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

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-alpha], 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

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-alpha], 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

Anti-LVS antibody titers of HK-LVS vaccinated BALB/cByJ mice do not correlate with survival.

<p>Sera from BALB/cByJ mice vaccinated with different doses of HK-LVS were individually analyzed six weeks after vaccination for anti-LVS total IgG. Mice were then challenged IP with a maximal lethal dose of LVS. Error bars depict standard deviation of the mean of triplicates samples tested in the ELISA. * indicates the antibody responses of the mice that eventually survived the lethal challenge. Results shown are from one representative experiment of two independent experiments of similar design and outcome.</p

Protein staining and reactivity with serum derived from vaccinated mice revealed differences among vaccine extracts.

<p>Twenty μg of protein extracts, prepared from whole LVS, HK-LVS, LVS-G, and LVS-R, were loaded on SDS-PAGE in reducing conditions and stained with Red Ponceau (Panel A). Following Ponceau staining, reactivity of protein extracts was analyzed by blotting the membranes with pooled sera derived from LVS vaccinated (Panel B) and from HK-LVS vaccinated BALB/cByJ mice (Panel C). Results shown are from one representative experiment of four independent experiments of similar design and outcome.</p

Humoral immune responses patterns to LVS-related vaccines differ between BALB/cByJ and C57BL/6J mice.

<p>Pooled sera from five mice for each vaccine group were obtained from BALB/cByJ mice (Panel A) and from C57BL/6J mice (Panel B) six weeks after vaccination, and analyzed for anti-LVS total IgG. Error bars depict standard deviation of the mean of triplicates samples tested in the ELISA. Results shown are from one representative experiment of four independent experiments of similar design and outcome.</p

Splenocytes from LVS-related vaccinated mice exhibit a hierarchy of control of intramacrophage LVS growth.

<p>BMMΦ from BALB/cByJ mice were infected with LVS (Macs), and co-cultured with splenocytes obtained from naïve or vaccinated BALB/cByJ mice (Panel A), and from naive or vaccinated BKO mice (Panel B), as indicated. After two days of co-culture, BMMΦ were washed, lysed, and plated to evaluate the recovery of intracellular bacteria. Values shown are the mean numbers of CFU/ml ± SD of viable bacteria for triplicate samples. Results shown are from one representative experiment of seven (using splenocytes of BALB/cByJ mice) or four (using splenocytes of BKO mice) independent experiments of similar design and outcome. Brackets indicate a significant difference (<i>P</i> < 0.05) between the recoveries of bacteria in co-cultures. There were no significant differences between the recovery of bacteria from co-cultures using LVS-immune cells and LVS-G-immune cells (Panel A) and the recovery of bacteria from co-cultures using LVS-G-immune cells and LVS-R-immune cells (Panel B).</p

Effect of differential vaccination on survival against lethal challenge with LVS.

<p>Three to five BALB/cByJ, BKO, or C57BL/6J mice per group, for a total of 33–35, 14–15, or 15–17 mice per vaccine group, respectively, were vaccinated I.D. with the indicated vaccines, and then challenged 4–6 weeks later with a lethal dose of 10⁶ LVS I.P. Values represent combined results of seven, three, or four-five independent experiments for the BALB/cByJ, BKO, or C57BL/6J mice, respectively. Values in parenthesis indicate ranges of survival among experiments.</p><p>Effect of differential vaccination on survival against lethal challenge with LVS.</p

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

<div><p>In the last decade several new vaccines against <i>Francisella tularensis</i>, 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 <i>in vitro</i> 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 <i>in vitro</i> 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 <i>F</i>. <i>tularensis</i>, and illustrate the complexity necessary to define a comprehensive set of correlates.</p></div

Relative gene expression of potential correlates in co-cultures using splenocytes from differentially vaccinated BALB/cByJ, BKO, and C57BL/6J mice.

<p>Values indicate median fold change of the indicated genes, compared to naive cells; values derived from analyses of splenocytes of BALB/cByJ, BKO, and C57BL/6J mice were calculated from seven, four, and four independent experiments, respectively.</p><p>Relative gene expression of potential correlates in co-cultures using splenocytes from differentially vaccinated BALB/cByJ, BKO, and C57BL/6J mice.</p