Mayfield, PA, United States
Mayfield, PA, United States

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Konecna K.,University of Hradec Kralove | Konecna K.,Charles University | Hernychova L.,University of Hradec Kralove | Reichelova M.,Veterinary Research Institute | And 6 more authors.
Proteomics | Year: 2010

The facultative intracellular bacterium Francisella tularensis is the causal agent of the serious infectious disease tularemia. Despite the dynamic progress, which has been made in last few years, important questions regarding Francisella pathogenicity still remain to be answered. Generally, secreted proteins play an important role in pathogenicity of intracellular microbes. In this study, we investigated the protein composition of the culture filtrate proteins of highly virulent F. tularensis subsp. tularensis, strain SCHU S4 and attenuated F. tularensis subsp. holarctica, live vaccine strain using a comparative proteomic analysis. The majority of proteins identified in this study have been implicated in virulence mechanisms of other pathogens, and several have been categorized as having moonlighting properties; those that have more than one unrelated function. This profiling study of secreted proteins resulted in the unique detection of acid phosphatase (precursor) A (AcpA), β-lactamase, and hypothetical protein FTT0484 in the highly virulent strain SCHU S4 secretome. The release of AcpA may be of importance for F. tularensis subsp. tularensis virulence due to the recently described AcpA role in the F. tularensis escape from phagosomes. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Spurgers K.B.,U.S. Army | Alefantis T.,Vital Probes, Inc. | Peyser B.D.,U.S. Army | Ruthel G.T.,U.S. Army | And 8 more authors.
Molecular and Cellular Proteomics | Year: 2010

An assessment of the total protein composition of filovirus (ebolavirus and marburgvirus) virions is currently lacking. In this study, liquid chromatography-linked tandem mass spectrometry of purified ebola and marburg virions was performed to identify associated cellular proteins. Host proteins involved in cell adhesion, cytoskeleton, cell signaling, intracellular trafficking, membrane organization, and chaperones were identified. Significant overlap exists between this data set and proteomic studies of disparate viruses, including HIV-1 and influenza A, generated in multiple cell types. However, the great majority of proteins identified here have not been previously described to be incorporated within filovirus particles. Host proteins identified by liquid chromatography-linked tandem mass spectrometry could lack biological relevance because they represent protein contaminants in the virus preparation, or because they are incorporated within virions by chance. These issues were addressed using siRNA library-mediated gene knockdown (targeting each identified virion-associated host protein), followed by filovirus infection. Knockdown of several host proteins (e.g. HSPA5 and RPL18) significantly interfered with ebolavirus and marburgvirus infection, suggesting specific and relevant virion incorporation. Notably, select siRNAs inhibited ebolavirus, but enhanced marburgvirus infection, suggesting important differences between the two viruses. The proteomic analysis presented here contributes to a greater understanding of filovirus biology and potentially identifies host factors that can be targeted for antiviral drug development. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.


Bergmann-Leitner E.S.,U.S. Army | Chaudhury S.,U.S. Army | Steers N.J.,Us Military Hiv Research Program | Sabato M.,Vital Probes, Inc. | And 4 more authors.
PLoS ONE | Year: 2013

Vaccine development efforts will be guided by algorithms that predict immunogenic epitopes. Such prediction methods rely on classification-based algorithms that are trained against curated data sets of known B and T cell epitopes. It is unclear whether this empirical approach can be applied prospectively to predict epitopes associated with protective immunity for novel antigens. We present a comprehensive comparison of in silico B and T cell epitope predictions with in vivo validation using an previously uncharacterized malaria antigen, CelTOS. CelTOS has no known conserved structural elements with any known proteins, and thus is not represented in any epitope databases used to train prediction algorithms. This analysis represents a blind assessment of this approach in the context of a novel, immunologically relevant antigen. The limited accuracy of the tested algorithms to predict the in vivo immune responses emphasizes the need to improve their predictive capabilities for use as tools in vaccine design.


Roset M.S.,CONICET | Fernandez L.G.,CONICET | DelVecchio V.G.,Vital Probes, Inc. | Briones G.,CONICET
Infection and Immunity | Year: 2013

Brucella is an intracellular bacterial pathogen that causes the worldwide zoonotic disease brucellosis. Brucella virulence relies on its ability to transition to an intracellular lifestyle within host cells. Thus, this pathogen must sense its intracellular localization and then reprogram gene expression for survival within the host cell. A comparative proteomic investigation was performed to identify differentially expressed proteins potentially relevant for Brucella intracellular adaptation. Two proteins identified as cyclophilins (CypA and CypB) were overexpressed in the intracellular environment of the host cell in comparison to laboratorygrown Brucella. To define the potential role of cyclophilins in Brucella virulence, a double-deletion mutant was constructed and its resulting phenotype was characterized. The Brucella abortus δcypAB mutant displayed increased sensitivity to environmental stressors, such as oxidative stress, pH, and detergents. In addition, the B. abortus δcypAB mutant strain had a reduced growth rate at lower temperature, a phenotype associated with defective expression of cyclophilins in other microorganisms. The B. abortus δcypAB mutant also displays reduced virulence in BALB/c mice and defective intracellular survival in HeLa cells. These findings suggest that cyclophilins are important for Brucella virulence and survival in the host cells. ©2013, American Society for Microbiology.


Marchesini M.I.,CONICET | Connolly J.,Vital Probes, Inc. | Delpino M.V.,CONICET | Baldi P.C.,CONICET | And 3 more authors.
PLoS ONE | Year: 2011

Choloylglycine hydrolase (CGH, E.C. 3.5.1.24) is a conjugated bile salt hydrolase that catalyses the hydrolysis of the amide bond in conjugated bile acids. Bile salt hydrolases are expressed by gastrointestinal bacteria, and they presumably decrease the toxicity of host's conjugated bile salts. Brucella species are the causative agents of brucellosis, a disease affecting livestock and humans. CGH confers Brucella the ability to deconjugate and resist the antimicrobial action of bile salts, contributing to the establishment of a successful infection through the oral route in mice. Additionally, cgh-deletion mutant was also attenuated in intraperitoneally inoculated mice, which suggests that CGH may play a role during systemic infection other than hydrolyzing conjugated bile acids. To understand the role CGH plays in B. abortus virulence, we infected phagocytic and epithelial cells with a cgh-deletion mutant (Δcgh) and found that it is defective in the internalization process. This defect along with the increased resistance of Δcgh to the antimicrobial action of polymyxin B, prompted an analysis of the cell envelope of this mutant. Two-dimensional electrophoretic profiles of Δcgh cell envelope-associated proteins showed an altered expression of Omp2b and different members of the Omp25/31 family. These results were confirmed by Western blot analysis with monoclonal antibodies. Altogether, the results indicate that Brucella CGH not only participates in deconjugation of bile salts but also affects overall membrane composition and host cell internalization. © 2011 Marchesini et al.


Delvecchio V.G.,Vital Probes, Inc. | Sabato M.A.,Vital Probes, Inc. | Trichilo J.,Vital Probes, Inc. | Dake C.,Vital Probes, Inc. | And 2 more authors.
Critical Reviews in Immunology | Year: 2010

Proteomics permits the large-scale and high-throughput analysis of proteins and has become a powerful tool with which to study the pathogenic mechanisms of bacteria. It not only provides a metabolic snapshot at a particular moment in the life of a pathogen, but can also determine where a protein resides, its function, whether it is secreted, and its interactions with other proteins, including those of the host. Comparative proteomics can yield important information on the differences between attenuated and pathogenic organisms and whether a protein is conserved among various strains. Our laboratory has utilized traditional and novel techniques to investigate the global and subproteomes of Bacillus anthracis as they relate to vaccine and therapeutic development. Recently, our efforts have focused on the use of mass spectrometry for B-cell epitope discovery and identification of components of a pathogen that interact with host proteins. Development of vaccines and therapeutics based on proteomic data in combination with novel adjuvants and delivery systems will be presented. © 2010 by Begell House, Inc.


Grant
Agency: Department of Defense | Branch: Army | Program: SBIR | Phase: Phase II | Award Amount: 627.14K | Year: 2010

Malaria caused by Plasmodium falciparum results in serious illness and, if untreated, often leads to death. Although a number of candidate vaccines have progressed to clinical trials the efficacy rate of those vaccines was much lower than ideal. New methods for simultaneous presentation and immune stimulation of malarial antigens are needed in order to rapidly progress promising antigens into efficacious vaccines. Platforms that present antigen to the immune system in a particulate manner that mimics the structure of a natural pathogen may improve the effectiveness of a vaccine. Prior work has demonstrated that Vital Probes, Gene-Mediated Inactivated Vaccine (GeMI-Vax) platform in E. coli combined with malaria antigens results in a protective immune response in mouse models of malaria. The GeMI-Vax production method gently inactivates bacteria that are engineered to express malaria protein antigens. The resulting particle-based vaccine has inherent adjuvant activity and appropriately presents vaccine antigens and stimulates the immune system. Phase I SBIR work resulted in successful expression of malaria antigens in E. coli and Shigella. In Phase II, Shigella-GeMI-Vax expressing malaria antigens will be produced and tested in animal efficacy models. Demonstration of efficacy will lead to follow-on research and development efforts towards testing in human clinical trials.


Grant
Agency: Department of Defense | Branch: Defense Advanced Research Projects Agency | Program: SBIR | Phase: Phase II | Award Amount: 974.29K | Year: 2012

Rapid Virulence Factor Discovery System (RVFDS) is virulence factor-discovery platform in which all of the genes of a pathogen are expressed generating a random expression library (REL). The REL is then screened for the presence of potential virulence factors with an array of pull-down assays that employ components of the host"s extracellular matrix, mucus, and basement membrane as bait. The host bait proteins interact with and capture pathogen prey proteins that may play a role in the pathogenic process. The potential virulence factors are then identified by mass spectrometry and proteomic methodologies. RVFDS can also define the immunome or complete repertoire of immunogenic proteins of a pathogen using antibodies from convalescent patients as bait in pull-down assays. Presently investigations into the host-pathogen relationship are severely hampered by the fact that the pathogen is grown under laboratory conditions that simulate the host environment, and thus, many of the virulence factors that are expressed in response to the true host environment are overlooked. RVFDS circumvents this overwhelming challenge because virtually all of the open reading frames of a pathogen are expressed and are easily and rapidly identified.


Grant
Agency: Department of Defense | Branch: Defense Advanced Research Projects Agency | Program: SBIR | Phase: Phase I | Award Amount: 99.89K | Year: 2011

Bacteria employ a vast array of virulence factors that enable them to confront and damage the host cell during the infectious process. Identification of these virulence factors is essential to understanding pathogenisis and defining targets for future development of more efficacious next-generation therapeutic agents and vaccines. Thus, there is an urgent need for a rapid, relatively inexpensive, comprehensive, high-throughput platform for the discovery of virulence factors. To this end, Vital Probes, Inc. has developed Rapid Virulence Factor Discovery System (RVFDS) which expresses virtually all of the open reading frames of a pathogen which can then be screened for interaction with host components. This proteomic-based system circumvents the drawbacks of laboratory-grown conditions, host environment simulations, and isolation of pathogens from biological specimens or host anatomical sites in the identification of the virulence factors. A typical RVFDS experiment can be accomplished in less than a month. This proposal intent to assess the feasibility of RVFDS to identify the novel virulence factors of methicillin-resistant Staphylococcus aureus (MRSA) which is the causal agent of a wide range of human diseases. This system is intended to not only feed VPI"s product pipeline with new target candidates for therapeutic and vaccine development but to also provide this discovery platform as a service to outside companies, government research institutions, and academic institutions.


Trademark
Vital Probes, Inc. | Date: 2011-06-14

Vaccines.

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