Extraction and Biological Evaluation of the Membrane Vesicles of Mycobacterium tuberculosis (CRBIP7.11) as Adjuvant and Vaccine Candidate

Abstract

Background: Membrane vesicles are non-viable structures released by pathogenic bacteria. They contain numerous antigenic materials from the bacterial outer membrane, making them attractive targets for use as vaccine antigens. The membrane vesicles are related to the virulence because of their capacity to concentrate immunomodulatory molecules and toxins. Objective: In the present study, we examined the membrane vesicles of Mycobacterium tuberculosis as adjuvant and vaccine candidate. Methods: Mycobacterium tuberculosis standard strain CRBIP7.11 was cultured at 37°C in Loewenstein Johnson (LJ) media for 3-4 weeks. To confirm the species, standard microbiological and biochemical tests were performed. After preparation of membrane vesicles, the amount of protein in membrane vesicles was measured by SDS-PAGE and Nanodrop. To analyze the integrity and morphology of extracellular vesicles, transmission electron microscopy was used. The lipopolysaccharide was determined using the Limulus Amebocyte Lysate (LAL) kit. Results: The total mass of vesicular fraction was 4.8 mg. SDS-PAGE showed protein bands in the approximate regions of 35, 40, 70, and 90 kDa. The amount of membrane vesicles total protein was 1.26 and 1.29 mg/mL. Transmission electron microscopy analysis of pellets revealed that the extracted vesicles are 50-200 nm in size. Also, LaL test showed negative results (values were less than 300 IU). Conclusions:The results of the present study give important evidence that actively released mycobacterial vesicles are delivery instruments for immunologically active molecules involved in mycobacterial virulence.