12_ANIMAL CELL TECHNOLOGY_707

548

dose T7(3)TK plasmid) were positive for the presence of HSV-TK DNA sequence
(Table 2). The positive samples originated from three different animals, two samples
were derived from auxilliary lymph nodes and one from spleen. The signals from all
three samples were weak in comparison to the positive control spiked DNA samples
indicating the presence of less than 10 plasmid copies in each sample. The weak
amplification of the HSV-TK sequence in replicates suggested that the HSV-TK
sequence was present at levels close to the level of assay sensitivity. All other DNA
samples from group 3 animals were negative for the presence of HSV-
TK DNA as determined by PCR analysis for the HSV-TK gene sequence.

Confirmation of the positive status of the 3 DNA samples from group 3 animals was
performed by Southern blot hybridisation using a labelled HSV-TK specific probe. All
DNA samples which tested positive by PCR were confirmed as positive for the
presence of HSV-TK DNA sequence as determined by Southern blot hybridisation.

Conclusion

We have demonstrated the ability to determine the biodistribution of a gene therapy
plasmid in tissues of treated animals by using a PCR strategy followed by Southern
blot hybridisation using primers homologous to the therapeutic gene. Only 3/42
tissues isolated from animals treated with high dose levels of gene therapy plasmid
were confirmed as positive for the HSV-TK DNA sequence. Furthermore the target
sequence was detected at extremely low levels (< 10 plasmid copies) demonstrating
the extreme sensitivity of the methodology employed.

References

1. Mullis, K.B. and Faloona, F.(1987) Specific synthesis of DNA in vitro via a polymerase- catalysed chain
reaction. Methods in Enzymol. 155.335-350.

2. Saiki, R. K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.G. and Erlich,
H.A.(1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase.
Science 239. 487-491.

3. Sambrook, J., Fritsch, E. and Maniatis, T. (1989) Molecular cloning: A laboratory manual, 2nd Ed.,
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

Covance Laboratories Ltd
Otley Road, Harrogate, North Yorkshire, HG3 1PY
United Kingdom

Tel: +44 (0) 1423 500011 Fax: +44 (0) 1423 569595
Website: http:\\www.covance.com

SESSION ON :

DEVELOPMENTS FOR IMMUNOLOGICALS AND VACCINES

The vaccine field is the oldest domaine of animal cell technology because this technology
started with the use of primary and diploid cells for the production of viral vaccines. Still
today many new and important developments are going on in this field.
Principally four different subchapters were treated in this session: development of new
classical vaccines, development of subunit vaccines, research on new adjuvant systems,
and development and use of new cytokines for modifying and modulating the immune
system.
In detail, new classical vaccines (= infection of cells with viruses for the production of
viral vacines) are still developed today in those fields, where no vaccines exist actually or
where old technologies, like the use of embryonated eggs, are still in use. New
developments in the production of influenza virus on MDCK in cell culture and in the use
of serum-free media for virus production were presented as well as the optimisation of
production methods for HSV-2 and live measles vaccines.
A lot of vaccines cannot be produced in a classical way, wherefore subunit vaccines are
produced and use, however, whose efficacy (i.e.: immunogenicity) relies largely on the
adjuvant systems used. Another new approach is based on the use of recombinant
suicidal DNA/RNA. Upon vaccination the gene(s) encoding for the relevant antigen(s) is
transiently expressed within the cells of the host, leading therefore to an activation of the
immune system.
Animal cell technology can also be used for the production of non-viral vaccines. This
can be achieved by infecting animal cells with parasites (e.g. for the production of
Cowdria ruminantium on goat endothelial cells) or by a subunit approach where the
relevant antigen is produced by a recombinant baculovirus - insect cell culture system.
The modulation of the immune system by cytokines is of the same importance as the
vaccination approach. This can be achieved by using interferons and gangliosides, for
instance. In this context, the in vitro immunization has to be mentioned, which is a very
important step for the development of human monoclonal antibodies which are useful for
diagnosis and treatment.
In conclusion, although this session contained only about 10% of all papers, it presented
a good reflect of the actual research and developments in this field

549

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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

THE PRODUCTION OF INFLUENZA VIRUS BY IMMOBILISED MDCK CELLS

D. Looby, J. Tree, A. Talukder, K. Hayes, H. House, G. Stacey

1 CAMR, Porton Down, Salisbury, Wiltshire, UK, SP1OJG
2 Department of Biomedical Science, University of Newcastle-upon -Tyne,

INTRODUCTION

Currently most human influenza vaccines are produced in embryonated hens eggs, however
there is now considerable interest in the development of tissue culture based processes. The
main reasons for this are : 1) ready supply of substrate for virus growth particularly during a

pandemic1, 2) virus passaged in tissue culture is more representative of the natural isolate 2,,
and 3) vaccine produced in tissue culture provides better protection than egg derived vaccine
2. Currently the cell culture systems of choice for large scale manufacturing of influenza
vaccine are batch processes based on either roller bottles or solid microcarriers3, however the
production of high titre influenza virus from a continuously perfused solid microcarrier
process has also been reported 4. Fixed bed reactors based on the immobilisation of cells
within porous carriers are increasingly being used for the production of biologicals from
adherant and suspension cells. In this study we describe the production of influenza virus

from MDCK cells immobilised in a fixed bed perfusion bioreactor. The potential advantages
of this approach are: 1) high immobilised cell density leading to high virus titres, 2) cells are
protected from the effects of fluid mechanical shear thus potentially preventing premature
sloughing of infected cells from the carriers which tends to happen in solid microcarrier
processes, and 3) the fixed bed system is scaleable e.g. an industrial process for recombinant
protein production has been scaled up to 30 litre bed volume without any loss in performance.
Data is presented on the kinetics of influenza virus production in the fixed bed system and
yields are compared to roller bottle and embryonated hens egg processes.

AIMS

• To establish a fixed bed perfusion process for the production of influenza virus
• To compare the performance of the fixed bed process to egg and roller bottle

processes

MATERIALS AND METHODS

Cell Line:
The cell line was MDCK. CCL34 (ATCC)

Virus:
The virus strain was Influenza A/ PR/8/34 (N1BSC, UK)

Media:
The cell growth media was High glucose DMEM supplemented with 10% FCS. The virus
maintenance media was high glucose DMEM supplemented with trypsin (25 mg/1) and extra
glucose (1.5g/L).

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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

552

Analysis:
Glucose was measured with a YSI Glucose/Lactate analyser. Virus titre was determined by
both haemagglutination and plaque assay.

Fixed Bed Process:
The fixed bed culture system5 consisted of a reactor filled with 100 ml porous borosilicate
glass carriers and a media reservoir (1 litre working volume). The fixed bed was inoculated

with approximately cells. The cells were grown until steady state was achieved as

indicated by a levelling off of the glucose consumption rate i.e.after approximately 6 days .
The cell growth media was removed and the fixed bed of immobilised cells was washed three
times with 100 ml PBS, then infected with virus by filling the bed with 100 ml of virus
suspension (MOI 0.05 approximately). 0.9 litre of virus growth media were added to the
reservoir, and after an incubation period of two hours (virus adsorption phase) media
recirculation was started (20 linear cm per min): for batch virus production the culture was
harvested at 50 hours: for continuous perfusion virus production, the virus maintenance media
was perfused (after 24 hours) at a rate of 1 litre per day until virus production ceased (120
hours). Samples were taken daily and assayed for glucose and stored at for virus assay.

Roller bottle process
Confluent roller bottles were washed 3 times with PBS then infected at an MOI of 0.05 per

cell in 10 ml virus growth media. After one hour incubation (to allow the virus to adsorb) the
media volume was increased to 100 ml. Samples were taken daily and assayed for glucose and

stored at virus assay.

553

Total harvest volume produced: fixed bed perfusion = 4.75 litres, fixed bed batch = 1.0 litre
and roller bottle = 0.1 litre.
Typical virus yield in eggs =

• A fixed bed perfusion process producing a total of PFU from a 100 ml fixed

bed has been established this is approximately equivalent to the production from 10 roller

bottles or 200 embryonated hens eggs (Table 1).

554
• Batch virus production in the fixed bed process produced more virus as determined by

plaque assay than continuously perfused virus production (fig 1).

• The potential for large scale production of influenza virus from immobilised cells in a fixed
bed perfusion system with batch virus production has been demonstrated.

REFERENCES
1 WHO report: Influenza vaccines: prospects for production from viruses grown in cell

culture , Geneva, Switzerland (1995)
2 Robertson, J.S., Cook, P., Attwell, A.M., and Williams, S.P. (1995). Replicative

advantage in tissue culture of egg - adapted influenza virus over tissue culture derived
virus: implications for vaccine manufacture.Vaccine. 13 , 1583 -1588,
3 Brands, R., van Scharrenburg, G.J.M. and Palache, A.M. (1997). Production
of Influenza virus in cell cultures for vaccine preparation . In: Animal Cell
Technology (Eds M.J.T. Carrondo et al.) Kluwer Academic Publishers pp 165-167.
4 Merten, O.W., Hannoun, C., Manuguerra, J.C., Ventre F and Petres. S., (1996).
Development of influenza subunit vaccine produced using mammalian cell technology.
In: Novel Srategies in Design and Production of Vaccines (Eds S. Cohen and A.
Shafferman) Plenum Press, New York. pp 141 -151
5 Looby, D. and Griffiths, J.B. (1988). Fixed bed porous glass sphere (porosphere)
bioreactors for animal cell culture. Cytotechnology 1, 339 - 346.

Acknowledgements
This work was supported by a grant from the Department of Health, UK.

SERUM-FREE GROWN MDCK CELLS : AN ALTERNATIVE FOR
INFLUENZA VACCINE VIRUS PRODUCTION

N. KESSLER, G. THOMAS, L. GERENTES, AND M.AYMARD

Laboratoire de Virologie, Faculté de medecine Grange-Blanche,
8 avenue Rockefeller, 69373 Lyon cedex 08, FRANCE

Abstract

Adaptation of MDCK cells to serum-free conditions was performed using UltraMDCK

medium (BioWHITTAKER).

Growth properties and karyotype stability of MDCK cells were monitored over a one

year period of cultivation in Ultra-MDCK . Scaling up of adapted cells to spinner culture

was achieved using several porous / non porous microcarriers.

Adapted MDCK cells were tested for their suitability to replicate influenza A and B

viruses, starting from egg and cell isolated strains and isolates.
Replication of influenza viruses in serum-free adapted MDCK cells, under standard

conditions (1µg/ml trypsin) was analysed using several tests : hemagglutinin (HA) and

neuraminidase (NA) activities , infectious titre, immunofluorescence of infected cells.

Replication of influenza viruses in serum-free adapted MDCK cells, in absence of

trypsin, was also analysed. All A and B influenza viruses were successfully adapted to

replicate to high titres without addition of exogenous proteolytic

enzyme, and production of trypsin-independent viruses was obtained as well in static

flasks as under agitated conditions.

1. Introduction

Production of influenza vaccine viruses is traditionally performed in embryonated
hens’eggs, but several constraints in such a technology generated an urgent need to
develop alternative cell culture systems (WHO memorendum, February 1995). Due to
potential contamination of sera with microorganisms, it is essential to cultivate cells in
serum-free media (SFM) and to evaluate the effects of such culture conditions on
influenza virus replication. With most of cells, the presence of trypsin is required during
influenza virus infection, in order to cleave the hemagglutinin precursor and to induce
multicycle virus replication; nevertheless, previous data (Rott et al, 1984) mentioned X-
31 (H3N2) virus multicycle replication in MDCK cells in absence of trypsin.
Consequently, in order to eliminate a potential source of microbial contamination in the
final product, we analysed the suitability of MDCK cells adapted to serum-free
conditions, to replicate influenza viruses in absence of trypsin.

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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

556

2. Materials and Methods

MDCK cells (BioWhittaker) were grown either in FBS supplemented EMEM
(abbreviation : EMEM cells) or in serum-free UltraMDCK medium (BioWhittaker),
(abbreviation U-MDCK cells).
Cell cultivation was performed either in static flasks or in spinner under agitated
conditions, using several microcarriers.

Karyotype analysis of cells was performed using a modification of the GTG
banding procedure described by Seabright (1971).

Influenza viruses were selected at once, on the basis of their type/subtype and
isolation substrate (embryonated hen’s eggs or MDCK cells). Viruses were as following:

Virus infection conditions were as following :

1) Standard infection conditions in presence of trypsin

2) Trypsin-free conditions

Virus production was estimated using the following tests : hemagglutinin and

neuraminidase activities, infectious titre in MDCK cells (TCID50/ml), indirect

immunofluorescence and hematoxylin-eosin staining of infected cells, plaque assays in

MDCK cells with and without trypsin in agar overlay.

Virus analysis was performed in terms of PAGE profile and of antigenic profile

of the hemagglutinin after introduction of viruses in hemagglutination inhibition test

with monoclonal antibodies and specific polyclonal anti-sera.

3. Results and discussion

3.1. MDCK CELL CULTIVATION IN SERUM-FREE ULTRA-MDCK MEDIUM

3.1.1. Static flasks

MDCK cells previously grown in FBS supplemented EMEM (EMEM cells) , were

transferred directly to Ultra-MDCK medium and then serially cultivated over a more

than one year period of time in that environment. Ten serial passages were necessary to

cells for a complete adaptation to their new serum-free environment, as shown by a

significant decrease (student’s t test) in the doubling time of cells between p1 and p11

post transfer. Mean doubling time of adapted U-MDCK cells was shown identical to

control EMEM cells, i.e, day.

3.1.2. Karyotype analysis of U-MDCK cells
In order to verify the genetic stability of MDCK cells whih were adapted to grow in

Ultra-MDCK medium, chromosome number distribution of U-MDCK cells was

557
monitored at regular intervals of time during their long term cultivation in absence of
serum and then compared to control MDCK cells as mentioned in the ATCC catalogue.
No significant modification was detected in the chromosome distribution after 75
passages in Ultra-MDCK and the modal number was found similar to control cells : 79
versus 78 chromosomes.

3.1.3. U-MDCK cell cultivation on microcarriers
When tested for growth on microcarriers, EMEM cells and U-MDCK cells exhibited
significant differences; indeed, EMEM cells were capable to spread and grow on porous
uncoated microcarriers while U-MDCK cells could not. Nevertheless, U-MDCK cells
were efficiently cultivated on two different kinds of microcarriers : porous, collagen
coated microcarriers and non porous, uncoated microcarriers. In both cases, cell
confluency was observed four days post seeding, but cell yield was shown consistently
higher on non porous , uncoated microcarrier than on porous, collagen coated one , i.e.

These results are very interesting, when
considering a potential industrial utilization of Ultra-MDCK medium for viral vaccine
production.

3.2. INFLUENZA VIRUS PRODUCTION IN U-MDCK CELLS UNDER STANDARD
CONDITIONS

Capacity of U-MDCK cells to serve as a potential substrate for influenza virus
production was demonstrated by analyzing virus progeny collected through ten serial
passages of A and B viruses, under different MOI (Multiplicity of Infection), in presence
of All different influenza viruses replicated to high titre in U-MDCK
cells irrespective of : virus type/subtype, isolation substrate,
passage number of viruses in U-MDCK cells and strains or isolates. In addition, virus
replication was shown 1) consistently higher in U-MDCK cells than in EMEM control
cells 2) as efficient in U-MDCK cells as in standard embryonated eggs (Table 1).

Immunofluorescence (anti NP monoclonal antibodies) and hematoxylin-eosin staining
studies confirmed the high sensitivity of U-MDCK cells to influenza virus infection.

558
3.3 INFLUENZA VIRUS PRODUCTION IN U-MDCK CELLS UNDER TRYPSIN-
FREE CONDITIONS

If considering a potential utilization of U-MDCK cells for influenza vaccine virus
production, it would be interesting not to work in presence of a proteolytic enzyme, in
order to eliminate a potential source of viral and microbial contamination.

3.3.1. Selection of trypsin-independent influenza viruses
Trypsin-independent viruses were selected by serial passages in U-MDCK cells, in

absence of trypsin and under selected MOI. Such a selection was performed, starting

from three different kinds of viruses : 1) viruses after five passages with trypsin, 2)

viruses after ten passages with trypsin and 3) viruses grown in allantoic fluid.

All A and B influenza viruses were efficiently selected and serially replicated to high

titre in absence of exogenous enzyme; The best results were

observed when selection started from Three selection profiles

emerged as a function of the easiness with which adaptation to trypsin-free conditions

was performed but, with the exception of B viruses which adapted quite immediately, it
was impossible to attribute the two other profiles to a specific A subtype.

3.3.2. Biological, structural and antigenic properties of selected trypsin-independent

influenza viruses
Trypsin-independent influenza viruses replicated efficiently in U-MDCK cells, as shown

by to HA ratio analysis of A and B viruses grown in either U-MDCK cells or
embryonated eggs ; for example 2.5×103 and 0.7×103 for A/Vic/36/88 (H1N1) viruses

grown in U-MDCK and eggs respectively, 2) by comparative plaque efficiency of

control viruses and selected trypsin-independent viruses when tested with / without
trypsin in agar overlay. In absence of trypsin, control viruses gave small size plaques
(0.5-1 mm in diameter) while selected, trypsin-independent viruses gave plaques whose
diameter (2-3 mm) was shown similar to that of control viruses in presence of trypsin.
It is interesting to notice, that the hemagglutinin of trypsin-independent viruses was

likely cleaved at the stage of entry in cells, since PAGE of all different A and B viruses
clearly confirmed a very high amount of HA0 precursor when compared with virus
grown in trypsin or produced in eggs. Such a cleavage of influenza virus hemagglutinin
at entry in cells, was previously mentioned by Boycott et al (1994) when A/WSN/33
virus was grown in MDBK cells without addition of trypsin. The comparative analysis
(figure 1) of antigenic reactivity (HI test) of the hemagglutinin of both, control and
trypsin-independent viruses with anti-HA monoclonal antibodies and polyclonal
antisera, showed significant differences which were consistent with conformational
differences in HA; indeed, HI titre of some MAbs (H240 and H375) differed up to five
dilutions when reacted with viruses obtained with/without trypsin.

559

3.3.3. Production of trypsin-independent influenza viruses in U-MDCK cells grown on
microcarriers
Finally, selected trypsin-independent A and B influenza viruses were produced in U-
MDCK cells grown on microcarriers (porous/non porous) under rod-stirred conditions.
Virus harvest was performed after 3-4 days as a function of virus type/subtype and titres
were shown consistently higher when using non porous, uncoated microcarriers in
comparison with porous, collagen coated ones : 20480-40960 HAU/ml and 2560-5120
HAU/ml respectively as mean values; as a consequence, mean production yield of A and
B influenza viruses was 5 to 10 times higher when using the former. Studies are now in
progress, in order to correlate this phenomenon with potential differences in the level of
differentiation of U -MDCK cells as a function of microcarriers.
In view of these data, MDCK cells grown in serum-free Ultra-MDCK medium appeared
full of promise as an alternative substrate for influenza virus production in presence as
well as in absence of trypsin.
Acknowledgements
This work was supported by a grant from Boehringer Ingelheim Bioproducts
Partnership.

4. References

Boycott, R., Klenk, H.D., and Ohuchi, M. (1994) Cell tropism of influenza virus mediated by hemagglutinin
activation at the stage of virus entry, Virology 203, 313-319.2
Rott, R., Orlich, M., Klenk, H.D., Wang, M.I., Skehel, J.J., Wiley, D.C.0984) Studies on the adaptation of
influenza viruses to MDCK cells, EMBO 3, 3329-3332.
Seabright, M.A. (1971) Rapid banding technique for human chromosomes, Lancet ii: 971-972.

EVALUATION OF THE NEW MEDIUM (MDSS2N), FREE OF SERUM AND
ANIMAL PROTEINS, FOR THE PRODUCTION OF BIOLOGICALS.

H. KALLEL1, P. PERRIN2 and O.-W, MERTEN3.
1 Institut Pasteur de Tunis.
13, Place Pasteur. B.P. 74. 1002 Tunis Belvèdère. Tunisie.
2,3 Institut Pasteur. Laboratoire des Lyssavirus2; Laboratoire
de Technologie cellulaire3.
25, rue du Docteur Roux. 75 724 Paris Cedex 15. France.

Abstract: The development of media free of serum and animal protein is of utmost
importance for increasing the safety of biologicals produced for therapy and
vaccination. In order to reduce the risk of contamination, we have modified the serum
free medium MDSS2, a very efficient serum free medium for the production of various
biologicals including experimental vaccines using different cell lines (Merten et al.

Cytotechnology 14 (1994), 47), by replacing the animal derived products by plant
extracts.
This new serum and animal protein free medium (MDSS2N), can be efficiently used
for biomass production of various cell lines : BHK-21/BRS cells, adapted to MDSS2N
medium, grew slightly slower in this medium versus in the
old formulation), whereas the growth of Vero cells was not influenced by this
modification Cultures of
Vero cells on microcarriers in stirred tank reactor did not show any differences with

respect to the growth rate when the old and the new formulation of MDSS2 were used.

A and were observed when MDSS2 and MDSS2N were

used respectively. The use of MDSS2N for cell culture and the production of various
biologicals like rabies virus will be discussed.

1. Introduction

The use of serum containing media for the production of biologicals has many
disadvantages like varying quality, high cost, risks of contamination with mycoplasma,
virus, BSE agent, etc. Thus today, different serum free media had been formulated and
are used for the culture of various cell lines. However, most of these media still contain
animal derived proteins.

To overcome this problem, we developed a medium, frée of serum and any animal
protein, (MDSS2N). This medium is essentially based on MDSS2, a serum free
medium, which we have previously developed and used for the culture of different cell
lines (Merten et al. (1994) ; Perrin et al. (1995)).

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562

In this work, we present the evaluation of this new medium, to support the growth of
various cells lines, like BHK-21/BRS and Vero cells. In addition, we have evaluated
the ability of MDSS2N medium to sustain the production of biologicals. In this
context, rabies virus production by BHK-21/BRS cells grown in MDSS2N medium, is
studied as a model.

2. Materials and Methods

Cell lines Vero cells and BHK-21/BRS cells, derived from BHK-21 C13 and adapted
to suspension growth, were used (Perrin et al. (1995)).

Virus strain : The rabies virus PV-Paris/BHK-21 and PV-Paris/BHK-21/BRS were
used to infect the BHK-21/BRS cells (Perrin et al. (1995)).

Culture Medium : DMEM (Axcell Biotechnologies 36213 SPM) + 5% FCS (Hyclone
A-1115-L), MDSS2 (Axcell Biotechnologies 39615) and MDSS2N (Axcell
Biotechnologies 39615N) were used.

Growth assay : Cell culture assays were performed at in 25 flasks, at an
initial concentration ranging between and cells/ml. Samples were taken
daily to determine cell concentration. The assays were performed in duplicate.

Virus production : BHK-21/BRS cells were infected by two strains of rabies virus (PV-
Paris/BHK-21 and PV-Paris/BHK-21/BRS) at a cell concentration of cells/ml
with a MOI of 0. 1/cell. Virus production was performed at in 50 ml agitated
tubes containing 10 ml of the medium to be tested. Samples were taken daily to
determine cell concentration, virus titre and glycoprotein content. The assays were
performed in duplicate.

Bioreactor cultures : The cultures were performed in a 2 litres bioreactor (1.6 1
working volume) equipped with a spin filter fixed on the axes for
the retention of cells growing on microcarriers. The following conditions were

applied: air-saturation, and agitation =45

rpm.
Cells were grown on microcarriers : Superbead (Flow 60-085-12) or Cytodex 1
(Pharmacia 17-0448-01) at a concentration of 2.5 g/1, 5 g/1 and 6.25 g/1. The cultures
were performed in perfusion mode. Perfusion was started when the cell density was
about

Cell counting : For BHK-21/BRS cells growing in clumps, 0.5 ml of a trypsin/versene
solution (50/50 mixture of a 0.1% trypsin-solution in PBS with a 0.04% versene-
solution in PBS) was added to 0.5 ml of a cell suspension. After incubation at for
10 minutes, cells were stained with trypan blue (0.2% in PBS) and counted. Vero cells

were washed with PBS then trypsinized at for 5 to 10 minutes and counted. For

563

Vero cells grown on microcarriers, they were treated with 0.5 ml 0.1M citric acid
containing 0.1% crystal violet then incubated at for at least one hour, the released

cell nuclei were counted.

Rabies virus titration : Virus titres were determined according to a modified RFFIT
method (Smith et al. (1973)) and expressed in Fluorescent Focus Units per ml
(FFU/ml).

Glycoprotein titration : rabies glycoprotein content was determined by EL1SA using
polyclonal antibodies (Perrin et al. (1990)).

3. RESULTS AND DISCUSSION

3.1. Growth in serum and animal protein free medium :

3.1.1. BHK-21/BRS cells:
MDSS2N has been tested for supporting the growth of BHK-21/BRS cells in
comparison to MDSS2. Tests have been performed in T-flasks. The results obtained

show that the maximal cell density of BHK-21/BRS cells obtained was
cells/ml and cells/ml, for MDSS2N and MDSS2, respectively (figure 1). The
average specific growth rates were 0.02 and in MDSS2N and MDSS2,
respectively. These results indicate that BHK-21/BRS cells exhibit similar growth

kinetics in the new protein-free medium MDSS2N as in the previously developed

medium MDSS2.

3.1.2. Vero cells:

MDSS2N has been also evaluated for its ability to sustain the growth of Vero cells in
static (T-flasks) and agitated (micro-carrier in bioreactor) cultures.
After 3 successive subcultures of Vero cells in MDSS2N and MDSS2, the maximal cell
density reached was and in MDSS2N and MDSS2,

564

respectively (figure 2). With regard to the average specific rate, we obtained 0.0153
and in MDSS2N and MDSS2, respectively. These results show that Vero
cells growth is equal in both media tested. MDSS2N supports Vero cells growth as

MDSS2 does.

For the reactor cultures different cell densities have been obtained, when three different
media were used. The maximal cell densities were and
when DMEM + 5% FCS, MDSS2, and MDSS2N, respectively, were
used (figure 3A). These differences were essentially due to the concentration of the
microcarriers used. By comparing the average specific growth rates for these three
cultures, it becomes evident that the cultures in both serum-free media showed a
slightly higher growth rate than the culture done in DMEM + 5% FCS (table 1). In
addition, by comparing the cell of microcarriers, it is evident that the

differences between the three media used are very low with respect to the
The cell number per increased rather parallelly (figure 3B), and
the fact that the culture done in MDSS2N stopped earlier was due to the fact that the

available surface was limited and saturated at 116 h and that this culture was a
conducted as a batch and not as a perfusion culture.

565

3.2. Rabies virus production :
To evaluate the capacity of the new medium MDSS2N for the production of biologicals
like vaccines, we infected BHK-21/BRS cells (grown in MDSS2N or MDSS2) with two
strains of rabies virus : PV-Paris/BHK-21/BRS (a strain adapted to MDSS2) and PV-
Paris/BHK-21 (a strain non adapted to MDSS2).
The results obtained shown in figures 4A and 4B, indicate that the behaviour of BHK-
21/BRS cells infected with PV-Paris/BHK-21 or PV-Paris/BHK-21/BRS was relatively

566

equal in both media. In both cases, we observed a decrease of the cell density from

to 5 days post infection. The maximal virus titres
obtained in MDSS2N, after the infection by the strain PV-Paris/BHK-21, was

(see table 2). In MDSS2, virus titres were 2.4 times higher. With regard
to virus production after infection by PV-Paris/BHK-21/BRS strain, we obtained

and in MDSS2N and MDSS2, respectively.
Figures 4A and 4B also show that by using the non adapted strain (PV-Paris/BHK-21),
the levels of glycoprotein, obtained at the end of the culture, were 330 ng/ml and 960
ng/ml, in MDSS2N and MDSS2, respectively. The specific productivity of glycopro-
tein was also lower in MDSS2N than in MDSS2 (table 3).

• : The values correspond to the ratio of final glycoprotein concentration
cell density before infection ×107

: «immune» glycoprotein level was calculated by subtracting «the soluble»
glycoprotein content from total glycoprotein. Soluble glycoprotein content was
measured after centrifuging the medium at the end of the culture, at 40 000 rpm at
and for 2 hours. Then its glycoprotein content was determined by ELISA.

567

On the other hand, the levels of glycoprotein obtained after the infection of cells by
PV-Paris/BHK-21/BRS strain were 1700 and 3800 ng/ml, in MDSS2N and MDSS2,
respectively (see figures 4A and 4B). MDSS2N seems to be less favourable for rabies
virus production than MDSS2, this is probably due to the non adaptation of the strain
PV-Paris/BHK-21/BRS to cells grown in the new serum free MDSS2N.
Specific productivity of glycoprotein obtained in MDSS2N was cells
whereas the production rate in MDSS2 was cells (see table 3). These
values are comparable to the standard roller process and to those obtained by Merten et
al. (1994). The normal productivity of the standard roller process ranged from 8 to 12
µg /107 cells.
The comparison of the «immune» glycoprotein levels obtained in the different
conditions studied (see table 3) shows that the percentage of the «immune»
glycoprotein is slightly higher after the infection with PV-Paris/BHK-21/BRS strain in

568

MDSS2N or MDSS2, than after the infection with the strain PV-Paris/BHK-21.
Nevertheless, MDSS2N seems to enhance the soluble glycoprotein production.

4. CONCLUSION

MDSS2N supported BHK-21/BRS cells growth as well as MDSS2 did. Vero cells also
exhibited similar growth in MDSS2N and MDSS2. Continuous cultures performed in
perfusion mode of Vero cells, have also shown that cell growth was not affected by the
medium used.
Rabies virus production seems to be influenced by the nature of the medium tested.
MDSS2N induces virus and glycoprotein productions but lower than MDSS2 does.
This is probably due to the non adaptation of rabies virus strains to cells grown in
MDSS2N. However, the results obtained are very encouraging. Further experiments
will be run to optimise rabies virus production in MDSS2N in batch and perfusion
mode.

5. REFERENCES

Merten, O.-W., Kierulff, 1, Castignolles, N. and Perrin, P. (1994) Evaluation of the
new serum free medium (MDSS2) for the production of different : use of various cell
lines, Cytotechnology 14, 47-59.

Perrin, P., Madhusudana, S., Gontier-Jallet, C., Petres, S., Tordo, N. and Merten, O.-
W. (1995) An experimental rabies vaccine produced with a new BHK-21 suspension
cell culture process : use of serum-free medium and perfusion-reactor system, Vaccine
13, 1244-1250.

Perrin, P., Morgeaux, S. and Sureau, P. (1990) In vitro rabies vaccine potency
appraisal by ELISA : advantages of the immunocapture method with a neutralizing
anti-glycoprotein monoclonal antibody, Biologicals 18, 321-330.

Smith, J. S., Yager, P. A. and Baer, G. M. (1973) A rapid tissue culture test for
determining rabies neutralizing antibody, in M. M. Kaplan and H. Koprowsky (eds.).

Laboratory Techniques in Rabies, WHO, Geneva/CH, pp. 354-357.

PRODUCTION OF HIGH TITRE DISABLED INFECTIOUS SINGLE CYCLE
(DISC) HSV-2 FROM A MICROCARRIER CULTURE

T.A. Zecchini, R.J. Smith.
Cantab Pharmaceutical Research Ltd.,
184 Cambridge Science Park, Cambridge. U.K. CB4 4GN

1. Introduction

Disabled Infectious Single Cycle (DISC) HSV, is a genetically inactivated HSV-2 from which the
viral glycoprotein H (gH) gene has been deleted (1). Modified in this way, the release of the virus
from the cell is not blocked. However, in non-complementing cells, the lack of glycoprotein H
prevents the virus from re-infecting new cells, thereby blocking any further viral spread. The DISC
virus can only replicate and be propagated in a gH complementing cell line, which in our case is the
modified Vero cell line CR2C9.

The aim of this work was to evaluate the production of DISC HSV-2 in a microcarrier based
culture system, compared to a roller bottle culture process.

Anchorage-dependant cell lines (Vero, MRC5) have been propagated at low cell con-centrations
to produce human viral vaccines (2). Griffiths et at (3) have also

demonstrated HSV-2 production from cells cultured on Cytodex microcarriers, yielding virus
titres similar to those expected from a roller bottle system.

The work set out to determine the conditions for the optimum growth of cells and ultimately,

production of high titre virus. Production of the virus was initially determined in small-scale
(1 litre) cultures. Scale-up of the production system was then demonstrated at the 15 litre scale.

2. Small scale production – 1 litre scale

2.1 METHODS

After extensive investigation, Cytodex 1 was selected as the
most suitable microcarrier for DISC HSV-2 production. The growth

medium used was Dulbecco’s Modified Eagle Medium (DMEM)
supplemented with 5% Foetal Bovine Serum. Cultures were inoculated

at approximately 10 CR2C9 cells per microcarrier (Fig 1) and were
maintained at 37°C throughout the growth period. Cell growth was

monitored by counting nuclei released from a sample incubated in
0.1M citric acid containing 0.1% (w/v) crystal violet (4). Glucose and

lactate concentrations were controlled by partial media changes on days

2 and 3 of the growth period.

Prior to infection with DISC HSV-2 the cell cultures were washed with
complete Dulbecco’s PBS to reduce contaminating FBS levels. The
culture medium for the infection stage was serum free (DMEM).
Confluent microcarriers (Fig 2), approximately 100 hours post inoculation,

were infected with DISC HSV-2 at an MOI of 0.01. The temperature
during the infection period was decreased and maintained at 34°C

Infected cells (Fig 3) were harvested from the microcarriers
approximately 60-72 hours post infection by removal of the media
followed by the addition of hypotonic saline. The detached cells were
separated from Cytodex 1 by filtration through a sterilisable

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570

The DISC HSV-2 virus was then released from the cells using a low
pressure disruption technique (Fig 4). Virus titres were calculated
using an in-house TCID50 assay with subsequent conversion to

2.2 CULTURE RESULTS

Typically we have achieved total pfu from a 1 litre
culture, with approximately a 20 hour period during which the culture
can be harvested without loss of titre achieved. From an equivalent
number of 25 roller bottles we routinely achieve total pfu
(Table 1). Therefore the two systems yield similar DISC HSV-2 titres.

3. Large scale production – 15 litre scale

3.1 METHODS

Cultures were inoculated at a density of approximately 10 CR2C9 cells
per microcarrier and 5g of The growth period was

maintained at a temperature of The growth medium used was
DMEM with 25mM HEPES (5% FBS). Cell growth was monitored by

counting released nuclei as for the small scale cultures. Glucose and

lactate concentrations were controlled by partial media changes on
days 2,3,4 and 5 of the growth period. The culture was maintained for
an additional 48 hour period compared to the small scale cultures in
order to provide a maximal cell density at the point of infection.

Prior to infection with DISC HSV-2 the cell cultures were washed with
complete Dulbecco’s PBS to reduce contaminating FBS levels. The
culture medium for the infection stage was changed to serum free DMEM
and the temperature was decreased to Cultures were infected with
DISC HSV-2 after approximately 140-160 hours at an MOI of 0.01

The infected cells were harvested from the microcarriers
approximately 60-72 hours post infection with DISC HSV-2 by the
addition of hypotonic saline. The detached cells were separated from
the microcarriers by filtration through a sterilisable mesh. The
low pressure disruption technique (Fig 4) was again used to release
DISC HSV-2 virus from the cells. Virus titres were calculated using

an in-house assay with subsequent conversion to

571

3.2 CULTURE RESULTS

From this size culture we would expect to achieve approximately total DISC HSV-2 pfu
(Table 1). However, this culture produced total DISC HSV-2 pfu. This high titre was able

to be recovered over a sustained period of at least 9 hours. Our results, therefore, compare very
favourably with the expectations from roller bottle cultures and the results achieved from the 1 litre

cultures.

4. Summary
Cytodex 1 microcarriers can be used for the production of our DISC HSV-2 virus. Yields
achieved were comparable to those obtained in roller bottle culture systems.
The process can be scaled up demonstrating increased yields in the 15 litre culture compared to
those obtained in a I litre culture. DISC HSV-2 titres for the 15 litre culture exceeded the
anticipated titre for the equivalent number of roller bottles.
A 15 litre culture has been shown to produce an equivalent amount of virus as
roller bottles. This reduces the labour necessary, minimising costs of materials and process time.
The virus is produced in a single batch culture with one set of aseptic manipulations.

References

1. M.E.G. Boursnell, C. Entwisle. D. Blakeley, C. Roberts, I.A. Duncan, S.E. Chisholm, G.M. Martin, R. Jennings, D.Ni
Challanain, I. Sobek and C.S. McLean (1997).
A genectically inactivated Herpes Simplex virus type 2 (HSV-2) vaccine provides effective protection against primary
and recurrent HSV-2 disease.
J. Infect. Dis; 175: 16-25.

2. L. Fabry, B. Baijot, E. D'Hondt, M. Duchene (1989).
High density microctirrier cell culture for viral vaccine production.
Advances in animal cell biology and technology for bioprocesses; 361 - 365.

3. B. Griffiths, B. Thornton, 1. McEntee (1980).
Production of Herpes viruses in microcarrier cultures of human diploid and primary cluck fibroblast cells.
Eur. J. Cell Biol; 22: 606,

4. K.K. Sanford, W.R. Earle, V.J Evans et al (1951).
J. Nat. Cancer Inst; 11 : 773 - 795.

5. Glas-Col Apparatus Company,
711 Hulman Street, P.O. Box 2128, Terre Haute, IN 47802, USA

NEW FORM OF THE LIVE MEASLES VACCINE FOR ORAL
ADMINISTRATION

E.A. NECHAEVA. E.A. KASHENTSEVA, A.P. AGAFONOV,
N.A. VARAKSIN, T.G. RYABICHEVA, A.P. KONSTANTINOV,
V.N. BONDARENKO, T.D. KOLOKOLTSOVA, I.V. KITS,
T.YU. SEN’KINA, N.V. ZHILINA
Research Institute of cell cultures. State Research Centre of Virology and
Biotechnology <Vector>, Koltzovo, Novosibirsk region, Russia

1. Introduction
The live measles vaccine based on measles virus strain Leningrad-16 (L-16) and
Japanese quails embryonic fibroblasts are now used in Russia for the prophylaxis of
measles. The vaccine possesses high immunogenicity and epidemiological efficacy, low
reactogenicity, and meets the WHO’s requirements to live measles vaccines. However,
the primary cell culture that lacks standard biological and genetic characteristics is
used in the production of this vaccine, and its administration does not exclude a
possibility of HIV and hepatitis infection during vaccination. In addition, production of
the vaccine requires considerable expenses at the stages of preparation and sterilization
of ampules, pouring of the preparation, sealing, preparation and control of the solvent.
In this connection, the development of oral forms of the measles vaccine is a topical
problem, since oral immunization is the most simple, sparing, untraumatic, and
physiologically based method of population vaccination. Besides, penetration of the
antigen in lymphoid tissue of the digestive tract and formation of specific secretory
immunoglobulins, which arc an additional protective factor at the level of infection
“entrance”, are characteristic of oral immunization.
The goal of this work was to study the immunogenicity and innocuousness of
encapsulated form of the live measles vaccine in measles virus-sensitive model, rhesus
monkeys.

2. Materials and methods
Measles virus strain L-16, isolated in Russia in 1960 from the blood of a measles-
infected child [1], was used for vaccine production. The Japanese quails embryonic

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© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

574

fibroblasts (JQEF) culture from lcukosis-free farm of the SRC VB “Vector” were used
as cell substrates.
JQEF culture were multiply infected with suspension of the measles virus at a dose of
0.01-0.05 1g TCID50/cell. Sterile virus-containing liquid from different bottles with
specific activity not less than 5.0 1g TCE50/0.5 ml was united, supplemented with
stabilizer, and lyophilized. The dry virus-containing material was placed in solid
gelatine capsules, the capsules were then covered with acid-resistant layer.
In accordance with the requirements of the State Pharmacopoeia, the vaccine samples
were certified according to their physical-chemical characteristics, innocuousness for
laboratory animals, and contents of foreign microorganisms [2]. Specific activity of the
measles virus was determined by its cytopathic effect in Vero cell culture; contents of
foreign viruses, mycoplasmas. and mycobacteria was estimated with techniques
approved by the Pharmacopoeial Committee of Russia [3].
Immunogenic characteristics of the vaccine were tested in monkeys (rhesus macaques)
by oral introduction of the encapsulated form of the vaccine at a dose of 100,000
TCE50 of measles virus and administration of injection form of the vaccine at a dose of

20,000 TCID50 of measles virus. Blood was collected form the animals on days 0, 1, 3,
5, 7, 9, 14, 21, 28, and 48 post immunization. Humoral immunity was assayed by
hemagglutination retardation reaction (HARR) with 2 AU of purified measles virus and
EL1SA [4, 5]. Virus-specific IgA in monkey saliva was determined by technique

described in [6] using modified EL1SA.

3. Results and discussion
Experimental batches of encapsulated form of the live measles vaccine were produced
by the technique which we developed. Specific activity of the vaccine batches amounted
to 3.5-4.5 1g TCID50 of measles virus per capsule. The vaccine samples did not
contain any pathogenic bacteria, mycoplasmas, cytopathogenic and hemoadsorbing
viruses. Capsules with the vaccine with covering did not dissolve in an acid medium

over and dissolved in a weakly alkaline medium over 1-1.5h.
Trials of the encapsulated vaccine in monkeys demonstrated marked specific changes
of immune system in the experimental animals.
The data on virusemia and humoral and secretory immunity in monkeys immunized
with different forms of measles vaccine are listed in Table 1.

Measles virus was revealed in blood mononuclears on days 7-9 post immunization.
While estimating secretory immunity, specific IgA at titers 1:8-1:16 were detected in
saliva samples of all monkeys immunized with the capsulated form of the vaccine and
were absent in animals vaccinated with the measles vaccine subcutaneously.
Application of the vaccine also resulted in the induction of the specific antibodies in all
animals, as determined by HARR and ELISA. The increase in antibody titers was
recorded starling from day 21 post immunization; 8-16-fold growth in antibody tilers,
by days 28-48. Increased proliferative activity of lymphocytes after their stimulation
with measles virus antigen was recorded in all animals; by days 28-48, this index

575
significantly exceeded the initial level of lymphocyte activity (day 0). Thus, a clone of
the cells that responded with increased proliferation to the encounter with the measles
antigen was formed in these animals.

Thus, the studies performed demonstrated that the encapsulated form of the live
measles vaccine caused stimulation of humoral, secretory, and cell-mediated immunity
in the vaccinated animals, which manifested itself in the induction of antibodies in
blood serum and saliva and formation of the clone of memory cells which acquired the
ability to transform into blastic forms on the repeated in vitro encounter with the
antigen.
4. Conclusion
The technology developed in the SRC VB “Vector” made it possible to create the
encapsulated form of the live measles vaccine for oral administration. The vaccine no
contains pathogenic bacteria, alien viruses, mycoplasmas, and in the studied
parameters meets the requirements of the national control institution of Russia to
measles vaccines. Immunization of monkeys with the encapsulated form of the vaccine
of measles viruses results in the induction of humoral, secretory, and cell-mediated
immunity.

5. References

1 Taros, Yu. L. (1967) An attempt of direct isolation of measles virus from monolayer cultures of guinea-pig
kidney tissue and chicken embryonic fibroblasts, Voprosy Virusologii 4, 399-402

2. The State Pharmacopoeia oft he USSR (1987), Moscow.
.1 Pharmacopoeial Article 42-179 VS-94 (1987), Dry cultural live vaccine.
4. Voller A., Bidwell, D., and Bartlett, A. (1976) Microplate enzyme immunoassays for the immunodiagnosis

of virus infections. In: Rose, N.R. and Friedman, II. (eds.). Manual of clinical immunology Washington, pp.
506-512.
5. Norrby, E. and Gollmar, Y. (1975) Identification of measles virus-specific hemolysis inhibiting antibodies
separate from hemagglutinin-inhihiting antibodies, J. Infection and Immunity 11, 231-239.
6. Fridman M. (1982) Radioimmunoassay for detection of virus-specific IgA antibodies in saliva, J. Immunol.
Methods 54, 203-205.

STUDY OF LENINGRAD-16 VACCINE STRAIN OF MEASLES VIRUS
REPRODUCTION IN CELL CULTURES PERSPECTIVE FOR
BIOTECHNOLOGY

E.A. NECHAEVA, T.N. GETMANOVA, T.Y. SEN’KINA,
N.D. YURCHENKO
Research Institute of cell cultures, State Research Centre of Virology
and Biotechnology <Vector> Koltzovo, Novosibirsk region, Russia

1. Introduction
L-16 strain measles virus and primary cell culture of Japanese quail embryo fi-
broblasts (QEF) are used in Russia for live measles vaccine production. Some het-
erogeneity of this culture and the possibility of contamination during the embryo
tripsinization gave rise for search of another more safe and uniform cell substrate.
During the last few decades much attention was paid to the use of human diploid
cell cultures in immunologicals production, because they are characterized by
normal karyotypes and uniformity, have no tumorogenecity, can be subjected to
kryoconservation [1-3]. Human diploid cell culture L-68 could be just the same
one to improve the technique of live measles vaccine production.
The aims of this study were to compare the main characteristics of virus produc-
tion and morphogenesis in infected cell cultures which are the most promising for
biotechnology, using equal conditions of cell cultivation and infection; to esti-
mate the sensibility of L-68 diploid cell to measle virus infection depending on the
passage level number; to receive data allowing to choose the most promising cell
substrates for improving the current measles vaccine technology.

2. Materials and methods.
Cell cultures. Vero cells (an African green monkey continuous cell line) was ob-
tained from Flow Laboratories. Continuous cell line 4647 produced from the kid-
ney of an Afrikan green monkey, was obtained from the Institute of Virology of
Russian Academy of Medical Sciences. Primary cell culture of quail embryo fi-
broblasts (QEF) was produced from 10-day-old embryos of Japanese quails certi-
fied by the Slate Institute of Standardization and Control of Medical and Biologi-
cal Preparations (SISC). Diploid cell culture L-6H, produced from the lung of 11-

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578

week-old human embryos, was obtained from the Research Institute of Viral
Preparations of Russian Academy of Medical Sciences, then certified according to
the WHO system by SISC and used at the 21st, 23rd and 29th passage level.
Measles virus. Vaccine strain Leningrad-16 measles virus, certified as a working
bank for live measles vaccine production by SISC, was used. Confluent cell
monolayers grown on glass cover slips were inoculated at a multiplicity of infec-
tion of 0,001 TCD50\cell. Pared control and infected samples were prepared for
electron microscopy daily from 1 to 10-13 days post infection. Cultural liquid
samples were tested for virus specific activity by titration on Vero cell culture.

3. Results and discussion
Electron microscopy data revealed that all cell cultures under study were free from
any contaminants.
After inoculation an active virus reproduction took place in the cell cytoplasm
with development of typical changes in cell ultrastructure (4). The surface com-
partments of the infected cell cytoplasm contained the short, brunched and diluted
cysternas of endoplasmatic reticulum. The density of the ground substances of the
cytoplasm was increased by diffuse implantation of ribosomes. Within the infected
cells numerous viral nucleocapsids assembled and formed inclusions; sometimes
nucleocapsids were found in cell nucleus, too. Cytoplasmic nucleocapsids were
thicker and covered by "fuzzy" coat, while those located in the nucleus had a
"smooth" surface. Giant multinuclear cells and syncytia were scattered over the
monolayer. In Vero cell culture the infection had the most active character: syn-
cytia were detected in 3 days post infection and reached the maximum size. In
QEF culture the infected cells were revealed more later (in 5-6 days), multinuclear
cells were formed in 7 days, syncytia and viroplasma had smaller sizes. Cell culture
L-68 studied at different passage had no differences in virus morphogenesis.
Measles virus released from the cell surface by budding, accumulated in intracellu-
lar space, revealed the typical form and structure and ranged from 150 to 300 nm
in diameter.
Specific virus activity measurement data were in accordance with results of viro-
genesis ultrastructural study.
All investigated cultures were characterized by high values of the virus output,
which confirmed their high sensitivity level. At the same lime they differed from
each other in terms of determination of the time of reaching the maximum titres
and the time of keeping them steady (Table),
The earliest virus cytopathic effect and long period of the titers keeping steady
were observed in the studied continuous and diploid cell cultures. Diploid cell
culture L-68 was able to produce high amounts of measles virus on passages
21, 23 and 29 and to keep its output steady for a long period even in the experi-
mental conditions without the cultural medium replacing. Taking into account the
genetic uniformity, lack of contaminants, availability for mass production and
safety of using L-68 cell culture as the diploid one, the results obtained indicate
the promising character of the diploid culture studied.

579

4. Conclusion
Cell cultures under study promising for biotechnology provided the high virus out-
put hut differed from each other by the moment of reaching the maximum titers
and the time of keeping them steady at the same conditions of cultivation and in-
oculation. Human diploid cell culture L-68 possessed high productivity and sensi-
tivity to measles virus infection, and could be used to improve the technique of live
measles vaccine production. There were no differences in the measles virus mor-
phogenesis and production level between various passages of the human diploid
cell culture L-68, which allowed to use this culture in the passage level range rec-
ommended by the WHO for immunologicals production.
5.References

1. Hayflick, L. (1989) History of cell substrates used for humanbiologicals, Develop. Biol. Stand. 70, 143-
146

2. Mirchamsy, B.H., Shafyi, A., Nazari, P., Ashtiani, M.P., Sassani, A. (1988) Evaluation of live mea-
sles vaccine prepared in human diploid cells for reimmunization, Epidemiol. and Infect. 101, 431-443.

3. Wood, D. and Minor, P . D . (1990) Use of human diploid cells in vaccine production, Biologicals
18, 143-146.

4. Dubois-Dulg, N., Holmes, H.V., Rentier, B. (1984) Assembly of Enveloped RNA Viruses, Springer-
Verlag, Wien.

RECENT ADVANCES WITH NEW VACCINE ADJUVANTS: FROM
PRECLINICAL TO CLINICAL DEVELOPMENT

T. VOSS
SmithKline Beecham Pharmaceuticals
Rue de 1’Institut 89
B-1330 Rixensart, Belgium

Abstract: Subunit, and recombinant subunit approaches to vaccine design have long
been recognised for their potential safety. However, their efficacy (i.e.:
immunogenicity) largely relies on the adjuvant systems used. The recent understanding
of the role of effector cell mediated immunity in disease prevention and control have
opened the way to an unprecedented search for novel adjuvant systems able to induce
such cellular responses.
This presentation will describe novel adjuvant systems developed at SmithKline
Beecham Biologicals, and their use in preclinical as well as clinical development of
vaccines against genital herpes, RSV, malaria and other diseases, as well as for the
immunotherapeutic treatment of chronic diseases such as chronic HBV or cancer.

Discussion

Aunins: You said your GD antigen is produced in CHO cells and it is
glycosylated. You have stressed the adjuvants, but I was
wondering if you had seen any effects of the glycosylation structure
on the antigenicity of the GD itself?

Voss: We characterised the immunogenicity of our truncated glycosylated
GD with monoclonal antibodies. We are quite sure, compared to
the native glycoprotein present on the virus, that we have not seen
differences using this technology.

Aunins: You have stressed CMI response, yet one of your criteria for
clinical trial inclusion was sero-negativity. Why did you pick sero-
negativity as opposed to an indicator of pre-existing cellular
immunity?

Voss: Sero-negativity was chosen because you want to be able to assess
in detail the CMI responses induced by the vaccine. So in the phase
II trial we had sero-positive and sero-negative individuals, but you
will see a clear induction of this response only in the sero-negatives.
For the efficacy trial it is clear because you need sero-negative
subjects to be potentially infected during the course of the study

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582 Do you have any experience with your adjuvants to obtain
Vazquez: responses against tumour associated antigen of a carbohydrate
Voss: nature?
Vazquez:
Voss: We have an on-going cancer programme, but I cannot discuss the
Keck: results here. With carbohydrate antigens we do have data
indicating that the adjuvants do work with that type of antigen.
Voss:
Wurm: The big problem is to obtain these antigens with helper because
they do not produce a memory.
Voss:
So you may want to couple these antigens to carriers to provide
help.

The field of adoptive immunotherapy has been around for many-
years, particularly the tumour infiltration lymphocyte, or CTL. In
most of the cases, particularly melanoma and ovarian carcinoma,
the results have not been that satisfactory. A number of
laboratories, including NIH and Anderson, are getting into antigen
presentation approach to this. Have you done any work on the in
vitro or in vivo expansion of TIL cells or other immune cells?

No. Our strategy is focused on adjuvant with sub-unit approaches.

A question about your strategy in the phase III trials. You have
already vaccinated some 10,000 patients, or candidates, in phases I
and II. What is the rate of infection when you have a partnership
between HSV positive and negative individuals? Also, what is the
time frame during which normal infection would occur in this
instance, and what are the numbers that you need to vaccinate to
get statistically relevant data?

We are expecting efficacy results next year. The study was initiated
in 1995 which gives us a period of 3 years. We have incorporated
about 800 couples in the multi-centre study. I cannot tell you the
exact rate of infection, but our statisticians told us that we need to
include that number of patients, and that time period, to get a
statistically relevant outcome.

VACCINATION WITH RECOMBINANT SUICIDAL DNA/RNA

P. BERGLUND1 M. FLEETON1, C. SMERDOU1,
I. TUBULEKAS2, B.J. SHEAHAN3, G.J. ATKINS4 AND
P. LILJESTRÖM1,5
Microbiology and Tumorbiology Center1 and Center for
Biotechnology2, Karolinska Institute, Stockholm, Sweden.
Department of Veterinary Pathology, University College3 and
Department of Microbiology, Moyne Institute of Preventive
Medicine, Trinity College4, Dublin, Ireland
Department of Vaccine Research, Swedish Institute for Infectious
Disease Control, Stockholm, Sweden 5

1. Introduction

Efficient protection from viral disease has traditionally relied on the use of
live attenuated vaccines, such vaccines being potent in generating broad
humoral and cellular immunity. However, use of these vaccines has been
hampered for reasons of biosafety. In contrast, killed or subunit vaccines are
safe, but usually inadequate with regard to efficacy. Simplistically, design of
novel vaccines should combine the efficacy of live or attenuated vaccines
and the safety of subunit vaccines. A vaccine resulting in antigen
presentation which mimics that during natural infection by the cognate
pathogen would appear have the highest probability of success. The strategy
is to achieve (i) effective protective immunity, (ii) a high population
response, (iii) prolonged duration of immunity, (iv) high level of safety, (v)
stable vaccine preparations which easily can be produced on large scale at
low cost, (vi) easy to administer.

During the last few years, the mechanisms by which antigens are
presented by the major histocompatibility complex (MHC) class I and class
II pathways (7, 11, 12, 15, 29), the role of natural killer (NK) cells (13, 14,
22) and the roles of cytokines and co-stimulatory factors in determining the
balance of the immune response (10, 24, 30), have become better
characterized. This knowledge has suggested ways whereby design of new
generation vaccines may be approached, and many of these approaches have
turned to use gene technology and viral vectors.

Many different viral vectors have been used to express antigens in vivo in
pursuit of new vaccines (8, 25, 27, 28). However, a problem is the expression

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of a large number of vector derived antigens which may negatively influence
the specific immune response, and safety still remains an issue with many of
these vectors. To circumvent these problems, immunization using naked
DNA has recently been tested by a number of groups (9, 23).

We have used a novel strategy of vaccine design and here describe
vectors which are based on a self-replicating, suicidal, recombinant RNA,
originating from Semliki Forest virus (SFV). In this system, genes encoding
relevant antigens are cloned into vectors which upon vaccination will
transiently express the antigen within the cells of the host. The SFV system
allows efficient delivery of vaccines as infectious virus particles or as naked
nucleic acids (NA).

2. A Vector System Based On An Alphaviral Replicon

Alphaviruses such as Semliki Forest virus (SFV) are positive strand RNA
viruses which have a very broad host range and infect mammalian, insect,
reptile and even fish cells (31). Due to the organization of the RNA genome
(Fig. 1), the viral RNA is on its own able to initiate productive replication
when introduced into a cell, be it by normal infection or by transfection. The
viral replicase will produce both new full-length RNA molecules but also a
subgenomic RNA species which encodes for the structural proteins of the
virus. Within a few days, the replication of the viral RNA will lead to cell
death.

585

2.1 BASIC EXPRESSION VECTORS

For creating a general expression vector a cDNA copy of the SFV genomic

RNA was modified by deleting the region for the structural genes and by
replacing it by a polylinker for insertion of foreign sequences (20, 21).
Later, other modifications of this strategy has allowed use of a variety of
different strategies for expressi,ng genes or gene fragments (Fig. 2) (1, 6, 16-
19, 32). RNA produced in vitro by the SP6 RNA polymerase can be directly

transfected into cells for expression of the cloned sequences.

2.2 A HELPER SYSTEM

Since transfection of cells is a rather inefficient process, a helper vector
system was developed in which the structural genes of SFV were provided in
trans (4, 20). When co-transfected with the formed recombinant vector RNA,
co-replication resulted in production of infectious recombinant SFV (Fig.
2). Such particles only carry recombinant RNA molecules, since the very
stringent packaging sequence of the virus is absent from the helper
molecules. In consequence, infection by such recombinant SFV will result in

only one round of virus RNA replication and expression of the foreign

sequences, while new virions cannot be formed in the absence of the
structural genes of the virus.

2.3 DNA/RNA VECTORS

To allow direct injection of DNA into host cells, yet another vector system
was created. In this case the SP6 RNA polymerase promoter was changed to

the cytomegalovirus (CMV) immediate early promoter. Thus, when the
plasmid is introduced into a cell, it will be transported to the nucleus where it
will be transcribed by the host RNA polymerase II (Fig. 2). After transport
to the cytoplasm this positive-strand RNA will be translated to produce the
viral replicase which then drives the further replication of the RNA molecule
and eventual production of the messenger encoding the foreign sequences.
As is the case in all three strategies, replication and expression of the foreign
sequences is transient and leads to cell death within a few days.

586

587

3. Vaccination

The properties of the SFV replicon makes it potentially very promising for
vaccine design: (i)Vigorous RNA replication combined with efficient
translation leads to production of large amounts of antigen; (ii) The SFV
RNA functions directly as a mRNA, which allows naked nucleic acid
delivery; (iii) The RNA is cytoplasmically self-replicating, with no risk of
integration into the chromosome. Replication leads to cell death which
further underlies the safety of the system and circumvents possible problems
related to tolerance; (iv) Large and multiple genes can be expressed
simultaneously. This multisubunit, multiepitope expression possibility
alleviates the need to define epitopes and/or haplotypes; (v) SFV has a broad
host range and infects all animal cell types, including non-replicating cells;
(vi) SFV is not wide spread, and therefore there is no preexisting immunity
against the vector; (vii) Vector structural proteins are not made escaping an
immune response against the vector itself.

3.1 INFLUENZA MODEL

The SFV vaccine approach has been most extensively tested in the influenza

model. The first experiments utilized injection of naked RNA encoding the

nucleoprotein of influenza A virus. Although naked RNA is expected to be

quite unstable, good immune responses could be generated (33). From a

more practical point of view, recombinant SFV particles or the SFV
DNA/RNA vector approach would be of better value for vaccination. Both

modalities of immunization have been tested in mice and shown that high

titers of antibodies to influenza HA and NP can be generated and that the
antibody levels stay high for very long times. Similarly, strong cellular
immunity is also achieved with prolonged memory (2, 34). When comparing
conventional DNA vaccine vectors to the SFV DNA/RNA vectors, we found
that to achieve a 100% take significantly less DNA was required when using

the SFV vector that using the conventional one. Moreover, despite the

transient expression of the antigen from the SFV vector, frequency of

precursor CTLs were much higher than when conventional DNA vectors

were used. The reason for this is not known at present, but may be due to

induction of when the SFV RNA replicates in the host tissue (5).

When mice vaccinated with either recombinant SFV or with SFV

DNA/RNA vectors were challenged with influenza A virus, protection against

death and disease was observed

588

3.2 FLAVIVIRUS MODEL

Recombinant SFV particles expressing Louping ill (LIV) flavivirus antigens
prM, E (spike proteins) and NS1 (nonstructural protein) were also used to
immunize mice. Again, strong humoral and cellular responses could be
measured, including IgA, when a mucosal (intranasal) route was used. When
mice immunized either intraperitoneally or intranasally were challenged with
lethal doses of LIV, protection was observed. Interestingly, the best
protection was obtained when only the NS1 gene was expressed, indicating
that cellular immunity plays a significant role in protection from flavivirus
infection. Most importantly, neuronal degeneration, present in all challenged
mice that showed clinical signs, were absent in all mice that survived
challenge.

3.3 HIV/SIV MODEL

In a previous study employing recombinant SFV for vaccination of pigtail
macaques, expression of the SIV envelope gp160 protein of PBj14 showed
induction of Env-specific antibodies. When the macaques were challenged
with a lethal dose of PBj14 SIV virus, the highly virulent character of this
isolate resulted in killing of 75% of the control (unimmunized) animals
within two weeks, whereas the animals which had received the SFV vaccine
were completely protected (26).

In a subsequent monkey trial animals were immunized with recombinant
SFV expressing the Env of HIV-1. After vaccination and boosts the animals
were challenged with an extremely high dose (10.000 MID100) of SHIV-4, a
hybrid virus expressing the Env from HIV and the Gag-Pol from SIV. This
model only allows measurement of viremia and cannot lead to AIDS. While
all animals were infected the vaccinated animal group showed significant
reduction in viral load, despite the high challenge dose (3).

4. References

1. Atkins, G. J., B. J. Sheahan, and P. Liljeström. 1996. Manipulation of the Semliki
Forest virus genome and its potential for vaccine construction. Mol. Biotechnol. 5:33-
38.

2. Berglund, P., M. Fleeton, and P. Liljeström. 1997. Immunization with recombinant
Semliki Forest virus generates long-term cytotoxic T lymphocyte and humoral responses
and protective immunity. Submitted.

3. Berglund, P., M. Quesada-Rolander, P. Putkonen, G. Biberfeld, R. Thorstensson, and P.
Liljeström. 1997. Outcome of immunization of cynomolgus monkeys with recombinant

589

Semliki Forest virus encoding human immunodeficiency virus type 1 envelope protein
and challenged with a high dose of SHIV-4 virus. AIDS Res.Hum. Retroviruses, In press.
4. Berglund, P., M. Sjöberg, H. Garoff, G. J. Atkins, B. J. Sheahan, and P. Liljeström.
1993. Semliki Forest virus expression system: production of conditionally infectious
recombinant particles. Nature Biotechnol. 11:916-920.
5. Berglund, P., C. Smerdou, M. Fleeton, I. Tubulekas, and P. Liljeström. 1997.
Vaccination with self-amplifying suicidal DNA induces protection against influenza.
Submitted.
6. Berglund, P., I. Tubulekas, and P. Liljeström. 1996. Alphaviruses as vectors for gene
delivery. Trends Biotechnol. 14:130-134.
7. Busch, R., and E. D. Mellins. 1996. Developing and shedding inhibitions: how MHC
class II molecules reach maturity. Curr. Opin. Immunol. 8:51-58.
8. Caruso, M., K. Pham-Nguyen, Y. L. Kwong, B. Xu, K. I. Kosai, M. Finegold, S. L. Woo,
and S. H. Chen. 1996. Adenovirus-mediated interleukin-12 gene therapy for metastatic
colon carcinoma. Proc. Natl. Acad. Sci. USA. 93:11302-11306.
9. Donnelly, J. J., J. B. Ulmer, J. W. Shiver, and M. A. Liu. 1997. DNA vaccines. Annu.
Rev. Immunol. 15:617-648.
10. Grewal, I. S., and R. A. Flavell. 1996. A central role of CD40 ligand in the regulation of

T-cell responses. Immunol. Today. 17:410-414.
11. Groettrup, M., A. Soza, U. Kuckelkorn, and P.-M. Kloetzel. 1996. Peptide antigen

production by the proteasome: complexity provides efficiency. Immunol. Today.
17:429-435.
12. Koopmann, J.-O., G. J. Hämmerling, and F. Momburg. 1997. Generation, intracellular
transport and loading of peptides associated with MHC class I molecules. Curr. Opin.
Immunol. 9:80-88.
13. Kos, F. J., and E. G. Engleman. 1996. Immune regulation: a critical link between NK
cells and CTLs. Immunol. Today. 17:174-176.
14. Lanier, L. L. 1997. Natural killer cell receptors and MHC class I interactions. Curr. Opin.
Immunol. 9:126-131.
15. Lehner, P. J., and P. Cresswell. 1996. Processing and delivery of peptides presented by
MHC class I molecules. Curr. Opin. Immunol. 8:59-67.
16. Liljeström, P. 1994. Alphavirus expression systems. Curr. Opin. Biotechnol. 5:495-
500.
17. Liljeström, P. 1996. Alphavirus Vectors for Gene Delivery. In I. D. Dubé and M. Cantley
(ed.), Gene Delivery Systems - An OECD Review, 109-118.
18. Liljeström, P. 1995. Recombinant self-replicating RNA vaccines, p. 173-180. In G.
Gregoriadis, B. McCormack, and A. C. Allison (ed.), Vaccines: New Generation
Immunological Adjuvants, vol. 282. Plenum Press, New York.
19. Liljeström, P., and H. Garoff. 1994. Expression of proteins using Semliki Forest virus
vectors, p. 16.20.1-16.20.16. In F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore,
J. A. Smith, J. G. Seidman, and K. Struhl (ed.), Current Protocols in Molecular Biology,
vol. 2. Greene Publishing Associates and Wiley Interscience, New York.
20. Liljeström, P., and H. Garoff. 1991. A new generation of animal cell expression vectors
based on the Semliki Forest virus replicon. Nature Biotechnol. 9:1356-1361.
21. Liljeström, P., S. Lusa, D. Huylebroeck, and H. Garoff. 1991. In vitro mutagenesis of a
full-length cDNA clone of Semliki Forest virus: the 6,000-molecular-weight membrane
protein modulates virus release. J. Virol. 65:4107-4113.
22. Lopez-Botet, M., L. Moretta, and J. Strominger. 1996. NK-cell receptors and recognition
of MHC class I molecules. Immunol. Today. 17:212-214.

590

23. McClements, W. L., M. E. Armstrong, R. D. Keys, and M. A. Liu. 1996. Immunization
with DNA vaccines encoding glycoprotein D or glycoprotein B, alone or in
combination, induces protective immunity in animal models of herpes simplex virus-2
disease. Proc. Natl. Acad. Sci. USA. 93:11414-11420.

24. Mosmann, T. R., and S. Sad. 1996. The expanding universe of T-cell subsets: Th1, Th2
and more. Immunol. Today. 17:138-146.

25. Moss, B. 1996. Genetically engineered poxviruses for recombinant gene expression,
vaccination, and safety. Proc. Natl. Acad. Sci. USA. 93:11341-1138.

26. Mossman, S., F. Bex, P. Berglund, J. Arthos, S. P. O'Neil, D. Riley, D. H. Maul, C.
Bruck, P. Momin, A. Burny, P. N. Fultz, J. I. Mullins, P. Liljeström, and E. A. Hoover.
1996. Protection against lethal SIVsmmPBj14 disease by a recombinant Semliki Forest
virus gp160 vaccine and by gp 120 subunit vaccine. J. Virol. 70:1953-1960.

27. Palese, P., H. Zheng, O. G. Engelhardt, S. Pleschka, and A. Garcia-Sastre. 1996.
Negative-strand RNA viruses: genetic engineering and applications. Proc. Natl. Acad.
Sci. USA 93:11354-11358.

28. Paoletti, E. 1996. Applications of pox virus vectors to vaccination: an update. Proc.
Natl. Acad. Sci. USA. 93:11349-11353.

29. Pieters, J. 1997. MHC class II restricted antigen presentation. Curr. Opin. Immunol.
9:89-96.

30. Shearer, G. M., and M. Clerici. 1997. Vaccine strategies: selective elicitation of cellular
or humoral immunity? Trends Biotechnol. 15:106-109.

31. Strauss, J. H., and E. G. Strauss. 1994. The alphaviruses: Gene expression, replication,
and evolution. Microbiol. Revs. 58:491-562.

32. Tubulekas, I., P. Berglund, M. Fleeton, and P. Liljeström. 1997. Alphavirus expression
vectors and their use as recombinant vaccines - a minireview. Gene. 190:191-195.

33. Zhou, X., P. Berglund, G. Rhodes, S. E. Parker, M. Jondal, and P. Liljeström. 1994. Self-
replicating Semliki Forest virus RNA as recombinant vaccine. Vaccine. 12:1510-1514.

34. Zhou, X., P. Berglund, H. Zhao, P. Liljeström, and M. Jondal. 1995. Generation of
cytotoxic and humoral immune responses by nonreplicative recombinant Semliki Forest
virus. Proc. Natl. Acad. Sci. USA. 92:3009-3013.

Discussion 591
Hauser:
Liljeström: You mentioned the translational enhancer. Could you briefly state
where it is located and the functional mechanism?
Wurm:
Liljeström: The enhancer is actually overlapping the first 34 amino acid
residues of the capsid gene. The structure proteins of the alpha
viruses are made as a polyprotein. It is one open-reading frame
which then self-cleaves into individual sub-units. It is a secondary
structure with a stem loop and we think it stores the ribosomes.
You cannot transfer it to a normal cDNA gene to enhance it. It has
to be in the context of the alpha virus RNA.

You mentioned, as an advantage of the viral approach, the suicide
mechanism. Is it something you think about as a FDA requirement,
or what is your real argument about this?

The argument about the conventional DNA vaccine is that you may
have two disadvantages, which have not turned out to be true.
One is induction of tolerance. Because of long term low
expression levels of the antigen, you would induce tolerance
against the vaccine. The other is that you would integrate the
DNA into the chromosome. In cell culture we know that we can
easily make stable cell lines and certainly DNA goes into the
chromosomes, but does it have a carcinogenic effect? We do not
know. I do not think that it will be a problem in conventional DNA
vaccines. However, who can tell whether in 40 years someone will
get a cancer from a paediatric vaccine. So I am trying to sell my
model by the fact that we get rid of the nucleic acids.

GROWTH OF GOAT ENDOTHELIAL CELLS FOR THE PRODUCTION OF A
VETERINARY VACCINE

MIRANDA P. M.1 MOREIRA J.L.1, CARRONDO M. J. T.1,2
1 - Inst. Biol. Exp. Tecn. /Inst. Tecn. Quim. Biol, Ap. 12, 2780 Oeiras, Portugal
2 - Lab. Eng. Bioq., FCT/UNL, 2825 Monte da Caparica, Portugal

Abstract

Goat jugular vein endothelial cells (cje), involved on the in vivo regulation of a large number of
important physiological processes, are essential for the in vitro production of candidate
heartwater vaccines based on its infection with Rickettsia Cowdria ruminantium.
The main purpose of this work is the definition of the best culture system and its operational
conditions for mass production of cje cells.The scale up of monolayer cultures was performed in
cell growth surfaces from 9.6 to 6320 cm.2. The optimal inoculum concentration was 2*104
cells/cm2, leading to a maximum cellular density of 6 *104 viable cells/cm2 and specific growth
rate of 0.014 h-1, independent upon the scale.
These cells were also grown in 250 cm3 spinner flasks operated as batch cultures and several
operational variables were studied: agitation rate, inoculum concentration, support type and
concentration. The maximum cell concentration 9.5* 105 cells/cm3 was achieved at 40 rpm (with
a specific growth rate of 0.023 h-1).

1. Introduction

As described by many authors, the culture of endothelial cells often present changes in
morphology [1,2,4], microstructure, size [6, 7] and biochemical activities [6] depending on the
culture system and particular shear stress. The bacteria Rickettsia Cowdria ruminantium
parasites in vivo the vascular endothelial cells of wild and domestic ruminants, provoking a
disease (Heartwater) that is endemic in Sub-Saharan Africa and Caribbean [5]. Since a vaccine
against the disease requires the production of the bacteria and this is, in vitro, a specific parasite
of endothelial cells [3], the problem associated with the man production of these cells has to be
overcome. Thus, the main goal of this work was the cultivation of endothelial cells under
different culture systems and its comparison regarding cell man production. Other problems are
the poor ability for the cells to proliferate in vitro and an uncontrollable phenotypic variability
that led to many difficulties for culturing endothelial cells.

2. Materials and Methods

Cje cells isolated from goat were obtained from Dr. Dominique Martinez (CIRAD/EMVT,
Guadeloupe, France).
Tissue cultured flasks, 6 well plates, Triple T-flasks, and Cell Fáctories were supplied by Nunc
(Roskilde, Denmark) and Roller Bottles from Corning Costar (Badhoevedor, The Netherlands).
The cells were maintained in Dulbecco’s Modified Eagle’s medium (DMEM), supplemented
with 10% (v/v) foetal bovine serum (FBS), 2 mM L-Glutamine, 1% (v/v) Streptomycin /

593

O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 593-595.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

594

Penicillin (all final concentrations and from Life technologies, Glasgow, UK). Bioreaction
studies were performed in 250 spinner-flasks (Wheaton). Cell number was evaluated using
the Trypan Blue dye exclusion method and a hemacytometer (Brand, Wertheim/Main,
Germany).

3. Stirred Tank Studies

The growth of cje 102 cells was performed in different types of microcarriers (Figure
1) with an inoculum concentration of 105 cells/cm3. Cytodex 3 was the microcarrier that led to
the highest cellular

concentration, Cultispher G also

led to a good cell growth (but

lower than cytodex 3) when
comparing with the other
microcarriers tested.

The growth of cje cells in all

types of supports, specially the
non porous, is limited by an
aggregation process induced on
the surface before confluency,
leading to cell death since these

cells are strictly anchorage

dependent.

The strength of this phenomena seems to be cell line dependent (data not shown). During the

development of this work the cell growth was highly dependent upon both the inoculum

concentration and the support concentration. In order to investigate the optimal operational

conditions, a two step approach was used by varying one variable (inoculum or support

concentration) maintaining the other constant. The best ratio is 2 grams/l of

Cytodex 3 per of

inoculum (data not shown).

In a second step the inoculum

concentration was varied from

maintaining the ratio (i.e.; The

Cytodex 3 concentration was
concomitantly varied from 2 to
The results are

presented on figure 2.

The maximum cell
concentration was obtained with

6 g/l of cytodex 3 and

of inoculum.

The specific growth rate was
, and the maximum cell

concentration achieved was (viability always higher than 95%). Similar values

were obtained for other cje cells with the some origin (data not shown). An alternative to stirred

595

tanks and the cell immobilised on the surface of
supports are static cultures, which are operated in the
absence of physical stresses. This work was
performed in a large range of culture growing areas,

from 10 to 6320
The initial part ofthe work involve the optimisation of
the culture conditions: the optimal inoculum
concentration of this leading to a

maximum cell density of viable and a
specific growth rate of (data not shown). 900

Roller bottles were also tested, the optimal
operational conditions being 100 ml of liquid media, an inoculum of and a

rotational rate of 12 rph.

4. Scale-up in different culture systems

As presented in figure 3, the maximum cell concentration achieved in the different culture
systems is quite similar (average of 5 to Also the specific cell growth rate is

independent upon the scale

5. Conclusions

It is possible to grow goat jugular vein endothelial cells both in static and stirred cultures. In
static the optimal inoculum concentration is this leading to 5 to 6 and a
specific growth rate of not depending on the scale In stirred tanks (250
the optimal culture conditions are: 40rpm, of Cytodex 3 and an inoculum of
leading to at a specific growth rate of

6. References

1. Ballerman B.J. & Ott M.J. (1995) Adhesion and differentiation of endothelial cells by exposure to chronic shear
stress: A vascular graft model. Blood Purif; 13:125-134

2. Barbee K. A. (1995) Changes in surface topography in endothelial monolayers with time at confluence: influence on
subcellular shear stress distribution due to flow. Biochem. Cell Biol. 73: 501-505

3. Bezuidenhout J.D. (1987) The present state of Cowdria ruminantium cultivation in cell lines. Onderstepoort J. Vet.
Res., 54: 205-210

4. Davies P.F., Remuzzi A , Gordon E.J., Dewey C.F., Gimbrone M.A. (1986) Turbulent fluid sheai stress induces
vascularendothelialturnover invitro. Proc Natl. Acad. Sci. USA83: 2114-2117

5. Kobold A.M., Martinez D, Camus E., Jongejan F. (1992) Distribution of Heartwater in the Caribbean determined on
the basis of detection of antibodies to the conserved 32-Kilodalton Protein of Cowdria ruminantium J. Clin.
Microbiology 1870-1873

6. Levesque M.J., Sprague E.A., Schwartz C.J., Nerem R.M. (1989) The influence of shear stress on cultured vascular
endothelial cells: the stress response ofan anchorage-dependent mammalian cell Biotec. Progress 5, 1: 1-8

7. Ott, M. J., Ballermann, B. J. (1995) Shear stress-conditionated, endothelial cell-seeded vascular grafts: improved cell
adherence in response to in vitro shear stress. Surgery 3 : 334-339.

7. Acknowledgements

The authors acknowledge the financial support from the European Union (CTT-634, DG XIII, VALUE) and PED1P
Medida 5.3, Acção C (Projecto JT1)
The authors are grateful to Dr. D. Martinez - CIRAD/EMVT, (Guadaloupe), Dr. Paul Bensaid - CIRAD/EMVT,
(Montepelier) for the supply of the cells and Ms. Rosário Clemente (1BET) for technical support

EXPRESSION IN INSECT CELLS OF THE MAJOR PARASITE
ANTIGEN ASSOCIATED WITH HUMAN RESISTANCE TO
SCHISTOSOMIASIS

Argiro L.(1),Doerig C.(1),Liabeuf S.(2),Bourgois A.(1),
Romette J.L.(2)*

(1) U399 INSERM, UFR médecine La Timone, F 13385 Marseille Cedex 5
(2) DISP/UPR 9039 CNRS, CESB Case 925, F 13288 Marseille Cedex 9

Introduction
Glyceraldehyde-3-Phosphate Dehydrogenase (G3PDH) is a key enzyme in the glycolytic
metabolism and the production of energy . This probably explains why G3PDH was
evidenced as a major therapeutical target in several parasitic diseases ; either as a vaccine
candidate or as a target for chemotherapeutic treatments . Schistosoma mansoni G3PDH
(Sm 37-G3PDH) is one of the main schistosome vaccine candidate (Wright et al ,1991 ;
Bergquist, 1995) . The production of a recombinant Sm37-G3PDH has been performed
to evaluate if this molecule is able to induce a protective immunity in animals and
eventually in humans . The cDNA coding for Sm37-G3PDH has been cloned and
sequenced (Dessein et al , 1988)(Goudot-Crozel et al, 1989) . In addition five B-cell and
two T-cell epitopes have been localized on the molecule among which a major B-cell
epitope has been evidenced. Different expression systems have been evaluated in respect
with the production yield and the recombinant protein quality . Most of them have led to
either a high production of insoluble material (Bacteria) or to an inactive enzyme (Yeast).
A large amount of soluble rSm37-G3PDH with an excellent bio-reactivity have been
obtained using the baculovirus-insect cell system .
Construction of the transfer vector.

Sm37-G3PDH cDNA was PCR-amplified from Sm37-lgt 11 clone prepared as
previously described (Goudot-Crozel et al. 1989) with primers containing restriction sites:

-Eco RI: 5’-CCGAATTCATGTCGAGAGCAAAG-3’
-Not I: 5’-GCGGCCGCTTATGCATGGTCGAC-3’)
The amplified products were run on a 1.2% agarose gel electrophoresis and the
1.1 kB band was purified with the QiaQuick gel extraction kit (Qiagen). Ligation products
were transfected in JM109 competent bacteria (Promega) and recombinant plasmide was
extracted from positive colony by Wizard maxipreps (Promega). The purified fragment
encoding for Sm37-G3PDH was digested with Eco RI and Not I and inserted into the
pAcHLT-A baculovirus transfer vector (Pharmingen, BaculoGold System) This vector is
a derivative of the pAcSGl vectors family which contain a six histidine tag to express
recombinant protein as a polyhistidine-containing fusion protein . The presence of this N
terminus tag allows the protein to be purified from a cleared cell lysate based on the high
affinity of the nickel-nitrilo triacetic acid resin ( Qiagen NiNTA) for the protein carrying
the 6X His tag . The protein bound to the resin can be eluted under very mild conditions.

597

O.-W. Merten el al. (eds.), New Developments and New Applications in Animal Cell Technology, 597-600.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

598

This pAcHLT-A /Sm37 plasmide was entirely sequenced on a Pharmacia Biotech
apparatus.

PThroediuncseticotncelol flinreecSopmodboipntaenrta baculovirus Sf9 , was cultured in TNM-FH medium
frugiperda ,
(Sigma) supplemented with 10 % feotal calf serum (Sigma) at 27 °C . Following the
BaculoGold system protocole the insect cells were co-transfected with the BaculoGold
DNA and the recombinant transfer vector . BaculoGold DNA is a modified wild
baculovirus DNA which contains a lethal deletion and cannot develop into viable virus by
itself . Recombination between the flanking regions of the polyhedrin gene from the
transfer vector and modified wild type baculovirus DNA therefore results in 100 %
recombinant baculovirus DNA.
The recombinant virus stock was amplified by a single large culture of Sf 9 insect cells . A
high titer virus stock was harvested

CrGom3PpaDraHtiveEsxtpudrieessswioenre achieved using the multiplicity of infection (MOI) and the host
cell density at the time of the infection as variables. The results obtained demonstrated a
significant impact of those parameters on the cell productivity. The highest level of
expression was found with an high cell density and a low MOI. (5 to 10 times more than
with high MOI and low cell density)
Similar results have been recently confirmed by J D Yang and co-workers (JD Yang et al
1T9h9e6)i.nsect cells derived from Trichoplusia ni , BTI-TN-5B1-4 were cultivated in
suspension and in serum free medium EXCELL 405 (JRH Sciences) . An inoculum was
produced in 300 mL spinner flasks (INTEGRA Biosciences) up to a cell density of
2.6X10E6 cells per mL . Then the inoculum was infected with the recombinant
baculovirus , MOI = 0.1 , and incubated under low stirring speed condition during 3 hrs .
After incubation the infected cell culture was transferred in a 3 liter working volume
bioreactor (Cytoflow ,INCELTECH - France) and diluted with fresh medium down to a
cell density of 5X10E5 cells per mL . In order to prevent the cells to aggregate 0.1% V/V
Pluronic P68(Sigma) was added to the medium. The rSm37-G3PDH production was
followed in real time evaluating the enzyme activity in the 3 mL samples collected from the
bulk solution .
An average of 130 mg / L of rSm37-G3PDH was routinely obtained.

Product concentration was evaluated by following the G3PDH activity according to the
Ferdinand method (1964)

599
Biochemical characterizations
The open reading frame encodes a protein of 338 amino acids. The translation product of

the gene has a deducted molecular mass of 36,589 Da. The purified protein synthesized in

baculovirus-insect cells system migrated in denaturing conditions at a position,relative to
standard markers of approximatively 37 kDa, indicating that the full length protein has
been produced. G3PDH isolated from others organisms has been described as a tetrameric
assembly where each monomer has its own active site. The purified rSm37-G3PDH was
eluted from a gel filtration column together with protein standards showing a molecular
weight of approximatively 150 kDa indicating that most likely a tetrameric form of the
protein has been extracted. This will be confirmed by cristallographic studies under
investigation.
The optimum pH was found at pH 9.2 using the DL-glyceraldehyde-3-phosphate as a
substrate.
Specific activity of the purified enzyme of 25 unit/mg was obtained.
Immunological characterizations
The fraction corresponding to the purified rSm37-G3PDH was clearly identified in
western blot by an Alkaline phosphatase-NiNTA conjugate and by an antibody anti-
Sm37-5 antibody mouse serum followed by an anti-mouse IgG-AP conjugate. Sm37-5
contains the major Sm37 B cell epitope. These results confirm that the purified molecule is
the rSm37-G3PDH

In addition, rSm37-G3PDH was recognized by sera from S.mansoni infected subjects
and its enzymatic activity was strongly inhibited by them:

-the anti-Sm37-G3PDH IgG levels in sera from 77 adolescents living in a
S.mansoni endemic area were evaluated. Most sera (63) contained anti-Sm37-G3PDH
IgG(mean=9.8 microg ;S.E.M.=1.4).

-the capacity of these sera to specifically inhibit the enzimatic activity of the
purified rSm37-G3PDH was checked. A strong inhibition (78% as an average) was
observed.

600

Conclusion
We have produced and purified a functional and active recombinant S.mansoni G3PDH .
To the best of our knowledge, this report is the first to describe the quantitative production
of biologically active rS.mansoni G3PDH . Given that IgG antibodies to the S.mansoni
G3PDH are associated with resistance to infection in human, the biologically active
rSm37-G3PDH may prove important as a component of an anti S.mansoni vaccine.
Recombinant antigens isolated under nondenaturating conditions which retain biological
activity as in this case should resemble the natural parasite antigen on the opposite to
inactive or denaturated forms.
With the availability of biologically active recombinant antigen, we are now in a position
to test the effectiveness of rSm37-G3PDH in vaccination and protection experiments as
well as to examine the type of immune responses stimulated by this antigen, alone or in
combination with other molecule, in animal models.

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G. and Silberklang, M. (1996) Biotechnol. Bioeng., 52, 696-706.

MODULATION OF CD4 EXPRESSION ON HELPER T LYMPHOCYTES AND
U937 CELLS BY GANGLIOSIDE GM3 AND ITS DERIVATIVES

D. HEITMANN, P. BUDDE*, J. FREY*, J. LEHMANN AND J. MÜTHING
Institute of Cell Culture Technology, University of Bielefeld, P.O. Box
100131, 33501 Bielefeld, Germany
*Department of Biochemistry, University of Bielefeld

Abstract

Ganglioside GM3 and a variety of chemically modified derivatives (e.g. lyso-GM3, de-N-
acetyl-GM3, GM3-amides) have been assayed for their potential upon CD4 modulation on
human peripheral blood lymphocytes and cells from the monocytic cell line U937.
The data presented show specific ganglioside-mediated CD4 down-regulation from minor
effects of about 14% fluorescence reduction to strong effects with a maximum reduction of
92% depending on oligosaccharide and ceramide composition of the assayed compounds.

1. Introduction

Gangliosides, sialylated glycosphingolipids (GSLs), are ubiquitous compounds of
mammalian cell membranes. Gangliosides and some of their derivatives are known to exert
various important biological functions [1-3].
The CD4 antigen is a 55 kD transmembrane glycoprotein present on T cells and cells of the
monocyte/macrophage lineage. CD4 participates in the interaction with MHC class II
molecules and is known as the receptor for HIV [4].
Recently, it was shown that gangliosides reduce surface expression of CD4 by inducing
CD4-internalization and degradation [5]. In this study, HPLC purified ganglioside GM3
(the major GSL of human serum and lymphocyte membranes), a variety of its natural
descendants (GM1, GD3) and a panel of chemically modified derivatives (Fig. 1) were
employed to gain some insight into the structure-function relationship underlying gangliosi-
de-induced CD4 internalization on T lymphocytes and U937 cells.

2. Materials and Methods

Natural gangliosides, sialic acids, neutral GSL
GM3 from CHO cells, GM1 from human brain and LacCer from human granulocytes were
isolated and purified by standard procedures [6]. GM3(NeuAc) and GM3(NeuGc) were
separated by anion exchange chromatography using TMAE (trimethylaminoethyl)-Fracto-

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O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 601-605.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

602

gel (Merck, Germany) [7]. Bovine GD3 was purchased from Pallmann GmbH (München,

Germany), NeuAc and NeuGc from Sigma (Deisenhofen, Germany).

Chemically modified GM3

GM3-ester and -amides were prepared from GM3(NeuAc) according to Lanne et al. [8],

and GMS-derivatives Dl to D5 according to Nores et al. [9]. Permethylation of

GM3(NeuGc) was carried out as previously described [10] and compounds were

structurally characterized by FAB-MS [11].

Lymphocytes. U937 cell cultures

Human peripheral blood lymphocytes were isolated by Lymphoprep (Nycomed Pharma,

Oslo, Norway) density gradient centrifugation of whole blood. The CD4-positive human

monocytic cell line U937 was cultivated in RPMI 1640 medium containing 10% FCS.

Prior to incubation with gangliosides, cells were washed three times and resuspended in

serum-free RPMI 1640.

Antibodies

Murine monoclonal anti-human CD4 antibody was purchased from DAKO (Hamburg,

Germany); DTAF-conjugated goat anti-mouse antibody was obtained from Dianova

(Hamburg, Germany).

Flow cytometry

Prior to labeling with anti-CD4 antibody at +4 °C for 45 min, human peripheral blood

lymphocytes and U937 cells were incubated with natural gangliosides,

derivatives and related compounds (Fig. 1) with concentrations of 100 µM and 400 µM in

serum-free RPMI 1640 at 37 °C for 60 min. After staining with DTAF-conjugated

secondary antibody at +4 °C for 30 min, fluorescence intensity was quantified by FACS-

analysis (FacSORT, Becton Dickinson, Heidelberg, Germany).

3. Results and Discussion

Highly purified gangliosides, GM3 derivatives and related compounds (Fig. 1) were

proved for their potential upon down-modulation of CD4 expression on U937 cells (Fig. 2)
and T lymphocytes (Fig. 3).
Low CD4 down-regulation
Only minor effects (max. 14%) were observed employing GM3 precursor LacCer, derivati-
ves with modified sialic acid (GM3 derivative D1, GM3-methylester, GM3-amide), perme-
thylated GM3 and sole sialic acids.
Moderate CD4 down-modulation

CD4 down-modulation ranging from 25% to 65% on U937 cells and from 13% to 40% on
T lymphocytes was observed after treatment of cells with native monosialogangliosides.
The type of sialic acid (NeuAc, NeuGc) as well as the oligosaccharide moiety chain length
seem to be of minor importance with regard to modulatory capacity.
Strong CD4 down-modulation

Strong effects concerning CD4 down-regulation occured by employment of disialoganglio-
side GD3 and GM3 derivatives D2, D3, D4 and D5 with a maximum reduction of 92% on
U937 cells after incubation with D5, and 31% and 60% on T lymphocytes after incubation
with D5 and GD3, respectively. Structural requirements for these effects seem to be either
a disialic character (GD3) or the loss of the fatty acid residue (GM3 derivatives D2, D3,

603

D4 and D5). In the latter case, N-acetylation of the sialic acid and/or the sphingosine
moiety do not markedly influence the modulatory efficacy.

604

Suggested structure-Function relationship
Results concerning compounds with structural modifications in the oligosaccharide moiety
(chain length, type, number and modifications of sialic acids) indicate the importance of