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Expression of a human anti-rabies virus monoclonal antibody in tobacco cell

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Article  in  Biochemical and Biophysical Research Communications · July 2006

DOI: 10.1016/j.bbrc.2006.03.219 · Source: PubMed

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 Biochemical and Biophysical Research Communications 345 (2006) 602–607
BBRC
www.elsevier.com/locate/ybbrc

Expression of a human anti-rabies virus monoclonal antibody

in tobacco cell culture q
Loı̈c Stéphane Girard a, Marzena Jolanta Fabis a, Maryse Bastin b, Didier Courtois b,
Vincent Pétiard b,*, Hilary Koprowski a,*
a
Thomas Jefferson University, 1020 Locust Street, Biotechnology Foundation, Philadelphia, PA 19107, USA
b
Centre de Recherche Nestlé-Tours, 101 Avenue G. Eiffel, BP 49716, 37097 Tours Cedex 2, France

Received 15 March 2006

Available online 19 April 2006

Abstract

A Nicotiana tabacum cv. Xanthi cell culture was initiated from a transgenic plant expressing a human anti-rabies virus monoclonal
antibody. Within 3 months, plant cell suspension cultures were established and recombinant protein expression was examined. The anti-
body was stably produced during culture growth. ELISA, protein G purification, Western blotting, and neutralization assay confirmed
that the antibody was fully processed, with association of light and heavy-chains, and that it was able to bind and neutralize rabies virus.
Quantification of antibody production in plant cell suspension culture revealed 30 lg/g of cell dry weight for the highest-producing cul-
ture (0.5 mg/L), 3 times higher than from the original transgenic plant. The same production level was observed 3 months after cell cul-
ture initiation. Plant cell suspension cultures were successfully grown in a new disposable plastic bioreactor, with a growth rate and
production level similar to that of cultures in Erlenmeyer flasks.
Ó 2006 Elsevier Inc. All rights reserved.

Keywords: Antibody; Disposable plastic bioreactors; Plant cell culture; Rabies; Recombinant; Xanthi

Plant-based systems are increasingly used for the pro-
duction of recombinant proteins, because of several advan-
tages over other production systems, such as the ability to
carry out necessary post-translational modifications not
available in bacterial systems, as well as greater safety
and lower production costs than those provided by ani-
mal-based systems.
Plant-based technology has recently been extensively
reviewed, with a full description of plants commonly used,
a listing of proteins expressed thus far [1–5], and the poten-
tial economic superiority of these systems for the recombi-
q
Abbreviations: cv., cultivar; DW, dry weight; ER, endoplasmic
reticulum; FW, fresh weight; HC, heavy-chain; LC, light-chain; mAb,
monoclonal antibody.
*
Corresponding authors. Fax: +33 0 2 47 49 14 14 (V. Pétiard); +1 215
923 6795 (H. Koprowski).
E-mail addresses: vincent.petiard@rdto.nestle.com (V. Pétiard),
hilary.koprowski@jefferson.edu (H. Koprowski).
nant protein production [5,6]. Protein stability during
production and storage is an important determinant of
the production cost. Indeed, degradation by proteases
can be avoided by the addition of a ‘‘Lys-Asp-Glu-Leu’’
(KDEL) signal to the protein sequence, which leads to
storage of the heterologous protein inside the endoplasmic
reticulum (ER). This ER targeting also results in a
glycosylation pattern closer to the human one, by prevent-
ing addition of plant-specific glycans, fucose, and xylose
to the protein [7] thus avoiding a potential source of
unwanted immunogenicity in the recombinant protein
while injected [8].
Many proteins relevant in human disease treatment have
already been expressed in plant-based systems (reviewed in
[2,9]), mainly in the form of edible vaccines [10,11] and
monoclonal antibodies (mAb) [5,12]. A safe and cost-effec-
tive source of mAb is particularly desirable in the treatment
of human rabies virus infection, since current protocols rely
heavily on anti-rabies immunoglobulins prepared mainly

0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2006.03.219
 L.S. Girard et al. / Biochemical and Biophysical Research Communications 345 (2006) 602–607
from horse serum; not only are such immunoglobulins
expensive to prepare, accounting for their limited supply
worldwide, but they present the risk of cross-contamina-
tion with known or unknown horse pathogens [13]. Con-
sidering that rabies virus exposure causes an economic
burden in the US of about $1 billion per year for vaccina-
tion and immunoglobulin administration [14], a plant-
based system of immunoglobulin production, as recently
developed in tobacco plants [15], is attractive. However,
mAb expression levels in field plants vary depending on
plant maturation stage and climatic influences [16 and per-
sonal observations], whereas plant cell cultures are based
on undifferentiated cells growing in a fully controlled envi-
ronment, ensuring a constant level of expression and pre-
venting contamination of the environment.
Here, we demonstrate for the first time the expression of
a human anti-rabies virus glycoprotein mAb in plant cell
culture. A low-cost biomass scale-up was performed with
a new disposable bioreactor.
Materials and methods
Plant material
A stable (T3) transgenic tobacco plant (Nicotiana tabacum cv. Xanthi)
expressing an anti-rabies virus mAb [15] was used and is designated herein
as R12. Briefly, the heavy-chain (HC) sequence, fused with a sequence
encoding the KDEL ER retention signal, is placed under control of the
cauliflower mosaic virus 35S promoter with duplicated upstream B
domains (Ca2p); the light-chain (LC) sequence is under the control of the
potato proteinase inhibitor II promoter (Pin2p) [15].
A well-established wild-type BY2 tobacco cell suspension (N. tabacum)
was used for comparison to newly established tobacco cell suspensions.
Plant cell culture
Callus induction. Primary explants were cut from 3-week-old wild-type
(WT) or R12 transgenic seedlings and placed on Murashige and Skoog-
based medium, supplemented with 1 mg/L naphthalene acetic acid
(NAA), 0.1 mg/L kinetin, 30 g/L sucrose, and 8 g/L agarose (MAK),
under photoperiod conditions (16 h light/8 h dark), at 25 °C. The medium
was autoclaved before explants for 20 min at 115 °C. Resulting calli were
subcultured monthly on Petri dishes.
Initiation of suspension culture. Randomly picked calli (20 g) were
inoculated into 50 mL MAK liquid medium and placed under photope-
riod conditions at 25 °C on an orbital shaker (100 rpm). After 2 weeks,
both transformed and non-transformed tobacco cell lines were grown in
250-mL Erlenmeyer flasks containing 100 mL MAK medium and sub-
cultured every 20 days at an initial density of 40 g of cells [fresh weight
(FW)]/L.
Protein extraction and analysis
Protein extraction, SDS–PAGE, and Western blotting were carried out
as described [17], with the following modifications. Twenty-day suspension
cultures were filtered (50-lm nylon mesh) to separate cells and medium.
Cell disruption was performed on 1 g FW of cells, using a stainless-steel
mixer mill (Retsch, Model MM301, Newtown, PA) at 30 hits/s, for 2 min.
The resulting mash was homogenized with 300 lL DB2X buffer (40 mM
Tris–HCl, pH 8.0, 20% glycerol, 2% SDS, and 4% b-mercaptoethanol)
containing 0.1% Triton X-100 and a complete protease inhibitor mixture
(Roche Diagnostics).
For Western blot analysis, HC and LC were detected with goat
anti-human antibodies [Fc c- and F(ab 0 )2 fragment specific, respectively,
603
1/10,000 dilution] conjugated to horseradish peroxidase (Jackson Immu-
noResearch). Whole-plant-derived mAb was used as a positive control.
Suspension culture in disposable bioreactors
A new type of plastic bag-based bioreactor (Nestlé Research Center,
Tours) was used to scale-up the cell culture from 500 mL to 10 L. After
sterilization of the disposable bioreactor, sterile medium was inserted into
the bioreactor and cells were inoculated [40 g/L (FW)].
Small-scale purification of plant cell-derived antibodies
Lyophilized cells [100 g dry weight (DW), equivalent to 1.45 kg FW]
were mixed with 1.5 L of extraction buffer (20 mM sodium phosphate, pH
7.2) and disrupted using a French press. Soluble proteins were recovered
by centrifugation (6000g, 20 min, 4 °C) and filtered on fiberglass (from
2.7- to 0.22-lm pore sizes). Cross-filtration on a 30-kDa membrane was
used to concentrate the supernatant to 300 mL. Soluble protein extracts
were purified with an Äkta purifier equipped with a HiTrapä protein G
high-performance affinity column (Amersham Biosciences) according
to the manufacturer’s recommendations. mAb was eluted with 0.1 M
glycine–HCl (pH 2.7) and pH was neutralized with 1 M Tris–HCl (pH
9.0).
ELISA
Binding of transgenic mAb to rabies virus was assessed by ELISA as
described [18], with the following modifications: 3 lg/mL of ERA rabies
virus was used to coat the plates, followed by loading with 95 lL of serial
3-fold dilutions of soluble extracted proteins obtained from 7 mg of
lyophilized plant cell extract disrupted in 1 mL of sodium phosphate
extraction buffer. Human anti-rabies virus mAb (1 lg/mL) was used as a
positive control. Recombinant bound mAb were detected by successive
additions of biotin-conjugated anti-human IgG1 (1:1000; BD PharMin-
gen, San Diego, CA), avidin alkaline phosphatase-conjugated antibody
(Sigma), and p-nitrophenyl phosphate (Sigma) as substrate. Absorbance
was read at 405 nm.
In vitro neutralization assay
Neutralizing activity of purified antibodies was assessed using a stan-
dard 20-h rapid fluorescent focus inhibition test (RFFIT) as described [18].
Briefly, diluted purified mAb produced by plant cell culture was incubated
with rabies virus (CVS-11, CVS-N2C or ERA). Baby hamster kidney
(BHK) cells were added to plates, incubated, and fixed. FITC-anti-rabies
nucleoprotein monoclonal globulin (Centocor, Malvern, PA) was added
and plates were examined under a fluorescence microscope to detect
infected cells. The original mAb produced in human hybridoma cells was
used as positive control. Both mAb were used at the same concentration to
compare their neutralization ability.
Results
Morphological features of plant cell culture
Primary calli initiated from WT tobacco seedlings and
R12 transgenic seedlings expressing human anti-rabies
virus mAb were grown on MAK medium and after 3 weeks
appear morphologically similar (Fig. 1A). The yellow color
turned to green after 3 months. Western blot analysis of
eight WT and eight R12 calli revealed HC and LC protein
production only in the R12 calli (data not shown).
Three weeks after inoculation, WT and R12 suspension
cultures appeared healthy in MAK medium (Fig. 1B), with
the same yellow color and the same morphology as a stabi-
604 L.S. Girard et al. / Biochemical and Biophysical Research Communications 345 (2006) 602–607
Fig. 1. Morphological features of calli and plant cell suspension cultures grown in MAK medium. (A) Three-week-old calli, obtained by callogenesis on
Nicotiana tabacum (cv. Xanthi) plant leaves grown on MAK medium (left, wild-type calli; right, R12 calli). (B) Comparison of wild-type (left) and R12
transformed (right) 10-day-old Nicotiana tabacum cv. Xanthi suspension cell culture. (C) Filtered cells from 20-day-old plant cell suspension. Note the
greenish color and clumped growth.
lized N. tabacum BY2 culture. One month later, cells began
to grow as clumps and turned greenish (Fig. 1C); cell sus-
pension cultures retained these features during further
subcultures.
Heavy- and light-chain expression in plant suspension
cultures and pellets
Western blot analysis (Fig. 2B) of soluble protein cell
extracts from five R12 suspension cultures and their
remaining pellets (after a second extraction) revealed HC
and LC bands migrating at 55 and 30 kDa, respectively.
The LC appeared to be expressed as two different forms
(Fig. 2B, lanes 2 and 4). Two other bands were also
detected in the pellet at 60 and 75 kDa (Fig. 2B). The
60-kDa band appears to be a dimer of LC since this band
was not detected by HC-specific antibody (data not
shown). The 75-kDa band might be an LC–HC dimer
resulting from partial denaturation of the mAb. None of
these specific bands was detected in WT soluble protein
Fig. 2. Analysis of protein extracts of WT and R12 cells by SDS–PAGE
stained with Coomassie blue (A) and Western blotting to detect human
anti-rabies virus monoclonal antibody light- and heavy-chains (B). Lanes
1 and 2 correspond to soluble protein extracts of wild-type and R12 cells
from suspension cell cultures, respectively; lanes 3 and 4 correspond to
proteins extracted from wild-type and R12 remaining pellets, respectively.
extracts (Fig. 2B, lanes 1 and 3), loaded at a similar
protein concentration as shown by SDS–PAGE and
Coomassie blue staining (Fig. 2A). Analysis of four succes-
sive extractions from the R12 remaining pellet revealed the
presence of HC and LC bands (data not shown). Expres-
sion of heterologous proteins was observed in all R12
suspensions.
Increased monoclonal antibody production in plant cell
suspension culture
ELISA results for mAb expression in the transgenic
plant [15] indicated levels at 3 lg/g leaf FW, corresponding
to 9 lg/g leaf DW (assuming 70% water content in
leaves), whereas similar analysis in plant cell suspensions
using lyophilized cells indicated 10 lg/g DW for four sus-
pension cell cultures and 30 lg/g DW for the highest-yield
culture.
In vitro neutralizing activity of monoclonal antibodies
The neutralization activity of mAb produced by plant
cell cultures was as efficient as the mAb produced by
hybridoma cells in a standard neutralization assay against
cell culture-adapted rabies virus strains (CVS-11, CVS-
N2C, and ERA). The same quantity of each mAb was
required to neutralize virus.
Kinetics of plant cell growth and monoclonal antibody
expression
Both WT and R12 cell suspensions were grown for 30
days and examined every 4 days for fresh weight (data
not shown) and dry weight (Fig. 3A). A well-established
N. tabacum BY2 suspension culture was used for compar-
ison. WT and R12 cell cultures showed similar growth
curves, with no exponential phase, starting from 4.5 g/L
 L.S. Girard et al. / Biochemical and Biophysical Research Communications 345 (2006) 602–607
Fig. 3. Monoclonal antibody production in plant cell cultures grown in
Erlenmeyer flasks. (A) Growth curve of wild-type (-h-) and R12 (-j-)
Nicotiana tabacum cv. Xanthi, and wild-type Nicotiana tabacum BY2 (-d-).
Data are the average of four samples and error bars correspond to the
resulting standard deviation. (B) Western blot detection of human anti-
rabies virus LC expressed in R12 cells with respect to growth curve.
and reaching 18 g/L final DW in 20 days. The BY2 cell cul-
ture reached 14 g/L in 10 days, starting from 0.6 g/L.
Western blot analysis of HC (data not shown) and LC
(Fig. 3B) expression in R12 cells revealed the same level
of expression at all time points examined. ELISA of extra-
cellular medium precipitated with 70% ammonium sul-
phate showed no significant difference between WT and
R12 suspension culture with respect to the presence of
mAb chains, and Western blot revealed only the LC in
the R12 medium, as a monomer (30 kDa) and a dimer
(60 kDa).
Bioreactor scale-up of monoclonal antibody production
Cells were grown for 15 days in Erlenmeyer flasks or a
disposable plastic bioreactor and harvested for analysis
of biomass and mAb expression. In both systems, biomass
production was on average of 13 g/L DW. mAb expression
as determined using ELISA was also comparable, with
10 lg/g DW in flasks and 13 lg/g DW in the bioreactor.
Small-scale purification of plant-produced monoclonal
antibodies
R12-produced mAb was purified on a protein G col-
umn, and flowthrough, washing step, and elution fractions
were examined for the presence of the HC and LC by Wes-
tern blotting (Fig. 4). WT cells were used as negative con-
trol. No HC or LC was detected in the WT sample or in the
flowthrough or the washing step phases of the R12 extract,
confirming the high specificity of the column. Both LC and
HC were detectable in the elution phase, in the same range
of intensity, and at the same molecular weight position as
605
Fig. 4. Western blot analysis of human anti-rabies virus monoclonal
antibody light- and heavy-chains in tobacco cell extracts after protein G
purification. FT, flowthrough; W, washing step; ELU, elution step; T+,
positive control (mAbP purified from plant on protein A column).
mAb purified from whole plants (Fig. 4). From 100 g
DW of cells, 250 lg of mAb was collected, which represents
25% of the measured production.
Discussion
In an effort to increase the safety and reproducibility of
human anti-rabies virus mAb production in plant-based
systems, a transgenic tobacco plant expressing this recom-
binant protein was grown as an undifferentiated cell cul-
ture. WT and R12 calli were rapidly obtained on MAK
medium, and LC and HC expression was readily detected
in R12 calli by Western blotting. All calli were morpholog-
ically healthy and identical, indicating that mAb expression
does not adversely affect cell metabolism. Moreover, LC
and HC expression proved that both the Ca2p and Pin2p
promoters are also functional in undifferentiated plant
cells.
Six weeks later, plant cell cultures were initiated in
MAK medium and examined for mAb production. Expres-
sion of the mAb appeared to remain constant throughout
the growth periods, with no effect on cell growth rate.
However, the presence of clumps, the lack of an exponen-
tial growth phase, and the slow growth rate indicated the
need for improved growth conditions. Indeed, N. tabacum
BY2 cells are about 10 times more efficient than Xanthi
cells for biomass production, reflecting the decades of opti-
mization of BY2 cell growth conditions while Xanthi cells
were rarely used. Despite the relatively low growth rate of
Xanthi cell cultures, a biomass scale-up was performed
with a new disposable plastic bioreactor and showed simi-
lar growth rate and mAb expression levels to those in
Erlenmeyer flask cultures. Disposable bioreactors up to a
size of 150 L for biomass production of BY2 cell culture
also showed similar results (personal communication).
These data demonstrate that inexpensive and easy-to-use
disposable bioreactors are suitable for biomass production
and for the production of recombinant proteins.
Analysis of the mAb concentration from plant cell sus-
pension cultures by ELISA revealed a production level as
606 L.S. Girard et al. / Biochemical and Biophysical Research Communications 345 (2006) 602–607
high as and even higher than that in whole transgenic
plants (up to 30 lg/g DW compared to 9 lg/g DW, respec-
tively). The production level in plant cells is probably high-
er than 30 lg/g DW since several successive extractions
revealed some mAb remaining in the pellet, probably
trapped in the ER. Since the ELISA used is based on rabies
virus recognition, the plant cell-produced mAb was pre-
sumably fully processed, i.e., with LC and HC association,
and functional, i.e., able to bind the virus. This resulting
mAb was also as effective as human hybridoma cell-pro-
duced mAb in neutralizing three different rabies virus
strains, preventing infection of BHK cells. This result
showed that undifferentiated plant cells were able to pro-
duce mAb with the same neutralization activity as whole
transgenic plants, also compared to human hybridoma
cell-produced mAb [15].
The glycosylation pattern of the mAb and several other
characteristics, such as immunogenicity, remain to be
examined. However, a high mannose-glycosylation like
that for whole-plant-produced mAb is expected due to
the KDEL ER-retention signal [7,8]. This hypothesis is
supported by the presence in Western blots of both LC
and HC at the same molecular weight (30 and 55 kDa,
respectively) as the glycosylated form of mAb produced
by the whole plant [8]. The LC might present two glycosyl-
ation patterns since a second band was observed. The
KDEL signal expressed on HC also prevented mAb secre-
tion into the medium. The small amount of mAb detected
in the R12 medium might be released by dying cells. Only
LC was detected outside of the cells, probably secreted via
the ‘‘default pathway’’ when not bound to HC.
The amount of human anti-rabies virus mAb recovered
from 100 g DW of R12 cells was 4-fold less than in pilot
experiments, probably due to mAb precipitation during
the long extraction process [17]. In the present study, the
main compounds responsible for mAb precipitation are
likely the polyphenols, as described by Haslam [19]. These
compounds form aggregates, possibly corresponding to the
dark precipitates observed in the supernatant even within a
few minutes after filtration through 0.22-lm filters. These
compounds imposed multiple filtration steps, several times
during the purification, increasing the processing time to
4 h. Another phenomenon has been described [20,21],
whereby 80% of a mAb dissolved in an Erlenmeyer flask
with pure Murashige and Skoog-medium can ‘‘disappear’’
in less than 2 h by anchoring onto the glassware wall. Such
adsorption of the mAb might also explain the low recovery.
Further studies are needed to overcome these obstacles to
optimal mAb recovery.
Plant cell culture is a developing technology for recom-
binant protein production and presents great potential in
competing with other production systems. Many features
remain to be optimized such as the expression level of the
product and the purification processes. Currently, the plant
cell culture system is more suitable for the production of
recombinant proteins needed in small quantity and pre-
senting high activity.
Acknowledgments
We thank Dr. K. Ko for providing seedlings and mAb
controls, and Dr. D.C. Hooper for fruitful discussion.
We also thank C. Brimer, R. Kean, P. Hilaire, and B.
Terrier for technical assistance. This work was supported
by a grant from USDA to Biotechnology Foundation (to
H.K.).
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