Influenza and SARS-CoV-2 are respiratory viruses that represent a continual threat to the health of many Americans, and will likely account for 100,000 deaths and many times that number of hospitalizations in the US alone each year. The available vaccines are inadequate, with the recent flu vaccine estimated to be only 40% effective and while the RNA vaccines for SARS-CoV-2 have been remarkable at preventing severe illness or death, they do not prevent reinfection in many people. Many with pre-existing conditions or immune deficiencies (e.g., obesity, diabetes) are vulnerable, which leads to suppressed response to vaccine or increased risk of severe outcome upon infection. Thus, the need to understand and improve both vaccines and the response to them is urgent. The Stanford CCHI has been a leader in understanding influenza vaccination and infection, and very quickly developed complementary expertise during the pandemic, making great strides in both understanding SARS-CoV- 2 infection and vaccine responses, and also in developing new technologies that promise even greater advances. These range from Dr. Wang’s insights in the effects of antibody glycosylation and lung inflammation, to Dr. Barnes seminal work on the structures of antibodies to SARS-CoV-2 antigens. Work that Dr. Barnes will carry further by designing and testing novel flu and SARS-Cov-2 antigen constructs. Dr. Khatri has also developed bioinformatic methods that have revealed conserved gene signatures regarding the immune response to viruses, and which he will use to define the cells and mechanisms that underlie these signatures. The clinical core under Dr. Chinthrajah will recruit and vaccinate obese and diabetic patients at risk for severe COVID and Influenza illness, and the results will be analyzed by Dr. Wang and also by the Human Immune Monitoring core under Dr. Maecker. Dr. Davis will head the Technology Development project which will continue his development of immune organoids and the use of spleen organoids particularly to characterize both existing and novel vaccine responses, and also attempt to reconstruct immune responses to vaccination and infection using skin and lung organoids from the same donors. Organoid responses to vaccination or infection will be validated against data in human subjects using our collection of influenza and SARS-CoV-2 specific T and B cell probes, as well as through TCR and BCR sequence analysis. Organoid processing and banking will be handled by the clinical core and all projects will make use of this resource, which will be especially useful in testing particular immunogens for their ability to make the desired antibodies, or the use of gene editing to identify key loci mediating particular effects. Immune organoids and the networks we propose to create will give us the ability to test hypotheses and define mechanisms in an entirely human system. The overall theme of this submission is to continue our efforts to understand Influenza and SARS-CoV-2 vaccination and infection using a broad range of approaches, integrating molecular, structural and bioinformatic methods, in vivo with unique cohorts and in vitro with organoids.
Visit our website at http://iti.stanford.edu.
Our U19 Center consists of three Cores and four Projects
ADMINISTRATIVE CORE
DAVIS, MARK MORRIS, Core Lead
The administrative core of the Stanford CCHI has been highly successful in the past almost twenty 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 effectiveness of its administrative core. Dr. Mark Davis will remain in his role as overall Pl of the Stanford CCHI and as the core lead for this core. He will also chair the executive committee consisting of the other project and core leaders (Drs. Wang, Barnes, Khatri, Maecker and Chinthrajah) who 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. Jasprina Noordermeer and Ms. Michele King will be assisting Dr. Davis to monitor overall progress, provide support to the administrative leadership team, and coordinate center activities and collaborations. They will be joined by Dr. Molly Miranda, who will oversee data management and sharing and upload of data to lmmPort according to the procedures outlined in the Data Management and sharing Plan.
CLINICAL CORE
CHINTHRAJAH, R. SHARON, Core Lead
The objective of our Cooperative Center for Human Immunology (CCHI) is to use the analysis of vaccine induced immunity and natural infections to Influenza and Sars COV-2 as a model for defining adaptive and innate immune mechanisms and antiviral protection in 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 HIMC Core and provide matched de-identified clinical data for analysis. 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 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. 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. Biospecimens (blood) and clinical data 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.
HUMAN IMMUNE MONITORING CENTER (HIMC) CORE
MAECKER, HOLDEN T., Core Lead
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.
REGULATION of ANTIBODY-MEDIATED EFFECTOR FUNCTIONS
WANG, TAIA, Project Lead
lgG perform immunomodulatory signaling via lgG Fe-Fey receptor (FcyR) interactions that trigger different effector functions according to the balance of activating to inhibitory (A/I) signals. Non-neutralizing, FcyR- mediated effector functions can be critical for optimal protection in many infectious diseases, including both influenza viruses and SARS-CoV-2. Studies over the past decade have shown tremendous heterogeneity in lgG Fe domains across individuals, with Fe domain structure (both protein and glycan components) being one key determinant of effector functions that are engaged during an infection. Yet, it is clear from clinical studies showing variable efficacy of monoclonal antibody (mAb) therapeutics with homogenous Fe domains that Fe domain structure is not the sole determinant of antibody effector function in vivo. The functional capacity of effector cells is almost certainly a critical determinant of lgG activity in vivo, but this has not been defined. This proposal addresses key unanswered questions in immunity mediated by broad, FcyR-dependent antibodies against influenza viruses and SARS-CoV-2: 1) how much heterogeneity exists in the functional capacity of human effector cells and is function altered by influenza virus or SARS-CoV-2 vaccination or infection, 2) how do obesity and diabetes – states that confer high risk during influenza virus or SARS-CoV-2 infections – impact effector cell function 3) can the functional capacity of hypo- or hyperresponsive effector cells be “tuned” using lgG engineering strategies. We will address these questions in experiments that include key collaborations with Project 2 (Barnes), Project 3 (Khatri) and the Technology Project (Davis). Designed immunogen baits via Project 2 will be used to pull out broadly reactive, anti-influenza and SARS-CoV-2 lgG from polyclonal antisera to study the effector functions recruited by these antibodies. Effector cells will furthermore be subject to transcriptomics before and after treatment with lgG immune complexes to define correlates for responses through collaboration with Project 3. In collaboration with the Technology Project we will use spleen, tonsil, and lung organoids to test the hypothesis that effector cell functions can be “tuned” using engineered lgG immune complexes. Finally, to follow up on our observation from humans that effector cell function is heterogeneous across individuals, we will use different collaborative cross mouse strains to test the hypothesis that the ability of broad, non-neutralizing anti-influenza mAbs to protect is correlated with the functional capacity of their effector cells (collaboration between Dr. Taia Wang and Dr. David Schneider). Collectively, this work, alongside the other projects proposed, helps to establish a rigorous immunological foundation for factors underlying protection against influenza virus and SARS-CoV-2.
LEVERAGING HUMAN ORGANOID MODELS for the DEVELOPMENT of BROAD INTERVENTIONS to MITIGATE EMERGENT RESPIRATORY VIRAL PANDEMICS
BARNES, CHRISTOPHER O’NEIL, Project Lead
Human respiratory viruses not only contribute to substantial morbidity and mortality worldwide but also pose a massive pandemic threat. This broad range of viruses includes human coronavirus (hCoVs), influenza A, and human metapneumoviruses (HMPV). Vaccines are among the most powerful means for mitigating viral epidemics but require significant neutralizing antibody breadth to maximize the probability of effectiveness against unknown viral threats. While a number of components of a protective immune response could be targeted to form the basis of a broadly protective coronavirus vaccine, neutralizing antibodies are generally accepted to be a key component of protective immunity. Although development of effective first-generation SARS-CoV-2 vaccines that induced protective antibody responses against severe illness has proceeded with unprecedented speed and seasonal influenza A (IAV) vaccines continue to offer protection, their effectiveness against emergent seasonal variants (e.g., Omicron) and, importantly, against other potential zoonotic viruses is less likely. Indeed, risk of zoonotic spillover events of divergent CoVs, such as the recently documented cases of human transmission from a canine alpha-CoV and a porcine delta-CoV that led to flu-like symptoms in infected Haitian children, highlights the need for innovative approaches to identify countermeasures targeting highly-conserved sites on CoV spikes shared among the Orthocoronavirinae subfamily. Project 2 will prioritize the development of immunogens that elicit neutralizing antibodies that can be measured in vitro. We will synergize with other Project members to test as many immunization strategies as possible in human organoid models, where the multifaceted functions of antibodies, effector cells, and host factors can be used to inhibit viral replication in an exposed host. Specifically, this proposal will focus on three interrelated aims: 1) identify the structural correlates of broad and potent antibody neutralization against conserved viral epitopes in CoV-S and IAV-HA trimers, 2) design multivalent immunogens that stimulate cross-reactive immune responses in human organoid model systems, and 3) provide insights into the molecular mechanisms of feedback inhibition and immune imprinting that limit diversification of B cell responses. Overall, this proposal will employ immunology, bioinformatics, structural biology, protein engineering and immune system models to discover how to elicit antibodies capable of neutralizing a broad range of emerging pandemic threat RNA viruses. The results will inform our understanding of broadly-protective anti-CoV and anti-lAV immunity in recovered and vaccinated individuals and will inform ongoing and future vaccine efforts.
IDENTIFYING and UNDERSTANDING the MECHAMISMS for INCREASED RISK of SEVERE INFECTION in OBESITY and DIABETES
KHATRI, PURVESHKUMAR, Project Lead
In Project 3, we propose to develop a next-generation of hybrid experimental framework that overcomes the limitations of single-cohort studies by leveraging heterogeneity between datasets and accelerates in vitro hypothesis testing through human organoids (in collaboration with the Technical Project). In building this hybrid framework, we will develop novel computational methods to predict which genes in which cell types should be knocked out (or knocked in) and what downstream genes and pathways will change as a result. We will demonstrate the successful development of this hybrid framework by identifying the mechanisms underlying the transcriptome signatures we have identified for predicting vaccine response to influenza and predicting the risk of severe outcome in patients with viral infection. In collaboration with Project 1 and 2, we will identify how different antibodies relate to protective and detrimental host responses to viral infections and vaccinations. To achieve these goals, we will create the largest bulk and single-cell transcriptome database of viral infections and vaccinations to date, which we estimate will include >20,000 bulk transcriptome profiles from ~100 cohorts and >10,000,000 single-cell RNA-seq profiles from ~2,000 samples. We will perform systems immunology analysis using the advanced statistical and machine learning methods and computational frameworks developed in the Khatri lab applied to these large amounts of bulk and single-cell transcriptome data. These computational frameworks will leverage biological, clinical, and technical heterogeneity in these data to identify and refine immune signatures (genes, proteins, cell types). Because this process typically identifies hundreds or thousands of genes, we will apply several methods we have developed to reduce this list of genes, including greedy forward search. We will also use statistical deconvolution and disease trajectory inference to reduce the number of genes, while still be able to identify underlying pathways, cell types, and mechanisms. Finally, we will employ systematic ablation-based methods to infer directional interactions between genes in immune cell types in which they occur. Using these inferred directed associations, we will pose hypotheses that will be investigated using organoids in collaboration with Dr. Satpathy and the Technical Project. We will derive a pan-virus conserved host response gene signature and perform a similar analysis for vaccination, obesity, and diabetes datasets to derive respective gene signature. We will confirm the immune cell types that preferentially express these genes and understand whether a change in transcriptome or a change in cell proportion or both lead to observed signatures. We will identify overlapping immune signatures between infection, vaccination, obesity, and diabetes, which will further identify detrimental and protective host responses associated with increased or decreased risk of severe outcome. We will also identify host responses associated with vaccination (e.g., antibody titers, T cell responses).
ADVANCING IMMUNE ORGANOID TECHNOLOGY and ORGANOID COMBINATIONS
DAVIS, MARK MORRIS, Project Lead
In this technology development project, we want to continue the significant progress that we made in this last granting period and take advantage of the many opportunities that we see to make human immune organoids an indispensable system for understanding human immune responses to vaccination and infection. Fortunately, we have made several major advances in our organoid work recently, especially our focus on spleen organoids that we obtain through our collaboration with Donor Network West, which now regularly supplies us with both spleens, skin, blood and lung fragments from a given donor. We have found that spleens are much more diverse in cell types than tonsils and are easier to get responses from both RNA vaccines and novel ones(to most individuals) such as Yellow Fever Vaccine. We will use them in Aim 1. to optimize the protocols for flu and SARS- CoV-2 RNA vaccines, and specifically to determine how well organoid responses compare with available studies in live humans, using our available reagents for T and B cell responses. An even deeper exploration of the BCR and TCR repertoire will be achieved by sequence analysis, which the Boyd and Davis groups are very experienced at. In Aim 2 we will focus on getting complete T and B cell responses with the Yellow Fever vaccine, currently producing only IgM antibodies. In Aim 3, we will take advantage of promising results we have obtained showing that a highly multimerized peptide-MHC reagent (60-mer) can stain specific T cells in fixed sections, as can a reagent for specific B cells. These would be a very valuable reagents to interrogate specific lymphocytes in the most standard type of clinical specimen. In Aim 4, we are teaming up with Drs. Kuo and Blish to use lung and spleen organoids from the same donors to see any form of cooperativity when we infect the lungs with influenza or SARS-CoV-2. Since we can see infiltration and rejection with co-incubated skin and tonsils/spleen from unmatched donors, we are confident that there will cooperativity, as we expect antigen loaded dendritic cells will migrate from infected lungs and trigger a response in nearby spleen organoids. Lastly, in Aim 5 we suspect that there are distinct differences in the immune responses of inbred mice and humans, and so have recently started make mouse spleen organoids so that we can analyze their vaccine responses alongside human spleens. With the advanced single cell analysis tools available it should be relatively straightforward to identify differences. We can then follow up those differences with gene editing using CRISPR-Cas9, as we have done recently. We will also serve as a resource for all three of the main projects, using gene editing and other methods with organoids for Dr. Wang and Khatri’s projects, as well has interrogating spleen organoids from high and low BMI individuals in collaboration with those projects and for quickly testing novel immunogens with Dr. Barnes.
