ABSTRACT
Traditionally, successful vaccines rely on specific adaptive immunity by activating lymphocytes with an attenuated pathogen, or pathogen subunit, to elicit heightened responses upon subsequent exposures. However, recent work with Mycobacterium tuberculosis and other pathogens has identified a role for "trained" monocytes in protection through memory-like but non-specific immunity. Here, we used an in vitro co-culture approach to study the potential role of trained macrophages, including lung alveolar macrophages, in immune responses to the Live Vaccine Strain (LVS) of Francisella tularensis. F. tularensis is an intracellular bacterium that replicates within mammalian macrophages and causes respiratory as well as systemic disease. We vaccinated mice with F. tularensis LVS and then obtained lung alveolar macrophages, or derived macrophages from bone marrow. LVS infected and replicated comparably in both types of macrophages, whether naïve or from LVS-vaccinated mice. LVS-infected macrophages were then co-cultured with either naïve splenocytes, splenocytes from mice vaccinated intradermally, or splenocytes from mice vaccinated intravenously. For the first time, we show that immune (but not naïve) splenocytes controlled bacterial replication within alveolar macrophages, similar to previous results using bone marrow-derived macrophage. However, no differences in control of intramacrophage bacterial replication were found between co-cultures with naïve macrophages or macrophages from LVS-vaccinated mice; furthermore, nitric oxide levels and interferon-gamma production in supernatants were largely comparable across all conditions. Thus, in the context of in vitro co-cultures, the data do not support development of trained macrophages in bone marrow or lungs of mice vaccinated with LVS intradermally or intravenously. IMPORTANCE: The discovery of non-specific "trained immunity" in monocytes has generated substantial excitement. However, to date, training has been studied with relatively few microbes (e.g., Mycobacterium bovis Bacille Calmette-Guérin, a live attenuated intracellular bacterium used as a vaccine) and microbial substances (e.g., LPS), and it remains unclear whether training during infection is common. We previously demonstrated that vaccination of mice with Francisella tularensis Live Vaccine Strain (LVS), another live attenuated intracellular bacterium, protected against challenge with the unrelated bacterium Listeria monocytogenes. The present study therefore tested whether LVS vaccination engenders trained macrophages that contributed to this protection. To do so, we used a previous in vitro co-culture approach with murine bone marrow-derived macrophages to expand and study lung alveolar macrophages. We demonstrated that alveolar macrophages can be productively infected and employed to characterize interactions with LVS-immune lymphocytes. However, we find no evidence that either bone marrow-derived or alveolar macrophages are trained by LVS vaccination.
ABSTRACT
Identifying suitable animal models and standardizing preclinical methods are important for the generation, characterization, and development of new vaccines, including those against Francisella tularensis. Non-human primates represent an important animal model to evaluate tularemia vaccine efficacy, and the use of correlates of vaccine-induced protection may facilitate bridging immune responses from non-human primates to people. However, among small animals, Fischer 344 rats represent a valuable resource for initial studies to evaluate immune responses, to identify correlates of protection, and to screen novel vaccines. In this study, we performed a comparative analysis of three Fischer rat substrains to determine potential differences in immune responses, to evaluate methods used to quantify potential correlates of protection, and to evaluate protection after vaccination. To this end, we took advantage of data previously generated using one of the rat substrains by evaluating two live vaccines, LVS and F. tularensis SchuS4-ΔclpB (ΔclpB). We compared immune responses after primary vaccination, adaptive immune responses upon re-stimulation of leukocytes in vitro, and sensitivity to aerosol challenge. Despite some detectable differences, the results highlight the similarity of immune responses to tularemia vaccines and challenge outcomes between the three substrains, indicating that all offer acceptable and comparable approaches as animal models to study Francisella infection and immunity.
ABSTRACT
Francisella tularensis, the causative agent of tularemia, is classified as Tier 1 Select Agent with bioterrorism potential. The efficacy of the only available vaccine, LVS, is uncertain and it is not licensed in the U.S. Previously, by using an approach generally applicable to intracellular pathogens, we identified working correlates that predict successful vaccination in rodents. Here, we applied these correlates to evaluate a panel of SchuS4-derived live attenuated vaccines, namely SchuS4-ΔclpB, ΔclpB-ΔfupA, ΔclpB-ΔcapB, and ΔclpB-ΔwbtC. We combined in vitro co-cultures to quantify rodent T-cell functions and multivariate regression analyses to predict relative vaccine strength. The predictions were tested by rat vaccination and challenge studies, which demonstrated a clear relationship between the hierarchy of in vitro measurements and in vivo vaccine protection. Thus, these studies demonstrated the potential power a panel of correlates to screen and predict the efficacy of Francisella vaccine candidates, and in vivo studies in Fischer 344 rats confirmed that SchuS4-ΔclpB and ΔclpB-ΔcapB may be better vaccine candidates than LVS.
