Engineered red blood cells (RBCs) expressing viral receptors could be used therapeutically as viral traps as RBCs lack nuclei and other organelles required for viral replication. Here we show that the combination of a powerful erythroid-specific expression system and transgene codon optimization yields high expression levels of the HIV-1 receptors CD4 and CCR5, as well as a CD4-glycophorin A (CD4-GpA) fusion protein on enucleated RBCs. Engineered RBCs expressing CD4 and CCR5 were efficiently infected by HIV-1, but CD4 or CD4-GpA expression in the absence of CCR5 was sufficient to potently neutralize HIV-1 in vitro. To facilitate continuous large-scale production of engineered RBCs, we generated erythroblast cell lines stably expressing CD4-GpA or ACE2-GpA fusion proteins, which produced potent RBC viral traps against HIV-1 and SARS-CoV-2. Our results suggest that this approach warrants further investigation as a potential treatment against viral infections.

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