Why use L.gg?
Lactobacillus rhamnosus GG (LGG) is a GRAS probiotic recognized by the US FDA . It robustly adheres to and persists on both intestinal and upper-respiratory mucosae, where it forms a living barrier by competitively excluding pathogens from binding sites and nutrients. These properties, together with an extensive genetic toolkit (shuttle vectors, inducible/constitutive promoters, genome editing protocols), make L.gg an exceptional, safety-validated chassis for engineering an intranasal spray that delivers antiviral biologics directly at the nasal portal of respiratory infections.
Why use scfvs?
scFv—the minimal antibody unit built from VH and VL linked by a peptide—keeps full antigen-binding power while offering low MW, deep tissue penetration, short half-life and negligible immunogenicity: ideal for bacterial secretion antivirals.
Influenza A’s two surface antigens—hemagglutinin (HA) for entry and neuraminidase (NA) for release—are validated drug targets. We therefore selected the scFvs of MEDI8852 (anti-HA stalk) and 1G01 (pan-NA inhibitor). Secreted together from our engineered strain, they block both viral invasion and egress, affording dual-mechanism, strain-spanning protection.
How to neutralize influenza A virus?
Sensing System
Design Rationale
The system envisions an extracellular sensing module connected to an intracellular signaling module via a linker region. An engineered binding moiety situated outside the cell surface is intended to recognize a viral antigen on Influenza A particles, initiating a conformational response, transmitting the membrane-spanning signal to the inside of the cell. The intracellular signaling component is positioned to respond to the conformational cue, creating a cascade that influences downstream processes.
KxYKxGKxW signal peptide domain-containing protein
The signal peptide KxYKxGKxW is a protein-directed sequence that guides the directional transport of newly synthesized proteins to the cell membrane or extracellular environment [1].
eGFP
This part contains the DNA sequence for the EGFP protein. It can be placed behind any promoter of your choice and expressed with ease. Within the scope of our project this protein was expressed as a control. It is a highly fluorescent protein and could be easily analysed to reveal the workings of your promoter or other constructs.
scfv-MEDI8852
scFv-MEDI8852 broadly neutralizes multiple subtypes of influenza A virus by specifically binding to a conserved epitope on the hemagglutinin stem, recognizing the virus and blocking its fusion with host cells.
two-component system histidine kinase PnpS
The two-component system histidine kinase, as a constitutive element in Lactobacillus rhamnosus, undergoes a conformational change and becomes strongly activated upon receiving an interfering signal transmitted through the hinge region. It then releases a phosphate group, transferring the signal to the downstream response regulator protein in the two-component system, thereby initiating downstream signaling pathways.
Final Design
When InfluenzaA Virus Binds to ScFv :
① Transmembrane domains → Conformational changes
② Intracellular domain → Pnps Autophosphprylation
Phosphate group → transfers to PnpR → Activates downstream → Antiviral effector geneexpression
Response System
Design Rationale
The Response Module, serving as the execution core of the FluBlocker system, is designed to convert viral signals received by the Sensor Module into efficient, specific neutralizing antibody secretion actions. When viral binding signals trigger a phosphorylation cascade within the intracellular two-component system (TCS), the activated Response Regulator (RR) specifically initiates the Phob-inducible promoter, driving transcription of the entire downstream operon. We selected a highly efficient RBS to ensure translation efficiency and employed its naturally optimized signal peptide to guide the newly synthesized proteins into the secretion pathway. This enables the efficient release of mature single-chain antibodies scFv-MEDI8852 and scFv-1G01 into the extracellular space. The former targets the conserved stem region of influenza virus hemagglutinin (HA) to block viral entry[1], while the latter binds to the active site of neuraminidase (NA) to inhibit viral release[2]. Together, they form a dual-blocking mechanism against the viral life cycle. Simultaneously, the integrated mCherry reporter gene provides real-time fluorescent signals, visually indicating system activation status. This comprehensive design not only enables rapid and precise targeting of influenza viruses but also establishes an expandable engineering platform for future adaptation to other pathogens.
Biological Part Chosen
Final Design
Suicide System
Design Rationale
Temperature Sensitive System
The human nasal cavity is a narrow ecological niche whose temperature (≈ 30-34 °C) is lower than that of the lower respiratory tract (≥ 37 °C)[5]. If our probiotic strain is accidentally aspirated into the lungs or is released into the environment, uncontrolled proliferation could lead to either systemic pathology or environmental colonisation. We therefore need a two-tiered genetic firewall that senses the physical boundary between “permitted” and “forbidden” habitats and executes airreversible self-destruction programme once the boundary is crossed.
The design is split into two orthogonal kill switches:
Biological Part Chosen
Temperature-sensitive module
Blue Light-sensitive module
Final Design
Reference
[1]Ali SO, Takas T, Nyborg A, Shoemaker K, Kallewaard NL, Chiong R, Dubovsky F, Mallory RM. Evaluation of MEDI8852, an Anti-Influenza A Monoclonal Antibody, in Treating Acute Uncomplicated Influenza. Antimicrob Agents Chemother. 2018 Oct 24;62(11):e00694-18. doi: 10.1128/AAC.00694-18.
[2]Madsen A, Dai YN, McMahon M, Schmitz AJ, Turner JS, Tan J, Lei T, Alsoussi WB, Strohmeier S, Amor M, Mohammed BM, Mudd PA, Simon V, Cox RJ, Fremont DH, Krammer F, Ellebedy AH. Human Antibodies Targeting Influenza B Virus Neuraminidase Active Site Are Broadly Protective. Immunity. 2020 Oct 13;53(4):852-863.e7. doi: 10.1016/j.immuni.2020.08.015. Epub 2020 Sep 24. PMID: 32976769; PMCID: PMC7572813.
[3]Kallewaard NL, Corti D, Collins PJ, Neu U, McAuliffe JM, Benjamin E, Wachter-Rosati L, Palmer-Hill FJ, Yuan AQ, Walker PA, Vorlaender MK, Bianchi S, Guarino B, De Marco A, Vanzetta F, Agatic G, Foglierini M, Pinna D, Fernandez-Rodriguez B, Fruehwirth A, Silacci C, Ogrodowicz RW, Martin SR, Sallusto F, Suzich JA, Lanzavecchia A, Zhu Q, Gamblin SJ, Skehel JJ. Structure and Function Analysis of an Antibody Recognizing All Influenza A Subtypes. Cell. 2016 Jul 28;166(3):596-608.
[4]Stadlbauer D, Zhu X, McMahon M, Turner JS, Wohlbold TJ, Schmitz AJ, Strohmeier S, Yu W, Nachbagauer R, Mudd PA, Wilson IA, Ellebedy AH, Krammer F. Broadly protective human antibodies that target the active site of influenza virus neuraminidase. Science. 2019 Oct 25;366(6464):499-504.
[5]Wiesmiller K, Keck T, Leiacker R, Lindemann J. Simultaneous in vivo measurements of intranasal air and mucosal temperature. Eur Arch Otorhinolaryngol. 2007 Jun;264(6):615-9. doi: 10.1007/s00405-006-0232-6. Epub 2007 Jan 20. ；Lindemann J, Leiacker R, Rettinger G, Keck T. Nasal mucosal temperature during respiration. Clin Otolaryngol Allied Sci. 2002 Jun;27(3):135-9. doi: 10.1046/j.1365-2273.2002.00544.x.
[6]Sharma P, Mondal K, Kumar S, Tamang S, Najar IN, Das S, Thakur N. RNA thermometers in bacteria: Role in thermoregulation. Biochim Biophys Acta Gene Regul Mech. 2022 Oct;1865(7):194871. doi: 10.1016/j.bbagrm.2022.194871. Epub 2022 Aug 28.
[7]Naik RR, Kirkpatrick SM, Stone MO. The thermostability of an alpha-helical coiled-coil protein and its potential use in sensor applications. Biosens Bioelectron. 2001 Dec;16(9-12):1051-7. doi: 10.1016/s0956-5663(01)00226-3.
[8]Ohlendorf R, Vidavski RR, Eldar A, Moffat K, Möglich A. From dusk till dawn: one-plasmid systems for light-regulated gene expression. J Mol Biol. 2012 Mar 2;416(4):534-42. doi: 10.1016/j.jmb.2012.01.001. Epub 2012 Jan 8. Erratum in: J Mol Biol. 2014 Jan 24;426(2):500. PMID: 22245580.