In vivo targeting of
peptides to antigen-presenting cells by use of agonistic anti-CD40
monoclonal antibodies has been used successfully as an immune response enhancing strategy. When tested in chickens, the antibody-guided platform was capable of inducing specific
IgG production within 1 week postimmunization. However, use of this method beyond its initial conception as a
vaccine delivery tool has not been fully exploited. In this study,
Clostridium perfringens alpha-toxin was used as a model microbial toxin for
epitope mapping by using the antibody-guided immunization method to generate a panel of
antibodies against specific, regions of the toxin in an attempt to identify crucial determinants on the toxin which, once bound, would hinder downstream toxicity. Alpha-toxin, which possesses both hemolytic and
phospholipase C (PLC) enzymatic activities, has long been known to be one of the key destructive etiological agents of necrotic
enteritis disease in poultry. Previous attempts to identify crucial
antigenic determinants on the toxin mediating its enzymatic activities have been performed using expensive and labor-intensive site-directed mutagenesis techniques. To create a panel of
antibodies, 23 short candidate alpha-toxin
peptide regions were selected in silico using
B-cell epitope prediction algorithms in the public domain and were custom synthesized to load onto the antibody-guided complex for immunization in birds for
antisera production.
Peptide-specific antibody responses were generated against all candidate neutralizing
epitopes and used for in vitro toxin neutralization tests.
Antisera against all 23
peptides were able to neutralize the toxin's hemolytic activity, with neutralization titers ranging from 80 to 320, but none were effective in blocking PLC. The novel approach of antibody-guided immunization introduces a new, inexpensive method for polyclonal
IgG production and de facto identification of neutralizing
epitopes in microbial toxins and
enzymes within 2 weeks from in silico analysis of a putative target sequence.