Abstract
The objective of this study was to evaluate a new recombinant chimeric vaccine against porcine reproductive and respiratory syndrome virus (PRRSV). The subunit vaccine, PRRSFREE, from Reber Genetics, Taiwan, Republic of China, is based on a plasmid containing a detoxified Pseudomonas exotoxin carrying open reading frame (ORF) 7, 1b, and 5 and 6 chimeric subunits of types 1 and 2 PRRSV. Pigs were injected intramuscularly with 2.0 mL of the vaccine at 21 and 42 d of age, according to the manufacturer’s recommendation. At the age of 63 d the pigs were inoculated intranasally with either type 1 or type 2 PRRSV. Regardless of the genotype of the challenging PRRSV, the vaccinated challenged pigs had significantly lower (P < 0.05) mean rectal temperature, respiratory score, lung lesion score, and amount of PRRSV antigen within areas of interstitial pneumonia, along with overall lower levels of viremia due to type 1 or type 2 PRRSV compared with the unvaccinated challenged pigs. The vaccinated challenged pigs also had significantly higher (P < 0.05) numbers of interferon-γ secreting cells compared with the unvaccinated challenged pigs. This study demonstrated that the new vaccine provides protection against respiratory disease from heterologous types 1 and 2 PRRSV challenge in growing pigs.
Résumé
L’objectif de la présente étude était d’évaluer un nouveau vaccin recombinant chimérique contre le virus du syndrome reproducteur et respiratoire porcin (VSRRP). Le vaccin sous-unitaire, PRRSFREE, de Reber Genetics, Taïwan, République de Chine, est basé sur un plasmide contenant une exotoxine de Pseudomonas détoxifiée portant les cadres de lecture ouverts (ORF) 7, 1b, et 5 et 6 unités chimériques des types 1 et 2 du VSRRP. Les porcs ont été injectés par voie intramusculaire avec 2,0 mL du vaccin à 21 et 42 j d’âge, selon les recommandations du manufacturier. À l’âge de 63 j les porcs ont été inoculés par voie intra-nasale avec le type 1 ou le type 2 du VSRRP. Indépendamment du génotype du VSRRP utilisé pour l’infection défi, des données significativement moindres (P < 0,05) étaient notées chez les porcs vaccinés et infectés comparativement aux animaux non-vaccinés et infectés quant à la température rectale moyenne, le pointage respiratoire, le pointage de lésions pulmonaires, et la quantité d’antigènes du VSRRP dans les sites de pneumonie interstitielle, de même que pour le degré de virémie dû au type 1 ou au type 2 du VSRRP. Les porcs vaccinés et challengés avaient également une quantité significativement plus grande (P < 0,05) de cellules secrétant de l’interféron-γ comparativement aux porcs non-vaccinés et infectés. Cette étude démontre chez des porcs en croissance que le nouveau vaccin fourni une protection contre les problèmes respiratoires causés par une infection défi hétérologue avec les types 1 et 2 du VSRRP.
(Traduit par Docteur Serge Messier)
Introduction
Porcine reproductive and respiratory syndrome (PRRS) was first reported in 1987 in the United States and had become widespread throughout Europe by 1991 (1). During the mid-1990s PRRS was also reported in Asian countries (2–4). The most economically important disease facing the global swine industry, PRRS occurs in 2 forms: reproductive failure in pregnant sows and respiratory disease in pigs of all ages (5). The causative agent is the PRRS virus (PRRSV), an enveloped positive-strand RNA virus classified in the order Nidovirales, family Arteriviridae, and genus Arterivirus (6). The PRRSV genome contains at least 9 open reading frames (ORFs), which encode the viral replicases (ORF1a and 1b) and 7 structural proteins (ORF2a, 2b, 3, 4, 5, 6, and 7) (6).The virus falls into 2 distinct genotypes, referred to as type 1 (European) and type 2 (North American), which are genetically and antigenically distinct (7–9).
Asian PRRSV isolates were of type 2 until 2000 (10). Early that year, type 1 PRRSV emerged in Asian countries (11–13). Currently both genotypes are circulating in Asian pig farms. Therefore, protection by vaccine against both genotypes is an important clinical issue in many Asian countries because of a lack of cross-protection by current vaccines against heterotypic PRRSV (14–16). A new recombinant chimeric PRRS vaccine (PRRSFREE PRRS subunit vaccine) was introduced into the Taiwanese market in 2012 by Reber Genetics (Taiwan, Republic of China), which claimed that it protected against both genotypes. Since the current needs in a PRRS vaccine are better safety and better efficacy against both genotypes (17), this new vaccine fit perfectly with the demands of swine producers. Theoretically, the vaccine has the potential to be clinically useful in controlling coinfection by both genotypes. However, there had been no reports on scientific studies in peer-reviewed publications that demonstrated protection of this vaccine against either PRRSV genotype. The objective of this study, therefore, was to evaluate the efficacy of the new vaccine in controlling respiratory disease in growing pigs challenged with heterologous types 1 and 2 PRRSV on the basis of clinical, immunologic, virologic, and pathological evaluations under experimental conditions.
Materials and methods
Vaccine
The recombinant chimeric PRRS vaccine used in this study is based on a plasmid containing a detoxified Pseudomonas aeruginosa exotoxin and ORF7, ORF1b, ORF5, and ORF6 chimeric subunits of type 1 and type 2 PRRSV (18). The epitopes of viral ORF7 and ORF1b are made from the conserved regions of PRRSV and other arteriviruses [GenBank (National Center for Biotechnology Information, Bethesda, Maryland, USA) no. X53459 for Equine arteritis virus, M96262 for PRRSV, U15146 for Lactate dehydrogenase-elevating virus, and U63121 for Simian hemorrhagic fever virus; US patent no. 7,595,054 B2]. Viral ORF5 and ORF6 are made from type 1 PRRSV (GenBank no. CAA63493.1) and type 2 PRRSV (GenBank ACG52416.1), respectively. The ORF1b gene encodes the key enzymes for PRRSV RNA synthesis, which is essential for genome replication and synthesis (19). The PRRSV subunit antigens encoded by ORF5 through ORF7 have been reported to confer immunogenicity (20,21). The GP5 protein encoded by ORF5 and the M protein encoded by ORF6 can induce neutralizing antibodies (22,23). The efficacy of PRRSV DNA vaccination is significantly enhanced by coexpression of the GP5 and M proteins as heterodimers (24). Therefore, a new recombinant chimeric vaccine containing ORF1b and ORFs 5 to 7 is a good choice to control PRRSV infection.
Virus inocula
The type 1 PRRSV SNUVR090485, a pan-European subtype 1 (GenBank no. JN315686), and the type 2 PRRSV SNUVR090851, lineage 1 (GenBank no. JN315685), were used as inocula. The type 1 virus was isolated from lung samples from an aborted fetus from southwestern Gyeonggi Province in 2009 (25). The type 2 virus was isolated from lung samples from newly weaned pigs in Chungcheung Province in 2010 (26). The 2 viruses have 59% ORF5 nucleotide identity.
Experimental design
A total of 108 colostrum-fed, cross-bred, conventional piglets were purchased at 14 d of age from a commercial PRRSV-free farm. All the piglets were negative for PRRSV, porcine circovirus 2 (PCV2), and swine influenza virus according to routine serologic testing, and all the piglets were negative for types 1 and 2 PRRSV viremia according to real-time polymerase chain reaction (PCR) (27).
The pigs were divided into 6 groups (18 pigs per group) by means of random number generation with Excel (Microsoft Corporation, Redmond, Washington, USA) (Table I). Those in groups Vac/Ch1, Vac/Ch2, and Vac/UnCh were injected intramuscularly in the right side of the neck with 2.0 mL of PRRSFREE (lot F4001) at 21 and 42 d of age, according to the manufacturer’s recommendation. The pigs in groups UnVac/Ch1, UnVac/Ch2, and UnVac/UnCh were injected intramuscularly in the same anatomic location with 2.0 mL of phosphate-buffered saline (0.01 M, pH 7.4) at the same age. At the age of 63 d the pigs in groups Vac/Ch1 and UnVac/Ch1 were inoculated intranasally with 3 mL of tissue culture fluid containing 105 50% tissue culture infective doses (TCID50)/mL of type 1 PRRSV (SNUVR090485, 2nd passage in alveolar macrophages). The pigs in groups Vac/Ch2 and UnVac/Ch2 were inoculated intranasally with 3 mL of tissue culture fluid containing 105 TCID50/mL of type 2 PRRSV (SNUVR090851 strain, 2nd passage in MARC-145 cells). The pigs in the UnVac/UnCh group remained unvaccinated and unchallenged and served as negative controls. The pigs in each group were housed separately within the facility. Blood samples and nasal swabs were collected at −35, −28, −21, −14, −7, 0, 3, 7, 10, 14, and 21 d after challenge. Subsets of pigs were sedated by an intravenous injection of sodium pentobarbital and then euthanized by electrocution at 7, 14, and 21 d after challenge as previously described (28). Tissues were collected from each pig at necropsy. All of the methods had been approved by the Seoul National University Institutional Animal Care and Use and Ethics Committee.
Table I.
Experimental design for evaluation of a new recombinant chimeric vaccine against porcine reproductive and respiratory syndrome virus (PRRSV) in pigs vaccinated (Vac) or not vaccinated (UnVac) at 21 and 42 d of age and then challenged at 63 d of age with type 1 (Ch1) or type 2 (Ch2) PRRSV or not challenged (UnCh)
| Group | Vaccinated | Challenged with PRRSV type 1 or 2 |
|---|---|---|
| Vac/Ch1 | Yes | Type 1 |
| UnVac/Ch1 | No | Type 1 |
| Vac/Ch2 | Yes | Type 2 |
| UnVac/Ch2 | No | Type 2 |
| Vac/UnCh | Yes | No |
| UnVac/UnCh | No | No |
Clinical observation
The pigs were monitored weekly for physical condition and scored daily for clinical severity of respiratory disease from 0 (normal) to 6 (severe dyspnea and abdominal breathing) (29). Rectal temperature was recorded daily at the same time by the same personnel.
Quantification of PRRSV RNA
RNA was extracted from the blood samples and nasal swabs to determine the numbers of genomic RNA copies for types 1 and 2 PRRSV by real-time PCR as previously described (27,30). Sterile polyester swabs (Fisher Scientific, Pittsburgh, Pennsylvania, USA) had been used to reach deeply into the turbinates to swab the nasal mucosa of both nostrils. The swabs had been stored in 5-mL plastic tubes (Fisher Scientific) containing 1 mL of sterile saline solution.
Antibody studies
Serum from the blood samples was tested with the commercial PRRSV enzyme-linked immunosorbent assay (ELISA) HerdChek PRRS X3 Ab test (IDEXX Laboratories, Westbrook, Massachusetts, USA). Samples were considered positive for PRRSV antibody if the sample/positive (S/P) ratio was greater than or equal to 0.4, according to the manufacturer’s instructions.
Tests were also done to determine the titers of serum virus-neutralizing (VN) antibody against the challenge strains of PRRSV as previously described (31). The presence or absence of virus-specific cytopathic effect in each well was recorded after incubation for 7 d. Serum samples were considered to be positive for VN antibody if the titer was greater than 2.0 (log2) (32).
Interferon gamma studies
The numbers of PRRSV-specific interferon gamma (IFN-γ)-secreting cells (IFN-γ-SCs) were determined in peripheral blood mononuclear cells (PBMCs) by enzyme-linked immunospot assay (ELISPOT) as previously described (30,33,34).
The phenotypes of the CD4+CD8+IFN-γ+cells in PBMCs were analyzed as described elsewhere (31,35) by means of flow cytometry with the use of 3 monoclonal antibodies: CD4a against swine antigen conjugated with R-phycoerythrin (SouthernBiotech, Birmingham, Alabama, USA), CD8a conjugated with fluorescein isothiocyanate (SouthernBiotech), and IFN-γ conjugated with Alexa Fluor 647 (BD Biosciences, San Diego, California, USA).
Lung-tissue studies
The scores for macroscopic and microscopic lung lesions were analyzed morphometrically as previously described (29). In-situ hybridization for the detection and differentiation of types 1 and 2 PRRSV nucleic acids in the lung tissue was done, and the results were analyzed morphometrically as previously described (26).
The PRRSV was isolated from lung tissues with the use of alveolar macrophages for type 1 PRRSV and MARC-145 cells for type 2 PRRSV as previously described (29).
Statistical analysis
All real-time PCR and VN antibody data were transformed to log10 and log2 values, respectively. Summary statistics were then calculated for all of the groups to assess the overall quality of the data, including normality. The continuous data (for rectal temperature, PRRSV RNA, and serologic and PBMC findings) were analyzed by repeated-measures analysis of variance (ANOVA) for each time point; when significance was revealed, a 1-way ANOVA was done to determine the significance of individual between-group differences. Discrete data (scores for clinical respiratory disease, macroscopic and microscopic lung lesions, and PRRSV antigen) were analyzed by Mann–Whitney tests. Chi-square and Fisher’s exact test were applied to evaluate the proportions of viremic pigs. A value of P < 0.05 was considered significant.
Results
The pigs in the UnVac/Ch1 group had significantly higher (P < 0.05) mean rectal temperatures (38.7°C to 39.7°C) than the pigs in the Vac/Ch1, Vac/UnCh, and UnVac/UnCh groups 4 d after challenge (Figure 1A). The pigs in the UnVac/Ch2 group had significantly higher (P < 0.05) mean rectal temperatures (39.5°C to 40.4°C) than the pigs in the Vac/Ch2, Vac/UnCh, and UnVac/UnCh groups on days 2 to 6 d after challenge (Figure 1A). The mean respiratory scores were significantly higher (P < 0.05) in the pigs in the UnVac/Ch1 group than in the pigs in the Vac/Ch1 group on days 5, 6, and 7 after challenge (Figure 1B). The mean respiratory scores were significantly higher (P < 0.05) in the pigs in the UnVac/Ch2 group than in the pigs in the Vac/Ch2 group on days 3, 4, 5, 6, 7, 8, 9, 12, and 13 after challenge (Figure 1B). The negative-control pigs (UnVac/UnCh) maintained normal temperatures without respiratory signs throughout the experiment.
Figure 1.
Mean rectal temperature (A) and mean respiratory score (B) of pigs vaccinated (Vac) or not vaccinated (UnVac) with a new recombinant chimeric vaccine against porcine reproductive and respiratory syndrome virus (PRRSV) and then challenged with type 1 (Ch1) or type 2 (Ch2) PRRSV or not challenged (UnCh). Symbols as follows: Vac/Ch1 (
), UnVac/Ch1 (
), Vac/Ch2 (
), UnVac/Ch2 (
), Vac/UnCh (
), and UnVac/UnCh (⋄). Variation is expressed as the standard deviation. a,b Denote significant differences (P < 0.05) between Vac/Ch1 and UnVac/Ch1a and between Vac/Ch2 and UnVac/Ch2b on the same day after challenge.
Genomic copies of the type 1 PRRSV were detected in the serum of the pigs in the Vac/Ch1 and UnVac/Ch1 groups after inoculation with the virus, the number of copies being significantly lower (P < 0.05) in the former compared with the latter on days 10, 14, and 21 after challenge. The pigs in the Vac/Ch1 group also had a significantly lower (P < 0.05) number of genomic copies of type 1 PRRSV in the nasal swabs taken on day 21 after challenge compared with the pigs in the UnVac/Ch1 group (Figure 2A). Genomic copies of the type 2 PRRSV were detected in the serum of the pigs in the Vac/Ch2 and UnVac/Ch2 groups after inoculation with the virus, the number of copies being significantly lower (P < 0.05) in the former compared with the latter on days 10 and 21 after challenge. The pigs in the Vac/Ch2 group also had a significantly lower (P < 0.05) number of genomic copies of type 2 PRRSV in the nasal swabs taken on day 21 after challenge compared with the pigs in the UnVac/Ch2 group (Figure 2B).
Figure 2.
Mean numbers of genomic copies of type 1 PRRSV (A) and type 2 PRRSV (B) in the serum (
and
) and nasal samples (
and
) from pigs in the Vac/Ch1 (
and
), UnVac/Ch1 (
and
), Vac/Ch2 (
and
), and UnVac/Ch2 (
and
) groups. Variation and superscript letters (a,b) as for Figure 1.
The prevalence of viremia among the pigs is summarized in Table II. No type 1 PRRSV was detected in any serum or nasal sample from the type 2 PRRSV-challenged pigs and vice versa. No type 1 or type 2 PRRSV was detected in the serum and nasal samples from the negative-control pigs throughout the experiment.
Table II.
Prevalence of PRRSV viremia, mean lung lesion score, and mean score for number of PRRSV-positive cells per unit area of lung within lesions after challenge, as determined by in-situ hybridization for the detection and differentiation of types 1 and 2 PRRSV nucleic acids in the lung tissue
| Group and day after challenge | Number of pigs with PRRSV viremia | Score for lung lesions, mean ± standard deviation | Score for number of PRRSV-positive cells, mean ± standard deviation | |||
|---|---|---|---|---|---|---|
|
|
|
|
||||
| Type 1 | Type 2 | Macroscopic | Microscopic | Type 1 | Type 2 | |
| Vac/Ch1 | ||||||
| 7 | 18/18 | 0/18 | 52.5 ± 8.8 | 1.92 ± 2.4 | 21.7 ± 8.0 | 0 |
| 14 | 6/12 | 0/12 | 26.6 ± 5.1 | 0.94 ± 0.4a | 9.3 ± 5.6a | 0 |
| 21 | 1/6 | 0/6 | 5.0 ± 5.4 | 0.19 ± 0.19a | 7.4 ± 3.5a | 0 |
| UnVac/Ch1 | ||||||
| 7 | 18/18 | 0/18 | 54.1 ± 10.6 | 2.19 ± 2.4 | 23.1 ± 2.0 | 0 |
| 14 | 10/12 | 0/12 | 30.0 ± 8.9 | 1.63 ± 0.64a | 18.5 ± 2.7a | 0 |
| 21 | 5/6 | 0/6 | 11.6 ± 18.3 | 0.63 ± 0.37a | 12.3 ± 3.1a | 0 |
| Vac/Ch2 | ||||||
| 7 | 0/18 | 18/18b | 6.6 ± 11.6 | 2.58 ± 0.17b | 0 | 39.1 ± 13.2 |
| 14 | 0/12 | 12/12b | 53.3 ± 4.0b | 2.27 ± 0.68 | 0 | 29.8 ± 4.2b |
| 21 | 0/6 | 4/6b | 20.0 ± 8.9 | 1.47 ± 0.16b | 0 | 18.8 ± 8.0b |
| UnVac/Ch2 | ||||||
| 7 | 0/18 | 18/18b | 83.3 ± 2.5 | 3.08 ± 0.27b | 0 | 41.6 ± 8.5 |
| 14 | 0/12 | 12/12b | 68.3 ± 11.2b | 2.83 ± 0.52 | 0 | 41.1 ± 8.9b |
| 21 | 0/6 | 6/6b | 30.8 ± 8.0 | 2.47 ± 0.41b | 0 | 33.4 ± 13.0b |
| Vac/UnCh | ||||||
| 7 | 0/18 | 0/18 | 0 | 0 | 0 | 0 |
| 14 | 0/12 | 0/12 | 2.0 ± 2.5 | 0.1 ± 0.16 | 0 | 0 |
| 21 | 0/6 | 0/6 | 3.0 ± 2.8 | 0 | 0 | 0 |
| UnVac/UnCh | ||||||
| 7 | 0/18 | 0/18 | 0 | 0 | 0 | 0 |
| 14 | 0/12 | 0/12 | 0 | 0 | 0 | 0 |
| 21 | 0/6 | 0/6 | 2.0 ± 3.2 | 0.2 ± 0.17 | 0 | 0 |
Significant difference (P < 0.05) between the Vac/Ch1 and UnVac/Ch1 groups on the same day after challenge.
Significant difference (P < 0.05) between the Vac/Ch2 and UnVac/Ch2 groups on the same day after challenge.
The pigs in all 6 groups were seronegative for antibodies against PRRSV at the time of the 1st vaccination against PRRS, at 3 wk of age (−35 d after challenge). Antibodies specific for PRRSV were detected by ELISA in the pigs in the 3 vaccinated groups from day 3 after challenge onward. The titers were significantly higher (P < 0.05) in the Vac/Ch1 and Vac/Ch2 groups than in the UnVac/Ch1 and UnVac/Ch2 groups from days 3 to 21 after challenge (Figure 3). Anti-PRRSV antibody was not detected in the negative-control (UnVac/UnCh) pigs at any time in the experiment.
Figure 3.
Mean sample/positive (S/P) ratios of anti-PRRSV antibodies in the serum of the pigs. Symbols, variation, and superscript letters (a,b) as for Figure 1.
The titers of specific VN antibody against type 1 PRRSV were significantly higher (P < 0.05) in the Vac/Ch1 group than in the UnVac/Ch1 and Vac/UnCh groups at 14 and 21 d after challenge (Figure 4A). Similarly, the titers of specific VN antibody against type 2 PRRSV were significantly higher (P < 0.05) in the Vac/Ch2 group than in the UnVac/Ch2 and Vac/UnCh groups at 14 and 21 d after challenge (Figure 4B). Among the negative-control pigs VN antibody was not detected (titer < 2 log2) at any time in the experiment.
Figure 4.
Mean titers of virus-neutralizing (VN) antibody specific for type 1 (A) or type 2 (B) PRRSV in the serum of the pigs. Symbols, variation, and superscript letters (a,b) as for Figure 1.
After stimulation by vaccination the number of PRRSV-specific IFN-γ-SCs per 106 PBMCs reached an average of 22.2 ± 18.9 in the pigs in the Vac/Ch1 group and 29.6 ± 8.6 in the pigs in the Vac/Ch2 group at −7 d after challenge. Upon challenge with PRRSV the numbers of PRRSV-specific IFN-γ-SCs per 106 PBMCs increased gradually and reached an average of 61 ± 20.2 in the pigs in the Vac/Ch1 group and 96 ± 37.6 in the pigs in the Vac/Ch2 group at 21 d after challenge. The pigs in the Vac/Ch1 group produced significantly higher (P < 0.05) numbers of challenging type 1 PRRSV-specific IFN-γ-SCs from −14 to 21 d after challenge compared with the pigs in the UnVac/Ch1 group (Figure 5A). Similarly, the pigs in the Vac/Ch2 group produced significantly higher (P < 0.05) numbers of challenging type 2 PRRSV-specific IFN-γ-SCs from −14 to 21 d after challenge compared with the pigs in the UnVac/Ch2 group (Figure 5B). The mean number of challenging PRRSV-specific IFN-γ-SCs remained at basal levels (< 20 per 106 PBMCs) in the negative-control pigs throughout the experiment.
Figure 5.
Mean numbers of interferon gamma (IFN-γ)-secreting cells (IFN-γ-SCs) specific for type 1 (A) or type 2 (B) PRRSV among peripheral blood mononuclear cells (PBMCs) from the pigs. Symbols, variation, and superscript letters (a,b) as for Figure 1.
The pigs in the Vac/Ch1 group had significantly higher (P < 0.05) proportions of CD4+CD8+IFN-γ+ cells among the PBMCs than the pigs in the UnVac/Ch1, Vac/UnCh, and UnVac/UnCh groups at 21 d after challenge (Figure 6A). Similarly, the pigs in the Vac/Ch2 group had significantly higher (P < 0.05) proportions of CD4+CD8+IFN-γ+ cells than the pigs in the UnVac/Ch2, Vac/UnCh, and UnVac/UnCh groups at 21 d after challenge (Figure 6B).
Figure 6.
Proportions of CD4+CD8+IFN-γ+cells specific for type 1 (A) or type 2 (B) PRRSV among the PBMCs at 21 d after challenge in the pigs in the Vac/Ch1 (
), UnVac/Ch1 (
), Vac/Ch2 (
), UnVac/Ch2 (
), Vac/UnCh (
), and UnVac/UnCh (□) groups. Variation and superscript letters (a,b) as for Figure 1.
Lung lesions were observed in pigs from the challenged groups (Vac/Ch1, UnVac/Ch1, Vac/Ch2, and UnVac/Ch2). Macroscopic lung lesions were mottled or diffusely tan. The affected lungs often failed to collapse, the parenchyma being firmer and heavier than that of the lungs from the negative-control pigs. The mean scores for the macroscopic lung lesions were significantly lower (P < 0.05) for the pigs in the Vac/Ch2 group than for the pigs in the UnVac/Ch2 group 14 d after challenge (Table II). Microscopic lung lesions were characterized by interstitial pneumonia with thickened alveolar septa and increased numbers of interstitial macrophages and lymphocytes. The mean scores for the microscopic lung lesions were significantly lower (P < 0.05) for the pigs in the Vac/Ch1 group than for the pigs in the UnVac/Ch1 group 14 and 21 d after challenge. Similarly, the mean scores for the microscopic lung lesions were significantly lower (P < 0.05) for the pigs in the Vac/Ch2 group than for the pigs in the UnVac/Ch2 group 7 and 21 d after challenge (Table II). No microscopic or macroscopic lung lesions were detected in the pigs in the Vac/UnCh and UnVac/UnCh groups throughout the experiment.
Nucleic acids from PRRSV were detected exclusively within the cytoplasm of macrophages and pneumocytes. Positive cells typically exhibited a dark brown to black reaction product by in-situ hybridization (Figure 7). The mean number of type 1 PRRSV-positive cells per unit area of lung was significantly lower (P < 0.05) in the pigs in the Vac/Ch1 group (Figure 7A) than in the pigs in the UnVac/Ch1 group (Figure 7B) at 14 and 21 d after challenge (Table II). Similarly, the mean number of type 2 PRRSV-positive cells per unit area of lung was significantly lower (P < 0.05) in the pigs in the Vac/Ch2 group (Figure 7C) than in the pigs in the UnVac/Ch2 group (Figure 7D) at 14 and 21 d after challenge (Table II). Type 1 PRRSV-positive cells were not detected in the lungs of the pigs in the Vac/Ch2 and UnVac/Ch2 groups, and vice versa. No PRRSV-positive cells were observed in lung sections from the pigs in the Vac/UnCh and UnVac/UnCh groups.
Figure 7.
Results of in-situ hybridization for the detection of type 1 (A and B) and type 2 (C and D) PRRSV nucleic acids in the lungs of pigs in the Vac/Ch1 (A), UnVac/Ch1 (B), Vac/Ch2 (C), and UnVac/Ch2 (D) groups 14 d after challenge.
The type 1 PRRSV isolated by cell culture from pigs in the Vac/Ch1 group (6 pigs at 7 d after challenge, 5 pigs at 14 d, and 3 pigs at 21 d) and the UnVac/Ch1 group (6 pigs at 7 and 14 d and 4 pigs at 21 d) was confirmed by sequencing to be identical to the type 1 PRRSV in the challenge stock; no type 2 PRRSV was isolated from pigs in these 2 groups. The type 2 PRRSV isolated by cell culture from pigs in the Vac/Ch2 group (6 pigs at 7 d after challenge, 5 pigs at 14 d, and 3 pigs at 21 d) and the UnVac/Ch2 group (6 pigs at 7, 14, and 21 d) was confirmed by sequencing to be identical to the type 2 PRRSV in the challenge stock; no type 1 PRRSV was isolated from pigs in these 2 groups. No type 1 or type 2 PRRSV was isolated from the pigs in the unchallenged groups.
Discussion
The results of the present study demonstrate that the new commercial recombinant chimeric PRRS vaccine provides protection against challenge with heterologous types 1 and 2 PRRSV. Vaccination of pigs with this new vaccine results in reduction of viremia levels, lung lesions, and types 1 and 2 PRRSV antigens within lung lesions from those observed in pigs challenged but not vaccinated. Our results agree with previous findings that vaccination of pigs with recombinant vaccines resulted in partial protection, with reduction in type 1 and type 2 PRRSV viremia and lung lesions (36,37). In contrast, respiratory disease was exacerbated in pigs challenged with PRRSV after vaccination with a recombinant GP5 subunit PRRSV vaccine produced in Escherichia coli (38). We have no clear explanation about the conflicting results, but they may be due to differences in viral glycoprotein and adjuvant between the 2 vaccines. The latter vaccine contains only GP5, which is encoded by ORF5, contains the main neutralizing epitope (22,39,40) of both type 1 (41,42) and type 2 (43,44) PRRSV isolates, and is responsible for immunodominant T-cell epitopes that stimulate IFN-γ-SCs (45). Vaccination with proteins GP5 and M of PRRSV confers a certain degree of protection (36,39,40). In a previous study, mice vaccinated with a detoxified Pseudomonas exotoxin-based human papillomavirus subunit vaccine exhibited a significant increase in the numbers of virus-specific CD4+ and CD8+ T-cells compared with mice treated with the subunit vaccine alone (46). Similarly, in the present study the recombinant chimeric PRRS vaccine increased the number of CD4+CD8+IFN-γ+ cells, which constitute the majority of IFN-γ-SCs (33). Therefore, it is possible that the Pseudomonas exotoxin plays an important role in enhancing the cellular immune response. Likewise, pigs vaccinated with a killed virus have been shown to have CD4+CD8+ T-cells that were preferentially recalled upon exposure to the live virus, which suggested the presence of virus-specific memory cell pools (47).
Selection of an appropriate challenge virus is critical to evaluating the efficacy of a vaccine. The challenge strains of types 1 and 2 PRRSV are virulent, causing interstitial pneumonia. In particular, the type 1 challenge virus used in this study is more pathogenic than any other Korean type 1 PRRSV isolates examined (25,26,48). In addition, several peer-reviewed publications describing the efficacy of PRRS vaccine reported using the same challenge strains (16,30,49,50). The recombinant chimeric PRRS vaccine augments anamnestic virus-specific VN and IFN-γ responses after a wild-type virus challenge, which contributes to viral clearance, as happens with inactivated PRRS vaccines (32,47,51).Vaccination of pigs with the recombinant chimeric PRRS vaccine is not able to attenuate peak viremia at 7 d after challenge but thereafter reduces the levels of types 1 and 2 PRRSV viremia consistently up to 21 d after challenge. In spite of the variable VN antibody titers and numbers of IFN-γ-SCs in vaccinated animals, viral clearance from the blood coincides with the induction of VN antibodies and IFN-γ-SCs.
The cocirculation of PRRS viruses of both genotypes, 1 and 2, is an important clinical issue in some parts of the world, most notably in East Asia, including Korea. The protection afforded by the recombinant chimeric PRRS vaccine against types 1 and 2 PRRSV is clinically significant because a commercial divalent vaccine against types 1 and 2 PRRSV is not yet available. Moreover, concurrent vaccination of pigs against types 1 and 2 PRRSV protects growing pigs against respiratory diseases caused by type 1 PRRSV but not against those caused by type 2 PRRSV (50). From the aspect of safety, inactivated PRRS vaccines are preferred over live-attenuated vaccines because a live-attenuated vaccine virus has been shown to revert to virulence under field conditions (52). The results of the present study demonstrate that, with the use of an optimized selection of PRRSV subunits in combination with a Pseudomonas exotoxin carrier, a recombinant chimeric PRRS vaccine can induce VN antibodies and IFN-γ-SCs and offer protection upon heterologous types 1 and 2 PRRSV challenge. Thus, this novel recombinant chimeric PRRS vaccine is a promising alternative to control both type 1 and type 2 PRRSV infection effectively and safely.
Acknowledgments
This research was supported by contract research funds (grant 550-20140103) from the Research Institute for Veterinary Science, College of Veterinary Medicine, and by the BK 21 Plus Program (grant 5360-20150100) for Creative Veterinary Science Research from Seoul National University.
References
- 1.Wensvoort G, Terpstra C, Pol JM, et al. Mystery swine disease in The Netherlands: The isolation of Lelystad virus. Vet Q. 1991;13:121–130. doi: 10.1080/01652176.1991.9694296. [DOI] [PubMed] [Google Scholar]
- 2.Kweon C-H, Kwon B-J, Lee H-J, et al. Isolation of porcine reproductive and respiratory syndrome virus (PRRSV) in Korea. Korean J Vet Res. 1994;34:77–83. [Google Scholar]
- 3.Zhou L, Yang H. Porcine reproductive and respiratory syndrome in China. Virus Res. 2010;154:31–37. doi: 10.1016/j.virusres.2010.07.016. [DOI] [PubMed] [Google Scholar]
- 4.Shimizu M, Yamada S, Murakami Y, et al. Isolation of porcine reproductive and respiratory syndrome (PRRS) from Heko-Heko disease of pigs. J Vet Med Sci. 1994;56:389–391. doi: 10.1292/jvms.56.389. [DOI] [PubMed] [Google Scholar]
- 5.Zimmerman JJ, Benfield DA, Dee SA, et al. Porcine reproductive and respiratory syndrome virus (porcine arterivirus) In: Zimmerman JJ, Karriker LA, Ramirez A, Schwartz KJ, Stevenson GW, editors. Diseases of Swine. 10th ed. Ames, Iowa: Wiley–Blackwell; 2012. pp. 461–486. [Google Scholar]
- 6.Snijder EJ, Kikkert M, Fang Y. Arterivirus molecular biology and pathogenesis. J Gen Virol. 2013;94:2141–2163. doi: 10.1099/vir.0.056341-0. [DOI] [PubMed] [Google Scholar]
- 7.Dea S, Gagnon CA, Mardassi H, Milane G. Antigenic variability among North American and European strains of porcine reproductive and respiratory syndrome virus as defined by monoclonal antibodies to the matrix protein. J Clin Microbiol. 1996;34:1488–1493. doi: 10.1128/jcm.34.6.1488-1493.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Murtaugh MP, Elam MR, Kakach LT. Comparison of the structural protein coding sequences of the VR-2332 and Lelystad virus strains of the PRRS virus. Arch Virol. 1995;140:1451–1460. doi: 10.1007/BF01322671. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Nelsen CJ, Murtaugh MP, Faaberg KS. Porcine reproductive and respiratory syndrome virus comparison: Divergent evolution on two continents. J Virol. 1999;73:270–280. doi: 10.1128/jvi.73.1.270-280.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Cheon D-S, Chae C. Antigenic variation and genotype of isolates of porcine reproductive and respiratory syndrome virus in Korea. Vet Rec. 2000;147:215–218. doi: 10.1136/vr.147.8.215. [DOI] [PubMed] [Google Scholar]
- 11.Chen N, Cao Z, Yu X, et al. Emergence of novel European genotype porcine reproductive and respiratory syndrome virus in mainland China. J Gen Virol. 2011;92:880–892. doi: 10.1099/vir.0.027995-0. [DOI] [PubMed] [Google Scholar]
- 12.Nam E, Park C-K, Kim S-H, Joo Y-S, Yeo S-G, Lee C. Complete genomic characterization of a European type 1 porcine reproductive and respiratory syndrome virus isolate in Korea. Arch Virol. 2009;154:629–638. doi: 10.1007/s00705-009-0347-3. [DOI] [PubMed] [Google Scholar]
- 13.Thanawongnuwech R, Amonsin A, Tatsanakit A, Damrongwatanapokin S. Genetic and geographical variation of porcine reproductive and respiratory syndrome virus (PRRSV) in Thailand. Vet Microbiol. 2004;101:9–21. doi: 10.1016/j.vetmic.2004.03.005. [DOI] [PubMed] [Google Scholar]
- 14.Labarque GG, Nauwynck HJ, van Woensel PAM, Visser N, Pensaert MB. Efficacy of an American and a European serotype PRRSV vaccine after challenge with American and European wild-type strains of the virus. Vet Res. 2000;31:97. [Google Scholar]
- 15.van Woensel PAM, Liefkens K, Demaret S. Effect on viraemia of an American and a European serotype PRRSV vaccine after challenge with European wild-type strains of the virus. Vet Rec. 1998;142:510–512. doi: 10.1136/vr.142.19.510. [DOI] [PubMed] [Google Scholar]
- 16.Kim T, Park C, Choi K, et al. Comparison of two commercial type 1 porcine reproductive and respiratory syndrome virus (PRRSV) modified live vaccines against heterologous type 1 and type 2 PRRSV challenge in growing pigs. Clin Vaccine Immunol. 2015;22:631–640. doi: 10.1128/CVI.00001-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Renukaradhya GJ, Meng X-J, Calvert JG, Roof M, Lager KM. Inactivated and subunit vaccines against porcine reproductive and respiratory syndrome: Current status and future direction. Vaccine. 2015;33:3065–3072. doi: 10.1016/j.vaccine.2015.04.102. [DOI] [PubMed] [Google Scholar]
- 18.Yang H-P, Wang T-C, Wang S-J, et al. Recombinant chimeric vaccine composed of PRRSV antigens and truncated Pseudomonas exotoxin A (PE-K13) Res Vet Sci. 2013;95:742–751. doi: 10.1016/j.rvsc.2013.05.003. [DOI] [PubMed] [Google Scholar]
- 19.Fang Y, Snijder EJ. The PRRSV replicase: Exploring the multi-functionality of an intriguing set of nonstructural proteins. Virus Res. 2010;154:61–76. doi: 10.1016/j.virusres.2010.07.030. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Barfoed AM, Blixenkrone-Møller M, Jensen MH, Bøtner A, Kamstrup S. DNA vaccination of pigs with open reading frame 1–7 of PRRS virus. Vaccine. 2004;22:3628–3641. doi: 10.1016/j.vaccine.2004.03.028. [DOI] [PubMed] [Google Scholar]
- 21.Plana-Duran J, Mourino M, Viaplana E, et al. New strategies in the development of PRRS vaccines. Subunit vaccines and selflimiting vectors, based on defective coronaviruses. Vet Res. 2000;31:41–42. [Google Scholar]
- 22.Gonin P, Pirzadeh B, Gagnon CA, Dea S. Seroneutralization of porcine reproductive and respiratory syndrome virus correlates with antibody response to the GP5 major envelope glycoprotein. J Vet Diagn Invest. 1999;11:20–26. doi: 10.1177/104063879901100103. [DOI] [PubMed] [Google Scholar]
- 23.Yang L, Frey ML, Yoon KJ, Zimmerman JJ, Platt KB. Categorization of North American porcine reproductive and respiratory syndrome viruses: Epitope profiles of the N, M, GP5 and GP3 proteins and susceptibility to neutralization. Arch Virol. 2000;145:1599–1619. doi: 10.1007/s007050070079. [DOI] [PubMed] [Google Scholar]
- 24.Jiang Y, Fang L, Xiao S, et al. Immunogenicity and protective efficacy of recombinant pseudorabies virus expressing the two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus. Vaccine. 2007;25:547–560. doi: 10.1016/j.vaccine.2006.07.032. [DOI] [PubMed] [Google Scholar]
- 25.Han K, Seo HW, Oh Y, Kang I, Park C, Chae C. Pathogenesis of Korean type 1 (European genotype) porcine reproductive and respiratory syndrome virus in experimentally infected pigs. J Comp Pathol. 2012;147:275–284. doi: 10.1016/j.jcpa.2011.12.010. [DOI] [PubMed] [Google Scholar]
- 26.Han K, Seo HW, Oh Y, Kang I, Park C, Chae C. Comparison of the virulence of European and North American genotypes of porcine reproductive and respiratory syndrome virus in experimentally infected pigs. Vet J. 2013;195:313–318. doi: 10.1016/j.tvjl.2012.06.035. [DOI] [PubMed] [Google Scholar]
- 27.Wasilk A, Callahan JD, Christopher-Hennings J, et al. Detection of U.S., Lelystad, and European-like porcine reproductive and respiratory syndrome viruses and relative quantitation in boar semen and serum samples by real-time PCR. J Clin Microbiol. 2004;42:4453–4461. doi: 10.1128/JCM.42.10.4453-4461.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Beaver BV, Reed W, Leary S, et al. 2000 Report of the AVMA Panel on Euthanasia. J Am Vet Med Assoc. 2001;218:669–696. doi: 10.2460/javma.2001.218.669. [DOI] [PubMed] [Google Scholar]
- 29.Halbur PG, Paul PS, Frey ML, et al. Comparison of the pathogenicity of two US porcine reproductive and respiratory syndrome virus isolates with that of the Lelystad virus. Vet Pathol. 1995;32:648–660. doi: 10.1177/030098589503200606. [DOI] [PubMed] [Google Scholar]
- 30.Park C, Seo HW, Han K, Kang I, Chae C. Evaluation of the efficacy of a new modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine (Fostera PRRS) against heterologous PRRSV challenge. Vet Microbiol. 2014;172:432–442. doi: 10.1016/j.vetmic.2014.05.030. [DOI] [PubMed] [Google Scholar]
- 31.Yoon IJ, Joo HS, Goyal SM, Molitor TW. A modified serum neutralization test for the detection of antibody to porcine reproductive and respiratory syndrome virus in swine sera. J Vet Diagn Invest. 1994;6:289–292. doi: 10.1177/104063879400600326. [DOI] [PubMed] [Google Scholar]
- 32.Zuckermann FA, Garcia EA, Luque ID, et al. Assessment of the efficacy of commercial porcine reproductive and respiratory syndrome virus (PRRSV) vaccines based on measurement of serologic response, frequency of gamma-IFN-producing cells and virological parameters of protection upon challenge. Vet Microbiol. 2007;123:69–85. doi: 10.1016/j.vetmic.2007.02.009. [DOI] [PubMed] [Google Scholar]
- 33.Meier WA, Galeota J, Osorio FA, Husmann RJ, Schnitzlein WM, Zuckermann FA. Gradual development of the interferon-gamma response of swine to porcine reproductive and respiratory syndrome virus infection or vaccination. Virology. 2003;309:18–31. doi: 10.1016/s0042-6822(03)00009-6. [DOI] [PubMed] [Google Scholar]
- 34.Diaz I, Mateu E. Use of ELISPOT and ELISA to evaluate IFN-gamma, IL-10 and IL-4 responses in conventional pigs. Vet Immunol Immunopathol. 2005;106:107–112. doi: 10.1016/j.vetimm.2005.01.005. [DOI] [PubMed] [Google Scholar]
- 35.Ferrari L, Martelli P, Saleri R, et al. Lymphocyte activation as cytokine gene expression and secretion is related to the porcine reproductive and respiratory syndrome virus (PRRSV) isolate after in vitro homologous and heterologous recall of peripheral blood mononuclear cells (PBMC) from pigs vaccinated and exposed to natural infection. Vet Immunol Immunopathol. 2013;151:193–206. doi: 10.1016/j.vetimm.2012.11.006. [DOI] [PubMed] [Google Scholar]
- 36.Bastos RG, Dellagostin OA, Barletta RG, et al. Immune response of pigs inoculated with Mycobacterium bovis BCG expressing GP5 and M protein of porcine reproductive and respiratory syndrome virus. Vaccine. 2004;22:467–474. doi: 10.1016/s0264-410x(03)00572-3. [DOI] [PubMed] [Google Scholar]
- 37.Qiu H-J, Tian Z-J, Tong G-Z, et al. Protective immunity induced by a recombinant pseudorabies virus expressing the GP5 of porcine reproductive and respiratory syndrome virus in piglets. Vet Immunol Immunopathol. 2005;106:309–319. doi: 10.1016/j.vetimm.2005.03.008. [DOI] [PubMed] [Google Scholar]
- 38.Prieto C, Martinez-Lobo FJ, Diez-Fuertes F, Aguilar-Calvo P, Simarro I, Castro JM. Immunisation of pigs with a major envelope protein sub-unit vaccine against porcine reproductive and respiratory syndrome virus (PRRSV) results in enhanced clinical disease following experimental challenge. Vet J. 2011;189:323–329. doi: 10.1016/j.tvjl.2010.07.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39.Pirzadeh B, Dea S. Monoclonal antibodies to the ORF5 product of porcine reproductive and respiratory syndrome virus define linear neutralizing determinants. J Gen Virol. 1997;78:1867–1873. doi: 10.1099/0022-1317-78-8-1867. [DOI] [PubMed] [Google Scholar]
- 40.Pirzadeh B, Dea S. Immune response in pigs vaccinated with plasmid DNA encoding ORF5 of porcine reproductive and respiratory syndrome virus. J Gen Virol. 1998;79:989–999. doi: 10.1099/0022-1317-79-5-989. [DOI] [PubMed] [Google Scholar]
- 41.Plagemann PG. GP5 ectodomain epitope of porcine reproductive and respiratory syndrome virus, strain Lelystad virus. Virus Res. 2004;102:225–230. doi: 10.1016/j.virusres.2004.01.031. [DOI] [PubMed] [Google Scholar]
- 42.Wissink EH, van Wijk HA, Kroese MV, et al. The major envelope protein, GP5, of a European porcine reproductive and respiratory syndrome virus contains a neutralization epitope in its N-terminal ectodomain. J Gen Virol. 2003;84:1535–1543. doi: 10.1099/vir.0.18957-0. [DOI] [PubMed] [Google Scholar]
- 43.Ostrowski M, Galeota JA, Jar AM, Platt KB, Osorio FA, Lopez OJ. Identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus GP5 ectodomain. J Virol. 2002;76:4241–4250. doi: 10.1128/JVI.76.9.4241-4250.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Plagemann PG. The primary GP5 neutralization epitope of North American isolates of porcine reproductive and respiratory syndrome virus. Vet Immunol Immunopathol. 2004;102:263–275. doi: 10.1016/j.vetimm.2004.09.011. [DOI] [PubMed] [Google Scholar]
- 45.Vashisht K, Goldberg TL, Husmann RJ, Schnitzlein W, Zuckermann FA. Identification of immunodominant T-cell epitopes present in glycoprotein 5 of the North American genotype of porcine reproductive and respiratory syndrome virus. Vaccine. 2008;26:4747–4753. doi: 10.1016/j.vaccine.2008.06.047. [DOI] [PubMed] [Google Scholar]
- 46.Liao CW, Hseu TH, Hwang J. Fusion protein vaccine by domains of bacterial exotoxin linked with a tumor antigen generates potent immunologic responses and antitumor effects. Cancer Res. 2005;65:9089–9098. doi: 10.1158/0008-5472.CAN-05-0958. [DOI] [PubMed] [Google Scholar]
- 47.Piras F, Bollard S, Laval F, et al. Porcine reproductive and respiratory syndrome (PRRS) virus-specific interferon-γ+ T-cell responses after PRRS virus infection or vaccination with an inactivated PRRS vaccine. Viral Immunol. 2005;18:381–389. doi: 10.1089/vim.2005.18.381. [DOI] [PubMed] [Google Scholar]
- 48.Han K, Seo HW, Park C, et al. Comparative pathogenicity of three Korean and one Lelystad type 1 porcine reproductive and respiratory syndrome virus (pan-European subtype 1) isolates in experimentally infected pigs. J Comp Pathol. 2013;149:331–340. doi: 10.1016/j.jcpa.2013.03.001. [DOI] [PubMed] [Google Scholar]
- 49.Park C, Choi K, Jeong J, Chae C. Cross-protection of a new type 2 porcine reproductive and respiratory syndrome virus (PRRSV) modified live vaccine (Fostera PRRS) against heterologous type 1 PRRSV challenge in growing pigs. Vet Microbiol. 2015;177:87–94. doi: 10.1016/j.vetmic.2015.02.020. [DOI] [PubMed] [Google Scholar]
- 50.Park C, Choi K, Jeong J, Kang I, Park S-J, Chae C. Concurrent vaccination of pigs with type 1 and type 2 porcine reproductive and respiratory syndrome virus (PRRSV) protects against type 1 PRRSV but not against type 2 PRRSV on dually challenged pigs. Res Vet Sci. 2015;103:193–200. doi: 10.1016/j.rvsc.2015.10.011. [DOI] [PubMed] [Google Scholar]
- 51.Vanhee M, Delputte PL, Delrue I, Geldhof MF, Nauwynck HJ. Development of an experimental inactivated PRRSV vaccine that induces virus-neutralizing antibodies. Vet Res. 2009;40:63. doi: 10.1051/vetres/2009046. [DOI] [PubMed] [Google Scholar]
- 52.Nielsen HS, Oleksiewics MB, Forsberg R, Stadejek T, Botner A, Storgaard T. Reversion of a live porcine reproductive and respiratory syndrome virus vaccine investigated by parallel mutations. J Gen Virol. 2001;82:1263–1271. doi: 10.1099/0022-1317-82-6-1263. [DOI] [PubMed] [Google Scholar]