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Highly pathogenic avian influenza causes severe economic losses and is a potential threat to public health. Better knowledge of the mechanisms of chicken response to the novel types of vaccines against avian influenza might be helpful in their successful implementation into poultry vaccination programs in different countries. This work presents a comprehensive analysis of gene expression response elicited in chicken spleens by a combined DNA/recombinant protein prime/boost vaccination compared to DNA/DNA and protein/protein regimens. All groups of vaccinated chickens displayed changes in spleen transcriptomes in comparison to the control group with 423, 375 and 212 identified differentially expressed genes in protein/protein, DNA/DNA and DNA/protein group, respectively. Genes with most significantly changed expression belong to immune-related categories. Depending on a group, a fraction of 15–34% of up-regulated and a fraction of 15–42% of down-regulated immune-related genes are shared by all groups. Interestingly, the most upregulated genes encode β-defensins, short peptides with antimicrobial activity and immunomodulatory functions. Microarray results were validated with RT-qPCR method, which confirmed differential regulation of the selected immune-related genes. Immune-related differentially expressed genes and metabolic pathways identified in this work are compared to the available literature data on gene expression changes in vaccinated and non-vaccinated chickens after influenza infection.Chickens were prime/boost immunised with DNA and/or protein of H5 HA.Spleen transcriptome was analysed one week after the booster.Analysis revealed comprehensive response of the immune system.Selected microarray data were validated with qRT-PCR.It is the first gene expression data for anti-influenza vaccinated uninfected chickens.