Download Dengue Fusion Proteins as Vaccine Candidates

Polymeric Immunoglobulin G Scaffold (PIGS) Dengue Fusion Proteins as Vaccine Candidates
St George’s University of London
Dr Miyoung Kim
June 17, 2015
Dengue: a potentially lethal disease that affects 50-100 million
people a year
Aedes mosquito
Epidemiology
Transmitted to humans
Asia
Dengue
Virus
Africa
South America
Dengue clinical symptoms
* Dengue fever
* Dengue hemorrhagic fever
* Dengue shock syndrome
Oceania
There are currently no licensed vaccines and substantial vector control
efforts have not stopped its rapid emergence and global spread.
Vaccine challenge:
• There are four antigenically distinct serotypes of dengue virus (65-70% homology)
• An effective tetravalent dengue vaccine could be achieved by using a combination
of all 4 serotypes or by using a consensus sequence from the 4 serotypes
Currently leading vaccine candidate: Sanofi Pasteur’s vaccine
Tetravalent dengue vaccine:
live attenuated yellow fever virus delivering each of the 4 serotypes/antigens
Phase 2: year 2012
The vaccine was partially effective but failed to protect against serotype 2;
however, due to small sample size, the vaccine was allowed to enter a phase
III trial
Phase 3: year 2014
Sanofi Pasteur announces a successful phase III trial in Latin
America. Plans to apply for license in 2015.
Currently leading vaccine candidate: Sanofi Pasteur’s vaccine
Difficult and expensive to make
Raise some safety issues
 An alternative, cheaper and safer vaccine
would be desirable and this is the main
objective in our approach
Aim of the study
 E (domain I, II, III) : viral attachment, entry to host cells
 EDIII domain: critical epitopes that elicit serotype-specific Abs
E domain III (ED III) of dengue virus is poorly immunogenic and needs an
appropriate adjuvant and multiple immunizations



To generate a cE vaccine based on a fusion protein with polymeric
IgG Scaffold (cE-PIGS)
To demonstrate the adjuvanticity of the cE-PIGS fusion protein
through Fcγ receptor binding and complement activation
To demonstrate immunogenicity of cE-PIGS in mice
Schematic cE-PIGS
cE
CH 1 H
C H3
C H2
μtp
mIgG2a
Cys237,240,242
Cys274
Cys340
Cys390
Cys456
Cys : Intra-monomeric disulfide bonds
Cys239,242
Cys274
Cys340
Cys390
Cys456
hIgG1
Cys575
: Inter-monomeric disulfide
bonds. May form a disulphide
bridge with J-chain
Antigen-Presenting Cell
Dengue cE
Complement receptor
mediated phagocytosis
FcR binding
C1q binding
Fc receptor mediated
phagocytosis
June 17, 2015
Partial CγH1
H
CγH2
CγH3
μ-tp
: IgM tail-piece (tp) that participates
in polymerization through a
penultimate cysteine residue
Plant expression of cE-PIGS
pEAQ
pTRAk.2
SAR 35x2/TEV5’UTR
pA35S
35/RNA2 5’ UTR RNA2 3’ UTR
J chain
: small protein that links
up monomeric IgM or
IgA molecules into higher
structures
cE-mPIGS
cE-hPIGS
Monomer
IgM
Hexamer
+ J chain
Pentamer
IgA
Expression of mouse cE-mPIGS
in WT-Nicotiana benthamiana
Reducing
Non-reducing
α -Mouse
α -Dengue
Expression of human cE-PIGS in ∆XF-Nicotiana
benthamiana
Reducing
Non-reducing
α-Human
α-Dengue
Verification of cE-hPIGS∆XF glycovariant
mPIGS in Wild type
N.benthamiana
hPIGS in ∆XF N. benthamiana
(absence of xylose & α-1,3-fucose)
(Steinkellner, BOKU, Vienna)
Reducing
α -human
α-plant glycan
Analysis of purified cE-PIGS fusion protein
on SDS-PAGE
cE-mPIGS
cE-hPIGS
* SIgA: 385 kDa
Non-reducing
Polymer: ~480 kDa
Dimer: 160 kDa
Monomer: 80kDa
Reducing
Single chain
cE-PIGS formation determined by Zetasizer
measurements
IgG
150 kDa
PIGS
80 kDa
Functional characterization by C1q complement binding assay
cE-mPIGS
cE-hPIGS
Monocyte binding by FACS analysis
Mouse J774
Human U937
Immunization protocol
Balb/c mice
(mPIGS)
Human CD64 Tg mice
(hPIGS)
0W
2W
4W
1st-immunization
2nd -immunization
3rd-immunization
Subcutaneous injection of PIGS
PIGS injected with/without Alum
Antibody & T cell response analyzed
Analysis of mPIGS-induced IgG responses, their neutralization potential
Cellular immune responses of mPIGS
Cytokine response
T cell proliferation
Evaluation of hPIGS specific IgG responses in sera of CD64 mice
bias
 Skewing towards
TH1 (IgG2a)
 Mixed TH1/TH2
Summary and conclusions

Human and mouse IgM-like poly-IgG scaffold (PIGS) molecules incorporating
consensus E domain III of dengue envelope glycoprotein (cE) have been generated
and expressed in tobacco plants

Both human and mouse cE-PIGS bound to C1q component of complement and to Fcγ
receptor-bearing cells

In vivo studies confirmed:

Bacterially expressed cE is not immunogenic without an adjuvant;

cE-PIGS induced a strong dengue cE specific IgG response in the absence of
adjuvant which was equivalent to cE with alum.

cE-mPIGS induced cellular immune responses in immunised mice above the
controls.

Initial studies showed neutralization of DV4 by mPIGS-immunised sera

Future studies will focus on further characterisation of induced immune responses and
neutralization of all 4 dengue serotypes
Acknowledgments
Dr J.R. del Valle
Pro. J. K-C. Ma
Dr R. Reljic
Dr C. van Dolleweerd
Dr M. Paul
Mrs T Guerra
Mr Gil Diogo