Review Article

VIRAL IMMUNOLOGY Volume X, Number X, 2015 ª Mary Ann Liebert, Inc. Pp. 1–11 DOI: 10.1089/vim.2015.0027

Immune Responses to Virulent and Vaccine Strains of Infectious Bronchitis Viruses in Chickens Rajesh Chhabra,1,2 Julian Chantrey,1 and Kannan Ganapathy1

Abstract

Infectious bronchitis (IB) is an acute and highly contagious chicken viral disease, causing severe economic losses to poultry producers worldwide. In the last few decades, infectious bronchitis virus (IBV) has been extensively studied, but knowledge of immune responses to virulent or vaccine strains of IBVs remains limited. This review focuses on fundamental aspects of immune responses against IBV, including the role of pattern recognition receptors (PRRs) in identification of conserved viral structures and the role of different components of innate immunity (e.g., heterophils, macrophages, dendritic cells, acute phase protein, and cytokines). Studies on adaptive immune activation and the role of humoral and cellular immunity in IBV clearance are also reviewed. Multiple interlinking immune responses are essential for protection against virulent IBVs, including passive, innate, adaptive, and effector T cells active at mucosal surfaces. Although the development of approaches for chicken transcriptome and proteome analyses have greatly helped the understanding of the underlying genetic mechanisms for immunity, there are still major knowledge gaps, such as the role of mucosal and cellular responses to IBVs. In view of recent reports of emergent IBV variants in many countries, there is renewed interest in a more complete understanding of poultry immune responses to both virulent and vaccine strains of IBVs. This will be critical for developing new vaccine or vaccination strategies and other intervention programs.

noticed, usually persisting for 5–7 days and diminishing within 10–14 days (89). Within 3 days of infection, chickens can also appear depressed, with a marked decrease in weight gain and food consumption (81). Mortalities can vary between 0% and 82%, depending on the strain of the virus involved and the birds’ age and immune status (55). Some strains of IBV are nephropathogenic and can produce necrotizing interstitial nephritis with high mortality in young chickens (55). IBV has a single-stranded RNA enveloped by four structural proteins: the spike (S), membrane glycoproteins (M), a small membrane envelope protein (E), and the internal nucleocapsid protein (N) (96). The S protein consists of two subunits: S1, which forms the globular head of the spike, and S2, which anchors the S protein in the membrane. The S1 subunit induces the release of neutralizing and hemagglutination inhibiting (HI) antibodies IBV (53), while the N protein reportedly induces cell-mediated immunity (94). Moreover, vaccination of chickens with the recombinant S1 (rS1), rN, or H120 has also been reported to be associated with induction of a cellular immune response as demonstrated by in vitro chicken interferon (ChIFN)-c production by splenocytes of vaccinated birds (72). An antibody response to

Introduction

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nfectious bronchitis virus (IBV) causes a highly contagious infection in chickens that is frequently followed by opportunistic bacterial infections, which promotes more severe diseases and may eventually lead to ranging economic losses for the farm and abattoir (55). IBV has an incubation period of 24–48 h, and viral spread occurs rapidly among chickens in a flock by aerosol and mechanical means, resulting in widespread infection and severe economic losses to poultry producers. Chickens are the most significant natural hosts of IBV, though other species such as geese, ducks, and pigeons may also play a role in the spread of IBV strains throughout the world (5). IBV replicates primarily in the chicken respiratory tract, but also grows in the kidney, oviduct, and various other organs, sometimes producing lesions (1,7,39). While chickens of all ages are susceptible, very young chicks exhibit more severe respiratory signs and much higher mortality compared with older birds (89). IBV respiratory infection causes characteristic, but not pathognomonic, signs such as gasping, coughing, tracheal rales, and nasal discharge (89). Edematous, inflamed periorbital tissue and swollen sinuses may occasionally be 1 2

University of Liverpool, Institute of Infection & Global Health, School of Veterinary Science, Neston, United Kingdom. College Central Laboratory, Lala Lajpat Rai University of Veterinary & Animal Sciences (LUVAS), Hisar, India.

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the M protein has also been reported in chicks vaccinated with either live or inactivated IBV (53). IBV is characterized by the capacity to evolve both by spontaneous mutation and by genetic recombination, mainly in the hypervariable regions of the S gene (95). Vaccination has been the predominant prevention strategy, but IBV’s ability to evolve has reduced vaccine effectiveness and protection (15). In order to develop better control and prevention strategies, the immune response to IBV needs to be better characterized. There have been few studies investigating the innate immune response to IBV in chicken, limiting knowledge on this subject. Although innate factors such as heterophils, macrophages, natural killer (NK) cells, and complement and pattern recognition receptors (PRRs) have been suggested to be involved in regulating the immune response to IBV, many questions remain to be addressed. The chicken immune response to IBV has mainly concentrated on the humoral response to IBV vaccination. IBVspecific antibody levels in serum, lachrymal fluid (tears), and nose and trachea secretion were measured using an enzymelinked immunosorbent assay (ELISA), virus neutralization (VN), or HI tests (14,15,31,38). In general, serum antibody levels do not correlate with protection, but local antibodies are believed to contribute in the protection of respiratory tract epithelium (87). Cytotoxic T cell (CTL) and cytokine responses in chickens have also been investigated during IBV infections and have been shown to correlate with early decreases in infection and clinical signs (82,86,92). In addition, increased expression of CTL-associated genes such as granzymes and perforin have been reported after IBV vaccination (47,78) and found to be significantly correlated with tracheal protection against homologous IBV challenge (78). Different aspects of immunity to IBV have been previously reviewed (8,15,88). This review aims to update and summarize some aspects of immune responses against IBV and to highlight areas for further research that may enhance the understanding of the immune responses to virulent and vaccine strains of IBVs. Innate Immunity

The innate immune response is the first line of defense and comprises a set of mechanisms, molecules, and cells that often nonspecifically target invasive pathogens as they enter the potential host. This can be achieved through physical barriers provided by the skin and mucous membranes, soluble factors such as complements and acute phase proteins, antimicrobial peptides, and leukocyte subsets such as heterophils, macrophages, NK cells, mast cells, basophils, and eosinophils. One of the first reactive components of innate immunity against IBV infection is hyperplasia of goblet cells and alveolar mucous glands, leading to seromucous nasal discharge and catarrhal exudates in the trachea (75). Five days post-infection (dpi), when a loss of cilia, epithelial degenerative changes, and depletion of goblet cells and alveolar mucus glands have occurred, other immunological components become activated (75). PRRs

PRRs are present on the cytoplasic surfaces of immune cells, such as dendritic cells (DCs), macrophages, lymphocytes, and several nonimmunological cells such as endothelial cells,

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mucosal cells, and fibroblasts. These cells rapidly recognize infectious agents through PRRs such as Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), and NOD-like receptors (NLRs). TLRs are one of the primary mediators of the innate immune system, recognizing conserved structures in a broad range of pathogens. In chickens, the currently known TLRs are TLR-1 LA, TLR-1 LB, TLR-2A, TLR-2B, TLR-3, TLR-4, TLR-5, TLR-7, TLR-15, and TLR-21 (97). An increase in TLR-3 mRNA expression IBV-M41 strain at 3 dpi has been reported (104), and its function in viral immunology is wellestablished (65,69,104). In another study, TLR-1 LA, TLR-1 LB, TLR-2, TLR-3, and TLR-7 gene expressions were all significantly upregulated in the tracheal epithelial cells of 3-week-old chickens immunized with attenuated IBVMassachusetts (IBV-Mass) by intranasal inoculation (45). These studies have not shown whether host responses are elicited against IBV strains in lung tissue, though the lung is also a target organ for IBV. This was attempted in a more recent study (60), where 6-day-old SPF chickens were intratracheally infected with a Connecticut strain of IBV, and thereafter an upregulation of TLR-3 and TLR-7 mRNA was identified in the trachea and lung. Conversely, downregulation in the expression of TLR-3, along with IL-1b and IFN-c, was also observed in the early phase (12 h post-infection [hpi]) of viral replication. This early delay in induction of innate responses following infection was thought to be due to an increase in IBV genome load and worsening histopathological lesions in the trachea and lungs of IBV-infected chickens. The chicken TLR21 is a functional homologue of mammalian TLR-9, and after stimulation with deoxyoligonucleotides containing CpG motifs, it induces NFj-B production, leading to enhanced transcription of a number of cytokines (12). A decrease in viral load has been reported after treatment of 18-day-old embryos with deoxyoligonucleotides containing CpG motifs prior to inoculation with IBV (25). Melanoma differentiationassociated protein 5 (MDA 5) expression levels were reported to be significantly increased in chicken kidney tissue after nephropathogenic IBV infection, suggesting a role for chicken MDA5 against IBV infection (19). In a more recent study, it has been shown that in vitro virulent IBV infection leads to a significant induction of IFN-b transcription through an MDA5- dependent activation of the IFN response (61). Current and future research in the field of avian immunology will allow a better understanding of the precise role and signaling mechanisms of PRRs following vaccination or virulent IBV infection. Heterophils

Activation of the innate immune response leads to the recruitment of phagocytic heterophils and macrophages. Heterophils form the polymorphonuclear cell population of the chicken, and are the primary phagocytic cells that are first recruited to the infection site during the early stages of IBVinduced inflammation in chickens, along with other lymphocytes in the Harderian gland (HG) and trachea of IBV-infected tissues (75,91,100). The dramatic increase in heterophil numbers during IBV infection from 24 to 72 hpi has been reported in experimental studies by respiratory lavage analysis (35). Although heterophils lack the expression of MHC class I and II molecules seen in other chicken innate immune cells, they are

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highly phagocytic and express most of the TLRs found in chickens (13). Heterophils have been proposed to be responsible for the destruction of IBV-infected cells during initial infection by phagocytosis and oxidative lysozomal enzyme release (36). This was supported by another study (45), where an upregulation in the gene expression of heterophil cathepsin S and bactericidal permeability-increasing protein (BPI) was detected in IBV-infected chicks. These antimicrobial cytotoxic molecules are released by heterophil degranulation to degrade and neutralize pathogens. The important role for heterophils in the chicken immune response is also evident, as heterophildepleted chickens infected with IBV showed more severe nasal exudation compared with controls (91). In the tracheal epithelium, however, heterophils did not reduce virus replication and worsened the severity of lesions (91). There have been few publications investigating heterophil– IBV interactions in chickens, and further immunological characterization is needed in order to identify their importance during the early stages of IBV infection. Macrophages

Macrophage and monocyte chemotaxis to the area of infection is regulated by the production of chemokines by the other innate immune leucocytes. Information on the functional roles of macrophages in response to IBV infection is scarce. It is clear that virus-induced cytolytic necrosis accounts for many of the pathological changes that are observed in IBV infections (24). However, an increase in the number of macrophages was found in spleens of chickens inoculated with IBV-M41 from 1 to 7 days post-vaccination (dpv) (24). Similarly, infiltration of macrophages into the tracheal and bronchial lumen of the IBV-M41-infected chickens has also been reported when collecting respiratory lavage fluid between 24 and 96 hpi (35). A rapid influx of macrophages within hours post infection was also seen in the lung after individual or mixed avian pathogenic Escherichia coli (APEC) and IBV infections (71). These findings were confirmed in a more recent study (60), where macrophage numbers within the lungs and tracheas of chickens infected with Conn strain IBV were found significantly increased at 24 hpi compared with uninfected controls, suggesting that respiratory macrophages may play an important role in limiting the replication of IBV within respiratory tissues. An upregulation in the gene expression of monocyte and macrophage signaling molecules has also been reported, including Spi-1/ PU.1, glia maturation factor GMF-b, and macrophage colony stimulating factor receptor M-CSFR in IBV-Mass-infected chicks (45). Spi-1 induces macrophage differentiation; GMF stimulates NF-jB, GM-CSF, and CD4-/CD8+ cell differentiation; and M-CSF is key for macrophage linage development (40). Unlike certain other avian viral respiratory infections, IBV was initially reported to have no effect on macrophagemediated phagocytosis of serum opsonized E. coli (76). Macrophages isolated from peripheral blood or the respiratory tracts of chickens infected with IBV-M41 showed effective phagocytic activity or bactericidal function for E. coli in vitro (76). However, IBV was later shown to lower the bactericidal activity of peripheral blood mononuclear cells (PBMCs) and splenocytes, though their phagocytic capacity and recruitment remained unaffected (2).

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Collectively, these studies indicate that IBV can alter certain components of the innate immune system to facilitate secondary bacterial infections. However, more work is required to investigate the importance of macrophages’ functional roles in innate immune responses such as phagocytosis, cytokine and chemokine secretion, and antigen presentation. This would help the development of antigen-specific adaptive immune responses or modulate disease severity during IBV (either attenuated or virulent) infections. DCs

DCs are members of the mononuclear phagocytic system. Innate activation of a variety of these cell subsets can increase their capacity to interact with T cells, the most important of these being the DC subsets (50). DCs play an important role in the initiation of the adaptive immune response, being the only cells that can activate naive T cell subsets by virtue of their high levels of major histocompatibility (MHC) molecules and co-stimulatory activity (e.g., expressing CD80/86) (50). Still, little is known about the function and migration of chicken DCs (105). In recent years, with the emergence of new chicken DCs markers, it has become increasingly possible to culture chicken DCs in vitro (34,105). However, there is currently no publication available on the nature of IBV interactions with chicken DCs. Hence, further characterization and studies on this cell may yield important immunological mechanisms conferring protection against virulent or vaccine strains of IBVs. NK cells

NK cells are cytotoxic lymphocytes that play a key role in the early defense against viral infections. As opposed to in mammals, NK cells are found in very low numbers in the spleen or peripheral blood of birds (42). The role of NK cells in IBV infection has not been studied extensively. It was initially reported that an IBV vaccine designated as the Holland strain, Mass serotype, had caused no alterations in NK cell activity (103). However, it was later shown that infection of chickens with IBV-M41 induced rapid NK cell activation in the lung and blood (102). Five-week-old chickens receiving IBV-M41 via intratracheal and intranasal routes showed an increase of CD107+CD3- NK cells in the lungs only at 1 dpi, whereas the change of NK cells in PBMCs levels was biphasic with an increase at 1 and 4 dpi but not 2 dpi. Taken together, such observed differences in NK cell activation could be due to the virulence differences between the vaccine strain and virulent IBV-Mass strains. Again, further work is required in order to characterize the role of NK cells and their activation kinetics during IBV infection and vaccination. Acute phase proteins

In general, inflammatory cytokines and chemokines stimulate the production of acute phase proteins (APP) in chickens, including aI acid-glycoprotein (AGP), mannose-binding lectin (MBL), transferrin-ovotransferrin (OVT), fibrinogen, and Creactive protein. AGP has various biological activities, including immunomodulation of the inflammatory response, and its concentration has been recorded to increase during IBV infection (4). MBL plays a role in the innate immunity against IBV through an acute phase response, whereby it is able to

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activate complement and inhibit the propagation of the virus in the trachea (58). MBL has also been found to be associated with increased disease severity after IBV infection, as lower levels of MBL in serum lead to increased replication of IBV in chickens (59). Another recent study by Kjaerup et al. (62) showed that MBL has the capability to bind to IBV in vitro and is involved in the immune response to IBV vaccination. In their study, two inbred lines (L10H and L10L) selected on the basis of high or low MBL serum concentrations, respectively, were vaccinated against IBV with and without the addition of the MBL ligands mannan, chitosan, and fructooligosaccharide (FOS). The addition of MBL ligands to the IBV vaccine, especially FOS, enhanced the production of IBV-specific IgG antibody production in L10H chickens but not L10L chickens after the second vaccination, suggesting that MBL is involved in the immune response to IBV vaccination. Elevated levels of OVT in serum, which is a moderate APP, have also been documented after being challenged with IBV (106). Though there are limited reports on APP estimation after IBV infection, the available data suggest that combining estimation of concentration of serum levels of acute phase mediators as biomarkers for the assessment of inflammatory processes associated with IBV could be an additional tool to study immunopathogenesis of IBV infection. Complement system

The complement system, as a component of innate immunity, is immediately ready to target and eliminate virus particles and to interact with the surface of virus-infected cells (6). The trachea of IBV-Mass-infected chickens demonstrated an increase in the gene expression of C1q, C1s, anaphylatoxin C3a receptor, and complement C4, and a downregulation in factor H (an inhibitor of the complement system) (45). C1q and C4b mediate opsonization and the activation of the classical pathway of the complement system, while C3a and C4a elicit local inflammatory responses to limit infection (45). Although the complement gene profile is activated by IBV, more work is still required to characterize the precise mechanisms of complement during IBV infection. Possibilities include it being be a functional bridge between innate and adaptive immune responses to allow an integrated host defense to IBV. Cytokines

Cytokines are crucial regulators of the immune system (both innate and adaptive), and bind to specific cell surface receptors to initiate cascades of intracellular signaling for specific cell functions (64). The infection of chicken with attenuated IBVMass elicited the gene expression of IL-1b (10.8-, 5.5-, 2.1-, and 2.5-fold for 1, 3, 5, and 21 dpi), IL-10R2 (6.9-, 2.4-, and 2.3-fold for 1, 3, and 12 dpi), and common cytokine receptor g (increased 2.4-, 4.0-, 4.4-, and 2.9-fold from 1 to 8 dpi) to bind IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 in the tracheal tissue (45). IBV infection has previously been found to induce interferons in the trachea, lung, and at lower levels in the plasma, kidney, liver, and spleen (82). The role of the proinflammatory cytokine, IL-6, has been investigated for its contribution to nephritis in the two genetic line of chicken regarding the susceptible S-line and a more disease-resilient HWL line infected with the T strain of IBV (4). Although IL-6 mRNA levels were elevated in both bird lines’ kidneys at 4 dpi, these levels were

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20 times higher in S line chickens than they were in HWL chickens. Furthermore, serum IL-6 levels were found to be three times higher in S-line chickens compared with HWL chickens after IBV infection, suggesting that IL-6 may play a role in IBV-induced nephritis. Differential immune responses of chickens have also been reported to two IBVs with different genotypes, KIIa and ChVI (56). In chickens infected with KIIa genotype, at 7 dpi in the trachea and 9 dpi in the kidney, simultaneous peaks occurred in the number of virus copies and the upregulation of mRNA levels of pro-inflammatory cytokines (IL-6 and IL-1b) and lipopolysaccharide-induced tumor necrosis factor (TNF)-a factor (LITAF). This appeared to contribute to the scale of pathophysiologic effect in the chickens. Alternatively, chickens infected with ChVI genotype showed comparatively mild upregulation in pro-inflammatory cytokines mRNA expression. In a recent report, after infection with IBV-M41 strain, an early (3 dpi) upregulation of proinflammatory cytokines IL6 and IL-1b was reported. This coincided with the highest viral loads and microscopic tracheal lesions, indicating a role for both of these cytokines with high virus loads and the development of tracheal lesions (79). In another study, a disorder was hypothesized in the expression of eggshell components in laying hens after IBV infection, linked with the expression of these proinflammatory cytokines (77). A significant downregulation in the relative expression of IFNc and IL-1b mRNA has also been noted within the trachea during the initial phase of Conn strain IBV infection compared with uninfected controls. Conversely, IL-1b mRNA showed a sharp increase in expression as the IBV infection progressed (60). The transcriptomics study of the host kidney, in response to IBV infection, revealed that viral infection contributed to differential expression of 1777 genes, of which 876 were upregulated and 901 downregulated compared with those of control chickens, and103 were associated with immune and inflammatory responses (19). In this study, increased expression of IL-6, IL-18, IL-10RA, IL-17RA, CCL4, CCL20, CCL17, and CCL19 was found after IBV infection. However, chemokine (C-X-C motif) ligand 12 expressions were found to be decreased. The studies involving IBV sensitivity to ChIFN have shown conflicting results. Initially, Holmes and Darbyshire (52) reported that none of the six strains of IBV examined were sensitive to ChIFN as tested in cultures of chick embryo tracheal rings. In contrast, Otsuki et al. (83) reported that all 10 strains of IBV tested were sensitive to the action of IFN in chicken kidney cells (CKC). Later, type I interferon, IFN-a, was reported to inhibit IBV (Beaudette or Gray strains) replication both in vitro (CKC) and in vivo (86). Intravenous or oral administration of IFN-a prevented IBV-related respiratory clinical signs by delaying the onset of the disease and decreasing the severity of illness (86). Moreover, IBV stimulated chicken IFN-c production in leukocytes of both infected birds and uninfected birds (3). This stimulation was reduced when IBV was inactivated, but IFN-c production remained elevated compared with unstimulated cells (3). Recently, an increased expression of IFN-c was reported in the lungs of IBV-M41-infected birds at 2–4 dpv and in the PBMCs within 1 dpv (102). In a 2014 study, IFN-c was upregulated in the trachea after infection with IBV-M41, which implicated viral infection in the tracheal pathology of nonimmune challenged chickens (79).

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Further studies are needed to characterize the actual contributions of these pro-inflammatory cytokines in IBV immunopathogenesis, as well as analyzing the correlation between immunopathogenesis and disease progression. Despite the need for measuring the cytokines directly, the data provided by transcriptome analysis and from gene expression by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) are also very useful because they can statistically be processed and used to identify correlations with the scores of pathological changes and/or inflammation induced by IBV in target organs of chickens. However, as biological processes are typically driven by proteins, mRNA expression measurements through the use of RT-PCR, rather than protein expression measurements, are often thought of as a proxy for functional pathway changes, which involve changes in protein concentrations or expression. To address this, more commercially available chicken cytokine antibodies, ELISA, or Luminex platforms should be developed in order to characterize further the role of cytokines during IBV infection and vaccination. Chemokines and other factors for immune cell trafficking

Chemokines orchestrate the migration of cells during immune surveillance. Attenuated IBV-Mass stimulated the gene expression of CXCR4 (2.1-, 2.1-, 3.0-, and 2.2-fold for 1, 3, 5, and 12 dpi), CCR6 (2.0-, 2.5-, and 2.2-fold for 1, 3, and 5 dpi), chemokine-like receptor 1/CHEMR23 (3.0- and 2.9fold for 1 and 3 dpi) in chicken tracheas (45). In addition, integrin b2 (CD18) was also upregulated in the tracheas of chickens following attenuated IBV-Mass infection, which indicates the activation of leukocyte chemotaxis and therefore chemokines (45). Matrix metalloproteinase (MMPs) levels were also increased, implicating the migration of immune cells, possibly T cells, in the trachea of IBV-infected chicks (45). Most recently, an association of CpG-ODNs induced changes in cytokine/chemokine gene expression has been found to coincide with suppression of IBV replication in chicken lungs (26). The data showed that significant differential suppression of IL-6 gene expression and upregulation of IFN-c, IL-8 (CXCLi2), and MIP-1b genes was associated with inhibition of IBV replication in lung tissue from embryos, which were pre-treated with CpG ODN. Chemotaxis and chemokines are crucial in the local inflammatory response to infection and therefore more work is needed to characterize the role of chemokines in immune response against IBV. Apoptosis

The induction of apoptosis represents one of the major components of the host antiviral responses, limiting virus replication by rapid induction of cell death following infection of target cells. Nonetheless, viruses establish intricate and complex interactions with the host to regulate apoptosis to ensure a successful replication cycle that allows the production and spread of virus progeny to neighboring cells (49). With the identification of the phenomenon of apoptosis, it was theorized that this process of controlled cell death might also be considered as an innate response induced to counteract the viral infection (17). Studies showed that IBV Beaudette induced cell

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cycle arrest and apoptosis in infected Vero cells (66,67). It has been demonstrated that IBV induces apoptosis in late-stage infected cells and proapoptotic (e.g., Bax and Bak) and antiapoptotic proteins (e.g., Bcl-2 and Bcl-XL) from the Bcl-2 family that modulate IBV-induced apoptosis have been identified (66,67,108,109). In a recent study involving the role of unfolded-protein response (UPR) sensor IRE1a in IBV Beaudette-induced apoptosis, IBV was found to induce ER stress in infected cells and activated the IRE1a-XBP1 pathway at late stage of infection (37). Moreover, Cong et al. (19) concluded in their study that apoptosis is a nonspecific defense mechanism against IBV infection by premature lysis of infected cells, thereby aborting viral multiplication. Adaptive Immunity

Augmenting innate immune mechanisms, adaptive immunity allows the activation of antigen-specific effector mechanisms including B cells, T cells, macrophages, and the production of leucocytic memory cells, which play a significant role in antiviral immunity against IBV. Humoral Immunity

Research on the humoral immune response to IBV has been extensively studied since the first detection of the virus. Upon immunostimulation, B cells differentiate into plasma cells and secrete antibodies in either the presence or absence of T helper cells. Chickens develop a humoral response to IBV infection, which can be continually measured in serum by ELISA, HI, or VN serological tests (89). The measurement of different classes of IBV-specific antibodies in particular tissues, after an IBV infection, has also gained importance. Antibody kinetics

IgG (also known as IgY in chickens) is the major circulating immunoglobulin, and the kinetics of the IgG response to IBV is very different to that of the IgM response. Anti-IBV IgG can be detected in serum within 4 dpi, peaking at about 21 dpi with the high titer possibly remaining for many weeks (73). Thus, the primary IgG response lasts much longer than the IgM response does. After two vaccinations, serum IgG levels were much higher than those observed in the primary response, but they follow a similar pattern to that observed after the primary vaccination (73). Moreover, in vitro stimulation of chicken PBMCs and splenocytes with IBV activate memory B cells to secrete antibodies at 21 dpi (85). Memory B cells secreting IBV antibody (IgG) were detected by ELISPOT assays in both PBMCs and splenocytes collected from chicks after 3–7 dpi infected with IBV Gray strain (85). Studies by Dhinakar Raj and Jones (90) reported that IgG antibody content was highest in lachrymal fluid (tears) at 7 dpi and was still detectable at 23 dpi. However, significant levels of IgG antibody were also present in oviduct washes at 7 and 23 dpi. IBV IgA antibodies have been detected in the lamina propria, tracheal washes, and between epithelial cells in the trachea of IBV-infected chickens (57,75). IBV-specific IgA antibodies have also been demonstrated in lachrymal fluid, which correlated with resistance to IBV reinfection (23,30,41,99). IBV-specific IgA antibodies in lachrymal fluid were initially detected 10 days after vaccination with live

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attenuated Ark DPI-type IBV vaccine. However, no further significant increase was noticed for IgA after subsequent challenges with Ark-IBV isolate AL/4614/98, clarifying the probable role of lachrymal fluid neutralizing antibodies at the time of challenge. This stimulation of the IgA antibody response upon challenge acts to reduce the potency of IBV infection (57). Raj and Jones (88) reported that lachrymal fluid showed the highest IgA antibody concentration on 7 dpi, but this decreased to an insignificant level by 17 dpi. In addition, ocular vaccination with a live-attenuated H120 vaccine induced IgA-positive plasma cells in the HG at 14 dpv (29,30). Alternatively, after ocular vaccination with a low passaged, mildly pathogenic Ark-type IBV isolate, IBV-specific IgA secreting cells increased at 9 dpv in the HG. Moreover, IBVspecific IgA was found in cecal tonsils at 14 dpv, representing a delay in the IgA response at this site compared with that in the HG (101). A recent study demonstrated that ocular vaccination with live attenuated Ark IBV vaccine induced higher IgA antibodies (in lachrymal fluid and plasma) in the primary IBV response, while the memory response is dominated by IgG antibodies. Therefore, lower mucosal IgA antibody levels are observed upon secondary exposure to IBV, which may contribute to increased susceptibility of host epithelial cells to reinfection by IBV and the persistence of the Ark serotype (80). Role of antibodies in protection

Maternally derived antibodies can provide a short-lived resistance to IBV (21,27). Newly hatched chicks would therefore need to develop specific humoral responses to counter IBV infections. However, the precise role of antibody in the control of IBV infections remains controversial. Several studies have shown that circulating antibody titers do not highly correlate with protection from IBV infection. In their first report, Raggi and Lee (87) demonstrated a lack of correlation between infectivity, serological response, and challenge with IBV vaccine. Gough and Alexander (43) reported no correlation between HI antibody titers and susceptibility to challenge, as measured by re-isolation of virus from the trachea. Nevertheless, other studies have demonstrated that humoral immunity plays an important role in disease recovery and virus clearance. Cook et al. (21) reported that following IBV infection, bursectomized chicks showed more severe and longer-lasting illness than intact chicks did. The viral titers in tissues were also higher and lasted longer in bursectomized chicks compared with normal chicks (20), though no difference in mortality was observed (20). Similar earlier results were also reported using cyclophosphamide (immunosuppressive agent)–treated chickens, which showed severe clinical signs and more severe histopathological renal lesions due to the prolonged persistence of IBV compared with controls (16). It has also been reported that high titers of humoral antibodies correlate well with the absence of virus re-isolation from the kidneys and genital tract (44,70,107), and protection against a drop in egg production (10). Later, IBV-specific antibodies were suggested to be involved in limiting IBV spread by viremia from the trachea to other susceptible organs, including the kidneys and oviduct (89). In general, serum antibody levels do not closely correlate with tissue protection, but local antibodies may contribute to the protection of the respiratory tract (54,87). Thus, antibodies at mucosal surfaces could contribute to this protec-

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tion as IBV enters through this site and initially replicates in the HG and trachea. However, the role that local antibodies play in preventing the reinfection is not clear. Some studies have reported that local antibodies play a role in protecting the respiratory tract principally in the prevention of reinfections (48,51,99) and that the HG contributes to the protective local immunity (23,28,48,99,101). Alternatively, Gelb et al. (41) found that some chickens with high tear IBV antibody titers were susceptible to IBV and that some chickens with low tear titers were protected, suggesting that mechanisms other than antibody-mediated immunity in tears are important in viral clearance following infection. Isotype studies of antibodies involved in protection showed that after vaccination of broilers with IBV vaccine (H120 and D274 combination), the groups with 50% positive sera in IBV-specific IgM at 10 dpv had better protection against IBV-M41challenge (33). However, most groups of broilers with low levels of IBV-specific IgM had a low or moderate level of protection against IBV-M41 challenge (33). It has been demonstrated that IBV-specific IgG responses were less protective against IBV than against IBV-specific IgA antibodies found in tears (99). Furthermore, IBV-specific IgA antibodies were first detected in tears and later in serum, which suggests that IgA is important in neutralizing IBV at mucosal surfaces and is thought to play a role in the control of IBV locally (30,41). In a study involving transcriptome analysis of tracheal samples from chickens ocularly vaccinated with attenuated IBV-Mass, a marked decreased was reported in expression of IgA at 8 and 12 dpi. Conversely, up to a sevenfold increase in the expression of IgG at 5 and 8 dpi was demonstrated, concluding that IgA might not be important in protection against IBV infection of the upper respiratory tract, whereas locally produced IgG, after a secondary immunization, provided effective protection against IBV by neutralizing this virus (45). This was also supported by a study that showed a significant correlation between the levels of lachrymal antiIBV IgG at 5 dpi in chickens vaccinated with the full dose of H120 and tracheal protection against homologous IBV challenge (78). In addition, a recent study also corroborates this hypothesis, which reported that secondary antibody response at mucosal sites (lachrymal secretion) induced by attenuated IBV vaccines are dominated by an early and high production of anti-IBV IgG in re-immunized chickens (80). There are many factors that complicate the studies of the mechanism and duration of the immune response to IBV, including the multiple serotypes, the variation in virulence observed among strains, and different manifestations of the IBV infection. IBV vaccination studies have mainly focused on humoral immune responses by monitoring effective protection. However, the lack of adequate correlation between IBV specific-antibody secretion and resistance to challenge suggests that while antibodies are important in recovery from IBV infection, other immunological features are also involved. The mechanisms that further enhance T cell immunity in particular should be investigated in order to improve the efficacy of current IBV vaccination regimes for protection against the broad range of IBV variants. Cellular Immunity

Our understanding of immunity against IBV is deficient, especially the roles of cell-mediated responses (22). Several

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studies have examined the CTL responses in chickens to IBV infections and its correlation with early decreases in infection and clinical signs (86,92). CTL activity was dependent on the major histocompatibility complex (MHC), and lysis was mediated by CD8+CD4- cells. In addition, the passive transfer of IBV infection–induced ab T lymphocytes bearing CD8+ antigens protected naive chicks challenged with IBV (93). Antigen-specific CTL activity was detected at 3 dpi and reached its peak level at 10 dpi in chickens infected with nephropathogenic Gray strain of IBV (92). Throughout this period, acute IBV infection was contained, and detectable IBV was neutralized. Viral titers were decreased in the lungs and kidneys of IBV-infected chickens, and this was followed by a decline in CTL activity 10 dpi (92). Such memory T cells persisted up to at least 10 weeks in PBMCs post-infection with IBV (84). However, the duration of these memory T cells in tissues such as lungs, kidneys, spleens, and trachea needs to be further examined in chickens. During the course of experimental viral infection, Kotani et al. (63) assessed the immunophenotyping of mononuclear cells in the tracheal mucosa. The CTLs were found to be significantly increased in the tracheal mucosa after 3 or 4 dpi, peaking at 5 dpi and then decreasing to baseline levels by 14 dpi. So, these infiltrating CTLs at the trachea mucosa were proposed to be involved in the clearance of IBV at this site in an early phase of infection. Effector CD8 + T cells were shown to be important in limiting acute IBV infection (18). Experimental transfer of immune CD8 + T cells to chicks, prior to FBV infection, demonstrated that these viral primed ab T lymphocytes could protect chicks from acute infections (18,84,93). The kinetics of viral load in the lungs and kidneys of IBV (Gray strain)– infected chicks correlated with the level of IBV-specific CTL activity of effector cells isolated from spleen of the same infected chicks (18). Such IBV-specific CTL activity was dependent on the S and N proteins of IBV (18). Furthermore, the CD8 + T cell response in the blood and spleen occurred prior to the serum IgG antibody response to IBV (68), supporting the hypothesis that the CTL response correlated with decreased viral load and an improved clinical response. In vitro studies have shown that CTL activity also induced IBV (Gray strain)– infected cell lysis. This lysis was dependent on the concentration of effector T lymphocytes, mostly CD8+, with lower effect from CD4+ cells (18). CD4+ lymphocytes’ minimal effect was suggested to be possibly due to either CD4+/CD8+ or CD4+/CD8- (18). In another study, donor CD8+ memory T cells protected recipient chicks from acute IBV infection for the first 4 dpi, and showed mild clinical illness at 5 dpi (84). In addition, adoptive transfer of CD4 + T cells did not appear to be important in initially containing IBV infection in chickens (93). However, the low levels of protection in IBV-infected chicks that received CD4 + T cells were suggested to be due to contamination by CD8 + T cells (84). The first IBV-specific T cell epitope in the IBV nucleoprotein (IBV N71–78) from an IBV H52 strain was described by Boots et al. (9). This study revealed that only the S1 and N proteins of IBV generated cytotoxic T cell responses, not the M protein of IBV. Later, the whole N protein and its carboxyterminal region were reported to induce a CTL response, but not its amino terminal region (94). This was further supported by the work of Guo et al. (46) wherein increased numbers of

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CD4+ and CD8 + T cells in the PBMCs were found after vaccination of chickens with a DNA vaccine containing the sequence of N protein. In addition, immunization of chickens with a DNA vector expressing the granulocyte macrophagecolony stimulating factor and S1 subunit of the spike protein, resulted in high antibody levels in serum, lymphocyte proliferation (including CD4+ and CD8 + T cells in PBMCs), and reduced the severity in clinical signs and mortality rate after challenge (98). Cytotoxic enzymes, granzymes and perforin, which are secreted by cytotoxic cells such as CTLs, cd T cells, NK cells, and so on, play an important role in the cytotoxic activity induced following exposure to IBV (102,104). Transcriptome analysis of tracheas at 3 dpi with IBV-Mass revealed induction of genes involved in cytotoxicity such as granzyme-A precursor, Fas, CD3 d and c chain, MHC II, and TLR-2. This expression pattern could be due to cytotoxic T cell responses, as suggested by the authors (104). More recently, after infection with IBV-M41, a late (7 dpi) induction of CD8aa and granzyme homolog A mRNA has been reported to be associated with highest scores of viral load and microscopic lesions in trachea, suggesting a role of both these cytotoxic enzymes and virus load on the development of tracheal lesions (79). In addition, one study showed that the levels of expression of these genes evaluated at mucosal sites correlated significantly with the parameters of tracheal pathologic changes (viral load, histopathology, and cilliostasis) against homologous IBV challenge in a vaccine dose-dependent manner (78). In summary, CD8 + T cells play a dominant role in the early stages of IBV infection in poultry. However, CD4 + T cells and B cells could be more critical for long-term virus control. Traditional vaccine approaches have focused on inducing neutralizing antibodies against IBV that neutralize cell receptors on the virus, but the occasional inability of these antibodies to cross-react with heterotypic viruses limit the efficacy of such vaccines in providing broad-spectrum protection. Future work concentrating on the identification of conserved epitopes that induce specific CD8 + T cell responses and phenotypic markers that define effector and memory CD8 + T cells are vital for the development of universal vaccines against IBV, which continually changes to produce a large number of regional and global variants. Maternal Derived Antibodies (Passive Immunity)

Passive immunity was initially suggested by Broadfoot et al. (11) in 1954 to protect young chicks from the development of abnormal oviducts when challenged with virulent field strains of IBV. IgG antibodies were detected in serum and respiratory mucus of new hatched chicks that passed from vaccinated hens, via the yolk, to the progeny (48). In another study, chicks hatched with high levels of maternally derived antibodies were also found to have excellent protection (>95%) against IBV challenge (IBV-Mass strain) at 1 day of age but not at 7 days (>30%) (74). This protection significantly correlated with levels of local respiratory antibody and not with serum antibody. Neither group of chicks produced IBV-specific antibodies when vaccinated with live IBV-Mass vaccine at 1 day of age by the intraocular route (74). Live vaccination of 1-day-old chicks induced a rapid decline in maternally derived antibodies due to binding and partial neutralization of vaccine viruses (74). Moreover, de

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Wit et al. recently challenged 6- or 10-day-old chicks and reported that maternally derived IBV (D388 serotype, QX genotype) neutralizing antibodies provide partial protection against tracheal damage and a high protection against viral replication in the chicks’ kidneys (32). Such protection was achieved in SPF layer breeders with a combination of heterologous live and inactivated IBV vaccines to boost their production of IBV inherited maternally derived antibodies (32). The prime boost strategies, using live vaccines followed by inactivated vaccines, are being followed in the field. This protects the laying or breeder hens from disease throughout the laying period and also leads to the transfer of high levels of antibody into progeny. Though early protection of chicks against IBV by these maternally derived antibodies is very important, more work is required to devise an effective strategy that could be used to vaccinate young chicks in the face of these high antibody levels. Conclusions

This review revealed that despite abundant studies on IBV, there has been no concerted effort in analyzing these data to account for the induction of immunity against virulent or by live and inactivated IBV vaccine viruses. The kinetics and importance of systemic anti-IBV mechanisms have been well studied, but our understanding of the role of mucosal and cellular immune response remains limited. The innate immunity against this virus is generally not well characterized, though in recent years, an increasing number of publications on this aspect has sparked further interest. With continuous emergence of new IBV genotypes globally and strategic use of currently available live vaccines to confer higher and broader protection against these variants, it is essential to improve our understanding of the immune responses to IBVs. Though much is still unknown about the underlying immune mechanism induced by IBV live vaccine viruses, it is clear that the protection conferred relies on multiple interrelating immune responses: innate, adaptive, and effector T cells, especially at mucosal surfaces. Author Disclosure Statement

No competing financial interests exist. References

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Address correspondence to: Dr. K. Ganapathy School of Veterinary Science Institute of Infection and Global Health Leahurst Campus University of Liverpool Neston Cheshire, CH64 7TE United Kingdom E-mail: [email protected]