Models Derived fromIn VitroAnalyses of Spleen, Liver, and Lung Leukocyte Functions Predict Vaccine Efficacy against the Francisella tularensis Live Vaccine Strain (LVS)

Abstract

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.

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

“…The mediators identified included cytokines and other mediators known to play mechanistic roles, such as gamma interferon (IFN-␥) and tumor necrosis factor alpha (TNF-␣) (13,14), and those that were subsequently found to play critical roles, such as interleukin-6 (IL-6) (17) and IL-12 receptor ␤2 (IL-12r␤2) (18), thus validating the approach. In addition, when the quantification of multiple selected markers was combined in multivariate analyses, the resulting models were significantly correlated with survival (19). However, our studies also found that the expression of potential correlates of protection varied slightly among mouse strains (12), routes of injection (19), and, importantly, the tissue sources of immune cells (19).…”

“…In addition, when the quantification of multiple selected markers was combined in multivariate analyses, the resulting models were significantly correlated with survival (19). However, our studies also found that the expression of potential correlates of protection varied slightly among mouse strains (12), routes of injection (19), and, importantly, the tissue sources of immune cells (19).…”

“…Cocultures of LVS-infected bone marrow-derived macrophages (BMM) with single-cell suspensions of splenocytes or PBLs derived from the vaccinated and nonvaccinated groups were performed according to a previously described protocol (16,19,29). Lymphocytes were harvested after 48 h and assessed for viability and for changes in cell surface phenotype by flow cytometry.…”

“…The genes chosen were those previously identified to be potential predictors of vaccine efficacy on the basis of in vitro restimulation of Francisella-immune splenocytes (16,19). As shown in Fig.…”

“…Semiquantitative analyses of gene expression were performed using selected sets of primers/probes, chosen from among those that best reflected the hierarchy of in vivo vaccine efficacy (16,19). The values shown are the median fold changes of expression of the indicated genes in immunized mice compared to their levels of expression in naive mice calculated from three independent experiments.…”