Construction of Synthetic Probiotic Bacteria for In Situ Delivery of Anti-SARS-CoV-2 Nanobodies

SARS-CoV-2 viral infection can be inhibited by blocking the interaction between the viral spike protein and the human receptor angiotensin-converting enzyme 2 (hACE2). The development of specific spike inhibitors using nanobodies, the antigen-binding region of llamas' antibodies, arose as a promising therapeutic method against SARS-CoV-2. However, one limitation of nanobodies is that they cannot be used directly in the human body due to their susceptibility to degradation. Bacteria-based delivery systems provide site-specific targeted action that can circumvent nanobody degradation. Here, we report the development of a genetically modified bacterium expressing anti-SARS-CoV-2 nanobodies that can inhibit the interaction between the hACE2 receptor and the receptor-binding domain (RBD) of the spike protein. Lactococcus lactis, a human symbiont probiotic bacterium, was selected to express nanobodies attached to their cell surface. Our data shows that FLAG-tagged anti-SARS-CoV-2 nanobodies were detected on the cell surface of recombinant L. lactis strains by flow cytometry and immunofluorescence without permeabilization. Furthermore, nanobodies are functional and can bind the RBD region from the spike protein in a dose-dependent manner. Inhibition of the hACE2-RBD interaction in cellular assays was quantified using a pseudotype lentivirus that mimics SARS-CoV-2 in an adaptation of the neutralization assay. Our results suggested that the recombinant bacteria can inhibit viral infectivity in more than 50% compared with a control without bacteria in a neutralization assay. These outcomes suggest that the engineered strain can be used in the future as a new therapeutic tool in COVID-19 prevention.