TLR4 agonist activity of Alcaligenes lipid a utilizes MyD88 and TRIF signaling pathways for efficient antigen presentation and T cell differentiation by dendritic cells.

Alcaligenes faecalis was previously identified as an intestinal lymphoid tissue-resident commensal bacteria, and our subsequent studies showed that lipopolysaccharide and its core active element (i.e., lipid A) have a potent adjuvant activity to promote preferentially antigen-specific Th17 response and antibody production. Here, we compared A. faecalis lipid A (ALA) with monophosphoryl lipid A, a licensed lipid A-based adjuvant, to elucidate the immunological mechanism underlying the adjuvant properties of ALA. Compared with monophosphoryl lipid A, ALA induced higher levels of MHC class II molecules and costimulatory CD40, CD80, and CD86 on dendritic cells (DCs), which in turn resulted in strong T cell activation. Moreover, ALA more effectively promoted the production of IL-6 and IL-23 from DCs than did monophosphoryl lipid A, thus leading to preferential induction of Th17 and Th1 cells. As underlying mechanisms, we found that the ALA-TLR4 axis stimulated both MyD88- and TRIF-mediated signaling pathways, whereas monophosphoryl lipid A was biased toward TRIF signaling. These findings revealed the effects of ALA on DCs and T cells and its induction pattern on signaling pathways.

Chemically Synthesized Alcaligenes Lipid A as an Adjuvant to Augment Immune Responses to Haemophilus Influenzae Type B Conjugate Vaccine.

We previously identified Alcaligenes spp. as a commensal bacterium that resides in lymphoid tissues, including Peyer's patches. We found that Alcaligenes-derived lipopolysaccharide acted as a weak agonist of Toll-like receptor four due to the unique structure of lipid A, which lies in the core of lipopolysaccharide. This feature allowed the use of chemically synthesized Alcaligenes lipid A as a safe synthetic vaccine adjuvant that induces Th17 polarization to enhance systemic IgG and respiratory IgA responses to T-cell-dependent antigens (e.g., ovalbumin and pneumococcal surface protein A) without excessive inflammation. Here, we examined the adjuvant activity of Alcaligenes lipid A on a Haemophilus influenzae B conjugate vaccine that contains capsular polysaccharide polyribosyl ribitol phosphate (PRP), a T-cell-independent antigen, conjugated with the T-cell-dependent tetanus toxoid (TT) antigen (i.e., PRP-TT). When mice were subcutaneously immunized with PRP alone or mixed with TT, Alcaligenes lipid A did not affect PRP-specific IgG production. In contrast, PRP-specific serum IgG responses were enhanced when mice were immunized with PRP-TT, but these responses were impaired in similarly immunized T-cell-deficient nude mice. Furthermore, TT-specific-but not PRP-specific-T-cell activation occurred in mice immunized with PRP-TT together with Alcaligenes lipid A. In addition, coculture with Alcaligenes lipid A promoted significant proliferation of and enhanced antibody production by B cells. Together, these findings suggest that Alcaligenes lipid A exerts an adjuvant activity on thymus-independent Hib polysaccharide antigen in the presence of a T-cell-dependent conjugate carrier antigen.

Chemically Synthesized Alcaligenes Lipid A Shows a Potent and Safe Nasal Vaccine Adjuvant Activity for the Induction of Streptococcus pneumoniae-Specific IgA and Th17 Mediated Protective Immunity.

Effective and safe vaccine adjuvants are needed to appropriately augment mucosal vaccine effects. Our previous study demonstrated that lipopolysaccharide (LPS) from Peyer's patch resident Alcaligenes stimulated dendritic cells to promote the production of mucosal immunity-enhancing cytokines (e.g., IL-6 and BAFF), thus enhancing antigen-specific immune responses (including IgA production and Th17 responses) without excessive inflammation. Here, we chemically synthesized Alcaligenes lipid A, the biologically active part of LPS, and examined its efficacy as a nasal vaccine adjuvant for the induction of protectively immunity against Streptococcus pneumoniae infection. Mice were nasally immunized with pneumococcal surface protein A (PspA) as a vaccine antigen for S. pneumoniae, together with Alcaligenes lipid A. Alcaligenes lipid A supported the generation of high levels of PspA-specific IgA and IgG responses through the augmentation of germinal center formation in the nasopharynx-associated lymphoid tissue and cervical lymph nodes (CLNs). Moreover, Alcaligenes lipid A promoted PspA-specific CD4+ Th17 responses in the CLNs and spleen. Furthermore, neutrophils were recruited to infection sites upon nasal infection and synchronized with the antigen-specific T and B cell responses, resulting in the protection against S. pneumoniae infection. Taken together, Alcaligenes lipid A could be applied to the prospective adjuvant to enhance nasal vaccine efficacy by means of augmenting both the innate and acquired arms of mucosal immunity against respiratory bacterial infection.

Impaired mucociliary motility enhances antigen-specific nasal IgA immune responses to a cholera toxin-based nasal vaccine.

Nasal mucosal tissues are equipped with physical barriers, mucus and cilia, on their surface. The mucus layer captures inhaled materials, and the cilia remove the inhaled materials from the epithelial layer by asymmetrical beating. The effect of nasal physical barriers on the vaccine efficacy remains to be investigated. Tubulin tyrosine ligase-like family, member 1 (Ttll1) is an essential enzyme for appropriate movement of the cilia on respiratory epithelium, and its deficiency (Ttll1-KO) leads to mucus accumulation in the nasal cavity. Here, when mice were intra-nasally immunized with pneumococcal surface protein A (PspA, as vaccine antigen) together with cholera toxin (CT, as mucosal adjuvant), Ttll1-KO mice showed higher levels of PspA-specific IgA in the nasal wash and increased numbers of PspA-specific IgA-producing plasma cells in the nasal passages when compared with Ttll1 hetero (He) mice. Mucus removal by N-acetylcysteine did not affect the enhanced immune responses in Ttll1-KO mice versus Ttll1-He mice. Immunohistological and flow cytometry analyses revealed that retention time of PspA in the nasal cavity in Ttll1-KO mice was longer than that in Ttll1-He mice. Consistently, uptake of PspA by dendritic cells was higher in the nasopharynx-associated lymphoid tissue (NALT) of Ttll1-KO mice than that of Ttll1-He mice. These results indicate that the ciliary function of removing vaccine antigen from the NALT epithelial layer is a critical determinant of the efficacy of nasal vaccine.

Clostridium perfringens enterotoxin-based protein engineering for the vaccine design and delivery system.

Clostridium perfringens is a major cause of food poisoning worldwide, with its enterotoxin (CPE) being the major virulence factor. The C-terminus of CPE (C-CPE) is non-toxic and is the part of the toxin that binds to epithelial cells via the claudins in tight junctions; however, C-CPE has low antigenicity. To address this issue, we have used protein engineering technology to augment the antigenicity of C-CPE and have developed a C-CPE-based vaccine against C. perfringens-mediated food poisoning. Moreover, C-CPE has properties that make it potentially useful for the development of vaccines against other bacterial toxins that cause food poisoning. For example, we hypothesized that the ability of C-CPE to bind to claudins could be harnessed to deliver vaccine antigens directly to mucosa-associated lymphoid tissues, and we successfully developed a nasally administered C-CPE-based vaccine delivery system that promotes antigen-specific mucosal and systemic immune responses. In addition, our group has revealed the roles that the nasal mucus plays in lowering the efficacy of C-CPE-based nasal vaccines. Here, we review recent advances in the development of C-CPE-based vaccines against the major bacterial toxins that cause food poisoning and discuss our C-CPE-based nasal vaccine delivery system.

Impaired airway mucociliary function reduces antigen-specific IgA immune response to immunization with a claudin-4-targeting nasal vaccine in mice.

Vaccine delivery is an essential element for the development of mucosal vaccine, but it remains to be investigated how physical barriers such as mucus and cilia affect vaccine delivery efficacy. Previously, we reported that C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) targeted claudin-4, which is expressed by the epithelium associated with nasopharynx-associated lymphoid tissue (NALT), and could be effective as a nasal vaccine delivery. Mice lacking tubulin tyrosine ligase-like family, member 1 (Ttll1-KO mice) showed mucus accumulation in nasal cavity due to the impaired motility of respiratory cilia. Ttll1-KO mice nasally immunized with C-CPE fused to pneumococcal surface protein A (PspA-C-CPE) showed reduced PspA-specific nasal IgA responses, impaired germinal center formation, and decreased germinal center B-cells and follicular helper T cells in the NALT. Although there was no change in the expression of claudin-4 in the NALT epithelium in Ttll1-KO mice, the epithelium was covered by a dense mucus that prevented the binding of PspA-C-CPE to NALT. However, administration of expectorant N-acetylcysteine removed the mucus and rescued the PspA-specific nasal IgA response. These results show that the accumulation of mucus caused by impaired respiratory cilia function is an interfering factor in the C-CPE-based claudin-4-targeting nasal vaccine.