# Defining molecular mechanisms of combination adjuvants: a systems immunology, transcriptomics and imaging approach

> **NIH NIH U01** · UNIVERSITY OF CALIFORNIA-IRVINE · 2023 · $592,289

## Abstract

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 unprecedent...

## Key facts

- **NIH application ID:** 10573197
- **Project number:** 5U01AI160397-03
- **Recipient organization:** UNIVERSITY OF CALIFORNIA-IRVINE
- **Principal Investigator:** David Huw Davies
- **Activity code:** U01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $592,289
- **Award type:** 5
- **Project period:** 2021-03-05 → 2025-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10573197

## Citation

> US National Institutes of Health, RePORTER application 10573197, Defining molecular mechanisms of combination adjuvants: a systems immunology, transcriptomics and imaging approach (5U01AI160397-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10573197. Licensed CC0.

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