Kathryn Sykes

Arizona State University, Phoenix, Arizona, United States

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Publications (30)154.98 Total impact

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    ABSTRACT: Tularemia is a potentially fatal bacterial infection caused by Francisella tularensis, and is endemic to North America and many parts of northern Europe and Asia. The outer membrane lipoprotein, Flpp3, has been identified as a virulence determinant as well as a potential subunit template for vaccine development. Here we present the first structure for the soluble domain of Flpp3 from the highly infectious Type A SCHU S4 strain, derived through high-resolution solution nuclear magnetic resonance (NMR) spectroscopy; the first structure of a lipoprotein from the genus Francisella. The Flpp3 structure demonstrates a globular protein with an electrostatically polarized surface containing an internal cavity-a putative binding site based on the structurally homologous Bet v1 protein family of allergens. NMR-based relaxation studies suggest loop regions that potentially modulate access to the internal cavity. The Flpp3 structure may add to the understanding of F. tularensis virulence and contribute to the development of effective vaccines. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Structure 05/2015; 23(6). DOI:10.1016/j.str.2015.03.025 · 6.79 Impact Factor
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    ABSTRACT: The capA gene (FTT0807) from Francisella tularensis subsp. tularensis SCHU S4 encodes a 44.4 kDa integral membrane protein composed of 403 amino acid residues that is part of an apparent operon that encodes at least two other membrane proteins, CapB, and CapC, which together play a critical role in the virulence and pathogenesis of this bacterium. The capA gene was overexpressed in Escherichia coli as a C-terminal His6-tagged fusion with a folding reporter green fluorescent protein (frGFP). Purification procedures using several detergents were developed for the fluorescing and membrane-bound product, yielding approximately 30 mg of pure protein per liter of bacterial culture. Dynamic light scattering indicated that CapA-frGFP was highly monodisperse, with a size that was dependent upon both the concentration and choice of detergent. Circular dichroism showed that CapA-frGFP was stable over the range of 3 to 9 for the pH, with approximately half of the protein having well-defined α-helical and β-sheet secondary structure. The addition of either sodium chloride or calcium chloride at concentrations producing ionic strengths above 0.1M resulted in a small increase of the α-helical content and corresponding decrease in the random coil content. Secondary structure predictions based on the analysis of the sequence indicate that the CapA membrane protein has two transmembrane helices with a substantial hydrophilic domain. The hydrophillic domain is predicted to contain a long disordered region of 50 to 60 residues, suggesting that the increase of alpha helical content at high ionic strength could arise due to electrostatic interactions involving the disordered region. CapA is shown to be an inner membrane protein and predicted to play a key cellular role in the assembly of polysaccharides.
    Biochemistry 03/2014; 53(12). DOI:10.1021/bi401644s · 3.01 Impact Factor
  • Biophysical Journal 01/2014; 106(2):688a. DOI:10.1016/j.bpj.2013.11.3806 · 3.97 Impact Factor
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    Biophysical Journal 01/2014; 106(2):193a. DOI:10.1016/j.bpj.2013.11.1142 · 3.97 Impact Factor
  • Cancer Research 08/2013; 73(8 Supplement):469-469. DOI:10.1158/1538-7445.AM2013-469 · 9.28 Impact Factor
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    Kurt Whittemore · Kathryn Sykes
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    ABSTRACT: The immune system responds to tumor cells. The challenge has been how to effectively use these responses to treat or protect against cancer. Toward the goal of developing a cancer vaccine, we are pursuing methodologies for the discovery and testing of useful antigens. We present an array-based approach for discovering these B cell antigens by directly screening for specific host-sera reactivity to lysates from tumor-derived cDNA expression libraries. Several cancer-specific antigens were identified, and these are currently being validated as potential candidates.
    Human Vaccines & Immunotherapeutics 07/2013; 9(10). DOI:10.4161/hv.25634 · 3.64 Impact Factor
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  • Shen Luhui · Kathryn Sykes · Stephen Albert Johnston
    Cancer Research 06/2012; 72(8 Supplement):1570-1570. DOI:10.1158/1538-7445.AM2012-1570 · 9.28 Impact Factor
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    Michael J McGuire · Stephen A Johnston · Kathryn F Sykes
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    ABSTRACT: The success of new sequencing technologies and informatic methods for identifying genes has made establishing gene product function a critical rate limiting step in progressing the molecular sciences. We present a method to functionally mine genomes for useful activities in vivo, using an unusual property of a member of the poxvirus family to demonstrate this screening approach. The genome of Parapoxvirus ovis (Orf virus) was sequenced, annotated, and then used to PCR-amplify its open-reading-frames. Employing a cloning-independent protocol, a viral expression-library was rapidly built and arrayed into sub-library pools. These were directly delivered into mice as expressible cassettes and assayed for an immune-modulating activity associated with parapoxvirus infection. The product of the B2L gene, a homolog of vaccinia F13L, was identified as the factor eliciting immune cell accumulation at sites of skin inoculation. Administration of purified B2 protein also elicited immune cell accumulation activity, and additionally was found to serve as an adjuvant for antigen-specific responses. Co-delivery of the B2L gene with an influenza gene-vaccine significantly improved protection in mice. Furthermore, delivery of the B2L expression construct, without antigen, non-specifically reduced tumor growth in murine models of cancer. A streamlined, functional approach to genome-wide screening of a biological activity in vivo is presented. Its application to screening in mice for an immune activity elicited by the pathogen genome of Parapoxvirus ovis yielded a novel immunomodulator. In this inverted discovery method, it was possible to identify the adjuvant responsible for a function of interest prior to a mechanistic study of the adjuvant. The non-specific immune activity of this modulator, B2, is similar to that associated with administration of inactivated particles to a host or to a live viral infection. Administration of B2 may provide the opportunity to significantly impact host immunity while being itself only weakly recognized. The functional genomics method used to pinpoint B2 within an ORFeome may be more broadly applicable to screening for other biological activities in an animal.
    Proteome Science 01/2012; 10(1):4. DOI:10.1186/1477-5956-10-4 · 1.88 Impact Factor
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    ABSTRACT: Burkholderia are highly evolved Gram-negative bacteria that primarily infect solipeds but are transmitted to humans by ingestion and cutaneous or aerosol exposures. Heightened concern over human infections of Burkholderia mallei and the very closely related species B. pseudomallei is due to the pathogens' proven effectiveness as bioweapons, and to the increased potential for natural opportunistic infections in the growing diabetic and immuno-compromised populations. These Burkholderia species are nearly impervious to antibiotic treatments and no vaccine exists. In this study, the genome of the highly virulent B. mallei ATCC23344 strain was examined by expression library immunization for gene-encoded protective antigens. This protocol for genomic-scale functional screening was customized to accommodate the unusually large complexity of Burkholderia, and yielded 12 new putative vaccine candidates. Five of the candidates were individually tested as protein immunogens and three were found to confer significant partial protection against a lethal pulmonary infection in a murine model of disease. Determinations of peripheral blood cytokine and chemokine profiles following individual protein immunizations show that interleukin-2 (IL-2) and IL-4 are elicited by the three confirmed candidates, but unexpectedly interferon-γ and tumor necrosis factor-α are not. We suggest that these pathogen components, discovered using genetic immunization and confirmed in a conventional protein format, will be useful toward the development of a safe and effective glanders vaccine.
    Frontiers in Microbiology 11/2011; 2:227. DOI:10.3389/fmicb.2011.00227 · 3.94 Impact Factor
  • Cancer Research 01/2011; 70(8 Supplement):4767-4767. DOI:10.1158/1538-7445.AM10-4767 · 9.28 Impact Factor
  • Cancer Research 01/2011; 70(8 Supplement):4768-4768. DOI:10.1158/1538-7445.AM10-4768 · 9.28 Impact Factor
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    ABSTRACT: To meet the growing demand for synthetic genes more robust, scalable and inexpensive gene assembly technologies must be developed. Here, we present a protocol for high-quality gene assembly directly from low-cost marginal-quality microarray-synthesized oligonucleotides. Significantly, we eliminated the time- and money-consuming oligonucleotide purification steps through the use of hybridization-based selection embedded in the assembly process. The protocol was tested on mixtures of up to 2000 oligonucleotides eluted directly from microarrays obtained from three different chip manufacturers. These mixtures containing <5% perfect oligos, and were used directly for assembly of 27 test genes of different sizes. Gene quality was assessed by sequencing, and their activity was tested in coupled in vitro transcription/translation reactions. Genes assembled from the microarray-eluted material using the new protocol matched the quality of the genes assembled from >95% pure column-synthesized oligonucleotides by the standard protocol. Both averaged only 2.7 errors/kb, and genes assembled from microarray-eluted material without clonal selection produced only 30% less protein than sequence-confirmed clones. This report represents the first demonstration of cost-efficient gene assembly from microarray-synthesized oligonucleotides. The overall cost of assembly by this method approaches 5¢ per base, making gene synthesis more affordable than traditional cloning.
    Nucleic Acids Research 10/2010; 38(19):e180. DOI:10.1093/nar/gkq677 · 9.11 Impact Factor
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    ABSTRACT: Identification of highly immunogenic antigens is critical for the construction of an efficacious subunit vaccine against Chlamydia pneumoniae infections. A previous project used a genome-wide screen to identify 12 protective C. pneumoniae candidate genes in an A/J mouse lung disease model (Li et al. [14]). Due to insufficient induction of Th1 immunity, these genes elicited only modest protection. Here, we used the Escherichia coli heat-labile enterotoxin as a Th1-enhancing genetic adjuvant, and re-tested these 12 genes, in parallel with six genes identified by other investigators. Vaccine candidate genes cutE and Cpn0420 conferred significant protection by all criteria evaluated (prevention of C. pneumoniae-induced death, reduction of lung disease, elimination of C. pneumoniae). Gene oppA_2 was protective by disease reduction and C. pneumoniae elimination. Four other genes were protective by a single criterion. None of the six genes reported elsewhere protected by reduction of lung disease or elimination of C. pneumoniae, but three protected by increasing survival.
    Vaccine 12/2009; 28(6):1598-605. DOI:10.1016/j.vaccine.2009.11.046 · 3.49 Impact Factor
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    ABSTRACT: The licensed smallpox vaccine, comprised of infectious vaccinia, is no longer popular as it is associated with a variety of adverse events. Safer vaccines have been explored such as further attenuated viruses and component designs. However, these alternatives typically provide compromised breadth and strength of protection. We conducted a genome-level screening of cowpox, the ancestral poxvirus, in the broadly immune-presenting C57BL/6 mouse as an approach to discovering novel components with protective capacities. Cowpox coding sequences were synthetically built and directly assayed by genetic immunization for open-reading frames that protect against lethal pulmonary infection. Membrane and non-membrane antigens were identified that partially protect C57BL/6 mice against cowpox and vaccinia challenges without adjuvant or regimen optimization, whereas the 4-pox vaccine did not. New vaccines might be developed from productive combinations of these new and existing antigens to confer potent, broadly efficacious protection and be contraindicated for none.
    Virology 09/2009; 395(1):97-113. DOI:10.1016/j.virol.2009.09.008 · 3.28 Impact Factor
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    ABSTRACT: A new method for biolistic delivery of nucleic acids using a combination of cationic micro- and nanoparticles is reported. The new method is simpler to perform than the conventional calcium/spermidine-based formulations and shows 11-fold improved nucleic acid binding capacity and dose-dependent performance both for in vitro and in vivo applications relative to either the conventional preparation or our recently reported direct cationic microparticle method. These features may enable higher throughput gene delivery and genetic immunization programs and open new venues for the biolistic delivery method.
    Molecular Pharmaceutics 09/2009; 6(6):1927-33. DOI:10.1021/mp900156h · 4.79 Impact Factor
  • Kathryn Sykes
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    ABSTRACT: Despite its young life, genetic immunization (GI) has seen both fame and infamy. Initially under the limelight because of its appeal as a simple method of delivering vaccines, some experiments displayed disappointing immune potency and, consequently, excitement dimmed. Newer focus on the flexibility of GI, afforded by its foundation in molecular biology, has recently rekindled activity in the field. Approached as a recombinant DNA technology, deficiencies become addressable. Unlike any other subunit vaccine modality, such as protein or carbohydrate, DNA is chemically simple, stable, consistent, easily amplified and the base material of a vast array of bioconstruction and biocontrol techniques. GI provides scientists with a simple platform for merging other disciplines, such as molecular biology, biochemistry, genetics, chemistry, informatics and microbiology, into the development of superior vaccine products.
    Expert Review of Vaccines 12/2008; 7(9):1395-404. DOI:10.1586/14760584.7.9.1395 · 4.22 Impact Factor
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    Sergei Svarovsky · Alexandre Borovkov · Kathryn Sykes
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    ABSTRACT: Here we report preparation and properties of positively charged gold microparticles, and their use for biolistic DNA delivery. Micron-sized gold microparticles were modified by building self-assembling polyethylenimine monolayers on their surfaces, which enabled their electrostatic interaction with negatively charged molecules such as DNA. One milligram of the surface-modified microparticles was able to bind directly to up to 3.5 microg of DNA, exceeding the 1 microg/mg limit of the conventional protocols. The binding showed no apparent dependency on DNA purity, size, or conformation. The interaction occurred over a broad range of pH values and salt concentrations, and was stable throughout the standard protocol for biolistic cartridge preparation. At the standard 1 microg dose, biological activity of the DNA biolistically delivered on the charge-modified gold was 25% higher than that delivered on conventional microparticles. Loading the charge-modified gold with more DNA stimulated proportionally higher gene expression. The charge-modified gold can be also used for delivery of small biological molecules such as siRNA. Tissue culture cells biolistically transfected with a LUC+ specific siRNA showed 80% reduction of Luc expression relative to those cells transfected with an irrelevant siRNA. Along with its superior properties as a DNA delivery vehicle, charge-modified gold offers a unique opportunity to deliver various DNA formulations in addition to traditional naked DNA.
    BioTechniques 12/2008; 45(5):535-40. DOI:10.2144/000112991 · 2.75 Impact Factor
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    ABSTRACT: An inbred A/J mouse respiratory challenge model was validated for vaccine testing against Chlamydia (C.) pneumoniae and used to screen the C. pneumoniae genome for vaccine candidates by expression library immunization (ELI). Biolistic delivery of genetic vaccine constructs elicited Th2-like immunity that was associated with inefficient elimination of C. pneumoniae. Delivery by injection elicited protective Th1-like responses. Since biolistic delivery of pools of ORFs was used in first round screening, the screen presumably selected against potent immunogens. Nevertheless, it was sufficiently accurate to identify three weakly protective antigens among all putative C. pneumoniae ORFs. The results suggest ELI discovery of highly protective C. pneumoniae vaccine candidates requires tight control of the Th1 immunity elicited by the genetically delivered library of test antigens.
    Vaccine 05/2006; 24(15):2917-27. DOI:10.1016/j.vaccine.2005.12.035 · 3.49 Impact Factor
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    ABSTRACT: We report the results of a general protocol that was used to screen the whole genome of Chlamydophila abortus, type strain B577 (formerly Chlamydia psittaci strain B577), in a mouse pneumonia model. Genetic immunization was used to functionally test the genes of C. abortus as vaccines in a mouse challenge system. Nine gene fragments were isolated that conferred protection, with five protecting as effectively as the live-vaccine positive control. Bioinformatics approaches were unable to reconstruct isolation of these antigens. These results suggest that pathogen genomes can be functionally screened for vaccine candidate antigens in a mouse model to reveal new classes of vaccine candidate antigens that may have therapeutic efficacy across host species, disease manifestations, and delivery platforms.
    Vaccine 05/2005; 23(23):3016-25. DOI:10.1016/j.vaccine.2004.12.013 · 3.49 Impact Factor

Publication Stats

389 Citations
154.98 Total Impact Points

Institutions

  • 2008–2015
    • Arizona State University
      • • Center for Innovations in Medicine
      • • School of Life Sciences
      Phoenix, Arizona, United States
  • 1999–2005
    • University of Texas Southwestern Medical Center
      • • Department of Internal Medicine
      • • Medical School
      Dallas, Texas, United States