Differential Growth of Francisella tularensis, Which Alters Expression of Virulence Factors, Dominant Antigens, and Surface-Carbohydrate Synthases, Governs the Apparent Virulence of Ft SchuS4 to Immunized Animals

The gram-negative bacterium Francisella tularensis (Ft) is both a potential biological weapon and a naturally occurring microbe that survives in arthropods, fresh water amoeba, and mammals with distinct phenotypes in various environments. Previously, we used a number of measurements to characterize Ft grown in Brain-Heart Infusion (BHI) broth as (1) more similar to infection-derived bacteria, and (2) slightly more virulent in naïve animals, compared to Ft grown in Mueller Hinton Broth (MHB). In these studies we observed that the free amino acids in MHB repress expression of select Ft virulence factors by an unknown mechanism. Here, we tested the hypotheses that Ft grown in BHI (BHI-Ft) accurately displays a full protein composition more similar to that reported for infection-derived Ft and that this similarity would make BHI-Ft more susceptible to pre-existing, vaccine-induced immunity than MHB-Ft. We performed comprehensive proteomic analysis of Ft grown in MHB, BHI, and BHI supplemented with casamino acids (BCA) and compared our findings to published “omics” data derived from Ft grown in vivo. Based on the abundance of ~1,000 proteins, the fingerprint of BHI-Ft is one of nutrient-deprived bacteria that—through induction of a stringent-starvation-like response—have induced the FevR regulon for expression of the bacterium's virulence factors, immuno-dominant antigens, and surface-carbohydrate synthases. To test the notion that increased abundance of dominant antigens expressed by BHI-Ft would render these bacteria more susceptible to pre-existing, vaccine-induced immunity, we employed a battery of LVS-vaccination and S4-challenge protocols using MHB- and BHI-grown Ft S4. Contrary to our hypothesis, these experiments reveal that LVS-immunization provides a barrier to infection that is significantly more effective against an MHB-S4 challenge than a BHI-S4 challenge. The differences in apparent virulence to immunized mice are profoundly greater than those observed with primary infection of naïve mice. Our findings suggest that tularemia vaccination studies should be critically evaluated in regard to the growth conditions of the challenge agent

Rickettsial pathogen inhibits tick cell death through tryptophan metabolite mediated activation of p38 MAP kinase

Summary: Anaplasma phagocytophilum modulates various cell signaling pathways in mammalian cells for its survival. In this study, we report that A. phagocytophilum modulates tick tryptophan pathway to activate arthropod p38 MAP kinase for the survival of both this bacterium and its vector host. Increased level of tryptophan metabolite, xanthurenic acid (XA), was evident in A. phagocytophilum-infected ticks and tick cells. Lower levels of cell death markers and increased levels of total and phosphorylated p38 MAPK was noted in A. phagocytophilum-infected ticks and tick cells. Treatment with XA increased phosphorylated p38 MAPK levels and reduced cell death in A. phagocytophilum-infected tick cells. Furthermore, treatment with p38 MAPK inhibitor affected bacterial replication, decreased phosphorylated p38 MAPK levels and increased tick cell death. However, XA reversed these effects. Taken together, we provide evidence that rickettsial pathogen modulates arthropod tryptophan and p38 MAPK pathways to inhibit cell death for its survival in ticks

Immunization against arthropod protein impairs transmission of rickettsial pathogen from ticks to the vertebrate host

Abstract Human anaplasmosis caused by Anaplasma phagocytophilum is one of the most common tick-borne diseases in the United States. The black-legged ticks, Ixodes scapularis, vector and transmit this bacterium to humans. In this study, we provide evidence that targeting I. scapularis membrane-bound organic anion transporting polypeptide 4056 (IsOATP4056) with an anti-vector vaccine affects transmission of A. phagocytophilum from ticks to the vertebrate host. Anaplasma phagocytophilum induces expression of IsOATP4056 in ticks and tick cells. Increased membrane localization of IsOATP4056 was evident in A. phagocytophilum-infected tick cells. Treatment with high dose (10 µg/ml) but not low dose (5 µg/ml) of EL-6 antibody that targets the largest extracellular loop of IsOATP4056 showed cytotoxic effects in tick cells but not in human keratinocyte cell line (HaCaT). Passive immunization, tick-mediated transmission and in vitro studies performed with mice ordered from two commercial vendors and with tick cells showed that EL-6 antibody not only impairs A. phagocytophilum transmission from ticks to the murine host but also aids in the reduction in the bacterial loads within engorged ticks and in tick cells by activation of arthropod Toll pathway. Furthermore, reduced molting efficiency was noted in ticks fed on EL-6 antibody-immunized mice. Collectively, these results provide a good candidate for the development of anti-tick vaccine to target the transmission of A. phagocytophilum and perhaps other tick-borne pathogens of medical importance

Integration of Sustainable Innovation in Research and Development Related to Natural Gas Processing

Abstract Considering current changes in supply and demand of the oil and gas value chain, it is essential to embed sustainability measures in the business. Research and development plays an important role in achieving the sustainability in natural gas processing. Hence, this work indicates how the sustainability elements economics, environment and social can be embedded in the management of research and development portfolio. A natural gas R&amp;D model is proposed for GASCO from the sustainability angle. Moreover, it will be shown how during the execution of research projects the sustainability aspect needs to taken into account at each stage, from concept definition till deployment of the technology. Several technologies are identified that can be further researched or evaluated for their application in natural gas processing, to eventually enhance the sustainability of current operations.</jats:p

Abstract 3993: Th1 cytokines regulate apoptotic cell death and HER family RTK expression in murine and human breast cancer lines

Abstract A recent neoadjuvant vaccine trial to treat early breast cancer demonstrated powerful induction of Th1 immunity against the HER-2, complete pathologic responses in over 18% of subjects, and for many subjects, evidence of down-regulated HER-2 expression on residual disease. To explain these observations, we investigated the action of archetypical Th1 cytokines (TNF-α + IFN-γ) on both murine and human breast cancer cell lines that varied in the surface expression of HER-family receptor tyrosine kinases. We found that most tumor cell lines were sensitive to dual Th1 cytokines as evidenced by lower metabolic activity (alamar blue assay), lower proliferation, and enhanced apoptosis (AnnexinV/PI staining and TUNEL assay) as well as a reversible inhibition of surface expression of HER proteins. Apoptotic cell death was accompanied by demonstrated increases in activated caspase-3. Furthermore, the pharmacologic caspase-3 activator, procaspase-activating compound (PAC-1), mimicked both the killing effects and HER-2 suppressive activities of Th1 cytokines, while the caspase 3/7 inhibitor ((5-[(S)-(+)-2-(Methoxymethyl)pyrrolidino]sulfonylisatin), prevented cytokine-induced HER-2 downregulation. These studies therefore demonstrated that many of the in vivo effects of vaccination (apparent tumor cell death and loss of HER-2 expression) could be replicated in vitro using only the principle Th1 cytokines. These findings are consistent with the notion that IFN-γ and TNF-α work in concert to mediate some of the biological effects of therapeutic Th1-polarizing vaccination through the induction of a caspase 3-dependent cellular death mechanism. Citation Format: Prachi M. Namjoshi, Lori Showalter, Brian Czerniecki, Gary K. Koski. Th1 cytokines regulate apoptotic cell death and HER family RTK expression in murine and human breast cancer lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3993.</jats:p

Evaluation of an outbred mouse model for Francisella tularensis vaccine development and testing.

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 the tools to assess these vaccines. Tularemia laboratory research has historically relied primarily upon a small number of inbred mouse strains, but the utility of such findings to outbred animals may be limited. Specifically, C57BL/6 mice are more susceptible than BALB/c mice to Ft infection and less easily protected against challenge with highly virulent type A Ft. Thus, depending on the inbred mouse strain used, one could be misled as to which immunogen(s)/vaccine will ultimately be effective in an outbred human population. Accordingly, we evaluated an outbred Swiss Webster (SW) mouse model in direct comparison to a well-established, inbred C57BL/6 mouse model. Mucosal vaccination with the live, attenuated Ft LVS superoxide dismutase (sodB) mutant demonstrated significantly higher protection in outbred SW mice compared to inbred C57BL/6 mice against Ft SchuS4 respiratory challenge. The protection observed in vaccinated outbred mice correlated with lower bacterial density, reduced tissue inflammation, and reduced levels of pro-inflammatory cytokine production. This protection was CD4+ and CD8+ T cell-dependent and characterized by lower titers of serum antibody (Ab) that qualitatively differed from vaccinated inbred mice. Enhanced protection of vaccinated outbred mice correlated with early and robust production of IFN-γ and IL-17A. Neutralizing Ab administered at the time of challenge revealed that IFN-γ was central to this protection, while IL-17A neutralization did not alter bacterial burden or survival. The present study demonstrates the utility of the outbred mouse as an alternative vaccination model for testing tularemia vaccines. Given the limited MHC repertoire in inbred mice, this outbred model is more analogous to the human in terms of immunological diversity

Outbred SW mice are better protected against bacterial replication and tissue damage following <i>Ft</i> SchuS4 challenge, as compared to that of inbred C57BL/6 mice.

SW and C57BL/6 female mice were immunized i.n. with 1x103 CFU of attenuated Ft LVS sodB mutant. Mice were then challenged on day 21 i.n. with 94 CFU of Ft SchuS4. Lungs (A), BALF (B), and spleen (C) were analyzed for bacterial burdens on 1, 3, 7, 10, 14, and 21 days post-challenge. Each point represents the mean +/- SE of 3 mice sacrificed per time point, these data are representative of two independent experiment of total of six mice, *p p sodB-vaccinated mice prior to Ft SchuS4 challenge (day 0) and at day 10 post-infection are shown in panel (D). Inflammatory foci in the lungs are denoted by a star (*) and necrosis by an (→). BALF total protein levels (E) and serum LDH (F) were quantified on indicated days post-challenge. Each bar represents mean ± SE (error bar) of two independent experiments with a total of six mice per group, *p p p x-axis.</p

Early and strong Th1/Th17-mediated cytokine responses in outbred SW versus inbred C57BL/6mice were produced following <i>Ft</i> SchuS4 challenge.

SW and C57BL/6 female mice were immunized and challenged as described in Fig 2. Lungs (A & D), BALF (B & E), and spleen (C & F) were then analyzed for the indicated cytokines on 1, 3, 7, 10, and 14 days post-challenge. Each point represents the average of 3 mice. Data shown are representative of two independent experiments. All vaccinated inbred mice died, as indicated by Ψ on the x-axis, *p p p < 0.001.</p

Outbred SW mice exhibited rapid clearance of <i>Ft sodB</i> mutant and significantly lower Ab response compared to that of inbred C57BL/6 mice.

SW and C57BL/6 female mice were immunized as described in Fig 2. On days 1, 3, 7, 10, 14, and 21 bacterial loads (CFUs) in lungs (A) and spleen (B) were evaluated. Each point represents the average of 3 mice sacrificed per time point, *p p Ft specific total Ab, IgG, IgA, and IgG2c titers by ELISA. Data are represented as the mean ± SE (8 vaccinated and 4 unvaccinated mice per group) and are combined results from two independent experiments (C). Whole cell lysates of wild type Ft LVS and clpB-mutant SchuS4 grown in BHI broth were resolved by SDS-PAGE and transferred to a nitrocellulose membrane (D). Sections of the membrane, each containing one lane of LVS and one lane of SchuS4, were probed with one of the indicated 4 sera pools prior to chemiluminescent development. To account for the higher titer of specific Ab in the vaccinated, C57/BL6 mice (~500 vs ~250, Fig 4C), sera from SW mice were used in this case at a dilution of 1:1,000, whereas sera from the C57BL/6 mice were used at a dilution of 1:2,000. The ~18 kDa band visible in all lanes is an endogenously-biotinylated Ft protein (likely AccB) detected by the streptavidin-HRP conjugate used during development of western blots. Except for differences in the primary antibody, all the membranes shown were developed in parallel under identical conditions. *p p p < 0.001.</p

CD4⁺ and CD8⁺ T cells and IFN-γ are essential for surviving virulent <i>Ft</i> infection in outbred SW mice.

SW female mice were immunized and challenged as described in Fig 2. Vaccinated mice were treated with anti-CD4+, anti-CD8+, or isotype control Ab on days -4, -1, 2, 5, and 8 relative to Ft SchuS4 infection and monitored for survival (A). Alternatively, mice were treated with anti-IFN-γ, anti-IL-17A, or isotype control Ab on days -1, 1, 3, and 5 relative to Ft SchuS4 infection and monitored for survival (B). Ft SchuS4 burdens in the lungs and spleens of naïve or vaccinated SW mice treated with depleting Ab specific for IFN-γ or IL-17A (n = 3 mice per group, day 5 post 40 CFU Ft SchuS4 in challenge) (C and D). Panels A & B represents combined results from two independent experiments with a total of 10 mice/ group, panel C is representative of two independent experiments. *p p p < 0.001.</p