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      Exploitation of stable nanostructures based on the mouse polyomavirus for development of a recombinant vaccine against porcine circovirus 2

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          Abstract

          The aim of this study was to develop a suitable vaccine antigen against porcine circovirus 2 (PCV2), the causative agent of post-weaning multi-systemic wasting syndrome, which causes significant economic losses in swine breeding. Chimeric antigens containing PCV2b Cap protein sequences based on the mouse polyomavirus (MPyV) nanostructures were developed. First, universal vectors for baculovirus-directed production of chimeric MPyV VLPs or pentamers of the major capsid protein, VP1, were designed for their exploitation as vaccines against other pathogens. Various strategies were employed based on: A) exposure of selected immunogenic epitopes on the surface of MPyV VLPs by insertion into a surface loop of the VP1 protein, B) insertion of foreign protein molecules inside the VLPs, or C) fusion of a foreign protein or its part with the C-terminus of VP1 protein, to form giant pentamers of a chimeric protein. We evaluated these strategies by developing a recombinant vaccine against porcine circovirus 2. All candidate vaccines induced the production of antibodies against the capsid protein of porcine circovirus after immunization of mice. The candidate vaccine, Var C, based on fusion of mouse polyomavirus and porcine circovirus capsid proteins, could induce the production of antibodies with the highest PCV2 neutralizing capacity. Its ability to induce the production of neutralization antibodies was verified after immunization of pigs. The advantage of this vaccine, apart from its efficient production in insect cells and easy purification, is that it represents a DIVA (differentiating infected from vaccinated animals) vaccine, which also induces an immune response against the mouse polyoma VP1 protein and is thus able to distinguish between vaccinated and naturally infected animals.

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          Most cited references52

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          Virus-like particles as a highly efficient vaccine platform: Diversity of targets and production systems and advances in clinical development

          Highlights ► Virus-like particles (VLPs) are a class of recombinant subunit vaccines. ► VLPs resemble native viruses but lack infectious genetic material. ► VLPs are promising vaccines due to strong immunogenicity and safety. ► VLPs can be produced in prokaryotic or eukaryotic expression systems, or in vitro. ► VLP-based vaccine candidates targeting many diseases are in clinical development.
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            Experimental reproduction of postweaning multisystemic wasting syndrome in pigs by dual infection with Mycoplasma hyopneumoniae and porcine circovirus type 2.

            The objectives of this study were to investigate the interactions between Mycoplasma hyopneumoniae and porcine circovirus type 2 (PCV2) and to establish a model for studying the pathogenesis of and testing intervention strategies for the control of PCV2-associated porcine respiratory disease complex (PRDC). Sixty-seven pigs were randomly assigned to four groups. Group 1 (n=17) pigs served as controls, group 2 (n=17) pigs were inoculated with M. hyopneumoniae, group 3 (n=17) pigs were dual infected with M. hyopneumoniae and PCV2, and group 4 (n=16) pigs were inoculated with PCV2. Pigs were inoculated intratracheally with M. hyopneumoniae at 4 weeks of age followed by intranasal inoculation with PCV2 at 6 weeks of age. Dual-infected pigs had moderate dyspnea, lethargy, and reduced weight gain. The overall severity of macroscopic lung lesions, PCV2-associated microscopic lesions in lung and lymphoid tissues, and the amount of PCV2-antigen associated with these lesions were significantly (P <0.05) higher in dual-infected pigs compared with all other groups. Four of 17 (23.5%) dual-infected pigs had decreased growth rate and severe lymphoid depletion and granulomatous lymphadenitis associated with high amounts of PCV2-antigen consistent with postweaning multisystemic wasting syndrome (PMWS). PCV2-antigen in lung tissue was most often associated with M. hyopneumoniae-induced peribronchial lymphoid hyperplasia, suggesting that this is an important site for PCV2 replication in the lung. This study indicates that M. hyopneumoniae potentiates the severity of PCV2-associated lung and lymphoid lesions, increases the amount and prolongs the presence of PCV2-antigen, and increases the incidence of PMWS in pigs.
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              Genomic analysis of PCV2 isolates from Danish archives and a current PMWS case-control study supports a shift in genotypes with time.

              Porcine circovirus type 2 (PCV2) is the primary cause of Postweaning Multisystemic Wasting Syndrome (PMWS) in pigs. PCV2, however, is found in both PMWS-affected herds and non-affected herds. The objective of this study was to clarify if PCV2 genome nucleotide sequences isolated from pigs from PMWS-affected herds and non-affected herds cluster phylogenetically in two separate groups. All isolates (45) belonged to PCV2 group 1 and shared a nucleotide sequence identity of 99.4-100% indicating a very homogeneous PCV2 population in Denmark. Phylogenetic analysis of the PCV2 isolates revealed no distinctive clustering of case- and control-herds suggesting that there is no link between PCV2 sequences and herd disease status. The appearance of only PCV2 group 1 isolates in this study (isolates from 2003/2004) led us to determine if PCV2 nucleotide sequences had changed in Denmark over time. Interestingly, all PCV2 isolates from before the first outbreak of PMWS (2001) belonged either to a new PCV2 group identified for the first time in this study and named group 3 (isolates from 1980, 1987 and 1990) or PCV2 group 2 (isolates from 1993 and 1996). The shift from PCV2 group 2 to 1 was confirmed on a more global scale by placing all full genome PCV2 sequences submitted to GenBank from 1997 to 2006 in either of the groups by phylogenetic analysis. The analysis showed that the shift happened in 2003 or even earlier. This may indicate that PCV2 group 1 is a more adapted form of PCV2 and possibly could be more pathogenic.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: Investigation
                Role: MethodologyRole: SupervisionRole: Validation
                Role: Formal analysisRole: Investigation
                Role: MethodologyRole: Supervision
                Role: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: Validation
                Role: ConceptualizationRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                18 September 2017
                2017
                : 12
                : 9
                : e0184870
                Affiliations
                [1 ] Charles University, Faculty of Science, Prague, Czech Republic
                [2 ] Dyntec spol s r.o., Terezín, Czech Republic
                Sun Yat-Sen University, CHINA
                Author notes

                Competing Interests: We have the following interests: IP is an employee of Dyntec spol. s r.o. All the authors declared that no competing interest exist. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials. Prepared nanostructure, Var C, is registered under the name “Vaccine based on chimeric protein nanoparticle against Porcine circovirus 2” as Design and utility model n. 29310, 2016, by Industrial Property Office, Czech Republic. National (Czech) patent application N. 2016 - 37 "Vakcína založená na proteinové chimerické nanočástici proti prasečímu cirkoviru 2" (Vaccine based on chimeric protein nanostructure against porcine circovirus 2) Announced patent application ( https://isdv.upv.cz/webapp/webapp.pts.det?xprim=10173520&lan=cs&s_majs=&s_puvo=&s_naze=&s_anot=), pending. There are no further patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

                Author information
                http://orcid.org/0000-0003-0219-506X
                Article
                PONE-D-17-14449
                10.1371/journal.pone.0184870
                5602543
                28922413
                f4bb1f56-6600-49f1-ac74-b6159a150367
                © 2017 Fraiberk et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 13 April 2017
                : 3 September 2017
                Page count
                Figures: 9, Tables: 0, Pages: 23
                Funding
                Funded by: Dyntec spol. s r. o.; www.dyntec.cz
                Award Recipient :
                Funded by: Technology Agency of the Czech Republic, www.tacr.cz
                Award ID: TA03010700
                Award Recipient :
                Funded by: Specific University research grant, http://www.cuni.cz/UKEN-65.html
                Award ID: SVV-2017-260426
                Funded by: Ministry of Education, Youth and Sports of CR within the National Sustainability Program II (Project BIOCEV-FAR), www.msmt.cz
                Award ID: LQ1604
                Funded by: Ministry of Education, Youth and Sports of CR, www.msmt.cz
                Award ID: ‘‘BIOCEV’’(CZ.1.05/1.1.00/02.0109)
                This work was supported by Technology Agency of the Czech Republic, TACR, project no. TA03010700, www.tacr.cz (MF, JF, ADM, IP); Specific University Research Grant of the Ministry of Education, Youth and Sports of CR, project no. SVV-2017-260426, www.msmt.cz (MF); National Sustainability Program II (Project BIOCEV-FAR) and by the project ‘‘BIOCEV’’ the Ministry of Education, Youth and Sports of CR, project no. LQ1604 and CZ.1.05/1.1.00/02.0109, www.msmt.cz (MF, JF); and Dyntec spol. s r.o., www.dyntec.cz (IP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. IP is an employee of Dyntec spol s r.o. Dyntec spol. s r.o. provided support in the form of salary for IP, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific role of this author is articulated in the “author contributions section”.
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