Adjuvant Discovery via a High Throughput Screen using Human Primary Mononuclear Cells

Abstract

SummaryWe describe a methodology utilizing high-throughput and high-content screening for novel adjuvant candidates that was used to screen a library of ∼2,500 small molecules via a 384-well quantitative combined cytokine and flow cytometry immunoassay in primary human peripheral blood mononuclear cells (PBMCs) from 4 healthy adult study participants. Hits were identified based on their induction of soluble cytokine (TNF, IFNγ and IL-10) secretion and PBMC maturation (CD 80/86, Ox40, and HLA-DR) in at least two of the four donors screened. From an initial set of 197 compounds identified using these biomarkers—an 8.6% hit rate—we downselected to five scaffolds that demonstrated robust efficacy and potencyin vitroand evaluated the top hit, vinblastine sulfate, for adjuvanticityin vivo. Vinblastine sulfate significantly enhanced murine humoral responses to recombinant SARS-CoV-2 spike protein, including a four-fold enhancement of IgG titer production when compared to treatment with the spike antigen alone. Overall, we outline a methodology for discovering immunomodulators with adjuvant potential via high-throughput screening of PBMCsin vitrothat yielded a lead compound within vivoadjuvanticity.MotivationVaccines are a key biomedical intervention to prevent the spread of infectious diseases, but their efficacy can be limited by insufficient immunogenicity and nonuniform reactogenic profiles. Adjuvants are molecules that potentiate vaccine responses by inducing innate immune activation. However, there are a limited number of adjuvants in approved vaccines, and current approaches for preclinical adjuvant discovery and development are inefficient. To enhance adjuvant identification, we developed a protocol based onin vitroscreening of human primary leukocytes.