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Francisella tularensis (Ft) is a biothreat agent for which there is no FDA-approved human vaccine. Currently, there are substantial efforts underway to develop both vaccines and improved tools to assess these vaccines. Ft expresses distinct sets of antigens (Ags) in vivo as compared to those expressed in vitro. Importantly, Ft grown in brain-heart infusion medium (BHIM) more closely mimics the antigenic profile of macrophage-grown Ft when compared to Mueller-Hinton medium (MHM)-grown Ft. Thus, we predicted that when used as a live vaccine BHIM-grown Ft (BHIM-Ft) would provide better protection, as compared to MHM-Ft. We first determined if there was a difference in growth kinetics between BHIM and MHM-Ft. We found that BHIM-Ft exhibited an initial growth advantage ex vivo that manifests as slightly hastened intracellular replication as compared to MHM-Ft. We also observed that BHIM-Ft exhibited an initial growth advantage in vivo represented by rapid bacterial expansion and systemic dissemination associated with a slightly shorter mean survival time of naive animals. Next, using two distinct strains of Ft LVS (WT and sodB), we observed that mice vaccinated with live BHIM-Ft LVS exhibited significantly better protection against Ft SchuS4 respiratory challenge compared to MHM-Ft-immunized mice. This enhanced protection correlated with lower bacterial burden, reduced tissue inflammation, and reduced pro-inflammatory cytokine production late in infection. Splenocytes from BHIM-Ft sodB-immunized mice contained more CD4+, effector, memory T-cells, and were more effective at limiting intracellular replication of Ft LVS in vitro. Concurrent with enhanced killing of Ft LVS, BHIM-Ft sodB-immune splenocytes produced significantly higher levels of IFN-γ and IL-17A cytokines than their MHM-Ft sodB-immunized counterparts indicating development of a more effective T cell memory response when immunizing mice with BHIM-Ft. [ABSTRACT FROM AUTHOR]

Fc gamma receptor IIB (FcγRIIB) is the only Fc gamma receptor (FcγR) which negatively regulates the immune response, when engaged by antigen- (Ag-) antibody (Ab) complexes. Thus, the generation of Ag-specific IgG in response to infection or immunization has the potential to downmodulate immune protection against infection. Therefore, we sought to determine the impact of FcγRIIB on immune protection against Francisella tularensis (Ft), a Category A biothreat agent. We utilized inactivated Ft (iFt) as an immunogen. Naïve and iFt-immunized FcγRIIB knockout (KO) or wildtype (WT) mice were challenged with Ft-live vaccine strain (LVS). While no significant difference in survival between naïve FcγRIIB KO versus WT mice was observed, iFt-immunized FcγRIIB KO mice were significantly better protected than iFt-immunized WT mice. Ft-specific IgA in serum and bronchial alveolar lavage, as well as IFN-γ, IL-10, and TNF-α production by splenocytes harvested from iFt-immunized FcγRIIB KO, were also significantly elevated. In addition, iFt-immunized FcγRIIB KO mice exhibited a reduction in proinflammatory cytokine levels in vivo at 5 days after challenge, which correlates with increased survival following Ft-LVS challenge in published studies. Thus, these studies demonstrate for the first time the ability of FcγRIIB to regulate vaccine-induced IgA production and downmodulate immunity and protection. The immune mechanisms behind the above observations and their potential impact on vaccine development are discussed. [ABSTRACT FROM AUTHOR]

The development of persistent antibiotic resistance by human methicillin-sensitive Staphylococcus aureus (MSSA) strains and substantial association with poly-N-acetyl glucosamine (PNAG) in biofilms is reported in this investigation. Sixteen of 31 MSSA strains under study were found to have developed resistance to one or more antibiotics, with four strains, two of which did not produce biofilms, showing resistance to cefoxitin, undetectable by mecA amplification. Antibiotic resistance displayed by 13/14 biofilm-forming S. aureus isolates remained persistent for 4 weeks prior to reverting back to the original antibiotic susceptibility, prompting a suggestion of determining antibiograms for clinical S. aureus isolates subcultured from biofilms developed in vitro as well as planktonic subcultures prepared from the site of infection. While there was correlation of antibiotic resistance with biofilm formation confirming previous reports, this is the first time that persistence of the biofilm-associated antibiotic resistance by S. aureus as planktonic cells is reported. Among the two methods used for assessment of biofilm formation, the tissue culture plate (TCP) method revealed that almost all strains were strong or moderate biofilm producers whereas only 19/31 strains were biofilm producers using the Congo Red agar (CRA) method indicating the superiority of the TCP method in detecting biofilm producers. We also observed no association between biofilm formation and major capsule types. However, substantial, although not absolute, association of biofilm formation with PNAG was observed, warranting continued identification of additional surface-associated polysaccharide and/or protein antigens associated with biofilm formation for development of an effective vaccine against S. aureus infections regardless of capsular phenotype. [ABSTRACT FROM AUTHOR]

Francisella tularensis ( Ft ) is a gram-negative intercellular pathogen and category A biothreat agent. However, despite 15 years of strong government investment and intense research focused on the development of a US Food and Drug Administration-approved vaccine against Ft , the primary goal remains elusive. This article reviews research efforts focused on developing an Ft vaccine, as well as a number of important factors, some only recently recognized as such, which can significantly impact the development and evaluation of Ft vaccine efficacy. Finally, an assessment is provided as to whether a US Food and Drug Administration-approved Ft vaccine is likely to be forthcoming and the potential means by which this might be achieved.