The vexing problem with PRRS immunity
Porcine reproductive and respiratory syndrome (PRRS) is an economically important disease of swine characterized by abortion, stillbirth and weak-born pigs. In its non-reproductive form, this syndrome affects younger pigs more severely than older animals, which clinically manifested by reduced growth rate, feed efficiency and pneumonia that can be made more severe by co-infection with other pathogens (Thacker, 2004; Klinge et al., 2009). The etiologic agent for this disease is an RNA virus (PRRSV) that belongs to the family Arteriviridae, which targets macrophages for infection (Wensvoort et al., 1991; Collins et al., 1992). PRRSV exhibits a significant degree of genetic diversification (Murtaugh et al., 1998; Key et al., 2001; Goldberg et al., 2003). There are two genotypes of PRRSV, denoted Types 1 (European type) and 2 [North American (NA) type]. In North America, Type 2 PRRSV is the dominant genotype in the field, which based on a comprehensive collection of ORF5 sequences, Type 2 PRRSV has been classified into nine distinct lineages (Shi et al., 2010). Notably, over the last 10 years a major shift in the genetic composition of Type 2 PRRSV has occurred in the Midwest USA as a result of multiple introductions of virus from Canada (Shi et al., 2013). These viruses are now endemic and have gradually replaced the original local virus populations that belong lineages 6 to 9 (USA-like), and have been replaced predominantly by lineage 1 (Canadian-like) viruses. Included in lineage 1 are the notorious PRRS viruses with an RFLP 1-8-4 and 1-22-2 types, which, as compared to the original USA-like lineages 6- 9, appear to be more virulent, grow to larger titers in pigs and are more readily shed in aerosols (Shi et al., 2013).
Under experimental conditions, the currently available modified live virus (MLV) vaccine against this pathogen has been shown to provide immunized pigs adequate protection from subsequent challenge with a non-genetically divergent (homologous) strain (Lager et al., 1997a, b), and partial protection against infection by a genetically divergent (heterologous) strains (Osorio et al., 1998; Lager et al., 1999; Mengeling et al., 2003a, b). Based on the steady increase in the prevalence of PRRS in the commercial swine operations in the United States as reported by the USDA APHIS as well as the results from experimental vaccination and challenge studies (Halbur, 2003), it can be reasonably stated that the overall level of protective immunity provided to swine by currently available PRRS modified live virus vaccines in commercial settings can be considered inadequate. This situation can be attributed to the apparent lack of development of sufficiently strong levels of protective immune effector mechansism elicited by either wild-type and modified live virus vaccines. This situation is evidenced by the fact that the infection of pigs with wild type PRRSV (Nelson et al., 1994; Loemba et al., 1996; Vezina et al., 1996; Yoon et al., 1995; Albina et al., 1998b; Gonin et al., 1999) or their vaccination with a live attenuated form of this virus (Labarque et al., 2000; Ostrowski et al., 2002) elicits an exuberant production of non-neutralizing antibodies. In contrast, a transient T cell mediated PRRSV-specific lymphoproliferative response is detected at 4 weeks post infection and lasts an additional 9 (Bautista and Molitor, 1997) to 14 weeks (Lopez-Fuertes et al., 1999). Moreover, during this time interval, limited quantities of IFN−γ secreting cells (SC) are generated (Meier et al., 2003; Xiao et al., 2004). Interestingly, in the absence of additional antigenic stimulation this polarity reverses within the ensuing 5 months, as manifested by a decreasing antibody response and a gradual increase in the intensity of the IFN−γ response (Meier et al., 2003). The initial antibody-dominated immune response is not the result of insufficient antigenic stimulation, since neither the inclusion of a commercial adjuvant during primary vaccination (Meier et al., 2003) nor booster immunizations of previously heavily vaccinated pigs (Bassaganya-Riera et al., 2004) enhances virus-specific cell-mediated immunity (CMI). Thus, PRRSV seems to inherently stimulate an imbalanced immune response characterized by an abundant virus-specific non-neutralizing antibody response and a limited, but potentially protective, T helper (Th) 1–like IFN−γ response (Murtaugh et al., 2002). Remarkably, a significant variability within the swine population in regards to their innate and adaptive immune responses to PRRSV has been observed (Xiao et al., 2004; Royaee et al., 2004). This variability is likely responsible for the inconsistency of the clinical outcomes seen upon challenging either naïve or previously immunized pigs with virulent PRRSV (Labarque et al., 2003; Mengeling et al., 2003a,b). Thus, the ultimate outcome of the interaction between this virus and its host will be determined by the ability of the host to overcome the inherent PRRS virus propensity to prevent the timely development of protective innate or adaptive immunity capable of inhibiting its infectious process.
Definition of PRRSV protective immunity
For a clinician the raison d'être for studies on the innate and adaptive immune response to a virus is develop strategies to elicit protective immune responses. In the case of PRRSV, the identification of the immunologic mechanism(s) responsible for mediating protective immunity against PRRSV poses a significant challenge for several reasons. First, while vaccination can decrease the duration and magnitude of viremia following an experimental challenge (van Woensel et al., 1998; Verheije et al., 2003), the reduction in viremia is not necessarily associated with a commensurate amelioration in the severity of other clinical parameters associated with PRRSV infection, such as a lessened rate of weight gain, fever, respiratory distress or virus transmission to sentinel pigs (Nodelijk et al, 2001; Labarque et al., 2003; Mengeling et al., 2003a). In studies conducted by van der Linden et al. (2003), a similar lack of correlation between viremia and clinical signs was also noted when two different age groups of non-immune pigs were infected with PRRSV. In theses studies a greater frequency of viremia with an accompanying higher virus titer was found in younger animals (2 months of age) as compared to older pigs (6 months of age), while the later exhibited more severe clinical signs. The difficulty of deciphering PRRSV biology is further revealed by the marked degree of variability and irreproducibility of consecutive trials conducted by the same investigators (Labarque et al., 2003; Mengeling et al., 2003a,b).
In the case of pseudorabies virus a positive association between protection from disease and the intensity of the IFN−γ response as well as virus neutralizing antibodies was established (Zuckermann et al., 1998; 1999; van Rooij et al., 2004). However, in the case of PRRSV, the association between protective immunity and humoral and/or cellular immune adaptive immune responses elicited by vaccination has been more difficult to establish in the respiratory form of the disease a similar relationship concerning PRRSV may or may not be observed (Meier et al., 2004). Thus, the identification of immunologic mechanism(s) responsible for mediating protective immunity against PRRSV will require studies that monitor both the humoral and cellular immune response to vaccination (Meier et al, 2003, 2004), and measure the extent of reproductive failure and pneumonia caused by this virus as a measurement of the degree of protection (Lager et al., 1999). Notably, in recent, related field studies we have noticed a positive correlation between the reduction of abortion/still births in sows and the relative frequency of PRRSV-specific IFN−γ SC in their blood (Lowe et al., 2004), although this correlation is not always evident (Lowe et al., 2005)
Studies by Osorio et al. (2002) have already shown that virus-neutralizing antibodies are capable of providing sterilizing immunity against PRRSV-induced reproductive failure. Strategies designed to shift the bias of the initial reaction to PRRSV from the strong elicitation of non-neutralizing antibody production towards a greater Th1-like immune response including the development of virus neutralizing antibodies are worth exploring since they could conceivably lead to the development of an improved vaccine against this pathogen. The need to develop such methodology is made palpable by the unusual kinetics of the immune response to this virus, as evidenced by the lack of a marked increase in the cell-mediated immune response upon vaccination and subsequent challenge with virulent virus. The IFN−γ response to PRRSV therefore appears to be determined at the time of the first exposure to this pathogen and is only minimally affected by re-exposure. In addition, similar limited changes in the IFN−γ response have been observed upon challenge of vaccinated pigs with wild-type virus (Foss et al., 2002) or booster immunization with MLV vaccine (Meier et al., 2003). Remarkably, the T-cell proliferative response to PRRSV was also not increased by a booster immunization in pigs that had previously been repeatedly exposed to a MLV vaccine, but rather appeared to be suppressed as compared to that elicited by the same vaccine in naïve pigs (Bassaganya-Riera et al., 2004). The mechanism responsible for this unusual effect is currently unknown, but might be related to the persistence of the virus in lymphoid tissues associated with the site of infection (Xiao, et al., 2004). Such sustained presence of virus could adversely affect a subsequent response due to an inherent yet unknown biological property of PRRSV. Clearly, further studies will be required to clarify how the outcome of a PRRSV infection is influenced by the intensity of the IFN−γ response mediated by memory T cells as well as by virus-neutralizing antibodies.
The innate immune response to PRRSV
One characteristic of PRRSV infection that probably contributes to the retarded development of a specific cell-mediated immune response is the apparent lack of an adequate IFN−α response to the viral infection. Usually, virus-infected cells secrete type I IFN and the released cytokine interacts with a subset of naïve T cells to promote their conversion into virus-specific IFN−γ SC (Cella et al., 2000; Cousens et al. 1999; Kadowaki et al., 2000; Biron, 2001; Levy et al., 2003). In contrast, the IFN−α response to exposure to PRRSV is nearly non-existent. Production of IFN−α in the lungs of pigs acutely infected with PRRSV was either almost undetectable, or 159-fold lower than that induced by another pathogen, porcine respiratory coronavirus (PRCV) (Buddaert et al. 1998; van Reeth et al., 1999). Such lack of efficient stimulation of IFN−α production by a pathogen would be expected to have a significant impact on the nature of the host’s adaptive immune response, since IFN−α up-regulates IFN−γ gene expression, and thus controls the dominant pathway that promotes the development of adaptive immunity, namely, T cell-mediated IFN−γ responses and peak antiviral immune defenses (Cousens et al. 1997; Levy et al., 2003). In this regard, it has become evident that the link between innate and adaptive immunity in viral infections occurs through the interaction of dendritic cells with type I interferon (Montoya et al., 2002; Though, 2004) and the dendritic-cell controlled polarization of T-cell function (Kapsenberg, 2003). The production of IFN−α by plasmacytoid dendritic cells (pDCs) has an autocrine effect that promotes their functional and phenotypic activations- events necessary for their optimal expression of co-stimulatory molecules and subsequent ability to cause naïve T cells to differentiate into IFN−γ -SC (Cella et al., 2000; Kadowaki et al., 2000; Fitzgerald- Bocarsly et al., 2002; Montoya et al., 2002; Honda et al., 2003). Presumably, PRRSV is a poor inducer of IFN−α production by pDCs since unlike transmissible gastroenteritis virus (Charley et al., 1990; Nowacki et al., 1993) and type−α CpG oligonucleotides (Guzylack-Piriou et al., 2004) it fails to stimulate the secretion of IFN−α from cultured porcine PBMC (Albina et al., 1998a; Calzada-Nova, et al., 2010). Direct examination of the outcome of the interaction of PRRSV with porcine pDCs will likely reveal important information on the immunobiology of this virus, especially since this virus is susceptible to the antiviral effects of IFN−α (Albina et al., 1998a).
Using mouse models of anti-viral immunity it has been shown that in the absence of IFN−α/β production, the cytokine IL-12 (Orange and Biron 1996) can increase the virus-specific IFN−γ production by T cells (Cousens et al. 1999). Thus, two alternative routes (IL-12- or type I IFN-dependent) can lead to an adaptive Th 1 cell-mediated immune response with potent antiviral effects (Biron, 2001). According to a scenario involving the presence of less than a requisite amount of IFN−α , IL-12 could provide the necessary impetus for the development of an anti-viral IFN−γ response. In this regard, IL-12 mRNA has been detected in porcine macrophages infected with PRRSV (Thanawongnuwech et al., 2001), and transiently in the lungs of PRRSV-infected pigs (Chung and Chae, 2003). However, this pathogen is also apparently a poor stimulator of IL-12 production, since a negligible quantity of IL-12 mRNA or protein was produced by porcine PBMC exposed in vitro to PRRSV (Royaee et al., 2004; Calzada-Nova et al., 2010).
Approaches to improve the stimulation of protective immunity to PRRS virus
To compensate for the apparent inadequate innate cytokine stimulation elicited by the infection of pigs with PRRSV, novel adjuvants have been used during immunization. The administration of IL-12 in combination with a live or killed PRRSV vaccine resulted in an increased lymphoproliferative response to this virus (Wee et al., 2001), as well as an enhanced the host IFN−γ response to a modified live PRRSV vaccine (Foss et al., 2002). Similarly, the injection of IFN−α provided exogenously in the form of an expressible cDNA (pINA) was found to exert an adjuvant effect on the vaccine-induced IFN−γ response to PRRS virus (Meier et al., 2004). Remarkably, no significant alteration in the development of the humoral immune response has observed as a result of either of these treatments. Thus, even with such interventions at the initiation of PRRSV immunization, the usual rapid onset of anti-PRRSV antibody production and delayed appearance of VN antibodies (Labarque et al., 2000; Ostrowski et al., 2002; Meier et al., 2003) still occurs. We have observed that the provision of IFN−α cDNA has a more pronounced and sustained effect on the intensity of the cell-mediated immune response (Meier et al., 2004) . Likewise, the introduction of a known inducer of IFN−α production in pigs, poly I:C (Derbyshire and Lesnick, 1990), during vaccination was found to temporarily amplify the quantities of PRRSV-specific IFN−γ SC, but is not as efficient as the IFN−α encoding plasmid at enhancing the IFN−γ response to the vaccine. The observation that the inclusion of either IL-12 and IFN−α during immunization increased the intensity of the IFN−γ response to PRRSV validates the proposed role of these two innate cytokines in directing the in vivo differentiation of swine Th1 cells, and helps explain the poor virus-specific IFN−γ response that normally develops as a result of the exposure of pigs to PRRSV (Meier et al., 2003; Xiao et al., 2004). It should be noted that the efficacy of a PRRS MLV vaccine is improved by simply producing the vaccine virus in a porcine alveolar macrophage rather than in simian cells (Calzada-Nova et al., 2012). Clearly, regarding the quality and effectiveness of the protective immunity elicited by a PRRS virus biologic there is room for improvement. Consistent with our previously postulated notion of an important role of IFN-α in eliciting protective immunity against PRRSV (Royaee et al., 2004; Meier et al., 2004) in recent studies we found that the PRRS live virus vaccine strain G16X developed in our laboratory, which is capable of eliciting a significant IFN-α response in vivo, stimulates the development of significant levels of protective immunity against highly virulent NA type PRRSV isolates that are genetically divergent (heterologous) to the vaccine. In these studies, the novel PRRS live virus vaccine G16X, which was derived from a naturally non-virulent PRRS virus and belongs to NA lineage 5, was capable of eliciting adequate levels of protective immunity against the genetically divergent and high virulence PRRSV isolate NADC20, which as an atypical (ATP) isolate belonging to lineages 8, as well as against a more modern lineage 1 (Canadian-like) isolate with an RFLP 1-22-2 that was isolated from a severe 2011 outbreak of PRRS in a breeding herd located in the American Midwest (Zuckermann et al., unpublished). The ability of this novel vaccine virus to provide adequate levels of protective immunity to heterologous strains is consistent with the growing evidence that the degree of genetic homology of PRRSV ORF5 between the challenge strain and the vaccine is not predictive of the degree of protective immunity elicited (Prieto et al., 2008), but rather, as we have postulated, has more to do with the type of host cell in which the vaccine is prepared (Calzada-Nova et al., 2012), as well the biological properties of the vaccine.It is apparent that infection or vaccination with PRRSV elicits in swine an immune response that is insufficient to provide satisfactory protective immunity from the field virus. PRRS virus elicits an immune response that is characterized by an abundance of non-neutralizing antibodies and a paucity of IFN−γ SC. The molecular pathway responsible for generating this type of immunity is unknown at this time, but based on our studies (Meier et al., 2004) it likely involves the limited induction of IFN−α and IL-12 production and/or inherent structural elements of the virus that promote such a response. We propose that the strong humoral immunity bias of the host response to PRRSV is mostly responsible for the difficulties in the development of a vaccine deemed effective in the field.