Mol Biotechnol (2015) 57:101–110 DOI 10.1007/s12033-014-9806-5
RESEARCH
Development of a Single-Replicon miniBYV Vector for Co-expression of Heterologous Proteins Alex Prokhnevsky • Tarlan Mamedov • Brett Leffet Rahila Rahimova • Ananya Ghosh • Vadim Mett • Vidadi Yusibov
•
Published online: 4 October 2014 Ó Springer Science+Business Media New York 2014
Abstract In planta production of recombinant proteins, including vaccine antigens and monoclonal antibodies, continues gaining acceptance. With the broadening range of target proteins, the need for vectors with higher performance is increasing. Here, we have developed a single-replicon vector based on beet yellows virus (BYV) that enables codelivery of two target genes into the same host cell, resulting in transient expression of each target. This BYV vector maintained genetic stability during systemic spread throughout the host plant, Nicotiana benthamiana. Furthermore, we have engineered a miniBYV vector carrying the sequences encoding heavy and light chains of a monoclonal antibody (mAb) against protective antigen (PA) of Bacillius
anthracis, and achieved the expression of the full-length functional anti-PA mAb at *300 mg/kg of fresh leaf tissue. To demonstrate co-expression and functionality of two independent proteins, we cloned the sequences of the Pfs48/45 protein of Plasmodium falciparum and endoglycosidase F (PNGase F) from Flavobacterium meningosepticum into the miniBYV vector under the control of two subgenomic RNA promoters. Agroinfiltration of N. benthamiana with this miniBYV vector resulted in accumulation of biologically active Pfs48/45 that was devoid of N-linked glycosylation and had correct conformation and epitope display. Overall, our findings demonstrate that the new BYV-based vector is capable of co-expressing two functionally active recombinant proteins within the same host cell.
A. Prokhnevsky T. Mamedov B. Leffet R. Rahimova A. Ghosh V. Mett V. Yusibov (&) Fraunhofer USA Center for Molecular Biotechnology, 9 Innovation Way, Suite 200, Newark, DE 19711, USA e-mail:
[email protected]
Keywords Beet yellows virus Plant viral vector Plant expression system Transient expression Heterologous proteins Monoclonal antibody Subunit vaccine
Present Address: T. Mamedov Department of Agricultural Biotechnology, Akdeniz University, Pınarbas¸ ı Mh., 07985 Antalya, Turkey
Introduction
Present Address: R. Rahimova Institut de Recherche en Cancerologie de Montpellier, 208 rue des Apothicaires, 34298 Montpellier Cedex 5, France Present Address: A. Ghosh Siemens Healthcare Diagnostics Inc., 500 GBC Drive, Newark, DE 19702, USA Present Address: V. Mett Cleveland BioLabs, Inc., 73 High Street, Buffalo, NY 14203, USA
Co-expression of more than one target gene is important for developing many human health products including vaccines and therapeutics. Several expression systems, including mammalian and insect cells as well as plants, have been used to produce such target proteins [1–4]. Among currently used approaches, the plant systems for transient expression of targets, particularly those based on plant RNA virus vectors, are gaining increased popularity due to simple and fast engineering, high levels of target expression, scalability, enhanced safety, and low cost. With all advantages that plant RNA virus vectors offer, simultaneous production of multiple targets in plants using current vectors remains to be a challenge. Over the last two
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Fig. 1 a Organization of the BYV vector including the open reading frame carrying sequences of two polypeptide chains of an antibody. Promoters and restriction sites used for cloning of a heavy chain (Hc) and a light chain (Lc) of an antibody are indicated in the lower panel. LB left border of T-DNA, 35S cauliflower mosaic virus 35S promoter, 1a the sequence encoding methyltransferase and RNA helicase, 1b the sequence encoding methyltransferase, RNA helicase and RNAdepended RNA polymerase, p6 the 6 kDa protein, HSP70h a homolog of heat shock protein 70, p64 a 64 kDa protein responsible for virion
assembly, CPm minor coat protein, CP coat protein, p20 BYV longdistance transport factor, p21 BYV silencing suppressor, R ribozyme, NOS nopaline synthase terminator, RB right border of T-DNA, GLRaV2 grapevine leaf roll-associated virus 2, BYSV beet yellow stunt virus. b Scheme of T-DNA with the miniBYV vector carrying the sequences of Hc and Lc of an anti-PA mAb. LB left border of T-DNA, 2En-35S cauliflower mosaic virus 35S promoter with dual enhancers, R ribozyme, NOS nopaline synthase terminator, RB right border of T-DNA, PA protective antigen of B. anthracis
decades, efforts have been made to overcome this challenge, including the engineering and use of hybrid [5], homologous [6], heterologous [7], and defective plant RNA [8] virus vectors. Although these efforts have led to significant technological improvements, engineering of a vector that can accommodate multiple targets remains a desirable goal. To circumvent shortcomings of current vectors, we have developed a single-component viral vector based on beet yellows virus (BYV) for expression of one or two proteins. BYV is a positive-strand RNA virus that belongs to the family Closteroviridae and the genus Closterovirus. The 15.5 kb genome of BYV encodes 10 open reading frames (ORFs) [9]. Two BYV proteins, methyltransferase and helicase and RNA-depended RNA polymerase (RdRp) encoded by ORFs 1a and 1b (Fig. 1), are responsible for virus replication [10, 11]. Five proteins, p6, HSP70h, p64, minor coat protein (CPm), and coat protein (CP), are responsible for virus cell-to-cell movement [12–15]. p20 is the virus long-distance transport factor [16], and p21 is the BYV silencing suppressor [17].
Previously, expression of green fluorescent protein (GFP) introduced into the BYV genome between CPm and CP coding sequences [18] and into a binary vector [16] was reported. In addition, to achieve accurate processing of viral RNA, hammerhead ribozyme sequences were introduced into the binary vector immediately downstream of the BYV sequences [16]. Here, we describe designing and engineering of the improved BYV-based vector that allows for efficient co-expression of either two target proteins that are required to assemble (e.g., heavy chain [Hc] and light chain [Lc] of a monoclonal antibody [mAb]) or two independent proteins within the same plant cell using Agrobacterium-mediated vector delivery.
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Materials and Methods Construction of BYV and miniBYV Vectors The binary vector p35S-BYV-GFP, previously described by Prokhnevsky et al. [16], was used for engineering of the
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full-size BYV vector carrying Hc and Lc of a mAb against protective antigen (PA) of Bacillius anthracis that has been described previously [19]. Briefly, a multiple cloning site (MCS) containing unique sequences recognized by PacI, AscI, BsrGI, and NheI was introduced between the CPm and CP genes of BYV. Using the MCS, two heterologous closteroviral CP subgenomic promoters (SGPs) were introduced into the BYV genome: the grapevine leaf rollassociated virus 2 (GLRaV2) CP SGP, nucleotides 12,706–13,083 (GenBank accession No. AF039204) [20] and the beet yellow stunt virus (BYSV) CP SGP, nucleotides 8,226–8,616 (GenBank accession No. U51931) [21]. The promoters were synthesized by GeneArt division of Life Technology Inc. (Carlsbad, CA). The coding sequences of Hc and Lc of the anti-PA mAb, containing the pathogenesis-related protein 1a (PR-1a) signal peptide sequence (MGFVLFSQLPSFLLVSTLL LFLVISHSCRA), were PCR-amplified from plasmids as described previously [22]. Hc was amplified using primers containing PacI/AscI restriction sites: 50 primer, GCG CTT AAT TAA AAT GGG ATT TGT TCT CTT TTC ACA ATT G and 30 primer, TAT AGG CGC GCC TCA TCA CTT TCC AGG AGA AAG AGA C. For Lc (kappa) amplification, primers containing BsrGI/NheI restriction sites were used: 50 primer, TCG ATG TAC AAA ATG GGA TTT GTT CTC TTT TCA C and 30 primer, TGA CGC TAG CTT ATC AGC ACT CTC CCC TG. As a mock control, the sequence of GFPc3 was cloned into the BYV genome using PacI/NheI restriction sites. The resulting constructs, BYV-PA-HcLc and BYV-GFPc3, were transformed into Agrobacterium tumefaciens strain GV3101. For construction of miniBYV vectors, the portion of the BYV genome including the BYV CP SGP following the MCS was synthesized by GeneArt and introduced into the BYV genome using the unique SnaBI/BstEII restriction sites. The GLRaV2 CP SGP was introduced using AscI/ BsrGI restriction sites, resulting in the miniBYV2 vector. To obtain the miniBYV1 vector, we substituted the GLRaV2 CP SGP with the BYSV CP SGP and confirmed the substitution by sequencing. To remove canonical splicing sites from BYV replicase, we employed the SplicePredictor software from the Center for Bioinformatics and Biological Statistics, Iowa State University, based on the algorithm described by Brendel et al. [23]. Arabidopsis thaliana was used as a template for splicing sites prediction. High-scoring donor and acceptor splicing sites have been determined within the BYV replicase sequence. To avoid a potential splicing event, we mutated the high-scoring canonical acceptor splicing site within the BYV replicase sequence by substituting adenosine by cytosine in the nucleotide position 2,219. This also resulted in elimination of the donor site in the position 3,606. The mutation was confirmed by sequencing.
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The DNA sequences encoding Hc and Lc were then cloned into both miniBYV2 and miniBYV1 as described for the BYV vector, and the resulting constructs were introduced into A. tumefaciens strain GV3101. The miniBYV2 vector was also used for co-expression of Pfs48/45 from Plasmodium falciparum (GenBank accession number: AAL74351) and PNGase F from Flavobacterium meningosepticum (GenBank accession number: J05411). The design of Pfs48/45 (Pfs48F1) and PNGase F sequences has been described previously [24]. In the current study, the sequences encoding Pfs48F1 and PNGase F were PCR-amplified and cloned under the control of the BYV CP SGP using PacI/AscI restriction sites and under the control of the GLRaV2 CP SGP using BsrGI/NheI restriction sites, respectively. Alternatively, the gene encoding Pfs48F1 was cloned in the miniBYV2 vector using PacI/NheI restriction sites. All plasmids had sequences confirmed and were introduced into A. tumefaciens strain GV3101. Plant Growth Conditions and Infiltration Procedure Nicotiana benthamiana plants were grown with an 11/13 h photoperiod at 25 °C and 50 % humidity. A. tumefaciens harboring a clone of interest was grown overnight at 28 °C in LB media in the presence of 50 mg/L kanamycin and rifampicin. The overnight culture of Agrobacterium was induced for 2 h in MMA media (10 mM MgCl2, 10 mM MES, pH 5.85, and 150 lM of acetosyringone) before plant infiltration. In addition, the silencing suppressor P1/ HC-Pro from turnip mosaic virus was co-introduced into plants to improve target expression level [25–27]. Twelve six-week-old N. benthamiana plants were manually infiltrated by syringe with a mixture of Agrobacteria carrying the expression plasmid and silencing suppressor at a ratio of OD600 1.0 and 0.2. Western Blot and ELISA Analyses of Protein Expression Leaf samples from N. benthamiana systemically infected with the BYV vector carrying the Hc and Lc genes of the anti-PA mAb were harvested at 30, 32, 34, 36, and 39 days post infiltration (dpi). The expression levels of Hc and Lc in the presence or absence of a reducing agent (Dithiothreitol) were analyzed by Western blotting [28] using the protein A-purified anti-PA83 mAb [22] as a standard. Samples from leaves infiltrated with miniBYV1 and miniBYV2 vectors carrying Hc and Lc of the anti-PA mAb were analyzed by Western blotting at 6–13 dpi. For detection of total IgG, a horseradish peroxidase (HRP)labeled goat anti-human IgG antibody (Jackson Immune Research, West Grove, PA) at the dilution 1:5,000 was
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used. For detection of Hc and Lc, goat anti-Fc and antikappa HRP-conjugated antibodies (Bethyl Laboratories Inc., Montgomery, TX), respectively, were used. MagicMarkTM XP Western Protein Standard (Life Technology Inc.) was used to estimate molecular weight of target proteins. Molecular expression levels were calculated using the GeneGnome5 gel imaging and analysis systems (Synoptics Inc., Frederick, MD). Samples from the leaves infiltrated with the miniBYV2 construct carrying the Pfs48F1 and PNGase F genes were taken at 6, 8, and 10 dpi, and Pfs48F1 expression was analyzed by Western blotting [28] using a primary antitetra-histidine (anti-4xHis) mouse mAb (Cat. No. 34670; Qiagen, Valencia, CA) and a secondary goat anti-mouse HRP-labeled antibody (Bio-Rad, Hercules, CA). The expression of PNGase F was confirmed by Western blot analysis using a rabbit anti-FLAG primary mAb (SigmaAldrich, St. Louis, MO) followed by a goat anti-rabbit HRP-labeled secondary antibody (Bio-Rad). Samples from systemic leaves were also analyzed by enzyme-linked immunosorbent assay (ELISA). In a 96-well MaxiSorp plate (NUNC, Rochester, NY), 100 lL per well of 5 lg/mL recombinant PA (Biological Laboratories Inc, Campbell, CA) in phosphate buffered saline (PBS) were plated, and the plate was incubated overnight at 4 °C. After incubation, the plate was washed with PBS-T buffer and blocked with 0.5 % I-block (Applied Biosystems, Foster City, CA) in PBS for 1 h at room temperature (RT). After blocking, 200 lL per well of serial dilutions of the systemic leaf clarified extract in PBS was added and incubated for 2 h at slow rotation. I-block in PBS served as a negative control and a human anti-PA mAb (IQ Therapeutics, Groningen, Netherlands) was used as a positive control. After the incubation, the wells were washed and incubated for 1 h with a 1:10,000 dilution of a goat antihuman HRP-labeled antibody as described above. Finally, 20 lL/well of the substrate (o-phenylenediamine dihydrochloride [OPD], Sigma-Aldrich) were added followed by incubation for 15–30 min at RT away from the light. The reaction was stopped by adding 50 lL of 5 M H2SO4 per well. The plate was scanned using a spectrophotometer, and data were analyzed using the Softmax software (Molecular Devices, Sunnyvale, CA). Purification of Glycosylated and Deglycosylated Recombinant Pfs48F1 from N. benthamiana Purification of the glycosylated and deglycosylated Pfs48F1 recombinant proteins produced using the miniBYV2 vector in N. benthamiana plants were performed using immobilized metal ion affinity chromatography (IMAC) as described previously [24]. Briefly, for purification of the in vivo deglycosylated recombinant Pfs48F1
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protein, 50 g of plant material infiltrated with miniBYV2Pfs48F1 or miniBYV2-Pfs48F1-PNGase F was homogenized in 150 mL of extraction buffer (50 mM sodium phosphate buffer, pH 8.0, 0.5 M NaCl, 20 mM Imidazole, and 1 mM Dieca) and incubated with 0.5 % Triton X-100 (final concentration) for 20 min at 4 °C with stirring. After incubation, the lysate was centrifuged at 48,0009g for 40 min, and crude extract was filtered through Miracloth and loaded onto a 5-mL HisTrap FF column (Cat. No. 17-5255-01; GE Healthcare, Waukesha, WI) followed by washing with 15 volumes of 50 mM sodium phosphate buffer, pH 8.0, 0.5 M NaCl, 20 mM Imidazole. Proteins were eluted with 50 mM sodium phosphate buffer, pH 8.0, 0.5 M NaCl, and 100 mM Imidazole. The eluted fraction was concentrated and dialyzed against PBS, pH 7.5. Comparative ELISA for Evaluation of Pfs48F1 Glycosylation Recognition of deglycosylated and glycosylated forms of Pfs48F1, produced in N. benthamiana using the miniBYV2 vector, by a rat mAb raised against epitope III of P. falciparum surface protein Pfs48/45 [29, 30] was assessed by ELISA similar to that described previously [24]. Briefly, 96-well MaxiSorp plates (NUNC, Rochester, NY) were coated with an anti-4xHis mAb (Qiagen) in PBS at 50 lL/ well (5 lg/mL) overnight at 4 °C. After blocking with 0.5 % I-block in PBS, desired amounts (1–1,000 ng) of the deglycosylated and glycosylated forms of Pfs48F1 were added and incubated for 2 h at RT. After washing plates, 50 mL (2 mg/mL in I-block) of the mAb against epitope III of Pfs48/45 were added and incubated for 2 h at RT. Bound antibodies were detected using a HRP-conjugated goat anti-rat polyclonal antibody (Bio-Rad, 1:25,000 in I-block) and visualized at 490 nm using OPD as a substrate.
Results Evaluation of the Ability to Co-express Heterologous Proteins Using the Engineered BYV-Based Vector In this study, we designed a BYV vector for co-expression of two ORFs and engineered a construct encoding Hc and Lc of a mAb against PA of B. anthracis. The sequences of Hc and Lc were cloned into the BYV vector inserted into a binary vector encoding the BYV genome, between the CPm and CP sequences (Fig. 1a). As a result, the sequences of Hc and Lc were cloned under control of the BYV CP and GLRaV2 CP SGPs, respectively, whereas the BYSV CP SGP controlled the BYV CP ORF (Fig. 1a; sequencing data not shown).
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Fig. 2 a A symptomatic leaf of N. benthamiana systemically infected by BYV carrying two chains of an antibody at 34 dpi. b Time course of total IgG expression in systemic leaves analyzed using nonreducing SDS-PAGE and Western blotting. Numbers in the lower
panel indicate protein expression levels in mg/kg of fresh leaf tissue. Ni non-inoculated N. benthamiana leaves, MW molecular weight in kDa. An arrow indicates the position of the assembled anti-PA mAb
To evaluate accumulation of the assembled anti-PA mAb and ability for expression of two chains of an antibody using the recombinant BYV vector, we manually coinfiltrated 5-week-old N. benthamiana leaves with cultures of Agrobacterium carrying the BYV vector encoding Hc and Lc of the anti-PA mAb and Agrobacterium carrying a binary vector encoding a silencing suppressor, P1/HC-Pro, from turnip mosaic virus [27], at the ratio of 1.0:0.2 OD600. At 30 dpi, first systemic symptoms of BYV infection (clearing veins) were detected in all 12 infiltrated plants, and more severe symptoms developed by 36 and 39 dpi (Fig. 2a). Western blot analysis performed at 30, 32, 34, 36, and 39 dpi on samples from systemic leaves of coinfiltrated plants demonstrated the highest level of assembled IgG expression, 49 ± 12 mg/kg of fresh leaf tissue weight, at 36 dpi (Fig. 2b). Some antibody degradation was observed at 34 and 36 dpi. At 30 and 32 dpi, the antibody expression level in systemic leaves was low, and at 39 dpi, when plant tissue was dying from infection, no assembled antibody was detected (Fig. 2b). In addition, an ELISA analysis was performed to verify the expression level of the assembled anti-PA mAb in systemically infected leaves. As demonstrated by ELISA, the expression level of the functional anti-PA mAb in the systemic leaves was 68 ± 29 mg/kg of fresh leaf tissue weight at 36 dpi. The difference in the expression levels of the assembled anti-PA mAb measured by Western blot and ELISA was not significant (data not shown).
closteroviral silencing suppressor p21 [31] and instead used a binary vector pCB-302 carrying P1/HcPro, a silencing suppressor from turnip mosaic virus, by co-infiltration [25, 27]. We cloned the Hc and Lc genes into this single miniBYV replicon for expression of the anti-PA mAb. The resulting vector, miniBYV2 (Fig. 1b), contained heterologous closteroviral promoters, BYV CP and GLRaV2 CP SGPs, engineered for co-expression of two foreign genes, that controlled the Hc and Lc sequences, respectively, as in the BYV vector (Fig. 1a). Evaluation of the expression levels of Hc and Lc of the anti-PA mAb was performed as described in ‘‘Materials and Methods’’ section. The highest expression levels of Hc and Lc of the anti-PA mAb in the infiltrated N. benthamiana were observed at 13 dpi and were 73 ± 22 and 37 ± 16 mg/kg, respectively (Fig. 3a and data not shown). No Hc or Lc of the anti-PA mAb were detected in non-inoculated N. benthamiana plants (Fig. 2b) and mock infiltrated (BYV-GFPc3) plants (data not shown). To increase the amount of initial transcript synthesized from the miniBYV vector, the cauliflower mosaic virus (CaMV) 35S promoter with dual enhancers was inserted upstream of the miniBYV sequence. Furthermore, to avoid potential splicing events, the high-scoring canonical acceptor splicing site within the BYV replicase sequence was mutated (see ‘‘Materials and Methods’’ section for details), which also made the donor site at 3,606 irrelevant. The final size of the T-DNA insert carrying the miniBYV2 vector sequence with Hc and Lc of the anti-PA mAb was 13,746 bp. To examine the expression levels of Hc and Lc from the miniBYV2 vector, we infiltrated 5-week-old N. benthamiana plants. Leaf disks were taken at 7, 9, 11, and 13 dpi, and mAb expression was analyzed by Western blotting (Fig. 3b). The results demonstrated expression of 240 ± 51 mg/kg for Hc at 7 dpi, which is three times greater than the expression achieved with the parental, non-
Engineering and Optimization of miniBYV Vectors and Co-expression of Heterologous Proteins To reduce the time of target expression and increase the yield of target proteins, we engineered a miniBYV replicon by removing all the BYV genes that are not necessary for the virus replication [11]. In addition, we removed a weak
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Fig. 3 a Expression levels of Hc (1) and Lc (2) of an anti-PA mAb using the miniBYV vector at 13 dpi analyzed using SDS-PAGE under reducing conditions. Numbers in the lower panel indicate protein expression levels in mg/kg of fresh leaf tissue. Ni non-inoculated N. benthamiana leaves, MW molecular weight in kDa, PA protective antigen. b Expression levels of Hc (1) and Lc (2) of an anti-PA mAb using the miniBYV2 vector at 7 dpi, analyzed using SDS-PAGE under reducing conditions. Numbers in the lower panel indicate protein expression levels in mg/kg of fresh leaf tissue. MW molecular weight in kDa, PA protective antigen. c (1) Expression levels of Hc
and a Lc of an anti-PA mAb using the miniBYV1 vector at 7 dpi, analyzed using SDS-PAGE under reducing conditions. Detection of Hc and Lc was performed under reducing conditions in the same membrane. Anti-Fc and anti-kappa antibodies were used for probing as described in ‘‘Materials and Methods’’ section. Protein expression levels of Hc and Lc in mg/kg of fresh leaf tissue are shown in the upper and lower panels, respectively. (2) The expression level of the assembled IgG under non-reducing conditions. Protein expression level in mg/kg is shown in the lower panel. MW molecular weight in kDa
modified miniBYV vector (73 ± 22 mg/kg at 7 dpi). The expression level of Lc (118 ± 29 mg/kg) was more than 40 % lower compared with Hc. Thus, the addition of the dual-enhancer CaMV 35S promoter to the miniBYV vector and mutation of the splicing sites resulted in a 3-fold increase in expression for both Hc and Lc of the anti-PA mAb compared with the parental full-length BYV vector. To balance the expression levels of Lc and Hc of the anti-PA mAb, we substituted the GLPRaV2 CP SGP by a
closely related BYSV CP SGP [21] (sequencing data not shown). The resulting vector, miniBYV1, was introduced into N. benthamiana leaves using infiltration with Agrobacterium. As shown by Western blotting at 7 dpi, Hc and Lc were expressed from the miniBYV1 vector within the same plant cells at equal amounts (298 ± 35 and 297 ± 62 mg/kg for Hc and Lc, respectively; Fig. 3c-1). The expression level of the assembled anti-PA mAb at 7 dpi was 302 ± 41 mg/kg (Fig. 3c-2).
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Fig. 4 a Time course of in vivo deglycosylation of Pfs48F1 coexpressed with PNGase F using the miniBYV2 vector. b Expression of PNGase F at 6 and 8 dpi. Glyc Pfs48F1, Ni non-inoculated N. benthamiana leaves, MW molecular weight in kDa
Co-expression of Pfs48F1 with Bacterial PNGase F Using an Optimized miniBYV Viral Vector As reported in a previous study using different viral and nonviral vectors, a biologically active deglycosylated Pfs48F1 protein from P. falciparum, containing seven predicted N-linked glycosylation sites (N23, N104, N163, N177, N227, N272, and N276), was co-expressed in plants in the presence of bacterial endoglycosidase F (PNGase F) from the Gram-negative bacterium F. meningosepticum [24]. To achieve a uniform deglycosylation profile of the Pfs48F1 protein, both sequences should be delivered into the same host cell. Using the miniBYV2 vector, we have co-expressed endoplasmic reticulum (ER)-targeted Pfs48F1 and PNGase F, purified Pfs48F1 using IMAC, and evaluated its biological activity in vitro. As shown in Fig. 4a, the electrophoretic mobility of glycosylated Pfs48F1 (the control Pfs48F1 protein expressed in plants without PNGase F) was different compared with the non-glycosylated form of Pfs48F1 co-expressed with PNGase Fusing the miniBYV2 vector. Time-course experiments determined that the peak of Pfs48F1 expression was at 8 dpi. The expression of PNGase F was also confirmed by Western blotting using a rabbit anti-FLAG mAb (Fig. 4b). The PR-1a signal peptide was present at the N-terminus of Pfs48F1 and PNGase F for their transient expression in N. benthamiana, and the ER retention signal, KDEL, was present at the C-terminus in both the Pfs48F1 and PNGase F sequences to provide the same intracellular compartmentalization for the antigen and enzyme. To test whether miniBYV2 produced functionally active recombinant Pfs48F1, the purified glycosylated and deglycosylated forms of Pfs48F1 were evaluated for recognition by the epitope-specific mAb III, as described previously [24]. As shown in Fig. 5, mAb III recognized the deglycosylated form of Pfs48F1 more than 3.5-fold stronger than the glycosylated form of the same protein. These results are consistent with the data reported in the previous study [24], where glycosylated and deglycosylated Pfs48F1 was produced using co-infiltration of N. benthamiana with the tobacco mosaic virus (TMV)-based
Fig. 5 Comparative ELISA analysis of the glycosylated and deglycosylated forms of Pfs48F1 produced using the miniBYV2 vector. Recognition of the glycosylated and deglycosylated Pfs48F1 by a rat mAb against epitope III of Pfs48/45 was assessed by ELISA as described in ‘‘Materials and Methods’’ section. Values shown represent the optical density (OD) at 490 nm
viral vector pGRD4-PNGase F and the binary vector pBI121-Pfs48F1. Here, we have demonstrated the feasibility of the production of subunit vaccines without N-linked glycosylation in plants. These findings open a new opportunity for co-expression of target and a modifying enzyme using the miniBYV viral vector system.
Discussion In the current study, we have engineered a novel closteroviral single-replicon viral vector based on BYV for coexpression of two recombinant proteins. The vector has successfully been used to express Hc and Lc of the anti-PA
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mAb under the control of homologous and heterologous closteroviral promoters in N. benthamiana, and functional antibody assembly in systemically infected N. benthamiana leaves was confirmed by ELISA analysis using a recombinant PA protein. Furthermore, to decrease the expression time and increase the expression level, we have generated miniBYV vectors by removing all genes that are not necessary for virus replication. The miniBYV expression system allowed for the co-expression of Hc and Lc of the assembled anti-PA mAb within the same plant host cells as well as for regulation of the expression levels of Lc and Hc using different heterologous closteroviral promoters, the BYV CP and BYSV CP promoters, respectively. Using this approach, we produced 302 mg/kg of fresh leaf weight of the assembled anti-PA mAb. Closteroviral vectors are not common tools used in plant-based biotechnology [32, 33]. In contrast, vectors based on TMV have been widely used by different biotechnology organizations (e.g., Kentucky BioProcessing, Owensboro, KY, USA; Icon Genetics [a subsidiary of Nomad Bioscience GmbH], Halle, Germany; and Fraunhofer USA Center for Molecular Biotechnology, Newark, DE, USA) as production platforms for plant-based subunit vaccine candidates (for review, see [5]. Yet, co-expression of subunit vaccines or Hc and Lc of mAbs using one or two competing TMV vectors is challenging because of viral recombination or very low target expression [6, 7]. To overcome the problem, Giritch et al. [34] designed an expression system (magnICON) consisting of two noncompeting vectors based on TMV and potato virus X, carrying Hc and Lc of a mAb. Using the magnICON vectors, Grohs et al. [35] achieved the expression of a mAb, trastuzumab, with the yield of 43 mg/kg of fresh leaf tissue. However, analysis of purified trastuzumab demonstrated the presence of an Hc degradation product indicating the presence of a non-assembled antibody. In a different study, Roy et al. [36] co-expressed Lc and Hc using a two-component TMV-based vector system (dRTV) that comprises an artificial defective RNA and a helper vector. A multi-component system based on the bipartite genome of cowpea mosaic virus (CPMV) was also employed for mAb production. As reported by Sainsbury and Lomonossoff [37], co-infiltration of one RNA1 encoding CPMV replicase and two RNA2 (or delRNA2) encoding Hc and Lc resulted in expression of the assembled C5-1 mAb. Recently, the geminiviral bean yellow dwarf virus-based expression system has also been developed and used for the production of an anti-Ebola mAb [38, 39]. In these studies, the yield of mAbs ranged between 0.2 and 0.5 g/kg of fresh leaf tissue weight, depending on the vector used, antibody sequence and method of protein measurement [34, 37, 38]. Viral and non-viral systems based on co-infiltration of two or more components vary in
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antibody expression and assembly. Giritch et al. [34] reported that the expression level of Lc of a mAb was much higher compared with Hc. For the dRT-V system that utilized two TMV-based vectors [36], it remained unclear if the two vector components move from cell to cell to produce an assembled mAb. Furthermore, the usage of a twocomponent system in general and viral replicons in particular may result in assembly of either non-functional mAbs such as Lc–Lc and/or Hc–Hc dimers or multimers or partially assembled mAbs containing two Hc and one Lc [40, 41]. These non-functional antibodies may interfere with evaluation of mAb expression level, and mAb purification, yield, and functionality. Garabagi et al. [42]. employed binary vectors containing modified promoters and terminators for expression of trastuzumab in the presence of P19, a silencing suppressor from tomato bushy stunt virus. The using of P19 at the concentration of OD600 = 0.2 increased the expression of the mAb 15-fold. The expression level of an antibody can also be increased by positioning gene(s) of interest closer to the 30 end of the viral vector [43] or using different heterologous promoters. However, our attempt to increase Hc expression by inserting Hc sequence closer to the 30 terminus under the control of the BYSV CP promoter in the miniBYV vector was not successful (data not shown). Localization of the CP gene in the proximity of the 30 terminus of the viral genome is common for the family Closteroviridae [9]. The BYV CP promoter is the strongest BYV promoter, and the promoter efficiency does not depend on positioning [44]. This phenomenon allows for using different closteroviral CP promoters independently from positioning of the gene of interest within the miniBYV genome for regulation of expression. According to the phylogenetic tree analysis for closterovirus CPs, the BYSV CP gene is most closely related to the BYV CP [21], suggesting that the expression level of Lc of the anti-PA mAb could be increased using the BYSV CP SGP. The expression levels of assembled plant-produced recombinant antibodies using different viral vectors depends on the viral vector/vectors systems, detection methods, or the sequence of particular immunoglobulins. Several approaches have been used in the literature for evaluation of antibody expression levels, including Coomassie staining of polyacrylamide gel, Western blots, and ELISA [34, 37, 38]. Unfortunately, no single standard method has been developed for evaluation of recombinant protein expression in plant tissues. Therefore, we used two independent approaches based on Western blot and ELISA to determine the expression levels of the assembled recombinant anti-PA mAb and deglycosylated Pfs48F1 produced in plants using the miniBYV vector. To confirm co-expression and functionality of two proteins within the same plant host cell, Pfs48F1 from the
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protozoan malaria parasite P. falciparum, containing seven predicted N-linked glycosylation sites, was chosen as a target sequence, and endoglycosidase F (PNGase F) from the Gram-negative bacterium F. meningosepticum was expressed as a modifying enzyme. The major difference between the same proteins produced in different eukaryotic recombinant expression systems lies in their different posttranslational modifications, glycosylation in particular. Glycosylation significantly affects folding, trafficking, stability, biological activity, and immunogenicity of recombinant pharmaceutical proteins produced in eukaryotic expression systems [45–49]. To avoid potential adverse effects of plant-specific glycans on immunogenicity of plantderived subunit vaccines or therapeutic proteins in humans, strategies are being developed to produce recombinant antigens with engineered N-glycosylation [47]. The native P. falciparum Pfs48/45 protein is not glycosylated. Recently, a strategy of enzymatic deglycosylation of proteins in vivo by co-introducing bacterial PNGase F, during transient expression in plants, has been reported [24, 50]. In this strategy, both ER-targeted bacterial PNGase F and the malaria vaccine candidate Pfs48F1 were transiently coexpressed in N. benthamiana plants via co-agroinfiltration, using pGRD4-PNGase F and pBI121-Pfs48F1 constructs, resulting in successful deglycosylation of Pfs48F1 in vivo. Here, the Pfs48F1 and PNGase F sequences were cloned into the miniBYV2 vector under the control of the BYV CP promoter and the GLRaV2 CP promoter, respectively. We have demonstrate that Pfs48F1 was deglycosylated when Pfs48F1 and PNGase F were co-expressed using the miniBYV2 vector, suggesting that PNGase F was functionally active. In addition, the deglycosylated form of Pfs48F1 produced using miniBYV2 had a similar size (Fig. 3a in [24] and Fig. 4 in this study) on SDS-PAGE to that of deglycosylated Pfs48F1 produced using pGRD4-PNGase F and pBI121-Pfs48F1 constructs, and showed significantly enhanced rates of recognition by a conformation-specific mAb raised against epitope III [29] of the native Pfs48/45 protein [24]. The experiments describing the development of the miniBYV vector were performed at the small plant expression scale. Using the large-scale approach would open a great opportunity to the future application of the miniBYV vector in molecular farming and the production of plant-based vaccines and therapeutics. For example, the new miniBYV vector could be used for co-expression of several virus-like particles [51], bacterial non-glycosylated proteins, molecular complexes, or targets and molecular chaperons. Furthermore, the miniBYV vector could be employed for antibody and therapeutic protein production in transgenic plants or plant cell cultures using an Agrobacterium-free inducible approach [52]. The ability to manipulate expression levels of recombinant proteins using different promoters
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in the miniBYV vector system opens new horizons for the production of plant-derived pharmaceuticals. Acknowledgments The authors would like to thank Dr. Valerian Dolja of Oregon State University for the p35S-BYV-GFP plasmid and M. Levikova for assistance with ELISA. The authors are grateful to Dr. Stephen J. Streatfield for critical reading of the manuscript and Dr. Natasha Kushnir for editorial assistance. Conflict of interest
The authors declare no conflict of interest.
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