Enhanced and Prolonged Immunogenicity in Mice of Thermally Stabilized Fatty Acid-Conjugated Vaccine Antigen

Background/Objectives: Influenza vaccines require good thermal stability without the need for refrigerator storage. Although the fatty acid-conjugated hemagglutinin (Heg) vaccine antigen provides good stability in both solid and liquid states, its therapeutic effectiveness must be validated in vivo. This study aimed to investigate the immunogenicity of the thermally stabilized Heg-oleic acid conjugate (HOC) and compare it with native Heg as a reference. Method: To evaluate HOC immunogenicity, an enzyme-linked immunosorbent assay was used to measure hemagglutinin inhibition (HI) titers, serum IgG antibody titers (IgG1, IgG2a), and cytokine secretion levels (IFN-γ, IL-4) in BALB/c mice after intramuscular (IM) injection. Results: Thermally stabilized HOC induced higher and more sustained serum IgG1 and IgG2a responses than the native Heg vaccine antigen. IgG1 is typically associated with a Th2 response, whereas IgG2a is associated with a Th1 response. HOC appeared to enhance both responses, inducing a more balanced immune response. Moreover, HOC antigens stimulate broader immune responses, suggesting stronger and longer-lasting immune memory. The cytokine levels of IFN-γ (2.8-fold) and IL-4 (6-fold) were significantly increased in the HOC-immunized group compared to the Heg group. IFN-γ, a cytokine that activates the Th1 immune response, demonstrated the enhanced ability of HOC to induce a Th1 response. IL-4, a cytokine that promotes the Th2 response, indicated that HOC also strongly induced a Th2 response. The thermal stability of HOC antigens was crucial for maintaining their structural integrity, enabling the continuous exposure to the stable antigen without denaturation. This allows immune cells to recognize stable antigens efficiently and form long-term immune memory. Conclusions: The stability of HOC antigens enhanced the antigen processing efficiency of antigen-presenting cells (APCs) and stimulated immune responses. The fatty acid-conjugated vaccine antigen could provide improved storage stability but also enhance immunogenic efficacy compared to the native antigen, supporting its potential for further applications.