P6195Nanoliposomal anti-PCSK9 vaccine induces long-term and safe protection against atherosclerosis in C57BL/6 mouse

Abstract

Background Proprotein convertase subtilisin/kexin 9 (PCSK9) can increase plasma levels of low-density lipoprotein (LDL) cholesterol (LDL-C) through reducing protein levels of liver LDL receptor (LDLR). PCSK9 inhibition has been found to efficiently alleviate hypercholesterolemia. Purpose In the present study, we prepared nanoliposomal anti-PCSK9 vaccine and evaluated its protective effects against hypercholesterolemia and atherosclerotic plaque formation in C57BL/6 mice fed on atherogenic diet. Methods Nanoliposome formulation containing 1,2-Dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), 1,2-Dimyristoyl-sn-glycero-3-phosphorylglycerol (DMPG) and cholesterol (Chol) were prepared using the lipid-film hydration method, and used as the vaccine delivery system. Immunogenic peptide called Immunogenic Fused PCSK9-Tetanus (IFPT) was exhibited on the surface of nanoliposome carriers by using DSPE-PEG-Maleimide lipid (L-IFPT) and adsorbed to Alum adjuvant (L-IFPTA+). The prepared vaccine formulations IFPT, L-IFPTA+, IFPTA+, IFPT, and empty liposome as the negative control were subcutaneously administrated four times with a bi-weekly interval in C57BL/6 mice fed on atherogenic diet. Protein levels of liver LDLR, lipid profile, plasma levels of anti-PCSK9 antibody, plasma concentration of PCSK9 protein, and effect of anti-PCSK9 antibody on PCSK9-LDLR interaction were measured in the vaccinated mice. Aortic arch atherosclerotic lesions were studied using hematoxylin and eosin staining. To determine inflammatory response, splenic CD4+producing IL-4 and IFN-γ were evaluated using flow cytometry analysis. Results Among formulated vaccines, the L-IFPTA+vaccine could generate the highest IgG anti-PCSK9 antibody in the vaccinated hypercholesterolemic mice. The generated anti-PCSK9 antibodies inhibited interaction of PCSK9 with LDLR through targeting plasma PCSK9, whereby protein levels of liver LDLR was elevated in the vaccinated mice. As reveled by lipid profile analysis, liposomal vaccines, with more extent the L-IFPTA+vaccine, could significantly decrease plasma TC and LDL-C in hypercholesterolemic mice. Of note, L-IFPTA+ vaccine could reduce size and severity of atherosclerotic lesion in the aorta arch. Importantly, long-term (48 weeks) follow-up of hypercholesterolemic vaccinated mice revealed that L-IFPTA+vaccine could induce a long-lasting humoral immune response against plasma PCSK9, which was accompanied with a significant decrease of TC and LDL-C (Figure). Additionally, Anti-inflammatory Th2 cells and IL-4 cytokine were significantly elevated in splenic cells isolated from hypercholesterolemic vaccinated mice. Figure 1 Conclusions L-IFPTA+vaccine can promote long-lasting, functional and safe anti-PCSK9 antibodies in hypercholesterolemic C57BL/6 mice, revealing long-term protective effect against hypercholesterolemia and atherosclerosis.

Pre-Clinical Evaluation of the Nanoliposomal antiPCSK9 Vaccine in Healthy Non-Human Primates

“…Notably, despite the previously observed efficient therapeutic [22,26] and preventive [27] effects of the L-IFPTA vaccine for reducing hypercholesterolemia and atherosclerotic plaque progression in hyperlipidemic C57BL/6 mice, the present study showed a non-significant reduction in serum lipid indices in healthy monkeys. Of note, another anti-PCSK9 vaccine formulation prepared using virus-like particle technology was reported to decrease serum LDL-C in an approximately similar but significant percentage (by 10-15%) in healthy non-human primates [16].…”

“…These findings support our previous studies on BALB/c and C57BL/6 mice immunized by the L-IFPTA vaccine that showed similar results [ 21 , 22 , 26 , 27 ]. Of note, provoking antibody production in the two different species, rodents and non-human primates, is not surprising.…”

Impact of PCSK9 Immunization on Glycemic Indices in Diabetic Rats

“…The LDL-lowering effect of the liposomal anti-PCSK9 vaccine has been also found in our other preclinical studies where the preventive [ 37 , 38 ] and the therapeutic [ 39 , 40 ] effects in hypercholesterolemic mice as well as the safety of the vaccine in healthy nonhuman primates [ 49 ] were evaluated. Such an effect has been similarly reported by the studies using other PCSK9 inhibiting vaccines, including the AFFITOPE®-based anti-PCSK9 vaccine [ 50 , 51 ], a human recombinant protein-based anti-PCSK9 vaccine [ 52 ], and a vaccine comprised of virus-like particles displaying PCSK9 peptides [ 53 ].…”