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Principal Investigator: David Huw Davies
Organization: UNIVERSITY OF CALIFORNIA-IRVINE
Fiscal Year: 2021
Award: $610,134
Funding agency: National Institute of Allergy and Infectious Diseases
PROJECT SUMMARY/ABSTRACT
In this proposal we will define molecular and cellular mechanisms of different combinations of known adjuvant
components MPLA (a TL4 agonist), CpG (a TLR9 agonist), agonists of cGAS-STING and NOD1/2 pathways,
and a squalene-in-water emulsion (AddaVAX™). Our overall Aim is to provide a detailed analysis of every
combination of these using high throughput in vitro and in vivo assays, followed an in-depth analysis of two
combination adjuvants using live cell imaging and single-cell mRNA sequencing of draining lymph nodes after
vaccination.
Initially, 96-well based in vitro assays of innate and adaptive immune system activation will be used to profile
different adjuvants components, both individually, and in different combinations and concentrations. These
assays will comprise: 1) activation of TLR, NOD and STING signaling pathways using primary dendritic cells
(DCs), T and B cells from reporter transgenic mice; 2) in vitro activation of naïve B cells to monitor their
differentiation into plasma cells and class switching. We anticipate some of the adjuvant combinations will have
synergistic effects that differ from the sum of the effects when used individually. Next, based on performance in
vitro a subset of combination adjuvants and their individual components will be evaluated as adjuvants for model
vaccine antigens (influenza hemagglutinin H1, filovirus (EBOV) glycoprotein and SARS-CoV-2 spike) in vivo in
mice. Immunogenicity metrics will comprise: 1) antibody dynamics and durability, isotype, avidity and breadth of
cross-reactivity using protein microarrays; 2) flow cytometry of antigen-specific B cells to assess differentiation
and cross-reactivity; 3) T cell recall assays to define Th1/Th2/Th17 cytokine profiles; 4) neutralization by sera of
live influenza, SARS-CoV-2 and VSV-pseudotyped with EBOV glycoprotein. This will provide a comprehensive
cellular and molecular profile associated with each combination adjuvant.
Two combination adjuvants (and their individual components for comparison) will be selected for a deep analysis
using: 1) transgenic mice that allow Ca2+ fluxes in live CD4 T and B cells and DCs to be visualized using 2-
photon microscopy. Combined with techniques of whole tissue imaging, we will monitor adjuvant-driven T cell
and DC mobilization, motility and interactions in live draining lymph nodes; 2) using single-cell RNAseq
technology (10x Genomics Inc) of cells in draining lymph nodes, we will define cell composition and phenotype,
cellular interactions and spatial organization. We will perform a deep analysis in Year 1 on the combination
adjuvant CpG/MPLA + AddaVAX (TLR9 and TLR4 agonists in a squalene-in-water emulsion) since we have
already shown this is a powerful combination adjuvant. A second combination adjuvant will be selected for deep
analysis based on data generated in the in vitro and in vivo assays described herein. Together these
complementary deep approaches will provide an unprecedented level of molecular and cellular detail of two
highly effective combination adjuvants. Overall, we anticipate these data will help guide the future design of
vaccines where the immune response required can be tuned according to the particular pathogen in question.
Terms: <2-photon><2-photon microscopy><2019 novel corona virus><2019 novel coronavirus><2019-nCoV><Acute Poliomyelitis><Adaptive Immune System><Adjuvant><Agonist><Algorithms><Antibodies><Antibody titer measurement><Antigen-Presenting Cells><Antigens><Assay><Avidity><B blood cells><B cell><B cell differentiation><B cell repertoire><B cells><B lymphocyte differentiation><B pertussis infection><B-Cell Activation><B-Cells><B-Lymphocytes><B-cell><B. pertussis infection><Bacterial DNA><Behavioral><Bioassay><Biologic Assays><Biological Assay><Blood Plasma Cell><Bordetella pertussis infection><CD4 Cells><CD4 Positive T Lymphocytes><CD4 T cells><CD4 helper T cell><CD4 lymphocyte><CD4+ T-Lymphocyte><CD4-Positive Lymphocytes><COVID-19 virus><COVID19 virus><Calcium><Cell Body><Cell Communication and Signaling><Cell Locomotion><Cell Migration><Cell Movement><Cell Signaling><Cell Surface Antigens><Cell Wall><Cells><Cellular Migration><Cellular Motility><Childhood><Class Switching><Class Switchings><CoV-2><CoV2><Computer Analysis><Data><Dendritic Cells><Diphtheria><Disease><Disorder><EBOV><Ebola><Ebola virus><Ebola-like Viruses><Emulsions><FLJ11330><Filoviridae><Filovirus><Flow Cytofluorometries><Flow Cytofluorometry><Flow Cytometry><Flow Microfluorimetry><Flow Microfluorometry><Future><Gene Expression><Generations><Genes><Genomics><Germinal Center><Glycoproteins><Grippe><Health><Heterogeneity><Homing><Homolog of Drosophila TOLL><Human><Hydrogen Oxide><IFN-regulatory factor 3><IRF-3 protein><IRF3><IRF3 gene><Image><Imaging Procedures><Imaging Technics><Imaging Techniques><Immune><Immune response><Immune system><Immunes><Immunity><Immunochemical Immunologic><Immunoglobulin Class Switching><Immunoglobulin Class Switchings><Immunologic><Immunologic Surface Markers><Immunological><Immunological Surface Markers><Immunological response><Immunologically><Immunologics><Immunology><In Vitro><Individual><Infection><Influenza><Influenza HA><Influenza Hemagglutinin><Interferon Regulatory Factor 3><Intracellular Communication and Signaling><Isotype Switching><Isotype Switchings><Link><Lipopolysaccharides><Longitudinal Studies><Measles><Measures><Mice><Mice Mammals><Microscopy><Modeling><Modern Man><Modernization><Molecular><Molecular Fingerprinting><Molecular Profiling><Monitor><Motility><Murine><Mus><NF-Kb-Activating Kinase Gene><Oils><Outcome><Pathway interactions><Pattern><Pattern recognition receptor><Performance><Pertussis><Phenotype><Phosphorylation><Plasma Cells><Plasmacytes><Polio><Poliomyelitis><Property><Protein Biochips><Protein Chips><Protein Microarray><Protein Microchips><Protein Phosphorylation><Reaction><Recombinant Proteins><Reporter><Rubeola><SARS corona virus 2><SARS-CoV-2><SARS-CoV2><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><SCmRNAseq><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome related corona virus 2><Signal Pathway><Signal Transduction><Signal Transduction Systems><Signaling><Squalene><Stimulator of Interferon Genes><Structure of germinal center of lymph node><Sum><Surface Antigens><System><T-Cell Activation><T-Cells><T-Lymphocyte><T2K><T4 Cells><T4 Lymphocytes><TBK1><TBK1 gene><TLR4><TLR4 gene><Techniques><Technology><Testing><Tissue imaging><Toll Homologue><Transgenic Mice><Transgenic Organisms><VSV><Vaccination><Vaccine Adjuvant><Vaccine Antigen><Vaccine Design><Vaccines><Veiled Cells><Vesicular Stomatitis Virus><Vesicular stomatitis Indiana virus><Viral><Virus><Water><Whooping Cough><Wuhan coronavirus><accessory cell><acquired immune system><activated B cells><adaptive immune response><adaptive immunity><allergic/immunologic body system><allergic/immunologic organ system><antibody titering><base><biological signal transduction><cGAMP STING><cGAMP-STING><cGAMP/STING><cGAS/STING><cell motility><computational analyses><computational analysis><computer analyses><coronavirus disease 2019 virus><cross reactivity><cytokine><design><designing><draining lymph node><ebolavirus><flow cytophotometry><flu HA><flu hemagglutinin><global gene expression><global transcription profile><hCoV19><host response><imaging><imaging approach><imaging based approach><immune system response><immunogen><immunogenicity><immunoresponse><in vitro Assay><in vivo><infected with B pertussis><infected with B. pertussis><infected with Burkholderia pertussis><influenza viral HA><influenza viral hemagglutinin><influenza virus HA><influenza virus hemagglutinin><interdisciplinary approach><interstitial><live cell image><live cell imaging><live cellular image><live cellular imaging><long-term study><longitudinal outcome studies><longterm study><microbial><molecular biomarker><molecular marker><molecular profile><molecular signature><morbilli><multidisciplinary approach><nCoV2><new approaches><novel approaches><novel strategies><novel strategy><pathogen><pathway><pediatric><plasmocyte><recruit><regional lymph node><response><scRNA-seq><secondary lymph organ><secondary lymphatic organ><secondary lymphoid organ><single cell RNA-seq><single cell RNAseq><single cell mRNA sequencing><single-cell RNA sequencing><thymus derived lymphocyte><toll-like receptor 4><tool><transcriptome><transcriptomics><transgenic><two photon excitation microscopy><two photon microscopy><two-photon><vaccine efficacy>