Influenza remains a continual threat to health of many Americans, resulting in 9.2-35.6 million illnesses, 140,000-710,000 hospitalizations, and 12,000-56,000 deaths annually since 2010 (CDC), as well as the ever-present likelihood of a devastating pandemic that could kill millions. Recent work has also suggested that a universal flu vaccine may be possible, although there is considerable uncertainty about how to achieve this. This renewal application seeks to leverage some of the important methodologies and data that we have developed in this past granting period to understand at a much deeper level what constitutes broad and effective B and T cell responses against influenza so as to better inform next generation vaccine efforts. Specifically, we have developed a unique tonsil organoid system that we can expose to a flu vaccine and produce high affinity antibodies several days to a week later. This gives us an ability to manipulate and test vaccine constructs and adjuvants in a fully human system in order to find the best way to trigger broadly neutralizing antibodies. In addition, we have developed powerful new T and B cell repertoire analysis methods that will allow us to productively analyze large flu- specific TCR and Ig data sets to identify which specificities or other correlates contribute the most to protection or amelioration of diseases in challenge studies. We also plan to further analyze influenza vaccine responses in pregnant women, the elderly and twins, to test various hypotheses that we have developed in our previous work in the CCHI. Lastly we will take advantage of recent advances by the Nolan lab in imaging tissue sections with large numbers of different antibodies to analyze the cellular organization the tonsil organoids with time during a flu vaccine response and after different interventions.

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Our U19 Center consists of four Cores and three Projects:


The administrative core of the Stanford CCHI has been highly successful in the past fifteen years in its leadership and coordination of center activities, management of finances and resources, and facilitation of intra-center collaboration and scientific support. The continued success of the Stanford CCHI will rely upon the continued success of its administrative core. Dr. Mark Davis will remain in his role as overall PI of the CCHI and as the core lead for the administrative core. He will be assisted by Dr. Scott Boyd and Dr. Kari Nadeau, and together with the other project and core leaders will be responsible for the overall organization, management, decision-making, and periodic evaluations within the Stanford CCHI. In addition, they will oversee resource allocation, protection of intellectual property in conjunction with Stanford’s Office of Technology Licensing, and the involvement of other institutional resources. Dr. Robert DiFazio and Ms. Michele King will continue assisting Dr. Davis to monitor overall progress, provide support to the administrative leadership team, and coordinate center activities and collaboration. They will be joined by Dr. Weiqi Wang, who will oversee data sharing and upload of data to ImmPort.


The objective of our Cooperative Center for Human Immunology (CCHI) is to use the analysis of vaccine- induced immunity to influenza 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 the research projects and scientific cores, 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 de-identified clinical data 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. One of the goals of the Clinical Core is to make the necessary IRB submissions for clinical studies, recruit and enroll adults and children into the protocols, assure that participant 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 participant reimbursement payments will be provided by the core. We will provide centralized clinical data management for research projects and other cores using an electronic database with electronic data entry and implement a quality management plan to assure data integrity. These data will then be made available to the research projects and scientific cores for final data analysis. Blood, lymph node tissue, aspirates, and tonsils will be collected by the Clinical Core staff according to the specifications of each clinical study protocol and delivered either to the CTRU laboratory or directly to research project staff.


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 influenza infection and vaccination and compare with other 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, and (2) analyze data from public repositories including the NIAID-funded ImmPort and the NCBI Gene Expression Omnibus for the Projects for hypothesis testing. The Bioinformatics Core will directly work with all Projects to address their need for robust bioinformatics techniques. The Bioinformatics Core will integrate the immune profiling data generated by all Projects with those available from public repositories, to enable multi-cohort integrated analysis. The Bioinformatics Core will work closely with 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.


The Human Immune Monitoring Center (HIMC) has been a comprehensive resource for immunological assays at Stanford for over ten years. The HIMC Core will leverage this facility and its robust infrastructure to provide biobanking, assays, and data organization services to the CCHI U19. Coordinating with the Clinical Core, blood samples will be processed for serum, RNA, and DNA, and biobanked according to well-optimized, standard procedures. Together with the existing inventory of >5000 specimens from previous CCHI and HIPC studies, these samples will be distributed to CCHI projects as needed, using an existing online portal to search for and request specific samples, with approval from an oversight committee. State-of-the-art, standardized immune assays will also be applied to the CCHI samples, including CyTOF mass cytometry, multiplexed Luminex cytokine analysis, and whole blood RNAseq, to provide comprehensive immunological data. As required for specific CCHI projects, custom Luminex panels and single-cell TCRseq assays will also be performed. Finally, the HIMC Core will integrate data from all HIMC assays using the online database, Stanford Data Miner (SDM). Here the assay data will be mapped to clinical and demographic information, for easy access and downloading by the Informatics Core, or by the Administrative Core, for upload to ImmPort. Existing scripts allow formatting of data from SDM into ImmPort templates for samples, persons, and assay results. The HIMC Core will create a valuable database of clinical specimens and comprehensive immunological data that will not only serve the needs of the CCHI U19 projects, but many other projects for years to come.


Influenza is a serious public health issue; vulnerable populations, including young children and the elderly, are especially at risk of influenza-related morbidity and mortality. Due to antigenic drift and shift of the virus as well as poor vaccine efficacy in older people, current immunization efforts fall substantially short of providing protection to the population. Research toward developing a universal influenza vaccine have been hindered by a lack of methods to model the human adaptive immune response. In this context, we have recently developed a tonsil organoid system using discarded human tonsil cells from sleep apnea patients that recapitulates at least some of the key features of an adaptive immune response against influenza, including high affinity antibodies specific for Influenza antigens and the HA molecule. We believe that this fully human system will be an ideal platform to explore and manipulate the anti-flu response in humans. In Aim 1, we will identify the minimal cellular requirements to develop protective influenza-specific T and B cell responses using these organoids. In Aim 2 we will investigate the immunomodulatory effects of adjuvants, particularly whether they influence the specificity, diversity or affinity of the influenza response. In Aim 3 we will manipulate the expression of particular genes that are likely to be important in the antibody and T cell responses and which address specific hypotheses-such as does AID play a major role in this response with respect to the specific antibodies that are generated in this system? Other genes that might alter the affinity or glycosylation pattern of the antibodies will also be investigated, as well as at least one that characterizes a uniquely flu specific response (CD38) and is expressed in germinal centers. In Aim 4 we combine computational modeling with nanoparticle and virosome stimulation of these organoids, test hypotheses about the optimal density of HA head vs stem constructs in order skew the antibody response towards broadly neutralizing, high affinity antibodies. These data could significantly aid the formulation of new vaccine strategies for the much hoped for universal flu vaccine.


Human adaptive responses to influenza vaccination and infection are complex, and can be impaired in a variety of conditions, including pregnancy and advanced age, for reasons that are still unclear. We will carry out extensive analyses of lymphocyte phenotypic repertoires, including those of NK cells, and the BCR and TCR sequence repertoires expressed by influenza-specific B cells and T cells, in clinical cohorts designed to include key vulnerable populations, pregnant women and the elderly, known to have impaired responses to influenza vaccination or infection. To evaluate potential specific defects in stages of germinal center reactions, and the proliferation and selection of influenza-specific lymphocytes that eventually become detectable in the blood, we will carry out serial fine-needle aspirations (FNAs) from draining lymph nodes after vaccination, and in tonsils from individuals receiving intranasal vaccine. Paired lymph node and blood data should enable detection of defects in responses that have previously not been accessible to study in humans. We will use this approach to identify key changes in immune responses with adjuvant (MF59) in influenza vaccination for the elderly, and correlate these with resultant antibody quality. An additional goal of this Project will be to leverage prior extensive influenza-specific TCR and BCR sequencing efforts in the Davis, Boyd and Robinson labs, as well as public data, to assemble databases of confirmed influenza-specific receptor sequences to test specific hypotheses. With new computational approaches, we will identify and validate convergent or public receptors that share sequence features indicating that they bind similar epitopes. We will test whether the frequency, diversity, or particular epitope targets of such receptor sequences can predict vaccine responses, and, more importantly, protection against live influenza viral challenge. Two different influenza viral challenge cohorts will be used to validate our TCR and BCR predictors of vaccine protection. This project will provide better understanding of age-related and pregnancy-related alterations in influenza vaccine responses, as well as new knowledge about vaccine responses in secondary lymphoid tissues including lymph nodes and tonsils. In the longer term, working within this U19, Project 2 will contribute to global efforts to improve quantitative and predictive understanding of the human immune system. This knowledge should help to shape strategies for improving and testing the next generation of influenza vaccines to prevent future epidemics and pandemics. Many of these new approaches may also provide a template for developing more effective vaccines in general.

NOLAN, GARRY P, Project Lead

New technologies such as mass cytometry have greatly expanded our ability to deepen our understanding of the complexity of lymphocytes and related populations. The Stanford CCHI group has been particularly attuned to the potential in following the lead of the Nolan lab in exploiting this technology, with ground-breaking studies of T cells, NK cells, and cancer immunology. But a clear need has been for high dimensional methods to interrogate tissue sections in a wide variety of circumstances. This inspired several complementary efforts by Dr. Nolan and his group, specifically MIBI, which uses metal labeled antibodies in a high-resolution format, and CODEX (CODetection by inDEXing), a multi- parameter fluorescence-based imaging technology adaptable to most standard three-color fluorescence microscopes, and currently capable of sensitively and quantitatively measuring more than 60 markers in a single tissue. CODEX extends the deep phenotyping capabilities of multi-parameter flow cytometry while enabling the associated spatial context of a multitude of cell types, including rare cell types implicated in disease mechanisms. To achieve this high-parameter capability, antibodies against target epitopes are each tagged with unique DNA oligonucleotides and iterative cycles of imaging and removal of corresponding tags is performed to collect single cell proteomic measurements across all parameters. We will deploy CODEX for deep phenotyping of the 2D and 3D architecture of tonsil organoids. Recognizing a growing international biomedical and pharmaceutical interest in imaging applications to immunology, vaccine and drug development, this Technology Development Project will extend the current features of CODEX to deep phenotypic profiling of tonsil tissue architecture before and after exposure to influenza vaccine. Specifically, the Davis lab has developed a unique tonsil organoid system that can be exposed to a flu vaccine with subsequent production of high affinity antibodies several days to a week later. The versatility of this organoid system provides an unprecedented opportunity to modify and test influenza vaccine constructs and adjuvants in a fully human system and determine how best to trigger production of broadly neutralizing influenza antibodies, a goal toward generating a universal vaccine. We will extract feature data with this unprecedentedly deep data for the understanding of wholesale and minor tissue alterations that occur in response to influenza vaccine challenge—enabling a first ever map of “tissue-omics” at the single cell level for the influenza vaccine response. We will also take advantage of the various manipulations that will be employed in Project 1 on this organoid system in order to gauge their effects on the organization of these cells and use this to formulate hypotheses regarding the significance of particular cellular groupings that we see in tonsils, which we refer to as “neighborhoods”.