Protective immunity against porcine circovirus 2 by ... - Semantic Scholar

Journal of General Virology (2008), 89, 1857–1865

DOI 10.1099/vir.0.2008/000125-0

Protective immunity against porcine circovirus 2 by vaccination with ORF2-based DNA and subunit vaccines in mice Hui-Gang Shen,1,2 Ji-Yong Zhou,1,2 Zhen-Yu Huang,1 Jun-Qing Guo,1,2 Gang Xing,1 Jia-Ling He,1 Yan Yan1,2 and Li-Yang Gong1,2 Correspondence Ji-Yong Zhou [email protected]

Received 5 January 2008 Accepted 20 March 2008

1

Laboratory of Virology and Immunology, Institute of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310029, PR China

2

State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University, Hangzhou 310003, PR China

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.

INTRODUCTION Post-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 2008/000125 G 2008 SGM Printed in Great Britain

et al., 2000; Nawagitgul et al., 2000; Truong et al., 2001; Zhou et al., 2005a) and a protein involved in PCV2induced apoptosis (Liu et al., 2005), 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 ORF2encoded Cap protein was the major immunogen, whilst the ORF1-encoded Rep protein was only weakly immunogenic. Kamstrup et al. (2004) subsequently investigated the potential of a DNA vaccine encoding the PCV2 Cap protein in mice and demonstrated the production of antibody. On the other hand, Fenaux et al. (2003, 2004) focused on a chimeric PCV1–PCV2, which was shown to induce specific antibody responses and protection against PCV2 in pigs. Recent studies on live virus vectors have shown that a recombinant pseudorabies virus (Ju et al., 2005; Song et al., 2007) and adenovirus (Wang et al., 2006, 2007) expressing Cap protein can elicit specific humoral and/or lymphocyte proliferation responses in mice and/or pigs, encouraging further studies of PCV2 vaccines. 1857

H.-G. Shen and others

However, the mechanisms underlying the immune response and protection against PCV2 have not yet been fully characterized. The purpose of this study was to characterize the protective immune response against PCV2 using a DNA plasmid expressing ORF2 (pORF2) and recombinant Cap protein as PCV2 DNA and subunit vaccines, respectively. Our data suggest that CD8+ T cells and recall virus-neutralizing (VN) antibody responses correlating with Cap-specific IgG2a may play important roles in developing protective immunity against PCV2 infection.

METHODS Cells, virus, protein and mice. A PCV-free PK-15 cell line was

maintained in minimal essential medium (Gibco) supplemented with 10 % heat-inactivated fetal bovine serum (FBS; Gibco). The virulent PCV2 isolate HZ0201 was originally isolated from pigs with naturally occurring PMWS (Zhou et al., 2006) and serially passaged 15 times in PK-15 cells. The PCV2 Cap protein was generated as a subunit vaccine as described previously (Zhou et al., 2005a). Eight-week-old female BALB/c mice were purchased from Shanghai Laboratory Animal Center (Chinese Academy of Sciences, Shanghai, China) and raised in automatic extrusion-independent venting isolation cages (Fengshi Laboratory Animal Equipment Co.).

recorded daily for 6 weeks, following which all mice were euthanized and their spleens collected for pathological analysis. Lymphocyte proliferation assay. Proliferation of splenocytes in the

immunized mice was determined using a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) test as described previously (Zhou et al., 2005b). In brief, the spleens were excised aseptically from the mice at 8 weeks p.i. in order to prepare single-cell suspensions (46106 cells ml–1) in RPMI 1640 containing 5 % FBS; the suspensions were applied to each well of a 96-well culture plate (100 ml per well). Wells containing no cells were used as blank controls. The PCV2 Cap protein diluted in RPMI 1640 with 5 % FBS was then added at a final concentration of 1 mg ml–1 (100 ml per well) to stimulate the splenocytes. After incubation at 37 uC in 5 % CO2 for 48 h, 20 ml MTT (5 mg ml–1; Sigma) was added to each well and the cells were incubated for 4 h. The cells were lysed by adding 100 ml lysis buffer (10 % SDS, 0.01 M HCl) to each well. After incubation for 20 h, the absorbance of each well was measured at 570 nm. The stimulation index (SI) was calculated using the following formula: SI5(Avaccine2Ablank)2(Acontrol – Ablank). Flow cytometric analysis (FCM). The cultured and stimulated splenocytes (106 cells) were resuspended in 50 ml PBS and incubated with 50 ml diluted (1 : 200) FITC-conjugated anti-mouse CD4 (L3T4;

BD Biosciences) and R-phycoerythrin-conjugated anti-mouse CD8a (Ly-2; BD Biosciences) mAbs at a concentration of 0.5 mg per 106 cells in an ice bath. After 20 min incubation, the cells were washed and analysed using a BD-LSR cytofluorimeter using CellQuest software (BD Biosciences).

Construction and preparation of an ORF2-based DNA vaccine.

The entire ORF2 was amplified from the genomic DNA of PCV2 isolate HZ0201 (GenBank accession no. AY188355) using specific forward (59-GCGGTCGACTCATTAAGGGTTAAGTGGG-39) and reverse (59-TATACGCGTTTATGACGTATCCAAGGAGG-39) primers, and was subcloned into the mammalian expression vector pCI-neo (Promega) to construct the recombinant expression plasmid pORF2. To determine the expression of Cap protein in eukaryotic cells, PCV-free PK-15 cells were transfected with pORF2 using Lipofectin reagent (Invitrogen). At 48 h post-transfection, the cells were fixed and examined using an indirect immunofluorescence assay (IFA) with murine monoclonal antibody (mAb) against Cap protein (Zhou et al., 2005a) and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (KPL, Kirkegaard and Perry Laboratories) as the primary and secondary antibodies, respectively. Finally, the pORF2 plasmid was purified using an EndoFree Plasmid Giga kit column (Qiagen) for use as a DNA vaccine.

Detection of Cap-specific IgG, IgG1 and IgG2a antibodies. An

Experimental design and sample collection. Eighty mice were

Detection of PCV2-neutralizing antibodies. A virus neutralization

divided randomly into eight groups of ten mice each. The mice in the four vaccine groups were injected intramuscularly in the quadriceps with 100 mg pORF2 plasmid or Cap protein prepared in 100 ml PBS as follows. The mice in the pORF2 and Cap groups received vaccination with pORF2 plasmid or Cap protein, respectively, three times every 2 weeks, whilst those in the pORF2/Cap or Cap/pORF2 group were primed with pORF2 plasmid (or Cap protein) and boosted with doses of Cap protein (or pORF2 plasmid) twice every 2 weeks. In the four control groups, the pCI-neo vector and crude lysate of E. coli strain BL21 transformed with pGEX-4T-1 were used as substitutes for pORF2 plasmid and Cap protein, respectively. After the first immunization, mouse serum samples were withdrawn from the retro-orbital sinus every 2 weeks until the end of the experiment for antibody detection and viraemia evaluation. At 8 weeks after the first immunization (p.i.), five mice from each group were euthanatized for flow cytometric analysis (FCM) and a lymphocyte proliferation assay. At 16 weeks p.i., the remaining five mice from each group were challenged intraperitoneally with 0.2 ml PCV2 inoculum (105.75 TCID50 ml21). Clinical signs in the challenged mice were

test was performed as described previously (Zhou et al., 2005a) with some modifications. In brief, the test sera were inactivated by heating at 56 uC for 30 min. An equal volume of PCV2 HZ0201 (106.0 TCID50 ml–1) and serial twofold dilutions (1 : 20 to 1 : 20 480) of the sera were mixed and incubated at 37 uC for 1 h. The serum/ virus mixture was added to 96-well microtitre plates containing semiconfluent monolayers of PCV-free PK-15 cells at 10 ml per well at a ratio of 1 : 10 using two wells per serum dilution. The plates were subsequently incubated at 37 uC for 48 h and finally screened by IFA as described above. The serum titres were determined as the reciprocal of the highest serum dilution resulting in ¢70 % fluorescent focus reduction in the infected cell cultures viewed under a fluorescent microscope.

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indirect ELISA (Shang et al., 2008) was performed to detect the titres of total IgG, IgG1 and IgG2a antibodies against PCV2 Cap protein. In brief, 96-well plates (Nunc) were coated with 100 ml PCV2 Cap protein (1 mg ml–1) in 0.05 M Tris/HCl (pH 8.5) and left at 4 uC overnight. The plates were then blocked with 5 % skimmed milk, and 100 ml serially twofold-diluted mouse serum samples (lowest dilution 1 : 64) were added and incubated at 37 uC for 60 min. The bound antibodies were detected by horseradish peroxidase-conjugated goat anti-mouse IgG, IgG1 or IgG2a antibody (diluted 1 : 6000; Southern Biotechnology Associates). Tetramethylbenzidine (Sigma) was used as a chromogen for colour development and absorbance was measured at 450 nm. Antibody titres were defined as the reciprocal of the highest dilution of sample for which the absorbance was at least twice that of the control serum sample run on the same plate. The data were presented as the log2 value of the titre.

Pathological analysis. The spleens of mice were collected at

6 weeks post-challenge (p.c.) and processed using conventional histopathological methods. Briefly, the spleens were fixed in 10 % neutral-buffered formalin solution, sectioned and stained with haematoxylin and eosin (H&E). Microscopic changes were Journal of General Virology 89

Protective immunity against PCV2 determined by comparing the splenic tissues of the challenged mice with those of the control-group mice. The intensity of lesions was estimated as the frequency of abnormal spleen follicles, which is the ratio between the number of the follicles with lymphoid depletion and histiocytic infiltration and the total number of follicles counted in the spleen section of each mouse. Quantitative real-time PCR for evaluation of viraemia. Viral

DNA levels in the mouse serum samples collected after PCV2 challenge were determined using a quantitative real-time PCR. Total DNA was extracted from 10 ml serum by using a UNIQ-10 virus DNA minikit according to the manufacturer’s instructions (Sangon). A forward (59-TGTAGTATTCAAAGGGCACAGAGC-39) and a reverse (59-CGGATATACTATCAAGCGAACCAC-39) primer were used to amplify a 130 bp fragment from the ORF2 of PCV2. The PCR contained a final concentration of 16 SYBR Premix Ex Taq (TaKaRa), 0.2 mM each primer and DNA equivalent to that of 1 ml serum as template. All reactions were carried out in duplicate on a RealPlex4 (Eppendorf). The program consisted of one cycle at 95 uC for 1 min and 40 cycles at 95 uC for 5 s and 60 uC for 20 s, and was followed by a melting curve analysis to analyse specificity. Serial dilutions of plasmid pORF2 were used to obtain a standard curve. The numbers of virus copies for each sample were presented as the mean value of duplicate reactions. The detection limit of this assay was 103 copies ml–1. Statistical analysis. Statistical analysis was performed by one-way analysis of variance using SPSS version 12.0 (SPSS). Results were considered to be statistically significant for P,0.05.

RESULTS In vitro expression of PCV2 Cap protein in PK-15 cells The recombinant plasmid pORF2 was identified using PCR and restriction enzyme digestion and was confirmed by DNA sequencing. In vitro expression of Cap protein was analysed by transient transfection followed by an IFA. PK15 cells transfected with pORF2 were recognized by mAb to Cap at 48 h post-transfection and the expressed Cap protein was found to localize in the nuclei of cells (Fig. 1), as observed in PCV2-infected cells (Allan et al., 1998b), suggesting that the mammalian expression vector pORF2 mimics PCV2 expression of Cap protein in vivo.

Cap-induced cellular immune responses Mice splenocytes were collected at 8 weeks p.i. and their lymphoproliferative responses to Cap protein were assayed (Table 1). The SI values of all the vaccinated mice were significantly higher than those of the control mice (P,0.01). Although mice in the Cap group exhibited the strongest responses to Cap protein, there was no significant difference among the responses of mice in the various vaccine groups (P.0.05). The proportions of CD4+ and CD8+ cells in the stimulated splenocytes were determined by FCM. As shown in Table 1, significant upregulation of CD4+ cells was elicited in all of the vaccine groups compared with the control group (P,0.01). With regard to the expression of CD8+ cells, only the pORF2 (P,0.01) and pORF2/Cap (P,0.05) groups showed significant increases in CD8+ cells. These data indicated that the pORF2 plasmid is superior to Cap protein in inducing CD8+ cells, whilst both had a comparable capability to trigger CD4+ cells. Total IgG and isotypes of serum antibody against PCV2 Cap protein The titres for total IgG against PCV2 Cap protein in the vaccinated mice were determined using an indirect ELISA. All mice primed with protein in the Cap and Cap/pORF2 groups seroconverted at 2 weeks p.i., whilst those in the pORF2/Cap and pORF2 groups seroconverted at 4 and 10 weeks p.i., respectively. As shown in Fig. 2, the Cap and pORF2/Cap protocols induced higher IgG titres with peaks of 18.2±0.8 and 17.0±1.7 log2 at 10 and 12 weeks p.i., respectively, compared with that of the pORF2 (14.8±1.3 log2) and Cap/pORF2 (15.3±2.2 log2) vaccines. A notable decrease in the antibody titre was observed at 8 weeks p.i. in the Cap/pORF2 group mice (Fig. 2). These data clearly showed that the Cap protein elicited more effective antibody responses in ELISA than the pORF2 plasmid. We analysed the IgG1/IgG2a isotypes against PCV2 Cap protein in order to determine the profile of antibody responses induced by different protocols. The pORF2 plasmid stimulated significantly higher IgG2a antibodies Table 1. PCV2 Cap-specific lymphoproliferative response and FCM analysis of mice splenocytes Significant differences between the vaccine and control groups are indicated: *, P,0.01; D, P,0.05. Group

Fig. 1. In vitro expression of PCV2 Cap protein. PCV-free PK-15 cells were transfected with pORF2 (a) or pCI-neo (b), fixed at 48 h post-transfection and detected using an IFA with mAb against Cap protein. http://vir.sgmjournals.org

Control pORF2 Cap Cap/pORF2 pORF2/Cap

SI

CD4+ cells (%)

CD8+ cells (%)

1.00±0.00 1.54±0.32* 1.63±0.14* 1.50±0.11* 1.51±0.20*

9.52±3.68 15.36±1.58* 15.89±1.65* 14.89±1.59* 16.50±1.67*

11.85±1.81 15.01±2.60* 12.80±0.99 13.50±1.32 14.31±1.51D

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respectively, in the Cap/pORF2 group (Fig. 3c). Statistically, the IgG1 titres were significantly higher than those of IgG2a at 2–4 weeks p.i. for Cap/pORF2 and at 4– 8 weeks p.i. for pORF2/Cap (P,0.05); they subsequently became non-significant (Fig. 3c, d). These results suggested that immunization with the pORF2 plasmid or Cap protein alone induced Th1 or Th2 responses, respectively; based on this, IgG2a was produced as a consequence of Th1 cell activation and IgG1 as a result of Th2 cell activation (Mosmann & Coffman, 1989b).

Fig. 2. Kinetics of total IgG production against PCV2 Cap protein at various times p.i. or p.c. The results are shown as means±SD (n55). Mice were challenged at 16 weeks p.i.

than IgG1 from 10 to 16 weeks p.i. (P,0.05), with a peak IgG2a titre of 14.0±1.5 log2 at 10 weeks (Fig. 3a). In contrast, the Cap protein induced significantly higher IgG1 levels than IgG2a levels from 2 to 16 weeks p.i. (P,0.01), with a peak IgG1 titre of 17.8±0.8 log2 at 6 weeks (Fig. 3b). With regard to heterologous vaccinations, peak IgG1 (16.0±0.8 log2) and IgG2a (14.8±2.1 log2) titres appeared at 12 weeks p.i. in the pORF2/Cap group (Fig. 3d), whilst peak IgG1 (13.5±1.7 log2) and IgG2a (14.3±1.0 log2) titres appeared at 16 and 14 weeks,

VN antibody to PCV2 and its correlation with IgG isotypes The ability of mice sera to neutralize PCV2 infection was detected using a virus neutralization test together with an ELISA. Prior to virus challenge, no detectable VN antibodies were observed in mice in any group (data not shown). After challenging the mice with PCV2 strain HZ0201 at 16 weeks p.i., strong recall VN antibody responses were observed in the pORF2, Cap/pORF2 and pORF2/Cap groups at 2 weeks p.c., with a seropositivity rate of 100 % and mean titres of 640, 805 and 960, respectively (Fig. 4). The VN antibody titres in the pORF2 and pORF2/Cap groups increased steadily and peaked at 6 weeks p.c., whereas those in the Cap/pORF2 group peaked at 2 weeks p.c. and then decreased gradually (Fig. 4). In contrast, mice in the Cap group exhibited

Fig. 3. Cap-specific IgG isotypes induced in the pORF2 (a), Cap (b), Cap/pORF2 (c), pORF2/Cap (d) and control (e) groups. The results are shown as means±SD (n55). The letters ‘a’ and ‘b’ above columns indicate a significantly higher IgG isotype with values of P,0.05 and P,0.01, respectively, at the same time in a group. Mice were challenged at 16 weeks p.i. 1860

Journal of General Virology 89

Protective immunity against PCV2

Table 2. Isotype analysis and VN antibody titres of individual mice in the pORF2 and Cap groups at 2 weeks p.c.

Fig. 4. PCV2-neutralizing antibody responses after PCV2 challenge. Letters above the column indicate statistical differences among the groups at the same time. Shared letters indicate no significant difference (P.0.05), whilst different letters indicate significant differences (P,0.05).

weak recall responses with mean VN antibody titres of 16, 28 and 16 at 2–6 weeks p.c. For PCV2-infected control mice, the VN antibody titres were only detected in a few mice at 4 (one mouse) and 6 (two mice) weeks p.c. Statistically, the VN antibody titres of the pORF2 and pORF2/Cap groups were higher than those in the Cap group at 2–6 weeks p.c. (P,0.05) (Fig. 4). These results showed that the protocol using the plasmid was more effective than that with the Cap protein alone in inducing recall PCV2-neutralizing antibody responses in mice. To investigate the relationship of VN antibodies and IgG isotypes, we compared the different antibody titres following virus challenge. At 2–6 weeks p.c., the Cap group mice exhibited higher IgG and IgG1 (P,0.05) antibodies than the pORF2 group mice, whilst the pORF2 group mice presented higher VN (P,0.05) and IgG2a antibodies than the Cap group mice (data not shown), implicating a probable correlation between the IgG2a and VN antibodies. We then analysed the antibody titres in individual mice. As shown in Table 2, mouse no. 12 with a IgG2a titre of 15 log2 generated a VN titre of 640, whilst mouse no. 23 with IgG1 titre of 16 log2 only generated a VN titre of 20. These results suggested that the neutralizing activity of the antibodies was associated with Cap-specific IgG2a rather than with IgG1.

Group*

Mouse no.

IgG1D

IgG2aD

VN titred

pORF2

11 12 13 14 15 21 22 23 24 25

15 12 11 6 10 16 15 16 14 18

17 15 16 11 11 13 12 13 14 14

1280 640 1280 0 0 20 0 20 20 20

Cap

*Mice were challenged at 16 weeks p.i. DCap-specific IgG1 and IgG2a titres were detected by ELISA and expressed as log2 values. dVN activity was analysed in a twofold dilution series starting at 1 : 20.

in the challenged control mice was 38.6±5.8 %, whereas that of the microscopic lesions in the immunized mice was significantly less compared with the control (P,0.01), with mean values of 10.6±8.5 % for the pORF2 group, 24.7±7.2 % for the Cap group, 13.0±8.4 % for the Cap/ pORF2 group and 9.7±7.0 % for the pORF2/Cap group (Fig. 5b). The frequency of lesions was significantly higher for the Cap group compared with the other vaccine groups (P,0.01). In addition to the histopathological evaluation, we also quantified the serum viral DNA loads of all PCV2inoculated mice using real-time PCR. The occurrence of viraemia and the mean numbers of PCV2 genomic copies are presented in Table 3. All of the challenged control mice were positive for PCV2 viraemia. Compared with the challenged control mice, those in the vaccine groups exhibited a reduction in the level of viraemia with significantly decreased numbers of PCV genomic copies (P,0.01) at 2–6 weeks p.c. For the different immunization protocols, the Cap group exhibited the highest PCV2 loads (P,0.05), whilst the pORF2/Cap group showed the lowest PCV2 loads throughout the post-challenge period.

Histopathological examination and evaluation of viraemia To investigate the protective efficacy of different vaccine protocols, we compared clinical symptoms and pathological changes in the challenged mice. No clinical signs or gross lesions were observed in the mice challenged with virulent PCV2. As shown in Fig. 5(a), lymphocyte depletion, histiocytic infiltration and infrequent apoptotic cells were found in the splenic corpuscles of the PCV2infected control mice. In contrast, after PCV2 challenge, the extent of the spleen lesions in the immunized mice was obviously smaller, occupying only a small part of the follicles. The frequency of the microscopic lesions (Fig. 5b) http://vir.sgmjournals.org

DISCUSSION PCV2 is the primary aetiological agent of PMWS. In this study, we characterized the protective immunity of mice against PCV2. Previous studies have reported mice as a model for PCV2 infection (Kiupel et al., 2001; Liu et al., 2006). Although two reports (Quintana et al., 2002; Wang et al., 2006) have described that PCV2 inoculation failed to induce murine lesions, this difference was considered to be related to inoculum dosage and route of administration (Quintana et al., 2002). In the present study, intraperitoneal inoculation of PCV2 successfully produced lesions 1861


Fig. 5. Microscopic lesions in the spleens of PCV2-challenged mice. (a) Mice were challenged with the virulent PCV2 at 16 weeks p.i. and the presence of splenic lesions was determined by conventional methods of histopathology at 6 weeks p.c. (i) No abnormal follicles were seen in the spleen of unchallenged mice. (ii) Significant depletion of lymphocytes within follicles (arrows) was observed in the spleen of PCV2-inoculated mice. (iii) Infiltration of histiocytes in the splenic follicle of a PCV2-inoculated mouse (arrows). (iv) Apoptotic cells (arrows) in the spleen follicle of a PCV2-inoculated mouse. (v) Mild lymphocyte depletion within a follicle (arrow) in the spleen of a Cap-immunized mouse inoculated with virulent PCV2. Cells were stained with H&E. Bars, 80 mm (i, ii, v); 20 mm (iii, iv). (b) Frequencies of abnormal spleen follicles at 6 weeks p.c. Shared letters above columns indicate no significant difference (P.0.05), whilst different letters indicate significant differences among the groups (P,0.05) (n55).

in mice. Furthermore, we chose changes in the spleen as the criteria of pathological analysis, as these are a distinct feature of PCV2 infection. The observed abnormal follicles with lymphocyte depletion and histiocyte infiltration resembled lesions found in PMWS-affected pigs (Chianini et al., 2003; Sarli et al., 2001), as well as those in PCV2inoculated mice (Kiupel et al., 2001; Liu et al., 2006); therefore, mice were deemed suitable models for this study. A recent in vivo study reported that B cells may be the primary site of PCV2 replication during early infection (Yu

et al., 2007), although in vitro studies have shown that PCV2 can replicate in primary porcine hepatocytes (Hirai et al., 2006) and that PCV2 particles can persist without replication in dendritic cells and porcine alveolar macrophages as well as in monocytes (Gilpin et al., 2003; Vincent et al., 2003). Thus, the target cells for PCV2 replication have not been clearly identified thus far. With regard to PCV2 immunization, no data regarding lymphocyte subsets in cellular immune responses have been recorded recently, although ORF2based vaccines against PCV2 have been reported previously (Blanchard et al., 2003; Fan et al., 2008; Fenaux et al., 2003;

Table 3. Viraemia in mouse serum measured by quantitative PCR Different superscripts within columns represent significantly different PCV2 loads for each week p.c. (P,0.05). A load value of 0 is equal to ,103 copies ml21 (the threshold of sensitivity of the quantitative PCR). Group

pORF2 Cap Cap/pORF2 pORF2/Cap Control

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No. of mice with viraemia/no. tested (mean log PCV2 load±SD) at the following weeks p.c. 2

4

2/5 (4.8±4.1)* 4/5 (5.7±4.5)D 2/5 (5.2±4.3)* 1/5 (4.8±4.1)* 5/5 (7.0±5.9)d

1/5 (3.9±3.3)* 3/5 (6.2±5.5)D 3/5 (4.6±3.9)* 1/5 (3.3±2.7)* 5/5 (7.6±6.6)d

6 1/5 3/5 1/5 0/5 5/5

(3.2±2.6)* (4.4±3.5)D (3.7±3.0)* (0)* (6.3±5.4)d


Protective immunity against PCV2

2004; Ju et al., 2005; Kamstrup et al., 2004; Song et al., 2007; Wang et al., 2006, 2007). In the present study, we investigated the Cap-specific lymphoproliferative activity and splenocyte phenotypes induced by different immunization protocols. Although the ORF2-based DNA and subunit vaccines were equally efficient in eliciting lymphoproliferative responses and CD4+ cells, the pORF2 plasmid was superior to the Cap protein in inducing CD8+ cells. Correspondingly, protocols using pORF2 exhibited a higher efficacy than those with subunit vaccine alone in the protection assay (Table 3, Fig. 5). These results imply that CD8+ cells, which are associated with virus-specific cytotoxicity, may contribute to protective immunity against PCV2 infection. Indeed, cytotoxic T-lymphocyte responses are considered to be the effector mechanisms required for protective immunity of a variety of intracellular infections, such as malaria (Doolan et al., 1996), human immunodeficiency virus infection (Ogg et al., 1998) and simian immunodeficiency virus infection (Schmitz et al., 1999). PCV2 Cap protein is related to the induction of PCV2neutralizing antibody (McNeilly et al., 2001; Zhou et al., 2005a). To date, the protective mechanism of humoral immunity against PCV2 infection has not been fully characterized. It is generally accepted that PCV2-specific antibodies are associated with protection, as field evidence suggests that the decrease in antibodies contributes to the development of PMWS (Allan & Ellis, 2000; Allan et al., 1998b; McIntosh et al., 2006). However, in some cases, the serum antibody level does not appear to influence PMWS occurrence (Carasova et al., 2007; McIntosh et al., 2006). During the PCV2 infection period in this experiment, the IgG2a antibody in the pORF2-immunized mice exhibited higher titres than in the Cap-vaccinated mice (Fig. 3). Correspondingly, we found that the tendency of VN antibody to protect against PCV2 infection coincided with Cap-specific IgG2a but not with total IgG or IgG1 (Figs 3 and 4, Table 2). Therefore, it is reasonable to suggest that VN antibody against PCV2 is composed mainly of IgG2a antibody against Cap protein, and that this Cap-specific neutralizing IgG2a antibody plays an important role in protecting against PCV2 infection. In fact, VN antibodies against dengue virus (Simmons et al., 2001) and herpes simplex virus type 1 (McKendall & Woo, 1988) have also been shown to comprise primarily IgG2a antibody in mice. A recent study reported that recombinant adenovirus expressing the Cap protein generated VN antibody titres ranging from 1 : 8 to 1 : 17 in mice (Wang et al., 2006), and all plasmids encoding Cap protein with different subcellular localizations induced VN antibody titres of ,1 : 10 in mice (Fan et al., 2008). Surprisingly, in our experiment, no VN antibody against PCV2 was detected in the immunized mice before virus challenge. This may have been due to the threshold of the virus neutralization test used in our study, which failed to detect VN titres of ,1 : 20. Therefore, a method for improving the induction of VN antibody against PCV2 requires further study. http://vir.sgmjournals.org

Th1 cells, which produce interleukin (IL)-2 and gamma interferon (IFN-c), induce the activation of macrophages, delayed-type hypersensitivity and the production of IgG2a (Abbas et al., 1996; Mosmann & Coffman, 1989a). Unmethylated CpG motifs in the plasmid are one of the pathogen-associated molecular patterns that tend to drive Th1 immune responses via Toll-like receptor 9 (Hemmi et al., 2000). Presently, at least one CpG-containing sequence has been identified as a potent inducer of IFN-a production in PCV2 ORF2 (Hasslung et al., 2003). In contrast, Th2 cells, which predominantly produce IL-4, IL5, IL-10 and IL-13, promote the development of eosinophilia as well as the generation of IgG1 and IgE antibody isotypes (Abbas et al., 1996; Mosmann & Coffman, 1989a). Based on the analysis of IgG1 and IgG2a in the immunized mice, our data showed that the pORF2 plasmid and Cap protein induced Th1- and Th2-biased immune responses, respectively, which is similar to reports on other viruses described previously (Ruitenberg et al., 2000; Sin et al., 1999; Tanghe et al., 2001; Zakhartchouk et al., 2005). Histopathological examination and real-time PCR results, particularly in the protection assay, also showed that mice immunized using the pORF2, pORF2/Cap and Cap/pORF2 protocols had milder microscopic lesions and lower serum PCV2 loads than the Cap-immunized mice. These results suggest that the Th1-biased immune response and possible cytokines conferred by the pORF2 plasmid benefit PCV2 control. It has been shown that immunity associated with the Th1 response is essential for cytotoxic T lymphocyte production. CD4+ Th1 cells mediate the killing of organisms responsible for a variety of intracellular infections through the production of IFN-c (Del Prete, 1998; Ogg et al., 1998; Seder & Hill, 2000). In summary, we have characterized the protective immunity against PCV2 by ORF2-based DNA and subunit vaccines in mice. Our data revealed that Cap-specific CD8+ cells and VN antibody responses, which generally correlated with Cap-specific IgG2a, play crucial roles in protecting against PCV2 infection. To our knowledge, this is the first report of a comparative study involving the cellular and humoral immune responses and protective immunity against PCV2. Our results provide insight for further research on host protective immunity against PCV2 infection.

ACKNOWLEDGEMENTS This work was supported by grants from the National Natural Science Foundation of China (30625030, 30370052) and from Zhejiang Provincial Department of Science and Technology (2003C12012), China.

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