Amino acid mutations in the capsid protein produce novel porcine circovirus type 2 neutralizing epitopes

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Abstract

Porcine circovirus type 2 (PCV2) is associated with postweaning multisystemic wasting syndrome in pigs. A monoclonal antibody (mAb) 8E4 against the PCV2 capsid protein has the capacity to neutralize the virus. However, this mAb can only react with some PCV2a strains (LG, CL, and JF2; mAb 8E4-positive strains), but does not cross-react with some PCV2b strains (YJ and JF; mAb 8E4-negative strains). In the present study, site-directed mutagenesis was performed targeting the external amino acids of the capsid proteins, which are different between mAb 8E4-positive and -negative strains. A mutation of arginine to alanine at position 59 in the capsid protein of strain JF allowed the mutant to be recognized and neutralized by mAb 8E4. Likewise, mutations of arginine to alanine at position 59 together with alanine to threonine at position 60 in the capsid protein of the YJ strain resulted in a gain of neutralization and recognition by mAb 8E4. Here, we demonstrated that the amino acids at positions 59 and 60 in the capsid protein of PCV2 participate in the formation of conformational neutralizing epitopes and mutations at positions 59 or 59/60 result in novel neutralizing epitopes of mAb 8E4-negative strains. This study provides valuable information for further in-depth mapping of the conformational neutralizing epitopes, clarification of antigenic differences among PCV2 strains, and development of a useful vaccine candidate for control of PCV2-associated diseases.

Introduction

Porcine circovirus (PCV) is the smallest virus that replicates autonomously in mammalian cells and two types have been characterized, PCV1 and PCV2. PCV1 is a persistent contaminant of porcine kidney (PK)-15 cell lines and is not considered to be pathogenic (Tischer et al., 1986), whereas PCV2 is the causative agent of several economically important porcine circovirus-associated diseases (Gillespie et al., 2009), of which postweaning multisystemic wasting syndrome in weaned pigs (Kennedy et al., 2000, Albina et al., 2001, Bolin et al., 2001) and reproductive failure in sows (Park et al., 2005, Rose et al., 2007, Madson et al., 2009, Saha et al., 2010) have been experimentally reproduced with PCV2 alone.

The covalently closed circular PCV2 genome consists of 1766–1769 nucleotides (nt) and contains at least three open reading frames (ORFs) (Huang et al., 2011a, Mankertz et al., 2004, Liu et al., 2005, Liu et al., 2006). ORF1 codes for the non-structural replication-associated protein Rep and its spliced frame-shifted variant Rep’ (Cheung, 2003, Mankertz et al., 2003). ORF2 codes for the only structural capsid protein (Nawagitgul et al., 2000) and the capsid protein is involved in immunogenicity. The non-structural ORF3 protein has been associated with apoptosis in vitro and viral pathogenesis in vivo (Liu et al., 2005, Liu et al., 2006).

The PCV2 capsid protein consists of 233–235 amino acids (aa) (Nawagitgul et al., 2000, Olvera et al., 2007, Huang et al., 2011a) and several antigenic domains have been identified on the PCV2 capsid protein as revealed by porcine polyclonal antibodies (Mahé et al., 2000, Truong et al., 2001) or mouse monoclonal antibodies (mAbs) (Lekcharoensuk et al., 2004, Shang et al., 2009). At least five overlapping conformational epitopes of the PCV2 capsid protein, within residues 47–85, 165–200, and 230–233, have been mapped in chimeric PCV1 and PCV2 (Lekcharoensuk et al., 2004). Conformational epitopes at residues 231–233 recognized by mAbs with neutralizing activity against PCV2 have been identified in transfected PK-15 cells (Shang et al., 2009).

Phylogenetic analysis distinguishes three genotypes of PCV2 (a, b, and c) (Segalés et al., 2008). PCV2a and PCV2b are found in many countries, whereas PCV2c has only been found in Denmark (Dupont et al., 2008). Recently, epidemiological studies in many countries have linked a shift from infection with PCV2a to PCV2b (Dupont et al., 2008, Cheung et al., 2007, Gagnon et al., 2007, Wiederkehr et al., 2009). Genotype- and cluster-specific domains on the capsid protein have also been identified using mouse mAbs generated against genotype PCV2a or PCV2b (Cheung et al., 2007, Cheung and Greenlee, 2011, Saha et al., 2012a, Huang et al., 2011a). However, mouse mAbs directed against PCV2 have shown some differences in reactivity with different PCV2 strains (Lefebvre et al., 2008, Shang et al., 2009).

Several authors have described mAbs with neutralizing activity against the PCV2 capsid protein (McNeilly et al., 2001, Lekcharoensuk et al., 2004, Zhou et al., 2005, Lefebvre et al., 2008, Shang et al., 2009). Recently, Huang et al. (2011a) demonstrated that PCV2 isolates (PCV2a/LG, PCV2a/CL, and PCV2a/JF2) were recognized and neutralized by mAb 8E4 using the immunoperoxidase monolayer assay (IPMA), but PCV2 isolates (PCV2b/YJ, PCV2b/JF, and PCV2b/SH) could not been recognized or neutralized by mAb 8E4. Further experiments demonstrated that one amino acid substitution, alanine (A) for arginine (R) at position 59 (A59R) in the capsid protein of PCV2a (CL, LG, and JF2) strains, completely inhibited the reactivity of three PCV2a strains with mAb 8E4. However, an R  A substitution at position 59 (R59A) in the capsid protein of PCV2b/YJ did not induce recognition or neutralization by mAb 8E4. Therefore, we investigated which aa change(s) in the capsid protein of PCV2b (YJ and JF) strains caused the switch from IPMA-negative and neutralization-negative (IPMA-N-) to IPMA-positive and neutralization-positive (IPMA+N+) with mAb 8E4. Hence, the aim of this study was to identify specific aa change(s) in the capsid protein responsible for the switch from IPMA-N- to IPMA+N+ using mAb 8E4 in PCV2b (YJ and JF) strains.

Section snippets

Cells, viruses and antibodies

A porcine kidney PK-15 cell line free of PCV1 contamination that is highly permissive for PCV2 replication was maintained in a minimum essential medium (MEM) (Invitrogen, Grand Island, NY, USA) supplemented with 5% heat-inactivated fetal bovine serum (FBS) (Invitrogen) at 37 °C under an atmosphere of 5% CO2. Strains PCV2a/CL (which could be neutralized by mAb 8E4), PCV2b/JF, and PCV2b/YJ (which could not be neutralized by mAb 8E4) were kept in our laboratory and were selected as the control

Analysis of ORF2 from different PCV2 strains to identify the aa that likely affect the conformational epitope of mAb 8E4

The similarities between the capsid proteins of three different strains (PCV2a/CL, PCV2b/YJ and PCV2b/JF) used in this study were determined using pairwise alignments and the Clustal W algorithm (www.clustal.org/) based on the methods of Huang et al. (2011a). The results revealed a high amino acid identity of the capsid protein among isolates of PCV2a (≥95.7%) and PCV2b (≥96.6%), respectively, while there was only 88–90.2% amino acid identity of the capsid protein between PCV2a and PCV2b. On

Discussion

In the present study, we demonstrated that single aa mutations in the PCV2 capsid protein result in a switch from IPMA-N- to IPMA+N+. For the mAb 8E4 analysis, the R  A mutation at position 59 in the capsid protein of PCV2b/JF resulted in a complete gain of recognition and neutralization. This clearly showed that single aa change in the capsid protein may switch the neutralization phenotype of PCV2, hence the aa at position 59 seems to play a critical role in the neutralization capacity of mAb

Conflict of interest statement

None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.

Acknowledgments

This study was supported by the National High Technology R&D program (863) of China (no. 2011AA10A208) and the Public Welfare Special Funds for Agricultural Scientific Research (no. 201203039) and the National Natural Science Foundation of China (no. 31101837), the State Key Laboratory of Veterinary Biotechnology (no. SKLVBP201203).

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