisation. The role of IFITM proteins during alphaviral infection of human cells and viral counteraction strategies remain largely unexplored. Here, we characterized the impact of IFITM proteins and variants on entry and spread of Chikungunya virus (CHIKV) and Mayaro virus (MAYV) in human cells, and provide first evidence for a CHIKV-mediated antagonism of IFITM proteins. IFITM1, 2 and 3 restricted infection at the level of alphavirus glycoprotein-mediated entry, both in the context of direct infection and during cell-to-cell transmission. Relocalization of normally endosomal IFITM3 to the plasma membrane resulted in the loss of its antiviral activity. rs12252-C, a naturally occurring variant of IFITM3 that has been proposed to associate with severe influenza in humans, restricted CHIKV, MAYV and influenza A virus infection as efficiently as wild-type IFITM3. Finally, all antivirally active IFITM variants displayed reduced cell surface levels in CHIKV-infected cells involving a posttranscriptional process mediated by one or several non-structural protein(s) of CHIKV.

microbiology↗

bioRxiv2020

IFITM proteins promote SARS-CoV-2 infection in human lung cells

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) are thought to restrict numerous viral pathogens including severe acute respiratory syndrome coronaviruses (SARS-CoVs). However, most evidence comes from single-round pseudovirus infection studies of cells that overexpress IFITMs. Here, we verified that artificial overexpression of IFITMs blocks SARS-CoV-2 infection. Strikingly, however, endogenous IFITM expression was essential for efficient infection of genuine SARS-CoV-2 in human lung cells. Our results indicate that the SARS-CoV-2 Spike protein interacts with IFITMs and hijacks them for efficient viral entry. IFITM proteins were expressed and further induced by interferons in human lung, gut, heart and brain cells. Intriguingly, IFITM-derived peptides and targeting antibodies inhibited SARS-CoV-2 entry and replication in human lung cells, cardiomyocytes and gut organoids. Our results show that IFITM proteins are important cofactors for SARS-CoV-2 infection of human cell types representing in vivo targets for viral transmission, dissemination and pathogenesis and suitable targets for therapeutic approaches.

microbiology↗

bioRxiv2020

Cell-intrinsic Innate Immune Responses against Chikungunya Virus in a Human Ex Vivo Synovial Fibroblast Model

In recent years, (re-)emerging arboviruses including Chikungunya virus (CHIKV) and Mayaro virus (MAYV) have caused growing concern due to expansion of insect vector ranges. No protective vaccine or specific antiviral strategies are currently available. Long-term morbidity after CHIKV infection includes debilitating chronic joint pain, which has associated health and economic impact. Here, we analyzed the early cell-intrinsic response to CHIKV and MAYV infection in primary human synovial fibroblasts. This interferon-competent cell type represents a potential source of polyarthralgia induced by CHIKV infection. Synovial fibroblasts from healthy and osteoarthritic donors were similarly permissive to CHIKV and MAYV infection ex vivo. Using RNA-seq, we defined a CHIKV infection-induced transcriptional profile with several hundred interferon-stimulated and arthralgia-mediating genes upregulated. Type I interferon was both secreted by infected fibroblasts and protective when administered exogenously. IL-6 secretion, which mediates chronic synovitis, however, was not boosted by infection. Single-cell RNA-seq and flow cytometric analyses uncovered an inverse correlation of activation of innate immunity and productive infection at the level of individual cells. In summary, primary human synovial fibroblasts serve as bona-fide ex vivo primary cell model of CHIKV infection and provide a valuable platform for studies of joint tissue-associated aspects of CHIKV immunopathogenesis.

microbiology↗

bioRxiv2020

Mono-ADP-ribosylation by ARTD10 restricts Chikungunya virus replication by interfering with the proteolytic activity of nsP2

A subset of intracellular mono-ADP-ribosyltransferases diphtheria toxin-like (ARTDs, aka mono-PARPs) is induced by type I interferons. Some of these mono-ARTDs feature antiviral activity while certain RNA viruses, including Chikungunya virus (CHIKV), encode mono-ADP-ribosylhydrolases, suggesting a role for mono-ADP-ribosylation (MARylation) in host-virus conflicts. CHIKV expresses four non-structural proteins (nsP1-nsP4), with nsP3 containing a macrodomain that hydrolyzes and thereby reverses protein MARylation in vitro and in cells. This de-MARylation activity is essential as hydrolase inactivating mutations result in replication defective virus. However, the substrates of MARylation during CHIKV infection are unknown and thus it is unclear how the macrodomain contributes to virus replication and how mono-ARTD-dependent MARylation confers antiviral immunity. We identified ARTD10 and ARTD12 as restriction factors for CHIKV replication in a catalytic activity-dependent manner. CHIKV replication requires processing of the non-structural polyprotein nsP1-4 by the nsP2-encoded protease and the assembly of the four individual nsPs into a functional replication complex. Expression of ARTD10 and ARTD12 resulted in a reduction of processed nsPs. Similarly, MAR hydrolase inactive CHIKV replicon mutants revealed a decrease in processed nsPs, comparable to an nsP2 protease defective mutant. This suggested that the macrodomain contributes to nsP2 protease activity. In support, a hydrolase-deficient virus was complemented by a protease-deficient virus. We hypothesized that MARylation regulates the proteolytic function of nsP2. Indeed, we found that nsP2 is MARylated by ARTD10. This inhibited nsP2 protease activity, thereby preventing polyprotein processing and consequently virus replication. This inhibition was antagonized by the MAR hydrolase activity of nsP3. Together, our findings provide a mechanistic explanation for the need of the viral MAR hydrolase for efficient replication of CHIKV. Author SummaryInfectious diseases still pose major health threats. Especially fast evolving viruses find ever new strategies to manipulate the immune response. With climate warming and increased human mobility vector-borne pathogens like Chikungunya virus (CHIKV) spread and cause world-wide epidemics. Beyond the acute phase, CHIKV patients regularly suffer from chronic rheumatism. This entails a decline in life quality and an economic burden. To date no drugs are approved and the mode of pathogenesis remains elusive. Here we describe a mechanistic function of the CHIKV nsP3 macrodomain. We found that the viral nsP2 is mono-ADP-ribosylated interfering with its auto-proteolytic function. The nsP3 macrodomain removes this modification and restores the protease activity that is essential for replication. Because macrodomains are highly conserved they might represent broad antiviral targets.

microbiology↗