Biological and Immunological Evaluation of <italic>Neisseria meningitidis</italic> Serogroup A Outer Membrane Vesicle as Vaccine Candidates

Abstract

Background: Neisseria meningitidis Serogroup A, is a major cause of bacterial meningitidis outbreaks in Africa and the Middle East. While polysaccharide vaccines have been available for many years, these vaccines have many disadvantages including the induction of T-cell independent responses which do not induce memory responses. Objectives: Thus to overcome this problem, in this research outer membrane vesicle (OMV) containing PorA was extracted and evaluated by biological and immunological methods. Materials and Methods: OMVs were extracted with deoxycholate and EDTA, and purification was performed by sequential ultracentrifugation. Physicochemical properties of extracted OMVs were analyzed by electron microscopy and SDS-PAGE. The toxicity of LPS content in its was assayed by LAL test. The Presence of PorA as a major component of OMV was confirmed by western blot. To study antibodies synthesis after immunization with OMV, ELISA method was used. Also serum bactericidal assay (SBA) was performed to determine the serum bactericidal activity against N. meningitidis serogroup A. Results: The results revealed that the content of protein extracted was 0.1 mg/mL. The electron microscopy showed that intactness of the vesicle in these preparation ranged more than 70%. The SDS-PAGE showed that PorA as a major immunological part of outer membrane vesicle was located in 35-40kDa. LAL test showed that the endotoxin activity was around 126EU/mL which is safe for using. The ELISA test revealed that the IgG total titer was elevated after the first injection. SBA indicates that bactericidal antibodies rise after the second dose of booster. Conclusions: The results showed that the extracted OMVs were conformationally stable, and there were no pyrogenic determinants in OMV. Also the results showed that the OMV elicited high level of specific antibodies against N. meningitidis serogroup A. These results indicate that the OMV obtained here, can be used as a meningococcal vaccine after further investigation.

Evaluation of protective immunity responses against pneumococcal PhtD and its C-terminal in combination with outer-membrane vesicles as adjuvants

“…In brief, OMVs were extracted using 0.1 M Tris-HCl, 10 mM EDTA and 0.5 % w/v deoxycholate (pH, 8.6). Purified OMVs were produced by sequential centrifugations at 20 000 g for 30 min and final centrifugation at 125 000 g for 2 h. The sediment of OMVs was homogenized in phosphate-buffered saline (PBS; pH, 7.2) and thiomersal (100 mg l −1 ) was added as a preservative in the OMV batch used in this study [25][26][27][28]. The concentration of OMV proteins was then evaluated using a NanoDrop spectrophotometer at 280 nm and PorA protein, as one of the most important immunological components in meningococcal serogroup B OMV, was analysed with SDS-PAGE on 12 % gel.…”

Extraction and biological evaluation of Mycobacterium kansasii extracellular vesicles as a vaccine candidate against mycobacterial pulmonary infections

“…Firstly, 0.1 M Tris-HCl (Sigma, USA), pH 8.6± 0.1 and 10 mM EDTA (Sigma, UK) solution, secondly, 0.1 M Tris-HCl, pH 8.6± 0.1 and 10 mM EDTA and 100% (w/v) deoxycholate (Merck, Darmstadt, Germany) solution and thirdly, 0.1 M Tris-HCl, pH 8.6± 0.1 and 10 mM EDTA and 0.5% w/v deoxycholate solution [12,13]. After deactivation of the bacterium for 2 h at 80°C, the cells were harvested by centrifugation at 1000 x g twice for 30 min.…”

“…The Nano-drop protein concentration method uses absorbance at 280 nm (NanoDrop™ 1000 Spectrophotometer, ThermoScientific Co. USA) [13]. Determination of EVs proteins by Bradford Coomassie brilliant blue assay was confirmed by measuring absorbance at 590 nm [14].…”

“…[13] Electron microscopy The intactness of the vesicles was checked by Scanning Electron Microscopy (SEM). For this purpose, field emission scanning electron microscope was used (FE-SEM; HITACHI S-4160 model with 5 nm image resolution, 20-30000 x magnification and maximum accelerating voltage of 30 kV).…”

“…First, a small amount of suspension sample was coated with gold and then was observed with the electron microscope. The Limulus Amebocyte Lysate (LAL) assay Endotoxin safety was evaluated by LAL test (Pierce® LAL Chromogenic Endotoxin Quantitation Kit, Thermo Fisher Scientific Co. USA; 88282), according to the manufacturer's instructions [13].…”