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RSV vaccine using recombinant

F protein ?
Sophie Nallet
Laboratory of Cellular Biotechnology
Iwo Knig, Nicole Westerfeld, Lucia Baldi, Mario Amacker,
David Hacker, Christiane Zaborosch, Rinaldo Zurbriggen, Florian Wurm
50 years of vaccine research
RSV is a worldwide burden (64 million infections/yr,
160000 deaths/year)
RSV was first isolated in 1956 (Morris et al.).
No licenced vaccine available
RSV weakly immunogenic
Enhanced disease associated with a formalin-inactivated
vaccine (Kapikian et al, 1969, Kim et al, 1969)
Subunit vaccine promising
F and G proteins induce neutralizing Ab (Walsh et
al,1987)
How to produce RSV-F ?
Production of recombinant RSV-F (rRSV-F) in
mammalian cells by Transient Gene Expression (TGE)
Correct folding, assembly, and post-translational
modifications
Scalable and simple process
Rapid and inexpensive
Production of viral RSV-F (vRSV-F)
low viral titers in cell culture
biosafety problems
Objectives
1. Production of rRSV-F by transient gene
expression in mammalian cells.
2. Scale up of the manufacturing process of
rRSV-F for animal studies.
3. rRSV-F in virosomes : Animal experiments.
Establish a manufacturing process for rRSV-F :
RSV-F : a trimeric membrane glycoprotein
Morton et al, 2003
1 100 200 300 400 500
F1 F2
SP FP TM
S-S
Proteases
cleavage
sites
F0
aa
Production of rRSV-F by transient gene
expression Proof of principle
RSV-F synthetic
sequence-
optimized cDNA
RSV
Transfect
mammalian
cells
Express
rRSV-F
Purify rRSV-F
Plasmid
Formulation of rRSV-F in virosomes -
Principle
Purified
rRSV-F
+
Empty virosome
=
rRSV-F integrated
in virosomes
Pevion Biotech

Hemagglutinin
Neuraminidase
DNA
Transfecting
agent
3h
Production
medium
2-3 d
Cell harvest
Purification
Transfection Production
Orbital shaking
1. Production of rRSV-F by transient gene
expression in mammalian cells
1. Production of rRSV-F by transient gene
expression in mammalian cells
Cell line
DNA amounts
Transfecting agent/DNA ratio
Medium for transfection
Medium for production
Temperature
Time of harvest
Addition of chemicals
Expression plasmid
Cell seeding density
Aeration
0
10
20
30
40
r
R
S
V
-
F

[
m
g
/
L
]
Cell seeding density
DNA and Transfecting Agent amounts
Optimal conditions yielded 30 mg/L of rRSV-F
in 48h in HEK-293E cells.
low cell density
high cell density
2x x
a 2a 3a a 2a 4a 6a
DNA
Transfecting Agent
[g/mL]
1. Production of rRSV-F by transient gene
expression in mammalian cells
30 mg/L at 10-mL scale
Is it achievable at a larger scale ?
Disposable bioreactors:
- Single use
- Reduced cross-
contamination
- Simple use
- Simplified validation
- Rapid set up
- No cleaning
- Cost-effective
- Orbital shaking
12 mg of purified rRSV-F were produced for
animal studies out of 1.3 L of cell culture.
ELISA
45
21
kDa
v
R
S
V
-
F
r
R
S
V
-
F
F1
F2
Reducing SDS-PAGE
2. Scale up of the manufacturing process of
rRSV-F for animal studies
0
5
10
15
20
25
30
1 2 3
Time [days]
r
R
S
V
-
F

[
m
g
/
L
]
Immunization of BALB/c mice
7.5 g RSV-F/dose
rRSV-F-virosome i.m.
0 3 6 8
Week
Pre-
Immunization
Influenza
RSV-F RSV-F Bleeding
3. rRSV-F in virosomes : Animal experiments
0
20
40
60
80
100
1:137.5 1:275 1:550 1:1100 1:2200
Serum dilution
%

i
n
h
i
b
t
i
o
n
Neutralization of RSV by BALB/c mice sera
Animal challenge in cotton rats has been performed
using rRSV-F formulated in virosomes and data
analysis is ongoing.
vRSV-F
rRSV-F
3. rRSV-F in virosomes : Animal experiments
Conclusion
1. We developed a scalable process for the
production of rRSV-F by transient gene expression
in mammalian cells.
2. Transient gene expression allowed the rapid
production of pure rRSV-F for animal studies.
3. rRSV-F in virosomes induces neutralizing
antibodies in BALB/c mice.
Go forward with transient technology
Transient gene expression :
for animal studies
for clinical trials ?
for production ?
Acknowledgements
Rinaldo Zurbriggen
Nicole Westerfeld
Mario Amacker
Lucia Baldi
David Hacker
Florian Wurm
Christiane Zaborosch
Iwo Knig
KTI/CTI projects