Ultrahigh-potent and broadly neutralizing anti-CD4 trimeric nanobodies inhibit HIV-1 infection by inducing CD4 conformational alteration

Abstract Despite substantial progress in antiretroviral therapy (ART) effectively suppressing HIV-1 replication in the bloodstream, a cure for HIV remains elusive. Existing antiviral drugs pose limitations, including lifelong medication, frequent administration, side effects, and viral resistance, necessitating novel HIV-1 treatment approaches. CD4, the primary receptor for HIV-1 entry into host cells, was once a prime target for drug or vaccine development. However, challenges, such as the potency and breadth of neutralization and cytotoxicity associated with anti-CD4 antibodies, hindered progress. Nevertheless, Ibalizumab, the sole approved CD4-specific antibody for HIV-1 treatment, reignited interest in exploring alternative anti-HIV targets, emphasizing CD4's potential value for effective drug development. Here, we investigated anti-CD4 nanobodies, with a focus on Nb457 isolated from a human CD4-immunized alpaca. Nb457 displayed remarkable ultra-high potency and broad-spectrum activity against HIV-1, surpassing Ibalizumab's efficacy. Importantly, Nb457 showed no impact on CD4 + T cell function, akin to Ibalizumab. Strikingly, engineered trimeric Nb457 nanobodies, Nb 457 -Nb HSA -Nb 457 , achieved 100% inhibition against live HIV-1, outperforming Ibalizumab and parental Nb457. Structural analysis revealed that Nb457 binding induced a CD4 conformational change, impeding viral entry. Molecular Dynamics simulations elucidated the structural basis for the complete inhibition of HIV-1 by Nb 457 -Nb HSA -Nb 457 . Furthermore, Nb457 exhibited significant therapeutic efficacy against HIV-1 infection in humanized mouse models. In conclusion, our study highlights ultra-potent anti-CD4 nanobodies as a compelling source of new HIV-1 therapeutics. The development of Nb457-based drugs holds the potential to revolutionize clinical HIV-1 treatment, providing a powerful tool in the battle against this persistent global health challenge.