Bacterial strains, media, and growth conditions
MRSA strains SA0831 (clinical isolate from a CF patient from Dr. S.E. Birket, University of Alabama-Birmingham), AMT0204 and AMT0559, along with methicillin-sensitive S. aureus (MSSA) strains ANT0186, AMT0557 (clinical isolates from Dr. R.K. Ernst, University of Maryland-Baltimore) were grown at 37 °C in tryptic soy broth (TSB, Difco Laboratories) or on trypticase soy agar. For animal challenge experiments, SA strain SA0831 was grown at 37 °C in TSB to an A600 1.2. Bacteria were collected from the liquid cultures by centrifugation, washed once with phosphate-buffered saline (PBS), and resuspended in PBS to the desired CFU dose, depending upon which animal model was to be used. To enumerate the CFU, the suspensions were serially diluted and plated on Mannitol Salt Agar (MSA) followed by incubation at 37 °C overnight. For PA animal infections, the mucoid P. aeruginosa strain mPA08-31 was streaked onto Pseudomonas isolation agar (PIA) and incubated overnight at 37 °C. The following day, 20 ml of LB was inoculated with several isolated colonies of PA and incubated overnight at 37 °C with shaking at 180 rpm. A 200 µl aliquot from the overnight culture was inoculated into 20 ml of LB and grown at 37 °C with 250 rpm shaking the A600 reached ∼0.3. PA were collected by centrifugation, washed once, resuspended in PBS, and diluted to desired CFU dose.
Protein preparation
L-PaF was made as previously described12. Briefly, E. coli Tuner cells expressing L-PaF/His-Tag PcrH were grown in TB (terrific broth) media with chloramphenicol (34 μg/ml) using a fed-batch mode in a 10 L bioreactor (Labfors 5, Infors USA Inc., MD). The culture temperature was maintained at 30 °C and protein expression was induced by adding IPTG to 1 mM when the culture reached an A600 ∼25. After 3 h, the bacteria were collected and processed for purification. The L-PaF/His-Tag PcrH was captured on an IMAC column followed by Q anion exchange chromatography. Lauryldimethylamine oxide (LDAO) was added to a final concentration of 0.1% to release the HT-PcrH. The protein solution was passed over a final IMAC column with the L-PaF passing through the column. L-PaF was dialyzed into PBS with 0.05% LDAO and stored at −80 °C.
NEAT2 (N2) was produced using a codon-optimized gene for IsdB residues 351–458 (from IDT) and cloned in-frame into pET15b to have a His-tag and thrombin cleavage site using Gibson assembly. His-N2 was then expressed in E. coli BL21 (DE3) cells and grown in LB broth at 37 °C to an A600 = 0.8. His-N2 was then expressed for 3 h at 37 °C followed by adding IPTG to 1 mM. The culture was then centrifuged and the cell pellet resuspended in lysis buffer (50 mM Tris-HCl (pH 8.0), 0.1 M NaCl, 2 mM MgCl2, 10 mM imidazole, 1 mM PMSF, lysozyme (2 mg/ml)), lysed by sonication, and the lysate clarified by centrifugation. His-N2 was initially isolated from the clarified lysate using nickel-NTA chromatography and then purified to homogeneity by passage over a HiLoad 26/600 Superdex 200 size exclusion column. LPS levels were determined using a NexGen PTS with EndoSafe cartridges (Charles River Laboratories, Wilmington, MA). All proteins had LPS levels <5 Endotoxin units/mg protein based on analysis using an Endosafe system.
Preparation of L-PaF/ME/N2 formulations
ME contains 10 mM histidine, pH 6, 5% sucrose, 2% squalene, 0.5% PS80, which was prepared as previously described1. Briefly, squalene and polysorbate 80 were mixed to achieve a homogenous oil phase. Using a high-speed mixer, 40 mM Histidine (pH 6) and 20% sucrose were added to the oil phase and mixed a followed by six passes through a microfluidizer to generate 4XME. To make the protein/ME formulations, the protein was added to the ME with a final concentration of 0.67 mg/ml, vortexed, and allowed to incubate overnight at 4 °C.
Mice and immunizations
All animal protocols were reviewed and approved by the University of Missouri Institutional Animal Care and Use Committee Practices (protocol 38241). Six-to eight-week-old female C57BL/6 mice (n = 20/group) (Charles River Laboratories, Wilmington, MA) were used for all experiments. Prior to vaccination, the following were prepared in 30 µl volumes: PBS, 20 µg N2, 1 μg L-PaF + 1 µg N2 in ME (L-PaF/ME/N2 1 µg), 1 μg L-PaF + 10 µg N2 in ME (L-PaF/ME/N2 10 µg), and 1 μg L-PaF + 20 µg N2 in ME (L-PaF/ME/N2 20 µg). For immunizations, mice were anesthetized using isoflurane and vaccine formulations administered IN as previously described1. Immunizations were on days 0, 14, and 28. Blood was collected prior to each vaccination and at days 42 and 56 from the retro-orbital sinus using sterile glass capillary tubes under proper anesthesia. For MRSA pre-exposure, mice were anesthetized using isoflurane and MRSA (1 × 107 CFU/30 µl) was administered IN three times with two-week intervals. After last pre-exposure mice were observed for 30 days followed by immunization using the same method as for the naïve mice. The mouse experiment scheme is graphically described in Supplementary Fig. 1.
Rabbits and Immunization
The rabbit animal protocol was also reviewed and approved by the University of Missouri Institutional Animal Care and Use Committee Practices (protocol 38241). We used 4–5 month-old female rabbits (n = 8/group), weighing 2.0–2.8 kg (Envigo, Indianapolis, IN). The following vaccine formulations were prepared in 500 µl volumes: PBS, 30 μg L-PaF + 50 µg N2 in ME (L-PaF/ME/N2 50 µg), 30 μg L-PaF + 100 µg in ME (L-PaF/ME/N2 100 µg). For immunizations, rabbits were anesthetized by parenteral administration of ketamine-xylazine (ketamine 30 mg/kg, xylazine 3 mg/kg body weight). The vaccine formulations were administered IN using MAD atomizers (Teleflex, Morrisville, NC) on days 0, 14, and 28. Blood was collected prior to each vaccination and at days 42 and 56 from the marginal ear vein using a 23-gauge needle and syringe under proper anesthesia. For MRSA pre-exposure, anesthetized rabbits were IN administered 1 × 107 CFU/rabbit two times with a 4-week interval. After the last pre-exposure, rabbits were observed for 30 days then immunized following the methods used for naïve rabbits. The rabbit experimental scheme is graphically described in Supplementary Fig. 2.
Antigen-specific ELISA
Antibody titers specific for PcrV, PopB, and N2 were determined by ELISA, as described previously12. Briefly, 96-well plates coated with PcrV, Pop,B or N2 (1 μg/ml in PBS) were blocked overnight with 10% milk (Santa Cruz Biotechnology, Dallas, TX) in PBS. Each well was incubated with serum samples from mice or rabbit for 1 h at 37 °C. After washing the plates with PBS-Tween (0.05%), secondary antibody was added and incubated for 1 h at 37 °C. IgG and IgA titers were determined using HRP-conjugated goat anti-mouse IgG or goat anti-mouse IgA (Southern Biotech, Birmingham, AL) or goat anti-rabbit IgG or goat anti-rabbit IgA (Thermo Fisher Scientific, Waltham, MA) respectively. IgG subtype titers in immunized mice serum were also determined using HRP-conjugated goat anti-mouse IgG1, IgG2a, IgG3 (Southern Biotech, Birmingham, AL). 3,3′,5,5′-Tetramethylbenzidine substrate was added, and the reaction was stopped with H3PO4. Endpoint titers were calculated and represented as Log10 ELISA units per mL (EU/ml).
Opsonophagocytic killing assay
OPK was performed as described with some modifications26. Briefly, mouse neutrophils were isolated from bone marrow by MojoSort™ Mouse Neutrophil Isolation Kit (Biolegend, San Diego, CA). Overnight culture of MRSA was diluted 1:100 in TSB and grown to A600 = 0.6. The MRSA was washed, resuspended in PBS, and incubated with complement-inactivated L-PaF/ME/N2 or PBS immunized mouse sera at 37 °C for 20 min. MRSA/serum mix was added to 105 mouse neutrophils at a multiplicity of infection (MOI) of 1:0.5 in the presence of 10% (vol/vol) baby rabbit complement. Following incubation at 37 °C for 1 h with agitation at 250 rpm, samples were plated on MSA for CFU enumeration. For PA OPK, overnight cultures of PA were diluted 1:200 in LB and grown to A600 = 0.3. PA was washed, resuspended in PBS, and incubated with complement-inactivated L-PaF/ME/N2 or PBS immunized mouse sera at 37 °C for 30 min. PA/serum mix was added to 105 mouse neutrophils an MOI of 1:0.5 in the presence of 10% (vol/vol) baby rabbit complement. Following incubation at 37 °C for 1 h with agitation at 250 rpm, samples were plated on PIA for CFU enumeration.
IL-17A and IFN-γ ELISpot
Immunized mouse lungs were extracted and processed to single-cell suspensions according to the manufacturer’s specifications (Miltenyi Biotec, Gaithersburg, MD). Lung cells (1 × 106 cells/well) were incubated for 24 h at 37 °C in the presence of 5 μg/ml PcrV, PopB, N2, or PBS, in plates coated with antibodies against IL-17A and IFN-γ for a colorimetric assay as per manufacturer’s specifications (ImmunoSpot, Cleveland, OH). The IL-17A and IFN-γ secreting cells were quantified using a CTL ImmunoSpot reader. Biological negative controls were maintained as PBS mice group while technical negative controls were cells without any protein treatment.
Cytokine determinations
Lung cells were incubated with 10 μg/ml PcrV, PopB, N2, or PBS for 48 h at 37 °C. Supernatants were collected and analyzed with U-PLEX kits for cytokines: IFN-γ, IL-2, IL-6, IL-22, IL-17A, and TNF-α (Meso Scale Discovery, Rockville, MD). Cytokine concentrations were determined using an MSD plate reader with associated analytical software (Meso Scale Discovery, Rockville, MD).
Challenge studies
Mouse challenge study
For MRSA challenge, the MRSA strain SA 0831 was prepared as described above. On day 56, mice (n = 10) were anesthetized by isoflurane and IN challenged with 1 × 109 CFU/30 µl MRSA. Clinical body scores were documented as follows: 1: Bright, alert, reactive, shiny hair coat, no piloerection; 2: Quiet, alert, reactive, early-stage piloerection, mild dehydration; 3: Quiet, not very reactive, mild piloerection, moderate dehydration; 4: Minimally reactive, severe piloerection, dull, dirty hair, hunched posture, respiratory distress. Mice were monitored twice a day for weight loss and health scores for 14 days. Mice were euthanized if their weight loss exceeded 25% of their original weight for more than 72 h. All remaining mice were euthanized on day 14 post-challenge.
For the PA challenge, the PA strain mPA08-31 was prepared as above. On day 56, mice (n = 5) were anesthetized by isoflurane and IN challenged with 4 × 107 CFU/30 µl PA. Mice were monitored twice a day for weight loss and health scores for two days. Clinical body scores have been documented as previously described. On day 2 post-infection, mice from each group were euthanized, the lungs were processed, and CFU/lung enumerated on PIA plates.
Rabbit challenge study
Both MRSA and PA were grown following the previously described protocol. On day 56, to establish MRSA or PA mediated pneumonia in the rabbit model, a 1.5 ml PBS containing 1 × 109/ml MRSA SA0831 strain51 or 1.5 ml PBS containing 9 × 107/ml PA mPA08-31 strain28 was delivered directly into the lungs of anesthetized New Zealand white outbred rabbits through a 2.5 mm pediatric endotracheal tube as described52. Post challenge, rabbits were monitored every 8 h for the first 48 hpi (hours post infection) and two times daily thereafter. Body weights were measured daily. Body weight loss was calculated for each individual animal using this formula: [100-[((Initial weight – Final weight) / Initial weight) × 100]]. Clinical body scores have been documented as follows: 1: Bright, alert, reactive 2: Quiet, alert, reactive, mild dehydration; 3: Quiet, not very reactive, moderate dehydration, moderate respiratory distress; 4: Minimally reactive, severe respiratory distress, loss of body temperature, loss of activity. Rabbits reached body score 3 or high were euthanized immediately by intravenous administration of a lethal overdose of pentobarbital and survivors were euthanized at 144 hpi following the same method. Lungs were removed aseptically from all the euthanized rabbits, weighted, homogenized and CFU enumerated on MSA plates for MRSA and PIA plates for PA.
Histological analysis
Following euthanasia, the left lung was harvested and fixed by gravity instillation through the bronchus with 10% formalin. For each left lung, a single section from the upper left lobe was stained with hematoxylin and eosin (H&E) and analyzed in a semiquantitative manner by a single investigator blinded to the experimental groups. Images were taken using Leica DM5500 microscope.
Statistical analysis
GraphPad Prism 9.0.1 was used for graphs and statistical analysis. Survival curves were generated using the Kaplan-Meier method, Log-rank (Mantel-Cox) tests were used for survival tests. Mann-Whitney U test (two tailed) was used for comparison between two groups. Kruskal-Willis test with Dunn’s multiple comparison was used for comparison between three or more groups. P values < 0.05 are considered statistically significant. *P < 0.05; **P < 0.01; ***P < 0.001. P values > 0.05 were mentioned otherwise.
