A virus-like particle-based bivalent PCSK9 vaccine lowers LDL-cholesterol levels in non-human primates
Abstract
Elevated low-density lipoprotein cholesterol (LDL-C) is an important risk factor in the development of atherosclerotic cardiovascular disease (ASCVD).
Inhibitors of proprotein convertase subtilisin/kexin type 9 (PCSK9), a negative regulator of LDL-C metabolism, have emerged as promising approaches for reducing elevated LDL-C levels.
Here, we evaluated the cholesterol-lowering efficacy of virus-like particle (VLP) based vaccines that target epitopes found within the LDL receptor (LDL-R) binding domain of PCSK9.
In both mice and non-human primates, a bivalent VLP vaccine targeting two distinct epitopes on PCSK9 elicited strong and durable antibody responses and lowered cholesterol levels.
In macaques, a VLP vaccine targeting a single PCSK9 epitope was only effective at lowering LDL-C levels in combination with statins, whereas immunization with the bivalent vaccine lowered LDL-C without requiring statin co-administration. These data highlight the efficacy of an alternative, vaccine-based approach for lowering LDL-C.
Introduction
Cardiovascular disease (CVD) is the leading cause of global mortality, responsible for ~19 million deaths in 20201. A major risk factor for atherosclerotic cardiovascular disease (ASCVD) is elevated plasma levels of low-density lipoprotein cholesterol (LDL-C)2,3. Correspondingly, reducing levels of circulating LDL-C can lower the risk of ASCVD4,5. Statins are the most commonly prescribed medication for lowering LDL-C levels. Statins, which inhibit 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase, an enzyme in the cholesterol biosynthetic pathway, can decrease both LDL-C levels and the risk of cardiovascular events6, but their effectiveness in lowering LDL-C varies amongst individuals. As many as 20% of patients are hypo-responsive to statin use7,8. Although statins are generally well-tolerated, their use can be associated with serious adverse effects, including myopathy and liver toxicity9. These limitations have prompted the development of non-statin lipid-lowering therapies that target other pathways involved in LDL-C metabolism10.
LDL-C is removed from circulation by the low-density lipoprotein receptor (LDL-R), which is most abundantly expressed in the liver. Once LDL-C interacts with LDL-R the complex is endocytosed. The low pH of the endosome causes the complex to dissociate, allowing LDL-C to be metabolized by the cell and LDL-R to be recycled back to the plasma membrane where it can continue to remove circulating LDL-C. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serum-associated secretory protein that directly inhibits the recycling of LDL-R. PCSK9 binds to the extracellular domain of LDL-R and this strong interaction mediates its lysosomal degradation upon endocytosis11. Thus, by inhibiting LDL-R recycling, PCSK9 mediates higher levels of circulating LDL-C. Genetic mutations that affect PCSK9 activity can have profound effects on LDL-C levels. Patients with gain of function (GOF) mutations that increase the affinity of PCSK9 for LDL-R can develop autosomal dominant hypercholesterolemia (ADH); these patients exhibit high levels of LDL-C and early onset ASCVD12. Conversely, patients with loss of function (LOF) mutations in PCSK9 exhibit low LDL-C levels and a decreased risk of cardiovascular events13,14. Importantly, human LOF mutations are not associated with any adverse consequences. Thus, PCSK9 has become a major therapeutic target for lowering circulating LDL-C and preventing ASCVD.
Several different strategies for inhibiting PCSK9 activity have been developed, including monoclonal antibodies (mAbs), small interfering RNA (siRNA), macrocyclic peptides, base editing, and vaccines15. Currently, there are three FDA-approved PCSK9 therapies that are effective at reducing LDL-C levels in combination with statins. Evolocumab (Repatha®) and alirocumab (Praluent®) are anti-PCSK9 mAbs that lower LDL-C levels by as much as 50-60%16,17,18. Inclisiran (Laqvio®) consists of liposomes encapsidating an siRNA cargo that prevents production of PCSK9 in the liver. Inclisiran is similarly effective to mAbs, reducing LDL-C by 40-60%19, but unlike mAbs, which need to be administered every 2–4 weeks, Inclisiran has long-lasting effects and only needs to be administered twice a year. However, both mAb- and siRNA-based PCSK9 inhibitors are expensive; alirocumab and evolocumab cost approximately US$6000 annually and inclisirin costs US$3250 per dose20, leading some to question the cost effectiveness of these drugs21,22,23. Because of their expense, anti-PCSK9 therapies are mostly used as secondary prevention in patients who do not achieve sufficiently low LDL-C levels using statins alone24.
Vaccines are another promising approach for modulating PCSK9 activity. Vaccines have several potential advantages over other therapeutic approaches; they are relatively inexpensive to produce, which could reduce patient costs, and they will likely require fewer doses, potentially increasing patient compliance. Although the immunological mechanisms of self-tolerance normally restrict the ability to induce antibody responses against self-antigens such as PCSK9, these mechanisms can be efficiently overcome by displaying self-antigens at high density on the surface of nanoparticle-based vaccine platforms, such as virus-like particles (VLPs)25,26,27. VLP-based vaccines targeting self-antigens have been evaluated in human clinical trials, and this approach has been shown to be safe and able to induce high titer antibody responses28,29. In previous work, we engineered VLP-based vaccines that displayed different linear peptides from human PCSK9 that were predicted to interact with LDL-R30. We identified several vaccine candidates that induced high titer anti-PCSK9 antibody responses and reduced total cholesterol levels in immunized mice. In this study, we have engineered and combined VLP-based vaccines that display two species-specific linear peptides from PCSK9 to test their efficacy to induce anti-PCSK9 antibody responses and reduce cholesterol levels in multiple animal models. Our results support the use of a bivalent VLP-based vaccine targeting two epitopes of PCSK9 to effectively lower LDL-C levels without requiring co-administration of statins.
Results
Evaluating the immunogenicity and efficacy of single and bivalent PCSK9 vaccines in the LDLR+/− mouse model
We previously showed that VLP-based vaccines targeting linear epitopes from human PCSK9 (hPCSK9) elicit high-titer anti-PCSK9 antibodies and lower cholesterol levels in mice30. Although the amino acid sequences of hPCSK9 and mouse PCSK9 (mPCSK9) are highly conserved, there are several differences within the epitopes we targeted that potentially could affect the anti-PCSK9 activity of induced antibodies (Fig. 1). In addition, our original study did not assess the effectiveness of bivalent (combination) vaccines targeting multiple PCSK9 epitopes. To address these issues, we produced VLPs displaying mPCSK9 peptides that correspond to the hPCSK9 epitopes that, when displayed on VLPs, most potently decreased cholesterol levels in Balb/c mice30. These epitopes (amino acids 153–163 and 207–223) are located on the face of PCSK9 that is involved in LDL-R binding (Fig. 1). Peptides representing mPCSK9153–163 and mPCSK9207–223 were synthesized and then conjugated onto Qß bacteriophage VLPs using a bifunctional chemical cross-linker. Each peptide was displayed on VLPs at high valency, ~360 peptides per VLP (data not shown).
To evaluate the immunogenicity and cholesterol-lowering activity of mPCSK9-targeted vaccines, we utilized mice heterozygous for the Ldlrtm1Her mutation which express low levels of functional LDL-R and have elevated serum cholesterol31. We had evaluated vaccines targeting the homologous epitopes from hPCSK9 previously30; to confirm that VLPs displaying mPCSK9 epitopes were similarly able to elicit anti-PCSK9 antibody responses, a small group of LDLR+/− mice (n = 6) were immunized three times with 5 µg doses of mPCSK9153-163 VLPs, mPCSK9207-223 VLPs, or, as a negative control, wild-type Qß VLPs. In addition, a larger group of mice (n = 18; with a similarly sized group of control mice) were immunized with a bivalent PCSK9 vaccine, which consisted of a mixture of 5 µg of mPCSK9153-163 VLPs and 5 µg of mPCSK9207-223 VLPs. The immunogenicity of each vaccine was evaluated by measuring antibody titers to the target PCSK9 peptides and to full-length recombinant mPCSK9. As is shown in Fig. 2, both individual and bivalent VLP-based vaccines elicited high titer IgG antibody responses against mPCSK9153-163, mPCSK9207-223, and full-length mPCSK9. Although the bivalent vaccine elicited slightly lower anti-peptide antibody titers than the individual vaccines that specifically targeted each epitope (Fig. 2a, b), it generated similarly strong anti-mPCSK9 IgG antibody titers (Fig. 2c).
To evaluate the cholesterol-lowering effects of the PCSK9 vaccines in the LDLR+/− mouse model, we measured the total cholesterol levels of vaccinated and control groups prior to the first immunization and three weeks following the third immunization.
As we previously reported in mice immunized with VLPs displaying an analogous hPCSK9 epitope30, mice immunized with mPCSK9207-223 VLPs had significantly reduced total cholesterol levels (~25% lower) relative to control mice immunized with Qß VLPs (Fig. 2d). The bivalent vaccine (Fig. 2e) also significantly lowered total cholesterol levels by a similar percentage. Although immunization with mPCSK9153-163 VLPs decreased total cholesterol levels relative to controls, this effect was not statistically significant.
Link: https://www.nature.com/articles/s41541-023-00743-6
Authors:
Alexandra Fowler, Koen K. A. Van Rompay, Maureen Sampson, Javier Leo, Jennifer K. Watanabe, Jodie L. Usachenko, Ramya Immareddy, Debbie M. Lovato, John T. Schiller, Alan T. Remaley & Bryce Chackerian
Date: Published: 28 September 2023
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