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 ...

Research

JoVE Journal

Immunology and Infection

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