Download Use of virus-like particles for therapeutic vaccination

Use
of virus-like
particles
therapeuticParticles
vaccination
Vaccines
Based
on for
Virus-Like
Regulation
of anti-viral
cell responses
against
addiction
andBother
chronic diseases
1
• The great success of the HBV and in
particular
HPV vaccines
the
Use of virus-like
particles for demonsrates
therapeutic vaccination
Regulation
of anti-viral
cell responses
addiction
andBother
chronic diseases
poweragainst
of VLPs
• All inacticated viruses are essentially VLPs
• Do we need anything more to say?
2
Harnessing the Immune System
Clinical Use
Antibodies
• Prophylactic Vaccines
• Self-specific Antibodies
• Allergies
• Drug-specific Antibodies
T Cells
• Cancer
• Chronic Viral Infections
3
4cLD0056_screenshow
Mechanism of action of classical vaccines
A selection of currently used vaccines and their
effector mechanisms
Pathogen
Pox
Polio
Hepatitis B
Hepatitis A
Measles
Rubella
Mumps
Tetanus
Pertussis
Tuberculosis
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Effector Mechanism
Antibody
Antibody
Antibody
Antibody
Antibody
Antibody
Antibody
Antibody
Antibody
T cells
Types of vaccines currently used and/or in development
• Live attenuated (eg Polio Sabine or Rubella)
• Inactivated (eg Polio Salk or Hepatitis A)
• Isolated pathogen-components (eg bacterial conjugate
vaccines, Tetanus, Diptheria, Pertussis (toxins))
• Recombinant (eg Hepatitis B, detoxified version of Diphteria)
• DNA
5
Safety of the various vaccine types
Safety/Specificity
The “less alive” the safer
Live attenuated
Inactivated
Isolated pathogencomponents
6
Recombinant
proteins
(DNA)
Immunogenicity of the various vaccine types
Immunogenicity
The “less alive” the less immunogenic
Live attenuated
Inactivated
Isolated pathogencomponents
7
Recombinant
proteins
DNA
Challenge
The problem that has to be solved
Make Attenuated Pathogens as Safe as Recombinant Proteins
or
Make Recombinant Proteins as immunogenic as Attenuated Pathogens
8
Make Attenuated Pathogens as safe as Recombinant Proteins
Ways to attenuate pathogens
• Specific deletions in pathogen genome (eg virulence factors) rather than passaging
• Recombinant versions of “safe” live vectors (eg yellow fewer vaccine that expresses
dengue fever proteins (Aventis Pasteur))
• Replications-incompetent vaccines (eg Alphavirus-based vaccines)
• Reassortant viruses (chimeras between human and non-human viruses)
• Attenuation of pathogens by making them more immunogenic (eg IL-2 expressing
vaccinia virus; listeriolysin expressing BCG)
9
Make Attenuated Pathogens as safe as Recombinant Proteins
Immunogenicity
•
However:
Degree of Attenuation/Safety
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•The danger to hit immunocompromised people (Polio in AIDS patient,
Tuberculosis Vaccine in IL-12-deficient patients)
• Reversion to virulent form (All Polio cases in Europe are vaccine-induced)
• If attenuation is based on impaired replication, there is a strong in vivo
pressure for the reversion to more virulent forms (Polio ÅÆ HIV)
• Even minimal side-effects are not tolerated (Rotashield)
Challenge
The problem that has to be solved
Make Attenuated Pathogens as safe as Recombinant Proteins
or
Make Recombinant Proteins as immunogenic as Attenuated Pathogens
11
B cell responses
The immunology behind it
12
B cell responses
How can we optimze B cell responses?
13
B cell responses
Optimizing Th cell responses
14
B cell responses
Optimizing Th cell responses: But which Th cells
15
TH1
IL-12, IFNγ
TH2
IL-4
Treg
TGFβ/IL-10
THfo
IL-21
B cell responses
Optimizing Th cell responses
3 strategies for enhancing Th cell responses
1) Formation of a depot Æ Long-lived T cell stimulation
2) Targeting dendritic cells
3) Activation of dendritic cells
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B cell responses
Depot formation
Typical Adjuvants forming a depot:
•
•
•
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Chemical precipitates (Alum)
Oil-in-Water/Water-in-oil (IFA, Montanide MF59 etc)
Protein precipitates (IC31)
B cell responses
Targeting dendritic cells
Typical adjuvants targeting dendritic cells
Æ Make them particulate
•
•
•
•
•
•
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Liposomes
Virosomes
Microparticles
ISCOMs/Iscomatrix
IC31
VLPs
B cell responses
Activating dendritic cells
Typical adjuvants activating dendritic cells
•
•
•
•
•
•
•
•
•
19
CpGs, IC31 (TLR9)
MPL (TLR4)
RNA (TLR7/8)
TLR-2,3,4,5 ligands
Peptidoglycan (nods)
Alum (Nalp3)
QS21
Saponin
Heat-shock proteins
B cell responses
20
B cell responses
• T help
• Epitope density of Ag
• Complement
• Toll-like receptor ligands
21
Scientific
Rational
Science 262, 1448-1451
22
Organization:
high
low
absent
Antibody response
+++
+
-
Induction of auto-antibodies
+++
-
-
Role of epitope densitiy and organisation
How important is Antigen-repetitiveness on a
viral surface?
Which is the minimal epitope density we need for effective crosslinking of the B-cell receptor?
Study of antigen density versus antigen amount
Antigen
HBc subunit
crosslinker
Antigen
HBc subunit
crosslinker
23
.
Role of Epitope Densitiy & Organization
Antigen organization is critical for IgG responses
10
v. low
low
medium
high
8
high
6
medium
4
low
v. low
2
0
d7
d18
d25
d50
d81
days after immunisation
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d126
d220
Role of epitope densitiy and organisation
Why is epitope reptitiveness so important
Our immune system exploits structural features
for the discrimination of self and foreign.
Viruses have small genomes and therefore
consist of a small number of proteins.
Viruses cannot help but have to express
highly repetitive and highly ordered arrays of antigens on their surface
25
B cell responses
• T help
• Epitope density of Ag
• Complement
• Toll-like receptor ligands
26
Role of epitope densitiy and organisation
Role of CD21 in B cell activation versus isotype
switch
The model: CD21 enhances BCR-signalling, increasing
B cell activation
27
Role of epitope densitiy and organisation
Role of Antigen Organization and CD21
for IgG responses
IgG-titers in CD21 -/-
7
6
5
4
3
2
1
0
High
Medium
Low
d14
d46
days after immunisation
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d95
E LIS A tite r (-log2 , 4 0 x
pre dilute d s e ra )
ELISA titer (-log2, 40 x
prediluted sera)
IgG-titers in Controls
7
6
5
4
3
2
1
0
High
Medium
d14
d46
days after immunization
d95
Role of epitope densitiy and organisation
B cell response
CD21 reduces the number of BCRs that need to be
engaged
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+CD21
no CD21
Nr of cross-linked BCRs
B cell responses
Optimal Vaccines should
An Optimal Vaccine should be:
• Particulate (DC Targeting)
• Repetitive (BCR receptor cross-linking)
• Fix Complement (CD19/CD21)
• Contain TLR-ligands or alike for DC and/or B cell activation
• Economical and GMP-compatible Production
• Stable
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Virus-Like Particles Based on RNA-Phages
VLPs derived from bacteriophages are:
• Particulate Æ Target dendritic cells
• Contain RNA Æ Activate dendritic cells
• Are highly repetitive
• Fix complement via natural Abs plus
alternative pathway
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In addition, they are
• very stable
• cheap to produce (3 g/l Bacterial Culture)
4cLD0056_screenshow
Generating Repetitive Antigen Arrays
Induction of Antibodies
antigen
N
N
O
N
O
N
O
O
O
O
N
N
O
N
O
N
O
O
O
O
N
N
N
N
O
O
O
O
O
Cys
O
N
N
O
O
O
O
O
O
N
N
O
O
O
N
N
N
N
O
O
O
N
N
O
N
N
O
O
O
N
O
O
O
O
O
O
O
O
O
N
O
N
O
N
O
O
N
N
O
O
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diameter = 30 nm
N
N
O
O
O
O
N
N
MW = 379.36
N
O
O
SMPH chemical linker
From Mice to Men
High Antibody Titers in Both Species
Mouse
Human
Antibody titer (Endpoint)
Antibody titer (OD50)
10000
1000
Factor 100
100
10
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10000
1000
100
10
Peptide
Adjuvants
100000
none
Peptide
Alum
PeptidePeptideProtein Carrier VLP
Alum
none
10μg
i.m.
50μg
i.m.
10μg
s.c.
50μg
s.c.
Antibody Response in Humans
Anti-Nicotine Ab Responses
1000000
Antibody titer
100000
10000
1000
100
10
1
0
1
2
3
4
5
month
34
Months after Immunization Against Nicotine
6
If You`re in a Rush
High Antibody Levels are Reached in a few
weeks
100 μg, weekly regimen
100 μg, biweekly regimen
20000
ELISA titer
Anti-nicotine IgG response
25000
100μg, monthly regimen (Ph. II)
p<0.0001
15000
10000
5000
0
0
1
2
3
4
5
6
7
weeks
35
8
9
10
11
12
13
Factor 10.2 increase
in antibody titers
Influenza A-Virus
Structure and Classification of Strains
Classification of Influenza A
Strains:
Hemagglutinin (HA): H 1-16
Neuraminidase (NA): N 1-9
e.g. H3N2 ; H5N1
Human isolates of the last
century:
- H1-3,
- N1-2
- RNA Virus, 8 segments – 10 Proteins
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Universal Influenza A Vaccine
Variability of HA versus M2 in Influenza A
major
pandemics
Antigenic “drift” and “shift” from 1918 - 2000
1918
1957
1968
Representative portion of HA
1918
1957
1968
Complete extracellular domain of M2
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Preclinical Testing of VLP-M2
Generation of M2-Immunodrug
Antigen
Linker
Carrier
(M2 peptide)
(SMPH)
(AP205)
O
N
N
Cys
N
O
O
SLLTEVETPIRNEWGCRCNDSSD-GGGC
38
O
O
O
O
Lys
Preclinical Testing of VLP-M2
Induction of M2 specific antibodies
M2 specific titers
anti-M2 IgG
40'000
30'000
20'000
10'000
0
d14
d21
Strong immune responses against M2 are induced by VLP-M2
without adjuvant
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Preclinical Testing of VLP-M2
Survival and „Fever“ Post 4xLD50 Dose
Survival
„Fever“
(Strain: PR8 H1N1 – mouse adapted)
Æ VLP-M2 vaccinated mice are protected from a lethal challenge with Influenza A
40
Isotype-Switching
ssRNA
ELISA titer (-log2 x 40)
IgG2a in mice and IgG1 in humans
6
5
4
3
2
1
0
Mice
IgG1
IgG2a
IgG2b
IgG3
OD at 1:50 dilution
Antibody Isotype
41
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Human
IgG1
IgG2
IgG3
IgG4
Isotype-Switching
Does RNA in Qb Play a Role?
Digestion of RNA using RNAse
42
Isotype-Switching
TLRs drive isotype switching
ELISA titer (-log2 x 40)
VLP/ssRNA
6
5
4
3
2
1
0
IgG1
IgG2a
IgG2b
IgG3
Antibody Isotype
ELISA titer (-log2 x 40)
VLP no RNA
6
5
4
3
2
1
0
IgG1
IgG2a
IgG2b
Antibody Isotype
43
IgG3
Isotype-Switching
Toll-like recpetor 7 is Essential for Qb/ssRNA
10000
Qb/ssRNA
IgG2a
1000
100
TLR7+/-
TLR7-/-
TLR7+/-
TLR7-/-
10000
IgG1
1000
100
44
No IgG2a in TLR7-/- mice
Influenza M2
Survival: Isotypes are the key
100
with RNAÆIgG2a
% survival
80
AP205-M2
60
no RNAÆIgG1
AP205-M2
40
20
0
0
2
4
6
8
10
12
14
days after infection
45
16
18
20
22
TLR7 signalling regulates IgA (and IgG2a) responses against Qβ-VLP
Mucosal Ab levels (BAL)
IgG2a
IgA
1000
IgG2a ELISA Titer
IgA ELISA Titer
100
10
10
1
1
wild type
46
100
wild type
(empty VLP)
TLR7+/+
TLR7-/-
Myd88-/-
wild type
wild type
(empty VLP)
TLR7+/+
TLR7-/-
Myd88-/-
CD8+ T cells
Induction of CD8+ T cells
Induction of Cytotoxic T cells
TLR ligands make all the difference!
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CD8+ T cells
Induction of CD8+ T cells
p33-VLP
7 days
48
CD8+ T cells
+ RNA
VLPPGp33
VLP
25
20
15
10
5
0
C57BL/6
49
+CpG
Empty
% p33 specific CD8 T cells
Induction of CD8+ T cells
VLPCpG- VLPPGp33
p33
VLP
MyD88-/-
VLPCpGp33
Malaria vaccine
Introduction: Life cycle of Plasmodium falciparum
50
Malaria vaccine
Introduction: MSP119
51
ƒ
MSP1 is expressed as 200 kDa protein -> several proteolytic processing
steps -> 42 kDa C-terminus -> further proteolysis -> 19 kDa C-terminus
ƒ
MSP119 and MSP142 are leading vaccine candidates against blood stages of
p. falciparum.
ƒ
MSP119 is highly conserved compared with other regions of the MSP1 antigen
ƒ
Characterization of the natural immune response of individuals from areas of
endemic malaria led to the conclusion that MSP119 is most probably the target
of a protective immune response
ƒ
Under certain conditions, recombinant MSP119 can induce immunity in
monkeys and in mice.
ƒ
A high antibody titer appears to be essential
Malaria vaccine
MSP1.19: Structure
N
C
Morgan WD et al., J. Mol. Biol. (1999) 289, 113-122
52
Malaria-vaccine
Conformation of coupled MSP1.19 ?
Quadruple ELISA, anti-MSP1, mAb 2F10
1:100
0.14
1:300
HRP
Y
Y
5
OD (450 nm)
0.12
Y
Y
0.06
1:243000
1:729000
0.04
1:2187000
1:6561000
1:19683000
MSP1-Qb
Qb
MSP1 alone
Quadruple ELISA, anti-MSP1, mAb 12.10
1:100
0.8
1:300
0.7
1:900
0.6
1:2700
0.5
1:81000
0.4
1:243000
0.3
1:729000
0.2
1:2187000
0.1
1:6561000
0
1:19683000
MSP1-Qb
Qb
MSP1 alone
antigen
53
no antigen
antigen
OD (450 nm)
5) Goat anti-mouse IgG HRP
4) Mouse anti-MSP1
3) MSP1-Qb [Qb]=[1 mg/ml] (titration)
2) Rabbit anti-Qb polyclonal serum
1) Goat anti-rabbit IgG
1:81000
0
2
1
1:2700
0.08
0.02
4
3
1:900
0.1
no antigen
Malaria-vaccine
Anti-MSP119 antibody isotypes in mice
ELISA titer (-log3 x
100)
anti-MSP1.19 Isotypes, d35
6
5
4
3
2
1
0
IgG1
IgG2a
MSP1-Qb
MSP1-Qb, Alum
Antigen injected
54
MSP1,Alum
Concusions
Not if, but which VLPs to take?
•
Efficient Production
•
Preferably NOT derived from a human pathogen
- Interference with diagnostic kits
- Potential interference with vaccine against the
the pathogen and/or pathogen-induced disease
(Immunopathology)
•
Not too small (a high enough number of epitopes
needs to be coupled for optimal B cell responses)
•
55
Packaging of natural TLR-ligand