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

Abstract

Vaccines 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. We 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-alpha, IFN-gamma; and IL10) 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 four scaffolds that demonstrated robust efficacy and potency in vitro and evaluated the top hit, vinblastine sulfate, for adjuvanticity in 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 PBMCs in vitro that yielded a lead compound with in vivo adjuvanticity.

From hit to vial: Precision discovery and development of an imidazopyrimidine TLR7/8 agonist adjuvant formulation

“…Our study also has some limitations, including (i) mice may not accurately reflect in vivo effects in humans and future studies may benefit from porcine and/or nonhuman primate studies, (ii) while TLRs 7 and 8 appear to be important to PVP-037’s action, we did not assess additional pathways that may contribute to activity such as the inflammasome ( 40 ), (iii) future functional studies including pathogen challenge could further assess adjuvant effects on vaccine efficacy, (iv) immunization studies were undertaken at relatively short timepoints, such that future studies are needed to assess the impact of PVP-037 on durability of immune responses, and although we focused on Ab responses as important correlates of protection against influenza and SARS-CoV-2 ( 41 , 42 ), (v) further studies should assess T cell responses, as cellular immunity provides an important layer of protection, along with toxicity studies on stable formulations. Last, although we screened molecules based on NF-κB and cytokine expression, additional endpoints such as costimulatory marker expressions (e.g., CD80, CD86, and OX40) are potentially valuable for future phenotypic HTS campaigns ( 43 , 44 ).…”

Next-Generation Adjuvants: Applying Engineering Methods to Create and Evaluate Novel Immunological Responses

“…When attempting to replicate human physiology, primary cells and organoids are perhaps the closest available choice. Recent studies have successfully performed high-throughput screens on primary human peripheral blood mononuclear cells (PBMCs) [ 98 , 99 ]. PBMCs are non-trivial to source, but recent miniaturization of assays allows for a larger number of experiments to be performed for the same number of cells [ 100 ].…”

Comprehensive immunoprofiling and systematic adjuvant comparisons for identifying suitable vaccine: Adjuvant pairings

“…The high failure rate is due to a lack of defined in vitro correlates of in vivo adjuvanticity. Multiplex assay platforms that expand the number of cytokines being assessed partially address the issue, 6 but this approach still relies on a limited number of parameters that may not necessarily correlate with in vivo adjuvanticity and can be prohibitively expensive when scaling up an adjuvant screening campaign.…”