Recombinant truncated gD glycoprotein from E.coli as a thermostable and protective subunit vaccine candidate against ILTV in chickens

Infectious laryngotracheitis virus (ILTV) is a major respiratory pathogen in poultry, and current vaccines pose safety risks or offer limited protection. To develop safer alternatives, we designed four truncated fragments of ILTV glycoproteins gB and gD (designated gBa, gBb, gB, and gD) based on bioinformatic analysis. The gBb and gD were successfully expressed in Escherichia coli (E.coli), and gD also in a baculovirus system. Three recombinant proteins (E.coli-gBb, E.coli-gD, and Bac-gD) showed favorable thermal stability and strong immunogenicity, as confirmed by NanoDSF and ELISPOT assays. SPF chickens were immunized with Freund's adjuvant-formulated E.coli-gBb, E.coli-gD, E.coli-gBb + gD, or Bac-gD proteins, followed by challenge with the virulent ILTV strain HB201806. Post-vaccination evaluation by ELISA and flow cytometry revealed that E.coli-gD induced the most robust humoral and cellular immune responses. Compared to the challenged control group, all immunized groups exhibited attenuated clinical symptoms and reduced mortality. Furthermore, at the peak of infection, viral loads in the trachea and Harderian glands were reduced to varying extents, with the E.coli-gD group demonstrating the greatest protective efficacy. These results indicate that recombinant gD, particularly the E.coli-expressed form, is a thermally stable, highly immunogenic, and protective subunit vaccine candidate for ILTV control in poultry.