Shiho Kurokawa

Comprehensive Gene Expression Profiling of Peyer’s Patch M Cells, Villous M-Like Cells, and Intestinal Epithelial Cells

Separate populations of M cells have been detected in the follicle-associated epithelium of Peyer’s patches (PPs) and the villous epithelium of the small intestine, but the traits shared by or distinguishing the two populations have not been characterized. Our separate study has demonstrated that a potent mucosal modulator cholera toxin (CT) can induce lectin Ulex europaeus agglutinin-1 and our newly developed M cell-specific mAb NKM 16-2-4-positive M-like cells in the duodenal villous epithelium. In this study, we determined the gene expression of PP M cells, CT-induced villous M-like cells, and intestinal epithelial cells isolated by a novel approach using FACS. Additional mRNA and protein analyses confirmed the specific expression of glycoprotein 2 and myristoylated alanine-rich C kinase substrate (MARCKS)-like protein by PP M cells but not CT-induced villous M-like cells. Comprehensive gene profiling also suggested that CT-induced villous M-like cells share traits of both PP M cells and intestinal epithelial cells, a finding that is supported by their unique expression of specific chemokines. The genome-wide assessment of gene expression facilitates discovery of M cell-specific molecules and enhances the molecular understanding of M cell immunobiology.

Secretory IgA-mediated protection against V. cholerae and heat-labile enterotoxin-producing enterotoxigenic Escherichia coli by rice-based vaccine.

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 heat-labile 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 CT- or 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 CTB-specific SIgA-mediated longstanding protection against V. cholerae– or LT-ETEC–induced diarrhea.

Nanogel-Based PspA Intranasal Vaccine Prevents Invasive Disease and Nasal Colonization by Streptococcus pneumoniae

ABSTRACT To 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.

Rice-based oral antibody fragment prophylaxis and therapy against rotavirus infection

Rotavirus-induced diarrhea is a life-threatening disease in immunocompromised individuals and in children in developing countries. We have developed a system for prophylaxis and therapy against rotavirus disease using transgenic rice expressing the neutralizing variable domain of a rotavirus-specific llama heavy-chain antibody fragment (MucoRice-ARP1). MucoRice-ARP1 was produced at high levels in rice seeds using an overexpression system and RNAi technology to suppress the production of major rice endogenous storage proteins. Orally administered MucoRice-ARP1 markedly decreased the viral load in immunocompetent and immunodeficient mice. The antibody retained in vitro neutralizing activity after long-term storage (>1 yr) and boiling and conferred protection in mice even after heat treatment at 94°C for 30 minutes. High-yield, water-soluble, and purification-free MucoRice-ARP1 thus forms the basis for orally administered prophylaxis and therapy against rotavirus infections.

A Rice-Based Oral Cholera Vaccine Induces Macaque-Specific Systemic Neutralizing Antibodies but Does Not Influence Pre-Existing Intestinal Immunity

We previously showed that oral immunization of mice with a rice-based vaccine expressing cholera toxin (CT) B subunit (MucoRice-CT-B) induced CT-specific immune responses with toxin-neutralizing activity in both systemic and mucosal compartments. In this study, we examined whether the vaccine can induce CT-specific Ab responses in nonhuman primates. Orally administered MucoRice-CT-B induced high levels of CT-neutralizing serum IgG Abs in the three cynomolgus macaques we immunized. Although the Ab level gradually decreased, detectable levels were maintained for at least 6 mo, and high titers were rapidly recovered after an oral booster dose of the rice-based vaccine. In contrast, no serum IgE Abs against rice storage protein were induced even after multiple immunizations. Additionally, before immunization the macaques harbored intestinal secretory IgA (SIgA) Abs that reacted with both CT and homologous heat-labile enterotoxin produced by enterotoxigenic Escherichia coli and had toxin-neutralizing activity. The SIgA Abs were present in macaques 1 mo to 29 years old, and the level was not enhanced after oral vaccination with MucoRice-CT-B or after subsequent oral administration of the native form of CT. These results show that oral MucoRice-CT-B can effectively induce CT-specific, neutralizing, serum IgG Ab responses even in the presence of pre-existing CT- and heat-labile enterotoxin-reactive intestinal SIgA Abs in nonhuman primates.

Oral MucoRice expressing double-mutant cholera toxin A and B subunits induces toxin-specific neutralising immunity.

Rice-expressed cholera toxin B (CTB) subunit is a cold-chain-free oral vaccine that effectively induces enterotoxin-neutralising immunity. We created another rice-based vaccine, MucoRice, expressing nontoxic double-mutant cholera toxin (dmCT) with CTA and CTB subunits. Western-blot analysis suggested that MucoRice-dmCT had the shape of a multicomponent vaccine. Oral administration of MucoRice-dmCT induced CTB- but not CTA-specific serum IgG and mucosal IgA antibodies, generating protective immunity against cholera toxin without inducing rice-protein-specific antibody responses. The potency of MucoRice-dmCT was equal to that of MucoRice-CTB vaccine. MucoRice has the potential to be used as a safe multicomponent vaccine expression system.

Distinct fucosylation of M cells and epithelial cells by Fut1 and Fut2, respectively, in response to intestinal environmental stress

The intestinal epithelium contains columnar epithelial cells (ECs) and M cells, and fucosylation of the apical surface of ECs and M cells is involved in distinguishing the two populations and in their response to commensal flora and environmental stress. Here, we show that fucosylated ECs (F-ECs) were induced in the mouse small intestine by the pro-inflammatory agents dextran sodium sulfate and indomethacin, in addition to an enteropathogen derived cholera toxin. Although F-ECs showed specificity for the M cell-markers, lectin Ulex europaeus agglutinin-1 and our monoclonal antibody NKM 16-2-4, these cells also retained EC-phenotypes including an affinity for the EC-marker lectin wheat germ agglutinin. Interestingly, fucosylation of Peyers patch M cells and F-ECs was distinctly regulated by α(1,2)fucosyltransferase Fut1 and Fut2, respectively. These results indicate that Fut2-mediated F-ECs share M cell-related fucosylated molecules but maintain distinctive EC characteristics, Fut1 is, therefore, a reliable marker for M cells.

Nanogel-based pneumococcal surface protein A nasal vaccine induces microRNA-associated Th17 cell responses with neutralizing antibodies against Streptococcus pneumoniae in macaques

We previously established a nanosized nasal vaccine delivery system by using a cationic cholesteryl group-bearing pullulan nanogel (cCHP nanogel), which is a universal protein-based antigen-delivery vehicle for adjuvant-free nasal vaccination. In the present study, we examined the central nervous system safety and efficacy of nasal vaccination with our developed cCHP nanogel containing pneumococcal surface protein A (PspA-nanogel) against pneumococcal infection in nonhuman primates. When [18F]-labeled PspA-nanogel was nasally administered to a rhesus macaque (Macaca mulatta), longer-term retention of PspA was noted in the nasal cavity when compared with administration of PspA alone. Of importance, no deposition of [18F]-PspA was seen in the olfactory bulbs or brain. Nasal PspA-nanogel vaccination effectively induced PspA-specific serum IgG with protective activity and mucosal secretory IgA (SIgA) Ab responses in cynomolgus macaques (Macaca fascicularis). Nasal PspA-nanogel-induced immune responses were mediated through T-helper (Th) 2 and Th17 cytokine responses concomitantly with marked increases in the levels of miR-181a and miR-326 in the serum and respiratory tract tissues, respectively, of the macaques. These results demonstrate that nasal PspA-nanogel vaccination is a safe and effective strategy for the development of a nasal vaccine for the prevention of pneumonia in humans.

RNAi suppression of rice endogenous storage proteins enhances the production of rice-based Botulinum neutrotoxin type A vaccine.

Mucosal vaccines based on rice (MucoRice) offer a highly practical and cost-effective strategy for vaccinating large populations against mucosal infections. However, the limitation of low expression and yield of vaccine antigens with high molecular weight remains to be overcome. Here, we introduced RNAi technology to advance the MucoRice system by co-introducing antisense sequences specific for genes encoding endogenous rice storage proteins to minimize storage protein production and allow more space for the accumulation of vaccine antigen in rice seed. When we used RNAi suppression of a combination of major rice endogenous storage proteins, 13 kDa prolamin and glutelin A in a T-DNA vector, we could highly express a vaccine comprising the 45 kDa C-terminal half of the heavy chain of botulinum type A neurotoxin (BoHc), at an average of 100 μg per seed (MucoRice-BoHc). The MucoRice-Hc was water soluble, and was expressed in the cytoplasm but not in protein body I or II of rice seeds. Thus, our adaptation of the RNAi system improved the yield of a vaccine antigen with a high molecular weight. When the mucosal immunogenicity of the purified MucoRice-BoHc was examined, the vaccine induced protective immunity against a challenge with botulinum type A neurotoxin in mice. These findings demonstrate the efficiency and utility of the advanced MucoRice system as an innovative vaccine production system for generating highly immunogenic mucosal vaccines of high-molecular-weight antigens.

In Vivo Molecular Imaging Analysis of a Nasal Vaccine That Induces Protective Immunity against Botulism in Nonhuman Primates

Nasal administration is an effective route for a needle-free vaccine. However, nasally administered Ags have the potential to reach the CNS directly from the nasal cavity, thus raising safety concerns. In this study, we performed real-time quantitative tracking of a nasal vaccine candidate for botulism, which is a nontoxic subunit fragment of Clostridium botulinum type A neurotoxin (BoHc/A) effective in the induction of the toxin-neutralizing immune response, by using 18F-labeled BoHc/A–positron-emission tomography, an in vivo molecular imaging method. This method provides results that are consistent with direct counting of [18F] radioactivity or the traditional [111In]-radiolabel method in dissected tissues of mice and nonhuman primates. We found no deposition of BoHc/A in the cerebrum or olfactory bulb after nasal administration of 18F-labeled BoHc/A in both animals. We also established a real-time quantitative profile of elimination of this nasal vaccine candidate and demonstrated that it induces highly protective immunity against botulism in nonhuman primates. Our findings demonstrate the efficiency and safety of a nasal vaccine candidate against botulism in mice and nonhuman primates using in vivo molecular imaging.