Immunotherapy against angiotensin II receptor ameliorated insulin resistance in a leptin receptor-dependent manner.

The angiotensin II type 1 receptor (AT1R) signaling pathway is reported to modulate glucose metabolism. Targeting AT1R, our group invented ATRQß-001 vaccine, a novel immunotherapeutic strategy to block the activation of AT1R. Here, we evaluated the therapeutic efficacy of ATRQß-001 vaccine in insulin resistance, and investigated the mechanism. Our results showed that ATRQß-001 vaccine and specific monoclonal antibody against epitope ATR-001 (McAb-ATR) decreased fasting serum insulin concentration and improved glucose and insulin tolerance in ob/ob mice. These beneficial effects were verified in high-fat diet-induced obese mice. McAb-ATR activated insulin signaling in skeletal muscle and insulin-resistant C2C12 myotubes without affecting liver or white adipose tissue of ob/ob mice. Mechanistically, the favorable impact of McAb-ATR on insulin resistance was abolished in db/db mice and in C2C12 myotubes with leptin receptor knockdown. AT1R knockdown also eradicated the effects of McAb-ATR in C2C12 myotubes. Furthermore, McAb-ATR treatment was able to activate the leptin receptor-mediated JAK2/STAT3 signaling in skeletal muscle of ob/ob mice and C2C12 myotubes. Additionally, angiotensin II downregulated the leptin signaling in skeletal muscle of ob/ob and diet-induced obese mice. We demonstrated that ATRQß-001 vaccine and McAb-ATR improved whole-body insulin resistance and regulated glucose metabolism in skeletal muscle in a leptin receptor-dependent manner. Our data suggest that immunotherapy targeting AT1R is a novel strategy for treating insulin resistance.

A bivalent antihypertensive vaccine targeting L-type calcium channels and angiotensin AT1 receptors.

BACKGROUND AND PURPOSE: Hypertension has been the leading preventable cause of premature death worldwide. The aim of this study was to design a more efficient vaccine against novel targets for the treatment of hypertension. EXPERIMENTAL APPROACH: The epitope CE12, derived from the human L-type calcium channel (CaV 1.2), was designed and conjugated with Qß bacteriophage virus-like particles to test the efficacy in hypertensive animals. Further, the hepatitis B core antigen (HBcAg)-CE12-CQ10 vaccine, a bivalent vaccine based on HBcAg virus-like particles and targeting both human angiotensin AT1 receptors and CaV 1.2 channels, was developed and evaluated in hypertensive rodents. KEY RESULTS: The Qß-CE12 vaccine effectively decreased the BP in hypertensive rodents. A monoclonal antibody against CE12 specifically bound to L-type calcium channels and inhibited channel activity. Injection with monoclonal antibody against CE12 effectively reduced the BP in angiotensin II-induced hypertensive mice. The HBcAg-CE12-CQ10 vaccine showed antihypertensive effects in hypertensive mice and relatively superior antihypertensive effects in spontaneously hypertensive rats and ameliorated L-NAME-induced renal injury. In addition, no obvious immune-mediated damage or electrophysiological adverse effects were detected. CONCLUSION AND IMPLICATIONS: Immunotherapy against both AT1 receptors and CaV 1.2 channels decreased the BP in hypertensive rodents effectively and provided protection against hypertensive target organ damage without obvious feedback activation of renin-angiotensin system or induction of dominant antibodies against the carrier protein. Thus, the HBcAg-CE12-CQ10 vaccine may provide a novel and promising therapeutic approach for hypertension.

Immunotherapy of Endothelin-1 Receptor Type A for Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a chronic fatal disease. The treatment of PAH is less than ideal and the control is far from satisfactory worldwide. Vaccination provides a promising approach for treatment of PAH. OBJECTIVES: This study sought to find a vaccine against endothelin-1 (ET-1) receptor type A (ETAR) for treating PAH. METHODS: The ETRQß-002 vaccine was screened and the specific antibodies against epitope ETR-002 belonging to the second extracellular loop of ETAR (including the polyclonal and monoclonal antibody) were produced. The effect of the antibodies on Ca2+-dependent signal transduction events was investigated. In vivo, ETRQß-002 vaccine was used to vaccinate monocrotaline (MCT)- and Sugen/hypoxia-induced pulmonary hypertension animals. The monoclonal antibody (mAb) against ETR-002 was also injected into the PAH animals. The effect of ETRQß-002 vaccine on pulmonary hypertension and remodeling of pulmonary arterioles and right ventricle (RV) was carefully evaluated. Further, the possible immune-mediated damage was detected in normal vaccinated animals. RESULTS: ETR-002 peptide has perfect immunogenicity and ETRQß-002 vaccine could induce strong antibody production. In vitro, the anti-ETR-002 antibody bound to ETAR and inhibited Ca2+-dependent signal transduction events, including extracellular signal-regulated kinase phosphorylation and elevation of intracellular Ca2+ concentration induced by ET-1. In vivo, both ETRQß-002 vaccine and the mAb significantly decreased the RV systolic pressure up to 20 mm Hg and 10 mm Hg in MCT-exposed rats and Sugen/hypoxia-exposed mice, respectively. Also, ETRQß-002 vaccine/mAb obviously ameliorated pathological remodeling of pulmonary arterioles and hypertrophy of the RV in PAH animals. Additionally, no significant immune-mediated damage was detected in vaccinated animals. CONCLUSIONS: ETRQß-002 vaccine/mAb attenuated remodeling of pulmonary arterioles and RV in MCT- and Sugen/hypoxia-induced PAH animals and decreased RV systolic pressure effectively through diminishing the pressure response and inhibiting signal transduction initiated by ET-1. ETRQß-002 vaccine/mAb may provide a novel and promising method for PAH treatment.

ATRQß-001 Vaccine Prevents Experimental Abdominal Aortic Aneurysms.

Background We have developed a peptide vaccine named ATRQß-001, which was proved to retard signal transduction initiated by angiotensin II (Ang II). Ang II was implicated in abdominal aortic aneurysm (AAA) progression, but whether the ATRQß-001 vaccine would prevent AAA is unknown. Methods and Results Ang II-infused ApoE-/- mice and calcium phosphate-induced AAA in C57BL/6 mice were used to verify the efficiency of ATRQß-001 vaccine in AAA. Results demonstrated that the vaccine effectively restrained the aneurysmal dilation and vascular wall destruction of aorta in both animal models, beyond anti-hypertensive effects. In Ang II-induced AAA vascular sections, Immunohistochemical staining showed that the vaccine notably constrained vascular inflammation and vascular smooth muscle cell (VSMC) phenotypic transition, concurrently reduced macrophages infiltration. In cultured VSMC, the anti-ATR-001 antibody inhibited osteopontin secretion induced by Ang II, thereby impeded macrophage migration while co-culture. Furthermore, metalloproteinases and other matrix proteolytic enzymes were also found to be limited by the vaccine in vivo and in vitro. Conclusions ATRQß-001 vaccine prevented AAA initiation and progression in both Ang II and calcium phosphate-induced AAA models. And the beneficial effects were played beyond decrease of blood pressure, which provided a novel and promising method to take precautions against AAA.

A Therapeutic Peptide Vaccine Against PCSK9.

Vaccination provides a promising approach for treatment of hypercholesterolemia and improvement in compliance. In this study, the appropriate virus-like particle (VLP)-peptide vaccines targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) were screened. The screening criteria of target peptides were as follows: (1) located in catalytic domain of PCSK9, or regulating the binding of PCSK9 and LDL receptors (LDLR); (2) having low/no-similarity when matched with the host proteome; (3) possessing ideal antigenicity and hydrophilicity; (4) including the functional mutation site of PCSK9. It was found that mice vaccinated with VLP -PCSK9 peptide vaccines, especially PCSK9Qß-003 vaccine, developed high titer IgG antibodies against PCSK9. PCSK9Qß-003 vaccine obviously decreased plasma total cholesterol in both Balb/c mice and LDLR+/- mice. Also, PCSK9Qß-003 vaccine decreased plasma PCSK9 level and up-regulated LDLR expression in liver. Additionally, PCSK9Qß-003 vaccine injection was associated with significant up-regulation of sterol-regulatory element-binding protein-2 (SREBP-2), hepatocyte nuclear factor 1α (HNF-1α), and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in LDLR+/- mice. No obvious immune injury was detected in vaccinated animals. The PCSK9Qß-003 vaccine, therefore, may be an attractive treatment approach for hypercholesterolemia through decreasing cholesterol and regulating lipid homeostasis.

Immune Response of A Novel ATR-AP205-001 Conjugate Anti-hypertensive Vaccine.

We developed a virus-like particle (VLP)-based therapeutic vaccine against angiotensin II receptor type 1, ATR-AP205-001, which could significantly reduce the blood pressure and protect target organs of hypertensive animals. In this study, we focused on the immunological effect and safety of the VLP-based vaccine. By comparing to the depolymerized dimeric vaccine ATR-Dimer-001, we found that ATR-AP205-001 reached subcapsular sinus of lymph node shortly after administration, followed by accumulation on follicle dendritic cells via follicle B cell transportation, while ATR-Dimer-001 vaccine showed no association with FDCs. ATR-AP205-001 vaccine strongly activated dendritic cells, which promoted T cells differentiation to follicular helper T cells. ATR-AP205-001 vaccine induced powerful germinal center reaction, which was translated to a boost of specific antibody production and long-lasting B cell memory, far superior to ATR-Dimer-001 vaccine. Moreover, neither cytotoxic T cells, nor Th1/Th17 cell-mediated inflammation was observed in ATR-AP205-001 vaccine, similar to ATR-Dimer-001 vaccine. We concluded that ATR-AP205-001 vaccine quickly induced potent humoral immunity through collaboration of B cells, follicular dendritic cells and follicular helper T cells, providing an effective and safe intervention for hypertension in the future clinical application.

Vaccination against type 1 angiotensin receptor prevents streptozotocin-induced diabetic nephropathy.

UNLABELLED: Recently, our group has developed a therapeutic hypertensive vaccine against angiotensin (Ang) II type 1 receptor (AT1R) named ATRQß-001. To explore its potential effectiveness on streptozotocin-induced diabetic nephropathy, male Sprague Dawley rats were randomly divided into two groups: a control and a diabetic model. After 1 week, the diabetic rats were divided into four subgroups (each with 15 rats) for 14-week treatments with saline, olmesartan, ATRQß-001, and Qß virus-like particle (VLP), respectively. In addition to lower blood pressure, ATRQß-001 vaccination ameliorated biochemical parameter changes of renal dysfunction, mesangial expansion, and fibrosis through inhibiting oxidative stress, macrophage infiltration, and proinflammatory factor expression. Furthermore, ATRQß-001 vaccination suppressed renal Ang II-AT1R activation and abrogated the downregulation of angiotensin-converting enzyme 2-Ang (1-7), similar to olmesartan treatment, while no obvious feedback activation of circulating or local renin-angiotensin system (RAS) was only observed in vaccine group. In rat mesangial cells, the anti-ATR-001 antibody inhibited high glucose-induced transforming growth factor-ß1 (TGF)-ß1/Smad3 signal pathway. Additionally, no significant immune-mediated damage was detected in vaccinated animals. In conclusion, the ATRQß-001 vaccine ameliorated streptozotocin-induced diabetic renal injury via modulating two RAS axes and inhibiting TGF-ß1/Smad3 signal pathway, providing a novel, safe, and promising method to treat diabetic nephropathy. KEY MESSAGES: Overactivation of RAS plays a crucial role in the development of the DN. Our aim was to verify the effectiveness of ATRQß-001 vaccine in STZ-induced DN. The ATRQß-001 modulated two RAS axes and inhibited TGF-ß1/Smad3 signal pathway. The vaccine therapy may provide a novel, safe, and promising method to treat DN.

ATRQß-001 vaccine prevents atherosclerosis in apolipoprotein E-null mice.

OBJECTIVE: Angiotensin II (AngII) type 1 receptor (AT1R) blockers have been proved to reduce atherosclerosis. Previously, we have invented ATRQß-001 vaccine which showed a desirable blocking effect for AT1R. The purpose of this study was to investigate whether ATRQß-001 vaccine would prevent atherosclerosis in apolipoprotein E-null (ApoE-/-) mice. METHODS: Male ApoE-/- mice were administered with ATRQß-001 vaccine, Qß virus-like particles, valsartan or vehicle over a period of 24 weeks. In vitro, human coronary artery endothelial cells preincubated with the anti-ATR-001 antibody, the neutralization antibody or valsartan for 2  h, were treated with AngII for 24  h. Histological stain and molecule biology methods were used to assess the atheroprotective effect of the vaccine. RESULTS: ATRQß-001 vaccine significantly reduced the lesion area and promoted the stability of atherosclerotic plaque. Meanwhile, macrophage infiltration as well as the expressions of adhesion molecules and monocyte chemoattractant protein-1 was obviously decreased in the ATRQß-001 vaccine group. Additionally, the vaccine markedly reduced the apoptosis in the lesions of the ApoE-/- mice. In vitro, the anti-ATR-001 antibody inhibited endothelial apoptosis induced by AngII. Furthermore, ATRQß-001 vaccine exhibited a dramatical attenuation in the expressions of lectin-like oxidized low-density lipoprotein receptor-1 and AT1R in the aortic. More importantly, compared with the valsartan group, no obvious feedback of the plasma renin-angiotensin system was elicited in the vaccine group. CONCLUSION: The results demonstrated that ATRQß-001 vaccine reduced the progression of atherosclerosis in ApoE-/- mice without obvious feedback of renin-angiotensin system.