Inhibition of Innate Immune Responses Is Key to Pathogenesis by Arenaviruses

Mammalian arenaviruses are zoonotic viruses that cause asymptomatic, persistent infections in their rodent hosts but can lead to severe and lethal hemorrhagic fever with bleeding and multiorgan failure in human patients. Lassa virus (LASV), for example, is endemic in several West African countries, where it is responsible for an estimated 500,000 infections and 5,000 deaths annually. There are currently no FDA-licensed therapeutics or vaccines available to combat arenavirus infection. A hallmark of arenavirus infection (e.g., LASV) is general immunosuppression that contributes to high viremia. Here, we discuss the early host immune responses to arenavirus infection and the recently discovered molecular mechanisms that enable pathogenic viruses to suppress host immune recognition and to contribute to the high degree of virulence. We also directly compare the innate immune evasion mechanisms between arenaviruses and other hemorrhagic fever-causing viruses, such as Ebola, Marburg, Dengue, and hantaviruses. A better understanding of the immunosuppression and immune evasion strategies of these deadly viruses may guide the development of novel preventative and therapeutic options.     

The Essential,Nonredundant Roles of RIG-I and MDA5 in Detecting and Controlling West Nile Virus Infection

Virus recognition and response by the innate immune system are critical components of host defense against infection. Activation of cell-intrinsic immunity and optimal priming of adaptive immunity against West Nile virus (WNV), an emerging vector-borne virus, depend on recognition by RIG-I and MDA5, two cytosolic pattern recognition receptors (PRRs) of the RIG-I-like receptor (RLR) protein family that recognize viral RNA and activate defense programs that suppress infection. We evaluated the individual functions of RIG-I and MDA5 both in vitro and in vivo in pathogen recognition and control of WNV. Lack of RIG-I or MDA5 alone results in decreased innate immune signaling and virus control in primary cells in vitro and increased mortality in mice. We also generated RIG-I^−/− × MDA5^−/− double-knockout mice and found that a lack of both RLRs results in a complete absence of innate immune gene induction in target cells of WNV infection and a severe pathogenesis during infection in vivo, similar to findings for animals lacking MAVS, the central adaptor molecule for RLR signaling. We also found that RNA products from WNV-infected cells but not incoming virion RNA display at least two distinct pathogen-associated molecular patterns (PAMPs) containing 5′ triphosphate and double-stranded RNA that are temporally distributed and sensed by RIG-I and MDA5 during infection. Thus, RIG-I and MDA5 are essential PRRs that recognize distinct PAMPs that accumulate during WNV replication. Collectively, these experiments highlight the necessity and function of multiple related, cytoplasmic host sensors in orchestrating an effective immune response against an acute viral infection.     

DEAD-box RNA解旋酶家族在天然免疫应答信号通路中的作用

病毒入侵宿主细胞时,宿主细胞启动抑制病毒复制的免疫机制.同样,病毒也会利用多种手段去逃避先天免疫感应机制的监测以及宿主细胞对外来者的降解,同时还会操纵宿主细胞为自身的增殖提供便利.DEAD-box解旋酶家族是一类存在于宿主细胞中的功能蛋白,它们在转录、剪接、mRNA的合成和翻译等多种细胞过程中起着关键作用.该家族成员拥...     

Modeling Innate Antiviral Immunity in Physiological Context

An effective innate antiviral response is critical for the mitigation of severe disease and host survival following infection. In vivo, the innate antiviral response is triggered by cells that detect the invading pathogen and then communicate through autocrine and paracrine signaling to stimulate the expression of genes that inhibit viral replication, curtail cell proliferation, or modulate the immune response. In other words, the innate antiviral response is complex and dynamic. Notably, in the laboratory, culturing viruses and assaying viral life cycles frequently utilizes cells that are derived from tissues other than those that support viral replication during natural infection, while the study of viral pathogenesis often employs animal models. In recapitulating the human antiviral response, it is important to consider that variation in the expression and function of innate immune sensors and antiviral effectors exists across species, cell types, and cell differentiation states, as well as when cells are placed in different contexts. Thus, to gain novel insight into the dynamics of the host response and how specific sensors and effectors impact infection kinetics by a particular virus, the model system must be selected carefully. In this review, we briefly introduce key signaling pathways involved in the innate antiviral response and highlight how these differ between systems. We then review the application of tissue-engineered or 3D models for studying the antiviral response, and suggest how these in vitro culture systems could be further utilized to assay physiologically-relevant host responses and reveal novel insight into virus-host interactions.     

Arenavirus Coinfections Are Common in Snakes with Boid Inclusion Body Disease

Recently, novel arenaviruses were found in snakes with boid inclusion body disease (BIBD); these form the new genus Reptarenavirus within the family Arenaviridae. We used next-generation sequencing and de novo sequence assembly to investigate reptarenavirus isolates from our previous study. Four of the six isolates and all of the samples from snakes with BIBD contained at least two reptarenavirus species. The viruses sequenced comprise four novel reptarenavirus species and a representative of a new arenavirus genus.     

The strepsipteran endoparasite Xenos vesparum alters the immunocompetence of its host, the paper wasp Polistes dominulus

It is unexplained how strepsipteran insects manipulate the physiology of their hosts in order to undergo endoparasitic development without being entrapped by the innate immune defences of the host. Here we present pioneering work that aimed to explore for the first time several components of the cellular and humoral immune response among immature stages of the paper wasp Polistes dominulus, in both unparasitized insects and after infection by the strepsipteran endoparasite Xenos vesparum. We carried out hemocyte counts, phagocytosis assays in vitro and antibacterial response in vivo. On the whole, hemocyte load does not seem to be drastically affected by parasitization: a non-significant increase in hemocyte numbers was observed in parasitized wasps as respect to control, while the two dominant hemocyte types were present with similar proportions in both groups. On the other hand, phagocytosis was significantly reduced in hemocytes from parasitized wasps while the antibacterial response seemed to be less effective in control. These somewhat unexpected results are discussed, along with the implications of a multiple approach in immune response studies.     

Cell entry by human pathogenic arenaviruses

The arenaviruses Lassa virus (LASV) in Africa and Machupo (MACV), Guanarito (GTOV) and Junin viruses (JUNV) in South America cause severe haemorrhagic fevers in humans with fatality rates of 15–35%. The present review focuses on the first steps of infection with human pathogenic arenaviruses, the interaction with their cellular receptor molecules and subsequent entry into the host cell. While similarities exist in genomic organization, structure and clinical disease caused by pathogenic Old World and New World arenaviruses these pathogens use different primary receptors. The Old World arenaviruses employ α-dystroglycan, a cellular receptor for proteins of the extracellular matrix, and the human pathogenic New World arenaviruses use the cellular cargo receptor transferrin receptor 1. While the New World arenavirus JUNV enters cells via clathrin-dependent endocytosis, evidence occurred for clathrin-independent entry of the prototypic Old World arenavirus lymphocytic choriomeningitis virus. Upon internalization, arenaviruses are delivered to the endosome, where pH-dependent membrane fusion is mediated by the envelope glycoprotein (GP). While arenavirus GPs share characteristics with class I fusion GPs of other enveloped viruses, unusual mechanistic features of GP-mediated membrane fusion have recently been discovered for arenaviruses with important implications for viral entry.     

Mycobacterium tuberculosis Strains Are Differentially Recognized by TLRs with an Impact on the Immune Response

Tuberculosis associates with a wide spectrum of disease outcomes. The Beijing (Bj) lineage of Mycobacterium tuberculosis (Mtb) is suggested to be more virulent than other Mtb lineages and prone to elicit non-protective immune responses. However, highly heterogeneous immune responses were reported upon infection of innate immune cells with Bj strains or stimulation with their glycolipids. Using both in vitro and in vivo mouse models of infection, we here report that the molecular mechanism for this heterogeneity may be related to distinct TLR activations. Among this Mtb lineage, we found strains that preferentially activate TLR2, and others that also activate TLR4. Recognition of Mtb strains by TLR4 resulted in a distinct cytokine profile in vitro and in vivo, with specific production of type I IFN. We also uncover a novel protective role for TLR4 activation in vivo. Thus, our findings contribute to the knowledge of the molecular basis underlying how host innate immune cells handle different Mtb strains, in particular the intricate host-pathogen interaction with strains of the Mtb Bj lineage.     

Coverage of Related Pathogenic Species by Multivalent and Cross-Protective Vaccine Design: Arenaviruses as a Model System

Summary: The arenaviruses are a family of negative-sense RNA viruses that cause severe human disease ranging from aseptic meningitis to hemorrhagic fever syndromes. There are currently no FDA-approved vaccines for the prevention of arenavirus disease, and therapeutic treatment is limited to the use of ribavirin and/or immune plasma for a subset of the pathogenic arenaviruses. The considerable genetic variability observed among the seven arenaviruses that are pathogenic for humans illustrates one of the major challenges for vaccine development today, namely, to overcome pathogen heterogeneity. Over the past 5 years, our group has tested several strategies to overcome pathogen heterogeneity, utilizing the pathogenic arenaviruses as a model system. Because T cells play a prominent role in protective immunity following arenavirus infection, we specifically focused on the development of human vaccines that would induce multivalent and cross-protective cell-mediated immune responses. To facilitate our vaccine development and testing, we conducted large-scale major histocompatibility complex (MHC) class I and class II epitope discovery on murine, nonhuman primate, and human backgrounds for each of the pathogenic arenaviruses, including the identification of protective HLA-restricted epitopes. Finally, using the murine model of lymphocytic choriomeningitis virus infection, we studied the phenotypic characteristics associated with immunodominant and protective T cell epitopes. This review summarizes the findings from our studies and discusses their application to future vaccine design.     

RIG-I and dsRNA-Induced IFNβ Activation

Except for viruses that initiate RNA synthesis with a protein primer (e.g., picornaviruses), most RNA viruses initiate RNA synthesis with an NTP, and at least some of their viral _pppRNAs remain unblocked during the infection. Consistent with this, most viruses require RIG-I to mount an innate immune response, whereas picornaviruses require mda-5. We have examined a SeV infection whose ability to induce interferon depends on the generation of capped dsRNA (without free 5′ tri-phosphate ends), and found that this infection as well requires RIG-I and not mda-5. We also provide evidence that RIG-I interacts with poly-I/C in vivo, and that heteropolymeric dsRNA and poly-I/C interact directly with RIG-I in vitro, but in different ways; i.e., poly-I/C has the unique ability to stimulate the helicase ATPase of RIG-I variants which lack the C-terminal regulatory domain.     

MyD88-Dependent Signaling Contributes to Host Defense against Ehrlichial Infection

The ehrlichiae are small Gram-negative obligate intracellular bacteria in the family Anaplasmataceae. Ehrlichial infection in an accidental host may result in fatal diseases such as human monocytotropic ehrlichiosis, an emerging, tick-borne disease. Although the role of adaptive immune responses in the protection against ehrlichiosis has been well studied, the mechanism by which the innate immune system is activated is not fully understood. Using Ehrlichia muris as a model organism, we show here that MyD88-dependent signaling pathways play a pivotal role in the host defense against ehrlichial infection. Upon E. muris infection, MyD88-deficient mice had significantly impaired clearance of E. muris, as well as decreased inflammation, characterized by reduced splenomegaly and recruitment of macrophages and neutrophils. Furthermore, MyD88-deficient mice produced markedly lower levels of IL-12, which correlated well with an impaired Th1 immune response. In vitro, dendritic cells, but not macrophages, efficiently produced IL-12 upon E. muris infection through a MyD88-dependent mechanism. Therefore, MyD88-dependent signaling is required for controlling ehrlichial infection by playing an essential role in the immediate activation of the innate immune system and inflammatory cytokine production, as well as in the activation of the adaptive immune system at a later stage by providing for optimal Th1 immune responses.     

A Sendai Virus-Derived RNA Agonist of RIG-I as a Virus Vaccine Adjuvant

The innate immune system is responsible for recognizing invading pathogens and initiating a protective response. In particular, the retinoic acid-inducible gene 1 protein (RIG-I) participates in the recognition of single- and double-stranded RNA viruses. RIG-I activation leads to the production of an appropriate cytokine and chemokine cocktail that stimulates an antiviral state and drives the adaptive immune system toward an efficient and specific response against the ongoing infection. One of the best-characterized natural RIG-I agonists is the defective interfering (DI) RNA produced by Sendai virus strain Cantell. This 546-nucleotide RNA is a well-known activator of the innate immune system and an extremely potent inducer of type I interferon. We designed an in vitro-transcribed RNA that retains the type I interferon stimulatory properties, and the RIG-I affinity of the Sendai virus produced DI RNA both in vitro and in vivo. This in vitro-synthesized RNA is capable of enhancing the production of anti-influenza virus hemagglutinin (HA)-specific IgG after intramuscular or intranasal coadministration with inactivated H1N1 2009 pandemic vaccine. Furthermore, our adjuvant is equally effective at increasing the efficiency of an influenza A/Puerto Rico/8/34 virus inactivated vaccine as a poly(I·C)- or a squalene-based adjuvant. Our in vitro-transcribed DI RNA represents an excellent tool for the study of RIG-I agonists as vaccine adjuvants and a starting point in the development of such a vaccine.     

NLRC5 regulates MHC class I antigen presentation in host defense against intracellular pathogens

NOD-like receptors (NLRs) are a family of intracellular proteins that play critical roles in innate immunity against microbial infection. NLRC5, the largest member of the NLR family, has recently attracted much attention. However, in vitro studies have reported inconsistent results about the roles of NLRC5 in host defense and in regulating immune signaling pathways. The in vivo function of NLRC5 remains unknown. Here, we report that NLRC5 is a critical regulator of host defense against intracellular pathogens in vivo. NLRC5 was specifically required for the expression of genes involved in MHC class I antigen presentation. NLRC5-deficient mice showed a profound defect in the expression of MHC class I genes and a concomitant failure to activate L. monocytogenes-specific CD8⁺ T cell responses, including activation, proliferation and cytotoxicity, and the mutant mice were more susceptible to the pathogen infection. NLRP3-mediated inflammasome activation was also partially impaired in NLRC5-deficient mice. However, NLRC5 was dispensable for pathogen-induced expression of NF-κB-dependent pro-inflammatory genes as well as type I interferon genes. Thus, NLRC5 critically regulates MHC class I antigen presentation to control intracellular pathogen infection.     

Polyfunctional CD4+ T cell responses to a set of pathogenic arenaviruses provide broad population coverage

Background

Several arenaviruses cause severe hemorrhagic fever and aseptic meningitis in humans for which no licensed vaccines are available. A major obstacle for vaccine development is pathogen heterogeneity within the Arenaviridae family. Evidence in animal models and humans indicate that T cell and antibody-mediated immunity play important roles in controlling arenavirus infection and replication. Because CD4⁺ T cells are needed for optimal CD8⁺ T cell responses and to provide cognate help for B cells, knowledge of epitopes recognized by CD4⁺ T cells is critical to the development of an effective vaccine strategy against arenaviruses. Thus, the goal of the present study was to define and characterize CD4⁺ T cell responses from a broad repertoire of pathogenic arenaviruses (including lymphocytic choriomeningitis, Lassa, Guanarito, Junin, Machupo, Sabia, and Whitewater Arroyo viruses) and to provide determinants with the potential to be incorporated into a multivalent vaccine strategy.

Results

By inoculating HLA-DRB1*0101 transgenic mice with a panel of recombinant vaccinia viruses, each expressing a single arenavirus antigen, we identified 37 human HLA-DRB1*0101-restricted CD4⁺ T cell epitopes from the 7 antigenically distinct arenaviruses. We showed that the arenavirus-specific CD4⁺ T cell epitopes are capable of eliciting T cells with a propensity to provide help and protection through CD40L and polyfunctional cytokine expression. Importantly, we demonstrated that the set of identified CD4⁺ T cell epitopes provides broad, non-ethnically biased population coverage of all 7 arenavirus species targeted by our studies.

Conclusions

The identification of CD4⁺ T cell epitopes, with promiscuous binding properties, derived from 7 different arenavirus species will aid in the development of a T cell-based vaccine strategy with the potential to target a broad range of ethnicities within the general population and to protect against both Old and New World arenavirus infection.