(M1030-01-05) Evaluating the Immunomodulatory Efficacy of an Ethanolic Spray-Dried Extract of Spirulina

Purpose: Adjuvants for vaccine formulations are an essential component of effective, robust, and long-lasting vaccine products. Despite this, there are only a handful of FDA-approved vaccine adjuvants on the market. Our lab has been researching herbal products and their component compounds to use as adjuvants in cancer vaccines. This study highlights the immunostimulatory efficacy of an ethanolic spray-dried extract of the blue-green micro- algae Spirulina (arthrospira platensis) to stimulate the innate and adaptive immune system. Dendritic cells (DC3.2) [innate immunity] were treated at varying concentrations from 200 μg - 1 μg to find effective and non-toxic dose, whereas T-cells [adaptive immunity] were co-cultured with dendritic cells in presence of Spirulina at specific concentrations (50 and 100 μg) to measure affinity.

Methods: Dendritic cells (DC3.2) were incubated with ethanolic and ethanolic spray-dried extracts of Spirulina at different concentrations ranging from 200 μg-1 μg. Cell expression outcomes (MHC-I/MHC-II) and viability were the primary outcomes. Nitric oxide (NO) release and cytokine secretion (IL-12p70, IL-1β, TNF-α) responses were measured for all treated groups [Eg: MHC-I response in Figure 1]. Ovalbumin was used as the protein candidate for all groups and Resiquimod and/or MPLA was used as the positive control. Once a safe concentration was established with reproduced studies, a 72-hour co-culture study involving DC3.2s (dendritic cells) and RF33.70 (T-cells) both syngeneic to a C57BL/6 mouse model was conducted. Primary outcomes for this co-culture included CD8+ cytotoxic T-cell activation, viability, and cytokine secretion from the supernatant [Eg: CD8+ expression response in Figure 2].

Results: Dendritic cells showed a significantly higher, dose-dependent stimulation of MHC-I when treated with the ethanolic extract of Spirulina with concentrations between 100 to 40 μg as compared to Ova, or the positive controls. Viability for these concentrations were similar between all groups. The spray-dried spirulina showed a 4-fold increase in MHC-I stimulation, at concentration as low as 25 μg, and no loss in viability. No detectable IL-12p70 response was observed and positive control showed a superior NO release than the Spirulina groups. In the co-culture experiment, Spirulina showed a significantly higher CD8+ expression at both 100 and 50 μg as compared with the resiquimod and MPLA positive controls. Combinations of Spirulina with the positive controls consistently showed significantly higher cytotoxic CD8+ T-cells.  All treatment groups containing either the 50 or 100 μg concentrations of Spirulina showed Il-12p70 and IFN-γ secretion.

Conclusion: Adjuvants that are efficacious immune stimulators for cancer antigens are a critical need for the advancement of cancer therapy. Spirulina has shown immense early potential for both innate and adaptive cell activation through surface marker over-expression and cytokine secretion in a cytotoxic T-cell priming pathway. Future studies will investigate three other organic sources of Spirulina to understand the differences in components and efficacy based on source variables. We will then optimize the best Spirulina extract to use as an adjuvant candidate, into a cancer vaccine formulation.