GM-CSF-adjuvanted Newcastle disease virus-vectored bivalent vaccine elicits enhanced dual immunity against Newcastle disease and infectious bursal disease in chickens

Infectious bursal disease (IBD) and Newcastle disease (ND) are major infectious diseases that endanger poultry. Despite current vaccination efforts, both diseases still occur worldwide. We have developed bivalent vaccines capable of simultaneously preventing ND and IBD, which does not produce mutant IBDV. Using reverse genetics, we constructed a recombinant Newcastle disease virus (NDV) vector based on the common vaccine strain Clone30 to express the host-protective immunogen VP2L of the IBDV strain and chicken granulocyte-macrophage colony-stimulating factor (GM-CSF). The IBDV-encoded VP2L protein and chicken GM-CSF gene were inserted into different positions of the NDV full-length cDNA in various forms to achieve high-level expression. We successfully rescued the ND-AI bivalent vaccines (rClone30-VP2L(P/M)-GM-CSF(P/M), rClone30-VP2L(NP)-GM-CSF(P/M), rClone30-VP2L(P/M)-GM-CSF(NP) and rClone30-VP2L-IRES-GM-CSF(P/M)). The ND-AI bivalent vaccines maintained genetic stability after at least three consecutive passages in chicken embryos and was confirmed to express VP2L and GM-CSF proteins. The replication titers of the ND-AI bivalent vaccines in chicken embryos and cell cultures were comparable to those of the parental NDV strain rClone30. To assess the immunogenicity of the ND-AI bivalent vaccines, it was administered to 14-day-old commercial chicken chicks, and immune responses were continuously monitored for four weeks post-vaccination. By day 10 post-vaccination, the hemagglutination inhibition (HI) antibody titers of the recombinant NDV vaccine had far exceeded the theoretical protective threshold (4 log2) and remained at high levels for 28 days. Additionally, the levels of IBDV-specific antibodies in the ND-AI bivalent vaccines rapidly increased and remained at high titers for 14 days. Concurrently, the proliferation responses of B cells, CD4 + , and CD8 + T cells were enhanced, and the protein expression levels and mRNA transcription levels of inflammatory factors significantly increased. In summary, the ND-AI bivalent vaccines can stimulate the body to produce a stronger immune response, demonstrating its potential as a vaccine for IBD and ND. Additionally, the addition of GM-CSF can further enhance the immune response.