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

Summary

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

Abstract

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 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.

Introduction

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.

Protocol

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

1. Preparation of the MRSA HI antigen protein

1. Obtain IsdB and Hla clones from commercial sources (see Table of Materials), perform polymerase chain reaction (PCR) amplification to amplify IsdB and <....

Results

The protocol for preparing the nanoemulsion adjuvant vaccine and in vitro and in vivo tests of this vaccine was evaluated. TEM, AFM, and DLS 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 i.......

Discussion

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.......

Materials

List of materials used in this article

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