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A commercial PCV2a-based vaccine significantly reduces PCV2b transmission in experimental conditions
Vaccine xxx (2016) xxx–xxx
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A commercial PCV2a-based vaccine significantly reduces PCV2b transmission in experimental conditions N. Rose ⇑, M. Andraud, L. Bigault, A. Jestin, B. Grasland Anses, Laboratoire de Ploufragan-Plouzané, BP 53, 22440 Ploufragan, France Université Bretagne Loire, Rennes, France
a r t i c l e
i n f o
Article history: Received 14 March 2016 Received in revised form 20 May 2016 Accepted 1 June 2016 Available online xxxx Keywords: Porcine Circovirus type 2 Vaccination Transmission Reproduction number
a b s t r a c t Transmission characteristics of PCV2 have been compared between vaccinated and non-vaccinated pigs in experimental conditions. Twenty-four Specific Pathogen Free (SPF) piglets, vaccinated against PCV2 at 3 weeks of age (PCV2a recombinant CAP protein-based vaccine), were inoculated at 15 days postvaccination with a PCV2b inoculum (6 105 TCID50), and put in contact with 24 vaccinated SPF piglets during 42 days post-inoculation. Those piglets were shared in six replicates of a contact trial involving 4 inoculated piglets mingled with 4 susceptible SPF piglets. Two replicates of a similar contact trial were made with non-vaccinated pigs. Non vaccinated animals received a placebo at vaccination time and were inoculated the same way and at the same time as the vaccinated group. All the animals were monitored twice weekly using quantitative real-time PCR and ELISA for serology until 42 days post-inoculation. The frequency of infection and the PCV2 genome load in sera of the vaccinated pigs were significantly reduced compared to the non-vaccinated animals. The duration of infectiousness was significantly different between vaccinated and non-vaccinated groups (16.6 days [14.7;18.4] and 26.6 days [22.9;30.4] respectively). The transmission rate was also considerably decreased in vaccinated pigs (b = 0.09 [0.05–0.14] compared to b = 0.19 [0.11–0.32] in non-vaccinated pigs). This led to an estimated reproduction ratio of 1.5 [95% CI 0.8 – 2.2] in vaccinated animals versus 5.1 [95% CI 2.5 – 8.2] in non-vaccinated pigs when merging data of this experiment with previous trials carried out in same conditions. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Porcine Circovirus type 2 (PCV2) is the primary agent of PCV2systemic disease (PCV2-SD), also called Post-weaning Multisystemic Wasting Syndrome (PMWS). Following the first descriptions in North-America in the mid-90s, the disease became worldwide spread and had devastating consequences in the pig sector. For several years and before vaccine commercialization, only modifications of farm practices based on known risk factors of clinical expression of PCV2-SD were available to tackle the disease [1–3]. It was further shown that some of those practices had a direct impact on reduction of PCV2 propagation within the population of a swine farrow-to-finish operation, decreasing massive exposure of young piglets to the virus [4]. From 2006, commercial vaccine solutions were made available on the market. Based on different principles (inactivated PCV2, PCV2 capsid protein produced in a vector or chimeric inactivated PCV1 virus including PCV2 ORF2), ⇑ Corresponding author at: Nicolas ROSE, Anses, Laboratoire de PloufraganPlouzané, PO Box 53, F22440 Ploufragan, France. E-mail address: [email protected] (N. Rose).
the efficacy of those vaccines, prepared with strains of PCV2a genogroup, has been described in several experimental [5] or field studies [6–11]. Generally, commercial vaccines have been shown to reduce viraemia, viral load in tissues and lymphoid lesions [12–14]. Efficacy towards improvement of the clinical situation in a PMWS or PCV2-SD context has been also documented [15,16]. Moreover, their efficacy was not compromised by challenge with heterologous genotype of PCV2, i.e. PCV2b [14] or more recently mutants from PCV2b [17]. Beside PCV2-SD, the involvement of the virus in subclinical infections and other syndromes such as Porcine Respiratory Disease complex, reproductive disorders or enteric diseases has been more and more documented [18]. The efficacy of PCV2 vaccination towards such PCV2 subclinical infections has also been shown through the improvement of growth performances and reduction of clinical and lesional consequences [19,20]. A meta-analysis based on 66 field trials showed that among the 4 commercial vaccines available on the market, no significant differences in efficacy (based on productivity measurement and mortality rates) could be found [21]. In field conditions and in a context of chronically infected herds, the whole piglet population is generally vaccinated at the age of
http://dx.doi.org/10.1016/j.vaccine.2016.06.005 0264-410X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Rose N et al. A commercial PCV2a-based vaccine significantly reduces PCV2b transmission in experimental conditions. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.06.005
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three weeks to homogenize the immune status of all animals as regards PCV2. Given the observed reduction of viremia in vaccinated pigs, one might expect a significant reduction of PCV2 transmission in vaccinated pigs. The objective was therefore to assess the efficacy of vaccination with a commercial vaccine regarding transmission of PCV2b in an experimental model simulating spread of PCV2b in a fully vaccinated population. Those data would provide quantitative estimates of the PCV2 transmission rate as well as the reproduction number (R) in vaccinated pigs to be compared to basic estimates already available [22,23].
2. Materials and methods
PCV2b (Genbank number: AF201311) suspension (105 TCID50/ ml). Pigs in room five were not infected (Fig. 1). Clinical symptoms were recorded on a daily basis. The PCV2 statuses of the inoculated and contact pigs were monitored twice a week respectively until 42 days post-inoculation (dpi) using realtime qPCR [24] in sera to assess the PCV2 genome load. Pigs were declared as infected as soon as PCV2 genome load was detected in the serum. In addition, PCV2 antibodies were monitored weekly with an ELISA test [25]. The experiment ended 42 days post inoculation when all the piglets were euthanized after anaesthesia with intravenous injection of 1 g/50 kg liveweight of NesdonalÒ (Merial, Lyon, France) followed by exsanguination. The protocol has been approved by the ethic committee registered under the number #16 by the French Ministry of Research.
2.1. Animals and experimental design 2.2. Quantification of PCV2 genome by real-time PCR The experiment was carried out in our air-filtered-level-3 biosecurity-facilities with 21 day-old specific pathogen free (SPF) piglets free from PCV2 and PCV1 and without maternal antibodies to PCV. Five different rooms were used. Rooms 1 to 4 contained two separate pens with solid partitions between each other. Only one pen was used in room 5. Each pen housed 8 pigs (4 + 4). Piglets in rooms 1 to 3 received 1 ml intramuscular injection of INGELVACÒ CircoFLEXTM at day 0. Non vaccinated piglets in room 4 received a placebo (1 ml intramuscular injection of PBS) at the same day, while pigs in room 5 were kept as a negative control group. At day 15, i.e. at the age of 36 days, four pigs in each pen in rooms 1–4 were selected and inoculated with PCV2b. Piglets to be inoculated were randomly chosen in each pen and grouped together within a pen for inoculation and thereafter distributed with their original corresponding contact pen mates 24 h postinoculation. The inoculated piglets were inoculated/injected at day 15 with 6 ml (5 ml intratracheal + 1 ml intramuscular) of a
DNA was extracted from 200 ll of each tested serum using the Wizard SV96 genomic DNA purification system (Promega, Madison, Wisconsin, USA) according to the manufacturer’s instructions. Elution was performed with 250 ll of sterile H2O and 5 ll of this extraction corresponding to 4 ll of serum, were used as template for PCV2 TaqMan PCR. Controls during DNA extraction, have been carried out by replacing serum with PBS, every five samples in order to check any PCV2 contamination. The number of PCV2 genome copies was assessed by a real-time PCR based on TaqMan technology as described previously [24]. For each PCR run, a positive control obtained from lymph nodes tissue from PCV2-infected pig, was included. Four negative controls were also introduced by replacing the DNA sample by purified water, with two negative controls close to the positive control and the other two negative controls at the end of the plate. The lack of amplification was also checked by PCV2-quantitative PCR with
Fig. 1. Experimental scheme.
Please cite this article in press as: Rose N et al. A commercial PCV2a-based vaccine significantly reduces PCV2b transmission in experimental conditions. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.06.005
N. Rose et al. / Vaccine xxx (2016) xxx–xxx
the negative controls of the DNA extraction. Samples were processed by duplicate. The limit detection of the technique is <103 PCV2 copies/ml.
Serum samples were tested for PCV2 antibodies by an ELISA based on the recognition of a recombinant PCV2 capsid protein/ GST fused protein and a GST (Glutathione S-transferase) protein [25]. Samples with an OD (Optical Density) ratio (OD ratio between ORF2-GST and GST alone) higher than 1.5 were considered positive for PCV2 antibodies (sensitivity: 0.98 and specificity: 0.95, taking IPMA [Immunoperoxidase Monolayer Assay] as a reference). 2.4. Statistical analysis Transmission parameters have been estimated in vaccinated and non-vaccinated pigs from results of the incidence of infection by the challenge virus in contact pigs between two sampling dates (interval Di = [ti, ti+1] of duration di). In this interval, the individual probability to escape infection is defined by pi = exp(di b pi), b being the transmission rate or the number of infected pigs by an infectious pig per day and pi the proportion of infectious pigs during the interval Di. Pigs were qualified as infectious from the midinterval-time prior to their first positive PCV2 genome result until their peak of serological responses. Parameters were estimated using Bayesian inference from the likelihood of the model [26]. Let DIj ¼ ½t Ij ; tIj þ1 denote the time interval during which the first positive serum sample was detected in pig j. The contribution of contact animal j in pen k to the likelihood, i.e. the probability for its first positive serum to stand in the interval DIj ¼ ½tIj ; t Ij þ1 is:
L ðDIj ; p
k ðkÞ w ; E jb;
between vaccinated and non-vaccinated groups at each sampling date using a Kruskal–Wallis test. 3. Results
2.3. Serology
ðjÞ
3
cÞ ¼
( Ij i X Y i¼1
) ðkÞ pl1 ð1
pki Þf Lat ðtIj
t i ; cÞ ;
l¼1
and the full likelihood is therefore:
LðDI ; pw ; Ejb; cÞ ¼
Nc Y LðjÞ ðDI ; pw ; Ejb; cÞ;
3.1. Clinical data No significant clinical signs were observed after vaccination. In the 3 vaccinated groups as well as in the 4th non-vaccinated group, only punctual (no more than 3 consecutive days) hyperthermia (>40 °C) were detected post-inoculation with no difference between groups. As regard growth parameters, there was no impact of the vaccination on average daily weight gain (ADWG) in the 15 days following vaccination which remained around 350 g/day and homogeneous between piglets. PCV2 infection did not have any impact on growth performance either. 3.2. Infection data The control pigs in room 5 remained negative (PCR and serology) throughout the experiment. The PCV2 viral genome load in blood was significantly lower in vaccinated pigs than in nonvaccinated ones (>1 log reduction) both in inoculated (Fig. 2a) and contact (Fig. 2b) piglets after the peak of viremia (p < 0.001, global comparison on the whole kinetic accounting for repeated measurement with time). All animals remained seronegative before infection even vaccinated ones which had received a oneshot vaccination 15 days earlier (Fig. 3). However, a faster antibody response was observed in vaccinated-inoculated pigs (rooms 1 to 3) compared with non-vaccinated inoculated piglets (Fig. 3a). Indeed, the OD ratio of the ELISA test was significantly higher in vaccinated-inoculated pigs than in non-vaccinated ones at 14 dpi (p < 0.001) (Fig. 3a). In contact piglets, seroconversion of the first piglets was observed 21 days post inoculation for vaccinated piglets whereas it was also delayed in non-vaccinated piglets with the first seroconversions observed at 35 dpi and OD ratio significantly lower at 14 (p < 0.01), 21 (p < 0.001) and 28 dpi (p < 0.05) (Fig. 3b).
j¼1
where N c is the total number of contact pigs. The latency period being relatively short in regards to the infectious period [22], we modeled the latency assuming an exponential distribution, for which the rate parameter (c), corresponding to the inverse of the average duration of latency period, was estimated. The transmission rate and parameter governing the distribution of the latency were estimated by Bayesian inference using Monte Carlo Markov Chain (MCMC) with uninformative uniform prior distributions. Three independent chains were run with initial values randomly drawn from the prior distribution. 50000 iterations were performed including 10% of burnin phase and with a thinning interval of 10 iterations. The duration of the infectious period has been modeled by a gamma distribution with A and B as shape and scale parameters respectively, estimated by maximum likelihood and corresponding confidence intervals were estimated by non-parametric bootstrap. All statistics and models were programmed using the R software [27]. Parameter estimates from MCMC analysis were compared between vaccinated and non-vaccinated groups using t-test. For duration of infectiousness, survival distributions were compared using a Cox-proportional hazard regression model. Viral genome load data were compared between vaccinated and non-vaccinated piglets using a mixed linear regression model which takes into account repeated measurements with time from the peak of viral genome load until the end of the experiment [28]. Mean OD ratios from the serological test were compared
3.3. Transmission parameters Convergence of MCMCs was assessed through visual inspection and conventional diagnostic tests. Heidelberger and Geweke diagnostics failed to reject the convergence hypothesis, which was also supported by Gelman-Rubin test based on three independent chains with a potential scale reduction factor (PSRF) equal to 1.0. For non-vaccinated piglets, estimations were based on the two replicates contemporary to this experiment as well as on the compilation of data of this experiment with those from a previous trial in non-vaccinated pigs with the same inoculation virus and the same contact structure [23]. The infection incidence was decreased in vaccinated pigs compared with non-vaccinated ones (Fig. 4). Estimation of the parameters governing the infection dynamics highlighted a significantly shorter latency period in vaccinated compared with nonvaccinated piglets (p < 0.001). Indeed, the number of days between infection and detection of the viral genome in serum was reduced by more than one day in vaccinated piglets (Table 1).The transmission rate was found 2- and up to 3-fold lower (p < 0.001) in vaccinated animals when data were compared to the contemporary non-vaccinated group or to the merged data (current and historical experimental data) on non-vaccinated pigs respectively (Table 1). Moreover, vaccinated animals also exhibited a duration of infectiousness 10 days shorter (p < 0.001) than in non-vaccinated piglets when all data from non-vaccinated piglets were grouped
Please cite this article in press as: Rose N et al. A commercial PCV2a-based vaccine significantly reduces PCV2b transmission in experimental conditions. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.06.005
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Fig. 2. Mean PCV2 viral genome load in PCR positive inoculated vaccinated (n = 24) and non-vaccinated (n = 8) piglets (a) and contact vaccinated (n = 24) and non-vaccinated (n = 8) piglets (b).
together (Table 2, merged data). Reduction of both parameters in vaccinated pigs led to an estimate of the reproduction number (R) significantly lower than in non-vaccinated piglets (1.5 versus 5.1, p < 0.001) while remaining higher than 1 (Table 2). 4. Discussion The classical vaccination-challenge experiments carried out to determine the clinical protection after infection and the level of viremia often showed that vaccination could not prevent infection or virus excretion [9,29] but they do not preclude an effect in terms of reduction of the spread of the virus within a vaccinated population. Quantitative evaluation of a vaccine efficacy on reducing transmission of an infectious agent has often been evaluated in experimental conditions setting up transmission experiments as for example Aujeszky disease virus [30], Actinobacillus pleuropneumoniae [31] or PRRS virus [26,32–34]. The present experiment focused on PCV2 infection dynamics characterization within homogeneous vaccinated or nonvaccinated populations. We showed the efficacy of a genotype abased commercial vaccine on reduction of transmission of a PCV2b strain, which is also representative of the strains involved in PCV2
systemic disease in densely pig production area such as Brittany, France. Piglets have been monitored individually twice weekly and on a long term (42 days post inoculation), which is pivotal for this virus because of long lasting viraemia and the possibility of transmission to susceptible contact pigs at least until 28 days post-infection [22]. In SPF inoculated but non vaccinated pigs, clinical and virological data were close to other results obtained previously with PCV2-only inoculation, i.e. subclinical infection with a persisting viral genome load slowly decreasing after the peak of viremia [23]. The observed duration of viremia was consistent with the data obtained in this previous experiment on unvaccinated SPF pigs with also similar viral genome load attained both in inoculated and contact piglets and a similar serological response [23]. Vaccinated pigs evidenced a faster rise in PCV2 antibodies (from 10 days post-inoculation) whereas the latter were not detectable in the 15 days following vaccination. The earlier seroconversion in vaccinated piglets post-inoculation could be due to the previous exposure to PCV2 vaccine antigen (boost response). However, the absence of detectable antibodies in PCV2 vaccinated piglets during 21 days post-vaccination could of course be related to an interference between the serological ELISA test (ORF2 based protein) and
Please cite this article in press as: Rose N et al. A commercial PCV2a-based vaccine significantly reduces PCV2b transmission in experimental conditions. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.06.005
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Fig. 3. Evolution of the serological response (ELISA ORF2-based serological test) in inoculated vaccinated (n = 24) and non-vaccinated (n = 8) piglets (a) and contact vaccinated (n = 24) and non-vaccinated (n = 8) piglets (b). Significant differences at each sampling time are indicated as such: ⁄⁄⁄p < 0.001, ⁄⁄p < 0.01, ⁄p < 0.05.
the composition of the commercial vaccine used (recombinant CAP protein expressed in baculovirus). Moreover, it has been shown that the level of IgG produced after vaccination was different according to the vaccine technology without being prejudicial to efficacy [35]. In this study, a weaker antibody response was observed 21 days post-vaccination compared with inactivated or chimeric vaccines. In the present experiment and in the non-vaccinated pigs, R0 estimate was in the same range as estimates obtained previously with the same experimental setting (within-pen transmission, 4 ⁄ 4 trial) although the precision was limited due to the 2 replications only for this category. However, when merging data together, we obtained an average R0 of 5.1 which is consistent with previously published estimates for this virus [22,23]. The duration of infectiousness was significantly shortened in our experiment in vaccinated piglets: 16.6 versus 26.6 days for non-vaccinated pigs. The impact of vaccination on viral genome load shed and duration of viremia has been shown previously by Fort et al. [36] and is therefore confirmed with the present results. This reduction of viremia persistence has a direct impact on the reproduction number estimate and was confirmed thereby with a significant decrease of the viral genome load in both inoculated and contact vaccinated piglets once the peak of a viremia was attained. Moreover, the statistical relationships between viremia magnitude and the evolution of the transmission rate with time evidenced previously [22], was also illustrated in the present study by the reduction in intensity of replication in vaccinated animals also associated with a significant decrease of the mean transmission rate.
In this experiment that mimicked a field situation where piglets are simultaneously vaccinated at the age of three weeks and thereafter exposed to PCV2, a reduction of both parameters (transmission rate and duration of infectiousness) led to a reduction of the reproduction number which didn’t fell below 1 but was still significantly (p < 0.001) lower than in non-vaccinated piglets. It comes out that in these experimental conditions, the vaccine would lead to a limited infection process in an important fraction of vaccinated pigs exposed to vaccinated shedding animals. These results reflect the dual effect of vaccination cumulating a reduction of susceptibility to infection along with limited transmission ability of the virus to contact animals. 5. Conclusion Vaccination of piglets against PCV2 decreased the virus transmission in experimental conditions. This work showed that vaccination of SPF piglets with a PCV2 commercial vaccine based on a recombinant genotype-a cap protein expressed in baculovirus provided a significant protection to the animals challenged with a PCV2b virus. It decreased the viral genome load and significantly reduced transmission in sentinel pigs. Both duration and transmission intensity (transmission rate 3 times lower than in nonvaccinated piglets) were decreased in vaccinated animals. The reproduction number estimated in vaccinated piglets includes 1 in the confidence interval, which suggests that the propagation of the virus would be expected to be considerably decreased within a vaccinated population but not fully abolished. Complementary experiments would be worth carrying out involving a PCV2a chal-
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Fig. 4. Individual virological data: PCV2 viral genome load in blood (log(number of PCV2 genome copies/ml)) in controls, vaccinated and non-vaccinated, inoculated and contact pigs. Shaded areas highlight PCR positive results with light grey: viral genome load between 103 and 104 copies/ml; medium grey: between 104 and 105 copies/ml; dark grey: between 105 and 106 copies/ml and black: >106 copies/ml. –: not analyzed, : dead.
Please cite this article in press as: Rose N et al. A commercial PCV2a-based vaccine significantly reduces PCV2b transmission in experimental conditions. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.06.005
N. Rose et al. / Vaccine xxx (2016) xxx–xxx Table 1 Estimation of transmission parameters (latency duration [1/c] and transmission rate [b] MCMC estimation, 3 chains, 50000 iterations, 5000 burnin iterations).
Vaccined (n = 48 pigs) Non-Vaccined (n = 16 pigs) Non-Vaccined (2 trialsa, n = 48 pigs) a
Average duration of latency period (days)
Transmission rate b (day1)
4.2 5.1 6.6
0.09 0.28 0.19
(2.4; 6.3) (2.9; 9.4) (4.7; 9.6)
(0.05; 0.14) (0.11; 0.66) (0.11; 0.32)
Merged with data from Andraud, Grasland [23].
Table 2 Estimates of the duration of the infectious period and reproduction number.
Vaccined (n = 48 pigs) Non-Vaccined (n = 16 pigs) Non-Vaccined (2 trialsa, n = 48 pigs) a
Duration of infectious period (days)
Reproduction Number R
16.6 24.8 26.6
1.48 6.94 5.05
(14.7; 18.4) (18.2; 31.4) (22.9; 30.4)
(0.77;2.24) (0.42;15.05) (2.46; 8.19)
Merged with data from Andraud, Grasland [23].
lenge virus to assess whether the reduction in transmission would be more effective in the case of a more homologous strain. Further work is needed to assess if this vaccine efficacy could also be transposed to field conditions especially because of frequent vaccination of young piglets after weaning while having generally high levels of maternal antibodies. Competing interests The authors declare that they have no competing interests. Authors’ contribution NR conceived, coordinated the study and participated in the animal experiment and the data analyses and drafted the manuscript. MA developed the mathematical model and participated in the data analyses, LB analyzed the PCV2 samples and interpreted the results, AJ coordinated the laboratory work, BG supervised the PCV2-related laboratory work and participated in the coordination of the study. All co-authors revised the manuscript and approved the final submitted version. Acknowledgements This work was partly supported by the Boehringer Ingelheim PCV2 award (2009) and the Conseil Départemental des Côtes d’Armor. The authors acknowledge R. Cariolet and A. Keranflec’h for the excellent management of the experiment. References [1] Rose N, Larour G, Le Diguerher G, Eveno E, Jolly JP, Blanchard P, et al. Risk factors for porcine post-weaning multisystemic wasting syndrome (PMWS) in 149 French farrow-to-finish herds. Prev Vet Med 2003;61:209–25. [2] Lopez-Soria S, Segalés J, Rose N, Viñas MJ, Blanchard P, Madec F, et al. An exploratory study on risk factors for postweaning multisystemic wasting syndrome (PMWS) in Spain. Prev Vet Med 2005;69:97–107. [3] Woodbine KA, Medley GF, Slevin J, Kilbride AL, Novell EJ, Turner MJ, et al. Spatiotemporal patterns and risks of herd breakdowns in pigs with postweaning multisystemic wasting syndrome. Vet Rec 2007;160:751–62. [4] Andraud M, Rose N, Grasland B, Pierre JS, Jestin A, Madec F. Influence of husbandry and control measures on porcine circovirus type 2 (PCV-2) dynamics within a farrow-to-finish pig farm: a modelling approach. Prev Vet Med 2009;92:38–51.
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[32] Velthuis AGJ, De Jong MCM, De Bree J, Nodelijk G, Van Boven M. Quantification of transmission in one-to-one experiments. Epidemiol Infect 2002;128: 193–204. [33] Nodelijk G, De Jong MCM, Van Leengoed LAMG, Wensvoort G, Pol JMA, Steverink PJGM, et al. A quantitative assessment of the effectiveness of PRRSV vaccination in pigs under experimental conditions. Vaccine 2001;19: 3636–44. [34] Pileri E, Gibert E, Soldevila F, García-Saenz A, Pujols J, Diaz I, et al. Vaccination with a genotype 1 modified live vaccine against porcine reproductive and respiratory syndrome virus significantly reduces viremia, viral shedding and
transmission of the virus in a quasi-natural experimental model. Vet Microbiol 2015;175:7–16. [35] Seo HW, Lee J, Han K, Park C, Chae C. Comparative analyses of humoral and cell-mediated immune responses upon vaccination with different commercially available single-dose porcine circovirus type 2 vaccines. Res Vet Sci 2014;97:38–42. [36] Fort M, Sibila M, Pérez-Martin E, Nofrarias M, Mateu E, Segalés J. One dose of a porcine circovirus 2 (PCV2) sub-unit vaccine administered to 3-week-old conventional piglets elicits cell-mediated immunity and significantly reduces PCV2 viremia in an experimental model. Vaccine 2009;27:4031–7.
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Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
A commercial PCV2a-based vaccine significantly reduces PCV2b transmission in experimental conditions N. Rose ⇑, M. Andraud, L. Bigault, A. Jestin, B. Grasland Anses, Laboratoire de Ploufragan-Plouzané, BP 53, 22440 Ploufragan, France Université Bretagne Loire, Rennes, France
a r t i c l e
i n f o
Article history: Received 14 March 2016 Received in revised form 20 May 2016 Accepted 1 June 2016 Available online xxxx Keywords: Porcine Circovirus type 2 Vaccination Transmission Reproduction number
a b s t r a c t Transmission characteristics of PCV2 have been compared between vaccinated and non-vaccinated pigs in experimental conditions. Twenty-four Specific Pathogen Free (SPF) piglets, vaccinated against PCV2 at 3 weeks of age (PCV2a recombinant CAP protein-based vaccine), were inoculated at 15 days postvaccination with a PCV2b inoculum (6 105 TCID50), and put in contact with 24 vaccinated SPF piglets during 42 days post-inoculation. Those piglets were shared in six replicates of a contact trial involving 4 inoculated piglets mingled with 4 susceptible SPF piglets. Two replicates of a similar contact trial were made with non-vaccinated pigs. Non vaccinated animals received a placebo at vaccination time and were inoculated the same way and at the same time as the vaccinated group. All the animals were monitored twice weekly using quantitative real-time PCR and ELISA for serology until 42 days post-inoculation. The frequency of infection and the PCV2 genome load in sera of the vaccinated pigs were significantly reduced compared to the non-vaccinated animals. The duration of infectiousness was significantly different between vaccinated and non-vaccinated groups (16.6 days [14.7;18.4] and 26.6 days [22.9;30.4] respectively). The transmission rate was also considerably decreased in vaccinated pigs (b = 0.09 [0.05–0.14] compared to b = 0.19 [0.11–0.32] in non-vaccinated pigs). This led to an estimated reproduction ratio of 1.5 [95% CI 0.8 – 2.2] in vaccinated animals versus 5.1 [95% CI 2.5 – 8.2] in non-vaccinated pigs when merging data of this experiment with previous trials carried out in same conditions. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Porcine Circovirus type 2 (PCV2) is the primary agent of PCV2systemic disease (PCV2-SD), also called Post-weaning Multisystemic Wasting Syndrome (PMWS). Following the first descriptions in North-America in the mid-90s, the disease became worldwide spread and had devastating consequences in the pig sector. For several years and before vaccine commercialization, only modifications of farm practices based on known risk factors of clinical expression of PCV2-SD were available to tackle the disease [1–3]. It was further shown that some of those practices had a direct impact on reduction of PCV2 propagation within the population of a swine farrow-to-finish operation, decreasing massive exposure of young piglets to the virus [4]. From 2006, commercial vaccine solutions were made available on the market. Based on different principles (inactivated PCV2, PCV2 capsid protein produced in a vector or chimeric inactivated PCV1 virus including PCV2 ORF2), ⇑ Corresponding author at: Nicolas ROSE, Anses, Laboratoire de PloufraganPlouzané, PO Box 53, F22440 Ploufragan, France. E-mail address: [email protected] (N. Rose).
the efficacy of those vaccines, prepared with strains of PCV2a genogroup, has been described in several experimental [5] or field studies [6–11]. Generally, commercial vaccines have been shown to reduce viraemia, viral load in tissues and lymphoid lesions [12–14]. Efficacy towards improvement of the clinical situation in a PMWS or PCV2-SD context has been also documented [15,16]. Moreover, their efficacy was not compromised by challenge with heterologous genotype of PCV2, i.e. PCV2b [14] or more recently mutants from PCV2b [17]. Beside PCV2-SD, the involvement of the virus in subclinical infections and other syndromes such as Porcine Respiratory Disease complex, reproductive disorders or enteric diseases has been more and more documented [18]. The efficacy of PCV2 vaccination towards such PCV2 subclinical infections has also been shown through the improvement of growth performances and reduction of clinical and lesional consequences [19,20]. A meta-analysis based on 66 field trials showed that among the 4 commercial vaccines available on the market, no significant differences in efficacy (based on productivity measurement and mortality rates) could be found [21]. In field conditions and in a context of chronically infected herds, the whole piglet population is generally vaccinated at the age of
http://dx.doi.org/10.1016/j.vaccine.2016.06.005 0264-410X/Ó 2016 Elsevier Ltd. All rights reserved.
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three weeks to homogenize the immune status of all animals as regards PCV2. Given the observed reduction of viremia in vaccinated pigs, one might expect a significant reduction of PCV2 transmission in vaccinated pigs. The objective was therefore to assess the efficacy of vaccination with a commercial vaccine regarding transmission of PCV2b in an experimental model simulating spread of PCV2b in a fully vaccinated population. Those data would provide quantitative estimates of the PCV2 transmission rate as well as the reproduction number (R) in vaccinated pigs to be compared to basic estimates already available [22,23].
2. Materials and methods
PCV2b (Genbank number: AF201311) suspension (105 TCID50/ ml). Pigs in room five were not infected (Fig. 1). Clinical symptoms were recorded on a daily basis. The PCV2 statuses of the inoculated and contact pigs were monitored twice a week respectively until 42 days post-inoculation (dpi) using realtime qPCR [24] in sera to assess the PCV2 genome load. Pigs were declared as infected as soon as PCV2 genome load was detected in the serum. In addition, PCV2 antibodies were monitored weekly with an ELISA test [25]. The experiment ended 42 days post inoculation when all the piglets were euthanized after anaesthesia with intravenous injection of 1 g/50 kg liveweight of NesdonalÒ (Merial, Lyon, France) followed by exsanguination. The protocol has been approved by the ethic committee registered under the number #16 by the French Ministry of Research.
2.1. Animals and experimental design 2.2. Quantification of PCV2 genome by real-time PCR The experiment was carried out in our air-filtered-level-3 biosecurity-facilities with 21 day-old specific pathogen free (SPF) piglets free from PCV2 and PCV1 and without maternal antibodies to PCV. Five different rooms were used. Rooms 1 to 4 contained two separate pens with solid partitions between each other. Only one pen was used in room 5. Each pen housed 8 pigs (4 + 4). Piglets in rooms 1 to 3 received 1 ml intramuscular injection of INGELVACÒ CircoFLEXTM at day 0. Non vaccinated piglets in room 4 received a placebo (1 ml intramuscular injection of PBS) at the same day, while pigs in room 5 were kept as a negative control group. At day 15, i.e. at the age of 36 days, four pigs in each pen in rooms 1–4 were selected and inoculated with PCV2b. Piglets to be inoculated were randomly chosen in each pen and grouped together within a pen for inoculation and thereafter distributed with their original corresponding contact pen mates 24 h postinoculation. The inoculated piglets were inoculated/injected at day 15 with 6 ml (5 ml intratracheal + 1 ml intramuscular) of a
DNA was extracted from 200 ll of each tested serum using the Wizard SV96 genomic DNA purification system (Promega, Madison, Wisconsin, USA) according to the manufacturer’s instructions. Elution was performed with 250 ll of sterile H2O and 5 ll of this extraction corresponding to 4 ll of serum, were used as template for PCV2 TaqMan PCR. Controls during DNA extraction, have been carried out by replacing serum with PBS, every five samples in order to check any PCV2 contamination. The number of PCV2 genome copies was assessed by a real-time PCR based on TaqMan technology as described previously [24]. For each PCR run, a positive control obtained from lymph nodes tissue from PCV2-infected pig, was included. Four negative controls were also introduced by replacing the DNA sample by purified water, with two negative controls close to the positive control and the other two negative controls at the end of the plate. The lack of amplification was also checked by PCV2-quantitative PCR with
Fig. 1. Experimental scheme.
Please cite this article in press as: Rose N et al. A commercial PCV2a-based vaccine significantly reduces PCV2b transmission in experimental conditions. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.06.005
N. Rose et al. / Vaccine xxx (2016) xxx–xxx
the negative controls of the DNA extraction. Samples were processed by duplicate. The limit detection of the technique is <103 PCV2 copies/ml.
Serum samples were tested for PCV2 antibodies by an ELISA based on the recognition of a recombinant PCV2 capsid protein/ GST fused protein and a GST (Glutathione S-transferase) protein [25]. Samples with an OD (Optical Density) ratio (OD ratio between ORF2-GST and GST alone) higher than 1.5 were considered positive for PCV2 antibodies (sensitivity: 0.98 and specificity: 0.95, taking IPMA [Immunoperoxidase Monolayer Assay] as a reference). 2.4. Statistical analysis Transmission parameters have been estimated in vaccinated and non-vaccinated pigs from results of the incidence of infection by the challenge virus in contact pigs between two sampling dates (interval Di = [ti, ti+1] of duration di). In this interval, the individual probability to escape infection is defined by pi = exp(di b pi), b being the transmission rate or the number of infected pigs by an infectious pig per day and pi the proportion of infectious pigs during the interval Di. Pigs were qualified as infectious from the midinterval-time prior to their first positive PCV2 genome result until their peak of serological responses. Parameters were estimated using Bayesian inference from the likelihood of the model [26]. Let DIj ¼ ½t Ij ; tIj þ1 denote the time interval during which the first positive serum sample was detected in pig j. The contribution of contact animal j in pen k to the likelihood, i.e. the probability for its first positive serum to stand in the interval DIj ¼ ½tIj ; t Ij þ1 is:
L ðDIj ; p
k ðkÞ w ; E jb;
between vaccinated and non-vaccinated groups at each sampling date using a Kruskal–Wallis test. 3. Results
2.3. Serology
ðjÞ
3
cÞ ¼
( Ij i X Y i¼1
) ðkÞ pl1 ð1
pki Þf Lat ðtIj
t i ; cÞ ;
l¼1
and the full likelihood is therefore:
LðDI ; pw ; Ejb; cÞ ¼
Nc Y LðjÞ ðDI ; pw ; Ejb; cÞ;
3.1. Clinical data No significant clinical signs were observed after vaccination. In the 3 vaccinated groups as well as in the 4th non-vaccinated group, only punctual (no more than 3 consecutive days) hyperthermia (>40 °C) were detected post-inoculation with no difference between groups. As regard growth parameters, there was no impact of the vaccination on average daily weight gain (ADWG) in the 15 days following vaccination which remained around 350 g/day and homogeneous between piglets. PCV2 infection did not have any impact on growth performance either. 3.2. Infection data The control pigs in room 5 remained negative (PCR and serology) throughout the experiment. The PCV2 viral genome load in blood was significantly lower in vaccinated pigs than in nonvaccinated ones (>1 log reduction) both in inoculated (Fig. 2a) and contact (Fig. 2b) piglets after the peak of viremia (p < 0.001, global comparison on the whole kinetic accounting for repeated measurement with time). All animals remained seronegative before infection even vaccinated ones which had received a oneshot vaccination 15 days earlier (Fig. 3). However, a faster antibody response was observed in vaccinated-inoculated pigs (rooms 1 to 3) compared with non-vaccinated inoculated piglets (Fig. 3a). Indeed, the OD ratio of the ELISA test was significantly higher in vaccinated-inoculated pigs than in non-vaccinated ones at 14 dpi (p < 0.001) (Fig. 3a). In contact piglets, seroconversion of the first piglets was observed 21 days post inoculation for vaccinated piglets whereas it was also delayed in non-vaccinated piglets with the first seroconversions observed at 35 dpi and OD ratio significantly lower at 14 (p < 0.01), 21 (p < 0.001) and 28 dpi (p < 0.05) (Fig. 3b).
j¼1
where N c is the total number of contact pigs. The latency period being relatively short in regards to the infectious period [22], we modeled the latency assuming an exponential distribution, for which the rate parameter (c), corresponding to the inverse of the average duration of latency period, was estimated. The transmission rate and parameter governing the distribution of the latency were estimated by Bayesian inference using Monte Carlo Markov Chain (MCMC) with uninformative uniform prior distributions. Three independent chains were run with initial values randomly drawn from the prior distribution. 50000 iterations were performed including 10% of burnin phase and with a thinning interval of 10 iterations. The duration of the infectious period has been modeled by a gamma distribution with A and B as shape and scale parameters respectively, estimated by maximum likelihood and corresponding confidence intervals were estimated by non-parametric bootstrap. All statistics and models were programmed using the R software [27]. Parameter estimates from MCMC analysis were compared between vaccinated and non-vaccinated groups using t-test. For duration of infectiousness, survival distributions were compared using a Cox-proportional hazard regression model. Viral genome load data were compared between vaccinated and non-vaccinated piglets using a mixed linear regression model which takes into account repeated measurements with time from the peak of viral genome load until the end of the experiment [28]. Mean OD ratios from the serological test were compared
3.3. Transmission parameters Convergence of MCMCs was assessed through visual inspection and conventional diagnostic tests. Heidelberger and Geweke diagnostics failed to reject the convergence hypothesis, which was also supported by Gelman-Rubin test based on three independent chains with a potential scale reduction factor (PSRF) equal to 1.0. For non-vaccinated piglets, estimations were based on the two replicates contemporary to this experiment as well as on the compilation of data of this experiment with those from a previous trial in non-vaccinated pigs with the same inoculation virus and the same contact structure [23]. The infection incidence was decreased in vaccinated pigs compared with non-vaccinated ones (Fig. 4). Estimation of the parameters governing the infection dynamics highlighted a significantly shorter latency period in vaccinated compared with nonvaccinated piglets (p < 0.001). Indeed, the number of days between infection and detection of the viral genome in serum was reduced by more than one day in vaccinated piglets (Table 1).The transmission rate was found 2- and up to 3-fold lower (p < 0.001) in vaccinated animals when data were compared to the contemporary non-vaccinated group or to the merged data (current and historical experimental data) on non-vaccinated pigs respectively (Table 1). Moreover, vaccinated animals also exhibited a duration of infectiousness 10 days shorter (p < 0.001) than in non-vaccinated piglets when all data from non-vaccinated piglets were grouped
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Fig. 2. Mean PCV2 viral genome load in PCR positive inoculated vaccinated (n = 24) and non-vaccinated (n = 8) piglets (a) and contact vaccinated (n = 24) and non-vaccinated (n = 8) piglets (b).
together (Table 2, merged data). Reduction of both parameters in vaccinated pigs led to an estimate of the reproduction number (R) significantly lower than in non-vaccinated piglets (1.5 versus 5.1, p < 0.001) while remaining higher than 1 (Table 2). 4. Discussion The classical vaccination-challenge experiments carried out to determine the clinical protection after infection and the level of viremia often showed that vaccination could not prevent infection or virus excretion [9,29] but they do not preclude an effect in terms of reduction of the spread of the virus within a vaccinated population. Quantitative evaluation of a vaccine efficacy on reducing transmission of an infectious agent has often been evaluated in experimental conditions setting up transmission experiments as for example Aujeszky disease virus [30], Actinobacillus pleuropneumoniae [31] or PRRS virus [26,32–34]. The present experiment focused on PCV2 infection dynamics characterization within homogeneous vaccinated or nonvaccinated populations. We showed the efficacy of a genotype abased commercial vaccine on reduction of transmission of a PCV2b strain, which is also representative of the strains involved in PCV2
systemic disease in densely pig production area such as Brittany, France. Piglets have been monitored individually twice weekly and on a long term (42 days post inoculation), which is pivotal for this virus because of long lasting viraemia and the possibility of transmission to susceptible contact pigs at least until 28 days post-infection [22]. In SPF inoculated but non vaccinated pigs, clinical and virological data were close to other results obtained previously with PCV2-only inoculation, i.e. subclinical infection with a persisting viral genome load slowly decreasing after the peak of viremia [23]. The observed duration of viremia was consistent with the data obtained in this previous experiment on unvaccinated SPF pigs with also similar viral genome load attained both in inoculated and contact piglets and a similar serological response [23]. Vaccinated pigs evidenced a faster rise in PCV2 antibodies (from 10 days post-inoculation) whereas the latter were not detectable in the 15 days following vaccination. The earlier seroconversion in vaccinated piglets post-inoculation could be due to the previous exposure to PCV2 vaccine antigen (boost response). However, the absence of detectable antibodies in PCV2 vaccinated piglets during 21 days post-vaccination could of course be related to an interference between the serological ELISA test (ORF2 based protein) and
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Fig. 3. Evolution of the serological response (ELISA ORF2-based serological test) in inoculated vaccinated (n = 24) and non-vaccinated (n = 8) piglets (a) and contact vaccinated (n = 24) and non-vaccinated (n = 8) piglets (b). Significant differences at each sampling time are indicated as such: ⁄⁄⁄p < 0.001, ⁄⁄p < 0.01, ⁄p < 0.05.
the composition of the commercial vaccine used (recombinant CAP protein expressed in baculovirus). Moreover, it has been shown that the level of IgG produced after vaccination was different according to the vaccine technology without being prejudicial to efficacy [35]. In this study, a weaker antibody response was observed 21 days post-vaccination compared with inactivated or chimeric vaccines. In the present experiment and in the non-vaccinated pigs, R0 estimate was in the same range as estimates obtained previously with the same experimental setting (within-pen transmission, 4 ⁄ 4 trial) although the precision was limited due to the 2 replications only for this category. However, when merging data together, we obtained an average R0 of 5.1 which is consistent with previously published estimates for this virus [22,23]. The duration of infectiousness was significantly shortened in our experiment in vaccinated piglets: 16.6 versus 26.6 days for non-vaccinated pigs. The impact of vaccination on viral genome load shed and duration of viremia has been shown previously by Fort et al. [36] and is therefore confirmed with the present results. This reduction of viremia persistence has a direct impact on the reproduction number estimate and was confirmed thereby with a significant decrease of the viral genome load in both inoculated and contact vaccinated piglets once the peak of a viremia was attained. Moreover, the statistical relationships between viremia magnitude and the evolution of the transmission rate with time evidenced previously [22], was also illustrated in the present study by the reduction in intensity of replication in vaccinated animals also associated with a significant decrease of the mean transmission rate.
In this experiment that mimicked a field situation where piglets are simultaneously vaccinated at the age of three weeks and thereafter exposed to PCV2, a reduction of both parameters (transmission rate and duration of infectiousness) led to a reduction of the reproduction number which didn’t fell below 1 but was still significantly (p < 0.001) lower than in non-vaccinated piglets. It comes out that in these experimental conditions, the vaccine would lead to a limited infection process in an important fraction of vaccinated pigs exposed to vaccinated shedding animals. These results reflect the dual effect of vaccination cumulating a reduction of susceptibility to infection along with limited transmission ability of the virus to contact animals. 5. Conclusion Vaccination of piglets against PCV2 decreased the virus transmission in experimental conditions. This work showed that vaccination of SPF piglets with a PCV2 commercial vaccine based on a recombinant genotype-a cap protein expressed in baculovirus provided a significant protection to the animals challenged with a PCV2b virus. It decreased the viral genome load and significantly reduced transmission in sentinel pigs. Both duration and transmission intensity (transmission rate 3 times lower than in nonvaccinated piglets) were decreased in vaccinated animals. The reproduction number estimated in vaccinated piglets includes 1 in the confidence interval, which suggests that the propagation of the virus would be expected to be considerably decreased within a vaccinated population but not fully abolished. Complementary experiments would be worth carrying out involving a PCV2a chal-
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Fig. 4. Individual virological data: PCV2 viral genome load in blood (log(number of PCV2 genome copies/ml)) in controls, vaccinated and non-vaccinated, inoculated and contact pigs. Shaded areas highlight PCR positive results with light grey: viral genome load between 103 and 104 copies/ml; medium grey: between 104 and 105 copies/ml; dark grey: between 105 and 106 copies/ml and black: >106 copies/ml. –: not analyzed, : dead.
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N. Rose et al. / Vaccine xxx (2016) xxx–xxx Table 1 Estimation of transmission parameters (latency duration [1/c] and transmission rate [b] MCMC estimation, 3 chains, 50000 iterations, 5000 burnin iterations).
Vaccined (n = 48 pigs) Non-Vaccined (n = 16 pigs) Non-Vaccined (2 trialsa, n = 48 pigs) a
Average duration of latency period (days)
Transmission rate b (day1)
4.2 5.1 6.6
0.09 0.28 0.19
(2.4; 6.3) (2.9; 9.4) (4.7; 9.6)
(0.05; 0.14) (0.11; 0.66) (0.11; 0.32)
Merged with data from Andraud, Grasland [23].
Table 2 Estimates of the duration of the infectious period and reproduction number.
Vaccined (n = 48 pigs) Non-Vaccined (n = 16 pigs) Non-Vaccined (2 trialsa, n = 48 pigs) a
Duration of infectious period (days)
Reproduction Number R
16.6 24.8 26.6
1.48 6.94 5.05
(14.7; 18.4) (18.2; 31.4) (22.9; 30.4)
(0.77;2.24) (0.42;15.05) (2.46; 8.19)
Merged with data from Andraud, Grasland [23].
lenge virus to assess whether the reduction in transmission would be more effective in the case of a more homologous strain. Further work is needed to assess if this vaccine efficacy could also be transposed to field conditions especially because of frequent vaccination of young piglets after weaning while having generally high levels of maternal antibodies. Competing interests The authors declare that they have no competing interests. Authors’ contribution NR conceived, coordinated the study and participated in the animal experiment and the data analyses and drafted the manuscript. MA developed the mathematical model and participated in the data analyses, LB analyzed the PCV2 samples and interpreted the results, AJ coordinated the laboratory work, BG supervised the PCV2-related laboratory work and participated in the coordination of the study. All co-authors revised the manuscript and approved the final submitted version. Acknowledgements This work was partly supported by the Boehringer Ingelheim PCV2 award (2009) and the Conseil Départemental des Côtes d’Armor. The authors acknowledge R. Cariolet and A. Keranflec’h for the excellent management of the experiment. References [1] Rose N, Larour G, Le Diguerher G, Eveno E, Jolly JP, Blanchard P, et al. Risk factors for porcine post-weaning multisystemic wasting syndrome (PMWS) in 149 French farrow-to-finish herds. Prev Vet Med 2003;61:209–25. [2] Lopez-Soria S, Segalés J, Rose N, Viñas MJ, Blanchard P, Madec F, et al. An exploratory study on risk factors for postweaning multisystemic wasting syndrome (PMWS) in Spain. Prev Vet Med 2005;69:97–107. [3] Woodbine KA, Medley GF, Slevin J, Kilbride AL, Novell EJ, Turner MJ, et al. Spatiotemporal patterns and risks of herd breakdowns in pigs with postweaning multisystemic wasting syndrome. Vet Rec 2007;160:751–62. [4] Andraud M, Rose N, Grasland B, Pierre JS, Jestin A, Madec F. Influence of husbandry and control measures on porcine circovirus type 2 (PCV-2) dynamics within a farrow-to-finish pig farm: a modelling approach. Prev Vet Med 2009;92:38–51.
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Please cite this article in press as: Rose N et al. A commercial PCV2a-based vaccine significantly reduces PCV2b transmission in experimental conditions. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.06.005