Effects of Food Intake on Biomarker Activity in Normal Humans Taking Immulina

The COVID-19 pandemic highlighted the need for safe, complementary approaches to enhance host antiviral defenses - a need that persists for seasonal influenza despite widespread vaccination and the availability of antiviral drugs. Immulina®, a bioassay-standardized botanical extract derived from spirulina (Limnospira fusiformis, formerly Arthrospira platensis), is a promising candidate for this host-targeted strategy. The investigators' research has identified Braun-type lipoproteins as the main immunostimulatory compounds that activate Toll-like receptor 2 (TLR2) and established a foundation of preclinical and clinical data through an NIH-funded Botanical Dietary Supplement Research Center grant (U19AT010838, 2020-2026). In the U19 project, Immulina® increased natural killer (NK) cell activity in a substantial minority of healthy participants, but the basis for this heterogeneous response remains unknown.

The investigators' preliminary mechanistic findings suggest a previously unrecognized explanation for this variability: Braun-type lipoproteins responsible for Immulina®'s immunostimulatory activity may be susceptible to loss of biological activity under postprandial gastrointestinal (GI) conditions. The investigators' preliminary in vitro studies demonstrated a progressive reduction in Immulina®'s TLR2-stimulating activity following exposure to fed-state simulated intestinal fluid containing pancreatic enzymes, including lipase, suggesting that the intestinal environment following a meal may promote enzymatic reduction or inactivation of these lipoproteins. In the investigators' U19 clinical trials, the timing of Immulina® administration relative to meals was not controlled, potentially exposing participants to different gastrointestinal conditions that may have contributed to variability in biological activity and immunological responses. Accordingly, investigators will explore the effects of fed versus fasting administration on the activity of Immulina® through mechanistic in vitro experiments and in a defined human study population, guided by the following hypothesis: Dietary status associated with food intake influences the TLR2-stimulating activity of Immulina®, thereby contributing to variability in biomarkers of antiviral resilience and potentially affecting vaccine-specific antibody responses. The investigators will examine this hypothesis by pursuing the following specific aims:

1. Characterize Immulina® (authentication, standardization, and capsule performance) and evaluate its gastrointestinal stability and functional bioaccessibility under simulated fed- and fasted-state conditions to inform optimal oral administration.

   A. Confirm botanical authentication, bioassay-based standardization, and capsule disintegration and dispersion performance of the clinical encapsulated product to ensure consistency with previously characterized material that was used in the U19 project.

   B. HEK-Blue™ hTLR2 cells will be used as a functional bioactivity detector to quantify the collective TLR2-stimulating activity of Braun-type lipoproteins released from Immulina® and retained following exposure to simulated fasted- and fed-state gastric and intestinal conditions. Intestinal studies will also compare pancreatin-mediated loss of activity with that induced by individual purified enzymes to identify specific contributors (e.g., lipase) to activity loss. These studies will identify gastrointestinal conditions most likely to preserve Immulina® activity and establish rational administration criteria for the proposed clinical studies.

2. Reproduce and refine the immunological findings of the U19 study by determining whether Immulina® dose and administration relative to meals influence biomarkers of antiviral resilience during four weeks of supplementation. Participants will receive Immulina® at daily doses of 400 or 800 mg, administered either two hours before breakfast following an overnight fast or simultaneously with breakfast. Changes in NK cell activity and antiviral cytokine profiles (IFNα, IFNγ, IL-2, IL-10, and IL-12) will be evaluated after two and four weeks of supplementation relative to baseline. These findings will help establish whether controlling administration conditions improves the consistency of immunological responses observed in the U19 study.
3. Examine the effects of pretreatment with Immulina® on vaccine-specific antibody titers after routine immunization for influenza. The investigators will compare vaccine-specific antibody titers four weeks after participants receive routine influenza after consuming daily Immulina® or placebo for four weeks prior to immunization.

Impact. The investigators' previous U19 project established a favorable safety profile for Immulina® in a diverse population of more than 400 participants and demonstrated substantial increases in NK cell activity among a subset of participants. Successful completion of the proposed research will determine the reproducibility of this biomarker response and identify administration conditions that could optimize the immune antiviral resilience. By addressing important knowledge gaps identified during the U19 project, these studies will provide the mechanistic and clinical information needed to refine Immulina® administration protocols to focus and refine the design of a larger clinical trial evaluating its potential to enhance antiviral resilience.