The overall focus of the Immunization against Multidrug-resistant Pathogens: Activating T Cell Immunity Center of Excellence for Translational Research (IMPACT-CETR) is to advance promising multicomponent vaccines for Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae. These are among the most important bacterial pathogens that cause severe clinical disease and death and yet are becoming increasingly resistant to the most effective antibiotics.
The overarching goal of this IMPACT-CETR is to harness the collaborative team’s complementary expertise in immunology, bacteriology, bioinformatics, primatology, vaccine development, and antigen-adjuvant formulations to achieve three deliverables: 1) novel multicomponent vaccines optimized for bacterial proteins and/or polysaccharides that elicit broad and potent serotype-independent protection against S. aureus, P. aeruginosa, and K. pneumoniae infections, including with resistant and MDR clinical isolates; 2) defined key mechanisms of host protection and biomarkers of vaccine efficacy; and 3) nonhuman primate (NHP) models to characterize immunogenicity and surrogate markers of protection. The proposed vaccine components are well-characterized, some are chemically defined, and all are designed for feasible scale-up and manufacture. The three research projects in this CETR are bonded by the theme that tissue-resident memory T cell responses, particularly tissue-resident Th17 cells, are critical for protective vaccines against these pathogens. The translational research projects will develop countermeasures to prevent/reduce disease caused by key resistant and MDR bacterial pathogens.
Projects
Project 1 addresses a multicomponent S. aureus vaccine formulated with conserved protein antigens using the new and innovative Multiple Antigen Presenting System (MAPS) platform wherein fusion proteins of the avidin derivative rhizavidin with conserved S. aureus protein antigens are complexed to a biotinylated polysaccharide to generate protective B- and T-cells. Project 2 addresses a multicomponent P. aeruginosa MAPS vaccine based on the serotype-independent P. aeruginosa biofilm polysaccharide Psl and its critical lipid epitope, combined with conserved protein antigens including the Th17-eliciting antigen PopB. Project 3 addresses a quadrivalent K. pneumoniae vaccine based on conserved proteins that elicit protective Th17 cells and antibodies. Vaccine candidates will be tested in wild-type and transgenic and/or knock-out mice and in non-human primates. These projects will be supported by an administrative core and three scientific cores that focus on bioinformatics, transgenic mouse models for mechanistic studies, and NHP studies. The expected milestone for each project is the development of a preclinical data package that would pave the way for subsequent IND applications and clinical trials.
Staphylococcus aureus vaccines (Research Project 1)
The goal of Research Project 1 in the “Immunization against Multidrug-resistant Pathogens: Activating T Cell Immunity” Center of Excellence for Translational Research (IMPACT-CETR) is to develop a multicomponent Staphylococcus aureus vaccine formulated with conserved antigens using the new and innovative Multiple Antigen Presenting System (MAPS) platform. Staphylococcus aureus is a major cause of community-acquired and healthcare-associated infections, including skin and soft tissue infections, surgical site and wound infections, pneumonia, bacteremia, and endocarditis. In particular, the emergence of methicillin-resistant S. aureus (MRSA) is associated with significantly increased mortality and morbidity. Previous vaccine development efforts against S. aureus have been focused exclusively on the generation of antibodies to the pathogen; all attempts in clinical trials have failed. Recent evidence strongly suggests that T-cell-mediated immunity, rather than antibodies, is required for host defense against S. aureus. This team at BCH developed MAPS, a novel vaccine platform to generate highly immunogenic protein and polysaccharide complexes via strong affinity coupling of biotin and the avidin-like protein rhizavidin. MAPS promotes the generation of antibodies and multipronged systemic and tissue-resident T-cell responses to the target protein antigens. A candidate S. aureus MAPS (SA MAPS) vaccine targets four conserved staphylococcal proteins and confers broad and enhanced protection in mouse models of S. aureus infection or colonization, including in models where antibody-based vaccines fail to protect. Published studies showed that SA MAPS-induced cellular immunity, more specifically, the tissue-resident memory T cells (TRMs), play a primary role in mediating immune defense against S. aureus in the skin and on mucosal surfaces. The objective of this project is to advance this candidate vaccine by characterizing the cellular and molecular mechanisms of protection, identifying surrogate markers that correlate with protection, and evaluating immunological responses in NHPs. Aim 1 is to elucidate SA MAPS vaccine-induced adaptive cellular defense network in skin tissues. Single-cell spatial profiling analysis and evaluation in genetically modified mouse stains will be used to define the spatial organization, molecular signatures, and intercellular interaction and signaling of SA MAPS-induced TRMs in skin tissues after S. aureus infection and to identify specific T-cell populations or signaling pathways that are responsible for protection. Aim 2 will evaluate potential surrogate markers that correlate with protection following SA MAPS immunization. Aim 3 will evaluate the immunogenicity of the SA MAPS vaccine in NHPs, refine the surrogate markers identified in Aim 2, characterize the vaccine-induced cellular defense network in skin, and compare the findings from NHPs and mice. Successful completion of this project will result in the characterization of cell-mediated adaptive host defense mechanisms and comprehensive preclinical data in mice and NHPs to support the advancement of this vaccine to future Phase 1 clinical studies.
Pseudomonas aeruginosa vaccines (Research Project 2)
The goal of Research Project 2 in “Immunization against Multidrug-resistant Pathogens: Activating T Cell Immunity” Center of Excellence for Translational Research (IMPACT-CETR) is to develop a multicomponent Pseudomonas aeruginosa MAPS vaccine based on the serotype-independent P. aeruginosa biofilm polysaccharide Psl and its critical lipid epitope, combined with conserved protein antigens including the Th17-eliciting antigen PopB. Hospital-acquired P. aeruginosa respiratory infections are a significant threat, due in part to their prevalence, increasing rates of antibiotic resistance, and the relative dearth of new antibiotics in the development pipeline. Vaccine strategies to elicit opsonophagocytic killing (OPK) antibodies to the bacterial lipopolysaccharide or surface proteins have had varied success, but none has been broadly protective or FDA-approved for human use. This team showed that Th17 cells (CD4 helper T cells secreting the cytokine IL-17) confer antibody-independent protection against P. aeruginosa in mouse models of pneumonia, including a neutropenic model. Intranasal vaccination with the P. aeruginosa protein PopB mixed with the Th17 adjuvant curdlan stimulates protective Th17 responses in mice. Adding the OprF/I fusion protein to PopB improves protective efficacy against pneumonia in mice, and PopB induces IL-17 secretion in human whole blood taken after P. aeruginosa infection. A vaccine containing the biofilm polysaccharide Psl was constructed using the Multiple Antigen-Presenting System (MAPS) platform, which links biotinylated Psl with a pneumococcal carrier protein fused to the avidin homolog rhizavidin. This candidate elicits OPK antibodies to Psl after active immunization in rabbits and passive immunization in mice; a recently described critical lipid epitope on Psl is retained and immunogenic. A MAPS vaccine containing PopB induces high Th17 responses after subcutaneous immunization of mice with alum as adjuvant. The hypotheses of this project are that: 1) site-selective coupling of vaccine protein antigens to Psl using MAPS will optimize immune responses to T and B cell epitopes; and 2) combining PopB with antigens that induce protective antibodies (Psl, the type 3 secretion system protein PcrV, and the OprF/I fusion protein) will improve the protective efficacy of PopB both in potency and in broadness of protection, with pulmonary tissue-resident memory (TRM) Th17 cells being critical for maximal protection against pneumonia. Aim 1 is to use MAPS to select the optimal combination and composition of vaccine antigens and adjuvants, and test naturally occurring vs synthetic Psl, both linked to PopB-containing fusion proteins. Studies will measure protective efficacy in mice for mucosal and systemic antibodies and T-cell responses. Aim 2 will identify mechanisms of protection using immunophenotyping and single-cell transcriptomics in transgenic mice deficient in antibody and Th17 responses. Aim 3 will test the immunogenicity of the lead vaccine candidate in nonhuman primates. Successful completion of this project will provide preclinical data to support the advancement of this vaccine to future Phase 1 clinical studies.
Klebsiella pneumoniae vaccines (Research Project 3)
The goal of Research Project 3 in the “Immunization against Multidrug-resistant Pathogens: Activating T Cell Immunity” Center of Excellence for Translational Research (IMPACT-CETR) is to develop a quadrivalent Klebsiella pneumoniae vaccine based on conserved proteins that elicit protective Th17 cells and antibodies. K. pneumoniae is the third leading cause of hospital-acquired pneumonia and the second leading cause of bloodstream infections, with a high incidence of serious infections in patients who are immunocompromised individuals (e.g., diabetics, solid organ transplant recipients) or who require mechanical ventilation. This team was the first group to identify lymphocyte-derived IL-17 as a major cytokine mediating lung immunity to Gram-negative bacteria such as K. pneumoniae. This confirmed that Th17 cells provide immunity against clades of organisms and thus may serve as major targets for novel and effective vaccine strategies. Advancing a vaccine candidate into the clinic requires optimizing adjuvant-antigen combinations, and routes of delivery, that generate Th17 responses in the nose/lung. Mucosal delivery of recombinant OMPs from serotype 2 K. pneumoniae, along with the unique LT-based adjuvant LTA1, confers serotype-independent Th17 immunity against hypervirulent K1 K. pneumoniae. Four K. pneumoniae OMP candidate antigens are immunogenic in mice and elicit both Th17 and IgA/IgG antibody responses. The objective of this project is to identify the best delivery route and the best adjuvant to prevent both lung infection and bacteremia in pre-clinical murine and non-human primate (NHP) models. The hypothesis is that the development of multivalent OMP combinatorial vaccine with LTA1 adjuvant will elicit mucosal Th17 memory cells that confer serotype-independent immunity to K. pneumoniae. Aim 1 is to compare immunization routes and adjuvants of the lead quadrivalent antigen formulation in murine models. Aim 2 is to test the role of humoral and type 17 immunity in protection in wild-type mice and transgenic/knock-out models of transplant immunosuppression. Aim 3 is to test immunogenicity and vaccine effect in an established NHP model of K. pneumoniae infection. Successful completion of this project will provide important evidence in support of a data package for a potential FDA IND application for a quadrivalent K. pneumoniae vaccine that can move forward to human clinical trials.
Bioinformatics Core
The Bioinformatics Core of the “Immunization against Multidrug-resistant Pathogens: Activating T Cell Immunity” Center of Excellent for Translational Research (IMPACT-CETR) is part of the research computing team at Boston Children’s Hospital (BCH) and will be available to help with the experimental design, data collection, analysis, integration, and visualization for any kind of biomedical research. As a scientific core of the IMPACT-CETR, we will provide consulting, software, tools, training and best practices to foster research and collaboration internally and externally. We have completed over 250 bioinformatics projects including 81 single-cell and spatial transcriptomics projects from 41 BCH divisions/programs, which leads to over 20 publications over last 4 years. Several publications were published in high-impacted journal such as Science Translational Medicine, Proceedings of the National Academy of Sciences, Journal of Clinical Investigation, and the American Journal of Hematology. With access to two High Performance Computing Clusters at BCH, which has 92 nodes, 6912 CPUs, 88 GPUs, 2 FPGAs, 34 TB RAM, 10 PB replicated, high-performance storage, 6 PB solid state storage and a Slurm queueing system and BioGrids, an academic software platform at Harvard University, we have the capability to support the three Research Projects of the IMPACT-CETR, including general biostatistics as well as single-cell RNA-seq and spatial transcriptomics analyses.
Nonhuman Primate Core
The goal of Scientific Core 2, the Nonhuman Primate Core of the “Immunization against Multidrug-resistant Pathogens: Activating T Cell Immunity” Center of Excellence for Translational Research (IMPACT-CETR), is to provide and perform studies with Macaca mulatta (Rhesus macaques) for vaccine candidates developed by the research projects. Nonhuman primates (NHP) are widely used as a prospective disease model for pathogenesis studies induced by experimental infection and as means to produce host response for immunogenicity and protective efficacy of vaccine candidates. Scientific Core 2, located at the Tulane National Primate Research Center, will focus on the judicious and thoughtful use of rhesus macaques in the proposed studies, and will address the technical rubrics that may be encountered during vaccine development and testing. It will provide the required significant planning, expert involvement, and thoughtful performance of requisite steps to ensure the best use of a critically precious scientific resource. Aim 1 is to characterize host immune response and prevailing pathophysiology of baseline infection with either phenotypic or resistant strains of the selected bacterial pathogens in immunocompetent, naïve rhesus macaques. Aim 2 is to evaluate immunogenicity and efficacy of vaccine candidates in NHPs. Scientific Core 2 will generate expert test systems for subsequent vaccine candidate evaluation and may yield novel tractable strategies for future medical countermeasure development and optimization.
Transgenic Mouse Core
Genetically engineered mice have been essential tools to study the function of immune genes in mammals. To support the overall goals of the “Immunization against Multidrug-resistant Pathogens: Activating T Cell Immunity” Center of Excellence for Translational Research (IMPACT-CETR) and to control for the gut microbiota that could influence results, we have centralized breeding of mice in the Transgenic Mouse Core. The Transgenic Mouse will supply genetically engineered mice for the research projects. The overall coordination of breeding animals will be through this core. Specific Aims are: 1) To monitor and maintain genetically engineered lines for the projects. 2) To coordinate breeding and shipment demands with the project leaders. 3) To provide assay support for genotyping as well as tissue specific recombination for conditional knockout lines.