CYTOKINES PROFILE INDUCED IN PERIPHERAL BLOOD MONONUCLEAR CELLS OF PIG AFTER STIMULATION IN VITRO WITH DIFFERENT CPG-ODN

Congreso; LXXI REUNIÓN SAI; 2023

We developed an innovative strategy for formulating vaccine components, which involves the formulation of antigen and CpG-ODN with a nanostructure formed by self-assembly of 6-O-ascorbyl palmitate (Coa-ASC16). Previously, we demonstrated that mice immunized with the OVA/CpG-ODN/Coa-ASC16 nanoformulation elicited antigen-specific antibody and cellular responses superior to those induced by an aqueous solution of OVA and CpG-ODN. CpG-ODNs, due to their chemical structures and immunostimulatory effects, can be categorized into three classes: A, B, and C. Furthermore, the immunostimulatory activity triggered by CpG-ODNs varies among different species. Our goal is to validate the CpG-ODN/Coa-ASC16 adjuvant strategy in the porcine species.To identify which CpG-ODN elicits the best response in pigs, peripheral blood from Large White x Landrace pigs was collected from the jugular vein in vacutainer tubes containing 18mg of EDTA K2. Peripheral blood mononuclear cells (PBMC) were isolated through FICOLL gradient separation and incubated at 37°C in an environment with 5% CO2 for 12 hours with four different types of CpG-ODN: 2395, 1826, 1018, and 2007. Medium without CpG-ODN was employed as the control (baseline). The mRNA expression of cytokines and TLR9 was assessed through RT-qPCR. Our findings indicate that CpG-ODN 2395 (a class C CpG-ODN originally used for humans and mice) is capable of inducing a broad spectrum of innate immune response mediators, including IL-6, IL-12p40, TNFα, IFNβ, and IFNγ (p<0.05). In second place, CpG-ODN 1018 (a class B CpG-ODN employed in humans in HEPLISAV-B® vaccine and vaccines against SARS-CoV-2) stimulated the production of IFNβ and IFNγ (p<0.05) but the levels of these cytokines were lower compared to those induced by CpG-ODN 2395 (p<0.05). CpG-ODN 1018 also stimulated the production of TNFα and the level of this cytokine was higher than that observed when PBMC were stimulated with CpG-ODN 2395 (p<0.05). Conversely, the other types of class B CpG-ODN evaluated (CpG-ODN 1826, widely used in mouse, and CpG-ODN 2007, utilized in experimental research with chickens, cattle, and pigs) did not elicit PBMC activation under the analyzed conditions. Significantly, PBMC stimulated by CpG-ODN 2359 and 1018 exhibited a significant increase in TLR9 mRNA expression compared to the baseline condition (p<0.05). In summary, our study reveals that only CpG-ODN 2359 and 1018 stimulate in vitro the innate immune response in PBMC, albeit with a differential profile of cytokines. Based on these findings, we conclude that CpG-ODN 2395 is the most suitable candidate for formulation with Coa-ASC16 and should be investigated further in vivo as a vaccine adjuvant in pigs.