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.

High levels of autoantibodies against apoB100 p210 are associated with lower incidence of atrial fibrillation in women.

BACKGROUND AND OBJECTIVES: Atrial fibrillation (AF) is associated with inflammation, both systemically and in the atrial tissue. Oxidized low-density lipoprotein (LDL) is increased in patients with AF and is suggested to be one of the molecules that drives inflammation. Autoantibodies against oxidized LDL and apolipoprotein B100, the protein component of LDL, are linked to atherosclerotic disease. However, whether these autoantibodies are associated with occurrence of AF is not known. We investigated autoantibodies against oxidized apolipoprotein B100 peptides and incidence of AF in a large population-based cohort. METHODS: IgM and IgG against native and aldehyde-modified apoB100 peptides 210 (p210) and 45 were analyzed by enzyme-linked immunosorbent assay (ELISA) in 5169 individuals from the Malmö Diet and Cancer cohort. RESULTS: Seven hundred sixty-nine incident AF cases were recorded during a follow-up of 21.3 years. Individuals with high levels of IgM against native p210 at baseline had a lower risk of developing AF; however, the association did not remain after adjustment for age and sex. Women had higher levels of IgM against native p210 than men (0.70 ± 0.22 AU vs. 0.63 ± 0.21 AU, p < 0.001). The association of IgM against native p210 and AF was significantly different between sexes (p for interaction = 0.024), where females with high IgM against p210 had a lower risk for incidence of AF (hazard ratio [95% confidence interval] 4th versus 1st quartile: 0.67 [0.49-0.91]; p = 0.01) after adjusting for risk factors and comorbidities. CONCLUSION: These findings support an association of humoral autoimmunity with AF.

Autoantibodies Against Methylglyoxal-Modified Apolipoprotein B100 and ApoB100 Peptide Are Associated With Less Coronary Artery Atherosclerosis and Retinopathy in Long-Term Type 1 Diabetes.

OBJECTIVE: Methylglyoxal (MGO), a reactive aldehyde forming advanced glycation end products (AGEs), is increased in diabetes and recognized by the immune system, resulting in anti-AGE-specific autoantibodies. The association of these immune responses with macro- and microvascular complications in type 1 diabetes remains unclarified. We investigated associations between MGO-modified apolipoprotein B100 (apoB100) and apoB100 peptide 5 (MGO-p5) autoantibodies and coronary atherosclerosis and retinopathy in type 1 diabetes. RESEARCH DESIGN AND METHODS: IgM and IgG against MGO-apoB100 and MGO-p5 were measured by ELISA in plasma from 103 subjects with type 1 diabetes and 63 control subjects (Dialong study) and in a replication cohort of 27 subjects with type 1 diabetes (Oslo study). Coronary atherosclerosis was assessed by computed tomography coronary angiography or intravascular ultrasound. Retinopathy was classified by retinal photos. RESULTS: MGO-apoB100 IgM and MGO-p5 IgM levels were higher in subjects with diabetes with no coronary artery stenosis compared with subjects with significant stenosis (median [interquartile range]: 96.2 arbitrary units [AU] [71-126.8] vs. 54 AU [36.1-85.4], P = 0.003 for MGO-apoB100; and 77.4 AU [58-106] vs. 36.9 AU [28.9-57.4], P = 0.005 for MGO-p5). MGO-apoB100 IgM and MGO-p5 IgM were associated with less severe coronary stenosis after adjusting for confounders (odds ratio 0.2 [95% CI 0.05-0.6], P = 0.01; and 0.22 [0.06-0.75], P = 0.02). The inverse association of MGO-p5 IgM and coronary stenosis was confirmed in the replication cohort. Subjects with proliferative retinopathy had significantly lower MGO-apoB100 IgM and MGO-p5 IgM than those with background retinopathy. CONCLUSIONS: Autoantibodies against AGE-modified apoB100 are inversely associated with coronary atherosclerosis and proliferative retinopathy, suggesting vascular protective effects of these autoantibodies in type 1 diabetes.

Antibodies against apoB100 peptide 210 inhibit atherosclerosis in apoE-/- mice.

Atherosclerotic plaques are characterized by an accumulation and subsequent oxidation of LDL, resulting in adaptive immune responses against formed or exposed neoepitopes of the LDL particle. Autoantibodies against native p210, the 3136-3155 amino acid sequence of the LDL protein apolipoprotein B-100 (apoB100) are common in humans and have been associated with less severe atherosclerosis and decreased risk for cardiovascular events in clinical studies. However, whether apoB100 native p210 autoantibodies play a functional role in atherosclerosis is not known. In the present study we immunized apoE-/- mice with p210-PADRE peptide to induce an antibody response against native p210. We also injected mice with murine monoclonal IgG against native p210. Control groups were immunized with PADRE peptide alone or with control murine monoclonal IgG. Immunization with p210-PADRE induced an IgG1 antibody response against p210 that was associated with reduced atherosclerotic plaque formation in the aorta and reduced MDA-LDL content in the lesions. Treatment with monoclonal p210 IgG produced a similar reduction in atherosclerosis as immunization with p210-PADRE. Our findings support an atheroprotective role of antibodies against the apoB100 native p210 and suggest that vaccines that induce the expression of native p210 IgG represent a potential therapeutic strategy for lowering cardiovascular risk.

Anti-Apo B-100 Autoantibody is a Marker of Unstable Coronary Plaque.

AIMS: Cardiovascular diseases (CVD) are a global leading cause of mortality. However, few biomarkers are available to predict future coronary plaque rupture. We have recently demonstrated that low levels of anti-apolipoprotein B-100 autoantibody (anti-apo B-100 Ab) correlated with an increased CVD risk in Japanese patients with diabetes. In the present study, we examined the relationship between serum anti-apo B-100 Ab levels and coronary plaque characteristics in patients undergoing elective percutaneous coronary intervention (PCI). METHODS: We conducted iMAP®-intravascular ultrasound (IVUS) in 88 Japanese male patients undergoing elective PCI, and the five consecutive slices of IVUS images at the center of the most stenotic culprit lesion were used for identifying the plaque characteristics. The serum levels of anti-apo B-100 Ab against synthetic peptides (p45 or p210) were measured using a homemade enzyme-linked immunosorbent assay. RESULTS: Serum IgG levels of anti-apo B-100 Ab against both native p45 and p210 (IgG N-p45 and IgGN-p210) and malondialdehyde (MDA)-modified p45 and p210 (IgGMDA-p45 or IgGMDA-p210) showed a negative correlation with plaque burden in total male patients undergoing elective PCI. Additionally, both IgGN-p45 and IgGN-p210, but neither IgGMDA-p45 nor IgGMDA-p210, correlated negatively with necrotic and positively with fibrotic components of iMAP®-IVUS plaque characteristics in the patients with ＜1 month statin treatment before elective PCI ("statin-untreated" group). There was no significant correlation between anti-apo B-100 Ab and any plaque characteristics in the patients with statin treatment for 1 month or more before elective PCI ("statin-treated" group). CONCLUSION: Measuring serum levels of anti-apo B-100 Ab might be helpful in the evaluation of unstable coronary plaque in male CVD patients without statin treatment.

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.

Induction of HLA-A2 restricted CD8 T cell responses against ApoB100 peptides does not affect atherosclerosis in a humanized mouse model.

Cardiovascular diseases form the most common cause of death worldwide, with atherosclerosis as main etiology. Atherosclerosis is marked by cholesterol rich lipoprotein deposition in the artery wall, evoking a pathogenic immune response. Characteristic for the disease is the pathogenic accumulation of macrophages in the atherosclerotic lesion, which become foam cells after ingestion of large quantities of lipoproteins. We hypothesized that, by inducing a CD8 T cell response towards lipoprotein derived apolipoprotein-B100 (ApoB100), lesional macrophages, that are likely to cross-present lipoprotein constituents, can specifically be eliminated. Based on in silico models for protein processing and MHC-I binding, 6 putative CD8 T cell epitopes derived from ApoB100 were synthesized. HLA-A2 binding was confirmed for all peptides by T2 cell binding assays and recall responses after vaccination with the peptides proved that 5 of 6 peptides could induce CD8 T cell responses. Induction of ApoB100 specific CD8 T cells did not impact plaque size and cellular composition in HLA-A2 and human ApoB100 transgenic LDLr-/- mice. No recall response could be detected in cultures of cells isolated from the aortic arch, which were observed in cell cultures of splenocytes and mesenteric lymph nodes, suggesting that the atherosclerotic environment impairs CD8 T cell activation.

Rapid affinity chromatographic isolation method for LDL in human plasma by immobilized chondroitin-6-sulfate and anti-apoB-100 antibody monolithic disks in tandem.

Low-density lipoprotein (LDL) is considered the major risk factor for the development of atherosclerotic cardiovascular diseases (ASCVDs). A novel and rapid method for the isolation of LDL from human plasma was developed utilising affinity chromatography with monolithic stationary supports. The isolation method consisted of two polymeric monolithic disk columns, one immobilized with chondroitin-6-sulfate (C6S) and the other with apolipoprotein B-100 monoclonal antibody (anti-apoB-100 mAb). The first disk with C6S was targeted to remove chylomicrons, very-low-density lipoprotein (VLDL) particles, and their remnants including intermediate-density lipoprotein (IDL) particles, thus allowing the remaining major lipoprotein species, i.e. LDL, lipoprotein(a) (Lp(a)), and high-density lipoprotein (HDL) to flow to the anti-apoB-100 disk. The second disk captured LDL particles via the anti-apoB-100 mAb attached on the disk surface in a highly specific manner, permitting the selective LDL isolation. The success of LDL isolation was confirmed by different techniques including quartz crystal microbalance. In addition, the method developed gave comparable results with ultracentrifugation, conventionally used as a standard method. The reliable results achieved together with a short isolation time (less than 30 min) suggest the method to be suitable for clinically relevant LDL functional assays.

Long-Term Efficacy and Safety of Immunomodulatory Therapy for Atherosclerosis.

BACKGROUND AND AIMS: The long-term effect of immune tolerance has not been explored so far in atherosclerosis. In the present study, we assessed the effect of mucosal tolerance to a multi antigenic construct expressing three peptides from ApoB, HSP60, and outer membrane protein from Chlamydia pneumonia (AHC) for 30 weeks at every 6-week interval to understand the kinetics of immune modulation in disease progression. The safety profile of the molecule was also evaluated in mice. METHODS: Apobtm2SgyLdlrtm1Her/J mice (5-6 weeks) were orally dosed with multi antigenic construct (AHC) molecule on alternate days, followed by high-fat diet feeding to initiate atherosclerosis. RESULTS: Treated animals showed an efficient reduction in plaque growth and lipid accumulation at 6 weeks (49%, p < 0.01) and 12 weeks (42.3%, p < 0.01) which decreased to 29% (p = 0.0001) at 18 weeks and at later time points. Macrophage accumulation was significantly lower at all time points (53% at 12 weeks to 27% at 30 weeks). Regulatory T cells increased in the spleen following treatment until 12 weeks (week 0 (2.57 ± 0.18 vs. 6.36 ± 0.03, p = 0.02), week 6 (4.52 ± 0.2 vs. 8.87 ± 0.32, p = 0.02), and week 12 (8.74 ± 0.37 vs. 15.4 ± 0.27, p = 0.02)) but showed a decline later. A similar trend was observed with tolerogenic dendritic cells. We observed an increase in antibody levels to low-density lipoprotein and oxidized LDL at later stages. AHC molecule was found to be safe in acute and repeated dose toxicity studies. CONCLUSIONS: Our results suggest that immune tolerance to AHC protein by oral administration is able to provide efficient atheroprotection up to 18 weeks and moderately at later stages. Apart from immune regulatory cells, protective antibodies may also have a role in controlling atherosclerosis.

MMP-9/Gelatinase B Degrades Immune Complexes in Systemic Lupus Erythematosus.

Systemic Lupus Erythematosus (SLE) is a common and devastating autoimmune disease, characterized by a dysregulated adaptive immune response against intracellular antigens, which involves both autoreactive T and B cells. In SLE, mainly intracellular autoantigens generate autoantibodies and these assemble into immune complexes and activate the classical pathway of the complement system enhancing inflammation. Matrix metalloproteinase-9 (MMP-9) levels have been investigated in the serum of SLE patients and in control subjects. On the basis of specific studies, it has been suggested to treat SLE patients with MMP inhibitors. However, some of these inhibitors induce SLE. Analysis of LPR-/-MMP-9-/- double knockout mice suggested that MMP-9 plays a protective role in autoantigen clearance in SLE, but the effects of MMP-9 on immune complexes remained elusive. Therefore, we studied the role of MMP-9 in the clearance of autoantigens, autoantibodies and immune complexes and demonstrated that the lack of MMP-9 increased the levels of immune complexes in plasma and local complement activation in spleen and kidney in the LPR-/- mouse model of SLE. In addition, we showed that MMP-9 dissolved immune complexes from plasma of lupus-prone LPR-/-/MMP-9-/- mice and from blood samples of SLE patients. Surprisingly, autoantigens incorporated into immune complexes, but not immunoglobulin heavy or light chains, were cleaved by MMP-9. We discovered Apolipoprotein-B 100 as a new substrate of MMP-9 by analyzing the degradation of immune complexes from human plasma samples. These data are relevant to understand lupus immunopathology and side-effects observed with the use of known drugs. Moreover, we caution against the use of MMP inhibitors for the treatment of SLE.

Serum Anti-Apo B Antibody Level as Residual CVD Marker in DM Patients under Statin Treatment.

AIM: In the pathogenesis of atherosclerosis, autoantibodies have two-facedness of progression and protection. Previous reports have indicated that low autoantibody levels against apolipoprotein B-100 (apo B-100) could increase the risk of atherosclerotic cardiovascular diseases (CVD) in healthy subjects. In this study, we investigated the relationship between circulating anti-apo B-100 autoantibodies and the clinical parameters in Japanese diabetic patients with or without CVD. METHODS: We measured the serum levels of anti-apo B-100 autoantibodies against native and malondialdehyde (MDA)-modified p45 or p210 epitopes, as well as anti-apo E autoantibodies, using enzyme-linked immunosorbent assay. RESULTS: In patients with CVD, the circulating levels of IgG against native p45, MDA-modified p45, and MDA-modified p210 (IgGN-45, IgGMDA-45, and IgGMDA-210) were significantly lower than those in patients without CVD, whereas no difference was observed in anti-apo E autoantibody levels. In addition, IgMN-45, IgMMDA-45, and IgGMDA-45 were negatively correlated with LDL-C levels, whereas IgGN-45 and IgGN-210 were positively correlated with HbA1c levels. No correlation was observed between autoantibody levels and diabetic microangiopathy. In the statin-treated subgroup, IgGMDA-45 and IgGMDA-210 were significantly lower in patients with CVD than in those without CVD. CONCLUSION: Measurement of serum anti-apo B-100 autoantibodies can be useful for the evaluation of CVD risk in patients with diabetes receiving statin treatment.

Predictive Models for the Risk of Dyslipidemia in Adolescents with Essential Arterial Hypertension.

Predictive models of comorbidity, dyslipidemic disorders and essential arterial hypertension, in Russian adolescents aged 12 to 18 years (mean 15.48±1.53) were formulated with consideration for biochemical (lipid profiles) and genetic parameters (carrier state of gene polymorphic variants of apolipoprotein genes ApoA1 (-75G/A and +83C/T), ApoB (Ins/Del), ApoC3 (S1/S2), and ApoE (ε2/ε3/ε4). Significant prognostic risk factors for the mentioned comorbid pathologies were lipid metabolism parameters HDL-Ch, LDL-Ch, VLDL-Ch and carrier state of the +83T allele of the ApoA1 gene and Del allele of the ApoB gene. The obtained mathematical model is characterized by high predictive accuracy: the percentage of correct classification or the rate of correct assignment of each participant to the proper group was 96.33%.

Associations between circulating IgG antibodies to Apolipoprotein B100-derived peptide antigens and acute coronary syndrome in a Chinese Han population.

Objectives: Acute coronary syndrome (ACS) is the major cause of mortality worldwide and caused mainly by atherosclerosis of coronary arteries. Apolipoprotein B100 (ApoB100) is a major component of low-density lipoprotein (LDL) and its oxidation can trigger inflammation in vascular endothelial cells leading to atherosclerosis. The association between antibodies to ApoB100-derived antigens and atherosclerotic diseases has been studied in recent years, but the findings appear to be controversial. The present study developed an ELISA in-house with ApoB100-derived peptide antigens to circulating anti-ApoB100 IgG antibodies in patients with ACS. Methods: Fifteen ApoB100-derived peptide antigens (Ag1-Ag15) were designed to develop an in-house ELISA for the detection of circulating anti-ApoB100 IgG levels in 350 patients with ACS and 201 control subjects amongst a Chinese population. Binary logistic regression was applied to examine the differences in anti-ApoB IgG levels between the patient group and the control group with adjustment for a number of confounding factors; the correlation between anti-ApoB100 IgG levels and clinical characteristics was also tested. Results: Patients with ACS had significantly higher levels of plasma IgG for Ag1 (adjusted P<0.001) and Ag10 antigens (adjusted P<0.001). There was no significant increase in the levels of IgG to the other 13 antigens in these ACS patients. In the control group, anti-Ag10 IgG levels were positively correlated with age, high-density lipoprotein (HDL), and ApoA levels (P≤0.001 for all) and negatively correlated with blood triglyceride (TG) (P=0.008); in the patient group, anti-Ag10 IgG levels were positively correlated with LDL (P=0.003), and negatively correlated with ApoA (P=0.048) and systolic blood pressure (SBP) (P=0.036). The area under ROC (receiver operator characteristic) curve (AUC) was 0.612 (95% confidence interval (CI): 0.560-0.664; P<0.001) in anti-Ag1 IgG assay and 0.621 (95% CI: 0.569-0.672; P<0.001) in anti-Ag10 IgG assay. Conclusion: Circulating IgG for ApoB100-derived peptide antigens may be a useful biomarker of ACS, although anti-ApoB IgG levels were not associated with the coronary artery plaque burden characterized by the coronary Gensini score.

Regulatory CD4+ T Cells Recognize Major Histocompatibility Complex Class II Molecule-Restricted Peptide Epitopes of Apolipoprotein B.

BACKGROUND: CD4+ T cells play an important role in atherosclerosis, but their antigen specificity is poorly understood. Immunization with apolipoprotein B (ApoB, core protein of low density lipoprotein) is known to be atheroprotective in animal models. Here, we report on a human APOB peptide, p18, that is sequence-identical in mouse ApoB and binds to both mouse and human major histocompatibility complex class II molecules. METHODS: We constructed p18 tetramers to detect human and mouse APOB-specific T cells and assayed their phenotype by flow cytometry including CD4 lineage transcription factors, intracellular cytokines, and T cell receptor activation. Apolipoprotein E-deficient ( Apoe-/-) mice were vaccinated with p18 peptide or adjuvants alone, and atherosclerotic burden in the aorta was determined. RESULTS: In human peripheral blood mononuclear cells from donors without cardiovascular disease, p18 specific CD4+ T cells detected by a new human leukocyte antigen-antigen D related-p18 tetramers were mostly Foxp3+ regulatory T cells (Tregs). Donors with subclinical cardiovascular disease as detected by carotid artery ultrasound had Tregs coexpressing retinoic acid-related orphan receptor gamma t or T-bet, which were both almost absent in donors without cardiovascular disease. In Apoe-/- mice, immunization with p18 induced Tregs and reduced atherosclerotic lesions. After peptide restimulation, responding CD4+ T cells identified by Nur77-GFP (green fluorescent protein) were highly enriched in Tregs. A new mouse I-Ab-p18 tetramer identified the expansion of p18-specific CD4+ T cells on vaccination, which were enriched for interleukin-10-producing Tregs. CONCLUSIONS: These findings show that APOB p18-specific CD4+ T cells are mainly Tregs in healthy donors, but coexpress other CD4 lineage transcription factors in donors with subclinical cardiovascular disease. This study identifies ApoB peptide 18 as the first Treg epitope in human and mouse atherosclerosis.

An ApoB100-mimetic vaccine prevents obesity and liver steatosis in ApoE-/- mice.

BACKGROUND: Recently, a peptide vaccine (B4T) was developed that prevents high fat diet (HFD)-induced obesity and liver steatosis in wild type mice and appears to target an epitope present in ApoB100 but not ApoB48. Here, we ask whether B4T remains effective in ApoE knockout (ApoE-ko) mice, which exhibit a greatly increased ApoB48/ApoB100 ratio and develop atherosclerosis under HFD. METHODS: HFD-fed male ApoE-ko mice were injected with B4T or vehicle 3 times between 5 and 15 weeks of age. Until 45 weeks of age, they were regularly weighed and antibody titers determined. In the end, adiposity and organ histologies were examined. RESULTS: We find that in the ApoE-ko mice, B4T prevents HFD-induced body weight increases (p<0.01) to a comparable degree as previously shown in wild type mice. Also, liver steatosis was prevented as previously shown in wild type mice. By contrast, atherosclerotic plaque formation was not prevented in any of the vaccinated mice studied, in line with the observation that antibody production paralleled the weight reduction but largely preceded atherogenesis. CONCLUSION: The findings demonstrate effectiveness of B4T despite the increased ApoB48/B100 ratio, but argue against an effect on de novo plaque formation. At least under the current vaccination schedule, the obesity- and atherosclerosis-related roles of ApoB appear to be dissociable.

Identification of apoB-100 Peptide-Specific CD8+ T Cells in Atherosclerosis.

BACKGROUND: T cells are found in atherosclerotic plaques, with evidence supporting a potential role for CD8+ T cells in atherogenesis. Prior studies provide evidence of low-density lipoprotein and apoB-100 reactive T cells, yet specific epitopes relevant to the disease remain to be defined. The current study was undertaken to identify and characterize endogenous, antigen-specific CD8+ T cells in atherosclerosis. METHODS AND RESULTS: A peptide fragment of apoB-100 that tested positive for binding to the mouse MHC-I allele H2Kb was used to generate a fluorescent-labeled H2Kb pentamer and tested in apoE-/- mice. H2Kb pentamer(+)CD8+ T cells were higher in apoE-/- mice fed an atherogenic diet compared with those fed a normal chow. H2Kb pentamer (+)CD8+ T cells in atherogenic diet-fed mice had significantly increased effector memory phenotype with a shift in Vß profile. H2Kb pentamer blocked lytic activity of CD8+ T cells from atherogenic diet-fed mice. Immunization of age-matched apoE-/- mice with the apoB-100 peptide altered the immune-dominant epitope of CD8+ T cells and reduced atherosclerosis. CONCLUSIONS: Our study provides evidence of a self-reactive, antigen-specific CD8+ T-cell population in apoE-/- mice. Immune modulation using the peptide antigen reduced atherosclerosis in apoE-/- mice.

Relationship of Autoantibodies to MDA-LDL and ApoB-Immune Complexes to Sex, Ethnicity, Subclinical Atherosclerosis, and Cardiovascular Events.

OBJECTIVE: Modifications of lipid constituents within atherosclerotic lesions generate neoepitopes that activate innate and adaptive immune responses. We aimed to define the prevalence, distribution, and relationship of autoantibody titers of oxidized lipoproteins to subclinical atherosclerosis and major adverse cardiovascular events (MACE) in different ethnic groups. APPROACH AND RESULTS: IgG and IgM autoantibodies to malondialdehyde-modified low-density lipoprotein (MDA-LDL) and apolipoprotein B-100-immune complexes were measured in 3509 individuals (1814 blacks, 1031 whites, 589 Hispanics, and 85 no race identifier) from the Dallas Heart Study with median 10.5-year follow-up. Coronary artery calcium score, abdominal aortic plaque by magnetic resonance imaging, and MACE were quantified. IgG MDA-LDL and IgG and IgM apolipoprotein B-100-immune complexes were significantly different between groups, with blacks having the highest levels of IgG MDA-LDL and IgG apolipoprotein B-100-immune complexes and Hispanics having the highest levels of IgM apolipoprotein B-100-immune complexes (P<0.001 for all). IgGs tended to be higher and IgMs lower with age for all markers. In multivariable-adjusted binary logistic regression analysis, a doubling of IgG MDA-LDL levels was associated with prevalent coronary artery calcium score >10 Agatston units (odds ratio [95% confidence interval], 1.21 [1.07-1.36]; P=0.002). Multivariable-adjusted Cox regression analysis revealed that IgG MDA-LDL was independently associated with time to incident MACE in the entire group (hazard ratio [95% confidence interval], 1.76 [1.16-2.72]; P=0.009 for fourth versus first quartile). This effect was particularly prominent in black subjects (hazard ratio [95% confidence interval], 2.52 [1.39-4.57]; P=0.002). CONCLUSIONS: Autoantibodies to oxidized lipoproteins and immune complexes with apoB-100 lipoproteins vary significantly by sex, age, and ethnicity. Higher baseline IgG MDA-LDL titers independently associate with new MACE. These findings may contribute to the understanding of differences in ethnic-specific MACE events.

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.

Tailor-made approach for selective isolation and elution of low-density lipoproteins by immunoaffinity sorbent on silica.

Immunoaffinity procedure was developed for isolation of low density lipoprotein (LDL) from biological samples by using silica-derived immunoaffinity sorbent. Sorbent was prepared by immobilization of monoclonal anti-apoB-100 antibody onto macroporous silica particles, using carefully optimized binding chemistry. Binding capacity of the sorbent towards LDL was determined by batch extraction experiments with solutions of isolated LDL in phosphate-buffered saline, and found to be 8 mg LDL/g. The bound LDL fraction was readily released from the sorbent by elution with ammonia at pH 11.2. The total time needed for isolation procedure was less than 1 h, with LDL recoveries being essentially quantitative for samples containing less than 0.3 mg LDL/mL. With higher concentrations, recoveries were less favorable, most probably due to irreversible adsorption caused by LDL aggreggation. However, reusability studies with isolated LDL at concentration 0.2 mg/mL indicate that the developed immunoaffinity material may be used for multiple binding-release cycles, with minor losses in binding capacity. Finally, the sorbent was successfully applied to isolation of LDL from diluted plasma. Apart from its practical implications for LDL isolation, this study provides crucial insights into issues associated with LDL-sorbent interactions, and may be useful in future efforts directed to development of lipoprotein isolation approaches.