Development of modern immunizing agent against porcine circovirus type 2 infection based on chimeric VP1-PCV2bCap recombinant protein

doi:10.21203/rs.3.rs-2263216/v1

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Development of modern immunizing agent against porcine circovirus type 2 infection based on chimeric VP1-PCV2bCap recombinant protein

https://doi.org/10.21203/rs.3.rs-2263216/v1

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Porcine circovirus type 2 is the main causative agent of post-weaning multisystemic wasting syndrome, which affects the immune system of swine and causes widespread epidemics in livestock farms resulting in significant piglet mortality and economic losses every year. Although several commercial vaccines were developed, the efficiency and safety need to be improved. Therefore, we have engineered the chimeric complex containing PCV2bCap protein based on virus like particles (VLPs) and the mouse polyomavirus (MPyV) as VLPs represent modern and safe alternative of classical vaccine with high B cells stimulating activity. The ability of this complex to induce an immune response in both mouse and pig models in vivo were evaluated. Firstly, experimental mice were divided into 4 groups and immunized with sterile buffer and VP1-PCV2bCap with different adjuvants, the immune response was monitored for 10 weeks. Robust immune response was detected after the first immunization and gradually increased after the second and third dose, especially in mice immunized by recombinant protein with Emulsigen (10%) as an adjuvant. Subsequently, to confirm the vaccine efficacy in a target organism, 8-week-old piglets were immunized with VP1-PCV2bCap protein with Emulsigen (10%). The levels of anti-PCV2b specific IgG antibodies were significantly increased in piglets after the second immunization. Finally, strong neutralizing activity of these antibodies was confirmed in PK-15 cells infected with PCV2 Stoon 1010. VP1-PCV2bCap protein complex appears as a promising candidate vaccine for preventing disease associated with PCV2 infection in pigs.

circovirus

recombinant antigen

vaccination

chimeric protein

Porcine circovirus (PCV) is a small non-enveloped DNA virus common in pigs. Four types of PCV have been identified up to now: non-pathogenic PCV1, pathogenic forms PCV2 and PCV3 [1], and recently identified PCV4 [2]. PCV2 is strongly associated with the occurrence of post-weaning multisystemic wasting syndrome (PMWS) causing significant economic loss in the swine industry. PCV2 is a causal agent of porcine circovirus diseases (PCVD; term used in Europe) and porcine circovirus-associated diseases (PCVAD; term used in the US) [3], including subclinical infection, systemic disease or PMWS, porcine dermatitis and nephropathy syndrome (PDNS), porcine respiratory disease complex (PPDC), granulomatous enteric disease, reproductive disease, and necrotizing lymphadenitis [4, 5]. Among all, PMWS is a typical clinical manifestation of PCVD/PCVAD [6–8] characterized by poor weight gain, chronic wasting, dyspnea, diarrhea, ictus, etc. in 6 to 11 weeks old piglets [9] leading to morbidity and post-weaning mortality.

The genome of PCV2 contains 11 open reading frames (ORFs) [5], but only two of them encode proteins. ORF1 is highly conserved and encodes viral DNA replication-associated proteins whereas ORF2 encodes relatively variable capsid protein (Cap) [3]. According to the differences in ORF2, PCV2 is divided into five genotypes PCV2a, PCV2b, PCV2c, PCV2d, and PCV2e [10]. Capsid proteins play an important role in PCV2 immunogenicity [11, 12].

Cap protein is necessary for the formation and integrity of virion capsid, infection, and replication of the viruses. Amino acids of Cap protein in the position of 169–180 play a main role as a decoy epitope affecting production of non-neutralizing antibodies against PCV2 [13]. This epitope represents a basis of a serological assays predicting protective antibody response against PCV2 infection. So called dimerization domain in the position of 51–103, especially cysteine 108 that is responsible for the formation of disulfide bonds, plays another important role [14]. Deletion of cysteine 108 leads to failure in dimerization processes and Cap protein is then incapable of producing stable dimers [14].

The main molecular mechanisms causing circovirus diseases are still unknown. Several commercial vaccines, especially attenuated viral vaccines or subunit protein vaccines, were developed. These vaccines are effective against the specific genotype of PCV2, with cross-reactivity against other genotypes [15, 16]. Whereas in attenuated vaccines there is still a risk of the reversion to a virulent phenotype in vivo, virus like particles (VLPs) represent modern and safe alternative of classical vaccines. These particles have an immunostimulating effect on B cells leading to the proliferation and antibody production in the absence of adjuvants [17]. Stable forms of VLPs may serve as scaffolds for preparation of chimeric immunostimulating agents inducing strong humoral immune response [18].

In this study, we present a new approach for developing modern and safe immunizing agent based on VLP particles capable of inducing strong humoral immune response against PCV2 infection with significant neutralizing activity. Moreover, these chimeric antigens containing VP1-PCV2bCap protein sequences based on the mouse polyomavirus (MPyV) have a so-called DIVA (differentiating infected from vaccinated animals) vaccine potential, which can induce immune response against the mouse polyoma VP1 protein and distinguish between PCV2 naturally infected and vaccinated animals.

Insertion of sequence of porcine circovirus 2b (PCV2b) capsid protein into pFastBacI –VP1_DAɅ7 transfer vector

The PCV2bCap sequence was amplified with PCR primers containing BamHI restriction sites at the 5´ end and a His-tag (6 x His) and KpnI at the 3´end and inserted into pFastBacI –VP1_DAɅ7 transfer vector via BamHI and KpnI restriction sites as we described previously [18].

Recombinant baculovirus preparation

Recombinant baculoviruses were produced by Bac-to-Bac system according to the manufacturer’s instructions (ThermoFisher Scientific, USA). Briefly, E. coli DH10Bac containing a bacmid and helper vector were transformed by pFastBacI –VP1_DAɅ7 transfer vectors and positive bacterial colonies were isolated by PureLink™ HiPure Plasmid DNA Miniprep Kit (ThermoFisher Scientific, USA) and verified by PCR. Sf9 insect cells were transfected with bacmids by lipofection using Cellfectin^™ II Reagent (ThermoFisher Scientific, USA). Recombinant baculoviruses were harvested 72 h after transfection (as a viral stock V₀).

Expression and purification of chimeric protein from Sf9 insect cells

To generate high-titer viral stocks, 3×10⁷ Sf9 cells (Gibco, USA) growing in 10 ml of SFM900II medium (Gibco, USA) at 27.5°C were infected with V₀ at 0.1 MOI by shaking at 200 rpm. After 4 days of cultivation, the supernatant (V₁) was collected and another 3×10⁷ cells in 10 ml SFM900II medium were infected with V₁ at 10 MOI. Cell culture was cultivated for 4 days at 27.5°C in a shaking incubator at 200 rpm and the supernatant (V₂) was then collected. For VP1-PCV2bCap over-expression, 2 ml of V₂ at 50 MOI was used to infect 9×10⁸ cells in 300 ml SFM900II medium in 600 ml TPP® TubeSpin bioreactor bottles (TPP, Switzerland) and cultivated for 48 h with a shaking rate of 200 rpm. Sf9 cells were then harvested and the pellet was frozen at -80°C overnight. Pellet was resuspended in native lysis buffer (20 mmol l^− 1 Tris-HCl, 150 mmol l^− 1 NaCl, pH 7.6) supplemented with cOmplete^™, EDTA-free Protease Inhibitor Cocktail (Merck, USA) and lysed by Microfluidizer Processor Cell Disruptor Homogenizer (New Life Scientific, USA) cooled on ice. Cell lysate was centrifuged at 10.000 g for 30 min and supernatant was treated with 180 U DENARASE^R (c-LEcta GmbH, Germany) and 2 µl of 1 M MgCl₂ per ml of supernatant and incubated for 1 h at 37°C. Supernatant was then centrifuged at 10.500 g for 10 min and chimeric VP1-PCV2bCap protein was purified under native conditions using CoNTA agarose (Merck, USA) according to the manufacturer’s instructions. The cell lysate and purified protein were separated o 5 % native‐PAGE stained with Coomassie Brilliant Blue R‐250 or blotted onto polyvinylidene difluoride (PVDF) membrane (Millipore, USA). The PVDF membrane was blocked overnight wit 3 % non‐fat milk and developed with horseradish peroxidase‐conjugated anti-PCV2 Cap antibody (Exbio, Czech Republic) diluted 1:500 followed by a detection using GE Healthcare Amersham™ ECL™ Prime Western Blotting Detection Reagent (ThermoFisher Scientific, USA) and analyzed with Azure Biosystems C300 (Azure Biosystems, Inc.) and cSeries Capture Software. The same conditions were used for SDS-PAGE and Western blot analysis with horseradish peroxidase‐conjugated anti-His tag antibody (ThermoFisher Scientific, USA) diluted 1:1000 as a control.

Nano Gold labeling

30 µl of purified protein was incubated with 3 µl of IgG PCV2Cap H9 primary antibody (Exbio, Czech Republic) 1 h at 37° C. Suspension was supplemented with protein A conjugated 20-nm gold particles (Protein-A-Gold, Sigma-Aldrich, Germany) and incubated for 1 h at room temperature. The immuno-gold labeled protein aggregates were observed by Philips 208s Electron Microscope Morgagni (FEI, Czech Republic) at 18.000 x magnification and an accelerating voltage of 80 kV.

Asymmetrical flow field-flow fractionation (AF4)

The Eclipse separation system (Eclipse AF4, Wyatt Technology Corporation, Dernbach, Germany) was connected to an isocratic pump, degasser and autosampler with temperature control unit (Agilent 1260 Infinity II, Agilent Technology, Böblingen, Germany). The Short separation channel (SC 145 mm length) was equipped with a trapezoidal polyethylene terephthalate spacer (Wyatt Technology Corporation, Dernbach, Germany) with thickness of 350 µm (M350) and regenerated cellulose membrane (10 kDa). The separation system was connected to the multi-angle light scattering MALS detector (DAWN HELEOS II, Wyatt Technology Corporation) equipped with a red laser (662 nm) with QELS detector at angle 149°. UV-VIS detector (Agilent 1260 Infinity II, Agilent Technology, absorption wavelength 280 nm). Filtered (0.2 µm) and preserved (0.04% ProClin™ 300, Sigma-Aldrich, USA) PBS buffer pH 7.4 was used as carrier liquid. Additional filter (0.1 µm) was placed between the pump and the channel.

To obtain pentamers of chimeric VP1-PCV2bCap protein, eluate of the protein purified by CoNTA was separated according following conditions: Constant Detector flow 1.0 ml/min, 100 µl of sample was injected into the channel with 2.0 mL/min cross flow over 3 min in the focus inject mode (Inject flow was 0.2 mL/min) and further focused over 10 min with the same cross flow. The sample was eluted with a crossflow decreasing from 2.0 to 0.05 mL/ min over 40 min and then with a constant cross flow 0.05 mL/min over 15 min followed by 7 min of elution without applied cross flow.

Measurement was evaluated by the Astra software version 7.1.4.8 (Wyatt Technology Corporation). UV detector (280 nm) was used as a concentration source with UV Extinction coefficient 0.6670 ml/(mg∙cm). The MALS detector was calibrated using HPLC grade toluene. Detectors were normalized using 1 mg/ml BSA in PBS buffer. The size of the particles was determined as hydrodynamic radius from the QELS detector using Stokes-Einstein equation. From the MALS detector, using 14 detectors and using Debye second order model, the root-mean-square (RMS) radius was determined.

Immunization of animals

All animal experiments were conducted according to the principles enunciated in the Guide for the Care and Use of Laboratory Animals issued by the Czech Society for Laboratory Animal Science and reviewed by the local Ethic committee and approved by the Ministry of Agriculture of the Czech Republic (permit no. MZE-2334, 9487/2019-3). Water and standard pellet diet were given ad libitum.

Mice experiments were performed on 6-week-old female BALB/c mice (purchased from Laboratory Animal Breeding and Experimental Facility, MUNI, Czech Republic). Mice were divided into four groups (n = 5):

1. Group immunized with 50 µg purified VP1-PCV2bCap protein in sterile buffer (50 mmol l^− 1 Tris-HCl, 300 mmol l^− 1 NaCl, pH 7.4)

2. Group immunized with 50 µg of purified VP1-PCV2bCap protein in sterile buffer supplemented with 10% Emulsigen as adjuvant

3. Group immunized with 50 µg of purified VP1-PCV2bCap protein in sterile buffer supplemented with 50% Freund’s Incomplete Adjuvant

4. Control group injected only with a sterile buffer

To obtain VP1-PCV2bCap-specific hyperimmune serum, mice were immunized three times subcutaneously (s.c.) with a booster at 3 weeks and at 5 weeks after the first immunization. Blood was harvested before immunization, then at 3, 5, 7, and 10 weeks after the first immunization.

Piglet experiments were performed on 8–week-old piglets (purchased from Bioprodukt Knapovec a. s., Czech Republic). Piglets were divided into two groups:

4 piglets immunized with 50 µg of purified VP1-PCV2bCap protein in sterile Dublecco´s Phosphate Buffered Saline (Sigma-Aldrich) supplemented with 10% Emulsigen as adjuvant

2 control piglets injected only with a sterile Dublecco´s Phosphate Buffered Saline

Piglets were immunized twice intramuscularly (i.m.) with a booster applied at 3 weeks after the first immunization. Blood was harvested before immunization, 3 weeks, and 6 weeks after the first immunization.

ELISA analyses

Indirect ELISA analyses were realized by commercial kit INgezim Circo IgG according to the manufacturer’s instructions (Eurofins Technologies Ingensa, Spain). Briefly, collected sera were diluted in a ratio 1:400 (mice) or 1:200 (piglet) and incubated for 1 h at room temperature. After the 4-time washing, 100 µl of conjugate were pipetted to the each well of the plate and incubated for 30 min at room temperature [in the case of mice Goat-anti mouse IgG (Whole Molecule) Peroxidase Conjugate (Sigma-Aldrich, USA) in a dilution of 1:6000 was pipetted to the each well of the plate and incubated 2 h at room temperature]. After the 6-time washing, 100 µl of substrate was pipetted to the each well and incubated at room temperature. Reaction was stopped by 100 µl of stop solution and optical density was analyzed at 450 nm by Gen5™ Data Analysis Software (BioTek, USA).

Virus neutralization assay

PCV-free PK-15 cells (kindly provided by Gordon Allan, The Queen’s University Belfast, Belfast, Northern Ireland, UK) were maintained in Minimum Essential Medium (MEM, Biowest, France) supplemented with 10 % fetal bovine serum (FBS), 1 % penicillin, and 1 % streptomycin (all from Sigma-Aldrich) at 37 °C in humidify atmosphere containing 5% CO₂. Reference strain PCV2 Stoon 1010 (kindly provided by Gordon Allan, The Queen’s University Belfast, Belfast, Northern Ireland, UK) was passaged several times in PK-15 cells maintained in 2 % FBS, 1 % penicillin, and 1 % streptomycin culture medium for 72 h at 37 °C.

PK-15 cells were seeded into the IBIDI 8-well tissue culture threaded chambers (Ibidi, Germany) at a density of 2.5 × 10⁵ cells/ ml, with a volume of 300 μl/well in a complete medium. After 24 h, cell culture medium was removed, and cells were consequently pre-treated with 0.03 % nonionic surfactant Tween 20 in serum-free medium for 23 h. To allow attachment of antibodies to virus, diluted sera (1:2, 1:4, 1:8) of piglets 42 days post infection (dpi) immunized by 50 μg purified VP1-PCV2bCap protein with 10 % Emulsigen (group 1) or controls (group 2) were mixed in equal volume of 100 µl with viral inoculum (10^{– 3.2} TCID₅₀ . ml^-1) PCV2 Stoon 1010 and incubated for 1 h at RT. The mixture was then added to the pretreated cells with nonionic surfactant Tween 20 and incubated for 1 h at 37 °C with 5 % CO₂. Then the viral inoculum was washed off and PK-15 cells were further incubated for 72 h in cell culture medium with 2 % FBS [19]. The untreated and treated cells were fixed in 4 % v/v paraformaldehyde for 15 min at RT. PK-15 cells were permeabilized by 0.05 % Triton X-100 in PBS for 5 min at RT, washed three times with PBST₂₀ and blocked with 4 % bovine serum albumin in PBS for 30 min at 37° C. 125x diluted Alexa Fluor 647 conjugate primary antibodies (PCV2Cap H9, Exbio, Czech Republic) in blocking buffer (100 µl/well) were added to PK-15 cells and incubated for 1 h at 37°C. Cells were washed 3 times with PBST₂₀and the nuclei were stained with Hoechst (Enzo, USA) diluted 1:800 in PBS. PCV2-infected PK-15 cells were analyzed using the confocal fluorescence microscope Leica DMi8. PCV2 positive cells were counted and the result of virus neutralization assay was expressed as percentage of inhibition ratio of PCV2 infection, assay was performed in 3 independent experiments; 6 areas per well were counted.

Preparation of conjugate antibody

The monoclonal antibody PCV2Cap H9 (Exbio, Czech Republic) was labelled with the fluorescent probe Alexa Fluor™ 647 NHS Ester (Thermo Fisher Scientific, USA) according to the attached Protein Labeling Kit protocol. Briefly, fluorescence dye was dissolved in anhydrous dimethyl sulfoxide (DMSO) to the final concentration of 10 µg/µl. For labelling purposes, 500 µl of the monoclonal antibody PCV2Cap H9 at a concentration of 2 mg/ml in PBS was mixed with 50 µl of the 1M solution of sodium bicarbonate (pH 8.5). Then, 2 µl of the fluorescence dye solution was added to the final mixture. The reaction was carried out at RT and constant stirring for 1 h. After the conjugation, the unbound dye was removed using a spin column at 1.000 g for 2 minutes.

Statistical analysis

Statistical analyses were carried out using GraphPad Prism 8 software. Data were expressed as mean ± SEM. Statistical analyses of differences between groups were performed using Student’s t-test for comparison of two independent groups and one-way ANOVA with Bonferroni post hoc test for comparison of more than two groups. Values of P<0.05 were considered statistically significant.

Chimeric VP1-PCV2bCap protein was produced in Sf9 cells and purified under native conditions using CoNTA agarose as the large aggregates containing VP1 pentamers. Purified protein complex was analyzed by native-PAGE and Western blot analysis, detected by native anti-Cap antibody conjugated to horseradish peroxidase (HRP) (Fig. 1A). The molecular weight of VP1-PCV2bCap protein pentamer was 356 kDa - only band corresponding to a decamer of the chimeric protein was detected by native-PAGE, which is in agreement with Fraiberk et al. [18]. A large portion of the loaded sample of purified protein remained in the stacking gel identifying the presence of the aggregate complexes with a high molecular weight. To confirm the purity of the sample, protein eluted from the CoNTA was analyzed by SDS-PAGE and Western blot, detected by anti-His-tag antibody as a 70 kDa monomer of VP1-PCV2bCap protein (Fig. 1B).

Purified VP1-PCV2bCap protein aggregates were separated by Asymmetrical flow field-flow fractionation (AF4) to obtain fractions of pentamers and higher aggregates (Fig. 2). The presence of large protein aggregates and pentamers was confirmed by electron microscopy after the immuno-gold labeling with IgG PCV2Cap H9 antibody (Fig. 3).

Humoral immune response of mice immunized with purified VP1-PCV2bCap protein was analyzed at several time points after immunization with primary dose and two boosters (Fig. 5A). High levels of anti-PCV2b specific antibodies were detected in all groups already at 3 weeks after the first dose with the strongest immune response in mice immunized with VP1-PCV2bCap + Emulsigen (10%), as an adjuvant. This immune response was further increased after first booster (at 3 weeks) in mice immunized with VP1-PCV2bCap alone and VP1-PCV2bCap + Freund’s Incomplete Adjuvant (50%). Second booster applied at 5 weeks further enhanced the antibody levels in group of mice immunized with VP1-PCV2bCap + Emulsigen (10%), but not in the other groups. The levels of specific antibodies remained relatively stable for the next 5-weeks after the third immunization (Fig. 4B.). VP1-PCV2bCap protein in combination with Emulsigen (10%) as an adjuvant was shown to elicit the highest levels of antibodies, therefore this antigen was selected for verification of its efficiency in the target organism. Purified VP1-PCV2bCap + Emulsigen (10%) was then used for the prime/boost immunization of 8-week old piglets. Humoral immune response of piglets was monitored during 6 weeks (Fig. 5A). While the immune response after the first dose was poor, the levels of specific antibodies markedly increased after the booster applied at 3 weeks after the first dose (Fig. 5B). Production of effective neutralizing antibodies was further determined by neutralization assay. PCV2 virus inoculum was mixed with inactivated piglet sera collected 6 weeks after the first immunization dose and used for infection of porcine PK15 kidney cells. Infected cells treated with serum of the control group were used as a positive control. Non-infected cells were detected in samples treated with the mixture of the reference strain PCV2 Stoon 1010 virus neutralized with sera of piglets immunized by purified VP1-PCV2bCap protein supplemented with 10% Emulsigen as an adjuvant, non-treated PK-15 cells served as negative control (Fig. 6.).

Vaccination is one of the most effective control strategies for PCVD/PCVAD. Accessible commercial vaccines against PCV2 are based on the antigens from PCV2a with cross-reaction against PCV2b and PCV2d types [18, 19]. The efficacy of commercial vaccines against PCV2 has been verified in numerous studies demonstrating a high efficacy in both experimental and field conditions [20, 21]. However, the virus persists in immunized populations of pigs and causes subclinical infections with symptoms similar to PCVD/PCVAD. Therefore, novel and more effective vaccines need to be developed.

PCV2 Cap protein is the primary target for developing anti-PCV2 vaccines and serological diagnostic tests [14]. Several variants of recombinant Cap protein expressed in baculovirus expression systems have been developed for diagnostic purposes, such as ELISA assays [15], as well as for the production of subunit vaccines (e.g. Porcilis PCV, Schering-Plough/Merck; Circoflex, Boehringer Ingelheim; Circumvent, Intervent/Merck). Although in general, subunit protein vaccines are safe, their immunogenicity might be low [16]. In comparison with subunit protein vaccines, inactivated viruses can induce robust immune response and elicit high levels of serum antibodies against PCV2 infection [3]. The first commercial attenuated vaccine Circovac (Merial) represents an inactivated oil-adjuvanted vaccine with good immunogenicity. Chimeric PCV2 vaccines, for example, Suvaxyn (Fort Dodge Animal Health) or Fostera PCV (Pfizer Animal Health) represent another type of vaccination strategy. In our previous study, the experimental VarC chimeric vaccine containing mouse polyoma VP1 protein, additionally induces an immune response against VP1 protein apart from PCV2, and so may distinguish between vaccinated and naturally infected animals [18]. We used this strategy for the development of such antigen and further vaccination of mice in our experiments. VP1-PCV2bCap fusion protein was purified as pentamers and a complex of pentamers, generated by the interaction between two Cap-Cap proteins forming giant VP1 aggregates which leads to a complex chimeric protein structure. The regular morphology of VP1-Cap fusion protein VarC was described previously [18].

The minimal concentration of VP1-PCV2bCap protein necessary for the sufficient immune response was analyzed in mice immunized by purified VP1-PCV2bCap protein alone at a concentration of 10 µg and 25 µg per dose or in combination with Emulsigen (10%) (data not shown). Both doses moderately stimulated the humoral immune response after the first immunization but with no significant difference between each other. Stronger response was observed after the second immunization in groups of mice immunized by VP1-PCV2bCap protein alone and by a mixture containing Emulsigen. However, only the dose of 50 µg of VP1-PCV2bCap protein showed substantial effect (data not shown).

Chimeric VP1-PCV2bCap protein pentamers highly stimulated an immune response against PCV2 capsid protein after the second and third immunization in all immunized groups of mice as detected by the markedly increased levels of specific IgG antibodies. The levels of specific antibodies remained stable for the next 5 weeks after the second booster. We suppose that the robust immune response observed after the first immunization might be due to the large protein aggregates that greatly stimulate the immune system in immunized mice [22].

In contrast with mice, the dose of 50 µg of purified VP1-PCV2bCap protein in combination with Emulsigen (10%) only slightly increased levels of specific IgG antibodies in piglets after the first immunization. Surprisingly, a substantial increase in specific IgG antibodies was observed after the second immunization. These antibodies also had strong protective effect against PCV2 infection, which was confirmed by the neutralization assay. The mixture containing 50 µg of VP1-PCV2bCap protein in combination with Emulsigen (10%) as an adjuvant could be therefore used for the immunization of piglets. This vaccine candidate represents a promising vaccination strategy for piglet prevention against PCV2 infections.

In this study, we prepared and purified chimeric protein complex VP1-PCV2bCap that meets all requirements for quality and purity. VP1-PCV2bCap in combination with Emulsigen in prime/boost strategy showed the ability to elicit specific IgG antibodies against PCV2b in mice as well as in piglets as a target organism, therefore it has a high potential as recombinant protein vaccine against PCV2b infections.

Ethics approval and consent to participate

All the animal work was conducted according to the principles enunciated in the Guide for the Care and Use of Laboratory Animals issued by the Czech Society for Laboratory Animal Science and reviewed by the Ethical committee and approval by the Ministry of Agriculture of the Czech Republic (permit no. MZE-2334 and 9487/2019-3)

Consent for publication

All authors have read and agreed to the published version of the manuscript.

Competing interests

The authors declare no competing financial interest.
Availability of data and materials Not applicable
Funding

This work was supported by the Ministry of Education, Youth and Sports of Czech Republic, project "Recombinant Biotechnology and Immunotherapy Centre" CZ.02.1.01/0.0/0.0/16_025/0007397. We also thanks Czech Ministry of Agriculture, project RO0518 for financial support.
Authors' contributions

All authors: investigation; A.V. writing - original draft; R.H., M.F., I.P., V.B. writing - review & editing; I.P. R.H. A.V. methodology, supervision; A.V., V.B., R.H., P.K. figures; V.B. and R.H. statistics; J.M.and I.P. project administration,
Acknowledgements Not applicable

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No competing interests reported.

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Development of modern immunizing agent against porcine circovirus type 2 infection based on chimeric VP1-PCV2bCap recombinant protein

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Insertion of sequence of porcine circovirus 2b (PCV2b) capsid protein into pFastBacI –VP1_DAɅ7 transfer vector

Recombinant baculovirus preparation

Expression and purification of chimeric protein from Sf9 insect cells

Nano Gold labeling

Asymmetrical flow field-flow fractionation (AF4)

Immunization of animals

ELISA analyses

Results

Discussion

Conclusions

Declarations

References

Additional Declarations

Status:

Version 1

Development of modern immunizing agent against porcine circovirus type 2 infection based on chimeric VP1-PCV2bCap recombinant protein

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Insertion of sequence of porcine circovirus 2b (PCV2b) capsid protein into pFastBacI –VP1DAɅ7 transfer vector

Recombinant baculovirus preparation

Expression and purification of chimeric protein from Sf9 insect cells

Nano Gold labeling

Asymmetrical flow field-flow fractionation (AF4)

Immunization of animals

ELISA analyses

Results

Discussion

Conclusions

Declarations

References

Additional Declarations

Status:

Version 1

Insertion of sequence of porcine circovirus 2b (PCV2b) capsid protein into pFastBacI –VP1_DAɅ7 transfer vector