Construction of a chimeric multi-antigen fusion vaccine, EimeriaBig, and evaluation of immune response and protective effect in Eimeria necatrix.

Avian coccidiosis, especially infection caused by the highly pathogenic Eimeria necatrix, causes major economic loss in the poultry production. With the increasing limitations of anticoccidial drugs and live vaccines, there is an urgent need for novel, broad-spectrum control strategies. In this study, a reverse vaccinology workflow was used to screen the proteomes of five Eimeria species. Six highly conserved membrane protein families were selected: carbonic anhydrase (CA), mechanosensitive ion channel (MSC), cell division control protein 50 (CDC50), major facilitator superfamily transporter (MFS), ATP-binding cassette transporter (ABC) and lipid transfer protein (LTP). Antigenic fragments from these families were linked with EAAAK linkers to generate a chimeric multi-antigen fusion construct, EimeriaBig. Five prime-boost regimens were tested in chickens challenged with E. necatrix. The regimen of one protein prime followed by two DNA boosts showed the highest protection among the tested regimens, with moderate protective efficacy. Our results demonstrated that a heterologous strategy, comprising a recombinant protein prime (rEimeriaBig) followed by two DNA vaccine boosts (pcDNA3.1-EimeriaBig), conferred the best protection among the regimens tested, with moderate efficacy. In this group, cecal lesion score was reduced by 47.64 % and oocyst output was reduced by 49.75 %, and the anticoccidial index was 168.72. The same regimen produced higher antigen-specific IgY and increased transcription of IL-2, IFN-γ, and IL-10. These results support further optimization of this vaccine strategy for control of E. necatrix.