Identification of the immune enhancement effect of CpG ODN on periodontitis genetic vaccine by RNA sequencing
Authors: Zhang HuiJie , Zeng FengJiao , Yu Hang , Chen Bin , Zhou XiaoHong , Wang MingWei , Bai GuoHui 
Published: June 2022

Abstract
Background: Periodontitis is one of the main causes of tooth loss in adults. Early diagnosis and intervention can effectively control the progression of periodontitis. The research group constructed a periodontitis genetic vaccine in the early stage and improved the immunogenicity of the body by adding CpG oligonucleotide (CpG ODN). The purpose of this study was to analyse the potential mechanism by which CpG ODN adjuvants enhance the preventive effect of periodontitis genetic vaccines at the molecular level by transcriptome sequencing and to screen out key genes and signaling pathways for follow-up research.Methods: Nine 4- to 6-week-old male Sprague–Dawley (SD) rats were randomly divided into 3 groups, namely, the blank group, pVAX1-HA2-fimA control group, and pVAX1-HA2-fimA + CpG ODN experimental group. At the 1st, 2nd, and 4th weeks, the rats were immunized by intranasal instillation, and three groups of rats were sacrificed at the 7th week. After total RNA was extracted from the spleen, RNA-seq library construction and sequencing were performed. Genetic ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of differentially expressed genes (DEGs). The screened poor DEGs were transferred to the STRING database to establish a protein–protein interaction (PPI) network map. To study the effect of CPG ODN on periodontitis genetic vaccines, we used Cytoscape software to further draw the network map, and quantitative real-time PCR (qRT-PCR) was used to validation of key genes.Results: A total of 161 significantly differentially expressed genes were found between the CPG ODN group and the control group. GO analysis showed that the differentially expressed genes were mainly enriched in immune-related functions such as antibacterial humoral responses, defense responses to other organisms, and defense responses to bacteria. KEGG pathway analysis showed that the differentially expressed genes were mainly involved in Staphylococcus aureus infection, oxidative phosphorylation, spliceosome, and nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) receptor signaling pathways. The PPI network graph constructed with Cytoscape software contains 58 nodes and 155 edges. Based on GO, KEGG, and PPI network map, we screened five key genes, including Oas2, Irf 7, Np4, Defa11 and Camp. These candidate genes may play a key role in the immune mechanism of periodontitis gene vaccines. The verification of key genes by qRT–PCR showed that compared with the control group, the expression of the Oas2 and Irf7 genes in the experimental group was significantly upregulated, and the expression of the Np4, Defa11 and Camp genes was significantly downregulated.Conclusions: The results were consistent with the transcriptome sequencing analysis of the preventive effect of periodontitis gene vaccine based on transcriptome sequencing, and screened five key genes involved in the synergistic effect of CPG ODN adjuvant and periodontitis gene vaccine, including Oas2, Irf7, Np4, Defa11 and Camp, to understand the immune mechanism of periodontitis gene vaccine at the genetic level.