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22nd US-Japan Cholera Conference
Conference Report tects the cross-reacting major epitope essential for inducing protection. It has been shown to be capable of conferring immunity in chimpanzees. Lambert (University of Geneva) presented evidence for the utility of anti-idiotype vaccines against parasitic infections to avoid the various immune response escape mechanisms of different parasites. Possible advantages which he saw were that it can be used to influence immunoglobulin isotype, bypass genetic restriction and avoid adjuvants. However, there is a potential problem of idiotype-anti-idiotype complexes. Krhler (Roswell Park Memorial Institute) also saw a role for anti-idiotype vaccines in the case of non-responders whether this be due to genetic reasons, an immature immune system, immunosuppression or being immunocompromized (as in AIDS, tumour and cancer patients). Synthetic peptides were also discussed in terms of vaccines and as carriers for antigens. Arnon (Weizmann Institute of Science) presented results with two different peptides. The first consists of a very immunogenic peptide present in numerous H3 type strains of influenza virus. Antibodies to this peptide produce a 50% reduction specific for the H3 type. The second peptide corresponds to residues 50-64 of the B subunit of cholera toxin. Antibodies have significant neutralizing capacity against the toxin and its effects as well as against the related LT toxin of enterotoxigenic Escherichia coli. Another peptide corresponding to the GM1 ganglioside binding domain of the B subunit of cholera toxin has been shown (Reid, Tseng, Hooper and Brewer, Walter Reed Army Institute of Research) to serve as a carrier for synthetic antigens during in vitro immunization. Apparent molecular mimicry of a host antigen by a pathogen also poses a problem and the example presented was the heart muscle determinant shared by group A streptococci (Cunningham, University of Oklahoma Health Sciences Center; Stinson, State University of New York at Buffalo). During acute rheumatic heart disease (S. pyogenes) and subacute bacterial endocarditis (S. mutans) heart reactive, i.e. autoantibodies, are detected. The myosin antibodies are not related to streptococci and streptococcal antibodies cannot react with cardiac muscle. The cause is not understood, but two possibilities seem likely; either the nonspecific activation of self reactive B-lymphocytes by bacterial components or tissue damage may cause the exposure of normally hidden self antigens. Both lead to the production of autoantibodies. The Ernest Witebsky Memorial Lecture was presented by Dr Jonathon Uhr (University of Texas Health Science
158 Vaccine,Vol. 5, June 1987
Center) on the topic 'Immunotoxins: new pharmacologic agents for the treatment of cancer and immune dysfunction'. He discussed the construction and use of ricin-coupled specific-antibodies as a means of selectively eliminating cells carrying the corresponding antigen. It was suggested that FAB fragments are preferable to whole Ig molecules and proposed a scheme by which these immunotoxins could be produced. This involved constructing a hybrid gene consisting of VH CH coding region coupled via a D N A linker to the coding region of the A chain of ricin. If this was transfected into a hybridoma secreting L chain (VL CL) then the appropriate FAB-ricin molecule would be made. Results of a phase 1 trial, in which
similar molecules directed against a melanoma were used, gave promise. However, the period of regression of the tumour took weeks instead of the predicted few days. The diversity of technologies and approaches presented at the meeting gives hope for a wealth of effective vaccines against a wide range of diseases. All the lectures and poster abstracts presented at the meeting are to be published in mid-1987 by Longmans, UK (Eds H. Krhler and P.T. LoVerde).
P.A. Manning University of Adelaide, Australia
22nd US-Japan Cholera Conference 20-23 July 1986, Toyama, Japan The annual conferences (held alternately in the US and Japan) organized by the cholera panel of the US-Japan Cooperative Medical Science Program do not just discuss Vibrio cholerae but encompass a broad range of bacterial diarrhoeal pathogens. The areas covered include epidemiology, pathology and immunology, molecular biology and molecular genetics. Together they are aiming to present a coherent picture of the interactions of the bacterium with its host and the role of the molecules involved in pathogenesis and immunity. Such an understanding is clearly providing guidelines for vaccine development and treatment of the diseases. The number of Vibrio spp. and the variety of the diseases they produce was demonstrated by the report of vibrios associated with cholecystitis (Yamagistu, Toyama Medical and Pharmaceutical University) and the wealth of toxins found (Honda, Osaka University). These toxins include cholera-like toxins, haemolysin, thermostable direct haemolysin, heat stable toxin, shiga-like toxin and protease. The ecology of non-01 V. cholera and V. mimicus in various waters and fish and the seasonal variability was described (Kodama, Toyama Institute of Health). Also the role of factors such as chitin and its derivatives in maintaining viability of vibrios was discussed (Amako, Kyushu University). Several papers were presented which related to cholera vaccines or potential protective antigens which should be present in a vaccine. Kaper (Center for Vaccine Development) presented data on another cholera toxin deletion strain which was less reactogenic than previous strains. However, some residual
diarrhoea is still detected (1 out of 9 volunteers) and the factor(s) responsible for this are not known. Clemens (ICDDR, Bangladesh) revealed results of the 1985 field trial in Bangladesh of the oral B-subunit killed whole cell (BSWC) and killed whole cell (WC) cholera vaccines. Protection of 85% with BSWC and 58% with WC was reported. The question, however, must be raised as to whether this level of protection would be obtained in an unprimed population, Another approach to a cholera vaccine was described by Manning (University of Adelaide). This involves introducing V. cholerae surface antigens into attenuated salmonellae to be used as live oral vaccines. He described molecular cloning of genes for two antigens, an outer membranes protein and the O-antigen of the lipopolysaccharide. Antibodies to this latter antigen are highly protective in animal models. Interestingly, an apparently unrelated marine vibrio shares O-antigenic determinants with V. cholerae 01 (Hisatsune, Josai University). Another antigen relevant to vaccines may be timbriae, and Ehara (Nagasaki University) presented data strongly implicating timbriae as important for ~dherence and antibodies to fimbriae being protective. Cholera toxin may have other effects besides that on the adenylate cyclase system. Zinnaka (National Defense Medical College) demonstrated a suppressive effect on cellular immunity and Taguchi (Kyorin University School of Medicine) suggested that cholera toxin promotes intra-intestinal growth of V. cholerae in a model animal system. Also using an animal model, Miura (Keio University School of Medicine) showed that both somatostatin and loperamide almost completely inhibited the secre-
Conference Report tory diarrhoea induced by cholera toxin without affecting its protein kinase activity. Imagawa (Chemo-Sero-Therapeutic Research Institute) has examined properties of synthetic peptides corresponding to regions of the B-subunit of cholera toxin, antibodies to two peptides (30-40) and (41-50), inhibited the interaction of cholera toxin B-subunit with its receptor, GMl-ganglioside. It is this region which is thought to contain the GM1 binding site. Enterotoxigenic Escherichia coil have always been studied closely with V. cholerae because of the similarity of the heat labile toxin (LT) and cholera toxin. Fimbriae have long ago been shown to be essential for adherence in E. coli and so it was interesting to see Ehara's (Nagasaki University) results with timbriae of V. cholerae. Complementary to this is the knowledge in V. cholerae that antibodies to the O-antigen of the lipopolysaccharide are protective and Sack (Johns Hopkins University) has now shown this also holds true for a homologous challenge with enterotoxigenic E. CO//.
The production and release of LT in E. coli appears to differ from that of cholera toxin in V. cholerae. Factors affecting LT production and the mechanism(s) have been examined by Okaba (Kagawa Medical School). Elegant studies by Shimonishi (Osaka University) and Schoolink (Stanford University) have analysed the central active domain of the family of heat stable toxins (STs). This domain of 13 amino acids contain six cysteine residues forming three disulphide bridges.
These workers have now defined which cysteine residues are linked. Several comparative studies were reported. Yamamoto (Juntendo University) presented data on the evolution of the two families of enterotoxins (heat labile, LT, and heat stable, ST). Moseley (University of Washington, Seattle), compared the K88 and F41 adhesins (fimbriae) found on porcine enterotoxigenic E. coli. The synthesis of each of these adhesins involves several clustered genes and the DNA cross hybridizes. However, the position of the fimbrial structural gene differs between the two. No cross reaction was detected with the genes encoding K99. A fusion between the B subunit of heat labile toxin (LT) and heat stable toxin (ST) of E. coli has been constructed (Kupersztoch, University of Texas Health Science Center). It is proposed as an immunogen for inducing antibodies against both toxin types, but its synthesis gives problems in E. coil K-12. Significant advances are being made in defining the virulence determinants of shigellae. Yoshikawa (University of Tokyo) described an analysis of 304 Tn5 insertions in the 230 kb virulence plasmid of Sh. flexneri. These studies implicate three quite widely separated loci. Mutants in one of these, virG, would appear to be defective in escaping from phagosomes suggesting that virG is a membrane damaging agent such as a phospholipase. Kopecko (Walter Reed Army Institute of Research) and Watanabe (National Institute of Health, Tokyo) both described cloning of plasmid associated determinants involved in
Shigella pathogenesis. These include outer membrane proteins consistently associated with and necessary for this invasive phenotype and the form I Oantigen of Sh. sonnei. Several toxins and requirements for their production were described in detail. These include a cytotoxin from Campylobacter jejuni, (Guerrant, University of Virginia School of Medicine) distinct from Shiga or Clostridium difficile cytotoxins, an enterotoxin produced by Clostridium sordellii (Yamakawa, Kanazawa University), a new subtype of heat labile (LT) enterotoxin produced by enterotoxigenic E. coli (Tsuji, Osaka University) and a Vero toxin immunologically unrelated to Shiga-toxin and produced by E. coli 0157:H7 (Yutsudo, The University of Tokyo). Robertson (University of Kansas) described the characterization and requirements for the production of a heat-stable toxin (ST) produced by Yersinia enterocolitica. This toxin is produced in several forms probably due to inefficient cleavage during synthesis. Characterization and molecular cloning of the structural gene for the E1 Tor haemolysin of V. cholerae was described (Yamamoto, University of the Ryukyus School of Medicine). This protein shows unusual enterotoxic activity in ligated ileal loops. A number of diarrhoeal toxins were described which are probably produced by dinoflagellates associated with various shellfish (Yasumoto, Chiba University).
P.A. Manning University of Adelaide, Australia
Vaccine, Vol. 5, June 1987
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158 Vaccine,Vol. 5, June 1987
Center) on the topic 'Immunotoxins: new pharmacologic agents for the treatment of cancer and immune dysfunction'. He discussed the construction and use of ricin-coupled specific-antibodies as a means of selectively eliminating cells carrying the corresponding antigen. It was suggested that FAB fragments are preferable to whole Ig molecules and proposed a scheme by which these immunotoxins could be produced. This involved constructing a hybrid gene consisting of VH CH coding region coupled via a D N A linker to the coding region of the A chain of ricin. If this was transfected into a hybridoma secreting L chain (VL CL) then the appropriate FAB-ricin molecule would be made. Results of a phase 1 trial, in which
similar molecules directed against a melanoma were used, gave promise. However, the period of regression of the tumour took weeks instead of the predicted few days. The diversity of technologies and approaches presented at the meeting gives hope for a wealth of effective vaccines against a wide range of diseases. All the lectures and poster abstracts presented at the meeting are to be published in mid-1987 by Longmans, UK (Eds H. Krhler and P.T. LoVerde).
P.A. Manning University of Adelaide, Australia
22nd US-Japan Cholera Conference 20-23 July 1986, Toyama, Japan The annual conferences (held alternately in the US and Japan) organized by the cholera panel of the US-Japan Cooperative Medical Science Program do not just discuss Vibrio cholerae but encompass a broad range of bacterial diarrhoeal pathogens. The areas covered include epidemiology, pathology and immunology, molecular biology and molecular genetics. Together they are aiming to present a coherent picture of the interactions of the bacterium with its host and the role of the molecules involved in pathogenesis and immunity. Such an understanding is clearly providing guidelines for vaccine development and treatment of the diseases. The number of Vibrio spp. and the variety of the diseases they produce was demonstrated by the report of vibrios associated with cholecystitis (Yamagistu, Toyama Medical and Pharmaceutical University) and the wealth of toxins found (Honda, Osaka University). These toxins include cholera-like toxins, haemolysin, thermostable direct haemolysin, heat stable toxin, shiga-like toxin and protease. The ecology of non-01 V. cholera and V. mimicus in various waters and fish and the seasonal variability was described (Kodama, Toyama Institute of Health). Also the role of factors such as chitin and its derivatives in maintaining viability of vibrios was discussed (Amako, Kyushu University). Several papers were presented which related to cholera vaccines or potential protective antigens which should be present in a vaccine. Kaper (Center for Vaccine Development) presented data on another cholera toxin deletion strain which was less reactogenic than previous strains. However, some residual
diarrhoea is still detected (1 out of 9 volunteers) and the factor(s) responsible for this are not known. Clemens (ICDDR, Bangladesh) revealed results of the 1985 field trial in Bangladesh of the oral B-subunit killed whole cell (BSWC) and killed whole cell (WC) cholera vaccines. Protection of 85% with BSWC and 58% with WC was reported. The question, however, must be raised as to whether this level of protection would be obtained in an unprimed population, Another approach to a cholera vaccine was described by Manning (University of Adelaide). This involves introducing V. cholerae surface antigens into attenuated salmonellae to be used as live oral vaccines. He described molecular cloning of genes for two antigens, an outer membranes protein and the O-antigen of the lipopolysaccharide. Antibodies to this latter antigen are highly protective in animal models. Interestingly, an apparently unrelated marine vibrio shares O-antigenic determinants with V. cholerae 01 (Hisatsune, Josai University). Another antigen relevant to vaccines may be timbriae, and Ehara (Nagasaki University) presented data strongly implicating timbriae as important for ~dherence and antibodies to fimbriae being protective. Cholera toxin may have other effects besides that on the adenylate cyclase system. Zinnaka (National Defense Medical College) demonstrated a suppressive effect on cellular immunity and Taguchi (Kyorin University School of Medicine) suggested that cholera toxin promotes intra-intestinal growth of V. cholerae in a model animal system. Also using an animal model, Miura (Keio University School of Medicine) showed that both somatostatin and loperamide almost completely inhibited the secre-
Conference Report tory diarrhoea induced by cholera toxin without affecting its protein kinase activity. Imagawa (Chemo-Sero-Therapeutic Research Institute) has examined properties of synthetic peptides corresponding to regions of the B-subunit of cholera toxin, antibodies to two peptides (30-40) and (41-50), inhibited the interaction of cholera toxin B-subunit with its receptor, GMl-ganglioside. It is this region which is thought to contain the GM1 binding site. Enterotoxigenic Escherichia coil have always been studied closely with V. cholerae because of the similarity of the heat labile toxin (LT) and cholera toxin. Fimbriae have long ago been shown to be essential for adherence in E. coli and so it was interesting to see Ehara's (Nagasaki University) results with timbriae of V. cholerae. Complementary to this is the knowledge in V. cholerae that antibodies to the O-antigen of the lipopolysaccharide are protective and Sack (Johns Hopkins University) has now shown this also holds true for a homologous challenge with enterotoxigenic E. CO//.
The production and release of LT in E. coli appears to differ from that of cholera toxin in V. cholerae. Factors affecting LT production and the mechanism(s) have been examined by Okaba (Kagawa Medical School). Elegant studies by Shimonishi (Osaka University) and Schoolink (Stanford University) have analysed the central active domain of the family of heat stable toxins (STs). This domain of 13 amino acids contain six cysteine residues forming three disulphide bridges.
These workers have now defined which cysteine residues are linked. Several comparative studies were reported. Yamamoto (Juntendo University) presented data on the evolution of the two families of enterotoxins (heat labile, LT, and heat stable, ST). Moseley (University of Washington, Seattle), compared the K88 and F41 adhesins (fimbriae) found on porcine enterotoxigenic E. coli. The synthesis of each of these adhesins involves several clustered genes and the DNA cross hybridizes. However, the position of the fimbrial structural gene differs between the two. No cross reaction was detected with the genes encoding K99. A fusion between the B subunit of heat labile toxin (LT) and heat stable toxin (ST) of E. coli has been constructed (Kupersztoch, University of Texas Health Science Center). It is proposed as an immunogen for inducing antibodies against both toxin types, but its synthesis gives problems in E. coil K-12. Significant advances are being made in defining the virulence determinants of shigellae. Yoshikawa (University of Tokyo) described an analysis of 304 Tn5 insertions in the 230 kb virulence plasmid of Sh. flexneri. These studies implicate three quite widely separated loci. Mutants in one of these, virG, would appear to be defective in escaping from phagosomes suggesting that virG is a membrane damaging agent such as a phospholipase. Kopecko (Walter Reed Army Institute of Research) and Watanabe (National Institute of Health, Tokyo) both described cloning of plasmid associated determinants involved in
Shigella pathogenesis. These include outer membrane proteins consistently associated with and necessary for this invasive phenotype and the form I Oantigen of Sh. sonnei. Several toxins and requirements for their production were described in detail. These include a cytotoxin from Campylobacter jejuni, (Guerrant, University of Virginia School of Medicine) distinct from Shiga or Clostridium difficile cytotoxins, an enterotoxin produced by Clostridium sordellii (Yamakawa, Kanazawa University), a new subtype of heat labile (LT) enterotoxin produced by enterotoxigenic E. coli (Tsuji, Osaka University) and a Vero toxin immunologically unrelated to Shiga-toxin and produced by E. coli 0157:H7 (Yutsudo, The University of Tokyo). Robertson (University of Kansas) described the characterization and requirements for the production of a heat-stable toxin (ST) produced by Yersinia enterocolitica. This toxin is produced in several forms probably due to inefficient cleavage during synthesis. Characterization and molecular cloning of the structural gene for the E1 Tor haemolysin of V. cholerae was described (Yamamoto, University of the Ryukyus School of Medicine). This protein shows unusual enterotoxic activity in ligated ileal loops. A number of diarrhoeal toxins were described which are probably produced by dinoflagellates associated with various shellfish (Yasumoto, Chiba University).
P.A. Manning University of Adelaide, Australia
Vaccine, Vol. 5, June 1987
159