Expression Of HIV Neutralizing Antibody By RAAV And RAdv

Since the discovery of human immunodeficiency virus(HIV),developing safe and effective vaccines is considered as the key strategy in controlling and treating AIDS.Because of the high variability and the susceptibility to drug resistance of the virus,AIDS vaccine has not been developed successfully so far.The treatment of broadly neutralizing antibodies(bNAbs)have been an important method in AIDS research,and shown good results in the prevention and treatment of HIV / AIDS.But the passive immunotherapy with bNAbs is too expensive,its wide range of applications in clinical treatment is impractical.Therefore,to establish safe and efficient expression of neutralizing antibody gene carrier to make the antibody have a long-term high expression in the body has become one of the key means of neutralizing antibody treatment of AIDS.In the previous research,the ideal construction scheme of adenovirus and adeno-associated virus vector expression neutralizing antibody gene were pDC316/eLcH and pSNAV/eLc H respectively.In this study,the recombinant Adenovirus vector and recombinant adeno-associated virus vector that contain the heavy and light chain of neutralizing antibody N60-B1.1,which had good neutralizing activity against HIV B subtype and C subtype virus,were constructed.Then to immunize mice individually and in combination with different recombinant virus(rAdv-N60?rAAV-N60)in order to express the antibody in mice for long-term and effectively.In this study,the heavy and light chain genes of N60-B1.1 were constructed in the adenovirus shuttle expression vector pDC316/eLcH and adeno-associated virus shuttle expression vector pSNAV/eLcH to obtain complete recombinant plasmid.The recombinant plasmid was transfected into BHK cells,then screening Stable Cell Strain by G418 Pressure Screening.The results showed that six cell lines that could stably express HIV monoclonal neutralizing antibody N60-B1.1 were obtained.After several rounds of selection,one cell which express the highest antibody was selected for expanding the cells in large quantities,and the expression of the supernatant was collected and purified to obtain the antibody.Binding and neutralizing activity of antibody were tested by ELISA and micro-neutralization assays,the results showed that the expression of the antibodies retained the biological activity.The purified antibody can also serve as a positive control for subsequent experiments.Secondly,the recombinant adenovirus(rAdv)and recombinant adeno-associated virus(rAAV)vaccine expressing N60-B1.1 neutralizing antibody were constructed.The recombinant virus was tested in cell level,and the virus titer was determined.Then,BALB/C mice and SCID mice were immunized in combination and separately by the two recombinant viruses.The expression of antibody in mice serum was detected at different time points after immunization,and the binding ability of neutralizing antibody expressed in SCID mice serum was detected by ELISA.The results showed that,the neutralized antibody N60-B1.1 was successfully expressed in mice,and the expression of antibody was still detected after 20 weeks.The antibody had a good binding activity with HIV antigen,but the expression of BALB/c mice was significantly lower than the SCID mice.The reason may be due to the BALB/C mice generated immune response to the virus vectors.In summary,this study successfully constructed recombinant plasmid N60-pDC316/eLcH and N60-pSNAV/eLcH with expression of neutralizing antibody N60-B1.1.The cell lines which can stably expressing HIV neutralizing antibody N60-B1.1 were screened.The recombinant virus containing the antibody gene can express the neutralizing antibody in animals for a long time and provide some experimental basis for the development of future HIV antibody vaccine.