Development of a Sporebased Vaccine to Tetanus'

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Development of a Spore-based Vaccine to Tetanus.

• Nguyen Quynh Uyen
• School of Biological Sciences
• Royal Holloway University of London
• Supervisor Prof. Simon Cutting

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Second Generation Vaccines

• Mucosal immunity
• Delivery by non-parental route
• Robust with good storage properties

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Examples of 2nd Generation Vaccine Vehicles
under Development
Non-Living Living Liposomes Colonising
Bacteria Microparticles Salmonella
spp. Immune stimulating S. gordoni complexes
(ISCOMS) Lactobacillus spp. Bilosomes Cho
lera toxin B Non-colonising
Bacteria Lactococcus lactis Others Plants,
viruses
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Advantages of mucosal immunisation

• Triggering both systemic and local immunity
• Decreasing needles and healthcare people

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Disadvantages of mucosal immunisation

• Medium or high dose
• Requirement of mucosal adjuvant
• Potential tolerance

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Mucosal adjuvant

• Attenuated mutants of bacteria as carriers
• Salmonella spp., Shigella.
• Particles
• Liposomes
• Bacterial products with adjuvant properties
• ADP-ribosylating enterotoxins CT, LT and their B
  subunits
• DNA-based adjuvant unmethylated CpG motifs
• Immunostimulant monophosphoryl lipid A

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Advantages of Spore Delivery

• Ethical Non-pathogenic
• Storage Dormant stable robust,
• ideal for 3rd World
• Cost Production is large scale and
  cheap
• Route Oral / intra-nasal
• edible vaccine

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Life cycle of Bacillus subtilis
Sporulation
Vegetative Growth
Stage II
Starvation
MC
F
Binary Fission
VC
F
MC
VC
VC
MC mother cell F forespore VC
vegetative cell
Spore
Germination
Stage III-VII
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Fate of Spores
taken up by cells of lymphoid tissues e.g.
M-cells of Peyers patches
germinate
replicate
re-sporulate
germinate
Casula Cutting. 2002. Appl Environ Microbiol
682344-52 Hoa et al. 2001. Appl Environ
Microbiol 673819-23 Duc et al. 2004. Vaccine
221873-85
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Basic strategies
1. Spore Coat
2. Germinating spore
ORAL DELIVERY
Uptake of antigen by phagocytosis of spore or
release of soluble antigen to GALT
Germination in GALT
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• TTFC-Tetanus Toxin Fragment C
• Model Antigen
• CotB-TTFC Chimera
• N-
• Confirm expression
• Mucosal immunity

C
TTFC
CotB
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Vegetative Cell Expression
Strong evidence that a proportion of spores can
germinate in the GALT

• Develop vectors that allow high levels of
  expression in the vegetative cell and simplify
  cloning
• rrn0 promoter

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Aims of project

• 1/ To identify the optimal dose and dosing regime
  when TTFC is fused with CotB
• 2/ To enhance immune response by
• - Expression of TTFC in both spore coat and
  vegetative cell
• - Using LTB as an adjuvant.
• 3/ Proving germination and resporulation of
  spores in GIT through immune response

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TTFC-Specific IgG
Oral
Nasal
Object 1 Dosing regime using construct CotB-TTFC
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TTFC-Specific IgG at day 68 after first
immunisation
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TTFC-Specific IgG
Oral
Nasal
Object 2 Using constructs expressed TTFC in
spore coat and or in vegetative cell
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IgG1/IgG2a ratio
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TTFC-Specific IgG
Nasal
Oral
Object 2 Using LTB as adjuvant
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SDS-PAGE and Western blot of spores coat before
and after treating by simulated gastric fluid
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TTFC-Specific IgG
?
?
Object 3 Germination and resporulation of spores
in GIT
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Summary

• Spores germinate in the lumen of GI tract
• Germinated cells can grow and replicate briefly
• Spores enter the GALT (Peyers patches MLN)
• Spores germinate and resporulate in the GALT
• Spores elicit cell mediated and humoral immune
  responses