Biology subjects

Charneau, P.

Publications and source records attributed to Charneau, P..

8 recordsLinked to original sources

bioRxiv2025

Optimized pipeline for generating highly potent neutralizing Affitins: application to SARS-CoV-2 spike protein

Viral infections represent a global health threat, causing three million deaths annually. Antiviral strategies primarily target viral genome replication or prevent virus entry into host cells. While most FDA-approved antiviral drugs are small chemical molecules, protein-based therapies like monoclonal antibodies (mAbs) offer advantages such as broad and high neutralizing activities, and ability to recruit immune responses to enhance viral clearance. Additionally, alternative protein scaffolds with favorable properties have been developed to substitute or complement mAbs. We have developed Affitins, a novel class of small artificial affinity proteins (7 kDa) derived from hyperthermophilic archaea. Affitins, selected from high-diversity libraries ([~]1012 variants) against any target protein, are highly stable, easy to engineer, and cost-effective to produce. Here, we developed a pipeline to generate Affitins in different multimerization formats targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein through ribosome display selection and assessed their efficacy. The most potent candidates, dimerized or trimerized into 17-27 kDa proteins, displayed high thermal stability, strong binding affinities, and potent neutralization against various SARS-CoV-2 variants, with IC50 values as low as 32 pM. We also report the first hexameric Affitins, formed by dimerizing trimers through Fc fragment fusion ([~]106 kDa), achieving potent neutralization with an IC50 of 0.8 pM, ranking them among the most potent B1.351 SARS-Cov-2 neutralizing proteins. Our findings highlight Affitins as promising antiviral agents, demonstrating their versatility for engineering affinity proteins with higher valencies than that of antibodies while retaining a lower molecular weight, thereby expanding the toolkit for combating viral threats.

biochemistry↗

bioRxiv2022

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↗

bioRxiv2022

Potent Human Broadly SARS-CoV-2 Neutralizing IgA and IgG Antibodies Effective Against Omicron BA.1 and BA.2

Memory B-cell and antibody responses to the SARS-CoV-2 spike protein contribute to long-term immune protection against severe COVID-19, which can also be prevented by antibody-based interventions. Here, wide SARS-CoV-2 immunoprofiling in COVID-19 convalescents combining serological, cellular and monoclonal antibody explorations, revealed humoral immunity coordination. Detailed characterization of a hundred SARS-CoV-2 spike memory B-cell monoclonal antibodies uncovered diversity in their repertoire and antiviral functions. The latter were influenced by the targeted spike region with strong Fc-dependent effectors to the S2 subunit and potent neutralizers to the receptor binding domain. Amongst those, Cv2.1169 and Cv2.3194 antibodies cross-neutralized SARS-CoV-2 variants of concern including Omicron BA.1 and BA.2. Cv2.1169, isolated from a mucosa-derived IgA memory B cell, demonstrated potency boost as IgA dimers and therapeutic efficacy as IgG antibodies in animal models. Structural data provided mechanistic clues to Cv2.1169 potency and breadth. Thus, potent broadly neutralizing IgA antibodies elicited in mucosal tissues can stem SARS-CoV-2 infection, and Cv2.1169 and Cv2.3194 are prime candidates for COVID-19 prevention and treatment.

immunology↗

bioRxiv2022

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↗

bioRxiv2021

sgDI-tector: defective interfering viral genome bioinformatics for detection of coronavirus subgenomic RNAs

Coronavirus RNA-dependent RNA polymerases produce subgenomic RNAs (sgRNAs) that encode viral structural and accessory proteins. User-friendly bioinformatic tools to detect and quantify sgRNA production are urgently needed to study the growing number of next-generation sequencing (NGS) data of SARS-CoV-2. We introduced sgDI-tector to identify and quantify sgRNA in SARS-CoV-2 NGS data. sgDI-tector allowed detection of sgRNA without initial knowledge of the transcription-regulatory sequences. We produced NGS data and successfully detected the nested set of sgRNAs with the ranking M>ORF3a>N>ORF6>ORF7a>ORF8>S>E>ORF7b. We also compared the level of sgRNA production with other types of viral RNA products such as defective interfering viral genomes.

bioinformatics↗

bioRxiv2021

Full Brain and Lung Prophylaxis against SARS-CoV-2 by Intranasal Lentiviral Vaccination in a New hACE2 Transgenic Mouse Model or Golden Hamsters

Non-integrative, non-cytopathic and non-inflammatory lentiviral vectors are particularly suitable for mucosal vaccination and recently emerge as a promising strategy to elicit sterilizing prophylaxis against SARS-CoV-2 in preclinical animal models. Here, we demonstrate that a single intranasal administration of a lentiviral vector encoding a prefusion form of SARS-CoV-2 spike glycoprotein induces full protection of respiratory tracts and totally avoids pulmonary inflammation in the susceptible hamster model. More importantly, we generated a new transgenic mouse strain, expressing the human Angiotensin Converting Enzyme 2, with unprecedent brain permissibility to SARS-CoV-2 replication and developing a lethal disease in <4 days post infection. Even though the neurotropism of SARS-CoV-2 is now well established, so far other vaccine strategies under development have not taken into the account the protection of central nervous system. Using our highly stringent transgenic model, we demonstrated that an intranasal booster immunization with the developed lentiviral vaccine candidate achieves full protection of both respiratory tracts and brain against SARS-CoV-2.

microbiology↗

bioRxiv2020

Intranasal Immunization with a Lentiviral Vector Coding for SARS-CoV-2 Spike Protein Confers Vigorous Protection in Pre-Clinical Animal Models

To develop a vaccine candidate against COVID-19, we generated a Lentiviral Vector (LV), eliciting neutralizing antibodies against the Spike glycoprotein of SARS-CoV-2. Systemic vaccination by this vector in mice, in which the expression of the SARS-CoV-2 receptor hACE2 has been induced by transduction of respiratory tract cells by an adenoviral vector, conferred only partial protection, despite an intense serum neutralizing activity. However, targeting the immune response to the respiratory tract through an intranasal boost with this LV resulted in > 3 log10 decrease in the lung viral loads and avoided local inflammation. Moreover, both integrative and non-integrative LV platforms displayed a strong vaccine efficacy and inhibited lung deleterious injury in golden hamsters, which are naturally permissive to SARS-CoV-2 replication and restitute the human COVID-19 physiopathology. Our results provide evidence of marked prophylactic effects of the LV-based vaccination against SARS-CoV-2 and designate the intranasal immunization as a powerful approach against COVID-19. HighlightsA lentiviral vector encoding for Spike predicts a promising COVID-19 vaccine Targeting the immune response to the upper respiratory tract is key to protection Intranasal vaccination induces protective mucosal immunity against SARS-CoV-2 Lung anti-Spike IgA responses correlate with protection and reduced inflammation

immunology↗

bioRxiv2020

Remodeling of the core leads HIV-1 pre-integration complex in the nucleus of human lymphocytes.

Retroviral replication proceeds through obligate integration of the viral DNA into the host genome. To enter the nucleus, the viral DNA must be led through the nuclear pore complex (NPC). During HIV-1 cytoplasmic journey, the viral core acts like a shell to protect the viral genetic material from antiviral sensors and ensure an adequate environment for the reverse transcription. However, the relatively narrow size of the nuclear pore channel requires that the HIV-1 core reshapes into a structure that fits the pore. On the other hand, the organization of the viral CA proteins that remain associated to the pre-integration complex (PIC) during and after nuclear translocation, in particular, in human lymphocytes, the main target cells of HIV-1, is still enigmatic. In this study, we analysed the progressive organizational changes of viral CA proteins within the cytoplasm and the nucleus by immuno-gold labelling. Furthermore, we set up a novel technology, HIV-1 ANCHOR, which enables specific detection of the retrotranscribed DNA by fluorescence microscopy, thereby uncovering the architecture of the potential HIV-1 PIC. Thus, we revealed DNA- and CA-positive complexes by correlated light- and electron microscopy (CLEM). During and after nuclear translocation, HIV-1 appears as a complex of viral DNA decorated by multiple viral CA proteins remodelled in a "pearl necklace" shape. Thus, we observed how CA proteins reshape around the viral DNA to permit the entrance of the HIV-1 in the nucleus. This particular CA protein complex composed by the integrase and the retrotranscribed DNA leads HIV-1 genome inside the host nucleus to potentially replicate. Our findings contribute to the understanding of the early steps of HIV-1 infection and provide new insights into the organization of HIV-1 CA proteins during and after viral nuclear entry. ImportanceHow the reverse transcribed genome reaches the host nucleus remains a main open question related to the infectious cycle of HIV-1. HIV-1 core has a size of [~]100 nm, largely exceeding that of the NPC channel ([~]39 nm). Thus, a rearrangement of the viral CA proteins organization is required to achieve effective nuclear translocation. The mechanistic of this process remains undefined due to the lack of a technology capable to visualize potential CA sub-complexes in association with the viral DNA in the nucleus of HIV-1-infected cells. By the means of state-of-the-art technologies (HIV-1 ANCHOR system combined with CLEM), our study shows that remodeled viral complexes retain multiple CA proteins but not intact core or only a single CA monomer. These viral CA complexes associated with the retrotranscribed DNA can be observed in the outer and inner side of the NE, and they represent potential PIC. Thus, our study shed light on critical early steps characterizing HIV-1 infection, thereby revealing novel, therapeutically exploitable points of intervention. Furthermore, we developed and provided a powerful tool enabling direct, specific and high-resolution visualization of intracellular and intranuclear HIV-1 subviral structures.

microbiology↗