Background
Atherosclerotic cardiovascular disease (ASCVD) is the main cause of morbidity and mortality worldwide. ASCVD slowly progresses over the lifetime and becomes clinically manifest after several decades [
1]. Active vaccination through the administration of immunogenic antigens to elicit an individual’s immune system to generate a humoral immune response is a highly effective and long-lasting method that could be used for preventing or treating ASCVD, though clinical evidence is lacking at the moment. However, passive immunotherapies based on monoclonal antibodies (mAbs) have improved the treatment of many chronic diseases including ASCVD [
2,
3].
Low-density lipoprotein cholesterol (LDL-C) exerts a causal effect on atherosclerosis initiation and progression [
1]. Various LDL-lowering methods, particularly statin therapy, that decrease LDL-C through upregulation of the LDL receptor (LDLR) have been revealed to decrease the risk of ASCVD events proportional to the absolute reduction of LDL-C in numerous randomized trials [
1]. Although statin therapy is the most commonly used approach to treat hypercholesterolemia, there might be a relatively large number of patients that are statin-resistant or statin-intolerant and unable to achieve optimal LDL-C levels despite intensive statin therapy [
4,
5].
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an important LDLR regulator that is involved in the progression of autosomal dominant hypercholesterolemia (ADH) [
6]. Based on the recent mechanistic, genetic, and clinical evidence, PCSK9 has been identified as an important regulator of plasma LDL-C and as a therapeutic target [
7‐
9]. PCSK9 mAbs, in combination with statins and/or ezetimibe, effectively decrease LDL-C by up to 50–60% and have now been shown to decrease the risk of ASCVD events proportional to the absolute reduction in LDL-C in two large-scale outcomes trials [
3,
10,
11]. However, due to their relatively short in vivo half-life leading to frequent administration and high-cost, long-term clinical application of mAbs can be undesirable for many patients. Of note, growing evidence is emerging to suggest that the causal relationship of LDL-C with the risk of atherosclerosis is influenced by both the absolute magnitude and the total time of exposure to LDL-C [
1]. Importantly, as arisen from Mendelian randomization studies, long-term exposure to LDL-lowering therapy beginning in early adulthood is correlated with up to a three-fold higher reduction in the risk of ASCVD events per unit change in LDL-C when compared with short-term therapy with a statin primed later in life [
12]. Thus, initiation of LDL-lowering therapy earlier in life may inhibit or slow the development of atherosclerotic lesions and therefore could be an effective strategy to decrease the lifetime risk of ASCVD events. For this reason, the shift to primeval and long-lasting prevention of the atherosclerotic lesion formation by reducing LDL-C earlier in life will need new approaches that are safe, specific, well-tolerated, and easy to use [
13].
Therefore, an active vaccination targeted against circulating PCSK9 if given in early adulthood before developing mature atherosclerotic lesions has the potential to decrease lifetime exposure to plasma LDL-C and may consequently result in potentially strong reductions in the lifetime risk of ASCVD events. A number of PCSK9-based vaccine strategies for reducing LDL-C are under investigation in preclinical or clinical phases [
14‐
17]. Different approaches have in common that peptide vaccines can be chemically synthesized or produced by recombinant strategies. Hence, anti-PCSK9 vaccines can be produced at a much lower cost compared with the anti-PCSK9 antibodies that are required to be administrated regularly.
However, the peptide antigens generally show low immunogenicity and, therefore, need to be improved. Adjuvant systems are the effective approach commonly used in vaccine development for enhancing the immunogenicity of the peptide antigens. The peptide-based anti-PCSK9 vaccines prepared by other research groups contained adjuvants such as keyhole limpet hemocyanin (KLH) [
18], DNA [
14], and virus-like particles [
16], which strongly increased the immunogenicity, though there are some concerns regarding their safety and efficiency in human.
An appropriate strategy to elicit strong antibody responses against self-antigens is to display them in a repetitive and highly dense format [
19,
20]. Liposome nanoparticles are safe bilayer spherical vesicles that have been frequently used as adjuvant delivery systems in vaccine formulations. They are a feasible carrier for displaying antigens in peptide-based vaccines [
21]. Our previous study showed that negatively charged nanoliposomes as an adjuvant delivery system could efficiently provoke a humoral immune response against exposed antigens. It was found that vaccines containing PCSK9 peptide exposed on the nanoliposome surface could induce long-term, strong, safe, and specific antibodies against PCSK9 in BALB/c mice [
22]. Thus, the present study was aimed to evaluate the long-term therapeutic effect of nanoliposome-displayed PCSK9 as an alternative peptide-based vaccine for the treatment of hypercholesterolemia and atherosclerosis in C57BL/6 mice.
Methods
Construction and characterization of liposomal vaccine
The present studied liposomal vaccine engaging a peptide construct with immunogenic properties displayed on the surface of nanoliposome particles was fabricated as described in the following sections and as detailed in our previous report [
22].
Preparation of liposome nanoparticles
To prepare nanoliposomes, cholesterol (Chol), 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), and 1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol (DMPG) (Avanti Polar Lipid; Alabaster, USA) lipids were dissolved in chloroform at the molar ratios of 15:10:75, respectively. Lipid solution was dried to a thin lipid film under reduced pressure using rotary evaporation (Heidolph, Germany). The prepared lipid film was then freeze-dried (VD-800F, Taitech, Japan) overnight to completely remove the solvent. Subsequently, the dried lipids were hydrated with 10 mM HEPES buffer (pH 7.2) containing 5% dextrose, and vortexed and bath-sonicated to disperse completely in the buffer. To obtain small unilamellar vesicles (SUVs) with a uniform size of 100–200 nm, the multilamellar vesicles (MLVs) were sequentially extruded using a mini extruder (Avestin, Canada) with polycarbonate membranes of 600, 400, 200, and 100 nm pore size, respectively. Physical properties of the prepared nanoliposomes, including particle size (diameter, nm), polydispersity index (PDI), and surface charge of the nanoliposomal formulation, were determined using dynamic light scattering (DLS) technique on a Zetasizer (Nano-ZS, Malvern, UK) at the room temperature (RT). The morphology and structure of the manufactured nanoliposomes were also visualized using a Philips CM10 transmission electron microscope (TEM).
Assembling of immunogenic peptide
A PCSK9 epitope, inspired the peptide sequence from the AFFiRiS group [
18], was selected to induce antibodies targeting plasma circulating PCSK9. The selected peptide provides a B cell epitope, originally designed using
AFFITOME® technology [
23], that mimics an N-terminal sequence of the native PCSK9 bound to LDLR, and thereby can induce anti-PCSK9 antibodies inhibiting PCSK9/LDLR interaction. Furthermore, to provide a T helper cell epitope increasing the response of CD4
+ T cells, a tetanus toxin peptide [
24] was embedded in the PCSK9 fragment using a 2-lysine-spacer sequence. The peptide construct embracing PCSK9 and tetanus epitopes called immunogenic fused PCSK9-tetanus (IFPT) peptide (Additional file
1: Table S1) with a purity grade of > 95% was synthesized by ChinaPeptides Co., Ltd. (Shanghai, China).
Manufacturing of nanoliposomal IFPT vaccine
To expose IFPT peptide on the surface of the prepared liposome nanoparticles, it was linked to DSPE-PEG-Mal (1,2-distearoyl-
sn-glycero-3-phosphoethanolamine-
N-[maleimide (PEG)-2000])
lipid (Lipoid GmbH, Germany) (Additional file
1: Figure S1). To approve the linkage of DSPE-PEG-Mal and IFPT peptide, a thin-layer chromatography (TLC) method was employed. Afterward, the DSPE-PEG-IFPT micelles were prepared using the solvent evaporation method followed by hydration. To quantify the peptide content of the micelles, high-performance liquid chromatography (HPLC) analysis (Knauer; Berlin, Germany) using an isocratic mobile phase of (0.1% TFA in water)/(0.1% TFA in acetonitrile) at gradient ratios of 55/45 to 45/55 in 10 min, at a flow rate of 1 mL min
−1, was operated. The efficiency of the linkage in the assembled DSPE-PEG-IFPT micelles was estimated by subtracting the HPLC-measured amount of free peptide within the micelles from the amount of the initially used IFPT peptide. The IFPT peptide was displayed on the surface of nanoliposome particles using the post-insertion method (Additional file
1: Figure S2), in which the prepared DSPE-PEG-IFPT micelles (100 μg, based on the linked peptide) and nanoliposome formulation (1 mL) were mixed and then gently shaken at 45 °C for 3 h. Morphological and physical properties of the prepared liposomal IFPT (L-IFPT) were determined using DLS and TEM, respectively, as employed for free nanoliposomes. To finalize the vaccine package, the prepared L-IFPT formulation was adsorbed to 0.4% alum adjuvant (Sigma-Aldrich) at a 1:1 (v/v) ratio and stored at 4 °C under argon.
Animal study
Twenty 6–8-week-old male C57BL/6 mice (18 ± 4 g) were purchased from the Laboratory Animal Research Center of Pasteur Institute of Tehran, Iran. All animal handling procedures were carried out in strict accordance with the Animal Welfare guidelines approved by the Institutional Ethics Committee and Research Advisory Committee of the Mashhad University of Medical Sciences, Mashhad, Iran. The animals were housed in an air-conditioned room at a constant temperature of 22 ± 2 °C with 12:12-h light/dark cycle and fed a standard rodent diet and water ad libitum. At the end of the study, all animals were euthanized by intravenous injection (30 mg/kg) of thiopental sodium [
25,
26].
Induction of atherosclerotic lesion
Poloxamer 407 (P-407) is a non-ionic surfactant that through modulating activity of several key enzymes implicated in lipid metabolism and transport, including inhibition of cholesterol 7α-hydroxylase (C7αH), lipoprotein lipase (LPL), and hepatic lipase (HL) as well as inducing activity of cholesteryl-ester-transfer-protein (CETP) and lecithin cholesterol acyltransferase (LCAT), can induce hyperlipidemia in rodents [
27,
28]. P-407 is also reported to decrease protein expression of liver LDLR [
29,
30] and, along with the simultaneous ingestion of atherogenic diet, promote atherosclerosis lesions in either sex of C57BL/6 mice, similar to those formed in humans [
31]. Here, to make a mouse model of atherosclerosis, 20 male 6–8-week-old C57BL/6 mice, weighting 18–23 g, were used. In order to induce atherosclerotic lesion, the animals fed with an atherogenic diet (containing 20% fat, 1.5% cholesterol, and 0.5% cholic acid) were simultaneously treated every third day with an intraperitoneal injection of P-407 (0.5 g kg
−1) for a time period of 16 weeks [
31]. After 16 weeks, to confirm atherosclerotic lesion development, the aortic arc region was isolated from 5 randomly selected mice.
Vaccination plan
To determine the therapeutic effect of the prepared vaccine on hypercholesterolemia and atherosclerosis development, the study design was planned in such a way that, initially, mouse model of atherosclerosis (described above) was developed and after that immunization schedule was primed (Additional file
1: Figure S3). Twenty male C57BL/6 mouse model of atherosclerosis were randomly and equally divided into two groups, including vaccine group and free nanoliposome (negative control) group. The vaccine group was immunized four times subcutaneously with L-IFPTA
+ vaccine (200 μL in both the right and left flanks) in bi-weekly intervals [a modified method taken from Galabova’s study [
18]]. Simultaneously, the negative control group received free nanoliposomes (200 μL) via the subcutaneous route, four times in bi-weekly intervals.
The time point at which the first inoculation was performed in order to induce anti-PCSK9 antibody refers to week 0 (W0) when it was simultaneous with 14 weeks of atherogenic regimen. Two weeks after the first immunization (W2), mice were put on a chow diet. Three more boosters were then carried out at W2, W4, and W6. To evaluate anti-PCSK9 antibody titers and lipid profile, the blood was withdrawn 2 weeks after each vaccination and at the pre-defined time points during 16 weeks after the first vaccination (Additional file
1: Figure S3).
Measuring the plasma level of anti-PCSK9 peptide antibody
Plasma samples isolated from vaccinated hypercholesterolemic and the negative control mice were assayed by the ELISA method to measure the titers of the induced antibodies against PCSK9 peptide. Briefly, a 96-well Nunc-MaxiSorp plate was coated with PCSK9 peptide solution at the concentration of 5 μg/mL in 0.1 M NaHCO3 buffer (pH 9.2–9.4), through an overnight incubation at 37 °C. Afterward, a blocking buffer (1× PBS, 1% BSA) was incubated on the coated plate for 1 h at 37 °C to bock free binding sites. Diluted plasma (1:400 in 1× PBS/0.1% BSA/0.05% Tween-20) was added, serially diluted 1:4, and incubated for 1 h at 37 °C. Each ELISA plate contained a standard antibody as the internal control. For the detection, biotinylated anti-mouse IgG (H+L) (Sigma-Aldrich; 1:1000) in 1× PBS/0.1% BSA/0.1% Tween-20 was incubated for 1 h at 37 °C. As the next step, horseradish peroxidase coupled to streptavidin (Sigma-Aldrich) was added (30 min, 37 °C) followed by the addition of the substrate TMB (Sigma-Aldrich) (15 min, RT). The optical density (OD) at 450 nm was measured with a microwell plate reader (BioTek, Synergy 2 plate reader, VT, USA), and the titers were defined as the dilution factor referring to 50% of the maximal optical density (ODmax/2). The mean titers ± SEM of all animals per group are presented.
Plasma PCSK9 quantification
Plasma PCSK9 levels of hypercholesterolemic vaccinated and the negative control mice were quantified by CircuLex mouse PCSK9 ELISA (CircuLexTM, Cy-8078, MBL, Woburn, MA) according to the manufacturer’s instructions. Briefly, 100 μL of the diluted 1:100 plasma samples was incubated on a 96-well microplate for 1 h at RT. An HRP-conjugated anti-PCSK9 antibody was incubated for 1 h followed by the substrate reagent and stop solution, all at RT. OD was measured at 450 nm with the microwell plate reader. Finally, to determine the PCSK9 level, a standard curve provided by the supplier was defined.
Evaluation of mouse PCSK9 targeting
To assess targeting of mouse PCSK9 by vaccine-generated antibodies, the interaction of plasma antibodies with PCSK9 was determined. To this, a modified method of CircuLex mouse PCSK9 ELISA kit was employed. In brief, to detect PCSK9-bound plasma antibodies, HRP-conjugated anti-mouse IgG (H+L) (Sigma Aldrich; dilution 1:5000, incubated for 1 h at RT), instead of HRP-conjugated anti-PCSK9 antibody, was used. Subsequently, PCSK9-targeting plasma antibody of vaccinated mice was indicated using detected OD at 450 nm.
Inhibitory effect of the induced antibodies on PCSK9/LDLR interaction in vitro
The ability of vaccine-generated antibodies for inhibition of the PCSK9/LDLR interaction in vitro was evaluated using PCSK9-LDLR in vitro binding assay kit (CircuLex™, Cy-8150, MBL, Woburn, MA). The principle of the assay is based on the binding between recombinant His-tagged PCSK9 and recombinant epidermal growth factor-like repeat (EGF-A) domain of LDLR, which contains a binding site for PCSK9. A biotinylated anti-His tag monoclonal antibody (supplemented with the company) specifically targets recombinant His-tagged PCSK9 that has been trapped with recombinant EGF-A domain coated on the microplate surface. Therefore, when evaluating a candidate PCSK9 inhibitor, if it can target the EGF-A binding domain of PCSK9, it will decrease the reaction of a biotinylated anti-His tag monoclonal antibody with His-tagged PCSK9 and consequently decrease subsequent detected OD. Briefly, 100 μL/well of the plasma samples of vaccinated mice or vehicle control was added to a 96-well microplate pre-coated with a recombinant LDLR-EGF-A domain. Immediately after that, the reaction was initiated by adding a “His-tagged PCSK9” solution incubated for 2 h followed by adding a biotinylated anti-His tag monoclonal antibody for 1 h at RT. Then, HRP-conjugated streptavidin was coated for 1 h at RT followed by the substrate reagent and stop solution. In this method, the higher amount of PCSK9-LDLR interaction is associated with higher ELISA OD, in which at the presence of anti-PCSK9 antibody, this interaction is inhibited and consequently ELISA OD is decreased. A dose-response curve with appropriate serial dilutions of “His-tagged PCSK9 wild-type” solution was drawn to measure accurate inhibition percentage of test samples.
Plasma lipid quantification
Peripheral blood was collected, and total cholesterol (TC), direct LDL-C, very low-density lipoprotein cholesterol (VLDL-C), and triglyceride (TG) were measured using the Biosystems kits (Biosystems S.A., Barcelona, Spain).
Histological assessment of atherosclerosis
At the end of the study, mice were euthanized and atherosclerotic lesion size and severity were assessed in the aortic arc area. Briefly, the aortic arc identified by the appearance of aortic valve leaflets was cut and immersion-fixed in 10% buffered formalin. The formalin-preserved tissue was gradually dehydrated, embedded in paraffin, and serially cut into sections with 5 μm thickness and intervals of 50 μm. Then, the slides were de-paraffinized in p-xylene and rehydrated in ratios of ethanol (100, 80, 70, and 50%) and rinsed in water. To stain the histopathology slides, hematoxylin and eosin (H&E) staining was used. Briefly, slides were stained in hematoxylin for 5 min and rinsed with water. Afterward, the slides were counterstained in eosin and mounted in distyrene, tricresyl phosphate, and xylene (DPX). For the assessment of atherosclerotic lesion thickness and severity, the lesions were classified into an arbitrary scale of 1–4 [
32]:
trace, minimal thickening of subintima with little injury to arterial endothelium (early fatty streak);
grade 1, plaque containing foam cells less than half as thick as the media with some form of endothelial dysfunction (regular fatty streak);
grade 2, plaque at least half as thick as the media with accumulation of intracellular lipid, macrophages, and smooth muscle cells (mild plaque);
grade 3, plaque as thick as the media with an abundance of macrophages, smooth muscle cells, and connective tissue (moderate plaque); and
grade 4, plaque thicker than the media with evidence of large extracellular intimal lipid core, foam cells, and calcification in the lipid core (severe plaque). The lesions were checked out using light microscopy (Olympus BX 51 microscope) supplied with a digital camera under × 400 magnifications. The lesion area was analyzed with ImageJ software [
33].
Western blot analysis of liver LDLR
To evaluate the protein expression of mouse liver LDLR, western blot analysis and immunohistochemical staining were performed. Liver crude protein was extracted using RIPA lysis buffer 500 μL per mg tissue (Sigma-Aldrich) with sodium orthovanadate, phenylmethylsulfonyl fluoride, and proteinase inhibitors. SDS-PAGE 10% gel was loaded with 50 μg protein per lane. Blotted proteins were developed using an HRP-conjugated anti-LDLR goat IgG (R and D Systems, Minneapolis, MI) diluted in primary antibody diluent buffer. HRP-conjugated anti-tubulin (Sigma, St. Louis, MO) mouse mAb was used to normalize data. Bands were visualized by Gel Documentation System (Bio-Rad) and quantified by ImageJ software.
Immunohistochemical staining for liver LDLR
Mouse liver sections were fixed in 10% buffered formalin and embedded in paraffin. Sections of 10 μL were obtained using a microtome, cleared in xylol, and rehydrated. The unmasking procedure was performed by incubating the samples in an antigen retrieval solution (EDTA buffer; pH 9.0) at 99 °C for 40 min. After rinsing in PBS, the sections were incubated with blocking solution (15 μL goat serum in 1 mL PBS) for 30 min at RT and then without an additional rinse, immediately incubated with rabbit monoclonal anti-LDLR antibody (Abcam Inc., Cambridge, UK) at the dilution of 1:500 for 1 h at RT. The samples were then rinsed in PBS and incubated with goat anti-rabbit HRP-conjugated IgG antibody (Sigma-Aldrich Inc., St. Louis, MO) at the dilution of 1:200 for additional 60 min. To counterstain the samples, the nuclei were stained with hematoxylin. The sections were dehydrated and mounted with Entellan (Merck KGaA, Darmstadt, Germany).
Evaluation of inflammatory response using ELISpot method
At the end of the study, the vaccinated hypercholesterolemic mice, non-vaccinated hypercholesterolemic mice, and naive mice (five animals per group) were sacrificed and their splenocytes were aseptically isolated, homogenized, and then passed through a 70-μm cell strainer (BD Falcon). The red blood cells (RBCs) were lysed using ACK buffer (0.15 M NH4Cl, 1.0 M KHCO3, 0.1 mM Na2EDTA), and the RBC-depleted splenocytes were counted using trypan blue (Gibco). INF-γ and IL-10 cytokines were assayed using the Mouse INF-γ and IL-10 ELISpot Kit (Mabtech), respectively, according to the manufacturer’s protocol. Briefly, 100 μL/well of either 15 μg/mL anti-INF-γ or anti-IL-10 antibody solution (diluted in sterile PBS pH 7.4) was coated on 96-well PVDF plates (Millipore Corp.) through an overnight incubation at 4 °C. To block non-specific binding, 200 μL/well blocking buffer containing RPMI medium (Gibco) supplemented with 10% fetal calf serum (FCS) (Gibco) was incubated on the coated plates for 30 min at RT. The blocked plates were seeded with the isolated splenocytes (5 × 105 cells per well) and re-stimulated either with 20 μg/mL phytohemagglutinin (PHA) (Sigma-Aldrich) or supplemented RPMI medium and incubated for 24 h at 37 °C. Eventually, spot-forming cells (SFC) were detected according to the manufacturer’s guidelines, and the amount of INF-γ- and IL-10-producing cells was quantified by counting the number of spots per well using Kodak 1D image analysis software (Version3.5, Eastman Kodak, Rochester, NY). Values were expressed as the number (median-interquartile range) of anti-INF-γ and/or anti-IL-10 SFC per 106 cells.
Statistical analysis
To determine the significance of the difference among the groups, unpaired two-tailed Student’s t test and one-way ANOVA followed by Tukey’s post hoc test were employed (GraphPad Prism software, version 7, San Diego, CA). Data with p < 0.05 were considered to be statistically significant.
Discussion
mAb-based PCSK9 inhibitors can effectively lower high levels of LDL-C but their long-term clinical application necessitates frequent administration and high cost [
34]. To subdue such limitation, active vaccination with long-lasting effects can be a safe alternative to passive immunotherapy of hypercholesterolemia [
35,
36]. Active vaccination for PCSK9 inhibition has been investigated by different approaches. AFFiRiS group developed an AFFITOPE®-based anti-PCSK9 active vaccination approach using a PCSK9 peptide linked to KLH carrier, which reduced TC by 53% [
18] and was associated with the reduction of size and severity of atherosclerotic lesion in vaccinated compared with control mice [
17]. However, the results of phase I clinical trial revealed that the mentioned vaccine failed to mimic similar cholesterol-lowering effects in humans, as Bauer et al. reported a 13% reduction in cholesterol level of vaccinated subjects (Bauer et al., Communication at ESC Congress, 2018 Munich; unpublished data). This finding highlights the need for further development of the introduced PCSK9 vaccine. We are the first to exhibit anti-PCSK9 vaccine with a nanoliposomal adjuvant carrier (L-IFPTA
+ vaccine) that exerted long-lasting lipid-lowering effects associated with anti-atherosclerotic effects in C57BL/6 mice receiving a severe atherogenic regimen.
In the present developed atherosclerosis model, a considerable reduction of plasma lipids occurred after stopping the atherogenic regimen. Therefore, to detect the net lipid-lowering effect of L-IFPTA
+ vaccine, we included a negative control (free nanoliposomes or phosphate-buffered saline) without effects on plasma lipids as verified in a pilot study (Additional file
1: Figure S9). Hence, the reduced level of plasma lipids in the negative control group (Figs.
2 and
4) is due to the atherogenic diet discontinuation and not an effect of free nanoliposomes.
L-IFPTA
+ vaccine-induced functional antibodies that specifically bound to circulating PCSK9 and inhibited its interaction with LDLR and thereby increased expression of LDLR on the surface of liver cells leading to significant reductions in TC and (V)LDL-C accompanied by a reduction in the atherosclerotic lesion size in the aortic arch of hypercholesterolemic mice. Upon four vaccinations with L-IFPTA
+, plasma antibody titer reached peak levels, at which plasma levels of PCSK9 were significantly decreased by 58.5% in the vaccinated hypercholesterolemic mice compared with hypercholesterolemic mice received free nanoliposomes (negative control). When antibody titers reached maximum levels (week 8), plasma LDL-C was found to be at the minimum level, which was associated with a 51.7% decrease in the plasma levels of LDL-C. The long-term study showed that during 16 weeks after prime vaccination, plasma LDL-C levels significantly decreased in vaccinated hypercholesterolemic mice, when compared with the negative control. At the end of the study (week 16), L-IFPTA
+ vaccine decreased LDL-C by 42% relative to the priming time point, showing a statistically significant negative correlation of antibody titers and LDL-C levels. Such a cholesterol-lowering effect was accompanied by the reduction of atherosclerotic lesion size (39.13%,
p = 0.016) and severity (46%,
p = 0.003) in the aortic arch of vaccinated hypercholesterolemic mice compared with the negative control. To the best of our knowledge, only one other group [
17] reported that active vaccination against PCSK9 could reduce atherosclerosis development in APOE*3Leiden.CETP mice; however, we showed the atheroprotective effect of L-IFPTA
+ in a different murine model of severe atherosclerosis. Regarding the intense induced hypercholesterolemia in our model, therapeutic effect of the present vaccine has the potential to be translated in patients with high levels of plasma cholesterol, suggesting its ideal benefit for future clinical use.
Furthermore, in another approach, Fattori et al. evaluated human recombinant PCSK9, together with a DNA oligonucleotide as an adjuvant, and reported ~ 40% reduction of LDL-C in mice [
14]. Pan et al. developed a human PCSK9 vaccine in which PCSK9 peptides were displayed at high valency on the surface of a bacteriophage virus-like particle, leading to nearly 20% reduction of TC relative to controls [
16]. However, it is questionable and unclear whether KLH [
18], DNA [
14], or virus-like particles [
16] used in the aforementioned studies can be safe adjuvants for clinical practices. Hence, besides the higher cholesterol-lowering effect of L-IFPTA
+ vaccine in comparison with other published PCSK9-targeting vaccines, it can rise important advantages of using safe adjuvant due to approved safety of liposome nanoparticles [
37], and thereby provide a clear outlook for clinical use.
On the other hand, inflammation is found to play a key role in atherosclerosis progression and complications. There is growing evidence showing the pro-atherogenic role for IFN-γ-producing cells [
38]. The pro-inflammatory IFN-γ cytokine could promote and exacerbate atherosclerosis via affecting the lipid accumulation and foam cell formation in the vascular wall and altering the cellular structure in the plaque, such as effects on SMC proliferation and macrophage activation [
39]. In contrast, IL-10-producing cells could counteract IFN-γ production [
40]. We showed that in the hypercholesterolemic control mice, IFN-γ-producing splenic cells were increased when compared with the naive mice on a chow diet. L-IFPTA
+ vaccine was found to significantly decrease IFN-γ-producing splenic cells, compared with controls. It was also revealed that IL-10-producing splenic cells were significantly increased in vaccinated versus control mice. Our finding is in accordance with the report of AFFiRiS group that showed PCSK9-targeting vaccine could ameliorate systemic and vascular inflammation in the aorta in APOE*3Leiden.CETP mice [
17]. The results can somewhat confirm the anti-inflammatory effect of L-IFPTA
+ vaccine that could have contributed to the observed anti-atherosclerotic effects; however, assessment of inflammatory cell distribution in plaque is needed to definitely verify this effect. Furthermore, both IFN-γ and IL-10 cytokines are validated markers for systemic inflammation [
41], and therefore, the present findings can provide the implications regarding the immunological safety of the L-IFPTA
+ vaccine.
While the present study is the first to report the anti-atherosclerotic and cholesterol-lowering effect of a nanoliposome-based PCSK9 peptide vaccine, a number of limitations need to be acknowledged to guide future studies. These limitations include the need for the verification of results in additional models of atherosclerosis including the transgenic models of atherosclerosis such as ldlr-, aopE-, or pcsk9-deficient mice as well as in rhesus monkeys. Besides, long-term evaluations are also encouraged to provide more in-depth information on the impact of the introduced vaccine on the size, morphology, and composition of atherosclerotic plaques. The impossibility of evaluating the effect of L-IFPTA+ vaccine on the other atherogenic plasma factors, such as lipoprotein a (Lp(a)), due to the absence of the mentioned factor in rodents was another limitation that calls for future testing in specific genetically engineered models. Finally, to provide more information on the effect of L-IFPTA+ vaccine on atherosclerosis plaques, staining of plaque lipids using Oil Red O method could be useful.
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