Protective immunity against porcine circovirus 2 by vaccination with ORF2-based DNA and subunit vaccines in mice

Abstract

The protective immune response against porcine circovirus 2 (PCV2) infection in mice was characterized using flow cytometric analysis (FCM), assays of antibody (of different IgG isotypes) and viraemia, and histopathological examination. An open reading frame 2 plasmid (pORF2) and the capsid protein (Cap) of PCV2 were used as DNA and subunit vaccines, respectively. In FCM analysis, although pORF2 and Cap alone showed comparable efficacy in eliciting lymphoproliferative responses and Cap-specific CD4 + T cells, pORF2 was superior to the Cap protein in triggering CD8 + T cells. A virus neutralization assay showed that pORF2 evoked stronger recall virus-neutralizing (VN) antibody responses than the Cap protein on PCV2 challenge. Correspondingly, VN antibody kinetics coincided with those of Cap-specific IgG2a, but not with the kinetics of IgG and IgG1. Following virus challenge, real-time PCR and histopathological analysis confirmed that only low viral DNA loads and mild microscopic lesions appeared in pORF2-immunized mice. These findings indicate that CD8 + T cells and VN antibody responses correlating mainly with Cap-specific IgG2a play crucial roles in protecting against PCV2 infection, and that the protective immunity induced by the pORF2 plasmid is superior to that induced by the PCV2 Cap protein. INTRODUCTIONPost-weaning multisystemic wasting syndrome (PMWS), first recorded in pigs in Western Canada in 1991, has been described in pigs from many regions of the world (Allan et al., 1998b;Allan & Ellis, 2000;Clark, 1997;Wen et al., 2005;Zhou et al., 2006). PMWS affects pigs of 5-12 weeks of age, with a morbidity of 5-30 %; it is characterized by weight loss, dyspnoea and jaundice. Field and experimental evidence has revealed that PMWS-affected pigs may develop severe immunosuppression (Segales et al., 2004).Porcine circovirus 2 (PCV2) has been identified as the primary aetiological agent of PMWS (Allan et al., 1998a, b;Allan & Ellis, 2000;Ellis et al., 1998;Fenaux et al., 2002;Meehan et al., 1998). PCV2 is a non-enveloped, singlestranded, circular DNA virus with a diameter of 17 nm (Tischer et al., 1982). The PCV2 genome comprises 1767 or 1768 nt and is assumed to have 11 potential open reading frames (ORF1-11;Hamel et al., 1998;Zhou et al., 2006). Previous studies have demonstrated that ORF1, -2 and -3 encode a 35.7 kDa replication (Rep) protein involved in virus replication (Mankertz et al., 1998), a 27.8 kDa capsid (Cap) protein involved in PCV2 immunogenicity (Mahe et al., 2000; Nawagitgul et al., 2000;Truong et al., 2001;Zhou et al., 2005a) and a protein involved in PCV2-induced apoptosis , respectively.Immunization against PCV2 has been studied intensely and found to be the most effective strategy for protecting pigs against PCV2 infection. Based on comparison of the immunogenicity of the PCV2 Cap and Rep proteins, Blanchard et al. (2003) demonstrated that the ORF2-encoded Cap protein was the major immunogen, whilst the ORF1-encoded Rep protein was only weakly immunogenic. Kamstrup et al. (2004) subsequently...

Identification and Functional Analysis of the Novel ORF4 Protein Encoded by Porcine Circovirus Type 2

“…In previous studies, BALB/c mice inoculated with wPCV2 showed microscopic lesions similar to those found in wPCV2-infected pigs (20,26,51). Therefore, we used the mouse model in the present study to compare PCV2⌬ and wPCV2 infection in vivo.…”

“…Viral DNA levels in the mouse serum samples collected after wPCV2 or PCV2⌬ challenge were determined using quantitative real-time PCR as previously described (26). Briefly, total DNA was extracted from 100 l of serum by using a UNlQ-10 column virus genomic DNA isolation kit (Sangon Biotech Co., Ltd., Shanghai, China) by following the manufacturer's instructions.…”

“…One part, fixed in 10% neutral buffered formalin, was used for pathological and immunohistochemical (IHC) analyses. The pathological analysis was performed according to a previously described method (26). Briefly, formalin-fixed spleens were sectioned and stained with hematoxylin and eosin (H&E).…”

“…The PCV2-ORF3 polyclonal antibody (PAb) was a kind gift from Jimmy Kwang of the Animal Health Biotechnology Group, Temasek Life Sciences Laboratory, National University of Singapore (25). Eukaryotic expression plasmids pCI-neo-PCV2-ORF1, -ORF2, -ORF3 (23,26), and -ORF4, as well as the plasmids pEGFP-PCV2-ORF1, -ORF2, -ORF3, -ORF4, and pEGFP-PCV1-ORF4 (our unpublished constructs), were maintained in our laboratory. Seven-week-old specificpathogen-free (SPF) BALB/c mice were purchased from the Shanghai Laboratory Animal Center, Chinese Academy of Sciences, Shanghai, China.…”

Immune Responses of Piglets Immunized by a Recombinant Plasmid Containing Porcine Circovirus Type 2 and Porcine Interleukin-18 Genes

“…Total serum immunoglobulin G (IgG) specific for PCV2 was measured by enzyme-linked immunosorbent assay (ELISA) according to a previous described method (31). Briefly, ELISA plates were coated overnight at 4°C with PCV2 lysates as an antigen, and then blocked with 5% skim milk.…”