We will carry out deep sequencing of rearranged immunoglobulin (Ig) and T cell receptor (TCR) genes from lymphocytes in human subjects responding to several distinct viral (H1N1 influenza, varicella zoster, and measles/mumps/rubella) and bacterial (meningococcal) vaccines, as well as natural infections with H1N1influenza and varicella zoster. Our initial analysis will examine rearranged Ig and TCR repertoire in total peripheral blood lymphocyte populations. Subsequent analysis will define repertoire in subsets of B-cells and T-cells selected on the basis of their immunophenotype or antigen binding activity. These data will provide a fine-detailed view of the number, size, and receptor sequence features of expanded B and T cell clones arising during these human immune responses, and will be correlated with a variety of parallel serological and cellular functional immune assays from the Stanford Human Immune Monitoring Research (SHIMR) Center to enable detection of the characteristics of effective vaccination and the immune response to natural infection. The effects of patient age and genetic background on immune responses will also be assessed in a subset of experiments.
Our intertwined goals for the five-year period of this grant are:
- Characterize immune receptor populations in adults before and following immunization with influenza H1N1 vaccine and following natural infection by H1N1.
- Characterize immune receptor populations in adults before and following immunization with a conjugated bacterial polysaccharide vaccine (Menactra).
- Characterize immune receptor populations following vaccination against Varicella Zoster (in older adults) and measles virus (in children).
- Develop new quantitative analytical tools for high throughput immunome data and use these tools to characterize the clinical relevance of commonalities and differences in immune spectra amongst immunized individuals.
- Interface immune repertoire data with additional immunophenotyping data including HLA type, antibody binding affinity and specificity, target killing, cytokine production, signaling response spectra, and cellular surface marker distribution.
ADMINISTRATIVE CORE for Vaccination and infection: indicators or immunological health and responsiveness.
The Administrative Core of the Stanford Human Immune Monitoring Research (SHIMR) Center will oversee the conduct of the research projects, pilot projects, and scientific cores proposed here. The Administrative Core will be led by Dr. Mark Davis as Project Leader, with Drs. Ann Arvin and Harry Greenberg as co-Project Leaders; they will anchor the Executive Committee, with the other Project Leaders Drs. Goronzy, Maldonado, Gans, Utz, Koller, and Fire as members. In addition, two other senior investigators from Stanford’s School of Medicine will be recruited to serve on the Executive Committee on a rotating basis for two year terms. The Specific Aims of the Administrative Core are to:
- Implement administrative & leadership mechanisms that will facilitate communication and cooperation among the Stanford project leaders and with the consortium and investigators at other institutions to ensure a productive research effort;
- Monitor the progress of each of the Research and Pilot projects and their interactions with the scientific cores;
- Provide an efficient, centralized unit for the fiscal and administrative operation of SHIMR Center activities;
- Provide infrastructure support for Stanford SHIMR Center investigators to develop collaborative studies with other members of the consortium and research groups.
The purpose of the SHIMR Pilot Project Core is to support investigators with novel ideas or technologies relevant to the priority research topics in human immunology in the RFA-AI-09-040. These small grnats will provide funds to obtain pilot data as a foundation for subsequent application for extramural funding. The specific aims of this Core are:
1A. to solicit pilot project proposals onan annual basis within the Stanford research community,
1B. to review these proposals and to forward requests for funding for 1-3 projects per year to the Steering Committee,
1C. to monitor the progress of the Pilot Projects on a quarterly basis as well as the overall success of the program by tracking publications and extramural funding obtained on the basis of these awards.
Human Immune Monitoring Center (HIMC)
The role of the human immune monitoring core (HIMC, Core D) will be to (1) provide standardized, state-of-the art immune monitoring assays at the RNA, protein, and cellular level, to support research projects within the U19; (2) to test and develop new technologies for immune monitoring that may be useful in the context of the U19; and (3) to efficiently archive, report, and mine data from immune monitoring studies, so as to increase the value of the data and to assist in biomarker discovery.
- Specific Aim 1: Standardized, state-of-the-art immune monitoring assays. The HIMC has validated a set of immune monitoring assays that will be available to U19 research projects. These include genome-wide RNA microarrays, multiplex Luminex cytokine assays, immunophenotyping, phosphoepitope flow cytometry, CFSE proliferation assays, and intracellular cytokine staining. An ELISPOT reader is also available for readout of ELISPOT assays.
- Specific Aim 2: New technology for immune monitoring. The HIMC is evaluating multiple new platforms with potential for immune monitoring, including: isoelectric focusing analysis of phosphoproteins (CellBiosciences); chemiluminescent cytokine detection (MesoScale Discovery); biomolecular interaction analysis (ForteBio); multiplexed tetramer analysis; flow cytometry with time-of-flight mass spectrometry (CyTof); qPCR arrays on sorted cell populations (Fluidigm Biomark); and specialized microarrays for immunologically relevant genes and for pathogen detection (Agilent).
- Specific Aim 3: Databasing. The HIMC is implementing the use of collaborative online
The specific aims of this Genomics Core are:
The primary responsibility of the Genomics Core will be to provide high-throughput sequencing support to the program using the Roche/454 Genome Sequencer FLX Titanium platform to determine the sequence variability in Human Leukocyte Antigen related genes (HLA/MIC) and to interrogate the repertoire of rearranged immunoglobulin (Ig) and T cell receptor (TcR) loci in samples isolated from the vaccine studies. More specifically the core will design and provide solutions for sample preparation for sequencing, run the 454/sequencer, offer a comprehensive Laboratory Information Management System (LIMS) that will ensure sample tracking and data dissemination, and perform the primary analysis of the data.
- Amplify and Sequence HLA Class I and II exons from patients. Sample preparation and novel exon amplification protocols that have been developed at the Stanford Genome Technology Center will be used to amplify selected HLA/MIC target sequences to determine sequence polymorphisms. Single-plex amplified exons from each patient will be pooled together and re-amplified with barcoded primer sequences designed to be compatible with the 454/Sequencer. Up to 200 barcoded samples from individual participants in the vaccine studies will be pooled and sequenced in a single instrument run.
- Analyze Exon sequences to determine the haplotype of exons. After the completion of each sequence run, the Genomics Core will compare each sequence to available reference sequences of HLA genes in the public database using our in-house tools that run on high-performance computational platforms, build the consensus sequence, and determine the haplotype for each HLA allele using the Assign SBT program. Both sequencing data and analysis results will be deposited into a central database and rendered through user-friendly web pages that will be available to the consortium. This web site can be made public when the steering committee decides to disseminate this information to the research community.
- Analyze the sequences of VDJ recombination. An additional responsibility of the Genomics Core will be to provide high-throughput sequencing support for rearranged immunoglobulin (Ig) and T cell receptor (TcR) loci, analyze those sequences for VDJ usage, and search for biologically significant patterns of VDJ sequences. A similar web site and database like those developed for HLA genotyping will be developed for reviewing and sharing both sequence and sequence analysis results.databases for flow cytometry, Luminex, and microarray data, and is also evaluating data aggregation and mining programs such as Tibco Spotfire.
Core E: Clinical Core
The Clinical Core will be responsible for:
- coordinating protocol design and implementation to maximize opportunities for parallel evaluations across Research Projects 1, 2 and 3 with H1N1v, seasonal influenza vaccines, and natural influenza infection and with Research Project 4 using Zostavax vaccinations;
- obtaining human subjects approvals; and
- creating and managing the centralized database to record demographic and clinical data. This includes working with SRI International who will recruit twins from their Twin Research Registry as volunteers for some of the influenza and zoster vaccine studies. The Clinical Core will interface with the CTRU laboratory (part of the Stanford CTSA) which will receive blood specimens and carry out initial sample processing after which specimens will be delivered to the Human Immune Monitoring Core laboratory (HIMC, Core C) for banking, analysis, and distribution to the Research Project labs for specialized analyses. As the work proceeds, the Clinical Core database will contribute clinical data to the comparative analyses of results obtained from the individual Research Projects.
- Specific Aim 1: Protocol and regulatory support. The Clinical Core will assist in the design of the clinical studies, make the necessary IRB submissions, recruit and enroll participants into clinical protocols, assure that subjects’ rights are respected throughout the duration of the trial, ensure compliance with all IRB and NIH regulations regarding human subjects research and provide follow-up to assure collection of complete sets of data from all subjects.
- Specific Aim 2: Blood sample collection and initial processing. The Clinical Core will coordinate with the CTRU laboratory to provide them with freshly collected blood samples for initial sample processing and distribution of whole blood, separated peripheral blood cells, and serum or plasma to the HIMC.
- Specific Aim 3: Clinical data management support. The Clinical Core will provide centralized clinical data management using the Medrio electronic data capture (EDC) software system and coordinate with the Biostatistics Core (Core F) and Research Project 7 to provide coded HIPAA-compliant clinical data for correlation with the immune monitoring data (HIMC), specialized Research Project immune assays, repertoire analysis from the Genomics Core and Research Project 6, and HLA typing data from the Genomics Core.
Core F: Biostatistics
Vaccination and infection: indicators of immunological health responsiveness.
The Biostatistics Core of this project will be led by Professor Richard Olshen, who will be joined in supportive efforts by Professors Bradley Efron and Lu Tian. There will be high level statistical consulting on all projects and all investigators of SHIMR by these investigators. All data made available to these individuals will be anonymized compliant with HIPPA rules. Open source computer programs written in the popular R language http://www.r-project.org/ will be made available to SHIMR investigators. Stanford’s Data Coordinating Center (DCC) is the umbrella organization that will supervise writing these open source computer programs. In most instances the programs will call existing routines, available for downloading from CRAN http://cran.r-project.org/, though occasionally we will create the ingredient routines ourselves.