Generation in yeast of recombinant virus-like particles of porcine circovirus type 2 capsid protein and their use for a serologic assay and development of monoclonal antibodies

Abstract

BackgroundPorcine circovirus type 2 (PCV2) is considered to be an important emerging pathogen associated with a number of different syndromes and diseases in pigs known as PCV2-associated diseases. It has been responsible for significant mortality among pigs and remains a serious economic problem to the swine industry worldwide leading to significant negative impacts on profitability of pork production.ResultsIn this study we have demonstrated that PCV2 capsid (Cap) protein based virus-like particles (VLPs) were efficiently produced in yeast S. cerevisiae and induced production of monoclonal antibodies (MAbs) reactive with virus-infected cells. Moreover, PCV2 Cap VLPs served as a highly specific recombinant antigen for the development of an indirect IgG PCV2 Cap VLP-based ELISA for the detection of virus-specific IgG antibodies in swine sera. Four hundred-nine serum samples collected from pigs in Lithuania were tested for PCV2-specific IgG to determine the sensitivity and specificity of the newly developed ELISA in parallel using a commercial SERELISA test as a gold standard. From 409 tested serum samples, 297 samples were positive by both assays. Thirty-nine sera from 112 serum samples were determined as negative by SERELISA but were found to be positive both in the newly developed indirect IgG PCV2 Cap VLP-based ELISA and the PCR test.ConclusionsWe have demonstrated that S. cerevisiae expression system is an alternative to insect/baculovirus expression system for production of homogenous in size and shape PCV2 Cap protein-based VLPs similar to native virions. Yeast expression system tolerated native virus genes encoding PCV2 Cap protein variants as well as the codon-optimized gene. Moreover, yeast-derived PCV2 Cap VLPs were capable to induce the generation of PCV2-specific MAbs that did not show any cross-reactivity with PCV1-infected cells. The high sensitivity and specificity of the indirect IgG PCV2 Cap VLP-based ELISA clearly suggested that this assay is potentially useful diagnostic tool for screening PCV2–suspected samples.

Immunogenicity of plant‐produced porcine circovirus‐like particles in mice

“…The production and use of VLP‐based vaccines are advantageous compared to subunit protein(s), as they provide the efficacy of whole‐killed vaccines but do not have the safety concerns associated with production and inactivation of virulent live virus. VLPs made in insect‐, yeast‐ and bacterial cell‐based production systems are capable of eliciting humoural‐ and cell‐mediated immunity in experimental animal trials (Li et al ., ; Nainys et al ., ; Xi et al ., ). Live attenuated vaccines elicit strong immune responses but carry a risk of reversion to virulence, whereas inactivated vaccines are safer to use if not to make, but are generally not as effective (Chambers et al ., ).…”

Self-assembly into virus–like particles of the recombinant capsid protein of porcine circovirus type 3 and its application on antibodies detection

“…Some VLPs, such as Hepatitis B surface antigen (HBsAg) VLPs, human papillomavirus (HPV) VLPs and Malaria VLPbase vaccines have already been clinically used for the prevention of infectious diseases (Kim and Kim 2017a). In recent years, the technology of expressing capsid protein (Cap) of PCV2 and self-assembly into virus-like particles (VLPs) has advanced, and can be used in multiple recombinant protein expression systems including baculovirus, yeast and E. coli (Liu et al 2008;Nainys et al 2014;Wu et al 2016). Many commercial vaccines based on VLPs have effectively prevented the infection of PCV2 (Beach and Meng 2012).…”

“…The enzyme-linked immunosorbent assay (ELISA) based on VLPs is widely used to measure antibodies or the neutralizing epitopes (Almanza et al 2008;Chao et al 2019). PCV2 VLPs as coating antigens can detect serum neutralizing antibodies (SNAbs) in ELISA (Nainys et al 2014;Zhang et al 2016). Some reports show that PCV3 infection in pigs do not present any significant clinical signs or symptoms and wild boars may also have susceptibility (Franzo et al 2018b;Klaumann et al 2019;Zheng et al 2017), thus it is particularly important to establish an effective antibody detection method to assess the PCV3 infection in pigs.…”

“…The enzyme-linked immunosorbent assay (ELISA) based on VLPs is widely used to measure antibodies or neutralizing epitopes (Almanza et al 2008;Chao et al 2019). For example, PCV2 VLPs are used as coating antigens to be able to detect serum neutralizing antibodies (SNAbs) in ELISA (Nainys et al 2014;Zhang et al 2016). Currently, PCV3 Cap proteins harvested from insect cells or E. coli have been used as antigens to detect PCV3-specific antibodies in an ELISA (Deng et al 2018;Zhang et al 2019).…”

Establishment of a Rep′ protein antibody detection method to distinguish natural infection with PCV2 from subunit vaccine immunization

“…Furthermore, PCV2 competitive ELISA (c-ELISA), established with the specific PCV2 monoclonal antibody as a competitor, showed better sensitivity and specificity than indirect IFA [27]. In addition, there are many ELISA methods that use recombinant PCV2 Cap protein as a coating antigen [21,22,[28][29][30]. All of these findings indicate that ELISA is widely used.…”