The immunobiology of Japanese encephalitis virus

Japanese encephalitis virus is the leading cause of viral encephalitis in Asia. In spite of this, the immunological correlates for recovery from primary Japanese encephalitis virus (JEV) infection remain poorly defined. In this work, I have first established a mouse model for Japanese encephalitis in which a low-dose virus inoculum was administered subcutaneously into adult C57BL/6 mice. In this model, ~60% of the mice developed fatal encephalitis, concomitant with virus burden in the central nervous system (CNS). JEV infection induced a peak natural killer (NK) cell response on day 4 post-infection (pi), a peak CD8+ T cell response on day 7 pi, a peak anti-JEV IgM response on day 8 pi, and an increasing anti-JEV IgG responses from day 8 pi onwards. Second, to investigate the relative importance of immune effector cell populations, splenocyte transfer experiments, and JEV-challenge of mice with acquired or induced cellular deficiency were performed. Using mice lacking B cells ({u03BC}MT^-/-^ mice) and immune B cell transfer to wild-type mice, I find a critically important role for humoral immunity in preventing virus spread to the CNS. T cell help played an essential part in the maintenance of an effective antibody response necessary to combat the infection, since mice lacking major histocompatibility complex class II showed truncated IgM and blunted IgG responses, and the infection was uniformly lethal. On the other hand, NK cells were dispensable for protection against lethal Japanese encephalitis, while CD8+T cells, by itself, have no significant effect on the survival of JEV-infected mice. Third, cytolytic effector pathways and interferon gamma (IFN-y) help protect against lethal JE. Granule exocytosis and Fas-Fas ligand pathways of cytotoxicity act within the CNS to reduce disease severity, and show a redundancy in control of JEV infection. Only mice defective in both cell contact-dependent cytotoxic effector mechanisms display increased susceptibility to subcutaneous challenge with a low dose of JEV. Also, I demonstrate that IFN-y is critical in recovery from primary infection with JEV by a mechanism involving suppression of virus growth in the CNS, but not in extraneural tissues, and that T cells are the main source of the cytokine that promotes viral clearance from the brain. Fourth, the chemokine receptor, CCR5, serves as a host antiviral factor important against lethal JE. CCR5 deficiency resulted in increased mortality, and viral burden in the CNS. Absence of CCR5 did not only result in impaired trafficking of leucocytes into the brain, but also resulted in a multifaceted deficiency of cellular immune responses characterized by reduced NK cell activity, reduced CD8+ T cell activity, and low splenic cellularity. This result is consistent with a mechanism by which CCR5 expression enhances lymphocyte activation and thereby promotes host survival after JEV infection. Fifth, a novel immunodeficient mouse strain, Tuara, was established. Tuara mice displayed enhanced susceptibility to flavivirus infections, due to a loss-of-function mutation that was originally derived from an inbred knockout mouse strain, MHCII-A{u03B1}-/-. The gene of interest was segregated from the MHCII defect, and after customized sequence capture and re-sequencing, was localized within ~50 Mb region of Chromosome 1. Among the candidate list, a G>T transversion on mouse genome reference position 52,179,547 bp of STAT! gene was identified as the most likely causal mutation. Finally, towards application of the insights gained on the basic immunobiology of JE, vaccination experiments using 'new generation' JE vaccines were performed. They revealed a dominant role of humoral immunity in vaccine-mediated protection of mice from lethal challenge with JEV, and in cross-protection against related viruses belonging to the JE serocomplex. CD8+ T cells were not required for protection, while CD4+ T cells provided substantial helper function for the protective humoral immune responses induced by vaccination. These immune correlates for homologous and heterologous protection against JE serocomplex viruses were observed in vaccination with either a live attenuated vaccine (ChimeriVax-JE), or a candidate inactivated Vero cell culture-derived JE vaccine (ccJE) formulated with Advax{u2122}, a novel immunologically active polysaccharide adjuvant

Pivotal Role of Antibody and Subsidiary Contribution of CD8+ T Cells to Recovery from Infection in a Murine Model of Japanese Encephalitis

The immunological correlates for recovery from primary Japanese encephalitis virus (JEV) infection in humans and experimental animals remain poorly defined. To investigate the relative importance of the adaptive immune responses, we have established a mouse model for Japanese encephalitis in which a low-dose virus inoculum was administered into the footpads of adult C57BL/6 mice. In this model, ~60% of the mice developed a fatal encephalitis and a virus burden in the central nervous system (CNS). Using mice lacking B cells (μMT-/- mice) and immune B cell transfer to wild-type mice, we show a critically important role for humoral immunity in preventing virus spread to the CNS. T cell help played an essential part in the maintenance of an effective antibody response necessary to combat the infection, since mice lacking major histocompatibility complex class II showed truncated IgM and blunted IgG responses and uniformly high lethality. JEV infection resulted in extensive CD8+ T cell activation, judged by upregulation of surface markers CD69 and CD25 and cytokine production after stimulation with a JEV NS4B protein-derived H-2Db-binding peptide and trafficking of virus-immune CD8+ T cells into the CNS. However, no significant effect of CD8+ T cells on the survival phenotype was found, which was corroborated in knockout mice lacking key effector molecules (Fas receptor, perforin, or granzymes) of cytolytic pathways triggered by T lymphocytes. Accordingly, CD8+ T cells are mostly dispensable for recovery from infection with JEV. This finding highlights the conflicting role that CD8+T cells play in the pathogenesis of JEV and closely related encephalitic flaviviruses such as West Nile virus

Multiple migrations to the Philippines during the last 50,000 years

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The chemokine receptor CCR5, a therapeutic target for HIV/AIDS antagonists, is critical for recovery in a mouse model of Japanese encephalitis

Japanese encephalitis is a severe central nervous system (CNS) inflammatory disease caused by the mosquito-borne flavivirus, Japanese encephalitis virus (JEV). In the current study we have investigated the immune responses against JEV in mice lacking expression of the chemokine receptor CCR5, which functions in activation and chemotaxis of leukocytes during infection. We show that CCR5 serves as a host antiviral factor against Japanese encephalitis, with CCR5 deficiency markedly increasing mortality, and viral burden in the CNS. Humoral immune responses, which are essential in recovery from JEV infection, were of similar magnitude in CCR5 sufficient and deficient mice. However, absence of CCR5 resulted in a multifaceted deficiency of cellular immune responses characterized by reduced natural killer and CD8 T cell activity, low splenic cellularity, and impaired trafficking of leukocytes to the brain. Interestingly, adoptive transfer of immune spleen cells, depleted of B lymphocytes, increased resistance of CCR5-deficient recipient mice against JEV regardless of whether the cells were obtained from CCR5-deficient or wild-type donor mice, and only when transferred at one but not at three days post-challenge. This result is consistent with a mechanism by which CCR5 expression enhances lymphocyte activation and thereby promotes host survival in Japanese encephalitis

Requirement for Cyclic AMP/Protein Kinase A-Dependent Canonical NFκB Signaling in the Adjuvant Action of Cholera Toxin and Its Non-toxic Derivative mmCT

Cholera toxin (CT) is widely used as an effective adjuvant in experimental immunology for inducing mucosal immune responses; yet its mechanisms of adjuvant action remain incompletely defined. Here, we demonstrate that mice lacking NFκB, compared to wild-type (WT) mice, had a 90% reduction in their systemic and mucosal immune responses to oral immunization with a model protein antigen [Ovalbumin (OVA)] given together with CT. Further, NFκB−/− mouse dendritic cells (DCs) stimulated in vitro with CT showed reduced expression of MHCII and co-stimulatory molecules, such as CD80 and CD86, as well as of IL-1β, and other pro-inflammatory cytokines compared to WT DCs. Using a human monocyte cell line THP1 with an NFκB activation reporter system, we show that CT induced NFκB signaling in human monocytes, and that inhibition of the cyclic AMP—protein kinase A (cAMP-PKA) pathway abrogated the activation and nuclear translocation of NFκB. In a human monocyte-CD4+ T cell co-culture system we further show that the strong Th17 response induced by CT treatment of monocytes was abolished by blocking the classical but not the alternative NFκB signaling pathway of monocytes. Our results indicate that activation of classical (canonical) NFκB pathway signaling in antigen-presenting cells (APCs) by CT is important for CT's adjuvant enhancement of Th17 responses. Similar findings were obtained using the almost completely detoxified mmCT mutant protein as adjuvant. Altogether, our results demonstrate that activation of the classical NFκB signal transduction pathway in APCs is important for the adjuvant action of both CT and mmCT

Partial dysfunction of STAT1 profoundly reduces host resistance to flaviviral infection

The genetic basis for a dramatically increased virus susceptibility phenotype of MHC-II knockout mice acquired during routine maintenance of the mouse strain was determined. Segregation of the susceptibility allele from the defective MHC-II locus combined with sequence capture and sequencing showed that a Y37L substitution in STAT1 accounted for high flavivirus susceptibility of a newly derived mouse strain, designated Tuara. Interestingly, the mutation in STAT1 gene gave only partial inactivation of the type I interferon antiviral pathway. Accordingly, merely a relatively small impairment of interferon α/β signalling is sufficient to overcome the ability of the host to control the infection

Proteomic analysis of cholera toxin adjuvant-stimulated human monocytes identifies Thrombospondin-1 and Integrin-beta 1 as strongly upregulated molecules involved in adjuvant activity

Cholera Toxin (CT) as well as its related non-toxic mmCT and dmLT mutant proteins have been shown to be potent adjuvants for mucosally administered vaccines. Their adjuvant activity involves activation of cAMP/protein kinase A (PKA) signaling and inflammasome/IL-1 beta pathways in antigen presenting cells (APC). To get a further understanding of the signal transduction and downstream pathways activated in APCs by this group of adjuvants we have, employing quantitative proteomic analytic tools, investigated human monocytes at various time points after treatment with CT. We report the activation of three main biological pathways among upregulated proteins, peaking at 16 hours of CT treatment: cellular organization, metabolism, and immune response. Specifically, in the further analyzed immune response pathway we note a strong upregulation of thrombospondin 1 (THBS1) and integrin beta 1 (ITGB1) in response to CT as well as to mmCT and dmLT, mediated via cAMP/PKA and NFKB signaling. Importantly, inhibition in vitro of THSB1 and ITGB1 in monocytes or primary dendritic cells using siRNA abrogated the ability of the treated APCs to promote an adjuvant-stimulated Th17 cell response when co-cultured with peripheral blood lymphocytes indicating the involvement of these molecules in the adjuvant action on APCs by CT, mmCT and dmLT

Antibody responses in CCR5^+/+ and CCR5^−/− mice.

<p>Eight-week-old CCR5^+/+ and CCR5^−/− mice were infected i.v. with 10³ PFU of JEV, and serum samples were collected at the indicated time points. Anti-JEV IgM (<b>A</b>) and IgG (<b>B</b>) isotype antibody titers were determined by ELISA. The data presented are reciprocal mean endpoint titers representative of 4 mice per time point with the SEM indicated by error bars. (<b>C</b>) Neutralizing antibody titers determined by plaque reduction neutralization assay. The data presented are mean PRNT₅₀ titers representative of 5 mice per time point, and error bars indicate the SEM.</p