Transcript KR Presentation

Pneumolysin fusion proteins as
novel vaccine candidates against
experimental pneumococcal disease
Dr Kirsty Ross
Introduction
 Streptococcus
pneumoniae
 The burden of pneumococcal disease
 Current and novel vaccine development
 PATH project
 Serology
 Protection
from colonisation
 Protection from invasive disease
 Impact on host
Pneumococcal disease
•Non-invasive disease
–
Otitis media
– Sinusitis
•
Invasive disease
–
Pneumonia
– Bacteræmia
– Meningitis
• http://courses.cit.cornell.edu/biomi290/microscopycases/OtitisBiofilm/images/earDiagram.gif
• http://img.thebody.com/sfaf/2008/winter08_pneumonia1.jpg
Pneumococcal disease
•Non-invasive disease
–
Otitis media
– Sinusitis
•
Invasive disease
–
Pneumonia
– Bacteræmia
– Meningitis
• O’Brien et al Lancet 2009; 374: 893-902
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Streptococcus pneumoniae
Gram positive, -haemolytic, aerotolerant anaerobe
 Capsule-main
virulence factor
 Phosphorylcholine
 Pneumolysin
 Protein virulence factors
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• http://www.laek-rlp.de/pictures/pneumokokken.png
• Mitchell et al Nature Reviews Microbiology 1, 219-230 (December 2003)
Current vaccines
Pneumovax®
 Capsular
–
polysaccharide only
23 capsular types (1, 2, 3, 4, 5, 6B, 7F, 8,
9N, 9V, 10A, 11A, 12F, 14, 15B, 17F,
18C, 19F, 19A, 20, 22F, 23F & 33F)
– Not efficacious in children under 2
Current vaccines
Pneumovax®
 Capsular
polysaccharide only
–
23 capsular types (1, 2, 3, 4, 5, 6B, 7F, 8, 9N,
9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F,
19A, 20, 22F, 23F & 33F)
– Not efficacious in children under 2
Prevenar/Prevnar® (PCV7)
 Capsular
polysaccharide chemically
conjugated to CRM197
–
7 capsular types (4, 6B, 9V, 14, 18C, 19F &
23F)
– Versions with increased valency also available
with additional serotypes (1, 3, 5, 6A, 7F, &
19A)
– Efficacious in children under 2
Current vaccines
Pros
Pneumovax®
 Capsular
polysaccharide only
–
23 capsular types (1, 2, 3, 4, 5, 6B, 7F, 8, 9N,
9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F,
19A, 20, 22F, 23F & 33F)
– Not efficacious in children under 2
Prevenar/Prevnar® (PCV7)
 Major
decrease IPD caused by
vaccine serotypes (VT)
 Protein based conjugation improves
efficacy in children under 2
 Herd immunity
Cons
 Capsular
polysaccharide chemically
conjugated to CRM197
–
7 capsular types (4, 6B, 9V, 14, 18C, 19F &
23F)
– Increased valency versions also available with
additional serotypes (1, 3, 5, 6A, 7F, & 19A)
– Efficacious in children under 2
 Expensive
to produce
 Replacement disease with nonvaccine serotypes (NVT)
 Polysaccharide only vaccines require
boosters every 5-10 years
Next generation vaccines
 Subunit
–
based
Many potentially protective antigens have been identified using whole genome
analysis and signature tagged mutagenesis screens
Next generation vaccines
 Subunit
–
Many potentially protective antigens have been identified using whole genome
analysis and signature tagged mutagenesis screens
 Cheap
–
based
to manufacture
Protein based vaccines would exploit existing manufacturing technologies for high
expression levels of protein
Next generation vaccines
 Subunit
–
Many potentially protective antigens have been identified using whole genome
analysis and signature tagged mutagenesis screens
 Cheap
–
to manufacture
Protein based vaccines would exploit existing manufacturing technologies for high
expression levels of protein
 Ease
–
based
of distribution
Ideally would be freeze dried or lack a ‘cold chain supply’, which would simplify
delivery in developing countries
Next generation vaccines
 Subunit
–
Many potentially protective antigens have been identified using whole genome
analysis and signature tagged mutagenesis screens
 Cheap
–
to manufacture
Protein based vaccines would exploit existing manufacturing technologies for high
expression levels of protein
 Ease
–
based
of distribution
Ideally would be freeze dried or lack a ‘cold chain supply’, which would simplify
delivery in developing countries
 Mucosal
–
delivery
Avoid potential for contamination with blood-borne diseases and increase patient
acceptability
Next generation vaccines
 Subunit
–
Many potentially protective antigens have been identified using whole genome
analysis and signature tagged mutagenesis screens
 Cheap
–
to manufacture
Protein based vaccines would exploit existing manufacturing technologies for high
expression levels of protein
 Ease
–
based
of distribution
Ideally would be freeze dried or lack a ‘cold chain supply’, which would simplify
delivery in developing countries
 Mucosal
–
Avoid potential for contamination with blood-borne diseases and increase patient
acceptability
 Life-long
–
delivery
immunity
Ideally protection would be conferred for life
Concept behind PATH project:
Antigens fused to the N terminus of
pneumolysin (PLY) become immunogenic
QuickTime™ and a
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Concept behind PATH project:
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 eGFP
and PsaA were fused to PLY and 6PLY
–
One 200ng dose sufficient to stimulate systemic antigen specific IgG
– Antigens had to be fused to PLY to elicit a response
– Antigen specific IgA detected in nasal lavage and BALF
•
Douce et al Vaccine
Objectives
1.Clone and fuse four vaccine candidates (PsaA, PspA, PspC &
PhtD) from S. pneumoniae TIGR4 to PLY & 6PLY
2.Purify protein & vaccinate i.n. & s.c. to evaluate immune
response to fused antigens
3.Challenge vaccinated mice with homologous and heterologous
pneumococcal strains in models of colonisation & pneumonia
Cloning & protein purification
 Infusion
system
 Resulting constructs:
 For
further details, see Jiangtao Ma
Purified protein & native protein from cell lysates
 Pooled
antisera from animals vaccinated with PsaA6PLY
 Sera recognises both recombinant & native forms of each protein partner
Jiangtao Ma
•
•
Vaccinated with either a mixture of
single antigens or PLY
–
This was administered three times
intranasally at fortnightly intervals
•
•
•
Single antigens & PLY group
•
–
Mice were monitored for 72hrs using
the following:

Initial images taken using a Xenogen IVIS
200 using 5 min on large bin, FOV E

Tail bleeds for development of bacteræmia
–
Mice were culled at 72hpi and
processed for blood, brain, nasal wash, &
lungs.
This group received 8µg 10xMix6PLY
–
This was administered three times
intranasally at fortnightly intervals
•
S. pneumoniae colonisation
All mice were given ~ 107 cfu/10 µl of
Xen 10 A66.1, D39 or Xen 35 TIGR4 S.
pneumoniae
10xMixture6PLY group
•
S. pneumoniae invasion
All mice were given ~ 107 cfu/50 µl of
D39 S. pneumoniae
–
Mice were monitored for 72hrs using
the following:

Tail bleeds for development of bacteræmia
–
Mice were culled at 72hpi and
processed for blood, brain, nasal wash, &
lungs.
In conclusion
& 6PLY are capable of significant adjuvant activity at nanogram
doses
 PLY
In conclusion
& 6PLY are capable of significant adjuvant activity at nanogram
doses
 Fusing other antigens to the N terminus appears not to affect haemolytic
activity (max. 96kDa tested so far)
 PLY
In conclusion
& 6PLY are capable of significant adjuvant activity at nanogram
doses
 Fusing other antigens to the N terminus appears not to affect haemolytic
activity (max. 96kDa tested so far)
 High titres of antigen specific IgG and IgA are generated in response to
i.n. delivery
 PLY
In conclusion
& 6PLY are capable of significant adjuvant activity at nanogram
doses
 Fusing other antigens to the N terminus appears not to affect haemolytic
activity (max. 96kDa tested so far)
 High titres of antigen specific IgG and IgA are generated in response to
i.n. delivery
 Potential use as a platform delivery system for other antigens of interest
 PLY
In conclusion
& 6PLY are capable of significant adjuvant activity at nanogram
doses
 Fusing other antigens to the N terminus appears not to affect haemolytic
activity (max. 96kDa tested so far)
 High titres of antigen specific IgG and IgA are generated in response to
i.n. delivery
 Potential use as a platform delivery system for other antigens of interest
 PLY
The response to mixed fusion proteins included:
In conclusion
& 6PLY are capable of significant adjuvant activity at nanogram
doses
 Fusing other antigens to the N terminus appears not to affect haemolytic
activity (max. 96kDa tested so far)
 High titres of antigen specific IgG and IgA are generated in response to
i.n. delivery
 Potential use as a platform delivery system for other antigens of interest
 PLY
The response to mixed fusion proteins included:
 Increased clearance of three different serotypes of pneumococcus during
colonisation
In conclusion
& 6PLY are capable of significant adjuvant activity at nanogram
doses
 Fusing other antigens to the N terminus appears not to affect haemolytic
activity (max. 96kDa tested so far)
 High titres of antigen specific IgG and IgA are generated in response to
i.n. delivery
 Potential use as a platform delivery system for other antigens of interest
 PLY
The response to mixed fusion proteins included:
 Increased clearance of three different serotypes of pneumococcus during
colonisation
 Reduction in bacterial burden & protection from invasive disease caused
by a heterologous serotype
Thank you
–
Prof. Tim Mitchell
– Dr Gill Douce
– Jiangtao Ma
–
Dr Andrea Mitchell
– Ashleigh Holmes
– Catherine Dalziel
–
Carol McInally
– Jenny Herbert
– M. Kashif Mughal
– Matt Horsham
– Ryan Ritchie
– M. Sultan Al-Sharif
– M. Yahya Noori