Development of Functional and Molecular Correlates of Vaccine-Induced Protection for a Model Intracellular Pathogen, F. tularensis LVS

Abstract

In contrast with common human infections for which vaccine efficacy can be evaluated directly in field studies, alternative strategies are needed to evaluate efficacy for slowly developing or sporadic diseases like tularemia. For diseases such as these caused by intracellular bacteria, serological measures of antibodies are generally not predictive. Here, we used vaccines varying in efficacy to explore development of clinically useful correlates of protection for intracellular bacteria, using Francisella tularensis as an experimental model. F. tularensis is an intracellular bacterium classified as Category A bioterrorism agent which causes tularemia. The primary vaccine candidate in the U.S., called Live Vaccine Strain (LVS), has been the subject of ongoing clinical studies; however, safety and efficacy are not well established, and LVS is not licensed by the U.S. FDA. Using a mouse model, we compared the in vivo efficacy of a panel of qualitatively different Francisella vaccine candidates, the in vitro functional activity of immune lymphocytes derived from vaccinated mice, and relative gene expression in immune lymphocytes. Integrated analyses showed that the hierarchy of protection in vivo engendered by qualitatively different vaccines was reflected by the degree of lymphocytes' in vitro activity in controlling the intramacrophage growth of Francisella. Thus, this assay may be a functional correlate. Further, the strength of protection was significantly related to the degree of up-regulation of expression of a panel of genes in cells recovered from the assay. These included IFN-γ, IL-6, IL-12Rβ2, T-bet, SOCS-1, and IL-18bp. Taken together, the results indicate that an in vitro assay that detects control of bacterial growth, and/or a selected panel of mediators, may ultimately be developed to predict the outcome of vaccine efficacy and to complement clinical trials. The overall approach may be applicable to intracellular pathogens in general.

Monophosphoryl Lipid A Enhances Efficacy of a Francisella tularensis LVS-Catanionic Nanoparticle Subunit Vaccine against F. tularensis Schu S4 Challenge by Augmenting both Humoral and Cellular Immunity

“…Importantly, we found that immunization with LVS-VϩMPL increased cellular immunity as well; not only did it increase the number of IFN-␥-producing T cells and the level of secreted IFN-␥, but it also enhanced the ability of splenic T cells from LVS-VϩMPL-immunized mice to activate macrophages in vitro to kill or inhibit the intracellular replication of F. tularensis. The assay using splenocytes to control intramacrophage growth of F. tularensis has been shown by others to represent an excellent correlate of cell-mediated immunity (35).…”

“…Functional assays utilizing coculture of F. tularensis infected BMDM and splenocytes from immunized mice were performed as previously described (35,38). Briefly, bone marrow was harvested from immunologically naive mice and cultured for 1 week in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% conditioned medium from the LADMAC cell line as a source of macrophage colony-stimulating factor (M-CSF), as well as 10% fetal bovine serum (FBS) (Atlanta Biologicals, Flowery Branch, GA), 200 mM L-glutamine (Gibco, Grand Island, NY), and 10 mM HEPES buffer (Sigma).…”

“…3. Two weeks after final immunization, splenocytes were harvested from naive mice or mice that had been immunized with VϩMPL, LVS-V, or LVS-VϩMPL, and these cells were cocultured with F. tularensis LVS-infected bone marrow-derived macrophages (BMDM), as described previously (35,38). After 72 h of coculture, the nonadherent cells were analyzed by flow cytometry, using a gating strategy for discriminating subsets of T cells (Fig.…”

“…Previous studies have shown that treatment of macrophages with IFN-␥ results in macrophage activation and killing of intracellular organisms (35,38). Therefore, in the same coculture system, the ability of the splenocytes to control intramacrophage replication of F. tularensis LVS was analyzed.…”

T-bet Regulates Immunity to Francisella tularensis Live Vaccine Strain Infection, Particularly in Lungs

“…Single-cell suspensions prepared from spleens or lungs were stained for a panel of murine cell surface markers and analyzed using a Becton, Dickinson LSR II flow cytometer (San Jose, CA) and FlowJo software (Tree Star, Inc.), as previously described (3). Briefly, cells were washed and resuspended in PBS-2% FCS.…”