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& TCP-targeted Vaccine Development Background image from: http://www.genomenewsnetwork.org/articles/06_02/cholera_t

& TCP-targeted Vaccine Development Background image from: http://www.genomenewsnetwork.org/articles/06_02/cholera_trip.shtml. The Vibrio cholerae Toxin Co-Regulated Pilus. Overview. Epidemiology V. cholerae Biology Model of V. cholerae Pathogenicity Acquisition

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& TCP-targeted Vaccine Development Background image from: http://www.genomenewsnetwork.org/articles/06_02/cholera_t

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  1. & TCP-targeted Vaccine Development Background image from: http://www.genomenewsnetwork.org/articles/06_02/cholera_trip.shtml The Vibrio cholerae Toxin Co-Regulated Pilus

  2. Overview • Epidemiology • V. cholerae Biology • Model of V. cholerae • Pathogenicity Acquisition • Toxin Co-Regulated Pilus • Vaccine Development Image from: http://microvet.arizona.edu/Courses/MIC420/lecture_notes/vibrio/vibrio_cholera_em.gifElectron microscopic image of Vibrio cholerae: curved rods with polar flagellum

  3. Modes of Transmission • Water (infectious dose = 109) • Food (infectious dose = 103) • Person-to-person The 19th-century illustration depicting the spirit of death at a pump was taken from http://news.nationalgeographic.com/news/2004/06/0614_040614_tvcholera.html

  4. Clinical Manifestations www.who.int/entity/water_sanitation_health/dwq/en/admicrob6.pdf

  5. Microbiological & Molecular Methods of Detection • Microbiological culture-based methods using fecal or water samples • Rapid Tests • Dark-field microscopy • Rapid immunoassays • Molecular methods - PCR & DNA probes www.city.niigata.niigata.jp/ info/sikenjo/521s...

  6. Treating Cholera Sack, David, et al. 2004. Seminar: Cholera. The Lancet. 363: 223-233.

  7. Preventing Cholera: Vaccines • Orochol • Contains 2x108 viable cells of attenuated strain CVD 103-HgR in a lyophilized form • Oral immunization of children older than 2 • Subunit A of the cholera toxin (CT) has been removed • Dukoral • Protects against O1 Inaba and Ogawa, Classical & El Tor strains • Contains 1x10 heat/formalin killed cells of strain WC/rBS Image from: http://www.pharmeragroup.com/dukoralb.htm

  8. Epidemic Control Measures • Hygienic disposal of human waste • Adequate supply of water • Good food hygiene • Thoroughly cooking food • Eating food while it’s hot • Preventing cooked foods from contacting raw foods (including water or ice) • Avoiding raw fruits or vegetables • Washing hands after defecation & before cooking Sack, David, et al. 2004. Seminar: Cholera. The Lancet. 363: 223-233. http://www.who.int/mediacentre/factsheets/fs107/en/print.html

  9. Overview • Epidemiology • V. cholerae Biology • Model of V. cholerae Pathogenicity Acquisition • Toxin Co-Regulated Pilus • Vaccine Development Image from: http://microvet.arizona.edu/Courses/MIC420/lecture_notes/vibrio/vibrio_cholera_em.gifElectron microscopic image of Vibrio cholerae: curved rods with polar flagellum

  10. V. cholerae Basics V. cholerae • Gram-negative • 2 chromosomes • Polar monotrichous • Asporogenous • Curved rod • Ferments glucose, sucrose, and mannitol Heidelberg, J. F. et al. 2000. DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae Nature.406, 477-483.

  11. Toxigenic V. cholerae O139 O1 Division into 2 biotypes inaba ogawa hikojima A & C A & B (A little C) Antigens A, B, C Classification Scheme Division into 2 epidemic serotypes Classical El Tor Each O1 biotype can have 3 serotypes Division into ribotypes Designed using information presented in review by NS Crowcroft. 1994. Cholera: Current Epidemiology. The Communicable Disease Report. 4(13): R158-R163.

  12. V. cholerae Classification Scheme NON-TOXIGENIC TOXIGENIC I define Vibrios! I’m an O1 or O139 Strain I may not be O1, Or O139! (but I can still stir up trouble) www.forth.go.jp/tourist/ panf/cholerae.html

  13. Known Virulence Factors • Integrons • Toxins • CT • HA Protease • RTX Toxin • ACE and Zot • Adherence/Adhesins • Accessory Colonization Factors (ACF) • OmpU & other Omp Proteins - outer membrane proteins • Mannose-fucose-resistant cell hemagglutinin & Mannose sensitivev hemagglutinin (Faruque, 2002) • Toxin Co-regulated Pilus (TCP) Image from: http://www.amnh.org/nationalcenter/infection/01_mic/01a_bac.html

  14. Integrons & Antibiotic Resistance Gene Acquisition Pennisi, Elizabeth. Microbiology: Versatile gene uptake system found in cholera bacterium. 1998. Science. 280(5363): 521-522.

  15. The SXT Constin Integron & Antibiotic Resistance Genes Image modified from: Iwanga, Masaaki et al. 2004. Antibiotic resistance by a class I integron and SXT constin in Vibrio cholerae O1 strains isolated in Laos. Antimicrobial Agents & Chemotherapy. 48(7): 2364-2369.

  16. Changes in SXT Constin Prevalence • Between 1993 and 2000 V. cholerae susceptibility in Laos changed dramatically. • After a period without cholera in the mid-nineties, strains were increasingly resistant to multiple antibiotics. • Why? Maybe because after 1998 isolates tested positive for the presence of the SXT constin. Iwanga, Masaaki et al. 2004. Antibiotic resistance by a class I integron and SXT constin in Vibrio cholerae O1 strains isolated in Laos. Antimicrobial Agents & Chemotherapy. 48(7): 2364-2369.

  17. Class I Integron & SXT Constin in V. cholerae Antibiotic Resistance Iwanga, Masaaki et al. 2004. Antibiotic resistance by a class I integron and SXT constin in Vibrio cholerae O1 strains isolated in Laos. Antimicrobial Agents & Chemotherapy. 48(7): 2364-2369.

  18. CT TOXIN • CT is an A-B type toxin encoded by genes located on phage CTX • V. cholerae Toxicity • CT does the dirty work. The organism never invades the cell. • The catalytic A subunit activates cAMP production • High intracellular cAMP results in the secretion of chloride ions, bicarbonate and water. Lencer, Wayne. 2001. Microbes and Microbial Toxins: Paradigms for Microbial-Mucosal Interactions. V. cholerae: invasion of the intestinal epithelial barrier by a stably folded protein toxin. Am J Physiol. Gastrointest Liver Physiol. 280: G781-G786.

  19. CTX Pathogenicity Island Boyd, Fidelma & Matthew K. Waldor. 1999. Alternative Mechanism of Cholera Toxin Acquisition by Vibrio cholerae: Generalized Transduction of CTX by Bacteriophage CP-T1. Infection and Immunity. 67(11): 5898-5905.

  20. Overview • Epidemiology • V. cholerae Biology • Model of V. cholerae Pathogenicity Acquisition • Toxin Co-Regulated Pilus • Vaccine Development Image from: http://microvet.arizona.edu/Courses/MIC420/lecture_notes/vibrio/vibrio_cholera_em.gifElectron microscopic image of Vibrio cholerae: curved rods with polar flagellum

  21. Model for the Evolution of Pathogenic V. cholerae Faruque, Shah & G. Balakrish Nair. 2002. Minireview: Molecular Ecology of Toxigenic Vibrio cholerae.Microbiol. Immunol. 46(2): 59-66.

  22. So What’s Out There? Virulence Potential Faruque, Shah et al. 2004. Genetic diversity & virulence potential of environmental Vibrio cholerae population in a cholera endemic area. PNAS. 101(7); 2123-2129.

  23. Genetic Diversity & Virulence Potential Faruque, Shah et al. 2004. Genetic diversity & virulence potential of environmental Vibrio cholerae population in a cholera endemic area. PNAS. 101(7); 2123-2129.

  24. Overview • Epidemiology • V. cholerae Biology • Model of V. cholerae Pathogenicity Acquisition • Toxin Co-Regulated Pilus • Vaccine Development Image from: Jonson, Gunhild et al. 1992. Analysis & Expression of Toxin-Coregulated Pili in Classical and El Tor Vibrio cholerae O1 in vitro and in vivo. Infection & Immunity. 60(10): 4278-4284.

  25. Proposed Model of Tcp Proteins Manning, Paul. 1997. The tcp gene cluster of Vibrio cholerae.Gene. 192: 63-70.

  26. Classical & El Tor TCP Regions are Highly Conserved Divergence Is Observed: • Gene Sequence • Transcriptional Regulation • Amino Acid Sequence Picture from: Johnson, Gunhild et al. 1992. Analysis of Expression of Toxin-Coregulated Pili in Classical and El Tor Vibrio cholerae O1 in vitro and in vivo. Infection and Immunity. 60 (10): 4278-4284.

  27. Regulation of Virulence Gene Expression Cotter, Peggy & Victor DiRata. 2000. Bacterial Virulence Gene Expression: An Evolutionary Perspective. Annu. Rev. Microbiol. 54: 519-565.

  28. Classical & El Tor Biotypes Differ in Timing of tcpPH Transcription Murley, Yvette et al. 2000. Classical & El Tor Biotypes of Vibrio cholerae Differ in Timing of Transcription of tcpPH during growth in inducing conditions. Infection & Immunity. 68(5): 3010-3014.

  29. Distribution of Changes in TcpA Paul Manning. 1997. The tcp gene cluster of Vibrio cholerae.Gene. 192: 63-70.

  30. Classical & El Tor TcpA Primary Structure Divergence Boyd, Fidelma et al. 2002. Evolutionary & Functional Analysis of Variants of the TCP protein TcpA from Toxigenic Vibrio cholerae non-O1/nonO139 serogroup isolates. Microbiology. (148): 1655-1666

  31. Distribution of Polymorphic Sites in TcpA Secondary Structure Boyd, Fidelma et al. 2002. Evolutionary & Functional Analysis of Variants of the TCP protein TcpA from Toxigenic Vibrio cholerae non-O1/nonO139 serogroup isolates. Microbiology. (148): 1655-1666

  32. Distribution of Polymorphism in Tertiary TcpA Structure Boyd, Fidelma et al. 2002. Evolutionary & Functional Analysis of Variants of the TCP protein TcpA from Toxigenic Vibrio cholerae non-O1/nonO139 serogroup isolates. Microbiology. (148): 1655-1666

  33. Overview • Epidemiology • V. cholerae Biology • Model of V. cholerae Pathogenicity Acquisition • Toxin Co-Regulated Pilus • Vaccine Development Image from: http://microvet.arizona.edu/Courses/MIC420/lecture_notes/vibrio/vibrio_cholera_em.gifElectron microscopic image of Vibrio cholerae: curved rods with polar flagellum

  34. Serum Response to TCP and TCP+ Adjuvants Jia-Yan Wu, et al. 2001. Evaluation of Cholera Vaccines Formulated with Toxin-Co-regulated Pilin Peptide Plus Polymer Adjuvant in Mice.Infection & Immunity. 69(12): 7695-7702.

  35. Protection from O395 Challenge of Neonatal CD-1 Mice Evaluation of Cholera Vaccines Formulated with Toxin-Co-regulated Pilin Peptide Plus Polymer Adjuvant in Mice. 2001. Jia-Yan Wu, et al. Infection & Immunity. 69(12): 7695-7702.

  36. Significant IgA Response to TcpA in Humans with Cholera Muhammad Asaduzzman, et al. 2004. The Major Subunit of the Toxin-Coregulated Pilus TcpA Induces mucosal and Systemic Immunoglobin A Immune Responses in patients with Cholera Caused by Vibrio cholerae O1 and O139. Infection & Immunity. 72(8): 4448-4454.

  37. TcpA-specific IgA antibody responses in sera & feces of patients with cholera Muhammad Asaduzzman, et al. 2004. The Major Subunit of the Toxin-Coregulated Pilus TcpA Induces mucosal and Systemic Immunoglobin A Immune Responses in patients with Cholera Caused by Vibrio cholerae O1 and O139. Infection & Immunity. 72(8): 4448-4454.

  38. Conclusion • Vibrio TcpA vaccine development may be therapeutically important • Different from preventative vaccines • Slightly immunogenic with passive immunity conferred to neonatal mice • Cross reactivity between El Tor and Classical strains makes TcpA a good target • Characterizing environmental reservoirs of V. cholerae non-toxigenic strains is important! • CTX and easy transferability of toxicity • SXT constin and the transfer of antibiotic resistance • Currently non-toxic environmental isolates capable of colonizing mammalian intestines may become toxic!

  39. Questions? Heidelberg, J. F. et al. 2000. DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae Nature.406, 477-483.

  40. References • Asaduzzaman, Muhammad et al. 2004. The Major subunit of the toxin-coregulated pilus TcpA Induces Mucosal and Systemic Immunoglobin A Immune responses in Patients with Cholera Caused by Vibrio Cholerae O1 and O139. Infection & Immunity. 72(8): 4448-4454. • Boyd, Fidelma et al. 2002. Evolutionary & Functional Analyses of Variants of the Toxin-Co-Regulated Pilus Protein TcpA from Toxigenic Vibrio Cholerae Non-O1/Non-O139 Serotypes. Microbiology. 148: 1655-1666. • Cotter, Peggy. 2000. Bacterial Virulence Gene Regulation: an Evolutionary Perspective. Annu. Rev. Microbiol. 54:519-565. • Crowcroft, NS. 1994. Cholerae: Current Epidemiology. Communicable Disease Report. 4(13): R157-R163. • Faruque, Shah et al. 2004. Genetic diversity & virulence potential of environmental Vibrio Cholerae poplation in a cholera endemic area. PNAS. 101(7); 2123-2129. • Faruque, Shah. 2002. Molecular Ecology of Toxigenic Vibrio cholerae. Microbiol. Immunol. 46(2): 59-66. • Faruque, Shah et al. 1998. Epidemiology, Genetics & Ecology of Toxigenic Vibrio cholerae.Microbiology & Molecular Biology Reviews. 62(4): 1301-1314. • Iwanga, Masaaki et al. 2004. Antibiotic resistance by a class I integron and SXT constin in Vibrio Cholerae O1 strains isolated in Laos. Antimicrobial Agents & Chemotherapy. 48(7): 2364-2369. • Jonson, Gunhild et al. 1992. Analysis & Expression of Toxin-Coregulated Pili in Classical and El Tor Vibrio Cholerae O1 in vitro and in vivo. Infection & Immunity. 60(10): 4278-4284. • Manning, Paul. 1997. The tcp gene cluster of Vibrio Cholerae. Gene. 192: 63-70. • Murley, Yvette et al. 2000. Classical and El Tor Biotypes Differ in Timing of Transcription of tcpPH during Growth in Inducing Conditions. Infection & Immunity. 69(5): 3010-3014. • Wu, Jia-Yan et al. 2001. Evaluation of Cholera Vaccines Formulated with Toxin-Coregulated Pilin Peptide Plus Polymer Adjuvant in Mice. Infection & Immunity. 69(12): 7695-7702.

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