Identification of one critical amino acid that determines a conformational neutralizing epitope in the capsid protein of porcine circovirus type 2

Abstract

Background: Porcine circovirus type 2 (PCV2) is associated with post-weaning multisystemic wasting syndrome (PMWS) in pigs. Currently, there is considerable interest in the immunology of PCV2; in particular, the immunological properties of the capsid protein. This protein is involved in PCV2 immunogenicity and is a potential target for vaccine development. In this study, we identified one critical amino acid that determines a conformational neutralizing epitope in the capsid protein of PCV2.

Results: One monoclonal antibody (mAb; 8E4), against the capsid protein of PCV2, was generated and characterized in this study. 8E4 reacted with the genotype PCV2a (CL, LG and JF2) strains but not PCV2b (YJ, SH and JF) strains by an immunoperoxidase monolayer assay (IPMA) and a capture ELISA. Furthermore, the mAb had the capacity to neutralize PCV2a (CL, LG and JF2) strains but not PCV2b (YJ, SH and JF) strains. One critical amino acid that determined a conformational neutralizing epitope was identified using mAb 8E4 and PCV2 infectious clone technique. Amino acid residues 47-72 in the capsid protein of PCV2a/CL were replaced with the corresponding region of PCV2b/YJ, and the reactivity of mAb 8E4 was lost. Further experiments demonstrated that one amino acid substitution, the alanine for arginine at position 59 (A59R) in the capsid protein of PCV2a (CL, LG and JF2) strains, inhibited completely the immunoreactivity of three PCV2a strains with mAb 8E4.

Conclusions: It is concluded that the alanine at position 59 in the capsid protein of PCV2a (CL, LG and JF2) strains is a critical amino acid, which determines one neutralizing epitope of PCV2a (CL, LG and JF2) strains. This study provides valuable information for further in-depth mapping of the conformational neutralizing epitope, understanding antigenic difference among PCV2 strains, and development of a useful vaccine for control of PCV2-associated disease.

Figure 1

Schematic diagram of PCV2 ORF2 chimeras and mutants and their reactivity with PCV2-positive serum and mAb 8E4. (a) PCV2 clones rCL-ORF2 and rYJ-ORF2 contained the complete 233 aa sequence of PCV2a/CL-ORF2 and the 235 aa sequence of PCV2b/YJ-ORF2, respectively. Five chimeras that consisted of aa 47-72 (rCL-YJ-1), 80-94 (rCL-YJ-2), 110-154 (rCL-YJ-3), 190-210 (rCL-YJ-4) and 230-235 (rCL-YJ-5) of the PCV2a/CL capsid protein were replaced with corresponding regions of the PCV2b/YJ capsid protein. (b) For the second set of constructs, four mutants containing single amino acid mutations of PCV2a/CL-ORF2 at positions 51 (rCL-YJ-1-51), 57 (rCL-YJ-1-57), 59 (rCL-YJ-1-59) and 63 (rCL-YJ-1-63) were substituted for the corresponding amino acids of the PCV2b/YJ capsid protein at the same position. (c) PCV2 clones rLG-ORF2 and rJF2-ORF2 contained the complete 233 aa sequences of PCV2a/LG-ORF2 and the 236 aa sequences of PCV2a/JF2-ORF2, respectively. The PCV2 mutants rLG-YJ-1-59 and rJF2-YJ-1-59 contained a single amino acid mutation from alanine to arginine at position 59 (A59R) in the capsid protein of PCV2a/LG and PCV2a/JF2, respectively. The last mutant rYJ-CL-1-59 contained a single amino acid mutation of arginine for alanine at position 59 (R59A) in the capsid protein of PCV2b/YJ. The IPMA reactivity between each antibody and PK-15 cells transfected with each PCV2 construct is indicated next to each construct. The IPMA reactivity of the constructs in transfected PK-15 cells was demonstrated by PCV2-positive serum and mAb 8E4. +: Positive; -: Negative.

Figure 2

Analysis of immunoreactivity of mAb by western blot analysis. Purified virions of the PCV2a/LG strain were separated by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with mAb. Lane M: protein molecular weight markers; lane 1: mAb 8E4; lane 2: mAb 6F10 as a positive control; lane 3: SP2/0 supernatant as a negative control.

Figure 3

Reactivity of six PCV2 isolates with mAb 8E4 by the IPMA, serum neutralization assay and capture ELISA. (a) IPMA reactivity of PK-15 cells inoculated with PCV2a/LG (1 and 2), PCV2a/CL (3 and 4), PCV2a/JF2 (5 and 6), PCV2b/YJ (7 and 8), PCV2b/SH (9 and 10) and PCV2b/JF (11 and 12), against PCV2-positive serum and mAb 8E4. Odd numbers represent PCV2-positive serum, whereas even numbers show mAb 8E4. (b) The neutralizing activity of mAb 8E4 was expressed as the percentage reduction in the number of infected cells in comparison with negative control. A mean neutralizing activity of > 50% was considered to represent neutralization. Error bars represent the standard deviations. (c) For the capture ELISA, cultures of six PCV2 isolates, recPCV1/G and PK-15 cells were tested with HRP-conjugated 8E4. P/N > 2.1 was regarded as a positive result. Error bars represent the standard deviations.

Figure 4

Predicted amino acid alignment of the capsid protein of PCV2 strains used in this study. Boxes show residues that differ from the consensus. The consensus was used as the majority sequence for this alignment.

Figure 5

IPMA reactivity between mAb 8E4 and each chimera or mutant.(a) rCL-YJ-1; (b) rCL-YJ-2; (c) rCL-YJ-3; (d) rCL-YJ-4; (e) rCL-YJ-5; (f) rCL-YJ-1-51; (g) rCL-YJ-1-57; (h) rCL-YJ-1-59; (i) rCL-YJ-1-63; (j) rLG-YJ-1-59; (k) rJF2-YJ-1-59; (l) rYJ-CL-1-59.