Kunio Tochikubo

Recombinant cholera toxin B subunit acts as an adjuvant for the mucosal and systemic responses of mice to mucosally co-administered bovine serum albumin.

We examined the mucosal adjuvant activity of recombinant cholera toxin B subunit (rCTB) produced by Bacillus brevis carrying pNU212-CTB by intranasal or oral co-administration of bovine serum albumin (BSA). Intranasal administration stimulated a high level of BSA-specific serum IgG antibody response and BSA-specific IgA antibody responses in the nasal and pulmonary lavages. Oral administration induced a moderate level of BSA-specific serum IgG antibody and a low level of BSA-specific IgA antibody in the large intestinal washes. These results show that CTB alone can act as an intranasal or oral delivery carrier; it also has strong adjuvant properties for stimulating serum IgG and mucosal IgA immune responses to unrelated, non-coupled antigens after intranasal or oral co-immunization.

Mucosal immunization against hepatitis B virus by intranasal co-administration of recombinant hepatitis B surface antigen and recombinant cholera toxin B subunit as an adjuvant

Recombinant cholera toxin B subunit (rCTB) produced by Bacillus brevis carrying pNU212-CTB has been previously found to be a potent mucosal adjuvant to aluminium-non-adsorbed tetanus toxoid (nTT) and diphtheria toxoid (nDT) co-administered intranasally, and the possibility of needle-free inoculation of these vaccines with rCTB has been suggested. In this paper we examined the potentiality of rCTB as a mucosal adjuvant to aluminium-non-adsorbed yeast-derived recombinant hepatitis B surface antigen (rHBs) being a particulate antigen when administered intranasally with rCTB. In-house ELISA showed that a mixture of rHBs (1 or 5 microg) and rCTB (10 microg) elevated not only systemic responses but also mucosal immune responses at the nasal cavity, the lung, the saliva, the small intestine and the vagina against rHBs, and these could be further increased with higher doses of antigen. With antibody isotypes of IgG, there were equally high levels of serum HBs-specific IgG1, IgG2a and IgG2b antibodies and induction of mixed Th1- and Th2-type responses was considered to occur in combination of rHBs and rCTB. Serum anti-HBs titres in almost all mice obtained from sandwich EIA using a commercial kit were higher than 1000 milli-international units ml(-1) (mIU ml(-1)). These results show that rCTB is also very effective as a mucosal adjuvant for a particulate antigen like rHBs, as well as soluble antigens like nTT and nDT reported previously, suggesting the possibility of intranasal immunization with rHBs plus rCTB in humans.

Induction of systemic and mucosal antibody responses in mice immunized intranasally with aluminium-non-adsorbed diphtheria toxoid together with recombinant cholera toxin B subunit as an adjuvant

Nasal mucosal immunization is very attractive for vaccination to prevent various bacterial and viral infectious diseases because of induction of systemic and mucosal immune responses. The aim of the present study was to investigate the possibility of changing the immunization procedure of diphtheria toxoid (DT) from intramuscular or subcutaneous injection to intranasal administration. Intranasal immunization with aluminium-non-adsorbed diphtheria toxoid (nDT) together with recombinant cholera toxin B subunit (rCTB, 10 microg) induced, at a concentration of 5 Lf, high levels of serum DT-specific IgG antibody responses and high or moderate levels of the specific IgA antibody responses in all mice and only a slight level of the specific IgE antibody responses in some mice. Furthermore, sufficiently high diphtheria antitoxin titres more than 0.1 international units (IU) ml(-1) were obtained from mice which showed high levels of serum DT-specific IgG antibody responses. Under the same experimental conditions, induction of significant levels of mucosal DT-specific IgA antibody responses occurred in the nasal cavity, the lung, the saliva and vaginal secretions and the small and large intestines of all mice, although there were different titres between individual mice. Similar results were also obtained with rCTB-specific serum IgG and IgA and mucosal IgA antibody responses; serum rCTB-specific IgE antibody titres were not detected. These results show that intranasal administration of nDT with rCTB must be a very useful means for vaccination against diphtheria.

Systemic and mucosal immune responses of mice to aluminium-adsorbed or aluminium-non-adsorbed tetanus toxoid administered intranasally with recombinant cholera toxin B subunit.

For the purpose of changing the immunization procedure of tetanus toxoid from intramuscular or subcutaneous injection, which has been in practice for a long time, to intranasal administration, we examined systemic and mucosal immune responses of mice to aluminium-adsorbed tetanus toxoid (aTT) and aluminium-non-adsorbed tetanus toxoid (nTT) inoculated intranasally with recombinant cholera toxin B subunit (rCTB). Intranasal immunization with aTT induced, at a concentration of 0.5 Lf, high levels of TT-specific serum IgG antibody titres and moderate levels of TT-specific serum IgA antibody titres in the presence and absence of rCTB. Induction of high or moderate levels of mucosal TT-specific IgA antibody responses was observed with and without rCTB in the lung, the nasal cavity, the small and large intestines and the vagina. Generally speaking, the co-administration of aTT and rCTB showed higher mucosal TT-specific IgA antibody titres when compared with the administration of aTT alone. In case of intranasal administration of nTT, the dose of 5 Lf was necessary and stimulated, only in the presence of rCTB (10 micrograms), high levels of tetanus toxoid (TT)-specific serum IgG antibody responses in all mice examined and moderate or slight levels of TT-specific IgA antibody responses in the nasal, pulmonary and small and large intestinal lavages of a few mice. All mice intranasally immunized with aTT alone or nTT and rCTB escaped onset of tetanus. This is the first report concerned with the mucosal adjuvant activity of an aluminium compound. Judging from these results, intranasal administration of aTT with and without rCTB or nTT with rCTB appears to be a very useful means for a vaccination against tetanus with respect to ease, safety, certainty, low cost and no need for an injection needle.

Safety evaluation of recombinant cholera toxin B subunit produced by Bacillus brevis as a mucosal adjuvant.

Mucosal immune responses are known to play important roles in the establishment of protective immunity to microbial infections through mucosa. We examined the toxic effects of recombinant cholera toxin B subunit (rCTB) secreted by Gram-positive bacterium Bacillus brevis as a mucosal adjuvant. Incubation of guinea-pig peritoneal macrophages with cholera toxin (CT) or aluminium hydroxide gel (Al-gel) released a significantly higher activity of lactate dehydrogenase than did commercial natural CTB (CTB) or rCTB. Intraintestinal or intramuscular administration of CT, CTB or Al-gel caused severe histopathological reactions. CT also caused infiltration of neutrophils and irregular arrangement or partial loss of the respiratory epithelium. In addition, CT and CTB elicited vascular permeability-increasing effects. rCTB elicited no toxic effects to macrophages and no vascular permeability-increasing effects. Moreover, it is noticeable that no distinct local histopathological reactions were observed in the nasal cavity, the small-intestinal loop or the muscle given rCTB. These results suggest that, from a safety standpoint, rCTB is a useful candidate as mucosal vaccine adjuvant.

Immune responses in mice to intranasal and intracutaneous administration of a DNA vaccine encoding Helicobacter pylori-catalase

We previously reported that the intracutaneous injection of DNA vaccines encoding Helicobacter pylori heat shock proteins elicited specific immune responses, and led to reduced infection in mice. In this study, we constructed DNA vaccine encoding H. pylori-catalase (pcDNA3.1-kat) and investigated the immune responses to intranasal and intracutaneous administration of pcDNA3.1-kat. C57/BL6 mice were immunized intracutaneously with 10 microg of pcDNA3.1-kat or intranasally with 50 microg of pcDNA3.1-kat. Catalase-specific IgG antibody was detected in the sera of intranasal and intracutaneous immunized mice. Both intranasal and intracutaneous immunized mice were significantly protected from colonization by H. pylori and had significantly reduced degrees of gastritis. These results demonstrate that DNA vaccine encoding H. pylori-catalase can induce an immune response against H. pylori, and that intranasal immunization works as well as intracutaneous immunization.

Properties and Origin of Filamentous Appendages on Spores of Bacillus cereus

Some physical, chemical, and immunological properties of filamentous appendages and the exosporium on the spores of Bacillus cereus were examined for the purpose of elucidating the origin of filamentous appendages. The main components of both filamentous appendages and the exosporium were protein and their amino acid compositions were similar in point of a high content of glycine, alanine, threonine, valine, and acidic amino acids and a low content of basic and sulphur‐containing amino acids. Treatment with 1 N NaOH at 50 C solubilized the isolated appendages completely and the isolated exosporia partially. In both preparations the solubilized proteins consisted of highly acidic monomeric subunits with molecular weights between 2,000 and 5,000. Treatment of the spores with 2% 2‐mercaptoethanol at 37 C resulted in the isolation of long filamentous appendages without segmentation. When the spores were treated with 10% 2‐mercaptoethanol, there was partial destruction of the exosporium as well as detachment of the filamentous appendages. There was a common antigenic component in the exosporium and the tips of the filamentous appendages. Five strains of B. cereus having a common appendage antigen also had a common exosporium antigen, whereas six other strains had neither a common appendage antigen nor a common exosporium antigen. From these facts it was concluded that the filamentous appendages arose from the exosporium.

Gene Cluster for Assembly of Pilus Colonization Factor Antigen III of Enterotoxigenic Escherichia coli

ABSTRACT The assembly of pilus colonization factor antigen III (CFA/III) of enterotoxigenic Escherichia coli (ETEC) requires the processing of CFA/III major pilin (CofA) by a prepilin peptidase (CofP), similar to other type IV pilus formation systems. CofA is produced initially as a 26.5-kDa preform pilin (prepilin) and then processed to a 20.5-kDa mature pilin by CofP which is predicted to be localized in the inner membrane. In the present experiment, we determined the nucleotide sequence of the whole region for CFA/III formation and identified a cluster of 14 genes, includingcofA and cofP. Several proteins encoded bycof genes were similar to previously described proteins, such as the toxin-coregulated pili of Vibrio cholerae and the bundle-forming pili of enteropathogenic E. coli. The G+C content of the cof gene cluster was 37%, which was significantly lower than the average for the E. coli genome (50%). The introduction of a recombinant plasmid containing thecof gene cluster into the E. coli K-12 strain conferred CFA/III biogenesis and the ability of adhesion to the human colon carcinoma cell line Caco-2. This is the first report of a complete nucleotide sequence of the type IV pili found in human ETEC, and our results provide a useful model for studying the molecular mechanism of CFA/III biogenesis and the role of CFA/III in ETEC infection.

Relation between D‐Glucose and L‐ and D‐Alanine in the Initiation of Germination of Bacillus subtilis Spore

The rate of L‐alanine‐initiated germination of Bacillus subtilis spore was measured by both loss of heat resistance and loss of turbidity, and the effect of glucose on the germination response to a wide range of concentrations of the germinant was analyzed in the presence and absence of D‐alanine, an inhibitor. Glucose stimulated L‐alanine germination by means of a cooperative effect: glucose increased the affinity of L‐alanine by about 3‐fold and the rate of germination by about 1.3‐fold. However, glucose had little effect on the binding affinity of D‐alanine. The apparent binding constant of L‐alanine to the spore, which was determined by the next measurable event in the trigger reaction, was 1.2 × 10−5, that of D‐alanine was 6 × 10−6, and that of glucose was 5 × 10−5. The relation between the binding site for glucose and those for L‐ and D‐alanine on the spore is discussed. Effect of glucose analogs was also examined.

Germination-Initiation and Inhibitory Activities of L-and D-Alanine Analogues for Bacillus subtilis Spores

The ability of 33 compounds of L‐alanine analogues over a wide range of concentrations to initiate germination of Bacillus subtilis spores was determined, and the inhibitory activity against L‐alanine‐initiated germination was determined for the above compounds and 22 of their D‐, DL‐isomers. Nineteen L‐isomers were able to initiate the germination. The maximum germination rate and the apparent binding affinity of the germinant were obtained from concentration‐germination response curves. Not only D‐isomers but also L‐isomers of many alanine analogues showed inhibitory action on L‐alanine‐initiated germination. The apparent binding affinity of an inhibitor was calculated by Schilds method. D‐Alanine, D‐serine, glycine, D‐2‐amino‐n‐butyric acid, D‐cysteine, D‐norvaline, and D‐threonine were competitive inhibitors for the L‐alanine action. Analysis of the relation between the structure of the side chain of L‐, D‐alanine analogues and their apparent affinity suggested that there are separate binding portions, which differ in size and electrostatic nature, for germination and for inhibition on the receptor. Certain L‐alanine analogues had a dualistic property of initiating germination at low concentrations and inhibitory activity at higher concentrations, i.e., autoinhibition. The autoinhibitory phenomenon might be explained by the above postulation of the presence of separate binding portions for germination and for inhibition.