Small-scale spirulina culture
Spirulina strains were grown in liquid culture using SOT medium. For antibiotic selection, medium was supplemented with 70–100 μg ml–1 kanamycin or 2.5–5.0 μg ml–1 streptomycin. Culture volumes ranged from 3 to 100 ml. In preparation of strains for transformation or downstream processing, cultures were grown in Multitron incubators at 35 °C, 0.5% CO2, 110–150 μEi of light and shaking at 120–270 r.p.m., depending on culture volume. Long-term cultures were maintained by incubation in Innova incubators at 30 °C, atmospheric CO2, 50–110 μEi of light and shaking at 120 r.p.m.
Design of integrating vectors for spirulina transformation
For each genomic locus targeted for integration, PCR primers with a 18–20-base-pair (bp) overlapping sequence and a vector backbone were designed to amplify 1.0–1.5-kb DNA fragments from the 5'- and 3'-regions flanking the locus. These regions represented the left homology arm (LHA) and right homology arm (RHA), respectively. Gel-purified fragments were assembled with the linearized backbone vector, which contained a p15 origin and an E. coli ampicillin resistance marker, by Gibson assembly. Markers for antibiotic resistance in spirulina were cloned between the two homology arms of the plasmid.
Transformation of spirulina
Spirulina cultures were grown for 3 days in Innova to optical density (OD700 ml–1) 0.5–1.0. A cell volume of 50 ml was harvested by centrifugation for 10 min at 1,600× relative centrifugal force (RCF). Cells were washed once with SOT medium at room temperature then resuspended in 2 ml of SOT. A 30-μl aliquot of cells was mixed with 300 ng of plasmid DNA and incubated at room temperature for 3 h. Samples were transferred to 0.6 ml of SOT medium in 14-ml round-bottom tubes and incubated overnight at 25–30 °C under 50–100 μEi of fluorescent light on a light rack. Each tube received 2.4 ml of SOT with appropriate antibiotics and was incubated under Multitron conditions to start selection. For the first 20–30 days, culture medium was changed every 3–5 days. After 30 days, when the transformants were robustly growing, cells were diluted every 3–5 days to facilitate segregation.
Genotyping of transformed spirulina
Genomic DNA was prepared from spirulina cells by digestion with proteinase K. In brief, 0.2–0.5 OD700 of cells was washed once with sterilized water. A 30-μl sample of cell pellet was mixed with 120 μl of buffer EB (10 mM Tris-Cl pH 8.5). Proteinase K was added to samples at a final concentration of 0.2 mg ml–1. Samples were incubated at 56 °C for 1 h followed by 95 °C for 10 min, to deactivate proteinase K. Samples were centrifuged briefly in a microfuge to pellet cell debris. A 1-μl sample of the supernatant was used per genotyping PCR reaction. Specific integration of the transgenic cassette was determined by separate PCRs for each homology arm. For each PCR, one primer annealed to a genomic sequence outside the homology arm and the other to a region within the transgene. Segregation of the chromosome was assessed using a primer pair annealing to regions within the RHA and LHA. Segregation PCR yielded fragments of two different sizes: one from the wild-type allele and the other from the transgenic allele. Strains were considered fully segregated when no wild-type allele amplicon was observed.
To verify the sequence of the transgene, PCR was performed with genomic DNA to amplify the fragments, which includes the transgene, the homology arms and 500 bp flanking each homology arm. PCR products were separated by electrophoresis on an agarose gel, and amplified bands were gel extracted using the Qiagen Gel Extraction kit. Purified PCR products were sequenced to verify the integrated gene and surrounding sequences.
To exclude the possibility of cross-contamination with other strains, PCR was performed to check other loci used for integration of exogenous genes. PCR of genomic DNA using locus-specific primers was performed, and fragment size was analyzed by agarose gel electrophoresis. DNA fragments were gel extracted and characterized by Sanger sequencing. A strain was considered free of other spirulina strains if only wild-type loci were observed. Once strains were homozygous, the DNA sequence of the transgene was periodically reassessed by Sanger sequencing of PCR products. No DNA sequence variation of the transgene was observed, even after 3 years of continuous cultivation.
Transformation of barcoded integrating plasmids
To evaluate the number of individual successful integration events per transformation, a library of DNA barcodes was transformed into spirulina and quantified by next-generation sequencing (NGS). In brief, a 19 N barcode was cloned adjacent to an antibiotic marker (aadA) in a plasmid containing homology arms for integration. The barcode library was estimated to contain >8 × 107 transformants. The library was transformed into strain SP3 in triplicate, following the transformation method described above, and cultured with streptomycin. Spirulina samples were collected 22 and 28 days after transformation. Genomic DNA was extracted from spirulina and used in a PCR reaction to prepare ~320-bp amplicons of the barcoded regions for NGS analysis on a MiSeq (Illumina). Sequencing reads were filtered for quality and analyzed to minimize false positives. Counting only barcodes that were (1) present at both time points within a replicate, (2) unique to each replicate and (3) observed >30 times within a replicate yielded an estimated number of integration events of ~100–300.
Isolation of single spirulina filaments
From an actively growing spirulina culture, 200–500 filaments were spread on a SOT agar plate. Cells were allowed to settle on the plate for 1–2 h and examined under a dissection microscope. Well-separated single filaments were picked with a 1-ml pipette tip and transferred in 3 ml of SOT with appropriate antibiotics in a round-bottom tube. Typically, 10–20 single filaments were cultured in Innova for 15 days for propagation.
Determination of transgene copy number
To assess the copy number of an integrated transgene, three sets of primer/taqman probe pairs were designed to target three regions: an endogenous spirulina gene present at a single locus (cpcB), a promoter region present at both an endogenous and transgenic locus (that is, two chromosomal copies) and an exogenous region unique to the transgene. A synthetic g-block containing the three target loci plus flanking sequences was purchased from IDT as a calibrator. Real-time PCR was performed with the above primer/probes using genomic DNA from the transgenic spirulina strain and the g-block as templates. As controls, the parental spirulina strain and a second transgenic strain lacking template for the transgene-specific probes were tested. The relative copy number of the integrated transgene was calculated as the fold difference between transgene and endogenous genes using the △△Ct method. The experiment was repeated five times with three separate preparations of genomic DNA. The expected abundance ratio for the endogenous gene, promoter and exogenous gene was 1, 2 and 1 respectively.
Axenic strain isolation
To establish axenic spirulina strains, cells were washed with SOT medium on 10-μm filters to exclude small, unicellular bacteria, and single filaments were isolated from cells captured on the filter. Cells were grown to a density of 0.5–1.0 OD750 ml–1 in an Innova incubator with appropriate antibiotics. Cells were pelleted from 5-ml cultures by centrifugation for 10 min at 1,600× RCF. To maintain sterility, the following steps were performed in a laminar-flow hood. The cell pellet was resuspended in 1 ml of SOT and transferred to a 10-μm filter prewetted with SOT medium, which was removed by gravity filtration. Cells were washed with successive 1-ml aliquots of SOT medium until at least 200 ml of total medium had passed through the filter. The remaining cells were resuspended with 0.5 ml of SOT and transferred to a sterile Eppendorf tube. Filaments were counted under a microscope as above, and 200–500 were spread on a SOT agar plate. Single filaments were isolated as above. After ~15 days, 10 μl of culture was spread on LB plates without antibiotics. Plates were incubated for 3–5 days in an incubator at 37 °C. Filament cultures free of contaminants on the LB agar plates were then seeded in 10 ml of SOT with 2.5 g l–1 dextrose at a density of 0.1 OD750 ml–1. Cultures were grown in Multitron for 3 days. A 100-μl sample of the culture was plated on LB agar plates without antibiotics and incubated at 37 °C for 5 days. Cultures with no contaminants observed on either set of LB plates were considered axenic.
Culture of non-spirulina microbes
To culture non-spirulina microbes, a flask of spirulina culture was placed on bench for 3–5 h to allow cells to settle at the bottom of the flask. A 100-μl sample of supernatant was carefully pipetted and transferred to either LB or mixed LB/SOT agar plates. Plates were incubated at 25–30 °C on a light rack (60–70 μEi) for 5–7 days. Single colonies were streaked on fresh plates for between five and ten rounds. Cells from single colonies were spread on fresh plates to propagate for further experiments.
Identification of non-spirulina bacteria from spirulina cultures
To culture non-spirulina microbes, a flask of spirulina culture was placed on the bench for 3–5 h to allow cells to settle at the bottom of the flask. A 100-μl sample of spirulina-conditioned medium was transferred to either LB agar or mixed LB/SOT agar plates, which were then incubated at 25–30 °C on a light rack (60–70 μEi) for 5–7 days. Genomic DNA was extracted from bacterial samples following the extraction method described above. Highly conserved and degenerate 16 S and 23 S ribosomal DNA PCR primers (Supplementary Table 1) were used to amplify genomic DNA, following published protocols50,51 from samples derived from both LB and LB/SOT plates. PCR product libraries were subcloned and sequenced. The probable species of origin was identified by BLAST query for similar sequences in the NCBI database.
Markerless strain engineering
To create a platform for markerless integration, a parental strain containing a recombinant, non-native antibiotic marker was first generated. An integrating plasmid bearing homology arms for the D01030 (kmR) locus flanking an aadA gene for streptomycin resistance was transformed into wild-type spirulina. The integrating vector was designed to precisely replace the open reading frame (ORF) of D01030 with the sequence for aadA. This vector was transformed into spirulina strains UTEX (SP3) and NIES (SP7), generating strains SP205 and SP207, respectively. After transformation, strains were propagated for 2 months and confirmed to be fully segregated by genotyping. The strains were also challenged with kanamycin to demonstrate loss of native kanamycin resistance.
Verification of markerless spirulina strains
Clonal isolates of fully segregated strains were verified as follows: (1) qPCR to demonstrate a single transgene per genome (above); (2) sequencing of chromosomal DNA to verify the absence of mutations in the homology arms and inserted gene(s) (above); (3) PCR to demonstrate loss of parental integration locus allele and complete segregation to homozygosity of the transgene (above); (4) chromosome DNA sequence of the 16 S rDNA locus to verify strain identity as A. platensis (above); (5) sequencing of alternative insertion sites in chromosomal DNA to verify lack of strain contamination with other engineered spirulina strains (above); (6) PCR to demonstrate absence of the integrating DNA vector backbone, which should be eliminated during integration by homologous recombination (below); and (7) verification of streptomycin sensitivity and kanamycin resistance by antibiotic challenge.
The vector backbone sequences outside of the homology arms should not integrate into the genome and thus should be absent from spirulina genomic DNA. To exclude the possibility of nonspecific integration of the vector backbone DNA, PCR was performed with primer pairs targeting the ampicillin resistance gene and E. coli origin of replication. At no point were these fragments observed in spirulina, suggesting that there is no integration of the vector outside of the homology arms.
Construction of markerless transgenes for spirulina integration
To ease cloning of transgenes into spirulina, a standardized vector was built for markerless integration. This ‘destination’ vector included integrating homology arms for the kmR locus flanking an ORF for the native kmR gene and a terminator. The antibiotic marker was followed by a recombinant promoter–terminator pair for transgene expression. The promoter–terminator pair consisted of a constitutively active, native A. platensis promoter (600 bp upstream of the cpcB gene, named Pcpc600) and the terminator of the E. coli ribosomal RNA gene rrnB (named TrrnB). A pair of Batrachochytrium salamandrivorans restriction endonuclease sites between the promoter–terminator pair was used for Golden Gate cloning of protein coding sequences for transgenic expression. Protein coding sequences with compatible B. salamandrivorans sites were purchased from IDT and cloned into the destination vector using a Golden Gate Assembly Kit (NEB). Plasmid DNA was purified from E. coli by the QIAprep Spin Miniprep Kit (Qiagen) and transformed into spirulina strain SP205. The product of integration of this construct is genetically identical to the wild-type kmR locus, excepting the transgene (that is, no non-native antibiotic markers are present).
Purification of recombinant protein from spirulina
Recombinant aa682 was purified from spirulina by immobilized metal affinity chromatography (IMAC). In brief, a 10-ml pellet of spirulina cells from strain SP1182 was collected from 2 l of culture by centrifugation. The pellet was resuspended in a total volume of 35 ml with lysis buffer (50 mM sodium phosphate buffer pH 8.0, 500 mM NaCl, 20 mM imidazole) supplemented with Pierce Protease Inhibitor Minitablets (Thermo Scientific) and 1 mM phenylmethylsulfonyl fluoride (PMSF). The resuspension was passed through a French pressure cell twice to lyse the cells. Samples were kept on ice throughout. The insoluble fraction was pelleted by centrifugation at 5,000× RCF for 30 min. The partially clarified lysate was mixed with 2 ml of washed HisPur Ni-NTA Resin (Thermo Scientific) and incubated at 4 °C with gentle rocking for 2 h. The resin was gently pelleted by centrifugation at 500× RCF for 1 min, supernatant discarded and the resin resuspended in fresh lysis buffer. This process was repeated until the supernatant was clear. The resin was collected in a small column by gravity filtration, washed with 20 ml of lysis buffer and spirulina-expressed aa682 was eluted with lysis buffer containing 200 mM imidazole. Purified aa682 was further polished by separation on a Superdex 75 Prep Grade column on an ÄKTA Pure, yielding a single band by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) electrophoresis.
Preparation of spirulina lysates for analysis of soluble protein
Soluble lysates from spray-dried spirulina samples were prepared using a flash-freeze protocol. Dried spirulina biomass was resuspended in PBS containing Pierce Protease Inhibitor minitablets and 1 mM PMSF at a biomass concentration of 10–40 mg ml–1 in 1.7-ml Eppendorf tubes. Samples were mixed to resuspend biomass powder and flash-frozen in liquid nitrogen for 2–5 min. Resuspensions were transferred to a water bath at 37 °C for 2–10 min. Samples were well mixed by inversion once thawed. The flash-freeze procedure was repeated an additional two times. Biomass samples were then centrifuged at 15,000g at 4 °C for 15–30 min, and the soluble fraction was transferred to a separate tube for downstream applications.
Expression analysis of recombinant proteins in spirulina
Recombinant protein expression in spirulina was measured by capillary electrophoresis immunoassay (CEIA) using a Jess system (ProteinSimple), which was run as recommended by the manufacturer. In brief, dried biomass samples were diluted to a concentration of 0.2 mg ml–1 using water and a 5× master mix prepared from an EZ Standard Pack 1, in either reducing or nonreducing format (Bio-Techne). Purified protein controls used to generate standard curves were typically loaded at a range of concentrations from 0.5 to 20 μg ml–1. A 12–230-kDa Jess/Wes Separation Module (ProteinSimple) was used and 3 μl of each sample was loaded for 9 s. A mouse anti-His-tag antibody (GenScript) was diluted 1:100 and used as the primary detection antibody. An anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (ProteinSimple) was primarily used for chemiluminescent detection; fluorescently labeled anti-mouse antibodies (ProteinSimple) for infrared or near infrared fluorescence detection were used for some experiments. Data analysis was performed using the Protein Simple Compass software.
Expression, purification and biotinylation of E. coli-expressed proteins
Recombinant C. jejuni flagellin was expressed and purified from E. coli. A region of FlaA (sequence ID: WP_178888959.1) predicted to be soluble and exposed on the surface of flagella (amino acids 177–482) was cloned onto the C terminus of MBP in a pET28b E. coli expression vector. The vector was transformed into BL-21(DE3) cells and grown overnight at 37 °C on agar plates with kanamycin, and a single colony was used to inoculate a culture of LB medium containing kanamycin. Cells were grown overnight with shaking at 225 r.p.m. and 37 °C, back diluted to OD600 = 0.05 and grown at 37 °C until cells reached mid-log phase (OD600 = 0.4–0.6). Cells were induced with sopropyl-β-d-thiogalactoside and incubated with shaking at 16 °C overnight. The following day, cells were pelleted by centrifugation at 3,500× RCF for 20 min at 4 °C, resuspended in 30 ml of lysis buffer containing protease inhibitors and lysed in a Q700 Sonicator (Qsonica). The MBP–FlaA fusion was purified from the clarified lysate using Amylose Resin (NEB) according to the manufacturer’s recommendations, and purified protein was aliquoted and stored at –80 °C. Biotinylated MBP–FlaA protein was prepared using an EZ-Link NGS-PEG4-Biotin kit (Thermo Scientific) following the manufacturer’s guidelines.
VHHs expressed in E. coli used similar expression vectors and bacterial cells lines. Culturing, induction and lysis of E. coli expressing VHHs followed the same protocol as for FlaA expression. Purification of VHHs from lysates was performed by IMAC, following the purification protocol described for aa682.
The RBD antigen used with VHH-72 was kindly provided by R. Strong (Fred Hutchinson Cancer Institute).
ELISA binding assays
The half-maximal effective concentration (EC50) binding activity of VHHs as a purified protein, and in spirulina lysate, was measured by ELISA. High-binding, 96-well plates (Greiner Bio-one or NUNC MaxiSorp) were coated with antigen by the addition of 100 μl of 1–5 μg ml–1 recombinant protein (FlaA or RBD antigen) in carbonate-bicarbonate buffer (Sigma) to each well and incubation overnight at 4 °C. Plates were washed three times with 300 μl of PBS supplemented with 0.05% Tween-20 (PBS-T). Washed plates were blocked with 250 μl of PBS-T supplemented with 5% nonfat dry milk (PBS-TM) for 2 h at room temperature. Blocking solution was discarded, and 100 μl of sample containing VHH was added to each well. VHH samples were prepared by dilution of purified protein or spirulina extracts with PBS-TM, and samples in a dilution series were serially diluted with PBS-TM. Samples were incubated at room temperature for 1 h to allow binding of VHH to antigen. After incubation, plates were washed three times with 300 μl of PBS-T 3. Wash was discarded, 100 μl of primary antibody diluted with PBS-TM was added to each well and plates were incubated at room temperature for 1 h. A 1:10,000 dilution of either a mouse anti-His-tag antibody (GenScript) or rabbit anti-camelid VHH antibody cocktail (GenScript) was used as the primary antibody. After incubation, plates were washed three times with 300 μl of PBS-T, and 100 μl of a secondary antibody was added to each well. A 1:10,000 dilution of either HRP-conjugated goat anti-mouse antibody or HRP-conjugated donkey anti-rabbit antibody was used as the secondary antibody. Plates were incubated at room temperature for 30–45 min, then washed twice with PBS-T and once with PBS. Plates were developed using either a SeraCare KPL TMB Microwell Peroxidase Substrate System (Sera Care Life Sciences) or 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Scientific) following the manufacturer’s recommendations. Peroxidase activity was quenched after 5–10 min with 50 μl of either 1 M HCl or 2 M sulfuric acid. Absorbance at 450 nm (A450) was measured on an M2 plate reader (Molecular Devices, SoftMax Pro software). All samples were tested in duplicate. Data analysis was performed using Prism (GraphPad Software).
Kinetics binding analysis of VHHs
Kinetics binding measurements were performed by biolayer interferometry (BLI) using an Octet Red96e (Forte Bio). Biotinylated MBP–FlaA was loaded onto streptavidin biosensors at a loading concentration of 100 nM and loading time of 4 min. After loading, probes were allowed to reach a baseline equilibrium in kinetics buffer (PBS with 1% bovine serum albumin and 0.05% Tween-20) for 2 min. Association and dissociation were monitored for 20 and 140 s, respectively. Purified aa682 diluted with kinetics buffer was assayed at concentrations ranging from 1 μM to 10 nM; the 10-nM sample was excluded from analysis due to a weak signal. Two biosensors were used as references: a 0-nM aa682 control and a no-ligand control. Kinetics binding values were determined using Octet Data Analysis HT software (ForteBio). Curve fits were performed using a global fit across all concentrations of aa682 assuming a 1:1 binding model.
Epitope mapping of VHH–antigen interaction
Epitope mapping of the interaction between FlagV6 and flagellin was performed using phage-displayed peptide fragments derived from a ~300-amino-acid-soluble fragment of C. jejuni FlaA. A sliding window of 30 amino acid fragments, with a two-amino-acid interval along the length of FlaA, was designed as oligos for cloning into the phagemid pADL-23c (Antibody Design Labs). The peptide library was cloned into the BglI site of the phagemid by Gibson Assembly and transformed into DH5a E. coli, yielding >6 × 104 transformants. The phagemid library was cleaned up with QiaPrep Spin Minikit columns and transformed into electrocompetent TG1 cells (Lucigen). Phage production was induced with the pIII-deficient helper phage CM13d3 (Antibody Design Labs) to ensure polyvalent display of the peptide epitopes. Phage from an overnight culture in 2× YT medium was precipitated and washed following the manufacturer’s protocol. Wells of an ELISA plate were coated overnight with 100 μl of 1 μg ml–1 FlagV6 VHH in carbonate-bicarbonate buffer, washed with PBS-T and blocked with PBS-TM. The phage library was diluted with PBS-TM to a concentration of 1012 phages ml–1 and incubated at room temperature for 30 min. Phages were then panned for VHH binders by the addition of 100 μl of blocked phage to wells of the ELISA plate and incubation on a vibrating platform for 2 h at room temperature. Unbound phages were washed from wells with 6,300 μl of PBS-T. Bound phages were eluted at low pH by the addtion of 100 μl of 100 mM glycine pH 2.0 and incubation for 5 min with shaking. The elution buffer was neutralized with 40 μl of 2 M Tris pH 7.5 and used to reinfect phage-competent TG1 cells (Antibody Design Labs). Library amplification and panning were performed for two additional rounds. After the third round of panning, all phagemid-containing colonies were observed to contain the same peptide fragment by Sanger sequencing. Two independent replicates of the experiment yielded overlapping fragments that mapped to the D3 domain of flaA.
Flow cytometry of VHH binding to C. jejuni
Binding of spirulina-expressed VHHs to a pure culture of C. jejuni was measured by flow cytometry. An aliquot of lysate prepared from spray-dried spirulina biomass was incubated with an equivalent volume of 107 CFU ml–1 C. jejuni 81–176 for 1 h at 4 °C. After washing with PBS containing 2% fetal bovine serum (FBS), bacteria were incubated for 30 min with the anti-His-tag antibody (iFluor647, GenScript). Samples were washed with PBS containing 2% FBS, resuspended in 2% paraformaldehyde and acquired on an LSR Fortessa flow cytometer (BD Biosciences) using forward and side scatter parameters in logarithmic mode. Data were analyzed using either FlowJo (TreeStar) or FACS Diva software (BD Biosciences).
Motility inhibition assay
The motility-inhibitory activity of spirulina-expressed aa682 was measured by the motility of C. jejuni through soft agar. All C. jejuni cultures were performed in a tri-gas incubator at 40 °C under microaerobic conditions (5% O2, 10% CO2) unless otherwise stated. Glycerol stocks of C. jejuni were first streaked on Campy Blood Agar Blaser plates (Thermo Scientific) and grown for 48 h. Bacteria were then used to inoculate 0.4% soft agar Mueller–Hinton (MH) plates by stab and incubated for 48 h. A slice of agar from the leading edge of motility halos was used to inoculate 10 ml of MH broth. Liquid cultures were incubated under standard conditions for 72 h. A 20-μl spot of 5 mg ml–1 purified aa682 in PBS was added to the center of soft agar MH plates and allowed to fully adsorb into the agar. VHH spots were inoculated with 1 μl of OD600 = 0.03 of C. jejuni from the liquid culture. Samples and controls were set up in triplicate. Plates were incubated under standard conditions. The diameter of motility halos was periodically measured and used to calculate area.
Midscale production of spirulina biomass for preclinical trials
To prepare biomass for preclinical mouse trials, the scale of spirulina culture was increased and harvested biomass was spray-dried. Spirulina cultures were initially propagated in shake flasks in medium based on the standard cyanobacterial SOT medium under Multitron conditions. Shake flask cultures were used to inoculate airlift reactors, with medium modified by partial replacement of sodium bicarbonate with sodium carbonate such that initial culture pH was 9.8. Cells were grown at light levels of 500–2,500 μmol m–2 s–1, with temperature maintained at 35 °C. As the culture utilizes CO2 and grows, pH rises and thus CO2 is added to the airlift stream to maintain pH between 9.8 and 10. Cultures were inoculated at a concentration of 0.1–0.5 g l–1 biomass by ash-free dry weight, and harvested by filtration at 2–4 g l–1.
To prepare for spray-drying, the harvested biomass was rinsed with a dilute (0.1%) trehalose solution to remove excess media salts, concentrated again by filtration and then spray-dried in a centrifugal nozzle spray-dryer. Feed rate, air flow and inlet air temperature were controlled to maintain an outlet air temperature of 68–72 °C at the powder-separation hydrocyclone. Once collected from the hydrocyclone, the powder was sealed and stored in airtight, opaque mylar bags to prevent exposure to moisture or light. The powder was stored at room temperature.
Before use in animal trials, spirulina biomass was analyzed to confirm strain identity. Dried biomass was genotyped to confirm the presence of the correct transgene and the absence of contaminating sequences (above). CEIA and ELISA binding assays (above) were also performed to confirm expression and binding activity of spirulina-expressed VHH.
Prophylactic treatment of C. jejuni infection in two mouse models
Two independent mouse models were used to test the efficacy of spirulina-expressed VHHs in the treatment of C. jejuni infection. Animal experiments at the University of Virginia were performed according to institutional review board (IRB) protocols. Animal experiments performed at the IRB were in accordance with the Swiss Federal Veterinary Office guidelines and authorized by the Cantonal Veterinary Office. In a pilot experiment with the first model of C. jejuni infection, 2–4-week-old C57BL/6 male mice were fed a zinc-deficient diet52 before challenge. Animals were maintained according to institutional protocols and fed a regular diet with ad libitum water for 3 days. Animals were then started on the study diet for 10 days, after which water was replaced by water containing an antibiotic cocktail for 3 days to condition gut flora for C. jejuni colonization. Water was replaced with untreated, antibiotic-free water for 1 day before C. jejuni challenge. On day 0, mice were given an inoculum of 106 live C. jejuni cells (strain 81–176, resuspended in PBS) by oral gavage. Food and water were provided ad libitum throughout. Mice were given five doses of a spirulina resuspension before and after challenge. Spray-dried spirulina biomass was resuspended in PBS at a concentration of 10% (w/v). A 200-μl resuspension was delivered by oral gavage on days –1, 0, 1, 2 and 3 relative to challenge. Groups received either PBS (eight mice), SP227 treatment (four mice) or SP526 treatment (eight mice). Day-of-challenge dosing was administered 60 min before challenge. Food and water were withdrawn 30 min before treatment, then provided ad libitum. To assess efficacy, mice were monitored for symptoms of diarrhea, changes in weight and bacterial shedding in stool. Weight measurements were made daily for 7 days. Stool samples were collected on days 1, 3 and 7 post challenge.
A second experiment using the first model of infection involved a change of study diet and a reduced spirulina dose. Animals were fed a regional basic diet for 10 days, followed by 3 days of antibiotic treatment. Untreated water was provided for 1 day before C. jejuni challenge. On day 0, mice were given an inoculum of 106 live C. jejuni cells (strain 81–176, resuspended in PBS) by oral gavage. A control group of four mice received no C. jejuni (PBS only). Food and water were provided ad libitum throughout. Mice were given three doses of spirulina before and after challenge. On days –1, 0 and 1 relative to challenge, mice were orally gavaged with 200 μl of spirulina resuspension or control. Day-of-challenge dosing was administered 60 min before challenge. Food and water were withdrawn 30 min before treatment, then provided ad libitum. Spirulina resuspension was prepared at a concentration of 2% (w/v) in PBS. Groups of mice were treated with either PBS (eight mice), SP257 biomass (four mice) or SP526 biomass (four mice). To assess efficacy, mice were monitored for changes in weight, bacterial shedding in stool and levels of biomarkers in cecum. Weight measurements were made daily for 7 days. Stool samples were collected on days 2, 4, 6, 8 and 10 post challenge. On day 11, levels of LCN-2 and MPO were measured in stool and cecal contents by ELISA (DuoSet ELISA Mouse Lipocalin-2/NGAL, R&D Systems).
In the second model of C. jejuni infection, mice were orally treated with a range of spirulina concentrations to identify the minimally effective prophylactic dose of therapeutic. Three-week-old C57BL/6 female mice were housed, five per cage, under standardized conditions (20 ± 2 °C, 55 ± 8% relative humidity, 12/12-h light/dark cycle). Food and water were available ad libitum and mice were monitored daily. Mice were pretreated orally with 10 mg of vancomycin in 200 μl of PBS at 48, 24 and 12 h before spirulina administration. A single 400-μl dose of spray-dried spirulina resuspended in PBS was administered by oral gavage to mice 1.5 h before infection with C. jejuni 81–176 (108 CFU 200 μl–1 PBS). To monitor efficacy, mice were observed daily and stools were collected at 24, 48 and 72 h post infection. To monitor pathogen load, stools were resuspended and plated on MH agar plates containing 10 μg ml–1 vancomycin and trimethoprim.
Cecal polymorphonuclear neutrophils (PMNs) were measured by flow cytometry 72 h post infection. Mice were sacrificed and the cecum was removed, opened longitudinally, carefully separated from cecal content and washed twice with ice-cold PBS. The cecum was digested twice with RPMI and EDTA 5 mM for 30 min at 37 °C. Filtrated fragments were then digested in RPMI 5% FBS, 1 mg ml–1 collagenase type II and 40 μg ml–1 DNase I for 40 min. The filtered suspension, containing cecum lamina propria cells, was centrifuged for 5 min at 300g and resuspended in RPMI complete medium. Single-cell suspensions from cecal lamina propria were stained with labeled antibodies diluted in PBS with 2% FBS for 20 min on ice. The following mouse antibodies were used: APC-conjugated anti-CD11b diluted 1:200 (Biolegend) and PE-conjugated anti-GR1 diluted 1:200 (TONBO Bioscience). Samples were acquired on an LSR Fortessa (BD Biosciences) flow cytometer. Data were analyzed using FlowJo or FACS Diva software.
The inflammation status of mice was evaluated by measurement of fecal LCN-2 levels in fecal supernatants by ELISA (DuoSet ELISA Mouse Lipocalin-2/NGAL, R&D Systems). In brief, feces collected at sacrifice were resuspended at 0.01 g 100 μl–1 PBS, centrifuged for 10 min at 17,000g and diluted before performing ELISA according to the manufacturer’s instructions.
Large-scale, continuous culture of spirulina
Spirulina cultures were grown at large scale (250 l) in airlift reactors following protocols similar to the midscale reactors described above. Cultures were inoculated into the same media described above for midscale cultures, at a concentration of 0.1–0.5 g l–1 biomass by ash-free dry weight, grown under identical temperature and pH controls and harvested by filtration over stainless steel screens at 2–4 g l–1. A portion of the harvested culture was used to inoculate serial cultures, and the remaining harvested biomass was used for spray-drying as above. The dried powder was sealed and stored at room temperature in airtight, opaque mylar bags to prevent exposure to moisture or light.
Post collection, quality control of powder lots included determination of concentration of the 6x-His-tagged protein using CEIA performed on a Jess system (ProteinSimple). Specific ligand binding activity was determined on an Octet Red96e biolayer interferometry instrument (Forte Bio) using recombinant, biotinylated C. jejuni FlaA protein attached to streptavidin-coated biosensors. In addition, microbial characterization was performed with USP <61> and <62> and elemental impurities determined by USP <233>.
Long-term stability of dried spirulina biomass
Batches of SP1182 spray-dried biomass were stored at room temperature and collectively assessed for binding activity by ELISA. Duplicate biomass samples from each batch were resuspended in PBS, lysed by freeze–thaw extraction and clarified by centrifugation. The binding activity of aa682 present in lysates was determined by ELISA with a recombinant FlaA antigen as described above. Purified aa682 was used to generate a standard curve for binding activity by linear regression using Excel (Microsoft software). The standard curve was used to calculate the concentration of aa682 in SP1182 lysates. The percentage of expected VHH activity was determined by normalization of aa682 concentration in each lysate to an assumed concentration of 3% aa682 per unit biomass.
In vitro gastric protease digests of dried spirulina biomass
Spray-dried SP1182 biomass was exposed to simulated gastric fluid (SGF) to determine the stability of the aa682 present in spray-dried spirulina. A sample of spray-dried SP1182 biomass was resuspended in PBS at 30 mg ml–1. This resuspension was diluted 1:30 with prechilled SGF (50 mM citrate-phosphate buffer pH 3.0, 94 mM NaCl, 13 mM KCl pH 3.5 with 2,000 U ml–1 pepsin (MP Biomedicals)) and incubated in a water bath at 37 °C. Protease activity was neutralized by the addition of 50 mM NaOH and 1 mM PMSF. Samples were pelleted by centrifugation at 17,000g for 5 min. Biomass pellets were solubilized using 1× NuPAGE LDS sample buffer to a final biomass concentration of 1 mg ml–1 and heated at 90 °C on a heat block for 10 min. A similar process was used to assess the stability of purified aa682, omitting the centrifugation step.
The stability and activity of biomass-encapsulated aa682 after exposure to low-pH, simulated gastric buffer was assessed by CEIA and ELISA binding assay. Spray-dried SP1182 biomass was resuspended in either 50 mM bicarbonate buffer or citrate-phosphate buffer pH 3.0 with 1 mM PMSF. Samples were incubated in a water bath at 37 °C for 60 min. After incubation, biomass resuspensions were pelleted at 10,000 r.p.m. for 5 min. The supernatant was transferred to fresh tubes and stored at 4 °C. Pellets were resuspended in 1 ml of 50 mM bicarbonate buffer to a final biomass concentration of 30 mg ml–1 and incubated in a water bath at 37 °C for 30 min. Resuspensions were treated to three cycles of flash-freezing in liquid nitrogen, followed by thawing at 37 °C for extraction of soluble protein. After the final thawing, samples were pelleted using a refrigerated tabletop centrifuge for 30 min at 17,000g to separate soluble protein from insoluble cellular debris. The supernatant was used to measure the expression level and binding activity of aa682 by CEIA and ELISA, respectively.
In vitro protease digests with intestinal proteases
To measure intestinal protease resistance, SP1182 lysates were digested with trypsin and chymotrypsin, and VHH binding activity was assessed by ELISA. Total soluble extract was prepared from a resuspension containing 40 mg of dried SP1182 biomass per 1 ml of bis-tris buffer (20 mM bis-tris pH 6.0) by the freeze–thaw protocol described above. Two volumes of soluble extract were mixed with one volume of protease in bis-tris buffer and one volume of PBS, to yield a final digest concentration of 0.1 or 0.01 mg ml–1 of trypsin or chymotrypsin with a reaction pH of ~6.5. Digests were performed at 37 °C for 1 h with shaking at 900 r.p.m. on an Eppendorf Thermomixer. Protease activity was neutralized by the addition of an equivalent volume of 2 mM PMSF and 2× Pierce Protease Inhibitor Mini tablets (Thermo Scientific) in PBS. Binding activity of VHH to recombinant FlaA was measured by ELISA as described above.
Intact mass spectrometry
The mass of purified aa682 was analyzed with a 6230 TOF (Agilent) using an ACQUITY UPLC Protein BEH C4 VanGuard pre-column (Waters Corp.). The following settings were used: temperature, 30 °C; injection volume, 20 μl; mobile phase A of water with 0.1% formic acid (FA); mobile phase B of acetonitrile with 0.1% FA; sheath gas flow rate, 10 l min–1; sheath gas temperature, 350 °C; nebulizer pressure, 20 psig; gas flow rate, 10 l min–1; gas temperature, 325 °C; nozzle voltage, 2,000 V; Vcap, 4,000 V; and mass range, 400–3,200 m/z.
Peptide mapping by mass spectrometry
The peptide sequence of aa682 was confirmed by mass spectrometry of peptide fragments produced by protease digestion. A 50-μl sample of 1 mg ml–1 aa682 was reduced and denatured with 6 μl of 0.25 M DTT and 10 μl of 6 M guanidinium HCL for 30 min at 37 °C in the dark. The denatured sample was diluted with 60 μl of PBS and then digested with 12 μl of 1 mg ml–1 trypsin and chymotrypsin overnight at 37 °C. The digested product was prepared for LC–MS with 2 μl of 5% trifluoroacetic acid (TFA) and run on an UltiMate 3000 UHPLC system (Thermo Scientific) with an LTQ XL mass spectrometer (Thermo Scientific) using a CSH C18 Column (Waters Corp.) The following settings were used: temperature, 50 °C; injection, 40–50 μg sample; mobile phase A of water with 0.05% TFA; and mobile phase B of LC–MS acetonitrile with 0.05% TFA. Peptide mapping data were processed with PepFinder 2.0 software (Thermo Scientific).
First-in-human clinical trial
A phase 1 clinical trial was designed and conducted to assess the safety and tolerability of LMN-101. The study protocol and all its amendments were reviewed and approved by the Alfred Hospital Ethics Committee. Eligible, healthy volunteers aged 18–50 years were enrolled following informed consent. The study was performed in accordance with ICH guidelines and in compliance with all local and international requirements. Details of the study can be found at clinicaltrials.gov (ID: NCT04098263).
Reporting Summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
