porcine parvovirus 2 co-challenge

Vaccine xxx (xxxx) xxx

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Porcine circovirus type 2a or 2b based experimental vaccines provide protection against PCV2d/porcine parvovirus 2 co-challenge Tanja Opriessnig a,b,⇑, Anbu K. Karuppannan b, Patrick G. Halbur b, Jay G. Calvert c, Gregory P. Nitzel c, Shannon R. Matzinger d, Xiang-Jin Meng d a

The Roslin Institute and The Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian, Scotland, United Kingdom Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA, USA Veterinary Medicine Research & Development, Zoetis Inc., Kalamazoo, MI, USA d Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA b c

a r t i c l e

i n f o

Article history: Received 17 October 2019 Received in revised form 31 December 2019 Accepted 4 January 2020 Available online xxxx Keywords: Cross-protection Co-challenge Porcine circovirus (PCV) PCV2a PCV2b PCV2d Porcine parvovirus 2 (PPV2) Vaccination

a b s t r a c t With the discovery of Porcine circovirus type 2d (PCV2d) in the USA in 2012 and subsequent genotype shift from the previously predominant PCV2b to PCV2d in the face of widespread PCV2a vaccination, concerns over PCV2 vaccine efficacy were raised. The objective of this study was to evaluate the efficacy of two similarly produced PCV2 vaccines, one containing the PCV2a capsid and the other one containing the PCV2b capsid, in the conventional pig model against PCV2d/porcine parvovirus 2 (PPV2) co-challenge. A co-challenge was added since there is evidence that PPV2 may exacerbate PCV2 infection and since PCV2 only rarely causes disease in experimentally infected pigs, hence vaccine efficacy can be difficult to assess. In brief, sixty 3-week-old-pigs from a PCV2 seropositive farm without evidence of active virus replication (no PCV2 viremia, low antibody titers with no evidence of increase after two consecutive bleedings) were blocked by PCV2 antibody titer and then randomly divided into three groups with 20 pigs each, a non-vaccinated group (challenge control), a PCV2a vaccinated group (VAC2a) and a PCV2b vaccinated group (VAC2b). Vaccinations were done at 4 and again at 6 weeks of age. At 8 weeks of age, all pigs were challenged with a PCV2d strain via intranasal and intramuscular routes of inoculation followed by intramuscular administration of PPV2 one day later. PCV2 vaccination, regardless of PCV2 genotype, resulted in significantly higher humoral and cellular immunity compared to non-vaccinated challenge control pigs as evidenced by increased numbers of interferon (IFN) c secreting cells after PCV2d stimulation of peripheral blood mononuclear cells collected prior to challenge. Furthermore, PCV2a and PCV2b vaccinations both reduced PCV2d viremia and PCV2-associated pathological lesions. Under the study conditions, the PCV2a and PCV2b vaccine preparations each induced immune responses and clinical protection against a heterologous PCV2d/PPV2 co-challenge. Ó 2020 Elsevier Ltd. All rights reserved.

1. Introduction Porcine circovirus type 2 (PCV2) was first discovered in 1998 and has since been associated with several disease manifestations in pigs including systemic and respiratory diseases [1]. Today it is well documented that there are several PCV2 genotypes circulating in pig populations worldwide including PCV2a, PCV2b and PCV2d [2]. Vaccines to protect pigs against PCV2-associated disease, also referred as PCVAD, became available in 2002 in Europe and in North America following the devastating PCVAD outbreaks in

⇑ Corresponding author at: The Roslin Institute and The Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian, Scotland, United Kingdom. E-mail address: [email protected] (T. Opriessnig).

2004 through 2006. Within a short period of time, approximately 6–12 months, it is estimated that the previously unvaccinated and naturally PCV2-infected North American pig population reached a vaccination range of over 90% in growing pigs [3]. Unaware at that time that PCV2 isolates would have a different genetic makeup (genotypes), all vaccines that were manufactured and introduced onto the market contained the whole virus or the capsid of the prevalent PCV2 strain at the time, later designated as PCV2a genotype. Almost at the same time as the initiation of PCV2a vaccine use, researchers discovered the presence of a novel PCV2 type [4], later designated as PCV2b [5,6], which quickly replaced PCV2a in the global pig population to become the most widely prevalent PCV2 type. During 2012, only 6 years after the introduction of PCV2a-based vaccines, PCV2d was reportedly associated

https://doi.org/10.1016/j.vaccine.2020.01.013 0264-410X/Ó 2020 Elsevier Ltd. All rights reserved.

Please cite this article as: T. Opriessnig, A. K. Karuppannan, P. G. Halbur et al., Porcine circovirus type 2a or 2b based experimental vaccines provide protection against PCV2d/porcine parvovirus 2 co-challenge, Vaccine, https://doi.org/10.1016/j.vaccine.2020.01.013

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with apparent vaccine failures and subsequently become the predominant strain currently circulating in the global pig population [2]. There are concerns by some that the emergence of PCV2d could indicate a ‘‘leaky” or imperfect vaccine situation and that PCV2d could resemble a virus mutant capable of co-existing in vaccinated pig populations [7]. Hence, it is logical to assume that PCV2b- or PCV2d-based vaccines may offer superior protection to PCV2d infections compared to PCV2a-based vaccines. It is well established that the presence of co-infections are important for full expression of clinical PCVAD under experimental conditions [8]. Recently, several emerging parvoviruses including porcine parvovirus 2 (PPV2) or Ungulate tetraparvovirus 3, a member of the Tetraparvovirus genus in the Parvoviridae family, have been described in Asia, Europe and North America [9–11]. During PCV2 field investigations a high level of PPV2 viremia was detected in PCV2 positive pigs [12,13] possibly suggesting some interaction of these viruses. The objective of this study was to evaluate the efficacy of two experimental PCV2 vaccines of the same type, one based on PCV2a and the other based on PCV2b, in a PCV2d/PPV2 co-challenge model.

conducted under biosafety level 2 (BSL-2) conditions. The pigs were housed in two temperature-controlled rooms, each with dedicated clean entry and exit areas on opposite sides of the room allowing a unilateral flow of all personnel and leading to a common shower facility. Each room contained a single pen of approximately 30 m2, which was equipped with four nipple waterers and four self-feeders. Treatment groups were comingled in the rooms throughout the study, with each replicate room containing equal numbers of pigs from each treatment group. The pigs were fed an age appropriate feed ration free of antibiotics and animal proteins (Heartland Co-Op, Prairie city, IA, USA) and all pigs had ad libitum access to feed and water. 2.4. Experimental design

Sixty, 3-week old, mixed-gender crossbred pigs, were purchased from a commercial U.S. breeding herd with evidence of PCV2 circulation (antibodies were present in a portion of the sows but all sows screened were negative for PCV2 viremia) but with no history of PCVAD or PCV2 vaccination in breeding females. Blood was collected from the piglets on farm and at arrival 2 weeks later at the research facility and there was no evidence of increasing antibody levels.

The experimental design is outlined in Fig. 1. A complete randomized block design was used to assign equal proportions of challenge control (non-vaccinated, PCV2d/PPV2 challenged; n = 20) pigs, VAC2a (vaccinated twice with a PCV2a-based vaccine followed by PCV2d/PPV2 challenge; n = 20) pigs, and VAC2b (vaccinated twice with a PCV2b-based vaccine followed by PCV2d/ PPV2 challenged; n = 20) pigs to each of the two rooms. In addition to the three treatments described here, there were three other treatment groups (containing experimental vaccines) present in the study and used in the statistical analysis, but results of the experimental vaccines in these groups are not reported here. Initially the pigs were blocked by PCV2 antibody levels for allocation into treatment groups. After group allocation the pigs were blocked by treatment group and randomly allocated into one of two BSL-2 rooms each housing 30 pigs (10 VAC2a, 10 VAC2b and 10 challenge control pigs). After an acclimation period of one week, the pigs were vaccinated with an experimental lot of a chimeric PCV1/ PCV2a-based vaccine (VAC2a), an analogous experimental lot of a chimeric PCV1/PCV2b-based vaccine (VAC2b), or were left unvaccinated (challenge control). Two weeks later the pigs were revaccinated similarly. The research personnel were blinded to the treatment status by being provided identical bottles for each treatment with unique number identifiers prepared by a person unrelated to the study. At 8 weeks of age (2 weeks after second vaccination), all groups were challenged with PCV2d using intranasal and intramuscular routes of inoculation and one day later all pigs were challenge with a lung tissue homogenate containing PPV2 using the intramuscular route. All pigs were euthanized and necropsied at 21 days post PCV2 challenge (dpc).

2.3. Housing and feed

2.5. Sample collection

After weaning, the pigs were transported to the Livestock Infectious Disease Isolation Facility at the College of Veterinary Medicine, Iowa State University, Ames, Iowa, USA. The study was

Blood samples were collected from all pigs on a weekly basis until PCV2d/PPV2 co-challenge and again on dpc 5, 13 and 20 (Fig. 1). To obtain serum, BD VacutainerÒ tubes (Becton, Dickinson

2. Materials and methods 2.1. Ethical approval The experimental protocol of this study was approved by the Iowa State University Institutional Animal Care and Use Committee (approval number 3-16-8222-S) and the Iowa State University Institutional Biosafety Committee (approval number 16-D/I0005-A). Different types of environmental enrichment were provided to the pigs and they were regularly checked by veterinarians unrelated to the project personnel. 2.2. Animal source

Fig. 1. Experimental design and blood collections (B).

Please cite this article as: T. Opriessnig, A. K. Karuppannan, P. G. Halbur et al., Porcine circovirus type 2a or 2b based experimental vaccines provide protection against PCV2d/porcine parvovirus 2 co-challenge, Vaccine, https://doi.org/10.1016/j.vaccine.2020.01.013

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and Company, Franklin Lakes, NJ, USA) were used for blood collection, were centrifuged at 1500g for 8 min at 4 °C, and then the serum was aliquoted and stored at 80 °C until testing. To obtain peripheral blood mononuclear cells (PBMCs), 8–10 mL blood was collected from each pig before PCV2d challenge using BD VacutainerÒ CPTTM cell preparation tubes with sodium citrate (Becton, Dickinson and Company). Within 2 h of blood collection, the tubes were centrifuged at 1800g for 20 min at room temperature. The buffy coat was collected and re-suspended in phosphate-buffered saline (PBS). Cells were washed and centrifuged at 500g for 5 min at 4 °C, the supernatant was discarded, and the pellet was used immediately for the ELISpot assay. At necropsy at dpc 21, tissues were collected in 10% buffered formalin for further histopathological analysis and immunohistochemistry (IHC) specific for PCV2. 2.6. Clinical parameters evaluated The pigs were weighed at arrival when they were 3 weeks of age, at challenge at 8 weeks of age and at necropsy at 11 weeks of age and the average daily weight gain was calculated for the time of arrival to challenge and from the challenge to necropsy (Table 1). All pigs were examined every day for signs of illness such as lethargy, respiratory disease, inappetence and lameness for the duration of the study and any aberrations from normal parameters were recorded. 2.7. Vaccine preparation and vaccination The experimental vaccines were provided by Zoetis Inc., labeled ‘‘L0616LW05” (PCV2a) and ‘‘L0616LW06” (PCV2b) and stored at 4 °C until usage. Both vaccines contained an inactivated chimeric PCV1-2 virus, which consists of a PCV2 capsid gene replacing the homologous capsid gene in a PCV1 genome backbone as described [14]. Virus and adjuvant (MetaStimÒ) concentrations were similar in both vaccine preparations. VAC2a and VAC2b groups were vaccinated via the intramuscular route into the right neck area. Each vaccinated pig received 2 mL of the vaccine at 4 weeks of age and again at 6 weeks of age. After vaccination the pigs were examined once a day for a total of 3 days for potential adverse reactions such as redness or swelling at the injection site.

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To obtain PPV2 tissue homogenate for inoculation the colostrum-deprived (CD) pig model was used [16]. In brief, three CD piglets, confirmed free of PCV2 and PPV2 viremia, were inoculated by the intramuscular route with a PPV2 DNA positive lung tissue homogenate twice 13 days apart. The lungs used originated from a field case and were negative for other parvoviruses known to circulate in pigs and PCV2 as determined by PCR assays [12]. The CD piglets were euthanized three days after the second inoculation and ileum and mesenteric lymph nodes were further processed and homogenized. The resulting tissue homogenate was filtered through a 0.22 mM filter, heat inactivated at 56 °C for 30 min, mixed with antibiotic and anti-mycotic (ThermoFisher, Waltham, MA, USA) and kept on ice for 30 min. Each ml of the homogenate was estimated to contain 1.8  104 genomic PPV2 copy numbers as determined by quantitative real time PCR [10]. Each pig in this study was given 2 mL of the PPV2 positive tissue homogenate intramuscularly into the left neck area. 2.9. DNA extraction, detection and quantification of PCV2d viremia by real-time PCR The presence and amount of PCV2 DNA in serum was determined by a real-time PCR assay targeting ORF1 as described [17,18]. In brief, total nucleic acids from serum samples collected on dpc 5, 13 and 20 were extracted using the MagMAX-96 nucleic acid isolation kit (ThermoFisher, Waltham, MA, USA) on the automated KingFisher Flex System (ThermoFisher) according to the instructions of the manufacturer. The extracted DNA was used in the real-time PCR assay performed in an ABI7500 Fast PCR machine with a TaqMan Universal real-time PCR Master Mix (ThermoFisher) as described previously [18]. A cycle threshold (CT) of 38 was considered negative. Appropriate negative and positive controls were included in each PCR run. For the positive control, a laboratory dilution of the PCV2d challenge virus was prepared, and sterile water served as negative control. At terminating of the study a differential real-time PCR assay targeting ORF2 [18], capable of detecting and differentiating PCV2a, PCV2b and PCV2d, was done on all dpc 20 PCV2 DNA positive serum samples and on both vaccines used in this study. Selected PCR products from PCV2 PCR-positive serum samples were sequenced by using a conventional PCR covering the entire ORF2 as described previously [19] at the Iowa State University DNA Facility, Ames, IA, USA.

2.8. PCV2d/PPV2 co-challenge

2.10. ELISpot assay

For the virus challenge, a cell culture propagated PCV2d infectious virus stock (JX535296) [15] at approximately 104.6 50% tissue culture infectious dose (TCID50) per mL was used. In brief, each pig received 2 mL of the PCV2d stock intranasally by slowly dripping 1 mL into each nostril and 2 mL intramuscularly into the neck area. The amino acid sequence identity between the ORF2 capsid proteins in the PCV2d challenge virus and the vaccine viruses was 91.4% (VAC2a) and 94.4% (VAC2b).

At the time of PCV2d/PPV2 co-challenge, PBMCs were isolated, then washed twice in sterile calcium and magnesium free phosphate buffered saline (PBS / ) by centrifugation at 300 g for 10 min. The PCV2-specific interferon c (IFN-c) secreting PBMC was estimated with a commercial ELISpot kit (Porcine IFNgamma ELISpot Kit; R&D systems, Inc., Minneapolis, MN, USA). A total of 2.5  105 viable PBMCs in 100 lL of RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum were seeded into microplates and pretreated to capture IFN c (provided in the kit). The PBMCs were incubated for 36 h at 37 °C in a 5% CO2 incubator, with 150 lL/well of the PCV2d virus stock that was used for the challenge. Then the ELISPOT assay was performed according to the manufacturer’s instructions. Briefly, cells were washed and incubated with a biotinylated detection antibody and any unbound antibody was washed off. Subsequently, alkaline-phosphatase conjugated to streptavidin was bound to the biotinylated detection antibody. Any unbound enzyme was removed by washing and a substrate solution (BCIP/NBT) was added. A blue-black colored precipitate was formed at the sites of cytokine localization and appeared as spots,

Table 1 Average daily weight gain in grams ± SEM from each group during two experimental periods, arrival to PCV2d/PPV2 challenge (35 days) and PCV2d/PPV2 challenge to necropsy (21 days). Group

Pig No.

Arrival ? challenge 3–8 weeks of age

Challenge ? necropsy 8–11 weeks of age

Challenge control VAC2a VAC2b

20 20 20

369.5 ± 12.9A* 392.8 ± 14.6A 377.7 ± 14.5A

756.7 ± 23.8A 834.6 ± 24.9A 798.2 ± 28.5A

* Different superscripts (A,B) indicate significantly (P < 0.001) different group means for each period of time.

Please cite this article as: T. Opriessnig, A. K. Karuppannan, P. G. Halbur et al., Porcine circovirus type 2a or 2b based experimental vaccines provide protection against PCV2d/porcine parvovirus 2 co-challenge, Vaccine, https://doi.org/10.1016/j.vaccine.2020.01.013

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with each individual spot representing an individual secreting cell. The IFN-c spots were counted with an ELISpot reader (Autoimmun Diagnostika GmbH, Strassberg, Germany). 2.11. Detection of anti-PCV2 IgG Serum samples collected at days post-vaccination (dpv) 0, dpv 21, dpv 28 (corresponding to zero days post-challenge, dpc), and dpc 13 and dpc 20. These were tested for seroconversion to PCV2 IgG using a commercial indirect ELISA PCV2 kit (INgezim CIRCO IgG R.11.PCV.K1; Ingenasa, Madrid, Spain), which contains ELISA plates coated with PCV2b ORF2 protein, as instructed by the manufacturer. The test was considered valid if the optical density (OD)450 of the negative control was lower than 0.35 and if the OD450 of the positive control was higher than 0.7. A sample optical density (OD)450 value of 0.25 more than the negative control was considered as positive for PCV2-specific IgG antibodies, while an OD450 between 0.2 and 0.25 more than the negative control was considered as doubtful. 2.12. Necropsy and macroscopic lesions At dpc 21, all pigs were euthanized by intravenous pentobarbital sodium overdose (Fatal PlusÒ, Vortech Pharmaceuticals, LTD, Dearborn, MI, USA) and a necropsy was performed. Gross lesions were assessed by a veterinary pathologist (PGH) blinded to the treatment status of the pigs. The percent of the lung surface affected with consolidation was subjectively estimated for each lung lobe and the total percentage of pneumonia was calculated based on weighted proportions of each lobe relative to the total lung volume as previously described [20]. The size of superficial inguinal lymph nodes was scored as described [21]. Any other lesions were recorded and sections of lymph nodes (superficial inguinal, external iliac, mediastinal, tracheobronchial, and mesenteric), tonsil, spleen, kidney, liver, and small intestines (ileum) were collected, fixed in 10% neutralbuffered formalin, and routinely processed for histological examination. 2.13. Histopathology, immunohistochemical analysis and calculation of the overall lymphoid lesion score Microscopic lesions were assessed by a veterinary pathologist (TO) blinded to the treatment status. Lymph nodes, spleen, and tonsil were evaluated for presence and degree of lymphoid depletion and granulomatous replacement of follicles ranging from 0 (normal) to 3 (severe) [22]. Lung sections were scored for presence and severity of interstitial pneumonia, ranging from 0 (normal) to 6 (severe diffuse) [20]. Sections of ileum, liver and kidney were evaluated for the presence of granulomatous inflammation and scored from 0 (none) to 3 (severe). Immunohistochemistry (IHC) for detection of PCV2 antigen was performed on formalin-fixed and paraffin-embedded sections of lungs, lymph nodes, tonsil, and spleen from all pigs using a rabbit PCV2 polyclonal antiserum [23]. PCV2 antigen scoring was done by a veterinary pathologist (TO) blinded to the treatment status. Scores ranged from 0 (no signal) to 3 (more than 50% of lymphoid follicles contained cells with PCV2 antigen staining) [22]. The overall lymphoid lesion score was calculated as described previously [22]. In brief, a combined scoring system for each lymphoid tissue that ranged from 0 to 9 (lymphoid depletion score 0– 3; granulomatous inflammation score 0–3; PCV2 IHC score 0–3) was used. Pigs were classified as having no lesions (score of 0), mild (scores of 1, 2 or 3), moderate (scores of 4, 5 and 6) and severe (scores of 7, 8 and 9) lesions consistent with PCVAD.

2.14. Statistical analysis The statistical analysis was performed using JMP software (JMP version Pro 14, SAS Institute Inc., Cary, NC, USA). Summary statistics were calculated to assess the overall quality of data and all data except real-time PCR results were normally distributed. Analysis of variance (ANOVA) was used for cross-sectional assessment of the average daily weight gain (ADG) and PCV2 viremia. To reach normal distribution for real-time PCR results (copies per mL serum) data were log10 transformed prior to statistical analysis. The significance level was P < 0.05, followed by pairwise testing using the Tukey-Kramer adjustments to identify the groups that were different. Non-repeated measures of necropsy and histopathology data were assessed using non-parametric Kruskal -Wallis ANOVA. If a non-parametric ANOVA test was significant (P < 0.05), then Wilcoxon tests were used to assess the differences of pairs of groups. Fisher’s exact test was used to determine difference in prevalence levels among groups. The area under the curve (AUC) of PCV2 shedding for each group was calculated using the log transformed values of the individual pig viral loads over time. 3. Results 3.1. Vaccine reactions and confirmation of the correct vaccine PCV2 type Adverse vaccination reactions were not observed in any of the vaccinated pigs. At termination of the study, a routine PCV2 ORF2 differential PCR on the two vaccines used in the study confirmed the presence of a single PCV2 type, ORF2 of PCV2a in VAC2a or ORF2 of PCV2b in VAC2b, in each preparation. 3.2. Clinical signs and weight gain All pigs remained clinically unremarkable for the duration of the study. No significant difference in ADG was observed among treatments during the two periods from arrival to challenge (0– 35 dpv) and from challenge to necropsy day (0–21 dpc) (Table 1). However, after challenge, the ADG was numerically lowest in the challenge control group. 3.3. PCV2 cell-mediated immune (CMI) response Mean group PCV2d-specific IFN-c secreting cells in PBMCs, indicative of the level of cell-mediated immunity, are summarized in Fig. 2. Both vaccinated groups (VAC2a and VAC2b) had significantly higher levels of PCV2d specific IFN-c secreting cells compared to the challenge control group (Fig. 2).

Fig. 2. Interferon c secreting cells per million PBMC on the day of PCV2d/PPV2 challenge in the different treatment groups. Different superscripts (A,B) indicate significantly different group means.

Please cite this article as: T. Opriessnig, A. K. Karuppannan, P. G. Halbur et al., Porcine circovirus type 2a or 2b based experimental vaccines provide protection against PCV2d/porcine parvovirus 2 co-challenge, Vaccine, https://doi.org/10.1016/j.vaccine.2020.01.013

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3.4. Humoral immune responses against PCV2 While all pigs were PCV2 seropositive at the time of vaccination, all were negative for PCV2 DNA in serum suggesting the absence of active PCV2 replication but presence of passively acquired antibodies. All pigs from both vaccinated groups were anti-PCV2 antibody positive by dpv 21 with significantly (P < 0.05) higher titers compared to the challenge control group (Fig. 3) in which antibodies had waned to undetectable levels in 50% (10/20) of the group. At the time of PCV2d challenge, antibody titers did not differ between VAC2a and VAC2b groups. While all pigs in all groups were seropositive by dpc 20, the PCV2 antibody titers were significantly higher in vaccinated groups compared to unvaccinated challenge control pigs. The mean group anti-PCV2 log2 titers ± SEM are shown in Fig. 3. 3.5. PCV2 viremia after PCV2d/PPV2 co-challenge At dpc 5, 15% (3/20) VAC2a pigs, 15% (3/20) VAC2b pigs, and 30% (6/20) challenge control pigs were PCV2 viremic. By dpc 13, PCV2 DNA was detected in serum samples from 35% (7/20) of the VAC2a pigs, 25% (5/20) of the VAC2b pigs and in 100% (20/20) of the challenge control pigs. By dpc 20, while all challenge control pigs were viremic (20/20), PCV2 DNA was detected in only 5% (1/20) of the VAC2a pigs and in 10% (2/20) of the VAC2b pigs. The AUC was 12.0 for VAC2a, 6.2 for VAC2b and 40.0 for the challenge control group. The average duration of PCV2 viremia in PCV2-infected pigs was 0.6 ± 0.2 weeks for VAC2a pigs, 0.5 ± 0.2 weeks for VAC2b pigs and 2.3 ± 0.1 weeks for the challenge control pigs. The duration of PCV2 viremia was significantly (P < 0.001) longer in challenge control pigs compared to the two vaccinated groups. Mean log10 group PCV2 genomic copy numbers in serum are summarized in Fig. 4. The presence of PCV2d was confirmed in the challenged pigs by a differential PCR assay and ORF2 sequencing on selected PCV2 DNA positive serum samples collected at dpc 20. 3.6. Macroscopic lesion Lymph nodes were normal to mildly enlarged in most pigs in all groups, without differences among the groups. There were no other remarkable observations during necropsy. 3.7. Microscopic lesions, PCV2 antigen in lymphoid tissues, and overall PCVAD score There were no microscopic lung lesions in any of the pigs. Mild-to-severe lymphoid lesions characteristic of PCV2 infection

Fig. 3. Mean log2 group anti-PCV2 antibody titers at different time points before and after PCV2d/PPV2 challenge. Different superscripts (A,B) indicate significantly different group means.

Fig. 4. Mean group PCV2 DNA levels in serum at different days post PCV2d/PPV2 challenge. Different superscripts (A,B) indicate significantly different group means.

(lymphoid depletion and histiocytic replacement of follicles) were observed in the lymph nodes of 55% of the challenge control pigs (11/20). Furthermore, in this group 14/20 pigs had detectable PCV2 antigen in at least one lymph node. For the vaccinated groups, microscopic lesions in lymphoid tissues were focal (in a single lymph node) and mild (score of 1) and were present in 3/20 VAC2a pigs and 3/20 VAC2b pigs. PCV2 antigen was detectable in 1/20 VAC2a pigs (low amount of antigen in a single lymph node) and in 0/20 VAC2b pigs. The prevalence of PCV2 antigen in tissues was significantly lower (P < 0.001) in both vaccinated groups compared to the challenge control. The overall PCVAD score revealed 1/20 challenge control pigs with severe lesions, 4/20 pigs with moderate lesions, 6/20 pigs with mild lesions, and 9/20 challenge control pigs without PCV2associated lesions. Among vaccinated groups, mild lesions were seen in 3/20 VAC2a and in 3/20 VAC2b pigs, and normal tissues were found in the remaining 17/20 pigs in each group. 4. Discussion The objective of this study was to determine the relative contributions of PCV2a and PCV2b genotype vaccine formulations on cross-protective efficacy against challenge with a PCV2d genotype virus (currently the most prevalent genotype). Two inactivated and adjuvanted PCV1/PCV2 chimeric vaccines, one based on PCV2a and one based on PCV2b, were tested for their ability to protect conventional pigs against PCV2d challenge at 8 weeks of age. All pigs were co-challenged with PPV2 one day following the PCV2d challenge based on previous evidence that PPV2 may exacerbate PCVAD. The PPV2 seems to have had little or no effect on PCV2d challenge virulence (data not shown). However; pigs singularly infected with PCV2d and PPV2 would have been needed to fully assess this. PCV2 is highly prevalent globally, and the vast majority of swine herds are PCV2 seropositive. At the initiation of PCV2 vaccination in growing pigs, much discussion focused on the possible negative impact of passively acquired maternal antibodies on vaccine efficacy. This issue likely has only a marginal impact [24,25]. Field evidence based on 12 years of experience using PCV2 vaccination suggests that despite being widely used in 2–3-week-old antibody-positive pigs, vaccination has had a major positive impact on decreasing PCVAD. Nevertheless, some producers continue to check for PCV2 antibody levels to determine the ideal timing of vaccination specific for their farm. In this present study to better mimic a field scenario, we used PCV2 seropositive pigs, blocked the pigs by antibody level and assigned them to treatment groups, then blocked them by treatment group and assigned them to one of two rooms with an equal number of pigs from each treat-

Please cite this article as: T. Opriessnig, A. K. Karuppannan, P. G. Halbur et al., Porcine circovirus type 2a or 2b based experimental vaccines provide protection against PCV2d/porcine parvovirus 2 co-challenge, Vaccine, https://doi.org/10.1016/j.vaccine.2020.01.013

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ment group in each room. As demonstrated in Fig. 3, while the antibody levels in vaccinated pigs did not change and remained at a similar level until challenge, the antibody levels in the nonvaccinated pigs decreased and were significantly lower compared to vaccinated pigs at day post vaccination 21 and 28. After PCV2d challenge both the PCV2a and PCV2b based vaccines had an immediate booster effect and the vaccinated pigs showed a faster increase of antibody titers compared to the challenge control group. PCV2 viremia levels; however, were significantly higher in the challenge control group compared to the vaccinated groups. Overall, this study further confirms that passively acquired antibodies likely have little impact on PCV2 vaccine efficacy (i.e. viremia was significantly reduced in VAC2a and VAC2b groups compared to the challenge control pigs); however, an increase in anti-PCV2 antibodies may not occur without exposure to field virus and thus monitoring antibody levels to assess vaccine efficacy may not be useful in vaccinated herds. The vast majority of experimental and field PCV2 vaccine evaluations has been conducted with PCV2a-based, mainly commercially available vaccines followed by PCV2a [25–28], PCV2b [26,27,29] or PCV2d [18,27,30–33] challenge or natural exposure. Few studies have evaluated experimental PCV2b-based vaccines with subsequent PCV2b or PCV2d challenge, or experimental PCV2d-based vaccines with subsequent PCV2d challenge. An experimental live PCV1-2 vaccine containing the capsid gene of PCV2b (PCV1-2b) induced cross-protection against PCV2a and PCV2b in the pig model [34]. Similarly, an experimental PCV1-2b vaccine candidate protected pigs against PCV2b and PCV2d challenge [35]. Two experimental inactivated PCV1-2 vaccines containing the capsid antigen of PCV2b or PCV2d were tested against PCV2b challenge and were found effective [36]. In another study, a live PCV1-2 vaccine virus containing a capsid antigen created by DNA shuffling of PCV2a, PCV2b, PCV2c and PCV2d induced protective immunity against PCV2b and PCV2d challenge [37]. Furthermore, an experimental PCV2d vaccine using the PCV2d capsid gene in the alphavirus replicon expression system protected pigs against PCV2d challenge [18]. Very few studies have compared similar vaccines based on different PCV2 genotypes, side by side, against PCV2 challenge. In a previous study, our group compared two live PCV1-2 vaccines, one with a PCV2a capsid and the other with a PCV2b capsid in the concurrent PCV2, porcine reproductive and respiratory syndrome virus (PRRSV) and porcine parvovirus (PPV) challenge model [38]. In that study, while both vaccines reduced viremia and protected pigs from PCVAD lesions, the PCV2b-based vaccine had a slight advantage over the PCV2a-based vaccine as evidenced by a significantly lower viremia level. However, it needs to be noted that in the live PCV2b vaccine more pigs became PCV2b viremic after vaccination compared to the PCV2a vaccine group [38]. In this study, both vaccines were produced under very similar conditions by using the same method for inactivation, similar concentrations of vaccine antigen in each dose and the same adjuvant. Furthermore, the two vaccines were administered to two groups of pigs, which were co-mingled with unvaccinated pigs in the same environment. PCV2 vaccination, regardless of PCV2 genotype present in the vaccine preparations, resulted in statistically significant increases in anti-PCV2 antibody titers and an increased number of PCV2d antigen-specific IFN-c secreting cells indicative of a PCV2d specific cellular immune response. After PCV2d challenge, vaccination significantly reduced the virus load in serum, viremia duration and PCV2 antigen prevalence in lymphoid tissues compared to nonPCV2 vaccinated challenge control pigs. The high viremia in the challenge control pigs suggests a high level of virus shedding from these pigs throughout the duration of the study. We did not measure PCV2 shedding in this study due to the study design, a

complete randomized block design, where pigs across all treatment groups were comingled with direct contact. Under this scenario, pigs with high viremia were present in the pens and likely contributed to an overall high PCV2 load in the environment. This could result in PCV2 contamination during the sample collection process, making interpretation of such data difficult to impossible. In contrast, VAC2a and VAC2b pigs had declining trends in viremia from 13 dpc to 20 dpc. Furthermore, while 25% of the challenge control group had lesions comparable with moderate to severe PCVAD, lesions associated with PCV2 in pigs in the VAC2a and VAC2b groups fell into the normal (85%) or mild (15%) categories. Although both vaccines provided a similar high degree of cross-protective efficacy, neither vaccine alone was able to eliminate PCV2d viremia or completely prevent PCVAD. Since each of the vaccine viruses share a discreet subset of B-cell and T-cell epitopes with the challenge virus, we would predict that a bivalent vaccine containing both PCV2a and PCV2b components may have shown a higher degree of efficacy against a PCV2d challenge compared to monovalent PCV2 vaccines. Future studies will explore this hypothesis. In this study, PCV2 tissue loads were assessed by IHC rather than by quantitative real-time PCR for practical reasons: PCV2 antiserum was readily available, PCV2 IHC is an established method in our laboratory, and based on our past experiences there is in general a good correlation of PCV2-antigen presence and lesions [39]. Sometimes it can be challenging to compare PCV2 viral loads in tissues due to disseminated viral tissue distribution especially in subclinically infected pigs, the possibility of PCV2 DNA crosscontamination during sample processing, and the need to weigh tissues individually to obtain comparable results among others. Overall, the results of this study indicate that inactivated and adjuvanted chimeric PCV1/PCV2 vaccines based on PCV2a or PCV2b each provided similar levels of cross-protection against PCV2d challenge when tested as monovalent vaccines, as evidenced by seroconversion, development of PCV2d specific IFN-c secreting cells, reduced PCV2 viremia and reduced microscopic lesions in nursery pigs. Bivalent vaccines will be evaluated for increased protection in future studies. CRediT authorship contribution statement Tanja Opriessnig: Conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, supervision, visualization, writing - original draft, writing - review & editing. Anbu K. Karuppannan: Data curation, validation, investigation, methodology, writing - review & editing. Patrick G. Halbur: Conceptualization, data curation, funding acquisition, investigation, supervision, validation, writing - review & editing. Jay G. Calvert: Conceptualization, methodology, writing - review & editing. Gregory P. Nitzel: Conceptualization, methodology, writing - review & editing. Shannon R. Matzinger: Methodology, writing - review & editing. Xiang-Jin Meng: Methodology, writing - review & editing. Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: JGC and GPN are employees of Zoetis. Zoetis produces a PCV2a and a combined PCV2a/PCV2b vaccine. These authors recognize the presence of a potential conflict of interest and affirm that the information represented in this paper is original and based on unbiased observations. All other authors declare that they have no known competing or financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Please cite this article as: T. Opriessnig, A. K. Karuppannan, P. G. Halbur et al., Porcine circovirus type 2a or 2b based experimental vaccines provide protection against PCV2d/porcine parvovirus 2 co-challenge, Vaccine, https://doi.org/10.1016/j.vaccine.2020.01.013

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Acknowledgements The authors thanks Dr. Jianliang Li, Dr. Huigang Shen, Dr. Megan Hindman, Danielle Tapp and Ryan Kass for assistance with the animal work.

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Funding

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This work was supported by a Research Services Agreement with Zoetis Inc, Kalamazoo, MI, USA. We would also like to acknowledge Biotechnology and Biological Sciences Research Council (BBSRC) support of the Roslin Institute Strategic Programme Control of Infectious Diseases (BBS/E/D/20002173 and BBS/E/D/20002174).

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Appendix A. Supplementary material Supplementary data to this article can be found online at https://doi.org/10.1016/j.vaccine.2020.01.013.

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Please cite this article as: T. Opriessnig, A. K. Karuppannan, P. G. Halbur et al., Porcine circovirus type 2a or 2b based experimental vaccines provide protection against PCV2d/porcine parvovirus 2 co-challenge, Vaccine, https://doi.org/10.1016/j.vaccine.2020.01.013