The Study Of A Therapeutic Peptide Vaccine Against PCSK9

Part? The Study on Screening of Anti-PCSK9 Peptide Epitope and Its Function of VaccineBackgroud and Objective: The proprotein convertase subtilisin 9?PCSK9?has received much attention since its discovery in 2003.it can bind to the low density lipoprotein receptor?LDLR?on the surface of hepatocytes,the latter which is degraded by the endoplasmic reticulum / lysosomes pathway and can not be recirculated to the cell membrane,reducing LDLR and increasing LDL-C,thus this makes PCSK9 as a new target for the treatment of hypercholesterolemia and even atherosclerotic cardiovascular disease.So far,various of PCSK9 inhibitors are emerging,including monoclonal antibodies?m Abs?,antisense oligonucleotides,vaccines and so on,especially m Abs,while owing to its expensive price,short half-life and other disadvantages that limit the clinical application.The vaccine against PCSK9 is particularly important because of its low dosage,low cost of preparation,and low economic burden in the treatment of chronic diseases.The purpose of this study was to screen out a number of different short peptide epitopes targeting PCSK9and couple the short peptide to the vector as vaccines,immunizing the animals,and then verify whether there is a reduction in low-density lipoprotein cholesterol?LDL-C?and / or total cholesterol?TC?,thus providing a new means for the treatment of hypercholesterolemia.Methods: First,we used the relevant website and analysis software aiming at PCSK9 protein structure and amino acid sequence to screen a variety of peptides that may become immune epitopes.Second,we used the vaccine preparation technology we have obtained,respectively,to couple the screened short peptides with the carrier Q?-VLP through the heterobifunctional cross-linking agent Sulfo-SMCC?Pierce,USA?and identified the vaccine conjugation rate by SDS-PAGE gel electrophoresis.Again,the verification of the vaccines' effect.The six-week-old male Balb/c as experimental animals,the vaccines immunized on the back of animals by multi-point subcutaneous injection.Anti-PCSK9-specific antibody titers were measured after immunization.Then we detected total cholesterol?TC?,low density lipoprotein cholesterol?LDL-C?,triglyceride?TG?,high-density lipoprotein cholesterol?HDL-C?regularly.In addition,we examined the expression of LDLR in liver tissue both by WB and q RT-PCR.Results: Fifteen short peptides were screened as immunophenotypes,whose number of amino acids ranged from seven to thirteen.Only part of them prepared into vaccine could produce high titers of Anti-PCSK9 antibody titers and maintain at a high level after 3-4immunization in animals.Additionally,one of the vaccines,PCSK9Q?-003 vaccine,reduced blood lipid levels in experimental animals,especially LDL-C and TC,but did not influence TC and HDL-C.Furthermore,PCSK9Q?-003 vaccine significantly increased the expression of LDLR in liver tissue.Conclusions: Among the fifteen short peptide-coupled vaccines,the PCSK9Q?-003 vaccine significantly produced higher and stable antibody titers in Balb/c animals and reduced the LDL-C and TC.Therefore,the PCSK9Q?-003 vaccine may become a new method for the treatment of hypercholesterolemia.Part? Functional Analysis of PCSK9Q?-003 Vaccine in LDLR^+/-MiceAims: In order to further validate the lipid-lowering effect of the PCSK9Q?-003 vaccine in LDLR +/-mouse.Meanwhile,we investigated whether the PCSK9Q?-003 vaccine could modulate cholesterol metabolism.Finally,we aimed to verify the safety of the PCSK9Q?-003 vaccine to provide a novel strategy for hypercholesterolemia therapy.Methods: Six-week-old,male LDLR +/-mice were randomly divideded into two groups,namely,the control group?control?and the vaccine group?V150-157?.PCSK9Q?-003-specific antibody titers were measured on days 14,28,42,56 and 77 by ELISA.For the assessment of blood lipid,TC,LDL-C,TG and HDL-C were detected respectively.We detected serum total PCSK9 and free PCSK9 by ELISA and the expression of LDLR in liver tissue through WB and q RT-PCR.To assess cholesterol metabolism,we measured the expression of SREBP-2,HNF-1?and HMG-Co A reductase.In addition,we also investigated the safety of PCSK9Q?-003 vaccine by HE,Masson and PAS staining in kidney,by flow cytometry in fresh spleen tissue to detect the ratio of Th1,Th2 and Th17 cells and by biochemical method in blood to detect creatinine and urea nitrogen.Results: PCSK9Q?-003 vaccine could stimulate LDLR+/-mice to produce high anti-PCSK9-specific antibody titers.Meanwhile,PCSK9Q?-003 vaccine distinguishingly reduced blood lipid,especially LDL-C and TC,but did not affect the TC and HDL-C.In addition,the combination of statin therapy could decrease TC more obviously.PCK9Q?-003 vaccine increased the total serum PCSK9 and oppositely reduced the free PCSK9,which was a advantage compared with statins.Besides,the expression of LDLR in liver tissue both in gene and protein level were significantly increased by PCSK9Q?-003 vaccine.Moreover,the vaccine could increase the expression of SREBP-2,HNF-1? and HMG-Co A reductase as well.More importantly,the vaccine was found to be safe because no abnormalities were observed in related tests,although further assessments are needed to confirm this statement.Conclusions: This experiment showed that PCSK9Q?-003 vaccine reduced LDL-C and TC in LDLR +/-mice,and increased the expression of LDLR in the liver,and affected cholesterol metabolism.What's more,PCSK9Q?-003 vaccine did not bring damage in kidney,spleen and other aspects.Therefore,we infered that PCSK9Q?-003 vaccine could be a new therapy for hypercholesterolemia and even atherosclerotic cardiovascular disease.