Protocol to express anti-viral nanobodies and antigenic proteins anchored on the bacterial cell surface through the engineering of probiotic Escherichia coli Nissle 1917

Summary

Here, we present a protocol to express anti-viral nanobodies or antigenic proteins anchored on the bacterial cell surface through the engineering of probiotic E. coli Nissle 1917 (EcN). We describe steps for bacterial transformation, protein validation, outer membrane vesicle (OMV) isolation, and in vivo administration. We then detail procedures for immunological assessment through ELISA, neutralization assay, and flow cytometry analysis. This protocol can be adapted for different therapeutic targets beyond SARS-CoV-2.

For complete details on the use and execution of this protocol, please refer to Kamble et al.¹

Graphical abstract

Highlights

• •Steps for genetic engineering of probiotic E. coli Nissle 1917 for surface display
• •Instructions for outer membrane vesicle (OMV) isolation for nanobody transport
• •Oral dosing guidance for engineered EcN to safely induce targeted immune response
• •Procedure for functional assessment of pseudovirus neutralization post treatment

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we present a protocol to express anti-viral nanobodies or antigenic proteins anchored on the bacterial cell surface through the engineering of probiotic E. coli Nissle 1917 (EcN). We describe steps for bacterial transformation, protein validation, outer membrane vesicle (OMV) isolation, and in vivo administration. We then detail procedures for immunological assessment through ELISA, neutralization assay, and flow cytometry analysis. This protocol can be adapted for different therapeutic targets beyond SARS-CoV-2.

Before you begin

This protocol details the development of a dual-function, probiotic-based antiviral platform engineered from Escherichia coli Nissle 1917 (EcN) to provide both mucosal and systemic protection following oral administration as depicted in graphical abstract (1). Traditional antiviral therapies and vaccines typically require intravenous or intramuscular delivery of monoclonal antibodies or purified/inactivated antigens. Although effective, such approaches can be limited by high production and distribution costs, cold-chain dependency, needle-based administration, and insufficient induction of mucosal immunity. The emergence of rapidly evolving viral pathogens such as SARS-CoV-2, further underscores the need for adaptable, scalable platforms suitable for both prophylaxis and therapy. This protocol addresses these challenges through a modular bacterial engineering workflow that enables EcN to serve as an orally delivered, living antiviral and vaccine system. We describe two complementary strategies: 1. Passive immunization via bacterial surface display and outer membrane vesicle (OMV)- mediated delivery of antiviral nanobodies (eg., Ty1, VHH72), and 2. Active immunization via surface expression of viral antigens (e.g., SARS-CoV-2 Spike RBD) to elicit robust mucosal and systemic responses (Figure 1). Unlike approaches that focus solely on antigen exposure, this integrated platform enables immediate viral neutralization through nanobodies, while simultaneously promoting long-term immunity through antigen presentation. Oral administration eliminates needle-associated risks, supports self-delivery, and reliably induces mucosal immunity-an essential first-line defense against respiratory and gastrointestinal pathogens. We further report on the use of OMVs as a systemic delivery vehicle. Engineered EcN releases OMVs that transport nanobodies across mucosal barriers into distal tissues, including lungs and brain. Surface display modules are encoded using Intimin- or Lpp-OmpA–based anchoring systems, allowing flexible attachment of nanobodies or antigens, and enabling rapid redesign of the platform against other targets such as influenza or RSV(1). In vivo validation demonstrates that EcN-based constructs induce IgA and IgG responses, inhibit pseudovirus–ACE2 interactions, and recruit antigen-presenting and cytotoxic immune cells in mice establishing both immunogenicity and biological relevance. Overall, this protocol provides a customizable and scalable strategy for engineering EcN as a living antiviral delivery system. The approach integrates synthetic biology, immunology, and microbiome engineering, offering a versatile platform for next-generation mucosal vaccines and therapeutic probiotics targeting diverse and emerging pathogens.

Figure 1.

Dual-function engineering of E. coli nissle 1917 OMVs for viral neutralization and mucosal immunity

Schematic diagram of engineering Escherichia coli Nissle 1917 to surface express nanobodies and spike protein (Left panel). OMVs with nanobodies neutralize the virus and OMVs decorated with spike protein induce immune response at mucosal surfaces (Right panel).

Innovation

This protocol introduces a highly innovative platform by repurposing Escherichia coli Nissle 1917 (EcN), a safe and clinically used probiotic, into a living delivery system for both therapeutic nanobodies and viral antigens. The innovation lies in its dual strategy: passive immunization via nanobody-mediated viral neutralization and active immunization through antigen-driven adaptive responses. Unlike conventional vaccine protocols that focus exclusively on antigen exposure, this system integrates immediate protection with long-term immunity, establishing a more versatile and durable defense.

A second key innovation is the use of outer membrane vesicles (OMVs) secreted by engineered EcN as natural, biocompatible carriers. OMVs enable nanobody to transport across mucosal barriers and extend their biodistribution to systemic sites, including lungs and brain. This represents a major advance over traditional oral vaccines, which often struggle with antigen stability and delivery beyond the gut.

The protocol also features a modular cloning design, using interchangeable anchors such as Intimin and Lpp-OmpA for surface display. This modularity allows rapid customization for different targets, making the workflow adaptable to emerging pathogens like influenza, RSV, or future pandemic threats. Importantly, the oral route of administration promotes self-delivery, reduced costs, and improved accessibility, while also eliciting robust mucosal IgA responses that are critical for blocking viral entry at epithelial surfaces.

By merging synthetic biology, immunology, and probiotic engineering, this protocol pioneers a next-generation oral vaccine platform that is customizable, scalable, and translational. Its innovations address unmet needs in antiviral therapy, global vaccine accessibility, and mucosal immunity overcoming the key limitations of current approaches.

Institutional permissions

The protocols included in this manuscript utilize commercially available cells and reagents. This protocol can be easily adapted to the studies using probiotic bacteria to express nanobodies and antigenic proteins. Nevertheless, readers are reminded to follow all the necessary institutional guidelines for the use of such material. Experiments involving live animals must be approved by an Institutional Animal Care and Use Committee (IACUC).

Experimental methods involving recombinant DNA and engineered microbial strains were reviewed and approved by Institutional Biosafety Committee (IBC) of the University of Cincinnati. All animal experiments were conducted under protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Cincinnati (Protocol Number: 23-03-30-02). According to current regulations of the U.S. Department of Agriculture and Department of Health and Human Services, animals used in these experiments were maintained in an Association for Assessment and Accreditation of Laboratory Animal Care approved facility (Assurance #D16-00190).

Plasmid design

Timing: 1–2 days

Here, we describe the design and construction of modular plasmids for surface expression of nanobodies or antigenic proteins in EcN. This workflow (Table 1) enables users to generate customizable fusion constructs incorporating surface display anchors (e.g., Intimin, Lpp-OmpA or ClyA), flexible coning sites and optional detection elements such as FLAG or Strep II tags. The procedure begins with in silico sequence design, assembly of gene cassettes under appropriate regulatory elements (promoter, RBS, and terminator), and inclusion of optional protease cleavage sites (e.g., TEV) to facilitate downstream analysis.

Table 1.

Workflow timeline for bacterial transformation with timing

Step	Duration
Day 1: Streak and incubate	∼16 h
Day 2: Inoculation + culture	3–4 h
Cold washing and preparation	∼1.5 h
Aliquot and storage	∼15 min
Transformation	2.5 h

Following construct design, synthetic gene fragments are cloned into the desired plasmid backbone using restriction cloning or PCR-based assembly. The resulting plasmids are purified, quantified, and prepared for transformation into EcN. This modular strategy allows rapid adaptation to new nanobody targets or viral antigens, streamlining development of oral therapeutic or vaccine candidates.

• 1.
  Preparation of Synthetic DNA Constructs/Plasmids.

  • a.
    Select a plasmid backbone appropriate for your application (e.g., protein expression) from a source such as Addgene (https://www.addgene.org/).
  • b.
    Design your gene construct in silico using software like SnapGene® Viewer or DNA2.0. The construct should encode a nanobody or antigenic protein.
  • c.
    Choose an appropriate surface display anchor (e.g., Intimin, Lpp-OmpA, or ClyA) based on the display strategy.
  • d.
    Construct the fusion sequence with an N-terminal display tag (e.g., Intimin or Lpp-OmpA) followed by your gene of interest. Ensure the design includes multiple cloning sites (MCS) and relevant restriction sites for modular cloning.
  • e.
    Include a suitable promoter, ribosome binding site (RBS), Shine-Dalgarno (SD) sequence, and the proper start (ATG) and stop codons.
  • f.
    Incorporate epitope or purification tags (e.g., FLAG, Strep II) and optional cleavage sequences (e.g., TEV site) for downstream purification and detection.
  • g.
    Submit your finalized in silico design for synthesis to commercial providers such as GeneArt Synthesis (Thermo Fisher) or IDT.
• 2.
  Cloning and Assembly.

  • a.
    Upon receiving the lyophilized gene block or plasmid, dissolve in 1× TAE buffer.
  • b.
    Perform restriction digestion using 1 μg each of plasmid and insert:
  • c.
    Example Restriction Digest Setup (50 μL total):

    • i.
      DNA (1 μg): X μL.
    • ii.
      Restriction Enzyme 1: 1 μL.
    • iii.
      Restriction Enzyme 2: 1 μL.
    • iv.
      CutSmart Buffer (10×): 5 μL.
    • v.
      dH₂O: to 50 μL total volume.
  • d.
    Incubate digestion reaction at 37°C for 1 h to (16–24 hr).
  • e.
    Alternatively, perform PCR amplification of the geneblock using Phusion High-Fidelity DNA Polymerase with primers flanked by desired restriction sites.
• 3.
  Gel Purification and Quantification.

  • a.
    Resolve digested products by agarose gel electrophoresis.
  • b.
    Prepare 1% agarose gel in 1× TAE buffer with 3 μL SYBR Safe stain.
  • c.
    Melt agarose in microwave (∼1.5 min), pour into gel tray, allow to solidify.
  • d.
    Visualize bands using a UV-safe imager (e.g., Thermo Fisher).
  • e.
    Excise desired DNA bands and extract using a Gel Extraction Kit (e.g., NEB).
  • f.
    Quantify purified DNA using a Nanodrop or Qubit fluorometer.
• 4.
  Ligation.

  • a.
    Set up ligation reaction (20 μL total volume):

    • i.
      Vector (digested) 55ng (0.020 pmol): 3 μL.
    • ii.
      Insert (digested) 37.5 ng (0.059 pmol): 14 μL.
    • iii.
      T₄ DNA Ligase: 1 μL.
    • iv.
      T₄ Ligase Buffer (10×): 2 μL.
  • b.
    Gently mix the reactions by pipetting up and down and microfuge 3×.
  • c.
    Incubate ligation at 16°C for 16–24 hr.
  • d.
    Proceed to transformation using chemically competent E. coli Nissle 1917 cells (see following section).

Cell preparation

Timing: 1–2 days

• 5.Preparation of Chemically Competent E. coli Nissle 1917 Cells.

Here, we describe the preparation of chemically competent E. coli Nissle 1917 (EcN) for plasmid transformation. EcN cells are grown to mid-log phase, chilled, and washed with ice-cold CaCl₂ and glycerol to promote membrane permeability. The final suspension is aliquoted for immediate use or frozen at −80°C.

Note: Maintaining low temperature throughout is essential for preserving competency. This streamlined workflow provides reproducible, high-quality competent cells for downstream engineering applications.

Note: Prepare all required reagents and solutions, including sterile 0.1 mM CaCl₂ and 10% glycerol. Pre-chill the centrifuge to 4°C. Label and pre-cool sterile 50 mL Falcon tubes and 1.5 mL Eppendorf tubes on ice.

• 6.Protocol: Day 1: Bacterial Pre-Culture.

Streak out the Escherichia coli Nissle 1917 (EcN) on a LB-agar plate and incubate 16–24 hr. at 37°C.

• 7.
  Day 2: Growth and Competency Induction.

  • a.
    Inoculate a single EcN colony into 3 mL LB broth in a sterile culture tube.
  • b.
    Incubate 16–24 hr. at 37°C with shaking at 200 rpm.
  • c.
    The next morning, dilute 100 μL of the 16–24 hr. culture into 9.9 mL fresh LB broth in a 50 mL Falcon tube.
  • d.
    Incubate at 37°C with shaking at 200 rpm until the culture reaches an OD₆₀₀ of 0.7–0.9 (∼3–4 h).

Note: From this point on, all steps should be performed on ice or at 4°C to maintain cell competency.

• 8.
  Washing and Preparation.

  • a.
    Immediately place the culture on ice for 5 min.
  • b.
    Centrifuge at 2,500 rcf for 15 min at 4°C. Carefully discard the supernatant.
  • c.
    Resuspend the cell pellet in 10 mL ice-cold 0.1 mM CaCl₂. Centrifuge again at 2,500 rcf for 15 min.
  • d.
    Repeat this wash step with 5 mL ice-cold 0.1 mM CaCl₂.
  • e.
    Wash again with 2.5 mL ice-cold 0.1 mM CaCl₂.
  • f.
    Wash the pellet with 1 mL ice-cold 10% glycerol. Centrifuge as before.
  • g.
    Resuspend the final pellet in 1 mL ice-cold 10% glycerol.
• 9.
  Aliquoting and Storage.

  • a.
    Aliquot 50 μL per tube into pre-chilled 1.5 mL Eppendorf tubes.
  • b.
    Use freshly for transformation or store at −80°C for long-term use.

Pause point: Cells can be stored at −80°C with 25% Glycerol.

CRITICAL: Maintain starter cultures at an OD600 of 0.7–0.9 before preparing chemically competent cells or initiating downstream assays that rely on log-phase physiology. Deviations below OD600= 0.7 reduce competency and surface-display expression, while values above OD600=0.9 indicate entry into late log/stationary phase, which lowers transformation efficiency. For consistency, measure OD600 using a 1 cm pathlength cuvette and normalize CFU by plating when precise dosing is necessary.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Bacterial and virus strains
E. coli Nissle (EcN) 1917	Mutaflor	N/A
NEB DH5α cells	New England Biolabs	Cat# C2987H
Chemicals, peptides, and recombinant proteins
DYKDDDDK Tag Monoclonal Antibody (FG4R) HRP	Invitrogen (Thermo Fisher)	Cat# AB_2537626
Alexa Fluor®647 anti-mouse IgG2b	BioLegend	Cat# 406716
PE anti-mouse IgG2b	BioLegend	Cat# 406708
Purified anti-SARS-CoV-2 S protein S2 Antibody	BioLegend	Cat# 943202
Recombinant SARS-CoV-2 S protein S1+S2 (Carrier free)	BioLegend	Cat# 793706
Anti-ACE2 (E−11)	Santa Cruz Biotechnology Inc.	Cat# sc-390851
SARS-CoV-2 Spike Protein (RBD), mFc Tag	BPS Biosciences	Cat# 100684-2
ACE, His-Tag	BPS Biosciences	Cat# 11003-2
Anti-His-HRP	BPS Biosciences	Cat# 25011
Antibody for the detection of FLAG conjugated proteins (MOUSE) Monoclonal antibody DyLight 800 conjugated	Rockland	Cat# 200-345-383
COVID-19 vaccine mRNA SpikevaxTM	Moderna	Cat# 1234-021
β-actin antibody –MA5-15739-HRP	Invitrogen	Cat# AB_2537667
Streptavidin-HRP	Bio-Techne	Cat# DY998
LB broth	Thermo Fisher	Cat# 10855021
LB agar	Thermo Fisher	Cat# 22700041
CaCl2	Thermo Scientific	Cat# L13191.0I
Glycerol	Thermo Scientific	Cat# A16205.0F
Ampicillin	Sigma-Aldrich	Cat# A5354
Chloramphenicol	Sigma-Aldrich	Cat# C0378
Kanamycin	Sigma-Aldrich	Cat# K1377
Bacterial cell lysis buffer	Gold Bio	Cat# GB-177-100
LIVE/DEAD BacLight Bacterial Viability Kit	Invitrogen	Cat#L7012
Dithiothreitol (DTT)	Thermo Scientific	Cat# R0862
Brefeldin A (1000×) Solution	Invitrogen eBioscience (Thermo Fisher Scientific)	Cat#00-4526-51
Cell Stimulation Cocktail (500×) PMA-Ionomycin	Invitrogen eBioscience (Thermo Fisher Scientific)	Cat#00-4970-03
Paraformaldehyde	Milipore Sigma	Cat#30525-89-4
Ethylenediaminetetraacetic acid (EDTA)	Thermo Scientific	Cat# A10713.36
Lysozyme	Thermo Scientific	Cat# 89833
DNase	Thermo Scientific	Cat# EN0521
RNase	Thermo Scientific	Cat# EN0531
Triethylammonium bicarbonate (TEAB) buffer	Sigma-Aldrich	Cat# T7408
Tris-(2-carboxyethyl) phosphine (TCEP)	Sigma-Aldrich	Cat# C4706
Methylmethane-thiosulfonate (MMTS)	Sigma-Aldrich	Cat# 64306
Formic acid	Thermo Scientific	Cat# 270480250
RPMI media	Gibco	Cat# 11875093
Fetal Bovine Serum (FBS)	Gibco	Cat# A5256701
5% Penicillin-streptomycin	Gibco	Cat# 15070063
DMEM media	Gibco	Cat# 12491015
Instant Blue Coomassie	Abcam	Cat# ab119211
Pierce™ Silver stain kit	Thermo Fisher Scientific	Cat# 24612
Nitrocellulose membrane	GE Healthcare Life Sciences	Cat# GE10600002
Bovine serum albumin (BSA)	Thermo Scientific	Cat# B14
Tris-buffered saline (TBS)	Fisher Bioreagents	Cat# BP2471-100
Phusion High Fidelity polymerase	New England Biolabs	Cat# M0530S
DreamTaq DNA Polymerase	Thermo Scientific	Cat# EP0703
NEB restriction enzymes	New England Biolabs	Cat# B7001S
T4 DNA ligase	New England Biolabs	Cat# M0202S
Acetone	Thermo Scientific	Cat# L10407.0F
Glycine	Alfa Aesar	Cat# A13816.0C
PierceⓇ ECL Western Blotting Substrate	Thermo Scientific	Cat# 32106
Phosphate buffered saline (PBS)	Cytiva	Cat# SH30256.01
Formaldehyde	Fisher Brand	Cat# F79-1
Tritonx100	Fisher Bioreagents	Cat# BP151-100
Blocking buffer (3% BSA in 1×PBS with 0.1% TritonX100)	Invitrogen	Cat# 00-5523-00
Isoflurane	Covetrus	Cat# 11695-6777-2
Saline	Aspen	Cat# 46066-807-25
Carbon dioxide	N/A	N/A
Hank’s Balanced Salt Solution (HBSS)	Gibco	Cat# 14175-095
RBC Lysis buffer	Invitrogen	Cat# 00-4333-57
LIVE/DEAD Fixable Blue	Invitrogen	Cat# L23105
Anti Mo CD3 super Bright 600	eBioscience	Cat# 63-0037-42
Anti-MO CD4 Brilliant violet 500	eBioscience	Cat# 14-0041-82
Anti-Mo CD8a-Super Bright 702	eBioscience	Cat# 67-0081-82
Anti-Mo CD56 AlexaFluor 488	eBioscience	Cat# MHCD5620
Anti-Mo-CD11c PE	eBioscience	Cat# 12-0114-82
Anti-Mo CD45 PerCpCyanine5.5	eBioscience	Cat# 45-0459-42
AntiMO CD68-Brilliant violent786	eBioscience	Cat# 417-0681-82
Formalin	Carolina	Cat# 86-3533
Ethanol	Decon Laboratories	Cat# 2701
Critical commercial assays
Pierce BCA Protein Assay Kit	Thermo Scientific	Cat# 23225
Mouse Anti-SARS-CoV-2 Antibody IgG Titer Serologic Assay Kit	Acro Biosystems	Cat# RAS T023
SARS-CoV-2 (WT) Inhibitor Screening Kit (Spike RBD)	Acro Biosystems	Cat# EP-107
IgG ELISA kit	Thermo Fisher	Cat# BMS2091
CD8+ ELISA kit	MyBioSource	Cat# MBS2516313
CD4+ ELISA kit	MyBioSource	Cat# MBS2506108
IgA ELISA kit	Raybiotech	Cat# ELM-IGA-1
COVID-19 Pseudovirus Neutralizing Antibody Assay (Luciferase) Kit	Abnova	Cat# KA6152
Recombinant DNA
CJ23105 plasmid	This study	N/A
pIntimin-Ty1	This study	N/A
pLpp-OmpA-Ty1	This study	N/A
pIntimin-VHH72	This study	N/A
pLpp-OmpA-VHH72	This study	N/A
Software and algorithms
E. coli Nissle 1917 database	BioCyc	www.biocyc.org
Proteome discoverer v.2.4	Thermo Fisher	https://www.thermofisher.com/us/en/home/industrial/mass-spectrometry/liquid-chromatography-mass-spectrometry-lc-ms/lc-ms-software/multi-omics-data-analysis/proteome-discoverer-software.html?erpType=Global_E1
LFQ workflow	Thermo Scientific	https://www.thermofisher.com/us/en/home/industrial/mass-spectrometry/proteomics-mass-spectrometry/quantitative-proteomics-mass-spectrometry/label-free-quantitation.html
Sequest HT search algorithm	Thermo Scientific	https://docs.thermofisher.com/r/Proteome-Discoverer-3.1-User-Guide/en-US1330164619v1
Snap Gene Viewer4.1.8	SnapGene	https://www.snapgene.com/snapgene-viewer
FCS express	De Novo	https://denovosoftware.com/
ImageJ	ImageJ	https://imagej.net/ij/
Experimental models: Organism/strains
C57BL/6J mice (Mus musculus, 9 week old, male and female)	Jackson Laboratory	Cat# 000664
Experimental models: Cell lines
293h-ACE2	Abnova	Cat#KA6152
Other
BioPhotometer	Eppendorf (Marshall Scientific)	Cat# E-BP6131
Centrifuge	Eppendorf (Marshall Scientific)	Cat#EP-541-5R
Speed Vac Systems	Thermo Scientific	Cat# SRF110P1-115
Incubator	Fisher Scientific	Cat# 15-015-2633
Centrifuge 5415 R	Eppendorf (Marshall Scientific)	Cat# EP-5415R
Incubator Shaker	BT Lab Systems	Cat# BT921
96 well plates	Corning	Cat# 3841
NanoLC-MS/MS	Orbitrap Eclipse	Cat# FSN04-10000
ChemiDoc Imaging system	BioRad	Cat# 12003154
Vivaspin 20	Sartorius	Cat#VS2021
EnVision 2102 Multilabel Reader	Perkin Elmer	Cat# 25554
IVIS Spectrum In Vivo Imaging system	Perkin Elmer	Cat# CLS158738
Fluorescent microscope EnVision 2102	Leica Microsystems	Cat# M205 FA
8-chamber slide	Ibidi	Cat# 80826
LiCOR Biotech LLC C-Digit Blot Scanner	Li Cor Biotech LLC 360000 (Fisher Scientific)	Cat#NC2225395
Pierce Protein Concentrators	Thermo Fisher	Cat# 88517
SsoAdvanced Universal SYBR Green Supermix	BioRad	Cat#1725271
Amicon Ultra-0.5 Centrifugal Filter Unit	Millipore	Cat# UFC5010
C1000 Touch Thermal Cycler with Dual 48/48 Fast Reaction Module	BioRad	Cat#1851148
0.22μm PVDF	Thermo Fisher	Cat# EW-12917-17
Zetasizer Nano ZS	Malvern Instruments	Cat# UFC5010
Oral gavage	Fisher Scientific	Cat# 14-825-252
Microcentrifuge tubes	Axygen Malvern	Cat# MCT-060-AN/A
UV/Vis microplate spectrophotometer	Thermo Scientific	Cat# A51119600DPC
1-mL syringe	Becton Dickinson	Cat# 309628
Mouse Tracheal Catheters	SAI Infusion Technologies	Cat# TRA-03
Razor blades	American Line	Cat# 66-0412
AttuneNXT flow cytometer	Thermo Fisher Scientific	Cat# A28995CFR
DMi8 Widefield Fluorescence/Brightfield Microscope	Leica Microsystems	N/A
Micropipettes (2-1000 μL)	Milipore Sigma	Cat#EP3124000121-1EA
MicroAmp Endura Plate Optical 96-well Fast Clear Reaction plates	Applied Biosystems	Cat#A36930
Applied Biosystems Quant Studio 5 Real-Time PCR systems	Applied Biosystems	Cat#AB-QS5384
Invitrogen SuperScript IV VILO Master Mix	Invitrogen Thermo Fisher Scientific	Cat#11756050

Step-by-step method details

Molecular cloning of nanobodies and antigenic proteins: Transformation of E. coli Nissle 1917

Timing: ∼4 days

Here, we describe the transformation of EcN with plasmids encoding nanobodies or antigenic proteins using standard-heat-shock-based molecular cloning. Following plasmid introduction, transformed EcN are recovered, selected on antibiotic plate, and screened by colony PCR to verify correct insert incorporation. This workflow enables rapid generation of surface-display constructs for downstream characterization.

Note: Thaw the competent cells on ice and ensure the plasmid DNA is purified and quantified.

• 1.
  Transformation Steps.

  • a.
    Add 1–5 μL of plasmid DNA (10–100 ng) (pIntimin-Ty1 or pLpp-OmpA-Ty1 plasmids) to 50 μL of thawed competent EcN cells.
  • b.
    Gently mix by pipetting or tapping (do not vortex).
  • c.
    Incubate the mixture on ice for 30 min.
  • d.
    Heat shock the cells at 42°C for 45 s.
  • e.
    Immediately transfer the tubes back to ice for 2 min.
  • f.
    Add 950 μL of LB broth (no antibiotics) to each tube.
  • g.
    Incubate at 37°C for 1 h with shaking at 200 rpm to allow expression of antibiotic resistance.
  • h.
    Plate 100–200 μL of the transformation mixture onto LB agar plates containing the appropriate antibiotic.
  • i.
    Incubate plates 16–24 hr. at 37°C. Transformed colonies should be visible the next day. Use colony PCR or restriction digestion to confirm successful plasmid uptake.
• 2.
  Colony PCR Screening for Insert Confirmation.

  • a.
    Pick 3–6 single colonies using a sterile pipette tip or toothpick.
  • b.
    Transfer each colony onto a fresh LB agar plate (for backup) and into a PCR tube containing 20 μL PCR mix.
• 3.
  PCR Reaction Mix (per reaction):

  • a.
    Forward primer (10 μM): 0.5 μL.
  • b.
    Reverse primer (10 μM): 0.5 μL.
  • c.
    Template (single colony): directly inoculated.
  • d.
    dNTPs (10 mM): 0.5 μL.
  • e.
    10× Buffer: 2 μL.
  • f.
    Taq DNA polymerase: 0.2 μL.
  • g.
    dH₂O: to 20 μL total volume.
• 4.Run PCR under suitable conditions depending on primer annealing temperature and expected amplicon size.
• 5.Load PCR products onto a 1% agarose gel stained with SYBR Safe or ethidium bromide.
• 6.Confirm presence of insert by expected band size.

Note: Transformed EcN colonies should yield positive insert confirmation by colony PCR and sequencing. In Kamble et al., 2025 (Fig. 2A-B, Supplementary Fig. S2), validated constructs for nanobody and spike protein display are shown.

Note: Positive colonies can be cultured in 3–5 mL LB + antibiotic 16–24 h. for plasmid miniprep and restriction digest sequencing verification.

CRITICAL: It is critical to perform transformation at exactly 42°C for 45 s for highest efficiency of transformation. For uniform heat shock, add water in the headblock or set water bath temperature to 42°C prior to transformation and heat shock.

Surface display validation

Timing: ∼2 days

Here, we describe a fluorescence-based workflow to verify successful surface display of nanobodies or antigenic proteins on engineered E. coli Nissle 1917 using recombinant Spike binding followed by antibody staining. Display is assessed quantitatively by flow cytometry and qualitatively by fluorescence microscopy, enabling confirmation of antigen accessibility on intact, non-permeabilized bacteria.

• 7.Grow transformed EcN containing pIntimin-Ty1 or pLpp-OmpA-Ty1 plasmids using WT EcN as a control to OD₆₀₀= 0.6 to 0.8, using a 16–24 hr. cultures.
• 8.Harvest 1 mL of culture, wash with sterile PBS.
• 9.Incubate bacteria with recombinant spike protein (2 μg/mL) for 30 min at RT.
• 10.Wash and stain with Alexa Fluor-647 conjugated anti-spike antibody.
• 11.After staining with recombinant Spike and Alexa-647 anti-Spike antibody, successful surface display should produce a clear, reproducible fluorescence signal on intact (non-permeabilized) bacteria that is distinguishable from all negative controls. Analyze by Flow cytometry or Fluorescence microscopy.
• 12.
  Flow cytometry (recommended quantitative readout).

  • a.
    Acquire ≥10,000 events per sample; use FSC/SSC to gate bacteria, then gate singlets (FSC-A vs FSC-H) and viable cells if using viability dye such as LIVE/DEAD™ BacLight Bacterial Viability Kits (Cat No#L7012).
  • b.
    Controls required: wild-type EcN (no plasmid), isotype or secondary-only control, and unstained bacteria. Optionally include a positive display control (known surface-display construct).
  • c.
    Metrics to report: (%) positive cells (percent events above fluorescence threshold) and median fluorescence intensity (MFI). Set threshold using negative control (e.g., gate so <1%–2% of WT EcN are positive).
  • d.
    Successful outcome: a unimodal or clearly shifted population with ≥30% positive cells and ≥1.5–2.0-fold increase in MFI over WT/secondary control. Robust display often yields 40%–90% positive depending on anchor and induction conditions. Report mean ± SD from biological replicates.
• 13.
  Fluorescence microscopy (qualitative/spatial validation).

  • a.
    Image intact, non-permeabilized bacteria at 60–100× oil with appropriate filter for Alexa-647. Include DIC/phase contrast.
  • b.
    Successful outcome: membrane-localized fluorescence surrounding bacteria (ring pattern), not diffuse cytoplasmic signal. Fluorescence should be absent in WT and secondary-only controls. Capture multiple fields and quantify % fluorescent cells if possible. Representative fluorescence microscopy images are provided in Kamble et al., 2025 (Fig. 3, Supplementary information Fig. S4).

CRITICAL: Maintain bacteria on ice during staining to prevent internalization of artifacts.

OMV isolation

Timing: ∼2 days

Here, we outline a standardized OMV isolation workflow involving bacterial culture, removal of cells and debris, and sequential filtration and ultracentrifugation to enrich purified vesicles suitable for downstream biochemical and immunological analyses. This approach yields reproducible OMV preparations and can be readily adapted for different EcN variants or related Gram-negative species.

• 14.Grow engineered EcN and WT-EcN 16–24 hr. at 37°C and 200 rpm.
• 15.Inoculate 1% of this culture into 1L fresh LB broth and grow until OD600 of ∼1.5 Unit.
• 16.Remove bacteria from the cultures by centrifugation at 8000 × g, 4°C, 15 min.
• 17.Concentrate the supernatant using Pierce Protein Concentrators (30 kDa, ThermoFisher #88531).
• 18.70mL of the concentrated supernatant obtained following Pierce Protein Concentration process using 30 kDa filters can be further concentrated by ultracentrifugation at 91,000 × g for 4.0hr.
• 19.Pellet obtained after ultracentrifugation, resuspend in 1×PBS, pH 7.4, followed by washing with Amicon Ultra-0.5 Centrifugal Filter Unit (Millipore#UFC500308) filters to completely remove the residual media. Sterile filter the dispersion through 0.22μm PVDF syringe filters (Cole-Parmer#UX-06060-62).
• 20.Characterize OMV size and size distribution using Dynamic Light Scattering (Zetasizer Nano ZS, Malvern Instruments). Successful OMV preparations typically show a size distribution in the range of 50–200 nm, consistent with outer membrane vesicles. The majority of particles should fall within a single, narrow peak.
• 21.The polydispersity index (PDI) should ideally be <0.3, indicating a relatively uniform vesicle population. Broader peaks or PDI >0.3 may suggest protein aggregates, incomplete removal of cell debris, or heterogeneous vesicle populations.
• 22.Representative results are-mean diameter ∼100 nm, unimodal peak, and stable scattering intensity over time.
• 23.Quantify OMVs with a Pierce™ BCA Protein Assay kit (Themo Scientific), using BSA standards.
• 24.Isolated OMVs exhibit a unimodal size distribution around 100 nm with PDI <0.3 by DLS. Transmission electron microscopy (TEM) confirms vesicle morphology (Kamble et al., 2025, Fig. 4, Supplementary Fig. S7).

Note: OMVs can be aliquoted and stored at −80°C.

CRITICAL: Grow bacteria to OD600=∼1.5

Oral administration of engineered bacteria in mice

Timing: ∼5 days (treatment + recovery)

Here, we describe the oral delivery of engineered E. coli Nissle 1917 (EcN) to mice to evaluate colonization and host responses. This workflow details preparation of bacterial suspensions, daily gavage, and post-treatment monitoring. Proper technique and welfare considerations are essential to minimize stress and ensure consistent dosing.

Note: All animal procedures should follow institutional and national ethical guidelines. Oral gavage can induce stress; therefore, handling should be minimized, and refinements such as habituating mice to gentle restraint prior to experiments are recommended.

• 25.Prepare fresh cultures of engineered EcN at 10⁷ CFU/mL.
• 26.Gavage 100 μL (10⁶ - 10⁷ CFU) daily for 4 consecutive days.
• 27.Monitor mice for distress, weight loss and temperature change.
• 28.Mice tolerate daily gavage without significant weight loss or distress. Colonization of engineered EcN is confirmed by plating fecal samples as reported in Kamble et al., 2025 (Fig. 10, Supplementary Fig. S10).

CRITICAL: Oral gavage should only be performed by trained personnel to minimize distress and risk of injury. Use appropriately sized gavage needles (20–22 gauge, 25–38 mm, with rounded ball tip) based on mouse age and weight. Immobilize mice gently but securely to avoid aspiration. Gavage volume should not exceed 10 mL/kg body weight (typically ≤200 μL for adult mice) per administration to prevent gastric over-distension. Ensure solutions are at 24°C and free of bubbles. Mice should be monitored closely during and after gavage for signs of distress, weight loss, or aspiration. If repeated administration is required, rotate handlers and limit daily handling time to reduce stress.

Immune response analysis

Timing: ∼3–5 days

Here, we describe methods to evaluate humoral and cellular immune responses induced by engineered EcN following oral administration. This includes quantification of antigen-specific IgG and IgA by ELISA, assessment of neutralizing activity using a pseudovirus-luciferase reporter assay, and characterization of cytokine-producing T cell populations by flow cytometry.

Note: Together, these assays provide a comprehensive readout of mucosal and systemic immunity and allow functional validation of nanobody- or antigen-based interventions.

ELISA

• 29.Collect Blood, Feces, Intestines, and (Bronchoalveolar lavage) BALF samples.
• 30.Coat ELISA plates with Spike Protein.
• 31.Detect IgG and IgA with HRP-conjugated secondary antibodies.
• 32.ELISA shows robust induction of serum IgG and mucosal IgA against Spike antigen. Pseudovirus neutralization assays demonstrate reduced luciferase activity in EcN-treated mice compared to controls (Kamble et al., 2025, Fig. 5, Supplementary Fig. S6, Table S4).
• 33.
  Pseudovirus Neutralization.

  Note: This assay evaluates the ability of samples (OMVs, serum, purified antibodies, or engineered bacteria) to neutralize pseudoviruses carrying the SARS-CoV-2 spike protein and a luciferase reporter. The reduction in luciferase signal reflects inhibition of viral entry.

  • a.
    Preparation of virus–sample mixtures.

    • i.
      Prepare serial dilutions of test samples in assay medium as follows:
    • ii.
      Serum samples: prepare 3-fold serial dilutions starting at 1:20.
    • iii.
      Purified antibodies: prepare 2-fold serial dilutions starting at 10 μg/mL.
    • iv.
      OMVs: normalize concentration using protein content (BCA assay), then prepare 2-fold dilutions starting at 100 μg/mL protein equivalent.
    • v.
      Bacteria (EcN): normalize to OD₆₀₀ = 1.0 (∼10⁸ CFU/mL), then prepare 10-fold serial dilutions to achieve ∼10⁸–10⁵ CFU equivalents.
    • vi.
      Mix 50 μL of diluted sample with 10 μL of pseudovirus expressing luciferase.
    • vii.
      Incubate the mixture at or 24°C for 30 min to allow neutralization.
  • b.
    Preparation of target cells.

    • i.
      Seed 293T-hACE2 cells (∼1 × 10⁵ cells/well) in a 24-well plate and culture at 37°C for ∼4 h until cells adhere.
    • ii.
      Remove media, wash cells twice with 1× PBS, and detach with Cell Dissociation Media (HiMedia) for 5 min at 37°C.
    • iii.
      Neutralize dissociation with complete medium, centrifuge at 300 × g for 5 min, and resuspend to 2 × 10⁵ cells/mL.
  • c.
    Infection.

    • i.
      Add 60 μL of the virus–sample mixture (from Step A3) to the prepared cells.
    • ii.
      Incubate plates at 37°C for 48 h.
  • d.
    Luciferase readout.

    • i.
      Wash cells with 200 μL 1× PBS.
    • ii.
      Lyse cells with 100 μL Luciferase Cell Lysis Reagent, scrape, vortex 10–15 s, centrifuge at 12,000 × g for 30 s, and keep lysates on ice.
    • iii.
      In a 96-well plate, mix 10 μL lysate with 50 μL Luciferase Assay Reagent.
    • iv.
      Measure luminescence immediately using an EnVision 2102 Multilabel Reader (PerkinElmer).
    • v.
      Flow cytometry of splenocytes reveals increased IFNγ⁺ and TNFα⁺ T cell populations (Kamble et al., Fig. 16).

      CRITICAL: Always prepare matched negative and positive control samples on each ELISA plate (e.g., pre-immune serum and a known positive control). Report sample dilutions explicitly (serum 1:20 start; fecal extracts normalized by weight). Use triplicate wells and compute ED50 or endpoint titer using a consistent cutoff (mean of negative controls + 3× SD).

      CRITICAL: For pseudovirus neutralization assays, always verify pseudovirus infectivity on control wells prior to the assay and use technical duplicates. Normalize OMV concentration by protein (BCA) and include a cell-only control to quantify background luminescence.

      Note: All pseudovirus experiments described in this protocol should be performed under BSL-2 containment following institutional biosafety committee approval. Pseudoviruses used here (e.g., SARS-CoV-2 Spike-pseudotyped lentivirus expressing luciferase) are replication-deficient and lack the full viral genome, making them safe surrogates for viral entry studies. Nevertheless, they retain the potential for cell entry and transgene expression; therefore, standard BSL-2 precautions are required, including use of a Class II biosafety cabinet, PPE (lab coat, gloves, eye protection), and appropriate waste decontamination (e.g., bleach or autoclaving). Do not handle pseudoviruses in open culture areas or outside of approved BSL-2 spaces.

Flow cytometry

Timing: ∼2 days (cell preparation, stimulation, staining, acquisition, and analysis)

Here, we describe a flow cytometry workflow to quantify antigen-specific cellular immune responses following oral administration of engineered EcN. This includes isolation of splenocytes, ex-vivo stimulation for cytokine induction, surface and intracellular staining, and multiparametric analysis of innate and adaptive immune subsets.

Note: The assay enables detection of key effector populations-including CD4⁺ and CD8⁺ T cells, dendritic cells, macrophages, and NK cells as well as cytokine responses (e.g., IFNγ, TNFα), providing a comprehensive functional readout of cellular immunity induced by probiotic-based immunization.

• 34.
  Prepare single-cell suspensions of splenocytes.

  • a.
    Harvest spleens aseptically from treated or control mice.
  • b.
    Pass through a 70 μm strainer into cold RPMI with 2% FBS.
  • c.
    Lyse red blood cells using RBC lysis buffer, wash twice with PBS, and resuspend at 1 × 10⁶ cells per sample.
• 35.
  Stimulation for cytokine analysis.

  • a.
    For intracellular cytokine staining, stimulate cells with 50 ng/mL PMA + 500 ng/mL ionomycin in the presence of Brefeldin A (protein transport inhibitor) for 4–6 h at 37°C, 5% CO₂.
  • b.
    Unstimulated cells should be included as negative controls.
• 36.
  Surface staining.

  • a.
    Wash cells with FACS buffer (PBS + 2% FBS).
  • b.
    Stain with antibodies against surface markers: CD3 (T cells), CD4 (helper T cells), CD8 (cytotoxic T cells), CD11c (dendritic cells), CD68 (macrophages), CD56 (NK cells), and anti-Flag (for tracking bacteria or bacterial proteins).
  • c.
    Incubate for 30 min at 4°C, protected from light. Wash twice with FACS buffer.
• 37.
  Fixation and intracellular staining.

  • a.
    Fix cells with 2% paraformaldehyde, then permeabilize with saponin-based buffer.
  • b.
    Stain for intracellular cytokines (IFNγ, TNFα). Include isotype controls for gating.
• 38.Acquisition.

Run samples on a flow cytometer (e.g., Attune NXT or equivalent). Acquire ≥50,000 live events per sample. Use single-stain and fluorescence-minus-one (FMO) controls for compensation and gating.

• 39.
  Analysis.

  • a.
    Gating strategy: exclude debris (FSC vs SSC), select singlets (FSC-A vs FSC-H), and gate live cells (viability dye).
  • b.
    Subset into CD3⁺ T cells - CD4⁺ or CD8⁺; CD11c⁺ dendritic cells; CD68⁺ macrophages; CD56⁺ NK cells.
  • c.
    Quantify frequency and mean fluorescence intensity (MFI) of IFNγ⁺ and TNFα⁺ cells in each subset.
  • d.
    Compare treated vs. control groups. Successful assays yield clear separation of immune subsets and measurable induction of IFNγ and TNFα after stimulation. Engineered EcN-treated mice are expected to show increased cytokine-producing T cells and innate populations compared to controls.

CRITICAL: Cell viability and staining consistency are crucial for reliable flow cytometry data. Ensure gentle tissue dissociation and avoid prolonged stimulation or harsh centrifugation, which can damage cells and artificially elevate background cytokine signals. Maintain all samples on ice or at 4°C during staining to minimize metabolic activity and nonspecific marker expression. For intracellular cytokine staining, strictly control stimulation time (4–6 h) and Brefeldin A concentration overexposure can reduce cytokine detectability and cell recovery. Compensation and gating accuracy depend on properly prepared single-stain and fluorescence-minus-one (FMO) controls; failure to include these can lead to misidentification of rare cell populations. Finally, acquire a consistent number of live events (≥50,000 per sample) and use the same voltage and compensation settings across all groups to ensure data comparability.

Expected outcomes

This protocol enables the successful engineering of Escherichia coli Nissle 1917 (EcN) to express antiviral nanobodies or spike proteins on the bacterial surface and to package them into outer membrane vesicles (OMVs). Upon oral administration, these engineered EcN strains are expected to yield both mucosal and systemic immune responses. Key anticipated results include an efficient surface display. The transformed EcN colonies should express nanobodies (e.g., Ty1 or VHH72) or spike-RBD antigens fused with surface display anchors (e.g., Intimin, Lpp-OmpA), as confirmed via flow cytometry and/or fluorescence microscopy using tagged antibodies. This protocol will also be used for high-yield OMV isolation. Engineered EcN will secrete OMVs containing nanobodies or spike proteins, confirmed by nanoparticle size distribution (Zetasizer), protein quantification (BCA assay), and TEM imaging.

This protocol will also generate systemic and mucosal immunogenicity. Following oral gavage in mice, significant levels of serum IgG (systemic response) and faecal and bronchoalveolar lavage fluid (BALF) generated IgA (mucosal response) against the spike protein antigen were detectable by ELISA. Using this protocol, functional virus neutralization can be performed. Serum and OMVs isolated from treated mice are expected to reduce luciferase activity in a pseudovirus-ACE2 cell binding assay, indicating neutralizing activity. Flow cytometry analysis of splenocytes will show increased populations of activated CD4⁺ and CD8⁺ T cells, as well as increased production of cytokines (e.g., IFNγ⁺ and TNFα⁺) upon stimulation, confirming a cellular immune response. Mice are expected to tolerate the oral EcN treatment without significant weight loss, temperature changes, or clinical distress, supporting the platform’s suitability for in vivo use.

Quantification and statistical analysis

Statistical comparisons are performed via One-Way ANOVA with Tukey’s post-hoc test and significance set at p < 0.05. Graphs were generated using GraphPad Prism 8.0v.

Limitations

While this protocol provides a versatile platform for engineering Escherichia coli Nissle 1917 (EcN) as an oral therapeutic and vaccine delivery system, several limitations should be considered. Although EcN is a well-characterized probiotic, its use as a recombinant delivery vehicle in humans remains experimental, and long-term colonization dynamics, stability, and potential interactions with the native microbiome have not been fully defined. Additional preclinical safety and biodistribution studies will therefore be necessary before translational application.

A second limitation is the reliance on pseudoviruses and mouse models for functional validation. Pseudoviruses are safe surrogates for viral entry assays, but they do not recapitulate the full replication cycle, pathogenesis, or immune evasion strategies of live viruses. Similarly, murine models, while widely accepted in preclinical studies, differ from humans in gut physiology, microbiota composition, and immune responses. Thus, while engineered EcN blocked pseudovirus entry in vitro and elicited immune responses in mice, it remains uncertain whether the same protective effects will occur against authentic viral infections in humans. Importantly, no live virus challenge or disease model was tested, leaving the true therapeutic and prophylactic potential of this system unverified.

In addition, the efficiency of surface display and OMV secretion may vary depending on bacterial fitness, growth conditions, and host-specific factors, leading to variable outcomes. Oral delivery further introduces challenges such as gastric acidity and proteolytic degradation, which may reduce the stability and bioactivity of therapeutic proteins unless additional protective formulations are employed.

In summary, while this protocol establishes a promising foundation for probiotic-based vaccines and therapeutics, its translation to clinical application will require optimization, testing in live virus challenge models, and thorough safety evaluations in more human-relevant systems.

Troubleshooting

Problem 1 (step 1, h and i)

No colonies after transformation.

Potential solution

Lack of colonies after transformation could be a result of poor bacterial competency or a degraded plasmid. Prepare fresh competent cells and use freshly prepped plasmid.

Problem 2 (step 1, i)

Low transformation efficiency.

Potential solution

This could be caused by inadequate heat shock or ice incubation. Verify ice incubation is 30 min and heat shock is 42°C for 45 sec.

Problem 3 (step 1, i)

Pellet hard to resuspend.

Potential solution

Difficulty resuspending the pellet is most likely due to excessive or high-speed centrifugation or rough pipetting. Reduce spin speed or time and pipette gently.

Problem 4 (step 1, i)

Contamination on agar plates.

Potential solution

Contamination is due to poor sterile technique. Make sure to use sterile pipette tips and media.

Problem 5 (step 7)

Weak surface display signal expression.

Potential solution

Weak surface display signal expression might have been due to a low expression or plasmid loss. Reconfirm antibiotic selection and plasmid stability.

Problem 6 (step 10)

Low Signal.

Potential solution

Increase antibody concentration (e.g., 1:100 to 1:50), extend incubation to 45–60 min at 4°C, verify induction/promoter activity, confirm construct sequence and frame, check codon optimization.

Problem 7 (step 11)

High background.

Potential solution

Include blocking (1% BSA), lower antibody concentration, ensure thorough washes (3× PBS), use Fc-blocking if serum components used.

Problem 8 (step 11)

Unexpected Intracellular Staining.

Potential solution

Ensure no permeabilization and avoid detergents; if signal persists, the passenger may be mislocalized - test fractionation and redesign linker/anchor.

Problem 9 (step 20)

Low or inconsistent OMV yields.

Potential solution

Low OMV yield might be due to cell lysis during cell growth. Shortening the growth timing and verifying viability will ensure high OMV yield. Ensure cultures harvested at mid-log phase (OD₆₀₀=0.6–0.8). Use fresh growth media and maintain consistent shaking speed and temperature.

Problem 10 (step 21)

Poor OMV quality.

Potential solution

Cell lysis during cell growth might result in poor OMV quality. Shortening growth time and verifying cell viability will ensure high quality OMVs.

Problem 11 (step 25): Inconsistent bacterial concentration at 10⁷ CFU/mL

Variation in 16–24 hr. culture (16–24 hr) growth or inaccurate OD₆₀₀-to-CFU conversion.

Potential solution

Always calibrate OD₆₀₀ readings with a CFU plating curve for engineered EcN before the experiment. Prepare fresh cultures on each treatment day and avoid using overgrown stationary-phase cultures.

Problem 12 (step 26): Difficulty in gavaging mice (regurgitation, injury, or inconsistent dosing)

Improper gavage needle size or technique; stressed or inadequately restrained animals.

Potential solution

Use an appropriately sized, ball-tipped gavage needle (20G for adult mice). Train personnel on gentle restraint and ensure the needle follows the esophageal path without forcing. If regurgitation occurs, pause, allow recovery, and re-administer at a slower pace.

Problem 13 (step 27): Unexpected mouse distress (weight loss >15%, lethargy, and abnormal temperature)

Stress from repeated gavage or adverse reaction to engineered EcN.

Potential solution

Monitor daily body weight and clinical score. If weight loss exceeds 15% or signs of distress persist, discontinue treatment and consult veterinary staff. Ensure bacteria are endotoxin-minimized and avoid gavaging late in the day to reduce stress.

Problem 14 (steps 25–28): Low colonization or rapid clearance of EcN

Host microbiota competition or poor viability of administered bacteria.

Potential solution

Verify EcN viability by plating the inoculum before gavage. If colonization is essential, consider pretreatment with mild antibiotics to reduce competition, or administer higher doses (≥10⁸ CFU) while monitoring safety.

Problem 15

(Step 33, c) Weak or no pseudovirus infection signal.

Potential solution

Low pseudovirus titer or poor cell viability. Verify pseudovirus infectivity on control wells before neutralization assay. Check 293T-hACE2 cell health and ensure they are ∼70%–80% confluent at seeding.

Problem 16 (step 33, d): High background luminescence or variability between wells

Incomplete washing or uneven lysis.

Potential solution

Incomplete washing or uneven lysis. Perform gentle but thorough PBS washes before adding lysis buffer. Ensure consistent incubation times for lysis and equal pipetting of luciferase reagent.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Nalinikanth Kotagiri (kotaginh@ucmail.uc.edu).

Technical contact

Questions about the technical specifics of performing the protocol should be directed to and will be answered by the technical contact, Nitin S. Kamble (kamblens@ucmail.uc.edu).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate data or code.

Acknowledgments

We acknowledge Dr. Ken Greis, Michael Wyder, and Wendy Haffey from UC Proteomics Core for proteomics analysis, Betsy DiPasquale for preparing IHC and immunofluorescence samples, Chet Closson from Live Imaging core facility for fluorescent and confocal microscopic image acquisition, Xiangning Wang and Dr. Lisa Lemen for IVIS imaging, and the Laboratory Animal Medical Service Staff at the University of Cincinnati and the core facilities at the Cincinnati Children’s Hospital Medical Centre for the instrumental support. All schematics were constructed using BioRender and Inkscape. This work was supported by grants from NIH: R01HL168588 and R01CA279962; CDMRP: ME200246; and University of Cincinnati Office of Research and College of Pharmacy (to N.K.).

Author contributions

N.K. conceived the project, and N.S.K. designed the experiments. All the experiments were lead and conducted by N.S.K. N.S.K. and A.C. generated schematics. A.C., N.M., and K.K. helped with reviews. N.S.K. and N.K. analyzed the data and wrote the manuscript with feedback and review from all the authors.

Declaration of interests

N.S.K. and N.K. have filed a patent application with the US Patent and Trademark Office related to this work.