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

DOI record: { "DOI": "10.1007/s12602-025-10758-1", "ISSN": [ "1867-1306", "1867-1314" ], "URL": "http://dx.doi.org/10.1007/s12602-025-10758-1", "abstract": "<jats:title>Abstract</jats:title>\n <jats:p>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. <jats:italic>Lactococcus lactis</jats:italic>, 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 <jats:italic>L. lactis </jats:italic>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.\n</jats:p>", "alternative-id": [ "10758" ], "assertion": [ { "group": { "label": "Article History", "name": "ArticleHistory" }, "label": "Received", "name": "received", "order": 1, "value": "6 May 2025" }, { "group": { "label": "Article History", "name": "ArticleHistory" }, "label": "Accepted", "name": "accepted", "order": 2, "value": "27 August 2025" }, { "group": { "label": "Article History", "name": "ArticleHistory" }, "label": "First Online", "name": "first_online", "order": 3, "value": "11 September 2025" }, { "group": { "label": "Declarations", "name": "EthicsHeading" }, "name": "Ethics", "order": 1 }, { "group": { "label": "Competing interests", "name": "EthicsHeading" }, "name": "Ethics", "order": 2, "value": "The authors declare no competing interests." } ], "author": [ { "affiliation": [], "family": "Portero", "given": "Carolina E.", "sequence": "first" }, { "affiliation": [], "family": "Smith", "given": "Claire", "sequence": "additional" }, { "affiliation": [], "family": "Zhou", "given": "Yuxi", "sequence": "additional" }, { "affiliation": [], "family": "Marchán-Rivadeneira", "given": "M. Raquel", "sequence": "additional" }, { "affiliation": [], "family": "Wu", "given": "Shiyong", "sequence": "additional" }, { "affiliation": [], "family": "Han", "given": "Yong", "sequence": "additional" } ], "container-title": "Probiotics and Antimicrobial Proteins", "container-title-short": "Probiotics &amp; Antimicro. 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