# Application of a small molecule calcium influx inducer as a vaccine adjuvant: enhancing Th2-biased immune responses

## TL;DR

A new compound, 2G272, was found to act as a vaccine adjuvant by boosting Th2 immune responses with minimal side effects.

## Contribution

2G272 was identified as a novel adjuvant that enhances Th2-biased immune responses through calcium influx and extracellular vesicle release.

## Key findings

- 2G272 activates dendritic cells and increases CD80/CD86 expression.
- 2G272 stimulates T cell proliferation and induces IgG1 and Th2 cytokine responses.
- 2G272 shows a favorable safety profile with minimal systemic cytokine release.

## Abstract

Vaccines are highly effective in preventing the spread of communicable diseases and are critical to overall public health. As immune stimulants vaccine adjuvants augment the level and longevity of these protective responses. Seeking novel adjuvants using parallel high throughput screens and subsequent systematic structure–activity relationship studies we identified an analogue of a hit compound, 2G272, that in screening assays retained in vitro induction of calcium (Ca2+) influx, tetraspanin CD63 EV reporter activity and cell viability. Here, we further our analyses of the biological activity of 2G272 related its potential use as a vaccine adjuvant.

2G272 was tested for activation of murine bone marrow-derived dendritic cells (mBMDC) by flow cytometry for Ca2+ entry, levels of CD80 and CD86 expression, and in vitro stimulation of antigen-specific T cell proliferation. Cytokines and IgG responses from BALB/c mice injected with 2G272 as a single agent or as an adjuvant with ovalbumen were measured by ELISA.

2G272 triggered store-operated Ca2+ entry in mBMDC as well as increases in CD80 and CD86 surface expression. In co-culture experiments, this compound amplified the stimulation of cognate T cell proliferation. Intramuscular injection of 2G272 elicited minimal systemic cytokine and chemokine release. When used as an adjuvant with ovalbumen, 2G272 generated a significant antigen-specific IgG1 response with a higher splenocyte T helper 2 (Th2) cytokine response.

2G272 activated mBMDCs associated with EV release and a store-operated calcium entry response. Enhanced cognate T cell proliferation was mediated either through direct engagement with compound-stimulated mBMDCs or indirectly via immunostimulatory extracellular vesicles released by 2G272-activated mBMDCs. 2G272 elicited minimal systemic cytokine and chemokine release, demonstrating a promising safety profile. When used as an adjuvant in a murine vaccination model, 2G272 enhanced the IgG1 response with an associated T helper 2 cytokine profile. Hence this compound shows promise as an adjuvant if a Th2 response is beneficial or in combination with other agents to provide a balanced immune response in vaccines.