Assessment of Two Transgenic Tobacco Plant Varieties for the HBsAg-Specific Plantibody Production
O. Maylin La
Transgenic plants are an attractive alternative to produce
antibodies for the manufacturing of biologics. The main subject of this study
was to assess two non-commercial transgenic tobacco plant varieties (BHmN and
Habana 92) cultivated in zeolite and confined conditions for the production
of a hepatitis B virus surface antigen (HBsAg)-specific plantibody (PHB-01).
The plantibody molecule biochemical characterization and the assessment of the
immunopurification capacity of the antigen recognized by PHB-01-immunoadsorbents
were other subjects studied in this study to decide which variety would be more
suitable for PHB-01 large-scale production. As results, the BHmN variety allowed
obtaining 1.18 fold more biomass of leaves and up to 2 fold in the pantibody
yield with similar specificity and affinity constant by the HBsAg. The assessment
of the HBsAg immunopurification capacity of the PHB-01 immunoadsorbents produced
by these two non-commercial transgenic plant varieties did not showed significant
differences in terms of adsorption capacity (p = 0.2135), elution capacity (p
= 0.1239), recovery (p = 0.2655) and purity during 13 purification cycles. In
conclusion, the BHmN variety would be the most suitable variety for the production
of the PHB-01.
to cite this article:
Leonardo Gomez, Sigifredo Padilla, Alejandro Fuentes, Yoslaine Ruiz, Tatiana Gonzalez, Margarita Somoza, Lisette Lopez, Julio Sanchez, David Gavilan, Elio Espinosa, Yenisleydis Avila, Otto Mendoza, Yordanka Masforrol, Cristina Garcia, O. Maylin La and Rodolfo Valdes, 2013. Assessment of Two Transgenic Tobacco Plant Varieties for the HBsAg-Specific Plantibody Production. Journal of Agronomy, 12: 11-19.
Received: January 22, 2013;
Accepted: April 01, 2013;
Published: June 03, 2013
Several procedures have been developed to produce small and large quantities
of monoclonal antibodies (Kohler and Milstein, 1975).
The history of these procedures has evolved from ascitic fluid method, to transgenic
plants (Knazek et al., 1972; Brodeur
et al., 1984; Hiatt et al., 1989,
Veliz, 2002; Wei et al.,
2006; Loos et al., 2011; Srivastava
et al., 2011). Regarding to this, the capacity of plant cells to
express and correctly process complex proteins such as antibodies, the unlimited
biomass that can be achieved, the absence of cross-contaminating adventitious
agents and the potentially simple downstream process are seem as advantages
of this production alternative (De Jaeger et al.,
1999; Stoger et al., 2002; Hood
et al., 2002; Arntzen et al., 2005;
Malabadi and Nataraja, 2007; Behrooz
et al., 2008; Gomez et al., 2010).
For these reasons, the Center for Genetic Engineering and Biotechnology of
Havana, Cuba developed an industrial-scale process to produce a plantibody (PHB-01)
specific for the hepatitis B virus surface antigen (HBsAg) by transgenic plants.
This HBsAg specific plantibody was expressed in tobacco plants to be isolated
and employed as immunoreagent (matrixs ligand) to immunopurify the HBsAg,
which is used as the active pharmaceutical ingredient (HBsAg) of the Cuban vaccine
against Hepatitis B (Ramirez et al., 2003; Valdes
et al., 2003a; Pujol et al., 2005;
Padilla et al., 2009).
However, the plantibody yield obtained in the Habana 92 variety has been no
enough so far (Ramirez et al., 2003), aspects
that seems to be a common issue of this technology (Franklin
and Mayfield, 2005; Stoger et al., 2005,
2000; Decker and Reski, 2008;
Sainsbury and Lomonossoff, 2008; Vezina
et al., 2009; Hensel, 2011; De
Muynck et al., 2010).
Therefore, the main subject of this study was to assess two non-commercial
transgenic tobacco plant varieties (BhmN and Habana 92) cultivated in zeolite
and confined conditions for the production of a immunopurification capacity
of the antigen recognized by PHB-01-immunoadsorbents were other subjects studied
in this study to decide which variety would be more suitable for PHB-01 large-scale
MATERIALS AND METHODS
Transgenic plants: Transgenic tobacco plants expressing the anti-HBsAg
CB.Hep-1 monoclonal antibody (mAb) PHB-01 BHmN (clone 26) and PHB-01 H-92 (clone
23) (Ramirez et al., 2003) were used in this
study to compare the plantibody yield in BHmN and Habana 92 varieties, respectively.
Briefly, a tandem expression vector strategy to produce these molecules in these
Nicotiana tabacum varieties was employed. Clones 26 and 23 were chosen
among several clones for this study.
Chemical and mineralogical compositions of zeolite used in this study:
The chemical composition of the zeolite employed as substrate to cultivate both
variety plants was (% in weight): SiO2 (66.62), Al2O3
(12.17), Fe2O3 (2.08), CaO (3.19), MgO (0.77), Na2O
(1.53), K2O (1.20), PPI (11.02), for a total of 98.58. The ratio
of SiO2/Al2O3 (mol) was 9.29. The total cationic-exchange
capacity expressed as TCEC meq/100 g was 138.69 and for each element was Ca2+
94.48, Mg2+ 4.13, Na2+ 32.49 and for K+ 7.59.
Its mineralogical composition was Clinoptilolite (49%), Mordenite (12%), Montmorillonite
(Na, Ca) 0.33 (Al, Mg) 2 Si4O10 (OH) 2nH2O
(very few); Calcite, CaCO3 (very few); Quarz, SiO2 (very
few); Feldespast, Fe-aluminosilicates (undetermined). The hardness was 1.08±2.1
and results of chemical resistance (oxidability) was NaCl (3.13±1.9%),
HCl (3.39±0.5%), NaOH (2.50±1.3%), H2O (2.38±1.2%),
Cl2 (1.58±2.19%). The characteristics of main granular materials
were (Mean±CV): Particle density (2120.0±1.45 kg cm¯3), apparent
density (1021.0±1.76 kg cm-3), porosity (0.51±0.4)
and shape factor (0.64±1.97) (Marquez et al.,
Generation of seedlings: Nicotiana tabacum L., seeds were cultivated
in Turba Rubia (85% organic matter, 15% ashes, 0,26% total nitrogen, 300 mg
L-1 phosphorous anhydride, 144 mg L-1 magnesium oxide,
pH 5.5-6, 50% maximum humidity, TCEC 100-130 meq/100 g and 500-700 μS cm-1
conductivity) using the floating tray method (Cristanini,
1995). Seedlings were fertilized with 5 g L-1 of the Haifa-Chemical,
Israel 19-19-19 NPK-fertilizer solution at days 7th and 21st, respectively and
pruned at 28th day after seedtime. Next, the pruning was repeated every 48 h
up to the seedling transplant into the green-containment for producing vegetal
biomass rich in PHB-01 BHmN and PHB-01 H-92, respectively.
Production of transgenic plants: Both varieties of plants were growth
in 5 furrows at 8 plants by square meter in a green house under controlled conditions
using the zeolite employed in the seedling generation. Briefly, plants were
fertilized, dropping four times per day during 4 min, with the 50 g L-1
of the 19-19-19 NPK-fertilizer solution (Haifa-Chemical, Israel). The vol-vol
Magnisal and 19-19-19 NPK-fertilizer solution (Haifa-Chemical, Israel) were
used after of 4th week. After 9 weeks, plants were then harvested.
The estimation of the biomass production and weight of leaves, roots and stems
were done using all cultivated plants. (Pushkina et al.,
Purification of plantibody from transgenic tobacco plants: Nicotiana
tabacum plants were processing using a Fitzmill model D6A Comminutor (Fitzpatrick
Company, USA). Green fraction was then removed from the juice by centrifugation
at 1,500 rpm in a Rina 100F 800 bascket centrifuge (Riera Nadeu S.A, Spain)
and supernatants, still containing small amount of suspended green particles,
were heated. Next, heated supernatants were centrifuged in a Wetsfalia centrifuge
and submitted to an acid precipitation with 100 mM HCl. After that, the precipitated
supernatants were equilibrated with 2M Tris and centrifuged again to be applied
in a chromatography column loaded with ProSepvA Ultra. Elution fractions were
submitted to saline precipitation using Ammonium sulfate. This material was
centrifuged again and pellets were dissolved in 20 mM Tris/150 mM NaCl, pH 7.0.
Subsequently, a gel filtration chromatography using Sephadex G-25 coarse (Amersham-Biosciences,
Uppsala, Sweden) was performed for desalting the material before the plantibody
preparation sterile filtration.
Estimation of plantibody concentration by enzyme-linked immunosorbent assay:
A polystyrene microplate (Costar, Cambridge, USA) was coated with 10 μg
per well of HBsAg in 100 mM NaHCO3 buffer for 20 min at 5°C.
After this step, samples were added to the plate in 150 mM saline buffered solution
(PBS)/0.05% Tween 20 and incubated for 1 h at 37°C. Several washings were
done with PBS/0.05% Tween 20. The plate was incubated for 1 h at 37°C with
an IgG-horseradish peroxidase conjugate (Sigma Chemical Co., St. Louis, USA).
The reaction was then revealed using 100 μL well-1 of 0.05%
O-phenylendiamine (OPD) and 0.015% H2O2 in citrate buffer
(pH 5.0) and stopped with 50 μL well-1 of 1.25 M H2SO4.
The absorbance was measured in a Multiskan ELISA reader (Labsystems, Helsinki,
Finland) using a 492 nm filter (Leyva et al., 2007).
Protein determination: Protein concentration was determined following
the method described by (Lowry et al., 1951),
using bovine serum albumin as standard material. The curve range was from 10
to 100 μg mL-1 and the absorbance of samples was measured at
280 nm in a UV/Visible Ultrospec 2000 Spectrophotometer (Pharmacia Biotech,
SDS-PAGE and western-blott: The purity of the plantibody samples was
analyzed carrying out an electrophoresis on a 12.5% (w/v) SDS-polyacrylamide
gel (Laemmli, 1970) followed by a Coomassie blue staining.
Determination of plantibody affinity constant: The affinity constant
of plantibody molecules was determined by the method described by Beatty
et al. (1987). Microtiter plates were coated with the HBsAg and incubated
with plantibodies. Plates were sequentially incubated with a horseradish peroxidase-antibody
conjugate and the reaction was revealed using OPD as substrate and 0.015% H2O2
in citrate buffer; pH 5.0. Reaction was stopped by adding 50 mL of 2M H2SO4.
The plantibody amount adherent to the HBsAg was reflected by the enzyme product
measured by optical density at 492 nm using an ELISA reader (Labsystem, Helsinki,
Specificity assay: The peptide recognition assay was performed using
40 mg of peptides extracts Tenta Gel resin humid. This resin was washed with
purified water (3 times x 5 min) and washed again with 150 mM PBS (3 times x
5 min). Then, it was blocked with BSA 1% in 150 mM PBS for 1h. After that, the
resin was incubated with respective PHB-01 molecules in 400 μL (1 mg mL-1)
in BSA's dissolution to the 1% in 150 mM PBS (1 h). Next, a wash with 0.05%
Tween 20/150 mM PBS (5 times x 5 min) was done. Incubation with 400 μL
with an anti mouse IgG-phosphatase alkaline was performed (1:2000) for 1h, followed
by washings with 0.05% Tween 20/150 mM (5 times x 5 min). The resin was then
incubated with BCIP solution (5-bromo-4-cloro-3-indolil-fosfato) in 400 μL
of substrate solution for 30 min (until color appeared). Reaction was stopped
when pearls colored were washed with 150 mM PBS (5x5 min). The 37 peptide sequence
was TTSTGPCKTCTT. This corresponds with the peptide 20 of a
determinant of the HBsAg and is a positive control. Besides, RFSWLSLLVPFV (P36)
corresponds with the peptide 29 of HBsAg but no with a determinant and is a
negative control. On the other hand, the mAb anti CEA (anti-carcinogenic embrionary
antigen) was used as negative control of antibody.
Purification of the CB.Hep-1 mAb used as experimental control: Hybridoma
48/1/5/4 was grown as ascites tumor in specific pathogen free Balb/c mice. Animals
received 0.5 mL of mineral oil and 1x106 cells per animal were inoculated
into the animal peritoneal cavity in cell culture medium RPMI (Gibco, Grand
Island, USA). Abdominal taps were performed when marked abdominal distention
was observed. The purification process included clarification and filtration
of the ascites, affinity chromatography, using n-protein A SepharoseTM
4 fast flow loaded in a column BPG100/500 operated at 100 cm h-1.
Then, a gel-filtration chromatography, using Sephadex G-25 coarse (Amersham-Biosciences,
Uppsala, Sweden), was performed in a BPG200/750 column at 130 cm h-1
to exchange the buffer for allowing the antibody concentration in a Sartocon
alpha (SARTOCON Silice Cassettte) and the sterile filtration using capsules
of 0.45-0.2 μm SARTOBRAN P Sterile MidiCap of Sartorius Stedim Biotech,
Hepatitis B surface antigen production: The HBsAg was produced by fermentation
of a recombinant strain of Pichia pastoris (C-226) in saline medium supplemented
with glycerol and its expression was induced by methanol. Cells were centrifuged
and disrupted with 20 mM Tris-HCl (pH 8.0)/3 mM EDTA/300 mM NaCl/3M KSCN/10
g L-1 sucrose on a bed mill (KDL type: WAB, Basel, Switzerland).
The homogenate was precipitated by adding 1M HCl and centrifuged at 10000 xg
for 30 min. Supernatant was adsorbed on Hyflo Super Cell (a flux-calcined grade
of Celite filter-aid) and equilibrate to pH 4.0 under continuous stirring during
2 h. After washing step, HBsAg was eluted with 20 mM Tris/1M HCl/3 mM EDTA/100
g L-1 sucrose (pH 8.2) and semipurified material of 10-25% purity
was used as starting material for the immunoaffinity chromatography experiments
(Lucila et al., 1992).
Immunoaffinity matrix: Sepharose CL-4B (Amersham-Bioscences, Uppsala,
Sweden) was activated by the cianogen bromide (CNBr) method (Kohn
and Wilchek, 1984). The CB.Hep-1 mAb and plantibodies were coupled as recommended
by Amersham-Biosciences, Uppsala, Sweden (Gomez et al.,
2002) (Wimalasena and Wilson, 1991). The amount
of coupled antibody was determined by measuring the total protein before and
after the coupling reaction (about 4 mg mL-1 of gel for each immunogel).
Immunoaffinity chromatography: Gels were packed into PD-10 chromatography
columns with 7 mL of gel. (Amersham-Bioscences, Uppsala, Sweden) and equilibrated
with 20 mM Tris-HCl/3 mM EDTA, pH 7.8. Adsorption and elution flow rates were
20 and 35 cm h-1, respectively. Columns were loaded with a partially
purified HBsAg preparation in the equilibrium buffer containing 1M NaCl. After
washing, the bound antigen was eluted with 20 mM Tris/3M KSCN/3 mM EDTA, pH
7.0 and monitored at 280 nm.
Mathematical analysis: ANOVA tests were carried out using the Statgraphic
program (version 5.0) and p-values less than 0.05 were considered to be of statistical
RESULTS AND DISCUSSION
Since Hiatt et al. (1989), pioneered the expression
of immunoglobulin chains in tobacco plants, various portions of immunoglobulin
chains have been expressed heterologously, including single chain molecules,
Fab fragments, small immune proteins, IgGs and chimeric secretory IgAs (Hensel,
2011). In view of the growing demand for recombinant antibodies, production
capacity has been saturated, so there is a need for the development of attractive
alternative means of production to the classical mammalian expression systems
such as transgenic plants (Morrison et al., 1984;
Jones et al., 1986; Jain
et al., 2007; De Muynck et al., 2010).
In regard to the production of antibodies in transgenic plants, different levels
of plantibody yield have been obtained in different host species and organs.
However, the huge majority of examples showed an insufficient level of expression.
For instances, the maximum expression level obtained in leaves has been 325
μg g-1 fresh weight (FW) of Nicotiana bethamiana, 757
μg g-1 FW in Nicotiana tabacum (Sainsbury
and Lomonossoff, 2008; Vezina et al., 2009),
1.1 μg g-1 FW (H10 antibody) in Nicotiana tabacum (Villani
et al., 2009), 0.9 μg g-1 FW (CO17-1A) in Nicotiana
Bethamiana (Ko et al., 2005), 1 μg g-1
FW (cT84.66) in Nicotiana tabacum (Vaquero et
al., 1999), 8.5 μg g-1 FW (rAb29) in Nicotiana tabaccum
(Schillberg et al., 1999), 60 μg g-1
FW (MGR48) in Nicotiana tabacum (Stevens et al.,
2000) and 31 μg g-1 FW (BR55-2) in Nicotiana tabacum
(Brodzik et al., 2006). Similar situation was
described by (Ramirez et al., 2003) for the expression
of the PHB-01 in Nicotiana tabacum. That is why; authors sought to assess
the yield of the PHB-01 in two different tobacco plant varieties (BHmN and Habana
92), to decide the suitable condition for large-scale production.
The comparison of these two varieties of transgenic tobacco plant cultivated
in zeolite under confined conditions in terms of biomass yield and weight of
leaves, stems and plants demonstrated that the biomass yield (kg of leaves)
measured in the BHmN variety was 1.18 fold higher than in Habana 92 variety
(p = 0.0023).
||Picture of the BHmN (left) and Habana 92 (right) varieties
cultivated in zeolite under confined conditions
Similar increase was observed when the weight of stems (1.14 fold, p = 0.0470)
was measured. In consequence, the comparison of the whole weight of the plants
evidenced better results for the BHmN variety (304.02 g) against Habana 92 (258.65
g). The increase in this parameter was 1.17 fold (p = 0.0089) (Table
1). Other agronomic values estimated at 7 weeks of cultivation were (BHmN:)
foliar area 1666.1 cm2, height growth rate 0.085 cm day-1 and average
height 80.6 cm. (Habana 92:) foliar area 1174.2 cm2, height growth
rate 0.028 cm day-1 and average height 72.4 cm. In addition, BHmN
plants were smaller than Habana 92 plants but had more and bigger leaves than
Habana 92 plants (Fig. 1).
The plantibody expression level measured was 0.28% for BHmN and 0.11% for Habana
92, which represented 2.54 fold of increase. In this regards, authors must point
out that Total Soluble Protein (TSP) concentration was 10.98 and 13.07 mg mL-1
in the leaf extract of BHmN and Habana 92 varieties, respectively, which was
coincident with those values reporter by (Floss et al.,
2008). As consequence, results of the plantibody yield in the extract of
leaves was 67.62 mg kg-1 for BHmN and 33.67 mg kg-1 for
Habana 92, representing thus a net increase of 2.00 fold (Table
1), which was analogous with those reported by (Stevens
et al., 2000) and controversial with results reported by (Vaquero
et al., 1999; Villani et al., 2009).
In respect to results obtained after the purification of the plantibody molecules,
the yield of plantibodies after the purification process, expressed as mg of
plantibody per kg of processed leaves, was 33.6 (BHmN) and 10.59 (Habana 92).
That corresponded to a 3.17 fold increase (Table 1).
|| Results of the harvests of transgenic plants and purification
of the plantibody molecules
|| Purity of the plantibody molecules measured by SDS-PAGE under
reduction conditions (a) and HPLC-GF (b). (a). Line 1: Molecular Weight
Marker-091201: Bovine serum albumin 60 kDa, Ovoalbumin 41.07 kDa, Trypsin
inhibitor 20.6 kDa, Lisozime 12 kDa, Aprotinin 6,4kDa. Line 2: PHB-01. Line
3: PHB-01 BHmN (experiment 1). Line 4: PHB-01 BHmN (experiment 2). Line
5: PHB-01 BHmN (experiment 3). Line 6: Reference Material-020907 (CB.Hep-1
monoclonal antibody used as experimental control). (B). a) PHB-01 BHmN,
b) PHB-01 H-92
The purity of the purified molecules estimated by SDS-PAGE under reducing conditions
was 93% for BhmN and 92% for Habana 92, whereas 99.57 and 99.14% were values
of purity measured by gel-filtration-HPLC in both varieties, respectively (Fig.
2). Therefore, no differences were observed in this important parameter
between both varieties. In that sense, similar results were obtained by Valdes
et al. (2003b).
Another remarkable issue that has to be considered is the nicotine level in
the purified plantibody preparation.
|| Results of the assay performed to demonstrate the specificity
of the plantibody molecules by the HB
This is an important aspect for human health because after the nicotine absorption,
Adrenals glands activation is produced and a discharge of adrenaline produces
corporal stimulation and sudden discharge of glucose, blood pressure increase,
respiration and heart rate. The addictive potential is due to the dopamine secretion
produced on the regions of the brain that control placer sensations. The nicotine
decreases basal metabolism. The interaction with other substances including
drugs is possible using the same metabolic way of C-P450 in liver. In the brain,
part of the metabolites is transformed in intermediates (as nornicotine) that
could be neurotoxic, which acts on the nicotine cholinergic receptors of the
central nervous system (Andersson et al., 2003).
In such sense, the nicotine values measured in both purified plantibody preparations
were always lower than the detection limit of the technique (0.05 mg L-1)
and lower than 50 mg. This is of the minimum toxic doses of nicotine permitted
in humans (Andersson et al., 2003; Tuduri,
Results of assay designed to detect the epitope recognized by plantibody molecules
corroborated that both molecules recognized the same epitope into the a
determinant of the HBsAg molecule. This can be appreciated with the blue color
displayed in samples of PHB-01 BHmN, PHB-01 H-92 and CB.Hep-1 mAb corresponding
with the peptide 37 case. The absence of color in the anti-CEA mAb sample in
the peptide 37 validated the specificity of the assay. Results of the assays
with the peptide 36 revealed that PHB-01 BHmN, PHB-01 H-92 and CB.Hep-1 mAb
did not recognized an aminoacid sequence non-related with the a
determinant of the HBsAg (Fig. 3).
Table 1 shows the affinity constant of plantibody molecules
purified from both varieties. The affinity constant values obtained were 2.35x10-8
and 2.97x10-8, which no also corroborate the specificity of the molecules
but the strength of association with HBsAg.
Finally, immunoadsorbents of both plantibodies were performed at a ligand density
ranged 3-5 mg mL-1 of gel, because we have previously demonstrated
that there were not differences in the behavior of the CB.Hep-1 mAb immunoadsorbents
during the HBsAg purification in this range of ligand density (Gomez
et al., 2002). As Table 2 shows, there were not
statistical differences in the plantibody coupling efficiency (p = 0.2032),
immunoadsorbent ligand density (p = 0.0844), HBsAg adsorption capacity
(p = 0.609), HBsAg elution capacity (p = 0.1615) and HBsAg recovery
(p = 0.1383). In addition, the ligand linkage determined in the purification
cycles (1, 7, 8 and 10) was always below the limit established for this parameters
(3 ng IgG mg-1 HBsAg) and without significant differences (p =
0.8171) between them (Table 3).
|| Results of the comparison of the plantibody inmovilization
|| Results of the evaluation of the HBsAg immunopurification
Similar results were obtained when results of the plantibody-immunoadsorbents
where compared with those obtained with the immunoadsorbent prepared with the
CB.Hep-1 mAb used as positive control.
The BHmN variety allowed obtaining 1.18 fold more biomass of leaves that Habana
92 variety. The plantibody amount measured in the leaf extracts of the BHmN
variety plants was 2.54 fold higher than in the Habana 92 variety. The total
soluble concentration was similar between both varieties. The plantibody yield
measured in the leaf extracts of the BHmN variety plants was 2.0 fold higher
than in the Habana 92 variety. The recovery of the plantibody process purification
was 1.65 fold higher in BHmN experiments than in Habana 92. The plantibody yield
measured after the purification process was 3.17 fold higher in BHmN variety
than in the Habana 92 variety experiments. The purity of the plantibody molecules
measured by SDS-PAGE and HPLC-GF was always higher than 90% and statistically
similar between molecules purified from both varieties. Both plantibody molecules
recognized the same epitope on the HBsAg and with similar affinity constant.
The behaviour of immunoadsorbents of both plantibody molecules was statistical
equal in terms of plantibody coupling efficiency, plantibody ligand density,
HBsAg adsorption capacity, HBsAg elution capacity, HBsAg recovery and plantibody
leakage. Therefore, the replacement of the Habana 92 variety by BHmN variety
for the production of the HBsAg-specific plantibody for vaccine purpose has
been scientifically argued.
This study was supported by the Center for Genetic Engeneering and Biotechnology
of Havana and authors gratefully acknowledge to: Danay Callard of the Direction
of Agropecuary Research for his assistant on the obtaining of transgenic BHmN
variety and Yordan Issac, Yarusenky Lescaille, Adelma Perez and Yandiesky Lowery
for his assistant in the cultivation of two N. tabacum varieties.
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