Mouse Models of Heterologous Flavivirus Immunity: A Role for Cross-Reactive T Cells

Most of the world is at risk of being infected with a flavivirus such as dengue virus, West Nile virus, yellow fever virus, Japanese encephalitis virus, tick-borne encephalitis virus, and Zika virus, significantly impacting millions of lives. Importantly, many of these genetically similar viruses co-circulate within the same geographic regions, making it likely for individuals living in areas of high flavivirus endemicity to be infected with multiple flaviviruses during their lifetime. Following a flavivirus infection, a robust virus-specific T cell response is generated and the memory recall of this response has been demonstrated to provide long-lasting immunity, protecting against reinfection with the same pathogen. However, multiple studies have shown that this flavivirus specific T cell response can be cross-reactive and active during heterologous flavivirus infection, leading to the question: How does immunity to one flavivirus shape immunity to the next, and how does this impact disease? It has been proposed that in some cases unfavorable disease outcomes may be caused by lower avidity cross-reactive memory T cells generated during a primary flavivirus infection that preferentially expand during a secondary heterologous infection and function sub optimally against the new pathogen. While in other cases, these cross-reactive cells still have the potential to facilitate cross-protection. In this review, we focus on cross-reactive T cell responses to flaviviruses and the concepts and consequences of T cell cross-reactivity, with particular emphasis linking data generated using murine models to our new understanding of disease outcomes following heterologous flavivirus infection

A hydrogen peroxide-inactivated virus vaccine elicits humoral and cellular immunity and protects against lethal west nile virus infection in aged mice

West Nile virus (WNV) is an emerging pathogen that is now the leading cause of mosquito-borne and epidemic encephalitis in the United States. In humans, a small percentage of infected individuals develop severe neuroinvasive disease, with the greatest relative risk being in the elderly and immunocompromised, two populations that are difficult to immunize effectively with vaccines. While inactivated and subunit-based veterinary vaccines against WNV exist, currently there is no vaccine or therapy available to prevent or treat human disease. Here, we describe the generation and preclinical efficacy of a hydrogen peroxide (H(2)O(2))-inactivated WNV Kunjin strain (WNV-KUNV) vaccine as a candidate for further development. Both young and aged mice vaccinated with H(2)O(2)-inactivated WNV-KUNV produced robust adaptive B and T cell immune responses and were protected against stringent and lethal intracranial challenge with a heterologous virulent North American WNV strain. Our studies suggest that the H(2)O(2)-inactivated WNV-KUNV vaccine is safe and immunogenic and may be suitable for protection against WNV infection in vulnerable populations

Human and murine IFIT1 proteins do not restrict infection of negative-sense RNA viruses of the Orthomyxoviridae, Bunyaviridae, and Filoviridae families

UNLABELLED: Interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) is a host protein with reported cell-intrinsic antiviral activity against several RNA viruses. The proposed basis for the activity against negative-sense RNA viruses is the binding to exposed 5\u27-triphosphates (5\u27-ppp) on the genome of viral RNA. However, recent studies reported relatively low binding affinities of IFIT1 for 5\u27-ppp RNA, suggesting that IFIT1 may not interact efficiently with this moiety under physiological conditions. To evaluate the ability of IFIT1 to have an impact on negative-sense RNA viruses, we infected Ifit1(-/-) and wild-type control mice and primary cells with four negative-sense RNA viruses (influenza A virus [IAV], La Crosse virus [LACV], Oropouche virus [OROV], and Ebola virus) corresponding to three distinct families. Unexpectedly, a lack of Ifit1 gene expression did not result in increased infection by any of these viruses in cell culture. Analogously, morbidity, mortality, and viral burdens in tissues were identical between Ifit1(-/-) and control mice after infection with IAV, LACV, or OROV. Finally, deletion of the human IFIT1 protein in A549 cells did not affect IAV replication or infection, and reciprocally, ectopic expression of IFIT1 in HEK293T cells did not inhibit IAV infection. To explain the lack of antiviral activity against IAV, we measured the binding affinity of IFIT1 for RNA oligonucleotides resembling the 5\u27 ends of IAV gene segments. The affinity for 5\u27-ppp RNA was approximately 10-fold lower than that for non-2\u27-O-methylated (cap 0) RNA oligonucleotides. Based on this analysis, we conclude that IFIT1 is not a dominant restriction factor against negative-sense RNA viruses. IMPORTANCE: Negative-sense RNA viruses, including influenza virus and Ebola virus, have been responsible for some of the most deadly outbreaks in recent history. The host interferon response and induction of antiviral genes contribute to the control of infections by these viruses. IFIT1 is highly induced after virus infection and reportedly has antiviral activity against several RNA and DNA viruses. However, its role in restricting infection by negative-sense RNA viruses remains unclear. In this study, we evaluated the ability of IFIT1 to inhibit negative-sense RNA virus replication and pathogenesis both in vitro and in vivo. Detailed cell culture and animal studies demonstrated that IFIT1 is not a dominant restriction factor against three different families of negative-sense RNA viruses

Interferon regulatory factor 5-dependent immune responses in the draining lymph node protect against West Nile Virus infection

Upon activation of Toll-like and RIG-I-like receptor signaling pathways, the transcription factor IRF5 translocates to the nucleus and induces antiviral immune programs. The recent discovery of a homozygous mutation in the immunoregulatory gene guanine exchange factor dedicator of cytokinesis 2 (Dock2(mu/mu)) in several Irf5(−/−) mouse colonies has complicated interpretation of immune functions previously ascribed to IRF5. To define the antiviral functions of IRF5 in vivo, we infected backcrossed Irf5(−/−) × Dock2(wt/wt) mice (here called Irf5(−/−) mice) and independently generated CMV-Cre Irf5(fl/fl) mice with West Nile virus (WNV), a pathogenic neurotropic flavivirus. Compared to congenic wild-type animals, Irf5(−/−) and CMV-Cre Irf5(fl/fl) mice were more vulnerable to WNV infection, and this phenotype was associated with increased infection in peripheral organs, which resulted in higher virus titers in the central nervous system. The loss of IRF5, however, was associated with only small differences in the type I interferon response systemically and in the draining lymph node during WNV infection. Instead, lower levels of several other proinflammatory cytokines and chemokines, as well as fewer and less activated immune cells, were detected in the draining lymph node 2 days after WNV infection. WNV-specific antibody responses in Irf5(−/−) mice also were blunted in the context of live or inactivated virus infection and this was associated with fewer antigen-specific memory B cells and long-lived plasma cells. Our results with Irf5(−/−) mice establish a key role for IRF5 in shaping the early innate immune response in the draining lymph node, which impacts the spread of virus infection, optimal B cell immunity, and disease pathogenesis. IMPORTANCE Although the roles of IRF3 and IRF7 in orchestrating innate and adaptive immunity after viral infection are established, the function of the related transcription factor IRF5 remains less certain. Prior studies in Irf5(−/−) mice reported conflicting results as to the contribution of IRF5 in regulating type I interferon and adaptive immune responses. The lack of clarity may stem from a recently discovered homozygous loss-of-function mutation of the immunoregulatory gene Dock2 in several colonies of Irf5(−/−) mice. Here, using a mouse model with a deficiency in IRF5 and wild-type Dock2 alleles, we investigated how IRF5 modulates West Nile virus (WNV) pathogenesis and host immune responses. Our in vivo studies indicate that IRF5 has a key role in shaping the early proinflammatory cytokine response in the draining lymph node, which impacts immunity and control of WNV infection

Pattern recognition receptor MDA5 modulates CD8+ T cell- dependent clearance of west nile virus from the central nervous system

Many viruses induce type I interferon responses by activating cytoplasmic RNA sensors, including the RIG-I-like receptors (RLRs). Although two members of the RLR family, RIG-I and MDA5, have been implicated in host control of virus infection, the relative role of each RLR in restricting pathogenesis in vivo remains unclear. Recent studies have demonstrated that MAVS, the adaptor central to RLR signaling, is required to trigger innate immune defenses and program adaptive immune responses, which together restrict West Nile virus (WNV) infection in vivo. In this study, we examined the specific contribution of MDA5 in controlling WNV in animals. MDA5(−/−) mice exhibited enhanced susceptibility, as characterized by reduced survival and elevated viral burden in the central nervous system (CNS) at late times after infection, even though small effects on systemic type I interferon response or viral replication were observed in peripheral tissues. Intracranial inoculation studies and infection experiments with primary neurons ex vivo revealed that an absence of MDA5 did not impact viral infection in neurons directly. Rather, subtle defects were observed in CNS-specific CD8(+) T cells in MDA5(−/−) mice. Adoptive transfer into recipient MDA5(+/+) mice established that a non-cell-autonomous deficiency of MDA5 was associated with functional defects in CD8(+) T cells, which resulted in a failure to clear WNV efficiently from CNS tissues. Our studies suggest that MDA5 in the immune priming environment shapes optimal CD8(+) T cell activation and subsequent clearance of WNV from the CNS

Human And Murine Ifit1 Proteins Do Not Restrict Infection Of Negative-sense Rna Viruses Of The Orthomyxoviridae, Bunyaviridae, And Filoviridae Families

Interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) is a host protein with reported cell-intrinsic antiviral activity against several RNA viruses. The proposed basis for the activity against negative-sense RNA viruses is the binding to exposed 5'-triphosphates (5'-ppp) on the genome of viral RNA. However, recent studies reported relatively low binding affinities of IFIT1 for 5'-ppp RNA, suggesting that IFIT1 may not interact efficiently with this moiety under physiological conditions. To evaluate the ability of IFIT1 to have an impact on negative-sense RNA viruses, we infected Ifit1(-/-) and wild-type control mice and primary cells with four negative-sense RNA viruses (influenza A virus [IAV], La Crosse virus [LACV], Oropouche virus [OROV], and Ebola virus) corresponding to three distinct families. Unexpectedly, a lack of Ifit1 gene expression did not result in increased infection by any of these viruses in cell culture. Analogously, morbidity, mortality, and viral burdens in tissues were identical between Ifit1(-/-) and control mice after infection with IAV, LACV, or OROV. Finally, deletion of the human IFIT1 protein in A549 cells did not affect IAV replication or infection, and reciprocally, ectopic expression of IFIT1 in HEK293T cells did not inhibit IAV infection. To explain the lack of antiviral activity against IAV, we measured the binding affinity of IFIT1 for RNA oligonucleotides resembling the 5' ends of IAV gene segments. The affinity for 5'-ppp RNA was approximately 10-fold lower than that for non-2'-O-methylated (cap 0) RNA oligonucleotides. Based on this analysis, we conclude that IFIT1 is not a dominant restriction factor against negative-sense RNA viruses. IMPORTANCE Negative-sense RNA viruses, including influenza virus and Ebola virus, have been responsible for some of the most deadly outbreaks in recent history. The host interferon response and induction of antiviral genes contribute to the control of infections by these viruses. IFIT1 is highly induced after virus infection and reportedly has antiviral activity against several RNA and DNA viruses. However, its role in restricting infection by negative-sense RNA viruses remains unclear. In this study, we evaluated the ability of IFIT1 to inhibit negative-sense RNA virus replication and pathogenesis both in vitro and in vivo. Detailed cell culture and animal studies demonstrated that IFIT1 is not a dominant restriction factor against three different families of negative-sense RNA viruses.891894659476Division of Intramural Research, NIAID, NIHNIH grant [F32 AI112274][U54 AI057160][R01 AI104972][R01 AI104002

Murine norovirus infection does not cause major disruptions in the murine intestinal microbiota

BACKGROUND: Murine norovirus (MNV) is the most common gastrointestinal pathogen of research mice and can alter research outcomes in biomedical mouse models of inflammatory bowel disease (IBD). Despite indications that an altered microbiota is a risk factor for IBD, the response of the murine intestinal microbiota to MNV infection has not been examined. Microbiota disruption caused by MNV infection could introduce the confounding effects observed in research experiments. Therefore, this study investigated the effects of MNV infection on the intestinal microbiota of wild-type mice. RESULTS: The composition of the intestinal microbiota was assessed over time in both outbred Swiss Webster and inbred C57BL/6 mice following MNV infection. Mice were infected with both persistent and non-persistent MNV strains and tissue-associated or fecal-associated microbiota was analyzed by 16S rRNA-encoding gene pyrosequencing. Analysis of intestinal bacterial communities in infected mice at the phylum and family level showed no major differences to uninfected controls, both in tissue-associated samples and feces, and also over time following infection, demonstrating that the intestinal microbiota of wild-type mice is highly resistant to disruption following MNV infection. CONCLUSIONS: This is the first study to describe the intestinal microbiota following MNV infection and demonstrates that acute or persistent MNV infection is not associated with major disruptions of microbial communities in Swiss Webster and C57BL/6 mice

Murine norovirus infection does not cause major disruptions in the murine intestinal microbiota

Abstract Background Murine norovirus (MNV) is the most common gastrointestinal pathogen of research mice and can alter research outcomes in biomedical mouse models of inflammatory bowel disease (IBD). Despite indications that an altered microbiota is a risk factor for IBD, the response of the murine intestinal microbiota to MNV infection has not been examined. Microbiota disruption caused by MNV infection could introduce the confounding effects observed in research experiments. Therefore, this study investigated the effects of MNV infection on the intestinal microbiota of wild-type mice. Results The composition of the intestinal microbiota was assessed over time in both outbred Swiss Webster and inbred C57BL/6 mice following MNV infection. Mice were infected with both persistent and non-persistent MNV strains and tissue-associated or fecal-associated microbiota was analyzed by 16S rRNA-encoding gene pyrosequencing. Analysis of intestinal bacterial communities in infected mice at the phylum and family level showed no major differences to uninfected controls, both in tissue-associated samples and feces, and also over time following infection, demonstrating that the intestinal microbiota of wild-type mice is highly resistant to disruption following MNV infection. Conclusions This is the first study to describe the intestinal microbiota following MNV infection and demonstrates that acute or persistent MNV infection is not associated with major disruptions of microbial communities in Swiss Webster and C57BL/6 mice.http://deepblue.lib.umich.edu/bitstream/2027.42/112329/1/40168_2012_Article_7.pd

IRF-5-dependent signaling restricts Orthobunyavirus dissemination to the central nervous system

ABSTRACT Interferon (IFN)-regulatory factor 5 (IRF-5) is a transcription factor that induces inflammatory responses after engagement and signaling by pattern recognition receptors. To define the role of IRF-5 during bunyavirus infection, we evaluated Oropouche virus (OROV) and La Crosse virus (LACV) pathogenesis and immune responses in primary cells and in mice with gene deletions in Irf3 , Irf5 , and Irf7 or in Irf5 alone. Deletion of Irf3 , Irf5 , and Irf7 together resulted in uncontrolled viral replication in the liver and spleen, hypercytokinemia, extensive liver injury, and an early-death phenotype. Remarkably, deletion of Irf5 alone resulted in meningoencephalitis and death on a more protracted timeline, 1 to 2 weeks after initial OROV or LACV infection. The clinical signs in OROV-infected Irf5 −/− mice were associated with abundant viral antigen and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells in several regions of the brain. Circulating dendritic cell (DC) subsets in Irf5 −/− mice had higher levels of OROV RNA in vivo yet produced lower levels of type I IFN than wild-type (WT) cells. This result was supported by data obtained in vitro , since a deficiency of IRF-5 resulted in enhanced OROV infection and diminished type I IFN production in bone marrow-derived DCs. Collectively, these results indicate a key role for IRF-5 in modulating the host antiviral response in peripheral organs that controls bunyavirus neuroinvasion in mice. IMPORTANCE Oropouche virus (OROV) and La Crosse virus (LACV) are orthobunyaviruses that are transmitted by insects and cause meningitis and encephalitis in subsets of individuals in the Americas. Recently, we demonstrated that components of the type I interferon (IFN) induction pathway, particularly the regulatory transcription factors IRF-3 and IRF-7, have key protective roles during OROV infection. However, the lethality in Irf3 −/− Irf7 −/− (DKO) mice infected with OROV was not as rapid or complete as observed in Ifnar −/− mice, indicating that other transcriptional factors associated with an IFN response contribute to antiviral immunity against OROV. Here, we evaluated bunyavirus replication, tissue tropism, and cytokine production in primary cells and mice lacking IRF-5. We demonstrate an important role for IRF-5 in preventing neuroinvasion and the ensuing encephalitis caused by OROV and LACV

Defining new therapeutics using a more immunocompetent mouse model of antibody-enhanced dengue virus infection

With over 3.5 billion people at risk and approximately 390 million human infections per year, dengue virus (DENV) disease strains health care resources worldwide. Previously, we and others established models for DENV pathogenesis in mice that completely lack subunits of the receptors (Ifnar and Ifngr) for type I and type II interferon (IFN) signaling; however, the utility of these models is limited by the pleotropic effect of these cytokines on innate and adaptive immune system development and function. Here, we demonstrate that the specific deletion of Ifnar expression on subsets of murine myeloid cells (LysM Cre(+) Ifnar(flox/flox) [denoted as Ifnar(f/f) herein]) resulted in enhanced DENV replication in vivo. The administration of subneutralizing amounts of cross-reactive anti-DENV monoclonal antibodies to LysM Cre(+) Ifnar(f/f) mice prior to infection with DENV serotype 2 or 3 resulted in antibody-dependent enhancement (ADE) of infection with many of the characteristics associated with severe DENV disease in humans, including plasma leakage, hypercytokinemia, liver injury, hemoconcentration, and thrombocytopenia. Notably, the pathogenesis of severe DENV-2 or DENV-3 infection in LysM Cre(+) Ifnar(f/f) mice was blocked by pre- or postexposure administration of a bispecific dual-affinity retargeting molecule (DART) or an optimized RIG-I receptor agonist that stimulates innate immune responses. Our findings establish a more immunocompetent animal model of ADE of infection with multiple DENV serotypes in which disease is inhibited by treatment with broad-spectrum antibody derivatives or innate immune stimulatory agents