Sm-p80 B7 cloning and production

A soluble deletion variant, termed B7, [44 kDa monomer; amino acid residues 79 to 450 of P27730 (UniProt)] has been generated from the full-length Sm-p80 antigen in which the hydrophobic regions and portions not containing the catalytic triad required for enzymatic activity have been removed. The B7 protein was tested for its enzymatic activity using the enzymatic assay described below.

The coding sequence for Sm-p80 B7 was obtained from the Seattle Structural Genomics Center for Infectious Disease (SSGCID, Seattle, WA, USA). Constructs were designed by comparing secondary structure predictions calculated by XTALPRED²⁴ with multiple sequence alignments (MSA) of the Sm-p80 peptide sequence. MSA were generated by BLASTP search of the Protein Data Bank^25,26. N and C-terminal boundaries were determined based on locations of the Sm-p80 sequence that had poor sequence conservation and no secondary structure predicted. Structure-based alignments included proteins with at least 35% sequence identity and >300 length matching residues. Gene fragments covering the coding sequence were produced (IDT, Coralville, IA, USA) and cloned into the pET29a expression vector using an E. coli Turbo competent bacterium (New England Biolabs, Ipswich, MA, USA). Plasmids obtained from the successfully transformed bacteria were verified via Sanger sequencing. pET29a containing Sm-p80 B7 was then transformed into an E. coli production strain, HMS174(DE3) (Millipore Sigma, Burlington, MA, USA).

Sm-p80 B7 protein was produced by induction of the recombinant HMS174(DE3) bacterial culture at an OD600 of 0.5 with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) followed by further incubation at 25 °C for 24 h. The culture was then spun down at 14,000 × g, and the resulting pellet was resuspended for lysis in B-PER^TM (Thermo-Fisher Scientific, Waltham, MA, USA) at a ratio of 10 mL per 1 g of cell pellet. The solution was then freeze-thawed 3 times to facilitate bacterial cell lysis, followed by centrifugation at 14,000 × g to isolate the protein-containing inclusion bodies (IB) pellet. The pellet was then resuspended in 8 M Urea/20 mM Tris at 37 °C until solubilization of the IBs occurred. A nickel-nitrilotriacetic acid (Ni-NTA) resin was then added to the supernatant to bind the His-tagged Sm-p80 B7 protein, and the mixture was left rotating overnight at 4 °C. The resin-lysate was loaded into a gravity-flow column and the protein was then eluted off the column after a series of washes with gradually increasing concentrations of imidazole in 8 M Urea/20 mM Tris. Purified B7 was refolded by dialysis three times each into 10 volumes of 20 mM Tris pH7.4. Following the third dialysis, the protein was recovered, tested in the calpain assay for enzymatic activity, and found to be active. Fractions were run on an SDS-PAGE gel to identify those containing Sm-p80 B7, and those fractions were pooled, concentrated and then buffer exchanged into 25 mM HEPES, pH 7.5, 500 mM NaCl, 5% Glycerol. The final product was analyzed by reducing SDS-PAGE gel and protein concentrations were calculated by comparing lane intensity to a standard load of bovine serum albumin (BSA) using ImageJ software (NIH, Bethesda, MD, USA). The B7 protein produced exhibited >95% purity and <500 EU/mg endotoxin levels. Endotoxin was measured using the FDA approved EndoSafe PTS reader from Charles River Laboratory (Wilmington, MA, USA).

Activity assay of the Sm-p80 protease core (clone B7)

Following screening of a number of synthetic- (e.g., Suc-LLVY-AMC, Suc-LY-AMC, Edans-PLFAERK-Dab, Edans-ALGIGTIPPK-Dab, Edans-ALGIGTIPPK-Dab) and protein substrates (e.g. casein, gelatin and fibronectin), a suitable fluorescent substrate (Suc-LLVY-AMC) for B7 was identified. Concentrations of 0.01 µg/µl, 0.02 µg/µl, 0.06 µg/µl and 0.12 µg/µl of B7 were tested in the reaction mixture (20 mM Tris pH 7.1, 10 mM CaCl₂). The 0.12 µg /µl concentration provided the most consistent reactivity. The Ca²⁺ requirement for the assay reaction was titrated from 0.1 to 100 mM (mixtures at Ca²⁺ >20 mM, precipitated) and 5–10 mM Ca²⁺ was found to be the optimal for the reaction. B7 showed continued reaction progress up to 3 h in the presence of 5 mM Ca²⁺ and 0.1 mM Suc-LLVY-AMC and was thermo stable at 37 ^oC for the same duration of time. B7 prefers neutral pH conditions, activity at pH 7.0–7.5 with Ca²⁺ 5–10 mM is defined as 100%. A number of inhibitors in different concentrations in the B7-based reaction were tested including EDTA, E64-c, Cast-d1, Ac-Cast, calpeptin, leupeptin, AEBSF and Bortezomib. Leupeptin, an established cysteine protease inhibitor was identified as the most consistent inhibitor of the reaction. Based on the above-mentioned parameter optimization steps, a consistent and optimally performing assay procedure was developed.

Optimized B7-based assay

Enzymatic reactions contained the following: Reaction Buffer (20 mM Tris, 50 mM NaCl, pH 7.1); 12 µg B7 enzyme, 10 mM CaCl₂, 0.1 mM Suc-LLVY-AMC (AnaSpec, Fremont, CA or Millipore-Sigma, Burlington, MA) in a final volume of 100 µL. Leupeptin 200 µM was used as a positive inhibitor control. Reaction mixture was set in a 96 well clear bottom plate. Fluorescence measurements were taken using a SpectraMax® iD3 multimode plate reader (Molecular Devices, San Jose, CA). Fluorescence was measured for 3 h at 2-min intervals using the following parameters: Wavelengths for extinction and emission, 345 and 445 nm, respectively, with integration times of 140 seconds, PMT setting low, shaking enabled, constant temperature at 37 ^oC.

Inhibition of B7-based assay activity with rodent, nonhuman primate, and human sera vaccinated with Sm-p80 + GLA-SE vaccine

To study antibody-mediated inhibition of B7 activity, a battery of sera from different studies were used: murine¹⁴ and baboon sera⁷ from animals vaccinated with Sm-p80 + GLA-SE in dilutions ranging from 1:1000, 1:10,000 and 1:100,000. In addition, IgG purified from sera of baboons vaccinated with Sm-p80 + GLA-SE⁷ was used at varying concentrations (0.032–1.6 ng/µL). Similarly, human serum samples from participants vaccinated with Sm-p80 with and without GLA-SE in the non-endemic population of the USA (ClinicalTrials.gov Identifier: NCT05292391) and schisto-endemic population of Madagascar, Africa (Ethical Approval, Ministere De La Sante Publique, CERBM: IORG00001212 No 122MSANP/SG/AMM/CERBM) were included to determine the inhibition of B7-mediated protease activity.

Data analysis on protease activity inhibition

To quantify B7 enzyme inhibition, the assay from above was performed in the presence of a variety of serum samples. A minimum of three technical replicates were included for controls and experimental samples across all of the dilutions used. Raw data were collected and plotted as relative fluorescence units (RFUs) versus 3-h reaction time (Y-axis = fluorescence; X-axis = time). The slope value of the plot indicated the B7 activity in RFU/min. Data are presented as mean ± SE. Significance of fluorescence intensity data differences was evaluated using Student’s t test with GraphPad Prism™ Software version 10. P values < 0.05 were considered statistically significant.

To normalize the RFU, the background RFU of each sample was obtained as the average RFU value of the wells containing buffer plus substrate but not B7 and subtracted from the RFU value of corresponding samples. The normalized relative RFU activity in each sample was obtained by dividing these with the B7 RFU values, and percent activity was then calculated. The relative inhibition in samples was calculated by subtracting the normalized activity from 100. A percent enzyme inhibition activity curve relative to antibody sera titer dilutions or IgG concentrations was plotted. Sm-p80-specific antibody titers were directly related to inhibition of B7-based activity; high titers and lower sera dilutions exhibited higher inhibition of the B7-based activity.