Nanotechnology is an innovative method of freely controlling nanometre-sized materials. Recent outbreaks of mucosal infectious diseases have increased the demands for development of mucosal vaccines because they induce both systemic and mucosal antigen-specific immune responses. Here we developed an intranasal vaccine-delivery system with a nanometre-sized hydrogel ('nanogel') consisting of a cationic type of cholesteryl-group-bearing pullulan (cCHP). A non-toxic subunit fragment of Clostridium botulinum type-A neurotoxin BoHc/A administered intranasally with cCHP nanogel (cCHP-BoHc/A) continuously adhered to the nasal epithelium and was effectively taken up by mucosal dendritic cells after its release from the cCHP nanogel. Vigorous botulinum-neurotoxin-A-neutralizing serum IgG and secretory IgA antibody responses were induced without co-administration of mucosal adjuvant. Importantly, intranasally administered cCHP-BoHc/A did not accumulate in the olfactory bulbs or brain. Moreover, intranasally immunized tetanus toxoid with cCHP nanogel induced strong tetanus-toxoid-specific systemic and mucosal immune responses. These results indicate that cCHP nanogel can be used as a universal protein-based antigen-delivery vehicle for adjuvant-free intranasal vaccination.

4 of both GALT and nasopharynx-associated lymphoid tissue, which act as a major inductive site for Ag-specific mucosal immune responses (1, 2). Recently, we also identified M cells in the small intestinal villous epithelium, at effector sites far from the FAE, suggesting that Ag sampling via villous M cells may be responsible for induction of systemic Ag-specific immune responses, such as IgG production via the oral route (3). Still missing, however, were a characterization of the shared and distinctive traits of Peyer's patches (PPs) and villous M cells and a better understanding of the immunological nature of each.Recent comprehensive gene expression analyses using microdissected FAE or whole cells dissociated from the FAE identified genes specifically expressed by PP M cells (4 -6). Similar data, however, have not been available for villous M cells, in part because sufficient numbers of M cells are difficult to isolate from the surrounding intestinal epithelial cells (IECs). In mice, lectin Ulex europaeus agglutinin-1 (UEA-1) possessing affinity for ␣ (1, 2) fucose has been routinely used for the detection of such M cells (3, 7). UEA-1, however, does not alone suffice to identify M cells because it also reacts to goblet cells (3). Our laboratory has recently succeeded in distinguishing M cells from goblet cells by developing a mAb (NKM 16-2-4 mAb) that specifically reacts to murine PP and villous M cells but not goblet cells and IECs (8). Furthermore, our recent separate studies have demonstrated that oral administration of cholera toxin (CT) as mucosal adjuvant resulted in the induction of NKM 16-2-4 mAb ϩ and UEA-1 ϩ M-like cells, which have pocket structure and Ag uptake ability, in the duodenal villous epithelium (Terahara et al., submitted for publication). These recent advances in our understanding of M cells allowed us to define gene expression profiles capable of distinguishing PP M cells, CT-induced villous M-like cells, and IECs. Materials and Methods AnimalsBALB/c mice were purchased from Japan SLC. These mice were maintained under specific pathogen-free conditions in horizontal flow cabinets in our experimental animal facility at the University of Tokyo. Following a previously established protocol (9, 10), CT (List Biologic Laboratories) was dissolved in PBS (20 g/mouse) and then orally administered to BALB/c mice. Two days after CT administration, mice were used for experiments. All animal experiments were approved by the Animal Care and Use Committee of University of Tokyo. Lectins and Abs for the detection of M cellsThe following fluorescence-conjugated lectins and Abs were used for the identification of PP and villous M cells by FACS and histochemistry: PEconjugated UEA-1 (Biogenesis), rhodamine-conjugated UEA-1 (Vector

Cholera and enterotoxigenic Escherichia coli (ETEC) are among the most common causes of acute infantile gastroenteritis globally. We previously developed a rice-based vaccine that expressed cholera toxin B subunit (MucoRice-CTB) and had the advantages of being cold chain-free and providing protection against cholera toxin (CT)-induced diarrhea. To advance the development of MucoRice-CTB for human clinical application, we investigated whether the CTB-specific secretory IgA (SIgA) induced by MucoRice-CTB gives longstanding protection against diarrhea induced by Vibrio cholerae and heatlabile enterotoxin (LT)-producing ETEC (LT-ETEC) in mice. Oral immunization with MucoRice-CTB stored at room temperature for more than 3 y provided effective SIgA-mediated protection against CTor LT-induced diarrhea, but the protection was impaired in polymeric Ig receptor-deficient mice lacking SIgA. The vaccine gave longstanding protection against CT-or LT-induced diarrhea (for ≥6 months after primary immunization), and a single booster immunization extended the duration of protective immunity by at least 4 months. Furthermore, MucoRice-CTB vaccination prevented diarrhea in the event of V. cholerae and LT-ETEC challenges. Thus, MucoRice-CTB is an effective long-term cold chain-free oral vaccine that induces CTBspecific SIgA-mediated longstanding protection against V. choleraeor LT-ETEC-induced diarrhea. cholera toxin B subunit | mucosal vaccine | oral vaccine | plant-made vaccine | MucoRice

kTo establish a safer and more effective vaccine against pneumococcal respiratory infections, current knowledge regarding the antigens common among pneumococcal strains and improvements to the system for delivering these antigens across the mucosal barrier must be integrated. We developed a pneumococcal vaccine that combines the advantages of pneumococcal surface protein A (PspA) with a nontoxic intranasal vaccine delivery system based on a nanometer-sized hydrogel (nanogel) consisting of a cationic cholesteryl group-bearing pullulan (cCHP). The efficacy of the nanogel-based PspA nasal vaccine (cCHP-PspA) was tested in murine pneumococcal airway infection models. Intranasal vaccination with cCHP-PspA provided protective immunity against lethal challenge with Streptococcus pneumoniae Xen10, reduced colonization and invasion by bacteria in the upper and lower respiratory tracts, and induced systemic and nasal mucosal Th17 responses, high levels of PspA-specific serum immunoglobulin G (IgG), and nasal and bronchial IgA antibody responses. Moreover, there was no sign of PspA delivery by nanogel to either the olfactory bulbs or the central nervous system after intranasal administration. These results demonstrate the effectiveness and safety of the nanogel-based PspA nasal vaccine system as a universal mucosal vaccine against pneumococcal respiratory infection.

SummaryGut epithelial organoids are routinely used to investigate intestinal biology; however, current culture methods are not amenable to genetic manipulation, and it is difficult to generate sufficient numbers for high-throughput studies. Here, we present an improved culture system of human induced pluripotent stem cell (iPSC)-derived intestinal organoids involving four methodological advances. (1) We adopted a lentiviral vector to readily establish and optimize conditioned medium for human intestinal organoid culture. (2) We obtained intestinal organoids from human iPSCs more efficiently by supplementing WNT3A and fibroblast growth factor 2 to induce differentiation into definitive endoderm. (3) Using 2D culture, followed by re-establishment of organoids, we achieved an efficient transduction of exogenous genes in organoids. (4) We investigated suspension organoid culture without scaffolds for easier harvesting and assays. These techniques enable us to develop, maintain, and expand intestinal organoids readily and quickly at low cost, facilitating high-throughput screening of pathogenic factors and candidate treatments for gastrointestinal diseases.