Rapid Production of VLPs in Insect Cells

Katie Rogers
1
, Ellaine Mariano
2
, Courtney Rockwell
1
, Rebecca Idiart
2
,

Janelle
Muranaka
2
, Kristin Ostrow
2
, Tom Foti
1
,

Robert Whalen
2
1
Aldevron, 441 Charmany Drive, Madison WI 53719
2
Altravax, 552 Del Rey Avenue, Sunnyvale CA 94085
Methods
Results
Conclusions
References
Abstract
Virus-like particles (VLPs) of influenza virus have been proposed as
an equally safe and efficacious alternative to the conventional
reverse-genetic vaccine platform. VLPs can be produced in a variety
of cell culture systems including insect cell lines, which typically entail
use of the Baculovirus Expression Vector System (BEVS).
Generation of recombinant baculovirus can take up to 21 days in a
labor-intensive process not amenable to high-throughput applications.
Here we present a rapid, transient-transfection based alternative for
producing VLPs in Sf9 cells. Starting with recombinant expression
vectors, this approach produces VLPs in only 72 hours and facilitates
high-throughput screening of multiple variants. Compared to
baculovirus-mediated expression, we found that the transient
transfection-based system gave similar results with much less time
and effort.
Objectives
Express FLU-only (HA/NA/M1) VLPs and Chimeric (HA/NA/Gag)
VLPs in Sf9 cells.
Determine suitability of transient-transfection based expression
for VLP production in Sf9 cells.
Compare transient transfection to baculovirus for expression of
VLPs in Sf9 cells.
Harvest VLPs
Setting up Sucrose Gradients
! Carefully overlay 2 x 1ml of 60%, 50%, 40% and 30% sucrose solutions
in an ultracentrifuge tube.
! Carefully load the sample on top.
! Spin at 100k x g for 15 hours.
! After the spin, carefully recover 1 ml fractions starting from the top of the tube.
! Run 10 !l of each fraction on a Western Blot.
! Use 200 !l of each fraction on a Reichert Refractometer to take the refractive
index of the sample. Calculate the density of the samples by comparing the
refractive index to the equation derived from the standard curve determined
from the stock sucrose solutions (see below).
1
%sucrose in
TNE
Density (g/ml) determined by
weight of 10 ml solution
Refractive Index determined by
a Reichert Refractometer
(nD-TC, 22.4C)
0 1.004 1.3347
30 1.119 1.3774
40 1.151 1.3918
50 1.197 1.4060
60 1.225 1.4203
The standard curves for sucrose solutions freshly prepared by EMA on 5/26/10
y = 2.6129x " 2.4824
R = 0.9979
1.00
1.10
1.20
1.30
1.30 1.35 1.40 1.45
Refractive Index
D
e
n
s
ity
(
g
/
m
l)
Western Blot Sucrose Gradient
hr5 enhancer
IE
1
p
ro
m
o
te
r
IE1 t...n
a
to
r
Am
pR
3780 bp
pIEx-4 (Novagen)
72 h
h
r5

O
R
F 603

1629
A
m
p
R

lef-2
IE
1
p
r
o
m

pIExBac-3 (Novagen)
6802 bp
IE1term

Generate
baculovirus:
HA
NA
M1 or
Gag
120 h
Amplify
high-titer
baculovirus
Determine
Viral Titer
Co-infect
Sf9 cells:
HA baculo
NA baculo
M1 baculo or
Gag baculo
96 h
72 h
72 h
Transient Transfection Baculovirus
Clone HA, NA,M1, Gag
into pIEx transient
transfection vector
Clone HA, NA, M1, Gag
into pIEx/Bac baculovirus
transfer vector
HA/NA/Gag - Chimeric VLPs:
Conclusions
HA/NA/M1 - FLU only VLPs:
HA
M1
HA
M1
Baculovirus
Fraction #
Control
Western Blot Analysis
Transfection Baculovirus
HA
NA
Gag
+ + + + -
+ - + - -
+ - + - -
Anti-HA
30s exp
30s exp
Transfection Baculovirus
HA
NA
Gag
+ + + +
+ - + -
+ - + -
Anti-Gag
30s exp
Western Blot Analysis
Transfection Baculovirus
HA
NA
M1
+ + + +
+ - + -
+ - + -
Anti-HA
30s exp
6-well plates of Sf9 cells were either transfected with recombinant
pIEx vectors or infected with recombinant baculoviruses encoding
HA,NA,M1 or Gag. 72 hours post-transfection or infection,
supernatants were harvested by centrifugation at 2,500 x g. 2 ul of
each supernant was analyzed by western blot. Blots were probed
with anti-HA and anti-M1 antibodies.
6-well plates of Sf9 cells were either transfected with recombinant
pIEx vectors or infected with recombinant baculoviruses encoding
HA,NA,M1 or Gag. 72 hours post-transfection or infection,
supernatants were harvested by centrifugation at 2,500 x g. 2 ul of
each supernatant was analyzed by western blot. Blots were probed
with anti-HA and anti-Gag antibodies.
Fraction #
Control
HA
Gag
HA
Gag
Transfection
Baculovirus
Sucrose Gradient Analysis
Sucrose solutions at 60%, 50%, 40% and 30% were overlaid in ultracentrifuge tubes. Supernatants
from transfections or infections were loaded on top. Tubes were centrifuged at 100k x g for 15 hours.
1 ml fractions were collected. 10 ul of each fraction was run on western blot. Blots were probed with
anti-HA or anti-Gag antibody.
Sucrose Gradient Analysis
Sucrose solutions at 60%, 50%, 40% and 30% were overlaid in ultracentrifuge tubes. Supernatants
from transfections or infections were loaded on top. Tubes were centrifuged at 100k x g for 15 hours.
1-ml fractions were collected. 10 ul of each fraction was run on western blot. Blots were probed with
anti-HA or anti-M1 antibody.
Transient-transfection based expression of VLPs in insect cells takes only 72
hours starting from expression plasmids compared to >14 days for baculovirus
expression.
Expression levels for Chimeric VLPs are comparable between transient
transfection and baculovirus.
Expression levels for FLU only VLPs are lower from transient transfection than
from baculovirus. However, expression level would likely increase by optimizing
ratios of HA/NA/M1 in transfection.
Sucrose gradient confirms VLPs are formed in both transient transfection and
baculovirus systems.
The transient transfection system provides a rapid alternative to BEVS for
expression of VLPs.
Guarino, L. A. and Dong, W. (1994) Virology 200, 328.
Jarvis, D. L. et al. (1996) Protein Expr. Purif. 8, 191.
Loomis, K., et al. (2005) J. Struct. Funct. Gen. 6, 189.
Co-transfect
Sf9 cells:
pIEx-HA
pIEx-NA
pIEx-M1
or
pIEx-Gag
Transfection Baculovirus
HA
NA
M1
+ + + +
+ - + -
+ - + -
Anti-M1
Contact
rogers@aldevron.com
rockwell@aldevron.com
ellaine.mariano@altravax.com
kristin.ostrow@altravax.com
rebecca.idiart@altravax.com
visit us on the web at www.aldevron.com
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Designed for you.
Designed for you.
1.0
1.1
1.2
1.3
1 2 3 4 5 6 7 8 9
28kDa
36kDa
28kDa
36kDa
62kDa
98kDa
Viet HA1
(ng)
2
0
1
0
5 2
.
5
1
.
2
420s exp
420s exp
312.1s exp
460s exp
Transient transfection
Baculovirus infection
D
e
n
s
it
y

(
g
/
m
l)
Transfection
Viet HA1 or SIV Gag VLP
(ng)
2
0
1
0
5 2
.
5
1
.
2
376.8s exp
161s exp
420s exp
161s exp
1.0
1.1
1.2
1.3
1 2 3 4 5 6 7 8 9
Transient transfection
Baculovirus infection
D
e
n
s
it
y

(
g
/
m
l)