TLR4 Agonist CRX-527 Modulates Intracellular and Inflammatory Cytokine Expression in Lymphoid Tissues of BCG-MSP1C-Immunized Mice

Introduction: Due to the complexity of the malaria parasite’s life cycle, together with the limited knowledge of its intricate immunological response, this study was conducted. This study aims to evaluate the ability of a Toll-like receptor 4 (TLR4) agonist, CRX-527, to enhance the production of intracellular and inflammatory cytokines in lymphocyte-derived tissues of malaria vaccine candidates. Methods: A total of forty-eight male Balb/c mice was randomly divided into eight groups (n = 6 per group). Two groups of mice were immunized intraperitoneally with 2 × 106 colony-forming units (CFU) of parent Bacillus Calmette–Guérin (BCG) in 200 µl of 1 × phosphate-buffered saline–Tween 80 (PBS-T80) in the presence or absence of 0.5 mg/kg of CRX-527. Another two groups of mice were immunized with 2 × 106 CFU of Mycobacterium bovis Bacillus Calmette–Guérin expressing the C-terminus merozoite surface protein-1 of Plasmodium falciparum (BCG-MSP1C) in 200 µl of 1 × PBS-T80 in the presence or absence of 0.5 mg/kg of CRX-527. Meanwhile, another two groups of mice were immunized with 200 µl of 1 × PBS-T80, whereas the remaining two groups were immunized with 200 µl of lipopolysaccharide (LPS) in the presence or absence of 0.5 mg/kg of CRX-527, serving as control groups. The lymphoid tissues obtained from the spleen, lymph nodes, and liver were harvested from the euthanized mice and were cultured for 24 hours. Then, the supernatant was collected to detect the production of selected intracellular and inflammatory cytokines using a sandwich enzyme-linked immunosorbent assay (ELISA). Results: There was a significant increase (p < 0.05) in tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), interferon-gamma (IFN-γ), and interleukin-4 (IL-4) in the presence of CRX-527, with the highest levels observed in the spleen, followed by the lymph nodes and liver from the BCG-MSP1C groups, followed by the parent BCG-immunized groups. Conclusion: These findings demonstrate that the increased production of several intracellular and inflammatory cytokines by the developed malaria vaccine candidate (BCG-MSP1C) in the presence of CRX-527 plays an important role in triggering innate immune responses in lymphocyte-derived tissues.