Arch Virol (2014) 159:963–970 DOI 10.1007/s00705-013-1907-0
ORIGINAL ARTICLE
Production and immunogenicity of VP2 protein of porcine parvovirus expressed in Pichia pastoris Chunhe Guo • Zemin Zhong • Yumao Huang
Received: 19 August 2013 / Accepted: 24 October 2013 / Published online: 13 November 2013 Ó Springer-Verlag Wien 2013
Abstract Viral protein 2 (VP2) of porcine parvovirus (PPV) is the major viral structural protein and is responsible for eliciting neutralizing antibodies in immunized animals. In this study, we constructed and characterized a recombinant yeast vector encoding the VP2 protein, designated as pGAPZaA-VP2. The construct was confirmed by restriction enzyme digestion, PCR, and sequencing and then introduced into P. pastoris strain SMD1168 by electroporation. The expressed VP2 protein was analyzed by SDS-PAGE and western blot. Immunization of mice with the VP2 protein elicited a PPV-specific humoral immune response. Notably, a preparation of VP2 protein containing adjuvant induced a much better antibody response than VP2 alone. Clearly, the adjuvant strongly enhanced the immunogenicity of VP2. This study provides a foundation for the application of the VP2 protein in the clinical diagnosis of PPV and in vaccination against PPV in the future.
C. Guo State Key Laboratory of Biocontrol, Guangzhou Higher Education Mega Center, School of Life Sciences, Sun Yat-sen University, North Third Road, Guangzhou, Guangdong 510006, People’s Republic of China e-mail: [email protected] Z. Zhong Y. Huang (&) College of Veterinary Medicine, South China Agricultural University, Guangzhou, Guangdong 510642, People’s Republic of China e-mail: [email protected] Z. Zhong e-mail: [email protected]
Introduction Porcine parvovirus (PPV) was first isolated from sows in Germany by Mayr et al., but subsequently, it has been isolated and characterized from most regions of the world, including China, where the economic impact has been significant [23]. PPV is one of many pathogens responsible for reproductive failure, characterized by embryonic death, mummification, stillbirth, infertility, and delayed return to oestrus [21]. In addition to inducing reproductive failure, PPV also causes diarrhea, dermatitis, and respiratory system disease [1, 8, 15]. The infection occurs without any clinical symptoms in growing pigs; however, the virus can cross the placental barrier during pregnancy. Furthermore, PPV has gained importance as an agent that is able to increase the effects of porcine circovirus type 2 infection on clinical symptoms such as postweaning multisystemic wasting syndrome (PMWS) [1, 15], which causes large economic loss in the global swine industry [24]. PPV has been reported from many different countries [14, 32]. Although there is only one serotype of PPV, the virus has been classified into four clinical genotypes (biotypes) according to pathogenicity. The nonpathogenic NADL-2 strain, which is currently used as an attenuated vaccine, causes only limited viremia and, surprisingly does not cross the placental barrier in experimental infections [5]. In contrast, the NADL-8 strain, isolated from mummified and dead fetuses, can cause viremia and crosses the placenta to infect fetuses, leading to death [6]. The other two groups are the Kresse and IAF-A83 strains, which are associated with dermatitis and enteric diseases, respectively [25]. PPV is a member of the genus Parvovirus, subfamily Parvovirinae, family Parvoviridae. PPV has a single-stranded DNA genome of about 5 kb. PPV is a non-enveloped, minusstranded and negative-sense small DNA virus. The viral capsids are composed of VP1, VP2, and VP3. VP2, the major
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structural protein, can elicit PPV-specific neutralizing antibodies [22, 41]. Furthermore, it consists of an eight-stranded antiparallel b-barrel motif with four large insertions between b-strands; the insertions, which are called loops, have many B-cell epitopes. It has been shown that all of the epitopes that generate neutralizing antibody are within VP2 [16, 27]. Many systems have been used to express VP2, resulting in successful self-assembly of virus-like particles (VLPs) with morphology similar to that of native virions and identical hemagglutination activity compared with active PPV [21]. PPV VLPs are highly immunogenic and can protect breeding sows against reproductive failure after virulent virus challenge [3]. Luckow et al. [19] and Si et al. [34] reported the expression of PPV VP2 in insect cell-baculovirus systems, and both groups demonstrated a 64-kDa band by western blot analysis. Additionally, Rueda et al. [31] demonstrated that contaminant baculovirus could be inactivated while maintaining immunogenicity. There is no effective treatment against PPV infection, and it has been reported that vaccination is an effective tool for controlling disease caused by this virus [7, 26]. There are several types of vaccines against PPV, including attenuated, live, and inactivated vaccines. However, the attenuated vaccine leads to side effects such as anaphylaxis. Furthermore, it may revert to a pathogenic strain [4, 17]. The inactivated vaccine has deficiencies in specific cellular immune efficacy and cannot effectively generate humoral immunity against PPV [2]. In this report, the production and immunogenicity in mice of recombinant yeast expressing PPV VP2 protein was demonstrated, with the aim of providing an attractive candidate subunit vaccine to be used in prevention and control of the disease associated with PPV infection in the future.
Materials and methods Materials The PPV (strain NADL-2) grown in cell culture was donated by WENS Co. (Guangdong, China). Escherichia coli strain DH5a and pig sera positive for PPV antibody were prepared and stored in the laboratory, College of Veterinary Medicine, South China Agricultural University (Guangzhou, China). Pichia pastoris (P. pastoris) strain SMD1168, ZeocinTM, and the expression vector pGAPZaA were purchased from Invitrogen (Carlsbad, USA). The E.Z.N.A Tissue DNA Extraction Kit was obtained from Promega (Madison, USA).
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Kit according to the manufacturer’s protocol. The extracted DNA was then used as a template to amplify the VP2 fragment by PCR. Based on the sequences of PPV genes available in the GenBank database (no. NC001718), Primer Premier 5.0 software (www.PremierBiosoft.com) was used to design and synthesize a pair of primers. In order to express the native N-terminus of the VP2 protein, an XhoI restriction site was introduced to allow in-frame cloning into the a-factor secretion signal of the pGAPZaA expression vector, and a nucleotide sequence encoding the KEX2 cleavage site was placed upstream of the VP2 fragment. The sense-strand primer (50 -CCCTCGAGAAAAGAATGAGTGAAAATGTGG AACAAC-30 ) thus included an XhoI restriction site (underlined) and a KEX2 protease cleavage site (bold), and the antisense-strand primer (50 -TTGCGGCCGCCTAGTATAA TTTTCTTGGTATAAGTTGTG-30 ) included a NotI restriction site (underlined). Construction The 1740-bp PCR product and plasmid pGAPZaA were both digested with XhoI and NotI and then ligated with T4 DNA ligase to yield the construct. The construct, named pGAPZaA-VP2, was identified by PCR and restriction enzyme digestion and confirmed by sequencing. Transformation by electroporation First, 5-10 lg of recombinant plasmid pGAPZaA-VP2 was prepared and linearized with BlnI, and a small aliquot of the digest was used to confirm complete linearization by agarose gel electrophoresis. Competent yeast cells (80 ll) were mixed with 5-10 lg of linearized DNA (in 5-10 ll sterile distilled water). The cell mixture was transferred to an ice-cold 0.2-cm electroporation cuvette and kept on ice for 5 min and then pulsed at 1.5 kV, 25 lF, and 200 X using a Bio-Rad Gene Pulser. Ice-cold 1 M sorbitol (1 ml) was added to the cuvette immediately after electroporation as described in the instructions of the EasyselectTM Pichia Expression Kit (Invitrogen, CA, USA). Finally, aliquots of 10, 25, 50, 100 and 200 ll were spread on separate yeast extract peptone dextrose (YPD) sorbitol plates containing 100 lg/ml ZeocinTM in order to isolate ZeocinTM-resistant clones. Plates were incubated for 2 to 3 days at 30 °C until colonies had formed. Subsequently, PCR amplification of genomic DNA using primers specific for pGAPZaA confirmed the identity of the transformants. Expression of VP2 protein
PCR amplification PPV strain NADL-2 was grown in cell culture, and viral genomic DNA was extracted using a Tissue DNA Extraction
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Scale-up expression of VP2 protein was achieved by shaking the highest-expressing P. pastoris clone in 10 mL YPD at 30 °C and 250 revolutions per minute (rpm) until
Porcine parvovirus VP2 protein expressed in Pichia pastoris
the OD600 reached 2, and an aliquot of 0.1 ml of this overnight culture was used to inoculate 50 ml of YPD in a 250-ml flask which, was grown at 28-30 °C in an agitating incubator (250-300 rpm). At the time points indicated below, 1 ml of the expression culture was transferred to a 1.5-ml microcentrifuge tube. These samples were used to analyze expression levels and to determine the optimal time to harvest the samples. The supernatants were collected after expression for 0, 24, 48, 72 and 96 h, respectively, and concentrated by ultrafiltration. The concentrated supernatants were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The expression levels of VP2 protein were evaluated by scanning the intensity of the bands and quantitation using a Bradford Protein Assay Kit (Beyotime, China). P. pastoris strain SMD1168/pGAPZaA (SMD1168 transformed with pGAPZaA) was used as a negative control. Optimization In order to improve the expression levels of the VP2 protein, the clone that expressed the highest level of VP2 protein was chosen for optimization experiments. First, the proteasedeficient P. pastoris strain SMD1168 was selected. Second, high-copy transformants were screened on YPD sorbitol plates containing 100, 150, 200, 250, or 300 lg/ml ZeocinTM and appropriate concentrations of EDTA formaldehyde, or protease inhibitors were added to YPD medium to prevent VP2 protein degradation. Finally, to determine the effect of time on the level of VP2 protein expression, samples were taken at 0, 24, 48, 72, and 96 h, and their OD600 values and expression levels were measured. Proteins from the cells supernatant containing VP2 were separated by 10 % SDS-PAGE and transferred to a nitrocellulose (NC) membrane. P. pastoris strain SMD1168/ pGAPZaA was used as a negative control. The VP2 protein was detected using a pig polyclonal antiserum (diluted 1000-fold) against PPV and an HRP-labeled anti-pig secondary antibody (diluted 2000-fold) (Dingguo, Guangzhou, China). Stability of the VP2 product The supernatant containing VP2 was placed at 4 °C and room temperature for 7, 14, 21 and 28 days, and analyzed by SDS-PAGE to determine whether the VP2 protein was degraded. Immunization of mice with the VP2 product Thirty-six healthy female BALB/c mice without PPVspecific antibody were randomly divided into six groups, with six mice in each group. They were housed in a
965 Table 1 Immunization schedule for the different groups Group
Inoculum
Group 1
The VP2 expression supernatant
Group 2
The VP2 expression supernatant containing aluminium adjuvant
Group 3
The VP2 expression supernatant containing Montanide ISA 50Va
Group 4
Commercial inactivated PPV vaccineb (positive control)
Group 5
SMD1168/pGAPZaA (negative control 1)
Group 6
PBS (negative control 2)
Thirty-six healthy BALB/c female mice were randomly divided into six groups. Each group was injected intramuscularly with 100 ll of the immunogen shown below and boosted twice at 2-week intervals a
The imported adjuvant Montanide ISA 50V was purchased from SEPPIC (Paris, France)
b
The commercial inactivated PPV vaccine applied in the pig industry was obtained from Qilu Animal Health (Shandong, China)
positive-pressure room. The immunization schedule is summarized in Table 1. Each mouse was injected intramuscularly with 100 ll of immunogen and boosted twice at 2-week intervals. At 14, 28 and 42 days after primary immunization, blood samples were obtained and allowed to clot at 37 °C for 2 h, and serum was collected and inactivated at 56 °C for 30 min. Enzyme-linked immunosorbent assay (ELISA) was used to analyze PPV-specific antibodies as reported previously [10]. Serum ELISA antibody assay PPV-specific antibodies from the collected sera were assayed using a PPV ELISA Kit (Keqian, Wuhan, China) according to the manufacturer’s protocol. Briefly, serum samples pre-diluted 1:50 in PBS were added to 96-well plates coated with VP2 protein and then incubated for 2 h at 37 °C. After three washes with PBS, 100 ll of horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (Dingguo, Guangzhou, China) was added to each well. Reactions were read at 630 nm using an ELISA plate reader (BioTek) to verify specific binding to the VP2 protein. Lymphoproliferation assays Peripheral blood mononuclear cells (PBMCs) were isolated on the fourteenth day following each vaccination injection as described previously [39]. Blood was collected in 5 mM EDTA and used immediately for the preparation of PBMCs. Proliferation assays were performed as described previously [38]. Briefly, 2 9 105 PBMCs per well were
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Expression
Fig. 1 A. Amplification of the PPV VP2 gene by PCR. The amplified VP2 fragment and DNA marker were loaded into a 0.8 % agarose gel to examine their size. M, DL2000 DNA marker; lane 1, VP2 gene; lane 2, blank control. B. Identification of recombinant yeast plasmid. M, 250-bp DNA marker; lanes 1 and 2, products of recombinant plasmids pGAPZaA-VP2 digested with XhoI and NotI; lane 3, blank control
cultured in triplicate in 96-well plates. PBMC samples were collected several times from each mouse group. Cells were incubated at 37 °C in a humidified atmosphere containing 5 % CO2 for 4 days. Following incubation, 20 lL 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) (Invitrogen) was added to each well for at least 5 h, and positive controls were established with concanavalin A (Sigma). The optical density was read at 490 nm using an ELISA plate reader (BioTek). Statistical analysis All experiments were performed independently at least three times. Statistical analysis was performed using GraphPad Prism and SPSS Software, and differences were evaluated using Student’s t-test. The limit of significance was 0.05 for all comparisons.
Results PPV VP2 fragment and identification of a recombinant yeast plasmid The PPV VP2 gene was successfully amplified by PCR using extracted DNA from virus grown in cell culture as a template. As shown in Fig. 1A, the size was 1740 bp, as predicted. The VP2 fusion gene in the plasmid pGAPZaA was confirmed by PCR to be 1740 bp long. The recombinant plasmid pGAPZaA-VP2 was digested with XhoI and NotI, and a VP2 fragment with a length of 1740 bp was obtained, which is consistent with the expected fragment size, as shown in Fig. 1B. These results indicated that the recombinant plasmid had been successfully constructed. Furthermore, the sequence of the VP2 fragment was found to be identical to the published sequence from GenBank (no. NC001718).
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The VP2 protein was identified by SDS-PAGE and western blot. A specific protein band of about 64 kDa expressed in P. pichia SMD1168 could be observed by SDS-PAGE, as shown in Fig. 2A. In contrast, no target protein band was observed in SMD1168 transformed with pGAPZaA (a negative control). The VP2 protein was transferred to an NC membrane and the result showed that the target protein could be conjugated to the resin. A single band of 64 kDa was detected by western blot (Fig. 2B), but the band was not found in the negative control. Determination of the VP2 protein concentration The VP2 protein concentration was determined using a Bradford protein assay kit following the kit manual. Bovine serum albumin (BSA) was used as a standard protein (0.5 mg/ml). Cell supernatant containing VP2 was diluted 1:1 in PBS, and 200 ll of G250 staining solution was added to each well. A microplate reader (BioTek) was used to determine the A570 value, which was used to estimate the total protein in the supernatant and controls. A light density scanner was used to scan the SDS-PAGE gel to determine the percentage of the total protein that was present in the target band. These data were used to calculate the expression levels of the VP2 protein. A protein standard concentration curve was generated using various concentrations of BSA. The OD595 of the VP2 protein was 0.9479, 1.0851 and 1.0310 after expression for 48, 72 and 96 h, respectively, and the target protein was 96.77 % of the total protein, as determined by the light density scanner method. Using this information, expression levels of the VP2 protein were 414.56 mg/L, 595.76 mg/L and 524.28 mg/L, respectively. Thus, the highest expression level of the VP2 protein was 595.76 mg/L, and the optimal expression time was 72 h. Stability of the VP2 expression product The cell supernatant containing VP2 was placed at 4 °C and room temperature (RT) for 7, 14, 21 and 28 days and then analyzed by SDS-PAGE as shown in Fig. 3. The VP2 protein was not degraded at all when placed at 4 °C for as long as 28 days; however, it degraded slowly with time when placed at room temperature. PPV-specific serum antibodies To examine the antibody responses elicited by the VP2 protein, the levels of anti-PPV antibodies in serum samples from immunized mice were determined by ELISA, and the
Porcine parvovirus VP2 protein expressed in Pichia pastoris
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Fig. 2 A. SDS-PAGE analysis of the recombinant VP2 protein. M, prestained protein ladder; lane 1, SMD1168 transformed with pGAPZaA (negative control): lane 2, VP2 protein. B. Western blot analysis of the recombinant VP2 protein. M, prestained protein ladder; lane 1, SMD1168; lane 2, SMD1168 transformed with pGAPZaA; lane 3, VP2 protein
Fig. 3 Stability of the VP2 expression product. Cell supernatant containing VP2 was stored at 4 °C and RT for 7, 14, 21 and 28 days and then analyzed by SDS-PAGE. VP2 protein stored at -20 °C was used as a positive control
results are shown in Fig. 4. The ELISA results showed that the titer of PPV-specific antibodies in the mice inoculated with the VP2 protein increased over time and reached the peak 14 days after the third vaccination. In parallel, there were no detectable antibodies in either of the negative control groups, SMD1168/pGAPZaA or PBS. Furthermore, the experimental groups (VP2 protein, VP2 protein containing aluminium adjuvant and VP2 protein containing the imported adjuvant Montanide ISA 50V [SEPPIC, France]) induced significantly higher levels of anti-PPV IgG compared to the negative control groups (P \ 0.001), measured 28 days after the second immunization and 42 days after the third immunization. These results indicated that the VP2 protein could elicit an anti-PPV response, and the titer of PPV-specific antibodies was higher when adjuvant was used, but lower than that induced using the commercial PPV inactivated vaccine (Qilu Animal Health, China) (Table 1).
Fig. 4 PPV-specific antibody response in immunized mice by ELISA. The OD value of each well was read at 630 nm to detect specific binding to the VP2 protein. The standard error of the mean (SEM) is shown for three independent experiments. *, P \ 0.05; **, P \ 0.01; ***, P \ 0.001 (for the experimental groups of VP2, VP2 containing aluminium adjuvant and VP2 containing imported adjuvant Montanide ISA 50V compared to the negative control group of SMD1168/pGAPZaA). #, P \ 0.05; ##, P \ 0.01; ###, P \ 0.001 (for the experimental groups of VP2, VP2 containing aluminium adjuvant and VP2 containing the imported adjuvant Montanide ISA 50V compared to the negative control group of PBS). Statistical analysis was performed using SPSS and GraphPad Prism
Cellular immune response to PPV VP2 The ability of PPV VP2 to induce specific T-cell responses against PPV was examined using lymphoproliferation assays. PBMCs were collected from blood samples on day 14 after each vaccination. As shown in Fig. 5, significant specific lymphoproliferation was observed in the experimental
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Fig. 5 Lymphoproliferation assays in immunized mice. PBMCs were obtained on day 14 post-vaccination. The OD value of each well was read at 490 nm. The standard error of the mean (SEM) is shown for three independent experiments. *, P \ 0.05; **, P \ 0.01; ***, P \ 0.001 (for the experimental groups of VP2, VP2 containing aluminium adjuvant and VP2 containing imported adjuvant Montanide ISA 50V compared to the negative control group of SMD1168/ pGAPZaA). #, P \ 0.05; ##, P \ 0.01; ###, P \ 0.001 (for the experimental groups of VP2, VP2 containing aluminium adjuvant and VP2 containing imported adjuvant Montanide ISA 50V compared to the negative control group of PBS). Statistical analysis was performed using SPSS and GraphPad Prism
groups receiving VP2, VP2 plus aluminium adjuvant, and VP2 plus the imported adjuvant Montanide ISA 50V, as well as the positive control (shown in Table 1), but not in the negative control groups receiving SMD1168/pGAPZaA or PBS. Furthermore, the lymphoproliferation was greater at 28 days after the first booster vaccination than at 14 days after primary vaccination, and it reached a peak at this point. As a result, PPV VP2 induced mice to generate a specific cellular immune response, and the level was higher when administed with an adjuvant, especially the imported adjuvant Montanide ISA 50V.
Discussion PPV is found in almost all pig-breeding countries. Various PPV strains have been isolated from field samples from China [30, 33]. Vaccines play a key role in the control of viral diseases. PPV causes serious economic losses in the breeding industry. Developing a safe, effective, and inexpensive method for vaccine production is warranted. In China, the commercially available vaccines against PPV are mainly inactivated vaccines. These vaccines are expensive and have the additional hazard that it is necessary to handle large quantities of infectious PPV [21]. Furthermore, because of the minimal cross-reactivity between different strains, the commercially available vaccines show little neutralization
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activity against some of the new strains reported in the past decade [11, 40]. Economic and safety considerations, together with the practical limitations associated with low yields of PPV particles from in vitro cultures, led us to develop recombinant subunit vaccines against PPV. The advantages of recombinant subunit vaccines are several. For example, the pathogen can be entirely eliminated from the production of vaccine, which eliminates risks associated with the production, the risk of reversion to virulent genotypes, and the risk of incomplete inactivation of whole-cell vaccine. Subunit vaccines based on recombinant proteins can be poorly immunogenic due to incorrect folding of the protein of interest or poor antigen presentation to the immune system. VP2, the major capsid protein of PPV, has nine linear B-cell epitopes, but only peptides from the N-terminus of VP2 have been reported to induce PPV-specific antibodies [13, 35, 36]. The VP2 gene tends to mutate, especially at key positions, such as sites 378, 383, 436, and 565, which are all under positive selection. There is evidence that the amino acid changes at these sites may be favorable for the survival of PPV [9]. VP2 can self-assemble in vitro into VLPs, which can then be used as a vaccine or a diagnostic reagent to detect antibodies produced in response to PPV infection or vaccination [18]. PPV VP2 was previously expressed in E. coli with immunogenicity similar to that of native PPV VP2 [29]. This paper describes a method for producing the VP2 protein of PPV in a P. pichia expression system. The P. pastoris expression system has been developed into an excellent tool for the large-scale expression of proteins from various sources [37]. Its advantages are its ability to perform many of the posttranslational modifications of higher eukaryotes and to secrete high levels of heterologous proteins into the supernatant under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. The P. pastoris expression system also differs from bacterial systems in that the vector containing the desired gene is integrated into the genome during transformation (homologous recombination) [20]. Furthermore, the vector pGAPZaA does not require methanol for induction, high levels of which are toxic to cells, making pGAPZaA an extremely convenient expression vector [28]. In the present study, the VP2 protein was demonstrated to be functionally expressed in P. pastoris. In order to improve the expression levels of VP2, the protease-deficient P. pastoris strain SMD1168 was selected, high-copy transformants were screened at a high concentration of ZeocinTM, and appropriate concentrations of EDTA formaldehyde or protease inhibitors were added to prevent the target protein degradation. To optimize the expression conditions for the VP2 protein, we found that the D-glucose concentration was a key parameter affecting protein expression levels in the P. pastoris expression
Porcine parvovirus VP2 protein expressed in Pichia pastoris
system. While the expression levels of VP2 in P. pastoris increased with D-glucose concentration, excessively high D-glucose concentrations were unfavorable for protein expression. A similar observation was made by Johnson et al. [12]. To obtain VP2 with a native N-terminus, the gene encoding VP2 including the KEX2 cleavage site was cloned, and it was successfully secreted into the culture supernatant using the a-mating factor signal sequence. Thus, the a-factor signal sequence was efficient at secreting recombinant proteins into the culture medium, and the signal peptide was efficiently processed by the KEX2 protease of P. pastoris. In summary, we have established a procedure to produce the PPV VP2 protein using the P. pastoris expression system and have evaluated its immunogenicity in the mice. The VP2 protein was expressed at high levels (up to 595.76 mg/l). The use of adjuvants enhanced the PPVspecific antibody responses. We believed that the VP2 protein would be a useful diagnostic reagent and an attractive candidate vaccine to prevent and control the disease associated with PPV infection in pigs. Acknowledgment This work was supported by Guangdong Province Live Pig Industry Technology System of Modern Agriculture (F10021). Conflict of interest The authors declare that they have no competing interests. None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper.
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ORIGINAL ARTICLE
Production and immunogenicity of VP2 protein of porcine parvovirus expressed in Pichia pastoris Chunhe Guo • Zemin Zhong • Yumao Huang
Received: 19 August 2013 / Accepted: 24 October 2013 / Published online: 13 November 2013 Ó Springer-Verlag Wien 2013
Abstract Viral protein 2 (VP2) of porcine parvovirus (PPV) is the major viral structural protein and is responsible for eliciting neutralizing antibodies in immunized animals. In this study, we constructed and characterized a recombinant yeast vector encoding the VP2 protein, designated as pGAPZaA-VP2. The construct was confirmed by restriction enzyme digestion, PCR, and sequencing and then introduced into P. pastoris strain SMD1168 by electroporation. The expressed VP2 protein was analyzed by SDS-PAGE and western blot. Immunization of mice with the VP2 protein elicited a PPV-specific humoral immune response. Notably, a preparation of VP2 protein containing adjuvant induced a much better antibody response than VP2 alone. Clearly, the adjuvant strongly enhanced the immunogenicity of VP2. This study provides a foundation for the application of the VP2 protein in the clinical diagnosis of PPV and in vaccination against PPV in the future.
C. Guo State Key Laboratory of Biocontrol, Guangzhou Higher Education Mega Center, School of Life Sciences, Sun Yat-sen University, North Third Road, Guangzhou, Guangdong 510006, People’s Republic of China e-mail: [email protected] Z. Zhong Y. Huang (&) College of Veterinary Medicine, South China Agricultural University, Guangzhou, Guangdong 510642, People’s Republic of China e-mail: [email protected] Z. Zhong e-mail: [email protected]
Introduction Porcine parvovirus (PPV) was first isolated from sows in Germany by Mayr et al., but subsequently, it has been isolated and characterized from most regions of the world, including China, where the economic impact has been significant [23]. PPV is one of many pathogens responsible for reproductive failure, characterized by embryonic death, mummification, stillbirth, infertility, and delayed return to oestrus [21]. In addition to inducing reproductive failure, PPV also causes diarrhea, dermatitis, and respiratory system disease [1, 8, 15]. The infection occurs without any clinical symptoms in growing pigs; however, the virus can cross the placental barrier during pregnancy. Furthermore, PPV has gained importance as an agent that is able to increase the effects of porcine circovirus type 2 infection on clinical symptoms such as postweaning multisystemic wasting syndrome (PMWS) [1, 15], which causes large economic loss in the global swine industry [24]. PPV has been reported from many different countries [14, 32]. Although there is only one serotype of PPV, the virus has been classified into four clinical genotypes (biotypes) according to pathogenicity. The nonpathogenic NADL-2 strain, which is currently used as an attenuated vaccine, causes only limited viremia and, surprisingly does not cross the placental barrier in experimental infections [5]. In contrast, the NADL-8 strain, isolated from mummified and dead fetuses, can cause viremia and crosses the placenta to infect fetuses, leading to death [6]. The other two groups are the Kresse and IAF-A83 strains, which are associated with dermatitis and enteric diseases, respectively [25]. PPV is a member of the genus Parvovirus, subfamily Parvovirinae, family Parvoviridae. PPV has a single-stranded DNA genome of about 5 kb. PPV is a non-enveloped, minusstranded and negative-sense small DNA virus. The viral capsids are composed of VP1, VP2, and VP3. VP2, the major
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structural protein, can elicit PPV-specific neutralizing antibodies [22, 41]. Furthermore, it consists of an eight-stranded antiparallel b-barrel motif with four large insertions between b-strands; the insertions, which are called loops, have many B-cell epitopes. It has been shown that all of the epitopes that generate neutralizing antibody are within VP2 [16, 27]. Many systems have been used to express VP2, resulting in successful self-assembly of virus-like particles (VLPs) with morphology similar to that of native virions and identical hemagglutination activity compared with active PPV [21]. PPV VLPs are highly immunogenic and can protect breeding sows against reproductive failure after virulent virus challenge [3]. Luckow et al. [19] and Si et al. [34] reported the expression of PPV VP2 in insect cell-baculovirus systems, and both groups demonstrated a 64-kDa band by western blot analysis. Additionally, Rueda et al. [31] demonstrated that contaminant baculovirus could be inactivated while maintaining immunogenicity. There is no effective treatment against PPV infection, and it has been reported that vaccination is an effective tool for controlling disease caused by this virus [7, 26]. There are several types of vaccines against PPV, including attenuated, live, and inactivated vaccines. However, the attenuated vaccine leads to side effects such as anaphylaxis. Furthermore, it may revert to a pathogenic strain [4, 17]. The inactivated vaccine has deficiencies in specific cellular immune efficacy and cannot effectively generate humoral immunity against PPV [2]. In this report, the production and immunogenicity in mice of recombinant yeast expressing PPV VP2 protein was demonstrated, with the aim of providing an attractive candidate subunit vaccine to be used in prevention and control of the disease associated with PPV infection in the future.
Materials and methods Materials The PPV (strain NADL-2) grown in cell culture was donated by WENS Co. (Guangdong, China). Escherichia coli strain DH5a and pig sera positive for PPV antibody were prepared and stored in the laboratory, College of Veterinary Medicine, South China Agricultural University (Guangzhou, China). Pichia pastoris (P. pastoris) strain SMD1168, ZeocinTM, and the expression vector pGAPZaA were purchased from Invitrogen (Carlsbad, USA). The E.Z.N.A Tissue DNA Extraction Kit was obtained from Promega (Madison, USA).
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Kit according to the manufacturer’s protocol. The extracted DNA was then used as a template to amplify the VP2 fragment by PCR. Based on the sequences of PPV genes available in the GenBank database (no. NC001718), Primer Premier 5.0 software (www.PremierBiosoft.com) was used to design and synthesize a pair of primers. In order to express the native N-terminus of the VP2 protein, an XhoI restriction site was introduced to allow in-frame cloning into the a-factor secretion signal of the pGAPZaA expression vector, and a nucleotide sequence encoding the KEX2 cleavage site was placed upstream of the VP2 fragment. The sense-strand primer (50 -CCCTCGAGAAAAGAATGAGTGAAAATGTGG AACAAC-30 ) thus included an XhoI restriction site (underlined) and a KEX2 protease cleavage site (bold), and the antisense-strand primer (50 -TTGCGGCCGCCTAGTATAA TTTTCTTGGTATAAGTTGTG-30 ) included a NotI restriction site (underlined). Construction The 1740-bp PCR product and plasmid pGAPZaA were both digested with XhoI and NotI and then ligated with T4 DNA ligase to yield the construct. The construct, named pGAPZaA-VP2, was identified by PCR and restriction enzyme digestion and confirmed by sequencing. Transformation by electroporation First, 5-10 lg of recombinant plasmid pGAPZaA-VP2 was prepared and linearized with BlnI, and a small aliquot of the digest was used to confirm complete linearization by agarose gel electrophoresis. Competent yeast cells (80 ll) were mixed with 5-10 lg of linearized DNA (in 5-10 ll sterile distilled water). The cell mixture was transferred to an ice-cold 0.2-cm electroporation cuvette and kept on ice for 5 min and then pulsed at 1.5 kV, 25 lF, and 200 X using a Bio-Rad Gene Pulser. Ice-cold 1 M sorbitol (1 ml) was added to the cuvette immediately after electroporation as described in the instructions of the EasyselectTM Pichia Expression Kit (Invitrogen, CA, USA). Finally, aliquots of 10, 25, 50, 100 and 200 ll were spread on separate yeast extract peptone dextrose (YPD) sorbitol plates containing 100 lg/ml ZeocinTM in order to isolate ZeocinTM-resistant clones. Plates were incubated for 2 to 3 days at 30 °C until colonies had formed. Subsequently, PCR amplification of genomic DNA using primers specific for pGAPZaA confirmed the identity of the transformants. Expression of VP2 protein
PCR amplification PPV strain NADL-2 was grown in cell culture, and viral genomic DNA was extracted using a Tissue DNA Extraction
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Scale-up expression of VP2 protein was achieved by shaking the highest-expressing P. pastoris clone in 10 mL YPD at 30 °C and 250 revolutions per minute (rpm) until
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the OD600 reached 2, and an aliquot of 0.1 ml of this overnight culture was used to inoculate 50 ml of YPD in a 250-ml flask which, was grown at 28-30 °C in an agitating incubator (250-300 rpm). At the time points indicated below, 1 ml of the expression culture was transferred to a 1.5-ml microcentrifuge tube. These samples were used to analyze expression levels and to determine the optimal time to harvest the samples. The supernatants were collected after expression for 0, 24, 48, 72 and 96 h, respectively, and concentrated by ultrafiltration. The concentrated supernatants were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The expression levels of VP2 protein were evaluated by scanning the intensity of the bands and quantitation using a Bradford Protein Assay Kit (Beyotime, China). P. pastoris strain SMD1168/pGAPZaA (SMD1168 transformed with pGAPZaA) was used as a negative control. Optimization In order to improve the expression levels of the VP2 protein, the clone that expressed the highest level of VP2 protein was chosen for optimization experiments. First, the proteasedeficient P. pastoris strain SMD1168 was selected. Second, high-copy transformants were screened on YPD sorbitol plates containing 100, 150, 200, 250, or 300 lg/ml ZeocinTM and appropriate concentrations of EDTA formaldehyde, or protease inhibitors were added to YPD medium to prevent VP2 protein degradation. Finally, to determine the effect of time on the level of VP2 protein expression, samples were taken at 0, 24, 48, 72, and 96 h, and their OD600 values and expression levels were measured. Proteins from the cells supernatant containing VP2 were separated by 10 % SDS-PAGE and transferred to a nitrocellulose (NC) membrane. P. pastoris strain SMD1168/ pGAPZaA was used as a negative control. The VP2 protein was detected using a pig polyclonal antiserum (diluted 1000-fold) against PPV and an HRP-labeled anti-pig secondary antibody (diluted 2000-fold) (Dingguo, Guangzhou, China). Stability of the VP2 product The supernatant containing VP2 was placed at 4 °C and room temperature for 7, 14, 21 and 28 days, and analyzed by SDS-PAGE to determine whether the VP2 protein was degraded. Immunization of mice with the VP2 product Thirty-six healthy female BALB/c mice without PPVspecific antibody were randomly divided into six groups, with six mice in each group. They were housed in a
965 Table 1 Immunization schedule for the different groups Group
Inoculum
Group 1
The VP2 expression supernatant
Group 2
The VP2 expression supernatant containing aluminium adjuvant
Group 3
The VP2 expression supernatant containing Montanide ISA 50Va
Group 4
Commercial inactivated PPV vaccineb (positive control)
Group 5
SMD1168/pGAPZaA (negative control 1)
Group 6
PBS (negative control 2)
Thirty-six healthy BALB/c female mice were randomly divided into six groups. Each group was injected intramuscularly with 100 ll of the immunogen shown below and boosted twice at 2-week intervals a
The imported adjuvant Montanide ISA 50V was purchased from SEPPIC (Paris, France)
b
The commercial inactivated PPV vaccine applied in the pig industry was obtained from Qilu Animal Health (Shandong, China)
positive-pressure room. The immunization schedule is summarized in Table 1. Each mouse was injected intramuscularly with 100 ll of immunogen and boosted twice at 2-week intervals. At 14, 28 and 42 days after primary immunization, blood samples were obtained and allowed to clot at 37 °C for 2 h, and serum was collected and inactivated at 56 °C for 30 min. Enzyme-linked immunosorbent assay (ELISA) was used to analyze PPV-specific antibodies as reported previously [10]. Serum ELISA antibody assay PPV-specific antibodies from the collected sera were assayed using a PPV ELISA Kit (Keqian, Wuhan, China) according to the manufacturer’s protocol. Briefly, serum samples pre-diluted 1:50 in PBS were added to 96-well plates coated with VP2 protein and then incubated for 2 h at 37 °C. After three washes with PBS, 100 ll of horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (Dingguo, Guangzhou, China) was added to each well. Reactions were read at 630 nm using an ELISA plate reader (BioTek) to verify specific binding to the VP2 protein. Lymphoproliferation assays Peripheral blood mononuclear cells (PBMCs) were isolated on the fourteenth day following each vaccination injection as described previously [39]. Blood was collected in 5 mM EDTA and used immediately for the preparation of PBMCs. Proliferation assays were performed as described previously [38]. Briefly, 2 9 105 PBMCs per well were
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Expression
Fig. 1 A. Amplification of the PPV VP2 gene by PCR. The amplified VP2 fragment and DNA marker were loaded into a 0.8 % agarose gel to examine their size. M, DL2000 DNA marker; lane 1, VP2 gene; lane 2, blank control. B. Identification of recombinant yeast plasmid. M, 250-bp DNA marker; lanes 1 and 2, products of recombinant plasmids pGAPZaA-VP2 digested with XhoI and NotI; lane 3, blank control
cultured in triplicate in 96-well plates. PBMC samples were collected several times from each mouse group. Cells were incubated at 37 °C in a humidified atmosphere containing 5 % CO2 for 4 days. Following incubation, 20 lL 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) (Invitrogen) was added to each well for at least 5 h, and positive controls were established with concanavalin A (Sigma). The optical density was read at 490 nm using an ELISA plate reader (BioTek). Statistical analysis All experiments were performed independently at least three times. Statistical analysis was performed using GraphPad Prism and SPSS Software, and differences were evaluated using Student’s t-test. The limit of significance was 0.05 for all comparisons.
Results PPV VP2 fragment and identification of a recombinant yeast plasmid The PPV VP2 gene was successfully amplified by PCR using extracted DNA from virus grown in cell culture as a template. As shown in Fig. 1A, the size was 1740 bp, as predicted. The VP2 fusion gene in the plasmid pGAPZaA was confirmed by PCR to be 1740 bp long. The recombinant plasmid pGAPZaA-VP2 was digested with XhoI and NotI, and a VP2 fragment with a length of 1740 bp was obtained, which is consistent with the expected fragment size, as shown in Fig. 1B. These results indicated that the recombinant plasmid had been successfully constructed. Furthermore, the sequence of the VP2 fragment was found to be identical to the published sequence from GenBank (no. NC001718).
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The VP2 protein was identified by SDS-PAGE and western blot. A specific protein band of about 64 kDa expressed in P. pichia SMD1168 could be observed by SDS-PAGE, as shown in Fig. 2A. In contrast, no target protein band was observed in SMD1168 transformed with pGAPZaA (a negative control). The VP2 protein was transferred to an NC membrane and the result showed that the target protein could be conjugated to the resin. A single band of 64 kDa was detected by western blot (Fig. 2B), but the band was not found in the negative control. Determination of the VP2 protein concentration The VP2 protein concentration was determined using a Bradford protein assay kit following the kit manual. Bovine serum albumin (BSA) was used as a standard protein (0.5 mg/ml). Cell supernatant containing VP2 was diluted 1:1 in PBS, and 200 ll of G250 staining solution was added to each well. A microplate reader (BioTek) was used to determine the A570 value, which was used to estimate the total protein in the supernatant and controls. A light density scanner was used to scan the SDS-PAGE gel to determine the percentage of the total protein that was present in the target band. These data were used to calculate the expression levels of the VP2 protein. A protein standard concentration curve was generated using various concentrations of BSA. The OD595 of the VP2 protein was 0.9479, 1.0851 and 1.0310 after expression for 48, 72 and 96 h, respectively, and the target protein was 96.77 % of the total protein, as determined by the light density scanner method. Using this information, expression levels of the VP2 protein were 414.56 mg/L, 595.76 mg/L and 524.28 mg/L, respectively. Thus, the highest expression level of the VP2 protein was 595.76 mg/L, and the optimal expression time was 72 h. Stability of the VP2 expression product The cell supernatant containing VP2 was placed at 4 °C and room temperature (RT) for 7, 14, 21 and 28 days and then analyzed by SDS-PAGE as shown in Fig. 3. The VP2 protein was not degraded at all when placed at 4 °C for as long as 28 days; however, it degraded slowly with time when placed at room temperature. PPV-specific serum antibodies To examine the antibody responses elicited by the VP2 protein, the levels of anti-PPV antibodies in serum samples from immunized mice were determined by ELISA, and the
Porcine parvovirus VP2 protein expressed in Pichia pastoris
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Fig. 2 A. SDS-PAGE analysis of the recombinant VP2 protein. M, prestained protein ladder; lane 1, SMD1168 transformed with pGAPZaA (negative control): lane 2, VP2 protein. B. Western blot analysis of the recombinant VP2 protein. M, prestained protein ladder; lane 1, SMD1168; lane 2, SMD1168 transformed with pGAPZaA; lane 3, VP2 protein
Fig. 3 Stability of the VP2 expression product. Cell supernatant containing VP2 was stored at 4 °C and RT for 7, 14, 21 and 28 days and then analyzed by SDS-PAGE. VP2 protein stored at -20 °C was used as a positive control
results are shown in Fig. 4. The ELISA results showed that the titer of PPV-specific antibodies in the mice inoculated with the VP2 protein increased over time and reached the peak 14 days after the third vaccination. In parallel, there were no detectable antibodies in either of the negative control groups, SMD1168/pGAPZaA or PBS. Furthermore, the experimental groups (VP2 protein, VP2 protein containing aluminium adjuvant and VP2 protein containing the imported adjuvant Montanide ISA 50V [SEPPIC, France]) induced significantly higher levels of anti-PPV IgG compared to the negative control groups (P \ 0.001), measured 28 days after the second immunization and 42 days after the third immunization. These results indicated that the VP2 protein could elicit an anti-PPV response, and the titer of PPV-specific antibodies was higher when adjuvant was used, but lower than that induced using the commercial PPV inactivated vaccine (Qilu Animal Health, China) (Table 1).
Fig. 4 PPV-specific antibody response in immunized mice by ELISA. The OD value of each well was read at 630 nm to detect specific binding to the VP2 protein. The standard error of the mean (SEM) is shown for three independent experiments. *, P \ 0.05; **, P \ 0.01; ***, P \ 0.001 (for the experimental groups of VP2, VP2 containing aluminium adjuvant and VP2 containing imported adjuvant Montanide ISA 50V compared to the negative control group of SMD1168/pGAPZaA). #, P \ 0.05; ##, P \ 0.01; ###, P \ 0.001 (for the experimental groups of VP2, VP2 containing aluminium adjuvant and VP2 containing the imported adjuvant Montanide ISA 50V compared to the negative control group of PBS). Statistical analysis was performed using SPSS and GraphPad Prism
Cellular immune response to PPV VP2 The ability of PPV VP2 to induce specific T-cell responses against PPV was examined using lymphoproliferation assays. PBMCs were collected from blood samples on day 14 after each vaccination. As shown in Fig. 5, significant specific lymphoproliferation was observed in the experimental
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Fig. 5 Lymphoproliferation assays in immunized mice. PBMCs were obtained on day 14 post-vaccination. The OD value of each well was read at 490 nm. The standard error of the mean (SEM) is shown for three independent experiments. *, P \ 0.05; **, P \ 0.01; ***, P \ 0.001 (for the experimental groups of VP2, VP2 containing aluminium adjuvant and VP2 containing imported adjuvant Montanide ISA 50V compared to the negative control group of SMD1168/ pGAPZaA). #, P \ 0.05; ##, P \ 0.01; ###, P \ 0.001 (for the experimental groups of VP2, VP2 containing aluminium adjuvant and VP2 containing imported adjuvant Montanide ISA 50V compared to the negative control group of PBS). Statistical analysis was performed using SPSS and GraphPad Prism
groups receiving VP2, VP2 plus aluminium adjuvant, and VP2 plus the imported adjuvant Montanide ISA 50V, as well as the positive control (shown in Table 1), but not in the negative control groups receiving SMD1168/pGAPZaA or PBS. Furthermore, the lymphoproliferation was greater at 28 days after the first booster vaccination than at 14 days after primary vaccination, and it reached a peak at this point. As a result, PPV VP2 induced mice to generate a specific cellular immune response, and the level was higher when administed with an adjuvant, especially the imported adjuvant Montanide ISA 50V.
Discussion PPV is found in almost all pig-breeding countries. Various PPV strains have been isolated from field samples from China [30, 33]. Vaccines play a key role in the control of viral diseases. PPV causes serious economic losses in the breeding industry. Developing a safe, effective, and inexpensive method for vaccine production is warranted. In China, the commercially available vaccines against PPV are mainly inactivated vaccines. These vaccines are expensive and have the additional hazard that it is necessary to handle large quantities of infectious PPV [21]. Furthermore, because of the minimal cross-reactivity between different strains, the commercially available vaccines show little neutralization
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activity against some of the new strains reported in the past decade [11, 40]. Economic and safety considerations, together with the practical limitations associated with low yields of PPV particles from in vitro cultures, led us to develop recombinant subunit vaccines against PPV. The advantages of recombinant subunit vaccines are several. For example, the pathogen can be entirely eliminated from the production of vaccine, which eliminates risks associated with the production, the risk of reversion to virulent genotypes, and the risk of incomplete inactivation of whole-cell vaccine. Subunit vaccines based on recombinant proteins can be poorly immunogenic due to incorrect folding of the protein of interest or poor antigen presentation to the immune system. VP2, the major capsid protein of PPV, has nine linear B-cell epitopes, but only peptides from the N-terminus of VP2 have been reported to induce PPV-specific antibodies [13, 35, 36]. The VP2 gene tends to mutate, especially at key positions, such as sites 378, 383, 436, and 565, which are all under positive selection. There is evidence that the amino acid changes at these sites may be favorable for the survival of PPV [9]. VP2 can self-assemble in vitro into VLPs, which can then be used as a vaccine or a diagnostic reagent to detect antibodies produced in response to PPV infection or vaccination [18]. PPV VP2 was previously expressed in E. coli with immunogenicity similar to that of native PPV VP2 [29]. This paper describes a method for producing the VP2 protein of PPV in a P. pichia expression system. The P. pastoris expression system has been developed into an excellent tool for the large-scale expression of proteins from various sources [37]. Its advantages are its ability to perform many of the posttranslational modifications of higher eukaryotes and to secrete high levels of heterologous proteins into the supernatant under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. The P. pastoris expression system also differs from bacterial systems in that the vector containing the desired gene is integrated into the genome during transformation (homologous recombination) [20]. Furthermore, the vector pGAPZaA does not require methanol for induction, high levels of which are toxic to cells, making pGAPZaA an extremely convenient expression vector [28]. In the present study, the VP2 protein was demonstrated to be functionally expressed in P. pastoris. In order to improve the expression levels of VP2, the protease-deficient P. pastoris strain SMD1168 was selected, high-copy transformants were screened at a high concentration of ZeocinTM, and appropriate concentrations of EDTA formaldehyde or protease inhibitors were added to prevent the target protein degradation. To optimize the expression conditions for the VP2 protein, we found that the D-glucose concentration was a key parameter affecting protein expression levels in the P. pastoris expression
Porcine parvovirus VP2 protein expressed in Pichia pastoris
system. While the expression levels of VP2 in P. pastoris increased with D-glucose concentration, excessively high D-glucose concentrations were unfavorable for protein expression. A similar observation was made by Johnson et al. [12]. To obtain VP2 with a native N-terminus, the gene encoding VP2 including the KEX2 cleavage site was cloned, and it was successfully secreted into the culture supernatant using the a-mating factor signal sequence. Thus, the a-factor signal sequence was efficient at secreting recombinant proteins into the culture medium, and the signal peptide was efficiently processed by the KEX2 protease of P. pastoris. In summary, we have established a procedure to produce the PPV VP2 protein using the P. pastoris expression system and have evaluated its immunogenicity in the mice. The VP2 protein was expressed at high levels (up to 595.76 mg/l). The use of adjuvants enhanced the PPVspecific antibody responses. We believed that the VP2 protein would be a useful diagnostic reagent and an attractive candidate vaccine to prevent and control the disease associated with PPV infection in pigs. Acknowledgment This work was supported by Guangdong Province Live Pig Industry Technology System of Modern Agriculture (F10021). Conflict of interest The authors declare that they have no competing interests. None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper.
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