PhD_presentation_glenting

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


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?


Northern blot ConA specific probe


Sequence analysis of the ON and OFF situation of strain WCFS1

msa

104 bp

14 bp inverted repeats

Inversion does not affect promoter orientation


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?


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?


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

PhD_presentation_glenting

Documents

Transcript of PhD_presentation_glenting