Norihisa Goto

Local tissue irritating effects and adjuvant activities of calcium phosphate and aluminium hydroxide with different physical properties

Effects of calcium phosphate and aluminium hydroxide adjuvants with different physical properties were examined in guinea pigs for local histopathological reactions, electron-microscopical changes of macrophages and adjuvanticity on total IgG antibody response to subcutaneously administered ovalbumin (OVA) and tetanus toxoid (TT). Calcium phosphate gel (Ca-gel) induced active inflammatory reactions consisting of neutrophils (pseudoeosinophils) and foamy macrophages associated with many multinuclear giant cells for at least 4 weeks. Aluminium hydroxide gel (Al-gel) also elicited granulomatous inflammatory reactions consisting mainly of macrophages with foamy cytoplasm, small lymphocytes and giant cells at the injection sites for up to 8 weeks or longer. Severity of local tissue irritation due to calcium phosphate gel (Ca-gel) was similar to that due to Al-gel except for the duration of the inflammatory reactions. Calcium phosphate suspension (Ca-sus)-induced local reactions completely ceased by the 4th week, while aluminium hydroxide suspension (Al-sus)-induced reactions were seen up to the 8th week. Electron-microscopical observations showed that both Al-gel and Al-sus caused damage of macrophages. The adjuvant activity of Al-gel for OVA or TT was significantly stronger than that of any other adjuvant material, whereas those of Ca-gel and Ca-sus were not seen at a dose of 3 mg calcium phosphate per millilitre. Al-sus-TT at a dose of 3 mg aluminium hydroxide per millilitre induced very low levels of antibody. These results suggest that calcium phosphate adjuvant may not be an useful alternative to Al adjuvant.

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.

Studies on the toxicities of aluminium hydroxide and calcium phosphate as immunological adjuvants for vaccines

Aluminium hydroxide (Al) and calcium phosphate (Ca) have been used for many years as immunological adjuvants for biologicals. We investigated the toxic effects of both adjuvants with different physical properties. Al-gel elicited vascular permeability-increasing and toxic effects to macrophages (M phi), while its haemolytic effect was weak. Ca-gel elicited a significantly stronger haemolytic effect, but no other toxic effect. Incubation of M phi or polymorphonuclear leucocytes with Al-suspension resulted in the largest release of lactate dehydrogenase. Ca-suspension caused haemolysis of about 50% of that caused by Ca-gel.

Histopathological Studies of Reactions in Mice Injected with Aluminum‐Adsorbed Tetanus Toxoid

Histopathological observations of changes at the injection site in mice were made for 20 weeks after a single intramuscular (im) injection of aluminum hydroxide‐adsorbed tetanus toxoid (Alum‐TT), plain tetanus toxoid (TT), or aluminum adjuvant (Alum alone). Marked injury to muscle fibers and infiltration of neutrophils around the aluminum remnants were observed, and some microabscesses were occasionally formed, after im injection of Alum‐TT. Some mature alum‐granulomas were seen in the Alum‐TT group, while the Alum alone group had only a few immature alum‐granulomas. The TT group showed only slight injury to muscle fibers and acute inflammation at an early stage. In the Alum‐TT group, well‐developed lymphoid tissues and granulomatous lesions were still observable even 20 weeks after the injection; however, these changes had already diminished in the Alum alone group by that time. These findings could serve as good models for undesirable local reactions to aluminum‐adjuvanted vaccines.

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.

Relationship between Hemolytic Activity and Adsorption Capacity of Aluminum Hydroxide and Calcium Phosphate as Immunological Adjuvants for Biologicals

Aluminum hydroxide (Al) and calcium phosphate (Ca) gels have been used as vaccine adjuvants for many years. We investigated mechanism of the hemolytic activities of both adjuvant materials. The hemolytic activity of each gel depended on the gel dose. The adsorption capacities and the hemolytic activities of both adjuvants decreased as the concentration of phosphate increased in a gel‐washing solution. A positive correlation between the hemolytic activity and the adsorption capacity was found in Al‐gel. A disruptive effect of Ca‐gel on membrane of erythrocytes was shown by electron microscopy. Ca‐gel required more than 10 times as much pre‐adsorbed ovalbumin as did Al‐gel to inhibit the hemolysis. These results suggest that the hemolytic activity of both adjuvant materials depended mainly on the adsorption ability, and it may be useful to control the adsorption ability of adjuvants to reduce their hemolytic activity.

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.