Viral infection engenders bona fide and bystander subsets of lung-resident memory B cells through a permissive mechanism.

Lung-resident memory B cells (MBCs) provide localized protection against reinfection in respiratory airways. Currently, the biology of these cells remains largely unexplored. Here, we combined influenza and SARS-CoV-2 infection with fluorescent-reporter mice to identify MBCs regardless of antigen specificity. We found that two main transcriptionally distinct subsets of MBCs colonized the lung peribronchial niche after infection. These subsets arose from different progenitors and were both class switched, somatically mutated, and intrinsically biased in their differentiation fate toward plasma cells. Combined analysis of antigen specificity and B cell receptor repertoire segregated these subsets into "bona fide" virus-specific MBCs and "bystander" MBCs with no apparent specificity for eliciting viruses generated through an alternative permissive process. Thus, diverse transcriptional programs in MBCs are not linked to specific effector fates but rather to divergent strategies of the immune system to simultaneously provide rapid protection from reinfection while diversifying the initial B cell repertoire.

Meningeal macrophages protect against viral neuroinfection.

The surface of the central nervous system (CNS) is protected by the meninges, which contain a dense network of meningeal macrophages (MMs). Here, we examined the role of tissue-resident MM in viral infection. MHC-II- MM were abundant neonatally, whereas MHC-II+ MM appeared over time. These barrier macrophages differentially responded to in vivo peripheral challenges such as LPS, SARS-CoV-2, and lymphocytic choriomeningitis virus (LCMV). Peripheral LCMV infection, which was asymptomatic, led to a transient infection and activation of the meninges. Mice lacking macrophages but conserving brain microglia, or mice bearing macrophage-specific deletion of Stat1 or Ifnar, exhibited extensive viral spread into the CNS. Transcranial pharmacological depletion strategies targeting MM locally resulted in several areas of the meninges becoming infected and fatal meningitis. Low numbers of MHC-II+ MM, which is seen upon LPS challenge or in neonates, corelated with higher viral load upon infection. Thus, MMs protect against viral infection and may present targets for therapeutic manipulation.

INFRAFRONTIER quality principles in systemic phenotyping.

Improving reproducibility and replicability in preclinical research is a widely discussed and pertinent topic, especially regarding ethical responsibility in animal research. INFRAFRONTIER, the European Research Infrastructure for the generation, phenotyping, archiving, and distribution of model mammalian genomes, is addressing this issue by developing internal quality principles for its different service areas, that provides a quality framework for its operational activities. This article introduces the INFRAFRONTIER Quality Principles in Systemic Phenotyping of genetically altered mouse models. A total of 11 key principles are included, ranging from general requirements for compliance with guidelines on animal testing, to the need for well-trained personnel and more specific standards such as the exchange of reference lines. Recently established requirements such as the provision of FAIR (Findable, Accessible, Interoperable, Reusable) data are also addressed. For each quality principle, we have outlined the specific context, requirements, further recommendations, and key references.

Genetic, structural, and chemical insights into the dual function of GRASP55 in germ cell Golgi remodeling and JAM-C polarized localization during spermatogenesis.

Spermatogenesis is a dynamic process that is regulated by adhesive interactions between germ and Sertoli cells. Germ cells express the Junctional Adhesion Molecule-C (JAM-C, encoded by Jam3), which localizes to germ/Sertoli cell contacts. JAM-C is involved in germ cell polarity and acrosome formation. Using a proteomic approach, we demonstrated that JAM-C interacted with the Golgi reassembly stacking protein of 55 kDa (GRASP55, encoded by Gorasp2) in developing germ cells. Generation and study of Gorasp2-/- mice revealed that knock-out mice suffered from spermatogenesis defects. Acrosome formation and polarized localization of JAM-C in spermatids were altered in Gorasp2-/- mice. In addition, Golgi morphology of spermatocytes was disturbed in Gorasp2-/- mice. Crystal structures of GRASP55 in complex with JAM-C or JAM-B revealed that GRASP55 interacted via PDZ-mediated interactions with JAMs and induced a conformational change in GRASP55 with respect of its free conformation. An in silico pharmacophore approach identified a chemical compound called Graspin that inhibited PDZ-mediated interactions of GRASP55 with JAMs. Treatment of mice with Graspin hampered the polarized localization of JAM-C in spermatids, induced the premature release of spermatids and affected the Golgi morphology of meiotic spermatocytes.

Neutralization of SARS-CoV-2 and Intranasal Protection of Mice with a nanoCLAMP Antibody Mimetic.

Intranasal treatment, combined with vaccination, has the potential to slow mutational evolution of viruses by reducing transmission and replication. Here, we illustrate the development of a SARS-CoV-2 receptor-binding domain (RBD) nanoCLAMP and demonstrate its potential as an intranasally administered therapeutic. A multi-epitope nanoCLAMP was made by fusing a pM affinity single-domain nanoCLAMP (P2710) to alternate epitope-binding nanoCLAMP, P2609. The resulting multimerized nanoCLAMP P2712 had sub-pM affinity for the Wuhan and South African (B.1.351) RBD (KD < 1 pM) and decreasing affinity for the Delta (B.1.617.2) and Omicron (B.1.1.529) variants (86 pM and 19.7 nM, respectively). P2712 potently inhibited the ACE2:RBD interaction, suggesting its utility as a therapeutic. With an IC50 = 0.4 ± 0.1 nM obtained from neutralization experiments using pseudoviral particles, nanoCLAMP P2712 protected K18-hACE2 mice from SARS-CoV-2 infection, reduced viral loads in the lungs and brains, and reduced associated upregulation of inflammatory cytokines and chemokines. Together, our findings warrant further investigation into the development of nanoCLAMPs as effective intranasally delivered COVID-19 therapeutics.

Intranasal Multiepitope PD-L1-siRNA-Based Nanovaccine: The Next-Gen COVID-19 Immunotherapy.

The first approved vaccines for human use against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are nanotechnology-based. Although they are modular, rapidly produced, and can reduce disease severity, the currently available vaccines are restricted in preventing infection, stressing the global demand for novel preventive vaccine technologies. Bearing this in mind, we set out to develop a flexible nanovaccine platform for nasal administration to induce mucosal immunity, which is fundamental for optimal protection against respiratory virus infection. The next-generation multiepitope nanovaccines co-deliver immunogenic peptides, selected by an immunoinformatic workflow, along with adjuvants and regulators of the PD-L1 expression. As a case study, we focused on SARS-CoV-2 peptides as relevant antigens to validate the approach. This platform can evoke both local and systemic cellular- and humoral-specific responses against SARS-CoV-2. This led to the secretion of immunoglobulin A (IgA), capable of neutralizing SARS-CoV-2, including variants of concern, following a heterologous immunization strategy. Considering the limitations of the required cold chain distribution for current nanotechnology-based vaccines, it is shown that the lyophilized nanovaccine is stable for long-term at room temperature and retains its in vivo efficacy upon reconstitution. This makes it particularly relevant for developing countries and offers a modular system adaptable to future viral threats.

The mammalian ABC transporter ABCA1 induces lipid-dependent drug sensitivity in yeast.

ABCA1 belongs to the A class of ABC transporter, which is absent in yeast. ABCA1 elicits lipid translocation at the plasma membrane through yet elusive processes. We successfully expressed the mouse Abca1 gene in Saccharomyces cerevisiae. The cloned ABCA1 distributed at the yeast plasma membrane in stable discrete domains that we name MCA (membrane cluster containing ABCA1) and that do not overlap with the previously identified punctate structures MCC (membrane cluster containing Can1p) and MCP (membrane cluster containing Pma1p). By comparison with a nonfunctional mutant, we demonstrated that ABCA1 elicits specific phenotypes in response to compounds known to interact with membrane lipids, such as papuamide B, amphotericin B and pimaricin. The sensitivity of these novel phenotypes to the genetic modification of the membrane lipid composition was studied by the introduction of the cho1 and lcb1-100 mutations involved respectively in phosphatidylserine or sphingolipid biosynthesis in yeast cells. The results, corroborated by the analysis of equivalent mammalian mutant cell lines, demonstrate that membrane composition, in particular its phosphatidylserine content, influences the function of the transporter. We thus have reconstituted in yeast the essential functions associated to the expression of ABCA1 in mammals and characterized new physiological phenotypes prone to genetic analysis. This article is a part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).

Anti-HVEM mAb therapy improves antitumoral immunity both in vitro and in vivo, in a novel transgenic mouse model expressing human HVEM and BTLA molecules challenged with HVEM expressing tumors.

BACKGROUND: Tumor necrosis factor superfamily member 14 (TNFRSF14)/herpes virus entry mediator (HVEM) is the ligand for B and T lymphocyte attenuator (BTLA) and CD160-negative immune co-signaling molecules as well as viral proteins. Its expression is dysregulated with an overexpression in tumors and a connection with tumors of adverse prognosis. METHODS: We developed C57BL/6 mouse models co-expressing human (hu)BTLA and huHVEM as well as antagonistic monoclonal antibodies (mAbs) that completely prevent the interactions of HVEM with its ligands. RESULTS: Here, we show that the anti-HVEM18-10 mAb increases primary human αß-T cells activity alone (CIS-activity) or in the presence of HVEM-expressing lung or colorectal cancer cells in vitro (TRANS-activity). Anti-HVEM18-10 synergizes with antiprogrammed death-ligand 1 (anti-PD-L1) mAb to activate T cells in the presence of PD-L1-positive tumors, but is sufficient to trigger T cell activation in the presence of PD-L1-negative cells. In order to better understand HVEM18-10 effects in vivo and especially disentangle its CIS and TRANS effects, we developed a knockin (KI) mouse model expressing human BTLA (huBTLA+/+) and a KI mouse model expressing both huBTLA+/+/huHVEM+/+ (double KI (DKI)). In vivo preclinical experiments performed in both mouse models showed that HVEM18-10 treatment was efficient to decrease human HVEM+ tumor growth. In the DKI model, anti-HVEM18-10 treatment induces a decrease of exhausted CD8+ T cells and regulatory T cells and an increase of effector memory CD4+ T cells within the tumor. Interestingly, mice which completely rejected tumors (±20%) did not develop tumors on rechallenge in both settings, therefore showing a marked T cell-memory phenotype effect. CONCLUSIONS: Altogether, our preclinical models validate anti-HVEM18-10 as a promising therapeutic antibody to use in clinics as a monotherapy or in combination with existing immunotherapies (antiprogrammed cell death protein 1/anti-PD-L1/anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4)).

Control of CDH1/E-Cadherin Gene Expression and Release of a Soluble Form of E-Cadherin in SARS-CoV-2 Infected Caco-2 Intestinal Cells: Physiopathological Consequences for the Intestinal Forms of COVID-19.

COVID-19 is the biggest pandemic the world has seen this century. Alongside the respiratory damage observed in patients with severe forms of the disease, gastrointestinal symptoms have been frequently reported. These symptoms (e.g., diarrhoea), sometimes precede the development of respiratory tract illnesses, as if the digestive tract was a major target during early SARS-CoV-2 dissemination. We hypothesize that in patients carrying intestinal SARS-CoV-2, the virus may trigger epithelial barrier damage through the disruption of E-cadherin (E-cad) adherens junctions, thereby contributing to the overall gastrointestinal symptoms of COVID-19. Here, we use an intestinal Caco-2 cell line of human origin which expresses the viral receptor/co-receptor as well as the membrane anchored cell surface adhesion protein E-cad to investigate the expression of E-cad after exposure to SARS-CoV-2. We found that the expression of CDH1/E-cad mRNA was significantly lower in cells infected with SARS-CoV-2 at 24 hours post-infection, compared to virus-free Caco-2 cells. The viral receptor ACE2 mRNA expression was specifically down-regulated in SARS-CoV-2-infected Caco-2 cells, while it remained stable in HCoV-OC43-infected Caco-2 cells, a virus which uses HLA class I instead of ACE2 to enter cells. It is worth noting that SARS-CoV-2 induces lower transcription of TMPRSS2 (involved in viral entry) and higher expression of B0AT1 mRNA (that encodes a protein known to co-express with ACE2 on intestinal cells). At 48 hours post-exposure to the virus, we also detected a small but significant increase of soluble E-cad protein (sE-cad) in the culture supernatant of SARS-CoV-2-infected Caco-2 cells. The increase of sE-cad release was also found in the intestinal HT29 cell line when infected by SARS-CoV-2. Beside the dysregulation of E-cad, SARS-CoV-2 infection of Caco-2 cells also leads to the dysregulation of other cell adhesion proteins (occludin, JAMA-A, zonulin, connexin-43 and PECAM-1). Taken together, these results shed light on the fact that infection of Caco-2 cells with SARS-CoV-2 affects tight-, adherens-, and gap-junctions. Moreover, intestinal tissues damage was associated to the intranasal SARS-CoV-2 infection in human ACE2 transgenic mice.

Design, immunogenicity, and efficacy of a pan-sarbecovirus dendritic-cell targeting vaccine.

BACKGROUND: There is an urgent need of a new generation of vaccine that are able to enhance protection against SARS-CoV-2 and related variants of concern (VOC) and emerging coronaviruses. METHODS: We identified conserved T- and B-cell epitopes from Spike (S) and Nucleocapsid (N) highly homologous to 38 sarbecoviruses, including SARS-CoV-2 VOCs, to design a protein subunit vaccine targeting antigens to Dendritic Cells (DC) via CD40 surface receptor (CD40.CoV2). FINDINGS: CD40.CoV2 immunization elicited high levels of cross-neutralizing antibodies against SARS-CoV-2, VOCs, and SARS-CoV-1 in K18-hACE2 transgenic mice, associated with viral control and survival after SARS-CoV-2 challenge. A direct comparison of CD40.CoV2 with the mRNA BNT162b2 vaccine showed that the two vaccines were equally immunogenic in mice. We demonstrated the potency of CD40.CoV2 to recall in vitro human multi-epitope, functional, and cytotoxic SARS-CoV-2 S- and N-specific T-cell responses that are unaffected by VOC mutations and cross-reactive with SARS-CoV-1 and, to a lesser extent, MERS epitopes. INTERPRETATION: We report the immunogenicity and antiviral efficacy of the CD40.CoV2 vaccine in a preclinical model providing a framework for a pan-sarbecovirus vaccine. FUNDINGS: This work was supported by INSERM and the Investissements d'Avenir program, Vaccine Research Institute (VRI), managed by the ANR and the CARE project funded from the Innovative Medicines Initiative 2 Joint Undertaking (JU).

ATP-binding cassette transporter hallmarks tissue macrophages and modulates cytokine-triggered polarization programs.

Macrophages are central players in both lipid metabolism and innate immunity. Their determinant role in the pathogenesis of atherosclerosis is under the control of the ATP-binding cassette transporter (ABCA1), which by minimizing cellular lipid content, limits development of pro-inflammatory foam cells. Considering the differential contribution of monocyte subsets to the generation of vascular lesions we analyzed the immunophenotype of ABCA1-expressing cells in the myeloid lineage, by the combined use of flow cytometry and real-time quantitative RT-PCR. ABCA1 expression is limited to "non-inflammatory" Ly6C(lo) circulating monocytes and tissue-resident macrophages expressing markers of alternative activation. In ABCA1(-/-) peritoneal macrophages the transcriptional programs induced by LPS/IFN-gamma or IL-4 cytokines are altered and deviated phosphorylation patterns of STAT transcriptional regulators in response to stimuli are observed.

Functional implications of the influence of ABCA1 on lipid microenvironment at the plasma membrane: a biophysical study.

The ABCA1 transporter orchestrates cellular lipid homeostasis by promoting the release of cholesterol to plasmatic acceptors. The molecular mechanism is, however, unknown. We report here on the biophysical analysis in living HeLa cells of the ABCA1 lipid microenvironment at the plasma membrane. The modifications of membrane attributes induced by ABCA1 were assessed at both the outer and inner leaflet by monitoring either the lifetime of membrane inserted fluorescent lipid analogues by fluorescence lifetime imaging microscopy (FLIM) or, respectively, the membrane translocation of cationic sensors. Analysis of the partitioning of dedicated probes in plasma membrane blebs vesiculated from these cells allowed visualization of ABCA1 partitioning into the liquid disordered-like phase and corroborated the idea that ABCA1 destabilizes the lipid arrangement at the membrane. Specificity was demonstrated by comparison with cells expressing an inactive transporter. The physiological relevance of these modifications was finally demonstrated by the reduced membrane mobility and function of transferrin receptors under the influence of an active ABCA1. Collectively, these data assess that the control of both transversal and lateral lipid distribution at the membrane is the primary function of ABCA1 and positions the effluxes of cholesterol from cell membranes downstream to the redistribution of the sterol into readily extractable membrane pools.

ABCA1, from pathology to membrane function.

The ABCA1 transporter is the prototype of the A class of mammalian adenosine triphosphate binding cassette transporters and one of the largest members of this family. ABCA1 has been originally identified as an engulfment receptor on macrophages and, more recently, it has been shown to play an essential role in the handling of cellular lipids. Indeed by promoting the effluxes of membrane phospholipids and cholesterol to lipid-poor apoprotein acceptors, ABCA1 controls the formation of high-density lipoproteins and thus the whole process of reverse cholesterol transport. A number of additional phenotypes have been found in the mouse model of invalidation of the ABCA1 gene. In spite of their clinical diversity, they all are extremely sensitive to variations in the physicochemical properties of the cell membrane, which ABCA1 controls as a lipid translocator.