7 research outputs found

    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

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    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

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    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

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    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

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

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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 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

    Aerosol prime-boost vaccination provides strong protection in outbred rabbits against virulent type A Francisella tularensis.

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    Tularemia, also known as rabbit fever, is a severe zoonotic disease in humans caused by the gram-negative bacterium Francisella tularensis (Ft). While there have been a number of attempts to develop a vaccine for Ft, few candidates have advanced beyond experiments in inbred mice. We report here that a prime-boost strategy with aerosol delivery of recombinant live attenuated candidate Ft S4ΔaroD offers significant protection (83% survival) in an outbred animal model, New Zealand White rabbits, against aerosol challenge with 248 cfu (11 LD50) of virulent type A Ft SCHU S4. Surviving rabbits given two doses of the attenuated strains by aerosol did not exhibit substantial post-challenge fevers, changes in erythrocyte sedimentation rate or in complete blood counts. At a higher challenge dose (3,186 cfu; 139 LD50), protection was still good with 66% of S4ΔaroD-vaccinated rabbits surviving while 50% of S4ΔguaBA vaccinated rabbits also survived challenge. Pre-challenge plasma IgG titers against Ft SCHU S4 corresponded with survival time after challenge. Western blot analysis found that plasma antibody shifted from predominantly targeting Ft O-antigen after the prime vaccination to other antigens after the boost. These results demonstrate the superior protection conferred by a live attenuated derivative of virulent F. tularensis, particularly when given in an aerosol prime-boost regimen

    The O-Ag Antibody Response to Francisella Is Distinct in Rodents and Higher Animals and Can Serve as a Correlate of Protection

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    Identifying correlates of protection (COPs) for vaccines against lethal human (Hu) pathogens, such as Francisella tularensis (Ft), is problematic, as clinical trials are currently untenable and the relevance of various animal models can be controversial. Previously, Hu trials with the live vaccine strain (LVS) demonstrated ~80% vaccine efficacy against low dose (~50 CFU) challenge; however, protection deteriorated with higher challenge doses (~2000 CFU of SchuS4) and no COPs were established. Here, we describe our efforts to develop clinically relevant, humoral COPs applicable to high-dose, aerosol challenge with S4. First, our serosurvey of LVS-vaccinated Hu and animals revealed that rabbits (Rbs), but not rodents, recapitulate the Hu O-Ag dependent Ab response to Ft. Next, we assayed Rbs immunized with distinct S4-based vaccine candidates (S4ΔclpB, S4ΔguaBA, and S4ΔaroD) and found that, across multiple vaccines, the %O-Ag dep Ab trended with vaccine efficacy. Among S4ΔguaBA-vaccinated Rbs, the %O-Ag dep Ab in pre-challenge plasma was significantly higher in survivors than in non-survivors; a cut-off of >70% O-Ag dep Ab predicted survival with high sensitivity and specificity. Finally, we found this COP in 80% of LVS-vaccinated Hu plasma samples as expected for a vaccine with 80% Hu efficacy. Collectively, the %O-Ag dep Ab response is a bona fide COP for S4ΔguaBA-vaccinated Rb and holds significant promise for guiding vaccine trials with higher animals

    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

    Get PDF
    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
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