Biology Reference
In-Depth Information
3.4 Measurement
of Viral Productivity
It is important to analyze viral productivity when a new virus is
used. Good productivity of HAdV-5 can be obtained in HEK-293
cells as they express high levels of integrins and CAR receptor.
However, chimeric vectors lacking the HAdV-5 fiber protein may
not efficiently infect permissive HEK-293 cells, making vector
amplification inefficient and viral productivity per cell very low, as
it happens for chimeric HAdV-5/40 vectors [
9
].
(
)
×
(
)
TitervgmLTotal viralvolumemL
Num
/
(
)
=
(3)
Productivity vg
/
cell
berofcells used
(
)
×
(
)
TiterIUmLTotal viralvolumemL
Num
/
(
)
=
(4)
Productivity IU cell
/
berofcells used
3.5 Analysis
of Adenoviral
Replicative Cycle
Adenoviral replicative cycle represents the time elapsing between
the adenovirus entry and the progeny release from the cell. Since a
premature harvest leads to a poor production due to the insufficient
virion encapsidation and maturation, it is crucial to know the viral
cycle to achieve a high production yield. Similarly, in a late harvest
most of the particles may have been released to the culture medium,
leading to a harder purification and bigger material expenditure.
Therefore, to optimize the adenovirus production is important to
determine the optimal harvest time in which the viral particles are
mostly mature and still are inside the cells.
Interestingly, as a consequence of the role of the fiber in the
entry and trafficking of the virus particle towards the nucleus,
replacement of the fiber protein is enough to alter the length of the
virus life cycle [
9
]. For example, the cycle of the chimeric adenovi-
rus HAdV-5/40s is delayed 20-24 h compared to the HAdV-5
cycle. Therefore, before performing a large-scale production, the
harvest time for a new chimeric adenovirus should be empirically
optimized to maximize the yield.
1. Seed 150,000 HEK-293 cells per well of a 24-well plate. Add
500 μL of growing medium. Samples must be harvested at 24,
36, 40, 44, 48, 52, 56, and 60 h post-infection, with a mini-
mum of
n
= 3 wells per time point (
see
Note 9
).
2. Add 3.75 × 10
6
Infection Units (IU) from your pre-stock to
12.5 mL of infection medium. Mix by inversion. These values
allow to infect 25 wells at an MOI of 5 in a volume of 500 μL/
well.
3. Replace the growing medium with 500 μL of the medium-
virus mix generated in the previous step (
see
Note 10
).
4. Fifteen hours later, replace the medium with fresh infection
medium to remove the excess of virus used in the infection.
