The document summarizes the development of three types of LAB-based (lactic acid bacteria-based) vaccines described in a Ph.D. thesis. It describes (1) the development of a safer plasmid DNA vaccine using L. lactis that obtained comparable antibody responses to an E. coli vector but lower CD8+ T cell activation, (2) the production of the peanut allergen Ara h 2 in L. lactis and its authenticity, and (3) the use of LAB for antigen delivery by presenting antigens on their surface and through adhesion mechanisms aided by molecules like the mannose-binding adhesin.

1. Lactic Acid Bacteria in Vaccine Development
Ph.D. thesis
Jacob Glenting

2. 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

3. The concept of Vaccination
Edward Jenner (1749-1823)
Vaccination began with a folklore:
”From milk maids to protective vaccines against smallpox”

4. 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

5. 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

6. Concept of subunit vaccination (DNA/protein)
Recombinant protein production

7. Concept of live recombinant vaccine vehicles
 Safe microorganism as vaccine carrier
 Mimics natural infection
 Mucosal cross talk
 Main functions
1. Antigen presentation
2. Target appropriate tissue (Adhesion)
3. Codelivery of adjuvant
Adhesion
1
2
3
Carrier
Immune activation

8. 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

9. 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

10. 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

11. 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

12. 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

13. 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

14.  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

15.  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
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week 0 week 3 week 6 week 9 week 15 week 19
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IgGanti-gp120titers(A.U.)
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week 0 week 3 week 6 week 9 week 15 week 19
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108
IgGanti-gp120titers(A.U.)
L. lactis vs. E. coli expression vector
Antibody response Cytolotic CTL response

16. Figure 7
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L. lactis vs. E. coli expression vector
pLL120+/-CpGpEC120+/-CpG
pLL120+/-CpGpEC120+/-CpG
 In vitro stimulation of spleenocytes by plasmid DNA
 L. lactis based vectors induced higher IFN-γ and IL-6
 CpG enhanced the adjuvant effect
 DNase treatment abolished the adjuvant effect

17. 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

18. Concept of subunit vaccination (DNA/protein)
Recombinant protein production

19. 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?

20.  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 standard
rAra h 2
Nuclease
ELISA Ara h 2 specific Ab

21. 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

22. 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

23. LAB as live mucosal vaccines
1. Antigen delivery/presentation
2. Mucosal adhesion
3. Codelivery of adjuvant
Antigen
Adhesion
Immune activation
1
2
3
LAB

24. 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?

25. 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

26. LAB as live mucosal vaccines
1. Antigen delivery/presentation
2. Mucosal adhesion
3. Codelivery of adjuvant
Antigen
Adhesion
Immune activation
1
2
3
LAB
mannose

27. 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

28. 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:

29.  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

30. 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

31. A Mannose Specific Adhesin of Lactobacillus
plantarum
 Searching for mRNA secondary structures
Transcriptional arrest by hairpin structures
msa

32.  The Qs that arises
1. 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

33.  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

34. 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

35. 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