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 nave 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.
Vaccines have had a major impact on human health through the elimination of childhood diseases such as measles, diphtheria, whooping cough and polio. Safer, new generation vaccines are made from recombinant proteins instead of whole pathogens, but these require adjuvants to stimulate the immune system. Conventionally, only one type of adjuvant is used but they are more effective when combined. In this proposal we will used modern immunological tools to dissect the cellular and molecular pathways that underlie synergistic effect of adjuvants when combined.
