Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection

The influenza virus and its variants remains a serious public health threat in both developing and developed counties, accounting for ~35,000 deaths and hundreds of thousands hospitalized in the US alone in an average year. In this renewal application from the Cooperative Center for Human Immunology at Stanford University, we wish to leverage our considerable experience in analyzing the influenza vaccine response to address some fundamental questions and hypotheses regarding how the human immune system  responds  to  influenza  vaccination  or  infection.  We  also  wish  to  continue  advancing  immune monitoring technologies relevant to analyzing the human immune response, such Cytometry by Time-Of- Flight, or CyTOF, combinatorial peptide-MHC tetramers and new TCR and BCR repertoire and gene expression analysis methods that open up entirely new areas of investigation and ways to test hypotheses. The general theme of our proposal is to address specific questions regarding how the human immune system  develops  and  changes  in  young  children,  adolescents  and  young  adults.  While  there  are considerable challenges in monitoring the immune responses of infants and young children, new technologies, many of them developed here at Stanford, now make some very fundamental questions feasible to address. These include testing hypotheses about memory phenotype T cells, and of flu-specific effector and memory CD4+ T cells (P1), the influence of innate immune factors and CD4+ T cells on influenza virus infection in a novelex vivo system (P2), the development of the immunoglobulin, T cell receptor and NK repertoires in response to vaccination in different age groups from our twin cohort (P3), and advancing the capabilities of the CyTOF instrument (P4). These efforts will be very ably supported by our Administrative (A), Pilot (B), Clinical (C), Human Immune Monitoring (D), Genomics (E) and Bioinformatics (F) Cores. Most of these cores have been in place for four or more years and have provided vital and state-of the-art support for the research activities of the group. We are entering into an exciting period of time for human immunology and we think that the Stanford team will continue to be extremely productive.

In this project we propose to follow up on a number of observations that we have made concerning CD4+ T cells and how they may contribute to immune competence, especially in young children. Recently we found that healthy adults have an abundance of memory phenotype CD4+ T cells (which we refer to as TMPs) that are specific for viral antigens to which they have never been exposed. This is in contrast to newborns, where we find a similar frequency of specific T cells, but almost entirely naïve in phenotype. These TMPs in adults have many of the expected characteristics of authentic memory T cells, and we hypothesize that they are the consequence of microbial exposure to cross-reactive T cell receptors (TCR), confer some degree of immunological protection, and contribute to immunodominance and a fully capable immune system. In Aim 1, we will characterize the time course with which specific TMPs appear in infants and children and also analyze whether their rise is continuous, or spikes with major vaccinations, acquisition of a microbiome or disease exposure. For the purposes of comparison to children recruited here, we will also have age and gender matched samples from our collaborators in Bangladesh. In Aim 2, we will perform a critical test of whether and to what degree cells of this type participate in a vaccine response, by first analyzing the TCR repertoires of naïve and TMPs specific for the same Hepatitis B antigens and then seeing which TCRs in the same subject are expanded in the effector and then memory T pools after challenge with the vaccine. We will also use this same procedure to analyze the response to unique flu strain epitopes. Another effort will be to find original cross-reactive epitopes for selected TMPs using unique peptide-MHC display libraries, which we have constructed. Lastly, in Aim 3, we wish to follow up on our studies of flu specific effector and memory CD4+  T cells, where we see evidence of an influence on both antibody responses and specific CD8+  T cell levels. The most prominent of these T cells responding to flu vaccination have some of the characteristics of Follicular helper T cells and may be driving the responses of the other cells.   Using the novel ex vivo infection system describe

Xiaosong He, PhD

The goal of this study is to address the mechanistic basis for variable rates of protection induced by influenza (flu) vaccination, especially the heterovariant immunity with intranasally administered live attenuated flu vaccines (LAIV) in children and dec reased immunity with inactivated flu vaccines (IIV) in the elderly. In Specific Aim 1 we hypothesize that Infection of the upper respiratory tract (URT), the initial site of replication of natural (“wild type”, wt) flu and LAIV, triggers hierarchical transcriptional signatures in resident epithelial and i mmune cells that control viral replication and regulate subsequent immunity. This hypothesis will be tested with two approaches. In an ex vivo approach we will infect primary human nasal cells with wt or LAIV viruses and examine: (a) early transcriptional responses and viral replication levels in different epithelial and lymphocytic cell types; (b) the role of specific nasal resident cells in shaping early mucosal immune responses and restricting viral replication; and (c) the relationship between pre-existing adaptive immunity and local immune responses and viral replication. Transcriptional responses will be measured from bulk tissue, purified epithelial and lymphocyte cell subsets, and single infected and non-infected cells using qRT-PCR. These ex vivo results will be e xtended with an “in vivo” approach where we collect flocked nasal swabs from people undergoing wt flu infection or receiving LAIV, measure local transcriptional response and pr e-existing flu specific B/T cells and A b in the blood (in vaccinees) and examine their relationship with the level of viral replication. We will also test the hypothesis that such local antiviral transcriptional responses and v iral replication levels predict the subsequent flu specific immune responses. In Specific Aim 2 we hypothesize that wt flu and LAIV induce broader B cell response than IIV in terms of the diversity of clonally expanded Ig gene families and heterovariant Ab reactivity of flu-specific Ig repertoires, especially in young children undergoing the first or second exposure to flu antigens, in comparison to those in the flu-experienced individuals. We predict these early differences affect the evolution of B cell response to new flu exposure later in life. To test this hypothesis we will examine the plasmablast Ig repertoire in infected (wt or LAIV) vs. IIV exposed young children 1 to 4 years of age who are flu-naïve (or almost naïve) and c ompare them to their subsequent vaccination and y oung and el derly adult vaccinees, using a novel barcoding based amplification and sequencing technology as well as production and characterization of recombinant mAbs, with a focus on t heir homotypic vs. heterovariant reactivity and original antigenic sin that has profound effects on the vaccine efficacy in different age groups.

Influenza viruses are major respiratory tract pathogens that present significant risks to individuals of all ages, particularly during pandemics. These risks would be greatly amplified should the lethal avian influenza strains,  such  as  H5N1  and  H7N9,  become  capable  of  human-to-human  transmission,  or  if  a  new recombinant strain were engineered as a bioterror agent. Although vaccination can prevent influenza- associated morbidity and mortality, it does not do so in some people, even in healthy adults. This proposal seeks to leverage technological advances in the Robinson and Blish laboratories to build on findings from prior CCHI studies at Stanford that highlighted environmental exposures and the diversity and maturity of the lymphocyte repertoire as critical factors influencing vaccine responses. We hypothesize that prior environmental  exposures  influence  the  maturity  and  diversity  of  the  immune  repertoire  and responses to different vaccines and play a greater role than genetics in generating effective vaccine- induced immunity against influenza. To investigate this hypothesis, we will use technologies recently developed in the Robinson and Blish laboratories to comprehensively define the phenotypic and functional repertoires of B cells, T cells and NK cells responding to influenza vaccination. We will couple our unique ability to measure the diversity, clonality, and functions of T, B, and NK cell populations at the single-cell level with the resources, expertise, and emerging technologies provided by the other U19 Projects and Cores (e.g., blood samples and clinical data from an extensive twin cohort assembled by the Clinical Core; CyTOF provided by the HIMC and developed by Project 4). In Aim 1, we will evaluate B cell, T cell and NK cell responses to influenza vaccination in monozygotic and dizygotic twins to determine the role of genetics and environment in the response to vaccination, comparing concordance in monozygotic twin pairs to that in dizygotic twin pairs. In Aim 2, we will determine how the vaccination method influences lymphocyte diversity and maturity, by comparing the B, T, and NK cell repertoire and responsiveness between monozygotic twin pairs receiving intranasally administered live attenuated vaccine (LAIV) and those receiving parenterally administered inactivated flu vaccine (TIV).In Aim 3, we will perform an integrated analysis of the datasets from this and other U19 Projects and Cores to determine the optimal levels of immune diversity and maturity that predict effective vaccination. Thus, by dissecting an extensive twin cohort with state-of-the-art tools, we propose to deliver a singularly detailed, integrated picture of the mechanisms governing human immune responses to influenza vaccination.

Single cell mass cytometry facilitates high-dimensional, quantitative analysis of the effects of bioactive molecules  on  cell  populations  at  single-cell  resolution.  Datasets  are  generated  with  antibody  panels (upwards of 40) in which each antibody is conjugated to a polymer chelated with a stable metal isotope, usually in the Lanthanide series of the Periodic Table. The antibodies recognize surface markers that delineate cell types and intracellular signaling molecules demarcating multiple cell functions such as apoptosis, DNA damage and cell cycle. By measuring all these parameters simultaneously, the signaling state of an individual cell can be measured at the network “level”. Given the capabilities of mass cytometry, and recognizing a growing international biomedical and pharmaceutical interest in its application to immunology, diagnostics, and drug development, this Project will extend the current features of mass cytometry to nearly double the number of assayable channels through the creation of novel chelator-isotope pairings as well as new nanodots for highly sensitive detection of surface molecules.  Further, we will enable additional “virtual channels” that increase the number of parameters measured per cell to as many as 200 using advanced signal processing tools such as compressed sensing along with signature based labeling. Finally, we will adapt DNA based amplification techniques to allow for low expressed protein epitope events and RNA copy number measurements down to as few as 5 target antigens measured quantitatively per cell. As per prior years with our other mass Cytometry protocols and computational abilities, developing and perfecting these additional capabilities will greatly enable the other Projects within our U19 center and will serve as a basis for extending these capabilities to others in the biomedical community, including other U19 Centers.

The objective of our Cooperative Center for Translational Research on Human Immunology and Biodefense is to use the analysis of vaccine-induced and naturally acquired immunity to influenza A as a model for defining adaptive and innate immune mechanisms and antiviral protection in children and younger adults. The Clinical Core will be responsible for coordinating protocol design and implementation to maximize opportunities for parallel evaluations across Projects 1, 2, 3, obtaining human subjects approvals, and creating and managing the centralized database to record clinical data. The Clinical Core will coordinate distribution of relevant specimens to the participating laboratories and provide matched clinical data in coded form for analysis.  Centralizing these functions is particularly important for protocols that involve children, to allow for the most efficient use of small volume blood samples. As the work proceeds, the Clinical Core database will facilitate comparative analyses of results obtained from the individual Research Projects. The Specific Aims of the Clinical Core are Aim 1: Protocol and Regulatory Support. To make the necessary IRB submissions for clinical studies, recruit and enroll adults and children to receive influenza or HBV vaccines or children and adults who have acute influenza A infection into clinical protocols, assure that subject rights are respected throughout the duration of their trial participation and provide follow-up to assure collection of complete sets of data from all subjects. Randomization codes and subject reimbursement payments will be provided by the Core. Aim 2: Clinical Data Management and Quality Management. Provide centralized clinical data management for Research Projects and other Cores using an electronic database with electronic data entry; implement a quality management plan to assure data integrity. These data will then be made available to the Research Projects, Bioinformatics and Genetics Cores for final data analysis. Aim 3: Sample Collection and Distribution.  Blood and nasopharyngeal specimens will be collected by the Clinical Core phlebotomists or research nurses according to the specifications of each clinical study protocol, and delivered either to the CTRU laboratory or directly to Research Project staff.

By combining immune monitoring on multiple platforms in a single center, the Human Immune Monitoring Center (HIMC) has created an ideal environment for all aspects of human immunology studies, from sample processing and banking, to immune assessment assays, and data analysis and integration.  Of particular relevance to this U19, we have an efficient system for high capacity sample banking and retrieval, working closely with the Clinical and Translational Research Unit (CTRU) and clinical coordinators to ensure accurate labeling, sample tracking, and optimal storage and retrieval for assays. We have also standardized both basic and advanced immune monitoring platforms, from simple ELISA assays for determination of CMV and EBV antibody titers, to complex CyTOF mass cytometry assays.  In our first Specific Aim, we will process and bank PBMC, serum, and RNA for clinical specimens, in collaboration with the CTRU and Clinical Core. In our second Specific Aim, we will offer CMV and EBV antibody testing and hemagglutinin inhibition assays, provide DNA and RNA extraction services, and run CyTOF assays for CCHI projects. We have highly trained personnel in all of these areas to support the generation of optimized and standardized data.  Finally, in our third Specific Aim, we will offer data integration services through our online relational database, Stanford Data Miner (SDM). With newly developed capabilities, SDM will allow integration of data across the above assays and with relevant clinical variables.  It will also be able to mine these integrated data sets using on-board machine learning tools such as decision trees and association rules mining.

The overall objective of the proposed center CCHI is to investigate the adaptive immune response in influenza vaccination and immunity. The genomics core will serve two research projects (Projects 2 and 3), and the purpose of the core is to provide leading edge genomic techniques, tools, and equipment to aid in the completion of the research objectives. We will 1) provide research projects with convenient and rapid access to highly multiplexed qRT-PCR as well as next-generation sequencing (NGS) including immune repertoire sequencing; 2) provide a novel platform and methodology to enable studies of immune cell responses to infection and vaccination at single-cell resolution; 3) optimize the single-cell approaches for each research project; 4) bridge the gap between research projects and the bioinformatics core by turning raw samples into pre-processed data ready for in-depth statistical and analytical analysis; and 5) serve as a source of information to research projects with respect to genomic technology and tools. The specific aims of the genomics core are:

Specific Aim 1 – Provide access to genomics technologies. The Genomics Core will carry out NGS sequencing on the samples generated by the research projects, as well as provide access to the single-cell analysis platform. The core also has a variety of peripheral equipment for sequencing library preparation and quality control, which will also be available to the projects. The core can also process raw sequence data ready to be interfaced with the bioinformatics core’s analytical tools, if needed.

Specific Aim 2 – Optimize single-cell approaches for whole transcriptome analysis of immune cells. The methodology and pipeline for single-cell whole transcriptome profiling allows elucidation of the transcriptional profile for all genes from each single cell in an automated fashion without complex hands-on workflows. This platform can be coupled with either NGS for whole transcriptome analysis, or with highly multiplexed qRT-PCR for targeted gene expression analysis. The core will optimize this methodology to work with immune cells, which are smaller in size and lower in RNA content than the epithelial cells that were used to initially validate the technology.

The overall goal of the Bioinformatics Core is to use advanced bioinformatics tools for identification and improved understanding of the innate and adaptive immune response in vaccination and diseases. The Bioinformatics Core will utilize existing informatics platforms, and adapt them as needed, to achieve these goals. The goals of the Bioinformatics Core will (1) provide robust bioinformatics methods to analyze the data generated by Projects 1, 2 and 3, (2) serve data to the Projects for hypothesis testing, and (3) publish the data for public availability in repositories including the NIAID-funded ImmPort, and the NCBI Gene Expression Omnibus (GEO).

The Bioinformatics Core will directly work with all Projects to address their need for robust bioinformatics techniques. The Bioinformatics Core will create a central repository of the genomic and immune profiling data,  generated  by  all  Projects,  and  integrate  with  genomic  and  immune  profiling  data  from  public repositories,  to  enable  multi-cohort  integrated analysis.  Furthermore,  the  Bioinformatics  Core will  work closely with the Genomics Core and the Human Immune Monitoring Center (HIMC) for this purpose. This will enable participating Projects to maximally utilize the genomic, immune monitoring and clinical phenotypic data  sets  to  determine  functional  dependencies  among  the  measured  elements  and  to  direct  further biological validation of these putative dependencies.