Background
HIV-1 has been a global health problem for decades, and finding a cure remains a challenge. Recent studies have explored antibodies targeting various parts of HIV as alternative therapies. These antibodies bind the virus and enter the cell, where the complex is sensed and targeted for proteasomal degradation by the E3 ubiquitin ligase and antibody receptor TRIM21. Here we investigated the potential of this TRIM21 technology to develop intracellular immunity against HIV-1 infection. Our hypothesis is that intracellular recognition of HIV-1 by antibodies results in degradation of viral particles.
Methods
We designed nanobodies, based on llama antibodies, targeting the HIV-1 capsid protein p24, fused to the human IgG1 Fc domain. These p24 nanobody-Fc constructs were synthesized and produced by a mammalian expression system. Next, they were characterized by flow cytometry and tested for their functionality to bind p24 through an ELISA-based binding assay. Lastly, p24 nanobody-Fc constructs were stably expressed intracellularly in the U87-CD4-CCR5 cell-line. Engineered cells were infected with the HIV-1 strain NL4.3-BaL. Moreover, Fc-modified nanobodies were purified using column chromatography and transfected into cells previously infected with NL4.3-BaL. Quantification of p24 was done using ELISA and flow cytometry. Statistics were performed using two-way ANOVA.
Results
Our data show that our Fc-modified nanobodies recognize intact HIV-1 capsid and have a functional Fc domain. Notably, HIV-1 p24 capsid was neither detected intracellularly nor extracellularly after HIV-1 exposure in the p24 nanobody-Fc expressing cell-line, even though HIV-1 had integrated in the genome at levels comparable to control cells. These results demonstrate that the nanobodies are blocking production of new virus particles, rather than targeting HIV-1 during initial infection. In cells transfected with the purified nanobody a similar decrease of p24 was observed. These data strongly suggest that the Fc-modified p24 nanobody actively degrades p24 and thereby prevents new virus production.
Conclusions
To conclude, we have developed a novel p24 nanobody construct that efficiently prevents HIV-1 production. Intracellular production, as well as transfection of Fc-modified p24 nanobodies highly reduces p24 production levels in U87-CD4-CCR5 cells upon infection with HIV-1. This technology holds great promise for the engineering of therapeutic antibodies to prevent viral infections via targeting of host or viral proteins.