Branka Horvat

Linking innate and acquired immunity: divergent role of CD46 cytoplasmic domains in T cell–induced inflammation

CD46 is a widely expressed transmembrane protein that was initially identified as binding and inactivating C3b and C4b complement products. We used mice that were transgenic for one of two human CD46 isoforms that differ in their cytoplasmic domains (termed CD46-1 and CD46-2) to analyze the effect of CD46 stimulation on the immune response. We show here that CD46 can regulate inflammatory responses, either by inhibiting (CD46-1) or increasing (CD46-2) the contact hypersensitivity reaction. We found that engagement of CD46-1 or CD46-2 differentially affected CD8+ T cell cytotoxicity, CD4+ T cell proliferation, interleukin 2 (IL-2) and IL-10 production as well as tyrosine phosphorylation of Vav in T lymphocytes. These results indicate that CD46 plays a role in regulating the T cell–induced inflammatory reaction and in fine-tuning the cellular immune response by bridging innate and acquired immunity.

Mechanism of Measles Virus–Induced Suppression of Inflammatory Immune Responses

Measles virus (MV) causes profound immunosuppression, resulting in high infant mortality. The mechanisms are poorly understood, largely due to the lack of a suitable animal model. Here, we report that particular MV proteins, in the absence of MV replication, could generate a systemic immunosuppression in mice through two pathways: (1) via MV-nucleoprotein and its receptor FcgammaR on dendritic cells; and (2) via virus envelope glycoproteins and the MV-hemagglutinin cellular receptor, CD46. The effects comprise reduced hypersensitivity responses associated with impaired function of dendritic cells, decreased production of IL-12, and the loss of antigen-specific T cell proliferation. These results introduce a novel model for testing the immunosuppressive potential of anti-measles vaccines and reveal a specific mechanism of MV-induced modulation of inflammatory reactions.

Acute Hendra virus infection: Analysis of the pathogenesis and passive antibody protection in the hamster model

Hendra virus (HeV) and Nipah virus (NiV) are recently-emerged, closely related and highly pathogenic paramyxoviruses. We have analysed here the pathogenesis of the acute HeV infection using the new animal model, golden hamster (Mesocricetus auratus), which is highly susceptible to HeV infection. HeV-specific RNA and viral antigens were found in multiple organs and virus was isolated from different tissues. Dual pathogenic mechanism was observed: parenchymal infection in various organs, including the brain, with vasculitis and multinucleated syncytia in many blood vessels. Furthermore, monoclonal antibodies specific for the NiV fusion protein neutralized HeV in vitro and efficiently protected hamsters from HeV if given before infection. These results reveal the similarities between HeV and NiV pathogenesis, particularly in affecting both respiratory and neuronal system. They demonstrate that hamster presents a convenient novel animal model to study HeV infection, opening new perspectives to evaluate vaccine and therapeutic approaches against this emergent infectious disease.

iNKT cell development is orchestrated by different branches of TGF-beta signaling.

Invariant natural killer T (iNKT) cells constitute a distinct subset of T lymphocytes exhibiting important immune-regulatory functions. Although various steps of their differentiation have been well characterized, the factors controlling their development remain poorly documented. Here, we show that TGF-β controls the differentiation program of iNKT cells. We demonstrate that TGF-β signaling carefully and specifically orchestrates several steps of iNKT cell development. In vivo, this multifaceted role of TGF-β involves the concerted action of different pathways of TGF-β signaling. Whereas the Tif-1γ branch controls lineage expansion, the Smad4 branch maintains the maturation stage that is initially repressed by a Tif-1γ/Smad4-independent branch. Thus, these three different branches of TGF-β signaling function in concert as complementary effectors, allowing TGF-β to fine tune the iNKT cell differentiation program.

Nipah Virus Uses Leukocytes for Efficient Dissemination within a Host

ABSTRACT Nipah virus (NiV) is a recently emerged zoonotic paramyxovirus whose natural reservoirs are several species of Pteropus fruit bats. NiV provokes a widespread vasculitis often associated with severe encephalitis, with up to 75% mortality in humans. We have analyzed the pathogenesis of NiV infection, using human leukocyte cultures and the hamster animal model, which closely reproduces human NiV infection. We report that human lymphocytes and monocytes are not permissive for NiV and a low level of virus replication is detected only in dendritic cells. Interestingly, despite the absence of infection, lymphocytes could efficiently bind NiV and transfer infection to endothelial and Vero cells. This lymphocyte-mediated transinfection was inhibited after proteolytic digestion and neutralization by NiV-specific antibodies, suggesting that cells could transfer infectious virus to other permissive cells without the requirement for NiV internalization. In NiV-infected hamsters, leukocytes captured and carried NiV after intraperitoneal infection without themselves being productively infected. Such NiV-loaded mononuclear leukocytes transfer lethal NiV infection into naïve animals, demonstrating efficient virus transinfection in vivo. Altogether, these results reveal a remarkable capacity of NiV to hijack leukocytes as vehicles to transinfect host cells and spread the virus throughout the organism. This mode of virus transmission represents a rapid and potent method of NiV dissemination, which may contribute to its high pathogenicity.

Octamerization Enables Soluble CD46 Receptor To Neutralize Measles Virus In Vitro and In Vivo

ABSTRACT A chimeric fusion protein encompassing the CD46 ectodomain linked to the C-terminal part of the C4b binding protein (C4bp) α chain (sCD46-C4bpα) was produced in eukaryotic cells. This protein, secreted as a disulfide-linked homo-octamer, was recognized by a panel of anti-CD46 antibodies with varying avidities. Unlike monomeric sCD46, the octameric sCD46-C4bpα protein was devoid of complement regulatory activity. However, sCD46-C4bpα was able to bind to the measles virus hemagglutinin protein expressed on murine cells with a higher avidity than soluble monomeric sCD46. Moreover, the octameric sCD46-C4bpα protein was significantly more efficient than monomeric sCD46 in inhibiting virus binding to CD46, in blocking virus induced cell-cell fusion, and in neutralizing measles virus in vitro. In addition, the octameric sCD46-C4bpα protein, but not the monomeric sCD46, fully protected CD46 transgenic mice against a lethal intracranial measles virus challenge.

Experimental infection of squirrel monkeys with nipah virus

We infected squirrel monkeys (Saimiri sciureus) with Nipah virus to determine the monkeys’ suitability for use as primate models in preclinical testing of preventive and therapeutic treatments. Infection of squirrel monkeys through intravenous injection was followed by high death rates associated with acute neurologic and respiratory illness and viral RNA and antigen production.

Current Animal Models: Transgenic Animal Models for the Study of Measles Pathogenesis

Animal models are highly important to understand the pathologic mechanisms of viral diseases. Therefore, the lack of a suitable animal model has greatly hindered the research into the pathogenesis of measles. Identification of two human receptors for measles virus, CD46 and CD150 (SLAM) has opened new perspectives in this field. During the last decade, numerous transgenic animal models have been developed in order to humanize mice and use them to study measles infection and virus-host interactions. Despite their limitations, these models have provided remarkable insights in different aspects of measles infection, providing a better understanding of virus-induced neuropathology, immunosuppression, mechanisms of virus virulence, and contribution of innate and adaptive immune response in viral clearance. They should certainly continue to help in studies of the host and viral factors that are important in measles infection and in developing of new antiviral agents and measles virus-based vaccines. In addition, as CD46 serves as a receptor for two other human viruses, some of these models may also find an important application in the study of adenovirus and herpesvirus 6 infection. In this review, we describe different CD46 and CD150 transgenic models and detail their utilization in the study of various aspects of measles pathogenesis.

High Pathogenicity of Wild-Type Measles Virus Infection in CD150 (SLAM) Transgenic Mice

ABSTRACT Measles virus (MV) infection causes an acute childhood disease, associated in certain cases with infection of the central nervous system and development of a severe neurological disease. We have generated transgenic mice ubiquitously expressing the human protein SLAM (signaling lymphocytic activation molecule), or CD150, recently identified as an MV receptor. In contrast to all other MV receptor transgenic models described so far, in these mice infection with wild-type MV strains is highly pathogenic. Intranasal infection of SLAM transgenic suckling mice leads to MV spread to different organs and the development of an acute neurological syndrome, characterized by lethargy, seizures, ataxia, weight loss, and death within 3 weeks. In addition, in this model, vaccine and wild-type MV strains can be distinguished by virulence. Furthermore, intracranial MV infection of adult transgenic mice generates a subclinical infection associated with a high titer of MV-specific antibodies in the serum. Finally, to analyze new antimeasles therapeutic approaches, we created a recombinant soluble form of SLAM and demonstrated its important antiviral activity both in vitro and in vivo. Taken together, our results show the high susceptibility of SLAM transgenic mice to MV-induced neurological disease and open new perspectives for the analysis of the implication of SLAM in the neuropathogenicity of other morbilliviruses, which also use this molecule as a receptor. Moreover, this transgenic model, in allowing a simple readout of the efficacy of an antiviral treatment, provides unique experimental means to test novel anti-MV preventive and therapeutic strategies.

Mechanism for Active Membrane Fusion Triggering by Morbillivirus Attachment Protein

ABSTRACT The paramyxovirus entry machinery consists of two glycoproteins that tightly cooperate to achieve membrane fusion for cell entry: the tetrameric attachment protein (HN, H, or G, depending on the paramyxovirus genus) and the trimeric fusion protein (F). Here, we explore whether receptor-induced conformational changes within morbillivirus H proteins promote membrane fusion by a mechanism requiring the active destabilization of prefusion F or by the dissociation of prefusion F from intracellularly preformed glycoprotein complexes. To properly probe F conformations, we identified anti-F monoclonal antibodies (MAbs) that recognize conformation-dependent epitopes. Through heat treatment as a surrogate for H-mediated F triggering, we demonstrate with these MAbs that the morbillivirus F trimer contains a sufficiently high inherent activation energy barrier to maintain the metastable prefusion state even in the absence of H. This notion was further validated by exploring the conformational states of destabilized F mutants and stabilized soluble F variants combined with the use of a membrane fusion inhibitor (3g). Taken together, our findings reveal that the morbillivirus H protein must lower the activation energy barrier of metastable prefusion F for fusion triggering.