In the scope of the current FY 2008 NRI Program Area 44.0 Animal Protection and Biosecurity (A), high priority area 1c: Poultry: Avian Clostridium perfringens, our goal is to design and evaluate an in ovo delivered adenovirus vectored vaccine for the prevention or control of C. perfringens-associated morbidity and mortality in poultry. Our central hypothesis for the proposal claims that in ovo vaccination with an adenovirus-vectored C. perfringens enterotoxin vaccine capable of targeting antigen to professional antigen presenting cells and co-delivery of an IgA switch factor will improve mucosal IgA response and protection in chickens. The hypothesis will be tested through completion of the following specific aims: <OL> <LI> Test whether in ovo immunization with an adenovirus expressing an agonistic anti-CD40 single chain antibody fused to C. perfringens alpha-toxin enhances mucosal IgA secretion and protection of chickens upon challenge; <LI> Determine whether in ovo co-delivery of adenoviruses expressing the CD40-targeted C. perfringens alpha-toxin and the IgA switch factor, BAFF, improves mucosal IgA response and protection of chickens upon challenge.
The expected outcome is an adenovirus vectored vaccine for in ovo delivery that is able to prevent or control C. perfringens-associated morbidity and mortality in poultry. A positive outcome from this study will form the basis for testing whether inclusion of other C. perfringens toxins in the vaccine will confer broader protection. Knowledge generated from this study will be important for development of vaccines against other livestock diseases.
NON-TECHNICAL SUMMARY: Necrotic enteritis (NE) is an economically important enteric disease of chickens primarily caused by infection with the bacterium Clostridium perfringens type A (CPA). The acute form of NE in a broiler flock can account for losses of 1% per day, for several consecutive days during the last weeks of the rearing period. In addition, some strains of C. perfringens type A produce an enterotoxin that causes food-borne disease in humans. Because of the voluntary or legally required withdrawal of the use of certain in-feed antibiotic growth promoters with anti-clostridia activity, few tools and strategies are available for prevention and control of C. perfringens in poultry. Our goal is to design and evaluate a vaccine that will be delivered by injection in the egg ("in ovo") consisting of a non replicating, harmless adenovirus that contains the necessary genetic information to produce protective fragments of two CPA toxins attached to an antibody that will guide the vaccine to the antigen-presenting cells of the chicken embryo. This strategy, combined with the co-delivery of an immune activating factor (BAFF) is expected to enhance the production of C. perfringens toxin-specific IgA antibodies in the gut, thereby improving protection against avian necrotic enteritis. A positive outcome from this study will form the basis for testing whether inclusion of other C. perfringens toxins in the vaccine will confer broader protection. Knowledge generated from this study will be important for development of vaccines against other livestock diseases. <P>
APPROACH: Necrotic enteritis (NE) is suspected to occur under certain predisposing conditions when C. perfringens Type A, and to a lesser extent Type C multiply in excess and secrete toxins in a small intestine that already has had some damage. C. perfringens Type A produces several exotoxins,the main one being alpha-toxin, which is a phospholipase (PLC). Alpha-toxin has been implicated as the major cause of lesions associated with this disease, although recently a new toxin, netB, has also been a associated with avian necrotic enteritis. We intend to induce specific IgA responses against protective, non-toxic fragments of the C. perfringens alpha and netB toxin. For this purpose we will direct the active uptake of the recombinant toxin fragments by professional antigen presenting cells (APCs) through the well-characterized APC activation by CD40 signaling, simultaneously with the provision of recombinant BAFF, an IgA switch factor. We will amplify the sequence encoding the truncated toxin domains for fusion with a gene encoding an agonistic anti-CD40 single chain variable fragment (scFv) gene. This construct will be inserted in a recombinant non-replicating adenovirus. In addition, we intend to promote IgA production and enhance immunity by adenoviral co-delivery of an IgA switch factor, BAFF. We expect to detect improved IgA responses and protection against NE in birds vaccinated with the proposed vaccine. The adenoviral vectored vaccine will be administered in ovo to white leghorn chicken embryonating eggs to evaluate antibody responses and protection against challenge as previously described (Sharma and Burmester, 1982). We will use 20 eggs per group estimated based on the protective indices (PI) data of Sharma and Burmester (1982). In an initial set of experiments, we will determine the viral dose that produces optimal antibody responses in vivo, and whether an orally delivered boost is advantageous in terms of antibody response. The vaccination protocol that results in the optimal specific antibody response (a high mucosal titer of toxin-neutralizing IgA) will be repeated and birds will be challenged according to the McReynolds NE model. On day 25, the birds will be euthanized and samples will be collected and analyzed. Intestinal lesions will be scored as described by McReynolds et al.(2004). Vaccination followed by Clostridium challenge will be performed in triplicate trials. The same trials will be repeated with in ovo coadministration of BAFF and specific IgA responses and protection against challenge with C. perfringens will be monitored.