Tomoko Kohda

A novel M cell–specific carbohydrate-targeted mucosal vaccine effectively induces antigen-specific immune responses

Mucosally ingested and inhaled antigens are taken up by membranous or microfold cells (M cells) in the follicle-associated epithelium of Peyers patches or nasopharynx-associated lymphoid tissue. We established a novel M cell–specific monoclonal antibody (mAb NKM 16–2-4) as a carrier for M cell–targeted mucosal vaccine. mAb NKM 16–2-4 also reacted with the recently discovered villous M cells, but not with epithelial cells or goblet cells. Oral administration of tetanus toxoid (TT)– or botulinum toxoid (BT)–conjugated NKM 16–2-4, together with the mucosal adjuvant cholera toxin, induced high-level, antigen-specific serum immunoglobulin (Ig) G and mucosal IgA responses. In addition, an oral vaccine formulation of BT-conjugated NKM 16–2-4 induced protective immunity against lethal challenge with botulinum toxin. An epitope analysis of NKM 16–2-4 revealed specificity to an α(1,2)-fucose–containing carbohydrate moiety, and reactivity was enhanced under sialic acid–lacking conditions. This suggests that NKM 16–2-4 distinguishes α(1,2)-fucosylated M cells from goblet cells containing abundant sialic acids neighboring the α(1,2) fucose moiety and from non-α(1,2)-fucosylated epithelial cells. The use of NKM 16–2-4 to target vaccine antigens to the M cell–specific carbohydrate moiety is a new strategy for developing highly effective mucosal vaccines.

Characterization of the Neurotoxin Produced by Isolates Associated with Avian Botulism

Abstract Several varieties of birds are affected by type C botulism. We conducted neutralization tests of culture supernatants of isolates from cases of avian botulism. Whereas the toxin produced by isolates derived from mammalian botulism was neutralized only with type C antitoxin, the toxins of all isolates related to avian botulism were neutralized with both type C and D antitoxins. An analysis of nucleotide sequences with several strains revealed that the neurotoxin gene in the isolates from avian botulism comprises two thirds of the type C neurotoxin gene and one third of the type D neurotoxin gene. This indicates that the neurotoxin of avian isolates is a mosaic of type C and D neurotoxins. We prepared three sets of primers to differentiate the gene for the mosaic form from the conserved genes of type C and D neurotoxins. The results of polymerase chain reaction with these primers indicated that all avian botulism–related isolates and specimens possess the gene for the mosaic form of the neurotoxin. The toxins purified from avian and mammalian isolates exhibited the same degree of lethality in mice, but the former showed greater toxicity to chickens than the latter. These results indicate that the mosaic neurotoxin is probably a pathogenic agent causing some forms of avian botulism.

Characterization of the D/C mosaic neurotoxin produced by Clostridium botulinum associated with bovine botulism in Japan

Clostridium botulinum types C and D are related to avian and mammalian botulism. Bovine botulism occurred at various farms from 2004 to 2007 in Japan. Since culture supernatants of isolates from cases of bovine botulism were neutralized completely and partially with type D and C antitoxins, respectively, we attempted to confirm the nucleotide sequences of the neurotoxin gene in isolates. The neurotoxin gene comprised two-thirds of the type D neurotoxin gene and one-third of the type C neurotoxin gene, indicating that the neurotoxin of bovine isolates is a mosaic of type D and C neurotoxins, D/C mosaic neurotoxin. We prepared four sets of primers to differentiate the genes of the mosaic and authentic forms with PCR. The results showed that all bovine botulism-related isolates possess the gene for the D/C mosaic form. Pulsed-field gel electrophoresis analysis demonstrated that isolates from bovine botulism which had occurred between 2004 and 2007 were genetically homologous, except for the isolate from one area. We further examined the biological and antigenic properties of the D/C mosaic neurotoxin, which was found to exhibit the highest lethal activity in mice compared with other types of neurotoxins. In the D/C mosaic neurotoxin, three epitopes recognized by monoclonal antibodies that specifically react to and neutralize the toxin were located in the carboxyl-terminal domain of the heavy chain. These results indicate that D/C mosaic neurotoxin is a pathogenic agent causing bovine botulism and has unique characteristics different from other type C and D neurotoxins.

Sequence of the gene for Clostridium botulinum type B neurotoxin associated with infant botulism, expression of the C-terminal half of heavy chain and its binding activity

Previously, we demonstrated that the neurotoxin of strain 111 (111/NT) associated with type B infant botulism showed antigenic and biological properties different from that (Okra/NT) produced by a foodborne botulism-related strain, Okra. In this study, the neurotoxin genes of 111/NT and Okra/NT were amplified and the sequences determined. The nucleotide sequences of the genes for both neurotoxins possessed an open reading frame of 3873 bp that encoded 1291 amino acids. The identities of nucleotide sequences and amino acid sequences were 97.6% and 95.7%, respectively. The ratio of nonsynonymous to synonymous substitutions was 0.47. The amino acid substitutions between 111/NT and Okra/NT occurred mainly in the domain of the C-terminal half of heavy chain (H(C)) responsible for binding to its receptor complex of protein and ganglioside. To characterize the binding capability of the H(C), recombinant genes for the H(C) and two hybrid H(C) in which one half of Okra/NT was replaced by the homologous half of 111/NT were constructed and expressed in Escherichia coli. The binding activity of the recombinant H(C) of 111/NT to the protein receptor synaptotagmin II, in the presence of ganglioside GT1b, was 4.2-fold less than Okra/NT, consistent with the corresponding two NTs. The use of hybrid H(C) revealed that mutation of 23 residues in carboxy terminal half of H(C) (1029-1291) of Okra/NT could be attributed to the lower binding activity of 111/NT and thus the differences in binding affinity between the two BoNT/B.

Genetic Characterization of Clostridium botulinum Associated with Type B Infant Botulism in Japan

ABSTRACT The 15 proteolytic Clostridium botulinum type B strains, including 3 isolates associated with infant botulism in Japan, were genetically characterized by phylogenetic analysis of boNT/B gene sequences, genotyping, and determination of the boNT/B gene location by using pulsed-field gel electrophoresis (PFGE) for molecular epidemiological analysis of infant botulism in Japan. Strain Osaka05, isolated from a case in 2005, showed a unique boNT/B gene sequence and was considered to be a new BoNT/B subtype by phylogenetic analysis. Strain Osaka06, isolated from a case in 2006, was classified as the B2 subtype, the same as strain 111, isolated from a case in 1995. The five isolates associated with infant botulism in the United States were classified into the B1 subtype. Isolates from food samples in Japan were divided into the B1 and the B2 subtypes, although no relation with infant botulism was shown by PFGE genotyping. The results of PFGE and Southern blot hybridization with undigested DNA suggested that the boNT/B gene is located on large plasmids (approximately 150 kbp, 260 kbp, 275 kbp, or 280 kbp) in five strains belonging to three BoNT/B subtypes from various sources. The botulinum neurotoxin (BoNT) of Osaka05 was suggested to have an antigenicity different from the antigenicities of BoNT/B1 and BoNT/B2 by a sandwich enzyme-linked immunosorbent assay with the recombinant BoNT/B-C-terminal domain. We established a multiplex PCR assay for BoNT/B subtyping which will be useful for epidemiological studies of type B strains and the infectious diseases that they cause.

Identification of the receptor-binding sites in the carboxyl-terminal half of the heavy chain of botulinum neurotoxin types C and D

Botulinum neurotoxin (BoNT) binds to presynaptic neuronal cells and blocks neurotransmitter release. The carboxyl-terminal half of the heavy chain (H(C)) of the neurotoxin recognizes its specific receptor on the plasma membrane. We have previously demonstrated that BoNT/C binds to gangliosides GD1b and GT1b under physiological conditions, while BoNT/D interacts with phosphatidylethanolamine (PE). Here we report that the recognition sites for gangliosides and PE are present in the carboxyl-terminal domain of H(C). Chimeric mutants and site-directed mutants of BoNT/C-H(C) and BoNT/D-H(C) were generated and their binding activities evaluated. The chimeric H(C) that consisted of the amino-terminal half of BoNT/D-H(C) and the carboxyl-terminal half of BoNT/C-H(C) possessed activity similar to the authentic BoNT/C-H(C), suggesting that the carboxyl-terminal region of H(C) is involved in the receptor recognition of BoNT/C. Moreover, analysis using site-directed mutants indicated that the peptide motif W(1257)Ycdots, three dots, centeredG(1270)cdots, three dots, centeredH(1282) plays an important role in the interaction between BoNT/C and gangliosides. In contrast, we revealed that two lysine residues of BoNT/D-H(C) are involved in the formation of the critical binding site for receptor binding.

Botulinum toxin A complex exploits intestinal M cells to enter the host and exert neurotoxicity

To cause food-borne botulism, botulinum neurotoxin (BoNT) in the gastrointestinal lumen must traverse the intestinal epithelial barrier. However, the mechanism by which BoNT crosses the intestinal epithelial barrier remains unclear. BoNTs are produced along with one or more non-toxic components, with which they form progenitor toxin complexes (PTCs). Here we show that serotype A1 L-PTC, which has high oral toxicity and makes the predominant contribution to causing illness, breaches the intestinal epithelial barrier from microfold (M) cells via an interaction between haemagglutinin (HA), one of the non-toxic components, and glycoprotein 2 (GP2). HA strongly binds to GP2 expressed on M cells, which do not have thick mucus layers. Susceptibility to orally administered L-PTC is dramatically reduced in M-cell-depleted mice and GP2-deficient (Gp2−/−) mice. Our finding provides the basis for the development of novel antitoxin therapeutics and delivery systems for oral biologics.

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.

A Novel Neurotoxoid Vaccine Prevents Mucosal Botulism

The threat posed by botulism, classically a food- and waterborne disease with a high morbidity and mortality, has increased exponentially in an age of bioterrorism. Because botulinum neurotoxin (BoNT) could be easily disseminated by terrorists using an aerosol or could be used to contaminate the food or water supply, the Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases has classified it as a category A agent. Although clearly the development of a safe and effective mucosal vaccine against this toxin should be a high priority, essentially no studies to date have assessed mucosal immune responses to this disease. To bridge this gap in our knowledge, we immunized mice weekly for 4 wk with nasal doses of BoNT type A toxoid and a mutant of cholera toxin termed E112K. We found elevated levels of BoNT-specific IgG Abs in plasma and of secretory IgA Abs in external secretions (nasal washes, saliva, and fecal extracts). When mice given nasal BoNT vaccine were challenged with 4 × 103 LD50 of BoNT type A (BoNT/A) via the i.p. route, complete protection was seen, while naive mice given the same dosage died within 2 h. To further confirm the efficacy of this nasal BoNT vaccine, an oral LD50 was determined. When mice were given an oral challenge of 5 μg (2 × oral LD50) of progenitor BoNT/A, all immunized mice survived beyond 5 days, while nonimmunized mice did not. The fecal extract samples from nasally vaccinated mice were found to contain neutralizing secretory IgA Abs. Taken together, these results show that nasal BoNT/A vaccine effectively prevents mucosal BoNT intoxication.

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.