The Development Of Novel Virus-like Particle-based Bivalent Antihypertensive Vaccine HBcAg-CE12-CQ10

Part ? Construction of the novel bivalent antihypertensive vaccine Objective: The combination drug therapy is a common practice in the clinical treatment of hypertension,and existing antihypertensive vaccines work against each single target.In order to develop a more advantageous antihypertensive vaccine,we will try to construct a novel bivalent antihypertensive vaccine against two different targets.Methods: The recombinant protein was constructed by inserting the short peptide epitopes of human angiotensin II type 1 receptor and L-type calcium channel into the immunodominant region of the modified hepatitis B virus core antigen(HBc Ag).The recombinant protein was expressed in Escherichia coli,and the expressed product was identified by polyacrylamide gel electrophoresis.Recombinant virus-like particles HBc Ag-CE12-CQ10 were obtained by inclusion body renaturation and protein purification.Morphological structure of recombinant virus-like particles HBc Ag-CE12-CQ10 was identified by transmission electron microscopy.In addition,conjugated vaccines targeting human angiotensin type 1 receptor and L-type calcium channel antigen were prepared by chemical conjugation technology.Mice were immunized with these vaccines,respectively,and monoclonal antibodies against human angiotensin II type 1 receptor and L-type calcium channel were prepared by hybridoma technology.Recombinant virus-like particles HBc Ag-CE12-CQ10 were bound with the monoclonal antibodies,or commercialized antibodies against HBc Ag respectively to detect the display of exogenous epitopes on the surface of recombinant virus-like particles HBc Ag-CE12-CQ10.Results: The recombinant protein was highly expressed in E.coli,mainly in the form of inclusion bodies.Recombinant proteins were prepared by inclusion body renaturation and protein purification.The recombinant protein successfully self-assembled into empty spherical virus-like particles with a diameter of about 30 nanometers.Monoclonal antibodies against human angiotensin II type 1 receptor and L-type calcium channel and commercialized antibodies against HBc Ag were used to bind to recombinant virus-like particles respectively.It was found that recombinant virus-like particles bind well with monoclonal antibodies against exogenous epitopes,but not with commercialized antibodies against HBc Ag.Conclusion: A recombinant virus-like particle HBc Ag-CE12-CQ10 was successfully constructed based on HBc Ag virus-like particles.The recombinant virus-like particles can display the two inserted exogenous epitopes at the same time,and may become a new bivalent antihypertensive vaccine.Part ? Effectiveness of the novel bivalent antihypertensive vaccine Objective: We developed a bivalent antihypertensive vaccine.In this study,we will use this vaccine to immunize hypertensive animal models to study the immunogenicity and the antihypertensive effect of the bivalent vaccine.Furthermore,to study the renal protective effect of the bivalent vaccine,spontaneously hypertensive rats were treated with L-NAME to induce renal injury.Methods:(1)In the first hypertensive animal model,spontaneously hypertensive rats were randomly divided into blank control group,L-NAME group,L-NAME + Qbeta-CQ10 vaccine group,L-NAME + Qbeta-CE12 vaccine group,L-NAME + amlodipine + valsartan group,and L-NAME + bivalent vaccine group.Each vaccine group was immunized with the corresponding vaccines on days 0,14,28,56,84 and 98,respectively.L-NAME + amlodipine + valsartan group was given amlodipine and valsartan by gavage since day 14.All L-NAME treated rats were administered L-NAME in drinking water from day 84 to 112.The blood pressure and heart rates of the animals were measured regularly by a non-invasive tail-cuff method.Antibody titers were detected regularly by ELISA.Before the end of the experiment,animal urine was collected by metabolic cages,and the urine protein content was measured.At the end of the experiment,kidneys of the animals were examined by Masson staining and transmission electron microscopy,and the pathological changes of the kidneys were analyzed.At the same time,serum samples were taken to determine the serum creatinine and urea nitrogen levels.(2)In another hypertensive animal model,BALB/c mice were randomly divided into blank control group,Ang II group,Ang II + amlodipine + valsartan group,and Ang II + bivalent vaccine group.Ang II + amlodipine + valsartan group was given amlodipine and valsartan by gavage since the 14 th day.Vaccine group was immunized with the bivalent vaccine on the 0th,14 th and 28 th day,respectively.The mice of each Ang II treated group were implanted subcutaneously with osmotic minipumps filled with Ang II on the 15 th day under sterile conditions to induce a hypertensive model.Blood pressure,heart rates and antibody titers of the animals were measured regularly.Results:(1)After the immunizations of the bivalent vaccine,specific antibodies against angiotensin II type 1 receptor and L-type calcium channel were successfully induced in spontaneous hypertensive rats,and systolic blood pressure of the rats were reduced effectively,with a maximum decrease of 25 mm Hg.After booster immunizations,the antihypertensive effect induced by the bivalent vaccine is superior to that induced by conjugated vaccines at certain extent.(2)In ang II-induced hypertensive mice,the bivalent vaccine also induced specific antibodies against angiotensin II type 1 receptor and L-type calcium channel,respectively.And the bivalent vaccine also reduced the systolic blood pressure of BALB/c mice effectively,with a maximum decrease of 35 mm Hg.(3)After L-NAME treatment,a few spontaneously hypertensive rats died.The mortality rate of bivalent vaccine group was slightly lower than that of L-NAME group,although the difference was not statistically significant.Further,the bivalent vaccine could effectively alleviate the renal fibrosis and glomerular basement membrane thickening caused by L-NAME.Besides,the bivalent vaccine also alleviated the elevation of serum creatinine,urea nitrogen and 24-hour urinary protein levels.Conclusion: The bivalent vaccine can induce specific antibodies against angiotensin II type 1 receptor and L-type calcium channel,and reduce the blood pressure of hypertensive animals effectively.Compared with the conjugated vaccines,the bivalent vaccine can be advantageous in reducing the blood pressure of hypertensive animals after booster immunizations.Besides,the bivalent vaccine can ameliorate L-NAME-induced renal injury effectively.Part ? Safety of the novel bivalent antihypertensive vaccine Objective: The bivalent antihypertensive vaccine can induce specific antibodies against therapeutic targets,and reduce the blood pressure of hypertensive animals effectively.We will evaluate the safety of the bivalent vaccine from different perspectives.Methods:(1)Before the end of the study on spontaneously hypertensive rats in part II,heart function of the animals were examined by ultrasonic examinations.The hearts,kidneys,livers and lungs of the animals were collected for pathological assessments at the end of the study.In addition,the kidneys were examined by transmission electron microscopy to detect electron dense deposits,especially in the glomerular basement membrane and mesangial area.(2)In another study,seven-week-old male BALB/c mice were randomly divided into three groups: Day 0 group,Day 7 group and Day 21 group.On day 0,all mice were immunized subcutaneously with 200 micrograms of bivalent vaccine.Mice in the Day 0 group were killed on the same day,and mice in Day 7 group were killed on day 7.Mice in Day 21 group were immunized subcutaneously with 200 micrograms of bivalent vaccine on the 14 th day,and were killed on the 21 st day.After the mice were killed,the spleen and serum were collected respectively.Lymphocyte differentiation in the spleen was detected by flow cytometry,and the Ig G antibody subtypes produced in the serum of mice were detected by ELISA kit.(3)HEK293 cells that stably expressing h ERG potassium channel and Na V1.5 sodium channel were prepared and cultured,respectively.Monoclonal antibodies against L-type calcium channel were co-incubated with the above cells,respectively.The effects of the monoclonal antibodies on h ERG potassium channel current and Na V1.5 sodium channel current were detected by patch clamp technique.Results: Echocardiographic examinations showed that the bivalent vaccine had no effect on the cardiac function of animals.No obvious inflammatory cell infiltration was found in important organs and tissues,and no immunocomplex deposits were found in the glomerular basement membrane and mesangial area.After immunization with bivalent vaccine,the differentiation of Th1 cells in the spleen decreased significantly,and the differentiation of Tfh cells was increased,and Ig G1 antibodies were mainly produced in the serum.Patch clamp experiments showed that monoclonal antibodies against L-type calcium channel had no significant effect on h ERG potassium channel current or Na V1.5 sodium channel current.Conclusion: After immunization with the bivalent vaccine,no obvious inflammation occurred in important tissues and organs,and humoral immune responses against the therapeutic targets were mainly induced.The possibility of malignant arrhythmia was low,and the bivalent vaccine was basically safe.