M edium for Production ofBacterialExtracellularVesicles

The presentinvention relatesto a growth medium ormedia and the use and/or manufacture thereof for the production of bacterial extracellularvesicles.

Although the following description refersto agrowth medium ormedia for production of bacterial extracellular vesicles (BEVs) from the engineered bacterium Bacteroidesthetaiotaomicron (Bt),the person skilled in the artwill appreciate that the current invention can be applied to otherbacterialstrains,both native and engineered and isnotlimited to engineered Bt.

Bacterial extracellular vesicles (BEVs) are spherical nanostructures composed of membrane-derived lipid bilayers with a diameter of between 20 and 400 nm. BEVs generated by Gram-negative bacteria primarily consist of vesicles derived from the outer membrane containing phospholipids, outer membrane proteins, lipopolysaccharides and capsular polysaccharides with their lumen principally filled with periplasmic content [Schwechheimerand Kuehn, 2015].These components,which include microbe associated molecular pattern molecules,conferinherentpotentadjuvanticity on BEVswhich together with their natural temperature and chemical resistance and their straightforward isolation [Arigita etah,2014;Kanojia etah,2018; Carvalho et ah, 2019a] makes them well suited as vaccine delivery vehicles capable of enhancing the immunogenicity of protein/peptide antigens without the need for chemical adjuvants [Chen, 2010].The ability ofBEVsto interactwith,and be acquired by,mucosalepithelial and immune cells [Stentz et ah,2018; Cecil et ah,2019;Jones et ah, 2020,Durant et ah,2020]furtherenhances their suitability formucosal administration and the generation of local and systemic immunity [Miquel-Clopes etah,2019]. W e have engineered the Gram-negative bacterium Bacteroides thetaiotaomicron (Bt),a prominent member of the intestinal microbiota of all animals [Smith et ah, 2006; Human Microbiome Project Consortium, 2012],to incorporate virus-,bacteria and human-derived proteinsinto its'BEVs [Carvalho etal.2019 a and b].These engineered Bt OMVs have been used to protectthe gastrointestinalor respiratory tracts againstinfection,tissue inflammation and injury.

Itistherefore an aim ofthe presentinvention to provide an improved growth media forBEV generation;

It is a further aim of the present invention to provide a method of producing an improved growth media.

In a first aspect of the invention there is provided a bacterialgrowth medium said medium comprising a buffered aqueous solution of any source of carbon that can be utilised by Bt in addition to any one or any combination of;

NaCl orKC1,

(NH4) ₂ SO _4,

MgCl _2, cysteine, hemin orhematin orprotoporphyrin, vitamin B12,cobamide ormethionine.

In one embodimentthe medium includesiron.

Typically the methionine isL-methionine.

In a preferred embodimentprotoporphyrin IX isused.

Typically the bacterial growth medium includes a buffered aqueous solution ofa carbon source thatcan be utilised by Btin addition to any one orany combination of:

NaCl orKC1 (NH4) ₂ SO ₄ MgCl ₂

CaCl ₂

L-cysteine

Hemin orprotoporphyrin IX vitamin B12,cobamide orL-methionine

FeSO ₄

The growth medium can optionally include L-histidine and vitamin K3 (menadione).

Typically the carbon source utilised by Bt is a hydrocarbon-based carbon source. Further typically the hydrocarbon is a saccharide, polysaccharide or sugar.

In one embodiment of the invention the carbon source is glucose, maltotriose and/orxylose.

In a preferred embodiment of the invention the buffered aqueous solution includes KH ₂ PO ₄ salt buffer. Typically the solution is buffered to around pH 7. Further typically the pH of the solution is pH 7-7.5/

In a preferred embodimentthe pH ofthe solution is 7.2.

In a preferred embodimentthe medium comprises K ₂ HPO ₄ * 3 H ₂ O in deionised water.

In one embodiment,formedia utilising glucose asthe preferred carbon source ittypically comprises around;

15 mM NaCl,

8.5 mM (NH4) ₂ SO ₄ ,

30mM of glucose

Furthertypically,ifpresent,the medium comprises around; 0.1 mM MgCl ₂ , 50 μM CaCl ₂ ,

2 μM hemin or2-50 μM protoporphyrin IX,

100 nM vitamin B12or 100 nM of any cobamide or 400 μM L- methionine 2-4.1 mM L-cysteine hydrochloride,

10 μM FeSO ₄ * 7 H ₂ O.

Preferably 50 μM protoporphyrin IX isused. In one embodimentaround 0.2 mM L-histidine isused.

In one embodimentaround 6 μM vitamin K3 (menadione)isused.

In a second aspect of the invention there is provided a method of preparing a growth medium, said method including preparing a salt buffered aqueoussolution in deionised waterand adding glucoseto said autoclaved buffered solution.

Typically the carbon source isadded while the buffered solution is still hot. Further typically the carbon source containing solution is placed in an anaerobic cabinetto equilibrate.

In one embodiment any one or any combination of the following compounds are added to the carbon source containing solution; NaCl or KC1,

(NH4) ₂ SO ₄ ,

MgCl ₂ ,

CaCl ₂ ,

L-histidine, Hemin/ protoporphyrin IX, vitamin B12, vitamin K3 (menadione),

L-cysteine FeSO ₄ * 7 H ₂ O Typically once prepared the medium is left in an anaerobic cabinet to fully deoxygenate. In a preferred embodiment of the invention approximately 2.61 g of KH ₂ PO ₄ and 7.03 g of K ₂ HPO ₄ * 3 H ₂ O are added into 477 mL of deionised water and autoclaved. W hile the solution is still hot, the following solutions to a final concentration of 15 mM NaCl,8.5 mM (NH4) ₂ SO ₄ , preferred carbon source are added and placed in an anaerobic cabinetto equilibrate.

Typically the preferred carbon source is glucose. Further typically around 30mM glucose is added. Typically any one or any combination of the following solutions are added,to a finalconcentration of0.1 mM MgCl ₂ ,50 μM CaCl ₂ ,0.2 mM L-histidine, 2 μM hemin/ 2 μM protoporphyrin IX, 100 nM vitamin B12, 6 μM vitamin K3 (menadione), 4.1 mM L-cysteine and 1.4 μM FeSO ₄ * 7 H ₂ O).

Further typically, all stock solutions are prepared in advance and autoclaved or filter sterilised,asbelow:

• 1.5M NaCl(autoclaved).

• 0.85 M (NF14) ₂ SO ₄ (autoclaved). · 0.1M MgCl ₂ * 6 H ₂ O(autoclaved).

• 50mM CaC12 * 2 H ₂ O (autoclaved).

• 0.41M L-cysteine hydrochloride (filtered).

• 0.2M L-histidine and store at4°C (filtered).

• 3 mM hemin in 1M NaOH and store at4°C (autoclaved) or3 mM protoporphyrin IX

• 1 mM vitamin B12 and store at4°C (filtered).

• 6 mM vitamin K3 (menadione) dissolved in ethanolfiltered and stored at-20°C. • 1.4 mM FeSO ₄ * 7 H ₂ O and store in anaerobic cabinet (filtered).

• 3 M Glucose (filtered).

Specific embodiments ofthe invention are now described.

Introduction

Conventional vaccinesbased on the use ofattenuated orinactivated forms ofthe targetpathogen have successfully eradicated smallpox and rinderpestaswellas significantly reducing the burden ofmany otherinfectious diseasesthroughoutthe pastcentury.However,the time needed to identify vaccine targets,the high costofvaccine development and manufacture,and the limited production capacity, make these traditionalapproacheslessthan optimalin the rapid response to epidemics and pandemics [Rauch etah,2018]. Furthermore, these vaccines are usually delivered parenterally via injection,which makesmass immunisation costly particularly in resource-poordeveloping countries [W allis etal.2019].There is therefore a need forthe developmentofnew vaccinesthatare versatile,cost-effective,safe,and enable worldwide immunisation.To this end,variousnew vaccination technologieshave emerged including the use ofsynthetic protein and peptide antigens [Francis, 2017].Protein subunitvaccines are attractive because oftheir inherentsafety although they can suffer from poorimmunogenicity and high manufacturing costs [Shin,2020].To addressthese constraints,nanoparticle-based delivery technologies have been developed which includesnanoparticle sized extracellularvesicles naturally produced by bacteria.

Bacterialextracellularvesicles (BEVs) are sphericalnanostructures composed ofmembrane-derived lipid bilayerswith a diameterof between 20 and 400 nm. BEVs generated by Gram-negative bacteria primarily consist of vesicles derived from the outer membrane containing phospholipids, outer membrane proteins, lipopolysaccharides and capsular polysaccharides with their lumen principally filled with periplasmic content [Schwechheimer and

Kuehn, 2015] . These components, which include microbe associated molecular pattern molecules, confer inherent potent adjuvanticity on BEVs which together with their natural temperature and chemical resistance and their straightforward isolation [Arigita et ah, 2014; Kanojia et ah, 2018; Carvalho et ah, 2019a] makes them well suited as vaccine delivery vehicles capable of enhancing the immunogenicity of protein/peptide antigens without the need for chemical adjuvants [Chen, 2010] . The ability of BEVs to interact with, and be acquired by, mucosal epithelial and immune cells [Stentz et ah, 2018; Cecil et ah, 2019; Jones et ah, 2020, Durant et ah, 2020] further enhances their suitability for mucosal administration and the generation of local and systemic immunity [Miquel-Clopes et ah, 2019] .

We have engineered the Gram-negative bacterium Bacteroides thetaiotaomicron (Bt), a prominent member of the intestinal microbiota of all animals [Smith et ah, 2006; Human Microbiome Project Consortium, 2012], to incorporate virus-, bacteria and human-derived proteins into its' BEVs [Carvalho et al. 2019 a and b] . These engineered Bt OMVs have been used to protect the gastrointestinal or respiratory tracts against infection, tissue inflammation and injury. Here we describe the methods to implement secretion of vaccine antigens and other proteins into Bt BEVs for mucosal delivery.

2 Materials Unless stated otherwise, all solutions are prepared with ddH ₂ O with all reagents being stored at ambient (room) temperature (~20°C). 2.1 Synthetic gene design

Consists of:

1. Protein sequence (e.g. stalk of the hemagglutinin antigen of influenza A virus strain H5N1 [Valkenburg et al., 2016; Carvalho et al.

5 2019a]).

2. Secretion sequence signal (e.g. N-terminal sequence of Bt outer membrane protein A [OmpA] (BT_3852)).

3. Optional additional sequences (e.g. His-tag or FLAG-tag sequences).

10 4. Escherichia coli / Bacteroid.es shuttle expression vector sequence (e.g., pGH90 [Wegmann et al, 2013]). (see Note 1).

2.2 Cloning of synthetic gene

The required materials for the cloning of the conjugative plasmid

15 harbouring the gene of interest are described here.

2.2. 1 Generation of recombinant DNA

1. E. coli / Bacteroides shuttle expression vector.

2. Restriction enzymes (e.g. New England Biolabs).

20 3. Ligation buffers and enzyme (e.g. Fast-Link™ DNA Ligation Kit, Lucigen)

2.2.2 Transformation of comp etent cells

1. Crushed ice for thawing the MAX Efficiency® DH10B

25 competent cells (e.g. New England Biolabs). 2. S.O.C. medium (e.g. New England Biolabs).

3. LB medium for casting agar plates (e.g., LB agar powder, Invitrogen, prepare according to the manufacturer's instructions). 4. Shaking incubator.

5. Luria-Bertani (LB) medium for the dilution of the bacterial cell suspension (e.g. LB broth base (Invitrogen), prepare according to the manufacturer's instructions).

6. Agar plates containing Ampicillin (working concentration 100 μg/mL).

7. Piston pipettes (e.g. Gilson's PIPETMAN® Classic).

8. Sterile single-use pipette tips (e.g. Sarstedt).

9. 1.5 mL centrifuge tubes (e.g. Sarstedt).

10. Water bath .3 Screening of recombinants bacteria

1. Shaking incubator.

2. Benchtop centrifuge.

3. Small-scale plasmid isolation kit (e.g. QIAprep Spin Miniprep Kit, Qiagen).

4. Gel electrophoresis chamber and electrophoresis power supply.

5. Stained agarose gel (1 % (w/v) agarose, e.g. with EvaGreen® Dye, Biotium).

6. Piston pipettes (e.g., Gilson's PIPETMAN® Classic). 7. Sterile single-use pipette tips (e.g. Sarstedt).

8. DNA loading buffer (e.g. Gel Loading Dye, Purple (6x), New England Biolabs).

9. DNA ladder (e.g. 2 Log DNA Ladder, New England Biolabs).

2.3 Conjugative transfer of shuttle vector into Bt 1. E. coli donor strain containing shuttle vector carrying gene of interest.

2. E. coli helper strain, e.g. J53/R751 [Shoemaker et ah, 1986] .

3. 5 ml LB bottle with ampicillin 100 μg/mL.

4. 5 ml LB bottle with trimethoprim 200 μg/ mL.

5. BHIH agar for casting agar plates with no antibiotics or supplemented with gentamycin 200 μg/mL and/or erythromycin 5 μg/mL. See Section 2.5 for BHIH recipe and add 1.5 % (w/v) agar for casting plates.

6. Filter disc 0.45μm pore size, 25 mm (MF-Millipore).

7. Piston pipettes (e.g. Gilson's PIPETMAN® Classic).

8. Sterile single-use pipette tips (e.g. Sarstedt).

9. Falcon® 50 mL conical centrifuge tubes.

10. Tweezers.

11. Sterile 25 mL wide neck universal glass bottle.

2.4 Assessing protein expression and secretion into BEV

2.4. 1 Culture of Bt transconjugants

1. BHIH agar plates containing gentamycin 200 μg/mL and erythromycin 5 μg/ mL.

2. Sterile inoculation loop(s).

3. 20 mL BHIH containing erythromycin 5 μg/mL in Universal bottles.

4. Anaerobic cabinet.

5. Falcon® 50 mL conical centrifuge tubes.

6. Refrigerated centrifuge (e.g., 5810 R centrifuge, Eppendorf).

7. - 20°C freezer.

2.4.2 Cell total protein extraction 1. 0.2 M Tris-HCl, pH 7.2. 2. Piston pipettes (e.g. Gilson's PIPETMAN® Classic).

8. Sterile single-use pipette tips (e.g. Sarstedt).

3. Sonicator (e.g., QSonica Q55 Sonicator, Cole-Parmer).

4. Refrigerated centrifuge (e.g. 5810 R centrifuge, Eppendorf). 5. Bradford reagent (Bradford protein assay, Bio-Rad).

6. 96-well microplate.

7. Bovine serum albumin (e.g. Bio-Rad Protein Assay Standard II).

2.4.3 BEV total protein extraction

1. Piston pipettes (e.g., Gilson's PIPETMAN® Classic).

2. Sterile single-use pipette tips (e.g. Sarstedt).

3. 1 ml syringe.

4. 0.22 mm pore-size polyethersulfone (PES) membranes (Sartorius, Goettingen, Germany).

5. Centrifugal concentrator, 100 kDa molecular weight cut-off (e.g. Vivaspin 20, Sartorius, Gottingen, Germany).

6. Refrigerated centrifuge (e.g., 5810 R centrifuge, Eppendorf).

7. 0.2 M Tris-HCl, pH 7.2.

8. Sonicator (e.g., QSonica Q55 Sonicator, Cole-Parmer).

9. Bradford reagent (Bradford protein assay, Bio-Rad).

10 96-well microplate. 11. Bovine serum albumin (e.g., Bio-Rad Protein Assay

Standard II).

2.4.4 Protein Western Plotting / Antigen immunodetection 1. Gel electrophoresis equipment (e.g. XCell SureLock™ Mini-Cell

Gel tank, Thermofisher).

2. Protein gel (e.g. TEO-Tricine Precast Gels - RunBlue™, Abeam).

3. SDS sample loading buffer (e.g. LDS Sample Buffer LDS Sample Buffer (4X) - RunBlue™ (TEO-Tricine), Abeam). 4. Reducing agent (e.g. DTT Reducer (10X) - RunBlue™).

5. Antioxidant for protein electrophoresis (e.g. Antioxidant (800X)

- RunBlue™, Abeam).

6. Running buffer (e.g. Run Buffer (20X) - RunBlue™ (TEO- Tricine), Abeam). 7. Blotting equipment (e.g. XCell II™ Blot Module, Thermofisher).

8. Western blotting membrane (e.g. Immobilon® PVDF membranes, Thermofisher).

9. Pair of tweezers.

10. Tris-Glycine transfer buffer (25X) ( see Note 2). 11. Methanol.

12. Orbital shaker.

13. TBS buffer (50 mM TrisHCl; 150 mM NaCl; pH 7.5).

14. TBST buffer: TBS buffer with 0.05% Tween 20.

15. Blocking buffer: TBST with 5% non-fat dry milk 16. Chemiluminescent substrate (e.g. Clarity™ Western ECL

Substrate, BIO-RAD)

17. Primary antibody (e.g. 6x-His Tag monoclonal antibody, Invitrogen) used at a working concentration recommended by the manufacturer or by other providers if the antibody is not commercially available ( see Note 3).

18. Secondary antibody labelled with horse radish peroxidase (HRP) (Thermofisher) (see Note 4). 19. Imaging System (e.g. ChemiDoc MP, Bio Rad).

2.5 Bacteria medium-scale culture

1. BHIH medium: Dissolve 18.5 g of brain heart infusion in 0.5 litre of deionised water and add 0.75 μM of hemin/ 0.75 μM protoporphyrin IX. Autoclave the medium and leave for a minimum of 24 hours in an anaerobic cabinet to fully deoxygenate.

2. Bacteroides defined medium Plus (BDM+): Add 2.61 g of KH2PO4 and 7.03 g of K ₂ HPO ₄ * 3 H ₂ O into 477 mL of deionised water and autoclave. While the solution is still hot, add the following solutions to a final concentration of 15 mM NaCl, 8.5 mM (NH4) ₂ S04, preferred carbon source (e.g., 30mM of glucose) and place in the anaerobic cabinet to equilibrate for a minimum of 24 h. Add the following solutions to a final concentration of 0.1 mM MgCl ₂ , 50 μM CaCl ₂ , 0.2 mM L-histidine, 2 μM hemin/2 μM protoporphyrin IX, 100 nM vitamin B12, 6 μM vitamin K3 (menadione), 4.1 mM L-cysteine and 1.4 μM FeSCL * 7 H ₂ O). Leave the medium for a minimum of 24 h in the anaerobic cabinet to fully deoxygenate. All stock solutions are prepared in advance and autoclaved or filter sterilised, as below:

• 1.5M NaCl (autoclaved).

• 0.85 M (NH4) ₂ SO ₄ (autoclaved).

• 0.1M MgCl ₂ * 6 H ₂ O (autoclaved).

• 50mM CaC12 * 2 H ₂ O (autoclaved).

• 0.41M L-cysteine hydrochloride (filtered).

• 0.2M L-histidine and store at 4°C (filtered).

• 3 mM hemin in 1M NaOH and store at 4°C (autoclaved)/ 3 mM protoporphyrin IX.

• 1 mM vitamin B12 and store at 4°C (filtered).

• 6 mM vitamin K3 (menadione) dissolved in ethanol filtered and stored at -20°C.

• 1.4 mM FeSCL * 7 H ₂ O and store in anaerobic cabinet (filtered).

• Preferred carbon source (e.g., 3 M Glucose) (filtered). 3. Anaerobic cabinet.

4. Magnetic stirrer.

5. Sterile magnetic stirring bar. 6. Spectrophotometer (wavelength: 600 nm) with cuvette holder and cuvettes.

7. Refrigerated high speed floor centrifuge (e.g. J2-MI centrifuge, Beckman Coulter), including appropriate rotor (e.g. JA-10 fixed-angle rotor, Beckman Coulter) with 500 mL sealable centrifuge bottles (NalgeneTM PPCO or equivalent).

8. 0.2 pm polyethersulfone (PES) bottle top filter unit (500ml).

9. Membrane vacuum pump.

10. Sterile 500 mL bottles.

2.6 BEV isolation

1. Filtration cassette Vivaflow 50 R (100,000 MWCO, Hydrostat, model VF05H4, Sartorius).

2. Peristaltic pump for running the Vivaflow-unit (e.g., Masterflex economy drive peristaltic pump, Sartorius).

3. Sterile phosphate-buffered saline (PBS), pH 7.4 (see Note 5)

4. 0.22 pm polyethersulfone (PES) syringe filters.

5. 5 mL sterile syringes.

6. Falcon® 15 mL conical centrifuge tubes.

7. 1.5 mL sterile low-bind Eppendorf tubes.

8. Deionised water.

9. 0.5 M NaOH solution.

10. 10% (v/v) ethanol. 2.7 BE Vs purification

In this part of the chapter, the use of size-exclusion chromatography is described for the removal of remaining proteins. Two options are proposed: 2.7.1 and 2.7.2 for an increased resolution.

2. 7. / Routine purification

1. Piston pipettes (e.g. Gilson's PIPETMAN® Classic).

2. Sterile single-use pipette tips (e.g. Sarstedt).

3. qEVoriginal/35nm SEC columns (IZON)

4. Support to maintain column in a vertical position

5. E5 mL lo-bind Eppendorf tubes

6. Amicon Ultra 0.5 mL centrifugal filters (RC, l0kDa MWCO)

7. Sterile phosphate-buffered saline (PBS), pH 7.4.

8. 0.22 pm PES membrane syringe filter.

9. 1 ml sterile syringes

10. LB and BHIH agar plates

2. 7.2 High-resolution fractionation E Piston pipettes (e.g. Gilson's PIPETMAN® Classic).

2. Sterile single-use pipette tips (e.g. Sarstedt).

3. CL2-B Sepharose (Sigma-Aldrich-Aldrich).

4. Sterile PBS, pH 7.4.

5. Chromatography column (120 cm x 1 cm) (e.g. Econo-Column® Chromatography Column, Bio-Rad) in PBS. 6. Chromatography fraction collector.

7. UV spectrophotometer.

8. Vivaspin 20 centrifugal concentrator (100 kDa molecular weight cut-off, Sartorius). 9. 0.22 μm PES membrane syringe filter.

2.8 BEVs size and concentration analysis

1. Nanoparticle Analyzer (ZetaView TWIN Particle Tracking Analyzer instrument or equivalent).

2. Particle-free deionised water.

3. 1 or 5 mL sterile syringes.

2.9 Antigen localisation with proteinase K assay

1. Proteinase K.

2. Phenylmethanesulfonyl fluoride (PMSF).

3. Water bath.

4. Sodium Dodecyl Sulphate (SDS).

5. See also materials in Sections 2.4.3 and 2.4.4.

2.10 Antigen quantification

1. Recombinant antigen.

2. See also materials in Sections 2.4.3 and 2.4.4.

2.11 Imm unis a tion See materials in [Carvalho et al. 2019 a and b] .

2.12 Sample collection and antibody titration

See materials in [Carvalho et al. 2019 a and b] .

3 Methods

3.1 Synthetic gene design

The gene can be synthesized de novo using commercial gene synthesis services. The N-terminus of the protein of interest is fused in frame to the signal peptide of the product of BT_3852 (OmpA of Bt); MKKILMLLAFAGVASVASA. The chimeric protein sequence is tested in silico for cleavage of the OmpA signal sequence using http://www.cbs.dtu.dk/services/SignalP/. If unsuccessful, change or add amino acids as appropriate to the N-terminus of the gene of interest and downstream from the signal peptide sequence. To facilitate immunodetection and/or purification of the protein in downstream applications, a fusion tag can be added to the 3'-end of the gene. It is important that the coding sequence of the desired protein incorporates codon usage optimisation for expression in Bt which is usually provided as part of gene synthesis services). The desired target sequence is then integrated into an acceptor vector.

3.2 Cloning of gene of interest 3.2. 1 Generation of recombinant DNA 1. Digest the plasmid containing the synthetic gene with restriction enzymes to excise the gene from the vector carrying the synthetic gene (e.g. pEX-K168, Eurofins Genomics).

2. Digest the E. coli / Bacteroides shuttle vector pGH90 {see Section 2.1) ( see Note 6).

3. Ligate the gene into the digested pGH090 expression vector to allow translational fusion.

3.2.2 Transformation of competent cells

1. Prepare LB agar plates containing ampicillin.

2. Thaw one vial of competent MAX Efficiency® DH10B on ice.

3. Gently add 1 -5 mΐ of ligation mixture to the MAX Efficiency® DH10B.

4. Place the vial with the bacteria suspension on ice for 30 min.

5. Induce a heat-shock at 42°C for 30 s.

6. Incubate the bacteria on ice for an additional 5 min.

7. Add 950 μL S.O.C. Medium.

8. Incubate at 37°C with agitation (250 rpm).

9. Seed 100 μL of the diluted bacteria suspension onto agar plates containing ampicillin.

10. Incubate the plates for 16-18 h at 37°C.

3.2.3 Screening of cloned recombinant DNA

1. Pick individual colonies from agar plates and add each colony to a tube containing 5 mL LB medium and ampicillin.

2. Incubate the liquid cultures at 37°C and 250 rpm for 16-18 h.

3. Isolate plasmid DNA from each culture using a small-scale isolation kit (e.g. QIAprep Spin Miniprep Kit, Qiagen) according to the manufacturer's instructions. 4. Digest the plasmid DNA using the appropriate restriction enzymes (see Note 7)

5. Resolve digested DNA on a TBE gel ( see Note 8).

6. Confirm the identity of the plasmids containing the insert of the expected size by DNA sequencing using appropriate primers. .3 Conjugative transfer of shuttle vector into Bt

3.3. 1 Triparental mating procedure

1. Prepare BHIH agar plates either with or without gentamycin and erythromycin.

2. Grow cultures of the E. coli donor strain with ampicillin (containing the plasmid with the correct inserted sequence, see 3.2.3) and the E. coli helper strain with trimethoprim in 10 mL of LB, at 37°C, under agitation for 16-18 h. In parallel, grow culture of the Bt recipient strain in BHIH in an anaerobic cabinet at 37°C for 16-18 h.

3. Inoculate 10 mL of LB with 100 μL of the E. coli donor strain and the helper strain (no antibiotics added) cultures and incubate for 2 hours at 37°C with agitation (e.g., 200 rpm). In parallel, inoculate in 30 mL BHIH contained in a 50 mL Falcon® tube with 800 μL of the Bt culture and incubate for 2 hours at 37°C in an anaerobic cabinet.

4. Add donor and helper cultures to the Bt recipient in the Falcon® tube, mix by vortexing briefly and centrifuge at 2000g for 15 min at 20°C.

5. Remove the supernatant and resuspend cells in 100 μL of BHIH. Transfer cell suspension to the surface of a sterile 0.45 pm filter (Millipore) placed on a BHIH agar plate. Incubate the plate aerobically for 16-18 h at 37°C.

6. Transfer the filter to a sterile wide-necked Universal bottle and add lmL of BHIH and resuspend the bacterial conjugation mixture by vortexing thoroughly. Make serial dilutions and plate 100 μl of each dilution and the undiluted cell suspensions onto BHIH agar plates containing gentamycin (to prevent E. coli growth) and erythromycin (selection of Bt transconjugants). ssessing protein expression and secretion into BEVs . 1 Culture of Bt transconjugants

1. Pick 4 individual colonies and re-streak each one on separate BHIH agar plates containing gentamicin and erythromycin.

2. Incubate the plates anaerobically at 37°C for 48 h.

3. Inoculate bottles containing 20 mL of BHIH with each of the 4 isolated clones.

4. Incubate the bottles anaerobically at 37°C for 48 h.

5. Centrifuge the 20 mL of culture in Falcon® 50 mL tubes, at 6,000 g for 15 min, at 4°C.

6. Collect the supernatant.

7. Wash cell pellets once in PBS before storing at -20°C prior to analysis. Cell total protein extraction

1. Resuspend thawed cell pellet sin 250 μl of 0.2 M Tris-HCl (pH 7.2).

2. Disrupt the cells via sonication using eight 10 s pulses (amplitude, 6 micrometers), with 30 s pauses on ice between each pulse.

3. Cell extracts are obtained after centrifugation at 14,000 g for 30 min at 4°C and harvesting the supernatant.

4. Measure total protein concentration of each supernatant/sample using the Bio-Rad protein assay (Bio- Rad) according to the manufacturer's instructions using bovine serum albumin to generate a standard curve.

3.4.3 BEV total protein extraction 1. Filter the 20 mL supernatants through 0.22 mm pore-size PES membranes (Sartorius) to remove debris and cells.

2. Concentrate the supernatants by ultrafiltration (100 kDa molecular weight cut-off, Vivaspin 20, Sartorius) to a final volume of 250 μL. 3. Discard the filtrate.

4. Rinse the retentate with 20 mL of 0.2 M Tris-HCl, pH 7.2 and concentrated to 250 μL.

5. Collect the retentate and disrupt the vesicles via sonication using eight 10 s pulses (amplitude, 6 pm), with 30 s pauses on ice between each pulse.

6. Measure the total protein content and concentration using the Bio- Rad protein assay (Bio-Rad) according to the manufacturer's instructions using bovine serum albumin to generate a standard curve.

3.4.4 Protein Western Blotting / Antigen immunodetection

1. Add BEV and cell extracts obtained in Section 3.4.3 to loading buffer (4x) containing freshly prepared DTT (1 /10).

2. Load 7 μg of the total protein onto a 12% precast gel and separate by electrophoresis at 180 volts for 40 min.

3. Transfer the proteins from the gel onto a polyvinylidene difluoride (PVDF) membrane using the XCell II™ Blot Module or equivalent (according to the manufacturer's instructions) at 25 volts for 2 h in a solution containing Tris-Glycine transfer buffer (see Note 2) and methanol 20 % (v/v).

4. Incubate the membrane with blocking buffer by gently shaking for 30 min at 20°C using an orbital shaker.

5. Discard the blocking solution and incubate the membrane for 16—18 h at 4°C in blocking buffer containing primary antibody (Usually 1 : 1000 to 1 : 10000).

6. After washing 3 times with TBST, membranes are incubated with HRP-conjugated secondary antibody in blocking buffer for 1 h at 20°C.

7. After 3 washes with TBST, Enhanced Chemiluminescent substrate (ECL) is added to detect bound antibody.

3.5 Medium-scale bacterial culture and harvesting conditioned media

Initially BHIH was used as a standard medium for generating Bt BEVs (Carvalho et al., 2019 a and b). However, considering the need to exclude all animal derived products from medicine/therapeutic formulations to be used in humans, we have modified a chemically defined Bacteroides growth media (BDM) [Bryant et ah, 2017] for Bt BEV production.

1. Inoculate 10 mL of BHIH with a frozen stock for ~ 16h.

2. Inoculate 10 mL of BDM+ with 100 μl BHIH culture from step 1 for ~8 h.

3. Inoculate 500 mL of BDM+ with 0.5mL of the pre-inoculum for 17 h (starting OD ₆₀₀ ~ 0.005) with mild stirring.

4. Collect bacterial cultures at final OD ₆₀₀ 1.5-2.5.

5. Pre-cool centrifuge and canisters for 5 min at 4°C.

6. Decant the culture into two 2 Nalgene™ PPCO Centrifuge Bottles and centrifuge at 6037 g for 30 min at 4°C. 7. Filter-sterilize the supernatant with a 0.22 pm bottle top filter unit and transfer the filtrate into a sterile 500mL bottle ( see Note

9) ·

8. Samples are stored at 4°C prior to BEVs isolation for up to 24 h.

3.6 Isolation of BE Vs

During the filtration process, the membrane will retain any molecules > 100 kDa including BEVs (retentate) and concentrate them in the reservoir, whereas molecules < 100 kDa will be removed in the flow- through (filtrate) directly to the waste. BEVs isolation should be carried out at ambient (20-22°C) temperature.

Three procedures are used in a step wise manner for isolating BEVs.

3.6.4 Module rinsing

1. Set up the system as illustrated herein.

2. Place 400 mL of deionised water in the reservoir and pump the liquid through the system at an initial speed setting of 2 to remove air pockets and then increase the speed setting to 4-5 (200- 300 mL/min) until 400 mL has been run through the system. Collect filtrate in waste bin. Check for any leaks. 3.6.5 Sample concentration

1. Set up the module as illustrated herein. 2. Fill the reservoir with 500 ml filtrate.

3. Pump sample through the system. The initial recirculation speed setting is 2 for at least 1 cycle and then adjusted to setting 4-5 for sampling (200-300 mL/min). Maximum recirculation speed setting is 5. Reduce the speed for lower volumes to avoid foaming.

4. Concentrate the sample until there is ~5 mL left in the system.

5. Switch off the pump.

6. Pour 500 mL of PBS pH 7.4 into the reservoir and pump through the system at an initial speed setting of 2 to remove any air pockets and then increase the speed to setting 4-5.

7. Reduce the recirculation speed setting to 1-2 (20-40 mL/min) to avoid foaming.

8. Switch off the pump with 1 to 4 ml remaining in the system.

9. Set up the module as illustrated herein.

10. Start the pump at a speed setting of 1 -2 and carefully collect the concentrated samples in a 15 mL tube or Eppendorf (use a bigger tube than the volume to be collected to account for foam).

11. When no more concentrated sample is emerging from the tubing, switch off the pump.

12. Place 0.5-1 mL of PBS in the reservoir and pump through the system at speed setting 1 - 2 to flush out remaining BEVs. Collect in the original 15 mL tube or Eppendorf. Final volume collected depends on concentration of BEVs needed.

13. Switch off the pump.

14. Centrifuge at 15,000 g for 20 min at 4°C to remove any precipitate.

15. Filter-sterilize the supernatant using a 0.22μm syringe filter, collecting the filtrate in sterile 1.5 mL lo-bind Eppendorf tubes or 15 mL tube.

3.6.6 Module decontamination and washing 1. Set up the system as illustrated herein.

2. Place 400 mL of deionised water in the reservoir and pump liquid through the system at an initial speed setting of 2 to remove any air pockets and increase the speed setting to 4-5 (200-400 mL/min). Collect filtrate in waste bin.

3. When all the water has been filtrated, switch off the pump.

4. Set up the system as illustrated herein.

5. Place 250 mL of decontamination solution (250 mL 0.5 M NaOH) and pump it through the system at a speed setting of 3-4 (50- 100 mL/min). Allow to recirculate for a minimum of 20 min then switch off the pump.

6. Set up the system as illustrated herein.

7. Place 400 mL of deionised water in the reservoir and pump liquid through the system at an initial speed setting of 2 to purge any air pockets and increase the speed setting to 4-5. (200-400 mL/min).

8. Switch off the pump.

9. Set up the system as illustrated herein.

10. Place 250 mL of 10% EtOH in the reservoir and pump liquid through the system at an initial speed setting of 2 to purge any air pockets and increase the speed setting to 3-4 (50- 100 mL/ min).

11. When half of the 10% EtOH has been filtered, switch off the pump and dismantle the system module.

12. Store cassette membrane in 10% EtOH at 4°C to avoid contamination.

13. Rinse the reservoir with water and let it air dry. Reservoir and tubing are left to dry at 20°C.

BEV isolation waste is discarded in the sink drain.

3.7 BEVs purification Contaminants (e.g. proteins) of BEV preparations can be removed by size exclusion chromatography (SEC). We recommend using the method described in Section 3.7.1 for routine preparations and in 3.7.2 if increased resolution is needed, for instance, to size fractionate BEVs.

3. 7. / Routine purification

1. Prepare LB and BHIH agar plates

2. Bring the BEVs preparation, column buffer (PBS pH7.4) and column(s) to 20°C. Do not remove the column caps until operational temperature is reached.

3. Attach the column in a vertical position to the retort stand.

4. Carefully remove the column top-cap.

5. Attach a column reservoir (if available) and add 1.5x column volume of buffer (15 mL PBS)

6. Remove the bottom column cap and allow the buffer to run under gravity to waste.

7. If any buffer other than PBS is used, flush with at least 3 column volumes of the buffer (> 30 mL).

8. The column will stop flowing when the buffer has entered the loading frit.

9. Load 0.5-1 mL of isolated BEVs onto the loading frit.

10. Collect the void volume (3 mL) fraction(s) into 1.5 mL lo-bind Eppendorf tubes.

11. Allow the sample to completely run into the column.

12. Top up the reservoir with 15 mL buffer (PBS IX) and collect 0.5 mL-1 mL elution fractions.

13. For a loading volume of 1 mL of BEVs and collecting 0.5mL elution fractions, BEVs will elute in fractions 7-12 with proteins eluting in fractions 10-20. 14. After the eluted fractions have been collected, flush the column with 1.5 volumes of buffer (PBS IX, 15 mL) before loading another sample.

15. If storing the column, flush with buffer containing 20% ethanol or 0.05% sodium azide

16. Store the column at 4°C to avoid contamination.

17. Filter-sterilize the BEVs using a 0.22μm syringe filter, collecting the filtrate in sterile 1.5 mL lo-bind Eppendorf tubes or 15 mL tube. 18. Pool BEVs elution fractions and concentrate to desired volume using Amicon Ultra 0.5mL centrifugal filters (RC, lOkDa MWCO).

19. To check sterility of the BEVs preparation plate 100 μl onto LB agar plates at 37°C and 20°C and BDM+ Agar and BHIH agar in anaerobic cabinet for 48 h. Re-sterilise using a 0.22um syringe filter if any colonies are found.

3.7.2 High -re solution size fractionation see Note 10

3.8 BEVs size and concentration analysis

Size and concentration of isolated BEVs suspension is determined using nanoparticle tracking analysis (NT A) and suitable NTA devices. The protocol described below is for the ZetaView PMX-220 TWIN device from Particle Metrix GmbH.

1. Prepare instrument set up according to the manufacturer's instructions.

2. Inject 5-10 mL of 1 :250,000 100 nm calibration silica microspheres suspension to focus alignment. 3. Inject 3x10ml water using a 10 mL syringe; avoid injecting air bubbles.

4. Dilute aliquots of BEVs suspension in 1 : 1000 to 1 :20,000 in particle-free deionised water.

5. Inject 1 mL sample with a syringe.

6. Acquire size distribution video data using the following settings: temperature: 25°C; frames: 60; duration: 2 seconds; cycles: 2; positions: 11; camera sensitivity: 80 and shutter value: 100. The ZetaView NTA software (version 8.05.12) is used with the following post acquisition settings: minimum brightness: 20; max area: 2000; min area: 5 and trace length: 30.

3.9 Proteinase K assay

To establish if heterologous proteins are expressed in the lumen or on the surface of BEVs we use broad-spectrum proteinase K ( see Note 11). Proteinase K digests proteins exposed at the surface of BEVs but not in the lumen. Extracts of BEVs obtained in the presence or absence of Proteinase K samples and analysed by Western blotting using antibodies specific for the heterologous protein makes it possible to distinguish between surface- and lumen-expressed antigens.

1. Add 100 mg/L of proteinase K into intact 10 ¹¹ BEVs/mL or solubilised (in 1 % SDS) and incubate for lh in a water bath at 37 °C.

2. The activity of proteinase K is stopped by addition of 1 mM PMSF.

3. Load samples onto a 12% polyacrylamide gel and perform a Western blot following steps described in Section 3.4.4.

3.10 An tigen quan tifica ti on

The amount of antigen expressed in BEVs can be readily determined by Western blotting using serial dilutions of the recombinant antigen and comparing the intensity of the bands visualised on the blot to estimate the concentration of antigen in the BEVs using Image Lab Software (Bio-Rad).

1. Prepare serial dilutions of the recombinant antigen and of the isolated BEVs. 2. Load samples onto a 12% polyacrylamide gel and perform a

Western blot (Section 3.4.4).

3. Analyse the image of the blot using Image Lab software (Bio Rad) using the quantity tool.

3.11 Immunisation Details of immunisation protocols are provided in Carvalho et al.

2019 a and b. In short, animals receive a primary immunisation of filter sterilised BEVs {see Note 12) via the nasal or oral route with booster immunisations carried out 7-14 days late with an infectious challenge following after a further 7-10 days. At necrosis body fluids and tissues are harvested for downstream analyses of antibody and immune cell profiles and histopathology.

3.12 Sample collection and antibody profiles

Serum and bronchoalveolar lavage and saliva samples are routinely used in ELISAs to identify and quantify antigen-specific IgA and IgG antibodies as described in Carvalho et al. 2019 a and b. In short, the ELISAs include coating the plate with recombinant protein for 16h at 4°C. After washing and incubating with blocking solution, serial dilutions of samples are added and incubated for 16h at 4°C. After washing a secondary antibody conjugated to HRP is added for 1 h at 20°C. A chromogenic substrate is then added and absorbance at

450nm is recorded using a spectrophotometer. Tissue homogenates (e.g. salivary glands and lungs) can also be used in this antibody detection assay. Notes

1. One possibility is to use the plasmid-borne inducible gene expression system developed for B. thetaiotaomicron and based upon a Bt endogenous mannan-inducible promoter [Horn et al., 2016] . This system allows to create translational fusions and generate protein products with the possibility of adding a C- terminal poly-histidine tag. This system requires the use of a carbon source different from glucose in BDM formulations as glucose suppresses expression of the mannan-inducible promoter.

2. Tris-Glycine transfer buffer recipe (25X): Dissolve 18.2 g Tris Base (77861, Sigma-Aldrich-Aldrich) and 90.0 g of glycine (56406, Sigma-Aldrich-Aldrich) in 450 mL of deionized water. Mix well and adjust the volume to 500 mL with deionized water. The pH of the buffer is 8.3. Store the buffer at 20°C. The buffer is stable for 6 months at 25 °C.

3. A 6x-His Tag antibody should be used if the chimeric protein contains a His-tag sequence at its C-terminus. An alternative is to use antibodies raised against epitopes of the antigenic protein either commercially available or from other sources.

4. The secondary antibody can a be conjugated to with various enzyme, fluorescent proteins, biotin or to polymers. The secondary anti-Ig antibody must have specificity for the Igs present in species in which the primary antibody was raised.

5. Alternatively, the buffer solution can be e.g., Tris 50 mM pH 7.0-7.6 or HEPES 50 mM pH 7.0-7.6.

6. The digestion of the E. coli / Bacteroides shuttle vector pGH090 with Ncol and EcoRI is provided as an example. In the case of DNA sequence constraints or if the EcoRI restriction site cannot be removed from the internal sequence of the synthetic gene, other restrictions sites can be used for the design of the 3'-end of the fragment (e.g., Bam HI or SmaV) that are located downstream from the NcoI site of pGH090 can be used [Wegmann et ah, 2013] . As an example, we describe the cloning of the gene encoding H5F the hgh;y conserved stalk region of the hemagglutinin molecule of IAV strain H5N1 [VN/04:A/ VietNam/1203/04]) into the Bacteroides expression vector pGH090 [Carvalho et al. 2019] . The synthetic gene flanked with a 5'-end signal peptide sequence and a 3'-end His-tag sequence was designed to contain a BspHI restriction site at its 5'-end and an EcoRI restriction site at its 3'-end to enable DNA cloning. BspHI was chosen because a lysine residue follows the first amino acid methionine in the sequence of the signal peptide. Therefore, the lysine AAA/G codon which starts with an A and follows the ATG start codon is included in the BspHI (TCATGA) restriction sequence. BspHI restriction enzyme generates a cohesive end compatible with the Ncol cohesive end of the restricted pGH090 vector to allow translation fusion of the synthetic gene. TBE gel electrophoresis. Weigh out the appropriate mass of agarose into an Erlenmeyer flask. Agarose gels are prepared using a w/v percentage solution. Add running buffer (e.g., TBE (45 mM Tris-borate, 1 mM EDTA)) to the agarose-containing flask. Swirl to mix. Melt the agarose/buffer mixture most commonly by heating in a microwave. Allow the agarose to cool either on the benchtop or by incubation in a 65 °C. Add the DNA dye (e.g., EvaGreen® Dye, at the concentration recommended by the manufacturer). Place the gel tray into the casting apparatus. Place an appropriate comb into the gel mold to create the wells. Allow the agarose to set at room temperature. Remove the comb and place the gel in the gel chamber. Add loading dye to the DNA samples to be separated. Gel loading dye is typically made at 6X concentration (0.25% bromphenol blue, 0.25% xylene cyanol, 30% glycerol). Add enough running buffer to cover the surface of the gel. Slowly and carefully load the DNA sample(s) into the gel. Attach the leads of the gel box to the power supply. An appropriate DNA size marker should always be loaded along with experimental samples. Turn on the power. Run the gel until the dye has migrated an appropriate distance. Turn off the power supply and remove the lid of the gel chamber. Remove the gel from the gel tray and expose the gel to UV or blue light to visualise

DNA.

9. BEV sterility is confirmed by checking for the growth of any contaminating bacterial cells. Spread 100 μL of the filter- sterilized solution on BHIH agar plates, incubate in an anaerobic cabinet for 48 h at 37°C and confirming the absence of colonies.

10. To increase the resolution of the SEC procedure and obtain a better separation of vesicles of different sizes, the SEC can be performed using a 120 cm x 1 cm column (Econo-Column® Chromatography Columns, Bio-Rad) filled with 90 mL of CL2-

B sepharose (Sigma-Aldrich-Aldrich) [Durant et ah, 2020] . The absorbance of the fractions is measured at 280 nm and the first fractions displaying an absorbance peak are pooled. Pooled fractions are concentrated to 1 mL with a Vivaspin 20 centrifugal concentrator (100 kDa molecular weight cut off,

Sartorius) and the retentate is filtered through a 0.22 pm PES membrane (Sartorius). The concentration of the vesicles can then be determined using nanoparticle tracking analysis as described in Section 3.7.1. 11. If the protein is expressed on the surface of the BEV

Proteinase K will degrade it and the band will be absent on the immunoblot. If the protein is expressed in the lumen the band will still be evident. SDS-treatment of vesicles makes their luminal contents accessible to Proteinase K and serves as control for enzyme activity and confirmation of the protein being expressed in the lumen of BEVs.

12. The sterility of BEV suspensions stored at 4°C is examined by checking for growth of any contaminating bacterial cells prior to immunisation. Add 90 mΐ of sterile BHIH broth to 10 mΐ of BEV suspension and spread the 100 μL onto BHIH agar plates. Incubate in an anaerobic cabinet for 48 h at 37°C and check for the absence of colony.