Enteric bacterial infections are a significant global health concern, particularly in developing nations, where they disproportionately affect children. These diseases include typhoid fever (caused by Salmonella enterica serovar Typhi -S. Typhi-), invasive non-typhoidal Salmonella (iNTS; e.g., S. Enteritidis, S. Typhimurium), and shigellosis (caused by multiple Shigella serotypes). Limited knowledge of systemic and local gut immunity, including immunological correlates of protection, for human-restricted enteric pathogens (e.g., Salmonella and Shigella) continues to hamper vaccine development. Importantly, very limited information is available regarding immune responses to these enteric pathogens in children. To address these major shortcomings, it is imperative that studies involving human-restricted infectious agents are performed in children and adults. Thus, we are proposing under the central theme of “Mechanisms of Mucosal and Systemic Immunity to Vaccination and Infection with Enteric Pathogens in Children and Adults” to further our understanding of the protective immunological mechanisms that can be elicited systemically and in the gut microenvironment by using exclusively human based models with major human-restricted pathogens, i.e., Salmonella (S. Typhi, S. Typhimurium S. Enteritidis) and Shigella flexneri 2a. These studies will use unique specimens from clinical studies (Core 1) in which participants are immunized, or not, with parenteral or oral candidate vaccines against these major enteric bacterial pathogens, and, in some cases, followed by challenge with wild type organisms (Research projects -RP- 1-2). This will allow us to examine the mechanisms (e.g., epigenetic modifications) involved in the induction and maintenance of human adaptive immunity associated with protection. Furthermore, because of the critical importance of innate immunity elicited in the gut, we will use explant tissues and advanced in vitro models of the human gut mucosa (e.g., enteroids) to study the early crosstalk between epithelial cells and innate immunity and the epigenetic mechanisms at play in the gut microenvironment following exposure to Salmonella and Shigella (RP3). The mechanisms underlying immune responses will be studied using advanced technologies e.g., suspension mass cytometry, imaging mass cytometry (Core 3), EpiTOF, ATAC-seq, transcriptomics (RNA-seq), phosphoflow, and single-cell RNA-seq (RP1-3). Mechanistic studies using unique human specimens, gut organoids and explants will employ common methods of analysis and tools and the same wild type and attenuated bacterial strains. A world-class team of systems biologists (Core 2) will integrate immunological and omics data. The proposed strategy involves synergistic interactions among all RP’s and Cores supported by an interdisciplinary team of renowned scientists in immunology, clinical studies, genomics, epigenetics, bioinformatics, modeling, biostatistics and adjuvants. The long-term goal of this “Enteric CCHI” is to advance our knowledge of systemic and gut human immunity to accelerate the development of novel and better vaccines for enteric bacterial pathogens.
Enteric bacterial infections include different types of diarrheal illnesses caused by pathogenic Salmonella and Shigella which afflict major populations around the world, particularly in developing nations, disproportionally affecting children. The development of new and improved vaccines is hampered because we do not fully understand how the human immune system operates to protect children and adults from these infections. To address these problems, the central theme of our “Mechanisms of Mucosal and Systemic Immunity to Vaccination and Infection with Enteric Pathogens in Children and Adults” CCHI application is to further our understanding of the immune mechanisms that operate in both systemically and in the gut mucosal environment, by using unique human infection and vaccination models of Salmonella and Shigella.
Our U19 Center consists of four Cores and three Projects
ADMINISTRATIVE CORE
SZTEIN, MARCELO B., Core Lead
The Administrative Core for the Enteric Cooperative Centers on Human Immunology (Enteric CCHI) will be based at the Center for Vaccine Development and Global Health (CVD), University of Maryland, Baltimore (UMB). The key role of the Enteric CCHI Administrative Core is to comprehensively coordinate, manage, organize, facilitate, evaluate, and supervise investigators and institutions to ensure that highly productive research efforts are supported within a strong collaborative environment. The full purview of Administrative Core activities includes fiscal oversight, resource management and prioritization, communication, coordination, data management and sharing, protection of intellectual property, regulatory compliance, and involvement of University of Maryland, Baltimore institutional resources with the goal of efficiently supporting and facilitating research timelines and milestones for the successful implementation of CCHI objectives. The Enteric CCHI Administrative Core will be led by its Principal Investigator and Director, Marcelo B. Sztein, M.D., who will oversee coordination efforts among the Research Projects and Cores and will work to foster a strong collaborative environment. Additionally, the Enteric CCHI Oversight Committee will facilitate interactions and support the Principal Investigator in monitoring scientific research. A highly experienced administrative team at the CVD has successfully managed two previous award cycles of U19 CCHI (Dr. Sztein, PI). In addition, the administrative team has served as the lead for many other multi-site, multi-project grants and cooperative agreements. The breadth and depth of experience over such a diverse group of grants and contracts is indicative of the proficiencies of this highly competent Administrative Core, which is poised to manage the Enteric CCHI.
CLINICAL CORE
CHEN, WILBUR H., Core Lead
The specific objective of the Clinical Core is to provide human specimens for the studies outlined in Research Projects (RP) 1-3. These human specimens will supplement the existing extensive collection of unique banked specimens that are future-use consented and which will be available for use in RP1-3. These specimens include specimens from unique immunization and/or challenge studies conducted at Maryland, against S. Typhi, Non- Typhoidal Salmonella, and Shigella species. Of particular note, as specified in RFA-AI-22-069, support is requested to obtain additional human specimens only from clinical studies involving subjects who are receiving licensed vaccines. Blood and duodenal and/or terminal ileum biopsies, from subjects undergoing medically indicated endoscopy who consent to receive the Ty21a oral typhoid vaccine, will be collected under Protocol CVD 38000. Blood will also be collected from un-vaccinated controls (Blood Donor CVD 4000) and Ty21a vaccinated individuals (Blood Donor CVD 5000). The Clinical Core will provide the infrastructure to collect these unique human clinical specimens, which will supplement the already extensive existing specimen bank. The leadership of the clinical core are composed of two experienced physician-scientists who also provide patient care within the University of Maryland Medical Center (UMMC) or as part of the Faculty Practice Office of the University of Maryland School of Medicine (UMSOM). The clinical core is also staffed by experienced nurse s that have been trained in GCP and are well acquainted with the research protocols to be utilized for our studies. The resources being made available through the clinical core and the patient volumes (clinical activity) of the involved clinics should ensure that sufficient human specimens will be provided for the in-depth immunological studies proposed in this CCHI application.
SYSTEMS BIOLOGY and BIOSTATISTICS CORE
FORST, CHRISTIAN, Core Lead
The overall goal of the Systems Biology and Biostatistics Core is to provide service to advance the knowledge of systemic and gut human immunity in children and adults, by furnishing systems biology, bioinformatics, and biostatistics expertise to strengthen, support, and augment the research conducted in the Research Projects. The Systems Biology and Biostatistics Core aims to deliver expertise and service to perform integrative network modeling of multiscale, multi-OMICs data to identify key drivers of disease severity and host response to vaccination and infection. To reach this overall goal, the System Biology and Biostatistics Core will i) support the Research Projects within the proposed Enteric CCHI by integrating the rich multi-OMICs, immunological, imaging and clinical data generated within each project; ii) aid in validating the hypotheses generated in the Research Projects to further our understanding of the protective immunological mechanisms of mucosal and systemic immunity to vaccination and infection with enteric pathogens in children and adults; and iii) provide advanced Biostatistics expertise. Such an endeavor requires state-of-the art integrative network approaches to construct, analyze and validate multiscale networks of enteric bacterial infection and vaccination through integration of large-scale molecular, cellular, and pathophysiological data in an unbiased manner. Our approaches will incorporate these multi-dimensional data into mechanistic network models to predict outcomes of exposure to wild-type and vaccine-strain bacteria and/or epigenetic immune modulators or adjuvants. Specifically, we will develop and apply several novel differential analysis and multiscale network inference approaches to identify molecular signatures, coexpression modules and causal relationships that will be further employed to discover key regulators and pathways underlying vaccination and infection pertaining enteric pathogens. Such systems approaches are completely data-driven and present global and unbiased maps of regulatory relationships involving hundreds of thousands of interactions, with significantly improved power to uncover novel host- pathogen pathways and driver genes. For this purpose, the Systems Biology and Biostatistics Core will deliver the following: Aim 1, assembled large scale multi-omics datasets, identified molecular, clinical and immunological signatures and integrated bulk and single-cell data, in particular cell-type information and B-cell antibody repertoire; Aim 2, mechanistic molecular networks in response to vaccination and infection with enteric pathogens; Aim 3, validated multiscale multi-Omics networks; and Aim 4, biostatistics expertise to help in the development of optimal experimental designs. The Core’s leadership have demonstrated a strong track record of employing systems biology and biostatistics approaches, ensuring the success of the proposed aims. Therefore, we expect the service and expertise provided by the Systems Biology and Biostatistics Core will have a profound impact on the proposed Enteric CCHI to advance our knowledge of systemic and gut human immunity to accelerate the development of novel and better vaccines for enteric bacterial pathogens.
IMAGING MASS CYTOMETRY CORE
HO, WON JIN, Core Lead
The overall goal of the proposed CCHI U19 grant is to determine the mechanisms of mucosal immune responses to human-restricted enteric pathogens, S. typhi and Shigella, which to date remain significant global health concerns. To determine the limitations of current vaccine approaches and immune protection, we need to better understand the immunologic changes and the mechanisms thereof that are occurring systemically and locally within the gut mucosa. Leveraging our CCHI U19 team’s longstanding expertise in and unparalleled clinical infrastructure for evaluating the immune responses to these enteric pathogens, our proposed center seeks to understand the changes within major immune compartments, including T cells, B cells, and innate cells, upon clinical vaccinations and/or pathogen challenge. To accomplish this, we propose to phenotype the different immune cell subtypes and their functional states in-depth at the molecular level and interrogate how they are epigenetically regulated. Furthermore, to determine how the immune responses are orchestrated in situ within the mucosa, we propose to determine the spatial coordination among the immune cells in human mucosal biopsy samples and integrate this information with other molecular features. To enable synergy across all Research Projects, the Imaging Mass Cytometry Core will operate as a centralized service core, providing an imaging pipeline to be implemented on all biospecimens. The overarching purpose of the Imaging Mass Cytometry Core is, therefore, to generate high- parameter (30-40+ markers) spatial immune profiles with subcellular resolution for all three Research Projects. The Core will support the Research Project investigators with the expertise necessary to most optimally utilize imaging mass cytometry techniques. We established an efficient workflow to receive mucosal biopsy samples from University of Maryland and process them for imaging mass cytometry at Johns Hopkins. This Core will deliver the following: Aim 1, optimized and validated antibody panels suited for each Research Project along with the resulting multicolored quality-controlled images; and Aim 2, segmented single-cell data from the acquired images for compositional and neighbor/distance-based spatial analyses. The Core’s leadership and staff have demonstrated a strong track record of employing imaging mass cytometry to advance immunology studies, ensuring success of the proposed aims. The incorporation of our Core’s spatial analysis expertise will uniquely enhance the efforts of the proposed CCHI U19 to understand the immunologic barriers to effective protection and ultimately motivate new strategies for improved vaccine development.
MECHANISMS of INDUCTION and MAINTENANCE of SYSTEMIC and GUT MUCOSAL T CELL IMMUNITY to SALMONELLA an SHIGELLA FOLLOWING INFECTION and VACCINATION in CHILDREN and ADULTS
SZTEIN, MARCELO B., Project Lead
Enteric diseases caused by Salmonella (typhoidal and non-typhoidal -NTS-) and Shigella (multiple serotypes) remain a major health problem worldwide especially in developing nations. Invasive NTS (iNTS) has emerged in sub-Saharan Africa where it is causing devastating morbidity and mortality among young adults and children. While the development of effective vaccines with durable and broad-spectrum protection against these pathogens has become a priority, their development is limited by the lack of knowledge of systemic and local gut immunity including immunological correlates of protection (CoP), particularly in children. No vaccines are available for iNTS and Shigella. Despite considerable progress in our understanding of Salmonella and Shigella T cell mediated immunity (T-CMI), crucial gaps exist in our knowledge of the mechanisms associated with the induction and persistence of local and systemic antigen-specific T cells following vaccination and/or challenge in adults and children. Thus, we propose to address these gaps in knowledge and to uncover the mechanisms (e.g., epigenetics) of induction and persistence of Salmonella specific T-CMI responses to further our understanding of mechanisms of protective immunity using unique specimens from clinical studies (Core 1). Our overarching goal is to evaluate the mechanisms of induction and persistence of S. Typhi, iNTS and Shigella specific T-CMI responses in key T cell subsets, including effector/memory (TEM), stem cell like memory (TSCM), follicular helper (TFH) and regulatory cells (TREG) observed at baseline and soon and long-term after immunization and/or challenge with wt S. Typhi or Shigella and determine their association with protection. Importantly, we will study the mechanisms of induction and persistence in children (the primary population affected by S. Typhi and iNTS) vaccinated with the live oral attenuated typhoid vaccine Ty21a. We will also determine the mechanistic differences between adults challenged with wt S. Typhi or immunized with oral (Ty21a) or a parenteral Trivalent Salmonella conjugate Vaccine (TSCV). Additional studies will involve adults challenged with wt Shigella or following immunization with the candidate attenuated Shigella CVD1208S-122 strain. Finally, we will evaluate the mechanisms of induction and persistence of S. Typhi responsive T cells operating in the local mucosa by using terminal ileum and duodenal biopsies obtained from Ty21a immunized adult volunteers. Throughout these studies, we make use of advanced technologies (e.g., mass-cytometry, EpiTOF, ATAC-seq, RNA-seq, scRNA-seq and imaging mass cytometry (Core 3). All the data will be analyzed by using a sophisticated system biology approach (Core 2) in concert with data provided by other projects (RP2 and RP3). These studies will uncover critical information on the induction and durability of local and systemic antigen specific T-CMI revealing epigenetic and transcriptomic signatures that might lead to the identification of molecular correlates of protection that may inform future vaccine design for adults and children.
IMMUNE MECHANISMS REGULATING the DEVELOPMENT of SYSTEMIC and MUCOSAL ANTIGEN-SPECIFIC B CELLS AFTER VACCINATION and CHALLENGE in HUMANS
TOAPANTA, FRANKLIN R., Project Lead
Shigellosis and salmonellosis continue to be serious public health problems affecting primarily young children in developing countries; however, adults are also susceptible to these pathogens. The emergence of multi-drug and extensively drug-resistant Shigella and Salmonella strains has created a sense of urgency for the development of effective vaccines. However, the lack of information on the development of mucosal immunity has hindered novel vaccine design. To date, it is unclear to what extend the immunity measured in peripheral blood is representative of the gut mucosal immunity. Recent studies have shown that immunity in local tissues is critical for protection from disease. The recently described resident memory B cells are likely the first line of defense against re-infection at the tissue level. The overall goal of Research Project (RP2) is to characterize and compare the mechanisms that drive the development of systemic and mucosal B cell immunity to human- restricted enteric pathogens using state-of-the-art immunological and genomics methods. To this end, unique human specimens (Core 1) will be used including a) PBMC from volunteers immunized parenterally with conjugate vaccines against Salmonella (TSCV) and Shigella (SF2s-TT15); b) PBMC from volunteers immunized with oral vaccines against Salmonella (Ty21a) and Shigella (CVD 1208S-122); c) PBMC from S. flexneri 2a challenge studies; d) PBMC and terminal ileum biopsies from adult Ty21a vaccinees; and e) PBMC from children immunized with Ty21a. Our overall hypothesis is that antigen-specific mucosal B cell responses differ in magnitude and characteristics to those in peripheral blood and that oral immunization favors responses associated with protection and these responses are also age dependent. In Aim 1, we will characterize Shigella- and Salmonella-specific B cells in PBMC after parenteral and oral immunizations. Aim 2 will characterize B cell immunity (PBMC) after a wt Shigella challenge. Aim 3 will characterize Salmonella-specific B cell responses after oral vaccination at the systemic (PBMC) and local (gut mucosa) levels. Aim 4 will explore Salmonella- specific B cell responses (PBMC) in children (<18 yo). In all aims, the mechanisms involved will be studied by evaluating changes in phenotype, gene expression (transcriptome), epigenome (EpiTOF), and/or BCR- sequence profiles (LIBRA-seq) in bacteria-specific B cells after vaccination/challenge. Additionally, Aim 3 will incorporate imaging mass cytometry (Core 3) to study Salmonella-specific B cells and their interaction(s) with other immune cells at the tissue level. Bacteria-specific B cells will be identified using a unique set of reagents developed to study enteric pathogens (e.g., bacteria with fluorescent tags). The rich data sets will be analyzed, compared and integrated in collaboration with the Systems Biology and Biostatistics Core (Core 2). Data integration will include results from RP1-3, so we will provide unprecedented information on the development of immunity to human-restricted enteric pathogens to aid vaccine design and determine correlates of protection.
ROLE of EPIGENETICS and THEIR PHARMACOLOGICAL MODULATORS in INNATE INTESTINAL IMMUNITY and ADJUVANTICITY in HUMANS
MEZGHANNI, ROSANGELA, Project Lead
The intestinal immune system has evolved to tolerate most antigens while mounting an effective immune response to combat infections by pathogens. Epigenetics, defined as heritable changes in the genome without alteration of the DNA sequence, has been shown to play a critical role in regulating intestinal immunity. However, a better understanding of epigenetic modifications and how their pharmacological modulators affect these modifications is still lacking. In addition, the current approach to developing vaccines containing novel adjuvants is slow and needs improvement. Epigenetics offers an exciting opportunity towards the development of novel adjuvants by targeting small molecules as pharmacological modulators to reprogram the epigenetic landscape of innate immune memory, or trained immunity, which could confer enhanced resistance against a broad array of bacteria in humans. In Research Project 3, our overall goal is to investigate the role of epigenetic marks and their pharmacological modulators in intestinal immunity, specifically focusing on the function of epithelial cells, natural killer (NK), and innate T cells, such as Mucosal-associated invariant T (MAIT) cells, T-cell receptor gamma delta (TCR- ) cells, and natural killer T (NKT) cells, in response to two important enteric pathogens, Salmonella enterica serovar Typhi (S. Typhi) and Shigella flexneri 2a (S. flexneri). This study will build on our previous findings that MAIT subsets exhibiting specific cytokine pattern signatures were associated with protection against typhoid fever and that S. Typhi infection regulates changes in chromatin marks that depend on individual cell subsets. Additionally, compared to the placebo group, vaccination with the oral Ty21a typhoid vaccine resulted in increased levels of NK cells upon a second antigen encounter after experimental human challenge with wild-type S. Typhi, which is consistent with the presence of trained immunity driven by epigenetic control. We have assembled a unique set of human specimens and tools to test our hypotheses, including (i) cells obtained from peripheral blood of healthy individuals immunized with oral vaccines, (ii) cells isolated from the duodenum, terminal ileum, and colon, and (iii) their counterparts as in vitro culture models [explants and organoids] from adults and children.
