The Vibrio cholerae Toxin CoRegulated Pilus TCPtargeted Vaccine

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

Overview • • • Epidemiology V. cholerae Biology Model of V. cholerae Pathogenicity Acquisition Toxin Co-Regulated Pilus Vaccine Development Image from: http: //microvet. arizona. edu/Courses/MIC 420/lecture_notes/vibrio_cholera_em. gif. Electron microscopic image of Vibrio cholerae: curved rods with polar flagellum

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

Clinical Manifestations www. who. int/entity/water_sanitation_health/dwq/en/admicrob 6. pdf

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. jp/ info/sikenjo/521 s. . .

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

Preventing Cholera: Vaccines • Orochol – Contains 2 x 108 viable cells of attenuated strain CVD 103 Hg. R in a lyophilized form – Oral immunization of children older than 2 – Subunit A of the cholera toxin (CT) has been removed • Dukoral – Protects against O 1 Inaba and Ogawa, Classical & El Tor strains – Contains 1 x 10 heat/formalin killed cells of strain WC/r. BS Image from: http: //www. pharmeragroup. com/dukoralb. htm

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 http: //www. who. int/mediacentre/factsheets/fs 107/en/print. html Sack, David, et al. 2004. Seminar: Cholera. The Lancet. 363: 223 -233.

Overview • Epidemiology • V. cholerae Biology • Model of V. cholerae Pathogenicity Acquisition • Toxin Co-Regulated Pilus • Vaccine Development Image from: http: //microvet. arizona. edu/Courses/MIC 420/lecture_notes/vibrio_cholera_em. gif. Electron microscopic image of Vibrio cholerae: curved rods with polar flagellum

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.

Classification Scheme Toxigenic V. cholerae Division into 2 epidemic serotypes O 139 Division into 2 biotypes Classical El Tor Each O 1 biotype can have 3 serotypes inaba ogawa hikojima Division into ribotypes A&C A & B (A little C) Antigens A, B, C Designed using information presented in review by NS Crowcroft. 1994. Cholera: Current Epidemiology. The Communicable Disease Report. 4(13): R 158 -R 163.

V. cholerae Classification Scheme NON-TOXIGENIC I may not be O 1, Or O 139! (but I can still stir up trouble) TOXIGENIC I define Vibrios! I’m an O 1 or O 139 Strain www. forth. go. jp/tourist/ panf/cholerae. html

Known Virulence Factors • Integrons • Toxins – – CT HA Protease RTX Toxin ACE and Zot • Adherence/Adhesins – Accessory Colonization Factors (ACF) – Omp. U & 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/01 a_bac. html

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

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 O 1 strains isolated in Laos. Antimicrobial Agents & Chemotherapy. 48(7): 2364 -2369.

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 O 1 strains isolated in Laos. Antimicrobial Agents & Chemotherapy. 48(7): 2364 -2369.

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 O 1 strains isolated in Laos. Antimicrobial Agents & Chemotherapy. 48(7): 2364 -2369.

CT TOXIN • • CT is an A-B type toxin encoded by genes located on phage CTX V. cholerae Toxicity 1. CT does the dirty work. The organism never invades the cell. 2. The catalytic A subunit activates c. AMP production 3. High intracellular c. AMP 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: G 781 -G 786.

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-T 1. Infection and Immunity. 67(11): 5898 -5905.

Overview • Epidemiology • V. cholerae Biology • Model of V. cholerae Pathogenicity Acquisition • Toxin Co-Regulated Pilus • Vaccine Development Image from: http: //microvet. arizona. edu/Courses/MIC 420/lecture_notes/vibrio_cholera_em. gif. Electron microscopic image of Vibrio cholerae: curved rods with polar flagellum

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.

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.

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.

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 O 1 in vitro and in vivo. Infection & Immunity. 60(10): 4278 -4284.

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

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 O 1 in vitro and in vivo. Infection and Immunity. 60 (10): 4278 -4284.

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

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

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

Classical & El Tor Tcp. A Primary Structure Divergence Boyd, Fidelma et al. 2002. Evolutionary & Functional Analysis of Variants of the TCP protein Tcp. A from Toxigenic Vibrio cholerae non. O 1/non. O 139 serogroup isolates. Microbiology. (148): 1655 -1666

Distribution of Polymorphic Sites in Tcp. A Secondary Structure Boyd, Fidelma et al. 2002. Evolutionary & Functional Analysis of Variants of the TCP protein Tcp. A from Toxigenic Vibrio cholerae non. O 1/non. O 139 serogroup isolates. Microbiology. (148): 1655 -1666

Distribution of Polymorphism in Tertiary Tcp. A Structure Boyd, Fidelma et al. 2002. Evolutionary & Functional Analysis of Variants of the TCP protein Tcp. A from Toxigenic Vibrio cholerae non. O 1/non. O 139 serogroup isolates. Microbiology. (148): 1655 -1666

Overview • Epidemiology • V. cholerae Biology • Model of V. cholerae Pathogenicity Acquisition • Toxin Co-Regulated Pilus • Vaccine Development Image from: http: //microvet. arizona. edu/Courses/MIC 420/lecture_notes/vibrio_cholera_em. gif. Electron microscopic image of Vibrio cholerae: curved rods with polar flagellum

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.

Protection from O 395 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.

Significant Ig. A Response to Tcp. A in Humans with Cholera Muhammad Asaduzzman, et al. 2004. The Major Subunit of the Toxin-Coregulated Pilus Tcp. A Induces mucosal and Systemic Immunoglobin A Immune Responses in patients with Cholera Caused by Vibrio cholerae O 1 and O 139. Infection & Immunity. 72(8): 4448 -4454.

Tcp. A-specific Ig. A antibody responses in sera & feces of patients with cholera Muhammad Asaduzzman, et al. 2004. The Major Subunit of the Toxin-Coregulated Pilus Tcp. A Induces mucosal and Systemic Immunoglobin A Immune Responses in patients with Cholera Caused by Vibrio cholerae O 1 and O 139. Infection & Immunity. 72(8): 4448 -4454.

Conclusion • Vibrio Tcp. A 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 Tcp. A 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!

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

References • • • Asaduzzaman, Muhammad et al. 2004. The Major subunit of the toxin-coregulated pilus Tcp. A Induces Mucosal and Systemic Immunoglobin A Immune responses in Patients with Cholera Caused by Vibrio Cholerae O 1 and O 139. Infection & Immunity. 72(8): 4448 -4454. Boyd, Fidelma et al. 2002. Evolutionary & Functional Analyses of Variants of the Toxin-Co-Regulated Pilus Protein Tcp. A from Toxigenic Vibrio Cholerae Non-O 1/Non-O 139 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): R 157 -R 163. 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 O 1 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 O 1 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 tcp. PH 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.