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Interferon-γ (IFNγ) is an important mediator of cellular immune responses, but high systemic levels of this cytokine are associated with immunopathology. IFNγ binds to its receptor (IFNγR) and to extracellular matrix (ECM) via four positively charged C-terminal amino acids (KRKR), the ECM-binding domain (EBD). Across evolution, IFNγ is not well conserved, but the EBD is highly conserved, suggesting a critical function. Here, we show that IFNγ lacking the EBD (IFNγΔKRKR) does not bind to ECM but still binds to the IFNγR and retains bioactivity. Overexpression of IFNγΔKRKRin tumors reduced local ECM binding, increased systemic levels and induced sickness behavior, weight loss and toxicity. To analyze the function of the EBD during infection, we generated IFNγΔKRKRmice lacking the EBD by using CRISPR–Cas9. Infection with lymphocytic choriomeningitis virus resulted in higher systemic IFNγΔKRKRlevels, enhanced sickness behavior, weight loss and fatal toxicity. We conclude that local retention of IFNγ is a pivotal mechanism to protect the organism from systemic toxicity during prolonged immune stimulation.

Virus-like particles (VLPs) are highly suited platforms for protein-based vaccines. In the present work, we adapted a previously designed non-infectious adenovirus-inspired 60-mer dodecahedric VLP (ADDomer) to display a multimeric array of large antigens through a SpyTag/SpyCatcher system. To validate the platform as a potential COVID-19 vaccine approach, we decorated the newly designed VLP with the glycosylated receptor binding domain (RBD) of SARS-CoV-2. Cryoelectron microscopy structure revealed that up to 60 copies of this antigenic domain could be bound on a single ADDomer particle, with the symmetrical arrangements of a dodecahedron. Mouse immunization with the RBD decorated VLPs already showed a significant specific humoral response following prime vaccination, greatly reinforced by a single boost. Neutralization assays with SARS-CoV-2 spike pseudo-typed virus demonstrated the elicitation of strong neutralization titers, superior to those of COVID-19 convalescent patients. Notably, the presence of pre-existing immunity against the adenoviral-derived particles did not hamper the immune response against the antigen displayed on its surface. This plug and play vaccine platform represents a promising new highly versatile tool to combat emergent pathogens.

Systemic lupus erythematosus (SLE) is a multisystem inflammatory disease characterized by the production of various autoantibodies. The aim of this study was to investigate the presence of anti–ficolin‐2 antibodies in SLEpatients and to evaluate the association between the levels of these autoantibodies, clinical manifestations, and disease activity. This is a comparative study using a cohort of 165 SLEpatients and 48 healthy subjects. SLEpatients were further divided into 2 groups (low disease activity [SLEDisease Activity Index (SLEDAI) score ≤4, n = 88] and high disease activity [SLEDAIscore >4, n = 77]). Clinical manifestations were defined according to the physician in charge. Active lupus nephritis (LN) was documented by kidney biopsy. Detection of anti–ficolin‐2 antibodies was performed by enzyme‐linked immunosorbent assay. Levels of anti–ficolin‐2 autoantibodies were significantly higher in SLEpatients as compared to healthy subjects and associated with SLEDAIscore. They were found to be positive in 61 of 165 SLEpatients (37%). The presence of anti–ficolin‐2 antibodies was significantly related only to renal involvement, with a very high prevalence (86%) of anti–ficolin‐2 antibodies in SLEpatients with active LN. Patients with active proliferative LNhad significantly more positive anti–ficolin‐2 antibodies than those with nonproliferative LN. The combination of anti–ficolin‐2, anti–ficolin‐3, and anti‐C1q demonstrated a very high specificity (98%) for the diagnosis of active LN. Our results support the usefulness of anti–ficolin‐2 as a complementary serologic biomarker for the diagnosis of active lupus with renal manifestations.

Recombinant and neutrophil‐secreted endogenous M‐ficolin bind membrane CD43 and promote neutrophil polarization and adhesion. M‐ficolin specificity for sialylated ligands prompted us to investigate its interactions with the main membrane sialoprotein of human neutrophils, CD43. rM‐ficolin bound CD43 and prevented the access of anti‐CD43 mAb. Moreover, rM‐ficolin reacted exclusively with CD43 on Western blots of neutrophil lysate. We confirmed that M‐ficolin is secreted by fMLP‐activated neutrophils, and this endogenous M‐ficolin also binds to CD43 and competes with anti‐CD43 mAb. Anti‐CD43 antibody cross‐linking or fMLP resulted in M‐ficolin and CD43 colocalization on polarized neutrophils. The binding of rM‐ficolin to resting neutrophils induced cell polarization, adhesion, and homotypic aggregation as anti‐CD43 mAb. The M‐ficolin Y271F mutant, unable to bind sialic acid, neither reacted with neutrophils nor modulated their functions. Finally, rM‐ficolin activated the lectin complement pathway on neutrophils. These results emphasize a new function of M‐ficolin, different from ficolin pathogen recognition, i.e., a participation to neutrophil adhesion potentially important in early inflammation, as nanomolar agonist concentrations are sufficient to mobilize M‐ficolin to the neutrophil surface. This multivalent lectin could then endow the antiadhesive CD43, essentially designed to prevent leukocyte aggregation in the blood flow, with new adhesive properties and explain, at least in part, dual‐adhesive/antiadhesive roles of CD43 in neutrophil recruitment.

AbstractInnate immunity relies upon the ability of a variety of recognition molecules to sense pathogens through conserved molecular signatures that are often carbohydrates. Ficolins are oligomeric proteins assembled from collagen-like stalks and fibrinogen-like domains that have the ability to sense these molecular patterns on both pathogens and apoptotic cell surfaces. Three ficolins, termed L, H and M, have been identified in humans. They differ in their localization and concentration in extracellular fluids, their mode of secretion and their recognition properties. From a structural point of view, ficolins are assembled from basal trimeric subunits comprising a collagen-like triple helix and a globular domain composed of 3 fibrinogen-like domains. The globular domains are responsible for sensing danger signals whereas the collagen-like stalks provide a link with immune effectors. This review mainly focuses on the structure and recognition properties of the 3 human ficolins, as revealed by recent crystallographic analysis of their recognition domains. The ligand binding sites have been identified in the 3 ficolins and their recognition mechanisms have been characterized at the atomic level. In the case of M-ficolin, a structural transition at acidic pH disables the binding pocket, and thus likely participates in the functional cycle of this protein.Copyright © 2009 S. Karger AG, Basel

Sulfs represent a class of unconventional sulfatases which provide an original post-synthetic regulatory mechanism for heparan sulfate polysaccharides and are involved in multiple physiopathological processes, including cancer. However, Sulfs remain poorly characterized enzymes, with major discrepancies regarding their in vivofunctions. Here we show that human Sulf-2 (HSulf-2) harbors a chondroitin/dermatan sulfate glycosaminoglycan (GAG) chain, attached to the enzyme substrate-binding domain. We demonstrate that this GAG chain affects enzyme/substrate recognition and tunes HSulf-2 activity in vitroand in vivo. In addition, we show that mammalian hyaluronidase acts as a promoter of HSulf-2 activity by digesting its GAG chain. In conclusion, our results highlight HSulf-2 as a proteoglycan-related enzyme and its GAG chain as a critical non-catalytic modulator of the enzyme activity. These findings contribute to clarifying the conflicting data on the activities of the Sulfs.

Recombinant adenovirus is one of most efficient delivery vehicles for gene therapy. However, the initial enthusiasm for the use of recombinant adenovirus for gene therapy has been tempered by strong immune responses that develop to the virus and virus-infected cells. Even though recombinant adeno-viruses are replication-defective, they introduce into the recipient cell, together with the gene of interest, viral genetes that might lead to fortuitous recombination if the recipient is infected by wild-type adenovirus. We propose the use of a dodecahedron made of adenovirus pentons or penton bases as an alternative vector for human gene therapy. The penton is a complex of two oligomeric proteins, a penton base and fiber, involved in the cell attachment, internalization, and liberation of virus into the cytoplasm. The dodecahedron retains many of the advantages of adenovirus for gene transfer such as efficiency of entry, efficient release of DNA from endosomes, and wide range of cell and tissue targets. Because it consists of only one or two adenovirus proteins instead of the 11 contained in an adenovirus virion and it does not contain the viral genome, it is potentially a safer alternative to recombinant adenovirus.