Bimmi Shrestha

B Cells and Antibody Play Critical Roles in the Immediate Defense of Disseminated Infection by West Nile Encephalitis Virus

ABSTRACT West Nile virus (WNV) causes severe central nervous system (CNS) infection primarily in humans who are immunocompromised or elderly. In this study, we addressed the mechanism by which the immune system limits dissemination of WNV infection by infecting wild-type and immunodeficient inbred C57BL/6J mice with a low-passage WNV isolate from the recent epidemic in New York state. Wild-type mice replicated virus extraneuronally in the draining lymph nodes and spleen during the first 4 days of infection. Subsequently, virus spread to the spinal cord and the brain at virtually the same time. Congenic mice that were genetically deficient in B cells and antibody (μMT mice) developed increased CNS viral burdens and were vulnerable to lethal infection at low doses of virus. Notably, a ∼500-fold difference in serum viral load was detected in μMT mice as early as 4 days after infection, a point in the infection when low levels of neutralizing immunoglobulin M antibody were detected in wild-type mice. Passive transfer of heat-inactivated serum from infected and immune wild-type mice protected μMT mice against morbidity and mortality. We conclude that antibodies and B cells play a critical early role in the defense against disseminated infection by WNV.

Pan-viral specificity of IFN-induced genes reveals new roles for cGAS in innate immunity

The type I interferon (IFN) response protects cells from viral infection by inducing hundreds of interferon-stimulated genes (ISGs), some of which encode direct antiviral effectors. Recent screening studies have begun to catalogue ISGs with antiviral activity against several RNA and DNA viruses. However, antiviral ISG specificity across multiple distinct classes of viruses remains largely unexplored. Here we used an ectopic expression assay to screen a library of more than 350 human ISGs for effects on 14 viruses representing 7 families and 11 genera. We show that 47 genes inhibit one or more viruses, and 25 genes enhance virus infectivity. Comparative analysis reveals that the screened ISGs target positive-sense single-stranded RNA viruses more effectively than negative-sense single-stranded RNA viruses. Gene clustering highlights the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS, also known as MB21D1) as a gene whose expression also broadly inhibits several RNA viruses. In vitro, lentiviral delivery of enzymatically active cGAS triggers a STING-dependent, IRF3-mediated antiviral program that functions independently of canonical IFN/STAT1 signalling. In vivo, genetic ablation of murine cGAS reveals its requirement in the antiviral response to two DNA viruses, and an unappreciated contribution to the innate control of an RNA virus. These studies uncover new paradigms for the preferential specificity of IFN-mediated antiviral pathways spanning several virus families.

Role of CD8+ T Cells in Control of West Nile Virus Infection

ABSTRACT Infection with West Nile virus (WNV) causes fatal encephalitis more frequently in immunocompromised humans than in those with a healthy immune system. Although a complete understanding of this increased risk remains unclear, experiments with mice have begun to define how different components of the adaptive and innate immune response function to limit infection. Previously, we demonstrated that components of humoral immunity, particularly immunoglobulin M (IgM) and IgG, have critical roles in preventing dissemination of WNV infection to the central nervous system. In this study, we addressed the function of CD8+ T cells in controlling WNV infection. Mice that lacked CD8+ T cells or classical class Ia major histocompatibility complex (MHC) antigens had higher central nervous system viral burdens and increased mortality rates after infection with a low-passage-number WNV isolate. In contrast, an absence of CD8+ T cells had no effect on the qualitative or quantitative antibody response and did not alter the kinetics or magnitude of viremia. In the subset of CD8+-T-cell-deficient mice that survived initial WNV challenge, infectious virus was recovered from central nervous system compartments for several weeks. Primary or memory CD8+ T cells that were generated in vivo efficiently killed target cells that displayed WNV antigens in a class I MHC-restricted manner. Collectively, our experiments suggest that, while specific antibody is responsible for terminating viremia, CD8+ T cells have an important function in clearing infection from tissues and preventing viral persistence.

A Critical Role for Induced IgM in the Protection against West Nile Virus Infection

In humans, the elderly and immunocompromised are at greatest risk for disseminated West Nile virus (WNV) infection, yet the immunologic basis for this remains unclear. We demonstrated previously that B cells and IgG contributed to the defense against disseminated WNV infection (Diamond, M.S., B. Shrestha, A. Marri, D. Mahan, and M. Engle. 2003. J. Virol. 77:2578–2586). In this paper, we addressed the function of IgM in controlling WNV infection. C57BL/6J mice (sIgM−/−) that were deficient in the production of secreted IgM but capable of expressing surface IgM and secreting other immunoglobulin isotypes were vulnerable to lethal infection, even after inoculation with low doses of WNV. Within 96 h, markedly higher levels of infectious virus were detected in the serum of sIgM−/− mice compared with wild-type mice. The enhanced viremia correlated with higher WNV burdens in the central nervous system, and was also associated with a blunted anti-WNV IgG response. Passive transfer of polyclonal anti-WNV IgM or IgG protected sIgM−/− mice against mortality, although administration of comparable amounts of a nonneutralizing monoclonal anti-WNV IgM provided no protection. In a prospective analysis, a low titer of anti-WNV IgM antibodies at day 4 uniformly predicted mortality in wild-type mice. Thus, the induction of a specific, neutralizing IgM response early in the course of WNV infection limits viremia and dissemination into the central nervous system, and protects against lethal infection.

Ly6c+ “inflammatory monocytes” are microglial precursors recruited in a pathogenic manner in West Nile virus encephalitis

In a lethal West Nile virus (WNV) model, central nervous system infection triggered a threefold increase in CD45int/CD11b+/CD11c− microglia at days 6–7 postinfection (p.i.). Few microglia were proliferating, suggesting that the increased numbers were derived from a migratory precursor cell. Depletion of “circulating” (Gr1−(Ly6Clo)CX3CR1+) and “inflammatory” (Gr1hi/Ly6Chi/CCR2+) classical monocytes during infection abrogated the increase in microglia. C57BL/6 chimeras reconstituted with cFMS–enhanced green fluorescent protein (EGFP) bone marrow (BM) showed large numbers of peripherally derived (GFP+) microglia expressing GR1+(Ly6C+) at day 7 p.i., suggesting that the inflammatory monocyte is a microglial precursor. This was confirmed by adoptive transfer of labeled BM (Ly6Chi/CD115+) or circulating inflammatory monocytes that trafficked to the WNV-infected brain and expressed a microglial phenotype. CCL2 is a chemokine that is highly expressed during WNV infection and important in inflammatory monocyte trafficking. Neutralization of CCL2 not only reduced the number of GFP+ microglia in the brain during WNV infection but prolonged the life of infected animals. Therefore, CCL2-dependent inflammatory monocyte migration is critical for increases in microglia during WNV infection and may also play a pathogenic role during WNV encephalitis.

Infection and Injury of Neurons by West Nile Encephalitis Virus

ABSTRACT West Nile virus (WNV) infects neurons and leads to encephalitis, paralysis, and death in humans, animals, and birds. We investigated the mechanism by which neuronal injury occurs after WNV infection. Neurons in the anterior horn of the spinal cords of paralyzed mice exhibited a high degree of WNV infection, leukocyte infiltration, and degeneration. Because it was difficult to distinguish whether neuronal injury was caused by viral infection or by the immune system response, a novel tissue culture model for WNV infection was established in neurons derived from embryonic stem (ES) cells. Undifferentiated ES cells were relatively resistant to WNV infection. After differentiation, ES cells expressed neural antigens, acquired a neuronal phenotype, and became permissive for WNV infection. Within 48 h of exposure to an exceedingly low multiplicity of infection (5 × 10−4), 50% of ES cell-derived neurons became infected, producing nearly 107 PFU of infectious virus per ml, and began to die by an apoptotic mechanism. The establishment of a tractable virus infection model in ES cell-derived neurons facilitates the study of the molecular basis of neurotropism and the mechanisms of viral and immune-mediated neuronal injury after infection by WNV or other neurotropic pathogens.

CD8+ T Cells Require Perforin To Clear West Nile Virus from Infected Neurons

ABSTRACT Injury to neurons after West Nile virus (WNV) infection is believed to occur because of viral and host immune-mediated effects. Previously, we demonstrated that CD8+ T cells are required for the resolution of WNV infection in the central nervous system (CNS). CD8+ T cells can control infection by producing antiviral cytokines (e.g., gamma interferon or tumor necrosis factor alpha) or by triggering death of infected cells through perforin- or Fas ligand-dependent pathways. Here, we directly evaluated the role of perforin in controlling infection of a lineage I New York isolate of WNV in mice. A genetic deficiency of perforin molecules resulted in higher viral burden in the CNS and increased mortality after WNV infection. In the few perforin-deficient mice that survived initial challenge, viral persistence was observed in the CNS for several weeks. CD8+ T cells required perforin to control WNV infection as adoptive transfer of WNV-primed wild-type but not perforin-deficient CD8+ T cells greatly reduced infection in the brain and spinal cord and enhanced survival of CD8-deficient mice. Analogous results were obtained when wild-type or perforin-deficient CD8+ T cells were added to congenic primary cortical neuron cultures. Taken together, our data suggest that despite the risk of immunopathogenesis, CD8+ T cells use a perforin-dependent mechanism to clear WNV from infected neurons.

Innate and adaptive immune responses determine protection against disseminated infection by West Nile encephalitis virus.

WNV continues to spread throughout the Western Hemisphere as virus activity in insects and animals has been reported in the United States, Canada, Mexico, and the Caribbean islands. West Nile virus (WNV) infects the central nervous system and causes severe disease primarily in humans who are immunocompromised or elderly. In this review, we discuss the mechanisms by which the immune system limits dissemination of WNV infection. Recent experimental studies in animals suggest important roles for both the innate and the adaptive immune responses in controlling WNV infection. Interferons, antibody, complement components and CD8+ T cells coordinate protection against severe infection and disease. These findings are analyzed in the context of recent approaches to vaccine development and immunotherapy against WNV.

The Development of Therapeutic Antibodies That Neutralize Homologous and Heterologous Genotypes of Dengue Virus Type 1

Antibody protection against flaviviruses is associated with the development of neutralizing antibodies against the viral envelope (E) protein. Prior studies with West Nile virus (WNV) identified therapeutic mouse and human monoclonal antibodies (MAbs) that recognized epitopes on domain III (DIII) of the E protein. To identify an analogous panel of neutralizing antibodies against DENV type-1 (DENV-1), we immunized mice with a genotype 2 strain of DENV-1 virus and generated 79 new MAbs, 16 of which strongly inhibited infection by the homologous virus and localized to DIII. Surprisingly, only two MAbs, DENV1-E105 and DENV1-E106, retained strong binding and neutralizing activity against all five DENV-1 genotypes. In an immunocompromised mouse model of infection, DENV1-E105 and DENV1-E106 exhibited therapeutic activity even when administered as a single dose four days after inoculation with a heterologous genotype 4 strain of DENV-1. Using epitope mapping and X-ray crystallographic analyses, we localized the neutralizing determinants for the strongly inhibitory MAbs to distinct regions on DIII. Interestingly, sequence variation in DIII alone failed to explain disparities in neutralizing potential of MAbs among different genotypes. Overall, our experiments define a complex structural epitope on DIII of DENV-1 that can be recognized by protective antibodies with therapeutic potential.

Gamma Interferon Plays a Crucial Early Antiviral Role in Protection against West Nile Virus Infection

ABSTRACT West Nile virus (WNV) causes a severe central nervous system (CNS) infection in humans, primarily in the elderly and immunocompromised. Prior studies have established an essential protective role of several innate immune response elements, including alpha/beta interferon (IFN-α/β), immunoglobulin M, γδ T cells, and complement against WNV infection. In this study, we demonstrate that a lack of IFN-γ production or signaling results in increased vulnerability to lethal WNV infection by a subcutaneous route in mice, with a rise in mortality from 30% (wild-type mice) to 90% (IFN-γ−/− or IFN-γR−/− mice) and a decrease in the average survival time. This survival pattern in IFN-γ−/− and IFN-γR−/− mice correlated with higher viremia and greater viral replication in lymphoid tissues. The increase in peripheral infection led to early CNS seeding since infectious WNV was detected several days earlier in the brains and spinal cords of IFN-γ−/− or IFN-γR−/− mice. Bone marrow reconstitution experiments showed that γδ T cells require IFN-γ to limit dissemination by WNV. Moreover, treatment of primary dendritic cells with IFN-γ reduced WNV production by 130-fold. Collectively, our experiments suggest that the dominant protective role of IFN-γ against WNV is antiviral in nature, occurs in peripheral lymphoid tissues, and prevents viral dissemination to the CNS.