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

GIULIANA VEDELAGO; LUZ MARÍA PALACIOS; FEDERICO DANIEL RUIZ MORENO; CONSTANZA MARIN; SANTIAGO PALMA; GABRIEL MORÓN; FABRICIO ALUSTIZA; MARÍA INÉS CRESPO; BELKYS A. MALETTO

San Luis

Congreso; LXXI Reunión de la Sociedad Argentina de Inmunología; 2023

Sociedad Argentina de Inmunología

We developed an innovative strategy forformulating vaccine components, which involves the formulation of antigen andCpG-ODN with a nanostructure formed by self-assembly of 6-O-ascorbyl palmitate(Coa-ASC16). Previously, we demonstrated that mice immunized with theOVA/CpG-ODN/Coa-ASC16 nanoformulation elicited antigen-specific antibody andcellular 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 becategorized into three classes: A, B, and C. Furthermore, the immunostimulatoryactivity triggered by CpG-ODNs varies among different species. Our goal is tovalidate the CpG-ODN/Coa-ASC16 adjuvant strategy in the porcine species.Toidentify which CpG-ODN elicits the best response in pigs, peripheral blood fromLarge White x Landrace pigs was collected from the jugular vein in vacutainer tubes containing18mg of EDTA K2. Peripheral blood mononuclear cells (PBMC) were isolatedthrough FICOLL gradient separation and incubated at 37°C in an environment with5% 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 throughRT-qPCR. Our findings indicate that CpG-ODN 2395 (a class C CpG-ODN originallyused for humans and mice) is capable of inducing a broad spectrum of innateimmune 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 andvaccines against SARS-CoV-2) stimulated the production of  IFNβ and IFNγ (p<0.05) but the levels ofthese 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 levelof this cytokine was higher than that observed when PBMC were stimulated withCpG-ODN 2395 (p<0.05). Conversely, the other types of class B CpG-ODNevaluated (CpG-ODN 1826, widely used in mouse, and CpG-ODN 2007, utilized inexperimental research with chickens, cattle, and pigs) did not elicit PBMCactivation under the analyzed conditions. Significantly, PBMC stimulated byCpG-ODN 2359 and 1018 exhibited a significant increase in TLR9 mRNA expressioncompared to the baseline condition (p<0.05). In summary, our study reveals that only CpG-ODN2359 and 1018 stimulate in vitro the innate immune response in PBMC, albeitwith a differential profile of cytokines. Based on these findings, we concludethat CpG-ODN 2395 is the most suitable candidate for formulation with Coa-ASC16and should be investigated further in vivo as a vaccine adjuvant in pigs.@font-face{font-family:"Cambria Math";panose-1:2 4 5 3 5 4 6 3 2 4;mso-font-charset:0;mso-generic-font-family:roman;mso-font-pitch:variable;mso-font-signature:-536870145 1107305727 0 0 415 0;}@font-face{font-family:Calibri;panose-1:2 15 5 2 2 2 4 3 2 4;mso-font-charset:0;mso-generic-font-family:swiss;mso-font-pitch:variable;mso-font-signature:-469750017 -1073732485 9 0 511 0;}p.MsoNormal, li.MsoNormal, div.MsoNormal{mso-style-unhide:no;mso-style-qformat:yes;mso-style-parent:"";margin-top:0cm;margin-right:0cm;margin-bottom:8.0pt;margin-left:0cm;line-height:107%;mso-pagination:widow-orphan;font-size:11.0pt;font-family:"Calibri",sans-serif;mso-fareast-font-family:Calibri;mso-ansi-language:EN-US;mso-fareast-language:EN-US;}.MsoChpDefault{mso-style-type:export-only;mso-default-props:yes;font-size:11.0pt;mso-ansi-font-size:11.0pt;mso-bidi-font-size:11.0pt;font-family:"Calibri",sans-serif;mso-ascii-font-family:Calibri;mso-fareast-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:Calibri;mso-font-kerning:0pt;mso-ligatures:none;mso-ansi-language:EN-US;mso-fareast-language:EN-US;}.MsoPapDefault{mso-style-type:export-only;margin-bottom:8.0pt;line-height:107%;}div.WordSection1{page:WordSection1;}