Title: Development of a Sporebased Vaccine to Tetanus'
1Development of a Spore-based Vaccine to Tetanus.
- Nguyen Quynh Uyen
- School of Biological Sciences
- Royal Holloway University of London
- Supervisor Prof. Simon Cutting
2Second Generation Vaccines
- Mucosal immunity
- Delivery by non-parental route
- Robust with good storage properties
3Examples 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
4Advantages of mucosal immunisation
- Triggering both systemic and local immunity
- Decreasing needles and healthcare people
5Disadvantages of mucosal immunisation
- Medium or high dose
- Requirement of mucosal adjuvant
- Potential tolerance
6Mucosal 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
7Advantages 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
8Life 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
9Fate 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
10Basic 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
11- TTFC-Tetanus Toxin Fragment C
- Model Antigen
- CotB-TTFC Chimera
- N-
- Confirm expression
- Mucosal immunity
C
TTFC
CotB
12Vegetative 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
13Aims 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
14TTFC-Specific IgG
Oral
Nasal
Object 1 Dosing regime using construct CotB-TTFC
15TTFC-Specific IgG at day 68 after first
immunisation
16TTFC-Specific IgG
Oral
Nasal
Object 2 Using constructs expressed TTFC in
spore coat and or in vegetative cell
17IgG1/IgG2a ratio
18TTFC-Specific IgG
Nasal
Oral
Object 2 Using LTB as adjuvant
19 SDS-PAGE and Western blot of spores coat before
and after treating by simulated gastric fluid
20TTFC-Specific IgG
?
?
Object 3 Germination and resporulation of spores
in GIT
21Summary
- 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