A Toxin-Conjugated Recombinant Protein Targeting gp120 and gp41 for Inactivating HIV-1 Virions and Killing Latency-Reversing Agent-Reactivated Latent Cells

Application of the combination antiretroviral therapy (cART) has reduced AIDS to a manageable chronic infectious disease. However, HIV/AIDS cannot be cured because of the presence of latent reservoirs, thus calling for the development of antiretroviral drugs that can eliminate latency-reversing agent (LRA)-activated HIV-1 virions and latent cells. In this study, we conjugated a small-molecule toxin, DM1, to a gp120-binding protein, mD1.22, a mutated CD4 domain I, and found that mD1.22-DM1 could inactivate HIV-1 virions. However, it could not kill LRA-activated latent cells. We then designed and constructed a dual-targeting protein, DL35D, by linking mD1.22 and the single-chain variable fragment (scFv) of a gp41 NHR-specific antibody, D5, with a 35-mer linker. Subsequently, we conjugated DM1 to DL35D and found that DL35D-DM1 could inhibit HIV-1 infection, inactivate HIV-1 virions, kill HIV-1-infected cells and LRA-reactivated latent cells, suggesting that this toxin-conjugated dual-targeting recombinant protein is a promising candidate for further development as a novel antiviral drug with potential for HIV functional cure. IMPORTANCE Although HIV-1 replication was successfully controlled by antiretroviral drugs, cure strategy for HIV-1/AIDS is still lacking. The long-lived HIV reservoir is considered one of the major obstacles to an HIV/AIDS cure. CD4-PE40 was the first drug that designed to kill HIV-1 infected cells; however, lower efficiency and high immunogenicity have limited its further development. In this study, we designed several dual-targeting recombinant proteins DLDs by linking gp120-binding protein mD1.22 and gp41-binding antibody D5 scFv with different length of linkers. Among them, DL35D with 35-mer linker showed the best anti-HIV-1 activity. We further conjugated the DM1 toxin to DL35D to produce DL35D-DM1, which maintained DL35D's inhibitory and inactivation activity against cell-free HIV-1 strains. Most importantly, DL35D-DM1 could specifically kill HIV-1-infected cells and LRA-reactivated-latent infected cells, suggesting that it is a proper candidate for development as a novel antiviral drug for use in combination with an LRA for HIV functional cure.

Automated summary

In this study, a dual-targeting recombinant protein, DL35D, was designed and constructed to simultaneously target HIV-1 virus and latent cells. DL35D-DM1, produced by linking the DM1 toxin to DL35D, showed inhibitory and inactivation activity against HIV-1 infection and effectively killed HIV-1-infected cells and LRA-reactivated latent cells, making it a promising candidate for the development of a novel antiviral drug for potential HIV functional cure.