Efficacy and future prospects of commercially available and experimental vaccines against porcine circovirus type 2 (PCV2)

Virus Research 164 (2012) 33–42

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Virus Research journal homepage: www.elsevier.com/locate/virusres

Review

Efficacy and future prospects of commercially available and experimental vaccines against porcine circovirus type 2 (PCV2) Nathan M. Beach, Xiang-Jin Meng ∗ Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA

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Article history: Available online 7 October 2011 Keywords: Porcine circovirus type 2 (PCV2) PCV2 vaccine Porcine multisystemic wasting syndrome (PMWS) Porcine circovirus associated disease (PCVAD) Porcine circovirus disease (PCVD)

a b s t r a c t Porcine circovirus type 2 (PCV2) is the causative agent of an economically significant collection of disease syndromes in pigs, now known as porcine circovirus associated diseases (PCVADs) in the United States or porcine circovirus diseases (PCVDs) in Europe. Inactivated and subunit vaccines based on PCV2a genotype are commercially available and have been shown to be effective at decreasing mortality and increasing growth parameters in commercial swine herds. Since 2003, there has been a drastic global shift in the predominant prevalence of PCV2b genotype in swine populations, concurrently in most but not all cases with increased severity of clinical disease. Although the current commercial vaccines based on PCV2a do confer cross-protection against PCV2b, novel experimental vaccines based on PCV2b genotype such as modified live-attenuated vaccines are being developed and may provide a superior protection and reduce vaccine costs. In this review, we discuss the current understanding of the impact of PCV2 infection on the host immune response, review the efficacy of the currently available commercial PCV2 vaccines in experimental and field conditions, and provide insight into novel experimental approaches that are useful in the development of next generation vaccines against PCV2. © 2011 Elsevier B.V. All rights reserved.

Contents 1. 2.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Host responses to PCV2 infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Characteristics of clinical PCVAD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. PCV2 and host immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Route and spread of PCV2 infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Current commercial PCV2 vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Currently available commercial PCV2 vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Vaccine efficacy in experimental infection models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Vaccination strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. Effect of PCV2 vaccination on vertical transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2. Vaccination of sows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.3. Vaccination of growing pigs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Experimental PCV2 vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. RNA-based antiviral therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Modified live-attenuated PCV2 vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. DNA-based vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Vectored vaccines against PCV2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Marker PCV2 vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Abbreviations: CD/CD, caesarean-derived colostrum-deprived; dpi, days post-infection; MLV, modified live-attenuated vaccine. ∗ Corresponding author at: Department of Biomedical Sciences and Pathobiology, Virginia Polytechnic Institute and State University, 1981 Kraft Drive, Blacksburg, VA 24061-0913, USA. Tel.: +1 540 231 6912; fax: +1 540 231 3414. E-mail address: [email protected] (X.-J. Meng). 0168-1702/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2011.09.041

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N.M. Beach, X.-J. Meng / Virus Research 164 (2012) 33–42

1. Introduction Porcine circovirus (PCV) is a small, non-enveloped virus with a single-strand circular DNA genome in the family Circoviridae (Todd et al., 2005). Type 1 PCV (PCV1) was discovered in 1974 as a contaminant of a porcine kidney cell line PK-15 (Tischer et al., 1982, 1974). Although PCV1 is present in swine populations worldwide (Allan et al., 1994; Tischer et al., 1986), it does not cause overt clinical disease in pigs (Allan et al., 1995; Tischer et al., 1986). A variant strain of PCV, designated PCV type 2 (PCV2), was discovered in Canada in the mid-1990s as the causative agent of postweaning multisystemic wasting syndrome (PMWS) (Allan et al., 1998; Ellis et al., 1998; Meehan et al., 1998). PCV1 and PCV2 have similar genomic organization with two major ambisense ORFs flanking the origin of replication (Finsterbusch and Mankertz, 2009). ORF1 encodes two viral replication-associated proteins, Rep and Rep , by differential splicing (Cheung, 2003, 2004b; Mankertz and Hillenbrand, 2001; Mankertz et al., 2003). The Rep and Rep proteins bind to specific sequences within the origin of replication located in the 5 intergenic region, and both are essential for viral replication (Cheung, 2004a, 2006, 2007; Mankertz and Hillenbrand, 2001; Mankertz et al., 1997; Steinfeldt et al., 2001, 2006). ORF2 encodes the viral capsid protein, which has the ability to bind to the host cell receptor (Khayat et al., 2011; Lekcharoensuk et al., 2004; Mahe et al., 2000; Misinzo et al., 2006; Nawagitgul et al., 2000; Shang et al., 2009). The capsid of PCV2 is the primary immunogenic protein (Blanchard et al., 2003; Mahe et al., 2000) and thus has been the target for development of vaccines and serodiagnostic assays for tracking PCV2-specific immune responses (Huang et al., 2011a; Nawagitgul et al., 2002; Patterson et al., 2008, 2011a; Sun et al., 2010). PCV2 is considered to be one of the most economically important viral pathogens essentially in all major swine producing countries (Gillespie et al., 2009). In order to describe the collection of disease syndromes associated with PCV2 infection, the American Association of Swine Veterinarians approved the usage of the umbrella term “porcine circovirus-associated disease” (PCVAD), which includes wasting, increased mortality, respiratory signs, enteritis, reproductive failure, and porcine dermatitis and nephropathy syndrome (PDNS) (Opriessnig et al., 2007), although experimental evidence directly linking PCV2 infection to PDNS is still absent. In Europe, the term porcine circovirus disease (PCVD) is more frequently used in the literature (Segales et al., 2005a). Reproduction of severe clinical PCVAD in conventional pigs experimentally infected with PCV2 alone is uncommon (Tomas et al., 2008), and coinfection with other swine pathogens such as porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus (PPV), or Mycoplasma hyopnemoniae (MHYO) is usually required to induce the full-spectrum of clinical PCVAD (Allan et al., 2000; Ellis et al., 1999; Harms et al., 2001; Kennedy et al., 2000; Opriessnig et al., 2007, 2004; Rovira et al., 2002). However, infection of caesarean-derived, colostrum-deprived (CD/CD) or gnotobiotic pigs with PCV2 alone has resulted in severe clinical PCVAD and mortality (Allan et al., 2003, 2004; Beach et al., 2010b; Bolin et al., 2001; Gauger et al., 2011; Lager et al., 2007). Currently, at least three distinct genotypes of PCV2 have been recognized: PCV2a, PCV2b, and PCV2c (Cortey et al., 2011a; Dupont et al., 2008; Segales et al., 2008). PCV2a and PCV2b have both been associated with clinical PCVAD of varying degrees of severity (Allan et al., 2007; An et al., 2007; Ciacci-Zanella et al., 2009; Gauger et al., 2011; Lager et al., 2007; Madson et al., 2008; Opriessnig et al., 2006, 2008c), while PCV2c was reported only in a few non-diseased herds in Denmark (Dupont et al., 2008). Prior to 2003, PCV2a and PCV2b were both present in Europe and China, while only PCV2a was present in the United States and Canada (Allan et al., 2007; Chae

and Choi, 2010; Dupont et al., 2008). Since 2003, there has been a drastic global shift in the predominant prevalence of PCV2b in commercial swine populations, concurrently with increased severity of clinical PCVAD (Carman et al., 2008; Chae and Choi, 2010; Cheung et al., 2007; Ciacci-Zanella et al., 2009; Cortey et al., 2011b; Dupont et al., 2008; Gagnon et al., 2007; Lipej et al., 2005; Wang et al., 2009; Wiederkehr et al., 2009). Sequence variations between PCV2a and PCV2b are mostly found in the capsid gene, including a distinct “signature amino acid motif” (Cheung et al., 2007; Olvera et al., 2007). The current PCV2a-based vaccines have been shown to confer cross-protective immunity against PCV2b (Fort et al., 2008, 2009b; Opriessnig et al., 2009; Segales et al., 2009). However the antigenic profiles of PCV2a and PCV2b are not identical (Cheung et al., 2007; Dupont et al., 2008; Lefebvre et al., 2008; Shang et al., 2009), and it remains to be seen whether new vaccines based on the PCV2b genotype would provide superior protective immunity against PCV2b field strains compared to the currently available PCV2a-based vaccines. In this article, we will discuss the host responses to PCV2 infection, including characteristics of clinical disease and immunity. The efficacy of current commercial vaccines and the outlook of the novel experimental vaccines are also discussed. 2. Host responses to PCV2 infection 2.1. Characteristics of clinical PCVAD Clinical PCVAD is diagnosed by satisfaction of three criteria: (1) observation of at least one clinical manifestation of disease such as weight loss, increased mortality, or respiratory signs; (2) presence of hallmark PCV2-associated microscopic lesions in lymphoid tissues; and (3) detection of PCV2 antigen or nucleic acids at sites of lymphoid lesions using immunohistochemistry (IHC) or in situ hybridization (ISH) (Opriessnig et al., 2007). PCV2 infection is widespread in commercial swine herds worldwide (Puvanendiran et al., 2011), although clinical PCVAD is only observed in a small fraction of infected pigs. In several studies, clinically affected pigs were found to have higher serum viral loads (as determined by quantitative PCR) than similar non-diseased pigs (Brunborg et al., 2010; Dupont et al., 2009; Gauger et al., 2011; Grau-Roma et al., 2009; Harding et al., 2008; Olvera et al., 2004; Segales et al., 2005b). This increased serum viral load generally coincides with a lower humoral immune response, which often correlates with a lack of an effective neutralizing antibody response (Fort et al., 2007; GrauRoma et al., 2009; Meerts et al., 2006; Wallgren et al., 2009). There is also a correlation between PCV2 antigen scores and histological lesions in lymphoid tissues with disease severity (Harding et al., 2008; Krakowka et al., 2005; Segales et al., 2005b; Silva et al., 2011). In general, pigs that are able to mount a successful immune response limit PCV2 infection and avoid clinical disease, whereas those who have an impaired immune response develop severe clinical disease (Fort et al., 2007; Gauger et al., 2011; Meerts et al., 2006; Wallgren et al., 2009). 2.2. PCV2 and host immunity The activation of the host immune responses is one of the primary factors modulating disease progression in PCV2-infected pigs. Pigs that survive PCV2 infection generally have a strong humoral immune response. In experimentally infected pigs that do not develop clinical disease, PCV2 capsid-specific neutralizing antibodies are elicited within 10–28 days post-infection (dpi) (Fort et al., 2007; Meerts et al., 2006, 2005; Pogranichnyy et al., 2000). The appearance of serum neutralizing antibodies coincides with a decrease in serum viral load (Fort et al., 2007). However, in

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clinically diseased pigs, the level of neutralizing antibodies is significantly decreased, leading to uncontrolled infection (Fort et al., 2007; Meerts et al., 2006, 2005). Therefore, it is believed that induction of neutralizing antibodies is an essential feature of an effective vaccine against PCV2 and PCVAD. Activation of cell-mediated immunity, including interferongamma production, has been shown to be necessary for control of PCV2 infection (Fort et al., 2009a; Meerts et al., 2005). PCV2 infection has been shown to induce IFN-gamma secreting cells, possibly including CD4+ and CD8+ T-cells (Fort et al., 2010; Steiner et al., 2009). Pigs that fail to produce sufficient levels of IFN-gamma have lower levels of neutralizing antibodies and higher levels of viremia that are associated with clinical diseases (Meerts et al., 2005). PCV2 has been shown to induce IL-10 secretion in monocytic cells, which may lead to suppression of T-cell recall responses (Kekarainen et al., 2008). An increased level of IL-10 mRNA in the thymus of clinically diseased pigs has also been associated with T-cell immunosuppression (Darwich et al., 2003). Elevated levels of IL-10 have been observed in clinically diseased pigs (Stevenson et al., 2006), and in a single pig with long lasting viremia (Fort et al., 2009a). It is unclear why some pigs are able to efficiently clear PCV2 infection while others fail to mount an effective immune response and develop clinical diseases. 2.3. Route and spread of PCV2 infection It is known that PCV2 spreads from pig-to-pig in experimental studies (Chiou et al., 2011; Dupont et al., 2009). In cases of systemic infections, it can be expected that PCV2 can be shed in many body secretions. In naturally and experimentally infected pigs, PCV2 virus is shed in oral and nasal secretions, feces and urine (Patterson et al., 2011b,c; Segales et al., 2005b). The duration of virus shedding in pigs naturally infected with PCV2 at or before 13 days of age was shown to be at least 209 days post-farrowing (Patterson et al., 2011b) or 69 days after experimental PCV2 infection (Patterson et al., 2011c). Infectious PCV2 has also been detected in the colostrum of infected sows, which is capable of infecting naïve piglets (Gerber et al., 2011; Ha et al., 2009, 2010; Madson et al., 2009b; Shen et al., 2010a). Shedding of PCV2 in the semen of adult boars is a concern for breeding herds. Boars experimentally infected with PCV2 have been shown to shed virus for at least 90 dpi (Madson et al., 2008). It has also been shown that insemination of naïve sows with semen containing large amounts of infectious PCV2 can lead to reproductive failures (Madson et al., 2009c). It appears that the spread of PCV2 through insemination is dose-dependent, as semen containing lower levels of PCV2 failed to infect sows (Madson et al., 2009d). It is known that infected pregnant sows may spread PCV2 to the fetus via intrauterine transmission. In one study of clinically normal breeding herds, 40% of newborn piglets were viremic for PCV2 (Shen et al., 2010a). However, in the same study, another 46% of newborn piglets were seronegative with no viremia, suggesting that no intrauterine infection had occurred (Shen et al., 2010a). A comprehensive review of vertical transmission of PCV2 is available (Madson and Opriessnig, 2011). Given the various routes of PCV2 shedding, the environmental load of PCV2 can be an important indicator of herd health. A recent

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report has confirmed that PCV2 coinfection with PRRSV results in increased duration and amount of PCV2 shedding, which would also contribute to a higher environmental viral load (Sinha et al., 2011). Airborne spread between farms in high-density swine production areas is a potential biosecurity concern. High levels of PCV2 DNA have been detected in dust particles in the air of production facilities, up to 107 genome copies/m3 air (Verreault et al., 2010). There were some variations of the viral DNA levels in the air among several farms tested, indicating a variable environmental PCV2 load. Another study found PCV2 DNA in houseflies that were captured in 5 swine production facilities in 2007 (Blunt et al., 2011). The PCV2 DNA found in the flies was matched to those in fecal samples collected from pigs in each farm. It is likely that the flies simply serve as a mechanical vector facilitating PCV2 spread, and could be a good measure of environmental PCV2 load. Institution of a PCV2 vaccination program in the pigs resulted in an apparent decrease in environmental PCV2 load, as no fly was tested positive at the farms the following year (Blunt et al., 2011). 3. Current commercial PCV2 vaccines 3.1. Currently available commercial PCV2 vaccines There are several commercial vaccine products available for the prevention of PCVAD in swine herds (Table 1), and all of them are based on the PCV2a genotype. This review is only meant to serve as a general summary, as availability and license for use in commercial swine herds varies by countries. The Circovac® vaccine (Merial) is composed of an inactivated PCV2a virus, and is intended for use either in healthy piglets greater than 3 weeks of age or in healthy female pigs of breeding age. Vaccination of piglets with Circovac® requires a single intramuscular injection, while vaccination of gilts and sows requires two injections 3–4 weeks apart prior to breeding, followed by booster 2–4 weeks prior to farrowing. Subunit vaccines based on the PCV2a capsid protein expressed in baculovirus system are also commercially available for vaccination of healthy piglets: Ingelvac CircoFLEX® (Boehringer Ingelheim), Circumvent® (Intervet/Merck), and Porcilis® PCV (Schering-Plough/Merck). FosteraTM PCV (Pfizer Animal Health Inc.) has recently been introduced to the market, which is a reformulation of the product formerly known as Suvaxyn® PCV2 One DoseTM (Fort Dodge Animal Health Inc.). FosteraTM PCV vaccine is an inactivated attenuated chimeric virus in which the immunogenic capsid gene of PCV2a was cloned into the genomic backbone of the non-pathogenic PCV1. Vaccination of piglets greater than 3 weeks of age with FosteraTM PCV requires a single dose of intramuscular injection, and the vaccine prevents PCV2 viremia and infection. 3.2. Vaccine efficacy in experimental infection models The efficacy of commercially available PCV2 vaccines has been extensively tested in controlled challenge experiments in pigs. Due to the limited clinical diseases produced in conventional piglets infected with PCV2 alone, most vaccine efficacy studies have used challenge models that are co-infected with two or three swine pathogens. The use of coinfection challenge models has the

Table 1 Commercially available PCV2 vaccines. Vaccine

Manufacturer

Antigen

Usage

Circovac® Ingelvac CircoFLEX® Circumvent® Porcilis® PCV FosteraTM PCV (formerly Suvaxyn® PCV2 One DoseTM )

Merial Boehringer Ingelheim Intervet (Merck) Schering-Plough (Merck) Pfizer

Inactivated PCV2a virus PCV2a capsid protein PCV2a capsid protein PCV2a capsid protein Inactivated attenuated chimeric PCV1-2a virus

Breeding sows/piglets (>3 weeks old) Piglets >3 weeks old Piglets >3 weeks old Piglets >3 days old Piglets >3 weeks old

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advantage of more closely mimicking field conditions, in which several coinfecting swine pathogens are known to contribute to clinical PCVAD. Coinfection with PRRSV has been shown to increase the severity of PCV2 infection in pigs, resulting in increased shedding of PCV2 in oronasal secretions and feces (Allan et al., 2000; Rovira et al., 2002; Sinha et al., 2011). Vaccination of pigs with Suvaxyn® PCV2 One DoseTM followed by PCV2/PRRSV challenge at 28 days postvaccination (dpv) induced neutralizing antibodies while reducing lung lesions, fecal shedding, and PCV2 viral loads in serum and lymphoid tissues compared to unvaccinated pigs (Opriessnig et al., 2008a). Several studies have compared the efficacy of Suvaxyn® PCV2 One DoseTM , Circumvent® , and CircoFLEX® in various coinfection challenge models. Compared to unvaccinated pigs, each vaccine was shown to induce neutralizing antibodies while reducing serum viral loads and lymphoid lesions after challenge with PCV2, swine influenza virus (SIV), and PRRSV (Opriessnig et al., 2009). PRRSV infection status at the time of PCV2 vaccination did not alter vaccine efficacy (Sinha et al., 2010), while vaccination of PCV2-viremic pigs decreased viremia and the amounts of PCV2 antigen in lymphoid tissues after challenge with PCV2, PRRSV, and PPV (Shen et al., 2010b). Coinfection of MHYO and PCV2 has also been shown to cause clinical PCVAD in conventional pigs (Opriessnig et al., 2004), and used as a challenge model for testing the efficacy of commercial PCV2 vaccines. Vaccination of boars with Suvaxyn® PCV2 One DoseTM followed by PCV2 and MHYO challenge prevented severe clinical diseases, decreased serum viral load and reduced PCV2 shedding in feces and semen compared to unvaccinated boars (Opriessnig et al., 2011a). Pigs vaccinated with Suvaxyn® PCV2 One DoseTM or CircoFLEX® showed a decrease in serum PCV2 viral load, reduced lymphoid lesions, and increased weight gain compared to unvaccinated pigs (Kim et al., 2011). Various vaccine efficacy studies under controlled experimental conditions have clearly proven the utility of these commercially available vaccines against PCV2 infection. Using coinfection challenge models that have been shown to reproduce clinical PCVAD in conventional pigs, vaccination was shown to effectively reduce the ability of PCV2 to replicate to high systemic levels. The protective immunity conferred from the commercial vaccines is thus expected to protect swine herds from complex field conditions that may expose them to various coinfecting swine pathogens. 3.3. Vaccination strategies 3.3.1. Effect of PCV2 vaccination on vertical transmission Vertical transmission of PCV2 in commercial swine herds (Shen et al., 2010a) may complicate management of herd health. PCV2 shedding in the semen of mature boars is a concern for breeding herds. It has been shown that the ability of PCV2 to be transferred via artificial insemination is somewhat dependent on the semen viral load (Madson et al., 2009c,d). Therefore, the use of PCV2 vaccination to decrease viremia, systemic viral load and shedding in semen of boars is a strategy to limit the spread of PCV2 to breeding herds. It has been shown that the use of Suvaxyn® PCV2 One DoseTM vaccine in boars naturally and experimentally exposed to PCV2 did not affect semen characteristics or quality, while decreasing viremia and shedding of virus in the semen (Alberti et al., 2011; Opriessnig et al., 2011a). Boars given an inactivated PCV2 vaccine and subsequently challenged with PCV2 had a decreased viremia and semen viral load compared to unvaccinated boars (Seo et al., 2011). It is known that infected pregnant sows may transmit PCV2 to the fetus via intrauterine infection, thus it is important to limit the systemic PCV2 viral load prior to gestation. It has been shown that sows vaccinated with CircoFLEX® and subsequently challenged

with PCV2 or inseminated with PCV2-spiked semen were still able to spread PCV2 to their offspring (Madson et al., 2009a,b). PCV2 vaccination is effective in reducing systemic viral loads, but does not prevent intrauterine infection completely. However, reducing the exposure of piglets to high levels of PCV2 is expected to contribute to positive production parameters in commercial swine herds.

3.3.2. Vaccination of sows One strategy for the prevention of PCVAD in piglets is to vaccinate the breeding sows, reducing viremia and systemic PCV2 loads, and increasing PCV2-specific neutralizing antibodies in the colostrum. Passive immunity in the form of maternal antibodies has been shown to at least partially protect naïve piglets from PCV2 infection. In experimental studies, piglets with high levels of maternal antibodies had reduced incidence of viremia after PCV2 challenge compared to those with low levels of maternal antibodies, and protection against PCV2 infection conferred by maternal antibodies is titer-dependent: higher titers are generally protective, but low titers are not (McKeown et al., 2005; Opriessnig et al., 2008b; Ostanello et al., 2005). It has been shown in experimental conditions that dam vaccination with Circovac® can offer similar protection as vaccination of piglets (Opriessnig et al., 2010). Vaccination of pregnant sows with Circovac® has been shown to improve production parameters of their piglets in field conditions. In a farm with acute PMWS, Circovac® vaccination of sows alone decreased preweaning mortality and increased weight gain of their offspring (Pejsak et al., 2010). Circovac® administration in dams with subclinical PCV2 infection increased antibody responses and average daily weight gain, and decreased fattening period in their offspring (Kurmann et al., 2011). Vaccination of sows also reduces the quantity of virus transmitted from sow to piglets during gestation and pre-weaning periods. Circovac® administration in sows with natural PCV2 exposure decreased but did not eliminate viral shedding in the colostrum (Gerber et al., 2011).

3.3.3. Vaccination of growing pigs Vaccines are commonly administered in growing piglets, and have been shown to improve production parameters in pigs naturally exposed to PCV2. In a farm affected by PMWS, vaccination of piglets with Circovac® was shown to decrease postweaning mortality and increase average weight gain (Pejsak et al., 2010). Several studies have reported reduction in mortality, viral load and duration of viremia, and increased weight gain on farms affected by PCV2 after vaccination with CircoFLEX® (Fachinger et al., 2008; Kixmoller et al., 2008; Lyoo et al., 2011; Takahagi et al., 2010) or Suvaxyn® PCV2 One DoseTM (Lyoo et al., 2011; Segales et al., 2009). Similarly, Circumvent® and Porcilis® were both shown to reduce mortality, viremia, viral load, and increased weight gain in pigs naturally infected with PCV2 (Lyoo et al., 2011; Martelli et al., 2011; Takahagi et al., 2010). Vaccination of pigs with Porcilis® induced IFN-gamma secreting cells, a strong humoral immune response and neutralizing antibodies, though the presence of high levels of maternal antibodies was shown to interfere with vaccine efficacy (Fort et al., 2008, 2009b). In a different study, Suvaxyn® PCV2 One DoseTM was used to vaccinate pigs with maternal antibodies and challenged 28 days post-vaccination (dpv) with PCV2. Among pigs with maternal antibodies, those that were vaccinated had significantly decreased serum viral load and decreased lymphoid lesion severity after challenge compared to unvaccinated pigs (Opriessnig et al., 2008b), indicating that vaccination in the presence of maternal antibodies can still increase protection in growing piglets.

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4. Experimental PCV2 vaccines The current commercially available vaccines are all based on PCV2a genotype and all have very good efficacy, even though currently the PCV2b genotype is the predominant type infecting pigs worldwide. Subunit and inactivated virus vaccines have advantages of stability and safety, yet there are other vaccine technologies available that have been shown to stimulate anti-PCV2 immune responses and prevent PCV2 infection (Table 2). Some RNA-based therapies have also been shown to interfere with PCV2 infection and replication, and modified live-attenuated vaccines (MLV) are capable of stimulating both cell-mediated and humoral immune responses compared to inactivated or subunit products. DNA vaccines and other vector-based vaccines have the potential as alternative systems for the delivery of PCV2 antigens to the host. 4.1. RNA-based antiviral therapy Several different strategies that use RNA molecules to neutralize infectious PCV2 or otherwise interfere with the infection of PCV2 have been reported. RNA aptamers, which are RNA molecules that bind specifically to a target, have been shown to block the infectivity of PCV2 in vitro in a dose-dependent manner (Yoon et al., 2010). Small interfering RNA (siRNA) has also been used in vitro to successfully block the expression of PCV1 and PCV2 replicase proteins, resulting in a decrease of infectious virus production in infected cells (Sun et al., 2007). Short-hairpin RNA (shRNA) has also been shown to decrease PCV2 replication in vitro, as well as decreasing the amount of PCV2 antigens produced after PCV2 infection in a mouse model (Feng et al., 2008). However, the clinical implication of these RNA-based antiviral therapies against PCV2 is unclear due to the inherited problems of in vivo delivery and prohibitive costs. 4.2. Modified live-attenuated PCV2 vaccines In general, a MLV stimulates both cell-mediated and humoral immunity that could lead to a superior protection compared to the current subunit or inactivated vaccines. Attenuation of wildtype PCV2 through serial passages in cell culture is possible. It has been shown that, after 120 passages in PK-15 cells, two nucleotide mutations were identified in the capsid gene (Fenaux et al., 2004b). The passage 120 virus (P120) containing the P110A and R191S mutations in the PCV2 capsid enhanced the growth ability of PCV2 in vitro but attenuated the virus in vivo, suggesting that the P120 could be a potential candidate for a MLV (Fenaux et al., 2004b). A drawback of this strategy is the potential reversion of such a MLV to a pathogenic phenotype that can actually cause diseases in pigs.

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An alternative and more logical strategy to develop safer MLV is the use of PCV1 and PCV2 chimeric viruses. Despite the major phenotypic difference, the non-pathogenic PCV1 and the PCVADassociated PCV2 are genetically closely related viruses with similar genome organization (Finsterbusch and Mankertz, 2009). In fact, it has been shown that the replication factors of PCV1 and PCV2 are functionally interchangeable (Beach et al., 2010a; Mankertz et al., 2003). Chimeric viruses in which ORF1 or ORF2 have been swapped between PCV1 and PCV2 are infectious in vitro and in pigs (Beach et al., 2010a; Fenaux et al., 2003). A chimeric virus (PCV12a) containing the capsid of PCV2a in the genomic backbone of PCV1 was shown to be infectious and attenuated in pigs, and is the basis for the inactivated FosteraTM PCV (formerly Suvaxyn® PCV2 One DoseTM ) vaccines (Fenaux et al., 2004a). Several subsequent studies with the live chimeric PCV1-2a virus have demonstrated that the chimeric PCV1-2a is attenuated, immunogenic, and genetically stable while providing similar protection against PCV2a and PCV2b challenge as current commercial inactivated and subunit PCV2 vaccines (Fenaux et al., 2004a; Gillespie et al., 2008; Shen et al., 2010b). Another chimeric virus with the capsid of genotype PCV2b cloned in the backbone of PCV1 (PCV1-2b) has recently been developed and tested for potential use as a MLV against PCV2. The chimeric PCV1-2b virus was shown to be attenuated, with the absence of clinical disease and decreased viremia, reduced viral load and lesions in lymphoid tissues in CD/CD pigs compared to the wildtype PCV2b (Beach et al., 2010b). Conventional pigs vaccinated with the attenuated chimeric PCV1-2b virus were protected from challenges with PCV2a and PCV2b, with decreased incidence and severity of microscopic lesions in the lymphoid tissue and decreased viral load in lymphoid tissues and serum (Beach et al., 2010b). In a separate study, pigs vaccinated with the attenuated chimeric PCV1-2b virus were protected from a challenge in a triple coinfection model against PCV2b, PRRSV, and PPV (Opriessnig et al., 2011b). The chimeric PCV1-2b virus was shown to spread by contact to naïve pigs but remained attenuated, as PCV1-2b virus replication was not upregulated in the presence of PRRSV (Opriessnig et al., 2011b). Collectively, the studies with the experimental chimeric PCV1-2a and PCV1-2b MLVs indicate that chimeric PCV1-2a and PCV1-2b viruses are attenuated while inducing broadly protective immunity in conventional pigs, and thus may be appropriate for use as a MLV in swine herds. Unlike the traditional virus attenuation process through serial cell culture passages of wildtype PCV2, the chimeric PCV1-2 virus strategy essentially eliminates the concern of potential reversion of MLV. Although the current PCV2 commercial vaccines are all very effective, the use of a safe MLV in swine herds will reduce the vaccination cost while eliciting both cell-mediated and humoral protective immunity against PCV2.

Table 2 Experimental PCV2 vaccines. Class

Type

Reference

Modified live-attenuated vaccines

Cell culture attenuated PCV2a virus Chimeric PCV1-2a virus Chimeric PCV1-2b virus

Fenaux et al. (2004b) Fenaux et al. (2004a) Beach et al. (2010b)

DNA-based vaccines

Dimerized chimeric PCV1-2a genome pORF2 pORF2 (mouse model)

Fenaux et al. (2004a) Blanchard et al. (2003) Aravindaram et al. (2009), Kamstrup et al. (2004) and Shen et al. (2008)

Vectored vaccines

Bacteriophage lambda PCV2 epitope display Pseudorabies virus vector Adenovirus vector Bordetella bronchiseptica vector Yeast expression (mouse model) Pseudotype baculovirus (mouse model)

Gamage et al. (2009) and Hayes et al. (2010) Song et al. (2007) Wang et al. (2007) Kim et al. (2009) Bucarey et al. (2009) Fan et al. (2008)

Marker vaccine

Chimeric PCV1-2a with foreign epitope markers

Beach et al. (2011)

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4.3. DNA-based vaccines The use of PCV2 DNA to elicit specific immune responses has been studied in pig and mouse models. Direct injection of dimerized PCV2 genomic DNA into the liver, lymph nodes, or muscle of pigs results in an active PCV2 infection that is similar to inoculation with live infectious virus (Fenaux et al., 2002, 2003, 2004a). Intramuscular injection of plasmid DNA containing the dimerized chimeric PCV1-2a genome induced protective immunity in pigs (Fenaux et al., 2004a). Therefore, the plasmid DNA carrying the dimerized chimeric PCV1-2 genome could be administered to pigs in a DNA form as a MLV in pigs. One of the advantages of using plasmid DNA containing chimeric PCV1-2 genome as a MLV is the relative easy for scale-up production of large quantity of plasmid DNA and thus reducing the vaccine cost. Intramuscular administration of plasmid DNAs encoding individual PCV2 genes have also been shown to induce PCV2-specific immune responses in pigs and mice. A plasmid encoding the PCV2 capsid (pORF2) was shown to elicit a protective immune response in pigs (Blanchard et al., 2003). DNA vaccination has also been studied in the mouse model and “gene gun” administration of pORF2 in mice resulted in seroconversion to PCV2 capsid-specific antibodies (Kamstrup et al., 2004). In mice, administration of pORF2 was shown to be more effective at stimulating a PCV2-specific neutralizing antibody response than administration of capsid protein alone (Shen et al., 2008). Vaccination of mice with pORF2 was shown to decrease PCV2 viral load and microscopic lesions after challenge with PCV2 compared to unvaccinated controls (Shen et al., 2008). It has also been reported that concurrent administration of DNA encoding ORF1 or ORF3 with pORF2 interfered with the protective immunity conferred by pORF2 alone (Shen et al., 2009). Interestingly, a different study found that “gene gun” DNA vaccination of mice worked best when combining pORF2/pORF3 or pORF1/pORF2/pORF3 (Aravindaram et al., 2009). The future of these DNA-based PCV2 vaccines is unclear due to regulatory issues. However, the approval in the United States of the first USDA-licensed DNA vaccine against West Nile virus in horses opens the door for similar products against other viruses in the future. 4.4. Vectored vaccines against PCV2 Current commercial subunit vaccines are based on the capsid protein expressed in the baculovirus expression system, though other expression methods of antigen production and delivery have also been explored. Oral administration of PCV2 capsid protein expressed in yeast has been shown to elicit anti-PCV2 antibodies in mice (Bucarey et al., 2009). Also, display of specific immunodominant epitopes from the PCV2 capsid on the surface of bacteriophage lambda has been successfully used to vaccinate and elicit PCV2specific neutralizing antibodies in pigs (Gamage et al., 2009; Hayes et al., 2010). Several vectored vaccines using live infectious agents for the delivery of PCV2 antigens have been tested in mouse and pig models. A recombinant pseudotype baculovirus (rBV) capable of infecting mammalian cells was generated for the expression of PCV2 capsid protein in a mouse model (Fan et al., 2008). It was shown that the rBV induced PCV2-specific cell mediated immunity and a strong neutralizing antibody response in infected mice (Fan et al., 2008). A live recombinant pseudorabies virus (PRV) expressing the capsid protein of PCV2 was shown to be immunogenic in pigs, and elicited humoral immune responses against both PCV2 and PRV (Song et al., 2007). It is unclear if such a vector would ever be approved for use in swine herds especially in the United States since this would complicate the PRV eradication program. Similarly, a recombinant adenovirus expressing the capsid of PCV2 stimulated production of PCV2-specific neutralizing

antibodies in vaccinated pigs (Wang et al., 2007). After challenge with PCV2, vaccinated pigs had an increased weight gain and a decreased severity of microscopic lesions and incidence of viremia compared to unvaccinated pigs (Wang et al., 2007). An attenuated strain of the bacterium Bordetella bronchiseptica expressing the capsid of PCV2 was shown to induce serum antibodies and decrease PCV2 viral load in lymph nodes of vaccinated pigs challenged with PCV2 (Kim et al., 2009). Live-vectored vaccines have the potential to induce strong anti-PCV2 immune responses without the risk of possible PCV2 infection. However, there are concerns about potential pre-existing vector immunity that may interfere with vaccination and the potential reversion of the live vaccine vectors to a virulent form for the use of any vectored vaccines in the field. 4.5. Marker PCV2 vaccines The ubiquitous nature of PCV2 infection in swine herds warrants the development of a marker vaccine that can distinguish natural infection from vaccination. Vaccines engineered with novel antigenic epitopes may be used to serologically differentiate vaccinated from naturally infected pigs. Recently, an ELISA was developed for confirmation of vaccination status of pigs vaccinated with Porcilis® PCV (BacucheckTM , Intervet/Schering Plough/Merck) (Ladinig et al., 2011), and is currently available in Germany. BacucheckTM detects antibodies generated against “baculomarkers” present in Porcilis® PCV vaccine and may be used to certify vaccination of piglets (Ladinig et al., 2011). However, there are no similar products for confirmation of other commercial vaccine products. Expression of novel epitopes on the surface of infectious PCV2 and chimeric PCV1-2a viruses has been studied as an alternative approach for marker vaccine production. It has been shown that the PCV2 genome tolerates insertions of at least 27 amino acids at the C-terminus of the capsid protein, which is exposed on the surface of infectious virions (Beach et al., 2011). Additionally, live-attenuated chimeric PCV1-2a viruses displaying short epitope tags were shown to elicit PCV2-specific neutralizing antibodies as well as anti-epitope tag antibodies in infected pigs (Beach et al., 2011). This has subsequently been confirmed in a separate study, in which an 11 amino acid tag was successfully inserted in the C-terminus of the PCV2 capsid, and the resulting virus remains infectious and immunogenic in a mouse model (Huang et al., 2011b). Therefore, attenuated epitope-tagged PCV2 or chimeric PCV1-2 may be useful as a positive compliance marker MLV that would facilitate the tracking of the spread of vaccine virus in swine herds. 5. Conclusions All the current commercially available PCV2 vaccines have been shown to be effective at reducing clinical disease and improving production parameters in farms with PCV2 infection. While vaccination does not completely prevent infection or spread of PCV2, it has been shown to significantly reduce viremia, systemic viral loads and shedding, which results in a decrease in environmental load of the virus. Given the impact of coinfecting swine pathogens on herd health and production, it is important to vaccinate pigs to minimize herd exposure to PCV2. Although the current commercial vaccines based on the PCV2a genotype are effective, future generations of vaccines should be based on the PCV2b genotype due to continued predominant prevalence of PCV2b in the field. Novel PCV2b-based vaccine technologies, including compliance markers and a safe MLV, would enhance humoral and cell-mediated immunity while significantly reducing the cost of PCV2 vaccination programs.

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