Infectious Bursal Disease Virus as a Dual Vaccine Vector for Protection Against Poultry Infectious Diseases

NON-TECHNICAL SUMMARY: Infectious bursal disease (IBD) is generally controlled by killed or live attenuated vaccines in the poultry industry worldwide. However, outbreaks still occur due to the emergence of variant or very virulent strains. Development of an alternative but effective vaccine/vaccination strategy is, therefore, critical to the poultry heath. The genome of infectious bursal disease virus (IBDV) is small. A viral vector with small genome will minimize the interference with expression of the recombinant gene by expression of extraneous viral genes and allow rapid and easy engineering of constructs. Infectious bursal disease virus is a double-stranded RNA virus with a bi-segmented genome (segments A and B). The negatively charged C-terminus of viral protein VP3 is considered to act as a regulatory switch in capsid assembly. Deletion of VP3 fragment from IBDV segment A, therefore, would render the IBDV replication incompetent. Replication-defective IBDV will not release progeny virions in the host tissues. Because IBDV has a tropism for B lymphocytes, the delivery of vaccine recombinant genes to B cells will ensure optimal antigenic presentation. The IBDV vector can be utilized for a broad avian host range, including chickens, ducks, turkeys, and quail since IBDV causes infection to these avian species. The development of a reverse genetics system with highly efficient recovery of replication-defective virus in the host tissue is a critical feature for the practical use of an IBDV vector. Furthermore, there will be a broader application of the IBDV vector as a vaccine vector to a wide range of poultry infectious diseases, including newly emerging pathogens or strains such as AIV, providing the optimal biosecurity for poultry. The replication-defective IBDV vector can serve as a dual vaccine vector and provide protection against IBD and avian influenza (AI) in chickens and turkeys. Successful completion of the proposed project will have a significant impact on the well-being of poultry and production revenue of U.S. poultry industry.<P>

APPROACH: <OL> <LI> Construction of VP3-deleted IBDV viral vector: A reverse genetics method as described previously will be used to generate an IBDV viral vector for the transduction and expression of recombinant genes and as a vaccine vector. A fragment of the VP3 structural gene from IBDV (strain VE) segment A will be deleted and substituted with the GFP reporter gene. <LI>Pathogenicity and immunogenicity of VP3-deleted IBDV in chickens: Three groups of 3-week-old specific-pathogen-free (SPF) chickens will be orally inoculated with (i) GFP+.VP3-.IBDV, (ii) IBDV (strain VE), or (iii) negative control. Five chickens from each group will be taken at 0, 3, 7, 15, 22, and 35 days after inoculation. Histopathological evaluation of tissues will be performed. Viral tissue titers will be determined using IBDV Real-time RT-PCR. Serum will be taken from each bird for assessment of the humoral immune responses by ELISA and virus neutralization(VN)test, respectively. <LI> Pathogenicity and immunogenicity of VP3-deleted IBDV in turkeys: Three groups of 1-week-old commercial large white turkeys will be orally inoculated with (i) GFP+.VP3-.IBDV, (ii) IBDV (strain VE), or (iii) negative control. Five turkeys from each group will be taken at 0, 3, 5, 7, 14, 28, and 42 days after inoculation. Histopathological evaluation of tissues will be performed. Viral tissue titers will be determined using IBDV Real-time RT-PCR. Serum virus neutralization (VN) titers against IBDV will be determined. <LI> Molecular cloning of HA gene of AIV in VP3-deleted IBDV: Viral RNA will be extracted from allantoic fluid of embryonated chicken eggs infected with low pathogenicity avian influenza (LPAI) virus. Virus (HA+.VP3-.IBDV) particles will be recovered from the transfection of Vero cells with RNA transcripts produced from the segments A.HA+.VP3- and B clones and with complementing DNA plasmid encoding the VP243 polyprotein. Similar approach will be used to generate HA1+.VP3-.IBDV virus as vaccine. <LI> Protection efficacy of HA+.VP3-.IBDV or HA1+.VP3-.IBDV in chickens and turkeys: Five Groups of 3-week-old SPF chickens will be orally inoculated at day 0 and boosted at day 7 with (i) GFP+.VP3-.IBDV, (ii) IBDV, (iii) HA+.VP3-. IBDV, or (iv) negative control. At 14 days PI, one third of the chickens in each group will be intra-ocularly or intra-tracheally challenged with allantoic fluid containing 100 lethal dose (LD)50 of AIV of the same H7 subtype as the vaccine isolate (A/Chicken/Pennsylvania/97; H7N2), one third with IBDV(strain VE), and one third with sham virus challenge. Five chickens will be taken from each treatment group and their tissues (serum, thymus, spleen, bursa, cecal tonsil, lung, trachea, conjunctiva, and infra-orbital sinus) will be collected at 14, 21, and 35 days post challenge for assessment of vaccine protection. In another study, the same approach for chickens will be performed on 1-week-old turkeys to determine protection efficacy of HA+.VP3-.IBDV in turkeys. Similar protection efficacy studies will be also carried out for HA1+.VP3-.IBDV in chickens and turkeys.
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PROGRESS: 2006/10 TO 2007/09 <BR>
OUTPUTS: Calreticulin (CRT) has been shown to display peptide-binding activity and present newly synthesized MHC I with the antigenic peptides on the surface of the antigen presenting cells. The present study was conducted to determine whether the presentation of infectious bursal disease virus (IBDV) large segment protein (VP243) by DNA coding for IBDV VP243 gene could be enhanced by linking it to chicken CRT gene. DNA plasmid carrying chicken CRT gene (p-CRT), IBDV VP243 gene (p-VP243), or IBDV VP243 gene linked to chicken CRT gene (p-CRT-VP243) were constructed. COS-7 cells in 100 % confluency were transfected with 1 ug of each constructed DNA plasmid for 12, 24, and 48 hours. Transfected COS-7 cells were incubated with anti-IBDV monoclonal antibody (Mab), followed by fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG to stain IBDV VP243 on the cell surface as green fluoresence. COS-7 cells were subsequently permeated by methanol and incubated with anti-IBDV Mab again, followed by Texas red (TXRD)-conjugated goat anti-mouse IgG to stain intracellular VP243 as red fluorescence. The levels of expressed of IBDV VP243 protein on the cell surface (green fluorescence) or in the cytoplasm (red fluorescence) of COS-7 cells were determined by the quantity and intensity of fluorescent COS-7 cells in 10 consecutive fields at 400x magnification. The quantity of fluorescent cells were scored as 1 to 4 with 1 having the lowest numbers of fluorescent cells and the fluorescent intensity was categorized as 1 to 4 with increasing intensity. The levels of expressed IBDV VP243 protein on the surface of COS-7 cells transfected with p-CRT-VP243 were significantly higher (P<0.05) than those of COS-7 cells transfected with p-VP243 at 12, 24, and 48 hours after transfection, respectively. The levels of expressed IBDV VP243 protein on the surface of COS-7 cells transfected with p-CRT-VP243 for 48 hours were significantly higher (P<0.05) than those of COS-7 cells transfected with p-CRT-VP243 for 12 or 24 hours. The presentation of IBDV VP243 protein by DNA vaccination, thus, can be enhanced by DNA encoding IBDV VP243 gene linked to chicken CRT gene. This work was part of a graduate student M.S. dissertation and the information and knowledge generated has been shared with poultry health researchers in the U.S. and the collaborators, Dr. Y.M. Saif, The Ohio State University, and Dr. J.J. Jiambrone, Auburn University. PARTICIPANTS: Individuals: Dr. Ching Ching Wu, principal investigator/project director, is responsible for overall progress of the project, animal studies, molecular and immunological aspects of the studies, data analysis and interpretation, statistical analysis, and preparation and submision of progress reports and manuscripts. Dr. Tsang Long Lin, co-principal investigator, is responsible for clinical and pathological aspects of the studies and provide assistance in preparation and submision of progress reports and manuscripts. Collaborators and contacts: Dr. Y. M. Saif, Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH. Dr. J. J. Jiambrone, Department of Poultry Science, Auburn University, Auburn, AL. Training or professional development: Three post-doctoral scientists, two Ph.D. graduate students, one M.S. graduate students had been or are involved with the project for research training. <BR> TARGET AUDIENCES: Target audiences of this project include layer and broiler farmers in Indiana, layer and broiler farmers in the U.S., and poultry health researchers in the U.S. Research progress and up-to-date findings have been presented to and communicated with Indiana State Poultry Association, North Central Avian Diseases Conference, and American Association of Avian Pathologists.
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IMPACT: 2006/10 TO 2007/09<BR>
The advantage of DNA vaccination is to provide protection without infection to the animals and without contamination of the environment with the live viruses. Effective protection of chickens against infectious bursal disease (IBD) by DNA vaccination has been demonstrated by previous studies. However, multiple times of intramuscular injection with large dose of DNA are required. Calreticulin (CRT) has been shown to display peptide-binding activity and present newly synthesized MHC I with the antigenic peptides on the surface of the antigen presenting cells. The findings of the present study indicated that the presentation of IBD virus (IBDV) VP243 protein by DNA vaccination can be enhanced by DNA encoding IBDV VP243 gene linked to chicken CRT gene. Enhancement of IBDV VP243 protein presentation is expected to enhance protective immunity against IBDV infection. Thus, the vaccination strategy by priming the chickens with DNA vaccine encoding IBDV VP243 gene linked to chicken CRT gene has the potential to enhance immunity and protection of chickens against IBD for practical prevention and control of IBD in the field.
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PROGRESS: 2005/10/01 TO 2006/09/30<BR>
One-day-old chickens were intramuscularly injected with DNA plasmid coding for large segment gene of infectious bursal disease virus (IBDV) strain variant E (VE) (P/VP243/E) and followed by weekly intramuscular injection of killed infectious bursal disease (IBD) vaccine containing both standard and variant IBDV at one or two weeks old. Chickens were orally challenged with IBDV strain VE or standard challenge strain (STC) at 3 weeks old and observed for 10 days. Seven trials were carrying out and bursal lesion scores, bursa weight/body weight (B/B) ratios, protection efficacy, IBDV antigen in bursae, enzyme-linked immunosorbent assay (ELISA) titers to IBDV, and virus neutralization (VN) titers to IBDV were determined. Concanavalin (Con) A induced and IBDV antigen stimulated spleen or blood lymphocyte proliferation response was performed at 14, 21, 24, and 31 days after priming with P/VP243/E. Chickens primed with 50, 100, 200, or 400 ug of P/VP243/E at one day old and boosted with 0.5 ml of killed IBD vaccine at 1 or 2 weeks old had 80 to 100% protection against challenge by IBDV strain VE or 71 to 100% against challenge by IBDV strain STC. Chickens in the groups primed with P/VP243/E and boosted with killed vaccine had significantly higher (P<0.05) B/B ratios and significantly lower (P<0.05) bursal lesion scores than chickens in the challenge control (CC) groups and groups primed with saline or vector plasmid and boosted with killed IBD vaccine or only primed with P/VP243/E. No IBDV antigen was detected by immunofluorescent antibody assay (IFA) in bursae of chickens protected by the DNA vaccine prime-killed vaccine boost vaccination. Prior to challenge, chickens (21 days old) in the groups primed with P/VP243/E and boosted with killed IBD vaccine had significantly higher (P<0.05) ELISA and VN titers to IBDV and stimulation indices for ConA-induced or IBDV antigen stimulated spleen or blood lymphocyte proliferation assays than chickens in the CC groups or groups primed with vector plasmid or saline and boosted with killed IBD vaccine or only primed with P/VP243/E. The results indicated that chickens at 1 day old primed with DNA vaccine encoding large segment gene of IBDV and boosted with killed IBD vaccine at 1 or 2 weeks old can be adequately protected against challenge by homologous variant or heterologous classic IBDV.
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IMPACT: 2005/10/01 TO 2006/09/30<BR>
Effective protection of chickens against infectious bursal disease (IBD) by DNA vaccination has been shown to require multiple times of intramuscular injection with large dose of DNA carrying large segment gene of infectious bursal disease virus (IBDV) in previous studies. The findings of the present study demonstrated that chickens at 1 days old primed with DNA vaccine encoding large segment gene of IBDV and boosted with killed IBD vaccine at 1 or 2 weeks old can be adequately protected against challenge by homologous variant or heterologous classic IBDV without the usage of live virus vaccines, therefore, avoiding the contamination of the environment with the live viruses and preventing the reverted viral virulence. Thus, the vaccination strategy by priming the chickens with DNA vaccine and boosting with killed vaccine has the potential to enhance immunity and protection of chickens against IBD for practical application in the field situation.
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PROGRESS: 2004/10/01 TO 2005/09/30<BR>
Of concern in the control of infectious bursal disease virus (IBDV) infection is the steady progression of viral evolution over recent decades and the emergence of both new antigenic variants and strains with enhanced virulence. Effective control of infectious bursal disease (IBD) is contingent on the availability of rapid and efficient diagnostic procedures for identifying and differentiating IBDV strains within a flock. Currently there is no single-step assay available that will rapidly identify and differentiate IBDV strains. A real-time reverse transcription (RT) polymerase chain reaction (PCR) assay was developed utilizing dual-labeled fluorescent probes binding to IBDV VP4 sequence that are specific to the classical, variant or very virulent subtypes of IBDV. The assay was highly sensitive and could detect as little as 3 times 1o to the second to 3 times 10 to the third copies of viral template. Viral genomic copy number could be accurately assayed over a broad range of 7 to 8 logs of viral genome. The variant-sequence specific probe was found to be highly specific in detecting isolates classified as variant A, D, E, G and GLS-5, and did not react with classical strains. A total of 130 field and experimental variant strain isolates were tested using this assay. The classical-sequence specific probe also demonstrated high sensitivity and specificity, and positively detected a total of 87 STC isolates, both field and experimental isolates, whilst differentiating between isolates that were variant and classical strains. Rapid identification of viral strain is a primary concern to poultry flock health programs to ensure administered vaccines will protect against current strains of virus circulating in the flock. The ability to concurrently quantify virus titer is also of assistance in identifying the progression of disease outbreaks within the flock. A rapid, sensitive, assay such as real-time RT PCR is advantageous in the accurate diagnosis of IBDV flock status.
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IMPACT: 2004/10/01 TO 2005/09/30<BR>
Rapid identification of IBDV strains is a primary concern to poultry industry to ensure administered vaccines will protect against current strains of IBDV circulating in the flock. The ability to concurrently quantify IBDV titer is also of assistance in identifying the progression of disease outbreaks within the flock. The developed real-time RT PCR is advantageous in the rapid and accurate diagnosis of IBDV flock status for the effective control of IBD.
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PROGRESS: 2003/10/01 TO 2004/09/29<BR>
Previous studies from our laboratory demonstrated that chickens could be efficaciously protected by DNA vaccination against infectious bursal disease (IBD) with multiple doses of DNA vaccine carrying large segment gene of infectious bursal disease virus (IBDV). Co-administration of chicken IL-2 and large segment gene of IBDV plasmids was conducted to determine if enhanced protection of chickens against IBD by DNA-mediated vaccination could be achieved. One-day-old chickens were intramuscularly injected with DNA plasmid carrying large segment gene of IBDV strain VE or chicken IL-2 gene individually or both plasmids once, twice, or three times at weekly intervals. Chickens were orally challenged with IBDV strain VE at 3 weeks old and observed for 10 days. Chickens inoculated with two plasmids in the separate legs two times had 58.8% to 64.7% protection against IBD with significantly higher (P<0.05) bursal/body weight ratios and significantly lower (P<0.05) bursal lesion scores than those only receiving DNA plasmid carrying large segment gene of IBDV strain VE two times (37.5% protection). The results indicated that plasmid encoding chicken IL-2 gene or large segment of IBDV gene given at separate sites two times enhanced protection of chickens against IBD.
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IMPACT: 2003/10/01 TO 2004/09/29<BR>
Chicken cytokine gene (such as IL-2 gene) co-administered with large segment gene of IBDV has immunomodulatory effect and offers enhanced protection of chickens against IBDV infection by DNA vaccination that does not require live viruses and avoids reverted virulence. Thus, immunostimulating modulators, including chicken IL-2, has the potential to enhance immunity and protection of chickens against IBD by DNA vaccination for practical application in the field situation.