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
ABSTRACT Human immunodeficiency virus 1 (HIV-1) remains a global health burden affecting over 39 million people worldwide, with approximately one million new infections occurring each year. Sexual transmission remains the predominant route of HIV-1 acquisition, during which the virus crosses mucosal barriers and interacts with host immune cells to facilitate infection. Dendritic cells (DCs) contribute to systemic viral spread and seeding of reservoirs by efficiently capturing HIV-1, leading to infection and subsequent transmission of the virus to CD4+ T cells. Siglec-1 (CD169) is a key receptor involved in HIV-1 capture, and its blockade may help prevent viral transmission. Here, we used newly developed single-domain antibodies, also known as nanobodies, against Siglec-1 as a candidate inhibitor to limit HIV-1 transmission. Using a Siglec-1-overexpressing cell line, we demonstrated that these nanobodies specifically blocked Siglec-1-mediated HIV-1 binding and transmission. Extending these findings to a more physiologically relevant context, the anti-Siglec-1 nanobodies neither induced immune nor cellular activation in DCs, indicating a favorable safety profile for functional applications. Most notably, the anti-Siglec-1 nanobody 2C2 effectively blocked HIV-1 binding as well as infection of DCs. Moreover, both replication-dependent and replication-independent HIV-1 transmission by DCs was also abrogated by the nanobody. Our findings not only underscore the relevance of Siglec-1 in HIV-1 capture but also highlight the therapeutic potential of utilizing host-targeting strategies against infectious diseases. IMPORTANCE Sexual transmission is the main route of human immunodeficiency virus 1 (HIV-1) infection, and novel interventions are needed to prevent this crucial first step. Mucosal dendritic cells play a key role by capturing HIV-1 via attachment receptors, leading to dendritic cell infection and subsequent transmission to T cells, thereby facilitating viral spread and establishment of infection. Here, we show that small, highly specific nanobodies targeting the attachment receptor Siglec-1 strongly interfere with this early stage of HIV-1 transmission. Siglec-1 nanobodies prevented HIV-1 binding to and infection of dendritic cells, thereby blocking transmission to T cells without inducing unwanted immune activation. Together, these findings identify Siglec-1 nanobodies as promising interventions and support the development of host-directed nanobody-based strategies to reduce HIV-1 spread. Sexual transmission is the main route of human immunodeficiency virus 1 (HIV-1) infection, and novel interventions are needed to prevent this crucial first step. Mucosal dendritic cells play a key role by capturing HIV-1 via attachment receptors, leading to dendritic cell infection and subsequent transmission to T cells, thereby facilitating viral spread and establishment of infection. Here, we show that small, highly specific nanobodies targeting the attachment receptor Siglec-1 strongly interfere with this early stage of HIV-1 transmission. Siglec-1 nanobodies prevented HIV-1 binding to and infection of dendritic cells, thereby blocking transmission to T cells without inducing unwanted immune activation. Together, these findings identify Siglec-1 nanobodies as promising interventions and support the development of host-directed nanobody-based strategies to reduce HIV-1 spread.
S. Man, A. Affandi, H. Brink et al.· Journal of Virology· 0 citations
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
