Identification of Eimeria tenella sporozoite immunodominant mimotopes by random phage-display peptide libraries–a proof of concept study

Abstract

IntroductionCoccidiosis, caused by parasites of numerous Eimeria species, has long been recognized as an economically significant disease in the chicken industry worldwide. The rise of anti-coccidian resistance has driven a search for other parasite management techniques. Recombinant antigen vaccination presents a highly feasible alternative. Properly identifying antigens that might trigger a potent immune response is one of the major obstacles to creating a viable genetically modified vaccine.MethodsThis study evaluated a reverse immunology approach for the identification of B-cell epitopes. Antisera from rabbits and hens inoculated with whole-sporozoites of E. tenella were used to identify Western blot antigens. The rabbit IgG fraction from the anti-sporozoite serum exhibited the highest reactogenicity; consequently, it was purified and utilized to screen two random Phage-display peptide libraries (12 mer and c7c mer). After three panning rounds, 20 clones from each library were randomly selected, their nucleotide sequences acquired, and their reactivity to anti-sporozoite E. tenella serum assessed. The selected peptide clones inferred amino acid sequences matched numerous E. tenella proteins.Results and ConclusionsThe extracellular domain of the epidermal growth factor-like (EGF-like) repeats, and the thrombospondin type-I (TSP-1) repeats of E. tenella micronemal protein 4 (EtMIC4) matched with the c7c mer selected clones CNTGSPYEC (2/20) and CMSTGLSSC (1/20) respectively. The clone CSISSLTHC that matched with a conserved hypothetical protein of E. tenella was widely selected (3/20). Selected clones from the 12-mer phage display library AGHTTQFNSKTT (7/20), GPNSAFWAGSER (2/20) and HFAYWWNGVRGP (8/20) showed similarities with a cullin homolog, elongation factor-2 and beta-dynein chain a putative E. tenella protein, respectively. Four immunodominant clones were previously selected and used to immunize rabbits. By ELISA and Western blot, all rabbit anti-clone serums detected E. tenella native antigens.DiscussionThus, selected phagotopes contained recombinant E. tenella antigen peptides. Using antibodies against E. tenella sporozoites, this study demonstrated the feasibility of screening Phage-display random peptide libraries for true immunotopes. In addition, this study looked at an approach for finding novel candidates that could be used as an E. tenella recombinant epitope-based vaccine.

Discovery of Antigens and Cellular Mechanisms in the Protozoan Parasite Sarcocystis aucheniae Using Immunoproteomics

“…Micronemes undergo exocytosis by fusing with the plasma membrane at the apical end to discharge their contents of adhesin complexes and perforins that participate in critical events for parasitic life, including host cell invasion and egress and gliding motility (see below, [32]). Interestingly, both T. gondii MIC12 and E. tenella MIC4 were found to be strongly immunogenic when screening a protein microarray or a phage display library, respectively, with immune sera against the respective parasites [33,34]. Consistently, in the present study, S. aucheniae MIC12 displayed the highest antigenicity among the proteins of its corresponding immunoreactive band and was one of the proteins selected for epitope B identification (Table 1, Supplementary Table S1).…”

Protective Efficacy Induced by the Common Eimeria Antigen Elongation Factor 2 against Challenge with Three Eimeria Species in Chickens

“…Some researchers have proposed that peptide-based vaccines are composed of immunogenic epitopes of various antigens to generate highly specific immune responses [ 63 , 64 , 65 , 66 , 67 ]. Since cellular immunity plays a crucial role against avian coccidiosis, selecting peptides of T cell epitopes as vaccine candidates for development may effectively control the incidence of avian coccidiosis [ 68 , 69 , 70 , 71 , 72 ]. Therefore, T cell epitopes of several common antigens of chicken coccidia identified in our previous study, including EF2, can be selected for the construction of a multivalent epitope vaccine, which can be targeted to increase the level of cellular immunity induced by the vaccine and thus improve its protective efficacy.…”