The Study Of Constructing Virus-like Particles And Therapeutic Anti-hypertensive Vaccines

Preparation and Alteration of the Bacteriophage Virus-like Particle CarrierObjectives:We have successfully constructed the QP-2aa phage virus-like particle (Qβ-2aa VLP, diameter=30nm). Hypertensive vaccines emphasize on the induction of type2humoral immune response. The larger diameter carrier within the range of20-200nm may be more conducive to the induction of type2immune response. Meanwhile, VLP has a good presenting ability of internal wrapping. In order to enhance the effect of hypertension vaccines, construction of larger diameter VLPs and re-equipment of small diameter VLPs become very necessary. This study focuses on the construction of HK97VLP (66nm) and its chimera, and the internal modification of AP205VLP (30nm).Methods:HK97phage capsid protein genes (GP4and GP5) were synthesized by gene synthesis technology and amplified by RT-PCR. Constructing the dual expression plasmid pETDuet-HK97, simultaneous expression of gp4and gp5proteins were induced by IPTG, following the self-assembly of VLP. By using chromatographic techniques HK97VLP was purified and induced by in vitro acidification. The maturity and purity of HK97VLP were determined by SDS-PAGE. The shape and size were observed under a transmission electron microscope; GP5’gene was synthesized and the chimeric expression plasmid pETDuet-HK97-P was constructed. By using the similar technology program, HK97-P VLP chimera was constructed. Wide tpye HK97VLP carrier vaccine was prepared and was used to immunize SD rats. HK97-P VLP chimera was also used to immunize the animals. The AP205VLP capsid protein (CP) gene was synthesized and the expression plasmid pET28-AP205was constructed, IPTG induced CP expression and self-assembly. AP205VLP was purified and its purity was determined by SDS-PAGE. CpG ODN2006and ODNA151were packed into AP205VLP internal and the stimulation or inhibitory effect to SPMNC were detected.Results:We successfully built the HK97VLP wild-type expression plasmid pETDuet-HK97and chimera expression plasmid pETDuet-HK97-P, which both could complete self-assemble into the HK97VLP precursors. The diameter of precursors was54nm. With the successful expansion of the precursor in vitro acidification, the precusor is cross-linked into mature HK97VLP. The diameter of mature HK97VLP was66nm. The wild-type HK97VLP coupling vaccine and the chimeric vaccine both could generate potent auxiliary anti-peptide antibody production, and the former is slightly stronger than the latter. The expression plasmid pET28-AP205was constructed and AP205VLP was induced successfully. The molecular weight was14KD by SDS-PAGE. Under the electron microscope, the CP could complete the self-assembly into the spherical particles with a diameter of about30nm. The AP205VLP-CpG ODN2006parcel physical directly stimulates the generation of the y-IFN of spleen mononuclear cells, while wrapped ODNA151-AP205VLP significantly inhibited LPS-induced y-IFN production.Conclusion:We successfully constructed HK97VLP (diameter=66nm) and AP205VLP (diameter=30nm), and completed the building of HK97VLP chimera and the internal modification of AP205VLP. These work provides a more suitable carrier for biological target therapy and a new way of ODN delivery. The Study of Therapuetic Vaccines Against Human and Rat ReninObjectives:Hypertension vaccine as a novel bio-therapeutic approach has got double attention for the past few years. Renin as the initiation of RAS plays a critical role in hypertension, and is one of the ideal targets for developing hypertension vaccine. This study will design several epitopic peptides from renin, and screen one or several peptides to develop renin hypertension vaccine.Methods:According to the three-dimensional crystal structure of human renin, Six peptides (rR32, rR72, rR215; hR32, hR72, hR215) as belonging to potential epitopes of rat and human renin had been selected. The main choice criteria were based on:(1) the peptide sequences should include one of renin catalytic sites or the flap peptide,(2) matching peptides with the host proteome, choosing low/no-similarity peptide sequences;(3) having ideal antigenicity and hydrophilicity. The peptides were coupled to keyhole limpet hemocyanin and injected into SpragueDawley (SD) rats, spontaneously hypertensive rats (SHRs) and Wistar-Kyoto rats. The antisera titers and the binding capacity with renin were detected. The effect of the anti-peptides antibodies on plasma renin activity (PRA) and blood pressure was determined.Results:All peptides elicited strong antibody responses. The antibody titer ranged from1:32,000to1:80,000on day63. All antisera could bind with renin in vitro. The purificated immunoglobulin (IgG) aganist the rR32, hR32, rR72and hR72peptides reduced the PRA level to less than50%compared to the control IgG Complete cross-reactivity of the anti-rR32antibody and the anti-hR32antibody against human renin was confirmed. The epitopes rR32and hR32vaccines had no obvious effect on systolic blood pressure (SBP) of SD rats, while significantly decrease SBP of SHRs up to15mmHg (175±2vs.190±3mmHg, P=0.035;180±2vs.195±3mmHg, P=0.039). Additionally, no significant immune-mediated damage was detected in the vaccinated animals.Conclusion:In conclusion, we firstly provided an antigenic peptide hR32vaccine mimicking the32Asp catalytic site of human renin. Although the blood pressure decline was limited, we provide a novel and promising method for renin vaccine development. The Mechanism Study in ATRQP-001Hypertensive VaccineObjectives:In our previous study, ATRQP-001vaccine, a peptide (ATR-001) derived from human angiotensin II receptor type1(AT1R) conjugated with Qβ-2aa bacteriophage virus-like particles (Qβ-2aa VLP) was developed. The ATRQβ-001vaccine could significantly decrease the blood pressure of hypertensive animals and protect target organs, but the mechanism is unclear. For better understanding the effect of the ATRQβ-001vaccine and paving the way for future clinical application, we investigate the mechanism of the ATRQP-001vaccine.Methods:The ATRQβ-001vaccine was prepared by Sulfo-SMCC. In vivo, the ATRQβ-001vaccine was used to immunize spontaneous hypertensive rats (SHR) and angiotensin (Ang) II-induced hypertensive mice. The blood pressure and the antibody titer were detected by tail-cuff method and enzyme-linked immuno-sorbent assay respectively. Parameters of anterior wall, posterior wall and the internal diameter of heart in both left ventricular systolic and diastolic period were judged by ultrasound. At the end of the experiment, the conditions of rat cardiac hypertrophy and fibrosis was measured. The plasma renin-angiotensin system (RAS) components and the Ang II concentration of local tissues were detected by radioimmunoassay (RIA). The mRNA and the protein expression of renin and ATIR was determined by quantitative real-time polymerase chain reaction and western bloting. In vitro, the binding of the anti-ATR-001antibody with ATIR was demonstrated by immunocytochemistry, while the competitive binding with ATIR of the anti-ATR-001antibody and Ang II by RIA. Protein kinase C (PKC)-a translocation affected by the anti-ATR-001antibody was measured by immunocytochemistry. The phosphorylation level of extracellular signal-regulated kinase (ERKl/2) was detected in small arterial smooth muscle cells and HEK293cells expressing ATIR by western blotting. The confocal laser scanning microscope was used to record the effect of the anti-ATR-001antibody on intracellular calcium concentration elevation in HEK293cells by Ang II stimulation.Results:The ATRQβ-001vaccine significantly decreased the blood pressure of Ang II-induced hypertensive mice up to35mmHg (143±4vs178±6mmHg, P=0.005) and SHRs up to19mmHg (173±2vs192±3mmHg, P=0.003) and prevented remodeling of vulnerable hypertensive target organs. Though the expression of ATIR was significantly decreased, no obvious feedback activation of circulating or local RAS was observed in the ATRQβ-001vaccine group. Additionally, no significant immune-mediated damage was detected in vaccinated hypertensive and non-hypertensive animals. The half-life of the anti-ATR-001antibody was14.4days, surpassing that of existing chemical drugs. In vitro, the anti-ATR-001antibody specifically bound to ATIR and inhibited Ca2+-dependent signal transduction events, including PKC-a translocation, ERK1/2phosphorylation (72%decrease, P=0.013) and elevation of intracellular Ca2+(68%decrease, P=0.017) induced by Ang II, but without inhibiting Ang II binding to the receptor.Conclusion:The ATRQβ-001vaccine decreased the blood pressure of Ang II-induced hypertensive mice and SHRs effectively through diminishing the pressure response and inhibiting signal transduction initiated by Ang Ⅱ. The ATRQβ-001vaccine did not cause feedback activation of circulating and local RAS system, but may inhibit ATIR internalization and promote its degradation, thereby compared to other drugs, showed better protective effects of target organs.