- Email: [email protected]
Baculovirus-expressed muscovy duck reovirus σC protein induces serum neutralizing antibodies and protection against challenge
Vaccine 20 (2002) 3113–3122
Baculovirus-expressed muscovy duck reovirus C protein induces serum neutralizing antibodies and protection against challenge Gaëlle Kuntz-Simon a,1 , Philippe Blanchard b , Martine Cherbonnel a , André Jestin b , Véronique Jestin a,∗ a
b
Avian and Rabbit Virology, Immunology and Parasitology Unit, Poultry and Swine Research Laboratory, French Agency for Food Safety (AFSSA), Zoopˆole Les Croix, BP 53, 22440 Ploufragan, France Viral Genetics and Biosafety Unit, Poultry and Swine Research Laboratory, French Agency for Food Safety (AFSSA), Zoopˆole Les Croix, BP 53, 22440 Ploufragan, France Received 18 September 2001; received in revised form 15 March 2002; accepted 23 May 2002
Abstract Recombinant baculoviruses with C- or B-encoding gene from muscovy duck reovirus (DRV) were constructed. Western-blot analysis showed that C was more immunoreactive than B. Vaccination of SPF ducks with two injections, 3 weeks apart, of emulsions containing C or C + B elicited DRV-specific neutralizing antibodies. Following challenge, vaccination partially—or even totally in some cases— prevented the appearance of clinical symptoms. Moreover, immunization reduced the severity of reovirus-induced tenosynovitis and prevented pericarditis development during the course of the assay. Thus, DRV C, alone or co-expressed with B, appeared as a good candidate for vaccination of ducks (96/100 mots). © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Vaccine; Orthoreovirus; Avian reovirus
1. Introduction When infected by a reovirus, muscovy ducks (Cairina moschata) may develop a disease with mortality and a high level of morbidity. Moreover, reovirus-infected ducks often exhibit bacterial complications leading to poor performance and increased seizures at the slaughterhouse. The acute form of the disease, first described in South Africa in 1950 [1], is usually apparent in muscovy ducklings between 2 and 4 weeks of age, in France. The clinical signs include apathy, diarrhea, reluctance to move and hobbling. Macroscopically, dead animals can show fibrinous pericarditis, friable liver and marbled spleen [2,3,4]. Microscopic lesions have been detailed [4]. Among them, it has to be noticed that the synovial sheath of the leg tendons shows a typical exudative inflammation. The muscovy duck reovirus (DRV), first isolated in France in 1972 [2], belongs to the Orthoreovirus genus, Reoviridae family [3,5]. As other reoviruses, it is a very resistant virus and, therefore, control through sanitation and ∗
Corresponding author. Tel.: +33-2-96016258; fax: +33-2-96016263. E-mail address: [email protected] (V. Jestin). 1 Present address: AFSSA-Ploufragan, Swine Virology and Immunology Unit, Zoopˆole Les Croix, BP 53, 22440 Ploufragan, France.
isolation is not practical for commercial poultry production. The principal method of control should be by vaccination. Despite good experimental results, field attempts to prevent the infection with a DRV inactivated vaccine were unsuccessful [6]. Similarly, assays carried out in the pharmaceutical industry to obtain a DRV attenuated vaccine combining activity and safety have failed. On the other hand, vaccination of muscovy ducks with the chicken reovirus vaccine strain S1133 gave poor results in two successive trials carried out in our laboratory in 1982 [7] and 1990. Though both duck and chicken reoviruses share some common epitopes, as shown by cross-immunodiffusion gel assay and ELISA, cross-neutralization tests revealed that they are antigenically different (Jestin, unpublished results; [8]). Extensive sequences comparisons of genome segments from both species suggested that DRV segregates as a specified genogroup among avian reoviruses [5], confirming that chicken reovirus S1133 is not a good candidate for duck protection. Hence, the development of safe and efficient vaccine against DRV is still required in the field. For that purpose, we proposed to evaluate the immunogenic properties of some DRV proteins. Concerning other reoviruses, it has been shown that most protective antibodies are directed against external viral antigens, particularly those associated with cell attachment [9].
0264-410X/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 0 2 ) 0 0 2 6 4 - 5
3114
G. Kuntz-Simon et al. / Vaccine 20 (2002) 3113–3122
In case of avian reovirus isolated from chicken, and commonly abbreviated ARV, mouse monoclonal antibodies specific from three other proteins neutralized virus infectivity in cell cultures [10,11,12,13]. These proteins are the major outer capsid protein (B), the minor outer capsid protein (C) and the large surface protein (C). ARV B and C proteins induce group-specific neutralizing antibody, while protein C, an homotrimer displaying cell-binding activity [14,15] induces type-specific neutralizing antibodies [16]. Being functionally and structurally related to the protein 1 of mammalian reovirus (MRV) [17,18], ARV C is suggested to be localized at the vertices of the spikes [19]. An Escherichia coli-expressed fusion protein was used to study the immunogenicity of ARV C in chickens. The recombinant protein did not elicit detectable neutralizing antibodies in sera, but some protection against virus-induced mortality in 1-day-old chicks was shown when tested in passive immunization studies [20]. Intending to identify DRV proteins that could play a role in protective immunity, we previously reported the molecular characterization and expression of the DRV-89026 B-encoded gene, that shares 61% identity with its ARV counterpart [21]. We demonstrated the antigenicity of a baculovirus-expressed B protein, but were unable to reveal any immune response against DRV B protein in muscovy ducks immunized with the recombinant protein, nor protection against reovirus challenge. We reported elsewhere that DRV C protein is atypically encoded by the major open reading frame (ORF) of the smallest genome segment (S4) and not by segment S1 as usually reported for other orthoreoviruses [5]. Despite extensive sequence divergence, we showed the conservation of structural domains between DRV and ARV C proteins and suggested that DRV C could be functionally related to ARV C, and thus, could play an important role in the immune response occurring in infected birds. In the present study, we report the expression of proteins C and B from DRV-89330, a strain we demonstrated to be more pathogenic and immunogenic than the DRV-89026 strain we used in a previous trial [21]. We tested the C protein ability, alone or in combination with B, to induce neutralizing antibodies in SPF muscovy ducks and to protect the immunized birds against homologous experimental infection.
2. Materials and methods 2.1. Cells and virus Primary duck embryo fibroblasts (DEF) were prepared from SPF fertile muscovy duck eggs. Trypsinization of 14-day-old embryos was performed according to standard practice. The dispersed cells were cultured as monolayers in BHK medium supplemented with 5% fetal calf serum
(FCS) (Gibco BRL). Spodoptera frugiperda 9 (Sf9) cells were grown and maintained in suspension or monolayer cultures at 28 ◦ C using TNM-FH media supplemented with 5% FCS. Muscovy duck reovirus strain 89330 (DRV-89330) was isolated in 1989 from the liver, spleen and heart of sick muscovy ducks, after several passages via the chorio-allantoic membrane (CAM) of SPF muscovy duck eggs. The virus was cloned and titrated on fertile muscovy duck eggs. Titers were expressed as log10 50% embryo lethal dose (ELD50 ). Wild-type of Autographa californica nuclear polyhedrosis virus (AcNPV) and recombinant strains of BaculoGold® virus (Pharmingen) were propagated in Sf9 cells according to standard methods. 2.2. Polyclonal sera The anti-reovirus polyclonal serum used as a positive control in the present study was obtained from sacrificed SPF muscovy ducks experimentally infected with cloned DRV-89330. This serum was shown to be reovirus-specific as assessed by checking its lack of reactivity against seven other viral pathogens infecting the duck species. The reovirus-negative serum was obtained from SPF muscovy ducks. 2.3. Amplification of DRV-89330 σ C and σ B-encoding genes and construction of recombinant baculovirus transfer vectors DRV-89330 C protein being encoded by the DRV-89330 S4 segment (nucleotides 280 to 1089), its coding sequence was obtained by PCR with TaqGold® polymerase (Perkin-Elmer) using a recombinant S4 containing pMOS vector (pMOS/FG14) as the template [5]. The primers used were sigCup (5 -GAACGAATTCCACCTTCACC-3 ) and sigClo (5 -GCGGCGAATTCACTACCTCA-3 ), introducing EcoRI restriction sites (shown in italics) and corresponding in their S4 gene-specific sequences to nucleotides 260–279 and 1106–1087, respectively. After a denaturation step at 95 ◦ C for 12 min, the PCR comprises 30 cycles of denaturation at 95 ◦ C for 30 s, primer annealing at 56 ◦ C for 30 s and extension at 72 ◦ C for 1 min, followed by a final extension cycle at 72 ◦ C for 10 min. The resulting PCR products were subjected to digestion with EcoRI and ligated into dephosphorylated EcoRI-digested baculovirus transfer vector pVL1392 (Pharmingen). In case of B, the encoded ORF of DRV-89330 S3 segment (nucleotides 31–1134) was extracted from pMOS/AG5 by digestion with PstI and BamHI and ligated into PstI/BamHI-digested pVL1392 vector. The derivative plasmids, pVLC-KV7 and pVLB-AQ1, respectively were proof sequenced and selected for recombinant baculovirus production. pVLC-KV7 contained a silencing mutation in position 363 (T > C), as compared to S4 ORF2 consensus sequence.
G. Kuntz-Simon et al. / Vaccine 20 (2002) 3113–3122
2.4. Transfection and selection of recombinant baculoviruses Ten microgram of pVLC-KV7 and pVLB-AQ1 plasmids were each co-transfected in insect cells with 500 ng of BaculoGold® linearized baculovirus DNA (Pharmingen) using DOTAP transfection reagent (Boehringer Mannheim), according to the supplier’s instructions. Polyhedrin-negative recombinant progeny viruses were selected, agar-plaque purified and amplified in Sf9 cells. Working stocks of the selected BacGC and BacGB recombinant viruses were prepared from the virus stock passage #1 by propagation in Sf9 cells. 2.5. Characterization of recombinant proteins by SDS-PAGE and protein blot analysis Sf9 cells were infected with BacGC or BacGB at a multiplicity of infection of 10–20 p.f.u. per cell. Monolayers were harvested 72 h post-infection (p.i.), washed twice with phosphate-buffered saline (PBS) and finally suspended in TE 0.1× (Tris–HCl 1 mM, EDTA 0.1 mM). The DC Protein Assay (Bio-Rad) was used for determining protein concentration. 5.104 equivalent cells were diluted (v/v) in 2× loading buffer (100 mM Tris–HCl pH 6.8, 4% SDS, 5% -mercaptoethanol, 0.2% bromophenol blue, 20% glycerol). The mixture was heated for 5 min at 100 ◦ C, and proteins were resolved by SDS-PAGE 12%. They were then electroblotted onto a Protran NB nitrocellulose membrane (Schleicher & Schuell) using a semi-dry apparatus (Cera Labo). Total proteins transferred were revealed by incubating the membrane in 0.2% Ponceau S (Sigma) for 3 min at room temperature. After membrane scanning and subsequent destaining in water, it was blocked for 30 min in PBS containing 5% milk powder and 0.05% Tween 20. Filters were then incubated overnight at 4 ◦ C with the duck anti-reovirus polyclonal serum diluted 1/2000 in PBS containing 0.2% Tween 20 (PBST). After several washes with PBST, bound antibodies were hybridized to a rabbit anti-duck serum conjugated to horseradish peroxidase (Nordic Immunological Laboratories) for 90 min at room temperature. Conjugated antibodies were detected by a chemiluminescence reaction using the RenaissanceTM kit (NEN) and revealed by autoradiography. 2.6. Extensive protein expression and emulsions preparation In order to produce large scale of recombinant proteins, Sf9 cells were either infected with BacGC or co-infected with BacGC and BacGB, at a total m.o.i. of 1 to 2 p.f.u per cell. Briefly, 80 ml of Sf9 cell cultures containing 107 cells per ml were infected with appropriate volume of recombinant baculoviruses. After 1 h virus adsorption at room temperature with occasional agitation, the suspension
3115
cultures were transferred into spinner flasks and the volume adjusted to 500 ml. After 96 h incubation at 28 ◦ C with agitation at 80 rpm, the cells were numerated, harvested by centrifugation at 2500 rpm for 15 min at 4 ◦ C, washed twice with PBS and finally resuspended in PBS at a concentration of 4 × 107 cell equivalents per ml. The cells were lysed by freezing at −70 ◦ C and thawing. The total amount of proteins in the crude extracts was quantified by the DC protein assay (Bio-Rad) and the relative amount of baculovirus-expressed DRV-89330 C and/or B proteins was estimated from SDS-PAGE analysis performed as described above. Immunoreactivity of recombinant C and/or B proteins was checked by Western-blot analysis before each inoculation. The inocula were prepared immediately before use, as follows. For 1 ml emulsion to prepare, 235 l of crude lysate were mixed with 15 l of 20% Tween 80 for 30 min at 4 ◦ C with constant agitation. 750 l of a water-in-oil adjuvant routinely used for inactivated avian vaccines and kindly provided by Dr. F.-X. Le Gros (Merial), were then added to the suspension and mixed for 30 min at 4 ◦ C with constant agitation. The mixtures were then emulsified and stored on ice until inoculation to ducks. 2.7. Immunization of muscovy ducks with recombinant proteins and protection test One hundred and twelve 4-week-old SPF muscovy ducks were divided into four groups randomized according to sex and weight. They were maintained in four separated air-filtered rooms and fed a home-made duck starter and water ad libitum. Ducks from two groups received two injections 3 weeks apart of either C emulsion (VC group) or C + B emulsion (VC + B), respectively. The emulsions were inoculated subcutaneously in the side of the neck. Each male duck received 1 × 107 -infected cell equivalents in a final volume of 1 ml for the first injection and 1.5 × 107 cell equivalents in a final volume of 1.5 ml for the second injection. Each female duck was inoculated with 0.9 × 107 infected cell equivalents in a final volume of 0.9 ml for the first injection and 1.5 × 107 cell equivalents in a final volume of 1.5 ml for the second injection. Ducks from both vaccinated groups and from the challenge–control group (CC group) were challenged intramuscularly with 0.5 ml DRV-89330 strain containing 106.5 ELD50 /ml, 3 weeks after the second injection. In the last group, ducks were not vaccinated and not challenged (C group). The ducks were daily observed for clinical symptoms appearance. All of them were weighted at the start day (W0), 6 weeks later just before the challenge (W6), and 2 and 3 weeks post-infection (W8 and W9, respectively). The minimal normal weight gain of ducks from each sex was defined, at 2 and 3 weeks post-challenge, as the mean weight gain of male or female control ducks minus its standard deviation. Any dead duck was examined for macroscopic lesions. All
3116
G. Kuntz-Simon et al. / Vaccine 20 (2002) 3113–3122
the ducks still alive 3 weeks post-challenge were sacrificed and examined for macroscopic lesions. Clinical status was defined as “normal”, “subnormal”, “abnormal” and “dead”. “Normal” meant no symptoms, no macroscopic lesions and normal weight gain until sacrifice. “Subnormal” meant symptoms with insufficient weight gain but without macroscopic lesion. Finally, “abnormal” meant specific macroscopic lesions. 2.8. Sample collection Samples of heart, liver, spleen and gastrocnemius tendon were collected on 2–3 males and 2–3 females from each group, 2 and 3 weeks post-challenge (W8 and W9, respectively) and processed for examination of microscopic lesions. Each lesion type was classified as “mild”, “moderate”, “severe” and “very severe” and recorded from 0 to 4. A microscopic score was calculated as 100 × (number of ducks with lesions × lesion intensity)/number of ducks examined. Blood specimens were collected on the days when the ducks were immunized (first and second vaccine injection, at W0 and W3, respectively) or challenged (W6), and weekly after challenge (W7–W9). Before the challenge, sera were obtained from 5 males and 5 females from the control and the CC groups, and from 10 birds of each sex from both vaccinated groups (VC and VC + B). After the challenge, 10 sera were also collected from birds of each sex in the CC group. 2.9. Detection of antibodies by indirect immunofluorescence All the collected sera were analyzed for the presence of DRV-specific antibodies in an indirect immunofluorescence assay. Briefly, 103.5 ELD50 DRV-89330 strain was inoculated onto subconfluent DEF monolayers cultured in 96-well plates and allowed to adsorb for 2 h at 37 ◦ C before adding medium up to 200 l. After 72 h incubation at 37 ◦ C, the cells were fixed in a methanol–acetone (v/v) mixture for 25 min at −20 ◦ C. The fixed cells were washed with PBS, dried and stored at −20 ◦ C. The sera were diluted 1/10, 1/50 and 1/100 in PBS and incubated onto the fixed infected cells (50 l per well) for 30 min at 37 ◦ C. The cells were then washed three times with PBS and incubated for 30 min at 37 ◦ C in the presence of 50 l of rabbit anti-duck IgG (H+L) conjugated to fluorescent (Nordic Immunological Laboratories), diluted 1/30 in PBS containing Evans Blue (1/75). The plates were washed twice with PBS and the cells were finally stored in a mixture PBS–glycerol (v/v) at 4 ◦ C until observation under optic fluorescence microscopy. The presence of antibodies in each serum was classified as “negative”, “mild positive”, “positive” and “strongly positive” and recorded from 0 to 3, respectively. The antibody score (Ab score) was defined as (number of sera × level)/number of sera examined.
2.10. Seroneutralization test All the collected sera were incubated at 56 ◦ C for 30 min and checked for the presence of neutralizing antibodies using a microtiter neutralization assay [21]. Briefly, 104.5 ELD50 of DRV-89330 strain in 25 l BHK maintenance medium were mixed with 25 l of sera diluted 1/10 and 1/40 in PBS and the mixture was incubated at 37 ◦ C for 1 h. Virus incubated with a duck reovirus-negative polyclonal serum was used as a negative control. Ten microliter of the mixtures were inoculated onto DEF monolayers in 96-well plates and antibody-free viral particles were allowed to adsorb for 2 h at 37 ◦ C before adding medium up to 200 l. After 72 h incubation at 37 ◦ C, the cells were fixed and processed by an indirect immunofluorescence assay as described above, using the duck anti-reovirus polyclonal serum diluted 1/100 in PBS. Four sera from each group were serially diluted from 1/40 to 1/2560 in order to determine neutralization titers. These titers were expressed as the log 2 of the highest dilution of antibody which reduced the fluorescent plaque number by 50% of that of the control dishes. A geometrical mean neutralization titer was calculated for sera from each group. 2.11. Statistical analysis Parametric statistical tests were applied using the SYSTAT® 7.0 computer software package (SPSS Inc.). Normal distributions of weight in each group were verified using the one sample Kolmogorov–Smirnov test with Lilliefors option. Multiple comparisons of the weight data and repeated measures analyses were made using ANOVA procedure. Differences were judged significant at P ≤ 0.05. Yates corrected χ 2 -test was used for comparisons of clinical status, after having grouped together the subnormal, abnormal and dead ducks from each group.
3. Results 3.1. Expression and antigenicity of DRV-89330 σ C and σ B proteins The coding sequences of DRV-89330 C (S4 ORF2) and B (S3 ORF) were inserted into baculovirus transfer vectors and recombinant baculoviruses BacGC and BacGB were constructed, respectively. Analysis of crude extracts of BacGC or BacGB-infected Sf9 cells by SDS-PAGE revealed the presence of proteins of approximately 29 and 38–40 kDa, respectively (Fig. 1) which was consistent with the expected size of C and B [5]. It has to be noticed that the molecular weights of recombinant proteins were assessed by comparison with ECL marker proteins (Fig. 1B) rather than with the prestained protein standards, the colored dyes used to stain them leading to retention in gel (Fig. 1A). Fig. 1B shows the immunological detection of the expressed
G. Kuntz-Simon et al. / Vaccine 20 (2002) 3113–3122
3117
Fig. 1. Expression and antigenicity of DRV-89330 C and B proteins. (A) SDS-PAGE analysis. Total proteins were revealed by Ponceau S after electroblotting onto nitrocellulose membrane. Lane 1: ECL protein molecular weight marker (Amersham). Lanes 2 and 7: mock-infected Sf9 cells (37 g). Lanes 3 and 8: wild-type baculovirus AcNPV-infected Sf9 cells (27 g). Lanes 4 and 9: BacGB-330-infected Sf9 cells (18 g). Lanes 5 and 10: BacGC-330-infected Sf9 cells (18 g). Lane 6: full-range rainbow molecular weight marker (Amersham). The positions of DRV C, DRV B and polyhedrin protein (P) are indicated. (B) Western-blot analysis after incubation of the membrane with SPF duck serum. Lane 1: ECL protein molecular weight marker (Amersham). Lanes 2–5: hybridization with a negative SPF duck anti-reovirus serum. Lanes 6–9: hybridization with a duck anti-reovirus polyclonal serum.
C and B proteins using a duck anti-reovirus polyclonal serum (lanes 8 and 9). It appeared that C hybridized more strongly than B with antibodies present in the polyclonal serum, suggesting that C was more antigenic than B. No reovirus-specific protein was detected in lysates derived from mock or wild-type baculovirus-infected cells (Fig. 1B, lanes 6 and 7) nor when Western-blot analyses were performed using a negative anti-reovirus serum from duck (Fig. 1B, lanes 2–5). In order to produce DRV C and B in the same culture, we co-infected Sf9 cells with various ratio of BacGC and BacGB, simultaneously. Western-blot analyses showed
that C and B proteins have been co-expressed (Fig. 2, lanes 3–5). The C protein was clearly revealed in a dose-dependent manner, which was not so evident in case of protein B. The ratio 50/50 was chosen to co-infect Sf9 cells into spinner flasks for large scale productions. It has to be noted that when loading low amounts of C on the gel (Fig. 2), or extending the gel run, we revealed that C migrated as two distinct bands. One possible explanation is that some of the primary translation products of the S4 mRNA underwent a post-translational modification that affected the electrophoretic mobility of the expression products.
3118
G. Kuntz-Simon et al. / Vaccine 20 (2002) 3113–3122
Fig. 2. Co-expression of recombinant DRV-89330 C and B proteins in insect cells. Total Sf9 cell lysates were resolved by SDS-PAGE and analyzed by Western-blot using a duck anti-reovirus polyclonal serum. Lane 1: BacGC-infected Sf9 cells (10 g). Lane 2: BacGB-infected Sf9 cells (10 g). Lanes 3–5: BacGC + BacGB-infected Sf9 cells (10 g) in a ratio of 30/70 (lane 3), 50/50 (lane 4), 70/30 (lane 5). Lane 6: mock-infected cells (20 g). Molecular weights (kDa) come from full-range rainbow molecular weight marker (Amersham) loaded in SDS-PAGE (data not shown).
3.2. Evaluation of duck immunization 3.2.1. Clinical signs of ducks before challenge Before the challenge, no clinical signs were observed irrespective of the groups. Analysis of weights is reported in Table 1. It was confirmed that the four groups were strictly homogeneous at the start day (W0). Comparisons of weight gains before the challenge (W6–W0) revealed no statistically significant differences between vaccinated and control groups. Only weight gains of male ducks from both vaccinated groups differed from each other (Table 1). 3.2.2. Clinical signs of ducks after challenge After the challenge, all the ducks from the CC group presented clinical symptoms typical from the reovirus infection.
During the first week, the droppings liquefied and became greenish, tending to the diarrhea. Around 50%, males or females, presented problems on legs with reluctance to move. One female showed a fibrinous deposition inside the anterior chamber of the eye and another one dead 2 weeks p.i. The post-mortem examination revealed an important pericarditis, necrosis lesions in liver and an hypertrophy of spleen. Three females sacrificed at that time for organ sampling exhibited the same lesions, with a slightly lower intensity. These lesions were milder or absent in sacrificed males. Ducks from the control group showed neither clinical signs nor macroscopic lesions. This observation was also true for females from the VC group. Among males from this group, 6/15 of them exhibited some leg weakness 1 week p.i. However, this ratio was reduced to 2/15 2 weeks p.i and no more symptoms were observed at the end of the assay. Only one C vaccinated male showed mild pericarditis when sacrificed 3 weeks p.i. In the VC + B group, a few males and females showed leg weakness and macroscopic lesions were exceptional, consisting in very mild pericarditis. Weights of females and males belonging to the CC group were negatively affected by the challenge, as shown by the weight loss measured 2 weeks p.i. (Table 1). In addition, reduced gain weights were still observed 3 weeks p.i. in case of challenged females, as compared to the control group (Table 1). By contrast, vaccinated females gained as much weight as unchallenged control ones, 2 and 3 weeks p.i. Weight gains of vaccinated males were lower than that of control counterparts and similar to challenged ducks 2 weeks p.i. but no more statistical differences between the different groups could be evidenced 3 weeks p.i. Finally, total weight gain of males were not different irrespective of the treatment, whereas vaccinated females gained more weight than those from the CC group. As defined in Section 2, clinical status was calculated based on symptoms, weight gain and macroscopic lesions. The normal weight gain during the 3 weeks post-challenge was ≥58 g for the females and ≥215 g for the males. The clinical status of females from both VC and VC + B
Table 1 Weight gains of ducks during the course of the assay Sex
Groups
W0
Gain before challenge (W6–W0)
Female
Control CC VC VC + B
818a 816a 817a 820a
± ± ± ±
49 47 47 49
(n (n (n (n
= 14) = 14) = 15) = 13)
979a 974a 1045a 965a
Male
Control CC VC VC + B
1072a 1081a 1078a 1091a
± ± ± ±
83 80 78 70
(n (n (n (n
= 14) = 14) = 15) = 13)
1471ab 1591ab 1719a 1417b
± ± ± ±
Gain 2 weeks p.i. (W8–W6)
Gain 3 weeks p.i. (W9–W6)
76 (n = 14) 104 (n = 14) 112 (n = 15) 101 (n = 13)
41a −101b −4a 65a
± ± ± ±
99 (n = 14) 116 (n = 13) 130 (n = 15) 75 (n = 13)
104a 9b 103a 142a
± ± ± ±
46 51 64 85
± 300 (n = 14) ± 220 (n = 14) ± 388 (n = 15) ± 22 (n = 13)
275a −95b 24b 87b
± ± ± ±
112 384 289 167
= 14) = 14) = 15) = 13)
376a 166a 180a 329a
± ± ± ±
161 (n = 11) 49 (n = 11) 307 (n = 12) 162 (n = 10)
(n (n (n (n
(n (n (n (n
= 12) = 10) = 12) = 11)
Total gain (W9–W0) 1080ab ± 100 (n = 12) 997a ± 104 (n = 10) 1144b ± 120 (n = 12) 1102b ± 88 (n = 11) 1904a 1755a 1945a 1766a
± ± ± ±
176 404 336 231
(n (n (n (n
= 11) = 11) = 12) = 10)
Note: body weights at the start day (W0) and weight gains during the course of the assay are shown as means (±S.D.) in grams. The number (n) of ducks by group is indicated into brackets. See the text for the definition of groups. Weights or weight gains that were not found statistically different (P > 0.05) at a given time or period, respectively, are affected with the same letter. Statistical analyses have been performed comparing female and male weights independently (separately?).
G. Kuntz-Simon et al. / Vaccine 20 (2002) 3113–3122
fibrinous exudate (lesion h2) with infiltration of leukocytes. In two ducks, inflammatory cells progressed as far as the myocardium (lesion h3). Much more characteristic, severe changes in the leg tendons consisted in a strong inflammation (lesion t1), associated with an exudative inflammation of the synovial sheaths (lesion t2). The peritendenous tissues also had a dense diffuse infiltrate of lymphocytes and plasmocytes (lesion t3). Microscopic scores, based on lesion intensity as defined in Section 2, were calculated for each group and are reported in Table 3. Two weeks p.i. (W8), total microscopic scores of VC and VC + B groups were 3.4 and two times lower than the score obtained by the CC group, respectively. Most of the ducks from vaccinated groups did not present any typical lesion at the heart level, two ducks having only developed a discrete pericarditis (h1). Inflammation of the synovial sheaths (t2) was strongly reduced in the VC group, as compared to the CC group. Most of the ducks from the VC + B group developed lesions at the tendon area, but these lesions were strongly attenuated as compared to those observed in the CC group. Three weeks p.i. (W9), ducks from the CC group recovered from some heart lesions, resulting in a lower microscopic score as compared to the W8 score. By contrast, the VC group score increased between 2 and 3 weeks p.i., the tendon lesions becoming quasi-constant and being even classified as “severe” in some cases. The VC + B group microscopic score slightly decreased between 2 and 3 weeks p.i. Nevertheless, no significant pericarditis (h1) was still observed 3 weeks p.i. in neither vaccinated groups. Finally, it has to be noted that hyperplasia of lymphoid tissues of liver (l) and spleen (s) were generally more intensive in vaccinated groups than in the CC group, especially 2 weeks p.i.
Table 2 Clinical status post-challenge Sex
Group
n
Clinical status Normal
Subnormal
Abnormal
Dead
Female
Control CC VC VC + B
14 14 15 13
13 2 11 13
1 2 4 0
0 9 0 0
0 1 0 0
Male
Control CC VC VC + B
14 14 15 13
13 2 7 9
1 7 7 4
0 5 1 0
0 0 0 0
3119
Note: see the text for the definition of clinical status; n = number of ducks by group at the time of challenge.
groups, as well as clinical status of males from the VC+B group, were similar to that exhibited by ducks from control group, females and males, respectively (Table 2). None of these vaccinated animals was classified as “abnormal”, by contrast to 9/14 males and 5/14 females from the CC group. VC males displayed an impaired clinical status, with an increased number of ducks classified as “subnormal” and 1/15 classified as “abnormal”. This status was intermediate between that exhibited by male ducks from the VC group and that exhibited by male ducks from the CC group. 3.2.3. Histological assessment 2 and 3 weeks post-challenge Among the ducks from the control group that were examined for histological studies, no one developed relevant microscopic lesions. By contrast, ducks from the CC group developed histopathological lesions typically observed in reovirus-infected birds, with no significant differences between males and females. In the liver, it was observed small infiltrates of plasmocytes and lymphocytes in the portal region as well as an hyperplasia of the lymphoid follicles (lesion l). Hyperplasia of the lymphoid tissue was also observed in the spleen (lesion s). In the heart, pericarditis (lesion h1) consisted in an infiltration of the serous membrane by mononuclear inflammatory cells, notably lymphocytes and plasmocytes. It was associated with a
3.2.4. Analysis of anti-DRV antibodies in immunized versus non-immunized ducks before and after challenge As assessed by indirect immunofluorescence assay, the non-immunized animals did not exhibit any anti-DRV antibodies prior to the challenge and stayed negative in the control group during the course of the assay, whereas all the ducks from the CC group became seropositive after the challenge. Three weeks after the first injection (V1) of vaccine
Table 3 Histological assessment 2 and 3 weeks post-challenge (microscopic score) Date
Groups
Lesions h1
h2
h3
l
s
t1
t2
t3
Total
2 weeks p.i. (W8)
Control CC VC VC + B
0 150 8 17
0 100 0 0
0 50 0 0
0 17 42 58
0 75 1 167
0 8 0 0
0 133 17 67
0 83 17 0
0 617 183 308
3 weeks p.i. (W9)
Control CC VC VC + B
8 75 8 0
0 0 0 0
0 0 0 0
0 75 8 33
0 1 133 141
0 8 8 0
8 125 1 58
0 0 8 0
17 383 267 233
Note: see the text for the definition of groups and lesions.
3120
G. Kuntz-Simon et al. / Vaccine 20 (2002) 3113–3122
Fig. 3. Immunogenicity of DRV C, alone or in combination with DRV B. (A) Antibody scores (see the text for the definition) obtained from indirect immunofluorescence assay performed on all the sera collected during the assay. (B) Neutralization titres obtained from seroneutralization test performed on selected sera (see the text for more details). (䊉): C group; (䉬): CC group; (䉱): VC group; (䊏): VC + B group. V1: first injection; V2: second injection; C: challenge.
preparation, antibodies were detected in 100% of the sera collected from both VC and VC + B groups, with a slightly higher Ab score for the VC group. Three weeks after the second injection (V2), VC group antibody levels kept going increasing whereas VC + B group levels remained quite constant. After the challenge, the levels were quite similar for the three infected groups. All the sera we previously characterized as containing reovirus-specific antibodies were found to neutralize the DRV-89330 strain infectivity in DEF cultures, except those collected from both vaccinated groups 3 weeks after the first injection (V1). The mean neutralization titers are reported in Fig. 3B. After the booster (V2), sera from vaccinated groups acquired a detectable neutralizing activity. Following challenge, immunized birds exhibited a rapid and sustained neutralization activity in cell cultures. Non-vaccinated ducks also produced neutralizing antibodies after challenge, but in a lower range than vaccinated animals. The neutralization titer of the CC group sera increased with time post-challenge, reaching the same titers than sera from vaccinated birds 2 weeks p.i., but decreasing quickly.
4. Discussion In the present study, we showed that baculovirus-expressed DRV C was around 29 kDa weight, i.e. 10 kDa less than
ARVC, that is in accordance with the size we previously estimated from amino acid sequence analysis [5]. This data also confirmed those obtained by others from comparative analysis of immunoprecipitated proteins from duck and chicken reoviruses [8]. Indeed, this analysis had suggested that, although showing a basically similar polypeptide pattern, DRV greatly differed from ARV in the mobility of the C protein, DRV C migrating faster than its ARV counterpart. We showed that recombinant DRV C protein hybridized to antibodies present into a duck reovirus-specific polyclonal serum. This immunoreactivity was stronger than that of recombinant DRV B protein, suggesting the relative antigenic character of both proteins. Radioimmunoprecipitation assay would permit to confirm that hypothesis. Immunization of ducks with crude extracts of insect cells expressing C alone or in combination with B did not induce any adverse clinical sign. By contrast, males from the VC group have gained more weight than those from the VC + B group before the challenge. Knowing that ducks from the VC group received twice the amount of recombinant C protein, it could be hypothesized that high doses of DRV-89330 C may stimulate the birds growing up. Alternatively, it should be checked that DRV-89330 B protein did not alter the growing up, although we previously shown that DRV-89026 B does not induce any adverse sign [21]. Further experiments including repetition of the assay and histological assessment before challenge should answer this question. On the basis of clinical signs developed by ducks from the CC group post-infection, it appeared that DRV-89330 challenge effect was more severe on females than on males. In our trial, vaccination with DRV-89330 recombinant proteins significantly reduced the appearance of these clinical symptoms after challenge. This immunization was more efficient in females. No difference in weight gain or clinical status was observed between both vaccinated female groups after the challenge, these parameters being similar to those obtain for females from the control group. In case of vaccinated males, weight gains were not different from those from the CC group 2 weeks p.i. and final weight gains were similar for all the groups including unchallenged controls. In addition, the clinical status of males from the VC + B group was not statistically different from that of males from the control group. Histological analyses revealed that immunization of ducks with C resulted in a transient reduction in the severity of reovirus-induced tenosynovitis, the lesions becoming, however, apparent at 3 weeks p.i., roughly in half of the samples. Immunization with both C and B had a slower inhibitory effect on tenosynovitis development but finally less lesions of tendon sheaths were observed in this group in comparison with those observed in the VC group. However, despite some leg tendons affections, very mild pericarditis was observed in vaccinated bird, either from VC or VC + B groups, in contrast to that observed in challenged control ducks. Histological assessment also revealed
G. Kuntz-Simon et al. / Vaccine 20 (2002) 3113–3122
an hyperplasia of lymphoid tissues in liver and spleen of vaccinated birds. These effects could be related to the immunogenicity of the recombinant proteins. Immunization with C or C + B induced the production of antibodies early post the first injection, the Ab score we detected being slightly higher for the VC group. However, despite their different Ab scores, both groups elicited approximately the same neutralizing activity in sera post-boosting. Finally, it can be concluded that both vaccinated groups globally displayed the same level of protection against challenge. This raises the question of how relevant is the combination of C and/or B in inducing that protection. It has to be noted that duck immunization with DRV-89026 B alone had no effect on lesion development in a previous trial [21]. Indirect immunofluorescence assay was performed on sera obtained from this assay and revealed that DRV-89026 B did not elicit the production of reovirus-specific antibody in ducks (data not shown). DRV-89330 B protein sharing 95% identity with DRV-89026 counterpart [5], we assumed common functions for both polypeptides and did not test DRV-89330 B protein alone in the present study. Whether the co-expression of C and B had a synergetic effect remains to be investigated. In this assay, the double amount of C protein injected in the VC group could not be related to an increased inhibitory effect on lesion appearance or a higher antibody production. The explanation could be that in the range of concentration we used in the present assay, twice the dosage of C did not lead to improved protection. However, whether the protection level we achieved could be improved requires further assays. It might be possible to reach higher efficiency by increasing amounts of recombinant proteins to inject. Indeed, other immunization experiments, i.e. in sheep using a bluetongue virus protein subunit as a vaccine, or in chickens using baculovirus-derived avian influenza hemagglutinin, demonstrated that there is a direct relationship between the amount of immunogen administered and the effectiveness of immunization at prevention of clinical disease [22,23]. Moreover, C was here injected without having been purified, in a total amount of 2 mg of proteins. Although crude extracts of insect cells infected with wild-type baculovirus were previously shown to be without any effect on ducks [21], purification of C could also be effective to increase immune activity. Thus, further studies should be undertaken to confirm if there is a dose–response curve for this DRV-89330 C immunogen. Moreover, assay performed on 1-day-old ducks, including earlier challenge and processing until normal mean age for slaughter would permit to evaluate DRV C ability to induce a fully protective immune response, to determine if virus clearance can occur and to clarify is there is or not a difference between both immunogens, C or C + B. Both cell-mediated and humoral immune responses directed against mammalian reovirus 1 have been shown to take place [9]. Despite limited knowledge of duck
3121
immunology and a lack of specific typing reagents to use for the identification and purification of duck lymphoid cell subsets, more detailed studies should also be directed to characterize the immune response in ducks of different ages to a range of doses of recombinant C protein, alone or in combination with B. Moreover, other experiments are necessary to evaluate whether C or C + B can be more immunogenic when used together with other DRV outer capsid proteins. Indeed, computer-based analyses revealed that the overall secondary structure of DRV C comprises (i) a fibrous tail at the N-terminal part, organized as a three-stranded ␣-helical coiled-coil, (ii) the neck, a discrete intermediate region of -sheet immediately behind a short ␣-helical coiled-coil and finally (iii) the head, at the remaining carboxy-terminal one-third, a structurally complex globular domain [5]. Thus, other structural proteins can be required to ensure correct C protein folding to induce efficient antigen presentation and protective immunity. Intending to verify this hypothesis, we cloned and sequenced the DRV M2 segment in order to express the B baculovirus-recombinant protein (Kuntz-Simon et al., unpublished results). Finally, it has to be noted that the baculovirus expression system is an helpful tool to screen candidate genes for vaccinology purpose. However, for practical application in the bird vaccinology field, it is likely that other expression vectors may be employed such as avian poxvirus or herpesvirus [24–26]. Such vectors expressing DRV C, alone or co-expressed with other reovirus proteins could be tested in the duck species.
Acknowledgements We are grateful to Marie-Odile Le Bras for technical assistance in serological analyses. We are indebted to Yannick Morin, Louis Le Coq, Pierre Le Bihannic and Guy Jarnet (AFSSA, Ploufragan) for their contribution to the experimental work on ducks and to Marie Lagadic (Veterinary Histocytopathology Laboratory, Maisons-Alfort, France) for having performed histological analyses. We thank Dr. François-Xavier Le Gros (Mérial, France) for having kindly provided the adjuvant used in vaccination assay. This work was supported by a grant from the FEDER. References [1] Kaschula VR. A new virus disease of the muscovy duck (Cairina moschata Linn.) present in Natal. J South Afr Vet Med Assoc 1950;21:18–26. [2] Gaudry D, Charles J, Tektoff J. A new disease expressing itself by a viral pericarditis in Barbary ducks. C R Acad Sci 1972;274:2916–29. [3] Malkinson M, Perk K, Weisman Y. Reovirus infection of young muscovy ducks. Avian Pathol 1981;10:433–40. [4] Marius-Jestin V, Lagadic M, Le Menec Y, Bennejean G. Histological data associated with muscovy duck reovirus infection. Vet Record 1988;123:32–3.
3122
G. Kuntz-Simon et al. / Vaccine 20 (2002) 3113–3122
[5] Kuntz-Simon G, Le Gall-Reculé G, De Boisséson C, Jestin V. Muscovy duck reovirus C protein is atypically encoded by the smallest genome segment. J Gen Virol 2002;83:1189–1200. [6] Marius V. Prévention de la réovirose du canard de Barbarie à l’aide d’un vaccin à virus inactivé mis au point au laboratoire national de pathologie aviaire de Ploufragan. L’aviculteur 1983;437:65–7. [7] Marius V. Prévention des maladies virales des palmipèdes. Bull Group Tech Vet 1982;82:77–82 (Abis). [8] Heffels-Redmann U, Müller H, Kaleta EF. Structural and biological characteristics of reoviruses isolated from muscovy ducks (Cairina moschata). Avian Pathol 1992;21:481–91. [9] Weiner H, Fields B. Neutralization of reovirus: the gene responsible for the neutralization antigen. J Exp Med 1977;146:1305–10. [10] Takehara K, Kimura Y, Tanaka Y, Yoshimura M. Preparation and characterization of monoclonal antibodies against an avian reovirus. Avian Dis 1987;31:730–4. [11] Wickramasinghe R, Meanger J, Enriquez CE, Wilcox GE. Avian reovirus proteins associated with neutralization of virus infectivity. Virology 1993;194:688–96. [12] Reddy SK, Sy D, Silim A. Radioimmunoprecipitation of avian reovirus polypeptides using virus specific IgM and IgG murine monoclonal and chicken polyclonal antibodies. J Vet Meth 1993;42:13–22. [13] Shapouri MRS, Frenette D, Larochelle R, Arella M, Silim A. Characterization of monoclonal antibodies against reovirus strain S1133. Avian Pathol 1996;25:57–67. [14] Martinez-Costas J, Grande A, Varela R, Garcia-Martinez C, Benavente J. Protein architecture of avian reovirus S1133 and identification of the cell attachment protein. J Virol 1997;71:59–64. [15] Grande A, Rodriguez E, Costas C, Everitt E, Benavente J. Oligomerization and cell-binding properties of the avian reovirus cell-attachment protein C. Virology 2000;274:367–77. [16] Meanger J, Wickramasinghe R, Enriquez CE, Robertson MD, Wilcox GE. Type-specific antigenicity of avian reoviruses. Avian Pathol 1995;24:121–34.
[17] Shapouri MRS, Arella M, Silim A. Evidence for the multimeric nature and cell binding ability of avian reovirus 3 protein. J Gen Virol 1996;77:1203–10. [18] Lee PWK, Gilmore R. Reovirus cell attachment protein 1: structure–function relationships and biogenesis. In: Tyler KL, Oldstone, MBA, editors. Reoviruses I. Berlin: Springer, 1998. p. 137–53. [19] Reinisch KM, Nibert M, Harrison SC. Structure of the reovirus core at 3.6 Å resolution. Nature 2000;404:960–7. [20] Shapouri MRS, Arella M, Silim A. Immunogenicity of E. coli-expressed 3 protein of avian reovirus in chicken. Avian Pathol 1997;26:419–25. [21] Le Gall-Reculé G, Cherbonnel M, Arnauld C, Blanchard P, Jestin A, Jestin V. Molecular characterization and expression of the S3 gene of muscovy duck reovirus strain 89026. J Gen Virol 1999;80:195–203. [22] Huismans H, Van Der Walt NT, Cloete M, Erasmus BJ. Isolation of a capsid protein of bluetongue virus that induces a protective immune response in sheep. Virology 1987;157:172–9. [23] Crawford J, Wilkinson B, Vosnesensky A, Smith G, Garcia M, Stone H, et al. Baculovirus-derived hemagglutinin vaccines protect against lethal influenza infections by avian H5 and H7 subtypes. Vaccine 1999;17:2265–74. [24] Swayne DE, Garcia M, Beck JR, Kinney N, Suarez DL. Protection against diverse highly pathogenic H5 influenza viruses in chickens immunized with a recombinant fowlpox vaccine containing an H5 avian influenza hemagglutinin gene insert. Vaccine 2000;18:1088–95. [25] Rautenschlein S, Sharma JM, Winslow BJ, McMillen J, Junker D, Cochran M. Embryo vaccination of turkeys against Newcastle disease infection with recombinant fowlpox virus constructs containing interferons as adjuvants. Vaccine 2000;18:426–33. [26] Sakaguchi M, Nakamura H, Sonoda K, Okamura H, Yokogawa K, Hira K. Protection of chickens with or without maternal antibodies against both Marek’s and Newcastle diseases by one-time vaccination with recombinant vaccine of Marek’s disease virus type 1. Vaccine 1998;16:472–9.
Baculovirus-expressed muscovy duck reovirus C protein induces serum neutralizing antibodies and protection against challenge Gaëlle Kuntz-Simon a,1 , Philippe Blanchard b , Martine Cherbonnel a , André Jestin b , Véronique Jestin a,∗ a
b
Avian and Rabbit Virology, Immunology and Parasitology Unit, Poultry and Swine Research Laboratory, French Agency for Food Safety (AFSSA), Zoopˆole Les Croix, BP 53, 22440 Ploufragan, France Viral Genetics and Biosafety Unit, Poultry and Swine Research Laboratory, French Agency for Food Safety (AFSSA), Zoopˆole Les Croix, BP 53, 22440 Ploufragan, France Received 18 September 2001; received in revised form 15 March 2002; accepted 23 May 2002
Abstract Recombinant baculoviruses with C- or B-encoding gene from muscovy duck reovirus (DRV) were constructed. Western-blot analysis showed that C was more immunoreactive than B. Vaccination of SPF ducks with two injections, 3 weeks apart, of emulsions containing C or C + B elicited DRV-specific neutralizing antibodies. Following challenge, vaccination partially—or even totally in some cases— prevented the appearance of clinical symptoms. Moreover, immunization reduced the severity of reovirus-induced tenosynovitis and prevented pericarditis development during the course of the assay. Thus, DRV C, alone or co-expressed with B, appeared as a good candidate for vaccination of ducks (96/100 mots). © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Vaccine; Orthoreovirus; Avian reovirus
1. Introduction When infected by a reovirus, muscovy ducks (Cairina moschata) may develop a disease with mortality and a high level of morbidity. Moreover, reovirus-infected ducks often exhibit bacterial complications leading to poor performance and increased seizures at the slaughterhouse. The acute form of the disease, first described in South Africa in 1950 [1], is usually apparent in muscovy ducklings between 2 and 4 weeks of age, in France. The clinical signs include apathy, diarrhea, reluctance to move and hobbling. Macroscopically, dead animals can show fibrinous pericarditis, friable liver and marbled spleen [2,3,4]. Microscopic lesions have been detailed [4]. Among them, it has to be noticed that the synovial sheath of the leg tendons shows a typical exudative inflammation. The muscovy duck reovirus (DRV), first isolated in France in 1972 [2], belongs to the Orthoreovirus genus, Reoviridae family [3,5]. As other reoviruses, it is a very resistant virus and, therefore, control through sanitation and ∗
Corresponding author. Tel.: +33-2-96016258; fax: +33-2-96016263. E-mail address: [email protected] (V. Jestin). 1 Present address: AFSSA-Ploufragan, Swine Virology and Immunology Unit, Zoopˆole Les Croix, BP 53, 22440 Ploufragan, France.
isolation is not practical for commercial poultry production. The principal method of control should be by vaccination. Despite good experimental results, field attempts to prevent the infection with a DRV inactivated vaccine were unsuccessful [6]. Similarly, assays carried out in the pharmaceutical industry to obtain a DRV attenuated vaccine combining activity and safety have failed. On the other hand, vaccination of muscovy ducks with the chicken reovirus vaccine strain S1133 gave poor results in two successive trials carried out in our laboratory in 1982 [7] and 1990. Though both duck and chicken reoviruses share some common epitopes, as shown by cross-immunodiffusion gel assay and ELISA, cross-neutralization tests revealed that they are antigenically different (Jestin, unpublished results; [8]). Extensive sequences comparisons of genome segments from both species suggested that DRV segregates as a specified genogroup among avian reoviruses [5], confirming that chicken reovirus S1133 is not a good candidate for duck protection. Hence, the development of safe and efficient vaccine against DRV is still required in the field. For that purpose, we proposed to evaluate the immunogenic properties of some DRV proteins. Concerning other reoviruses, it has been shown that most protective antibodies are directed against external viral antigens, particularly those associated with cell attachment [9].
0264-410X/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 0 2 ) 0 0 2 6 4 - 5
3114
G. Kuntz-Simon et al. / Vaccine 20 (2002) 3113–3122
In case of avian reovirus isolated from chicken, and commonly abbreviated ARV, mouse monoclonal antibodies specific from three other proteins neutralized virus infectivity in cell cultures [10,11,12,13]. These proteins are the major outer capsid protein (B), the minor outer capsid protein (C) and the large surface protein (C). ARV B and C proteins induce group-specific neutralizing antibody, while protein C, an homotrimer displaying cell-binding activity [14,15] induces type-specific neutralizing antibodies [16]. Being functionally and structurally related to the protein 1 of mammalian reovirus (MRV) [17,18], ARV C is suggested to be localized at the vertices of the spikes [19]. An Escherichia coli-expressed fusion protein was used to study the immunogenicity of ARV C in chickens. The recombinant protein did not elicit detectable neutralizing antibodies in sera, but some protection against virus-induced mortality in 1-day-old chicks was shown when tested in passive immunization studies [20]. Intending to identify DRV proteins that could play a role in protective immunity, we previously reported the molecular characterization and expression of the DRV-89026 B-encoded gene, that shares 61% identity with its ARV counterpart [21]. We demonstrated the antigenicity of a baculovirus-expressed B protein, but were unable to reveal any immune response against DRV B protein in muscovy ducks immunized with the recombinant protein, nor protection against reovirus challenge. We reported elsewhere that DRV C protein is atypically encoded by the major open reading frame (ORF) of the smallest genome segment (S4) and not by segment S1 as usually reported for other orthoreoviruses [5]. Despite extensive sequence divergence, we showed the conservation of structural domains between DRV and ARV C proteins and suggested that DRV C could be functionally related to ARV C, and thus, could play an important role in the immune response occurring in infected birds. In the present study, we report the expression of proteins C and B from DRV-89330, a strain we demonstrated to be more pathogenic and immunogenic than the DRV-89026 strain we used in a previous trial [21]. We tested the C protein ability, alone or in combination with B, to induce neutralizing antibodies in SPF muscovy ducks and to protect the immunized birds against homologous experimental infection.
2. Materials and methods 2.1. Cells and virus Primary duck embryo fibroblasts (DEF) were prepared from SPF fertile muscovy duck eggs. Trypsinization of 14-day-old embryos was performed according to standard practice. The dispersed cells were cultured as monolayers in BHK medium supplemented with 5% fetal calf serum
(FCS) (Gibco BRL). Spodoptera frugiperda 9 (Sf9) cells were grown and maintained in suspension or monolayer cultures at 28 ◦ C using TNM-FH media supplemented with 5% FCS. Muscovy duck reovirus strain 89330 (DRV-89330) was isolated in 1989 from the liver, spleen and heart of sick muscovy ducks, after several passages via the chorio-allantoic membrane (CAM) of SPF muscovy duck eggs. The virus was cloned and titrated on fertile muscovy duck eggs. Titers were expressed as log10 50% embryo lethal dose (ELD50 ). Wild-type of Autographa californica nuclear polyhedrosis virus (AcNPV) and recombinant strains of BaculoGold® virus (Pharmingen) were propagated in Sf9 cells according to standard methods. 2.2. Polyclonal sera The anti-reovirus polyclonal serum used as a positive control in the present study was obtained from sacrificed SPF muscovy ducks experimentally infected with cloned DRV-89330. This serum was shown to be reovirus-specific as assessed by checking its lack of reactivity against seven other viral pathogens infecting the duck species. The reovirus-negative serum was obtained from SPF muscovy ducks. 2.3. Amplification of DRV-89330 σ C and σ B-encoding genes and construction of recombinant baculovirus transfer vectors DRV-89330 C protein being encoded by the DRV-89330 S4 segment (nucleotides 280 to 1089), its coding sequence was obtained by PCR with TaqGold® polymerase (Perkin-Elmer) using a recombinant S4 containing pMOS vector (pMOS/FG14) as the template [5]. The primers used were sigCup (5 -GAACGAATTCCACCTTCACC-3 ) and sigClo (5 -GCGGCGAATTCACTACCTCA-3 ), introducing EcoRI restriction sites (shown in italics) and corresponding in their S4 gene-specific sequences to nucleotides 260–279 and 1106–1087, respectively. After a denaturation step at 95 ◦ C for 12 min, the PCR comprises 30 cycles of denaturation at 95 ◦ C for 30 s, primer annealing at 56 ◦ C for 30 s and extension at 72 ◦ C for 1 min, followed by a final extension cycle at 72 ◦ C for 10 min. The resulting PCR products were subjected to digestion with EcoRI and ligated into dephosphorylated EcoRI-digested baculovirus transfer vector pVL1392 (Pharmingen). In case of B, the encoded ORF of DRV-89330 S3 segment (nucleotides 31–1134) was extracted from pMOS/AG5 by digestion with PstI and BamHI and ligated into PstI/BamHI-digested pVL1392 vector. The derivative plasmids, pVLC-KV7 and pVLB-AQ1, respectively were proof sequenced and selected for recombinant baculovirus production. pVLC-KV7 contained a silencing mutation in position 363 (T > C), as compared to S4 ORF2 consensus sequence.
G. Kuntz-Simon et al. / Vaccine 20 (2002) 3113–3122
2.4. Transfection and selection of recombinant baculoviruses Ten microgram of pVLC-KV7 and pVLB-AQ1 plasmids were each co-transfected in insect cells with 500 ng of BaculoGold® linearized baculovirus DNA (Pharmingen) using DOTAP transfection reagent (Boehringer Mannheim), according to the supplier’s instructions. Polyhedrin-negative recombinant progeny viruses were selected, agar-plaque purified and amplified in Sf9 cells. Working stocks of the selected BacGC and BacGB recombinant viruses were prepared from the virus stock passage #1 by propagation in Sf9 cells. 2.5. Characterization of recombinant proteins by SDS-PAGE and protein blot analysis Sf9 cells were infected with BacGC or BacGB at a multiplicity of infection of 10–20 p.f.u. per cell. Monolayers were harvested 72 h post-infection (p.i.), washed twice with phosphate-buffered saline (PBS) and finally suspended in TE 0.1× (Tris–HCl 1 mM, EDTA 0.1 mM). The DC Protein Assay (Bio-Rad) was used for determining protein concentration. 5.104 equivalent cells were diluted (v/v) in 2× loading buffer (100 mM Tris–HCl pH 6.8, 4% SDS, 5% -mercaptoethanol, 0.2% bromophenol blue, 20% glycerol). The mixture was heated for 5 min at 100 ◦ C, and proteins were resolved by SDS-PAGE 12%. They were then electroblotted onto a Protran NB nitrocellulose membrane (Schleicher & Schuell) using a semi-dry apparatus (Cera Labo). Total proteins transferred were revealed by incubating the membrane in 0.2% Ponceau S (Sigma) for 3 min at room temperature. After membrane scanning and subsequent destaining in water, it was blocked for 30 min in PBS containing 5% milk powder and 0.05% Tween 20. Filters were then incubated overnight at 4 ◦ C with the duck anti-reovirus polyclonal serum diluted 1/2000 in PBS containing 0.2% Tween 20 (PBST). After several washes with PBST, bound antibodies were hybridized to a rabbit anti-duck serum conjugated to horseradish peroxidase (Nordic Immunological Laboratories) for 90 min at room temperature. Conjugated antibodies were detected by a chemiluminescence reaction using the RenaissanceTM kit (NEN) and revealed by autoradiography. 2.6. Extensive protein expression and emulsions preparation In order to produce large scale of recombinant proteins, Sf9 cells were either infected with BacGC or co-infected with BacGC and BacGB, at a total m.o.i. of 1 to 2 p.f.u per cell. Briefly, 80 ml of Sf9 cell cultures containing 107 cells per ml were infected with appropriate volume of recombinant baculoviruses. After 1 h virus adsorption at room temperature with occasional agitation, the suspension
3115
cultures were transferred into spinner flasks and the volume adjusted to 500 ml. After 96 h incubation at 28 ◦ C with agitation at 80 rpm, the cells were numerated, harvested by centrifugation at 2500 rpm for 15 min at 4 ◦ C, washed twice with PBS and finally resuspended in PBS at a concentration of 4 × 107 cell equivalents per ml. The cells were lysed by freezing at −70 ◦ C and thawing. The total amount of proteins in the crude extracts was quantified by the DC protein assay (Bio-Rad) and the relative amount of baculovirus-expressed DRV-89330 C and/or B proteins was estimated from SDS-PAGE analysis performed as described above. Immunoreactivity of recombinant C and/or B proteins was checked by Western-blot analysis before each inoculation. The inocula were prepared immediately before use, as follows. For 1 ml emulsion to prepare, 235 l of crude lysate were mixed with 15 l of 20% Tween 80 for 30 min at 4 ◦ C with constant agitation. 750 l of a water-in-oil adjuvant routinely used for inactivated avian vaccines and kindly provided by Dr. F.-X. Le Gros (Merial), were then added to the suspension and mixed for 30 min at 4 ◦ C with constant agitation. The mixtures were then emulsified and stored on ice until inoculation to ducks. 2.7. Immunization of muscovy ducks with recombinant proteins and protection test One hundred and twelve 4-week-old SPF muscovy ducks were divided into four groups randomized according to sex and weight. They were maintained in four separated air-filtered rooms and fed a home-made duck starter and water ad libitum. Ducks from two groups received two injections 3 weeks apart of either C emulsion (VC group) or C + B emulsion (VC + B), respectively. The emulsions were inoculated subcutaneously in the side of the neck. Each male duck received 1 × 107 -infected cell equivalents in a final volume of 1 ml for the first injection and 1.5 × 107 cell equivalents in a final volume of 1.5 ml for the second injection. Each female duck was inoculated with 0.9 × 107 infected cell equivalents in a final volume of 0.9 ml for the first injection and 1.5 × 107 cell equivalents in a final volume of 1.5 ml for the second injection. Ducks from both vaccinated groups and from the challenge–control group (CC group) were challenged intramuscularly with 0.5 ml DRV-89330 strain containing 106.5 ELD50 /ml, 3 weeks after the second injection. In the last group, ducks were not vaccinated and not challenged (C group). The ducks were daily observed for clinical symptoms appearance. All of them were weighted at the start day (W0), 6 weeks later just before the challenge (W6), and 2 and 3 weeks post-infection (W8 and W9, respectively). The minimal normal weight gain of ducks from each sex was defined, at 2 and 3 weeks post-challenge, as the mean weight gain of male or female control ducks minus its standard deviation. Any dead duck was examined for macroscopic lesions. All
3116
G. Kuntz-Simon et al. / Vaccine 20 (2002) 3113–3122
the ducks still alive 3 weeks post-challenge were sacrificed and examined for macroscopic lesions. Clinical status was defined as “normal”, “subnormal”, “abnormal” and “dead”. “Normal” meant no symptoms, no macroscopic lesions and normal weight gain until sacrifice. “Subnormal” meant symptoms with insufficient weight gain but without macroscopic lesion. Finally, “abnormal” meant specific macroscopic lesions. 2.8. Sample collection Samples of heart, liver, spleen and gastrocnemius tendon were collected on 2–3 males and 2–3 females from each group, 2 and 3 weeks post-challenge (W8 and W9, respectively) and processed for examination of microscopic lesions. Each lesion type was classified as “mild”, “moderate”, “severe” and “very severe” and recorded from 0 to 4. A microscopic score was calculated as 100 × (number of ducks with lesions × lesion intensity)/number of ducks examined. Blood specimens were collected on the days when the ducks were immunized (first and second vaccine injection, at W0 and W3, respectively) or challenged (W6), and weekly after challenge (W7–W9). Before the challenge, sera were obtained from 5 males and 5 females from the control and the CC groups, and from 10 birds of each sex from both vaccinated groups (VC and VC + B). After the challenge, 10 sera were also collected from birds of each sex in the CC group. 2.9. Detection of antibodies by indirect immunofluorescence All the collected sera were analyzed for the presence of DRV-specific antibodies in an indirect immunofluorescence assay. Briefly, 103.5 ELD50 DRV-89330 strain was inoculated onto subconfluent DEF monolayers cultured in 96-well plates and allowed to adsorb for 2 h at 37 ◦ C before adding medium up to 200 l. After 72 h incubation at 37 ◦ C, the cells were fixed in a methanol–acetone (v/v) mixture for 25 min at −20 ◦ C. The fixed cells were washed with PBS, dried and stored at −20 ◦ C. The sera were diluted 1/10, 1/50 and 1/100 in PBS and incubated onto the fixed infected cells (50 l per well) for 30 min at 37 ◦ C. The cells were then washed three times with PBS and incubated for 30 min at 37 ◦ C in the presence of 50 l of rabbit anti-duck IgG (H+L) conjugated to fluorescent (Nordic Immunological Laboratories), diluted 1/30 in PBS containing Evans Blue (1/75). The plates were washed twice with PBS and the cells were finally stored in a mixture PBS–glycerol (v/v) at 4 ◦ C until observation under optic fluorescence microscopy. The presence of antibodies in each serum was classified as “negative”, “mild positive”, “positive” and “strongly positive” and recorded from 0 to 3, respectively. The antibody score (Ab score) was defined as (number of sera × level)/number of sera examined.
2.10. Seroneutralization test All the collected sera were incubated at 56 ◦ C for 30 min and checked for the presence of neutralizing antibodies using a microtiter neutralization assay [21]. Briefly, 104.5 ELD50 of DRV-89330 strain in 25 l BHK maintenance medium were mixed with 25 l of sera diluted 1/10 and 1/40 in PBS and the mixture was incubated at 37 ◦ C for 1 h. Virus incubated with a duck reovirus-negative polyclonal serum was used as a negative control. Ten microliter of the mixtures were inoculated onto DEF monolayers in 96-well plates and antibody-free viral particles were allowed to adsorb for 2 h at 37 ◦ C before adding medium up to 200 l. After 72 h incubation at 37 ◦ C, the cells were fixed and processed by an indirect immunofluorescence assay as described above, using the duck anti-reovirus polyclonal serum diluted 1/100 in PBS. Four sera from each group were serially diluted from 1/40 to 1/2560 in order to determine neutralization titers. These titers were expressed as the log 2 of the highest dilution of antibody which reduced the fluorescent plaque number by 50% of that of the control dishes. A geometrical mean neutralization titer was calculated for sera from each group. 2.11. Statistical analysis Parametric statistical tests were applied using the SYSTAT® 7.0 computer software package (SPSS Inc.). Normal distributions of weight in each group were verified using the one sample Kolmogorov–Smirnov test with Lilliefors option. Multiple comparisons of the weight data and repeated measures analyses were made using ANOVA procedure. Differences were judged significant at P ≤ 0.05. Yates corrected χ 2 -test was used for comparisons of clinical status, after having grouped together the subnormal, abnormal and dead ducks from each group.
3. Results 3.1. Expression and antigenicity of DRV-89330 σ C and σ B proteins The coding sequences of DRV-89330 C (S4 ORF2) and B (S3 ORF) were inserted into baculovirus transfer vectors and recombinant baculoviruses BacGC and BacGB were constructed, respectively. Analysis of crude extracts of BacGC or BacGB-infected Sf9 cells by SDS-PAGE revealed the presence of proteins of approximately 29 and 38–40 kDa, respectively (Fig. 1) which was consistent with the expected size of C and B [5]. It has to be noticed that the molecular weights of recombinant proteins were assessed by comparison with ECL marker proteins (Fig. 1B) rather than with the prestained protein standards, the colored dyes used to stain them leading to retention in gel (Fig. 1A). Fig. 1B shows the immunological detection of the expressed
G. Kuntz-Simon et al. / Vaccine 20 (2002) 3113–3122
3117
Fig. 1. Expression and antigenicity of DRV-89330 C and B proteins. (A) SDS-PAGE analysis. Total proteins were revealed by Ponceau S after electroblotting onto nitrocellulose membrane. Lane 1: ECL protein molecular weight marker (Amersham). Lanes 2 and 7: mock-infected Sf9 cells (37 g). Lanes 3 and 8: wild-type baculovirus AcNPV-infected Sf9 cells (27 g). Lanes 4 and 9: BacGB-330-infected Sf9 cells (18 g). Lanes 5 and 10: BacGC-330-infected Sf9 cells (18 g). Lane 6: full-range rainbow molecular weight marker (Amersham). The positions of DRV C, DRV B and polyhedrin protein (P) are indicated. (B) Western-blot analysis after incubation of the membrane with SPF duck serum. Lane 1: ECL protein molecular weight marker (Amersham). Lanes 2–5: hybridization with a negative SPF duck anti-reovirus serum. Lanes 6–9: hybridization with a duck anti-reovirus polyclonal serum.
C and B proteins using a duck anti-reovirus polyclonal serum (lanes 8 and 9). It appeared that C hybridized more strongly than B with antibodies present in the polyclonal serum, suggesting that C was more antigenic than B. No reovirus-specific protein was detected in lysates derived from mock or wild-type baculovirus-infected cells (Fig. 1B, lanes 6 and 7) nor when Western-blot analyses were performed using a negative anti-reovirus serum from duck (Fig. 1B, lanes 2–5). In order to produce DRV C and B in the same culture, we co-infected Sf9 cells with various ratio of BacGC and BacGB, simultaneously. Western-blot analyses showed
that C and B proteins have been co-expressed (Fig. 2, lanes 3–5). The C protein was clearly revealed in a dose-dependent manner, which was not so evident in case of protein B. The ratio 50/50 was chosen to co-infect Sf9 cells into spinner flasks for large scale productions. It has to be noted that when loading low amounts of C on the gel (Fig. 2), or extending the gel run, we revealed that C migrated as two distinct bands. One possible explanation is that some of the primary translation products of the S4 mRNA underwent a post-translational modification that affected the electrophoretic mobility of the expression products.
3118
G. Kuntz-Simon et al. / Vaccine 20 (2002) 3113–3122
Fig. 2. Co-expression of recombinant DRV-89330 C and B proteins in insect cells. Total Sf9 cell lysates were resolved by SDS-PAGE and analyzed by Western-blot using a duck anti-reovirus polyclonal serum. Lane 1: BacGC-infected Sf9 cells (10 g). Lane 2: BacGB-infected Sf9 cells (10 g). Lanes 3–5: BacGC + BacGB-infected Sf9 cells (10 g) in a ratio of 30/70 (lane 3), 50/50 (lane 4), 70/30 (lane 5). Lane 6: mock-infected cells (20 g). Molecular weights (kDa) come from full-range rainbow molecular weight marker (Amersham) loaded in SDS-PAGE (data not shown).
3.2. Evaluation of duck immunization 3.2.1. Clinical signs of ducks before challenge Before the challenge, no clinical signs were observed irrespective of the groups. Analysis of weights is reported in Table 1. It was confirmed that the four groups were strictly homogeneous at the start day (W0). Comparisons of weight gains before the challenge (W6–W0) revealed no statistically significant differences between vaccinated and control groups. Only weight gains of male ducks from both vaccinated groups differed from each other (Table 1). 3.2.2. Clinical signs of ducks after challenge After the challenge, all the ducks from the CC group presented clinical symptoms typical from the reovirus infection.
During the first week, the droppings liquefied and became greenish, tending to the diarrhea. Around 50%, males or females, presented problems on legs with reluctance to move. One female showed a fibrinous deposition inside the anterior chamber of the eye and another one dead 2 weeks p.i. The post-mortem examination revealed an important pericarditis, necrosis lesions in liver and an hypertrophy of spleen. Three females sacrificed at that time for organ sampling exhibited the same lesions, with a slightly lower intensity. These lesions were milder or absent in sacrificed males. Ducks from the control group showed neither clinical signs nor macroscopic lesions. This observation was also true for females from the VC group. Among males from this group, 6/15 of them exhibited some leg weakness 1 week p.i. However, this ratio was reduced to 2/15 2 weeks p.i and no more symptoms were observed at the end of the assay. Only one C vaccinated male showed mild pericarditis when sacrificed 3 weeks p.i. In the VC + B group, a few males and females showed leg weakness and macroscopic lesions were exceptional, consisting in very mild pericarditis. Weights of females and males belonging to the CC group were negatively affected by the challenge, as shown by the weight loss measured 2 weeks p.i. (Table 1). In addition, reduced gain weights were still observed 3 weeks p.i. in case of challenged females, as compared to the control group (Table 1). By contrast, vaccinated females gained as much weight as unchallenged control ones, 2 and 3 weeks p.i. Weight gains of vaccinated males were lower than that of control counterparts and similar to challenged ducks 2 weeks p.i. but no more statistical differences between the different groups could be evidenced 3 weeks p.i. Finally, total weight gain of males were not different irrespective of the treatment, whereas vaccinated females gained more weight than those from the CC group. As defined in Section 2, clinical status was calculated based on symptoms, weight gain and macroscopic lesions. The normal weight gain during the 3 weeks post-challenge was ≥58 g for the females and ≥215 g for the males. The clinical status of females from both VC and VC + B
Table 1 Weight gains of ducks during the course of the assay Sex
Groups
W0
Gain before challenge (W6–W0)
Female
Control CC VC VC + B
818a 816a 817a 820a
± ± ± ±
49 47 47 49
(n (n (n (n
= 14) = 14) = 15) = 13)
979a 974a 1045a 965a
Male
Control CC VC VC + B
1072a 1081a 1078a 1091a
± ± ± ±
83 80 78 70
(n (n (n (n
= 14) = 14) = 15) = 13)
1471ab 1591ab 1719a 1417b
± ± ± ±
Gain 2 weeks p.i. (W8–W6)
Gain 3 weeks p.i. (W9–W6)
76 (n = 14) 104 (n = 14) 112 (n = 15) 101 (n = 13)
41a −101b −4a 65a
± ± ± ±
99 (n = 14) 116 (n = 13) 130 (n = 15) 75 (n = 13)
104a 9b 103a 142a
± ± ± ±
46 51 64 85
± 300 (n = 14) ± 220 (n = 14) ± 388 (n = 15) ± 22 (n = 13)
275a −95b 24b 87b
± ± ± ±
112 384 289 167
= 14) = 14) = 15) = 13)
376a 166a 180a 329a
± ± ± ±
161 (n = 11) 49 (n = 11) 307 (n = 12) 162 (n = 10)
(n (n (n (n
(n (n (n (n
= 12) = 10) = 12) = 11)
Total gain (W9–W0) 1080ab ± 100 (n = 12) 997a ± 104 (n = 10) 1144b ± 120 (n = 12) 1102b ± 88 (n = 11) 1904a 1755a 1945a 1766a
± ± ± ±
176 404 336 231
(n (n (n (n
= 11) = 11) = 12) = 10)
Note: body weights at the start day (W0) and weight gains during the course of the assay are shown as means (±S.D.) in grams. The number (n) of ducks by group is indicated into brackets. See the text for the definition of groups. Weights or weight gains that were not found statistically different (P > 0.05) at a given time or period, respectively, are affected with the same letter. Statistical analyses have been performed comparing female and male weights independently (separately?).
G. Kuntz-Simon et al. / Vaccine 20 (2002) 3113–3122
fibrinous exudate (lesion h2) with infiltration of leukocytes. In two ducks, inflammatory cells progressed as far as the myocardium (lesion h3). Much more characteristic, severe changes in the leg tendons consisted in a strong inflammation (lesion t1), associated with an exudative inflammation of the synovial sheaths (lesion t2). The peritendenous tissues also had a dense diffuse infiltrate of lymphocytes and plasmocytes (lesion t3). Microscopic scores, based on lesion intensity as defined in Section 2, were calculated for each group and are reported in Table 3. Two weeks p.i. (W8), total microscopic scores of VC and VC + B groups were 3.4 and two times lower than the score obtained by the CC group, respectively. Most of the ducks from vaccinated groups did not present any typical lesion at the heart level, two ducks having only developed a discrete pericarditis (h1). Inflammation of the synovial sheaths (t2) was strongly reduced in the VC group, as compared to the CC group. Most of the ducks from the VC + B group developed lesions at the tendon area, but these lesions were strongly attenuated as compared to those observed in the CC group. Three weeks p.i. (W9), ducks from the CC group recovered from some heart lesions, resulting in a lower microscopic score as compared to the W8 score. By contrast, the VC group score increased between 2 and 3 weeks p.i., the tendon lesions becoming quasi-constant and being even classified as “severe” in some cases. The VC + B group microscopic score slightly decreased between 2 and 3 weeks p.i. Nevertheless, no significant pericarditis (h1) was still observed 3 weeks p.i. in neither vaccinated groups. Finally, it has to be noted that hyperplasia of lymphoid tissues of liver (l) and spleen (s) were generally more intensive in vaccinated groups than in the CC group, especially 2 weeks p.i.
Table 2 Clinical status post-challenge Sex
Group
n
Clinical status Normal
Subnormal
Abnormal
Dead
Female
Control CC VC VC + B
14 14 15 13
13 2 11 13
1 2 4 0
0 9 0 0
0 1 0 0
Male
Control CC VC VC + B
14 14 15 13
13 2 7 9
1 7 7 4
0 5 1 0
0 0 0 0
3119
Note: see the text for the definition of clinical status; n = number of ducks by group at the time of challenge.
groups, as well as clinical status of males from the VC+B group, were similar to that exhibited by ducks from control group, females and males, respectively (Table 2). None of these vaccinated animals was classified as “abnormal”, by contrast to 9/14 males and 5/14 females from the CC group. VC males displayed an impaired clinical status, with an increased number of ducks classified as “subnormal” and 1/15 classified as “abnormal”. This status was intermediate between that exhibited by male ducks from the VC group and that exhibited by male ducks from the CC group. 3.2.3. Histological assessment 2 and 3 weeks post-challenge Among the ducks from the control group that were examined for histological studies, no one developed relevant microscopic lesions. By contrast, ducks from the CC group developed histopathological lesions typically observed in reovirus-infected birds, with no significant differences between males and females. In the liver, it was observed small infiltrates of plasmocytes and lymphocytes in the portal region as well as an hyperplasia of the lymphoid follicles (lesion l). Hyperplasia of the lymphoid tissue was also observed in the spleen (lesion s). In the heart, pericarditis (lesion h1) consisted in an infiltration of the serous membrane by mononuclear inflammatory cells, notably lymphocytes and plasmocytes. It was associated with a
3.2.4. Analysis of anti-DRV antibodies in immunized versus non-immunized ducks before and after challenge As assessed by indirect immunofluorescence assay, the non-immunized animals did not exhibit any anti-DRV antibodies prior to the challenge and stayed negative in the control group during the course of the assay, whereas all the ducks from the CC group became seropositive after the challenge. Three weeks after the first injection (V1) of vaccine
Table 3 Histological assessment 2 and 3 weeks post-challenge (microscopic score) Date
Groups
Lesions h1
h2
h3
l
s
t1
t2
t3
Total
2 weeks p.i. (W8)
Control CC VC VC + B
0 150 8 17
0 100 0 0
0 50 0 0
0 17 42 58
0 75 1 167
0 8 0 0
0 133 17 67
0 83 17 0
0 617 183 308
3 weeks p.i. (W9)
Control CC VC VC + B
8 75 8 0
0 0 0 0
0 0 0 0
0 75 8 33
0 1 133 141
0 8 8 0
8 125 1 58
0 0 8 0
17 383 267 233
Note: see the text for the definition of groups and lesions.
3120
G. Kuntz-Simon et al. / Vaccine 20 (2002) 3113–3122
Fig. 3. Immunogenicity of DRV C, alone or in combination with DRV B. (A) Antibody scores (see the text for the definition) obtained from indirect immunofluorescence assay performed on all the sera collected during the assay. (B) Neutralization titres obtained from seroneutralization test performed on selected sera (see the text for more details). (䊉): C group; (䉬): CC group; (䉱): VC group; (䊏): VC + B group. V1: first injection; V2: second injection; C: challenge.
preparation, antibodies were detected in 100% of the sera collected from both VC and VC + B groups, with a slightly higher Ab score for the VC group. Three weeks after the second injection (V2), VC group antibody levels kept going increasing whereas VC + B group levels remained quite constant. After the challenge, the levels were quite similar for the three infected groups. All the sera we previously characterized as containing reovirus-specific antibodies were found to neutralize the DRV-89330 strain infectivity in DEF cultures, except those collected from both vaccinated groups 3 weeks after the first injection (V1). The mean neutralization titers are reported in Fig. 3B. After the booster (V2), sera from vaccinated groups acquired a detectable neutralizing activity. Following challenge, immunized birds exhibited a rapid and sustained neutralization activity in cell cultures. Non-vaccinated ducks also produced neutralizing antibodies after challenge, but in a lower range than vaccinated animals. The neutralization titer of the CC group sera increased with time post-challenge, reaching the same titers than sera from vaccinated birds 2 weeks p.i., but decreasing quickly.
4. Discussion In the present study, we showed that baculovirus-expressed DRV C was around 29 kDa weight, i.e. 10 kDa less than
ARVC, that is in accordance with the size we previously estimated from amino acid sequence analysis [5]. This data also confirmed those obtained by others from comparative analysis of immunoprecipitated proteins from duck and chicken reoviruses [8]. Indeed, this analysis had suggested that, although showing a basically similar polypeptide pattern, DRV greatly differed from ARV in the mobility of the C protein, DRV C migrating faster than its ARV counterpart. We showed that recombinant DRV C protein hybridized to antibodies present into a duck reovirus-specific polyclonal serum. This immunoreactivity was stronger than that of recombinant DRV B protein, suggesting the relative antigenic character of both proteins. Radioimmunoprecipitation assay would permit to confirm that hypothesis. Immunization of ducks with crude extracts of insect cells expressing C alone or in combination with B did not induce any adverse clinical sign. By contrast, males from the VC group have gained more weight than those from the VC + B group before the challenge. Knowing that ducks from the VC group received twice the amount of recombinant C protein, it could be hypothesized that high doses of DRV-89330 C may stimulate the birds growing up. Alternatively, it should be checked that DRV-89330 B protein did not alter the growing up, although we previously shown that DRV-89026 B does not induce any adverse sign [21]. Further experiments including repetition of the assay and histological assessment before challenge should answer this question. On the basis of clinical signs developed by ducks from the CC group post-infection, it appeared that DRV-89330 challenge effect was more severe on females than on males. In our trial, vaccination with DRV-89330 recombinant proteins significantly reduced the appearance of these clinical symptoms after challenge. This immunization was more efficient in females. No difference in weight gain or clinical status was observed between both vaccinated female groups after the challenge, these parameters being similar to those obtain for females from the control group. In case of vaccinated males, weight gains were not different from those from the CC group 2 weeks p.i. and final weight gains were similar for all the groups including unchallenged controls. In addition, the clinical status of males from the VC + B group was not statistically different from that of males from the control group. Histological analyses revealed that immunization of ducks with C resulted in a transient reduction in the severity of reovirus-induced tenosynovitis, the lesions becoming, however, apparent at 3 weeks p.i., roughly in half of the samples. Immunization with both C and B had a slower inhibitory effect on tenosynovitis development but finally less lesions of tendon sheaths were observed in this group in comparison with those observed in the VC group. However, despite some leg tendons affections, very mild pericarditis was observed in vaccinated bird, either from VC or VC + B groups, in contrast to that observed in challenged control ducks. Histological assessment also revealed
G. Kuntz-Simon et al. / Vaccine 20 (2002) 3113–3122
an hyperplasia of lymphoid tissues in liver and spleen of vaccinated birds. These effects could be related to the immunogenicity of the recombinant proteins. Immunization with C or C + B induced the production of antibodies early post the first injection, the Ab score we detected being slightly higher for the VC group. However, despite their different Ab scores, both groups elicited approximately the same neutralizing activity in sera post-boosting. Finally, it can be concluded that both vaccinated groups globally displayed the same level of protection against challenge. This raises the question of how relevant is the combination of C and/or B in inducing that protection. It has to be noted that duck immunization with DRV-89026 B alone had no effect on lesion development in a previous trial [21]. Indirect immunofluorescence assay was performed on sera obtained from this assay and revealed that DRV-89026 B did not elicit the production of reovirus-specific antibody in ducks (data not shown). DRV-89330 B protein sharing 95% identity with DRV-89026 counterpart [5], we assumed common functions for both polypeptides and did not test DRV-89330 B protein alone in the present study. Whether the co-expression of C and B had a synergetic effect remains to be investigated. In this assay, the double amount of C protein injected in the VC group could not be related to an increased inhibitory effect on lesion appearance or a higher antibody production. The explanation could be that in the range of concentration we used in the present assay, twice the dosage of C did not lead to improved protection. However, whether the protection level we achieved could be improved requires further assays. It might be possible to reach higher efficiency by increasing amounts of recombinant proteins to inject. Indeed, other immunization experiments, i.e. in sheep using a bluetongue virus protein subunit as a vaccine, or in chickens using baculovirus-derived avian influenza hemagglutinin, demonstrated that there is a direct relationship between the amount of immunogen administered and the effectiveness of immunization at prevention of clinical disease [22,23]. Moreover, C was here injected without having been purified, in a total amount of 2 mg of proteins. Although crude extracts of insect cells infected with wild-type baculovirus were previously shown to be without any effect on ducks [21], purification of C could also be effective to increase immune activity. Thus, further studies should be undertaken to confirm if there is a dose–response curve for this DRV-89330 C immunogen. Moreover, assay performed on 1-day-old ducks, including earlier challenge and processing until normal mean age for slaughter would permit to evaluate DRV C ability to induce a fully protective immune response, to determine if virus clearance can occur and to clarify is there is or not a difference between both immunogens, C or C + B. Both cell-mediated and humoral immune responses directed against mammalian reovirus 1 have been shown to take place [9]. Despite limited knowledge of duck
3121
immunology and a lack of specific typing reagents to use for the identification and purification of duck lymphoid cell subsets, more detailed studies should also be directed to characterize the immune response in ducks of different ages to a range of doses of recombinant C protein, alone or in combination with B. Moreover, other experiments are necessary to evaluate whether C or C + B can be more immunogenic when used together with other DRV outer capsid proteins. Indeed, computer-based analyses revealed that the overall secondary structure of DRV C comprises (i) a fibrous tail at the N-terminal part, organized as a three-stranded ␣-helical coiled-coil, (ii) the neck, a discrete intermediate region of -sheet immediately behind a short ␣-helical coiled-coil and finally (iii) the head, at the remaining carboxy-terminal one-third, a structurally complex globular domain [5]. Thus, other structural proteins can be required to ensure correct C protein folding to induce efficient antigen presentation and protective immunity. Intending to verify this hypothesis, we cloned and sequenced the DRV M2 segment in order to express the B baculovirus-recombinant protein (Kuntz-Simon et al., unpublished results). Finally, it has to be noted that the baculovirus expression system is an helpful tool to screen candidate genes for vaccinology purpose. However, for practical application in the bird vaccinology field, it is likely that other expression vectors may be employed such as avian poxvirus or herpesvirus [24–26]. Such vectors expressing DRV C, alone or co-expressed with other reovirus proteins could be tested in the duck species.
Acknowledgements We are grateful to Marie-Odile Le Bras for technical assistance in serological analyses. We are indebted to Yannick Morin, Louis Le Coq, Pierre Le Bihannic and Guy Jarnet (AFSSA, Ploufragan) for their contribution to the experimental work on ducks and to Marie Lagadic (Veterinary Histocytopathology Laboratory, Maisons-Alfort, France) for having performed histological analyses. We thank Dr. François-Xavier Le Gros (Mérial, France) for having kindly provided the adjuvant used in vaccination assay. This work was supported by a grant from the FEDER. References [1] Kaschula VR. A new virus disease of the muscovy duck (Cairina moschata Linn.) present in Natal. J South Afr Vet Med Assoc 1950;21:18–26. [2] Gaudry D, Charles J, Tektoff J. A new disease expressing itself by a viral pericarditis in Barbary ducks. C R Acad Sci 1972;274:2916–29. [3] Malkinson M, Perk K, Weisman Y. Reovirus infection of young muscovy ducks. Avian Pathol 1981;10:433–40. [4] Marius-Jestin V, Lagadic M, Le Menec Y, Bennejean G. Histological data associated with muscovy duck reovirus infection. Vet Record 1988;123:32–3.
3122
G. Kuntz-Simon et al. / Vaccine 20 (2002) 3113–3122
[5] Kuntz-Simon G, Le Gall-Reculé G, De Boisséson C, Jestin V. Muscovy duck reovirus C protein is atypically encoded by the smallest genome segment. J Gen Virol 2002;83:1189–1200. [6] Marius V. Prévention de la réovirose du canard de Barbarie à l’aide d’un vaccin à virus inactivé mis au point au laboratoire national de pathologie aviaire de Ploufragan. L’aviculteur 1983;437:65–7. [7] Marius V. Prévention des maladies virales des palmipèdes. Bull Group Tech Vet 1982;82:77–82 (Abis). [8] Heffels-Redmann U, Müller H, Kaleta EF. Structural and biological characteristics of reoviruses isolated from muscovy ducks (Cairina moschata). Avian Pathol 1992;21:481–91. [9] Weiner H, Fields B. Neutralization of reovirus: the gene responsible for the neutralization antigen. J Exp Med 1977;146:1305–10. [10] Takehara K, Kimura Y, Tanaka Y, Yoshimura M. Preparation and characterization of monoclonal antibodies against an avian reovirus. Avian Dis 1987;31:730–4. [11] Wickramasinghe R, Meanger J, Enriquez CE, Wilcox GE. Avian reovirus proteins associated with neutralization of virus infectivity. Virology 1993;194:688–96. [12] Reddy SK, Sy D, Silim A. Radioimmunoprecipitation of avian reovirus polypeptides using virus specific IgM and IgG murine monoclonal and chicken polyclonal antibodies. J Vet Meth 1993;42:13–22. [13] Shapouri MRS, Frenette D, Larochelle R, Arella M, Silim A. Characterization of monoclonal antibodies against reovirus strain S1133. Avian Pathol 1996;25:57–67. [14] Martinez-Costas J, Grande A, Varela R, Garcia-Martinez C, Benavente J. Protein architecture of avian reovirus S1133 and identification of the cell attachment protein. J Virol 1997;71:59–64. [15] Grande A, Rodriguez E, Costas C, Everitt E, Benavente J. Oligomerization and cell-binding properties of the avian reovirus cell-attachment protein C. Virology 2000;274:367–77. [16] Meanger J, Wickramasinghe R, Enriquez CE, Robertson MD, Wilcox GE. Type-specific antigenicity of avian reoviruses. Avian Pathol 1995;24:121–34.
[17] Shapouri MRS, Arella M, Silim A. Evidence for the multimeric nature and cell binding ability of avian reovirus 3 protein. J Gen Virol 1996;77:1203–10. [18] Lee PWK, Gilmore R. Reovirus cell attachment protein 1: structure–function relationships and biogenesis. In: Tyler KL, Oldstone, MBA, editors. Reoviruses I. Berlin: Springer, 1998. p. 137–53. [19] Reinisch KM, Nibert M, Harrison SC. Structure of the reovirus core at 3.6 Å resolution. Nature 2000;404:960–7. [20] Shapouri MRS, Arella M, Silim A. Immunogenicity of E. coli-expressed 3 protein of avian reovirus in chicken. Avian Pathol 1997;26:419–25. [21] Le Gall-Reculé G, Cherbonnel M, Arnauld C, Blanchard P, Jestin A, Jestin V. Molecular characterization and expression of the S3 gene of muscovy duck reovirus strain 89026. J Gen Virol 1999;80:195–203. [22] Huismans H, Van Der Walt NT, Cloete M, Erasmus BJ. Isolation of a capsid protein of bluetongue virus that induces a protective immune response in sheep. Virology 1987;157:172–9. [23] Crawford J, Wilkinson B, Vosnesensky A, Smith G, Garcia M, Stone H, et al. Baculovirus-derived hemagglutinin vaccines protect against lethal influenza infections by avian H5 and H7 subtypes. Vaccine 1999;17:2265–74. [24] Swayne DE, Garcia M, Beck JR, Kinney N, Suarez DL. Protection against diverse highly pathogenic H5 influenza viruses in chickens immunized with a recombinant fowlpox vaccine containing an H5 avian influenza hemagglutinin gene insert. Vaccine 2000;18:1088–95. [25] Rautenschlein S, Sharma JM, Winslow BJ, McMillen J, Junker D, Cochran M. Embryo vaccination of turkeys against Newcastle disease infection with recombinant fowlpox virus constructs containing interferons as adjuvants. Vaccine 2000;18:426–33. [26] Sakaguchi M, Nakamura H, Sonoda K, Okamura H, Yokogawa K, Hira K. Protection of chickens with or without maternal antibodies against both Marek’s and Newcastle diseases by one-time vaccination with recombinant vaccine of Marek’s disease virus type 1. Vaccine 1998;16:472–9.