Vaccination and Infection U19

In this proposal, we aim to use a systems biology approach to survey immune responsiveness across a range of different vaccines including influenza, both seasonal and the new pandemic H1N1v, Herpes zoster, and the bacterium Neisseria meningitidis.  In the case of influenza and Herpes zoster, we will also be able to directly compare the immune response to vaccines with those natural responses to the pathogen itself. These investigations will compare different age groups—children, young adults, and the elderly—to look for specific markers and assays of immune competence and especially those that might be common across different pathogens and age groups.  We will also survey the influenza vaccine responses of individuals with impaired or dysregulated immune systems in order to see how these might deviate from healthy individuals. We will employ a streamlined model in which clinical specimens from individuals exposed to different vaccines or infectious diseases will all be assayed for an array of basic immune functions in our Human Immune Monitoring Center and also for several “state of the art” assays such as our recently developed high throughput HLA sequencing technique, which is able to generate complete HLA haplotypes from hundreds of people simultaneously. We will also perform selected Immunoglobulin and T Cell Receptor repertoire analysis and combinatorial peptide-MHC tetramer analysis to efficiently search for informative T cell epitopes. We will employ advanced bioinformatics analysis such as our new cell type specific SAM algorithms (csSAM), which use cell subset information to achieve much higher sensitivity from whole blood gene expression data as well as developing new bioinformatics tools to interrogate our rich and complex data base across all projects, pilots and cores.

The 2009 influenza pandemic reiterates the urgency in developing improved influenza vaccines. This pandemic is caused by the influenza A/H1N1 variant strain (H1N1v), to which much of the human population has little pre-existing immunity. H1N1v vaccines have recently been approved for distribution in the fall. This very uncommon circumstance with the advent of a new pandemic influenza strain provides the opportunity to address several critical questions regarding the B cell immunity against influenza, a key determinant of protection against influenza infection. Traditional evaluation for B cell responses against influenza infection or vaccination has relied heavily on convalescent serum antibody assays, which may not represent the entire antibody response, especially mucosal antibody responses that are frequently of great importance for protecting against respiratory pathogens. Antibody responses are first mediated by activated B cells, or plasmablasts, which migrate through circulation to different target sites and become effector B cells, or plasma cells. Recently we have developed a comprehensive flow cytometric assay to define the patterns of multiple trafficking receptor expression on blood plasmablasts, including those with trafficking signals for the respiratory tree. We have also developed sensitive methods to collect and analyze the polyclonal antibodies secreted by the plasmablast population or its specific subsets. Taking advantage of these new assays, and the rare opportunity provided by the H1N1v pandemic, we will address the following specific aims to:  1.Analyze the trafficking receptor profiles imprinted on plasmablasts after natural infection with those induced by immunization with two different influenza vaccines; 2.  define the quantitative and qualitative differences in plasmablast-derived polyclonal antibody (PPAb) responses to acute infection vs. mucosal or systemic vaccination in different age groups;  3.  qualitatively and quantitatively compare the homotypic and heterosubtypic PPAb reactivity induced by wild type H1N1v infection vs. the PPAb reactivity induced by the two types of H1N1v influenza vaccine, as well as by seasonal influenza vaccines. Together these aims will define new immune indicators and provide new insights to the mechanisms of B cell response to influenza infection and vaccination.

Here we propose to characterize the responses of CD4+, CD8+ and gd T cells to the pandemic influenza H1N1v vaccination or infection in human volunteers. We will use both systems biology approaches as well as analyzing a variety of antigen specific responses with combinatorial peptide-MHC tetramers combined with single cell cytokine analysis, in which we can screen for hundreds of different epitopes and dozens of cytokines. In Aim 1. we will ask whether particular epitope or cytokine expression patterns correlate with a robust vaccine response, as measured by a microneutralization assay, and/or a granzyme B T cell assay, or in  the case of infected individuals, disease severity. With the  twin studies we will ask how closely does dominant epitope choice follow genetic identity and does that change with increasing age and exposure to infectious diseases. This will be complementary to the results obtained with the complete HLA genotyping being done by the Genomics Core, as we expect that HLA haplotypes will have a major influence on epitope selection and dominance. In Aim 2. we will characterize activation markers and cytokine expression in gd T cells, which recent evidence suggests influence antibody responses and help shape the responses of CD4+ and CD8+ T cells and other immune system components through the early production of IL-17.  In Aim 3 we will continue our now three year old longitudinal study of seasonal flu vaccination in young adults (20-30 yrs.) versus older cohorts (60-96 yrs), taking a systems biology approach with the help of the Human Immune Monitoring Core and the Biostatistics Core in which we correlate gene expression, cytokine stimulation and serum cytokines with parameters such as immune senescence to uncover new markers and mechanisms behind the failure of proper immune function in many older people. We have already identified a number of new markers of immune deficiency, including a decrease in AID expression in B cells and deficient responses to cytokines in many older people using phosphoflow analysis. Support by this mechanism will allow us to look extensively at year-on-year variations and early markers of immune senescence. Lastly, this same systems approach will be taken to compare H1N1v vaccination responses to actual infections, which should help us to understand the similarities and differences between the two and aid in the design of vaccines that are more protective.

The goal of Project 3 is to comprehensively characterize immune systems of patients who are known to have abnormal immune systems, and to compare their immune responses with those of normal subjects. We will challenge the immune system by vaccinating patients with influenza vaccines, characterizing the response of an abnormal immune system to this perturbation. Project 3 will study patients with Common Variable Immunodeficiency Disease (CVID) and Systemic Lupus Erythematosus (SLE), prototypic diseases of immune dysregulation. There are 3 aims: (i.) to develop and validate a multistrain influenza and H1N1v antigen microarray for profiling antibodies in vaccinated human subjects. We will clone and express major antigens from influenza strains, which will then be printed onto derivatized glass microscope slides. Arrays will first be validated using commercially-available monoclonal and polyclonal antibodies, then further validated using serum derived from normal subjects vaccinated with H1N1v and seasonal flu vaccines; (ii.) to compare the baseline function of the immune system in normal human subjects with the baseline function of the immune system in immunosuppressed patients. We will create a comprehensive database of immune function measurements in CVID and in SLE, comparing the responses with normal subjects (Core C), and vaccinated subjects studied in Projects 1-7. We will take advantage of the Stanford Immunologic and Rheumatic Disease Registry and Biospecimen Repository, and the Adult and Pediatric Immunodeficiency Clinics for access to clinical samples. (iii.) to compare the global response of the immune system in normal subjects with the response in immunosuppressed patients (CVID, mild vs severe SLE, and therapeutic immunosuppression) when immunized with seasonal and H1N1v influenza vaccines. We will test the hypothesis that a subset of healthy individuals has immune deficit(s) similar to those observed in patients with autoimmunity, immunodeficiency disorders, or who are immunosuppressed with drugs such as glucocorticoids. We further hypothesize that this subset of patients will have an abnormal response to vaccine challenge with H1N1v or seasonal flu vaccines.  Results of Project 3 may improve future vaccination strategies for patients with immune deficiencies, and may identify subsets of “normal” patients who are unlikely to respond to existing vaccine protocols.

Improving vaccination strategies and understanding and treatment of infectious diseases require identification and quantification of variables that determine the ability of the immune system to respond to an antigenic challenge. A particular challenge is the aging immune system where the ability to mount adaptive immune responses to vaccinations and viral infections declines. Variables that determine the outcome of an immune response and are sensitive to aging include the repertoire of antigen-specific T and B cells; the robustness of homeostatic control mechanisms; the ability of cells to migrate to the site of antigen exposure; the activation threshold of responding cell population; the availability of and responsiveness to nonspecific stimuli; and the ability of responding T and B cell population to proliferate and differentiate. We will perform immune profiling before and after VZV vaccination to identify signatures that correlate with successful T and B cell vaccine responses. The overreaching hypothesis is that comparison of vaccinated individuals of different age groups and patients with zoster reactivation will allow defining underlying mechanisms that can be used to target interventions to compensate for defective pathways. In Specific Aim 1, we will perform a twin study to examine the influence of age and inherited factors on frequency, phenotype and repertoire of VZV-specific T cells in naturally acquired VZV immunity. Specific Aims 2 and 3 propose a longitudinal study of the VZV vaccine response in different age groups. Specific aim 2 will focus on the early immune response to define the factors that correlate with a rapid and diverse T cell response, Specific Aim 3 proposes to identify T cell signatures at peak response that correlate with T cell memory development and antibody production. In Specific Aim 4, we identify patients with zoster reactivation in two at-risk groups, patients with the autoimmune diseases rheumatoid arthritis and systemic lupus erythematosus and patients older than 60 years. Signatures obtained in these patients during active herpes zoster infection will be compared to the vaccination-induced signatures.

Led by Dr. Daphne Koller, together with Drs. Robert Tibshirani and Atul Butte, this series of bioinformatics projects will focus on the development of computational methods for analyzing the cellular network underlying the immune system and for understanding how this network is perturbed by the response to vaccine.  This project will build on our successful development of new deconvolution software (csSAM) to extend it to the development of applications to other datasets in addition to whole blood gene expression such as serum cytokines and phosphoflow. Building on existing successful tools this team has previously developed, cell-specific deconvolved data will then be used to construct the regulatory network of each of the different cell-types present in peripheral blood as well as the inter-cellular network between them. Integrating the data collected from all of the other projects in this proposal, this project will develop computational tools for predicting vaccine responses, and for identifying common vaccine mechanisms and genetic correlates with response.  The learned models will provide novel scientific insights regarding the immune system, including: the relationships between genotype, gene expression, phosphoprotein levels, and phenotype; the interactions between genes within an immune cell; and the cytokine-mediated interaction between different immune cells.  All these will be used for the specification of human immune metrics. 
The specific aims for Project 5 are to develop:

• Aim 1: Methods for analysis of data derived from mixed samples;
• Aim 2: Methods for using diverse data to construct network models for immune system cells;
• Aim 3: Methods for understanding the factors affecting immune response; and to:
• Aim 4: Use systems-level analysis to identify causal polymorphisms that affect immune response.

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:

1. Characterize immune receptor populations in adults before and following immunization with influenza H1N1 vaccine and following natural infection by H1N1.
2. Characterize immune receptor populations in adults before and following immunization with a conjugated bacterial polysaccharide vaccine (Menactra).
3. Characterize immune receptor populations following vaccination against Varicella Zoster (in older adults) and measles virus (in children).
4. 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.
5. 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.

Core A

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.

Core B

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.

Core C

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

Core D

Genomics Core

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.

1. 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.
2. 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.
3. 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:

1. 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;
2. obtaining human subjects approvals; and
3. 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.

• Specific Aim 1:  Develop a microfluidics platform for automated sample processing for phospho-flow cytometry. Working with the Stanford Microfluidics Foundry, we will develop a microfluidics platform with magnetic retention and thermoelectric temperature control for working with peripheral blood leukocytes.  The chip will allow at least two independent stimulations of patient sample. We will then expand the chip architecture to allow at least 12 independent stimulations of patient sample on-chip. Fluidics control, thermal control, and magnetic retention systems will be fully automated by USB interface to computer.
• Specific Aim 2:  Demonstrate system can work with different types of patient material with stringent temporal control.  Validate for use with ficoll-separated PBMCs. Demonstrate system can work with whole blood. Demonstrate system can carry out short duration TCR stimulations with high precision and consistency.
• Specific Aim 3:  Investigate with high temporal resolution the changes in TCR signaling kinetics in healthy young and old individuals. Perform pilot experiments using tetramers for antigen-specific T cells.

Myeloid and plasmacytoid dendritic cells (DC) play a critical role in shaping the response of the immune system to infectious agents and vaccines. We and others have recently discovered the existence in humans of functionally distinct subsets of myeloid DC. These distinct populations of cells secrete different cytokines and appear to be able to selectively induce the formation of Th1, Th2, Th17 and Treg cells from naïve T cells. We hypothesize that the frequency and/or activity of these subsets may have a profound influence on the quality and potency of the immune response to infectious agents and vaccination. Ultimately, it may be possible to design vaccines that selectively target and activate a particular DC subset in order to achieve a desired response. Unfortunately, no cell surface markers currently exist that can distinguish these DC subsets from one another. The objective of this project is to address this problem through the development of a method that enables the identification and isolation of viable functionally distinct DC. Once this method is developed we will use it to assess the potential effects of subset frequency on the immune response to vaccination, as well as the effects of vaccination and infection on the frequency and activity of these subsets. 

Individual differences in host protection after vaccination is always a critical issue in developing effective vaccines. With the pandemic breakout of the Influenza A H1N1/09 virus in early 2009 (referred to here as H1N1v), new attention is being paid to how little we know about what governs the effectiveness of influenza vaccines and why there is such differential effectiveness in vaccinated individuals, especially the elderly. To better understand the mechanisms underlying vaccination effectiveness, we propose a pilot study to identify the differentially expressed/spliced genes that are associated with vaccination and effectiveness in the B cells of older and younger individual from the Ellison study (Project 2) by deep-sequencing the transcriptomes of vaccinated individuals and then correlating that data with the other datasets generated by the HIMC and projects 1 and 2. The identified gene expression signature of those individualsmaking a stronger response will be invaluable toward a better understanding of effective host response to the H1N1 vaccine (and other related vaccines), as well as future development of more effective vaccination.

Specific Aims

Here we propose to launch a pilot study to analyze the host response in individuals before and after vaccination and correlate the results with vaccine effectiveness. We will use RNA-Sequencing (RNA-Seq) to search for differential gene expression/splicing profiles that are associated with a seasonal flu (TIV) vaccination and its effectiveness. Specifically we will:

• Specific Aim 1:Use RNA-Seq to identify differentially expressed/spliced genes expressed in B cells from older and younger individuals from the Ellison study, starting with ten from the youngest group (20-30 years) and ten from the oldest group (80-96 years).   
• Specific Aim 2: Validate the differentially expressed genes/splicing variants identified in Specific Aim 1 in the remaining Ellison cohort of 70 individuals in the three age groups (20-30 years old, 60-79 years old and 80-96 years old) using quantitative RT-PCR...

These studies are expected to both identify the gene expression signature of protection and response to a seasonal influenza infection and may also provide insight into mechanisms of protection.

Human B cell responses to vaccination for seasonal and novel “epidemic” influenza virus are most often measured clinically and in research settings by subjecting patient serum or plasma immunoglobulins to hemagglutination inhibition (HAI) and viral replication inhibition assays. While these gold-standard assays can accurately determine changes in antibody titer associated with vaccination, it is not possible to obtain epitopespecific information from either assay. Novel technologies that provide information about the development of novel epitopes on influenza proteins targeted by human patient antibodies post-vaccination would significantly increase both the amount of diagnostic information available to clinicians and researchers, as well as the scope of influenza vaccine research. Here we propose to develop a new multiplex array platform in collaboration with Intel® that has the ability to detect individual human antibody epitopes on immunogenic regions of influenza proteins at single amino acid resolution. Using photolithographic printing technology, we will generate overlapping peptides that span up to a 60 amino acid region of influenza hemagglutinin (A/Brisbane/10/2007(H3N2) and (A/California/08/2009(H1N1)) that have been shown in our preliminary studies to contain epitopes recognized by antibodies in serum from vaccinated subjects. We will use this array to profile existing pre- and post-vaccine samples from 90 healthy human subjects who have been vaccinated with the Fluzone® 2009 seasonal influenza vaccine and 30 patients given the 2009 H1N1 “swine flu” influenza vaccine between September and November of 2009. We hypothesize that we will define novel antibody reactivity in post-vaccine samples that correlates with HAI assay and virus replication inhibition titers, and that we will define individual epitopes targeted in post-vaccine samples that are a direct result of vaccination. Single amino acid-level resolution of these epitopes will constitute a new and powerful tool that will aid clinicians and researchers in better measuring and understanding the human immune response to influenza vaccination.