Bioinformatics Analyses Determined the Distinct CNS and Peripheral Surrogate Biomarker Candidates Between Two Mouse Models for Progressive Multiple Sclerosis.

Previously, we have established two distinct progressive multiple sclerosis (MS) models by induction of experimental autoimmune encephalomyelitis (EAE) with myelin oligodendrocyte glycoprotein (MOG) in two mouse strains. A.SW mice develop ataxia with antibody deposition, but no T cell infiltration, in the central nervous system (CNS), while SJL/J mice develop paralysis with CNS T cell infiltration. In this study, we determined biomarkers contributing to the homogeneity and heterogeneity of two models. Using the CNS and spleen microarray transcriptome and cytokine data, we conducted computational analyses. We identified up-regulation of immune-related genes, including immunoglobulins, in the CNS of both models. Pro-inflammatory cytokines, interferon (IFN)-γ and interleukin (IL)-17, were associated with the disease progression in SJL/J mice, while the expression of both cytokines was detected only at the EAE onset in A.SW mice. Principal component analysis (PCA) of CNS transcriptome data demonstrated that down-regulation of prolactin may reflect disease progression. Pattern matching analysis of spleen transcriptome with CNS PCA identified 333 splenic surrogate markers, including Stfa2l1, which reflected the changes in the CNS. Among them, we found that two genes (PER1/MIR6883 and FKBP5) and one gene (SLC16A1/MCT1) were also significantly up-regulated and down-regulated, respectively, in human MS peripheral blood, using data mining.

Murine survival of infection with Francisella novicida and protection against secondary challenge is critically dependent on B lymphocytes.

Respiratory infection of mice with Francisella novicida has recently been used as a model for the highly virulent human pathogen Francisella tularensis. Similar to F. tularensis, even small doses of F. novicida administered by respiratory routes are lethal for inbred laboratory mice. This feature obviously limits study of infection-induced immunity. Parenteral sublethal infections of mice with F. novicida are feasible, but the resulting immune responses are incompletely characterized. Here we use parenteral intradermal (i.d.) and intraperitoneal (i.p.) F. novicida infections of C57BL/6J mice to determine the role of B cells in controlling primary and secondary F. novicida infections. Despite developing comparable levels of F. novicida-primed T cells, B cell knockout mice were much more susceptible to both primary i.d. infection and secondary i.p. challenge than wild type (normal) C57BL/6J mice. Transfer of F. novicida-immune sera to either wild type C57BL/6J mice or to B cell knockout mice did not appreciably impact survival of subsequent lethal F. novicida challenge. However, F. novicida-immune mice that were depleted of T cells after priming but just before challenge survived and cleared secondary i.p. F. novicida challenge. Collectively these results indicate that B cells, if not serum antibodies, play a major role in controlling F. novicida infections in mice.

Activities of Murine Peripheral Blood Lymphocytes Provide Immune Correlates That Predict Francisella tularensis Vaccine Efficacy.

We previously identified potential correlates of vaccine-induced protection against Francisella tularensis using murine splenocytes and further demonstrated that the relative levels of gene expression varied significantly between tissues. In contrast to splenocytes, peripheral blood leukocytes (PBLs) represent a means to bridge vaccine efficacy in animal models to that in humans. Here we take advantage of this easily accessible source of immune cells to investigate cell-mediated immune responses against tularemia, whose sporadic incidence makes clinical trials of vaccines difficult. Using PBLs from mice vaccinated with F. tularensis Live Vaccine Strain (LVS) and related attenuated strains, we combined the control of in vitro Francisella replication within macrophages with gene expression analyses. The in vitro functions of PBLs, particularly the control of intramacrophage LVS replication, reflected the hierarchy of in vivo protection conferred by LVS-derived vaccines. Moreover, several genes previously identified by the evaluation of splenocytes were also found to be differentially expressed in immune PBLs. In addition, more extensive screening identified additional potential correlates of protection. Finally, expression of selected genes in mouse PBLs obtained shortly after vaccination, without ex vivo restimulation, was different among vaccine groups, suggesting a potential tool to monitor efficacious vaccine-induced immune responses against F. tularensis. Our studies demonstrate that murine PBLs can be used productively to identify potential correlates of protection against F. tularensis and to expand and refine a comprehensive set of protective correlates.

Francisella tularensis Vaccines Elicit Concurrent Protective T- and B-Cell Immune Responses in BALB/cByJ Mice.

In the last decade several new vaccines against Francisella tularensis, which causes tularemia, have been characterized in animal models. Whereas many of these vaccine candidates showed promise, it remains critical to bridge the preclinical studies to human subjects, ideally by taking advantage of correlates of protection. By combining in vitro intramacrophage LVS replication with gene expression data through multivariate analysis, we previously identified and quantified correlative T cell immune responses that discriminate vaccines of different efficacy. Further, using C57BL/6J mice, we demonstrated that the relative levels of gene expression vary according to vaccination route and between cell types from different organs. Here, we extended our studies to the analysis of T cell functions of BALB/cByJ mice to evaluate whether our approach to identify correlates of protection also applies to a Th2 dominant mouse strain. BALB/cByJ mice had higher survival rates than C57BL/6J mice when they were immunized with suboptimal vaccines and challenged. However, splenocytes derived from differentially vaccinated BALB/cByJ mice controlled LVS intramacrophage replication in vitro in a pattern that reflected the hierarchy of protection observed in C57BL/6J mice. In addition, gene expression of selected potential correlates revealed similar patterns in splenocytes of BALB/cByJ and C57BL/6J mice. The different survival patterns were related to B cell functions, not necessarily to specific antibody production, which played an important protective role in BALB/cByJ mice when vaccinated with suboptimal vaccines. Our studies therefore demonstrate the range of mechanisms that operate in the most common mouse strains used for characterization of vaccines against F. tularensis, and illustrate the complexity necessary to define a comprehensive set of correlates.

Models derived from in vitro analyses of spleen, liver, and lung leukocyte functions predict vaccine efficacy against the Francisella tularensis Live Vaccine Strain (LVS).

Currently, there are no licensed vaccines and no correlates of protection against Francisella tularensis, which causes tularemia. We recently demonstrated that measuring in vitro control of intramacrophage bacterial growth by murine F. tularensis-immune splenocytes, as well as transcriptional analyses, discriminated Francisella vaccines of different efficacies. Further, we identified potential correlates of protection against systemic challenge. Here, we extended this approach by studying leukocytes derived from lungs and livers of mice immunized by parenteral and respiratory routes with F. tularensis vaccines. Liver and lung leukocytes derived from intradermally and intranasally vaccinated mice controlled in vitro Francisella Live Vaccine Strain (LVS) intramacrophage replication in patterns similar to those of splenocytes. Gene expression analyses of potential correlates also revealed similar patterns in liver cells and splenocytes. In some cases (e.g., tumor necrosis factor alpha [TNF-α], interleukin 22 [IL-22], and granulocyte-macrophage colony-stimulating factor [GM-CSF]), liver cells exhibited even higher relative gene expression, whereas fewer genes exhibited differential expression in lung cells. In contrast with their strong ability to control LVS replication, splenocytes from intranasally vaccinated mice expressed few genes with a hierarchy of expression similar to that of splenocytes from intradermally vaccinated mice. Thus, the relative levels of gene expression vary between cell types from different organs and by vaccination route. Most importantly, because studies comparing cell sources and routes of vaccination supported the predictive validity of this coculture and gene quantification approach, we combined in vitro LVS replication with gene expression data to develop analytical models that discriminated between vaccine groups and successfully predicted the degree of vaccine efficacy. Thus, this strategy remains a promising means of identifying and quantifying correlative T cell responses. IMPORTANCE Identifying and quantifying correlates of protection is especially challenging for intracellular bacteria, including Francisella tularensis. F. tularensis is classified as a category A bioterrorism agent, and no vaccines have been licensed in the United States, but tularemia is a rare disease. Therefore, clinical trials to test promising vaccines are impractical. In this report, we further evaluated a novel approach to developing correlates by assessing T cell immune responses in lungs and livers of differentially vaccinated mice; these nonprofessional immune tissues are colonized by Francisella. The relative degree of vaccine efficacy against systemic challenge was reflected by the ability of immune T cells, particularly liver T cells, to control the intramacrophage replication of bacteria in vitro and by relative gene expression of several immunological mediators. We therefore developed analytical models that combined bacterial replication data and gene expression data. Several resulting models provided excellent discrimination between vaccines of different efficacies.

Generation of protection against Francisella novicida in mice depends on the pathogenicity protein PdpA, but not PdpC or PdpD.

Previous results suggest that mutations in most genes in the Francisella pathogenicity island (FPI) attenuate the bacterium. Using a mouse model, here we determined the impact of mutations in pdpA, pdpC, and pdpD in Francisella novicida on in vitro replication in macrophages, and in vivo immunogenicity. In contrast to most FPI genes, deletion of pdpC (FnΔpdpC) and pdpD (FnΔpdpD) from F. novicida did not impact growth in mouse bone-marrow derived macrophages. Nonetheless, both FnΔpdpC and FnΔpdpD were highly attenuated when administered intradermally. Infected mice produced relatively normal anti-F. novicida serum antibodies. Further, splenocytes from infected mice controlled intramacrophage Francisella replication, indicating T cell priming, and mice immunized by infection with FnΔpdpC or FnΔpdpD survived secondary lethal parenteral challenge with either F. novicida or Francisella tularensis LVS. In contrast, deletion of pdpA (FnΔpdpA) ablated growth in macrophages in vitro. FnΔpdpA disseminated and replicated poorly in infected mice, accompanied by development of some anti-F. novicida serum antibodies. However, primed Th1 cells were not detected, and vaccinated mice did not survive even low dose challenge with either F. novicida or LVS. Taken together, these results suggest that successful priming of Th1 cells, and protection against lethal challenge, depends on expression of PdpA.

Once initiated, viral encephalitis-induced seizures are consistent no matter the treatment or lack of interleukin-6.

Theiler's murine encephalomyelitis virus infection of C57BL/6 mice results in acute behavioral seizures in 50% of the mice. Treatment of infected mice with minocycline or infection of interleukin (IL)-6-deficient chimeric mice results in a significant decrease in the number of mice developing seizures. However, in those mice that do develop seizures, the pathological changes (neuronal cell loss, inflammation [perivascular cuffing, gliosis, activated microglia/macrophages]), and the numbers of virus infected cells in minocycline-treated or IL-6-deficient chimeric mice are very similar. Therefore, once seizures develop, the pathological changes are consistent regardless of the treatment or genetic background.

Lack of correlation of central nervous system inflammation and neuropathology with the development of seizures following acute virus infection.

Infection of C57BL/6 mice by the intracerebral route with the Daniels (DA) strain of Theiler's murine encephalomyelitis virus (TMEV) resulted in acute behavioral seizures in approximately 50% of the mice. By titration, the viral dose correlated with the percentage of mice developing seizures; however, neuropathological changes were similar over the dose range, and viral clearance from the brains occurred uniformly by day 14 postinfection (p.i.). Other TMEV strains and mutants (GDVII, WW, BeAn 8386 [BeAn], DApBL2M, H101) induced seizures in C57BL/6 mice to various degrees. The BeAn strain and DApBL2M mutant were similar to the DA strain in the percentages of mice developing seizures and neuropathological changes and in the extent of infected cells. The GDVII and WW strains caused 100% mortality by days 5 and 6 p.i., respectively, at which time neuropathological changes and neuronal infection were extensive. The H101 mutant induced seizures and caused 100% mortality by day 7 p.i.; however, only minor neuropathological changes and few infected cells were observed. Thus, in H101 mutant infections, it appears that elevated levels of cytokines, rather than neuronal cell death, play the dominant role in seizure induction.

Interleukin-6, produced by resident cells of the central nervous system and infiltrating cells, contributes to the development of seizures following viral infection.

Cells that can participate in an innate immune response within the central nervous system (CNS) include infiltrating cells (polymorphonuclear leukocytes [PMNs], macrophages, and natural killer [NK] cells) and resident cells (microglia and sometimes astrocytes). The proinflammatory cytokine interleukin-6 (IL-6) is produced by all of these cells and has been implicated in the development of behavioral seizures in the Theiler's murine encephalomyelitis virus (TMEV)-induced seizure model. The assessment, via PCR arrays, of the mRNA expression levels of a large number of chemokines (ligands and receptors) in TMEV-infected and mock-infected C57BL/6 mice both with and without seizures did not clearly demonstrate the involvement of PMNs, monocytes/macrophages, or NK cells in the development of seizures, possibly due to overlapping function of the chemokines. Additionally, C57BL/6 mice unable to recruit or depleted of infiltrating PMNs and NK cells had seizure rates comparable to those of controls following TMEV infection, and therefore PMNs and NK cells do not significantly contribute to seizure development. In contrast, C57BL/6 mice treated with minocycline, which affects monocytes/macrophages, microglial cells, and PMNs, had significantly fewer seizures than controls following TMEV infection, indicating monocytes/macrophages and resident microglial cells are important in seizure development. Irradiated bone marrow chimeric mice that were either IL-6-deficient mice reconstituted with wild-type bone marrow cells or wild-type mice reconstituted with IL-6-deficient bone marrow cells developed significantly fewer behavioral seizures following TMEV infection. Therefore, both resident CNS cells and infiltrating cells are necessary for seizure development.