Antiretroviral therapy (ART) strategies against HIV-1 have been successful in suppressing HIV-1 replication and preventing disease progression. However, drug-resistance development, side-effects and life-long adherence of current drugs have driven the development of novel inhibitors against HIV-1. Inhibition of capsid production by targeting the highly conserved capsid protein (CA) has shown to be promising in blocking viral replication. Here, we developed a capsid-targeting biological composed of a single-domain Llama VHH that binds CA, fused to human IgG1 Fc to activate TRIM21-mediated degradation. This anti-capsid biological (aCA-Fc) efficiently bound CA. When expressed intracellularly aCA-Fc completely blocked HIV-1 production by degrading the capsid precursor Gag. Moreover, transfection of purified aCA-Fc during HIV-1 infection also resulted in degradation of Gag. aCA-Fc inhibited HIV-1 replication to similar extent as clinically approved capsid inhibitor lenacapavir (LEN). Interestingly, aCA-Fc didn't interfere with viral entry and reverse transcription, but eliminated newly produced HIV-1 Gag via TRIM21-mediated proteasomal degradation, even when added after infection. Gag production was restored by proteasome inhibition, introduction of the H433A mutation in the Fc domain and by TRIM21 knockdown using siRNA. These findings suggest that capsid-targeting biologicals, upon effective intracellular delivery, could serve as novel therapeutic strategies for viral suppression through intracellular protein degradation.
F. M. Stel, Charlotte E J Verkuijlen, E. Zijlstra-Willems et al.· Communications Biology· 1 citation
Antiretroviral therapy (ART) has proven effective in suppressing HIV-1 replication, but further development of HIV-1 inhibitors is continually driven by the challenge of drug resistance and viral adaptation. The HIV-1 capsid is a promising target for treatment due to its high sequence conservation as well as its crucial role in the viral life cycle. Recently, we have developed a novel capsid-targeting biologic that prevents HIV-1 replication by efficient degradation of newly synthesized capsid. Here, we have investigated the sensitivity to viral escape as well as the breadth of this biologic against HIV-1 subtypes. The capsid-targeting biologic efficiently blocked replication of different primary HIV-1 isolates, and continuous exposure of these viruses to the biologic resulted in viral breakthrough of two out of ten primary HIV-1 isolates tested. Notably, the breakthrough variants did not have amino acid changes in the nanobody epitope but primarily in the matrix region. The breakthrough variants remained sensitive to the biologic albeit to a lesser extent. In the absence of the biologic, breakthrough variants showed increased replication kinetics when compared to their parental virus, suggesting that adaption to the biologic is likely due to the increased viral production and that the target area of the biologic is too conserved for actual escape. This is further underscored by the broad specificity of the biologic as importantly the biologic blocked infection of different HIV-1 subtypes that occur worldwide (A, B, C, D, CRF01_AE, CRF02_AG). These results demonstrate the broad neutralization potential of anti-capsid biologics with a high barrier to resistance, making capsid-targeting inhibitors important for novel antiretroviral drug strategies worldwide.
F. M. Stel, E. Zijlstra-Willems, A. V. Nuenen et al.· International Journal of Mol...· 0 citations