Search

In Streptococcus pyogenes, proteins involved in determining virulence are controlled by stand-alone response regulators and by two-component regulatory systems. Previous studies reported that, compared to the parental strain, the yvqE sensor knockout strain showed significantly reduced growth and lower virulence. To determine the function of YvqE, we performed biofilm analysis and pH assays on yvqE mutants, and site-directed mutagenesis of YvqE. The yvqE deletion mutant showed a slower acid production rate, indicating that YvqE regulates acid production from sugar fermentation. The mutant strain, in which the Asp(26) residue in YvqE was replaced with Asn, affected biofilm formation, suggesting that this amino acid senses hydrogen ions produced by fermentative sugar metabolism. Signals received by YvqE were directly or indirectly responsible for inducing pilus expression. This study shows that at low environmental pH, biofilm formation in S. pyogenes is mediated by YvqE and suggests that regulation of pilus expression by environmental acidification could be directly under the control of YvqE., (© 2016 APMIS. Published by John Wiley & Sons Ltd.)

Bacterial DNA comprising palindromic sequences and containing unmethylated CpG is recognized by toll-like receptor 9 of plasmacytoid dendritic cells (pDCs) and induces the production of interferon-α and chemokines, leading to the activation of a Th1 immune response. Therefore, synthetic equivalents of bacterial DNA (CpG oligodeoxynucleotides) have been developed for clinical applications. They are usually phosphorothioated for in vivo use; this approach also leads to adverse effects as reported in mouse models.Mucosal vaccines that induce both mucosal and systemic immunity received substantial attention in recent years. For their development, phosphodiester-linked oligodeoxynucleotides, including the sequence of a palindromic CpG DNA may be advantageous as adjuvants because their target pDCs are present right there, in the mucosa of the vaccination site. In addition, the probability of adverse effects is believed to be low. Here, we review the discovery of such CpG oligodeoxynucleotides and their possible use as mucosal adjuvants.

Background: CpG oligodeoxynucleotides (ODNs), resembling bacterial DNA, are currently tested in clinical trials as vaccine adjuvants. They have the nuclease-resistant phosphorothioate bond; the immune responses elicited differ according to the CpG ODN sequence and vaccination method. To develop a CpG ODN that can induce plasmacytoid dendritic cell (pDC)-mediated T(H)1 immunity through the mucosa, we constructed phosphodiester G9.1 comprising one palindromic CpG motif with unique polyguanosine-runs that allows degradation similar to naturally occurring bacterial DNA., Methods: T(H)1 and T(H)2 immunity activation was evaluated by cytokine production pattern and T-bet/GATA-3 ratio in human peripheral blood mononuclear cells and mouse bone marrow cells. Adjuvanticity was evaluated in mice administered G9.1 with diphtheria toxoid (DT) through nasal vaccination., Results: G9.1 exhibited stronger IFN-α-inducing activity than A-class CpG ODN2216 and increased T-bet/GATA-3 ratio by enhancing T-bet expression. Nasally administered G9.1 plus DT induced DT-specific mucosal IgA and serum IgG, but not IgE, responses with antitoxin activity in C57BL/6 and BALB/c mice, possibly due to IFN/BAFF production. Induction of T(H)1, but not T(H)2-type Abs depended completely on pDCs, the first in vivo demonstration by CpG ODNs., Conclusions: G9.1 is a promising mucosal adjuvant for induction of pDC-mediated T(H)1 immunity.

Background: Mycobacterium bovis bacillus Calmette Guérin (BCG) vaccine, which has been inoculated to more than one billion people world-wide, has significant effect in preventing tuberculous meningitis and miliary tuberculosis (TB) in neonate and early childhood. However, BCG fails to adequately protect against pulmonary TB and reactivation of latent infections in adults. To overcome this problem, adequate booster is urgently desired in adult who received prior BCG vaccination, and appropriate animal models that substitute human cases would be highly valuable for further experimentation., Findings: The booster effect of the synthesized CpG oligomer (Oligo-B) on aged mice which had been primarily vaccinated with BCG at the age of 4-week old. The specific Th1 type reaction, production of interferon-γ, in response to TB antigens, purified protein derivatives (PPD) and protection against challenge with Mycobacterium tuberculosis (MTB) H37Rv decreased with increasing age and were not observed in 89-week old mice. In order to rejuvenate the Th1 type response against PPD and protection activity against MTB infection, Oligo-B, which is known to augment Th1 responses, was administered as a booster to 81-90-week old mice (late 50's in human equivalent) vaccinated with BCG at 4-week old. The boosting with Oligo-B increased the number of CD4+ CD44high CD62Lhigh, central memory type T cell. Furthermore, the Oligo-B boosting rejuvenated the ability of mice to protect against infection with MTB H37Rv., Conclusions: Th1-adjuvant CpG oligo DNA, such as Oligo-B, may be a promising booster when coupled with BCG priming.

Endotoxin contamination is a serious threat to the safety of parenteral drugs, and the rabbit pyrogen test has played a crucial role in controlling this contamination. Although the highly sensitive endotoxin test has replaced the pyrogen test for various pharmaceuticals, the pyrogen test is still implemented as the control test for most blood products in Japan. We examined the applicability of the endotoxin test to blood products for reliable detection and quantification of endotoxin. Nineteen types of blood products were tested for interfering factors based on spike/recovery of endotoxin by using 2 types of endotoxin-specific lysate reagents for photometric techniques. Interfering effects on the endotoxin test by the products could be eliminated by diluting from 1/2 to 1/16, with the exception of antithrombin III. However, conventional lysate reagents that also react with non-pyrogenic substances, such as (1-3)-β-D-glucan, produced results that were not relevant to endotoxin content or pyrogenicity. Our results showed that the endotoxin test would be applicable to most blood products if used with appropriate endotoxin-specific lysate reagents., (Copyright © 2010 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.)

The identification of a safe and effective adjuvant that is able to enhance mucosal immune responses is necessary for the development of an efficient inactivated intranasal influenza vaccine. The present study demonstrated the effectiveness of extracts of mycelia derived from edible mushrooms as adjuvants for intranasal influenza vaccine. The adjuvant effect of extracts of mycelia was examined by intranasal co-administration of the extracts and inactivated A/PR8 (H1N1) influenza virus hemagglutinin (HA) vaccine in BALB/c mice. The inactivated vaccine in combination with mycelial extracts induced a high anti-A/PR8 HA-specific IgA and IgG response in nasal washings and serum, respectively. Virus-specific cytotoxic T-lymphocyte responses were also induced by administration of the vaccine with extract of mycelia, resulting in protection against lethal lung infection with influenza virus A/PR8. In addition, intranasal administration of NIBRG14 vaccine derived from the influenza A/Vietnam/1194/2004 (H5N1) virus strain administered in conjunction with mycelial extracts from Phellinus linteus conferred cross-protection against heterologous influenza A/Indonesia/6/2005 virus challenge in the nasal infection model. In addition, mycelial extracts induced proinflammatory cytokines and CD40 expression in bone marrow-derived dendritic cells. These results suggest that mycelial extract-adjuvanted vaccines can confer cross-protection against variant H5N1 influenza viruses. The use of extracts of mycelia derived from edible mushrooms is proposed as a new safe and effective mucosal adjuvant for use for nasal vaccination against influenza virus infection., ((c) 2009 Wiley-Liss, Inc.)

Transcriptomics is an objective index that reflects the overall condition of cells or tissues, and transcriptome technology, such as DNA microarray analysis, is now being introduced for the quality control of medical products. In this study, we applied DNA microarray analysis to evaluate the character of Japanese encephalitis (JE) vaccines. When administered into rat peritoneum, Vero cell-derived and mouse brain-derived JE vaccines induced similar gene expression patterns in liver and brain. Body weights and blood biochemical findings were also similar after administration of the two vaccines. Our results suggest that the two JE vaccines are likely to have equivalent characteristics with regard to reactivity in rats.

CpG-DNA is currently attracting attention as an effective and safe vaccine adjuvant to prevent from microbial infections. In this report, we examined the effects of oligo B, which is a synthetic CpG-DNA, in mucosal administration of Bacillus Calmette-Guérin (BCG) and diphtheria toxoid (DT). Co-administration with oligo B enhanced BCG-induced delayed type hypersensitivity to purified protein derivative (PPD) in guinea pigs. The titers of anti-DT serum IgG, IgA and mucosal IgA antibodies induced by intranasal administration with DT plus oligo B were significantly higher than that with DT alone. In both C57BL/6 and BALB/c mice, intranasal administration of DT with oligo B induced enough level of antibodies to prevent onset of diphtheria. The analysis of antibody subclasses showed that intranasal administration of oligo B induced not only IgG1 but also IgG2a, IgG2c and IgA anti-DT antibodies. In contrast, there was no or little production of the anti-DT serum IgE. Taken together our data suggest that oligo B is a powerful adjuvant in mucosal immunization.

Vaccines differ from other pharmaceutical products. The quality and safety of batches are regulated to high standards by national regulatory authorities. Various quality control and safety tests have been developed, including the abnormal toxicity test (ATT), which is described in the World Health Organization (WHO) guidelines and in each country's pharmacopoeia. However, the criteria for abnormal results are not well defined in these guidelines. In addition, the animal grade to be used in ATT, classified on the basis of microbial colonization, was not designated in either guideline. In this study, we report a new and improved method of performing ATT, including statistical, histopathological analysis and hematological findings. It is based on the observation that there are body weight changes characteristic to each vaccine, and such standardized changes can be used as references for evaluating test vaccines. In addition, histopathological data are useful for determining vaccine quality and safety. Combined with histopathological examination, the improved ATT will be of great use for evaluating the consistency, quality and safety of different batches of vaccine. The results of these analyses were similar using either 'clean' or specific pathogen-free guinea pigs.

Although vaccines are routinely used to prevent infectious diseases, little is known about the comprehensive influences caused by vaccines. In this study, we showed, using comprehensive gene expression analysis, that pertussis vaccine affected many genes in multiple organs of vaccine-treated animals. In particular, lung was revealed to be the most suitable target to evaluate pertussis vaccine toxicity. The 13 genes identified from the analysis of vaccine-treated lung at day 1 showed a clear dendrogram corresponding to pertussis vaccine toxicity. Furthermore, quantitative analysis of these genes revealed a positive correlation between their respective expression levels and the degree of toxic effects observed in samples that had been treated with various doses of reference pertussis vaccines. The quantification of this 13 gene-set is an indicator of the vaccine toxicity-related reaction.

We propose that DNA microarray analysis can be used in the quality control of pandemic and endemic influenza vaccine. Based on the expression profiles of 76 genes in the rat lung one day after inoculation of influenza vaccine, we can distinguish whole-virion influenza vaccine (PDv: pandemic influenza vaccine and WPv: whole virion-particle vaccine) and sub-virion vaccine (HA vaccine) from saline. Among these 76 genes, we found genes up-regulated by influenza infection, as well as genes involved in the immune response, and interferon. Hierarchical clustering of each influenza vaccine by the expression profiles of these 76 genes matched data from current quality control tests in Japan, such as the abnormal toxicity test (ATT) and the leukopenic toxicity test (LTT). Thus, it can be concluded that DNA microarray technology is an informative, rapid and highly sensitive method with which to evaluate the quality of influenza vaccines. Using DNA microarray system, consistent with the results of the ATT and LTT, it was clarified that there was no difference in vaccine quality between PDv and WPv.

To develop an efficient nasal influenza vaccine, influenza A and B virus HA with rCTB as a mucosal adjuvant were administered to mice intranasally. Serum anti-HA IgG and IgA antibody responses for both HA vaccines were significantly increased in the presence of rCTB. Higher HI and neutralizing antibody titers and higher mucosal IgA antibody responses in the respiratory tract were detected when rCTB was added than without rCTB. When mice were immunized with HA vaccine with or without rCTB and challenged by intranasal administration of mouse-adapted pathogenic influenza A virus, all mice immunized with HA plus rCTB survived for seven days without any inflammatory changes in the lungs, while not all the mice immunized with HA without rCTB survived, and all of them had lung consolidations. These results demonstrate that intranasal co-administration of rCTB as a mucosal adjuvant with influenza virus HA is necessary not only for the induction of systemic and mucosal HA antibodies, but also for the protection of mice from morbidity and mortality resulting from virus infection.

Transcutaneous immunization (TCI) is a promising needle-free technique for vaccination. In this method, strong adjuvants, such as the cholera toxin, are generally crucial to elicit a robust immune response. Here, we showed that prolonged antigen presence on the skin of mice during TCI could effectively enhance the immune response. Substantial antigen-specific antibodies were produced in the sera of mice even after non-adjuvanted TCI when the antigen presence was for longer than 16 h. This non-adjuvanted TCI method was applied using the tetanus toxoid, and potent tetanus toxoid-specific antibodies were successfully induced in the sera of mice; they survived a lethal tetanus toxin challenge with no clinical signs. Thus, non-adjuvanted approach might be a possible option for TCI, and this method might improve the safety and practicality of transcutaneous vaccination.

Conventional animal tests such as leukocytosis promoting tests have been used for decades to evaluate toxicity of pertussis vaccine. Here, we examined gene expression in relation to the vaccine toxicity using a DNA microarray. Comparison of conventional animal test data with the DNA microarray-based gene expression data revealed a gene expression pattern highly correlated with leukocytosis in animals. Of 10,490 rat genes analyzed, two genes, alpha1-acid-glycoprotein (Agp) and hemopexin (Hpx), were found up-regulated by the toxin administration in a dose-dependent manner (assayed by a quantitative PCR based on the microarray). Variation of the gene expression was very small amongst the test animals, and the results were highly reproducible. These findings suggest that gene expression analysis of vaccine-treated animals can be used as an accurate and simple method of pertussis vaccine safety assessment.

Recombinant cholera toxin B subunit (rCTB) which is produced by Bacillus brevis carrying pNU212-CTB acts as a mucosal adjuvant capable of enhancing host immune responses specific to unrelated, mucosally co-administered vaccine antigens. When mice were administered intranasally with diphtheria-pertussis-tetanus (DPT) combination vaccine consisting of diphtheria toxoid (DTd), tetanus toxoid (TTd), pertussis toxoid (PTd), and formalin-treated filamentous hemagglutinin (fFHA), the presence of rCTB elevated constantly high values of DTd- and TTd-specific serum ELISA IgG antibody titres, and protective levels of diphtheria and tetanus toxin-neutralizing antibodies but the absence of rCTB did not. Moreover, the addition of rCTB protected all mice against tetanic symptoms and deaths. DPT combination vaccine raised high levels of serum anti-PT IgG antibody titres regardless of rCTB and protected mice from Bordetella pertussis challenge. These results suggest that co-administration of rCTB as an adjuvant is necessary for induction of diphtheria and tetanus antitoxin antibodies on the occasion of intranasal administration of DPT combination vaccine.

Recombinant cholera toxin B subunit (rCTB) is a safe and potent mucosal adjuvant. To gain insight into the mechanism underlying the adjuvant effect of rCTB, the effects of rCTB on cell-mediated immune responses of mice and guinea pigs were examined after intranasal administration of Mycobacterium bovis -bacillus Calmette-Guérin (BCG) with and without rCTB. Delayed-type hypersensitivity, for skin reactions in guinea pigs and for footpad swelling reactions in mice, to purified protein derivative (PPD) were enhanced by intranasal co-administration of BCG and rCTB, as compared to giving BCG alone to these animals. Moreover, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma production of spleen cells and antigen specific spleen cell proliferation, stimulated with PPD, were enhanced in the presence of rCTB. These results strongly suggest that rCTB enhances cellular as well as humoral immune responses.

Vaccination via a mucosal route is a very attractive means for immunization, because both local and systemic immune responses are inducible and vaccines can be administered easily and safely from infants to elderly persons. For developing widely applicable mucosal vaccines using recombinant cholera toxin B subunit (rCTB) as a safe adjuvant, we examined whether frequent nasal administrations of rCTB-containing same and different vaccines could induce antigen-specific immune responses without induction of systemic tolerance and suppression by pre-existing anti-rCTB immunity. Ten repetitive nasal administrations to mice of tetanus toxoid (TT) + rCTB or diphtheria toxoid (DT) + rCTB raised and maintained high levels of antigen- and rCTB-specific serum IgG including high levels of tetanus/diphtheria antitoxin titres and raised nasal, salivary, lung, vaginal and fecal secreted IgA, suggesting that the regimen did not induce systemic tolerance to TT/DT and rCTB. Mice successively received repetitive five doses of TT as the first antigen and subsequent five doses of DT as the second antigen, and vice versa, raised serum IgG to the second antigen at various levels including low but sufficient protective levels of antitoxin titres and induced mucosal IgA in the lungs, the vaginas and feces, but hardly in the nasal secretions and salivas. After an interval of 22 weeks between the dosage of the first and second antigens, mice induced serum IgG to the second antigen at high levels and mucosal IgA in all sites. In conclusion, anti-TT and -DT serum and mucosal antibody responses induced by repeated intranasal immunization using rCTB adjuvant lasted for a long period, and for improving the effectivity of vaccination, different rCTB-containing vaccines should be administered at appropriate intervals.

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., (Copyright 2002 Elsevier Science Ltd.)

Recombinant cholera toxin B subunit (rCTB) is a safe and potent mucosal adjuvant. As a clue to the mechanism of the adjuvant effect of rCTB, the profile of cytokines secreted in vitro by the mouse peritoneal macrophage (Mphi) treated with rCTB was examined. IL-1beta secretion, intracellular production, and expression of its mRNA of LPS-stimulated Mphi was greatly enhanced by treatment with rCTB. IL-1beta production in response to other microbial stimulators, such as Pansorbin, Sansorbin, insoluble peptidoglycan, and Taxol, was also potentiated by rCTB. Mphi pretreated with rCTB before 24 hr could maintain the ability to produce a high level of IL-1beta, suggesting that this ability may be involved in the adjuvant activity of rCTB on Mphi stimulation. The possibility of close association between rCTB and signal transduction of a Toll-like receptor family in Mphi is discussed.

24-Hydroxylated derivatives were synthesized in 24(R) and 24(S) forms by the convergent method as analogs related to 1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D(3). In the convergent synthesis, the A-ring fragment, synthesized from diethyl D-tartarate, and the C/D-ring fragments in 24(R) and 24(S) forms (vitamin D numbering), obtained from vitamin D(2) via the Inhoffen-Lythgoe diol, were coupled in moderate yields to give 1alpha,24(R),25-trihydroxy-2beta-(3-hydroxypropoxy)vitamin D(3) and 1alpha,24(S),25-trihydroxy-2beta-(3-hydroxypropoxy)vitamin D(3). In preliminary biological evaluations, 24-hydroxylation of 1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D(3) caused weakened affinity to vitamin D binding protein in vitro and less calcemic activity in vivo compared to the parent compound. While the affinity to vitamin D receptor in 24(R) epimer was sustained, the affinity in 24(S) epimer was less than that of the parent compound.

A-ring diastereomers of 1alpha,25-dihydroxy-22-oxavitamin D3 (OCT) (2), 3-epi-1alpha,25-dihydroxy-22-oxavitamin D3 (3-epiOCT) (3) and 1,3-diepi-1alpha,25-dihydroxy-22-oxavitamin D3 (1,3-diepiOCT) (4) were synthesized by the convergent method. In vitro binding affinity for rat vitamin D binding protein and calf-thymus vitamin D receptor, differentiation-inducing activity on HL-60 cells, and transcriptional activity of 3-epiOCT (3) and 1,3-diepiOCT (4) were evaluated in comparison with OCT (2), 1-epi-1alpha,25-dihydroxy-22-oxavitamin D3 (1-epiOCT) (5) and 1alpha,25-dihydroxyvitamin D3 (1).

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.

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 (Mphi). Cytokine production by non-immunized Mphi 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)-1alpha/beta or IL-6 production by Mphi, but combination of rCTB with lipopolysaccharide (LPS) enhanced both IL-1alpha/beta production. Conversely, CT plus LPS suppressed IL-1alpha/beta production more than LPS alone. Both rCTB and CT suppressed IL-12 secretion induced by interferon gamma (IFN gamma) 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 Mphi from that of CT may mean a difference between the mechanisms of rCTB and CT during the early stage of an immune response.

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.

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.

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.

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.

The amino acid sequence of a cytotoxic factor, CTF-I, isolated from the venom of the Japanese habu snake (Trimeresurus flavoviridis) has been determined through automatic phenylisothiocyanate degradation of the PE-protein and derived proteolytic peptides. CTF-I consists of 72 amino acids and contains an Arg-Gly-Asp sequence present in trigramin-like peptides isolated from other snake venoms. The primary structure of another cytotoxic factor, CTF-II, consisting of 75 amino acids, was deduced to comprise that of CTF-1 with an additional Glu-Leu-Leu-sequence at its N-terminal.

Hydrolysis of substance P and nine kinds of substance P analogs by angiotensin-converting enzyme highly purified from rat lung was examined by using amino-group fluorometry and high-performance liquid chromatography. The enzyme hydrolyzed substance P and several analogs, notwithstanding that they did not contain free C-terminal residues. The analyses of cleavage products separated by high-performance liquid chromatography indicated that the enzyme hydrolyzed substance P and its analogs mainly at the bond between Phe8-Gly9 and also at another bond, possibly between Gly9-Leu10, to a lesser extent by an endopeptidase action, followed by successive release of dipeptides by a dipeptidyl carboxypeptidase action. The analogs that had D-amino acid residues substituted at the presumed cleavage sites were scarcely hydrolyzed. It was further found that (Pyr6)-fragment (6-11) was hydrolyzed by the enzyme more efficiently than the other fragment-type analogs and was cleaved at a single bond by the endopeptidase activity of the enzyme. Therefore, this fragment was used as a substrate in order to characterized the endopeptidase activity of the enzyme by employing fluorometry. The activity was dependent on chloride ion, and was inhibited by captopril, MK-421, and EDTA. Thus, the endopeptidase activity of the enzyme showed properties similar to those of the dipeptidyl carboxypeptidase activity of the enzyme.

Ovalbumin (OA)-complexed guinea-pig IgG1 and IgG2 antibodies were found to bind to homologous polymorphonuclear leukocytes (PMNs). As these bindings are assumed to be mediated by certain Fc receptors (FcRs) for IgG1 and IgG2, the variety and properties of the FcRs on the cells were investigated by the use of two monoclonal antibodies to guinea-pig macrophage FcRs which were prepared by Shimamura T. et al., 1987 (Molec. Immun. 24, 67-74): VI A2 IgG1 to the FcR for IgG1 and IgG2 (FcR1,2) and VII A1 IgG1 to the FcR for IgG2 (FcR2). PMNs were shown to bind the Fab' of VI A2 IgG1 (VI A2 Fab') by flow cytofluorometry, suggesting that the cells possess a certain FcR which cross-reacts antigenically with macrophage FcR1,2. In fact, VI A2 Fab' inhibited completely the binding of OA-complexed IgG1 antibody to the cells. When the FcR was isolated by affinity chromatography on the F(ab')2 of VI A2 IgG1 coupled to Sepharose, it gave a 55,000 mol. wt band on sodium dodecylsulfate-polyacrylamide gel electrophoresis, as in the case of macrophage FcR1,2. The number of the FcR molecules per PMN cell was estimated to be 2 X 10(4) by measuring the binding of 125I-VI A2 Fab'. The binding of OA-complexed IgG2 antibody to PMNs was also inhibited with VI A2 Fab', but partially. This finding indicates that the FcR bound by VI A2 Fab' may be an FcR1,2 which is able to bind both OA-complexed IgG1 and IgG2 antibodies, and also that PMNs possess another FcR, namely FcR2 which binds IgG2 antibody alone. The Fab' of VII A1 IgG1 (VII A1 Fab'), on the other hand, did not exhibit any inhibitory activity on the bindings of OA-complexed IgG1 and IgG2 antibodies to PMNs. Since no evidence indicating the binding of VII A1 Fab' to PMN cells was obtained by flow cytofluorometry, the FcR2 of PMNs may be antigenically different from its macrophage counterpart. In conclusion, these results indicate that two distinct types of FcR for IgG isotypes exist on guinea-pig PMN cells: FcR1,2 similar to macrophage FcR1,2, and FcR2 distinct from macrophage FcR2.