Nasal Vaccination with a Fusion Protein of Hemagglutinin A Antigenic Region and Maltose-Binding Protein Elicits CD4⁺ T Cell Responses via Toll-Like Receptor 4

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Our previous study demonstrated that nasal immunization with the fusion protein of the 25-kDa antigenic hemagglutinin A antigenic region and maltose-binding protein (25k-hagA-MBP) elicited high levels of 25k-hagA-specific antibody responses in both systemic and oral mucosal areas. In this study, we further elucidated the immunogenicity of 25k-hagA-MBP using Toll-like receptor (TLR) 4 gene-deficient (TLR4^-/-) mice. When CD4⁺ T cells isolated from the spleen and cervical lymph nodes (CLN) of 25k-hagA-MBP-stimulated TLR4^+/+ mice were restimulated with 25k-hagA in vitro, significant proliferative responses were induced. In contrast, only low levels of CD4⁺ T cell proliferation were induced in restimulated cells isolated from TLR4^-/- mice. Analysis of cytokine responses showed that nasal administration of 25k-hagA-MBP to TLR4^+/+ mice produced significant levels of IL-6 and IL-10 in cells from the spleen and CLN. However, these cytokine responses were marginal in TLR4^-/- mice nasally immunized with 25k-hagA-MBP. These results suggest that nasal immunization with 25k-hagA-MBP induces 25k-hagA-specific CD4⁺ T cells via a TLR4-dependent pathway.