phd_presentation_glenting
TRANSCRIPT
Lactic Acid Bacteria in Vaccine Development
Ph.D. thesisJacob Glenting
Outline of presentation
1. Vaccines – From the pioneer studies to 3rd generation vaccines
2. Reasons for using Lactic Acid Bacteria (LAB) in vaccine development
3. Development of three types af LAB-based vaccines
4. Summary
The concept of Vaccination
Edward Jenner (1749-1823)
Vaccination began with a folklore:”From milk maids to protective vaccines against smallpox”
Building on Jenners principle - 1st generation vaccines
The next generation
Bacteria Virus
Perfect mimicry (architechture/ Needle free)
Genetic engineering facilitates defined attenuation
Risc of reversion
Attenuation
Building on Jenners principle - 2nd/3rd generation vaccines
The next generation
Bacteria Virus
By genetic engineering Isolated antigen or antigen-encoding genes Equip a safe carrier with foreign antigen
Isolation of subunits
Concept of subunit vaccination (DNA/protein)
Recombinant protein production
Concept of live recombinant vaccine vehicles
Safe microorganism as vaccine carrier
Mimics natural infection
Mucosal cross talk
Main functions1. Antigen presentation 2. Target appropriate tissue (Adhesion)3. Codelivery of adjuvant
Adhesion
1
23Carrier
Immune activation
Why use Lactic Acid Bacteria in vaccines
LAB use in manufacture of dairy products and is a natural constitutent of the human gut flora
Why use LAB for vaccines and jepordize their good name? Because the unique features :
History of safe use>>
– Safe cell factory
– Safe vehicle
Well developed tools for genetic engineering and production Gram Positive > efficient protein secretor Lack of endotoxins (problematic contaminant) Stimulates the immune system Adhere to mucosal surfaces
Keydrivers for thesis topic
Increasing demand for safe and effective vaccines
Applications of LAB in development of:
1. Subunit vaccines: Plasmid DNA and protein 2. Live vaccine vehicles
Principles of plasmid DNA vaccines- A 2-component system
Why change these elements?
Anatomy of the pDNA vaccineMicrobial cells as plasmid factories
E. coli
AntibioticR
Focus of DNA vaccine research (1993-)
Major focus areas in development of plasmid DNA vaccines Antigen discovery and optimization Delivery Mixed modality vaccines (combination treatment) Mixed with Adjuvants and cytokines
Less focus on The “non-antigen coding” genetic components:
plasmid back bone, and microbial host for plasmid production
Basic genetic components have changed very little since the beginning!
By using L. lactis we can develop a safer vaccine and shed light on the immunological mechanisms of DNA
L. lactis based plasmid vaccine production
Lactococcus lactis Safe organism
Contain no endotoxins
History in the production of fermented foods
Fermentation technology using synthetic medium
Plasmid Based on L. lactis genes only (food grade?)
Non-antibiotic selection system
(Threonine auxothrophy)
No antibiotic resistance genes
Minimized plasmid
L. Lactis vs. E. coli expression vector
Set of plasmids with identical HIV-1 BX08 gp120 expression unit +/- CpG
Plasmid backbone of different nature
L. lactis E. coli
In vitro expression of gp120 in human cells
Pair wise similar expression
CpG motif seems to down regulate expression
L. Lactis vs. E. coli expression vector
Intramuscular DNA immunization of mice (Week 0,9,15)
Similar antibody response E. coli induced more CTL responses
Addition of CpG motifs had no effect CpG increased specific CTL responses
Figure 3
102
week 0 week 3 week 6 week 9 week 15 week 19
103
104
105
106
107
108
IgG
anti
-gp1
20 t
iters
(A.U
.)
102
week 0 week 3 week 6 week 9 week 15 week 19
103
104
105
106
107
108
IgG
anti
-gp1
20 t
iters
(A.U
.) L. lactis vs. E. coli expression vector
Antibody response Cytolotic CTL response
Figure 7
0
100200
300
400
500
600
700
800
900
Day 0 Day 3
pg I
L-6
/ml
0
500
1000
1500
2000
2500
3000
3500
4000
pg I
FN
g/m
l
Day 0 Day 3
+ + + + + + + + + + + ++ + + +0
100200
300
400
500
600
700
800
900
Day 0 Day 3
pg I
L-6
/ml
0
500
1000
1500
2000
2500
3000
3500
4000
pg I
FN
g/m
l
Day 0 Day 3
+ + + + + + + + + + + ++ + + +
L. lactis vs. E. coli expression vector
pLL1
20+
/-CpG
pEC12
0+/-CpG
pLL1
20+
/-CpG
pEC12
0+/-CpG
In vitro stimulation of spleenocytes by plasmid DNA
L. lactis based vectors induced higher IFN- and IL-6
CpG enhanced the adjuvant effect
DNase treatment abolished the adjuvant effect
The L. lactis based DNA vaccine system
Conclusions
Alternative microbial production system
New backbone for plasmid DNA vaccines
Endotoxin and antibiotic-free production
Comparable induction of antibody response to E. coli based vector
CD8+ T-cells less activated by the L. lactis based vectors
Adjuvant properties of the L. lactis plasmid is potent
New experiments needed to explain the lower CTL induction
Concept of subunit vaccination (DNA/protein)
Recombinant protein production
The protein subunit vaccine
Widening the definition of vaccines
Allergen vaccines = natural allergen extracts
Batch-to-batch variations, undefined composition
”Allergenome” is being characterized
Opens for recombinant expression
Authenticity of recombinant allergen may be problematic
Need for suitable expression systems
Gram positive, secretable alternative: Lactococcus lactis AUTHENTICITY?
Biological equivalence to native Ara h2
Production of Ara h2 Peanut allergen in Lactococcus lactis
rAra h2 shows parallelisme to native Ara h2 by ELISA>> High Authenticity(?)
Peanut standardrAra h 2
Nuclease
ELISA Ara h 2 specific Ab
Conclusions
Efficient recombinant production of full length rAra h2 (Compared to E. coli)
Immunological analysis showed conserved IgG epitopes
Conformational features and IgE epitopes of rAra h2 largely unknown
….L. lactis can support: Production of allergens free of other native substances Development of hypoallergens
Production of Ara h2 Peanut allergen in Lactococcus lactis
Keydriver for thesis topic
Increasing demand for safe and effectice vaccines
Applications of LAB in development of:
1. Subunit vaccines: Plasmid DNA and protein 2. Live vaccine vehicles
LAB as live mucosal vaccines
1. Antigen delivery/presentation
2. Mucosal adhesion
3. Codelivery of adjuvantAntigen
Adhesion
Immune activation1
23LAB
Antigen presentation on LAB
LAB
Localisation of passenger protein
1. Surface associated
2. Intracellular
3. Extracellular – Free form
Bet v1 AnchorSpacerSP
Plasmid located expression unit:
Authenticity of birch pollen allergen?
Authenticity of surface bound Bet v1
Immunological activity of surface associated Bet v1
IgE inhibition assay (IgE from allergic patients)
•Complete neutralisation of Bet v1 reactive IgE•High authenticity of allergen produced on two lactobacilli strains LAB1 and LAB2
Bet v1
LAB1 LAB2
LAB as live mucosal vaccines
1. Antigen delivery/presentation
2. Mucosal adhesion
3. Codelivery of adjuvantAntigen
Adhesion
Immune activation1
23LAB
mannose
A Mannose Specific Adhesin of Lactobacillus plantarum
Mannose binding adhesin (Msa) in L. plantarum WCFS1 (Pretzer et al.,2005)
Screening LABs for affinity to mannose
Low affinity to mannose compared to L. plantarum strain 299v
Similar molecular mechanisms?
115 kDa
Almost identical sequence to msa of WCFS1
Lectin domain
A Mannose Specific Adhesin of Lactobacillus plantarum
Msa of strain 299v is responsible for adhesion to epithelial cells
Importance of Msa in mannose binding/adhesion:
Strain WCFS1 and 299v have identical msa genes but different phenotype
Heterogenous culture of strain WCFS1
Overnight culture - 2% showed strong mannose binding (ON) (n=1000)
Upregulated expression of msa?
A Mannose Specific Adhesin of Lactobacillus plantarum
Northern blot ConA specific probe
A Mannose Specific Adhesin of Lactobacillus plantarum
Sequence analysis of the ON and OFF situation of strain WCFS1
msa
104 bp
14 bp inverted repeats
Inversion does not affect promoter orientation
A Mannose Specific Adhesin of Lactobacillus plantarum
Searching for mRNA secondary structures
Transcriptional arrest by hairpin structures
msa
The Qs that arises1. With what mechanism?2. Why DNA rearrengements to control expression?
A Mannose Specific Adhesin of Lactobacillus plantarum
One recombinase can control several gene operons Instant response
2)
Strain 299v is in OFF configuration BUT has high Msa expression3)
Target for recombinases
1) msa
Conclusions Highly conserved msa in L. plantarum 299v and WCFS1 Different control of expression Caution: Surface molecules may change within a culture Importance of adhesins in vaccine delivery?
A Mannose Specific Adhesin of Lactobacillus plantarum
Summary
Developed a safer pDNA vaccine based on L. lactis and obtained information on adjuvant effect of DNA
LABs are suitable for production and delivery of allergens
Identified an important adhesion molecule In vitro studies on surface molecules are tricky
Acknowledgement
Bioneer Søren Madsen Helle Wium Ulla Poulsen Pernille Smith Annemette Brix Peter Ravn Hans Israelsen Bjørn Holst Astrid Vrang Anne Cathrine Simon S. Jensen Ole Cai Hansen Lars Pedersen
DHI Stephen Wessels Ann Detmer
SSI Anders Fomsgaard Gregers Gram Mette Thorn
Danish University Hospital Lars K. Poulsen
Biocentrum DTU Hanne Frøkiær
ALK Abello Mercedez Ferreras Jens Brimnes