Long-term generation of antiPCSK9 antibody using a nanoliposome-based vaccine delivery system

Background and aims: Proprotein convertase subtilisin kexin type 9 (PCSK9) is a liver secretory enzyme that controls plasma low-density lipoprotein cholesterol (LDL-C) levels through modulation of LDL receptor (LDLR). Inhibition of PCSK9 using monoclonal antibodies (mAbs) can efficiently lower plasma LDL-C. However, the relatively short half-life of mAbs necessitates frequent passive immunization, which is costly. These limitations can be circumvented by active immunization. Here, we evaluated the long-term antiPCSK9 antibody generation in BALB/c mice vaccinated with a nanoliposomal PCSK9-specific active vaccine. Methods: Negatively charged nanoliposomes were used as a vaccine delivery system and prepared via lipid-film hydration method. We constructed a peptide vaccine termed Immunogenic Fused PCSK9-Tetanus (IFPT) by linking a short PCSK9 peptide (as B cell epitope) to a tetanus peptide (as T cell epitope). The IFPT peptide was conjugated to the surface of nanoliposome carriers using a DSPE-PEG-Maleimide (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(PEG)-2000]) linker. Nanoliposomal IFPT (L-IFPT) construct was formulated with alum vaccine adjuvant (L-IFPTA(+)). To evaluate induction of antiPCSK9 antibody in vivo, BALB/c mice were subcutaneously inoculated four times in bi-weekly intervals with prepared vaccine formulations, including L-IFPT, L-IFPTA(+), IFPTA(+), IFPT, and empty liposomes as negative control. The long-term efficacy of antiPCSK9 antibodies was evaluated over 48 weeks after prime inoculation. Specificity of generated antiPCSK9 antibodies was assessed using ELISA method. To evaluate immunogenic safety, production of IL-4 and IFN-gamma, and population of CD8(+) and CD4(+) T cells in splenic cells isolated from the vaccinated mice were analyzed. Results: The L-IFPTA(+) vaccine was found to elicit the highest IgG antibody response against PCSK9 peptide in the vaccinated mice, when compared with the other vaccine formulations. Antibody titer analyses over 48 weeks post-prime vaccination revealed that the L-IFPTA(+) vaccine was able to stimulate a long-lasting humoral immune response against PCSK9 peptide, and thereby decrease plasma PCSK9. Generated antibodies could specifically target PCSK9 and thereby inhibit PCSK9-LDLR interaction. Analysis of splenic cells showed that the population of anti-inflammatory CD4(+) Th2 cells and production and secretion of IL-4 cytokine were increased in mice vaccinated with the L-IFPTA(+) vaccine, while population of inflammatory CD4(+) Th1 cell and cytotoxic CD8(+) T cells as well as production and secretion of IFN-gamma were not altered. Conclusions: The results indicate efficient activity of the tested nanoliposomal construct (L-IFPTA(+)) to induce humoral immune response against PCSK9 in BALB/c mice. L-IFPTA(+) vaccine can induce immunogenic-safe and long-term generation of antiPCSK9 antibodies in BALB/c mice.