& TCP-targeted Vaccine Development

1
The Vibrio cholerae Toxin Co-Regulated Pilus

• TCP-targeted Vaccine Development
• Background image from http//www.genomenewsnetwo
  rk.org/articles/06_02/cholera_trip.shtml

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/M
IC420/lecture_notes/vibrio/vibrio_cholera_em.gifEl
ectron 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/2004/06/06
14_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 its 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/M
IC420/lecture_notes/vibrio/vibrio_cholera_em.gifEl
ectron 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
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! Im 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/in
fection/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/M
IC420/lecture_notes/vibrio/vibrio_cholera_em.gifEl
ectron 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 Whats 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/M
IC420/lecture_notes/vibrio/vibrio_cholera_em.gifEl
ectron 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. 54519-565.
• 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.