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University of Veterinary Medicine Hannover Clinic for Poultry Molecular epidemiology of infectious bursal disease viruses and development of a microparticle based vaccine THESIS Submitted in partial fulfillment of the requirements for the degree DOCTOR OF PHILOSOPHY (PhD) at the University of Veterinary Medicine Hannover, Germany By Tamiru Negash Alkie (Arsi, Ethiopia) Hannover, Germany 2013 Supervisor: Prof. Dr. Silke Rautenschlein (Clinic for Poultry, University of Veterinary Medicine Hannover, Germany) Advisory Committee: Prof. Dr. Silke Rautenschlein Prof. Dr. Beatrice Grummer (Institute of Virology, University of Veterinary Medicine Hannover, Germany) PD Dr. Reimar Johne (Federal Institute for Risk Assessment, Berlin, Germany) 1st Evaluation: Prof. Dr. Silke Rautenschlein Prof. Dr. Beatrice Grummer PD Dr. Reimar Johne 2nd Evaluation: Prof. Dr. H. M. Hafez (Institute of Poultry Diseases, Faculty of Veterinary Medicine, Free University Berlin, Germany) Date of oral exam: 25 April, 2013 Dedicated to My wife, Eskedar Hailegebral & My children, Heldana and Yafet V Table of Contents Table of Contents ....................................................................................................... V Publications .............................................................................................................. VII List of abbreviations ................................................................................................... IX List of Figures .......................................................................................................... XIII List of Tables ........................................................................................................... XIV 1. Introduction ............................................................................................................. 1 2. Literature review ..................................................................................................... 4 2.1. IBDV genomic organization and functions ....................................................... 4 2.2. Pathobiology of IBDV ....................................................................................... 8 2.2.1. Pathogenetic mechanisms ........................................................................ 8 2.2.1.1. Polyprotein (PP) cleavage sites .......................................................... 8 2.2.1.2. Capsid and polymerase proteins......................................................... 8 2.2.1.3. Nonstructural protein ......................................................................... 10 2.2.2. IBDV host cell receptor and virus entry ................................................... 11 2.2.3. Pathogenesis of infectious bursal disease ............................................... 11 2.2.4. Clinical disease and pathology ................................................................ 14 2.3. Immune responses to IBDV ........................................................................... 16 2.3.1. Innate immunity ....................................................................................... 16 2.3.2. Humoral immunity.................................................................................... 17 2.3.3. Cellular immunity ..................................................................................... 18 2.4. Field evolution and molecular epidemiology of IBDV ..................................... 19 2.4.1. IBDV reassortment .................................................................................. 20 2.4.2. IBDV recombination................................................................................. 22 2.4.3. IBDV quasispecies and reversion to virulence ........................................ 24 2.5. Diagnostic methods........................................................................................ 25 2.5.1. Embryo inoculation .................................................................................. 25 2.5.2. In vitro virus propagation ......................................................................... 25 2.5.3. Immunological methods ........................................................................... 25 2.5.4. Molecular characterization ....................................................................... 26 2.6. Vaccines and vaccination against IBDV ......................................................... 28 VI 2.7. DNA vaccines ................................................................................................ 32 2.7.1. Stimulation of immune cells by DNA vaccines ......................................... 32 2.7.2. IBDV DNA vaccines................................................................................. 33 2.8. Adjuvants ....................................................................................................... 34 2.8.1. Cytokine adjuvants for avian viral vaccines ............................................. 35 2.8.1.1. Chicken IL-2 and IFN-γ as adjuvants for IBDV-DNA vaccines .......... 40 2.8.2. Toll like receptor (TLR) ligands as adjuvants ........................................... 40 2.9. Microparticulate vaccine and adjuvant carrier systems .................................. 42 2.9.1. Enhancing specific immunity by PLGA MPs ............................................ 45 3. Goals and objectives ............................................................................................ 47 4. Molecular evidence of very virulent infectious bursal disease virus in chickens in Ethiopia .................................................................................................................... 48 5. Mucosal application of cationic poly(D, L-lactide-co-glycolide) microparticles as carriers of DNA vaccine and adjuvants to protect chickens against infectious bursal disease ..................................................................................................................... 51 6. Discussion and conclusions.................................................................................. 81 6.1. Molecular epidemiology of IBDV field isolates ............................................... 81 6.2. Immune responses induced by candidate IBDV DNA vaccines and correlation to protection .......................................................................................................... 85 6.3. Do molecular adjuvants enhance protectivity of an IBDV DNA vaccine? ....... 86 6.4. Cationic PLGA MPs as particulate carriers for DNA vaccine and molecular adjuvants............................................................................................................... 88 6.5. Promise of cationic PLGA MPs in improving an IBDV DNA vaccine .............. 89 6.6. Concluding remarks and future perspectives ................................................. 92 7. Summary .............................................................................................................. 94 8. Zusammenfassung ............................................................................................... 96 9. References ........................................................................................................... 99 10. Acknowledgements .......................................................................................... 143 VII Publications Research articles: NEGASH, T., E. GELAYE, H. PETERSEN, B. GRUMMER u. S. RAUTENSCHLEIN (2012): Molecular evidence of very virulent infectious bursal disease virus in chickens in Ethiopia. Avian Dis 56 :605-610. NEGASH, T., M. LIMAN u. S. RAUTENSCHLEIN (Vaccine revised): Mucosal application of cationic poly(D, L-lactide-co-glycolide) microparticles as carriers of DNA vaccine and adjuvants to protect chickens against infectious bursal disease Oral presentations at scientific meetings: NEGASH, T., B. GRUMMER u. S. RAUTENSCHLEIN (2012): Molecular identification and differentiation of infectious bursal disease viruses (IBDVs) in field outbreaks. 82. Fachgespräch über Geflügelkrankheiten, Hannover, Germany; November 2012. VIII NEGASH, T.u. S. RAUTENSCHLEIN (2013): Does mucosal application of cationic poly(D, L-lactide-co-glycolide) (PLGA) microparticles as carriers of DNA vaccine and adjuvants enhance immunity against infectious bursal disease virus? 23rd Annual meeting of the society for virology, Kiel, Germany; March 2013. Poster presentations at scientific meetings: NEGASH, T., B. GRUMMER u. S. RAUTENSCHLEIN (2011): Molecular evidence of very virulent infectious bursal disease virus in chickens in Ethiopia. 21st Annual meeting of the society for virology, Freiburg, Germany; March 2011. NEGASH, T u. S. RAUTENSCHLEIN (2011): Development of new generation vaccination strategies against infectious bursal disease virus of chickens. 4th Graduate school day, Bad Salzdetfurth, Germany; November 2011. NEGASH, T. u. S. RAUTENSCHLEIN (2012): Mucosal application of cationic PLGA microparticles as carriers of DNA vaccines and adjuvants enhances viral clearance in SPF chickens after IBDV challenge. XIIth Avian immunology research group conference. Roslin institute, Edinburgh, UK August 2012. IX List of abbreviations aa Amino acids Abs Antibodies AC-ELISA Antigen capture ELISA Ag Antigen AIV Avian influenza virus aMPV Avian metapneumovirus APCs Antigen presenting cells BF bursa of Fabricius CAM Chorioallantoic membrane CD Cluster of differentiation CEFs Chicken embryo fibroblasts chIFN Chicken interferon cHsp90 Chicken heat shock protein 90 cIBDV Classical infectious bursal disease CMI Cell mediated immunity CMV Cytomegalovirus CpG-ODN CpG-deoxynucleoside CTAB Cetyltrimethylammonium bromide DCs Dendritic cells DNA Deoxyribonucleic acid dpi Days postinfection dsRNA Double stranded RNA ELISA Enzyme-linked immunosorbent assay FDCs Follicular dendritic cells GC Germinal center GILZ Glucocorticoid-induced leucine zipper GM-CSF Granulocyte-macrophage cell stimulating factor Gzm A Granzyme A HA Hemagglutinin X HI Hemagglutination inhibition HIV Human immunodeficiency virus HN Hemagglutinin-neuraminidase hVP2 Hypervariable region of VP2 HVT Herpesvirus of Turkeys IBD Infectious bursal disease IBDV Infectious bursal disease virus IBD-ICX IBD immune complex IBV Infectious bronchitis virus IFN-γ Interferon-γ Ig Immunoglobulin iIELs Intraepithelial lymphocytes IL-2 Interleukin-2 IL-2R IL-2 receptor ILTV Infectious laryngotracheitis virus IM Intramuscular iNOS Inducible NO synthase JAK/STAT Januskinase/signal transducers and activators of transcription MAB Maternal Ab mAbs Monoclonal Abs MDV Marek’s disease virus MHC Major Histocompatibility complex MPs Microparticles mRNA Messanger RNA ND Newcastle disease NDV Newcastle disease virus NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells NK cells Natural killer cells NO Nitric oxide XI nt Nucleotide OAS 20-50-oligoadenylate synthetase P Projection PAMPs Pathogen-associated molecular patterns PBL Peripheral blood lymphocytes PCR Polymerase chain reaction PEI Polyethylenimine PFN Perforin pi Postinfection PI3K)/Akt Phosphatidylinositol 3-kinase PKR Protein kinase R PLGA poly(D, L-lactide-co-glycolide) PP Polyprotein PRRs Pathogen recognition receptor pVP2 Precapsid VP2 qRT-PCR Quantitative RT-PCR rAdV Recombinant Adenovirus RFLP Restriction fragment length polymorphism rFPV Recombinant fowlpox virus RNA Ribonucleic acid RT-PCR Reverse transcriptase PCR SPF Specific pathogen free SRs Scavenger receptors SVPs Subviral particles Th1 T helper-1 TLRs Toll-like receptors tMRCA Time of the most recent common ancestor TNF-α Tumor necrosis factor-α Tregs Regulatory T-cells VDAC2 Voltage-dependent anion channel 2 VLPs Virus-like particles XII VNT Virus neutralization test VP2 Virus protein 2 vvIBDV Very virulent IBDV vvVP2 Very virulent VP2 XIII List of Figures Chapter 2 Fig. 1. Method of vaccine (DNA/protein) microencapsulation in PLGA microspheres using the w/o/w emulsion solvent evaporation method ........................................................................................... 44 Chapter 5 Fig. 1. Quantification of postchallenge chIFN-α mRNA expression levels in the bursa at 3 (A), and 7 dpi (B); and chIL-4 mRNA expression at 3 (C) and 7 dpi (D). .................................................................... 78 Fig. 2. Quantification of postchallenge intrabursal CD4+ T-cells at 3 (A), and 7 dpi (B); and CD8+ T-cells at 3 (C) and 7 dpi (D). ................. 80 XIV List of Tables Chapter 2 Table 1. Functions of IBDV proteins .............................................................. 7 Table 2. Examples of natural reassortant IBDV isolates ............................. 21 Table 3. Examples for natural recombinant IBDV isolates ........................... 23 Table 4. Experimental IBDV vaccines .......................................................... 31 Table 5. Improvement of the protective efficacy of avian viral vaccines after co-administration of plasmid encoded avian cytokines .......... 38 Table 6. Examples for avian cytokines delivered by live viral vectors as adjuvants for avian viral vaccines .................................................. 39 Chapter 5 Table 1. Experiment 1: Evaluation of protection conferred by IBDV DNA vaccinations in comparison to a live IBDV vaccine ........................ 70 Table 2. Experiment 2: Evaluation of protection induced by IBDV DNA vaccination in combination with molecular adjuvants ..................... 72 Table 3. Experiment 3: Study design showing experimental groups and the timing of vaccine and adjuvant application ............................... 74 Table 4. Experiment 3: Determination of the protective efficacy of MP based DNA vaccine and adjuvants after mucosal application to SPF chickens ................................................................................ 75 1 1. Introduction Immunosuppressive pathogens are major constraints of poultry production. They can affect the efficacy of the immune system, which leads to vaccination failure and increased susceptibility to many pathogens. Infectious bursal disease virus (IBDV) is a relevant immunosuppressive virus of chickens. It is a dsRNA virus targeting primarily the immature IgM+ B-cells residing in the bursa of Fabricius (BF), which is a primary lymphoid organ in avian species. Worldwide, the poultry industry has encountered heavy economic losses associated with very virulent (vv) IBDV strains during the last several years. These strains may cause high mortality in affected chicken flocks and severe immunosuppression that involves both innate and adaptive immune responses. During recent years, significant progress has been made to understand the molecular epidemiology of vvIBDV. After the emergence of the virus in Europe in the late 1980’s, it was reported to have spread to several poultry producing regions worldwide. By the mid-1990’s, almost 80% of the World Organization for Animal Health (OIE) member countries have reported the occurrence of vvIBDV based on pathological and molecular investigations from field outbreaks. A recent study addressing the global molecular epidemiology of IBDV from four continents including Africa showed that 60-76% of IBDV isolates were vvIBDVs. However, limited information is available regarding the molecular epidemiology of IBDV, particularly that of vvIBDV in Africa. Although an acute infectious bursal disease (IBD) infection was suspected in major field outbreaks involving commercial farms and breeding centers in Ethiopia in recent years, the nature and epidemiology of the virus remain unknown. Effective vaccination programs in combination with biosecurity are vital to control IBD. Inactivated vaccines are applied mainly to breeder flocks and may require multiple boosts to induce strong humoral immunity. Conversely, live attenuated vaccines are applied to layers and broilers and stimulate not only humoral immunity but also cell mediated immunity (CMI). Major challenges of live IBDV vaccines 2 include the possible risk of reversion to more virulent IBDVs and generation of chimeric viruses by exchange of viral segments between vaccinal and wild IBDV strains. New generation IBDV-subunit vaccines may circumvent some of these problems and provide protective levels of immunity. DNA vaccines may be good candidates due to their ability to elicit both humoral and cellular immunity. Nevertheless, they did not provide protective levels of immunity in many studies and may require well characterized adjuvants to enhance their immunogenicity and protection. A number of studies have demonstrated that co-administration of chicken interleukin-2 (chIL-2) and synthetic unmethylated oligodeoxynucleotides (ODN) containing cytosineguanosine in succession (CpG-ODN) improved IBDV DNA vaccine efficacy. CpGODN stimulates the host defense system by mimicking host invasion by pathogens to keep antigen presenting cells (APCs) in a state of alert. Generally, both molecular adjuvants have multifunctional immunoregulatory properties and are known to activate dendritic cells, macrophages, B- and T-cells and they can enhance the expression of costimulatory molecules by these cells. This may enhance Ag presentation and subsequent immunological responses to the co-administered IBDV DNA vaccines. Despite measurable progress that has been made in developing DNA vaccines, further improvements are required for possible large scale applications. Vaccines and adjuvants can be made more effective by employing appropriate particulate delivery systems to enhance the uptake and processing by APCs. Microparticles (MPs) prepared from biodegradable poly(D, L-lactide-co-glycolide) (PLGA) polymers are one of the most extensively characterized particulate carriers for delivering vaccines and adjuvants either parenterally or mucosally to enhance systemic and mucosal immunity. The goals of this study were to determine the molecular epidemiology of recent IBDV isolates in Ethiopia and furthermore to develop and improve IBDV DNA vaccines by using adjuvants and MPs as mucosal vaccine and adjuvant carriers. 3 The objectives of the first part of this study were to evaluate the pathogenicity of Ethiopian IBDV field isolates experimentally and to characterize their molecular nature by investigating the hypervariable region of the virus protein (VP) 2 (hVP2) and the 5` two thirds of VP1. The objectives of the second part of the thesis were to assess the immunogenicity and protective efficacy of candidate recombinant IBDV DNA vaccines that encode the vvIBDV-VP2 genes of a selected Ethiopian and reference IBDV strain; we included the molecular adjuvants, CpG-ODN and plasmid encoded chIL-2 to enhance protection. Furthermore, we evaluated cationic PLGA MPs as a carrier for mucosal delivery of the DNA vaccine and adjuvants to improve the efficaciousness of the IBDV DNA vaccine. 4 2. Literature review IBDV is one of the economically most important immunosuppressive viruses of chickens. IBDV is an Avibirnavirus and belongs to the family of Birnaviridae (BROWN 1989). Two serotypes of the virus have been described. Serotype 1 IBDV strains are pathogenic to chickens (MÜLLER et al. 2003; VAN DEN BERG et al. 2004), whereas serotype 2 strains are non-pathogenic (MCFERRAN et al. 1980). Serotype 1 IBDV isolates comprise the variant, calssical virulent and vvIBDV strains, which greately differ in their pathogenicity to chickens. Variant IBDVs do not cause mortality, whereas the classical strains cause up to 20% mortality (MÜLLER et al. 2003). vvIBDV causes mortality exceeding 50% in susceptible chickens (CHETTLE et al. 1989; BERG et al. 1991; MÜLLER et al. 2003). 2.1. IBDV genomic organization and functions IBDV is a non-enveloped virus with a bipartite dsRNA genome (DOBOS et al. 1979; MULLER et al. 1979). The main open reading frame of genome segment A encodes a polyprotein (PP) (NH2-pVP2-VP4-VP3-COOH) that is cleaved by the virus encoded protease into pre-capsid virus protein (VP)2, VP4 and VP3 within infected cells (BIRGHAN et al. 2000; LEJAL et al. 2000). The pre-capsid VP2 undergoes defined sequential C-terminal cleavage by VP4 (viral protease) (SANCHEZ u. RODRIGUEZ 1999), host protease (puromycin-sensitive aminopeptidase) (IRIGOYEN et al. 2012), and by the endopeptidase activity of VP2 (LUQUE et al. 2007; IRIGOYEN et al. 2009) to release the mature VP2 protein. Other small peptides released during the PP processing such as pep46 (46 aa) remain associated with the outer capsid (CHEVALIER et al. 2005), but can not be visualized in IBDV particles by X-ray crystallography (COULIBALY et al. 2005). The N-terminus moiety of pep46 bears a positively charged hydrophobic domain and may be responsible for virus penetration into the cytoplasm of infected cells (GALLOUX et al. 2007). The C-terminal moiety of pep46 may assist in the formation of larger pore sizes to enhance viral entry into infected cells (GALLOUX et al. 2010). 5 The VP2 crystal structure indicates three domains: the base (B), shell (S), and projection (P) domains (COULIBALY et al. 2005; GARRIGA et al. 2006; LEE et al. 2006). The B and S domains are formed by the conserved N-and C-terminal stretches of VP2. The P domain is the middle part containing the host cell receptor binding motifs and the hypervariable region of VP2 (hVP2). The hVP2 harbours antigenic major hydrophilic peak A (amino acid; aa 212-224) and B (aa 314-325) (SCHNITZLER et al. 1993) that form loops PBC (aa 219-224) and PHI (aa 316-324) (COULIBALY et al. 2005), respectively. The minor hydrophilic peak 1 (aa 248-254) and peak 2 (aa 279-290) in the hVP2 form loops PDE (aa 249-254) and PFG (aa 279284) (COULIBALY et al. 2005). The IBDV outer capsid is composed of a single shell of 260 trimeric spikes formed by the P domain of VP2 radially projected from the capsid, and organized in a T=13 icosahedral lattice (BOTTCHER et al. 1997). This organization is determined by the electrostatic interactions of precapsid VP2 with the VP3 C-terminal residues (SAUGAR et al. 2010). The expression of the mature VP2 alone as a recombinant protein forms dodecahedral T=1 subviral particles (SVPs) containing 20 VP2 trimers (CASTON et al. 2001; GARRIGA et al. 2006), whereas precapsid VP2 expression forms a tubular structure (CASTON et al. 2001). The last five acidic residues at the C-terminus of VP3 interact with pVP2 during particle morphogenesis for correct capsid assembly (CHEVALIER et al. 2004; SAUGAR et al. 2005; LUQUE et al. 2009). The sixteen C-terminal residues of VP3 interact with VP1 (TACKEN et al. 2002; MARAVER et al. 2003; GARRIGA et al. 2007). VP3 binding to the genomic dsRNA and VP1 forms the ribonucleoprotein complex (LUQUE et al. 2009). The VP5 protein is encoded by another open reading frame on segment A that partially overlaps with the 5’ end of the PP gene. It is a host membrane-associated and highly basic protein with a cytoplasmic N-terminus and an extracellular Cterminal domain (LOMBARDO et al. 2000). 6 Segment B encodes the polymerase (SAUGAR et al. 2010), which is present in the virion as a free protein or covalently linked to the 5′ ends of both genome segments (VPg) (MÜLLER u. NITSCHKE 1987). PAN et al. (2007) has recently characterized the VP1 crystal structure. VP1 has three domains: the N- terminus (aa 1-167), central polymerase (aa 168-658) and C-terminal (aa 659-878) regions. The N-terminus of VP1 is involved in protein priming as it possesses the putative guanylylation site (XU et al. 2004; PAN et al. 2007). The central polymerase domain folds into a right-hand shape (fingers-palm-thumb) structure. The five RNA polymerase motifs (C, A, B, D and E) are located in the palm region of the polymerase. Each of the aa motifs function during virus replication, for example, in nucleotide recognition and binding ( e.g. motifs A, B & F), phosphoryl group transfer (A & C), in a metal ion like Mn2+ and Mg2+ binding (C), nucleotide guidance to active sites (D), and primer gripping (E) (PAN et al. 2007). Motif C forms the polymerase active site (SHWED et al. 2002; PAN et al. 2007). The finger sub-domains contain polymerase motifs F and G that are involved in virus replication (PAN et al. 2007). The 5’ non-coding regions of both segments contain promoter elements (NAGARAJAN u. KIBENGE 1997) as well as 18S rRNA binding element and play roles in the initiation of virus replication (MUNDT u. MÜLLER 1995). No N-linked glycosylation of any of the virion proteins has been detected. The biological functions of the different proteins of IBDV are summarized in table 1. 7 Table 1: Functions of IBDV proteins Proteins Functions References VP2 Host receptor binding (OGAWA et al. 1998) Contains neutralizing epitopes (AZAD et al. 1987) Virulence determinant (BRANDT et al. 2001) Cell culture adaptation (MUNDT 1999) Apoptosis (FERNANDEZARIAS et al. 1997) VP3 Endopeptidase activity (IRIGOYEN et al. 2009) Chaperone activity (CHEVALIER et al. 2004) Antiapoptosis by interacting with PKR (BUSNADIEGO et al. 2012) Suppresses hosts RNA silencing mechanism (VALLI et al. 2012) VP4 Transcriptional activator (TACKEN et al. 2002) Forms ribonucleoprotein complex (LUQUE et al. 2009) Viral protein processing (viral protease) (BIRGHAN et al. 2000) Trans-activate VP1 synthesis (BIRGHAN et al. 2000) Suppresses type I IFN by interacting with (LI et al. 2013b) GILZ VP5 VP1 Early antiapoptotic effects (LIU u. VAKHARIA 2006) Late apoptotic effects (LI et al. 2012) Viral polymerase (SAUGAR et al. 2010) Virulence determinant (LE NOUEN et al. 2012) 8 2.2. Pathobiology of IBDV 2.2.1. Pathogenetic mechanisms Early studies revealed segment A as the sole determinant of IBDV virulence. Nevertheless, the molecular basis of IBDV pathogenicity likely depends on the synergism between both segments of the virus (BOOT et al. 2000; BRANDT et al. 2001; BOOT et al. 2005; ESCAFFRE et al. 2012). 2.2.1.1. Polyprotein (PP) cleavage sites To determine the molecular determinants of IBDV virulence, segment A and B of several strains were sequenced and analysed (XIA et al. 2008). vvIBDV isolates with aa substitutions adjacent to the pVP2 maturation site (aa 441–442), close to the VP2–VP4 (aa 512–513) and VP4–VP3 (aa 755–756) cleavage sites (BROWN u. SKINNER 1996; CHEVALIER et al. 2004; XIA et al. 2008) and at the C-terminus of VP3 (CHEVALIER et al. 2004a) were reported. An aa substitution at the predicted protease active-site of the VP4 gene of one of the European highly virulent strains UK661 was suspected to contribute to its virulence. Generally, these mutations may speed up the proteolytic activity of VP4, the processing and maturation of VP2, and the capsid assembly efficiency. This may enhance the replication of vvIBDV and lead to higher yields of virus particles in the host tissues (YAMAGUCHI et al. 1997b; CHEVALIER et al. 2004; XIA et al. 2008). 2.2.1.2. Capsid and polymerase proteins Exposed at the virion surface, VP2 contributes to IBDV virulence (YAMAGUCHI et al. 1996b; BOOT et al. 2000). Most vvIBDV isolates have key aa marker of virulence at the hVP2 region (BROWN et al. 1994; BROWN u. SKINNER 1996; YAMAGUCHI et al. 1997a; ISLAM et al. 2001). Specific aa residues responsible for tissue culture 9 adaptation, virulence and cell tropism have been mapped onto the VP2 gene using reverse genetics (MUNDT u. VAKHARIA 1996). Amino acid mutations at positions 253 (Q →H), 279 (D →N) and 284 (A →T) in the VP2 were generated by reverse genetics and completely attenuated the vvIBDV isolates. The modified viruses propagated well in cell-culture and showed reduced pathogenicity in chickens (LIM et al. 1999; MUNDT 1999; VAN LOON et al. 2002). The adaptation of vvIBDV strain OKYM to cell culture introduced comparable aa mutations at positions 279 (D →N) and 284 (A →T) (YAMAGUCHI et al. 1996b). These mutations were also detected in other cell culture-adapted classical and vvIBDV strains (YAMAGUCHI et al. 1996a). A single aa mutation at position 253 (H →Q/N) in VP2 markedly increased the virulence of an attenuated IBDV strain (JACKWOOD et al. 2008). Substantial molecular evidence showed the role of segment B in IBDV pathogenicity as well (LIU u. VAKHARIA 2004; BOOT et al. 2005). Field reassortant IBDV isolates comprising segment A of vvIBDV and segment B of attenuated strains show reduced pathogenicity under field conditions and when evaluated experimentally in susceptible chickens compared to typical vvIBDV isolates (LE NOUEN et al. 2006). This field observation of reduced pathogenicity was proven with a classical virulent IBDV that was genetically modified to contain the VP2 gene from a vvIBDV strain. This virus failed to cause morbidity and mortality in SPF chickens unless the genetically modified virus contained a typical segment B from vvIBDV (LIU u. VAKHARIA 2004). Recombinant IBDVs generated by exchanging different regions of VP1 of a vvIBDV with their counterparts of VP1 of an attenuated IBDV in a vvIBDV segment A background showed reduced virulence in SPF chickens (LE NOUEN et al. 2012). These viruses replicated to reduced virus titers in the bursa indicating the important role of VP1 in vvIBDV virulence. Putative virulence marker aa residues across the VP1 protein were predicted (YU et al. 2010). JACKWOOD et al. (2012) recently described the presence of aa motif TDN at positions 145, 146 and 147 in the VP1 gene of all vvIBDVs tested, and their absence in non-vvIBDV isolates. An IBDV strain designated as 94432 that maintained all virulence markers aa in its VP2 gene like other prototype vvIBDVs failed to cause mortality in chickens. The presence of 10 threonine (T) at position 276 in the exposed hydrophobic groove of the finger domain of VP1 has been then shown to contribute to the reduced pathogenicity. Exchanging this aa at position 276 from T →V restores the pathogenicity of the molecularly cloned virus (ESCAFFRE et al. 2012). 2.2.1.3. Nonstructural protein VP5 is believed to play an important role in IBDV pathogenicity; nonetheless it is not essential for viral replication (MUNDT et al. 1997). In vitro, the VP5 protein showed extensive accumulation within the plasma membrane of infected cells at later time points during IBDV replication (LOMBARDO et al. 2000). VP5 may have antiapoptotic effects on infected cells during early time points (LIU u. VAKHARIA 2006), and at the same time it may trigger apoptosis at later stages of IBDV replication in infected cells (YAO u. VAKHARIA 2001). Furthermore, it was suggested that VP5 plays a significant role in virus release from infected cells by triggering cell death (WU et al. 2009). IBDV infection induces the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway during the early phase of IBDV replication in DF-1 cells. The regulatory subunit of PI3K may suppress premature apoptosis of infected cells, which may sustain IBDV replication and production of large quantities of infectious virus progeny (WEI et al. 2011). When PI3K was inhibited most of the infected cells showed early apoptotic signatures (WEI et al. 2011). Voltage-dependent anion channel 2 (VDAC2) and VP5 (aa residues from 1-50) interaction later during IBDV infection may be responsible for the release of infectious virus particles from infected cells (LI et al. 2012). VDAC2 is a host molecule, which forms pores in the outer mitochondrial membrane and is involved in apoptosis (SHOSHAN-BARMATZ et al. 2010). Blocking VDAC2 by small interfering RNA inhibited IBDV-induced apoptosis, reduced virus release and virus titer. 11 2.2.2. IBDV host cell receptor and virus entry IBDV may bind to host cell proteins such as N-glycosylated polypeptide(s) expressed on the cell membrane of immature IgM+ B-cells during viral entry process (OGAWA et al. 1998; LUO et al. 2010). A pore forming peptide of the virus (pep46), which is associated with the outer capsid of the IBDV particle, may facilitate viral entry into the cytoplasm of infected cells (GALLOUX et al. 2007; GALLOUX et al. 2010). A lipid raft mediated endocytic mechanism was suggested based on the results of an in vitro study to support entry of attenuated IBDV to the cells (YIP et al. 2012). An in vitro binding assay showed the interaction of subviral particles (SVPs) derived from the mature VP2 with the chicken heat shock protein 90 (cHsp90) expressed on the surface of DF-1 cells (LIN et al. 2007). This binding of IBDV to cHsp90α was inhibited by cHsp90α interfering microRNAs that resulted in reduced virus titer (YUAN et al. 2012). The binding of VP2-SVP with α4β1 integrin suggested an integrin-binding domain in VP2 of IBDV, which was later confirmed that aa residues at positions 234236 are responsible (DELGUI et al. 2009). This integrin-binding motif is conserved in all IBDV strains. A single point mutation within this motif might completely abrogate the binding of SVPs to cells and virus infectivity (DELGUI et al. 2009). Immature Bcells have abundant α4β1 on their surfaces (ROSE et al. 2002). 2.2.3. Pathogenesis of infectious bursal disease Natural IBDV infection occurs by the oral route. Other mucosal routes have been demonstrated for experimental IBDV infection. The mononuclear phagocytic cells and lymphoid cells of the gut mucosa may serve as targets for IBDV infection and replication (MÜLLER et al. 1979). Infected macrophages transport the virus to the bursa of Fabricius (BF), the prime target organ for extensive IBDV replication in the cytoplasm of intrabursal IgM+ B-cells (KAUFER u. WEISS 1980; HIRAGA et al. 1994). Virus dissemination to other lymphoid organs such as to the thymus, bone marrow, spleen, Peyer's patches, cecal tonsils, and Harderian glands may take place 12 mainly during vvIBDV infection of susceptible chickens (ETERRADOSSI u. SAIF 2008). The cecal tonsils and bone marrow may serve as non-bursal lymphoid tissues supporting virus replication at later time points (ELANKUMARAN et al. 2002). As early as 48 hr pi, IBDV infection induces prominent inflammation in the BF. By day 3 to 4 pi, all bursal IgM+ B-cells are infected and show cytolytic changes (CHEVILLE 1967). vvIBDV strains such as UK661 can infect Bu-1+ cells and IgY+ B-cells in several lymphoid tissues indicating both immature and mature B-cells can be infected (WILLIAMS u. DAVISON 2005), whereas classical virulent and variant IBDVs mostly target immature B-cells. Between 7 and 21 dpi, IBDV infection results in significant reduction in the number of the B subpopulation compared to the A subpopulation of IgM+ B-cells as determined by flow cytometric analysis (PETKOV et al. 2009). These two phenotypes differed on their cell size and granularity as well as showed differential expression levels of Lewis(x), IgM, Bu-1b, and MUI78 surface antigens. It becomes apparent that macrophages are infected with IBDV and in vitro studies indicated rapid virus replication with altered in vitro phagocytic activity of such cells (KHATRI u. SHARMA 2009b). Other cells like the bone marrow-derived mesenchymal stem cells may be infected with IBDV (KHATRI u. SHARMA 2009b). The haemopoietically derived reticular cells, which reside in the antigen-trapping zone of the spleen (ellipsoid and periellipsoidal white pulp), were found to be more susceptible to IBDV. The reticular cells of mesenchymal origin, which reside in the bursal cortex, and periarteriolar lymphoid sheaths, germinal center (GC) and red pulp of the spleen were relatively resistant to IBDV (BIRO et al. 2011). Bursal follicular dendritic cells disappeared during IBDV infection probably due to lack of an intact Bcell microenvironment (JEURISSEN et al. 1998; KABELL et al. 2006). Generally, the sequellae of IBDV infections such as severity of clinical signs, organ lesions and immunosuppression correlate with the status of immunity, age and genetic background of affected chickens and with the virulence of the infecting virus strain (BERG 2000). SPF chickens infected with vvIBDV develop an earlier onset of mortality and more severe bursal lesions compared to broiler chickens with MAB and vaccinated chickens (HASSAN et al. 2002; ARICIBASI et al. 2010). Infections by 13 virulent IBDV resulted in an earlier onset and higher production of IFN-γ in the bursa of young SPF chickens compared to their older counterpart. Massive interferon-γ (IFN-γ) production from T-cells infiltrating the bursa during an acute IBDV infection (ELDAGHAYES et al. 2006; RAUW et al. 2007) further activates macrophages to release proinflammatory cytokines such as interleukin-6 (IL-6) and also nitric oxide (NO), which may aggravate bursal lesions (KIM et al. 1998). IFN-γ is suggested to be a potent apoptosis inducer in IBDV infected or adjacent healthy B-cells (LAM 1997; TANIMURA u. SHARMA 1998). A massive mast cell influx detected in the bursa of SPF chickens infected with vvIBDV may aggravate bursal lesions as typical indicators of acute hypersensitivity responses were observed in the bursa of such chickens (WANG et al. 2008; WANG et al. 2012a). These cytokine mediated bursal lesions may result in an early onset of severe immunosuppression in younger chickens (RAUTENSCHLEIN et al. 2007). Similar patterns of cytokine production and bursal lesions were detected in SPF layer type chickens infected with virulent IBDV compared to infection of age-matched 3 weeks old broiler type chickens (ARICIBASI et al. 2010). Bursal lesions with B-cell depletions lead to severe humoral immunosuppression (SHARMA et al. 1994; SHARMA et al. 2000). When susceptible neonatal chickens younger than two weeks of age are infected, they may lose the entire bursal B-cells, which result in permanent immunologic damage (HUDSON et al. 1975; WITHERS et al. 2005). Cytokine dysregulation may cause suppression of innate and cellular immunity (RAUW et al. 2007). This was elucidated as recombinant chicken IFN-γ (rchIFN-γ) inhibited an in vitro proliferation of naïve peripheral blood lymphocytes (PBL) or splenocytes (RAUW et al. 2007). Lymphocytes harvested from the blood and lymphoid organs of an IBDV infected or live IBDV vaccinated chickens showed reduced lymphoproliferative responses when stimulated by conventional mitogens (MAZARIEGOS et al. 1990; SHARMA et al. 2000). This inhibition has been detected during early time points after IBDV infection coinciding with an increased IFN-γ response. Recently, in vitro study described the interaction of IBDV VP4 with the chicken glucocorticoid-induced leucine zipper (cGILZ) that inhibited the transcription 14 of NF-κB with a subsequent suppression of the innate immunity (LI et al. 2013b). Others suggested T-cells with regulatory property may mediate cellular immunosuppression (KIM et al. 1998; SHANMUGASUNDARAM u. SELVARAJ 2011). IBDV infection induced mucosal immunosuppression denoted by reduced intraepithelial lymphocytes (iIELs) and their in vitro cytotoxicity activity (KUMAR et al. 1998). A reduced intestinal secretory IgA+ B-cells were also detected (WANG et al. 2009a). The immunological functions of both B- and T-cells may be restored during recovery of infected chickens (KIM et al. 1999; SHARMA et al. 2000). The mechanisms of Bcell functional restoration were described, whereas of T-cells remained unclear. Bursal stem cells, which survive IBDV-induced depletion, proliferate to generate new and larger bursal follicles. They became repopulated with IgM+ B-cells, and Bu-1+ cells expressing IgM or IgY (WILLIAMS u. DAVISON 2005) and dendritic-like cells (WITHERS et al. 2005). The B-cells in these follicles may undergo immunoglobulin (Ig) gene (hyper)conversion for Ig diversity and sustain specific immunological functions (WITHERS et al. 2005; WITHERS et al. 2006). Previous study indicated the strong expression of the Lex and chB1 genes in the recovering follicles as indicators of Ig gene (hyper)conversion (IVAN et al. 2001). Medullary B-cells surviving IBDV infection form small follicles, which lack Ig gene (hyper)conversion. Birds with only small follicles do not produce Abs against IBDV or other Ags, and such chickens may be in a state of permanent immunosuppression (WITHERS et al. 2006). 2.2.4. Clinical disease and pathology Chickens infected between 3 and 6 weeks of age develop the most severe clinical signs of IBD (ETERRADOSSI u. SAIF 2008). Susceptible chickens exposed to vvIBDV and classical virulent strains show a sudden onset of clinical disease within 2-3 days of exposure, characterized by severe depression and ruffled feathers (VAN DEN BERG et al. 2004). Chickens younger than 2 weeks of age and birds older than 6 weeks rarely develop clinical signs (VERVELDE u. DAVISON 1997). 15 Experimentally, broilers especially with higher MAB levels may not show clinical signs or mortality when infected with vvIBDV. Mortality peaks at 4 dpi with virulent strains. A rapid recovery after 5-7 dpi is a prominent feature of acute IBD (VAN DEN BERG et al. 2000a). In general viral shedding in the faeces of naturally infected or live vaccine vaccinated chickens can last up to 2 weeks and viral RNA can be detected by RT-PCR up to 4 weeks (KABELL et al. 2005). Age and immune status of infected chickens (SKEELES et al. 1979; ABDEL-ALIM u. SAIF 2001; IVAN et al. 2005; SAPATS et al. 2005), route of infection and nature of infecting viruses (WINTERFIELD et al. 1972; SKEELES et al. 1979; ELANKUMARAN et al. 2002) influence the development of clinical IBD and virus shedding. On the first few days after infection, an increased in bursa to body weight ratio is usually observed due to edematous bursae. Occasionally, extensive hemorrhage throughout the entire bursa has been observed in the case of vvIBDV. Compared with a moderately pathogenic strain of the virus, the vvIBDV strains caused a greater decrease in thymic weight index. Generally, hemorrhagic inflammation of the bursa is the main pathological feature of infection by virulent strains, whereas variant strains (e.g. GLS and E/Del) cause rapid bursal atrophy mostly without an inflammatory response (LAM 1997; TANIMURA u. SHARMA 1998). IBDV-induced lymphoid cell depletion is responsible for bursal atrophy as early as 7-8 dpi (CHEVILLE 1967). Histological bursal lymphoid depletions are comparable between vvIBDV and virulent IBDV infection in the early few days of infection (TANIMURA et al. 1995; TSUKAMOTO et al. 1995; INOUE et al. 1999; STOUTE et al. 2009), soon followed by heterophilic infiltration. A more virulent IBDV strains caused severe lymphoid depletions in the cecal tonsils, thymus, spleen, and bone marrow. The pathogenicity may correlate with lesion production in non-bursal lymphoid organs. As the inflammatory reaction subsides, cystic cavities develop in the medulla of affected bursal follicles followed by fibrosis in interfollicular areas (ETERRADOSSI u. SAIF 2008). Proliferation of the bursal epithelial layer produced a glandular structure of columnar epithelial cell containing globules of mucin. During recovery stage, scattered foci of lymphocytes appeared in the bursal follicles. 16 2.3. Immune responses to IBDV Apart from its immunosuppressive effects, IBDV infection in chickens activates all branches of the immune system. However, the level of activation varies depending on the virulence of infecting strains, age, immune status and genetic background of affected chickens. 2.3.1. Innate immunity The influx of macrophages, heterophils and mast cells in the bursa of Fabricius constitutes the early innate immune response to IBDV (KHATRI et al. 2005; PALMQUIST et al. 2006; RAUTENSCHLEIN et al. 2007; WANG et al. 2008). The influx of these cells may be mediated by chemokines (IL-8, iNOS) (KHATRI et al. 2005; ELDAGHAYES et al. 2006; PALMQUIST et al. 2006; RAUTENSCHLEIN et al. 2007; RAUW et al. 2007; RAUF et al. 2011a). Toll-like receptors (TLRs) such as TLR3 and TLR7 expressed by these inflammatory cells detect IBDV nucleic acids. Their mRNA expressions have been found upregulated during an acute IBDV infection (RAUF et al. 2011a; GUO et al. 2012). These interactions between IBDV and TLR3 or TLR7 have been shown to activate the interferon (IFN) system and also induced proinflammatory cytokines (IL-6, IL-1β, and IL-18) (RAUF et al. 2011a; GUO et al. 2012). The release of these cytokines was suggested to be tightly regulated by NF-κB, whereby its expression was found to be elevated in the bursa during the early phase of IBDV infection (KHATRI u. SHARMA 2006; GUO et al. 2012). The upregulation of IFN-α/β mRNA expression was reported in lymphoid organs and PBL of chickens experimentally infected with virulent IBDV (KIM et al. 1998; RAUF et al. 2011a; MAHGOUB et al. 2012). The expression levels of these cytokines in the bursa differ in relation to the virulence of infecting strains, genetic background and age of infected chickens (ELDAGHAYES et al. 2006; RAUTENSCHLEIN et al. 2007; ARICIBASI et al. 2010; RAUF et al. 2011a). IFNs may protect chickens against IBDV infection. This was verified experimentally whereby chickens pretreated with 17 recombinant chicken IFN-α/β and latter challenged with IBDV had reduced challenge virus replication and pathological lesions in the bursa (CAI et al. 2012). IBDV infection of chicken embryo fibroblasts (CEFs) resulted in upregulation of IFNinducible 20-50-oligoadenylate synthetase (OAS), IFN regulatory factors, IL-6 and IL8 mRNA expression (LI et al. 2007). The OAS and RNase L pathway interferes with viral infection through the cleavage of viral ssRNA, which is one of the recognized viral suppressor activities of OAS (MALATHI et al. 2007). Recombinant type I IFN pretreated CEFs resisted IBDV replication and resulted in reduced viral titer after infection (O'NEILL et al. 2010; CAI et al. 2012). Nitric oxide released by macrophages may constitute an early host defence against IBDV and promotes the killing of IBDV-infected and possibly virus-free cells (KHATRI et al. 2005; KHATRI u. SHARMA 2006; PALMQUIST et al. 2006; KHATRI u. SHARMA 2009a). 2.3.2. Humoral immunity Significant titers of systemic IBDV specific-Abs have been detected in the convalescent sera of chickens that are naturally or experimentally infected with IBDV (ETERRADOSSI u. SAIF 2008). All classes of Igs can be produced, but the Ab response may not protect chickens from antigenetically different IBDV strains. Neutralizing Abs are directed against the conformation dependent neutralizing epitopes of VP2 (FAHEY et al. 1991; SNYDER et al. 1992). Abs against VP3 (BECHT et al. 1988; FAHEY et al. 1991) and conformation-independent antigenic domains of VP2 (AZAD et al. 1987) are non-neutralizing. Live and inactivated IBDV vaccines may induce vigorous Ab responses in the first few weeks postvaccination (MAAS et al. 2001; ARICIBASI et al. 2010). Compared to cell culture derived strains, bursal and embryo derived strains induce higher neutralizing Ab titers (RODRIGUEZCHAVEZ et al. 2002). 18 Humoral immunity plays a significant role in protection against IBDV. Maternal antibody (MAB) provides passive protection in the first few weeks after hatch (ALNATOUR et al. 2004). MAB positive chickens developed significantly less bursal lesions than Ab negative chickens after IBDV challenge supporting the role of passive immunity in protection (HASSAN et al. 2002; ARICIBASI et al. 2010). MAB may interfere with the development of an active immune response after IBDV vaccination (RAUTENSCHLEIN et al. 2005a). Although Ab mediated immunity is crucial against IBDV, an important role of the cell mediated immunity (CMI) is suggested by several groups (RAUTENSCHLEIN et al. 2002a; YEH et al. 2002). 2.3.3. Cellular immunity During acute IBD, while bursal follicles are B-cell depleted, T-cells accumulate at the site of virus replication (TANIMURA u. SHARMA 1997; KIM et al. 1998; KIM et al. 2000; SHARMA et al. 2000). A notable influx of CD4+- and CD8+ T-cells was detected as early as 1 dpi and peaked at around 7 dpi (KIM et al. 2000). Although viral Ag was cleared by week 3 pi, T-cell influx and activation continued to week 12 pi. No T-cell depletion was detected from the bursa during IBDV infection. However, IBDV particles were detected in intrabursal T-cells (MAHGOUB et al. 2012). Infiltrating T-cells in the bursa show markers of activation such as upregulated IL-2, major histocompatibility complex (MHC) class II molecules, and IFN-γ mRNA expression (KIM u. SHARMA 2000; RAUW et al. 2007; RAUF et al. 2011b). T-cells are not only involved in bursal recovery by killing virus infected cells, but also contribute to bursal lesions. T-cell compromised SPF-chickens had the highest viral Ag load and milder inflammatory bursal lesions compared to T-cell intact birds after IBDV infection (RAUTENSCHLEIN et al. 2002a). T-cells infiltrating the bursa after IBDV infection expressed higher levels of the mRNA for cell membrane-disrupting proteins such as perforin (PFN) and granzyme A (Gzm A), and other cytolytic molecules such as the high mobility group proteins. PFN and Gzm mediated 19 cytotoxic activity may contribute to rapid viral clearance from the bursa (RAUF et al. 2011b). The role of T-cells in IBDV protection was supported in vaccination studies with T-cell or B-cell compromised chickens. Chickens depleted of functional T-cells either by neonatal thymectomy or Cyclosporin A treatment showed insufficient protection against IBDV challenge after immunization with an inactivated IBDV vaccine, whereas chickens with intact T-cells had significantly higher IBDV protection rates (RAUTENSCHLEIN et al. 2002b). Chickens with severely compromised Abproducing ability following treatment with cyclophosphamide were sufficiently protected against IBDV challenge despite the absence of detectable vaccine-induced Abs. This implies that T-cells may have role in protection (YEH et al. 2002). 2.4. Field evolution and molecular epidemiology of IBDV The first outbreak of infectious bursal disease (IBD) that had occurred in 1957 in a broiler farm near Gumboro, the Delaware area in the USA, was caused by the classical serotype 1 IBDV (COSGROVE 1962). The variant IBDV strains then emerged in the 1980’s in IBDV-vaccinated farms in the Delmarva area and were antigenetically different from the former isolates. In the late 1980’s, vvIBDV emerged in Europe (CHETTLE et al. 1989) and rapidly spread across continental Europe and Asia (LIN et al. 1993; SHCHERBAKOVA et al. 1998), Middle East (PITCOVSKI et al. 1998), South America (DI FABIO et al. 1999), and Africa (ZIERENBERG et al. 2000). IBDV undergoes genetic variation during its evolution to adapt to new hosts and to escape the host immune responses. Different biological mechanisms may play important roles for the emergence of novel viruses, particularly in segmented RNA viruses, such as IBDV. Early IBDV isolates frequently showed mutations at the major hydrophilic domains particularly in the loops PBC and PHI, which affected the antigenicity of the strains and induced vaccination failure (BAYLISS et al. 1990; HEINE et al. 1991; LANA et al. 1992; DORMITORIO et al. 1997). In the past few years, several field IBDV strains 20 isolated from different geographic areas showed aa substitutions at the minor hydrophilic domains mainly at position 254 (loop PDE) and 284 (loop PFG) (JACKWOOD u. SOMMER-WAGNER 2005; MARTIN et al. 2007; DURAIRAJ et al. 2011; JACKWOOD u. SOMMER-WAGNER 2011). Most of these viruses have been identified from areas where the viruses have been circulating for a long period of time (MARTIN et al. 2007). In the USA, one-third of the investigated field IBDV isolates (out of 300) failed to react with any of the described monoclonal abs (mAbs) that have been used to identify IBDV strains for the last 2 decades, which may reveal the circulation of new IBDV subtypes (DURAIRAJ et al. 2011). A new variant IBDV differing from the Delaware (Del E) variant of the Delmarva Peninsula was identified, which did not react with those mAbs (GELB et al. 2012). IBDV isolates, which contain epitopes of both variant and classical IBDVs in their VP2 genes were demonstrated, which can affect mAb reactivity (JACKWOOD 2012). This may provide an explanation for the increased antigenic and virulence diversity of the recent IBDV isolates. Atypical IBDVs, which harbour aa residues characteristics of variant, classical, and vvIBDV in their VP2 were characterized and showed atypical pathogenicity and reactivity patterns to most of the mAbs (MARTIN et al. 2007). 2.4.1. IBDV reassortment Genetic reassortment might be accountable for the emergence of vvIBDV in the late 1980’s in Europe (HON et al. 2006). The time of the appearance of the most recent common ancestor (tMRCA) of very virulent (vv) VP2 is approximated around 1960, whereas of vvVP1 around 1980 (HON et al. 2006), in which the latter coincided with the emergence of vvIBDV in the late 1980’s (CHETTLE et al. 1989). Thus a newly appeared vvVP1 from an unidentified avian reservoir was suggested to recombine with an already existing vvVP2 to evolve to the vvIBDV genotype, which then caused massive mortality in Europe (HON et al. 2006). This indicates the independent evolutionary history of the two segments of vvIBDV (ISLAM et al. 2001; LE NOUEN et al. 2006). Recently, several natural reassortant IBDV isolates were characterized 21 during field outbreaks (Table 2). The most common reassortant IBDVs contain segment A of vvIBDV and segment B from attenuated strains indicating the drawbacks of extensive application of live IBDV vaccines. Attempted experimental generation of reassortant viruses by co-infecting specific pathogen free (SPF) chickens with vvIBDV and attenuated serotype 1 IBDV has failed. The process of reassortment may be more complex in the field than expected and may involve the interactions of several factors: time, environment and vaccine pressure (WEI et al. 2008). Table 2: Examples of natural reassortant IBDV isolates Isolates Sources of segments Country Year References Segment A Segment B Unknown vvIBDV vvIBDV Europe 1980 (HON et al. 2006) SH95 vvIBDV Variant E China Unknown (SUN et al. 2003) 02015.1 vvIBDV Attenuated France Unknown (LE NOUEN et al. 2006) ZJ2000 Attenuated vvIBDV China 2000 (WEI et al. (2006) TL2004 Attenuated vvIBDV China 2004 (WEI et al. 2008) CA-K785 vvIBDV Serotype 2 USA 2009 (JACKWOOD et al. 2011) KZC-104 vvIBDV Attenuated Zambia 2004 (KASANGA et al. 2012) 22 2.4.2. IBDV recombination Natural homologous intragenic recombination is described for many animal viruses (LEE et al. 2013). The risk of live vaccines recombining to generate virulent natural recombinants have been well described, and disease outbreaks associated with these viruses have recently been described for infectious laryngotracheitis virus (ILTV) infections of chickens (LEE et al. 2012). Recombination may lead to antigenetically and genetically diverse IBDV populations and the emergence of novel vvIBDV groups (HON et al. 2008; HE et al. 2009a). It has the potential to alter the interactions of IBDV proteins and the orientation of the capsid domains preventing neutralization by pre-existing Abs, which lead to vaccine failure. Almost all IBDV recombinant viruses identified from field outbreaks are VP2 recombinants (Table 3). Intrasegment recombination was also detected in segment B of two vvIBDV strains possibly due to the recombination between two vvIBDV donors (HON et al. 2008). A recently isolated IBDV strain, GX-NN-L, has reassortant characteristics, whereby its segment A derived from vvIBDV, and segment B from an attenuated strain. But interestingly, segment B contains putative aa residues typical for vvIBDV isolates (CHEN et al. 2012a). An attenuated vaccinal strain, ViBursaCE, is suggested to be a potential recombinant whereby its segment A is a mosaic between variant (Variant E) and an attenuated French vaccine strain (Rhone-Merieux, strain-CT) (HON et al. 2008). 23 Table 3: Examples for natural recombinant IBDV isolates Isolates VP2 recombinants Country Year References SH-h hVP2 from vvIBDV (HLJ-5 strain) China Unknown (HON KSH/KK1 849VB et al. within segment A of an attenuated 2008; HE et al. (D78) strain 2009a) hVP2 from vvIBDV (SH.92 strain) Korea 1992/1997 (HON et al. within segment A from an 2008; HE et al. attenuated (D78) strain 2009a) Part of segment A from attenuated Belgium 1987 (D78), and the other part from (HON et al. 2008) vvIBDV (D6948) Several aa sequences at loop regions PBC and Venezuela 2001-2005 (JACKWOOD isolates PHI from classical IBDV & aa at PDE Colombia 2012) and PFG from variant IBDV Several aa sequences at loop regions PBC isolates from variant & PDE and PFG from Mexico 2004-2011 (JACKWOOD 2012) classical IBDV 157776 aa at positions 294 to 299 from Italy 2003 vvIBDV & residues from 222 to 279 (MARTIN et al. 2007) from an attenuated strain VP1 recombinants OE/G2 Segment B recombinant between Turkey Unknown OKYM & OA/G1 vvIBDV strains Harbin-1 Segment B recombinant between (SILVA et al. et al. 2012) China Unknown (HON HLJ-7 or HENAN & GZ/96 vvIBDV 2008; SILVA et strains al. 2012) 24 2.4.3. IBDV quasispecies and reversion to virulence The existence of RNA virus quasispecies may have a paramount contribution to virus evolution. An RNA virus population is made up of heterogeneous viruses, which share the consensus sequence but differ from each other by one or many mutations (DOMINGO et al. 1985). In IBDV vaccine and field strains, the quasispecies phenomenon has been described by real time RT-PCR and melting curve analysis (JACKWOOD u. SOMMER 2002; HERNANDEZ et al. 2006). Pre-existing selection pressure, for example, altered host immune status may favor one clone of a virulent virus to overwhelm the virus population to maintain its endemicity (MORIMOTO et al. 1998). Attenuated live IBDV vaccines are most frequently used to vaccinate commercial chickens. Reversion of these attenuated vaccinal strains to more virulent phenotypes under field and experimental conditions has been frequently reported (YAMAGUCHI et al. 2000; JACKWOOD et al. 2008) possibly due to a lack of IBDV polymerase fidelity during vaccine viral genome replication in the host cells. A tissue cultureadapted IBDV generated by reverse genetics from a vvIBDV strain reverted phenotypically and genotypically to the vvIBDV pathotype after inoculation into SPF chickens and maintained this pathotype afterwards (RAUE et al. 2004). Genetic reversion of vaccine strains is most likely to be one of the mechanisms that may contribute to the dissemination and persistence of virulent IBDV in the chicken population worldwide. 25 2.5. Diagnostic methods 2.5.1. Embryo inoculation The inoculation of bursal homogenates from IBDV infected chickens per the chorioallantoic membrane of 9-10 days old embryonated SPF chicken eggs is the most sensitive diagnostic method for virus isolation. The embryos die mostly within 35 days in the case of classical and very virulent viruses (HITCHNER 1970; ROSALES et al. 1989). Embryos may not die from infection by the variant viruses, yet show hepatic necrosis. 2.5.2. In vitro virus propagation Adaptation of IBDV field isolates to a cell culture system requires passages in embryonated chicken eggs and subsequent passages in cell culture system further attenuates the virus to the extent that these viruses do not induce bursal lesions (YAMAGUCHI et al. 1996a). The cell culture adapted viruses replicate in primary avian cells such as CEFs (SHI et al. 2009) and continuous cell lines of avian (QT35) and mammalian origins (Vero cells) (LUKERT u. DAVIS 1974). Compared to classic and variant strains, adaptation of the vvIBDV viruses to cell culture has been very difficult. 2.5.3. Immunological methods ELISA and virus neutralization test (VNT) can be used to determine IBDV Ab levels. Different IBDV-ELISA procedures have been described for routine diagnostic purposes (MARQUARDT et al. 1980; KECK et al. 1993). Polyclonal based Agcapture ELISA is more sensitive (HASSAN et al. 1996) compared to the mAb-based ones to detect IBDV Abs (LEE u. LIN 1992; VAN DEN BERG et al. 2004). 26 Commercially available ELISA systems use either intact viral particles or a mixture of recombinant VP2 based proteins coated plates. VNT is mostly used for research purposes (SKEELES et al. 1979). The VN titers accurately correlate with protection of chickens against IBDV (KNOBLICH et al. 2000). Only VNT allows to differentiate serotype 1 from serotype 2 IBDVs (JACKWOOD et al. 1985). Determination of the antigenic properties of IBDV field isolates is necessary when recurrent outbreaks are observed in poultry farms that had been previously vaccinated against IBDV. Although in vitro VN tests can be used for detection of antigenic differences between virus strains, in vivo cross protection studies are essential to determine the antigenicity of a virus and complete evaluation of host immune responses (JACKWOOD u. SAIF 1987). In vitro antigenicity is determined by the reaction patterns of IBDV isolates to panels of mAbs, which target conformation dependent neutralizing epitopes at the hydrophilic regions of VP2 (SCHNITZLER et al. 1993; BERG et al. 1996; ETERRADOSSI et al. 1997) using VNT or ELISA (SNYDER et al. 1988; VAKHARIA et al. 1994). By sequence analysis and sitedirected mutagenesis, aa residues, which influence the reactivity patterns of IBDVs with specific mAbs were identified in the past (LANA et al. 1992; LETZEL et al. 2007; ICARD et al. 2008; DURAIRAJ et al. 2011). Recombinant Abs developed from a chicken single chain variable Ab fragments (scFv) also differentiate IBDV isolates in a sandwich ELISA platform (SAPATS et al. 2005; SAPATS et al. 2006). 2.5.4. Molecular characterization The classical methods for molecular characterization and differentiation of IBDV field isolates include RT-PCR and restriction fragment length polymorphism (RFLP), nucleotide sequence analysis, and quantitative real time RT-PCR (qRT-PCR) (JACKWOOD 2004; WU et al. 2007a). The hypervariable region of VP2 (hVP2) of IBDV between aa residues 206 and 350 shows significant aa sequence variations. Sequencing of this region is used to categorize isolates into different pathogenic 27 strains (YAMAGUCHI et al. 1997b; JACKWOOD u. SOMMER-WAGNER 2005; JACKWOOD u. SOMMER-WAGNER 2007; SREEDEVI et al. 2007). In early studies, RT-PCR-RFLP analysis was performed mainly on hVP2 and seldom on VP1 to distinguish IBDV isolates (MEIR et al. 2001; JUNEJA et al. 2008). According to their restriction profiles, the viruses form molecular groups or genotypes (JACKWOOD et al. 2001). However, the limitation of this method is that viruses in a molecular group or with matching RFLPs may differ in their antigenic or virulence property and require sequence analysis. Direct sequencing of RT-PCR products of the hVP2 and 5’two thirds of VP1 may provide deeper insights into the nucleotide and predicted aa identity of strains and may determine mutations, recombination and reassortment in the viral genomes (LE NOUEN et al. 2005). The nucleotide sequences of VP2 and VP1 can be further used to construct phylogenetic trees to demonstrate genetic relationships between IBDV isolates (HON et al. 2006). A TaqMan qRT-PCR and melting curve analysis can be used to trace mutations in the hVP2 region (JACKWOOD et al. 2003). This method allows comparing sequences between field and vaccinal strains (JACKWOOD u. SOMMER 2002; GAO et al. 2007). It determines a single nucleotide polymorphism in VP2 (WU et al. 2007a). qRT-PCR quantifies viral load (MOODY et al. 2000). RT-loop-mediated isothermal amplification (RT-LAMP) is a rapid field test used as a screening method, particularly when direct nt sequencing facilities are unavailable (XU et al. 2009). The LAMP assay is based on the principle of autocycling strand displacement DNA synthesis performed by the Bst DNA polymerase and a set of two inner and two outer primers that recognize 6–8 regions of target DNA. LAMP has high specificity as the primers recognize six specific regions of the target amplicon (TSAI et al. 2012). 28 2.6. Vaccines and vaccination against IBDV Vaccination is widely used to prevent IBD outbreaks in the field. Most of the commercially available vaccines against IBDV are live attenuated and inactivated ones; recombinant and subunit vaccines have been licensed in some countries. Live vaccines are produced from classical and variant IBDV strains by passaging these viruses in tissue cultures or embryonated chicken eggs (YAMAGUCHI et al. 1996a; LASHER et al. 1997; JACKWOOD u. SOMMER-WAGNER 2011). They can be classified as mild, intermediate or intermediate plus vaccines based on the level of attenuation and residual virulence for SPF chickens (VAN DEN BERG et al. 2000a). The intermediate plus vaccines are regularly applied to protect chickens against vvIBDV challenges. The Deventer formula may help to determine the optimal time for IBDV vaccination to circumvent the neutralizing activity of MAB (DE WIT 1998). Live vaccines are favourable for mass application through drinking water and can induce strong humoral and cellular immunity (MÜLLER et al. 2003; MÜLLER et al. 2012). The proven reversion to virulence (YAMAGUCHI et al. 2000) and their residual immunosuppressive effects (RAUTENSCHLEIN et al. 2005b; RAUTENSCHLEIN et al. 2007) are major safety concern of their extensive field applications. Breeder vaccination by priming with live vaccines and boosting with inactivated oil-emulsion vaccines prior to laying ensures higher levels of MAB transfer to the progeny (MAAS et al. 2001; MÜLLER et al. 2012) and is applied in some countries. Commercially available IBD immune complex (IBD-ICX) vaccines are found to be safe and efficacious for in ovo and posthatch vaccination of broilers (HADDAD et al. 1997; GIAMBRONE et al. 2001; IVAN et al. 2005). They are prepared by combining an IBDV-hyperimmune serum with live intermediate plus IBDV (WHITFILL et al. 1995; JOHNSTON et al. 1997). The entrapment and retention of ICX on bursal follicular dendritic cells (FDCs) and on splenic FDCs in the germinal center were suggested as the immune enhancing mechanism of such vaccines (JEURISSEN et al. 1998). The viruses are released from the ICX when the levels of MAB declined to induce specific humoral immune responses that protect chickens against challenge 29 virus. A recombinant neutralizing Ab has been evaluated for formulation of an IBDICX vaccine (IGNJATOVIC et al. 2006). The protective effects of many recombinant IBDV vaccines were evaluated under experimental and field conditions. The polyprotein (PP), mature VP2 or immunogenic domains of VP2 of pathogenic IBDV strains were targeted to produce candidate vaccines: subunit, vectored, virus-like particles (VLPs) and chimeric virus particles. Some of these experimental vaccines are presented in table 4. An IBDV-VP2 subunit vaccine expressed in Pichia pastoris is licensed for commercial uses (PITCOVSKI et al. 2003). An E. coli expressed subunit vaccine has been evaluated under field conditions (RONG et al. 2007). The use of peptide epitope mimics, i.e. mimotopes as candidate IBDV vaccines have become promising strategy. Mimotopes are chemically synthesized and resembled the neutralizing epitopes of VP2. Their expression in prokaryotic expression vector resulted in a bioactive peptide that can induce significant neutralizing Abs and protection against IBDV challenge (WANG et al. 2007). These types of vaccines induce strong humoral immunity and always require adjuvants and multiple injections for inducing protective levels of neutralizing Abs. Many live vectored IBDV vaccines, which mimic natural infection have been developed and tested for efficacy. A live Newcastle disease virus (NDV) vectored VP2 vaccine has been experimentally evaluated (HUANG et al. 2004) and recently HVT-IBD vaccine was licensed for in ovo and posthatch vaccination of broilers and layers in various countries (BUBLOT et al. 2007; LE GROS et al. 2009). These vectored vaccines induce strong systemic neutralizing Ab levels and mucosal Abs, but pre-existing immunity for example against NDV-vector may affect their efficacy. Other IBDV-candidate vaccines include virus-like particles (VLPs). These vaccines lack viral genomes and are non-infectious. They preserve the native conformation of the capsid protein and present multiple copies of these immunogenic epitopes (BRUN et al. 2011). However, the expression systems determine the nature of the VLPs. The expression of IBDV PP by a recombinant vaccinia virus in mammalian 30 cells resulted in true VLPs (FERNANDEZ-ARIAS et al. 1998), whereas defective VLPs were detected when the PP was expressed in insect cells by a baculovirus (HU et al. 1999; KIBENGE et al. 1999; CHEVALIER et al. 2002). The main reason for the lack of true VLP formation in the yeast and insect cells may be the absence of the host protease, puromycin-sensitive aminopeptidase that is required for the processing of the PP (IRIGOYEN et al. 2012). The VP2 icosahedral capsid had been shown to induce the higher neutralizing Ab levels and better protection against IBDV challenge than the PP-derived structures and the VPX tubules (MARTINEZTORRECUADRADA et al. 2003). Candidate attenuated live IBDV vaccines generated by reverse genetics have been shown to induce strong protective immunity (BOOT et al. 2002; MUNDT et al. 2003; ZIERENBERG et al. 2004; QIN et al. 2010; GAO et al. 2011), but vaccinated chickens developed milder bursal lesions after a challenge study. These tailored chimeric IBDV vaccines were generated to contain VP2 regions of two different strains of serotype 1 IBDV (MUNDT et al. 2003; GAO et al. 2011) or were chimeric between segment A of serotype 1 and segment B of serotype 2 IBDVs (ZIERENBERG et al. 2004). A VP5 mutant IBDV vaccine induced better protection than its molecular cloned PP counterpart (QIN et al. 2010). BOOT et al. (2002) produced a chimeric virus containing the C-terminal serotype 2 VP3 inserted into genome segment A of serotype 1 IBDV. Nevertheless, the risk of reversion to virulence of these genetically modified viruses hinders their field applications (RAUE et al. 2004). Recombinant IBDV-VP2 vaccines may possibly be used as ‘’marker vaccines’’ (MÜLLER et al. 2012) by allowing the differentiation of infected from vaccinated animals (DIVA) by the detection of anti-VP3 Abs in naturally infected birds. 31 Table 4: Experimental IBDV vaccines Candidate Targeted immunogenic Expression system or Evaluation of immunity/protection vaccines domain vectors after challenge Subunit hVP2 Pichia pastoris 30% morbidity and mortality, IBDV Ag detected References (VILLEGAS et al. 2008) in the bursa N-terminal VP2 (aa 18–139) E. coli ↑ELISA-Ab titer, 100% protection from mortality, (PRADHAN et al. 2012) IBDV Ag detected in the bursa Mimotope E. coli ↑ELISA- and VN-Ab titer, 100% survival rate (WANG et al. 2007) VP2 Plants Seroconverted, 80% protection from mortality (WU et al. 2004; WU et al. 2007b) Chimeric virus Neutralizing epitope from the Bamboo mosaic virus ↑ ELISA-Ab titer, mild to moderate bursal lesions (CHEN et al. 2012b) particles PBC loop Mimotope polypeptide Human hepatitis B virus ↑ELISA- and VN-Ab titer, 100% survival rate (WANG et al. 2012c) VP2 Fowlpox virus 14% and 33% of the chickens protected from (TSUKAMOTO et al. 2000) Live vectored virus gross and histological lesions, respectively Marek’s disease virus 55% protection from bursal lesions, IBDV (TSUKAMOTO et al. 1999) infection was not prevented Live bacterial- VP2 Semliki forest virus Some levels of neutralizing Abs detected (PHENIX et al. 2001) Vaccinia virus VN-Ab titer demonstrated (ZANETTI et al. 2012) Avian adenovirus ↑VN-Ab titer, mortality up to 20% (FRANCOIS et al. 2004) T4 bacteriophage Ab detected, partial protection (CAO et al. 2005) E. coli Over 95% protection from mortality, (MAHMOOD et al. 2007) delivered seroconversion detected PP S. Typhimurium 73% protection from mortality and seroconversion detected (LI et al. 2006) 32 2.7. DNA vaccines Over the last two decades, recombinant DNA vaccines have been developed and evaluated for protection against several infectious and non-infectious diseases in humans and animals. These DNA vaccines have been shown to elicit cellular and humoral immunity. Whilst several experimental studies confirmed their protection in challenge model studies, in almost all cases the results are not transferable to a large scale commercial application. A plasmid based DNA vaccine against West Nile virus was licensed for horses (DAVIS et al. 2001) and against infectious hematopoietic necrosis virus for fish (GARVER et al. 2005). Normally DNA vaccines are applied intramuscularly. The intramuscular (IM) administration is, however, less efficient in inducing strong immunity. Plasmids are distributed systemically from the site of injection, and may then be degraded by the endonuclease activity of the host tissues (FAUREZ et al. 2010). In mammals, several other DNA vaccine delivery methods have been investigated to improve the immunogenicity of these candidate vaccines. Examples include parenteral administration with electroporation and gene gun delivery methods (YAGER et al. 2009; MURAKAMI u. SUNADA 2011). In the avian species, the IM route is the most frequently used one, however, mucosal (PETERS et al. 2004) and in ovo (PARK et al. 2009) routes have been used for DNA vaccine delivery. Very recently, the gene gun technology has been evaluated to administer DNA vaccine and the subsequent immunological responses (NIEDERSTADT et al. 2012). In ovo electroporation of exogenous plasmid DNA encoding green fluorescent protein (GFP) had been conducted to study the biological development of chicken embryos (FARLEY et al. 2011). 2.7.1. Stimulation of immune cells by DNA vaccines The eukaryotic expression cassette of a typical plasmid DNA vaccine consists of a transcriptional promoter (for example the cytomegalovirus intron A, CMV), the translatable gene and transcriptional terminator and polyadenylation signal sequence (for example the bovine growth hormone) (STEPENSON et al. 2008). Following 33 inoculation of the plasmid DNA by the IM route, the de novo production of the Ag mimics infection by a pathogen. The Ag will be efficiently presented by MHC class I and II molecules for the stimulation of humoral and cell mediated immunity. Two scenarios are proposed regarding the mechanisms by which the immune responses are mounted after IM application of DNA vaccines (PARDOLL u. BECKERLEG 1995). The APCs may be directly transfected and immunity is initiated in the context of MHC class I molecules. The intramuscularly applied plasmid DNA transfects mostly myocytes (ULMER et al. 1996). The Ag may be nibbled from the surface of myocytes by APCs (the so-called cross presentation) via the scavenger receptors (SRs) and presented in the context of MHC class II molecules to induce the desired Ag specific immunity (HARSHYNE et al. 2003). SRs are expressed on the surface of macrophages and dendritic cells and as secreted proteins. SRs have been described in chickens (HE et al. 2009b). The transfected muscle cells show increased expression of MHC class I and co-stimulatory molecules suggesting their roles in endogenous Ag presentation directly to CD8+ T-cells (SHIROTA et al. 2007; MARINO et al. 2011). 2.7.2. IBDV DNA vaccines A number of studies were conducted to evaluate the immunogenicity and protective efficacy of IBDV DNA vaccines following their IM administration to commercial broilers and SPF chickens. The VP2 or the PP gene of pathogenic IBDV strains were thus cloned into eukaryotic expression vectors as candidate DNA vaccines. Experimental studies demonstrated that IBDV-DNA vaccinated chickens may or may not develop neutralizing Abs (LI et al. 2003; SUN et al. 2005; HSIEH et al. 2010; CHEN et al. 2011c). Previously, it has been speculated that CMI may be involved in IBDV DNA vaccine-induced protection. This has been forwarded due to protection of challenged chickens in the absence of higher levels of Ab responses (CHANG et al. 2001; CHANG et al. 2003; KIM et al. 2004). In vitro and in vivo lymphoproliferative assays showed that IBDV DNA vaccines might induce significant T-cells responses (KIM et al. 2004; LI et al. 2006; HSIEH et al. 2007; MAHMOOD et al. 2007; HSIEH et 34 al. 2010) including memory T-cells in vaccinated chickens (HSIEH et al. 2007, 2010). Broiler chickens vaccinated with DNA vaccines showed increased T-cell responses indicating no interference of T-cell immunity with existing MAB (HSIEH et al. 2010). DNA vaccines applied intramuscularly induced only few prechallenge T-cells in the bursa as demonstrated immunohistochemically (CHEN et al. 2011c). These responses might provide some levels of protection presumably by clearing virus infected cells during challenge. However, the results of most challenge studies showed variable success of protection. Chickens vaccinated with these candidate DNA vaccines at higher doses (200 µg to 7 mg/bird) showed reduced clinical signs and bursal lesions after challenge (HSIEH et al. 2010). IBDV DNA vaccines evaluated after in ovo application and through mucosal routes conferred partial protection (PETERS et al. 2004; PARK et al. 2009). Generally, IBDV DNA vaccines showed good immunogenicity when administered at higher doses, however, this remains impractical for field applications. Several adjuvants and adjuvant formulations have been investigated to improve the immunogenicity and protection provided by these vaccines (section 2.8 and 2.9). 2.8. Adjuvants Vaccine adjuvants are compounds that can modulate the intrinsic immunogenicity of an Ag and elicit strong and long lasting immune responses without having any specific antigenic effect by themselves (VOGEL 1998). They can be derived from natural compounds such as minerals, microbial components or natural products of animal and plant origins (BEREZIN et al. 2010). Most inactivated and subunit avian vaccines are formulated as oil-in-water emulsion or in alum (SWAYNE et al. 2012). In this case, montanide was used as oil-in-water preparation for experimental and commercial vaccines. This vaccine emulsion has been shown to induce strong humoral responses with reduced reactogenicity at the injection sites (JANG et al. 2011; LIU et al. 2011). These adjuvants provide slow 35 release of Ags at the injection sites. Microbial components such as cholera and E. coli heat-labile toxins have been investigated for example with inactivated avian influenza vaccines as mucosal adjuvants in chickens and induced strong mucosal and systemic immunity (HIKONO et al. 2012; MILLER et al. 2012). These conventional adjuvants in general promote strong Ab responses, yet show limited ability to induce CMI. New classes of molecular adjuvants are required, which enhance immune responses by the new generation vaccines such as DNA vaccines. 2.8.1. Cytokine adjuvants for avian viral vaccines Cytokines are small soluble proteins secreted mainly by immune cells. They play pivotal roles in cell-to-cell signaling and thus control immune responses to pathogenic infections and vaccination (BAROUCH et al. 2004). Recently, several avian cytokines have been cloned and their biological activities have been investigated in vitro and in vivo. Over 23 interleukins, eight type I IFNs, IFN-γ, IFN-λ, and GM-CSF are characterized in chickens (KAISER et al. 2005; KARPALA et al. 2008; KAISER 2010). The functional domains of most of the avian cytokines (GU et al. 2010a; GU et al. 2010b; KIM et al. 2012), which interact with the receptors on the surface of the immune cells are sufficiently characterized (GU et al. 2010c; JEONG et al. 2012; KIM et al. 2012). The intracellular signalling cascades (JAK/STAT) that result in the modulation of the immune response have been well determined (KAMPA u. BURNSIDE 2002; OSINALDE et al. 2011). The multifunctional effects of these cytokines on the innate and adaptive immune system of the host include the activation and differentiation of APCs, B- and T-cell proliferation, and induction of the expression of costimulatory molecules (HILTON et al. 2002). Chickens inoculated with recombinant chIL-2 (HILTON et al. 2002) or plasmid encoded chIL-2/IL-15 (LILLEHOJ et al. 2001) developed an increase in the numbers of peripheral blood CD4+- and CD8+ T-cells. These cells expressed higher levels of the IL-2 receptor (IL-2R) (HILTON et al. 2002). Potent therapeutics and vaccine adjuvant effects of several avian cytokines were described for poultry 36 (LILLEHOJ et al. 2001; KAISER 2010; KUMAR et al. 2010). Cytokine adjuvants can be classified as Th1 promoting and include IL-2, IFN-γ, IL-12, IL-15, and IL-18. These cytokines can augment CMI. Th2 promoting cytokines like granulocyte-macrophage cell stimulating factor (GM-CSF) and IL-4 enhance humoral immunity (RAHMAN u. EO 2012). Simultaneous application of Th1 and Th2 cytokine adjuvants may result in balanced Th1/Th2 immune responses to an Ag. The major advantages of cytokine adjuvants are their suitability to be administered with DNA, killed, and live viral vaccines (Table 5 and 6). They may show synergistic effects when two or more of them are administered simultaneously. They can be delivered as recombinant cytokines (XIAOWEN et al. 2009), and have been shown to induce significant humoral immune responses when co-administered for example together with inactivated Newcastle disease (ND) and infectious bronchitis (IB) vaccines (DEGEN et al. 2005; WANG et al. 2006; HUNG et al. 2010). However, the major limitations of these recombinant cytokines as vaccine adjuvants are their extraordinary short in vivo half-life time. To circumvent this problem, cytokineencoding plasmids are utilized and co-administered with Ags (BAROUCH et al. 2004). Large numbers of plasmid encoded cytokines have been evaluated as DNA or conventional vaccine adjuvants for several viral Ags in chickens (Table 5). They may be cloned into a bicistronic eukaryotic expression vector together with the immunogenic target gene to enhance the protective levels of both humoral and cellular immunity (KUMAR et al. 2009). Alternatively, cytokine genes encoded by live viral vectors such as HVT, fowlpox (FPV) and adenovirus (Ad) vectors may enhance neutralizing Ab production of the co-administered vaccines (Table 6). Orally administered live attenuated S. Typhimurium that expressed IL-18 modulated both humoral and Th1-biased CMI against the co-delivered inactivated avian influenza vaccine (RAHMAN et al. 2012). Expression of chIL-2 as fusion protein with influenza neuraminidase VLPs has been shown to induce strong humoral immunity when applied together with inactivated influenza A (H3N2) vaccine (YANG et al. 2009). 37 Although the adjuvant properties are well recognized, none of the cytokine-based adjuvants are licensed so far. Appropriate methods of cytokine delivery still need to be developed. 38 Table 5: Improvement of the protective efficacy of avian viral vaccines after co-administration of plasmid encoded avian cytokines Cytokines Vaccines Evaluation of immunity/protection after challenge References IFN-γ Live cell-free HVT ↓tumor incidence after vMDV challenge (HAQ et al. 2011) NDV-DNA ↑HI-Ab titer, ↑T-cell proliferation (SAWANT et al. 2011) Live ND 44% of the live chicks had IgM after vaccination (DILAVERIS et al. 2007) Inactivated ND ↑HI-Ab, ↑↑IFN-γ, ↑↑CD8+ T-cells, 100% protection (HUNG et al. 2010) IL-18 against NDV ILTV-DNA ↑CD8+ T-cells, ↑IFN-γ, 80% protection against ILTV (CHEN et al. 2011b) AIV-DNA ↑HI-Ab, ↑CD4+ T-cells (LIM et al. 2012) IL -15 AIV-DNA ↑↑HI-Ab, ↑↑CD4+ T-cells (LIM et al. 2012) IL-4 NDV-DNA ↑↑↑HI-Ab, 40% survival after challenge with (SAWANT et al. 2011) vNDV GM-CSF IBV-DNA ↑ELISA-Ab, ↑T-cell proliferation, 86% protection (TAN et al. 2009) IL-18 IBDV-DNA ↑ELISA-Ab, ↑T-cell proliferation, ↑IL-4 & IFN-γ mRNA expression, 93% (LI et al. 2013a) protection from clinical signs & mortality, milder bursal lesions IL-6 IBDV-DNA ↑ELISA-Ab, 60% protection, milder bursal lesions (SUN et al. 2005) IBDV-DNA* ↑ELISA-Ab, 90% protection, milder bursal lesions (SUN et al. 2005) NDV-DNA is a bicistronic plasmid encoding the HN & F gene of NDV; ILTV-DNA represents a plasmid encoding glycoprotein B (gB) of ILTV; AIV-DNA encodes the HA of H5N1; IBV-DNA encodes S1 gene of IBV; IBDV-DNA represents a plasmid encoding VP2 of IBDV & IBDV-DNA* encodes the PP gene. IBDV= infectious bursal disease virus; AIV= avian influenza virus; ILTV= infectious laryngotracheitis virus; HVT= herpesvirus of Turkeys; NDV= Newcastle disease virus. ↑ denotes increase and ↓ denotes decrease. 39 Table 6: Examples for avian cytokines delivered by live viral vectors as adjuvants for avian viral vaccines Cytokine vectors Vaccines/target immunogen Evaluation of immunity/protection after challenge References rHVT-IL-2 HVT ↑VN-Ab, 43% protection against vMDV (TARPEY et al. 2007a) Live IBV ↑ELISA-Ab, 83% protection against vIBV, ↓ciliostasis (TARPEY et al. 2007b) rFPV-HN-IL-12 HN-NDV ↑↑HI-Ab, 100% protection against vNDV (SU et al. 2011) rFPV-IL-12 rFPV-HN-NDV ↑HI-Ab, ↑IFN-γ, 83% protection against vNDV (SU et al. 2011) rFPV-HA-IL-6 HA-H5N1 ↑HI-Ab, ↑T-cell proliferation, 80% protection against H5N1, (QIAN et al. 2012) ↓virus shedding rFPV-gB-IL-18 gB-ILTV ↑CD4: CD8+ T-cell ratio, 100% protection against vILTV (CHEN et al. 2011a) rFPV-HA-IL-18 rFPV-HA (H9N2) ↑HI-Ab,↑↑ T-cell proliferation, no viral shedding (CHEN et al. 2011a) rAd-GM-CSF-S1 S1-IBV ↑HI-Ab, ↑↑IFN-γ, 100% protection, mild renal lesions (ZESHAN et al. 2011) rAd-GM-CSF rAd-S1-IBV ↑HI-Ab, ↑CMI, 100% protection against IBV, mild renal (ZESHAN et al. 2011) lesions rFPV-IL-12 rFPV-VP2 ↑VN-Ab, 100% protection from challenge, some birds with (SU et al. 2011) bursal lesions rHVT-IL-2 represents recombinant HVT expressing IL-2; rFPV-HN-IL-12 coexpresses HN of NDV & IL-12; rFPV-HA-IL-6 coexpresses HA of H5N1 & IL-6; rFPV-gB-IL18 represents FPV co-expressing gB of ILTV & IL-18; rAd-GM-CSF-S1 co-expresses GM-CSF & S1of IBV; rFPV-IL-12 expresses IL-12; rFPV-HN-NDV expresses the HN gene of NDV; rFPV-HA (H9N2) expresses HA of H9N2; rAd-S1-IBV expresses S1 gene of IBDV; rFPV-VP2 expresses VP2 of IBDV. rFPV= recombinant fowlpox virus; ILTV= infectious laryngotracheitis virus; rHVT= recombinant herpesvirus of Turkeys; rAd= recombinant Adenovirus. ↑ denotes increase and ↓ denotes decrease. 40 2.8.1.1. Chicken IL-2 and IFN-γ as adjuvants for IBDV-DNA vaccines IL-2 is mainly secreted by T helper cells. Apart from its proliferative effects on T-cells and APCs (HILTON et al. 2002), IL-2 promotes growth of chickens (FORD et al. 2002). Co-administration of plasmid encoded chIL-2 together with plasmid encoded IBDV-VP2 or PP genes promoted IBD-specific neutralizing Ab production and T-cell responses. The vaccinated SPF chickens were partially protected against lethal virus challenge (HULSE u. ROMERO 2004; LI et al. 2004; KUMAR et al. 2009). The immune potentiating effect of IL-2 was found to be significant when cloned together with the VP2 gene into a bicistronic eukaryotic expression vector (KUMAR et al. 2009). In contrast to these findings, lack of adjuvant effects of plasmid-encoded chIL2 was demonstrated after in ovo application with an IBDV DNA vaccine (PARK et al. 2009). The co-administration of plasmids encoding chIFN-γ and IBDV-PP did not enhance the immune response and protection against IBDV (ROH et al. 2006). Repeated IM co-administration of chIFN-γ and PP encoding plasmids at the same injection sites was found to be immunosuppressive and challenged chickens developed more severe bursal lesions compared to the administration of the two plasmids at different sites (HSIEH et al. 2006). 2.8.2. Toll like receptor (TLR) ligands as adjuvants An antigen presenting cell (APC) uses its pathogen recognition receptors (PRRs) such as TLRs to recognize pathogen-associated molecular patterns (PAMPs), which include viral nucleic acids, bacterial DNA, and others (LI et al. 2010). For chickens, ten different types of TLRs expressed by APCs such as DCs (WU u. KAISER 2011), monocytes (HE et al. 2006), B-cells (ST PAUL et al. 2012d), thrombocytes (ST PAUL et al. 2012c), and heterophils (KOGUT et al. 2005), CD4+- and CD8+ T-cells (SHANMUGASUNDARAM u. SELVARAJ 2011; ST PAUL et al. 2012a) and erythrocytes (ST PAUL et al. 2013) have been described. TLR3, TLR7 (PHILBIN et al. 2005) and TLR21 are localized in the endosomes of APCs and sense viral and 41 bacterial nucleic acids, respectively. Core TLR signalling cascades will be initiated after the recognition of PAMPs, which in turn leads to activation and upregulation of the host defense genes such as type I IFNs, chemokines and cytokines (CIRACI u. LAMONT 2011). The cytokine responses can amplify the innate immune responses and activate adaptive immunity (CHEVRIER et al. 2011). In mice and humans, CpG-ODNs may reach to the endosomally located TLR9 through the lectin receptor referred to as DEC-205 (LAHOUD et al. 2012) or CD14 (BAUMANN et al. 2010). Granulin, which is a soluble protein, binds to CpG-ODN and may serve as co-factor to recruit CpG-ODN to TLR9 (MORESCO u. BEUTLER 2011; PARK et al. 2011). The chicken TLR21, which is functionally orthologous to the mammalian TLR9, recognizes synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs (CpG-ODN) or bacterial DNA that normally contains this motif (BROWNLIE et al. 2009; KEESTRA et al. 2010). The chicken bone marrowderived dendritic cells expressed higher DEC-205 levels in response to stimulation by lipopolysaccharide (WU et al. 2010), yet its role as receptor for CpG-ODN uptake in avian species remains unclear. Synthetic class B CpG-ODN is a single stranded short DNA sequence that contains phosphorothioated internucleotide linkage, which confer resistance of the DNA to degradation by nucleases (SESTER et al. 2000). They have been extensively investigated for their in vitro immunostimulatory properties in avian cells. They can activate avian APCs (HE et al. 2006; WU u. KAISER 2011) and B-cells (WATTRANG 2009) that responded to CpG-ODN stimulation through the production of NO, IL-1β, and IFN-γ (BHAT et al. 2010). Avian monocytes showed a strong Th1-biased immune response to stimulation by CpG-ODN and TLR3 agonist (Poly I: C) (HE et al. 2012a). CD4+ T-cells and B-cells isolated from the bursa responded to CpG-ODN through the production of IFN-γ (ST PAUL et al. 2012a). B-cells showed upregulation of transcripts associated with Ag presentation such as CD80 and MHC class II molecules (ST PAUL et al. 2012d). In vivo treatment of chickens with CpG-ODN promoted the upregulation of MHC class II and IFN-γ mRNA expression in the 42 spleen, indicating the role of CpG-ODN in enhancing Ag presentation (ST PAUL et al. 2011). Prophylactic effects of class B CpG-ODNs after in ovo or parenteral application have been reported against Eimeria (DALLOUL et al. 2004), E. coli (GOMIS et al. 2004) and S. Typhimurium (TAGHAVI et al. 2008) infections in chickens. High mRNA expression levels of proinflammatory cytokines, IFN-γ and chemokines in the spleen of chicken embryos inoculated with CpG-ODN at embryonation day 18 prior to IBV challenge has limited IBV replication (DAR et al. 2009). Chickens that had received CpG-ODN and were challenged with a low pathogenic avian influenza virus showed reduced virus shedding (ST PAUL et al. 2012b). Several synthetic class B CpG-ODNs have been investigated as vaccine adjuvants in mammals and avian species (MAHMOOD et al. 2006; DUTHIE et al. 2011; MALLICK et al. 2012). They have been shown to enhance the immunogenicity and efficacy of inactivated viral vaccines (WANG et al. 2009b; XIAOWEN et al. 2009) and recombinant poultry vaccines (MALLICK et al. 2012). Additional work showed that chickens intramuscularly inoculated with CpG-ODN and IBDV-VP2 DNA vaccine exhibited higher levels of protective immunity and survival following lethal virus challenge (WANG et al. 2003; MAHMOOD et al. 2006). IBDV DNA vaccine combined with CpG-ODN induced CMI as indicated by increased delayed hypersensitivity reactions (MAHMOOD et al. 2006; MAHMOOD et al. 2007). In contrast, ROH et al. (2006) reported that CpG-ODN had no significant effects on the immunogenicity of an IBDV PP DNA vaccine when co-administered intramuscularly to chickens. 2.9. Microparticulate vaccine and adjuvant carrier systems The hurdle of poor immunogenicity of DNA and peptide vaccines may be resolved through effective microparticulate vaccine carrier systems (SINGH et al. 2000; O'HAGAN et al. 2004b; FOGED 2011; DE TEMMERMAN et al. 2012). These carrier systems are mostly prepared from poly(D, L-lactide-co-glycolide) (PLGA), which is a 43 versatile biodegradable polymer used to deliver drugs, hormones, and vaccines in human medicine (LANGER et al. 1990). PLGA MPs undergo hydrolysis in vivo to non-harmful and non-toxic compounds that are readily excreted from the body (BENDIX 1998). The formulation of vaccines with PLGA MPs increased the persistence of Ag at the injection sites. MPs thus serve as tailored slow control release system for the Ags that are expressed in situ in myocytes. Ags are released either by diffusion or by hydrolysis of MPs and this quality of MPs may avoid the application of booster vaccinations (SINGH et al. 1997). They are suitable carriers to deliver multiple copies of different Ags to the same APCs. The targeted delivery of Ags to APCs by these MPs may have Ag dose sparing effects and is an advantage if large numbers of animals need to be vaccinated (O'HAGAN et al. 2006). At the same time the recruitment of large numbers of phagocytes to the sites of IM injection of PLGA MPs may allow Ag presentation by these cells following phagocytosis (DENIS-MIZE et al. 2003). PLGA MPs with a diameter of 0.5-10 µm can be readily phagocytosed by APCs (JILEK et al. 2005; O'HAGAN et al. 2006; JAIN et al. 2011) with the help of β2 integrins expressed on the surface of APCs (ROGERS u. BABENSEE 2011). This may be followed by an immediate increase in the levels of expression of costimulatory molecules (CD86) and NF-κB, indicating immune system stimulation by PLGA MPs (NICOLETE et al. 2011). The maturation process of APCs in mammals was further improved by delivery of DNA vaccine adsorbed onto the surface of positively charged PLGA MPs (JILEK et al. 2004; REDDY et al. 2012). The processes of microencapsulation or adsorption of vaccines and adjuvants are outlined in Fig.1. 44 1st homogenization 2nd homogenization Solvent evaporation DNA/protein solution PLGA solution (Aqueous phase) (Organic/oily phase) Surfactant solution Water-in-oil emulsion (Aqueous phase) Water-in-oil-in-water emulsion and MPs separation (W/O/W) PLGA MPs hardening Lyophilization & Lyophilized PLGA MPs ready to use Fig.1. Method of vaccine (DNA/protein) microencapsulation in PLGA microspheres using the w/o/w emulsion solvent evaporation method (SINGH et al. 1997; TINSLEYBOWN et al. 2000; FREITAS et al. 2005; OSTER u. KISSEL 2005). PLGA/polyethylenimine (PEI) is dissolved in dichloromethane. Surfactant contains polyvinyl alcohol to coat the outer PLGA matrix. This prevents MPs aggregation and facilitates their uptake by phagocytic cells by influencing the structure and hydrophobicity of MPs. PEI may be added to the oily phase to condense DNA and reduces DNA vaccine degradation during microencapsulation (ZHANG et al. 2008). Preferentially, DNA can be adsorbed onto cationic PLGA MPs to prevent the destruction of the supercoiled DNA structure of the plasmid during microencapsulation (OSTER et al. 2005). Cetyltrimethylammonium bromide (CTAB) is added to the surfactant solution to provide a positive surface charge to PLGA MPs to readily adsorb DNA (O'HAGAN et al. 2004a; SINGH et al. 2006). CTAB binding to the surface of MPs could be facilitated by addition of PEI in the oily/organic phase, 45 which neutralizes the anionic charges of the PLGA-matrix (OSTER et al. 2005). During surface adsorption, plasmid DNA is incubated with cationic PLGA MPs for adsorption through electrostatic interactions (OSTER et al. 2005; LIMAN et al. 2007). Vaccine adjuvants can be entrapped in or adsorbed onto PLGA MPs in similar ways. 2.9.1. Enhancing specific immunity by PLGA MPs Several early studies have shown the induction of strong immunological responses following parenteral or mucosal administration of microencapsulated protein or DNA vaccines against several infectious diseases including HIV-AIDS (O'HAGAN 2001; SINGH et al. 2001b; WILKHU et al. 2011). The IM administration of cationic PLGA MPs with an adsorbed hepatitis B virus DNA vaccine to mice was shown to induce significantly higher Ab levels, CMI, and protection compared to the naked DNA vaccine. This may probably be due to improved transfection of inflammatory phagocytic cells or due to persistent Ag expression from the transfected myocytes (HE et al. 2005). An intramuscularly delivered anti-foot and mouth disease DNA vaccine adsorbed onto cationic MPs induced a long-term immune response against challenge (REDDY et al. 2012). The combination of using electroporation before or after IM application of PLG-encapsulated plasmid DNA enhanced the expression of the Ag, which resulted in a more robust T-cell response (BARBON et al. 2010). Mucosal delivery of DNA vaccines entrapped in PLGA MPs or adsorbed onto the surface of cationic MPs have been shown to induce systemic and mucosal immunity in several animal models for different infectious diseases (SINGH et al. 2001b; WANG et al. 2011; BHOWMIK et al. 2012). MPs may prevent DNA vaccine degradation by mucosal enzymes (JONES et al. 1998). Recently, the usefulness of PLGA MPs as vaccine delivery systems to avian species was introduced (PEISER 2006; LIMAN et al. 2007). An oculonasally administered avian Metapneumovirus (aMPV) fusion protein-encoding plasmid DNA and the corresponding subunit vaccine by PLGA MPs in a prime-boost approach to turkeys induced mucosal and cellular immunity (LIMAN et al. 2007). 46 There is a need to further enhance the immunogenicity of MP-entrapped or adsorbed vaccines by additional adjuvants (O'HAGAN et al. 2006; FISCHER et al. 2009). While CpG-ODN shows extremely potent immunomodulating effects on innate and adaptive immune cells in vitro, it has become apparent that rapid in vivo degradation and an inefficient delivery to target cells hinder its application as a novel vaccine adjuvant. The adjuvant effects of several immunostimulatory molecules including CpG-ODN and cytokines in non-avian vaccines were found to be superior when they were administered adsorbed or microencapsulated in PLGA MPs compared to their applications in soluble forms (SINGH et al. 2001a; MALYALA et al. 2008; MALYALA et al. 2009; WANG et al. 2012b). The delivery of chIL-2 and Newcastle disease DNA vaccine in the form of nanoparticles improved immunity against NDV challenge (ZHANG et al. 2010). This formulation resulted in a higher HI-Ab titers and serum IFN-γ levels compared to the naked DNA vaccine (ZHANG et al. 2010). 47 3. Goals and objectives The present study was designed to understand the molecular epidemiology of IBDV in Ethiopia and to develop a new generation vaccine against IBDV. The objectives of the first part of the study include - To collect IBDV isolates from field outbreaks - To investigate their pathogenicity in vivo in chickens - To conduct genotypic characterization of the isolates and construct phylogenetic trees. The specific objectives in the second part of the study include - To construct IBDV DNA vaccines - To identify suitable molecular adjuvants - To improve the immunogenicity of the DNA vaccine by utilizing particulate vaccine delivery system - To enhance the protection conferred by MP-based DNA vaccine by delivering adjuvant loaded MPs 48 4. Molecular evidence of very virulent infectious bursal disease virus in chickens in Ethiopia Tamiru Negash, Esayas Gelaye, Henning Petersen, Beatrice Grummer and Silke Rautenschlein Avian Dis 2012 56:605-610. http://www.aaapjournals.info/doi/full/10.1637/10086-022012-ResNote.1 49 SUMMARY. Infectious bursal disease virus (IBDV) is an important immunosuppressive pathogen of chickens worldwide. The introduction and evolution of IBDV in most African countries, especially in Ethiopia remains unclear. We have investigated IBDV isolates obtained from commercial broilers, indigenous chickens and pullets. The hypervariable region of the virus protein (VP) 2 (hVP2) and the 5` two third of VP1 of eleven IBDV isolates were characterized by RT-PCR and further sequencing. All isolates were identified as very virulent (vv) IBDV based on the predicted amino acid (aa) sequences of the VP2 protein. Interestingly, the sequence analysis of the 5` two third of VP1 indicated that the Ethiopian IBDV strains have aa residues typical for vvIBDV and also for attenuated IBDV strains. Amongst all IBDV strains included in this study for phylogenetic comparison of VP2 nucleotide sequences, Ethiopian strains form a cluster within the vvIBDV lineage. We have shown that Ethiopian IBDV strains have mutations in the VP1 region. Their roles in IBDV virulence may require further in vivo studies. As depicted in this study, the nucleotide and aa sequence analysis of VP1 in addition to VP2 is necessary to obtain a clear picture of the molecular evolution of IBDV. Key words: chicken, Ethiopia, infectious bursal disease virus, molecular epidemiology 50 The extent of contribution from Tamiru Negash to this article is evaluated according to the following scale: A. has contributed to collaboration (0-33%). B. has contributed significantly (34-66%). C. has essentially performed this study independently (67-100%). 1. Design of the project including design of individual experiments C 2. Performing of the experimental part of the study C 3. Analysis of experiments C 4. Presentation and discussion of the study in article form C 51 5. Mucosal application of cationic poly(D, L-lactide-co-glycolide) microparticles as carriers of DNA vaccine and adjuvants to protect chickens against infectious bursal disease Tamiru Negasha, Martin Liman1, Silke Rautenschlein a* a Clinic for Poultry, University of Veterinary Medicine Hannover, Bünteweg 17, 30559 Hannover, Germany 1 Present address: AniCon Labor GmbH, Mühlenstraße 13 49685, Höltinghausen, Germany Vaccine (submitted) 52 Abstract Infectious bursal disease virus (IBDV) is an immunosuppressive virus of chickens. The virus protein (VP) 2 induces neutralizing antibodies, which protect chickens against the disease. The aim of this study was to develop a cationic poly(D, L-lactide-co-glycolide) (PLGA) microparticle (MP) based IBDV-VP2 DNA vaccine (MP-IBDV-DNA) for chickens to be delivered orally and by eye drop route. The tested IBDV-VP2 DNA vaccines were immunogenic for specific-pathogen-free chickens and induced an antibody response after intramuscular application. Coinoculation with a plasmid encoding chicken IL-2 (chIL-2) or CpG-ODN did not significantly improve protection against IBDV challenge. However, the application of a MP-IBDV-DNA vaccine alone or in combination with a delayed oral and eye drop application of cationic MP loaded with CpG-ODN or chIL-2 improved protection against challenge. The MP-IBDV-DNA-vaccinated chickens showed less pathological and histopathological bursal lesions, a reduced IBDV antigen load as well as T-cell influx into the bursa of Fabricius (BF) compared to the other groups (p<0.05). The addition of chIL-2 loaded MP improved challenge virus clearance from the BF as demonstrated by lower neutralizing antibody titers and reduced IL-4 and IFN-α mRNA expression in the bursa at 7 days postchallenge compared to the other challenged groups. Overall, the efficacy of the IBDV-DNA vaccine was improved by adsorption of the DNA vaccine onto cationic PLGA-MP, which also allowed mucosal application of the DNA vaccine. Key words: Adjuvant, chicken, DNA vaccine, IBDV, poly(D, L-lactide-co-glycolide) microparticles 53 Abbreviations ChIL-2= chicken interleukin 2; CpG-ODN = oligodeoxynucleotides (ODN) containing CpG; DNA= deoxyribonucleic acid; IBDV= infectious bursal disease virus; MP= microparticles; PLGA= poly(D, L-lactide-co-glycolide); Tregs= regulatory T-cells; VP2= virus protein 2 54 1. Introduction Infectious bursal disease virus (IBDV) causes an acute and economically important immunosuppressive disease in poultry worldwide. IBDV is an Avibirnavirus [1] with a bisegmented dsRNA genome [2], of which segment A encodes the virus protein 2 (VP2) responsible for the induction of neutralizing antibodies [3]. IBDV replicates in the gut-associated lymphoid tissues and macrophages. The main target cells are immature intrabursal B-cells [4]. IBDV causes severe lymphoid cell depletion in the bursa of Fabricius (BF) [4,5]. IBDV infection of young chickens is marked by severe mortality and immunosuppression, which leads to vaccination failure and increases susceptibility of chickens to other pathogens [6]. Live IBDV vaccines are widely applied to commercial chickens to control IBDV [7,8]. Nevertheless, these vaccines may revert to virulent strains [9], induce immunosuppression [8] and may undergo segment reassortment with field strains [10]. IBDV VP2 subunit vaccines have been developed [11,12], but production costs of these subunit vaccines are usually high. Recently a vector vaccine on the basis of herpesvirus of turkeys-expressing IBDV VP2 was licensed [13]. DNA vaccines against IBDV have been developed encoding-VP2 or the polyprotein gene of IBDV. These vaccines often conferred only partial protection [1416]. It was demonstrated that IBDV DNA vaccine efficacy can be improved by coadministering plasmid encoded chicken interleukin-2 (chIL-2) or CpG-ODN [17,18]. In mice and humans it was shown that DNA vaccination together with a delayed application of cytokine adjuvants may further improve the protective responses after DNA vaccination [19,20]. It is speculated that this strategy may reduce the expansion of immunosuppressive regulatory T-cells (Tregs) [20], which may otherwise suppress antigen specific T-cells. Microparticulate carriers for DNA vaccines may provide additional adjuvant effects and protect the DNA from degradation after mucosal application [21-23]. Poly(D, L-lactide-co-glycolide) (PLGA) is the polymer of choice for cationic microparticle (MP) preparation [24]. Targeted delivery and prolonged persistence of the DNA when coated onto cationic MP improve the duration of DNA-vaccine induced immunity [21,25]. The objective of our study was to develop and test the 55 efficacy of an improved cationic MP based IBDV-DNA vaccine to be delivered by mucosal routes to chickens in combination with a delayed mucosal delivery of cationic MP adsorbing chIL-2 or CpG-ODN. 2. Materials and Methods 2.1. Chickens Eggs from specific pathogen free (SPF) layer type chickens (VALO®, Lohmann LSL-LITE) were obtained from Lohmann Tierzucht (Cuxhaven, Germany). The hatched chickens were randomly distributed and maintained in different groups under isolated conditions in the animal facilities of the University of Veterinary Medicine Hannover. All animal experimentations were approved and carried out following the institutional guidelines for animal care. Feed and water was supplied ad libitum 2.2. Cloning and expression of VP2 and chIL-2 cDNAs of VP2 of the two very virulent (vv) IBDV strains 82Eth [26] and 89163/7.3 (provided by N. Eterradossi, AFSSA, Ploufragan, France) were synthesized from total RNA using Oligo(dT)20 SuperScriptâ„¢ III RT kit (Invitrogen). The RNA had been isolated from bursae of IBDV infected birds. The vvIBDV strains had been isolated from several chicken flocks that had experienced high mortality rate [26]. The sequence coding for chIL-2 was amplified from splenocytes of chickens, which were stimulated in vitro with Concanavalin (Con) A (5 µg/mL) for 6 hrs [27]. PCR was conducted with the TaKaRa Ex Taqâ„¢ Polymerase (Takara Bio Inc., Shiga, Japan) using the primers; GCCGGTACCGACGCAGCGATGACAAACCTGC-3’; CGGGCGGCCGCTGATCACCTTATGGCCCGGATTA-3’, GCCGGTACCGACGCAATGATGTGCAAAGTACTGATC-3’; CGGGCGGCCGCTGATTATTTTTGCAGATATCTCACAAA-3’). VP2f: VP2r: 5’- chIL-2f: and KpnI 5’5’- chIL-2r: and 5’NotI restriction sites were included (underlined in the primers). The PCR products were 56 double digested by FastDigest® NotI and FastDigest® KpnI (Fermentas GmbH, St. Leon-Rot, Germany) and purified with NucleoSpin® Extract II PCR clean up and Gel extraction kit (Macherey-Nagel, Düren, Germany). Double digested PCR products were cloned into the pCR3.1® eukaryotic expression vector (Invitrogen) and transformed into chemically competent TOP10F' E. coli (Invitrogen) following the guidelines of the manufacturer. The inserts were verified by sequence analysis. The expression of VP2 and chIL-2 was verified by immunofluorescence after transfection of chicken embryo fibroblasts (CEFs) with the respective plasmids (TransFectinâ„¢ lipid reagent; Bio-Rad, Hercules, California, USA). We used the following primary Abs: rabbit anti-IBDV polyclonal Ab [28], or VP2 mAb (provided by Egbert Mundt, FLI, Germany) and the mouse anti-chicken IL-2 mAb (AbD Serotec MorphoSys, Düsseldorf, Germany); and secondary antibodies: fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG or FITC-conjugated goat antimouse polyvalent Ab (Sigma-Aldrich, St. Louis, USA). VP2 and chIL-2 expressing plasmids were designated DNA-VP2/Eth82, DNA-VP2/7.3 and DNA-chIL-2. DNAvec represented control vector (pCR3.1). 2.3. chIL-2 bioactivity assay Splenocytes from SPF chickens were seeded (2x 105 cells/well) in complete RPMI medium (Biochrom AG, Berlin, Germany). Triplicates were stimulated with ConA (5 µg/mL), supernatants from DNA-chIL-2 or DNA-vec transfected CEFs at 41°C and 5% CO2 [18]. Lymphocyte proliferation was measured by MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Sigma, St. Louis, MO) colorimetric assay [29]. 2.4. Cationic MP preparation and DNA adsorption Cationic MP were prepared as described previously [25,30] with the following modifications. Three-hundred mg of PLGA Resomer® RG 753S (Boehringer Ingelheim, Ingelheim, Germany) and 30 mg of Polyethylenimine (PEI; Sigma-Aldrich) were dissolved in 3 mL dichloromethane (DCM; Sigma-Aldrich) and in 2 mL DCM, respectively. The two organic solutions were mixed and homogenized with 0.5 mL 57 PBS, and subsequently with 25 mL of 0.5% Cetyltrimethylammonium bromide (Boehringer Ingelheim, Ingelheim, Germany). Finally, the organic solvents were evaporated and the cationic PLGA-MP were washed with 5% sucrose and lyophilized. Plasmid DNAs were adsorbed onto the MP by incubating 100 mg of MP resuspended in distilled water (pH 5.0) with 1 mg of DNA as previously described [25,30]. MP were washed and lyophilized. Phosphorothioate backbone modified CpG-ODN (5'- TCGTCGTTGTCGTTTTGTCGTT-3'; CpG motifs underlined; Biomers.net, Ulm, Germany) was adsorbed onto the MP following the protocol of plasmid DNA adsorption. This particular CpG-ODN sequence has been selected based on its significant immunostimulatory properties on avian immune cells; while its non-CpG-ODN sequence has been shown to induce only marginal upregulation of transcripts associated with antigen presentation [31,32]. The MP adsorbed with the DNA-VP2/Eth82, DNA-chIL-2, CpG-ODN, DNA-vec and non-adsorbed MP were designated as VP2-MP, chIL-2-MP, CpG-MP, Vec-MP and Mock-MP. 2.5. Characterization of MP The diameter of the lyophilized MP was determined by the Grimm Aerosol Spectrometer (Grimm Technologies, Douglasville, USA). The MP adsorption efficiency for DNA was determined spectrometrically by subtracting the unbound DNA left in the supernatants at the end of the adsorption period from the amounts of DNA used for adsorption [25]. The resistance of the MP-adsorbed DNA to degradation by enzymes was determined by incubating the loaded MP with 15 U of DNAse I (Sigma). The integrity of the plasmid DNA was verified by gel electrophoresis [25]. A 24 hrs in vitro-release of the MP-adsorbed DNA was determined by incubating 10 mg loaded MP in PBS at 37°C. The amount of DNA released into the medium was determined spectrometrically. 58 2.6. In vitro phagocytosis of MP FITC-labeled bovine serum albumin (FITC-BSA) was microencapsulated as described previously [25]. Chicken mononuclear cells (MQ-NCSU) were grown in L15 Leibovitz-McCoy’s 5A modified medium and incubated with FITC-BSA-MP (250µg/well/mL) for 4 hrs at 41°C in a humidified 5% CO2 incubator. After washing, the MQ-NCSU cells were stained for MHC Class II surface antigen with an Rphycoerythrin (R-PE)-conjugated mouse anti-chicken MHC Class II Ab (B-L) (Southern Biotech, Biozol, Eching, Germany), and FITC-BSA-MP uptake and MHC class II staining was verified by immunofluorescence microscopy. 2.7. Antibody detection IBDV-specific antibodies were detected by a commercial IBDV-ELISA (Synbiotics IBD ProFLOK Plus®, San Diego, USA). Neutralizing Abs were also measured [33]. Briefly, two-fold serially diluted sera were mixed with 100 TCID50 of an intermediate IBDV strain. Chicken embryo fibroblasts (105/well) were added and the development of cytopathic effects was evaluated. Geometric mean titers (log2) per group are presented. 2.8. Histopathology and immunohistochemistry IBDV induced bursal lesion scores were determined as described before [34]. The mean bursal lesion score for each bird was determined from five microscopic fields (400x) and group mean computed. Bursal sections were immunohistochemically (IHC) stained for IBDV antigen following a previously published protocol [28]. The average number of IBDV antigen positive cells in the bursa of each bird was determined from five microscopic fields (400×). A maximum of 100 brown cells/field was counted. 2.9. Detection of cytokine mRNA A TaqMan real-time RT-PCR amplification of chIL-4 and IFN-α mRNA from bursa samples was performed using the AgPath-IDâ„¢ One-Step RT-PCR Reagent kit (Applied Biosystems®) and the detection and quantification was done by the 59 Mx3005PTM thermal cycler (STRATAGENE; Agilent Technologies Company). The relative transcript levels of these cytokines were normalized to the chicken 28S rRNA [35] and values are expressed as 35-CT. The primers, probes and amplification conditions were described previously [28,35]. 2.10. Intrabursal T-cells T-cell influx in the bursa after IBDV challenge was evaluated by immunohistochemistry. Cryosections from frozen bursa were stained for CD4+- and CD8+ T-cells. Mouse anti-chicken CD4 and CD8 mAbs (Southern Biotech, Eching, Germany) were used as primary Abs, and the anti-mouse IgG biotinylated Ab as a secondary antibody (Vector Laboratories, Burlingame, USA). T-cell quantification was conducted as described for IBDV Ag. 2.11. Experimental design 2.11.1. DNA vaccination (experiment 1) Three weeks old SPF chickens were assigned into 5 groups (n=14-16/group). Group 1, 2 and 3 were immunized into the thigh muscle (100 μg DNA/bird) with DNAVP2/Eth82, DNA-VP2/7.3 and DNA-vec, respectively (Table 1). Two boost DNA immunizations were conducted every two weeks. Group 4 received PBS intramuscularly. Group 5 was vaccinated orally with a commercially available chicken embryo adapted live IBDV vaccine strain (103EID)50/bird). Blood samples were collected weekly for Ab detection. Two weeks after the last vaccination each group was subdivided. One subgroup was challenged with the vvIBDV strain 89163/7.3 (103 ELD50/bird) by eye drop [36] and the other subgroup remain unchallenged. Birds were monitored for morbidity and mortality. At 3 and 10 days postinfection (dpi), 3-4 chickens/subgroup were sacrificed. Bursa to body weight (B/B) ratio was determined. Bursae were collected for histology and IBDV Ag detection. 2.11.2. DNA vaccination and adjuvant application (experiment 2) The vaccine groups (n=9-10/group; 3 weeks old SPF chickens) and descriptions are presented in Table 2. The route of vaccination, dosage, frequency, 60 challenge and sampling in this experiment was similar to the first experiment. Plasmid encoded chIL-2 (100 μg /bird) or CpG-ODN (25 µg/bird) were mixed with the DNA vaccine and delivered in one shot. The dosage for CpG-ODN and chIL-2 were based on results of previous studies [17,18]. Necropsy was done at 7 dpi. 2.11.3. Vaccination with cationic MP (experiment 3) The protective efficacy of the MP based IBDV DNA vaccine was tested after mucosal application to 3 days-old SPF chickens (n =10-12/group) intended to see if even in immunologically immature birds this regime may lead to a detectable immune responses. The vaccination protocol is summarized in Table 3. Briefly, chickens of all groups received 4.5 mg MP (3.5 mg orally and 1 mg by eye drop), except the CpG-MP group. One mg of VP2-MP or chIL-2-MP adsorbed 8.7 µg of DNAVP2/Eth82 or DNA-chIL-2. When CpG-ODN was administered, each chicken received 3 mg CpG-MP (2 mg orally and 1 mg by eye drop). One mg of CpG-MP adsorbed 5.6 µg CpG-ODN. Group 5 (G5) and 6 (G6) were administered respectively with chIL-2-MP and CpG-MP as an adjuvant two days after inoculation with VP2-MP. Two booster immunizations were administered every two weeks. Chickens were offfed for 2-3 hrs after MP administration. Two weeks after the last vaccination, chickens were orally challenged with an intermediate plus IBDV vaccine strain (≥2.0 log10 EID50/bird), which induce significant macroscopical and microscopical bursal lesions without high mortality rates [8]. Group 1 (G1) served as non-challenge control. At 3 and 7 dpi, 5-6 chickens were sacrificed and B/B ratio was determined. Bursae were collected for histology, Ag detection, cytokine mRNA analysis and T-cell evaluation. 2.12. Statistics ANOVA was used to analyze the data on lymphocyte proliferation and from experiment 3. Group means were compared with Tukey HSD pot-hoc test. KruskalWallis one-way nonparametric ANOVA was used in other experiments. Student’s ttest was used to analyze B/B ratios between subgroups. p<0.05 indicates significant differences. 61 3. Results 3.1. Cloning and expression of VP2 and chIL-2 Transfection studies revealed expression of VP2 and chIL-2 in CEFs (data not shown). Chicken lymphocytes stimulated with supernatants from CEFs, which had been transfected with DNA-chIL-2, showed a higher lymphoproliferative index (2.0±0.5) (p<0.05) than treatment with supernatants from vector-transfected cells (0.9±0.1). 3.2. Immunogenicity of DNA vaccines (experiment 1&2) At five weeks post intramuscular immunization, 75% (12/16) of the DNAVP2/Eth82 vaccinated chickens had developed IBDV-ELISA antibody titers of 2070±1565, comparably lower than the live virus vaccinated group (9734±1824). Only 40% of the DNA-VP2/7.3 (6/15) vaccine group had seroconverted at week 5 after immunization. Four out of eight DNA-VP2/Eth82 vaccinated bird showed depression and ruffled feathers after challenge. The vector and PBS groups showed severe clinical signs (Table 1). The DNA-VP2/Eth82 vaccinated group had a significantly lower mean bursal lesion score (p<0.05) and fewer intrabursal Ag positive cells compared to the other challenged groups, except the live vaccine group (Table 1) indicating only partial protection. DNA-chIL-2 or CpG incorporation did not improve the immunogenicity of the DNA vaccine (Table 2). 3.3. Characterization of cationic MP The prepared MP had diameters of 3.5-8.5 µm, and the FITC-BSA-MP were successfully phagocytized by MQ-NCSU cells (data not shown). The adsorption efficiency was 83-87% for DNA-VP2/Eth82 and DNA-chIL-2, and 56% for CpG-ODN, indicating the adsorption of 8.3-8.7 µg of plasmid DNA and 5.6 µg of CpG-ODN to 1 mg of cationic MP. Forty five percent of the adsorbed plasmid DNA and 60% of the CpG-ODN were released into the incubation medium after 24 hrs in vitro incubation at 37°C. The MP-adsorbed DNA was protected from degradation by DNAse I as the 62 supercoiled plasmid was preserved at the end of DNAse I degradation assay (data not shown). 3.4. Evaluation of the protective efficacy of the cationic MP-delivery strategy for IBDV-DNA vaccination (experiment 3) At 3 dpi, no difference was observed in the B/B ratios between groups (p>0.05). The chickens in G4 (VP2-MP), G5 (VP2-chIL-2-MP) and G6 (VP2-CpGMP) had a significantly higher B/B ratio at 7 dpi compared to the other challenged groups (Table 4) (p<0.05). At 7 dpi bursal lesions were lower in G4, 5, and 6 (p<0.05) compared to the G2 (Mock-MP+), 3 (Vec-MP), 7 (chIL-2-MP) and 8 (CpG-MP) groups (p<0.05) (Table 4). All VP2-MP groups had a relatively lower intrabursal Ag load compared to the other groups at 3 and 7 dpi (Table 4) (p<0.05). Chickens in G5 had the lowest intrabursal Ag load and three out of six chickens had cleared the virus at 7 dpi. Neutralizing antibodies were neither detected in prechallenge sera nor at 3 dpi in any group. Chickens vaccinated with VP2-ChIL-2-MP had the lowest postchallenge VN Ab titers at 7 dpi (Table 4). At 3 dpi no significant differences were detected in bursal IFN-α and IL-4 mRNA expression between groups (Fig. 1A & C). At 7 dpi the expression levels of chIFN-α mRNA were significantly lower in the bursae of G4, 5 and 6 (Fig. 1B) compared to other groups (p<0.05). Significant upregulation of IL-4 mRNA was observed in G2 and G3 at 7 dpi compared to G1, G4, and G5 (Fig. 1D). Higher numbers of intrabursal CD4+ T-cells were detected in the Mock-MP+ group compared to the other groups at 3 dpi (Fig. 2A; p<0.05). The number of CD8+ T-cells increased in all challenged groups at 3 dpi and G2, G3, G7 and G8 (Fig. 2C) had the highest number of CD8+ T-cells. At 7 dpi more CD4+ (Fig. 2B) and CD8+ Tcells (Fig. 2D) were recruited to the bursa of G2, 3, 7 and 8 (p<0.05) compared to the VP2-MP and VP2-MP plus adjuvant groups. 63 4. Discussion The aim of this study was to develop an improved MP based IBDV DNA vaccine and to further enhance the protection efficacy of this vaccine by including MP loaded with adjuvants. The VP2-DNA vaccines were shown to be immunogenic and conferred partial protection against challenge. Generally, DNA vaccinations are known to not induce significant Ab responses, but may provide partial protection against virulent IBDV [11,15,17,18]. Co-administration of CpG-ODN or plasmid encoded chIL-2 together with the IBDV-VP2 or polyprotein gene induced variable levels of humoral and cellular immunity. Yet, chickens mostly exhibited bursal atrophy after challenge [17,37,38], which were even more pronounced than in groups without adjuvants [39], coinciding with our observation. The characterization of the MP revealed appropriate physical properties for in vivo application [25, 30]. PEI incorporation into the MP may condense plasmid DNA on the MP surfaces and likely prevents DNA degradation by mucosal enzymes [23,30]. The mucosal delivery of the VP2-loaded MP (VP2-MP) in combination with a delayed mucosal application of chIL-2 or CpG-ODN loaded MP did not induce virus neutralizing Abs. We speculate that the DNA is slowly released from the MP, VP2 is expressed, processed and presented by APCs to culminate in a CTL response [40]. After IBDV challenge, bursal pathology and intrabursal Ag load and T-cell influx were reduced in all VP2-MP vaccinated groups. This provides the proof-ofconcept for the potential of MP to improve DNA vaccine efficacy. The lower antigen load in VP2-MP plus chIL-2-MP-inoculated chickens may explain the lower postchallenge neutralizing Ab titers, IL-4 and IFN-α mRNA expression possibly due to reduced virus replication or faster clearance in these birds compared to other groups. CpG-ODN and chIL-2 were shown to exert potent adjuvant effects when delivered by particulate carriers compared to their delivery in soluble forms [41,42]. Baden et al. [19] suggested that increased IL-2 receptor expression on antigen primed T-cells may be necessary for an optimal activation and proliferation of these 64 cells by IL-2. However, during simultaneous administration of an Ag and cytokine, the cytokine may simulate rather a broad cellular response with relatively fewer antigenspecific cells [19]. Tregs in mice and humans showed an increased level of toll like receptor (TLR) expression [43] and may expand to an extent dominating antigen specific CD4+ T-cells when CpG was administered simultaneously with a vaccine. Thus, the timing of TLR agonist (CpG-ODN) application may influence the outcome of vaccine-induced immune responses [44]. We hypothesize that in our vaccination model the delayed application of chIL-2 after an MP-DNA delivery may enhance the activation and proliferation of Ag specific T-cells in chickens before extensive proliferation of immunosuppressive Tregs may take place. Overall the results of this study indicate that DNA-vaccination against IBDV using MP as a delivery system in combination with molecular adjuvants may be an interesting alternative IBDV-vaccination approach, which may help to reduce the risks associated with the use of live vaccine strains. In future studies, we not only need to investigate additional routes of administration but also we need to elucidate the mechanism of protection and to what extent the different types of T-cells are involved in the control of IBDV challenge after MP-DNA-vaccination. 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Infect Immun 2012;80:1744-52. 70 Table 1 Experiment 1: Evaluation of protection conferred by IBDV DNA vaccinations in comparison to a live IBDV vaccineg Mean±SD Groups 1 2 3 4 5 Vaccines DNA-VP2/Eth82 DNA-VP2/7.3 DNA-vec PBS Live virus vaccine Challenge Morbidity Mortality B/B ratio Bursal lesion score Bursal antigen load 3 dpi 10 dpi 3 dpi 10 dpi 3 dpi 10 dpi - 0/8 0/8 3.8±0.9 3.7±0.9b 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 + 4/8 1/8 4.9±1.0 2.3±1.0a 0.3±0.1a 2.5±0.1a 65.0±9.0a 51.0±17.3a - 0/7 0/7 4.2±0.7 3.7±0.9b 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 a 1.6±0.2 0.0±0.0 + 5/8 1/8 6.2±1.7 2.1±1.6 - 0/7 0/7 3.9±1.5 4.4±0.3b a 4.0±0.0 b 3.5±0.1 b 0.0±0.0 c 71.0±13 a 0.0±0.0 c 65.0±17.3a 0.0±0.0 b 100±0.0b + 7/7 1/7 4.6±1.0 1.7±1.6 - 0/7 0/7 4.6±0.3 4.4±0.3b 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 + 7/7 2/7 4.9±1.0 1.7±1.7a 4.0±0.0c 4.0±0.0c 100±0.0b 100±0.0b - 0/8 0/8 3.3±0.6 3.4±1.5b 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 + 0/8 0/8 4.0±1.4 3.7±0.4b 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 4.0±0.0 100±0.0 71 g Three weeks old SPF chickens (n=14-16/group) were immunized by the IM route with DNA vaccines (DNA-VP2/Eth82, DNA- VP2/7.3) followed by two DNA booster immunizations (group 1-3). Group 5 was vaccinated orally with a commercially available chicken embryo adapted live IBDV vaccine strain (103EID)50/bird). Two weeks after the last vaccination groups were divided: one subgroup was challenged (+) with vvIBDV strain 89163/7.3. The other subgroup remained unchallenged (-).The vvIBDV strains had been isolated from several chicken flocks that had experienced high mortality rate [26]. Morbidity, mortality, B/B ratio and microscopic bursal lesions were determined. IBDV Ag load in the bursa was quantified by counting antigen positive cells in five microscopic fields/section. Histopathology of the bursae in affected groups included lymphoid depletion in the cortex and medulla. Different superscripts in a column show significant difference between challenged groups (Kruskal-Wallis test, p<0.05). Comparison of B/B ratios of challenged and non-challenged subgroups was done by Student’s t-test. p<0.05. 72 Table 2 Experiment 2: Evaluation of protection induced by IBDV DNA vaccination in combination with molecular adjuvantsh Groups Vaccine group Challenge Morbidity Mortality Mean±SD B/B ratio Bursal lesion score 1 DNA-vec+DNA-chIL-2 + 9/9 4/9 2.1±0.7 4.0±0.0b 2 DNA-vec+CpG + 9/9 4/9 2.4±0.4 4.0±0.0b 3 DNA-VP2/7.3 + 8/10 2/10 2.9±1.8 3.1±0.2a 4 DNA-VP2/7.3+DNA-chIL-2 + 10/10 5/10 3.3±2.2 4.0±0.0b 5 DNA-VP2/7.3+CpG + 10/10 4/10 2.7±0.2 3.5±0.5b 6 DNA-chIL-2 + 10/10 2/10 2.8±0.9 4.0±0.0b 7 CpG + 10/10 3/10 3.2±0.6 4.0±0.0b 8 DNA-vec + 9/9 4/9 2.8±0.9 4.0±0.0b 9 PBS - 0/10 0/10 3.5±0.7 0.0±0.0 10 PBS + 9/9 4/9 2.7±0.9 4.0±0.0b 73 h Three weeks old SPF chickens were randomly assigned into 10 groups (n=9-10/group) and each group received different treatments. To improve the immune responses to the IBDV DNA vaccine, DNA-chIL-2 and CpG-ODN as adjuvants were included. After two booster vaccinations, the chickens were challenged with vvIBDV strain 89163/7.3. Morbidity and mortality were recorded. B/B ratio and microscopic bursal lesions were determined at 7 dpi. Different superscripts in a column show significant difference between challenged groups. Kruskal-Wallis test, p<0.05. 74 Table 3 Experiment 3: Study design showing experimental groups and the timing of vaccine and adjuvant applicationi Groups Challenge Cationic MP administration at days postvaccination (dpv) 42 dpv 0 2 14 16 28 30 G1 (Mock-MP) - Mock-MP Mock-MP Mock-MP G2 (Mock-MP) + Mock-MP Mock-MP Mock-MP G3 (Vec-MP) + Vec-MP Vec-MP Vec-MP G4 (VP2-MP) + VP2-MP VP2-MP VP2-MP G5 (VP2-chIL-2-MP) + VP2-MP chIL-2-MP VP2-MP chIL-2-MP VP2-MP chIL-2-MP G6 (VP2-CpG-MP) + VP2-MP CpG-MP CpG-MP CpG-MP G7 (chIL-2-MP) + chIL-2-MP chIL-2-MP chIL-2-MP G8 (CpG-MP) + CpG-MP CpG-MP CpG-MP i VP2-MP VP2-MP In this experiment DNA vaccine and adjuvants were delivered through mucosal routes to 3-5 days old SPF chickens. The DNA vaccine and adjuvants were adsorbed separately onto cationic PLGA-MP. G5 and G6 were administered with chIL-2-MP and CpGMP, respectively two days after the chickens were inoculated with the DNA vaccine adsorbed onto the MP, both at the primary and booster vaccinations. G means group. 75 Table 4 Experiment 3: Determination of the protective efficacy of MP based DNA vaccine and adjuvants after mucosal application to SPF chickensj Mean±SD Groups Challenge VN titer (log2)/group B/B ratio 7 dpi 3 dpi 7 dpi Bursal lesion score Bursal antigen load 3 dpi 7 dpi 3 dpi 7 dpi 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 G1 (Mock-MP) - 0.0±0.0 3.7 ±0.9 3.59 ±0.2b G2 (Mock-MP) + 5.0±0.0a 4.9±0.9 1.65 ±0.2a 2.6±0.2a 4.0±0.0b 100 ±0.0d 100±0.0a G3 (Vec-MP) + 5.0±0.0a 4.0±0.6 1.65 ±0.3a 0.9±0.4b 4.0±0.0b 100±0.0d 100±0.0a G4 (VP2-MP) + 3.3±0.0b 3.5±1.2 2.15 ±0.5c 0.9±0.1b 2.1±0.3a 45±13c 22±8.5b G5 (VP2-chIL-2-MP) + 1.7±0.0c 4.2±0.6 2.75 ±0.3c 0.7±0.2b 1.9±0.1a 28±18c 9±2.1c G6 (VP2-CpG-MP) + 2.5±0.0d 3.8±0.5 2.85 ±0.6c 1.7±0.4c 1.8±0.2a 42±16c 25.0±8.9b G7 (chIL-2-MP) + 5.0±0.0a 4.1±0.9 1.47 ±0.3a 2.3±0.5a 4.0±0.0b 98±2.0d 100±0.0a G8 (CpG-MP) + 5.0±0.0a 4.3±1.3 1.96 ±0.4a 1.9±0.3c 4.0±0.0b 100±0.0d 100±0.0a 76 j Three to five days old SPF chickens (n=10-12/group) received 4.5 mg MP of DNA vaccine or chIL-2. When CpG group was administered, each chicken received 3 mg MP. Each milligram MP adsorbed either 8.7 µg of DNA (VP2 or chIL-2) or 5.6 µg CpG. G5 and G6 were administered with chIL-2-MP and CpG-MP two days after the chickens were inoculated with the DNA vaccine adsorbed to the MP. Two weeks after the last vaccination, chickens were orally challenged with an intermediate plus IBDV vaccine strain (≥2.0 log10 EID50/bird), which induce significant macroscopical and microscopical bursal lesions without high mortality rates (RAUTENSCHLEIN et al. 2005a). Different superscripts in a column show significant differences between challenged groups, while identical superscripts show no statistically significant differences between challenged and nonchallenged groups. ANOVA; p<0.05. IFN-alpha corrected mRNA expression (35-CT) 77 25.00 20.00 15.00 10.00 5.00 0.00 G1 (MockMP-) G2 (MockMP+) G3 (Vec- G4 (VP2- G5 (VP2- G6 (VP2MP) MP) chIL2- CpG-MP) MP) G7 (chIL2MP) G8 (CpGMP) G3 (Vec- G4 (VP2- G5 (VP2- G6 (VP2G7 MP) MP) chIL2- CpG-MP) (chIL2MP) MP) G8 (CpGMP) Experimental groups A IFN-alpha corrected mRNA expression (35-CT) 25 20 * 15 * 10 5 0 G1 (MockMP-) B * G2 (MockMP+) Experimental groups 78 IL-4 corrected mRNA expression (35-CT) 5 4 3 2 1 # # 0 G1 (MockMP-) G2 (MockMP+) G3 (Vec- G4 (VP2- G5 (VP2- G6 (VP2G7 G8 (CpGMP) MP) chIL2- CpG-MP) (chIL2MP) MP) MP) Experimental groups IL-4 corrected mRNA expression (35-CT) C 5 * * 4 3 2 1 0 G1 (MockMP-) G2 (MockMP+) G3 (Vec- G4 (VP2- G5 (VP2- G6 (VP2G7 G8 (CpGMP) MP) chIL2- CpG-MP) (chIL2MP) MP) MP) Experimental groups D Fig. 1. Quantification of postchallenge chIFN-α mRNA expression levels in the bursa at 3 (A), and 7 dpi (B); and chIL-4 mRNA expression at 3 (C) and 7 dpi (D). Chickens were challenged with an intermediate plus IBDV vaccine strain, which induce significant macroscopical and microscopical bursal lesions without high mortality rates [8] two weeks after the last booster vaccination. *indicates statistically significant differences between groups. # Ct value of 35 was used to calculate the relative gene expression in all samples. ANOVA, p<0.05. Group descriptions are presented in Table 3. A Average no. of CD4+ T cells/field 79 120 100 80 60 40 20 0 * G1 (MockMP-) G2 G3 (VecG4 (MockMP) (VP2MP+) MP) G5 (VP2chIL2MP) G6 (VP2CpGMP) G7 (chIL2MP) G8 (CpGMP) Experimental groups A Average no. of CD4+ T cells/field B 120 100 80 60 40 20 0 * * * * G1 G2 (Mock- (MockMP-) MP+) G3 (VecMP) G4 (VP2MP) G5 (VP2chIL2MP) G6 (VP2CpGMP) G7 G8 (chIL2- (CpGMP) MP) Experimental groups Average no. of CD8+ T cells/field 80 120 100 80 60 40 * 20 0 G7 G3 (Vec- G4 (VP2- G5 (VP2- G6 (VP2G2 (chIL2CpGMP) MP) chIL2(MockMP) MP) MP) MP+) G1 (MockMP-) Experimental groups Average no. of CD8+ T cells/field C D G8 (CpGMP) 120 * * * 100 80 60 40 20 D 0 G2 G1 (Mock- (MockMP+) MP-) G3 (VecMP) G4 (VP2MP) G5 (VP2chIL2MP) G6 (VP2CpGMP) G7 (chIL2MP) G8 (CpGMP) Experimental groups Fig. 2. Quantification of postchallenge intrabursal CD4+ T-cells at 3 (A), and 7 dpi (B); and CD8+ T-cells at 3 (C) and 7 dpi (D). Chickens were challenged with an intermediate plus IBDV vaccine strain, which induce significant macroscopical and microscopical bursal lesions without high mortality rates [8] two weeks after the last booster vaccination.*indicates statistically significant differences between groups (ANOVA, p<0.05). Group descriptions are presented in Table 3. 81 6. Discussion and conclusions Poultry pathogens change their nature in response to intensified poultry production and present complex challenges to the poultry health and productivity. Classical IBDV strains that emerged in the 1960s still cause mortality rates of 1% to 30% in susceptible chickens (MÜLLER et al. 2003). Very virulent IBDVs, which emerged in the late 1980s can cause mortality of up to 70% and induce severe immunosuppression (CHETTLE et al. 1989). Vaccination pressure may likely contribute to the emergence of highly pathogenic virus strains. Thus, understanding the molecular epidemiology of IBDVs in the field has been important for the design of preventive measures and for the development of a new generation of vaccines to control the spread of the virus. 6.1. Molecular epidemiology of IBDV field isolates Molecular approaches allow the identification and differentiation of IBDV strains circulating in chicken populations and associate recent and past isolates (LE NOUEN et al. 2005). The molecular epidemiology of IBDV has been studied in many geographical areas and IBDV evolution well documented. Particularly, serotype 1 IBDV strains have been circulating in many poultry operations in North and South America, Europe, Asia and in African countries (YAMAGUCHI et al. 1997a; ZIERENBERG et al. 2000; VAN DEN BERG et al. 2004; JACKWOOD u. SOMMER 2005; JACKWOOD u. SOMMER-WAGNER 2007; JUNEJA et al. 2008; HE et al. 2012b; KASANGA et al. 2012). In this study, several IBDV isolates were collected during field outbreaks in different types of poultry flocks in Ethiopia. Experimental inoculation of chickens with most of these isolates allowed us to confirm the pathogenicity of the viruses. Inoculated chickens showed significant bursal lesions and an extensive IBDV Ag load in the bursa. The characterization of the nucleotide (nt) sequences of the hVP2 and the 5’ two thirds of VP1 of 11 isolates demonstrated that the Ethiopian isolates showed 82 close homology to vvIBDV isolates from other countries. Based on a recent study, it is expected that, worldwide, about 60 to 76% of IBDV isolates are of vvIBDV genotype (JACKWOOD u. SOMMER-WAGNER 2007; HE et al. 2012b). The rest of the isolates are classical and variant strains based on their hVP2 characteristics (JACKWOOD u. SOMMER-WAGNER 2007; HE et al. 2012b). Although phenotypic characterization of IBDV in experimentally infected SPF chickens is the gold standard to determine IBDV virulence, genotypic features can support IBDV grouping (VAN DEN BERG et al. 2004). vvIBDV has shown a rapid global spread; even in the USA, where the classical and variant strains dominated field outbreaks for nearly five decades, vvIBDV emerged in 2008 (STOUTE et al. 2009). Almost all poultry producing regions report the co-existence of two or more strains of varied pathogenicity (JACKWOOD u. SOMMER-WAGNER 2007; HE et al. 2012b). Interestingly, only vvIBDV was identified in Ethiopia during the period of our study. A recent countrywide study reported IBDV seropositivity rates in backyard chickens to be close to 92% (CHAKA et al. 2012; JENBREIE et al. 2012). Detailed molecular studies of IBDV in this segment of the chicken population may provide further information about IBDV epidemiology. Phylogenetic approaches have been particularly useful for studying the origin and subsequent evolution of a virus. Based on the VP2 nt sequence, phylogenetic analysis revealed the formation of a cluster by Ethiopian IBDV strains; yet this cluster reclines in one major monophyletic lineage with other vvIBDVs. It was expected that vvIBDV strains of the same geographic origins may cluster together, yet some minor non-significant clustering outside the main cluster was reported among isolates of the same origins (CORTEY et al. 2012; SILVA et al. 2012). This may indicate a strong association between geographical origin and viral diversity. In contrast to this observation, vvIBDVs isolated from the major chicken producing regions of China showed very divergent phylogenetic clusters based on their VP2 sequences (HE et al. 2012b). This may highlight the circulation of vvIBDVs with diverse genetic mutations. Several recent IBDV field strains from USA did not form clusters with most of the IBDV VP2 sequences retrieved from databases, implying the virus is under 83 continuous evolution (DURAIRAJ et al. 2011). Despite vvIBDV isolates maintaining the identical virulence marker aa in their VP2 regions, they show differences in their pathogenicity to SPF chickens even under standardized experimental conditions. This suggests the importance of the role of VP1 in IBDV virulence (SILVA et al. 2012), which should be besides VP2 regularly included in the molecular analysis of IBDV strains. Normally, the VP1 gene of IBDV strains shows multiple phylogenetic lineages, additionally providing evidence of its contribution in IBDV pathogenicity (JACKWOOD et al. 2012). How the vvIBDV strains evolved in Ethiopia remains unclear. Literature suggests that international trade of live poultry and poultry products may facilitate the global spread of IBDV (COBB 2011). IBDV may spread through contaminated equipment (FLENSBURG et al. 2002; JACKWOOD u. SOMMER-WAGNER 2010). The high tenacity of the virus and its resistance to several disinfections and virucidal procedures may contribute to the rapid distribution of the virus (VAN DEN BERG et al. 2000b; GARRIGA et al. 2006). The risk of transmission via infected free living wild birds (JEON et al. 2008) can not be excluded as IBDV or IBDV-specific Abs were detected in other avian species and in backyard chickens (KASANGA et al. 2008). Almost all acute disease outbreaks in backyard chickens in developing countries remain undiagnosed. vvIBDV isolates from wild birds and backyard chickens were shown to be highly pathogenic for SPF chickens under experimental conditions and maintain virulence marker aa residues across their VP2 and VP1 genes (HERNANDEZ-DIVERS et al. 2008). Ethiopian IBDV isolates appear clonal and are very virulent. In the case of RNA viruses, biological events including genetic reassortment or recombination alter the phenotypes and genotypes of circulating viruses and compromise their genetic stability. Also the first vvIBDV that had caused severe disease outbreak in Europe was linked to the emergence of segment reassortant IBDV between a newly appeared IBDV, which combined its very virulent VP1 segment with an endemic IBDV bearing a very virulent VP2 segment (HON et al. 2006). Natural homologous intragenic recombination apart from reassortment may lead to new variants of IBDV 84 (HON et al. 2008; HE et al. 2009a). Likewise, Ethiopian strains in our study showed aa residues within the region of VP1 that are characteristic of attenuated as well as vvIBDV, so we can not rule out the possibility of recombination within this gene segment. While prototype vvIBDVs present glycine (G) at position 254 (loop PDE) of the minor hydrophilic peak 1 domain of the hypervariable region of VP2, we found a serine (S) residue in all Ethiopian vvIBDV isolates at this position. A serine (S) residue at this position was detected in most recent vvIBDV field isolates from Africa, Europe and Asia from chickens vaccinated with classical live IBDV vaccines (KASANGA et al. 2007; MARTIN et al. 2007). Recent study revealed that most other IBDV isolates show frequent aa exchange at this position as well (ICARD et al. 2008; DURAIRAJ et al. 2011). This indicates a significant role of this domain in determining IBDV antigenicity as demonstrated by previous studies (BAYLISS et al. 1990; HEINE et al. 1991; LANA et al. 1992; DORMITORIO et al. 1997). Also the crystal structure of the VP2 protein suggests selection pressure associated with the hydrophilic domains due to their location at the outermost exposed part of the VP2 protein (COULIBALY et al. 2005). A neutralizing Ab escape mutant virus at position 254 (S) was generated from Del-E IBDV by site-directed mutagenesis (JACKWOOD u. SOMMER-WAGNER 2011). Chickens vaccinated with a live Del-E vaccine and challenged with the mutant developed severe bursal lesions, whereas those vaccinated with Del-E and challenged with a homologous virus were fully protected (JACKWOOD u. SOMMERWAGNER 2011) showing the contribution of this particular aa residue in vaccination failure. Vaccination failure associated with the application of plaque purified and cloned live IBDV vaccines during and in subsequent IBD outbreaks in Ethiopia (personal communication with poultry producers and veterinarians) may be due to the lack of cross protection due to aa exchanges at the hydrophilic domains of field isolates compared to the vaccinal strains. Vaccines on the basis of IBDV quasispecies (JACKWOOD u. SOMMER 2002) may in contrast to a cloned live vaccine provide a 85 better cross protection in the face of heterologous IBDV field challenges counteracting at least in part the development of Ab escape mutants. 6.2. Immune responses induced by candidate IBDV DNA vaccines and correlation to protection Considering the significant economic losses associated with IBDV, the development and evaluation of new generation IBDV vaccines are important. With this goal, candidate DNA vaccines were produced from the newly characterized vvIBDV strains circulated in the chicken population in Ethiopia. In the current study, IM immunization of SPF chickens with DNA encoding IBDV-VP2 from two different vvIBDV strains induced humoral responses, although the Ab titers were lower compared to a commercial live IBDV vaccine. Multiple boosts could not significantly increase the Ab levels as confirmed by previous studies (LI et al. 2003). Similar investigations, however, reported that IM administration of large doses (10 mg/bird) of DNA encoding the IBDV polyprotein (PP) gene induced significantly higher Ab responses compared to the VP2 DNA vaccine (HSIEH et al. 2010). The oral delivery of live attenuated S. Typhimurium or E. coli that expressed the PP or VP2 induced higher Ab levels comparable to a conventional live IBDV vaccine and provided better protection (LI et al. 2006; MAHMOOD et al. 2007; HSIEH et al. 2010). Normally, DNA encoding the PP of vvIBDV induced higher neutralizing Ab responses and protection compared to PP originated from an attenuated strain (KIM et al. 2004). The highest Ab titers to IBDV DNA vaccines were detected when they were used for priming followed by boosters with killed or subunit vaccines (HSIEH et al. 2007; PARK et al. 2009; GAO et al. 2013). In our study, DNA-VP2 vaccinated chickens were protected to some extent against IBDV challenge. Earlier studies showed that DNA-PP vaccines were more effective to induce protection against challenge than the DNA-VP2 vaccines possibly due to higher IBDV-specific neutralizing Abs (LI et al. 2003; KIM et al. 2004; LI et al. 2004). The expression of PP in vivo by transfected muscle cells following IM application may 86 lead to the formation of virus-like particles (MARTINEZ-TORRECUADRADA et al. 2003). This may be possible because of correct processing of the PP by viral and host proteases that maintain the natural conformation of the immunodominant epitopes of VP2, thus their immunogenicity and protective efficacy. In most previous studies no complete protection against challenge was achieved by IBDV-DNA vaccination. Bursal atrophy, viral RNA or Ags were detected after challenge in the bursal follicles of recovered birds similar to our study (ZIERENBERG et al. 2000; CHANG et al. 2001; CHANG et al. 2003; KIM et al. 2004; LI et al. 2006; MAHMOOD et al. 2007; HSIEH et al. 2010). IBDV DNA vaccine induced complete protection possibly depends on the levels of both humoral and cellular immunity (CHEN et al. 2011c). 6.3. Do molecular adjuvants enhance protectivity of an IBDV DNA vaccine? Since ‘first-generations’ IBDV DNA vaccines are poorly immunogenic, and rarely generated detectable Ab levels, we next evaluated the inclusion of plasmids encoding chIL-2 or synthetic unmethylated CpG-ODN as adjuvants. Previous studies indicated the in vitro and in vivo immunoregulatory properties of chIL-2 and CpGODN on various cell populations of the immune system of avian species (HILTON et al. 2002; HE u. KOGUT 2003; ASIF et al. 2004; SHANMUGASUNDARAM u. SELVARAJ 2011). We wanted to extend these observations by utilizing these molecular adjuvants as IBDV DNA vaccine adjuvants in various formulations. With our vaccination protocol, which was similar to previously described ones, chIL-2 did not enhance the potency and protection conferred by the IBDV DNA vaccine. A lack of an adjuvant effect of a plasmid-encoded chIL-2 was previously demonstrated when it was applied in ovo with the IBDV DNA vaccine (PARK et al. 2009). Others had shown that the inclusion of IL-2 encoding DNA enhanced humoral and cellular immune responses induced by the DNA-VP2 vaccine; although protection varied greatly (HULSE u. ROMERO 2004; LI et al. 2004). The co-administration of DNA 87 encoding chIL-2 with the DNA-PP intramuscularly to SPF chickens induced an early and higher Ab response and protection against lethal IBDV compared to the chIL-2 plus DNA-VP2 group, which had the lowest Ab response (LI et al. 2004). The Ab responses in the chIL-2 plus DNA-VP2 group remained lower compared to the PP or VP2 alone group, but protection was comparably higher in the former group indicating stimulation of T-cell responses by IL-2 treatment (LI et al. 2004). IL-2 is known to promote a Th1-type response, and induces the proliferation and activation of T-cells and NK cells (HILTON et al. 2002). Higher IBDV-specific ELISA titers and CD4+ T-cell responses, and greater survival rates were demonstrated when broiler chickens were vaccinated with a bicistronic plasmid expressing chIL-2 and VP2 even by using a very low dose (50 µg/bird) of plasmid (KUMAR et al. 2009). One limitation of this bicistronic plasmid study may be the quality of the plasmid DNA. Immune modulating effects of lipopolysaccharide from lysed bacterial cell wall during plasmid DNA isolation additionally have contributed to the enhanced immunogenicity. The in vitro immunoregulatory properties of CpG-ODNs on avian APCs, B- and Tcells have been well investigated; yet their applications as avian vaccine adjuvants are very limited. Class B-CpG-ODN that has been used in previous in vitro and in vivo studies in avian species was shown to be well tolerated by chickens and they were used in our study. Despite a relatively high dose of CpG-ODN, DNA-VP2 vaccine did neither induce seroconversion nor protection in the vaccinated chickens. Co-administration of CpG-ODN at higher doses and frequency together with a DNAPP vaccine aggravated bursal lesions and mortality rates in the challenged chickens compared to the PP vaccine alone (ROH et al. 2006). No detectable prechallenge Abs and CMI were demonstrated, which was comparable to our findings (ROH et al. 2006). Others had shown increased IBDV-Ab and CMI responses, and protection against mortality by virulent IBDV when CpG-ODN was co-administered with a DNAVP2 vaccine; nevertheless clinically recovered birds showed prominent bursal lesions (WANG et al. 2003; MAHMOOD et al. 2006; MAHMOOD et al. 2007). 88 Previous mouse studies suggested that the co-administration of phosphorothioate backbone modified CpG-ODN mixed with a plasmid DNA vaccine may reduce gene expression of the plasmid (WEERATNA et al. 1998), which may have happened in our study. CpG-ODN stimulated mammalian and avian APCs have been shown to secrete higher levels of TNF-α and IFN-γ (CIRACI u. LAMONT 2011). It was suggested that in mice higher expression levels of TNF-α and IFN-γ at the site of inoculation together with the plasmid DNA preparation may affect the activity of the cytomegalovirus (CMV) promoter, which results in down-regulation of protein expression from genes encoded by the vector (QIN et al. 1997). Additionally, in mammals, CpG-ODN induces proliferation of regulatory T-cells (Tregs) (MOSEMAN et al. 2004). Tregs may dampen immunity induced by DNA vaccination, which has to be evaluated further in chickens. It seems that the in vivo adjuvant property of IL-2 and CpG-ODN depends on the nature of the vaccine, expression systems of IL-2, and vaccine dosage and formulations. Previous studies tested the adjuvant property of IL-2 in a co-inoculation protocol with IBDV DNA vaccines and only partial immune enhancing effects were demonstrated. The adjuvant effects of CpG-ODN may be better exploited when immunogens are administered prior to CpG-ODN application. This hypothesis had been evaluated in a pneumococcal vaccination model. Delayed CpG-ODN application improved the protection of mice against pneumococcus challenge compared to simultaneous administration of the vaccine and CpG-ODN (JENSEN et al. 2012). 6.4. Cationic PLGA MPs as particulate carriers for DNA vaccine and molecular adjuvants To improve the immunogenicity and protective efficaciousness of our candidate DNA vaccine further we have exploited particulate carrier systems for the vaccines and adjuvants. For this purpose cationic MPs were prepared following previously 89 published protocols, and their physical properties were characterized. Plasmid encoding the VP2 gene of IBDV or chIL-2, and CpG-ODN were adsorbed on preformed cationic MPs. These cationic PLGA MPs prepared with PEI had a diameter of 3-10 µm and are readily phagocytosed by APCs of mammalian and avian origin (OSTER et al. 2005; LIMAN et al. 2007). The adsorption efficacy of cationic PLGA MPs was found to be higher for plasmid DNAs than for CpG-ODN as shown in this study. One hundred percent adsorption efficacy for plasmid DNA was previously reported (OSTER et al. 2005). Cationic MPs prepared from PLGA Resomer® RG 503H could achieve 100% adsorption efficiency compared to PLGA Resomer® RG 753S as shown in our preliminary tests (data not shown). The former polymer induced rather highly aggregated MPs and may not be suitable to deliver Ags to APCs. Our in vitro DNA release studies confirmed release of 45% of adsorbed plasmid DNA and 60% of CpG-ODN in 24 hrs incubation period, which was lower compared to a previous report of 78% release of CpG-ODN (SINGH et al. 2001a). An in vitro DNAse I degradation assay demonstrated considerable DNA protection against enzymatic degradation when the DNA was bound to MPs (HE et al. 2005). It was speculated that inclusion of PEI during MPs preparation may condense DNA molecules on the surface of MPs, which protects the DNA from in vitro degradation (OSTER et al. 2005). Using mucosal or systemic routes, cationic MPs have been shown to induce strong immunity to various infectious diseases in animals including avian species (DENIS-MIZE et al. 2003; OTTEN et al. 2005; LIMAN et al. 2007; WANG et al. 2011). This provides circumstantial evidence that even under in vivo conditions MPs may protect DNA from degradation by enzymes. 6.5. Promise of cationic PLGA MPs in improving an IBDV DNA vaccine We evaluated the efficacy of IBDV DNA vaccine adsorbed onto MPs in SPF chickens following mucosal application. The adjuvant effects of chIL-2 or CpG-ODN adsorbed onto cationic MPs were assessed after mucosal priming with DNA vaccine to augment immune responses provoked by the DNA vaccine. 90 We used bursal pathology, IBDV Ag load in the bursa and intrabursal CD4+- and CD8+ T-cell counts as markers of protection against IBDV challenge. Chickens vaccinated with our MPs with adsorbed DNA-VP2 developed moderate bursal lesions, showed lower IBDV Ag load and less T-cell influx into the bursa compared to the challenged nonvaccinated controls, adjuvant and vector groups. Experimental infections of non-vaccinated susceptible SPF chickens with IBDV is expected to induce extensive intrabursal CD4+- and CD8+ T-cell influx and viral replication in the bursa (KIM et al. 2000). Our results indicate that even in the absence of detectable prechallenge systemic neutralizing Abs, DNA-VP2-MP vaccinated chickens controlled IBDV replication in the bursa. In this case, cell mediated immunity may be involved in rapid viral clearance and resulted in fewer IBDV Ag positive cells in the bursa. An increased endolysosomal pH that is suggested to occur after phagocytosis of the DNA loaded MPs may disrupt the endosomal membrane to facilitate DNA-MPs complex escape to the cytoplasm (XU u. SZOKA 1996) and support CD8+ T-cell stimulation (SHEN et al. 2006; HELSON et al. 2008; SCHLIEHE et al. 2011). More hydrophobic polymers showed delayed degradation within APCs in previous studies and may even enhance endosomal escape and induce stronger cellular immunity (XU u. SZOKA 1996). Evaluation of an HIV DNA vaccine delivered by cationic PLGA MPs through the IM route induced both humoral and cellular immune responses (OTTEN et al. 2005). This underlines the critical role of polymer nature, its hydrophobicity, nature of targeted gene and route of immunization for the phenotype and robustness of immune responses (OTTEN et al. 2005; ROMAN et al. 2008). Mucosal priming of turkeys with DNA encoding the fusion protein of aMPV adsorbed onto cationic PLGA followed by boosting with encapsulated fusion protein did not induce significant aMPV specific ELISA Abs, but elicited F protein-specific VN Abs. Partial protection against challenge was observed in this vaccination modality (LIMAN et al. 2007). Many studies have been published concerning the induction of neutralizing secretory Abs and cell mediated immunity at mucosal surfaces following oral and intranasal delivery of DNA vaccines loaded onto PLGA MPs (WANG et al. 2011). IgA is important in mucosal immunity. A DNA vaccine expressing βgalactosidase was shown to stimulate an IgA response after eye-drop inoculation in 91 chickens (RUSSELL u. MACKIE 2001). This aspects of mucosal immune responses in protection against challenge needs to be further validated particularly when DNA vaccines are being delivered by PLGA MPs. The delayed application of plasmid encoded chIL-2, which was delivered by cationic PLGA MPs at least by 48 hrs relative to VP2-MPs, did provide an increased level of protection compared to the VP2 loaded MPs alone. The lower level of postchallenge VN Abs, which also coincided with a low level of IL-4 and IFN-α mRNA expression, suggests rapid viral clearance before sufficient IBDV replication could take place to induce significant Ab levels. Previously, it was suggested that memory T-cells may play a significant role in rapid challenge virus clearance from the bursa of chickens vaccinated with DNA vaccines (CHEN et al. 2011c). Delayed IL-2 administration has been suggested to amplify DNA vaccine-provoked Ab and cellular immune responses in humans (BADEN et al. 2011). Effective IL-2 binding to its high-affinity IL-2Rα receptor after DNA vaccination may result in significant APC and T-cell proliferation. In a lymphocytic choriomeningitis virus infection model in mice, delayed application of IL-2 induced the proliferation of memory T-cells that resulted in rapid viral clearance (BLATTMAN et al. 2003). DNA vaccination preceding IL-2 application may minimize the immunosuppressive effects of Tregs as well (BRANDENBURG et al. 2008). Tregs suppress T-cell activation mainly by inhibiting IL-2 gene transcription in CD4+ T-cells (THORNTON et al. 2004). They do not produce their own IL-2, but may compete for the available IL-2 with effector CD4+ T-cells leading to decrease in the number of antigen responsive CD4+ T-cells. It has been of interest in our study to determine whether CpG-ODN may modulate a MP-IBDV DNA vaccine induced immune response. Our results show that CpG-ODN indeed improved the immune responses by enhancing the clearance of challenge virus from the bursa. Previously, it was demonstrated that the biologic effects of CpG-ODN could be significantly improved by using a delivery vehicle to target APCs instead of administering it in a soluble form (MALYALA et al. 2009). Our vaccination 92 protocol may have induced expansion of IBDV effector T-cells due to additional stimulation by CpG-ODN (LIU u. ZHAO 2007). 6.6. Concluding remarks and future perspectives We have investigated the molecular epidemiology of vvIBDV in Ethiopia and showed the clonality of vvIBDV strains circulating in the chicken population. While the information obtained from the nt sequences of the hVP2 of IBDV has been extremely valuable to understand the genetic and antigenic variability of IBDV, an additional VP1 characterization is necessary to determine IBDV evolution. Ethiopian IBDV isolates showed antigenic drift both at the VP2 and VP1 genes possibly through mutations or recombination. The antigenicity of Ethiopian IBDV isolates needs to be characterized for example by comparing their reaction patterns to panels of mAbs, which target the hydrophilic domains of hVP2 or by crossprotection studies with available live IBDV vaccines. By considering the new IBDV epidemiological situations new vaccination strategy against IBDV may be required. In spite of several years of experimental studies to validate candidate IBDV DNA vaccines, their commercial application remains elusive. Thus, a standard DNA vaccination protocol and optimization in terms of age at vaccination, dosage, intervals and frequency of DNA vaccination need to be empirically determined. Usage of proper adjuvants and improved vaccination strategies may enhance the protective efficacy of these vaccines. The efficaciousness of IBDV DNA vaccines can be possibly improved further by adsorbing the DNA vaccine onto cationic PLGA MPs that would allow mucosal application and additionally provide some adjuvant effects. The efficacy of this MPIBDV DNA vaccine was further potentiated by delayed application of CpG-ODN and chIL-2 DNA also adsorbed onto cationic MPs. 93 Further studies are required to elucidate the mechanism of protection in our vaccination protocol using cationic PLGA as delivery system. In particular, the extent to which T-cells and including Tregs are involved in the immune response and the distribution of the MP-DNA vaccine after mucosal application require further investigations. Alternatively, encapsulating CpG-ODN by W/O/W method and adsorbing DNA-VP2 on the surface of the MPs may mimic our vaccination protocol. The CpG-ODN can be released from the interior of the MPs after complete hydrolysis of the PLGA MPs to stimulate immune responses provoked by surface adsorbed DNA vaccine. Generally, the induction of higher IBDV-specific Ab titers requires a large dose of soluble DNA vaccine. Broilers with MAB require at least 25-50 times higher doses of DNA vaccine compared to SPF chickens for the induction of protective levels of immune responses due to MAB interference to developing humoral immunity (HSIEH et al. 2010). Our improved vaccination protocol may allow the administration of lower doses of IBDV DNA vaccine and adjuvants. 94 7. Summary Tamiru Negash Alkie Molecular epidemiology of infectious bursal disease viruses and development of a microparticle based vaccine Infectious bursal disease virus (IBDV) is an immunosuppressive virus of chickens with distribution across all continents. IBDV results in B-cell depletion primarily in the bursa of Fabricius, but other lymphoid tissues may be affected. Virus proteins (VP) 2 and VP1 contribute to the pathogenicity of IBDV, whereby VP2 as the main structural protein elicits neutralizing antibodies (Abs). The present study was designed to understand the molecular epidemiology of IBDV in Ethiopia and to develop a new generation IBDV DNA vaccine in combination with a slow release vaccine delivery system on the basis of biodegradable poly(D, L-lactideco-glycolide) (PLGA) microparticles (MPs). We characterized IBDV field isolates from different Ethiopian chicken flocks showing clinical IBD. In vivo experimental studies demonstrated that these isolates replicated extensively and induced significant bursal lesions in chickens. Molecular characterization by RT-PCR and sequencing of the hypervariable region of VP2 (hVP2) and the 5’ two-thirds of VP1 revealed close homology to very virulent (vv) IBDVs. Phylogenetic analysis of the VP2 region showed that all IBDV isolates had a vv genotype. Molecularly identified virulence marker amino acids (aa) at the hVP2 region have been well conserved in these strains. The VP1 region encoded aa residues typical for vvIBDV but also for attenuated IBDV strains. The implication on virulence mechanism needs further investigation. This molecular epidemiological study revealed vvIBDV as the major cause of severe losses in the different segments of poultry production in Ethiopia despite vigorous vaccination with classical live IBDV vaccines. Questions regarding vaccination failure need to be addressed in the future including appraisal of the vaccination strategy itself and the selection of vaccine strains. 95 Reversion of live IBDV vaccines to more virulent strains in the field justifies the development of new generation IBDV vaccines. In this study plasmid based IBDVDNA vaccines were developed encoding the VP2 gene of two different vvIBDV strains. Their immunogenicity and the consequent protection were evaluated. Significant numbers of specific pathogen free (SPF) chickens intramuscularly vaccinated with IBDV-DNA vaccines did seroconvert, and were partially protected against oral challenge with vvIBDV. DNA vaccinated chickens developed less severe bursal lesions compared to non-vaccinated ones. Co-administration of plasmid encoded chicken IL-2 (chIL-2) or CpG-ODN and the VP2-DNA vaccine neither enhanced seroconversion nor protection. Subsequently the DNA vaccine application was modified by adsorption of vaccine and molecular adjuvants onto PLGA based MPs. We investigated mucosal (oral and intranasal) application of the VP2-DNA loaded MP (VP2-MP) in combination with a delayed mucosal delivery of MP loaded either with chIL-2 or CpG-ODN. Chickens inoculated with the VP2-MP with or without adjuvants were significantly better protected against oral challenge compared to chickens inoculated with unloaded-MPs or with MPs loaded with adjuvants or vector only. VP2-MP-vaccinated chickens showed higher bursal to body weight (B/B) ratio, lower bursal lesion scores, reduced IBDV antigen load and less T-cell influx into the bursae compared to the different control groups. Rapid virus clearance particularly in the group inoculated additionally with chIL-2 loaded MPs lead to lower postchallenge virus neutralizing Ab titers, and lower IL-4 and IFN-α mRNA expression levels in the bursa. The results of this vaccination study provide proof-of-concept for further development and optimization of this vaccination platform as a possible alternative strategy to protect chickens against IBDV. 96 8. Zusammenfassung Tamiru Negash Alkie Molekulare Epidemiologie Infektiöser Bursitis Viren und Entwicklung einer Mikropartikel-basierten Vakzine Das Infektiöse Bursitis Virus (IBDV) ist ein immunsuppressives Virus, welches Hühner infiziert und eine Verbreitung über alle Kontinente aufweist. IBDV löst vorwiegend in der Bursa Fabricii aber auch in anderen lymphatischen Geweben BZell-Depletion aus. Das Virusprotein (VP) 2 und das VP1 tragen zur Pathogenität von IBDV bei. Neutralisierende Antikörper (Abs) werden gegen das Hauptstrukturprotein VP2 gebildet. Die vorliegende Studie hatte zum Ziel, die molekulare Epidemiologie von IBDV in Äthiopien zu untersuchen und eine neue Formulierung für eine IBDV-DNA-Vakzine, zu entwickeln, in dem diese an biologisch abbaubare Poly- (D, L-lactide-co-glycolide) (PLGA) Mikropartikel (MPs) adsorbiert und nach Applikation langsam freigesetzt werden kann. IBDV-Feldisolate aus verschiedenen erkrankten äthiopischen Hühnerbeständen wurden charakterisiert. In vivo Studien zeigten, dass diese Isolate stark replizierten und signifikante Läsionen in der Bursa infizierter Hühner hervorriefen. Molekulare Charakterisierung durch RT-PCR mit nachfolgender Sequenzierung der hypervariablen Region des VP2 (hVP2) und Zweidritteln des 5´Endes des VP1 zeigten große Homologien zu hochvirulenten (vv) IBDVs. Anhand phylogenetischer Analysen der VP2-Regionen wurden alle IBDV-Isolate dem hochvirulenten Genotyp zugeordnet. Molekulargenetisch identifizierte Aminosäuren (aa), die als Virulenzmarker dienen, waren in der hVP2-Region aller Stämme hochkonserviert. Die VP1-Region wies sowohl charakterliche aa-Sequenzen von vvIBDV als auch attenuierten Stämmen auf. Weitere Studien über die Bedeutung dieser aa-Positionen zur Klärung der Virulenzmechanismen sind nötig. Obwohl die Herden mit klassischen 97 Lebendvakzinen gegen IBDV geimpft waren, weisen die Ergebnisse dieser molekularepidemiologischen Studie auf die Bedeutung von vvIBDV als Ursache der großen wirtschaftlichen Verluste in den verschiedenen Geflügel-Produktionsebenen Äthiopiens hin. Daraus ergeben sich Fragen, wie durch neue Impfstrategien bzw. durch die Auswahl eines geeigneten Impfstammes ein Impfversagen zukünftig vermieden werden kann. Die Möglichkeit der Reversion von IBDV-Lebendvakzinen im Feld zu Stämmen höherer Virulenz, erfordert die Entwicklung einer neuen Generation von IBDVImpfstoffen. Es wurden zwei Plasmid basierte IBDV-DNA-Vakzinen hergestellt, die die VP2 Gene von zwei verschiedenen vvIBDVs kodierten. Die Immunogenität, sowie der hervorgerufene Impf-Schutz wurden evaluiert. Eine signifikante Anzahl an spezifisch-pathogenfreien Hühnern, die intramuskulär mit den IBDV-DNA-Vakzinen geimpft worden waren, serokonvertierten und waren partiell gegen eine orale Belastungsinfektion mit vvIBDV-Stämmen geschützt. Hühner, die mit der DNAVakzine geimpft wurden, entwickelten weniger Bursaläsionen im Vergleich zu nicht geimpften Tieren. Die gleichzeitige Gabe der DNA-Vakzine mit plasmidkodiertem Hühner IL-2 (chIL-2) oder CpG-ODN führte weder zu einer besseren Serokonversion noch zu einem besseren Schutz. Nachfolgend wurde die Applikation der DNAVakzine durch die Adsorption des Impfstoffes und molekularer Adjuvantien an PLGA basierte MPs modifiziert. Die orale und intranasale Schleimhautapplikation der VP2-DNA beladenen MP (VP2MP) wurde kombiniert mit einer verzögerten Applikation von mit chIL-2 oder CpGODN beladenen MPs. Hühner, die mit dem VP2-MP geimpft worden waren, waren unabhängig vom Einsatz von Adjuvantien signifikant besser gegen eine orale Belastungsinfektion geschützt als Hühner, die mit unbeladenen MPs, MPs mit Adjuvanz oder Vektor inokuliert worden waren. VP2-MP geimpfte Hühner zeigten ein besseres Bursa-Körpergewichtsverhältnis (B/B), einen geringeren Bursaläsionsscore, weniger IBDV-Antigen und eine geringere Anzahl an T-Zellen in der Bursa im Vergleich zu ungeschützten Tieren. 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SALEM (2008): Comparative study of poly (lactic-co-glycolic acid)-poly ethyleneimine-plasmid DNA microparticles prepared using double emulsion methods. J Microencapsul 25, 1-12 142 ZIERENBERG, K., H. NIEPER, T. P. VAN DEN BERG, C. D. EZEOKOLI, M. VOSS u. H. MULLER (2000): The VP2 variable region of African and German isolates of infectious bursal disease virus: comparison with very virulent, "classical" virulent, and attenuated tissue culture-adapted strains. Arch Virol 145, 113-125 ZIERENBERG, K., R. RAUE, H. NIEPER, M. R. ISLAM, N. ETERRADOSSI, D. TOQUIN u. H. MULLER (2004): Generation of serotype 1/serotype 2 reassortant viruses of the infectious bursal disease virus and their investigation in vitro and in vivo. Virus Res 105, 23-34 143 10. Acknowledgements The dedication and expertise given by my supervisor, Prof. Dr. Silke Rautenschlein, from the inception of the PhD research to the end of my PhD was amazing. I thank you very much for your encouragements in all circumstances and for the livable guidance to complete my work. The PhD supervisory committee members, Prof. Dr. Beatrice Grummer and PD Dr. Reimar Johne, without your continual advise and support, my PhD would not have been materialized. The role of Hannover Graduate School for Veterinary Pathobiology, Neuroinfectiology, and Translational Medicine (HGNI), University of Veterinary Medicine Hanover (TiHo), Germany, was also critical for my PhD work mainly through coordination, financing all my international conference fees and for providing an additional 3 months financial support. My very heartfelt acknowledgment directly goes to the German Academic Exchange Service (DAAD) for the provision of the scholarship, family allowances and for unreservedly providing all the necessary information during my PhD study. I express my appreciation to the PhD secretaries for providing valuable ideas during the PhD course work. Mrs. Maritta Ledwoch, from the International Office, you are so wonderful and I always feel comfortable when I visit your office and for the nice extracurricular dialogue which we had in the past. Likewise, the University of Gondar, Ethiopia is highly acknowledged for granting me a PhD study leave and the Institute of Biodiversity Conservation and Research for handling my sample export permit favorably. The National Veterinary Institute, Ethiopia equally shares my thanks for providing me some of the samples for my PhD research. 144 My very special thanks to Christine Haase for her excellent technical support; Sonja Bernhard, Sabrina Techel and Katja Stolpe for their excellent help in animal care and necropsy. The institute for parasitology, TiHo also deserves my thanks for allowing me to use some of the laboratory facilities. I am very pleased and would like to take this opportunity to pay tribute to every single member of the staff at the Clinic for Poultry, who have played a part in my research. I do not have words to express my thanks to Lydia Teske for your kindness and support in translating the summary part of my thesis into the German version. Dr. Henning Petersen, from the Clinic for Poultry, I thank you very much for structuring and formatting my thesis. Dr. Awoke, Mohammed Yusuf Adem and Bidir, thank you for handling my animal experiment at Gondar while I was processing my visa. No amounts of words express my love and respect to my wife, Eskedar Hailegebral, for providing me a lesson of patience in my life, also for sharing your mature political thoughts and for your untiring responsibility in managing our family. I know that it is stressful to handle the two very little kids. Eskedar you suffered, but never get disappointed we are decorated with the two blessings. In the front line of my PhD work is my lovely daughter Heldana, she is the fortitude of my life.
