| Background: Although highly active antiretroviral therapy(HAART)is effective in controlling HIV-1 infection,there are many problems such as poor medication compliance and drug resistance.Antibody-based strategies in HIV therapy has some potential advantages not associated with ART.Broadly neutralizing antibodies(b NAbs)can neutralize HIV-1 and prevent HIV-1 infection by specifically binding to HIV-1envelope glycoprotein gp120.Functional cure and even radical cure are very likely to play an important role and bring about subversive breakthroughs.However,the HIV infection situation in China is complex and changeable.At present,there is no rapid and reliable b NAbs screening method for domestic HIV epidemic strains,as well as for evaluation of affinity and neutralizing activities.Aim: The purpose of this study is to rapidly identify b NAbs that may have good affinity with domestic HIV-1 dominant strains by molecular docking,and further test their affinity and neutralizing ability.It is expected to serve as important guide for antibody and CAR-T immunotherapy focus on HIV-1 subtypes in China,and serve as a reference for the design of immunotherapy strategies for HIV-1 functional cure.Method: The molecular docking methods were used to identify b NAbs that show high affinity to HIV-1 gp120,which is prevalent in China.We expressed and purified four HIV-1 recombinant gp120 s from subtypes prevalent in China.Meanwhile,the b NAbs were cloned and transiently expressed in 293 F suspension cells under optimized conditions.The affinity of b NAbs binding to the gp120 s was measured by BLI assays.The backbone plasmid of HIV pseudovirus was further constructed and the packaged viruses were used to determine the neutralizing activity of b NAbs.Results: In this study,b NAbs targeting gp120 with high affinity to HIV-1 subtypes in China were successfully identified from the b NAbs discovered in the world: VRC01 antibody,N6 antibody,3BNC117 antibody;Three antibody expression plasmids and four gp120 expression plasmids were constructed.The 293 F suspension expression system was established and optimized for higher antibody yield in a defined,serum-free medium.The purified antibody and gp120 protein all have the purity more than >95%.Furthermore,we performed the BLI and neutralization test and confirmed that these 3antibodies have high affinity and neutralizing activity to HIV-1 subtypes prevalent in China.Conclusion: In this study,3 b NAbs were identified with better docking scores for domestic HIV-1 by molecular docking,and evaluated the affinity and neutralizing activity by BLI and neutralization test,respectively.The results showed that the three identified b NAbs had good affinity and broad-spectrum neutralizing activity against the four domestic HIV-1 epidemic strains,among which the N6 antibody had the best neutralizing effect.This finding provides an indication for the selection of the optimal b NAbs in the follow-up adoptive immunotherapy and CAR-T cell transfer therapy of the clinical trials,and provides a new prospective for the development of innovative HIV vaccine.The molecular docking approach to identify b NAbs also provides a new method for antibody drug discovery and development. |
