Characterization of Francisella tularensis Schu S4 defined mutants as live-attenuated vaccine candidates

Abstract

Francisella tularensis (Ft), the etiological agent of tularemia and a Tier 1 select agent, has been previously weaponized and remains a high priority for vaccine development. Ft tularensis (type A) and Ft holarctica (type B) cause most human disease. We selected six attenuating genes from the live vaccine strain (LVS; type B), F. novicida and other intracellular bacteria: FTT0507, FTT0584, FTT0742, FTT1019c (guaA), FTT1043 (mip) and FTT1317c (guaB) and created unmarked deletion mutants of each in the highly human virulent Ft strain Schu S4 (Type A) background. FTT0507, FTT0584, FTT0742 and FTT1043 Schu S4 mutants were not attenuated for virulence in vitro or in vivo. In contrast, Schu S4 gua mutants were unable to replicate in murine macrophages and were attenuated in vivo, with an i.n. LD50 > 105 CFU in C57BL/6 mice. However, the gua mutants failed to protect mice against lethal challenge with WT Schu S4, despite demonstrating partial protection in rabbits in a previous study. These results contrast with the highly protective capacity of LVS gua mutants against a lethal LVS challenge in mice, and underscore differences between these strains and the animal models in which they are evaluated, and therefore have important implications for vaccine development.

An In Vitro Co-culture Mouse Model Demonstrates Efficient Vaccine-Mediated Control of Francisella tularensis SCHU S4 and Identifies Nitric Oxide as a Predictor of Efficacy

“…Vaccination with LVS effectively prevents laboratory-acquired infection, but studies on volunteers have revealed that it affords only marginal protection against aerosol infection (Saslaw et al, 1961a,b; Burke, 1977). It was hypothesized that it would be possible to generate a superior F. tularensis vaccine and we and others have generated a number of targeted mutants of SCHU S4 and their efficacy has been evaluated using in vivo models with virulent F. tularensis strains (Kadzhaev et al, 2009; Conlan et al, 2010; Shen et al, 2010; Ryden et al, 2012; Santiago et al, 2015). In particular one mutant, Δ clpB , was found to confer superior efficacy to LVS, as demonstrated by survival after infection with SCHU S4 (Conlan et al, 2010).…”

Benzoxazoles, Phthalazinones, and Arylurea-Based Compounds with IMP Dehydrogenase-Independent Antibacterial Activity against Francisella tularensis

“…These observations further indicated that, in most cases, antibacterial activity does not result from the inhibition of FtIMPDH. Second, we determined the antibacterial activities of 10 compounds against the ΔguaB strain of F. tularensis Schu S4 (17), which lacks the gene encoding FtIMPDH and requires the presence of guanine for growth. These bacteria should be resistant to FtIMPDH inhibitors.…”

“…The MIC was defined as the lowest drug concentration at which the growth was inhibited at least 90% compared to that of the positive control. The ΔguaB strain (FTT1317c) (17), which lacks FtIMPDH, was always grown in the presence of guanine (10 g/ml). The MBC was determined by plating 5 l of culture from each well on a Mueller-Hinton agar (MHA) plate and incubating it at 37°C for 2 days.…”

“…IMP dehydrogenase (IMPDH) controls the guanine nucleotide pool, and hence proliferation, in virtually every organism. Deletion of guaB, the gene encoding IMPDH, decreases the virulence of F. tularensis Schu S4 by a factor of Ͼ10 7 when mice are infected via intranasal administration (17). This strain is also unable to replicate within macrophages.…”

Characterization of Schu S4 aro mutants as live attenuated tularemia vaccine candidates

“…We previously reported that vaccine candidates derived from subsp. tularensis with deletions in a series of targeted genes, including guaB and guaA genes, did not confer protection in the mouse model [9]. In this study, we have targeted another critical metabolic pathway by engineering three novel live attenuated vaccine candidates, Schu S4ΔaroC, Schu S4ΔaroD, and Schu S4ΔaroCΔaroD.…”