Name: evaluating the immune response of a nanoemulsion adjuvant vaccine against methicillin-resistant staphylococcus aureus (mrsa) infection
Uploaded: 2023-09-01T12:40:00
Description: 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

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 f...

<ol>
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S., Otto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenicity+and+virulence+of+Staphylococcus+aureus.">Pathogenicity and virulence of Staphylococcus aureus.</a> Virulence. 12 (1), 547-569 (2021).</li><li>Lakhundi, S., Zhang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methicillin-resistant+Staphylococcus+aureus:+molecular+characterization,+evolution,+and+epidemiology.">Methicillin-resistant Staphylococcus aureus: molecular characterization, evolution, and epidemiology.</a> Clinical Microbiology Reviews. 31 (4), e00020 (2018).</li><li>Mancuso, G., Midiri, A., Gerace, E., Biondo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+antibiotic+resistance:+the+most+critical+pathogens.">Bacterial antibiotic resistance: the most critical pathogens.</a> Pathogens. 10 (10), 1310 (2021).</li><li>Goull&#233;, J. 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V., Stylianou, A., Malamou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscopy+nanoindentation+method+on+collagen+fibrils.">Atomic force microscopy nanoindentation method on collagen fibrils.</a> Materials. 15 (7), 2477 (2022).</li><li>Zeng, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+immunodominant+epitope-specific+monoclonal+antibody+cocktail+improves+survival+in+a+mouse+model+of+Staphylococcus+aureus+bacteremia.">An immunodominant epitope-specific monoclonal antibody cocktail improves survival in a mouse model of Staphylococcus aureus bacteremia.</a> The Journal of Infectious Diseases. 223 (10), 1743-1752 (2021).</li><li>Roy, U., Kornitzer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heme-iron+acquisition+in+fungi.">Heme-iron acquisition in fungi.</a> Current Opinion in Microbiology. 52, 77-83 (2019).</li><li>Saeed, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+toxins+in+musculoskeletal+infections.">Bacterial toxins in musculoskeletal infections.</a> Journal of Orthopaedic Research. 39 (2), 240-250 (2021).</li><li>Xu, Q., Zhou, A., Wu, H., Bi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+in+vivo+evaluation+of+baicalin-loaded+W/O+nanoemulsion+for+lymphatic+absorption.">Development and in vivo evaluation of baicalin-loaded W/O nanoemulsion for lymphatic absorption.</a> Pharmaceutical Development and Technology. 24 (9), 1155-1163 (2019).</li><li>Singh, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoemulsion:+Concepts,+development+and+applications+in+drug+delivery.">Nanoemulsion: Concepts, development and applications in drug delivery.</a> Journal of Controlled Release. 252, 28-49 (2017).</li><li>Kadakia, E., Shah, L., Amiji, M. 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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 o...

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 stru...

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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 ...

The demonstrated protocol underlined the direct and effective experimental approach and the detailed steps to immune response effects of novel viral adjuvants. This protocol provides the research with not only a basically immune evaluation method for adjuvant vaccine but can also determines their mass stability. To begin, take the 10 milligrams per milliliter working solution of the MRSA HI antigen protein, and to it, sequentially add the three excipients, isopropyl myristate, polyoxyethylene castor oil, and propylene glycol, maintaining a mass ratio of one is to six is to three.Stir the mixture well, then gradually add sterilized pure water to reach a system volume of 10 milliliters. To prepare the nanoemulsion vaccine by low energy emulsification method, stir it at 500 rotations per minute and 25 degrees Celsius for approximately 20 minutes. Subsequently, the nanoemulsion vaccine containing one milligram per milliliter protein is obtained as a clear transparent liquid.For antigen coating of the enzyme linked immunosorbent assay or ELISA plate, dilute HI protein antigen to 10 micrograms per milliliter with coating solution, and add 100 microliters of the diluted antigen per well in the ELISA plate. Incubate the plate at four degrees Celsius overnight. After incubation, wash the plate with 300 microliters of PBST per well.Seal the wells with 300 microliters of the sealing solution per well for two hours at 37 degrees Celsius, then dilute three microliters of the mice serum from each experimental group 100 times by adding 297 microliters of PBST, and vortex the diluted serum. Add 100 microliters of diluted serum per well in the coated ELISA plate for each serum sample. Similarly, dilute the positive and negative control serums 100 times with PBST by adding four microliters of positive or negative control serum to 396 microliters of PBST, followed by vortex mixing.To perform double ratio dilution, add 200 microliters of the previously diluted experimental serum to the first row of the coated ELISA plate, then add 100 microliters of PBST to all wells, except those in rows one and 12. Next, adjust the pipette gun to 100 microliters and transfer 100 microliters of serum from the first row to the second row containing PBST. Mix the content in the second row 10 times using the pipette gun.Similarly, perform one is to two successive dilutions of the serum til row 11. Once done, discard 100 microliters of liquid from row 11. Add 100 microliters of diluted negative and positive serum to each corresponding well in row 12 following the sample template.Seal the ELISA plate with plastic wrap and incubate at 37 degrees Celsius for one hour. Meanwhile, dilute secondary antibody anti-mouse IgG HRP 10, 000 times by adding PBST. After the one-hour incubation, wash the ELISA plate with 300 microliters of PBST per well to clean the unbound antibodies, then add 100 microliters of the diluted secondary antibody to each well and incubate the plate at 37 degrees Celsius for 40 minutes.Wash the plate with 300 microliters of PBST per well, then add 100 microliters of TMB color development solution to each well. Place the plates at 37 degrees Celsius and away from light for 10 minutes. After which, terminate the reaction immediately with 50 microliters of two molar sulfuric acid.Detect the optical density or OD values at 415 nanometers using an enzyme marker within 15 minutes after termination. The physical characteristics of novel nanoemulsion vaccine were analyzed using transmission electron microscopy and atomic force microscopy. Dynamic light scattering was used to determine the particle size distribution and the zeta potential of the sample.SDS-PAGE and western blotting showed that the amount of antigen in the precipitate and supernatant did not significantly defer after centrifugation, indicating the vaccine was structurally intact, specific, and immunogenic. The nanoemulsion adjuvant vaccine significantly increased the total IgG, IgG1, and IgG2a antibody titers of the mice. The immunogenicity of the protein vaccine was significantly improved.The serum IgG2b OD values at 450 nanometers were highest when PBS was used at a one is to 5, 000 dilution. The lethal model of MRSA 252 mice reflected that the nanoemulsion vaccine showed a good protective effect and effectively inhibited bacterial infection with MRS A 252, improving the survival rate of infected mice. The preparation of nanoemulssion and the addition of serum to the plant are the most important.Also, where detection the antibody titer, one should pay attention to correct to a wider and liquid-based out. Besides adjuvants, this method can also be used for the immune evolution of other math arrays, such as plastics or delivery systems.

Research

JoVE Journal

Immunology and Infection

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