Evaluating the Immune Response of a Nanoemulsion Adjuvant Vaccine Against Methicillin-Resistant Staphylococcus aureus (MRSA) Infection

The animal experiments were conducted based on the manual on the use and care of experimental animals and were approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University. Female Balb/c mice, 6-8 weeks old, were used for the present study. The animals were obtained from commercial sources (see Table of Materials).

1. Preparation of the MRSA HI antigen protein

1. Obtain IsdB and Hla clones from commercial sources (see Table of Materials), perform polymerase chain reaction (PCR) amplification to amplify IsdB and Hla genes, and ligate their products to the PGEX-6P-2 plasmid (obtained commercially; see Table of Materials). Screen with the vector Xl/blue strain of Escherichia coli¹⁰.
2. Transform the positive clone into E. coli BL21 competent cells (see Table of Materials) and amplify with shark culture¹⁰.
3. Express the antigen and isolate after induction with isopropyl-β-D-1-mercaptogalactopyranoside and multimodal chromatography (MMC) isolate kits¹¹(see Table of Materials).
4. Characterize the antigen protein and purify it with a full-automation purifier¹¹.
   1. Affinity-purify gutathione S-transferase (GST)-tagged proteins from cleared lysates using a commercially available affinity chromatography resin (see Table of Materials).
   2. Purify the recombinant proteins by MMC¹¹.
   3. Remove the endotoxin by the Triton X-114 phase separation method¹¹. Briefly, mix 1% Triton X-114 with recombinant protein eluate at 0 °C for 5 min to ensure a homogenous solution, and incubate at 37 °C for 5 min to allow two phases to form.
5. Use the bacterial endotoxin test method¹¹ with Turtle kits (see Table of Materials) to detect the endotoxin concentration. The endotoxin for protein antigen is 0.06 EU/µg, lower than the international standard (1.5 EU/µg)¹⁰.
   1. Perform an interference test to eliminate the possible influence of the test product on endotoxin detection¹¹.
      NOTE: According to the Appendix 2020 of Chinese pharmacopeia¹², the endotoxin content should be less than 5 EU/dose. The sensitivity of Limulus lysate (λ0.25 EU/mL) and the maximum effective dilution ratio of 33.3 were calculated¹² based on MVD = cL/λ, where L is the limit value of bacterial endotoxin for the test article, c is the concentration of the test article solution, and when L is expressed in EU/m, c is equal to 1.0 mg/mL. λ is the labeled sensitivity (EU/mL) of the horseshoe crab reagent in the gel method.
6. Recombine the antigen (48 kDa, determined by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]) at 1.5 mg/mL in sterile water without endotoxin or phosphate buffered saline (PBS) (sodium dichlorate method)¹¹.
   ​NOTE: The protein peak time was 13.843 min, the purity was 99.4% (High-performance liquid chromatography method), and the protein was stored at -70 °C¹⁰. Genomic DNA extracted from S. aureus strain MRSA252 (see Table of Materials) was used as the PCR template. The IsdB gene was amplified using the forward primer 59-CGCGGATCCATGAATGGCGAAGCAAAAGCAG
   C-39 and the reverse primer 59-TTTTCCTTTTGCGG
   CCGCCTATGTCATATCTTTATTAGATTCTTC-39. The Hla gene was amplified using the forward primer PHLAF (GCGGATCCGATTCTGATATTAATATTAAAACC) and the reverse primer PHLAR (TAAGCGGCCGCTTAT
   CAATTTGTCATTTCTTC).

2. Preparation of the nanoemulsion vaccine

1. According to the mass ratio of 1:6:3(w/w), add three kinds of excipients in sequence (isopropyl myristate, polyoxyethylene castor oil, and propylene glycol; see Table of Materials) to the HI antigen protein solution (10 mg/mL) and stir well. Then, add gradually sterilized pure water to reach a final volume of 10 mL and stir at 500 rpm and 25 °C for approximately 20 min.
   NOTE: The nanoemulsion prepared in this protocol has a volume of 10 mL, and the masses of the three excipients are 0.34 g, 2 g, and 1 g, respectively. The protein solution here is the working solution, 10 times the concentration of the final solution.
2. Prepare the nanoemulsion adjuvant vaccine (1 mg/mL protein) by low-energy emulsification methods¹³ to obtain a clear and transparent liquid solution.
   ​NOTE: The blank nanoemulsion (BNE) was prepared by similar methods, except that water was replaced with HI. The usual emulsification method involves heating the outer and inner phases to approximately 80 °C for emulsification and then stirring and cooling them gradually. In this process, a large amount of energy is consumed, and finally, the emulsion is obtained.

3. Physical characterization and stability of the nanoemulsion adjuvant vaccine

1. Particle size, zeta potential, and polymer dispersion index characterization (PDI).
   1. Prepare 100 µL of the nanoemulsion adjuvant vaccine and dilute 50-fold with water for injection. Add 2 mL of sample for detection.
   2. Observe the particle sizes, zeta potential, and PDI at 25 °C using a Nano Zetasizer (ZS; see Table of Materials).
2. Transmission electron microscopy (TEM)
   1. Dilute 10 µL of nanoemulsion vaccine 50-fold with water, place on a carbon-coated 100-mesh copper grid, and stain with 1% phosphotungstic acid (see Table of Materials) for 5 min at room temperature.
   2. Remove excess phosphotungstic acid solution with filter paper.
   3. Observe the morphology under a transmission electron microscope (see Table of Materials). Examine whether the finished products are nearly stable and circular within the field of view of the electron microscope and whether they exhibit good dispersion.
3. Scanning electron microscopy (SEM)
   1. Dilute 10 µL of the nanoemulsion adjuvant vaccine 50-fold with water, drop 10 µL of prediluted samples on a silicon wafer, and place in a 37 °C thermostat-controlled incubator for 24 h.
   2. Spray the prepared platinum on the silicon for 40 s and cool to room temperature.
   3. Observe the morphological properties of the prepared samples under a high-resolution scanning electron microscope (see Table of Materials). Determine whether the samples are nearly stable, spherical, and well dispersed within the field of view under the electron microscope.
4. Morphological stability
   1. Place 1 mL of nanoemulsion vaccine samples in microcentrifuge tubes, and evaluate the stability of fresh samples by centrifuging for 30 min at 13,000 x g at room temperature (25 °).
   2. Observe the appearance of these fresh samples and then perform a thermodynamic stability test of six temperature cycles (one cycle: store at 4 °C for 48 h, 25 °C for 48 h, and 37 °C for 48 h).
   3. After centrifugation, allow the samples to stand for 15 min to determine whether there is a Tyndall effect (whether the sample is clear and transparent under light) and whether demulsification has occurred (shown by clear stratification after demulsification).
5. SDS-PAGE and western blot
   1. Shake the samples, mix a solution of 0.6 mL of absolute ethanol and 0.3 mL of vaccine, and centrifuge (13,000 x g, 30 min at room temperature, 25 °C).
   2. Transfer the supernatant of the solution to a clean tube and dissolve the precipitate with 0.3 mL of water. Obtain the supernatant and precipitate solutions (both blank nanoemulsion and vaccine) after treatment with absolute ethanol and distilled water solution, and perform the same 50-fold dilution.
   3. Mix 2.5 mL of separation gel A and 2.5 mL of separation gel B (see Table of Materials), add 50 µL of 10% ammonium persulfate, and quickly place the solution in the glass plate with a pipette. Mix 1 mL of concentrated gel A and 1 mL of concentrated gel B, add 20 µL of 10% ammonium persulfate into the glass plate, insert an appropriately sized comb, and wait for solidification.
   4. Add a total of 5 µL of 5x protein loading buffer solution to the diluted sample obtained in step 3.5.2, and boil the sample for 5 min.
   5. Add 10 µL of heated protein to the corresponding well, and add 5 µL of protein marker to the marker well.
   6. Connect the electrode, turn on the electrophoresis meter (see Table of Materials), and adjust the voltage to 300 V. Turn off the power when the electrophoresis reaches 1 cm away from the bottom of the PAGE rubber.
   7. Remove the gel and rinse with ddH₂O. Add Coomassie bright blue G-250 staining solution (see Table of Materials) for 10 min. Pour out the staining solution, and rinse the gel twice with ddH₂O.
   8. Add the commercially available decolorization solution and microwave the gel for 1 min. Shake the gel for 10 min, and repeatedly change the decolorization solution until the gel background is clear. Then, perform gel imaging (see Table of Materials).
      NOTE: The western blot was preprocessed, as described in steps 3.5.1-3.5.6.
   9. Soak three filter paper blocks with an electrophoresis transfer buffer. Soak the polyvinylidene fluoride (PVDF) membrane in methanol for 30 s and transfer with a semidry gel transfer instrument (two layers of filter paper, PVDF membrane, electrophoresis gel, and one layer of filter paper). Use a voltage of 23 V to transfer the membrane for 23 min.
   10. Remove the PVDF membrane and wash once with tris-buffered saline-Tween 20 (TBST) after transfer.
   11. Place the PVDF membrane in sealing solution (5% skimmed milk powder solution) and seal overnight at 4 °C.
   12. Remove the PVDF membrane and wash three times with TBST or 10 min each.
   13. Incubate with the primary antibody (from immunized mice with positive serum). Dilute the primary antibody¹¹ (see Table of Materials) to be tested 500-fold (100-fold) with TBST. Place the blocked PVDF membrane in the primary antibody and incubate at 37 °C for 1 h.
   14. Remove the PVDF membrane and wash three times with TBST for 10 min each.
   15. Incubate with the secondary antibody (see Table of Materials). Dilute alkaline phosphatase (AP)-labeled goat anti-mouse secondary antibody 5,000-fold with TBST. Place the PVDF membrane in the secondary antibody and incubate at 37 °C for 40 min.
   16. Remove the PVDF membrane and wash four times with TBST for 10 min each.
   17. Submerge the PVDF membranes in a mixture (just enough to soak the membrane) of the AP substrate nitro blue tetrazole (NBT) and BCIP (5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt) (see Table of Materials) for staining. A positive result is purple.
       ​NOTE: The results must be a clear purple band, and the sample strip and the molecular weight of the antigen should be at the same labeled position.

4. Assessment of antibody immune response to this vaccine after intramuscular administration

NOTE: The mice were immunized by intramuscular injection of the nanoemulsion vaccine following a published report¹¹. The mice were administered PBS as a negative control. At 1 week after three immunizations were completed, serum was collected from the mice¹¹. The serum levels of IgG and subclasses of IgG1, IgG2a, and IgG2b were quantitatively determined by enzyme-linked immunosorbent assay (ELISA).

1. Antigen coating: Dilute HI protein antigen to 10 µg/mL with coating solution and add to the ELISA plate at 100 µL/well. Incubate at 4 °C overnight.
   NOTE: The coating solution is prepared by dissolving 1.6 g of anhydrous sodium carbonate and 2.9 g of sodium bicarbonate in 1 L of deionized water.
2. Sealing: Wash the plate (three cycles, each cycle 300 µL). Add 300 µL of the sealing solution to each well, and seal the wells for 2 h at 37 °C.
   NOTE: The sealing solution is prepared by adding Tween-20 and bovine serum albumin (BSA) to PBS. The content of BSA in the PBST solution is 1%.
3. Serum dilution: Dilute the sample serum of mice 100 times with PBST (take 3 µL of serum and add it to 297 µL of PBST and vortex), and use 100 µL for each serum sample. Dilute the positive serum and negative control serum 100 times with PBST by adding 4 µL to 396 µL of PBST, then mixing by vortexing.
4. Double ratio dilution: Add 200 µL of the above diluted experimental serum to the first row of the coated ELISA plate, and add 100 µL of PBST to all wells except the first and 12^th rows. Perform a 1:2 dilution successively from the first row: adjust the pipette to 100 µL, transfer 100 µL from the first row to the second row, and mix with the pipette 10 times.
5. Dilute the serum to Row 11 at multiple ratios and discard 100 µL of liquid.
6. Add diluted negative and positive serum to the corresponding wells in Row 12 according to the sample template (see Table 1)-100 µL/well.
7. Seal the ELISA plates with plastic wrap and incubate at 37 °C for 1 h.
8. Dilute secondary antibody anti-mouse IgG-HRP (see Table of Materials) with PBST at 1:10,000.
9. Wash the plate (three cycles, each cycle 300 µL). Then, add 100 µL of the diluted secondary antibody to each well, and incubate the plate at 37 °C for 40 min.
10. Staining and termination: Wash the plate (five cycles, each cycle 300 µL). Add 3,3′,5,5′-tetramethylbenzidine (TMB) staining solution (100 µL; see Table of Materials) to each well. Place the plates at 37 °C for 10 min away from light, and then terminate the reaction immediately with 50 µL of 2 M H₂SO₄.
11. Detect the optical density (OD₄₅₀) value by an enzyme marker (read the OD₄₅₀ value within 15 min after termination).

5. Evaluation of the in vivo effects of the novel adjuvant vaccines

NOTE: The protective effect of this nanoemulsion vaccine was evaluated in a commercially obtained lethal MRSA252 mouse model (see Table of Materials). According to the previous results of our research group¹⁴, 1 x 10⁸ colony forming units (CFU)/mouse is the best dose to evaluate the protective effect of the lethal model of MRSA252 bacterial infection.

1. Strain recovery: Obtain a tube of frozen MRSA252, inoculate the bacteria on Mueller Hinton (MH) (A) plates (see Table of Materials) by the "three-line method", and culture overnight at 37 °C. Perform the steps of the "three-line method" as mentioned below:
   1. Holding the inoculation ring in the right hand, cauterize and cool it to obtain a ring of bacterial liquid.
   2. Hold the agar plate in the left hand (keeping the lid on the table) above the front left area of the flame of the alcohol lamp so that the plate faces the flame, preventing the bacteria from entering the air. With the right hand, move the inoculation ring of infected bacteria back and forth on the agar surface in a dense but nonoverlapping way, covering an area of approximately 1/5-1/6 of the whole plate, which is the first zone.
      NOTE: The inoculation ring and the agar should be in gentle contact at a 30°-40° angle; damaging the agar can be avoided by sliding with wrist force.
   3. Cauterize the excess bacteria on the inoculation ring (whether to cauterize or sterilize each area depends on the amount of bacteria in the specimen). After cooling, repeat the penultimate Rows 2-3 at the end and draw a second area (about 1/4 of the plate area).
   4. After the second zone is finished, cauterize the ring, and draw the third zone in the same way to fill the entire plate.
   5. After the lines are drawn, place the plate on the lid of the dish, mark the plate, and incubate in a 37 °C incubator for 18-24 h to observe the distribution of colonies on the agar surface. Focus attention on whether a single colony is isolated, and observe colony characteristics (such as size, shape, transparency, and pigmentation).
2. Bacterial activation: The next day, take two 50 mL shaker flasks and add 20 mL of MH (B) medium (see Table of Materials) to each. Pick two single colonies with a 10 µL pipette tip and add them to the shakers. Incubate the colonies overnight at 220 rpm at 37 °C.
3. Secondary activation: The next day, take two 50 mL shaker flasks, each containing 20 mL of MH (B) medium. Remove 200 µL of bacterial solution from the shaker containing the first activated bacteria, add to the two new flasks containing 20 mL of MH (B) medium, and incubate at 37 °C and 220 rpm for 5 h.
4. Collect the second activated bacterial solution in a 50 mL centrifuge tube, separate at 3,000 x g for 20 min, and discard the supernatant.
5. Resuspend the precipitated bacteria in 40 mL of saline.
6. Measure the OD₆₀₀ value of the bacterial solution and adjust to 1 with saline. At this time, the bacterial quantity of MRSA252 in our experiment was 2 x 10⁹ CFU/mL.
7. Dilute the bacterial solution with an OD₆₀₀ of 1 to a concentration of 1 x 10⁹ CFU/mL.
8. Randomly divide 48 Balb/c mice into four groups (n=12).
   NOTE: Group I: PBS group; Group II: BNE control group; Group III: naive antigen [protein] group; Group IV: nanoemulsion adjuvant vaccine group.
9. Anesthetize the mice by intraperitoneal injection of 100 mg/kg of 1% pentobarbital sodium.
10. Irradiate the mice with an infrared physiotherapy lamp (see Table of Materials) for approximately 1 min to cause vasodilation of the tail vein.
11. Challenge the mice with injection of the diluted bacterial solution (step 5.7) into the tail vein, 100 µL per mouse.
12. Place the mice under the infrared physiotherapy lamp until they can return to normal activity.
13. Observe and record the survival of mice every 12 h for 10 days.
14. Euthanize the mice that survived the attack by intraperitoneal injection of 100 mg/kg of 1% sodium pentobarbital.