DHVI Cores
Resources for Scientific Discovery
Message from DHVI Cores Scientific Director The DHVI has assembled a group of state-of-the-art instruments, techniques and services to support basic and translational research initiatives in vaccine immunology, immune reconstitution, host-pathogen interactions, emerging infectious diseases and biodefense. These comprehensive Shared Resources or Cores are available to DHVI investigators and the overall Duke community. In 2017, DHVI Core laboratories supported over 120 individual Principal Investigators at Duke and collaborating non-profit research institutions. Services were provided at cost to these investigators totaling $1.3M in fiscal year 2017. Investigators used over 186 unique grants, contracts and sponsored research awards to support their research efforts that accessed DHVI Core facilities. In addition to DHVI Core laboratories being a central repository of state-of-the-art instrumentation, we also see these facilities, and more specifically their Directors/Managers, as institutional thought leaders in their respective areas (e.g., crystallography, biocontainment, animal models, flow cytometry, immune monitoring etc.). The majority of our Core leaders have doctoral degrees in their respective fields and provide mentorship to the next generation of scientists through hands-on user training opportunities and consultative services. All DHVI Core facilities are live in the CoreResearch@Duke system; a centrally supported, enterprisewide, efficient, booking to billing web portal to support operation of all Duke University Shared Resources (coreresearch.duke.edu). For more information about individual core services, procedures for scheduling instrument time, requesting services and current pricing please go to shared-resources.dhvi.duke.edu.
Core metrics Fiscal Year 2017 (July 1, 2016 to June 30, 2017)
Gregory D. Sempowski
Professor in Medicine & Pathology
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186
DHVI Shared Resource Core Facilities
Scientific Director:
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Gregory D. Sempowski, PhD
Professor, Medicine and Pathology Director, Duke Regional Biocontainment Laboratory 919.684.3349 gregory.sempowski@duke.edu
Duke Departments/Divisions/ Centers/Institutes supported
120
DHVI, Administrative Manager
Darlene McCain, MS, CFM 919.684.3349Â darlene.mccain@duke.edu
Uniuqe Sponsored grants and contracts
Principal Investigators supported
2,800 Invoiced jobs
7,200
Hours provided on specialized instruments/equipment
Index
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01 02 03 07 09 11
Message from the Director Duke Human Vaccine Institute Duke Regional Biocontainment Laboratory Biomolecular Interation Analysis Macromolecular X-ray Crystallography Viral Genetic Analysis (Sequencing)
13 15 17 19 21
Accessioning Unit (Clinical Support) Flow Cytometry and Cell Sorting Immunology/Virology Quality Assurance Protein Production Facility Core Research @ Duke
Duke Human Vaccine Institute Our Values
The DHVI supports the development of vaccines and therapeutics through a commitment to Innovation, Collaboration and Excellence.
• Innovation through ingenuity and the pursuit of continuous improvement
• Collaboration through embracing change and cultivating diversity to achieve synergy • Excellence through a commitment to respect, integrity and safety
Our Mission
Will develop vaccines and therapeutics against diseases of global importance while training the next generation of scientists
DHVI Translational Pipeline Basic Science Research
Preclinical Research
Clinical Research
Proof of Concept in Human Phase I Trial
Clinical Research
Phase II - III in Human “Efficacy” Trial
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Duke Regional Biocontainment Laboratory
Mission To support basic research to develop drugs, diagnostics, and vaccines for emerging and reemerging infections and biodefense.
About Us The Duke Regional Biocontainment Laboratory (RBL) was built with funding from NIH (NIAID UC6AI058607) as part of a network of 12 regional and 2 national biocontainment laboratories. In 2007, the Duke RBL became the first of the regional labs to be commissioned for use. The Duke RBL provides state-of-the-art biocontainment facilities for BSL2, BSL3, and Select Agent research and has a comprehensive safety and operations program to support work in these facilities. The Duke RBL functions as a shared resource/core facility through its Immunology, Virology, and Microbiology units, which are available to Duke faculty and their collaborators as fee-for-service. These units can be utilized separately or all together to provide comprehensive study support.
Services Small Animal Models Support • Can accommodate ABSL2, ABSL3, and Select Agent small animal models (mouse, rabbit, ferret) • Live animal imaging using the IVIS system • Aerosol exposure Biocontainment Support • Hourly and monthly use of customizable BSL2/3 containment suites, decontamination (vaporized hydrogen peroxide) Safety/Operations Support • Training • Customized didactic and hands-on biosafety training for BSL2/3 and Select Agent research
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Duke RBL Research Programs
In addition to cost-recovery shared resource support, the Duke RBL receives direct funding through sponsored programs in the areas of: Safety / Biopreparedness Training • DIDRT – Duke Infectious Disease Response Training Program (NIH UH4-ES027072; PI: G. Sempowski) Vaccine/Therapeutic Development • Duke DARPA Pandemic Prevention Platform Program (P3) (DoDDARPA HR0011-17-2-0069; PI: G. Sempowski) • CETR – Center of Excellence in Translational Research (Immunology & Influenza Virology Core; NIH U19-AI109784; Duke PI: G. Sempowski) • VTEU – Vaccine Treatment and Evaluation Unit (Duke) (Immune Monitoring Core; NIH HHSN2722013000171; Duke PIs: E. Walter, G. Swamy, K. Weinhold, G. Sempowski) Host Response & Immune Monitoring • DOD Center for Long Term Follow-up of the Late Effects of Acute Radiation Exposure in Primates (Project 3: Immune Recovery; DoD W81XWH-15-1-0574; Duke PIs: B, Chen, G. Sempowski) • AC STI CRC – Atlantic Coast Sexually Transmitted Infections Cooperative Research Center (Host Response Monitoring Core; NIH U19-AI113170; Duke PIs: G. Sempowski, H. Staats) • RadCCORE – Radiation Countermeasures Center of Research Excellence (Immune Monitoring Core; NIH U19AI067798; Duke PIs: N. Chao, D. Kirsch, B, Sullenger, B. Chen, G. Sempowski) Proficiency Testing & Quality Assurance • EQAPOL – External Quality Assurance Program Oversight Laboratory (Luminex and Viral Diversity Programs; HHSN272201700061C; PI: T. Denny)
Microbiology
Coordinator: Rose Asrican, MS rosemarie.asrican@duke.edu 919.681.5480
The RBL Microbiology Unit is a collaborative research service component of the Duke Regional Biocontainment (housed in GHRB). The Unit maintains quality-controlled secure inventories of approved BSL2, BSL3, and Select Agent pathogens for RBL projects and collaborating investigators. RBL Microbiology focuses on minimum inhibitory concentration screening, colony forming unit assays, and bacterial and fungal challenge models in mice. To submit a service request, go to CoreResearch@duke and select DHVI RBL: Microbiology (D-0045).
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Immunology Regional Biocontainment Laboratory
Manager: Andrew Macintyre, PhD a.n.macintyre@duke.edu 919.668.4704
The RBL Immunology Unit is a service component of the Duke Regional Biocontainment Laboratory (housed in GHRB). In addition to providing access to specialized instrumentation for real-time PCR and Luminexbased multiplex assays, this unit assists investigators with design and implementation of molecular, protein, and cellular assays to quantify immune reconstitution or immune responses in vitro and in vivo. To submit a service request, go to CoreResearch@duke and select DHVI RBL: Immunology (D-0002).
Services Commercial Luminex assays (Magnetic or Non-magnetic) • Full service from sample to data (plasma, serum, cell supernatant, tissue homogenate, cerebrospinal fluid, lavage fluid, etc.) • Assay read only (BioPlex 200, FlexMap 3D, MagPix) • BSL2, BSL3, and Select Agents Sample Preparation • Plasma, serum, or PBMC prep from whole blood • Tissue specimen prep/homogenization • Nucleic acid extraction • Bead-based cell separation ELISA • Commercial kit • Custom antigen-specific endpoint ELISAs Real-time Thermocycler use Quantitative signal joint T Cell Receptor Excision Circle (sjTREC) analysis (mouse, human, NHP) TCRB sequencing (human)
Instrumentation • • • • •
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Luminex bead readers (BioPlex200, FlexMap 3D, MagPix) CFX96 real-time PCR detection system BioTek microplate washers BioTek Synergy H1 multi-mode microplate reader QIAcube sample prep systems
Virology
Manager: Charles McGee, PhD charles.mcgee@duke.edu 919.681.9824
The RBL Virology Unit is a service component of the Duke Regional Biocontainment Laboratory (housed in GHRB). The Virology Unit was created to provide world-class quality-controlled support for virus research requiring up to and including enhanced biosafety level 3 containment. In addition to a comprehensive portfolio of influenza assays and services, the RBL Virology Unit has expertise in work with alphaviruses, flaviviruses, poxviruses, rotavirus and vesicular stomatitis virus. To submit a service request, go to CoreResearch@duke and select DHVI RBL: Virology (D-0031).
Services Quality-controlled virus stock propagation • BSL2, BSL3 and Select Agent • Tissue culture propagation • Chicken egg propagation Viral load quantification • Plaque/focus forming unit assays • Tissue culture infectious dose 50% assays • Hemagglutination units assay Virus serology assays • Hemagglutination inhibition assay • MicroNeutralization assay • Plaque reduction neutralization test In vivo challenge models including sample procurement and processing from infected animals • Human influenza in ferrets • Mouse-adapted influenza in mice • Rabbitpox in rabbits Development of project-specific virology support assays • Track record working with flaviviruses, human cytomegalovirus, influenza viruses, human rotavirus, poxviruses, and vesicular stomatitis virus
CONTACT INFO Director: Assoc. Dir., Safety & Operations: Asst. Dir., Programs & Development: RBLINFO@duke.edu 919.684.3349
Gregory D. Sempowski, PhD T. Scott Alderman, MS Heather E. Lynch, PhD
Learn more here 66
Biomolecular Interation Analysis Mission The DHVI Biomolecular Interaction Analysis (BIA) Core Facility offers state-of-the-art Surface Plasmon Resonance (SPR) and Biolayer Interferometry (BLI) instruments, expertise, training, and support for label-free, real-time analysis of biomolecular interactions to basic and clinical researchers within the Duke Community.
Leadership / Experience Director: S. Munir Alam, PhD Munir Alam is a Professor of Medicine and Pathology at Duke University and the Founding Director of the DHVI BIA Core Facility. Dr. Alam is a principal investigator at DHVI and a recognized expert in biochemical and biophysical analyses of receptor-ligand interactions, including TCR-ligand and antibody-antigen binding, and HIV Envelope protein antigenicity characterization. He is Director of Protein Analytics at both the DHVI and its GMP facility, a Co-Investigator in the Duke CHAVI-ID program, and was the Proteomics Director for the Duke CHAVI program. Manager: Brian Watts, PhD Brian Watts is the BIA Core Facility Manager and has extensive experience in assay development and biophysical characterization of molecular interactions by SPR, ITC, DSC, NMR, AFM, and FPLC.
Services Binding Specificity and Screening • Yes/No Binding • Ranking of Binding Response Thermodynamics Epitope Mapping
Kinetics and Affinity • Direct Binding Kinetics • Steady State Affinity • In-solution Affinity • Avidity Assessment
Active Concentration • Surface Competition • Solution Inhibition Conformation Changes
Technology / Instruments Surface Plasmon Resonance In a typical SPR experiment, a soluble analyte is injected over a ligand-immobilized sensor chip. Interaction between the biomolecular species results in surface site population and an increase in SPR response. Upon injection completion, the bound analyte dissociates from the surface, reducing the SPR response. The surface can be regenerated to remove any residual analyte in preparation for the next injection. The kinetics (ka, kd) and affinity (Kd) of the interaction and/or the active analyte concentration can be calculated. Biacore systems are low sample consumption (< 25 µg) and compatible with a wide-range of biomolecules • Proteins, peptides, DNA/RNA, small molecules, carbohydrates, lipid membranes, micelles, liposomes, polymers, whole viruses, and whole cells • Crude cell culture lysates or clinical sera/bodily fluids Biomolecules can be functionalized on sensor chips via: • Covalent coupling • High-affinity capture (with any affinity tag) • Hydrophobic adsorption 7
DHVI BIA Core Facility Platforms Biacore 3000 • Standard platform • Simultaneously detects 4 interactions • 10 RU sensitivity
Biacore T200/S200 • LMW screening • Simultaneously detects 4 interactions • 1 RU Sensitivity • T200 available for independent use
Biacore 4000 • High-throughput analysis of clinically-derived or infectious samples • Supports 96- and 384-well plate racks • Simultaneously detects 12 interactions ForteBio Octet-RED96 • Biolayer Interferometry (BLI) • Ligand-coated biosensors in 96-well plate • Crude sample compatibility • Simultaneously detects 8 interactions
Reservations / Service Requests Sample Submission – Full-Service, All platforms • All experimentation, optimization, and analysis will be performed by Core Staff Independent Use – Self-Service, T200 only • Complete training in the independent operation of the Biacore T200 • Available 24/7/365 through CoreResearch@Duke Consultation • Assistance in experimental design and analysis/ interpretation of data • Contact Brian Watts to schedule an initial consultation and to discuss sample submission goals and/or training
To submit a service request, go to CoreResearch@duke and select DHVI: BIA Core (D-0029).
CONTACT INFO brian.watts@duke.edu 919.668.6995
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Macromolecular X-ray Crystallography Mission The DHVI Macromolecular X-ray Crystallography Core offers services in determining and publishing macromolecular crystal structures. We provide everything from protein expression through crystallization and structure determination. Facility staff can assist with any and all steps in the process as needed. We welcome inquiries from investigators associated with Duke as well as those associated with other research facilities.
Leadership / Experience Director: Nathan Nicely, PhD Nathan Nicely, Ph.D., is an Assistant Professor of Medicine with the Duke Human Vaccine Institute and Director of the Duke University X-ray Crystallography Core Facility. He has experience in diverse topics including immunology, enzymology, pathogenic metabolism, and oncology.
Services Protein production Crystallization trials Automated imaging of crystallization experiments X-ray diffraction and data collection Expert staff support Protein kinase A catalytic subunit threaded with Aspergillus fumigatus
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HIV antibody DH522.2
Technology / Instruments Rigaku MicroMax-007 • Micro focus rotating anode x-ray generator • VariMax HR (high resolution) optics • RAXIS IV++ image plate detector • Inverse phi axis for easy crystal mounting • X-stream 2000 cryogenic system • Plexiglass enclosure Minstrel HT UV • Every drop dual imaged in white and UV light • Fully automated operation with a manual mode • Inspection schedule of choice
Crystallization experiment setups • Phoenix robot dispenses 96-well screens all at once • Oryx4 robot dispenses proteins and proteinreagent mixtures • Drop volumes of 100-500 nL • Handles seeds
Reservations / Service Requests To submit a service request, go to CoreResearch@duke and select DHVI: X Ray Crystallography (D-0033).
CONTACT INFO crystallography@duke.edu 919.681.3917
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Viral Genetic Analysis Core (Sequencing) Mission The Duke Human Vaccine Institute Viral Genetic Analysis Core facility provides sequencing (NGS and Sanger) and genomic technologies to serve the needs of the Duke Human Vaccine Institute, Duke Center for AIDS Research, Duke University and external collaborators.
Leadership / Experience Director: Todd Bradley, PhD Dr. Bradley is a Medical Instructor in the Department of Medicine and became facility director in 2016. Dr. Bradley has over 10 years of experience in developing next-generation sequencing strategies to investigate mechanisms of cell biology and immune cell responses. Manager: Bhavna Hora, PhD Dr. Hora is a researcher and a core manager with over 17 years of experience in molecular biology, immunology, and virology. Dr. Hora has been managing the sequencing core for the past two years and has over 12 years of experience in HIV sequencing, SGA, Q-PCR, mutagenesis etc.
Services Next-Gen Sequencing • High throughput RNA/ DNA sequencing, library prep, NGS runs and data analysis 10X Genomics • The Chromium Single Cell 3’ Solution provides high-throughput, single cell expression measurements that enable discovery of gene expression dynamics and molecular profiling of individual cell types Sample and library QC • Quantification and quality control of samples and libraries Single Genome Amplification • Isolation and analysis of sequences for single nucleotide polymorphisms within the HIV-1 genome Sanger Sequencing • Sequence up to twenty-four 96-well plates with reads of up to 1000 base pairs per sample PCR Purification • Utilizing an innovative paramagnetic beads technology for high-throughput purifications of PCR amplicons Site Directed Mutagenesis • Single, multiple base, deletion mutants in a plasmid Drug Resistance Mutations • Population sequencing of viral samples to determine the DRMs in the sample Sequence Analysis of Viral Stocks Co-Receptor usage Assay • Determine coreceptor usage of a virus sample in NP2 cells by measuring p24
Technology / Instruments Single Cell Genomics The Chromium Single Cell 3’ Solution provides high-throughput, single cell expression measurements that enable discovery of gene expression dynamics and molecular profiling of individual cell types. 11
Next-generation (NGS) Sequencing on Illumina We offer services on Illumina MiSeq and NextSeq 500. Because of their similar chemistry, same library preparations can be used across these instruments, allowing us to tailor to researchers’ sequencing needs according to turn around time (few hours to three-day run) read length (75 bp single-end reads to 300 bp paired-end reads) and throughput (300 Mb/run to 500 Gb/run). Sample/ Library Quantification and Analysis
StepOne Plus (qPCR)
Qubit
Tapestation 2000
MiSeq
NextSeq
Sanger Sequencing
ABI 3730XL DNA
Biomek FXp Laboratory Automation workstation
Reservations / Service Requests The DHVI VGA Core offers 3 ways to utilize our facility to accomplish your research objectives: Sample submission • Researchers provide DNA/RNA or PCR amplicons and service is performed by Core staff (Sanger DNA sequencing, PCR amplicon purification and Illumina next-generation DNA/RNA sequencing, HIV co-receptor, subtype and drug resistance analysis) Intermediate use • For Illumina next-generation DNA/RNA sequencing submit prepared libraries and Core staff can QC, optimize and provide appropriate sequencing kits and generate raw data Independent use • Researchers can reserve and operate the instruments independently and provide all of the required reagents for Illumina Miseq, Illumina NextSeq, real-time qPCR, TapeStation, or Qubit fluorometer. Complete training in the independent operation of the Illumina and QC platforms is required First step • Contact Core personnel for consultation and a quote for your desired service. We will guide you through the process to achieve your research goal as fast and cost-effectively as possible Second step • To submit a service request, go to CoreResearch@duke and select DHVI: Viral Genetic Analysis Facility (D-0030).
CONTACT INFO todd.bradley@duke.edu bhavna.hora@duke.edu 919.668.1964
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Accessioning Unit (Clinical Support) Mission This Core provides support for human and large animal studies that fall within DHVIâ&#x20AC;&#x2122;s mission of performing research against infectious diseases that impact global health. The team provides support for developing protocols, recruiting participants of all ages and also for processing, storage, and retrieval of specimens obtained for these studies.
Leadership / Experience Director: Tony Moody, MD Tony Moody, MD is an Associate Professor in the Department of Pediatrics, Division of Infectious Diseases and Assistant Professor of Immunology at Duke University Medical Center. Dr. Moody is the Director of the Laboratory of B cell Immunotechnology at the Duke Human Vaccine Institute (DHVI) as well as the DHVI Chief Medical Officer. Specimen Processing Manager: Thad Gurley, MS Thad Gurley is the specimen processing manager of the Clinical Support Core and has 11 years of sample processing and storage experience with DHVI. Clinical Recruitment Manager: Amanda Stemke MPH, CCRP Amanda Stemke has served as manager of the Clinical Support Core for over 2 years and has 10 years of clinical research experience with DHVI.
Services Sample Processing and Storage Processing Training and Consultation Data Analysis Protocol Support and Development
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Technology / Instruments DHVI Specimen Processing/Storage Processing: Provide isolation of sera, plasma, and PBMCs from human whole blood samples Storage: Provide -80°C storage for sera and plasma and -200°C storage for PBMC samples (24/7/365 monitored) All processing and storage is completed under established SOPs that are overseen by the Quality Assurance for Duke Vaccine Immunogenicity Program (QADVIP).
Reservations / Service Requests For Protocol Support • (e.g., protocol development, interaction with participants to obtain information or specimens) Please contact Amanda Stemke, MPH and/or the facility director (Tony Moody, MD) to discuss project details. For Specimen Processing • (e.g., processing and storage of materials obtained from participants in a clinical study) Please contact Thad Gurley, MS and/or the facility director (Tony Moody, MD) to discuss project details. For researchers who wish to access both service pipelines • Please contact Amanda Stemke, MPH and/or the facility director (Tony Moody, MD) to discuss project details. To submit a service request, go to CoreResearch@duke and select DHVI: Clinical Support (D-0034).
CONTACT INFO thad.gurley@duke.edu 919.681.6498 amanda.stemke@duke.edu 919.684.6043
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Flow Cytometry and Cell Sorting Mission The DHVI Flow Cytometry Facility serves the flow cytometric needs of the Duke Human Vaccine Institute and researchers throughout the Duke Community. DHVI Flow offers support over the full range of a flow cytometry experiment, from instrument training, to panel design, to data analysis, and presentation.
Leadership / Experience Director: Derek W. Cain, PhD, CCy Dr. Cain obtained a PhD in Immunology from Duke University, and completed his postdoctoral work in Dr. John Cidlowski’s laboratory at the National Institute of Environmental Health Sciences (NIEHS). He joined the Duke Human Vaccine Institute in 2016. Dr. Cain’s research interests include characterizing T Follicular Helper cells following vaccination or after HIV infection.
Services Flow cytometric analysis • Independent use, 24/7 Flow cytometric sorting • Operator-assisted Consultation • Experiment design, analysis, and presentation Instrument training for independent use
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FlowJo® Site License Fluorofinder® Partnership • Build an antibody panel using the configuration of our cytometers • Access our panel repository through Fluorofinder® Groups feature
Technology / Instruments BD LSRFortessa (F01) • Independent use, 24/7 analyzer • 2 Lasers (488, 640 nm) • 2 scatter parameters, 8 fluorescent parameters BD Influx (N01) • Operator-assisted sorter • 5 Lasers (355, 405, 488, 532, 639 nm) • 2 scatter parameters, 23 fluorescent parameters • 6-way sorting, plate sorting, index sorting BD LSRII (L01 and L02) • Independent Use, 24/7 analyzers • 4 Lasers (405, 488, 532, 639 nm) • 2 scatter parameters, 18 fluorescent parameters • High throughput sampler BD FACS Aria II (A01 and A02) • Operator-assisted sorters • 4 Lasers (405, 488, 532, 639 nm) • 2 scatter parameters, 18 fluorescent parameters • 4-way sorting, plate sorting, index sorting • BSL2, 2+, 3 (Duke RBL)
Reservations / Service Requests Instrument Reservations • Can be made on our online calendar through CoreResearch@Duke Training Requests • Can be made through our website (see “Contact Info” below) Consultation • Email us for assistance with experiment design, analysis, and/or presentation (dhviflo@dm.duke.edu) To submit a service request, go to CoreResearch@duke and select DHVI: Flow Cytometry Facility (D-0059).
CONTACT INFO dhviflo@dm.duke.edu 919.684.4130
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Immunology/Virology Quality Assurance Mission To provide laboratory procedures and assays for HIV and Non-HIV clinical studies and trials under Good Lab Practices (GLP). These activities include non-human primate SIV viral load, HIV-1 RNA viral load, PBMC processing from leukopaks, HIV1 WB, sterility, mycoplasma and endotoxin testing, flow cytometry acquisition and Elutriation cell separation.
Leadership / Experience Director: Thomas Denny MSc, MPhil Mr. Denny is a Professor of Medicine in the Duke School of Medicine and Chief Operating Officer (COO) of the Duke Human Vaccine Institute (DHVI). He is also the Director and PI of the DHVI Immunology/Virology Quality Assurance Center (IVQAC), a CAP accredited / CLIA-approved, GCLP- compliant unit with ISO accreditations. Manager: Ambrosia Garcia, MS Ambrosia Garcia serves as the IVQAC Core Facility Manager for the last 7 years and has extensive experience in molecular assay development and implementation.
Services SIV Viral Load • Validated for EDTA rhesus plasma and requires 600 μL of sample. The lower limit of detection for this SIV assay is 250 copies/mL HIV-1 RNA Viral Load • HIV-1 RNA viral load is quantified using the Roche COBAS AmpliPrep/COBAS Taqman 48 system. It is a real-time PCR assay with a quantifiable range of 20 - 10,000,000 RNA copies/mL (HIV) HIV EIA Ab • HIV serology testing is determined qualitatively using the BioRad HIV-1, 2 + O EIA platform HIV-1 Western Blot (WB) • WB confirmatory assay is performed using a Bio-Rad clinical diagnostics kit. HIV acute disease staging (Fiebig staging based on results from both HIV-1 Ab and WB) Leukopak Processing • PBMCs are obtained, under GLP, through density gradient centrifugation and are assessed for viability and cell number, aliquoted, cryopreserved, and stored in vapor phase LN2 freezers. The approximate final PBMC cell yield of one Leukopak is 4.0 x109 cells Sterility Testing for Bacteria/Fungi • This qualitative test involves direct inoculation of the test sample into two different types of media: Tryptic Soy Broth (TSB), also commonly referred to as Soybean-Casein digest, and Fluid Thioglycollate Media (FTM). Inoculated media is monitored for microorganism growth
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Elutriation • Counterflow centrifugation procedure for separation of cell populations on a large scale. Separation is based on cell size, shape, and density to serve the analytic needs of investigators Sterility Testing for Mycoplasma • This qualitative test utilizes the MycoAlert® test kit to detect the presence of Mycoplasma Sterility testing for Endotoxin • Testing is done using the Limulus Ameobocyte Lysate (LAL) Pyrogent® plus single test vial gel clot assay. This qualitative assay has a sensitivity of 0.125 EU/mL
Technology / Instruments QIAsymphony SP
Avanti J-26XP SP
BD FACSCanto
COBAs AmpliPrep/TaqMan 48
Reservations / Service Requests Sample Submission • Full-service, all platforms • All experimentation, optimization, and analysis will be performed by Core Staff Independent Use • Self-Service, Becton Dickinson FACSCanto only • Complete training in the independent operation available through CoreResearch@Duke Consultation • Assistance in experimental design, analysis and interpretation of data • Contact Ambrosia Garcia to schedule an initial consultation and to discuss sample submission goals and/ or training To submit a service request, go to CoreResearch@duke and select DHVI: IVQA Center (D-0049).
CONTACT INFO ambrosia.garcia@duke.edu 919.684.5861
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Protein Production Facility Mission The Protein Production Facility (PPF) was established with a grant awarded by the Bill & Melinda Gates Foundation with the mission of providing high-quality, researchgrade recombinant proteins and antibodies to the members of the Collaboration for Aids Vaccine Discovery (CAVD). In July 2017, the PPF opened its doors as a DHVI Shared Resource Facility that is open to all Duke University researchers. The mission of the DHVI PPF is to provide high-quality proteins and antibodies for in vitro assays for basic research and clinical study immune monitoring and to facilitate immunogen discovery. The production of all proteins and antibodies is governed by a Quality Management plan with oversight by the Quality Assurance for Duke Vaccine Immunogenicity Program (QADVIP), Dukeâ&#x20AC;&#x2122;s Quality Assurance Unit. All proteins and antibodies are produced following Standard Operating Procedures (SOPs) and using Good Documentation Practices. Following production, proteins and antibodies undergo a quality control panel and are visualized by SDS-PAGE under reduced and non-reduced conditions and tested for sterility, Mycoplasma and endotoxin.
Leadership / Experience Director: James Peacock, PhD James obtained his PhD in Biology and Immnology from the University of North Carolina at Charlotte and has over five years of experience managing the production of antibodies and HIV1 envelope proteins. He has extensive experience in SOP and batch record development and ensures that the PPF maintains compliance with the Quality Management Agreement which has been established with Dukeâ&#x20AC;&#x2122;s Quality Assurance Unit. Manager: Cynthia Nagle, PhD Cynthia Nagle has over two years of experience as a Program Manager at DHVI and as the Program Manager for the Protein Production Facility. Cynthia interfaces with researchers and the PPF lab team to evaluate the costs and timelines for completing specific protein production requests and then works with Kathy to manages the production schedule in the lab to ensure timely delivery of products. Manager: Kathy Yarborough, MS Kathy Yarborough has six years of experience in the DHVI expressing proteins and antibodies by transient transfection. She is responsible for the workflow of projects in the PPF as well as quality control of all proteins and antibodies; SDS-PAGE, Western blots and testing for endotoxin.
Services Protein Production (1L and 4L transfections in 293 cells or CHO cells) Antibody/Fab Production (1L and 4L transfections in 293 cells) FPLC/SEC Endotoxin Testing (individual samples) Consulting for antibody and HIV-1 Env sequence modifications/design 19
Work Flow for Protein Production
Plasmids with the insert produced and purified
Eluted protein is buffer exchanged into PBS, filtered and aliquots made
Supernatants harvested and clarified
Transiently transfect 293 Incubated shaking 4 days in 8% CO2 at 37°C.
Purification
QC release testing performed prior to delivery Protein aliquots stored - 80°C until shipped
Technology / Instruments CO2 Incubators and Biosafety Cabinets for Project Segregation • Protein expression and available nickel, lectin, protein A, G or M, and Kappa select purification methods • Recombinant Viral Envelope Proteins, SOSIP trimers and Fc Receptors • Recombinant Immunoglobulin (Ab or Fab) • Endotoxin testing • FPLC/SEC
AKTA pure with Fraction Collector
QC Equipment
Endotoxin Assay
SOPs and Quality Management Plan
Reservations / Service Requests To submit a service request, go to CoreResearch@Duke and select DHVI: Protein Production Facility (D-0078).
CONTACT INFO cynthia.nagle@duke.edu 919.660.0352 james.peacock@duke.edu 919.613.6149
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2020
An enterprise wide, centrally supported, core booking to billing and biospecimen management system to support the research efforts of Duke’s shared resources and bio-bank repositories. There is a System Guide within the application that provides links to how-to and cheat sheet documents within Support@Duke.
Access The CoreResearch@Duke web site is coreresearch.duke.edu • You must have a valid Duke NetID account and password • Chrome, Internet Explorer, and Safari are the ONLY web browsers that are supported by CoreResearch@Duke at this time • Mozilla FireFox and Microsoft Edge are NOT supported web browsers • While other browsers may allow you to login to the application, certain screens will not display properly
Learn more here 21
"The DHVI Shared Resources and Cores bring together world-class people and innovative technology to accelerate our development of vaccines and therapeutics in a collaborative environment. These Units support research excellence in an ever-changing landscape of naturally occuring and deliberate public health threats." Barton F. Haynes, MD DHVI Director
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Cores and Shared Resources
The Duke RBL is located in the Global Health Research Building (GHRB) DHVI Core Facilities are located on the Duke Medical Center Campus (MSRB2, GSRB2, LSRC and GHRB) DHVI Core Administrative Offices are located in Global Health Research Building (GHRB) 909 S. LaSalle Street DUMC Box 103020, Durham, NC 27710
shared-resources.dhvi.duke.edu