This research was supported by No. 2021YFC2302603 of the National Key Research and Development Program of China, No. 32070924 and 32000651 of the NSFC, and No. 2019jcyjA-msxmx0159 of the Natural Science Foundation Project Program of Chongqing.

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
<ol>
	<li>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.</li>
	<li>Transform the positive clone into E. coli BL21 competent cells (see Table of Materials) and amplify with shark culture10.</li>
	<li>Express the antigen and isolate after induction with isopropyl-&#946;-D-1-mercaptogalactopyranoside and multimodal chromatography (MMC) isolate kits11(see Table of Materials).</li>
	<li>Characterize the antigen protein and purify it with a full-automation purifier11.
	<ol>
		<li>Affinity-purify gutathione S-transferase (GST)-tagged proteins from cleared lysates using a commercially available affinity chromatography resin (see Table of Materials).</li>
		<li>Purify the recombinant proteins by MMC11.</li>
		<li>Remove the endotoxin by the Triton X-114 phase separation method11. Briefly, mix 1% Triton X-114 with recombinant protein eluate at 0 &#176;C for 5 min to ensure a homogenous solution, and incubate at 37 &#176;C for 5 min to allow two phases to form.</li>
	</ol>
	</li>
	<li>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/&#181;g, lower than the international standard (1.5 EU/&#181;g)10.
	<ol>
		<li>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 (&#955;0.25 EU/mL) and the maximum effective dilution ratio of 33.3 were calculated12 based on MVD = cL/&#955;, 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. &#955; is the labeled sensitivity (EU/mL) of the horseshoe crab reagent in the gel method.</li>
	</ol>
	</li>
	<li>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. 
	&#8203;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 &#176;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).</li>
</ol>
2. Preparation of the nanoemulsion vaccine
<ol>
	<li>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 &#176;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.</li>
	<li>Prepare the nanoemulsion adjuvant vaccine (1 mg/mL protein) by low-energy emulsification methods13 to obtain a clear and transparent liquid solution. 
	&#8203;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 &#176;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.</li>
</ol>
3. Physical characterization and stability of the nanoemulsion adjuvant vaccine
<ol>
	<li>Particle size, zeta potential, and polymer dispersion index characterization (PDI).
	<ol>
		<li>Prepare 100 &#181;L of the nanoemulsion adjuvant vaccine and dilute 50-fold with water for injection. Add 2 mL of sample for detection.</li>
		<li>Observe the particle sizes, zeta potential, and PDI at 25 &#176;C using a Nano Zetasizer (ZS; see Table of Materials).</li>
	</ol>
	</li>
	<li>Transmission electron microscopy (TEM)
	<ol>
		<li>Dilute 10 &#181;L 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.</li>
		<li>Remove excess phosphotungstic acid solution with filter paper.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Scanning electron microscopy (SEM)
	<ol>
		<li>Dilute 10 &#181;L of the nanoemulsion adjuvant vaccine 50-fold with water, drop 10 &#181;L of prediluted samples on a silicon wafer, and place in a 37 &#176;C thermostat-controlled incubator for 24 h.</li>
		<li>Spray the prepared platinum on the silicon for 40 s and cool to room temperature.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Morphological stability
	<ol>
		<li>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 &#176;).</li>
		<li>Observe the appearance of these fresh samples and then perform a thermodynamic stability test of six temperature cycles (one cycle: store at 4 &#176;C for 48 h, 25 &#176;C for 48 h, and 37 &#176;C for 48 h).</li>
		<li>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).</li>
	</ol>
	</li>
	<li>SDS-PAGE and western blot
	<ol>
		<li>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 &#176;C).</li>
		<li>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.</li>
		<li>Mix 2.5 mL of separation gel A and 2.5 mL of separation gel B (see Table of Materials), add 50 &#181;L 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 &#181;L of 10% ammonium persulfate into the glass plate, insert an appropriately sized comb, and wait for solidification.</li>
		<li>Add a total of 5 &#181;L of 5x protein loading buffer solution to the diluted sample obtained in step 3.5.2, and boil the sample for 5 min.</li>
		<li>Add 10 &#181;L of heated protein to the corresponding well, and add 5 &#181;L of protein marker to the marker well.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Remove the PVDF membrane and wash once with tris-buffered saline-Tween 20 (TBST) after transfer.</li>
		<li>Place the PVDF membrane in sealing solution (5% skimmed milk powder solution) and seal overnight at 4 &#176;C.</li>
		<li>Remove the PVDF membrane and wash three times with TBST or 10 min each.</li>
		<li>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 &#176;C for 1 h.</li>
		<li>Remove the PVDF membrane and wash three times with TBST for 10 min each.</li>
		<li>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 &#176;C for 40 min.</li>
		<li>Remove the PVDF membrane and wash four times with TBST for 10 min each.</li>
		<li>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&#39;-indolyphosphate p-toluidine salt) (see Table of Materials) for staining. A positive result is purple. 
		&#8203;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.</li>
	</ol>
	</li>
</ol>
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).
<ol>
	<li>Antigen coating: Dilute HI protein antigen to 10 &#181;g/mL with coating solution and add to the ELISA plate at 100 &#181;L/well. Incubate at 4 &#176;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.</li>
	<li>Sealing: Wash the plate (three cycles, each cycle 300 &#181;L). Add 300 &#181;L of the sealing solution to each well, and seal the wells for 2 h at 37 &#176;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%.</li>
	<li>Serum dilution: Dilute the sample serum of mice 100 times with PBST (take 3 &#181;L of serum and add it to 297 &#181;L of PBST and vortex), and use 100 &#181;L for each serum sample. Dilute the positive serum and negative control serum 100 times with PBST by adding 4 &#181;L&#160;to 396 &#181;L of PBST, then mixing by vortexing.</li>
	<li>Double ratio dilution: Add 200 &#181;L of the above diluted experimental serum to the first row of the coated ELISA plate, and add 100 &#181;L of PBST to all wells except the first and 12th rows. Perform a 1:2 dilution successively from the first row: adjust the pipette to 100 &#181;L, transfer 100 &#181;L from the first row to the second row, and mix with the pipette 10 times.</li>
	<li>Dilute the serum to Row 11 at multiple ratios and discard 100 &#181;L of liquid.</li>
	<li>Add diluted negative and positive serum to the corresponding wells in Row 12 according to the sample template (see Table 1)-100 &#181;L/well.</li>
	<li>Seal the ELISA plates with plastic wrap and incubate at 37 &#176;C for 1 h.</li>
	<li>Dilute secondary antibody anti-mouse IgG-HRP (see Table of Materials) with PBST at 1:10,000.</li>
	<li>Wash the plate (three cycles, each cycle 300 &#181;L). Then, add 100 &#181;L of the diluted secondary antibody to each well, and incubate the plate at 37 &#176;C for 40 min.</li>
	<li>Staining&#160;and termination: Wash the plate (five cycles, each cycle 300 &#181;L). Add 3,3&#8242;,5,5&#8242;-tetramethylbenzidine (TMB) staining solution (100 &#181;L; see Table of Materials) to each well. Place the plates at 37 &#176;C for 10 min away from light, and then terminate the reaction immediately with 50 &#181;L of 2 M H2SO4.</li>
	<li>Detect the optical density (OD450) value by an enzyme marker (read the OD450 value within 15 min after termination).</li>
</ol>
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.
<ol>
	<li>Strain recovery: Obtain a tube of frozen MRSA252, inoculate the bacteria on Mueller Hinton (MH) (A) plates (see Table of Materials) by the &#34;three-line method&#34;, and culture overnight at 37 &#176;C. Perform the steps of the &#34;three-line method&#34; as mentioned below:
	<ol>
		<li>Holding the inoculation ring in the right hand, cauterize and cool it to obtain a ring of bacterial liquid.</li>
		<li>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&#176;-40&#176; angle; damaging the agar can be avoided by sliding with wrist force.</li>
		<li>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).</li>
		<li>After the second zone is finished, cauterize the ring, and draw the third zone in the same way to fill the entire plate.</li>
		<li>After the lines are drawn, place the plate on the lid of the dish, mark the plate, and incubate in a 37 &#176;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).</li>
	</ol>
	</li>
	<li>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 &#181;L pipette tip and add them to the shakers. Incubate the colonies overnight at 220 rpm at 37 &#176;C.</li>
	<li>Secondary activation: The next day, take two 50 mL shaker flasks, each containing 20 mL of MH (B) medium. Remove 200 &#181;L 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 &#176;C and 220 rpm for 5 h.</li>
	<li>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.</li>
	<li>Resuspend the precipitated bacteria in 40 mL of saline.</li>
	<li>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.</li>
	<li>Dilute the bacterial solution with an OD600 of 1 to a concentration of 1 x 109 CFU/mL.</li>
	<li>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.</li>
	<li>Anesthetize the mice by intraperitoneal injection of 100 mg/kg of 1% pentobarbital sodium.</li>
	<li>Irradiate the mice with an infrared physiotherapy lamp (see Table of Materials) for approximately 1 min to cause vasodilation of the tail vein.</li>
	<li>Challenge the mice with injection of the diluted bacterial solution (step 5.7) into the tail vein, 100 &#181;L per mouse.</li>
	<li>Place the mice under the infrared physiotherapy lamp until they can return to normal activity.</li>
	<li>Observe and record the survival of mice every 12 h for 10 days.</li>
	<li>Euthanize the mice that survived the attack by intraperitoneal injection of 100 mg/kg of 1% sodium pentobarbital.</li>
</ol>

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The authors declare they have no conflicts of interest in this work.

IsdB, a bacterial cell wall-anchored and iron-regulated surface protein, plays an important role in the process of obtaining heme iron15. Hla, alpha toxin, is among the most effective bacterial toxins known in MRSA, and can form pores in eukaryotic cells and interfere with adhesion and epithelial cells16. In our study, a novel recombination MRSA antigen protein (HI) was constructed and expressed with gene engineering technology based on the antigen genes of IsdB and Hla. Then, a nanoemulsion vaccine was developed by the low-energy emulsification method, and the stability and effectiveness of this vaccine were evaluated. For example, some characteristics, such as long-term stability, structure, shape, absorption, and effect in vivo, are greatly affected by the size of the emulsion droplet7. The results showed that particle size was an important factor affecting the transport of nanoemulsions in vivo and their phagocytosis by antigen-presenting cells17. Regarding cytophagocytosis, nanoparticles smaller than 1 &#181;m are more easily phagocytosed by dendritic cells (DCs) and macrophages and are the most likely to stimulate systemic immunity18.
Regarding particle transport, nanoparticles smaller than 100 nm are more likely to be transmitted through lymphatic vessels to enhance the immune effect; however, small nanoparticles infiltrate into blood vessels, while large nanoparticles slow their transport speed in lymphatic vessels19. Thus, the optimal particle size is in the range of 20-50 nm. Hence, it is critical to study the physical characteristics of nanoparticles, including shape, size, and zeta potential. TEM, with its high precision, has become an important and basic tool for understanding the properties of nanoemulsion adjuvant vaccines. Additionally, the technology has been widely applied in many fields for several years. Another nanomechanical characterization tool with 3D and nanoscale high-resolution information, AFM, was also used to evaluate the nanoemulsion vaccine13. Importantly, DLS, which simultaneously reveals both size and charge20, can provide key information, such as the aggregation state of nanoemulsion vaccine particles in solution. According to the literature, a high zeta potential value is indispensable for the physical and chemical stability of colloidal suspensions, as a large repulsive force often prevents aggregation caused by occasional collisions with adjacent nanoparticles. Therefore, in our research, we set up these schemes and evaluated their physical and chemical properties with TEM, AFM, and DLS.
Protein vaccines play an important role in the prevention and treatment of major infectious diseases21. Protein vaccines have developed rapidly in recent years, but serious challenges remain in immune efficacy and persistence. The fundamental reasons for this are poor protein stability, small distribution coefficient, short biological half-life of antigens, and high clearance rates in vivo22. In this protocol, the stability of the novel nanoemulsion vaccine was evaluated, and no antigen degradation was observed by SDS-PAGE or western blot.
A safe, effective, and marketable protein vaccine must be supplemented with an appropriate immune adjuvant to significantly enhance the immunogenicity of the protein antigen and the type of protective immune response20 to obtain the optimal vaccine immune protection. The antibody level stimulated by a protein vaccine is among the important indicators for the effective prevention and treatment of infection, and the antibody titer is an important index to evaluate whether the novel nanoemulsion adjuvant can improve the immunogenicity of the naked antigen. Novel immune adjuvants can significantly stimulate the body to enhance the humoral and cellular immune response levels, and enriching the types of immune response and immune protective efficacy of vaccines is an important development direction and the component of future research and the development of novel vaccines23. Therefore, in this protocol, antibody titers of mouse serum-specific IgG, IgG1, IgG2a, and IgG2b with the novel nanoemulsion adjuvant vaccine were detected. The lethal mouse model24 is the gold standard for evaluating vaccines, and in our protocol, a lethal MRSA252 mouse model was used to evaluate the protective effect. The results showed that the novel nanoemulsion adjuvant vaccine effectively improved the survival rate of Balb/c mice infected with MRSA.
This protocol is not sufficient in the stability evaluation of nanoemulsion vaccines. Circular dichroism is a suitable method if it can be applied to detect the spatial structure of vaccine proteins in a simple and fast method. Although the survival rate of mice tested by the attack is the gold standard for evaluating the in vivo test and effectiveness of the vaccine, it would be more convincing if the amount of bacterial colonization of the organs of mice after attack could be measured. Therefore, there are still some shortcomings in the present protocol to evaluate the immune response of the nanoemulsion adjuvant vaccine, and the above experiment needs to be supplemented in subsequent relevant experiments for evaluation.
In conclusion, it is necessary to establish a series of evaluations of physical properties, stability, specific immune response, and challenge protection. Completing these schemes will provide vital experimental fundamentals for the application and development of novel nanoemulsion adjuvant vaccines.

Methicillin-resistant Staphylococcus aureus (MRSA) is an opportunistic pathogen with one of the highest infection rates in an intensive care unit (ICU) wards1, cardiology departments, and burn departments worldwide. MRSA exhibits high rates of infection, mortality, and broad drug resistance, presenting great difficulties in clinical treatment2. In the Global Priority List of Antibiotic-Resistant Bacteria released by the World Health Organisation (WHO) in 2017, MRSA was listed in the most critical category3. A vaccine against MRSA infection is therefore urgently needed.
Aluminum adjuvant has been used for a long time, and the adjuvant auxiliary mechanism is relatively clear, safe, effective, and well tolerated4. Aluminum adjuvants are currently a widely used type of adjuvant. It is generally believed that antigens adsorbed on aluminum salt particles can improve the stability and enhance the ability of the injection site to uptake antigens, providing good absorption and slow release5. Currently, the main disadvantage of aluminum adjuvants is that they lack an adjuvant effect or exhibit only a weak adjuvant effect on some vaccine candidate antigens6. In addition, aluminum adjuvants induce IgE-mediated hypersensitivity reactions5. Therefore, it is necessary to develop novel adjuvants to stimulate a stronger immune response.
Nanoemulsion adjuvants are colloidal dispersion systems composed of oil, water, surfactants, and cosurfactants7. In addition, the adjuvants are thermodynamically stable and isotropic, can be autoclaved or stabilized by high-speed centrifugation, and can be formed spontaneously under mild preparation conditions. Several emulsion adjuvants (such as MF59, NB001-002 series, AS01-04 series, etc.) are currently on the market or in the clinical research stage, but their particle sizes are greater than&#160;160 nm8. Therefore, the advantages of nanoscale (1-100 nm) medicinal preparations (i.e., large specific surface area, small particle size, surface effect, high surface energy, small size effect, and macro quantum tunneling effect) cannot be fully exploited. In the present protocol, a novel adjuvant based on nanoemulsion technology with a diameter size of 1-100 nm has been reported to exhibit good adjuvant activity9. We tested the recombination subunit vaccine antigen protein HI (&#945;-hemolysin mutant [Hla] and Fe ion surface determining factor B [IsdB] subunit N2 active fragment fusion protein); a series of procedures were established to examine the physical properties and stability, evaluate its specific antibody response after intramuscular administration, and test the protective effect of the vaccine using a mouse systemic infection model.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5424-Small high speed centrifugeFA-45-24-11</td>
			<td>Eppendorf, Germany&#160;</td>
			<td>5424000495</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Corning Incorporated, USA</td>
			<td>CLS3922</td>
			<td></td>
		</tr>
		<tr>
			<td>AFM Dimension FastScan</td>
			<td>BRUKER, Germany&#160;</td>
			<td>null</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol lamp</td>
			<td>Shenzhen Yibaxun Technology Co.,China</td>
			<td>YBS-AA-11408</td>
			<td></td>
		</tr>
		<tr>
			<td>Balb/c mice&#160;</td>
			<td>Beijing HFK Bioscience Co. Ltd.&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BCIP/NBT</td>
			<td>Fuzhou Maixin Biotechnology Development Company,China</td>
			<td>BCIP/NBT</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rad 6.0 microplate reader</td>
			<td>Bio-Rad Laboratories Incorporated Limited Co., CA, USA</td>
			<td>null</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21 Competent Cell</td>
			<td>Merck millipore,Germany</td>
			<td>70232-3CN</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA-100G</td>
			<td>Sigma-Aldrich, USA</td>
			<td>B2064-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5810 R</td>
			<td>Eppendorf, Germany&#160;</td>
			<td>5811000398</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie bright blue G-250 staining solution</td>
			<td>MIKX,China</td>
			<td>DB236</td>
			<td></td>
		</tr>
		<tr>
			<td>Decolorization solution</td>
			<td>BOSTER,China</td>
			<td>AR0163-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Electro-heating standing-temperature cultivator HH-B11-420</td>
			<td>Shanghai Yuejin Medical Device Factory, China</td>
			<td>null</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis apparatus</td>
			<td>Beijing Liuyi Instrument Factory, China</td>
			<td>DYCZ-25D</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel image</td>
			<td>Tanon, USA</td>
			<td>null</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione-Sepharose Resin GST</td>
			<td>Mei5bio,China</td>
			<td>affinity chromatography resin</td>
			<td></td>
		</tr>
		<tr>
			<td>H2SO4</td>
			<td>Chengdu KESHI Chemical Co., LTD,China</td>
			<td>7664-93-9</td>
			<td></td>
		</tr>
		<tr>
			<td>HI recombinant protein</td>
			<td>Third Military Medical University,China</td>
			<td>110-27-0</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP -Goat Anti-Mouse IgG</td>
			<td>Biodragon, China</td>
			<td>BF03001</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP- Goat anti-mouse IgG1</td>
			<td>Biodragon, China</td>
			<td>BF03002R</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP- Goat anti-mouse IgG2a</td>
			<td>Biodragon, China</td>
			<td>BF03003R</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP- Goat anti-mouse IgG2b</td>
			<td>Biodragon, China</td>
			<td>BF03004R</td>
			<td></td>
		</tr>
		<tr>
			<td>Inoculation loop</td>
			<td>Haimen Feiyue Co.,LTD,China</td>
			<td>YR-JZH-1UL</td>
			<td></td>
		</tr>
		<tr>
			<td>IsdB and Hla clones</td>
			<td>Shanghai Jereh Biotechnology Co,China</td>
			<td>null</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl nutmeg (pharmaceutic adjuvant)</td>
			<td>SEPPIC, France</td>
			<td>null</td>
			<td></td>
		</tr>
		<tr>
			<td>isopropyl- &#946;-D-1-mercaptogalactopyranoside</td>
			<td>fdbio,China</td>
			<td>FD3278-1</td>
			<td></td>
		</tr>
		<tr>
			<td>LB bouillon culture-medium</td>
			<td>Beijing AOBOX Biotechnology Co., LTD,China</td>
			<td>02-136</td>
			<td></td>
		</tr>
		<tr>
			<td>Lnfrared physiotherapy lamp</td>
			<td>Guangzhou Runman Medical Equipment Co.,China</td>
			<td>7600</td>
			<td></td>
		</tr>
		<tr>
			<td>Low temperature refrigerated centrifuge</td>
			<td>Eppendorf, Germany&#160;</td>
			<td>null</td>
			<td></td>
		</tr>
		<tr>
			<td>Malvern NANO ZS</td>
			<td>Malvern Instruments Ltd., UK</td>
			<td>null</td>
			<td></td>
		</tr>
		<tr>
			<td>MH(A) medium</td>
			<td>Beijing AOBOX Biotechnology Co., LTD,China</td>
			<td>02-051</td>
			<td></td>
		</tr>
		<tr>
			<td>MH(B) medium</td>
			<td>Beijing AOBOX Biotechnology Co., LTD,China</td>
			<td>02-052</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro plate washing machine 405 LSRS</td>
			<td>Bio Tek Instruments,Inc Highland&#160; Park,USA</td>
			<td>null</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-TBC Compact Film Transfer Instrument</td>
			<td>BeiJingDongFangRuiLi Co.,LTD,China</td>
			<td>1658030</td>
			<td></td>
		</tr>
		<tr>
			<td>MMC packing</td>
			<td>TOSOH(SHANGHAI)CO.,LTD</td>
			<td>0022818</td>
			<td></td>
		</tr>
		<tr>
			<td>MRSA252</td>
			<td>USA, ATCC</td>
			<td>null</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop ultraviolet spectrophotometer</td>
			<td>Thermo Scientific, USA</td>
			<td>null</td>
			<td></td>
		</tr>
		<tr>
			<td>New FlashTM Protein any KD PAGE Protein electrophoresis gel kit</td>
			<td>DAKEWE, China</td>
			<td>8012011</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>biosharp, China</td>
			<td>null</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR, Amplifier</td>
			<td>Thermal Cycler, USA</td>
			<td>null</td>
			<td></td>
		</tr>
		<tr>
			<td>pGEX-target gene recombinant plasmid</td>
			<td>Shanghai Jereh Biotechnology Co,China</td>
			<td>B3528G</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphotungstic acid</td>
			<td>G-CLONE, China</td>
			<td>CS1231-25g</td>
			<td></td>
		</tr>
		<tr>
			<td>pipette</td>
			<td>Eppendorf, Germany&#160;</td>
			<td>3120000844</td>
			<td></td>
		</tr>
		<tr>
			<td>polyoxyethylated castor oil (pharmaceutic adjuvant)</td>
			<td>Aladdin, China</td>
			<td>K400327-1kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary antibody</td>
			<td>Laboratory homemade:from immunized mice with positive sera</td>
			<td>null</td>
			<td>See Reference 11 for details</td>
		</tr>
		<tr>
			<td>propylene glycol (pharmaceutic adjuvant)</td>
			<td>Sigma-Aldrich, USA</td>
			<td>P4347-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein Marker</td>
			<td>Thermo Scientufuc, USA</td>
			<td>26616</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF TRANSFER MEMBRANE</td>
			<td>Invitrogen,USA</td>
			<td>88518</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope</td>
			<td>JEOL,Japan</td>
			<td>JSM-IT800</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pentobarbital</td>
			<td>Merck,Germany</td>
			<td>Tc-P8411</td>
			<td></td>
		</tr>
		<tr>
			<td>Talos L120C TEM</td>
			<td>Thermo Fisher, USA</td>
			<td>null</td>
			<td></td>
		</tr>
		<tr>
			<td>TMB color solution</td>
			<td>TIAN GEN, China</td>
			<td>PA107-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Turtle kits</td>
			<td>Xiamen Bioendo Technology Co.,LTD</td>
			<td>ES80545</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Macklin, China</td>
			<td>9005-64-5</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluating the immune response of a nanoemulsion adjuvant vaccine against methicillin-resistant staphylococcus aureus (mrsa) infection

Nanoemulsion adjuvant vaccines have attracted extensive attention because of their small particle size, high thermal stability, and ability to induce validly immune responses. However, establishing a series of comprehensive protocols to evaluate the immune response of a novel nanoemulsion adjuvant vaccine is vital. Therefore, this article features a rigorous procedure to determine the physicochemical characteristics of a vaccine (by transmission electron microscopy [TEM], atomic force microscopy [AFM], and dynamic light scattering [DLS]), the stability of the vaccine antigen and system (by a high-speed centrifuge test, a thermodynamic stability test, SDS-PAGE, and western blot), and the specific immune response (IgG1, IgG2a, and IgG2b). Using this approach, researchers can evaluate accurately&#160;the protective effect of a novel nanoemulsion adjuvant vaccine in a lethal MRSA252 mouse model. With these protocols, the most promising nanoemulsion vaccine adjuvant in terms of effective adjuvant potential can be identified. In addition, the methods can help optimize novel vaccines for future development.

The protocol for preparing the nanoemulsion adjuvant vaccine and in vitro and in vivo&#160;tests of this vaccine was evaluated. TEM, AFM, and DLS&#160;were used to determine the important characteristics of the zeta potential and particle size on the surface of this sample (Figure 1). SDS-PAGE and western blotting showed that the amount of antigen in the precipitate and supernatant did not significantly degrade after centrifugation, which indicated that the vaccine was structurally intact, specific, and immunogenic (Figure 2). The nanoemulsion adjuvant vaccine significantly increased the total IgG, IgG1, and IgG 2a antibody titers. The immunogenicity of the vaccine was significantly improved (Figure 3A-C). The serum IgG2b OD values at 450 nm of the antigen group&#160;were the highest (dilute at 1:500 PBS)&#160;(Figure 3D). The lethal MRSA252 mouse model most directly reflected that the nanoemulsion adjuvant vaccine showed a good protective effect and effectively inhibited bacterial infection with MRSA252, improving the survival rate of infected mice (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65152/65152fig01v2.jpg" /> 
Figure 1: Physical characteristics of the novel nanoemulsion adjuvant vaccine. (A) Transmission electron micrograph. (B) Atomic force microscopy micrograph. (C) Size diameter and distribution. (D) Zeta potential and distribution. The figure is reproduced from Sun, H. W. et al. International Journal of Nanomedicine. 10, 7275-7290 (2015)11. Originally published by and used with permission from Dove Medical Press Ltd.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65152/65152fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65152/65152fig02.jpg" /> 
Figure 2: Physical stability of the novel nanoemulsion adjuvant vaccine. (A) Structural integrity of the antigenic proteins. (B) Structural specificity of the antigen protein. Lane 3: blank nanoemulsion-treated supernatant; Lane 4: blank nanoemulsion treatment precipitate; Lane 5: prestained marker; Lane 6: vaccine supernatant; Lane 7: vaccine precipitate; Lane 8: vaccine treatment supernatant; Lane 9: vaccine treatment precipitate; Lane 10: native protein agent; (A) and (B) mixed term. Clearly defined protein channels are marked by black squares. Black arrows indicate antigenic proteins. The figure is reproduced from&#160;Sun, H. W. et al.&#160;International Journal of Nanomedicine. 10,&#160;7275-7290 (2015)11.&#160;Originally published by and used with permission from Dove Medical Press Ltd.Sun et al.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65152/65152fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65152/65152fig03.jpg" /> 
Figure 3: IgG and its subgroup antibody levels after intramuscular injection with the nanoemulsion adjuvant vaccine. (A) Log2 value of the IgG titers. (B) 450 nm optical density of serum IgG1. (C) 450 nm optical density of serum IgG2a. (D) 450 nm optical density of serum IgG2b. The figure is reproduced from&#160;Sun, H. W. et al.&#160;International Journal of Nanomedicine. 10,&#160;7275-7290 (2015)11.&#160;Originally published by and used with permission from Dove Medical Press Ltd.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65152/65152fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65152/65152fig04.jpg" /> 
Figure 4: Protective effect in the lethal model of MRSA252 bacterial infection. The survival ratio of mice in response to systemic MRSA infection. The figure is reproduced from&#160;Sun, H. W. et al.&#160;International Journal of Nanomedicine. 10,&#160;7275-7290 (2015)11.&#160;Originally published by and used with permission from Dove Medical Press Ltd.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65152/65152fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: ELISA spiking sequence and dilution template. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65152/65152_Table 1.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Evaluating the Immune Response of a Nanoemulsion Adjuvant Vaccine Against Methicillin-Resistant Staphylococcus aureus MRSA Infection at JoVE.com

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

The present protocol prepares and evaluates the physical properties, immune response, and in vivo protective effect of a novel nanoemulsion adjuvant vaccine.

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

Nanoemulsion adjuvant vaccines have attracted extensive attention because of their small particle size, high thermal stability, and ability to induce ...

evaluating-immune-response-nanoemulsion-adjuvant-vaccine-against

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

1.7K Views.  Third Military Medical University. The present protocol prepares and evaluates the physical properties, immune response, and in vivo protective effect of a novel nanoemulsion adjuvant vaccine.

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

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1 Bacterial biofilms are encased within a matrix and attached to a surface.2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)3 or growing biofilms for visualization (flow cells)4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.
Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.6,7,8,9
The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.9,11,12,13

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1  Bacterial biofilms are encased within a matrix and attached ...

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the United States. Among soft-tissue infections, myositis, pyomyositis, and necrotizing fasciitis have been increasingly reported in association with MRSA arising from the community. To understand the interplay between MRSA and host immunity leading to more severe infection, the availability of animal models is critical, permitting the study of host and bacterial factors. Several infection models have been introduced to assess the pathogenesis of S. aureus during superficial skin infection. Here, we describe a subcutaneous infection model that examines the skin, subcutaneous, and muscle pathologies.

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA

Murine skin and soft tissue infection model is utilized for assessing the virulence function of methicillin resistant Staphylococcus aureus (MRSA) and the host immunological responses. Here, we presented a subcutaneous infection model for skin and soft tissue infection.

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus (MRSA) in Rat

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause of such syndromes with unacceptably high morbidity and mortality even with appropriate antimicrobial agent treatments4-6. The increase in infections due to methicillin-resistant S. aureus (MRSA), the high rates of vancomycin clinical treatment failures and growing problems of linezolid and daptomycin resistance have all further complicated the management of patients with such infections, and led to high healthcare costs7, 8. In addition, it should be emphasized that most recent studies with antibiotic treatment outcomes have been based in clinical settings, and thus might well be influenced by host factors varying from patient-to-patient. Therefore, a relevant animal model of endovascular infection in which host factors are similar from animal-to-animal is more crucial to investigate microbial pathogenesis, as well as the efficacy of novel antimicrobial agents. Endocarditis in rat is a well-established experimental animal model that closely approximates human native valve endocarditis. This model has been used to examine the role of particular staphylococcal virulence factors and the efficacy of antibiotic treatment regimens for staphylococcal endocarditis. In this report, we describe the experimental endocarditis model due to MRSA that could be used to investigate bacterial pathogenesis and response to antibiotic treatment.

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

Experimental rat endocarditis model due to methicillin-resistant S. aureus.

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Staphylococcal Cassette Chromosome mec (SCCmec) typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

Staphylococcal Cassette Chromosome mec (SCCmec) typing  is a very important molecular tool for understanding the epidemiology and  clonal strain ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

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

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The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus (MRSA) in Rat

Quantitative Measurement of the Immune Response and Sleep in Drosophila

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice