Antigen production
Protein vaccine antigens were produced recombinantly in E. coli at Genscript. The lukA and lukB genes for LukAB CC8Δ10C and LukAB RARPR-33 were cloned in a single expression vector behind a T7 promoter, and a non-cleavable N-terminal His-tag was added to LukA. The genes for the SpA* surface protein, MntC and ClfA were cloned in an expression vector behind a T7 promoter, and a cleavable N-terminal His-SUMO tag was added. All vaccine antigens were expressed in the E. coli cytosol and subsequently affinity purified from the supernatant of the whole cell lysate. After affinity purification of the LukAB RARPR-33 protein, the material was subjected to gel filtration to remove residual impurities. After affinity purification of the SpA* protein, MntC and ClFA, the cleavable His-SUMO tag was removed.
The wild-type LukAB toxins were produced in S. aureus. The lukA and lukB genes for LukAB toxins were cloned in a single expression vector behind a native lukAB promoter, and a non-cleavable N-terminal His-tag added to LukA. The toxins were purified from the S. aureus culture supernatant by affinity purification.
CP5 and CP8 were extracted from S. aureus biomass at Premas Biotech (Gurgaon, India) and conjugated to CRM197 (Reagent proteins, Pfenex) through thioether chemistry.
Adjuvants
Commercially available AS01B, which is provided as separate vial as part of the Shingrix vaccine (GSK) was used42. The liposome-based adjuvant contains 100 µg/ml 3-O-desacyl-4′-monophosphoryl lipid A (MPL), a TLR4 agonist, and 100 µg/ml QS-21, a natural saponin molecule. For animal studies, the adjuvant was mixed 1:1 (v/v) with antigens prior to vaccination, resulting in injection of a half human AS01B dose (25 µg MPL and 25 µg QS-21 in 500 µl). Liquid research grade GLA-SE, a synthetic TLR4 agonist in squalene oil-in-water emulsion, was manufactured by the Access to Advanced Health Institute (AAHI, Seattle, WA) formerly the Infectious Disease Research Institute (IDRI)43. The concentration of the GLA-SE stock was 250 µg/ml GLA, 10% SE. For preparation of the formulation with antigens, the GLA-SE stock was diluted with 10% SE and buffer (1.825% (v/v) glycerol and 25 mM ammonium phosphate pH 5.75) to 40 µg/ml GLA, 4% SE and mixed 1:1 (v/v) with the antigens to obtain a dose of 10 µg GLA, 2% SE for intramuscular vaccination. For intradermal vaccination, the GLA-SE stock was diluted with buffer to 100 µg/ml GLA, 4% SE and mixed 1:1 (v/v) with the antigens to obtain a 5 µg GLA dose with 2% SE.
Minipig immunization
Five to eight-month-old male Göttingen minipigs (Marshall Biosciences, North Rose, NY) were group-housed and maintained on a 12-hour light/dark cycle with access to water ad libitum. Animals were vaccinated by 3 injections separated by 3 weeks with antigen(s) combined with the adjuvant 30 min prior to each vaccination. On the morning of vaccination, fasted minipigs were sedated with Telazol (Zoetis, Parsippany-Troy Hills, NJ; 3.5–4 mg/kg) given intramuscularly and bled before each vaccination. For intramuscular injections, the vaccines were administered in a 500 µl volume in the left hind leg using a 23 G ¾” needle. For intradermal injections, the vaccines were administered in a 100 µl volume behind the ear using the Tropis® needle-free injection system (PharmaJet, Golden, CO). Surgery and infection occurred 3 weeks after the last immunization.
Deep seated SSI model
The deep-seated SSI model was described earlier8. On the morning of surgery, fasted minipigs were sedated with a mixture of ketamine (8–10 mg/kg) and dexmedetomidine (Dexdomitor; Zoetis, Parsippany-Troy Hills, NJ; 0.08 to 0.1 mg/kg) given intramuscularly, away from the surgical site. Once intubated, the animals were placed on isoflurane inhalant anesthesia and maintained for the duration of the surgery. Before surgery, animals received buprenorphine (0.02 to 0.05 mg/kg) intramuscularly away from the surgical site.
Surgery was performed on the left thigh whereby the muscle layer was exposed and a 5-mm bladeless trocar (ENDOPATH® Xcel, Ethicon Endo-Surgery; Guaynabo, Puerto Rico) was advanced to the depth of the femur. A bacterial challenge consisting of 20 µL inoculum (approx. 106 CFU S. aureus) was injected into the wound (top of femur) via a 6-inch MILA spinal needle (Mila International, Inc.; Florence, KY) through the trocar, which was then removed. After administration of the bacterial challenge, the muscle was closed with a silk suture, and the skin was closed with an absorbable polydioxanone suture. Eight days later while under sedation, minipigs were euthanized with a barbiturate. Samples were homogenized in saline using a Bead Ruptor Elite (Omni International; Kennesaw, GA, USA), then diluted and plated on tryptic soy agar plates using an Autoplate 5000 Spiral Plater (Spiral Biotech; Norwood, MA, USA). Plates were incubated 18–24 h at 37 °C, then read on a QCount colony counter (Spiral Biotech). Clearance of bacteria from the surgical site was our primary endpoint. We also examined body weight and body temperature of the animals. The animals appeared bright and alert following surgery/infection and there was no effect on body weight as all animals continued to gain weight after bacterial challenge. Body temperature was assessed, and all animals experienced an increase in body temperature following infection but generally this was resolved within 24 h, regardless of the treatment. All animal studies were reviewed and approved by the Janssen Spring House Institutional Animal Care and Use Committee and housed in an AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care International)-accredited facility.
Minipig SWI model
Fasted minipigs were sedated, intubated, and placed under anesthesia for the duration of the surgery. A full thickness incision 2 cm long and positioned 2 cm from the spine was made in the skin and 50 µl of the inoculum (5 × 106 CFU of S. aureus USA300) was pipetted into the incision. The incision remained open for the 8-days infection period. Skin was collected and homogenized. Tissue was collected from both edges and weighed and normalized to tissue weight. Samples were processed as described above for the SSI model.
Bacterial strains for in vivo challenge and growth conditions
Two clinical blood isolates strains of S. aureus were used and are described in Table 1: ST398 (OC 26263, MSSA) was used for the minipig challenge studies (ST398 strains typically colonize pigs but can cause disease in humans) and ST8/USA300 (OC26260, MRSA) were included because of their high global prevalence among healthcare-associated and community-acquired MRSA infections. Strains were grown in tryptic soy broth overnight prior to use.
Enzyme-linked immunosorbent assay
To determine total IgG in minipig serum, antigens were coated onto Nunc 384 -well Maxisorp plates (VWR, Amsterdam, Netherlands) for 1 h (LukAB, 1 µg/ml), or overnight (SpA*, 0.25 µg/ml), at 2 °C–8 °C. Plates were blocked for 1 h at room temperature (RT) with 2.5% (w/v) skimmed milk in PBS prior to washing and subsequent addition of serial dilution of serum. The sera were incubated for 1 h at RT. After washing, secondary antibody (rabbit antipig IgG horseradish peroxidase [HRP], Sigma Aldrich, St. Louis, MO, USA) was added at 1:10,000 dilution and incubated at RT for 1 h. After further washing, 3,3′,5,5′-tetramethylbenzidine was added to detect the HRP. The reaction was stopped after 30 min with 1 M sulfuric acid, and absorbance was read at 450 nm.
Cytotoxicity assays
To evaluate the cytotoxicity of LukAB protein complexes, freshly isolated primary human PMNs were intoxicated with S. aureus toxins. Prior to intoxication, all toxins were normalized to 100 µg/ml (per subunit) and then serially diluted 2-fold in 10 µl of 1× PBS. PMNs were isolated and normalized to 200,000 cells per 90 µl RPMI (10 mM HEPES + 0.1% HSA). 90 µl of PMNs were added to each well and incubated at 37 °C + 5% CO2 for 1 hr. 10 µl of CellTiter 96 Aqueous One Solution (CellTiter; Promega, Madison, WI, USA) was added, and the mixture was incubated at 37 °C in 5% CO2 for 1.5 h. PMN viability was assessed with a PerkinElmer EnVision 2103 Multilabel Reader at an absorbance of 492 nm. % Dead cells were calculated by subtracting out background (healthy cells + PBS) and normalizing to Triton X-100-treated cells which were set at 100% dead.
Toxin neutralization assays
Neutralization of LukAB toxin with sera from minipigs was described earlier8. In brief, THP1 cells were incubated for 2 h with serially diluted serum samples and a fixed concentration of each LukAB toxin. The supernatant was harvested, and the release of lactate dehydrogenase (LDH) was measured via a fluorometric reaction. The amount of LDH released in the supernatant is directly proportional to the damage inflicted by the toxin. A percentage cytotoxicity was calculated for each serum dilution. Toxin neutralization results were reported as IC50 values calculated from 4-PL curves of the cytotoxicity values, which indicate the half maximal inhibitory concentration effective in inhibiting the LukAB toxin function.
PBMC assays
Minipig blood was collected in cell preparation tubes (CPT) with sodium heparin. Within two hours after collection, tubes were centrifuged for 20 min at 1700 g at RT. The mononuclear cell layer was collected and washed twice with PBS (10 min at 300 g). Cells were frozen at concentration of 5 × 106 cells/ml in 10% DMSO, 25% FBS and 65% RPMI. After overnight storage at −80 °C, the cells were moved to liquid nitrogen until further use. For the T cell proliferation assay, cells were thawed at 37 °C and washed with IMDM with Glutamax + 10% FBS and penicillin-streptomycin. Cells were rested for 2 h at 37 °C, after which they were labelled with CellTrace CFSE (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s protocol. 2 × 105 PBMCs were stimulated per well, and depending on cell availability multiple wells were used per condition. PBMCs were stimulated for 4 days with 8 µg/ml LukAB RARPR-33 or SpA*. Medium only was used as a negative control. After 4 days, PBMCs were stained with conventional T cell markers and analyzed using a LSR Fortessa flow cytometer. For ELISpot assay, fresh PBMCs were isolated from the blood three weeks post the third immunization. 500,000 cells were stimulated for 18–20 h with 8 µg/ml LukAB RARPR-33 or medium in ELISpot plates (Mabtech, Nacka Strand, Sweden). ELISpot plates were revealed for IFN-γ and/or TNF-α secreting cells according to manufacturer’s protocol (Mabtech).
Statistical analysis
ANOVA with Dunnett post hoc test, or in case of censored values, a Tobit Model with a Bonferroni correction, was used to test statistical significance between multiple groups within one study. The model contained group and surgery date as explanatory factors. P values < 0.05 were considered significant.
