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Protection of swine against post-weaning multisystemic wasting


Vaccine 21 (2003) 4565–4575

Protection of swine against post-weaning multisystemic wasting syndrome (PMWS) by porcine circovirus type 2 (PCV2) proteins
P. Blanchard a, , D. Ma

hé a , R. Cariolet a , A. Keranec’h a , M.A. Baudouard b , P. Cordioli c , E. Albina d , A. Jestin a
a

c

Agence Franaise de Sécurité Sanitaire des Aliments (AFSSA) Ploufragan, Unité Génétique Virale et Biosécurité, BP 53, Zoop le Les Croix, Fr 22440 Ploufragan, France o b Laboratoire de Développement et d’Analyses (LDA22), Zoop le Le Sabot, BP 54, 22440 Ploufragan, France o Istituto Zooprolattico Sperimentale della Lombardia ed Emilia Romagna “B. Ubertini” (IZSLER), Brescia, Italy d Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), Elevage et Médecine Vétérinaire, TA 30/G, 34398 Montpellier Cedex 5, France Received 2 December 2002; received in revised form 5 June 2003; accepted 8 June 2003

Abstract Porcine circovirus type 2 (PCV2) is known to be associated with post-weaning multisystemic wasting syndrome (PMWS), a recently described disease of young pigs. Since no PCV2 vaccine was available so far, we have developed a specic PCV2 vaccine candidate. The Orf1-encoded replication protein and Orf2-encoded capsid protein of PCV2 were expressed and detected in either mammalian or insect expression systems. In a rst trial, Orf2 protein was found to be a major immunogen, inducing protection in a prime-boost protocol; the piglets received a rst injection with plasmids directing Orf2 protein and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression, followed by a second injection, a fortnight later, associated with baculovirus-expressed Orf2 protein. As evaluated by growth parameters, clinical signs (fever), seroconversion, the pigs were protected against a PCV2 challenge after vaccination. In a second trial, protection induced by a subunit vaccine was even better than the one induced by DNA vaccine, since PCV2 replication was completely inhibited. 2003 Elsevier Ltd. All rights reserved.
Keywords: Vaccine; Porcine circovirus type 2; PMWS

1. Introduction Post-weaning multisystemic wasting syndrome (PMWS) is a new disease affecting 8–13-week-old piglets, that was rst observed in Brittany in 1996 [1]. PMWS rst emerged in western Canada in 1991 [2], and similar disorders were subsequently observed in herds in European countries and in the United States and Asia [3–8]. Clinically, the disease is characterized by pallor, fever and progressive wasting, together with respiratory and digestive disorders. Morbidity rates of 5–30% have been reported in affected pigs [9]. Isolation of virus from tissues of affected pigs led to the identication of a porcine circovirus type 2 (PCV2), considered to be of etiological importance in PMWS [10–14]. Many studies have conrmed the pivotal role of PCV2 in experimental reproduction of the disease [15–19]. Moreover, co-infections with PCV2 and other viral agents, such as
Corresponding author. Tel.: +33-2-96-01-62-72; fax: +33-2-96-01-62-83. E-mail address: p.blanchard@ploufragan.afssa.fr (P. Blanchard). 0264-410X/$ – see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0264-410X(03)00503-6


PPV or PRRSV, under experimental conditions increased the severity of the disease, proving the importance of co-factors in the pathogenesis of PMWS [20–24]. Under farm conditions, the improvement of certain management practices can attenuate the effects of PMWS by reducing infection pressure [1,9]; however, PMWS still remains a major problem in the swine population, as the mortality is still considerable in some farms [40]. One of our approaches to the prophylaxis of PMWS involves the development of a vaccine in order to control PCV2. Our previous work allowed us to develop a reproducible experimental model of PMWS [17], that can be used to study various vaccine compositions. This report describes two vaccine protocols, using DNA and subunit vaccines, alone or in combination. DNA vaccine is a relatively new technology derived from the discovery by Wolff et al. [25] using the injection of pure plasmid DNA (“naked DNA”). Many studies have demonstrated the protective immunity induced by DNA against viral diseases in animal models [26,27]. In a previous survey, we showed that injection of plasmid

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DNA encoding pseudorabies virus (PRV) glycoproteins into pig muscles resulted in protective immunity against lethal infection [28]. Co-delivery of DNA encoding porcine cytokine like granulocyte-macrophage colony-stimulating factor (GM-CSF) increased the immune response and could exert an immuno-adjuvant effect [29]. Co-administration of GM-CSF and PCV2 genes was used in our DNA vaccine approach. In our previous study, we demonstrated the antigenic properties of Orf2-encoded capsid protein of PCV2 expressed by baculovirus [30,31]. The baculovirus expression system has become a powerful tool in the development of subunit vaccines. Many studies have demonstrated the immunogenic and protective properties conferred by proteins expressed in this system [32–34]. Baculovirus-recombinant PCV2-proteins were used in order to evaluate their immunogenic and protective properties. In the present study, the efcacy of protection induced by DNA and/or subunit vaccines was evaluated by growth parameters and clinical signs, such as fever, compared to non-vaccinated and challenged piglets. Seroconversion and viral dissemination were also analyzed. 2. Materials and methods 2.1. Cloning of PCV2 genes The Orf1 and Orf2 genes encoding PCV2-proteins (replication and capsid) were cloned into the pcDNA3.1 mammalian expression vector (Invitrogen) or the pVL1393 baculovirus transfer vector (Pharmingen), as described elsewhere [30]. Recombinant clones were sequenced to ensure that no mutation occurred. 2.2. Production and in vitro analysis of plasmids from mammalian expression vectors Plasmids from pcDNA3.1 encoding Orf1–PCV2 and Orf2–PCV2 were designated pOrf1 and pOrf2. A pcDNA3 plasmid expressing porcine granulocyte-macrophage colony-stimulating factor (designated pGM-CSF) was supplied by INRA (Jouy-en-Josas, France). The three plasmids were puried by EndoFree plasmid Giga kit columns (Qiagen, Hilden, Germany) and resuspended in PBS. The DNA concentration was estimated by spectrophotometry. pOrf1, pOrf2 and pGM-CSF expression were conrmed in transfected PK15 cells, either by IPMA for circovirus proteins [30], or by a bone marrow proliferation assay for porcine GM-CSF [29]. 2.3. Production and characterization of baculovirus-expressed proteins Two recombinant baculoviruses were obtained after co-transfection with transfer vectors and linearized Bacu-

loGold virus (Pharmingen), as described previously [30]: BacOrf1 expressed the Orf1–PCV2-protein and BacOrf2 expressed the Orf2–PCV2-protein. Sf9 cells were infected with wild-type Autographa californica nuclear polyhedrosis virus (AcNPV) or recombinant baculoviruses (BacOrf1 or BacOrf2) at a multiplicity of infection (moi) of 10 plaque-forming units (pfu) per cell. The cells were harvested 72 h post-infection (pi) and were washed twice with PBS, counted and resuspended in PBS at a concentration of 107 cell equivalents per ml. The cells were lysed by freezing at 70 C and thawing. Total proteins from 1.5 × 105 cells were separated on a 12% SDS–PAGE and transferred onto a nitrocellulose membrane (Bio-Rad). Immunoreactivity of recombinant BacOrf1 and BacOrf2 was checked by Western blot analysis with a swine anti-PCV2 antiserum. 2.4. Vaccination protocol The DNA vaccine preparation was composed of 200 g of each plasmid in a total volume of 1 ml in PBS pH 7.2. The protein vaccine was prepared immediately before use, as follows. For 2 ml of emulsion to be prepared, 500 l of crude lysate from each recombinant baculoviruses (BacOrf1, BacOrf2), alone or in association, were completed to 1 ml of PBS pH 7.2 and mixed with 1 ml of a water-in-oil adjuvant (Montanide IMS 1313 PR provided by Seppic). For the control, 1 ml of crude lysate from wild-type AcNPV was mixed under the same conditions. The preparations were emulsied and stored on ice until inoculation of the pigs. 2.4.1. Immunization of pigs according to a prime-boost protocol In this rst trial, 35 25-day-old SPF piglets were divided into ve groups of seven piglets randomized according to sex and weight in our facilities under strictly controlled conditions (level 3 biosecurity) [35]. Piglets from four groups received a rst intramuscular injection of DNA plasmid preparation on one side of the neck, followed by a second injection, 2 weeks later on the same side, completed by a third injection of recombinant protein emulsion on the opposite side. Pigs were challenged according to the experimental model described by Albina et al. [17], namely intratracheally with 105.2 TCID50 in a dose volume of 5 ml and intramuscularly with 104.5 TCID50 in 1 ml of a tissue preparation-based inoculum, 10 days after the second vaccine injection. In the fth group, piglets were not vaccinated and not challenged. The composition of the vaccines for the various groups of piglets is presented in Table 1. Clinical observations and rectal temperatures were recorded daily and the piglets were bled and weighed weekly. Duration of the pyrexic phase and relative daily weight gains (RDWG)/kg bodyweight were determined for 5 weeks after challenge. The clinical protection conferred in the vaccinated groups was evaluated with reference to the challenge–control (CC) group according to the

P. Blanchard et al. / Vaccine 21 (2003) 4565–4575 Table 1 Composition of the vaccines for the various groups of piglets in trial no. 1, and vaccination protocol Trial no. 1 (seven pigs per group) Challenge–control group (CC) Orf1-vaccine group Orf2-vaccine group Orf1&Orf2-vaccine group Injection 1 (DNA 25-day-old pigs) pcDNA3.1—200 g GM-CSF—200 g Orf1/PCV2—200 g GM-CSF—200 g Orf2/PCV2—200 g GM-CSF—200 g Orf1/PCV2—200 g Orf2/PCV2—200 g GM-CSF—200 g Without Injection 2 (DNA—recombinant proteins 39-day-old pigs) pcDNA3.1—200 g/GM-CSF—200 g AcNPV/107 cells with Montanide Orf1/PCV2—200 g/GM-CSF—200 g Orf1/PCV2—5 × 106 cells with Montanide Orf2/PCV2—200 g/GM-CSF—200 g Orf2/PCV2—5 × 106 cells with Montanide Orf1/PCV2—200 g/Orf2/PCV2—200 g GM-CSF—200 g Orf1/PCV2—5 × 106 cells/Orf2/PCV2— 5 × 106 cells with Montanide Without

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PCV2 challenge (49-day-old pigs) YES YES YES YES

Control group (C)

NO

Note: DNA vaccine was prepared in a total volume of 1 ml in PBS pH 7.2 and subunit vaccine was prepared in a total volume of 2 ml, 1 ml of cells in PBS pH 7.2 mixed with 1 ml of a water-in-oil adjuvant (Montanide). The PCV2 inoculum used for the challenge consisted of a ltered (0.45 m) tissue preparation and the piglets were infected intratracheally with 105.2 TCID50 in a dose volume of 5 ml and intramuscularly with 104.5 TCID50 in 1 ml.

experimental model previously described by Albina et al. [17]. 2.4.2. Comparison between DNA vaccine and subunit vaccine In the second trial, 64 4-week-old SPF piglets were divided into eight groups of eight piglets, randomized as described in the rst trial. In the DNA vaccine protocol, piglets received a rst intramuscular injection of DNA plasmid preparation, followed by a second injection, 2 weeks later. In the subunit vaccine protocol, piglets received a rst intramuscular injection of baculovirus-expressed protein emulsion, followed by a second injection, 2 weeks later. Piglets were challenged 11 days after the second injection, as described in the rst trial. The second trial was conducted in duplicate, a rst series of four groups was monitored for 5 weeks after challenge and a second series of four groups was subjected to early killing between 4 and 17 days after challenge. The composition of the vaccines of the various groups are presented in Table 2. In the rst series (groups CC1, DNA1, SU1 and C1), clinical observations and rec-

tal temperatures were recorded daily and the piglets were bled and weighed weekly. Duration of the pyrexic phase and relative daily weight gains/kg bodyweight were determined for 5 weeks after challenge. At necropsy, tissue samples (bronchial lymph nodes, tonsil) were collected for laboratory investigation. In the second series (groups CC2, DNA2, SU2, C2), two piglets from each group were killed and necropsied at days 4, 8, 14 and 17 post-infection (dpi) and tissue samples (bronchial, inguinal, mesenteric lymph nodes, tonsil, lung and ileum) were collected for laboratory investigations. 2.5. Laboratory investigations in the second trial 2.5.1. Detection of PCV2-antibodies by a liquid phase blocking ELISA (LPBE) The sera collected from the CC1, DNA1, SU1 and C1 groups just before vaccination and up to 4 weeks after challenge were analyzed for the presence of PCV2-specic antibodies. The ELISA protein test using the baculovirusexpressed Orf2 protein developed in our laboratory [31] could not be used, because of the cross-reaction with the

Table 2 Composition of the vaccines for the various groups of piglets in trial no. 2, and vaccination protocol Trial no. 2 (eight pigs per group) Challenge–control groups (CC1 and CC2) DNA-vaccine groups (DNA1 and DNA2) Injection 1 (28-day-old pigs) Without Orf1/PCV2—200 g Orf2/PCV2—200 g GM-CSF—200 g Orf1/PCV2—5 × 106 cells Orf2/PCV2—5 × 106 cells With Montanide Without Injection 2 (42-day-old pigs) Without Orf1/PCV2—200 g Orf2/PCV2—200 g GM-CSF—200 g Orf1/PCV2—5 × 106 cells Orf2/PCV2—5 × 106 cells With Montanide Without PCV2 challenge (53-day-old pigs) YES YES

Subunit-vaccine groups (SU1 and SU2)

YES

Control groups (C1 and C2)

NO

Note: DNA vaccine was prepared in a total volume of 1 ml in PBS pH 7.2 and subunit vaccine was prepared in a total volume of 2 ml, 1 ml of cells in PBS pH 7.2 mixed with 1 ml of a water-in-oil adjuvant (Montanide). The PCV2 inoculum used for the challenge consisted of a ltered (0.45 m) tissue preparation and the piglets were infected intratracheally with 105.2 TCID50 in a dose volume of 5 ml and intramuscularly with 104.5 TCID50 in 1 ml.

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same expression system. Serological analysis was therefore performed by a liquid phase blocking ELISA (LPBE), using monoclonal antibodies [36]. Briey, the reaction was optimized using two PCV2-specic monoclonal antibodies. One MAb was used as a catcher antibody, and a second peroxidase-conjugated MAb was used as a detector antibody. Non-puried PCV2, at an optimal concentration, and sera were pre-incubated and then transferred onto ELISA plates. Results were expressed as the percentage of inhibition produced by the serum compared to the non-inhibited reaction. Sera giving an inhibition higher than 50% at the initial screening dilution, set at 1/6, were considered to be positive sera. The titers were expressed as the log 10 of the highest dilution of antibody inducing 50% inhibition. A mean titer was calculated for the sera from each group. 2.5.2. PCR detection of PCV2 in tissue samples All tissue samples from the eight groups of piglets were kept frozen at 72 C prior to DNA extraction. DNA was extracted using the Qiagen Dneasy tissue kit according to the manufacturer’s recommendations. A previously described PCR technique for the detection of PCV2 was carried out [30]. Amplication reactions were performed in a 50 l mixture containing 0.4 M of each primer (forward: 5 -CCTGTCTACTGCTGTGAGTACCTTGT-3 and reverse: 5 -GCAGTAGACAGGTCACTCCGTTGTCC-3 ), 0.1 mM of each dNTP, 2.5 U Taq DNA polymerase (Boehringer) with the following cycling parameters: denaturation at 95 C, 5 min; 35 cycles (94 C, 30 s; annealing at 60 C, 30 s; 72 C, 2 min); nal elongation at 70 C, 10 min. Amplication generated 1779 bp DNA fragments, corresponding to the PCV2 genome. An internal control was added to each PCR reaction to reveal any false–negative samples. This internal control was produced and cloned from the PCV2 genome, and revealed a 428 bp fragment with the same primers. 2.6. Statistical analysis Parametric statistical tests were applied using the SYSTAT 9 computer software package (SPSS Inc.). Relative daily weight gains were analyzed by ANOVA procedure during the vaccination period and by the Kruskal–Wallis non-parametric test for the 5 weeks post-infection. The number of days when the rectal temperature was higher than 40.5 C was analyzed by the Yates χ2 -test. 3. Results 3.1. Identication of PCV2 major immunogen in a prime-boost pig immunization approach (trial no. 1) In trial no. 1, 25-day-old pigs were vaccinated with a DNA plasmid preparation, followed by a second injection

2 weeks later, completed by a recombinant protein preparation. Before the PCV2 challenge, 10 days later (D49), no clinical signs were observed in the ve groups during the vaccination period. Comparisons of the relative daily weight gain (D25–D49) of vaccinated pigs with that of control pigs (value = 1) did not reveal any statistically signicant difference between the groups. After an incubation period of 10–14 days after challenge, all piglets from the challenge–control (CC) group developed pyrexia and growth retardation. The number of days during which the rectal temperature exceeded 40.5 C and the relative daily weight gain compared to the control (C) group were recorded (Fig. 1a and b). In this group, the pyrexic phase and growth retardation were essentially observed during the second-week post-infection (duration of pyrexic phase = 1 ± 1.53 days and RDWG = 0.70 ± 0.23, P < 0.05) and the third-week post-infection (duration of pyrexic phase = 2.43 ± 1.72 days and RDWG = 0.38 ± 0.29, P < 0.01), compared to the C group (RDWG = 1), in which pyrexia and growth retardation were not observed. These results were in agreement with those reported by Albina et al. [17] during development of the experimental model of PMWS in SPF piglets. Several animals presented clinical symptoms (dyspnea, tremor, ataxia, rough hair-coat), and one animal developed wasting and was killed, for ethical reasons, 4 weeks postinfection. As shown in Fig. 1a, the duration of the pyrexic phase observed in the vaccinated groups (Orf1, Orf2 and Orf1&Orf2 groups) was shorter than in the CC group. In the Orf1 group, the difference was signicant (P < 0.05) only during the third-week post-infection compared to the CC group, whereas the Orf2 and Orf1&Orf2 groups showed a more signicant difference during the second(P < 0.01) and third-week post-infection (P < 0.001). Furthermore, in the Orf1 group, four piglets presented hyperthermia, whereas only two piglets in the Orf2 or Orf1&Orf2 groups showed a rectal temperature higher than 40.5 C. The relative daily weight gain was also better in vaccinated groups compared to the CC group (Fig. 1b). A signicant difference was observed during the second-week post-infection, while only the Orf2 and Orf1&Or2 groups presented a signicant difference during the third-week post-infection (P < 0.01 and <0.05, respectively). To evaluate clinical protection, we determined a GW3 index, corresponding to the difference in mean daily weight gain (expressed as a percentage of weight in kg per day) during the third-week post-challenge period between vaccinated pigs and challenge–control pigs (Table 3). These results conrmed a signicant enhancement of protection after vaccination with an Orf2-based preparation (DNA and recombinant protein), conrming Orf2 as a major protective immunogen. One piglet in the Orf1 group also showed severe symptoms of PMWS (dyspnea, ataxia, rough hair-coat, tremor, and wasting).

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Fig. 1. Number of days with rectal temperature higher than 40.5 C (a) and relative daily weight gains (RDWG)/kg body weight (b) of four vaccinated and challenged groups in trial no. 1. Columns represent the average values for each group, challenge–control group (CC), Orf1-vaccine group (Orf1), Orf2-vaccine group (Orf2) and Orf1&Orf2-vaccine group (Orf1&Orf2). No pyrexia was observed in the control group (C). The RDWG of vaccinated and challenged pigs is compared with that of the control pigs (value = 1). Bars represent standard deviations.

Table 3 Clinical protection of vaccinated groups after PCV2 challenge in trial no. 1 Groups of pigs Challenge–control group Orf1-vaccine group Orf2-vaccine group Orf1&Orf2-vaccine group MDWG third-week pi 0.9 1.29 1.99 1.5 ± ± ± ± 0.69 0.71 0.43 0.36 GW3 index +0.39 +1.09 +0.6 PMWS clinical symptoms Yesa (1) Yesa (1) No No

This piglet was nally killed, for ethical reasons, at 35 dpi. 3.2. Comparison between a DNA vaccine and a subunit vaccine (trial no. 2) In the second trial, the pigs were vaccinated at the age of 4 and 6 weeks with either an Orf1&Orf2-based plasmid preparation (groups DNA1 and DNA2), or an Orf1&Orf2-based protein preparation (groups SU1 and SU2). They were then PCV2 challenged 11 days after the second vaccine injection. Pigs in groups CC1 and CC2 (challenge–control) were not vaccinated, but were challenged and pigs in groups C1 and C2 (control) were not vaccinated and not challenged. No clinical signs were observed in any of the groups before the challenge. Only pigs vaccinated with the protein preparation presented hyperthermia just after vaccination. However, comparison of relative daily weight gain (D28–D53) of vaccinated pigs with that of control pigs (value = 1)

Note: GW3 index was determined after PCV2 challenge as the difference in mean daily weight gain (MDWG expressed as % kg per day ± S.D.) during the third-week post-challenge period between vaccinated pigs and challenge–control pigs. a Several animals showed clinical symptoms, such as dyspnea, tremor, ataxia, rough hair-coat, prostration. (1) One animal developed wasting and was killed for ethical reasons, 4 weeks (CC group) or 5 weeks (Orf1 group) post-infection. Statistical analysis was performed in comparison with the challenge–control group (P < 0.05). Statistical analysis was performed in comparison with the challenge–control group (P < 0.01).

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Fig. 2. Number of days with rectal temperature higher than 40.5 C (a) and relative daily weight gains (RDWG)/kg body weight (b) of the three challenged groups in trial no. 2. Columns represent the average values for each group, challenge–control group (CC1), DNA-vaccine group (DNA1), subunit-vaccine group (SU1). No pyrexia was observed in the control group (C1). The RDWG of challenged pigs is compared with that of the control pigs (value = 1). Bars represent standard deviations.

did not reveal any signicant difference between the groups. 3.2.1. Follow-up of the rst series of pigs (groups CC1, DNA1, SU1 and C1) for 5 weeks post-infection As in the rst trial, all piglets of the CC1 group developed pyrexia and growth retardation after challenge. The number of days during which the rectal temperature exceeded 40.5 C and the relative daily weight gain compared to the C group were recorded (Fig. 2a and b). In this group, the pyrexic phase and growth retardation were essentially increased during the third-week post-infection, 2.13 ± 1.25 days with a RDWG of 0.12 ± 0.21 (P < 0.001), respectively, compared to the C1 group, in which pyrexia and growth retardation were not observed (RDWG = 1). A signicant growth retardation was also observed during the rst- and second-week post-infection. However, clinical symptoms were less pronounced than during the rst trial (digestive disorders only). The difference of expression of PMWS in CC pigs in these two trials cannot be ascribed to

placebo vaccination with GM-CSF and Montanide applied to CC pigs in the rst trial. Indeed, clinical symptoms were also observed in other trials without placebo vaccination [17].
Table 4 Clinical protection of vaccinated groups after PCV2 challenge in trial no. 2 Groups of pigs MDWG third-week pi GW3 index +1.20 +1.54 PMWS clinical symptoms Fewa Fewb No

Challenge–control group 0.29 ± 0.48 DNA-vaccine group 1.49 ± 0.52 Subunit-vaccine group 1.83 ± 0.38

Note: GW3 index was determined after PCV2 challenge as the difference in mean daily weight gain (MDWG expressed as % kg per day ± S.D.) during the third-week post-challenge period between vaccinated pigs and challenge–control pigs. a A few animals showed clinical symptoms, only digestive disorders. b A few animals showed clinical symptoms, only prostration. Statistical analysis was performed in comparison with the challenge–control group (P < 0.001).

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Fig. 3. PCV2-specic antibodies elicited in the challenge–control group (CC1), DNA-vaccine group (DNA1) and subunit-vaccine group (SU1). The animals of the three groups were challenged 2 weeks after the booster. Data represent the mean serum titers (log 10) in each group determined as the highest dilution of antibody inducing 50% inhibition, using a liquid phase blocking ELISA. No PCV2-antibodies were detected in the control group (C1). The threshold of detection of this ELISA test was estimated at 0.78 log 10: (V1) st injection; (V2) second injection; (C) challenge. Bars represent standard deviations.

As shown in Fig. 2a, the duration of the pyrexic phase of the DNA1 group was longer than in the CC1 group (2.63 ± 1.19 days) during the third-week post-infection, whereas the SU1 group presented a shorter pyrexic phase during this period, signicantly different from that observed in the CC1 group (0.86 ± 1.21 days, P < 0.05). As shown in Fig. 2b, the DNA1 and SU1 groups presented a highly signicant difference in relative daily weight gain compared to the CC1 group during the third-week post-infection, with a RDWG of 0.64±0.24 and 0.79±0.16 (P < 0.001), respectively. In order to measure clinical protection, as in the rst trial, we determined the GW3 index during the third-week post-challenge period between vaccinated pigs and challenge–control pigs (Table 4). These

results showed a signicant level of protection against PCV2 challenge when the piglets were vaccinated with either the DNA vaccine ( GW3 = 1.2), or the subunit vaccine ( GW3 = 1.54). To evaluate the seroconversion of the pigs before and after challenge, a Liquid Phase Blocking ELISA was performed to detect PCV2-specic antibodies [36]. All piglets of the CC1 group seroconverted 2 weeks post-infection (Fig. 3), while no antibodies were detected in the C1 group. These results are in concordance with those previously obtained using our protein ELISA test [31]. On the other hand, all piglets of the DNA1 group seroconverted 1 week after challenge, while antibodies were detected in ve piglets from the SU1 group 2 weeks after the rst injection, and in all piglets just

Fig. 4. Detection of PCV2-DNA in tissue samples by PCR in the challenge–control group (CC2), DNA-vaccine group (DNA2) and subunit-vaccine group (SU2). The results are expressed as the percentage of positive PCR samples detected in the two pigs necropsied at 4, 8, 14 and 17 days post-infection (bronchial, inguinal, mesenteric lymph nodes, tonsil, lung and ileum). No PCV2 acid nucleic was detected in the control group (C2).

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before challenge. After challenge, the antibody level in SU1 group remained relatively constant and slightly lower than in the DNA1 and CC1 groups. These results show a better antibody response to the subunit-vaccine group compared to the DNA-vaccine group and no evidence of a boosting effect of the challenge in the former group compared to the latter. 3.2.2. Killing kinetic of the second series of pigs (groups CC2, DNA2, SU2 and C2) Two pigs per group of the second series were killed at days 4, 8, 14 and 17 post-infection (dpi) and DNA extracted from tissue samples was submitted to PCV2 PCR (six tissue samples per pig). PCR results are summarized in Fig. 4. At 4 dpi, PCV2 nucleic acid was detected in one sample (tracheobronchial lymph node) of one pig of each challenged group (CC2, DNA2 and SU2), as a more intense amplication signal was revealed in pig vaccinated with DNA. At 8 dpi, two pigs of the CC2 and DNA2 groups presented PCV2 positive samples (4/12 and 8/12, respectively), while no PCV2 nucleic acid was detected in samples of SU2 pigs. At 14 and 17 dpi, all tissue samples from the CC2 and DNA2 groups, excepted for lung (only 3/8), revealed PCV2 nucleic acid. Only one tracheobronchial tissue sample showed a very slight amplication signal at 14 dpi in the SU2 group. No PCV2 acid nucleic was detected in the C group. These results are in favor of viral neutralization in the subunit-vaccine group compared to the DNA-vaccine group, in which viral dissemination appears to be increased compared to the non-vaccinated and challenged group.

4. Discussion Post-weaning multisystemic wasting syndrome is now recognized as a major disease problem of economic importance in many pig-producing areas of the world [9,37]. There is no doubt that the expression of this disease is dependent on the presence of PCV2 [15–17,19], although other co-factors have been shown to be important in development of the disease [20,21,38,39]. Porcine PMWS is still causing signicant levels of mortality in many herds [40], despite application of recommendations regarding housing and herd management described by Madec et al. [1]. In the present study, we chose to develop a PCV2 vaccine, using our experimental model for PMWS occuring in growing piglets [17]. Vaccines are mainly composed of live, attenuated pathogens; however, there is a current tendency towards the development of non-replicating vaccines, such as subunit vaccines or DNA vaccines [41]. In our case, the difculty of producing high PCV2 viral titers in vitro led us to investigate both of these approaches. Many studies have already demonstrated the efcacy of DNA vaccines [28,29] and recombinant subunit vaccines produced in insect cells [33,34]. We have previously developed a reproducible experimental model of PMWS [17]. Using inocula from tissue homogenates, the disease was induced after an incubation

period of 12 days and was characterized by elevated rectal temperature (>40.5 C) for 7–11 days and growth retardation observed during the second and third weeks after challenge. This mild form of the disease was reproduced in 82% of inoculated piglets, while a characteristic severe wasting syndrome was induced in 7.2% of piglets. We used this inoculum for this study, under similar conditions of administration. Other studies have also enabled us to characterize the expression of the PCV2 major viral proteins (Orf1 and Orf2) and to analyze their immunological properties in either the mammalian system, or the baculovirus system [30]. We used both of these expression systems to evaluate the immunogenic and protective properties of PCV2-proteins. In our DNA vaccines approach, we chose the co-administration of GM-CSF and PCV2 genes. The GM-CSF gene, used as a genetic adjuvant, has been shown to be able of enhancing both T cell and B cell responses [29]. We have previously found in a PRV model, that co-delivery of GM-CSF gene with genes of interest signicantly increased the levels of IFN- , IL-2, and IL-4 mRNA, suggesting a Th1–Th2 bias on the immune response [28]. The molecular weights of baculovirus-expressed PCV2 Rep- and Cap-protein were shown to be about 34 and 28 kDa, respectively, in accordance with the size previously estimated from amino acid sequence analysis [12–14]. Swine anti-PCV2 serum also conrmed the specic immunoreactivity directed against the PCV2 capsid protein, as no reactivity was observed with the PCV1 capsid protein (data not shown). Expression of the two Rep and Cap-proteins of PCV2 using the baculovirus system was therefore conrmed, allowing their potential use as a subunit vaccine against PCV2 infection. During our two trials and based on comparison of the results obtained in the two control–challenge groups, we reproduced a mild form of PMWS, in concordance with the experimental model previously described by Albina et al. [17]. After an incubation period (10–14 days), almost all of the piglets presented an elevated rectal temperature (>40.5 C for 1–8 days) and growth retardation, essentially observed during the third-week post-challenge. Several animals showed clinical symptoms, such as dyspnea, tremor, ataxia, rough hair-coat; however, a characteristic severe wasting syndrome was induced in only one of the 15 challenge–control piglets. Another piglet presented wasting syndrome in the Orf1-vaccine group during the rst trial. This low incidence of severe PMWS has already been described by Albina et al. [17] and can be compared with the incidence of PMWS observed in well-managed farms, where only 5–10% of pigs develop severe wasting syndrome [9]. The efcacy of the protection conferred by the two major PCV2-proteins, Rep and Cap, alone or in combination, can be evaluated on the basis of growth retardation and fever parameters presented by piglets in the challenge–control group in trial no. 1. We used a prime-boost protocol, including a rst DNA vaccine followed by a DNA-booster and a subunit vaccine, 2 weeks later. In this trial, vaccination

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signicantly reduced the duration of the pyrexic phase, more efciently in the Orf2 and Orf1&Orf2-vaccine groups than in the Orf1-vaccine group. Moreover, during the third-week post-infection, only the Orf2 and Orf1&Orf2 groups showed a signicantly reduced growth retardation compared to the CC group, which was more pronounced in the Orf2 group. No difference was observed between the Orf2 group and the C group (non-challenged control group), while the Orf1&Orf2 group showed a signicant difference with the C group (P < 0.01) during the third week. These results were conrmed by a higher GW3 index in the Orf2 group than in the Orf1&Orf2 group (1.09 versus 0.6) during the third-week post-challenge. Although no signicant difference was observed between these two groups, the lower GW3 index in the Orf1&Orf2 group suggest the existence of a reduction effect of Orf1 addition on Orf2-vaccine efcacy. However, no clinical symptoms were observed in these two groups, while pigs in the Orf1 group presented typical PMWS symptoms (ataxia, rough hair-coat, wasting). We therefore showed that the Orf2-encoded capsid protein, used in a preparation-based DNA and subunit vaccine, constitutes the major immunogen to induce protection of piglets against a PCV2 challenge. In contrast, the Orf1-encoded replication protein appears to be only weakly immunogenic. These results differ from those reported for Chicken anemia virus (CAV), for which co-synthesis of VP1 (capsid protein) and VP2 are necessary to mount a protective immune response [33,42]. In our trial no. 2, we focused on the vaccine composition to compare the efcacy of the protection conferred by either a DNA vaccine or a subunit vaccine. Independently of the previous results, we decided to use the two associated major PCV2-proteins (Rep and Cap), in two injections. As in the rst trial, on the basis of growth retardation and fever parameters presented by piglets in the challenge–control group, we observed that the subunit vaccine signicantly reduced the duration of the pyrexic phase (P < 0.05) during the third-week post-infection, while the DNA vaccine did not have any effect on the duration of the pyrexic phase. All piglets of the DNA group presented hyperthermia higher than 40.5 C for one to 5 days versus three piglets of the subunit group for 1–3 days. In contrast, the two vaccinated groups (DNA and SU) showed a better growth, compared to the CC group. These results were conrmed by a high GW3 index, 1.20 and 1.54, respectively. The GW3 index obtained in trial no. 2 was higher than that obtained in trial no. 1. However, we cannot consider so signicant the difference in GW3 obtained in the various groups of animals (Orf2, Orf1&Orf2, DNA, subunit groups). In the case of conventional commercial vaccines against Aujeszky’s disease virus (ADV), the ofcial requirement is a minimum G7 index of 1.5. Although these two diseases are not comparable, the GW3 index obtained in the present study indicates a useful protection performance for the production of vaccines against a PCV2 challenge. To complete the results of trial no. 2 obtained by evaluation of rectal temperature and weight gain, we analyzed the

immune response of piglets from the vaccination period until 4 weeks after challenge, performed by a LPBE test, using monoclonal antibodies [36]. We obtained earlier seroconversion with the subunit vaccine 2 weeks after the rst injection, at the time of the second injection, while seroconversion was only observed after challenge with the DNA vaccine. Different antibody titers were elicited in the groups and were higher for DNA and CC groups after challenge, while the antibody titer of the subunit group remained lower, suggesting the absence of PCV2 replication and boosting effect after challenge. DNA vaccination was able to prime the antibody response since an earlier seroconversion was observed in the DNA group compared to CC group. However, this antibody formation was not early and high enough to preclude PCV2 replication and pathogenic effect. It is interesting to compare these results with those obtained by PCR evaluating viral dissemination after challenge. While the PCR results revealed signicant viral neutralization in the subunit group, the DNA group showed strong viral replication during the 3 weeks post-infection, comparable to that observed in the challenge–control group. These ndings are in favor of a better protection induced by the subunit vaccine, eliciting an early antibody response able to neutralize the virus, compared to the DNA-vaccine group. However, further analyses using a quantitative PCR method are necessary to evaluate PCV2 load, as, although PCV2 genome was detected in tissue samples of all piglets of the challenge–control group at the end of the experimental period (35 dpi), only ve piglets from the DNA group revealed the presence of PCV2 genome in tissue samples, suggesting more delayed viral neutralization. In addition, a recent study was performed to evaluate maternal antibody protection against PCV2 challenge, using a virus neutralizing assay (VNA) [43]. The authors showed a protective effect of these antibodies on PCV2 circulation; no active seroconversion was seen and some protection against antigen load in lymph nodes was observed. Our results, compared with these observations, tend to suggest that the subunit vaccine induced a Th2-like humoral response, based on neutralizing antibodies. It also appears interesting to compare these ndings to those reported on immunostimulation, since it has been experimentally demonstrated that activation of the immune system can play a role in the development of PMWS [39,44]. These studies were performed on PCV2 pre-infected piglets before non-specic vaccination, whereas our trials were carried out on vaccinated piglets before challenge. Under our conditions, we revealed PCV2 replication that was promoted in the case of DNA-vaccinated piglets and neutralized in the case of subunit-vaccinated piglets. The immune response elicited after either DNA vaccine or subunit vaccine therefore appears to modify PCV2 replication. DNA-vaccinated piglets also presented an intense pyrexic period after challenge although their growth was delayed to a lesser degree than that of non-vaccinated piglets. Further investigations will be necessary to assess how the nature of

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P. Blanchard et al. / Vaccine 21 (2003) 4565–4575 [2] Clark EG. Post-weaning multisystemic wasting syndrome. In: Proceedings of the American Association of Swine Practitioners. 1997. p. 499–501. [3] Allan GM, McNeilly F, Kennedy S, Daft B, Clarke EG, Ellis JA, et al. Isolation of porcine circovirus-like viruses from pigs with a wasting disease in the USA and Europe. J Vet Diagn Invest 1998;10: 3–10. [4] Allan GM, Mc Neilly F, Meehan BM, Kennedy S, Mackie DP, Ellis JA, et al. Isolation and characterisation of circoviruses from pigs with wasting syndromes in Spain, Denmark and Northern Ireland. Vet Microbiol 1999;66:115–23. [5] Kennedy S, Allan G, McNeilly F, Adair BM, Hughes A, Spillane P. Porcine circovirus infection in Northern Ireland. Vet Rec 1998;142:495–6. [6] Segalès J, Sitjar M, Domingo M, Dee S, Del Pozo M, Noval R, et al. First report of post-weaning multisystemic wasting syndrome in pigs in Spain. Vet Rec 1997;141:600–1. [7] Kiupel M, Stevenson GW, Mittal SK, Clark EG, Haines DM. Circovirus-like viral associated disease in weaned pigs in Indiana. Vet Pathol 1998;35:303–7. [8] Onuki A, Abe K, Togashi K, Kawashima K, Taneichi A, Tsunemitsu H. Detection of porcine circovirus from lesions of a pig with wasting disease in Japan. J Vet Med Sci 1999;61:1119–23. [9] Madec F, Eveno E, Morvan P, Hamon P, Blanchard P, Cariolet R, et al. Post-weaning multisystemic wasting syndrome (PMWS) in pigs in France: clinical observations from follow-up studies on affected farms. Livestock Prod Sci 2000;63:223–33. [10] Allan GM, Meehan B, Todd D, Kennedy S, McNeilly F, Ellis J, et al. Novel porcine circoviruses from pigs with wasting disease syndromes. Vet Rec 1998;142:467–8. [11] Ellis J, Hassard L, Clark E, Harding J, Allan G, Willson P, et al. Isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome. Can Vet J 1998;39:44–51. [12] Hamel AL, Lin LL, Nayar GP. Nucleotide sequence of porcine circovirus associated with postweaning multisystemic wasting syndrome in pigs. J Virol 1998;72:5262–7. [13] Meehan BM, McNeilly F, Todd D, Kennedy S, Jewhurst VA, Ellis JA, et al. Characterization of novel circovirus DNAs associated with wasting syndromes in pigs. J Gen Virol 1998;79:2171–9. [14] Morozov I, Sirinarumitr T, Sorden SD, Halbur PG, Morgan MK, Yoon KJ, et al. Detection of a novel strain of porcine circovirus in pigs with postweaning multisystemic wasting syndrome. J Clin Microbiol 1998;36:2535–41. [15] Ellis J, Krakowka S, Lairmore M, Haines DM, Bratanich A, Clark E, et al. Reproduction of lesions of postweaning multisystemic wasting syndrome in gnotobiotic piglets. J Vet Diagn Invest 1999;11:3–14. [16] Balasch M, Segales J, Rosell C, Domingo M, Mankertz A, Urniza A, et al. Experimental inoculation of conventional pigs with tissue homogenates from pigs with post-weaning multisystemic wasting syndrome. J Comp Pathol 1999;121:139–48. [17] Albina E, Truong C, Hutet E, Blanchard P, Cariolet R, L’Hospitalier R, et al. An experimental model for post-weaning multisystemic wasting syndrome (PMWS) in growing piglets. J Comp Pathol 2001;125:292–303. [18] Magar R, Larochelle R, Thibault S, Lamontagne L. Experimental transmission of porcine circovirus type 2 (PCV2) in weaned pigs: a sequential study. J Comp Pathol 2000;123:258–69. [19] Bolin SR, Stoffregen WC, Nayar GP, Hamel AL. Postweaning multisystemic wasting syndrome induced after experimental inoculation of cesarean-derived, colostrum-deprived piglets with type 2 porcine circovirus. J Vet Diagn Invest 2001;13:185–94. [20] Allan GM, McNeilly F, Ellis J, Krakowka S, Meehan B, McNair I, et al. Experimental infection of colostrum deprived piglets with porcine circovirus 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) potentiates PCV2 replication. Arch Virol 2000;145:2421–9.

the immune response presumably Th1-like versus Th2-like, can inuence the expression of PMWS. The objective of this study was to evaluate the efcacy of protection induced by vaccination of piglets against a PCV2 challenge. This is the rst report to relate the use of vaccines in PCV2 infection. Orf2-vaccine group (in a prime-boost protocol) and the subunit-vaccine group appeared to provide signicant protection against PCV2 infection. However, the subunit group still presented a signicant growth retardation compared to the control group at the third- and fourth-week post-infection (P < 0.05). We therefore demonstrated the essential role of the recombinant capsid protein produced by the baculovirus system in a vaccine concept against PCV2 infection. Further trials must be conducted in order to dene a better vaccine protocol. Prime-boost vaccination with DNA and recombinant baculovirus-expressed protein successfully enhances the immune response to other viruses, resulting in broad humoral and cell-mediated immune responses [45,46]. In the case of a vaccine against PCV2 infection, more detailed studies are necessary to characterize the immune response. However, previous studies have demonstrated induction of immune priming T and B cells with DNA vaccination in the pig model [28] and on the basis of the results obtained with our subunit vaccine in terms of seroconversion and clearance of virus following PCV2 challenge, we can predict a good efcacy of our subunit vaccine in a prime-boost approach, that would induce both antibodies and cell-mediated immunity.

Acknowledgements The authors are grateful to Bernard Beaurepaire and Gérard Bennevent for their contribution to the experimental work on pigs in the AFSSA facilities. We are also grateful to Daniela Bresciani (IZSLER) for LPBE serological analyses and to Régis Vinet (LDA22) for PCR analyses. We thank Stéphanie Bougeard for statistical analysis. We also thank Franois Lefèvre and Chandra Somasundaram (INRA, Jouy en Josas) for having kindly provided the plasmid encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) and Vincent Ganne (Seppic, France) for having kindly provided the Montanide adjuvant used in the protein vaccination assay. This work was supported by funds from the “Fonds Européens de Développement et de Restructuration” (FEDER 5b), the “Comité Régional porcin”, the “Région Bretagne”, and the European Commission (contract QLK2-CT-1998-00307).

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