(T1530-05-30) Discovery of Oral Vaccine Adjuvant via Bioanalytical HTS Assays

Purpose: Immunotherapy serves to modulate the host immune system to fight foreign pathogens or suppress autoimmunity. Conventional immunotherapies utilize parenteral injections, with limited utilization of oral delivery (e.g., polio vaccine). This administration, while effective, requires healthcare personnel for intradermal, intramuscular, or subcutaneous injection, which can present unique logistic challenges.¹ Additionally, traditional therapies induce systemic humoral immune response (IgG), but are less effective at inducing mucosal immune responses (IgA), which are critical to clear pathogens and reduce autoimmune activation in the lung and GI tract.^2,3 This study aims to discover and validate new compounds capable of selectively activating immune cells without triggering severe inflammation in the GI tract. Furthermore, we want to show these compounds are effective in modulating mucosal immunity and to create an efficient oral vaccine delivery platform.

Methods: High-throughput screening (HTS) of three drug libraries including FDA-approved compounds (FDA), soluble diversity (SDIV), and bioactives (BA) was employed for initial screening. Testing was done with RAW 264.7 Blue cells, utilizing a RAW-NF-kB promotor-SEAP (Secreted embryonic alkaline phosphatase) assay in 384-well plates. SEAP is used as a reporter to measure NF-kB activation to identify potential immune modulators. Quantification of SEAP in each well at 620nm was measured and compared to DMSO negative controls and lipopolysaccharide (LPS) positive controls to identify initial “hits”. These “hit” compounds were those that yielded at least a 3-fold increase in SEAP absorbance over controls. Subsequent dose- and time-dependent testing of “hit compounds was done to measure potency and kinetics. Finally, cytotoxicity testing was performed to evaluate upper limits of potential compound dosing.

Results: Initial screening yielded a total of 117 “hit” compounds across all three libraries screened (Table 1). In addition, several other compounds showed significant reductions in SEAP absorbance opposed to increases. Average minimum dose required for SEAP activation for “hits” was 5 µM. Time-dependent SEAP activation was greatest for “hits” and LPS controls at 0.5h, decreasing with longer exposure time. Dose-response reconfirmation revealed cytotoxicity at one or more concentration tested for 23 “hits”.

Conclusion: Initial discovery of potential new immunomodulatory drugs is crucial to develop increasingly potent and targeted immunotherapies. Expansion of traditional HTS screening with the inclusion of dose- and time-dependent activation helped to narrow “hit” compound selection criteria. With early candidates identified, we will proceed with further in vitro characterizations using immune cells (e.g., BMDC, macrophages). Ongoing testing will employ cytokine quantification via enzyme-linked immunosorbent assays (ELISAs) and immunophenotyping through flow cytometry analysis. In the larger picture, these compounds could serve as an oral vaccine adjuvant to overcome challenges associated with developing prophylactic and therapeutic regimes for GI tract infectious disease and autoimmune issues. Future work and the larger goal of this study will be to formulate these compounds into poly(lactic-co-glycolic) acid (PLGA) nanoparticles for use as an oral vaccine designed for GI delivery.

References: 1. J. N. Zuckerman, et al. The importance of injecting vaccines into muscle: different patients need different needle sizes. Bmj. 321 (2000), pp. 1237–1238.
2. A. T. Jones, et al., HIV-1 vaccination by needle-free oral injection induces strong mucosal immunity and protects against SHIV challenge. Nat. Commun. 10, 1–14 (2019).
3. J. Holmgren, et al. Mucosal immunity and vaccines. Nat. Med. 11, S45–S53 (2005).

Acknowledgements: This work was supported in part by a grant from CoBRE-Chemical Biology of Infectious Disease (NIH award number P20GM113117) (H.K). Personnel funding (G.A.F) provided by the Madison and Lila Self Graduate Fellowship.

Table 1. "Hit" compound distribution across screened libraries.

Figure 1. Scattergram of FDA (A) and SDIV (B) compounds on increasing SEAP activity against DMSO controls.

Figure 2. Scattergram of BA library compounds on SEAP activity against DMSO and LPS controls.