Introduction
Porcine reproductive and respiratory syndrome (PRRS) causes an annual economic loss of an estimated $664 million to the U.S. pork industry (Holtkamp and Kliebenstein, Reference Holtkamp and Kliebenstein2011). According to the Animal and Plant Health Inspection Service report of January 2009, 49.8% of unvaccinated pigs in the U.S. are seropositive to PRRSV. This is based on collective data from 94% of pork producers in 17 states, suggesting the widespread prevalence of PRRS in the U.S. Pigs in more than 50% of sites from all four regions in the U.S. (North, South, West Central and East Central) had PRRSV antibodies and approximately 60% of pig samples in northern and southern regions were positive for anti-PRRSV antibodies. PRRSV causes respiratory and reproductive disease (Wensvoort et al., Reference Wensvoort, Terpstra, Pol, Ter Laak, Bloemraad, De Kluyver, Kragten, Van Buiten, Den Besten, Wagenaar, Broekhuijsen, Moonen, Zetstra, de Boer, Tibben, de Jong, Van't Veld, Groenland, Van Gennep, Voets, Verheijden and Braamskamp1991; Collins et al., Reference Collins, Benfield, Christianson, Harris, Hennings, Shaw, Goyal, McCullough, Morrison, Joo, Gorcyca and Chladek1992; Christopher-Hennings et al., Reference Christopher-Hennings, Nelson, Hines, Nelson, Swenson, Zimmerman, Chase, Yaeger and Benfield1995), with major losses from reproductive failure in sows, including stillbirths, mummifications, weak born piglets and high preweaning mortality (Mengeling et al., Reference Mengeling, Lager and Vorwald1998; Meulenberg, Reference Meulenberg2000; Rowland, Reference Rowland2007). PRRSV is excreted from all the body secretions at low levels or perhaps intermittently in saliva, nasal secretions, urine, milk, colostrum and feces from infected pigs, and also in semen of infected boars (Rossow et al., Reference Rossow, Bautista, Goyal, Molitor, Murtaugh, Morrison, Benfield and Collins1994; Voicu et al., Reference Voicu, Silim, Morin and Elazhary1994; Albina, Reference Albina1997; Christopher-Hennings et al., Reference Christopher-Hennings, Nelson, Nelson, Rossow, Shivers, Yaeger, Chase, Garduno, Collins and Benfield1998; Wagstrom et al., Reference Wagstrom, Chang, Yoon and Zimmerman2001; Zimmerman, Reference Zimmerman2003). Under natural conditions in the absence of any intervention, transport and transmission of PRRSV by fomites and personnel occur between swine populations (Pitkin et al., Reference Pitkin, Deen and Dee2009). In addition, airborne transport of PRRSV may occur over long distances with no change in the infectivity of virus (Otake et al., Reference Otake, Dee, Corzo, Oliveira and Deen2010). Therefore, control of PRRSV transmission within and between swine herds is a major challenge to the swine industry.
PRRSV is divided broadly into two distinct genotypes, type I (European) and type II (North American). Each genotype contains several subtypes and strains, which are genetically highly diverse and display significant differences in their virulence and pathogenicity (Kim et al., Reference Kim, Lee, Johnson, Roof, Cha and Yoon2007). PRRSV field isolates within the North American genotype have genetic diversity ranging from 84 to 100%, based on the amino acid homologies of ORFs 2–6 relative to VR2332, a prototype North American strain of PRRSV (Kim et al., Reference Kim, Lee, Johnson, Roof, Cha and Yoon2007). Immunity to the initial infecting genotype of the PRRSV may therefore provide partial to no protection against reinfection, reflecting not only the complexity of the genetics but also immunological variation among strains (Botner et al., Reference Botner, Strandbygaard, Sorensen, Have, Madsen, Madsen and Alexandersen1997; Kimman et al., Reference Kimman, Cornelissen, Moormann, Rebel and Stockhofe-Zurwieden2009; Martelli et al., Reference Martelli, Gozio, Ferrari, Rosina, De Angelis, Quintavalla, Bottarelli and Borghetti2009; Li et al., Reference Li, Xue, Wang, Chen, Chen and Cao2010).
Currently, commercial modified live virus-PPRS (MLV-PRRS) and inactivated PRRSV vaccines are licensed for use; although the MLV-PRRS offers good protection versus homologous virus isolates, there continue to be questions regarding the vaccine efficacy against antigenically variant or heterologous field strains (Botner et al., Reference Botner, Strandbygaard, Sorensen, Have, Madsen, Madsen and Alexandersen1997; Mengeling et al., Reference Mengeling, Lager, Vorwald and Clouser2003a, Reference Mengeling, Lager, Vorwald and Koehlerb; Cano et al., Reference Cano, Dee, Murtaugh and Pijoan2007; Kimman et al., Reference Kimman, Cornelissen, Moormann, Rebel and Stockhofe-Zurwieden2009; Martelli et al., Reference Martelli, Gozio, Ferrari, Rosina, De Angelis, Quintavalla, Bottarelli and Borghetti2009). The efficacy of available killed PRRSV vaccines is inadequate to protect pigs against even genetically closely related PRRSV, and none of the current vaccines prevent respiratory infection and pig-to-pig transmission of PRRSV (Kimman et al., Reference Kimman, Cornelissen, Moormann, Rebel and Stockhofe-Zurwieden2009). Administration of either field isolates of PRRSV or vaccine including MLV-PRRS by the parenteral or intranasal route suppresses the innate natural killer (NK) cell cytotoxic function and IFN-α production (Albina et al., Reference Albina, Carrat and Charley1998; Renukaradhya et al., Reference Renukaradhya, Alekseev, Jung, Fang and Saif2010; Dwivedi et al., Reference Dwivedi, Manickam, Patterson, Dodson, Weeman and Renukaradhya2011b, Reference Dwivedi, Manickam, Binjawadagi, Linhares, Murtaugh and Renukaradhya2012).
The most efficient and rapid host response against viruses consists of production of type I IFNs (IFN-α/β), an essential part of the antiviral innate immune system (Thiel and Weber, Reference Thiel and Weber2008). The IFN production is triggered by the first contact of cells with the virus; the secreted IFN slows down or even blocks virus multiplication, aiding in establishment of an adaptive immune response (Thiel and Weber, Reference Thiel and Weber2008). The NK cell is a lymphocyte subset that can be activated to mediate significant levels of innate anti-viral cytotoxic activity and also produce high levels of IFN-γ and chemokines (Biron et al., Reference Biron, Nguyen, Pien, Cousens and Salazar-Mather1999; Gerner et al., Reference Gerner, Kaser and Saalmuller2009). Thus, innate immune response mediated through type I IFNs and NK cells is critical for induction of protective immunity. PRRSV experts collectively agree that only replicating PRRSV vaccines have the promise to reduce the PRRS incidence in the field (http://vetmed.illinois.edu/news/PRRSwhitepaper.pdf). Since the 1990s, development of a safe and effective vaccine that induces broad protective immunity to PRRS remains as a principal goal of researchers and the swine industry.
PRRSV-induced modulation in cytokine production
Swine are the only known hosts susceptible to PRRSV, and the virus enters the host through respiratory and reproductive mucosal surfaces. Primary permissive cells are the alveolar macrophages (Mɸs) and the virus also infects Mɸs present in pulmonary intravascular spaces, lymph nodes, thymus, heart, spleen, placenta and umbilical cord (Halbur et al., Reference Halbur, Miller, Paul, Meng, Huffman and Andrews1995; Rossow et al., Reference Rossow, Benfield, Goyal, Nelson, Christopher-Hennings and Collins1996; Duan et al., Reference Duan, Nauwynck and Pensaert1997; Lawson et al., Reference Lawson, Rossow, Collins, Benfield and Rowland1997; Beyer et al., Reference Beyer, Fichtner, Schirrmeier, Polster, Weiland and Wege2000). PRRSV elicits poor innate and adaptive immune responses (Albina et al., Reference Albina, Carrat and Charley1998; Van Reeth et al., Reference Van Reeth, Labarque, Nauwynck and Pensaert1999; Meier et al., Reference Meier, Galeota, Osorio, Husmann, Schnitzlein and Zuckermann2003; Renukaradhya et al., Reference Renukaradhya, Alekseev, Jung, Fang and Saif2010) associated with increased immune modulation (Read et al., Reference Read, Malmstrom and Powrie2000; Suradhat et al., Reference Suradhat, Thanawongnuwech and Poovorawan2003; Sakaguchi et al., Reference Sakaguchi, Wing, Onishi, Prieto-Martin and Yamaguchi2009; Dwivedi et al., Reference Dwivedi, Manickam, Patterson, Dodson, Murtaugh, Torrelles, Schlesinger and Renukaradhya2011a, Reference Dwivedi, Manickam, Patterson, Dodson, Weeman and Renukaradhyab), resulting in incomplete viral clearance and associated secondary microbial infections (Thanawongnuwech et al., Reference Thanawongnuwech, Young, Thacker and Thacker2001; Murtaugh et al., Reference Murtaugh, Xiao and Zuckermann2002a; Van Reeth et al., Reference Van Reeth, Van Gucht and Pensaert2002; Zimmerman et al., Reference Zimmerman, Benfield, Murtaugh, Straw, Zimmerman, D'Allaire, Taylor, Straw, Zimmerman and D'Allaire2006; Renukaradhya et al., Reference Renukaradhya, Alekseev, Jung, Fang and Saif2010). In infected pigs, PRRSV suppresses innate IFN-α secretion (Albina et al., Reference Albina, Carrat and Charley1998; Buddaert et al., Reference Buddaert, Van Reeth and Pensaert1998; Murtaugh et al., Reference Murtaugh, Xiao and Zuckermann2002a, Reference Murtaugh, Yuan, Nelson and Faabergb), and dampens the NK cell-mediated cytotoxicity (Jung et al., Reference Jung, Renukaradhya, Alekseev, Fang, Tang and Saif2009; Renukaradhya et al., Reference Renukaradhya, Alekseev, Jung, Fang and Saif2010; Dwivedi et al., Reference Dwivedi, Manickam, Binjawadagi, Linhares, Murtaugh and Renukaradhya2012).
Cytokine interleukin-10 (IL-10) possesses potent immunosuppressive properties and its production is necessary to dampen the virus-induced inflammatory response, which otherwise causes severe tissue damage and lethal disease (Sun et al., Reference Sun, Dodd, Moser, Sharma and Braciale2011; Trandem et al., Reference Trandem, Jin, Weiss, James, Zhao and Perlman2011a, Reference Trandem, Zhao, Fleming and Perlmanb). However, viruses evolved methods to evade the host immunity by promoting early secretion of IL-10 to antagonize the protective antiviral Th1 immune response (Suvas et al., Reference Suvas, Azkur, Kim, Kumaraguru and Rouse2004; Humphreys et al., Reference Humphreys, De Trez, Kinkade, Benedict, Croft and Ware2007; Gomez-Laguna et al., Reference Gomez-Laguna, Salguero, Barranco, Pallares, Rodriguez-Gomez, Bernabe and Carrasco2010; Avdic et al., Reference Avdic, Cao, Cheung, Abendroth and Slobedman2011; Lee et al., Reference Lee, Yeh, Lai, Wu, Su, Takada and Chang2011). Pig monocyte/Mɸs infected with four PRRSV isolates of varying clinical virulence were found to induce increased IL-10 gene expression associated with significantly reduced IFN-γ and TNF-α expression in vitro (Charerntantanakul et al., Reference Charerntantanakul, Platt and Roth2006). In pigs, a significant correlation was observed between PRRSV infection and expression of cytokine IL-10, and up-regulated IL-10 was associated with reduced expression of cytokines IFN-α, IFN-γ, IL-12 and TNF-α (Gomez-Laguna et al., Reference Gomez-Laguna, Salguero, Barranco, Pallares, Rodriguez-Gomez, Bernabe and Carrasco2010), indicating that immune responses generated in pigs to PRRSV are inadequate to completely clear the virus during the first 8–12 weeks post-infection. This dysregulated immune response has a strong impact on PRRSV pathogenesis and on severity of the disease. One possible approach to overcome the PRRSV-induced immunosuppression is to consider alternative routes of administration of vaccines such as intranasal delivery that could help to stimulate protective mucosal immunity, because a majority (>80%) of total body immune cells are present at mucosal sites (Czerkinsky and Holmgren, Reference Czerkinsky and Holmgren2010).
Administration of the MLV-PRRS vaccine intranasally mimics the patho-immunology of infection with the field isolates, with the induction of IL-10 and transforming growth factor β (TGF-β) in pig lungs and blood. However, if the commercial MLV-PRRSV is intranasally co-administered with a potent adjuvant, production of IL-10 and TGF-β is significantly reduced (Dwivedi et al., Reference Dwivedi, Manickam, Patterson, Dodson, Murtaugh, Torrelles, Schlesinger and Renukaradhya2011a). PRRSV-induced increased frequency of FoxP3-expressing T-regulatory cells (Tregs) has been implicated in mediating immunosuppression in pigs (Didierlaurent et al., Reference Didierlaurent, Goulding and Hussell2007). Infiltration of Tregs into infected pig lungs also contributes to secretion of high levels of IL-10 and TGF-β (Didierlaurent et al., Reference Didierlaurent, Goulding and Hussell2007). The role of Tregs in the establishment of chronic persistent diseases such as those caused by HIV, hepatitis C and B viruses, cytomegalovirus and Epstein–Barr virus have been reported (Kinter et al., Reference Kinter, Hennessey, Bell, Kern, Lin, Daucher, Planta, McGlaughlin, Jackson, Ziegler and Fauci2004; Vahlenkamp et al., Reference Vahlenkamp, Tompkins and Tompkins2005; Peng et al., Reference Peng, Li, Wu, Sun, Chen and Chen2008). Perhaps there are similar mechanisms that explain persistent infection of PRRSV in pigs, but this remains to be proved. However, our laboratory has reported that intranasal co-administration of MLV-PRRS with an adjuvant Mycobacterium tuberculosis whole cell lysate (M. tb WCL) significantly reduces the frequency of Tregs in the lungs, blood and the tracheobronchial lymph nodes (TBLNs) associated with a concomitant reduction in the production of IL-10 and TGF-β (Dwivedi et al., Reference Dwivedi, Manickam, Patterson, Dodson, Murtaugh, Torrelles, Schlesinger and Renukaradhya2011a). Overall, these results all support the immunosuppressive nature of PRRSV by induction of IL-10, TGF-β and Tregs (Read et al., Reference Read, Malmstrom and Powrie2000; Johnsen et al., Reference Johnsen, Botner, Kamstrup, Lind and Nielsen2002; Suradhat et al., Reference Suradhat, Thanawongnuwech and Poovorawan2003; Royaee et al., Reference Royaee, Husmann, Dawson, Calzada-Nova, Schnitzlein, Zuckermann and Lunney2004; Diaz et al., Reference Diaz, Darwich, Pappaterra, Pujols and Mateu2005; Kaser et al., Reference Kaser, Gerner, Hammer, Patzl and Saalmuller2008; Sakaguchi et al., Reference Sakaguchi, Wing, Onishi, Prieto-Martin and Yamaguchi2009; Silva-Campa et al., Reference Silva-Campa, Cordoba, Fraile, Flores-Mendoza, Montoya and Hernandez2009a, Reference Trandem, Zhao, Fleming and Perlmanb; Renukaradhya et al., Reference Renukaradhya, Alekseev, Jung, Fang and Saif2010; Wongyanin et al., Reference Wongyanin, Buranapraditkun, Chokeshai-Usaha, Thanawonguwech and Suradhat2010; Dwivedi et al., Reference Dwivedi, Manickam, Patterson, Dodson, Murtaugh, Torrelles, Schlesinger and Renukaradhya2011a, Reference Dwivedi, Manickam, Patterson, Dodson, Weeman and Renukaradhyab; Leroith et al., Reference Leroith, Hammond, Todd, Ni, Cecere and Pelzer2011).
Pigs infected with PRRSV (both low pathogenic and virulent strains) are known to be susceptible to secondary microbial (bacterial and viral) infections, probably due to a PRRSV-induced immunosuppressive response resulting in exacerbation of the respiratory disease (Done and Paton, Reference Done and Paton1995; Van Reeth, et al., Reference Van Reeth, Van Gucht and Pensaert2002; Renukaradhya et al., Reference Renukaradhya, Alekseev, Jung, Fang and Saif2010). In a co-infection study involving PRRSV and porcine respiratory coronavirus, a severe respiratory disease was associated with elevated levels of cytokines IL-6, IL-12, IL-10 and TGF-β, and reduced IFN-α and IFN-γ production with synergistically dampened NK cell-mediated cytotoxicity compared to infection by either single virus (Jung et al., Reference Jung, Renukaradhya, Alekseev, Fang, Tang and Saif2009; Renukaradhya et al., Reference Renukaradhya, Alekseev, Jung, Fang and Saif2010). Both live and inactivated PRRSV have the ability to up-regulate IL-10 gene expression in porcine peripheral blood mononuclear cells (PBMCs) (Suradhat et al., Reference Suradhat, Thanawongnuwech and Poovorawan2003). Even after the clearance of PRRS viremia, the virus continues to induce IL-10 production for a prolonged (>2 months) period of time (Drew, Reference Drew2000; Johnsen et al., Reference Johnsen, Botner, Kamstrup, Lind and Nielsen2002; Yoo et al., Reference Yoo, Song, Sun, Du, Kim and Liu2010; Dwivedi et al., Reference Dwivedi, Manickam, Patterson, Dodson, Murtaugh, Torrelles, Schlesinger and Renukaradhya2011a). Induction of IL-10 production in PBMCs and bronchoalveolar lavage fluid (BAL) cells by both North American and European strains of high and low virulence PRRSV was observed (Aasted et al., Reference Aasted, Bach, Nielsen and Lind2002; Suradhat and Thanawongnuwech, Reference Suradhat and Thanawongnuwech2003; Silva-Campa et al., Reference Silva-Campa, Cordoba, Fraile, Flores-Mendoza, Montoya and Hernandez2009a).
Induction of IL-10 by PRRSV is mediated by interaction between PRRSV proteins and Mɸs/DCs, as these cells are susceptible targets of PRRSV. The PRRSV N protein induces IL-10 production by pig PBMCs and alveolar Mɸs (Wongyanin et al., Reference Wongyanin, Buranapraditkun, Thanawonguwech, Roth and Suradhat2009; Yoo et al., Reference Yoo, Song, Sun, Du, Kim and Liu2010). In contrast, results of a study involving pigs of all ages infected with PRRSV suggested that virulent PRRSV infection and disease are markedly more severe and prolonged in younger piglets than in finishers or sows. This is because porcine innate and adaptive immune systems are not fully developed at birth and the prolonged infection may not be due to IL-10-mediated immunosuppression (Klinge et al., Reference Klinge, Vaughn, Roof, Bautista and Murtaugh2009). However, based on several other reports, the role of IL-10 in delaying the onset and dampening the PRRSV-targeted protective immunity is clearly evident. Thus, based on our recent study and other published reports eliciting a protective mucosal immune response with the aid of potent adjuvant/s appears to be one of the viable mechanisms for overcoming PRRSV vaccine-induced immunosuppressive response to achieve a better control over the viral clearance during outbreaks, especially in growing pigs.
PRRSV-induced modulation in the immune cell population
PRRSV-modulated cytokines have a role in altering the abundance of the immune cells that are essential for viral clearance. Following natural or experimental infection of pigs with PRRSV, the virus-specific T-cell response is delayed for 1–3 months post-infection (Bautista and Molitor, Reference Bautista and Molitor1999; Lopez Fuertes et al., Reference Lopez Fuertes, Domenech, Alvarez, Ezquerra, Dominguez, Castro and Alonso1999). Recently, we evaluated the abundance of the immune cells present in PRRSV (both virulent MN184 and vaccine strain VR2332) infected pigs at post-infection days (PID) 15, 30 and 60 in the lung parenchyma and TBLN (Dwivedi et al., Reference Dwivedi, Manickam, Patterson, Dodson, Weeman and Renukaradhya2011b; Manickam et al., unpublished data). We evaluated immune responses in the lungs and TBLN side-by-side, because PRRSV causes lung pathology in infected pigs and persists in the lymphoid tissues for long periods, up to 200 days. Generally, for effective viral clearance from the body, adequate adaptive immune responses have to be generated and the sites of initiation of such response are in the TBLN. Following infection of pigs with PRRSV strain MN184, the total T-lymphocyte population was gradually reduced over a period of time in the lungs, and by PID 60 it was 20% of the mock-control pigs. In contrast, the T-lymphocyte population was reduced 50% at PID 30 and returned to near normal levels by PID 60 post-infection in the TBLN. Similarly, in PRRSV VR2332-infected pigs the total T-lymphocyte population was reduced in the lungs but at a slower rate, and by PID 60, their population was 65% lower than in mock pigs. There were no appreciable changes in the lymphocyte population in TBLN of VR2332-infected pigs. Strikingly, the number of Tregs was increased by greater than 2.5-fold in the lungs, TBLN and PBMCs in pigs infected with either PRRSV strain, and the number of Tregs in these tissues remained high until PID 60 (Dwivedi et al., Reference Dwivedi, Manickam, Patterson, Dodson, Weeman and Renukaradhya2011b; Manickam et al., unpublished data).
Others have also reported that PRRSV infection significantly reduces the leukocyte population in blood, wherein the levels of total leukocytes, lymphocytes, monocytes and eosinophils, but not neutrophils, are lower for several days shortly after infection (Lohse et al., Reference Lohse, Nielsen and Eriksen2004; Che et al., Reference Che, Johnson, Kelley, Van Alstine, Dawson, Moran and Pettigrew2011). Such a decline in total peripheral blood leukocyte population during the early stage of PRRSV infection may undermine the immune functions in infected pigs, and promote susceptibility of pigs to secondary microbial infections. In addition, depending on the virulence of the PRRSV strain, infected pigs have varying degrees of modulation in the abundance of lymphocytes in the lungs. However, we have demonstrated that PRRSV-induced reduction in the lymphocyte populations at both mucosal and systemic sites could be restored to a physiological normal level when pigs were vaccinated intranasally using MLV-PRRS combined with M. tb WCL adjuvant prior to PRRSV challenge (Dwivedi et al., Reference Dwivedi, Manickam, Patterson, Dodson, Murtaugh, Torrelles, Schlesinger and Renukaradhya2011a).
PRRSV-induced modulation of antibody response
PRRSV-specific neutralizing antibodies (NAs) play a critical role in viral clearance and are considered as a major component of the protective immune response (Murtaugh et al., Reference Murtaugh, Xiao and Zuckermann2002a, Reference Murtaugh, Yuan, Nelson and Faabergb; Osorio et al., Reference Osorio, Galeota, Nelson, Brodersen, Doster, Wills, Zuckermann and Laegreid2002; Lopez and Osorio, Reference Lopez and Osorio2004; Lopez et al., Reference Lopez, Oliveira, Garcia, Kwon, Doster and Osorio2007). PRRSV infection instigates abundant production of antibodies in pigs of all ages (Loemba et al., Reference Loemba, Mounir, Mardassi, Archambault and Dea1996; Lemke et al., Reference Lemke, Haynes, Spaete, Adolphson, Vorwald, Lager and Butler2004), and this is due to virus-mediated polyclonal activation of B-cells (Drew, Reference Drew2000; Lamontagne et al., Reference Lamontagne, Page, Larochelle, Longtin and Magar2001). However, a majority of these secreted antibodies are antibodies to host proteins and PRRSV non-NAs (Lemke et al., Reference Lemke, Haynes, Spaete, Adolphson, Vorwald, Lager and Butler2004). Studies of the kinetics of humoral immune response induced in pigs experimentally infected intranasally with a North American strain (VR2332) of PRRSV identified that the earliest antibodies are directed against viral nucleocapsid (N) protein, followed by matrix (M) and surface glycoprotein (GP) 5 (Nelson et al., Reference Nelson, Christopher-Hennings and Benfield1994). Also, in MLV-PRRS vaccinated pigs, enhanced production of non-NAs, directed mainly against PRRSV-N protein and also against anti-neutralizing epitopes (decoys) of the virus, was detected (Yoon et al., Reference Yoon, Zimmerman, Swenson, McGinley, Eernisse, Brevik, Rhinehart, Frey, Hill and Platt1995; Lopez and Osorio, Reference Lopez and Osorio2004). PRRSV surface GP contains immunodominant epitopes A and B, wherein epitope A is a decoy epitope inducing an early and strong non-NA response (Osorio et al., Reference Osorio, Galeota, Nelson, Brodersen, Doster, Wills, Zuckermann and Laegreid2002; Ostrowski et al., Reference Ostrowski, Galeota, Jar, Platt, Osorio and Lopez2002). The neutralizing epitope B is surrounded by up to four N-linked glycosylation sites, and variant strains are lacking few of those important glycosylation sites that potentially promote or assist escape from antibody-mediated neutralization (Faaberg et al., Reference Faaberg, Hocker, Erdman, Harris, Nelson, Torremorell and Plagemann2006; Mateu et al., Reference Mateu, Diaz, Darwich, Casal, Martin and Pujols2006; Vu et al., Reference Vu, Kwon, Yoon, Laegreid, Pattnaik and Osorio2011).
Overall, the PRRSV-NA response is vague, weak and delayed (Mateu et al., Reference Mateu, Diaz, Darwich, Casal, Martin and Pujols2006; Mateu and Diaz Reference Mateu and Diaz2008). Moreover, due to the low fidelity of the PRRSV RNA-dependent–RNA polymerase, mutations are consistently introduced into the viral genome resulting in the emergence of antigenic heterogeneity in immunodominant epitopes recognized by both antibodies and T-cells (Meng, Reference Meng2000; Huang and Meng, Reference Huang and Meng2010). This plasticity of the PRRSV genome results in numerous “vaccine failures” in the field, posing constant challenges to swine veterinarians and producers.
Such genetic and antigenic variability supports the evolution of PRRSV variants in the field and may explain the need for a vaccine that can elicit immune response to other conserved epitopes present on the viral surface GP that induces broadly reactive immune response to heterologous PRRSV. We have demonstrated that, in pigs inoculated intranasally with MLV-PRRS along with M. tb WCL virus, specific total antibody response was reduced substantially by PID 30, and interestingly, the virus-specific NA response was significantly increased compared to pigs administered only MLV-PRRS intranasally. These results suggest that mucosal immunization with MLV-PRRS coupled with M. tb WCL adjuvant suppresses the virus-induced total antibody response and at the same time importantly enhances the required virus neutralization titers for better viral clearance.
Mucosal adjuvants and MLV-PRRS
Until 2010 there were no published studies on the development of a live PRRSV mucosal vaccine. This may be due to limited research undertaken to identify potent mucosal adjuvants for use with MLV-PRRS. However, the ability of PRRSV to rapidly modulate the host innate immune response suggests the need for a potent adjuvant that has the inherent capacity to overcome the PRRSV vaccine-induced immunosuppressive response, and at the same time promote virus-specific cell-mediated immunity. Therefore, in our attempt to search for a potent mucosal adjuvant to used with MLV-PRRS intranasally, we evaluated the elicited immune responses in the respiratory tract of pigs inoculated intranasally with a panel of candidate adjuvants. We chose a panel of nine bacterial preparations belonging to Mycobacterium, Vibrio and Streptococcus species, and all known to possess adjuvant properties in rodent models (Bonavida et al., Reference Bonavida, Katz and Hoshino1986; Foss and Murtaugh, Reference Foss and Murtaugh1999; Kuroki et al., Reference Kuroki, Morisaki, Matsumoto, Onishi, Baba, Tanaka and Katano2003; Barral and Brenner, Reference Barral and Brenner2007; Beetz et al., Reference Beetz, Marischen, Kabelitz and Wesch2007). Pigs were inoculated with adjuvants alone, an adjuvant with MLV-PRRS and MLV-PRRS alone intranasally; the pigs were euthanized on day 7 post-inoculation and immune responses were assessed. Based on PRRSV-specific mucosal and systemic immune responses mediated by a battery of candidate adjuvants, we chose three preparations for use along with MLV-PRRS in viral challenge studies, they were M. tb WCL, Cholera toxin B subunit (Sigma) and OK-432 (a product of Streptococcus pyogenes) (Dwivedi and Renukaradhya, unpublished data). Results of this study demonstrated that among these three adjuvants, only M. tb WCL significantly dampened the PRRSV-induced immunosuppressive response, in addition to inducing enhanced virus-specific innate and adaptive immune responses (Dwivedi et al., Reference Dwivedi, Manickam, Patterson, Dodson, Murtaugh, Torrelles, Schlesinger and Renukaradhya2011a, Reference Dwivedi, Manickam, Patterson, Dodson, Weeman and Renukaradhyab).
Heat-killed M. tb is an excellent candidate adjuvant used in the preparation of Freund's complete adjuvant (FCA) and has been used extensively in experimental animals (Roberts et al., Reference Roberts, Jeffery, Mitchell, Andrew, Boulnois, Feldman, Cole and Wilson1992). However, its use in humans and in food animals is constrained due to a severe granulomatous inflammatory reaction induced at the injection site. The adverse effect due to FCA results from toxic cell wall components of M. tb, such as mycolic acids, arabinogalactan and wax D (Bekierkunst Reference Bekierkunst1968). However, adjuvanticity of various purified components of M. tb have been evaluated individually with satisfactory results (Bekierkunst et al., Reference Bekierkunst, Yarkoni, Flechner, Morecki, Vilkas and Lederer1971; Harmala et al., Reference Harmala, Ingulli, Curtsinger, Lucido, Schmidt, Weigel, Blazar, Mescher and Pennell2002). Particularly, water soluble purified fraction (M. tb WCL) free from bacterial toxic cell wall components has been shown to possess superior adjuvanticity in rodents, guinea pigs and rabbits (White et al., Reference White, Coons and Connolly1955; Werner et al., Reference Werner, Maral, Floch, Migliore-Samour and Jolles1975; Srivastava, Reference Srivastava2002; Choudhary et al., Reference Choudhary, Mukhopadhyay, Chakhaiyar, Sharma, Murthy, Katoch and Hasnain2003; Bansal et al., Reference Bansal, Elluru, Narayana, Chaturvedi, Patil, Kaveri, Bayry and Balaji2010). In our recently published study, pigs that received M. tb WCL did not have any toxic symptoms or any adverse inflammatory response at the local inoculation site, and there was no fever (Dwivedi et al., Reference Dwivedi, Manickam, Patterson, Dodson, Murtaugh, Torrelles, Schlesinger and Renukaradhya2011a, Reference Dwivedi, Manickam, Patterson, Dodson, Weeman and Renukaradhyab). At present, it is not known what component of the M. tb WCL made this candidate adjuvant more potent than the other tested candidates in the respiratory tract of pigs that also received MLV-PRRS. A vector-based recombinant vaccine expressed in M. tb strain BCG containing PRRSV GP5 and M protein helped to reduce clinical PRRS disease. Immunized pigs challenged with virulent homologous PRRSV had decreased viremia and viral load in tissues, suggesting that a favorable immune response to PRRSV could be induced using M. tb preparation as an adjuvant (Bastos et al., Reference Bastos, Dellagostin, Barletta, Doster, Nelson, Zuckermann and Osorio2004).
Pigs vaccinated intranasally with MLV-PRRS along with M. tb WCL and challenged with the virulent heterologous PRRSV strain MN184 had significantly increased PRRSV-specific NA response compared to pigs vaccinated with MLV-PRRS without any adjuvant (Dwivedi et al., Reference Dwivedi, Manickam, Patterson, Dodson, Murtaugh, Torrelles, Schlesinger and Renukaradhya2011a). In addition, significantly higher levels of IFN-γ and IL-12 (P<0.05) and significantly lower amounts of IL-10 were detected in pigs given the combination of MLV-PRRS and M. tb WCL (Dwivedi et al., Reference Dwivedi, Manickam, Patterson, Dodson, Murtaugh, Torrelles, Schlesinger and Renukaradhya2011a). However, both groups of vaccinated pigs were viremic and the virus was not eliminated until day 60 post-challenge (albeit relatively less virus was detected in pigs that received the adjuvant). Interestingly, significantly reduced lung lesions with no clinical signs were associated with increased anti-PRRSV-specific Th1 immune response in pigs that received the adjuvanted MLV-PRRS vaccine; this indicates the benefits of intranasal vaccination of commercially available MLV-PRRS with M. tb WCL (Dwivedi et al., Reference Dwivedi, Manickam, Patterson, Dodson, Murtaugh, Torrelles, Schlesinger and Renukaradhya2011a, Reference Dwivedi, Manickam, Patterson, Dodson, Weeman and Renukaradhyab).
The study described above did not include a group of pigs inoculated with MLV-PRRS alone by the parenteral route for a side-by-side comparison (Dwivedi et al., Reference Dwivedi, Manickam, Patterson, Dodson, Weeman and Renukaradhya2011b). However, there have been several previous studies of pigs inoculated with MLV-PRRS by the parenteral route and challenged with heterologous PRRSV (Botner et al., Reference Botner, Strandbygaard, Sorensen, Have, Madsen, Madsen and Alexandersen1997; Mengeling et al., Reference Mengeling, Lager, Vorwald and Clouser2003a, Reference Mengeling, Lager, Vorwald and Koehlerb; Cano et al., Reference Cano, Dee, Murtaugh and Pijoan2007; Kimman et al., Reference Kimman, Cornelissen, Moormann, Rebel and Stockhofe-Zurwieden2009; Martelli et al., Reference Martelli, Gozio, Ferrari, Rosina, De Angelis, Quintavalla, Bottarelli and Borghetti2009), and one study performed during evaluation of mucosal adjuvanticity of a panel of adjuvants along with MLV-PRRS administered by the intranasal route (Dwivedi et al., Reference Dwivedi, Manickam, Patterson, Dodson, Weeman and Renukaradhya2011b, Dwivedi and Renukaradhya, unpublished data). Based on these studies, we strongly hypothesize that mucosal vaccination with a potent adjuvant such as M. tb WCL is advantageous in reducing the heterologous PRRSV-induced disease burden. However, similar studies using a large number of pigs under field conditions and in pigs of diverse genetic background are essential to advance this promising mucosal immunization approach to the field.
Mucosal surfaces and associated lymphoid tissues in the respiratory tract
Mucous membranes cover the largest surface area in the body, with 80% of total body immune cells present at mucosal surfaces and mucosa-associated lymphoid tissues (MALT) (Czerkinsky and Holmgren, Reference Czerkinsky and Holmgren2010). The MALT is a highly compartmentalized immune system that functions independently of the systemic immune system, including the skin-associated lymphoid tissues (Holmgren and Czerkinsky, Reference Holmgren and Czerkinsky2005). It is highly populated with phenotypically and functionally distinct B-cell, T-cell and accessory cell subpopulations comparable to systemic lymphoid tissues. Also, it possesses well-developed strong restrictions upon lymphoid cell recirculation between mucosal sites. MALT comprises anatomically defined lymphoid micro-compartments such as the Peyer's patches, the mesenteric lymph nodes, the appendix and solitary follicles in the intestine called the gut-associated lymphoid tissues (GALT); nasopharynx-associated lymphoid tissues (NALT), tonsils and adenoids at the entrance of the aerodigestive tract; and bronchus-associated lymphoid tissues (BALT) in the lower respiratory tract.
The NALT is spread over the nasal cavity, nasopharynx and the middle ear (Kiyono and Fukuyama, Reference Kiyono and Fukuyama2004; Canessa et al., Reference Canessa, Vierucci, Azzari and Vierucci2010; Randall, Reference Randall2010), which also includes tonsils that form the Waldeyer's ring. A Waldeyer's ring consists of five different tonsils made of lymphoepithelial tissue, and are present in a similar pattern in both humans and pigs at the oropharyngeal and nasopharyngeal openings (Davis, Reference Davis2001). Recently, we studied the abundance of various immune cell populations in the tonsils of pigs and detected T-lymphocytes (42%), NK cells (4%), myeloid cells (CD172+) (10%), dendritic cells (DCs) (3%) and γδ T cells (6%). Further, the population of subsets of T-lymphocytes include CD8+ T-cells (CD3+CD4−CD8+; 22%); memory/T-helper cells (CD3+CD4+CD8+, 16%); T-helper cells (CD3+CD4+CD8−; 47%); and FoxP3+ Tregs (CD4+CD25+ FoxP3+, 25%) (Renukaradhya et al., unpublished data). The MALT also contains diffuse accumulations of large numbers of lymphoid cells in the parenchyma of mucosal organs and exocrine glands which form the mucosal effector sites where immune responses are manifested. Considering the relative immunological similarities of humans and pigs, we expect comparable structure, distribution and function of immune cells present in the pig MALT.
Immune cells present at mucosal sites are involved in providing pathogen-specific mucosal immunity. Thus, effective mucosal delivery of vaccines has been gaining increased attention for the control of pathogens which primarily cause pathology and disease at mucosal sites, for example, influenza, parainfluenza, HIV, Rotavirus and PRRSV. Stimulating the immune system systemically (by parenteral injection) results mainly in systemic protection with low mucosal immune responses. In contrast, optimum stimulation of the mucosal immune system provides both mucosal and systemic immunity, resulting in inhibition of replication and/or colonization of infectious agents before they replicate and cause disease (Holmgren et al., Reference Holmgren, Czerkinsky, Lycke and Svennerholm1992; Etchart Wild et al., Reference Etchart, Wild and Kaiserlian1996; Singh et al., Reference Singh, Briones and O'hagan2001; Holmgren and Czerkinsky, Reference Holmgren and Czerkinsky2005). NALT and BALT contain T- and B-cell zones with an entire repertoire of accessory immune cells strategically located to orchestrate regional immune functions against airborne infections (Mann et al., Reference Mann, Acevedo, Campo, Perez and Ferro2009; Randall, Reference Randall2010). Immune cells at mucosal sites are primarily involved in the regulation of inflammatory responses, but at the same time guard the body against the entry of harmful pathogens. Therefore, it is difficult to elicit protective mucosal immunity using conventional vaccine preparations delivered directly to mucosal sites. However, administration of conventional vaccines in the presence of suitable adjuvants by the intranasal route has been shown to modulate the immune regulatory response with simultaneous upregulation of protective antibody and cell-mediated immune response (Shephard et al., Reference Shephard, Todd, Adair, Po, Mackie and Scott2003; Kamijuku et al., Reference Kamijuku, Nagata, Jiang, Ichinohe, Tashiro, Mori, Taniguchi, Hase, Ohno, Shimaoka, Yonehara, Odagiri, Tashiro, Sata, Hasegawa and Seino2008; Guillonneau et al., Reference Guillonneau, Mintern, Hubert, Hurt, Besra, Porcelli, Barr, Doherty, Godfrey and Turner2009; Dwivedi et al., Reference Dwivedi, Manickam, Patterson, Dodson, Weeman and Renukaradhya2011b). Therefore, mucosal vaccination offers an advantage in inducing immune protection against viruses that typically invade and cause disease at mucosal sites. The respiratory tract is a portal of entry for many viruses and it is a challenge for the mucosal immune system to completely clear the virus-infected cells. NALT, BALT, upper and lower respiratory tract draining lymph nodes such as the cervical and TBLNs, help in the clearance of pathogens by initiating specific adaptive immune responses. Considering the inherent advantages of potent mucosal immunity, researchers have been finding innovative strategies to develop effective mucosal vaccines.
Inducible BALT structures in pigs
BALT is a secondary lymphoid organ embedded in the walls of large airways and at airway bifurcations; it is similar to Peyer's patches in function (Sminia et al., Reference Sminia, Van Der Brugge-Gamelkoorn and Jeurissen1989). In addition to normally present BALT tissues, additional BALT structures in bronchi are inducible and are called “inducible BALT” (iBALT). iBALT is considered to be a tertiary lymphoid structure that develops either in response to microbial infection or under chronic inflammatory conditions (Tschernig and Pabst, Reference Tschernig and Pabst2000; Foo and Phipps, Reference Foo and Phipps2010), and persists for several months, even after clearance of pathogens (Moyron-Quiroz et al., Reference Moyron-Quiroz, Rangel-Moreno, Carragher and Randall2007). Both BALTs serve as principal mucosal inductive sites to initiate immune responses against pathogens (Mowat, Reference Mowat2003; Kiyono and Fukuyama, Reference Kiyono and Fukuyama2004; Holmgren and Czerkinsky, Reference Holmgren and Czerkinsky2005). Influenza-specific memory CD8 T-cells and plasma cells which contribute to the generation of effector T-cells and NAs, respectively, aid in clearance of both homologous and heterologous strains of influenza virus and were identified in iBALT (Moyron-Quiroz et al., Reference Moyron-Quiroz, Rangel-Moreno, Carragher and Randall2007). Pigs immunized intranasally with MLV-PRRS in combination with M. tb WCL and challenged with highly virulent heterologous strain (MN184) of PRRSV had increased formation of iBALT structures (Fig. 1). The formation and persistence of iBALT for a long period of time is dependent on the critical recruitment of DCs into the lungs (GeurtsvanKessel et al., Reference Geurtsvankessel, Willart, Bergen, Van Rijt, Muskens, Elewaut, Osterhaus, Hendriks, Rimmelzwaan and Lambrecht2009). In contrast, Tregs present in the lymph nodes draining the lung attenuate iBALT formation by inhibiting the activation and proliferation of lymphocytes and other unidentified populations of cells (Foo and Phipps, Reference Foo and Phipps2010). Consistent with that, there was an increased frequency of DC-rich fraction (CD172+ CD11c+ SLAII+ cells) in both blood and the lungs, and significantly reduced frequency of Tregs in TBLN and the lungs of pigs intranasally immunized (MLV-PRRS+M. tb WCL) and challenged with the MN184 strain of PRRSV (Dwivedi et al., Reference Dwivedi, Manickam, Patterson, Dodson, Weeman and Renukaradhya2011a). Interestingly, iBALT contains DCs in higher frequency in the T-cell zone compared to other lymphoid tissues and also DCs are scattered throughout the B-cell zone (Woodland and Randall, Reference Woodland and Randall2004). Thus, formation of increased iBALT structures in intranasally immunized pigs has the potential to induce effective mucosal immunity against PRRSV.
iBALT in pigs. Pigs were unvaccinated (mock) or vaccinated with MLV-PRRS plus Mtb WCL and challenged with virulent heterologous PRRSV strain MN184, and euthanized on day 15 post-challenge. Representative photographs of the lungs of pigs from the mock and the virus challenged groups are shown. Arrow indicates iBALT structures.
For the mucosal immune system to recognize microbes or vaccines, specialized epithelial cells called “microfold cells” (M-cells) located in follicle-associated epithelium overlying the MALT should capture and transport the antigens across the mucosal barrier (transcytosis). This will lead to subsequent uptake and processing by professional antigen-presenting cells (APCs), DCs, Mɸs and B-cells for initiation of specific adaptive immune response (Jepson et al., Reference Jepson, Clark, Foster, Mason, Bennett, Simmons and Hirst1996; Neutra et al., Reference Neutra, Mantis, Frey and Giannasca1999; Kraehenbuhl and Neutra, Reference Kraehenbuhl and Neutra2000; Neutra and Kozlowski, Reference Neutra and Kozlowski2006; Corr et al., Reference Corr, Gahan and Hill2008; Suzuki et al., Reference Suzuki, Sekine, Kataoka, Pascual, Maddaloni, Kobayashi, Fujihashi, Kozono and McGhee2008). APCs prime the naïve T-cells to clonally expand and differentiate into T-cell subsets (Th1, Th2, Th17, or Tregs). Subsequently, activated T-cells migrate to submucosal regions where they perform effector functions. In particular, particulate antigens have an inherent affinity for mucosal M-cells and also for underlying APCs (Brayden et al., Reference Brayden, Jepson and Baird2005). In pigs, M-cells are present (Gebert et al., Reference Gebert, Rothkotter and Pabst1994), and their frequency in follicle-associated epithelium is highly variable; they constitute greater than 20% of the cells, which is higher than in rodents (~10%) and humans (<5%) (Buda et al., Reference Buda, Sands and Jepson2005). Recently, an abundant population of M-cells in the nasopharyngeal mucosa of rodents was identified (Kim et al., Reference Kim, Sato, Fukuyama, Sagara, Nagatake, Kong, Goda, Nochi, Kunisawa, Sato, Yokota, Lee and Kiyono2011). M-cells express MHC class II molecules (Allan et al., Reference Allan, Mendrick and Trier1993), thus fulfilling many of the criteria for APCs (Brayden et al., Reference Brayden, Jepson and Baird2005). Overall, M-cell-targeted delivery of vaccines to successfully stimulate the mucosal immune system has become an innovative strategy to develop effective mucosal vaccines (Plotkin, Reference Plotkin2005).
Mucosal vaccination is an effective alternative to control infections
Research on mucosal vaccine development has demonstrated that it is possible to activate immune cells present in the MALT, using vaccine antigens co-administered with appropriate adjuvants to mucosal sites. This approach potentiates the generation of long-lasting memory immune response against specific pathogens. There are many potent adjuvants available but a majority of them lack the ability to augment the mucosal immune response. From among a few identified mucosal adjuvants the majority are of bacterial origin (reviewed in Lawson et al., Reference Lawson, Norton and Clements2011). Mucosal vaccines co-delivered with suitable adjuvants have proved to be effective in controlling many diseases, particularly against pathogens which cause disease primarily at mucosal sites. In this regard, there are several studies, for example: (a) gastrointestinal infections caused by Helicobacter pylori, Vibrio cholerae, enterotoxigenic Escherichia coli (ETEC), Shigella spp., Clostridium difficile, polio virus, rotaviruses and calici viruses; (b) respiratory infections caused by Mycoplasma pneumoniae, influenza virus, parainfluenza-3 virus, respiratory syncitial virus and PRRSV; (c) sexually transmitted genital infections caused by HIV, simian immunodeficiency virus, Chlamydia trachomatis, Neisseria gonorrhoeae and herpes simplex virus-1 (Ogra, Reference Ogra1984; Karron et al., Reference Karron, Wright, Hall, Makhene, Thompson, Burns, Tollefson, Steinhoff, Wilson, Harris, Clements and Murphy1995; Van der Poel et al., Reference Van Der Poel, Kramps, Quak, Brand and Van Oirschot1995; Imaoka et al., Reference Imaoka, Miller, Kubota, Mcchesney, Lohman, Yamamoto, Fujihashi, Someya, Honda, McGhee and Kiyono1998; Schmidt et al., Reference Schmidt, Wenzke, Mcauliffe, St Claire, Elkins, Murphy and Collins2002; Rodriguez et al., Reference Rodriguez, Troye-Blomberg, Lindroth, Ivanyi, Singh and Fernandez2003; Petrovsky and Aguilar, Reference Petrovsky and Aguilar2004; Holmgren and Czerkinsky, Reference Holmgren and Czerkinsky2005; Ichinohe et al., Reference Ichinohe, Kawaguchi, Tamura, Takahashi, Sawa, Ninomiya, Imai, Itamura, Odagiri, Tashiro, Chiba, Sata, Kurata and Hasegawa2007a, Reference Ichinohe, Tamura, Kawaguchi, Ninomiya, Imai, Itamura, Odagiri, Tashiro, Takahashi, Sawa, Mitchell, Strayer, Carter, Chiba, Kurata, Sata and Hasegawab; Kamijuku et al., Reference Kamijuku, Nagata, Jiang, Ichinohe, Tashiro, Mori, Taniguchi, Hase, Ohno, Shimaoka, Yonehara, Odagiri, Tashiro, Sata, Hasegawa and Seino2008; Guillonneau et al., Reference Guillonneau, Mintern, Hubert, Hurt, Besra, Porcelli, Barr, Doherty, Godfrey and Turner2009; Dwivedi et al., Reference Dwivedi, Manickam, Patterson, Dodson, Weeman and Renukaradhya2011b). The protective mucosal immunity against the above pathogens or their toxins has been more effective in reducing the disease burden than have conventional parenteral routes of immunization.
Activated innate immune response at mucosal sites plays a major role in mucosal immunity against enteric and respiratory infections (Mann et al., Reference Mann, Acevedo, Campo, Perez and Ferro2009). Intranasal administration of influenza and parainfluenza-3 vaccines with a potent adjuvant generated enhanced cytotoxic T-lymphocyte and central memory immune response to conserved internal viral proteins, resulting in cross-protective immunity against a wide range of heterologous influenza and parainfluenza viruses (Shephard et al., Reference Shephard, Todd, Adair, Po, Mackie and Scott2003; Kamijuku et al., Reference Kamijuku, Nagata, Jiang, Ichinohe, Tashiro, Mori, Taniguchi, Hase, Ohno, Shimaoka, Yonehara, Odagiri, Tashiro, Sata, Hasegawa and Seino2008; Guillonneau et al., Reference Guillonneau, Mintern, Hubert, Hurt, Besra, Porcelli, Barr, Doherty, Godfrey and Turner2009). Currently, a licensed nasal-spray flu vaccine (FluMist, MedImmune, Gaithersburg, Maryland, USA) containing cold-adapted, temperature-sensitive, live-attenuated influenza virus (LAIV) reassortant strains was found to be effective (Belshe et al., Reference Belshe, Lee, Walker, Stoddard and Mendelman2004). FluMist induces an immune response that closely resembles natural immunity compared to conventional injectable vaccines (Cox et al., Reference Cox, Brokstad and Ogra2004). In one large study among children aged 15–85 months, FluMist reduced the chance of influenza illness by 92% compared with placebo. In yet another study among adults vaccinated with FluMist vaccine, there were 19% fewer severe febrile respiratory tract illnesses, 24% fewer respiratory tract illnesses with fever, 23–27% fewer days of illness, 13–28% fewer lost work days, 15–41% fewer health care provider visits and 43–47% less use of antibiotics compared with placebo (http://pediatrics.about.com/cs/immunizations/a/flumist_qa.htm). Nasal or tonsillar immunization in humans results in protective mucosal antibody response in the upper respiratory tract and the lungs but not in the gastrointestinal tract (Johansson et al., Reference Johansson, Wassen, Holmgren, Jertborn and Rudin2001, Reference Johansson, Bergquist, Edebo, Johansson and Svennerholm2004). In addition, nasal immunization has been found to generate enhanced secretory-IgA and IgG antibody responses in the human reproductive tract mucosa (Kozlowski et al., Reference Kozlowski, Cu-Uvin, Neutra and Flanigan1997; Johansson et al., Reference Johansson, Wassen, Holmgren, Jertborn and Rudin2001; Nardelli-Haefliger et al., Reference Nardelli-Haefliger, Wirthner, Schiller, Lowy, Hildesheim, Ponci and De Grandi2003). This suggests that intranasal delivery of PRRSV vaccine in the presence of a suitable adjuvant has the potential to augment protective immunity against PRRS.
Mucosal PRRSV vaccination in pigs and its implications
The pig is an important food animal species, and it possesses a number of immunologically unique features, such as an inverted lymph node structure (Binns, Reference Binns1982), and an unusual route for lymphocyte circulation (Pabst and Binns, Reference Pabst and Binns1989). Porcine lymphocyte populations are also unusual and feature abundant CD4CD8 double positive T-cells (Zuckermann, Reference Zuckermann1999), NK (Yang and Parkhouse, Reference Yang and Parkhouse1996; Yang and Parkhouse, Reference Yang and Parkhouse1997) and γδ T-cells (Pabst and Binns, Reference Pabst and Binns1989; Yang and Parkhouse, Reference Yang and Parkhouse1996, Reference Yang and Parkhouse1997). In pigs, classical cytotoxic T-cells are fewer in number, but large numbers of lymphocytes are CD8α+ (NK, γδ T-cells, NKT cells) with a strong potential for innate cytotoxic activity.
PRRSV gains entry through respiratory and reproductive mucosal surfaces and also causes disease primarily at mucosal sites. Therefore, a protective mucosal vaccine to PRRSV may be an appropriate strategy to control PRRS outbreaks effectively. Since the development of MLV-PRRS in the 1990s, the vaccine has been delivered to pigs by the intramuscular route. MLV-PRRS induces both humoral and cell-mediated immune responses (Foss et al., Reference Foss, Zilliox, Meier, Zuckermann and Murtaugh2002; Meier et al., Reference Meier, Galeota, Osorio, Husmann, Schnitzlein and Zuckermann2003) associated with reduced lung lesions (Mengeling et al., Reference Mengeling, Lager, Vorwald and Clouser2003a, Reference Mengeling, Lager, Vorwald and Koehlerb) and reduced viremia in younger pigs challenged with a homologous virus (Foss et al., Reference Foss, Zilliox, Meier, Zuckermann and Murtaugh2002). However, incomplete protection induced by MLV-PRRS against reinfections and against genetically variant viruses indicates a need for alternate vaccination approaches, such as mucosal immunization, to effectively control PRRS.
Mucosal live PRRSV vaccines
Our research group and many others have demonstrated that, in both PRRSV infected and MLV-PRRS vaccinated pigs, the virus-specific cell-mediated immune responses are either delayed or dampened. The poor immune response is attributed to virus-induced immunosuppression (Johnsen et al., Reference Johnsen, Botner, Kamstrup, Lind and Nielsen2002; Suradhat et al., Reference Suradhat, Thanawongnuwech and Poovorawan2003; Royaee et al., Reference Royaee, Husmann, Dawson, Calzada-Nova, Schnitzlein, Zuckermann and Lunney2004; Diaz et al., Reference Diaz, Darwich, Pappaterra, Pujols and Mateu2005; Renukaradhya et al., Reference Renukaradhya, Alekseev, Jung, Fang and Saif2010). In a study to enhance the MLV-PRRS-induced cell-mediated immune response, pigs were administered four injections of recombinant porcine IL-1 and IL-6 together, IL-12 alone, or cholera toxin alone within 1 week of intramuscular administration of MLV-PRRS. Results indicated that IL-12 and cholera toxin have the potential to enhance immune response to MLV-PRRS (Foss et al., Reference Foss, Zilliox, Meier, Zuckermann and Murtaugh2002). However, there was no difference in the level of viremia between pigs that received vaccine with or without adjuvant followed by homologous PRRSV challenge. In addition, due to lack of data on NA titers and lack of information on heterologous PRRSV challenge in vaccinated pigs, it is difficult to assume that this vaccination strategy induces protective immunity. Moreover, we need an MLV-PRRS that permits differentiation of animals that have been infected by field viruses from animals that have been vaccinated (DIVA). The importance of marker (DIVA) vaccines has been recognized and such vaccines have been constructed, and companion diagnostic tests have been developed that allow infected animals to be distinguished from vaccinated animals (van Oirschot et al., Reference Van Oirschot, Rziha, Moonen, Pol and Van Zaane1986). Pseudorabies and bovine herpesvirus 1 DIVA vaccines have been demonstrated to reduce transmission of wild-type virus in populations of pigs and cattle in the field (van Oirschot et al., Reference Van Oirschot, Rziha, Moonen, Pol and Van Zaane1986; Kit, Reference Kit1990). The implementation of regional elimination projects has overtaken immunology and epidemiology research, necessitating the development of tests to differentiate infected and vaccinated animals (DIVA).
In our study, pigs vaccinated intranasally with MLV-PRRS along with Mtb WCL were challenged with a heterologous PRRSV strain MN184 (Kim et al., Reference Kim, Lee, Johnson, Roof, Cha and Yoon2007); we detected a significant rescue in body weight gain, reduced lung pathology, enhanced PRRSV NA titers and reduced viremia at early time points post-challenge (Dwivedi et al., Reference Dwivedi, Manickam, Patterson, Dodson, Murtaugh, Torrelles, Schlesinger and Renukaradhya2011a). Immunologically, in pigs intranasally vaccinated with M. tb WCL were compared to those vaccinated without adjuvant or unvaccinated and challenged with strain MN184; an increased frequency of Th cells, Th/memory cells, γδ T-cells, DCs and activated Th cells, and a reduced frequency of Tregs were detected at both mucosal and systemic sites. Further, reduced secretion of immunosuppressive cytokines (IL-10 and TGF-β) and up-regulation of the Th1 cytokine IFN-γ in the blood and the lungs were detected (Dwivedi et al., Reference Dwivedi, Manickam, Patterson, Dodson, Murtaugh, Torrelles, Schlesinger and Renukaradhya2011a). Similar and more robust immune responses were detected in pigs vaccinated using MLV-PRRS in combination with M. tb WCL and challenged with a homologous PRRSV VR2332 (Dwivedi et al., unpublished data).
PRRSV-specific lymphocyte responses in pigs vaccinated with M. tb WCL and challenged with strain MN184 or VR2332 were also generated against internal viral protein of PRRSV, indicated by significantly increased M protein-specific IFN-γ and IL-12 cytokine response in re-stimulated lung MNC and PBMCs (Dwivedi et al., Reference Dwivedi, Manickam, Patterson, Dodson, Murtaugh, Torrelles, Schlesinger and Renukaradhya2011a). This result has provided the evidence that PRRSV internal viral protein-specific epitopes-targeted response is augmented by intranasal vaccination of MLV-PRRS with M. tb WCL, and that could be responsible for increased cross-protective immune response. Our results are consistent with those from an intranasal influenza vaccination study in mice wherein adjuvanted live or inactivated influenza virus vaccine enhanced the virus-specific cytotoxic T-lymphocyte and central memory immune cell response against influenza internal viral proteins, resulting in cross-protective immunity (Guillonneau et al., Reference Guillonneau, Mintern, Hubert, Hurt, Besra, Porcelli, Barr, Doherty, Godfrey and Turner2009). Intranasal immunization with adjuvant α-GalCer and inactivated hemagglutinin vaccine of influenza derived from H1N1, H3N2 and H5N1 strains induce effective cross-protection against both heterologous and heterosubtypic influenza viral strains through invariant NKT cell-mediated immune activation in the lungs (Kamijuku et al., Reference Kamijuku, Nagata, Jiang, Ichinohe, Tashiro, Mori, Taniguchi, Hase, Ohno, Shimaoka, Yonehara, Odagiri, Tashiro, Sata, Hasegawa and Seino2008).
Previous studies related to passive protection provided by PRRSV (NAs indicated that: an NA titer of 1/16 protects pregnant sows against reproductive failure and also blocks transplacental infection (Osorio et al., Reference Osorio, Galeota, Nelson, Brodersen, Doster, Wills, Zuckermann and Laegreid2002); an NA titer of 1/8 or higher protects piglets against development of viremia; and NA titers of 1/32 provide sterilizing immunity (Lopez et al., Reference Lopez, Oliveira, Garcia, Kwon, Doster and Osorio2007)). Thus, vaccines capable of inducing NA titers of more than 1/16 should protect pigs from clinical disease and may help in PRRS clearance from the body. Pigs immunized intranasally with MLV-PRRS along with M. tb WCL had NA titers of more than 1/32 from day 7 until day 35 post-immunization (Dwivedi et al., Reference Dwivedi, Manickam, Patterson, Dodson, Weeman and Renukaradhya2011b). In contrast, pigs inoculated with MLV-PRRS alone (without any adjuvant), had NA titers of less than 1/16 at multiple time points post-immunization (Dwivedi et al., Reference Dwivedi, Manickam, Patterson, Dodson, Weeman and Renukaradhya2011b). In pigs that were immunized with MLV-PRRS plus M. tb WCL and challenged with the heterologous PRRSV MN184 strain, NA titer of more than 1/16 persisted until PID 60. This was associated with significantly reduced viral load in the blood of challenged pigs (Dwivedi et al., Reference Dwivedi, Manickam, Patterson, Dodson, Weeman and Renukaradhya2011b). Moreover, the enhanced NA titer in pigs which received MLV-PRRS plus M. tb WCL was associated with augmented Th1 and suppressed immunosuppressive responses.
Following intranasal vaccination of pigs with MLV-PRRS and no adjuvant, the numbers of virus-specific IFN-γ secreting cells were in the range of 50–100 cells per million PBMCs and they persisted until 10 weeks post-challenge (Dwivedi et al., Reference Dwivedi, Manickam, Patterson, Dodson, Murtaugh, Torrelles, Schlesinger and Renukaradhya2011a). In contrast, pigs vaccinated against Aujeszky's disease virus, had 200–300 IFN-γ secreting cells per million PBMCs (Meier et al., Reference Meier, Galeota, Osorio, Husmann, Schnitzlein and Zuckermann2003). In these pigs IFN-γ secreting cells were mainly CD4+CD8+ T-cells. This observation indicates that PRRSV induces relatively weak IFN-γ response in infected pigs. However, pigs immunized intranasally with MLV-PRRS plus M. tb WCL and challenged with PRRSV MN184 had greater than 300 IFN-γ secreting cells per million PBMCs, associated with greater than a 2-fold increase in the frequency of total CD4+CD8+ T-cells at day 15 post-challenge (Dwivedi et al., Reference Dwivedi, Manickam, Patterson, Dodson, Murtaugh, Torrelles, Schlesinger and Renukaradhya2011a). These results strongly indicate that immunization of pigs with MLV-PRRS in combination with a potent adjuvant administered by the intranasal route has the potential to induce enhanced IFN-γ response which could reduce PRRSV replication and dampen the severity of clinical PRRS.
Optimal production of reactive oxygen species (ROS) by immune cells is essential for antimicrobial activity (Martin and Edwards, Reference Martin and Edwards1993; Quinn and Gauss, Reference Quinn and Gauss2004; Acker, Reference Acker2005; Chvanov et al., Reference Chvanov, Petersen and Tepikin2005), and excess production of ROS causes apoptosis/necrosis of infected and bystander cells (Clutton, Reference Clutton1997; Vaughan, Reference Vaughan1997; Halliwell and Gutteridge, Reference Halliwell and Gutteridge2006). ROS are one of the important mediators of diffused PRRSV-mediated pathology in the lungs and lymph nodes of infected pigs (Sirinarumitr et al., Reference Sirinarumitr, Zhang, Kluge, Halbur and Paul1998). BAL cells and PBMCs in pigs intranasally vaccinated (MLV-PRRS plus M. tb WCL) and challenged with either PRRSV VR2332 or MN184 strain had higher levels of ROS production compared to mock control pigs; but the ROS levels in pigs that received MLV-PRRS plus M. tb WCL were significantly lower than in unvaccinated challenged animals (Binjawadagi et al., Reference Binjawadagi, Dwivedi, Manickam, Torrelles and Renukaradhya2011). This result suggests that M. tb WCL adjuvant effects optimally induced ROS production which helped to boost the immunity to PRRSV without causing lung pathology in virus-challenged pigs.
Enhanced antigen-specific proliferation of lung lymphocytes in vaccinated (MLV-PRRS plus Mtb WCL) MN184 challenged pigs, and increased antigen-specific proliferation of CD8+ T-cells in the blood of similarly vaccinated PRRSV VR2332 challenged pigs have been detected (Manickam et al., unpublished data). Nitric oxide (NO′) has been shown to inhibit the viral replication in influenza and herpes simplex virus type 1 infections (Croen, Reference Croen1993; Rimmelzwaan et al., Reference Rimmelzwaan, Baars, De Lijster, Fouchier and Osterhaus1999; Akerstrom et al., Reference Akerstrom, Mousavi-Jazi, Klingstrom, Leijon, Lundkvist and Mirazimi2005). Consistent with this, the level of NO′ was significantly higher in the lungs of intranasally vaccinated (MLV-PRRS plus M. tb WCL) virus-challenged pigs (Manickam et al., unpublished data). These results support the conclusion that elicited protective humoral and cell-mediated immune responses were associated with suppression of ROS-mediated lung damage and also enhanced NO′ production in pigs vaccinated with MLV-PRRS in combination with a potent adjuvant. Thus, it is possible to generate protective anti-PRRSV immunity against reinfections caused by genetically divergent strains of PRRSV.
Mucosal killed PRRSV vaccines
Although many reports demonstrated satisfactory immunity induced by MLV-PRRS in growing pigs, others reported reversion of the vaccine virus and the presence of reverted virulent PRRSV in unvaccinated sows maintained in the same farm premises (Madsen et al., Reference Madsen, Hansen, Madsen, Strandbygaard, Botner and Sorensen1998; Nielsen et al., Reference Nielsen, Oleksiewicz, Forsberg, Stadejek, Botner and Storgaard2001, Reference Nielsen, Botner, Bille-Hansen, Oleksiewicz and Storgaard2002). Also recombination between vaccine and field strains associated with revertant PRRSV strains that are pathogenic in sows and piglets has been reported (Nielsen et al., Reference Nielsen, Oleksiewicz, Forsberg, Stadejek, Botner and Storgaard2001; Mengeling et al., Reference Mengeling, Clouser, Vorwald and Lager2002; Murtaugh et al., Reference Murtaugh, Xiao and Zuckermann2002a, Reference Murtaugh, Yuan, Nelson and Faabergb; Li et al., Reference Li, Fang, Xu, Liu, Gao, Jiang, Chen and Xiao2009). In addition, identification of mutations in multiple vaccine-derived isolates in identical positions of the viral genome has suggested a strong parallel selective pressure on critical positions in the vaccine virus in the field (Nielsen et al., Reference Nielsen, Oleksiewicz, Forsberg, Stadejek, Botner and Storgaard2001). Vaccine-derived virus has been isolated from fetuses, stillborn and dead piglets, indicating the spread of live vaccine virus from the vaccinated growing pigs in the neighboring herd to non-vaccinated sows, and the involvement of vaccine virus in reproductive problems (Nielsen et al., Reference Nielsen, Botner, Bille-Hansen, Oleksiewicz and Storgaard2002). Also, vaccine-derived PRRSV isolates are common in swine populations (Key et al., Reference Key, Guenette, Yoon, Halbur, Toth and Meng2003), and some vaccine-derived field strains of PRRSV are actually causing diseases, at least under experimental conditions (Opriessnig et al., Reference Opriessnig, Halbur, Yoon, Pogranichniy, Harmon, Evans, Key, Pallares, Thomas and Meng2002). Therefore, safety of widely used MLV-PRRS remains questionable as the vaccine-derived mutated virus can spread to unvaccinated herds and increase the PRRS incidence by many-fold in the future. Since pregnant sows are not protected by vaccination, prevention of PRRSV infection in sows is a big challenge to swine producers. Control of PRRS in sows is very important because this prevents transmission of the virus to susceptible pigs through vertical and horizontal routes. Therefore, live PRRSV vaccine is not safe for use where growing pigs and pregnant sows are maintained in the same premises, and the use of live PRRSV vaccine in sows, gilts and breeding boars is not practiced. Unfortunately, none of the available killed PRRSV vaccines elicits protective immunity. All these limitations made research on the development of a cross-protective killed PRRSV vaccine a high priority.
Several attempts have been made to develop protective killed PRRSV vaccines using recombinant PRRSV protein-expressing plasmid DNA and inactivated PRRSV administered with or without adjuvant by a parenteral route, but the results are not satisfactory (reviewed in Charerntantanakul, Reference Charerntantanakul2009). Studies performed to determine the efficacy of killed PRRSV vaccine administered intranasally along with adjuvants CpG ODN (TLR-9 ligand) (Zhang et al., Reference Zhang, Tian and Zhou2007) and oligodeoxynucleotides containing synthetic immunostimulatory motifs (Zhang et al., Reference Zhang, Tian and Zhou2006) resulted in augmented PRRSV-specific antibody response, enhanced antigen-specific T-cell proliferation, and increased secretion of cytokines (IFN-γ and IL-6). It is possible to induce PRRSV-specific NA response when the vaccine virus undergoes proper inactivation processes, such as by UV irradiation or treatment with binary ethylenimine; these treatments help to preserve virus-NA-specific epitopes. In addition, such an inactivated vaccine candidate co-administered with incomplete Freund's adjuvant elicits a protective immune response to a homologous viral challenge (Vanhee et al., Reference Vanhee, Delputte, Delrue, Geldhof and Nauwynck2009; Darwich et al., Reference Darwich, Diaz and Mateu2010). It is noteworthy that the suppressive effect on IFN-α production was lost when the PRRSV was inactivated by UV light (Vanhee et al., Reference Vanhee, Delputte, Delrue, Geldhof and Nauwynck2009).
Oral immunization with PRRSV nucleocapsid as a genetic fusion with cholera toxin resulted in virus-specific local intestinal mucosal antibody response with undetectable immune response in vaginal secretions (Hyland et al., Reference Hyland, Foss, Johnson and Murtaugh2004). Therefore, the development of a protective killed PRRSV vaccine to replace widely used MLV-PRRS to eliminate issues with revertant vaccine viruses remains as a challenge to researchers and to the swine industry.
Lack of success in developing a protective killed PRRSV vaccine reflects the inability to properly present killed PRRSV antigens to the immune system, or like live PRRSV, killed virus may also be immunosuppressive. In addition, the antigenic mass present in the killed PRRSV vaccine may be an issue in inducing protective immunity, suggesting the need for potent adjuvants and/or novel delivery systems to improve the immune response to killed PRRSV vaccines. One promising area of technology developed over the past few years is nanotechnology based vaccine delivery, an important area of research in the 21st century (Panyam and Labhasetwar, Reference Panyam and Labhasetwar2003; Duncan, Reference Duncan2005; Nel et al., Reference Nel, Xia, Madler and Li2006). Nanoparticles offer the advantage of increasing the efficiency of drug and vaccine delivery, and also possess adjuvant properties (Gupta et al., Reference Gupta, Chang and Siber1998). In addition, due to the particulate nature of nanoparticles and the inherent ability of APCs to passively phagocytize particulate matter (Inaba et al., Reference Inaba, Inaba, Naito and Steinman1993), nanoparticles protect killed vaccine antigens from degradation by proteases present at mucosal surfaces and aid in pulse release of the vaccine (Gupta et al., Reference Gupta, Chang and Siber1998). The development of nanoparticle-based mucosal vaccines is gaining increased attention with respect to the induction of protective mucosal immunity against infectious pathogens. Therefore, nanoparticles may serve as appropriate vehicles to deliver killed vaccines to mucosal surfaces, and the presence of abundant APCs (Mɸs and DCs) at mucosal sites could facilitate uptake of nanoparticles and induce protective immunity against diseases like PRRS.
A killed influenza virus vaccine entrapped in nanoparticles containing E. coli heat labile toxin as an adjuvant administered intranasally to mice, rabbits and pigs elicited protective immunity (Singh et al., Reference Singh, Briones and O'hagan2001). Immune responses elicited in pigs by intranasal delivery of nanoparticles based vaccine was significantly better than those elicited by immunization by intramuscular administration of the vaccine (Singh et al., Reference Singh, Briones and O'hagan2001). Nanoparticles prepared from PLGA [poly (DL-lactide-co-glycolide)] containing hepatitis B, rotavirus, influenza or parainfluenza virus delivered to mucosal sites of mice generated protective immunity (Singh et al., Reference Singh, Briones and O'hagan2001; Shephard et al., Reference Shephard, Todd, Adair, Po, Mackie and Scott2003; Nayak et al., Reference Nayak, Panda, Ray and Ray2009; Thomas et al., Reference Thomas, Gupta and Ahsan2009). Biodegradable and biocompatible PLGA nanoparticles are free from any toxicity in animals and humans, they are safe for use in humans, and are approved by the U.S. Food and Drug Administration (Duncan, Reference Duncan2005; McNeil, Reference McNeil2005; Semete et al., Reference Semete, Booysen, Lemmer, Kalombo, Katata, Verschoor and Swai2010). The use of nanoparticles as a vaccine delivery system allows flexibility in the size, charge and surface properties of the formulations for targeted delivery of vaccine to mucosal immune cells (Lawson et al., Reference Lawson, Norton and Clements2011). At present our laboratory is working on a nanoparticles-based killed PRRSV vaccine to augment protective immunity to PRRSV. Our results are encouraging in reducing gross and microscopic lung pathology as well as in reducing PRRSV load in the lungs and blood of immunized, homologous and heterologous PRRSV challenged pigs (Dwivedi et al., manuscript in preparation).
Conclusion
The mucosal immune system in the pig respiratory tract is only partially understood compared to information known in rodents and humans. There is, therefore, a need for additional research aimed at improving our knowledge of the pig respiratory mucosal immune system. There are positive outcomes of mucosal vaccinology research in effective control of several respiratory viral pathogens, while a need exists for improved vaccines against PRRS, a porcine respiratory disease of major importance in the pig industry. It therefore seems important to conduct research aimed at adequately activating the pig respiratory immune system to generate protective immunity against PRRS. Importantly, experimental studies have demonstrated that it is possible to generate heterologous protection by effective mucosal immunization strategies. We note that protective mucosal immunity against a vaccine could be achieved only when the vaccine was co-administered with a suitable adjuvant and/or a delivery system; this is critical for overcoming the immune regulatory system at mucosal sites. Pilot studies performed by us on intranasal delivery of PRRSV vaccines in pigs, and by others in rodent models, to control other respiratory viral pathogens using both live and killed viral vaccines, have provided ample evidence of feasibility and effectiveness of mucosal vaccines. Thus, adaption of innovative strategies to control PRRS by mucosal immunization may help to control PRRS more effectively than by using conventional parenteral immunization.
