Optimized conditions for preserving stability and integrity of porcine circovirus type2 virus-like particles during long-term storage

Journal of Virological Methods 243 (2017) 146–150

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Optimized conditions for preserving stability and integrity of porcine circovirus type2 virus-like particles during long-term storage Naidong Wang a , Yan Zhang a , Xinnuo Lei a , Wanting Yu a , Yang Zhan a , Dongliang Wang a , Jiaxin Zhang a , Aibing Wang a , Lehui Xiao b , Ping Jiang c,∗ , Yi Yang a,∗ a Laboratory of Functional Proteomics (LFP) and Research Center of Reverse Vaccinology (RCRV), College of Veterinary Medicine, Hunan Agricultural University, Changsha 410128, China b College of Chemistry, Nankai University, Tianjin 300071, China c Key Laboratory of Animal Diseases Diagnostic and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China

a b s t r a c t Article history: Received 22 December 2016 Received in revised form 20 January 2017 Accepted 22 January 2017 Available online 25 January 2017 Keywords: Porcine circovirus type 2 (PCV2) Virus-like particles (VLPs) Osmolytes Stability

Although porcine circovirus type 2 (PCV2) virus-like particles (VLPs) have been successfully harvested from various protein expression systems, conditions to promote their stability and integrity during longterm storage have not been well defined since only the intact VLPs, instead of the monomeric capsid protein (Cap), can induce neutralizing antibodies in pigs in previous studies. In this study, freshly prepared PCV2 VLPs were stored in several media (various concentrations of NaCl, sorbitol, sucrose and trehalose) at three temperatures (4 ◦ C, −20 ◦ C and −80 ◦ C) and their stability and integration were evaluated after 7 month. Addition of 15% trehalose in storage buffer promoted long-term preservation of PCV2 VLPs. In contrast, storage buffer with 5% osmolytes (sucrose, trehalose and sorbitol) did not confer stabilization for long-term storage. These refined storage conditions for stabilization of PCV2 VLPs should enhance their use in vaccines. © 2017 Published by Elsevier B.V.

Porcine circovirus type 2 (PCV2), a widespread pathogen, causes various diseases and syndromes collectively termed PCV2-systemic disease (PCV2-SD)(Segales, 2012). Furthermore, co-infections of PCV2 and other pathogens also commonly cause subclinical infections in swine (Segales et al., 2013). Clinical signs of PCV2-SD are nonspecific and variable, and mainly include wasting, respiratory disease, jaundice and enteritis (Darwich and Mateu, 2012). Pigs infected with PCV2 have immunosuppression (Marco-Ramell et al., 2014), which causes financial losses, due to reduced production and mortality, as well as implementation of control and treatment strategies. The swine industry has benefited greatly from vaccination against PCV2. Several commercial subunit vaccines based on PCV2 capsid protein (Cap) have enhanced swine production, including increased average daily weight gain and reduced mortality, and reductions in PCV2 viremia, viral shedding and PCV2-associated lesions (Fraile et al., 2012; Opriessnig et al., 2014). Assembled empty virus-like particles (VLPs) are non-infectious particles iden-

∗ Corresponding authors. E-mail addresses: [email protected] (P. Jiang), [email protected] (Y. Yang). http://dx.doi.org/10.1016/j.jviromet.2017.01.021 0166-0934/© 2017 Published by Elsevier B.V.

tical to native virions, albeit without viral core genetic material. They are well suited for vaccine production and for investigation of viral infection mechanisms. The PCV2 VLPs are empty particles assembled from 60 monomers of the Cap, but lacking viral genomic DNA. Furthermore, ORF2 encodes viral Cap (28 kDa), the principal structural and immunogenic protein (Nawagitgul et al., 2000). Several commercial vaccines have used PCV2 VLPs (derived from sf9-baculovirus, E. coli or yeast) as their active component (Bucarey et al., 2009; Wu et al., 2012). Our group has frequently produced E. coli-derived PCV2 VLPs (and chimeric VLPs) for immunogenicity studies and an indirect IgG enzyme linked immunosorbent assay (Hu et al., 2016; Zhang et al., 2016). PCV2 Cap has two structural forms, one of which is highly organized VLPs assembled from 60 Cap subunits, and the other is a monomer. The effects of both forms on immunogenicity have been evaluated in previous studies (Trible et al., 2011, 2012a,b). Of note, the intact VLPs are capable of eliciting neutralizing antibodies (NAbs) against PCV2 infections in both experimental and field conditions. However, the Cap monomer fails to induce NAbs in swine and could not provide effective protection from PCV2 infections since a decoy epitope, hidden inside the PCV2 VLPs after assembly of the Cap, is exposed on the surface when the

N. Wang et al. / Journal of Virological Methods 243 (2017) 146–150

Cap is monomeric (Trible et al., 2012a,b). During long-term storage PCV2 VLPs may become unstable and dissembled. In many cases it was necessary to store VLPs for prolonged intervals. Therefore, it is important to optimize conditions to retain stability and integrity of VLPs during long-time storage. To this end, effects of buffers, NaCl concentrations (used to adjust ionic strength) and osmolytes (trehalose, sorbitol and sucrose) on stability (native-like non-aggregated state and morphology) of PCV2 VLPs at three temperatures over a 7-month period were evaluated with transmission electron microscopy (TEM). In the present study, the cap gene (GenBank: KF700357) of PCV2 was subcloned into bacteria expression vector (pET100/D-TOPO plasmid vector) and expressed in E. coli BL21 (DE3). The PCV2 VLPs were prepared and purified as described (Zhang et al., 2016). Then, purified VLPs were confirmed by TEM (CM100, Philips Electron Optics, Zurich, Switzerland) and dynamic light scatting (DLS, Malvern Zetasizer Nano ZS). Concentrations of VLPs were determined using the bicinchoninic acid (BCA) assay and diluted to 0.2 mg/ml with buffer A (0.1 M NaH2 PO4 2H2 O, 0.1 M Na2 HPO4 , 20 mM imidazole, 10 mM Tris base, 300 mM NaCl, 50 mM KCl, 2 mM MgCl2 , 0.1 M ammonium citrate, and 5% glycerol, pH 8.0) for assays described below. The PCV2 VLPs were dialyzed against PBS buffer (1.4 mM KH2 PO4 , 150 mM NaCl, 0.01 M Na2 HPO4 ·12H2 O, 2.7 mM KCl, pH 6.0) or buffer A to evaluate effects of buffers on VLPs stability. To evaluate effects of ionic strength on the stability of VLPs, PCV2 VLPs were dialyzed against buffer A containing 0, 0.1, or 0.5 M NaCl. To determine effects of various concentrations of osmolytes (sucrose, sorbitol or trehalose, Sigma-Aldrich, St. Louis, MO, USA) on the stability of VLPs, PCV2 VLPs were dialyzed against buffer A with 5 or 15% osmolyte concentrations, covering the range over which stability of other VLPs have been assessed (Kissmann et al., 2008; Peixoto et al., 2007). Finally, effects of temperatures (4, −20 and −80 ◦ C) for 1, 4 or 7-mo were determined after PCV2 VLPs were dialyzed against buffer A containing both of the most suitable NaCl concentrations and a range of osmolyte concentrations. The final protein concentration of PCV2 VLPs in these experiments was 100 ␮g/ml. Formation of PCV2 VLPs was verified by TEM (CM100, Philips Electron Optics, Zurich, Switzerland). The VLPs were fixed onto carbon-coated copper grids, stained with 2% uranyl acetate and visualized with TEM to confirm presence and stability of spherical particles of PCV2 VLPs. The PCV2 VLPs were graded on the basis of the average number of VLPs per three representative TEM images, as reported (Lynch et al., 2012). Those with diameters of approximately 17 nm and morphology similar to freshly purified PCV2 VLPs √ (Fig. 2A, D) were graded as stable ( ), whereas those with degradation or alteration (up to 50% and more, Fig. 2B, E; 100%, Fig. 2C, F) were graded as semi-unstable (±) and unstable (X), respectively. Additionally, VLPs not determined were designated nd. Pre- and post-dialysis samples of purified PCV2 Cap were visualized with TEM to confirm the presence and morphology of E. coli-derived PCV2 VLPs (Fig. 1A, B). After dialysis, substantial PCV2 VLPs were observed under TEM. As a control, only a few particles were present in the pre-dialysis sample. Furthermore, diameters of both pre- and post-dialysis samples were 1.8–2.0 and 17–20 nm, respectively (Fig. 1E, F), based on DLS. Therefore, we inferred that the purified PCV2 Cap were efficiently assembled into spherical VLPs with a diameter of approximately 17 nm in vitro after dialysis. To further assess morphology of the PCV2 VLPs, purified VLPs were diluted with buffer A (pH 8.0) to various concentrations (100, 50 and 20 ␮g/ml), and numbers of the PCV2 VLPs present in the frames of TEM were measured. The presence of VLPs with uniform spherical shapes (17–20 nm in diameter) was confirmed under various concentrations; average numbers (232 ± 18, 118 ± 10, 56 ± 6) of VLPs collected from three representative viewing frames had a linear association with various concentrations of VLPs (Fig. 2B–D).

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To assess effects of various buffers on the PCV2 VLPs stability, PBS and buffer A were selected for VLPs storage at 4 ◦ C for up to 7 mo. Based on TEM, VLPs morphology was similar to freshly purified PCV2 after 4 mo of storage in buffer A. However, in PBS, morphology was altered after 1 mo, and particles had disappeared after 4 mo (Fig. 2C). Therefore, buffer A was more suitable for long-term storage of PCV2 VLPs. It is well known that NaCl minimizes VLPs aggregation and promotes VLPs stability (Shi et al., 2005). To determine effects of NaCl on PCV2 VLP stability, various concentrations of NaCl were added to the PCV2 VLPs aliquots in buffer A and then their morphologies were examined by TEM after storage at 4 ◦ C for 1, 4 or 7 mo. Although there was no difference in the VLPs morphologies at 4 ◦ C for 4 mo, irrespective of NaCl concentration (Table 1), the number of VLPs particles in 0.5 M NaCl exceeded those in 0 or 0.1 M NaCl after 7 mo (Table 1, Fig. 2A); therefore, higher salt concentrations (i.e. 0.5 M NaCl) more effectively stabilized PCV2 VLPs for 7 mo at 4 ◦ C. Previous studies suggested that salt can affect structural stability of proteins in a variety of ways e.g. ion-specific interactions with a particular protein (Beauchamp and Khajehpour, 2012). Furthermore, high NaCl concentrations decreased detectable aggregations of the human papillomavirus (HPV) VLPs and increased VLPs stability during preparation and storage (Shi et al., 2005). Therefore, we inferred that optimization of salt concentrations may be necessary for effective stabilization of HPV VLPs in solution. We concluded that 0.5 M NaCl provided stabilization for long-term storage of the PCV2 VLPs (4–7 mo) at 4 ◦ C. That various concentrations of NaCl did not affect stability of EV71 VLPs during long term storage (Lin et al., 2014) might be related to surface charge of VLPs. The PCV2 VLPs were dialyzed against buffer A supplemented with 0.5 M NaCl and stored at various temperatures (4, −20 and −80 ◦ C). At all three temperatures, PCV2 VLPs retained normal morphology and size (17 ∼ 20 nm) at all-time points. However, particle number was dramatically decreased after 4 or 7 mo storage at low temperatures (−20 and −80 ◦ C; Table 1 and Fig. 2B), perhaps due to disassembly of some VLPs during storage or during freezing and thawing. The PCV2 VLPs stored in 15% sucrose, sorbitol or trehalose had no difference in morphology (relative to their original appearance) after storage for 1 mo at 4, −20 or −80 ◦ C (Table 1). Furthermore, samples stored in 15% sorbitol or trehalose had no change in morphology after storage for 4 mo at 4, −20 or −80 ◦ C (Table 1). Similarly, samples stored in 15% sucrose, sorbitol or trehalose had no change in morphology after 1, 4, or 7 mo at 4 ◦ C (Table 1). However, VLPs stored in 15% sucrose (Fig. 2E) or 15% sorbitol had degradation or morphological alteration (up to 50% and more) after 7 mo at −20 or −80 ◦ C, whereas those stored in 15% trehalose remained quite stable at −20 or −80 ◦ C (Table 1 and Fig. 2D). These findings corroborated previous reports that trehalose stabilizes HIV-1 Pr55 gag, Norwalk and rotavirus VLPs (Kissmann et al., 2008; Lynch et al., 2012; Peixoto et al., 2007). Furthermore, samples stored in 5% sucrose or 5% trehalose (Fig. 2F) were unstable after 4 and 7 mo at −20 or −80 ◦ C, respectively (Table 1). Samples stored in buffer A alone at 4 ◦ C had better stability than those stored in low osmolyte concentrations. The PCV2 VLPs stored in higher percentage (15%) osmolytes generally retained their original appearance at 4, −20 or −80 ◦ C for 4 mo (except for 15% sucrose after 4 mo at −80 ◦ C), indicating that they provided stability for the PCV2 VLPs. To date, commercial VLPs-based vaccines harvested from BEVS ® (e.g., Ingelvac CircoFLEX, Boehringer Ingelheim) against PCV2 infection have already been used to prevent PCV2-SD (Oh et al., 2014). Storage conditions (salt concentration, buffer and temperature) for PCV2 VLPs are important to further develop VLP-based vaccines, since they may affect structural integrity and stability of VLPs, consequently reducing immunogenicity. In the present study,

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Fig. 1. Electron micrographs (A–D) and size distributions of VLPs assembled from PCV2 Cap (E and F). (A) PCV2 Cap (Pre-dialysis, 100 ␮g/ml); (B–D) PCV2 Cap (post-dialysis, 100 ␮g/ml in B; 50 ␮g/ml in C and 20 ␮g/ml in D). Size distribution (DLS) of samples before (E) and after (F) dialysis.

Fig. 2. Electron micrographs of the PCV2 VLPs stored in various formulated media at 4 ◦ C, −20 ◦ C and −80 ◦ C for prolonged intervals. (A) PCV2 VLPs stored in buffer A + 0.5 M NaCl at 4 ◦ C for 7 mo; (B) PCV2 VLPs stored in buffer A + 0.5 M NaCl at −20 ◦ C for 4 mo; (C) PCV2 VLPs stored in PBS at 4 ◦ C for 4 mo; (D) PCV2 VLPs stored in buffer A + 0.5 M NaCl + 15% trehalose at −80 ◦ C for 7 mo; (E) PCV2 VLPs stored in buffer A + 0.5 M NaCl + 15% sucrose at −20 ◦ C for 7 mo; and (F) PCV2 VLPs stored in buffer A + 0.5 M NaCl + 5% trehalose at −20 ◦ C for 4 mo.

we compared effects of NaCl, sucrose, trehalose and sorbitol on stabilizing the PCV2 VLPs. Addition of 15% trehalose to buffer A significantly enhanced stabilization of the PCV2 VLPs for 7 mo at low temperature (−20 or −80 ◦ C), thereby facilitating maintenance of particle morphology without impairing stability during vaccine

production, storage and transportation. However, buffer A with 5% sucrose, trehalose (Fig. 2F) and sobitol resulted in a degradation or alteration after 1, 4, or 7 mo of storage (Table 1), indicating that only 5% of the osmolytes were not enough to stabilize PCV2 VLPs during long-term storage.

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Table 1 Summary of morphological observations of PCV2 VLPs under various storage conditions. Formulation medium

Temp (◦ C)

Buffer A PBS Citrate + 0 M NaCl Citrate + 0.1 M NaCl Citrate + 0.5 M NaCl

4 4 4 4 4 −20 −80 4 −20 −80 4 −20 −80 4 −20 −80 4 −20 −80 4 −20 −80 4 −20 −80

Buffer A + 0.5 M NaCl + 5% sorbitol

Buffer A + 0.5 M NaCl + 15% sorbitol

Buffer A + 0.5 M NaCl + 5% sucrose

Buffer A + 0.5 M NaCl + 15% sucrose

Buffer A + 0.5 M NaCl + 5% trehalose

Buffer A + 0.5 M NaCl + 15% trehalose

Storage time (mo) 1 √

4 √

± √ √ √ √ √ √

X √ √ √

± ± √ √ √ ± ± ± √ √ √ ± ± ± √ √ √

± ± ± ± ± √ √ √ ± X X √ √ ± ± X X √ √ √

7 ± nd ± ± √ ± X ± X X √ ± ± ± nd nd √ ± ± X nd nd √ √ √

√ , VLPs with diameters and morphology similar to freshly purified VLPs (stable). ±, up to 50% and more of the VLPs with degradation or alteration (semi-unstable). X, 100% of the VLPs with degradation or alteration (unstable). nd, not determined.

In addition, PCV2 VLPs were also stable in storage buffer A with 15% trehalose at room temperature (25 ◦ C) and 37 ◦ C for 1 mo (data not shown), similar to a previous report that EV71 VLPs had stable morphology after storage for 1 mo at 25 or 37 ◦ C (Lin et al., 2014), although VLPs had some evidence of degradation (i.e. aggregation after storage for 2 mo). These findings are relevant for vaccine transportation, as storage at room temperature would avoid the need for refrigeration (cold chain) during shipment of vaccines containing PCV2 VLPs. In summary, this study represented a valuable baseline for development of long term-storage stabilizer (buffer and osmolytes) for PCV2 VLPs-based vaccines. These storage conditions might greatly benefit vaccine preparation, storage and transportation, and ultimately improve efficacy of vaccines against PCV2 infection. Acknowledgments This project was supported by: General Program of National Natural Science Foundation of China (Grant Nos. 31270819 and 31571432); Hunan Provincial Natural Science Foundation of China (Grant No. 13JJ1022/S2013J5050/2015JC3097); Research Foundation of Hunan Provincial Education Department, China (Grant No. 15A086); the Planned Science and Technology Project of Hunan Province (2014FJ2011), Postgraduate Research and Innovation Project of Hunan Province (Grant Nos. CX2016B285 and CX2016B314) and a grant from the Ministry of Agriculture for swine diseases controlling techniques (CARS-36, to Dr. P. Jiang). References Beauchamp, D.L., Khajehpour, M., 2012. Studying salt effects on protein stability using ribonuclease t1 as a model system. Biophys. Chem. 161, 29–38. Bucarey, S.A., Noriega, J., Reyes, P., Tapia, C., Saenz, L., Zuniga, A., Tobar, J.A., 2009. The optimized capsid gene of porcine circovirus type 2 expressed in yeast forms virus-like particles and elicits antibody responses in mice fed with recombinant yeast extracts. Vaccine 27, 5781–5790. Darwich, L., Mateu, E., 2012. Immunology of porcine circovirus type 2 (PCV2). Virus Res. 164, 61–67.

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