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ABSTRACTWe previously identified potential correlates of vaccine-induced protection against Francisella tularensisusing 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. tularensisLive Vaccine Strain (LVS) and related attenuated strains, we combined the control of in vitroFrancisellareplication within macrophages with gene expression analyses. The in vitrofunctions of PBLs, particularly the control of intramacrophage LVS replication, reflected the hierarchy of in vivoprotection 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 vivorestimulation, 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. tularensisand to expand and refine a comprehensive set of protective correlates.

ABSTRACTStaphylococcus aureusis one of the most common etiological agents of community-acquired skin and soft tissue infection (SSTI). Although the majority of S. aureuscommunity-acquired SSTIs are uncomplicated and self-clearing in nature, some percentage of these cases progress into life-threatening invasive infections. Current animal models of S. aureusSSTI suffer from two drawbacks: these models are a better representation of hospital-acquired SSTI than community-acquired SSTI, and they involve methods that are difficult to replicate. For these reasons, we sought to develop a murine model of community-acquired methicillin-resistant S. aureusSSTI (CA-MRSA SSTI) that can be consistently reproduced with a high degree of precision. We utilized this model to begin to characterize the host immune response to this type of infection. We infected mice via epicutaneous challenge of the skin on the outer ear pinna using Morrow-Brown allergy test needles coated in S. aureusUSA300. When mice were challenged in this model, they developed small, purulent, self-clearing lesions with predictable areas of inflammation that mimicked a human infection. CFU in the ear pinna peaked at day 7 before dropping by day 14. The Th1 and Th17 cytokines gamma interferon (IFN-?), interleukin-12 (IL-12) p70, tumor necrosis factor alpha (TNF-a), IL-17A, IL-6, and IL-21 were all significantly increased in the draining lymph node of infected mice, and there was neutrophil recruitment to the infection site. In vivoneutrophil depletion demonstrated that neutrophils play a protective role in preventing bacterial dissemination and fatal invasive infection.

ABSTRACTFrancisella tularensis, a small gram-negative intracellular bacterium responsible for causing tularemia, is highly pathogenic and classified as a category A agent of bioterrorism. As for other intracellular pathogens, successful protective immune responses to Francisella tularensisrequire rapid and efficient induction of gamma interferon (IFN-γ) production. Studies using intracellular bacteria such as Listeria monocytogenesas well as Francisellasuggest that natural killer (NK) and T cells are important sources of IFN-γ. However, comprehensive characterization of specific sources of IFN-γ produced during Francisellainfection in vivo remains incomplete, and depletion of NK cells before infection of mice with the F. tularensislive vaccine strain (LVS) has little impact on the course or outcome of infection. In this study, we determined the cell subpopulations that respond quickly to intradermal F. tularensisLVS infection of mice by producing IFN-γ within hours to a few days. Splenic and liver lymphocytes were obtained from LVS-infected mice and analyzed for IFN-γ mRNA by reverse transcription-PCR, for intracellular cytokine expression by multiparameter flow cytometry, and for ex vivo production of IFN-γ protein by enzyme-linked immunosorbent assay. Cells producing IFN-γ were readily detectable by day 3 after infection, and numbers progressively increased through days 5 to 7. Importantly, the cell types responsible for IFN-γ production were much more varied than expected: these included not only NK cells and T cells, which might be predicted, but also other cells, including dendritic cells (DCs), “NK DCs,” NK T cells, and neutrophils. Most importantly, since RAG-1 knockout mice appeared to exhibit a frequency of IFN-γ-producing cells comparable to that of intact wild-type mice, early IFN-γ production by innate immune cells does not depend on the presence of T or B cells.

Regulatory T cells (TREGs) control the key aspects of tolerance and play a role in the lack of antitumor immune responses. Cyclophosphamide (CY) is a chemotherapeutic agent with a dose-dependent, bimodal effect on the immune system. Although a previous study demonstrated that CY reduces the number of TREGs, the mechanism involved in this process has yet to be defined. In this report, it is established that low-dose CY not only decreases cell number but leads to decreased functionality of TREGs. CY treatment enhances apoptosis and decreases homeostatic proliferation of these cells. Expression of GITR and FoxP3, which are involved in the suppressive activity of TREGs, is down-regulated after CY administration, though the level of expression varies depending on the time studied. This is the first report demonstrating that CY, in addition to decreasing cell number, inhibits the suppressive capability of TREGs. The relevance of the loss of suppressor functionality and the changes in gene expression are further discussed.

ABSTRACTCurrently, there are no licensed vaccines and no correlates of protection against Francisella tularensis, which causes tularemia. We recently demonstrated that measuring in vitrocontrol of intramacrophage bacterial growth by murine F. tularensis-immune splenocytes, as well as transcriptional analyses, discriminated Francisellavaccines 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. tularensisvaccines. Liver and lung leukocytes derived from intradermally and intranasally vaccinated mice controlled in vitro FrancisellaLive 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 vitroLVS 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.IMPORTANCEIdentifying and quantifying correlates of protection is especially challenging for intracellular bacteria, including Francisella tularensis. F. tularensisis 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 vitroand 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.