Satoshi Kozuka

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.

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.

Exosporia and Appendages of Spores of Bacillus Species

There have been several reports on the presence of exosporia and appendages on the spores of certain Bacillus species (1, 3, 4). Hachisuka and Kozuka (2) stated that examination for the presence of filamentous appendages on the surface of the exosporium will be a useful diagnostic test for classification of Bacillus cereus and Bacillus anthracis because the spores of B. cereus have these appendages. In this study it was demonstrated that it is possible to divide Bacillus species into four groups on the basis of the presence of the exosporia and the appendages on their spores. Twenty-five strains from 18 species of Bacillus were examined. B. alvei IAM 1258, B. badius IAM 11059, B. brevis IAM 1031, B. cereus IAM 1110, B. circulars IAM 1140, B. firmus IAM 1188, B. lentus IAM 11055, B. licheniformis IAM 11054, B. macerans IAM 1243, B. megaterium IAM 1166, B. polymyxa IAM 12075, B. sphaericus IAM 1286, B. subtilis IAM 1206, B. thiaminoyticus IAM 1034, and B. thuringiensis IAM 11056 and IAM 11064 were obtained from the Institute of Applied Microbiology, The Uni versityof Tokyo, Tokyo. B. alvei IFO 3343, B. brevis IFO 3331, B. macerans IFO 3490, B. polymyxa IFO 3020, and B. pumilus IFO 12102 were obtained from the In stitutefor Fermentation, Osaka. B. anthracis IID 501 was obtained from the Institute of Medical Science, The University of Tokyo, Tokyo. B. licheniformis AHU 1371 was obtained from the Faculty of Agriculture, Hokkaido University, Hokkaido. B. subtilis PCI 219 (ATCC 6633) and a B. terminalis strain, the designation of which is un known,were obtained from the Institute of Infectious Disease, The University of Tokyo, Tokyo, in 1953. Each strain was inoculated on a slant of beef extract agar (Eiken Co., Ltd., To kyo)and cultivated for 5 days at 37C to produce spores. After confirmation of spore formation with a phase contrast microscope, the spores were harvested in distilled water. The spore suspension was washed several times with distilled water by centrifugation (at 1,500•~g for 15min). Spore specimens placed on

Mucosal and systemic antibody responses against an acellular pertussis vaccine in mice after intranasal co-administration with recombinant cholera toxin B subunit as an adjuvant

To investigate the possibility of intranasal immunization with an acellular pertussis vaccine, groups of mice were administered intranasally with aluminium-non-adsorbed pertussis toxoid (PTd; 0.5 or 5 microg) and formalin-treated filamentous hemagglutinin (fFHA; 5 microg) with and without recombinant cholera toxin B subunit (rCTB; 10 microg) as a mucosal adjuvant. At a low concentration of PTd, the following things became clear: (1) earlier and higher elevation of serum anti-PTd and anti-FHA IgG antibody titres in the presence of rCTB than in its absence, (2) higher serum anti-PTd and anti-FHA IgG antibody titres than 200 and 100 ELISA units ml(-1) (EU ml(-1)) in all mice, respectively, in the presence of rCTB, which were obtained by calibration against a reference anti-pertussis mouse serum, and (3) in an intranasal challenge experiment with Bordetella pertussis, slightly more rapid elimination of the bacteria from the lungs of mice intranasally immunized in the presence of rCTB, suggesting the effectiveness of rCTB as a mucosal adjuvant. However, irrespective of rCTB and dose of PTd, mice which were immunized four times and sacrificed on day 35 developed high levels of anti-PTd serum IgG antibodies, high or moderate levels of anti-FHA serum IgG antibodies and mucosal anti-PTd IgA antibodies in the lungs; only a slight or no increase of anti-FHA mucosal IgA antibodies was observed in the lung. These facts suggested the immunogenicity and mucosal adjuvanticity of PTd, and therefore, the mucosal adjuvanticity of rCTB seemed to be inconspicuous. Moreover, the addition of rCTB induced higher anti-PTd serum IgE antibody responses than no addition of it depending on dose of PTd. These results show that dose of PTd included in an acellular pertussis vaccine had better be low as possible and the addition of rCTB may not be always necessary in case of this nasal vaccine alone unlike tetanus and diphtheria toxoids and hepatitis B virus vaccine reported before.

Cytokine Responses to Recombinant Cholera Toxin B Subunit Produced by Bacillus brevis as a Mucosal Adjuvant

We attempted to clarify the mechanism of the mucosal adjuvanticity of recombinant cholera toxin B subunit (rCTB), which is inherently uncontaminated with the holotoxin produced by Bacillus brevis and has a powerful mucosal adjuvant activity, on cytokine responses compared with that of cholera toxin (CT). rCTB had no ability to stimulate cyclic AMP formation in mouse peritoneal macrophages (Mφ). Cytokine production by non‐immunized Mφ cultured with rCTB or CT and by the spleen cells of mice co‐immunized intranasally with ovalbumin (OVA) and rCTB or CT was examined. rCTB alone did not induce interleukin (IL)‐1α/β or IL‐6 production by Mφ, but combination of rCTB with lipopolysaccharide (LPS) enhanced both IL‐1α/β production. Conversely, CT plus LPS suppressed IL‐1α/β production more than LPS alone. Both rCTB and CT suppressed IL‐12 secretion induced by interferon γ (IFN γ) plus LPS. IL‐2, IL‐4, IL‐5, and IL‐10 were secreted by mouse spleen cells restimulated with OVA after intranasal co‐administration of OVA together with rCTB, and in response to CT, the same cytokines were secreted. The different effect of rCTB on Mφ from that of CT may mean a difference between the mechanisms of rCTB and CT during the early stage of an immune response.

A New Test of Differentiation of Bacillus cereus and Bacillus anthracis Based on the Existence of Spore Appendages

There have been few reports concerning the existence of appendages on the exosporium of bacterial spores (1,3-5). Based on this fact, Hachisuka et al (3) suggested the possibility of differentiation between Bacillus cereus and other Bacillus species since spores of all strains of B. cereus examined by them had fine filamentous appendages on the surface of the exosporium. In this paper, it is reported that the spores of all strains of B. cereus which were collected from several institutes in Japan and other places have filamentous appendages but the spores of all collected strains of Bacillus anthracis do not, and it is suggested that the confirmation of the filamentous appendages on the surface of spore exosporia may be a useful test for the differentiation of B. cereus and B. anthracis. Eleven strains of B. cereus and fourteen strains of B. anthracis were examined. Strains lAM 1029, 1072, 1110, 1656, 1729, and No.2 of B. cereus were obtained from the Institute of Applied Microbiology, The University of Tokyo. Strains IFO 3001, 3131, and 3457 of B. cereus were obtained from the Institute for Fermentation. Strain Nagoya City H.R.I. of B. cereus was obtained from the Health Research Institute of Nagoya City. The T strain of B. cereus was obtained from the Department of Microbiology, Michigan State University. Strains IID 501-505 of B. anthracis were obtained from the Institute of Medical Science, The University ofTokyo. Strains Akashi, Nishinomiya, Sumoto and Mihara of B. anthracis were obtained from the Health Institute of Hyogo. Strains NIAH 1013-1015 and Vollum of B. anthracis were obtained from the National Institute of Animal Health, Ministry of Agriculture, Forestry and Fisheries. Strain Nishine of B. anthracis was obtained from the Iwate Medical College. Each strain was inoculated on a slant of beef-extracted agar (Eiken Co., Ltd.) and cultivated for 3 days for B. cereus or for 5 days for B. anthracis at 37 C to produce spores. Spores were suspended in distilled water and washed several times with distilled water by centrifugation (at 3,000 rpm for 15 min). Spore specimens placed on grids coated with Formvar-carbon film were shadowed with Pt-palladium and examined with a Hitachi HU-12 electron microscope at an accelerating voltage of 75 kV.