Preclinical Immunogenicity Evaluation Of AIDS Multiple Antigen Peptide Vaccine And Construction And Packaging RAAV8 Which Express HIV-1 Broadly Neutralizing Antibodies

AIDS is a major infectious disease with a very high fatality rate.The infection of human immunodeficiency virus(HIV)is the cause of AIDS.Because the wide and rapid spread of HIV,AIDS serious impacted the health of human.Currently,the main therapeutic method of AIDS is reducing the viral load of HIV as much as possible and rebuilding the immune function of patients.At present,although cocktail therapy can minimize the viral load in patients to,it still has many shortcomings,such as drug resistance,side effects and high mortality.Therefore,the development of AIDS vaccines has always been the focus in the research field of AIDS prevention.Researchers isolated hundreds of broadly neutralizing antibodies which have cross-protective activity from AIDS elite controllers.broadly neutralizing antibodies can neutralize many types of HIV and remove the viruses floating in blood or in the infected cells.Therefore,broadly neutralizing antibodies can effectively inhibit the virus and partly control the development of AIDS.Therefore,in-depth study of broadly neutralizing antibodies is one of the important directions for the development of AIDS vaccine in the future.What is more,active immunotherapy and passive immunotherapy are important strategies to design and develop HIV vaccines to produce broadly neutralizing antibodies.Part ?:Preclinical immunogenicity evaluation of branched peptide vaccine against AIDSIn our previous studies,we have screened and identified the linear epitope 4E10based on HIV-1 MPER.And then,four branched peptide of 4E10 epitope was used as the immunogen and supplemented by oil adjuvant.The 4E10-MAP4 immunogen can induce binding antibodies with titers of 1×10~4-1×10~5 in guinea pigs,and produce93%and 73%neutralization activities for C and BC HIV-1,respectively.On this basis,we determined the optimal immune strategies,which were 50 and 100?g peptide with aluminium hydroxide as adjuvant for mice and guinea pigs respectively.And the immune process was once every two weeks and four times of subcutaneous immunization.As the results showed,the antibody titer was 1×10~4-1×10~5.Based on these results,we entrusted Changchun Baike Company to prepare the AIDS4E10-MAP4 vaccine.The long-term stability and accelerated stability study of the vaccine were tested.It was verified that the quality of this vaccine met the requirements of the Pharmacopoeia.And the validity period of the vaccine preparation was determined to be 36 months under the condition of 2-8?,1 month under 20-25?and 36±1?for storage and transportation conditions with stable physical and chemical properties.In order to lay a foundation for the preclinical and clinical trials of 4E10-MAP4vaccine,we evaluated the immunogenicity of 4E10-MAP4 branched peptide vaccine before clinical trials in this study,which including serum positive conversion rate,antibody titer,neutralizing antibody activity and antibody expression in tissues.After detecting the binding antibodies levels in the serum of cynomolgus monkeys and BALB/c mice,the results have showed that the positive conversion rate of the experimental animals was over 80%.What is more,the activity of induced binding antibodies were very high.The antibody titer of some individuals was over 2×10~5,indicating the immunogenicity of this vaccine was strong.The antibodies in cynomolgus monkeys have low neutralizing activity.When the serum is diluted at1:30,the highest neutralizing percentage is 52%,and the ID50>30.However,the ID50of antibodies obtained from BALB/c mice is less than 30.In order to study the distribution of antibodies induced by branched peptide vaccine,BALB/c mice received single immunization.The distribution of binding antibodies in serum and tissues of BALB/c mice was detected at 1,7 and 21 days after immunization.Binding antibodies were detected in some tissues such as spleen and liver.Antibodies in tissues may play an important role in many aspects,such as neutralizing viruses,stimulating immune responses.Therefore,the preclinical immunogenicity evaluation of the vaccine was conducted in the above aspects.Part ?:Construction and packaging rAAV8 which express HIV-1 broadly neutralizing antibodiesBroadly neutralizing antibody 10E8 has stronger neutralizing ability and wider range of action than 4E10 antibody which targeted HIV-1 MPER.The results in the previous study of Huang,J.et al has showed that,10E8 has neutralizing activity over98%of selected viruses.Antibody NIH45-46,which can act with CD4 binding sites on HIV-1,and great neutralizing activities.The broadly neutralizing antibody PG9,which has a circular structure,can closely binds to the head of HIV-1 trimer and acts on the V1/V2 region of gp120 and has strong binding ability and strong neutralization activity.Therefore,in order to produce high concentration of broadly neutralizing antibodies in the body,we conducted passive immunotherapy.Adeno-associated virus type 8,which has less pre-existing immunity in humans,can mediate long-term expression of exogenous genes.Therefore,in part II of this study,we used rAAV8 as vector to mediate the expression of broadly neutralizing antibodies 10E8,NIH45-46and PG9.As result has shown,four types of recombinant adeno-associated viruses:rAAV-NIH45-46,rAAV-PG9 and rAAV-RFP,were successfully packaged and purified.Our study lays a foundation for the production of broadly neutralizing antibodies10E8,NIH45-46 and PG9 mediated by recombinant adeno-associated virus type 8.In the next plan of our study,we will detect the expression efficiency of antibodies in vitro.And determine the in vivo activities of rAAV8 using BALB/c mice.We hope to obtain broadly neutralizing antibodies against HIV mediated by recombinant adeno-associated virus type 8,which can be expressed in a long-term manner,and provide a basis for the exploration of passive immunotherapy for AIDS.This will also provide a new vaccine candidate against AIDS.In summary,we analyzed the immune effect of 4E10-MAP4 branched peptide vaccine based on active immunization to induce broadly neutralizing antibodies.As the results showed,this vaccine has good immunogenicity,and could induce the binding antibodies which can reduce the viral load in tissues and protect AIDS patients.What is more,our study provided new research directions and relevant data for preclinical trials of vaccine preparations.At the same time,in order to induce broadly neutralizing antibodies against HIV-1,the construction and packaging of 8recombinant adeno-associated virus could express neutralizing antibody were conducted.Our research in passive immunization laid foundation for mediating the production of broadly neutralizing antibodies against HIV-1,provided a view to developing candidate vaccines for AIDS vaccine by passive immunotherapy.