Production of porcine parvovirus virus-like particles using silkworm larvae

Journal Pre-proofs Full length article Production of porcine parvovirus virus-like particles using silkworm larvae Seung Hee Lee, Sung Min Bae, Won Seok Gwak, Soo Dong Woo PII: DOI: Reference:

S1226-8615(19)30492-3 https://doi.org/10.1016/j.aspen.2019.10.002 ASPEN 1447

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Please cite this article as: S.H. Lee, S.M. Bae, W.S. Gwak, S.D. Woo, Production of porcine parvovirus virus-like particles using silkworm larvae, Journal of Asia-Pacific Entomology (2019), doi: https://doi.org/10.1016/j.aspen. 2019.10.002

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Production of porcine parvovirus virus-like particles using silkworm larvae

Seung Hee Leea, Sung Min Baeb, Won Seok Gwakc and Soo Dong Wooc, *

a Institute

bKBNP

of Biotechnology, CJ Cheiljedang, Suwon 16495, Republic of Korea

Technology Institute, KBNP Inc., Anyang 14059, Republic of Korea

cDepartment

of Agricultural Biology, College of Agriculture Life & Environment Sciences, Chungbuk National

University, Cheongju 28644, Republic of Korea

Running title: Production of PPV-VLP in silkworm

*Corresponding author: Soo Dong Woo, Department of Agricultural Biology, College of Agriculture Life & Environment Sciences, Chungbuk National University, Cheongju 28644, Republic of Korea E-mail: [email protected]; Tel: +82-43-261-2553; Fax: +82-43-271-4414

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ABSTRACT Porcine parvovirus (PPV) is a significant causative agent of porcine reproductive failure, causing serious economic losses in the swine industry. PPV is a nonenveloped virus, and its capsid is assembled from three viral proteins (VP1, VP2, and VP3). The major capsid protein, VP2, is the main target for PPV neutralizing antibodies and vaccine development. In this study, PPV-VP2 protein was expressed in silkworm larvae, and its antigenicity and production were compared with those in B. mori cells (Bm5). The recombinant VP2 protein was expressed successfully in silkworm larvae and Bm5 cells with a size of approximately 64 kDa. The formation of virus-like particles (VLPs) by recombinant PPV-VP2 was confirmed through transmission electron microscopy. The recombinant PPV-VP2 protein assembled into spherical particles with diameters ranging from 20 to 22 nm. The antigenicity of PPV-VLPs was comparatively analyzed between Bm5 cells and silkworm larvae by ELISA, hemagglutination and hemagglutination inhibition assays. Consequently, it was confirmed that the PPV-VLPs produced in the silkworm larvae were more antigenic than VLPs produced in Bm5 cells. Therefore, it is expected that economical and effective vaccine development will be possible by mass production of PPV-VLPs in silkworm larvae.

Keywords: Porcine parvovirus, Virus-like particle, Silkworm larva, Recombinant PPV-VP2

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Introduction The baculovirus expression vector system (BEVS) is an effective and widely used method for the production of recombinant proteins in insect cells and larvae (Bae et al., 2017; Kidd and Emery, 1993; Kim et al., 2012; Morokuma et al., 2015). The most useful feature of BEVS is its ability to produce a specific recombinant protein with similar cotranslational and posttranslational modifications, including glycosylation, protein processing and phosphorylation, as those produced in as mammalian cells (Bae et al., 2013; Kato et al., 2010; Kato et al., 2012; Kidd and Emery, 1993). These advantages over prokaryotic expression systems make it possible to express multiple proteins simultaneously in a single infection and to obtain multimeric proteins sharing functional similarity with their natural analogs. Therefore, the expression of various animal and human pharmaceutically relevant proteins is currently being explored using BEVS for practical use (Cox, 2012; Cox and Hollister, 2009; Harper et al., 2006). Most research and commercialization of human and animal drugs involves vaccine development against various virus diseases (Blanchard et al., 2003; Cox, 2012; Fuenmayor et al., 2017; Harper, 2009). In particular, among various expression systems, BEVS has been reported to be most useful for the development of virus-like particle (VLP) vaccines (Fernandes et al., 2013; Liu et al., 2013). VLPs consist of viral proteins that self-assemble in much the same way as natural virus particles. VLPs are considered a highly safe vaccine agent because they do not replicate and are not infectious because they do not contain infectious genetic material (Ding et al., 2018; Fuenmayor et al., 2017; Lua et al., 2014; Ludwig and Wanger, 2007). Due to their morphological similarities, VLPS produce an immune response similar to that of natural viruses. Therefore, research and commercialization of VLP vaccines have recently been actively carried out to replace many existing traditional viral vaccines. In BEVS, cultured cells and insect larvae and pupae can be used for the production of recombinant proteins. Since the production of human interferon was reported in silkworm (Bombyx mori) larvae using recombinant B. mori nucleopolyhedrovirus (BmNPV) (Maeda et al., 1985), many recombinant proteins have been produced in silkworms (Kato et al., 2010; Usami et al., 2010). In particular, many eukaryotic proteins have been produced in silkworm larvae. The silkworm has been used as a biofactory for large-scale protein production due to its high protein production capacity, scalability and low-cost (Kato et al., 2010; Usami et al., 2010). This system has become attractive for use in the biomedical sciences. In 1994, the world's first feline interferon for feline calicivirus infection was developed as an antiviral drug for animals. Since then, many proteins involved in 3

animal diseases have been expressed using silkworm for the development of vaccines (Choi et al., 2000; Feng et al., 2011; Kato et al., 2010; Kato et al., 2012). In particular, recently, many researchers reported using recombinant BmNPV in silkworm larvae for the production of VLPs, as well as for the development of VLPbased vaccines in the vaccine industry (Kato et al., 2012; Kemp et al., 2012; Wang and Roden, 2013). Porcine parvovirus (PPV) is widespread in swine herds and causes reproductive failure in pregnant sows (van Leengoed et al., 1983; Streck et al., 2015). The infection occurs without clinical symptoms in adults; however, the virus can cross the placental barrier during the infection and cause fetal death, stillbirths and a return to estrus (Mészáros, et al., 2017). PPV is a member of the family Parvoviridae and is a small icosahedral nonenveloped virus of approximately 25 nm in diameter (Ji et al., 2017; Mayer et al., 1968). The genome of PPV is a negative-sense single-stranded DNA encoding two nonstructural proteins and three capsid proteins. Capsid proteins consist of three viral proteins, VP1, VP2, and VP3. The major structural protein, VP2, is the main target of neutralizing antibodies and can self-assemble into VLPs (Streck et al., 2015). Therefore, the VP2 protein is generally considered to be the major antigen for PPV vaccine development. PPV is also considered to be a cofactor of PCV2, and concurrent infections of porcine circovirus type 2 (PCV2) and PPV have been reported to increase disease and lesion severity compared to PCV2 single infections (Mészáros, et al., 2017). PCV2 is a major cause of postweaning multisystem wasting syndrome (PMWS), a disease with severe immunosuppressive effects in swine and is a globally economically important swine disease. Therefore, to effectively control PCV2 as well as PPV, it is necessary to develop an effective PPV vaccine. Previous studies have shown that parvovirus-like particles produced in insect cells show high antigenicity and good results in animal experiments (Antonis et al., 2006; Ji et al., 2017; Rueda et al., 2000; Sedlik et al., 1997; Zhang et al., 2014). However, the results have yet to be put to practical use. Therefore, in this study, PPV-VP2 was produced in silkworm larvae, which were superior to insect cells in terms of their productivity, activity and antigenicity. The results of this study are expected to accelerate the commercialization of vaccines using PPV-VLPs.

Materials and methods

Cells, viruses and insects 4

The B. mori cell line (Bm5) was maintained at 27 °C in TC-100 insect medium (WelGENE, Korea) supplemented with 10% fetal bovine serum. PPV-VRI-1 (GenBank accession number AY390557) was provided by Optipharm Inc. and BmNPV-K1 were used in this study. Routine cell culture maintenance and virus production procedures were performed according to published procedures (King and Possee, 1992). The silkworm, B. mori, was reared on an artificial diet as previously described (Gui et al., 2006).

Generation of recombinant virus The VP2 gene was amplified from the PPV genome with the primer set PPV-VP2-F (5’CCGCGGCCACCATGAGTGAAAATGTGGAAC-3’)/PPV-VP2-R

(5’-

TCTAGACTAGTATAATTTTCTTGGTATAAG-3’) and cloned into pMD20-T (Takara, Japan). The underlined nucleotides represent Sac II and Xba I restriction sites. The cloned PCR products were digested with Sac II and Xba I restriction endonucleases and subsequently cloned into the corresponding restriction sites of the pBmKSK3 vector (Choi et al., 2000) to generate pBmKSK3-VP2. Bm5 cells were cotransfected with a mixture of purified pBmKSK3-VP2, bBpGOZA DNA (Je et al., 2001), and Cellfectin II (Invitrogen, USA), according to the manufacturer’s instructions, to obtain the recombinant virus rBp-VP2. The recombinant virus was purified and propagated in Bm5 cells, as described previously (O’Reilly et al., 1992).

Preparation of recombinant protein sample The infection for the analysis of recombinant PPV-VP2 was carried out in 2 × 106 cells per 25 cm2 flask infected with virus at a multiplicity of infection (MOI) of 1. Virus-infected cells were collected at 3 days postinfection and washed with ice-cold phosphate-buffered saline (PBS). The cell lysate was prepared by incubating cells in 1 mL of lysis buffer (20 mM Tris-HCl, 500 mM NaCl, 1 mM EDTA, 0.1% Tween 20, pH 7.0, with a protease inhibitor cocktail (Sigma-Aldrich, USA)) for 30 min on ice followed by sonication. To prepare samples from silkworm larvae, 5th instar larvae were injected with virus at 1 × 105 PFU (20 μL/larva), and hemolymph and fat bodies were collected 3 days after injection. The hemolymph was collected in chilled tubes containing 1 mM DTT to prevent melanization. The collected hemolymph was centrifuged at 10,000 × g for 10 min to remove hemocytes and cell debris, and the supernatant was stored at -70 °C until further use. The collected fat body by dissection was homogenized in 10 volumes of lysis buffer for 30 min on ice followed by 5

sonication. The homogenate was centrifuged at 10,000 × g for 10 min. The supernatant was collected and stored at -70 °C until further use.

SDS-PAGE and Western blot analysis The prepared protein samples from cells and larvae were mixed with sample buffer, boiled for 5 min, and subjected to 12% SDS-PAGE. For Coomassie staining, gels were washed with deionized water and stained with Bio-Safe Coomassie Stain (Bio-Rad, CA, USA). For Western blot analysis, the protein samples were subjected to 12% SDS-PAGE and transferred to a nitrocellulose (NC) membrane (Pall Corp., NY, USA). The membranes were blocked in 5% milk in Tris-buffered saline containing 0.05% Tween 20 and were probed with a PPV-VP2 polyclonal antibody (Biorbyt, Cambridge, UK). The membranes were then incubated with a horseradish peroxidase-coupled anti-rabbit IgG antibody (Cell Signaling, Danvers, MA, USA), and the bound antibodies were detected using an enhanced chemiluminescence system (Merck Millipore, Burlington, MA, USA) according to the manufacturer’s instructions.

VLP purification VLP samples were prepared from recombinant virus-infected Bm5 cells and silkworm larvae. Each supernatant of Bm5 cells, hemolymph, and fat body was prepared by the above same method as the preparation of recombinant protein samples. In addition, hemolymph supernatant was diluted 10-fold with PBS and used for purification of VLPs. Each supernatant was adjusted to 20 % saturated ammonium sulfate at 4 °C for 1 hour. The precipitate was recovered by centrifugation at 12,000 ×g for 20 min and resuspended in PBS.

Energy filtering transmission electron microscopy Negative staining was performed to observe the PPV-VLPs. A plastic carbon-coated 400 mesh grid was hydrophilized using a plasma cleaner. The prepared sample was placed on the grid and allowed to stand for 5 6

min. To remove the PBS, a grid was placed on sterilized water using a 0.45 μm syringe filter and washed. The sterile water was removed, 2% uranyl acetate (pH 7.2) was added to the grid for negative staining and the remaining staining solution was removed using filter paper. After drying at room temperature for 10 min, the PPV-VLPs were observed on an energy filtering transmission electron microscope (Carl Zeiss Libera 120, Zeiss, Oberkochen, Germany).

Serum preparation For generation of rabbit PPV antisera, New Zealand White rabbits were immunized intramuscularly with PPV mixed with an equal volume of Freund's complete adjuvant (Sigma-Aldrich Chemical Co., St. Louis, MO, USA). Two subsequent injections were administered with antigens mixed with equal volumes of Freund's incomplete adjuvant at 2-week intervals beginning one week after the first injection. Blood was collected 3 days after the last injection with antigens only and centrifuged at 10,000 ×g for 10 min after clotting at 4 °C overnight. The supernatant antibodies were stored at -70 °C. To generate guinea pig PPV-VLP antisera, hemolymph collected silkworm larvae infected with rBp-VP2 was used to immunize guinea pig. The other processes for the immunizations were the same as the above methods.

Enzyme-linked immunosorbent assay (ELISA) The VLP samples used for ELISAs were prepared from 0.5 mL of Bm5 cells (1 x 106 cells/5 mL), 1 mg of fat body, and 1 μL of hemolymph. The PPV serum was diluted 1:1,000 with carbonate buffer in an ELISA plate and treated at 4 °C for 12 hrs to coat each well. The coating solution was then removed, and the samples were washed three times with PBS. Each VLP sample was diluted 1:10 to 1:100,000 and treated at 37 °C for 60 min. The plates were then washed three times and treated with a VP2 monoclonal antibody (VMRD Inc., USA) for 60 min at 37 °C. Then, a horseradish peroxidase-coupled anti-mouse IgG antibody (Cell Signaling, USA) was added and incubated for 60 min at 37 °C. After washing, 3,3’,5.5’-tetramethylbenzidine (TMB) substrate (1.2 mM, Sigma-Aldrich, USA) was added for 15 min. Then, 0.5 M sulfuric acid was added to stop the reaction, and the absorbance was measured on a microplate reader (Molecular Devices, Wokingham, UK) at a wavelength of 7

450 nm.

Hemagglutination and hemagglutination inhibition assays In the hemagglutination (HA) assay, the protein samples were serially diluted 2-fold, mixed with 1% guinea pig red blood cells and treated in a 96-well plate. After 30 min at room temperature, each well was checked to determine the HA titer. In the hemagglutination inhibition (HAI) assay, PPV-VLPs with 26 hemagglutination units (HAU) and PPV-VLP serum produced in guinea pigs were diluted 2-fold, and after 15 min, 1% guinea pig red blood cells were added. After 30 min at room temperature, the HAI titers were determined.

Ethics statement Animal experimental procedures were approved by the Committee on the Use and Care of Animals (Permit Number: CBNUA-878-15-01, Laboratory Animal Research Center, Chungbuk National University, Cheongju, Korea) and performed in accordance with the institutional guidelines.

Results and discussion

The production of recombinant PPV-VP2 protein Recombinant rBp-VP2, in which the PPV-VP2 gene was under the control of the polyhedrin promoter, was generated, and the expression of the VP2 gene was confirmed in Bm5 cells and silkworm larvae. SDS-PAGE analysis showed that a novel band of approximately 60 kDa, the size of the recombinant PPV-VP2 protein, was present in Bm5 cells and the larval fat body (Fig. 1A). However, the presence of PPV-VP2-specific bands was not observed in silkworm larval hemolymph due to the large amount of storage protein bands. However, Western blot analysis using a PPV-VP2 antibody showed a specific band of approximately 60 kDa in all samples (Fig. 1B). These results indicate that the recombinant PPV-VP2 protein was successfully produced by the recombinant virus rBp-VP2. 8

Porcine parvovirus, recently renamed Ungulate protoparvovirus 1, is considered to be one of the most important causes of reproductive failure in swine (Mészáros, et al., 2017). However, it has been little interest in developing new PPV vaccines because traditional inactivated vaccines have been used successfully to prevent PPV infection (Mészáros et al., 2017). The recent reports of several new PPV strains that are different from the PPV strain that has long been used in vaccines have increased the need for new vaccines (de Souza et al., 2019; Jó´zwik et al., 2009; Mészáros et al., 2017; Streck et al., 2015; Wang et al., 2019). The new vaccine approach is focused on the development of recombinant vaccines with increased immunogenicity that can be produced quickly in response to virus strain variation. The expression of VP2, which is the main structural protein of PPV, has been extensively studied because it can self-assemble into VLPs (Martínez et al., 1992). According to the recent reports, VP2-based PPV-VLPs can induce similar humoral and cellular immune responses to those of inactivated vaccines and have been shown to excel in the production of a cellular immune response (Antonis et al., 2006; Tian et al., 2019; Zhao et al., 2014). Therefore, it is increasingly possible to develop new PPV-VLP vaccines for various PPV strains with increased speed and efficiency using only VP2.

Comparison of VLP antigenicity Formation of PPV-VLPs by the recombinant PPV-VP2 was investigated in Bm5 cells and silkworm larvae. Electron microscopy showed that PPV-VLPs of icosahedral form with approximately 20-22 nm in diameter were formed in all samples (Fig. 2). This shape is similar to that of wild-type PPV, suggesting that humoral and cellular immunity could be effectively induced by PPV-VLPs. After the formation of PPV-VLPs was confirmed, the antigenicity was tested by sandwich ELISA using PPV serum and a VP2 antibody. The total sample from silkworm larvae was either 1 μL of hemolymph or 1 μg of fat body. The absorbance of all samples infected with the rBp-VP2 virus was much higher than that of the negative control (Table 1). In particular, samples prepared from Bm5 cells showed significant absorbance up to a ratio of 1:1000, while samples prepared from silkworm larvae showed significant absorbance up to a ratio of 1:100000. Although the amount of viral particles could not be measured directly and the amounts of samples used in this experiment were not absolutely comparable to each other, it is clear that the production of VLPs in silkworm larvae was higher than in Bm5 cells. The production of PPV-VLPs has been reported in various expression systems, but the production efficiency and antigenicity of silkworm larvae and cell lines have not 9

been comparatively evaluated thus far. To the best of our knowledge, this is the first result that the production of PPV-VLPs is superior to silkworm larvae than cell lines.

Immunization of guinea pigs The hemagglutination activity of the rBp-VP2-infected silkworm larvae was assessed by an HA assay using guinea pigs. In the HA assay, PPV-VLPs caused hemagglutination up to 218-220 HAU (Fig. 3A). In addition, an HAI assay was conducted using guinea pigs to investigate the immunogenicity and activity of the VLPs. The HAI result confirmed that the hemagglutination reaction was inhibited up to 1280-5120 HAU by PPV-VLP serum produced in guinea pigs (Fig. 3B). These results indicate that the serum from guinea pigs produced using PPV-VLPs from silkworm larvae could react effectively with PPV-VLPs. In addition, these results suggest that PPV-VLPs produced in silkworms may be more potent than those produced in cell lines, both quantitatively and antigenically, for vaccine development. Usami et al. (2011) reported the expression of 48 recombinant proteins using silkworms and insect cells. They clearly demonstrated that recombinant proteins were expressed to a greater extent in silkworm than in insect cells. In addition, in many studies, silkworm larvae have been reported to be highly efficient bioreactors for the production of recombinant proteins (Kato et al., 2010). Until recently, however, a comparative evaluation of insect cell lines and larvae for the production of VLPs using BEVS has not been performed. A similar study comparing Rous sarcoma virus (RSV)-gag VLP production in transgenic insect cell lines and silkworm larvae showed that silkworm larvae produce greater amounts of VLPs than insect cells (Doe et al., 2011). Our results also demonstrated that insect larvae are more effective than cell lines at producing VLPs.

Conclusions In this study, PPV-VLP production in silkworm larvae and cell lines was compared for the first time. We confirmed that PPV-VP2 expressed in silkworm larvae forms VLPs that are similar to the wild-type PPV. In addition, recombinant PPV-VLP production in silkworm larvae was superior to that in cell lines, and these VLPs showed high levels of antigenicity. Future studies, such as direct animal experiments on swine and mass production condition development in silkworm larvae, are needed. Our results confirm that PPV-VLP production in silkworm larvae is more industrially useful than that in cell lines. We expect that this method will 10

be sufficient for the development of new vaccines that can be rapidly developed and applied to protect against various PPV strains.

Conflict of interest No conflicts of interest are declared.

Acknowledgements This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2017R1D1A1B04036274) and conducted during the research year of Chungbuk National University in 2019.

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Figure legends

Fig. 1. Analysis of the recombinant PPV-VP2 in Bm5 cells and B. mori larvae. Bm5 cells and 5th instar larvae of B. mori were infected with virus at an MOI of 1 and 1 × 105 PFU, respectively. Virus-infected cells and larvae were collected at 3 days postinfection. Hemolymph and fat body samples were prepared from B. mori larvae. Protein samples were analyzed by 12% SDS-PAGE (A) and Western blot analysis with a PPV-VP2 polyclonal antibody (B). Arrows indicate the recombinant PPV-VP2.

Fig. 2. Electron microscopy analysis of PPV-VLPs negatively stained with 2% uranyl acetate. VLP samples were prepared from rBp-VP2-infected Bm5 cells (A), fat body (B), and hemolymph (C) of B. mori larvae.

Fig. 3. Hemagglutination analysis performed on guinea pig red blood cells. HA (A) and HAI (B) assays were performed using purified PPV-VLPs from B. mori larvae infected with rBp-VP2. In the HA assay, VLPs were diluted, guinea pig red blood cells were added, and the plate was imaged at 30 min. For HAI assay, VLPs and PPV-VLP serum produced in guinea pig was diluted, and after 15 min, guinea pig red blood cells were added. This assay was performed in duplicate. After 30 min, the plate was imaged. Arrows indicate HA or HAI activity.

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Table 1. Antigenicity comparison of PPV-VLPs produced in Bm5 cells and B. mori larvae. The VLP samples were prepared from Bm5 cells and B. mori larvae infected with recombinant rBp-PPV-VP2. Bm5 cell, fat body, and hemolymph of B. mori larvae infected or noninfected with BmNPV were used as negative controls.

Bm5 cells (0.5 mL / 5 mL)

B. mori – fat body (1 mg)

B. mori – hemolymph (1 μL)

Dilution Cells

BmNPV

rBp- VP2

Fat body

BmNPV

rBp-VP2

Hemolymph

BmNPV

rBp-VP2

1:10

0.1935

0.1215

0.8535

-

-

-

-

-

-

1:100

0.1460

0.1375

0.6030

0.3478

0.3325

0.8244

0.4598

0.3399

1.9746

1:1000

0.1465

0.1510

0.3795

0.3283

0.3241

1.2564

0.4123

0.3146

1.5675

1:10000

0.2185

0.1135

0.1930

0.3169

0.3030

1.1091

0.3803

0.3057

1.0584

1:100000

0.1490

0.1225

0.2240

0.3676

0.3289

0.6456

0.3564

0.3202

0.5769

1:1000000

-

-

-

0.3318

0.3169

0.3751

0.3755

0.3154

0.3458

19

20

Highlights 

Porcine parvovirus-like particles (PPV-VLPs) were successfully formed in silkworm larvae



PPV-VLP production in silkworm larvae was superior to cell line



PPV-VLP produced in silkworm larvae had high antigenicity

Fig. 1

21

22

23

Fig. 2

24

Fig. 3

25

26

Conflict of Interest Statement August 02, 2019

Title of Manuscript: Production of porcine parvovirus virus-like particles using silkworm larvae

Authors: Seung Hee Lee, Sung Min Bae, Won Seok Gwak and Soo Dong Woo

All the authors agreed the submission of this paper to Journal of Asia-Pacific Entomology and are responsible for its contents. Also, all the authors agreed that the corresponding author may act on their behalf regarding any subsequent processing of this paper.

Seung-Hee Lee Sung Min Bae Won-Suk Gwak 27

Soo Dong Woo

28