Identification of Major Histocompatibility Complex Class II Epitopes From Lyme Autoantigen Apolipoprotein B-100 and Borrelia burgdorferi Mcp4 in Murine Lyme Arthritis.

BACKGROUND: During infection with the Lyme arthritis (LA) pathogen Borrelia burgdorferi, T-cell responses to both host and pathogen are dysregulated, resulting in chronic infection and frequent development of autoimmunity. METHODS: To assess CD4+ T-cell epitopes presented during development of LA, we used an unbiased, immunopeptidomics approach to characterize the major histocompatibility complex (MHC) class II immunopeptidome in B burgdorferi-infected C57BL/6 (B6) mice, which develop mild, self-limiting LA, and infected B6 Il10-/- mice, which develop severe, persistent LA at 0, 4, and 16 weeks postinfection (22-23 mice per group). RESULTS: Peptides derived from proteins involved in adaptive T- and B-cell responses and cholesterol metabolism, including human Lyme autoantigen apolipoprotein B-100 (apoB-100), were enriched in infected Il10-/- mice; whereas peptides derived from proteins involved in neutrophil extracellular net formation were enriched in infected B6 mice. Presentation of apoB-100 peptides showed evidence of epitope expansion during infection. Of several identified B burgdorferi peptides, only 1, a methyl-accepting chemotaxis protein peptide Mcp4442-462, was immunogenic. CONCLUSIONS: ApoB-100, a human Lyme autoantigen, undergoes marked epitope expansion during LA development. The paucity of immunogenic B burgdorferi epitopes supports previous findings suggesting CD4+ T-cell responses are suppressed in murine LA.

Contribution of intestinal triglyceride-rich lipoproteins to residual atherosclerotic cardiovascular disease risk in individuals with type 2 diabetes on statin therapy.

AIMS/HYPOTHESIS: This study explored the hypothesis that significant abnormalities in the metabolism of intestinally derived lipoproteins are present in individuals with type 2 diabetes on statin therapy. These abnormalities may contribute to residual CVD risk. METHODS: To investigate the kinetics of ApoB-48- and ApoB-100-containing lipoproteins, we performed a secondary analysis of 11 overweight/obese individuals with type 2 diabetes who were treated with lifestyle counselling and on a stable dose of metformin who were from an earlier clinical study, and compared these with 11 control participants frequency-matched for age, BMI and sex. Participants in both groups were on a similar statin regimen during the study. Stable isotope tracers were used to determine the kinetics of the following in response to a standard fat-rich meal: (1) apolipoprotein (Apo)B-48 in chylomicrons and VLDL; (2) ApoB-100 in VLDL, intermediate-density lipoprotein (IDL) and LDL; and (3) triglyceride (TG) in VLDL. RESULTS: The fasting lipid profile did not differ significantly between the two groups. Compared with control participants, in individuals with type 2 diabetes, chylomicron TG and ApoB-48 levels exhibited an approximately twofold higher response to the fat-rich meal, and a twofold higher increment was observed in ApoB-48 particles in the VLDL1 and VLDL2 density ranges (all p < 0.05). Again comparing control participants with individuals with type 2 diabetes, in the latter, total ApoB-48 production was 25% higher (556 ± 57 vs 446 ± 57 mg/day; p < 0.001), conversion (fractional transfer rate) of chylomicrons to VLDL was around 40% lower (35 ± 25 vs 82 ± 58 pools/day; p=0.034) and direct clearance of chylomicrons was 5.6-fold higher (5.6 ± 2.2 vs 1.0 ± 1.8 pools/day; p < 0.001). During the postprandial period, ApoB-48 particles accounted for a higher proportion of total VLDL in individuals with type 2 diabetes (44%) compared with control participants (25%), and these ApoB-48 VLDL particles exhibited a fivefold longer residence time in the circulation (p < 0.01). No between-group differences were seen in the kinetics of ApoB-100 and TG in VLDL, or in LDL ApoB-100 production, pool size and clearance rate. As compared with control participants, the IDL ApoB-100 pool in individuals with type 2 diabetes was higher due to increased conversion from VLDL2. CONCLUSIONS/INTERPRETATION: Abnormalities in the metabolism of intestinally derived ApoB-48-containing lipoproteins in individuals with type 2 diabetes on statins may help to explain the residual risk of CVD and may be suitable targets for interventions. TRIAL REGISTRATION: ClinicalTrials.gov NCT02948777.

Lipoprotein(a): A Genetically Determined, Causal, and Prevalent Risk Factor for Atherosclerotic Cardiovascular Disease: A Scientific Statement From the American Heart Association.

High levels of lipoprotein(a) [Lp(a)], an apoB100-containing lipoprotein, are an independent and causal risk factor for atherosclerotic cardiovascular diseases through mechanisms associated with increased atherogenesis, inflammation, and thrombosis. Lp(a) is predominantly a monogenic cardiovascular risk determinant, with ≈70% to ≥90% of interindividual heterogeneity in levels being genetically determined. The 2 major protein components of Lp(a) particles are apoB100 and apolipoprotein(a). Lp(a) remains a risk factor for cardiovascular disease development even in the setting of effective reduction of plasma low-density lipoprotein cholesterol and apoB100. Despite its demonstrated contribution to atherosclerotic cardiovascular disease burden, we presently lack standardization and harmonization of assays, universal guidelines for diagnosing and providing risk assessment, and targeted treatments to lower Lp(a). There is a clinical need to understand the genetic and biological basis for variation in Lp(a) levels and its relationship to disease in different ancestry groups. This scientific statement capitalizes on the expertise of a diverse basic science and clinical workgroup to highlight the history, biology, pathophysiology, and emerging clinical evidence in the Lp(a) field. Herein, we address key knowledge gaps and future directions required to mitigate the atherosclerotic cardiovascular disease risk attributable to elevated Lp(a) levels.

A New Structural Model of Apolipoprotein B100 Based on Computational Modeling and Cross Linking.

ApoB-100 is a member of a large lipid transfer protein superfamily and is one of the main apolipoproteins found on low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) particles. Despite its clinical significance for the development of cardiovascular disease, there is limited information on apoB-100 structure. We have developed a novel method based on the "divide and conquer" algorithm, using PSIPRED software, by dividing apoB-100 into five subunits and 11 domains. Models of each domain were prepared using I-TASSER, DEMO, RoseTTAFold, Phyre2, and MODELLER. Subsequently, we used disuccinimidyl sulfoxide (DSSO), a new mass spectrometry cleavable cross-linker, and the known position of disulfide bonds to experimentally validate each model. We obtained 65 unique DSSO cross-links, of which 87.5% were within a 26 Å threshold in the final model. We also evaluated the positions of cysteine residues involved in the eight known disulfide bonds in apoB-100, and each pair was measured within the expected 5.6 Å constraint. Finally, multiple domains were combined by applying constraints based on detected long-range DSSO cross-links to generate five subunits, which were subsequently merged to achieve an uninterrupted architecture for apoB-100 around a lipoprotein particle. Moreover, the dynamics of apoB-100 during particle size transitions was examined by comparing VLDL and LDL computational models and using experimental cross-linking data. In addition, the proposed model of receptor ligand binding of apoB-100 provides new insights into some of its functions.

Pathogenic Autoimmunity in Atherosclerosis Evolves From Initially Protective Apolipoprotein B100-Reactive CD4+ T-Regulatory Cells.

BACKGROUND: Throughout the inflammatory response that accompanies atherosclerosis, autoreactive CD4+ T-helper cells accumulate in the atherosclerotic plaque. Apolipoprotein B100 (apoB), the core protein of low-density lipoprotein, is an autoantigen that drives the generation of pathogenic T-helper type 1 (TH1) cells with proinflammatory cytokine secretion. Clinical data suggest the existence of apoB-specific CD4+ T cells with an atheroprotective, regulatory T cell (Treg) phenotype in healthy individuals. Yet, the function of apoB-reactive Tregs and their relationship with pathogenic TH1 cells remain unknown. METHODS: To interrogate the function of autoreactive CD4+ T cells in atherosclerosis, we used a novel tetramer of major histocompatibility complex II to track T cells reactive to the mouse self-peptide apo B978-993 (apoB+) at the single-cell level. RESULTS: We found that apoB+ T cells build an oligoclonal population in lymph nodes of healthy mice that exhibit a Treg-like transcriptome, although only 21% of all apoB+ T cells expressed the Treg transcription factor FoxP3 (Forkhead Box P3) protein as detected by flow cytometry. In single-cell RNA sequencing, apoB+ T cells formed several clusters with mixed TH signatures that suggested overlapping multilineage phenotypes with pro- and anti-inflammatory transcripts of TH1, T helper cell type 2 (TH2), and T helper cell type 17 (TH17), and of follicular-helper T cells. ApoB+ T cells were increased in mice and humans with atherosclerosis and progressively converted into pathogenic TH1/TH17-like cells with proinflammatory properties and only a residual Treg transcriptome. Plaque T cells that expanded during progression of atherosclerosis consistently showed a mixed TH1/TH17 phenotype in single-cell RNA sequencing. In addition, we observed a loss of FoxP3 in a fraction of apoB+ Tregs in lineage tracing of hyperlipidemic Apoe-/- mice. In adoptive transfer experiments, converting apoB+ Tregs failed to protect from atherosclerosis. CONCLUSIONS: Our results demonstrate an unexpected mixed phenotype of apoB-reactive autoimmune T cells in atherosclerosis and suggest an initially protective autoimmune response against apoB with a progressive derangement in clinical disease. These findings identify apoB autoreactive Tregs as a novel cellular target in atherosclerosis.

Riboflavin deficiency alters cholesterol homeostasis partly by reducing apolipoprotein B100 synthesis in HepG2 cells.

Riboflavin deficiency led to lower blood cholesterol level and higher content of hepatic cholesterol in rats and the mechanisms are not clarified yet. We hypothesized that riboflavin deficiency might alter cholesterol homeostasis via apolipoprotein B100, one of the important proteins in cholesterol transport. To test this hypothesis, HepG2 cells were cultured in riboflavin-deficient media for 4 days to develop riboflavin deficiency. Compared to riboflavin-sufficient cells, the mRNA (0. 37 ± 0.04 vs 1.03 ± 0.29 relative expression level, n = 3) and protein expressions of apolipoprotein B100 (intracellular: 173.7 ± 14.4 vs 254.8 ± 47.2 µg/mg protein; extracellular: 93.8 ± 31.1 vs 161.6 ± 23.9 µg/mg protein; n = 3) were significantly reduced in riboflavin-deficient cells (P < 0.05). Endoplasmic reticulum oxidoreductin 1 and protein disulfide isomerase, two enzymes involved in the oxidative folding of apolipoprotein B100, were also lower remarkably in expression at both mRNA and protein levels. Meanwhile, intracellular cholesterol was increased (256.3 ± 17.1 µM/g protein vs 181.4 ± 23.9 µM/g protein, n = 4) and extracellular cholesterol decreased (110.0 ± 23.2 µM/g protein vs 166.2 ± 34.6 µM/g protein, n = 4) significantly in riboflavin-deficient cells (P < 0.05). Very low-density lipoprotein was also diminished (29.0 ± 6.1 µM/g protein vs 67.0 ± 11.0 µM/g protein, n = 4) in the culture media (P < 0.05). These findings suggest that riboflavin deficiency alters cholesterol homeostasis partly by reducing apolipoprotein B100 synthesis in HepG2 cells.

Separation of postprandial lipoproteins: improved purification of chylomicrons using an ApoB100 immunoaffinity method.

Elevated levels of triglyceride-rich lipoproteins (TRLs), both fasting and postprandial, are associated with increased risk for atherosclerosis. However, guidelines for treatment are defined solely by fasting lipid levels, even though postprandial lipids may be more informative. In the postprandial state, circulating lipids consist of dietary fat transported from the intestine in chylomicrons (CMs; containing ApoB48) and fat transported from the liver in VLDL (containing ApoB100). Research into the roles of endogenous versus dietary fat has been hindered because of the difficulty in separating these particles by ultracentrifugation. CM fractions have considerable contamination from VLDL (purity, 10%). To separate CMs from VLDL, we produced polyclonal antibodies against ApoB100 and generated immunoaffinity columns. TRLs isolated by ultracentrifugation of plasma were applied to these columns, and highly purified CMs were collected (purity, 90-94%). Overall eight healthy unmedicated adult volunteers (BMI, 27.2 ± 1.4 kg/m2; fasting triacylglycerol, 102.6 ± 19.5 mg/dl) participated in a feeding study, which contained an oral stable-isotope tracer (1-13C acetate). We then used this technique on plasma samples freshly collected during an 8 h human feeding study from a subset of four subjects. We analyzed fractionated lipoproteins by Western blot, isolated and derivatized triacylglycerols, and calculated fractional de novo lipogenesis. The results demonstrated effective separation of postprandial lipoproteins and substantially improved purity compared with ultracentrifugation protocols, using the immunoaffinity method. This method can be used to better delineate the role of dietary sugar and fat on postprandial lipids in cardiovascular risk and explore the potential role of CM remnants in atherosclerosis.

Prevalence of poor lipid control in patients with premature coronary artery disease.

BACKGROUND AND AIMS: Lipid goals have become more stringent in high risk patients. However, no studies have analyzed lipid control defined as the composite achievement of goals in low-density lipoprotein cholesterol (LDL-C), non-high-density lipoprotein cholesterol (Non-HDL-C) and apolipoproteinB-100 (ApoB-100), in patients with premature coronary artery disease (CAD). We aimed to analyze lipid control rates, and the associated factors with its poor achievement in patients with premature CAD. METHODS AND RESULTS: The study included 1196 patients with CAD diagnosed before 55 and 65 years old in men and women, respectively. The American Heart Association/American College of Cardiology (non-strict) and the American Association of Clinical Endocrinologists (strict) criteria were used to analyze lipid control rates. Sociodemographic, dietary-healthy and clinical characteristics of the patients were collected. Participants were 54 ± 8 years old, 19.7% were women, and median CAD evolution was 2.4 years. Non-strict and strict lipid control was achieved in 23.0% and 8.9% of the patients, respectively. Moreover, 46.5% and 62.8% of the patients did not achieve any lipid goal using both criteria. Sociodemographic data were not different among patients who achieved or not lipid control. Treatment adherence<85%, prescription of low- and moderate-intensity statins, and obesity were consistently associated with poor lipid control. CONCLUSIONS: Lipid control is suboptimal in patients with premature CAD. Low lipid-lowering treatment adherence, low prescription of high-intensity statins, and obesity were independently associated with poor lipid control. Novel preventive programs and more aggressive pharmacological intervention should be implemented in order to reduce the burden of premature CAD.

A monoclonal antibody to assess oxidized cholesteryl esters associated with apoAI and apoB-100 lipoproteins in human plasma.

Atherosclerosis is associated with increased lipid peroxidation, leading to generation of multiple oxidation-specific epitopes (OSEs), contributing to the pathogenesis of atherosclerosis and its clinical manifestation. Oxidized cholesteryl esters (OxCEs) are a major class of OSEs found in human plasma and atherosclerotic tissue. To evaluate OxCEs as a candidate biomarker, we generated a novel mouse monoclonal Ab (mAb) specific to an OxCE modification of proteins. The mAb AG23 (IgG1) was raised in C57BL6 mice immunized with OxCE-modified keyhole limpet hemocyanin, and hybridomas were screened against OxCE-modified BSA. This method ensures mAb specificity to the OxCE modification, independent of a carrier protein. AG23 specifically stained human carotid artery atherosclerotic lesions. An ELISA method, with AG23 as a capture and either anti-apoAI or anti-apoB-100 as the detection Abs, was developed to assay apoAI and apoB-100 lipoproteins that have one or more OxCE epitopes. OxCE-apoA or OxCE-apoB did not correlate with the well-established oxidized phospholipid-apoB biomarker. In a cohort of subjects treated with atorvastatin, OxCE-apoA was significantly lower than in the placebo group, independent of the apoAI levels. These results suggest the potential diagnostic utility of a new biomarker assay to measure OxCE-modified lipoproteins in patients with CVD.

Low-Density Lipoprotein-Reactive T Cells Regulate Plasma Cholesterol Levels and Development of Atherosclerosis in Humanized Hypercholesterolemic Mice.

BACKGROUND: Atherosclerotic cardiovascular disease is a chronic inflammatory process initiated when cholesterol-carrying low-density lipoprotein (LDL) is retained in the arterial wall. CD4+ T cells, some of which recognize peptide components of LDL as antigen, are recruited to the forming lesion, resulting in T-cell activation. Although these T cells are thought to be proatherogenic, LDL immunization reduces disease in experimental animals. These seemingly contradictory findings have hampered the development of immune-based cardiovascular therapy. The present study was designed to clarify how activation of LDL-reactive T cells impacts on metabolism and vascular pathobiology. METHODS: We have developed a T-cell receptor-transgenic mouse model to characterize the effects of immune reactions against LDL. Through adoptive cell transfers and cross-breeding to hypercholesterolemic mice expressing the antigenic human LDL protein apolipoprotein B-100, we evaluate the effects on atherosclerosis. RESULTS: A subpopulation of LDL-reactive T cells survived clonal selection in the thymus, developed into T follicular helper cells in lymphoid tissues on antigen recognition, and promoted B-cell activation. This led to production of anti-LDL immunoglobulin G antibodies that enhanced LDL clearance through immune complex formation. Furthermore, the cellular immune response to LDL was associated with increased cholesterol excretion in feces and with reduced vascular inflammation. CONCLUSIONS: These data show that anti-LDL immunoreactivity evokes 3 atheroprotective mechanisms: antibody-dependent LDL clearance, increased cholesterol excretion, and reduced vascular inflammation.

A clinically applicable adjuvant for an atherosclerosis vaccine in mice.

Vaccination with MHC-II-restricted peptides from Apolipoprotein B (ApoB) with complete and incomplete Freund's adjuvant (CFA/IFA) is known to protect mice from atherosclerosis. This vaccination induces antigen-specific IgG1 and IgG2c antibody responses and a robust CD4 T cell response in lymph nodes. However, CFA/IFA cannot be used in humans. To find a clinically applicable adjuvant, we tested the effect of vaccinating Apoe-deficient mice with ApoB peptide P6 (TGAYSNASSTESASY). In a broad screening experiment, Addavax, a squalene-based oil-in-water adjuvant similar to MF59, was the only adjuvant that showed similar efficacy as CFA/IFA. This was confirmed in a confirmation experiment for both the aortic arch and whole aorta analyzed by en face analysis after atherosclerotic lesion staining. Mechanistically, restimulated peritoneal cells from mice immunized with P6 in Addavax released significant amounts of IL-10. Unlike P6 in CFA/IFA, vaccination with P6 in Addavax did not induce any detectable IgG1 or IgG2c antibodies to P6. These data suggest that squalene-based adjuvants such as MF59 are good candidate adjuvants for developing a clinically effective atherosclerosis vaccine.

[Indicators of lipid exchange as early markers of development calcific aortic valve disease.]

A comprehensive study of lipidograms of 36 patients with initial signs of calcification of aortic semilunium was performed. It was determined that the content of apolipoprotein A-1 was significantly lower in comparison with the control groups, which requires further studies of the evaluation of the effect on the process of early development of calcification of drugs capable to increasing its content. If the concentration of apolipoprotein A-1 is less than 1,1 mg/dl and/or the apoB/apoA ratio is increased by more than 1, it is advisable to recommend dispensary follow-up with regular (no less than 1 time in 5 years) echocardiographic study with a detailed study of the functional state of the aortic valve.

Genetic Variation in 9p21 and the Plasma Proteome.

Single nucleotide polymorphisms (SNPs) in the noncoding region of 9p21 have been associated with cardiovascular disease (CVD), but the mechanisms by which these genetic variants contribute to the pathogenesis of CVD remain unknown since no annotated proteins are present in this region of DNA. The objective of the current study was to determine if 9p21 genotypes are associated with distinct plasma proteins in young adults. Subjects were 1611 young adults aged 20-29 years from the Toronto Nutrigenomics and Health Study (1098 females and 513 males). DNA was isolated from fasting blood to analyze four SNPs in 9p21 (rs2383206, rs10757274, rs10757278, and rs1333049). Abundant plasma proteins ( n = 54) were measured using liquid chromatography multiple reaction monitoring mass spectrometry. Analysis of covariance was used to determine differences in plasma proteins between genotypes. In Caucasians ( n = 524), four SNPs were associated with apolipoprotein E and two with haptoglobin-ß-chain concentration. In East Asians ( n = 388), three SNPs were associated with α-1b-Glycoprotein, two with apolipoprotein B-100, one with apolipoprotein E and one with haptoglobin-ß-chain concentration. In South Asians ( n = 117), one SNP was associated with apolipoprotein B-100 concentration. Our findings suggest that 9p21 genotypes may play a role in various pathophysiological pathways that contribute to risk of CVD in early adulthood that might be distinct among different ethnocultural groups.

Effect of atorvastatin on dyslipidemia and carotid intima-media thickness in children with refractory nephrotic syndrome: a randomized controlled trial.

BACKGROUND: Dyslipidemia is an important cardiovascular risk factor in steroid-resistant nephrotic syndrome (SRNS). Efficacy of statins for treatment of hyperlipidemia in children with SRNS is unclear. METHODS: This prospective, randomized, double-blind, placebo-controlled, parallel-group clinical trial enrolled 30 patients with SRNS, aged 5-18 years, with serum low-density lipoprotein cholesterol (LDL-C) levels between 130 and 300 mg/dl, to receive a fixed dose of atorvastatin (n = 15, 10 mg/d) or placebo (n = 15) by block randomization in a 1:1 ratio. Primary outcome was change in serum LDL-C at 12 months. Change in levels of other lipid fractions, carotid intima-media thickness (cIMT), flow-mediated dilation (FMD) of the brachial artery, and adverse events were also evaluated. RESULTS: At the end of 12 months, atorvastatin was not superior to placebo in reducing plasma LDL-C levels, median percentage reduction 15.8% and 9.5% respectively, in atorvastatin and placebo arms (n = 14 in each; P = 0.40). Apolipoprotein B levels significantly declined with atorvastatin in modified intention-to-treat analysis (P = 0.01) but not in the per-protocol analysis. There was no significant effect on other lipid fractions, cIMT and FMD. Adverse events were similar between groups. Change in serum albumin was negatively associated with change in serum LDL-C, very low-density lipoprotein cholesterol, total cholesterol, triglyceride, and apolipoprotein B (P < 0.001), irrespective of receiving atorvastatin, age, gender, body mass index, and serum creatinine. CONCLUSIONS: Atorvastatin, administered at a fixed daily dose of 10 mg, was not beneficial in lowering lipid levels in children with SRNS; rise in serum albumin was associated with improvement in dyslipidemia.

Novel association of TM6SF2 rs58542926 genotype with increased serum tyrosine levels and decreased apoB-100 particles in Finns.

A glutamate-to-lysine variant (rs58542926-T) in transmembrane 6 superfamily member 2 (TM6SF2) is associated with increased fatty liver disease and diabetes in conjunction with decreased cardiovascular disease risk. To identify mediators of the effects of TM6SF2, we tested for associations between rs58542926-T and serum lipoprotein/metabolite measures in cross-sectional data from nondiabetic statin-naïve participants. We identified independent associations between rs58542926-T and apoB-100 particles (ß = -0.057 g/l, P = 1.99 × 10-14) and tyrosine levels (ß = 0.0020 mmol/l, P = 1.10 × 10-8), controlling for potential confounders, in 6,929 Finnish men. The association between rs58542926-T and apoB-100 was confirmed in an independent sample of 2,196 Finnish individuals from the FINRISK study (ßreplication = -0.029, Preplication = 0.029). Secondary analyses demonstrated an rs58542926-T dose-dependent decrease in particle concentration, cholesterol, and triglyceride (TG) content for VLDL and LDL particles (P < 0.001 for all). No significant associations between rs58542926-T and HDL measures were observed. TM6SF2 SNP rs58542926-T and tyrosine levels were associated with increased incident T2D risk in both METSIM and FINRISK. Decreased liver production/secretion of VLDL, decreased cholesterol and TGs in VLDL/LDL particles in serum, and increased tyrosine levels identify possible mechanisms by which rs58542926-T exerts its effects on increasing risk of fatty liver disease, decreasing cardiovascular disease, and increasing diabetes risk, respectively.

Atheroprotective vaccination with MHC-II-restricted ApoB peptides induces peritoneal IL-10-producing CD4 T cells.

Although immunization with major histocompatibility complex (MHC) class II-restricted apolipoprotein B (ApoB) peptides has been shown to be atheroprotective, the mechanism is unclear. Here, we investigated CD4+ T cell populations in immunized atherosclerotic mice. Peptides (16-mers) from mouse ApoB, the core protein of low-density lipoprotein (LDL), were screened for binding to I-Ab by computer prediction and confirmed by radiolabeled peptide competition. Three new peptides, P101 (FGKQGFFPDSVNKALY, 5.5 nM IC50), P102 (TLYALSHAVNSYFDVD, 6.8 nM), and P103 (LYYKEDKTSLSASAAS, 95 nM), were tested in an atherosclerosis model (Apoe-/- mice on Western diet). Immunization with each of the three peptides (1 time in complete Freund's adjuvant subcuntaneously and 4 time in incomplete Freund's adjuvant intraperitoneally) but not with adjuvant alone showed significantly reduced atherosclerotic plaques in the aortic root by serial sections and in the whole aorta by en face staining. There were no differences in body weight, LDL cholesterol, or triglycerides. Peritoneal leukocytes from ApoB peptide-immunized mice, but not control mice, secreted significant amounts of IL-10 (150 pg/ml). Flow cytometry showed that peptide immunization induced IL-10 in 10% of peritoneal CD4+ T cells, some of which also expressed chemokine (C-C motif) receptor 5 (CCR5). Vaccination with ApoB peptides expanded peritoneal FoxP3+ regulatory CD4+ T cells and more than tripled the number of CCR5+FoxP3+ cells. Similar trends were also seen in the draining mediastinal lymph nodes but not in the nondraining inguinal lymph nodes. We conclude that vaccination with MHC class II-restricted autologous ApoB peptides induces regulatory T cells (Tregs) and IL-10, suggesting a plausible mechanism for atheroprotection.NEW & NOTEWORTHY Vaccination against apolipoprotein B (ApoB), the protein of LDL, attracts attention as a novel approach to prevent atherosclerosis. We discovered major histocompatibility complex class II-restricted ApoB peptides, which reduce atherosclerosis and induce IL-10-producing CD4+ T cells and chemokine (C-C motif) receptor 5 expression on regulatory T cells, suggesting that immunization with ApoB peptides inhibits atherosclerosis by inducing anti-inflammatory cytokines.

Vaccination against T-cell epitopes of native ApoB100 reduces vascular inflammation and disease in a humanized mouse model of atherosclerosis.

BACKGROUND AND OBJECTIVES: The T-cell response to low-density lipoprotein (LDL) in the vessel wall plays a critical role in atherosclerotic plaque formation and stability. In this study, we used a new translational approach to investigate epitopes from human apolipoprotein B100 (ApoB100), the protein component of LDL, which triggers T-cell activation. We also evaluated the potential of two selected native ApoB100 epitopes to modulate atherosclerosis in human ApoB100-transgenic Ldlr-/- (HuBL) mice. METHODS AND RESULTS: HuBL mice were immunized with human atherosclerotic plaque homogenate to boost cellular autoimmune response to tissue-derived ApoB100 epitopes. In vitro challenge of splenocytes from immunized mice with a library of overlapping native peptides covering human ApoB100 revealed several sequences eliciting T-cell proliferation. Of these sequences, peptide (P) 265 and P295 were predicted to bind several human leucocyte antigen (HLA) haplotypes and induced high levels of interferon (IFN)-γ. Vaccination of HuBL mice with these peptides mounted a strong adaptive immune response to native ApoB100, including high levels of epitope-specific plasma IgGs. Interestingly, P265 and P295 vaccines significantly decreased plaque size, reduced macrophage infiltration and increased IgG1 deposition in the plaques. Purified IgGs from vaccinated mice displayed anti-inflammatory properties against macrophages in vitro, reducing their response to LPS in a dose-dependent manner. CONCLUSION: We identified two specific epitopes from human native ApoB100 that trigger T-cell activation and protect HuBL mice against atherosclerosis when used in a vaccine. Our data suggest that vaccination-induced protective mechanisms may be mediated at least in part through specific antibody responses to LDL that inhibit macrophage activation.

A correlation between secondary structure and rheological properties of low-density lipoproteins at air/water interfaces.

The secondary structure of apolipoprotein B-100 is studied within the bulk phase and at the air/water interface. In these "in viro" experiments, infrared reflection absorption spectroscopy (IRRAS) study was performed at the air/water interface while circular dichroism (CD) was conducted in the bulk phase. In the bulk phase, the conformational structure containing a significant amount of ß-structure, whereas varying amount of α-helix, unordered structures, and ß-sheet were observed at the air/water interface depending on the low-density lipoprotein (LDL) film interfacial pressure. The present IRRAS results demonstrate the importance of interfacial pressure-induced structural conformations on the apoB-100. A correlation between the secondary structure of the apoB-100 protein and the monomolecular film elasticity at the air/water interface was also established. The orientation of apoB-100 with respect to the LDL film-normal was found to depend on the interfacial pressure exhibited by the monomolecular film. These results may shed light on LDL's pivotal role in the progression of atherosclerotic coronary artery disease as demonstrated previously by clinical trials.

Role of apolipoprotein B100 and oxidized low-density lipoprotein in the monocyte tissue factor induction mediated by anti-ß2 glycoprotein I antibodies.

OBJECTIVE: The objective of this paper is to elucidate the not yet known plasma molecule candidates involved in the induction of tissue factor (TF) expression mediated by ß2GPI-dependent anticardiolipin antibody (aCL/ß2GPI) on monocytes. METHODS: Human serum incubated with FLAG-ß2GPI was applied for affinity chromatography with anti- FLAG antibody. Immunopurified proteins were analyzed by a liquid chromatography coupled with mass spectrometry (LC-MS). TF mRNA induced by the identified molecules on monocytes was also analyzed. RESULTS: Apolipoprotein B100 (APOB) was the only identified serum molecule in the MS search. Oxidized LDL, containing APOB as well as ox-Lig1 (a known ligand of ß2GPI), was revealed as a ß2GPI-binding molecule in the immunoprecipitation assay. TF mRNA was markedly induced by oxidized LDL/ß2GPI complexes with either WBCAL-1 (monoclonal aCL/ß2GPI) or purified IgG from APS patients. The activities of lipoprotein-associated phospholipase A2, one of the component molecules of oxidized LDL, were significantly higher in serum from APS patients than in those from controls. CONCLUSION: APOB (or oxidized LDL) was detected as a major ß2GPI binding serum molecule by LC-MS search. Oxidized LDL/aCL/ß2GPI complexes significantly induced TF expressions on monocytes. These data suggest that complexes of oxidized LDL and aCL/ß2GPI may have a crucial role in the pathophysiology of APS.