Dear Andres,

The following covers the answer to your question:

FEMS Immunol Med Microbiol. Author manuscript; available in PMC 2015 May 11.

Published in final edited form as:

FEMS Immunol Med Microbiol. 2012 Dec; 66(3): 436–444.

doi: 10.1111/1574-695X.12010

PMCID: PMC4426994

NIHMSID: NIHMS413557

Pluronic P85 enhances the efficacy of outer membrane vesicles as a subunit vaccine against Brucella melitensis challenge in mice

Neeta Jain-Gupta,1 Araceli Contreras-Rodriguez,2 Ramesh Vemulapalli,3 Sharon G. Witonsky,4 Stephen M. Boyle,1 and Nammalwar Sriranganathan1,*

Abstract

Brucellosis is the most common zoonotic disease worldwide and there is no vaccine for human use. Brucella melitensis Rev1, a live attenuated strain, is the commercial vaccine for small ruminants to prevent B. melitensis infections but has been associated with abortions in animals. Moreover, strain Rev1 is known to cause disease in humans and cannot be used for human vaccination. Outer membrane vesicles (OMVs) obtained from B. melitensis have been shown to provide protection similar to strain Rev1 in mice against B. melitensis challenge. In the present work we tested the efficacy of Pluronic P85 as an adjuvant to enhance the efficacy of Brucella OMVs as a vaccine. P85 enhanced the in vitro secretion of TNF-α by macrophages induced with OMVs and P85. Further, P85 enhanced the protection provided by OMVs against B. melitensischallenge. This enhanced protection was associated with higher total IgG antibody production but not increased IFN-γ or IL-4 cytokine levels. Moreover, P85 alone provided significantly better clearance of B. melitensis compared to saline vaccinated mice. Further studies are warranted to find the mechanism of action of P85 that provides non-specific protection and enhances the efficacy of OMVs as a vaccine against B. melitensis.

Materials and Methods

OMV Purification and characterization

OMVs were obtained from B. melitensis 16M as previously described (Gamazo, et al., 1989) but with the following modifications. Briefly, stock cultures of B. melitensis 16M were streaked on tryptic soy agar (TSA) plates and incubated for 48 h. Using cotton swabs, the bacterial culture was transferred to TSA slants containing 0.7% yeast extract and allowed to grow for 48 h. Next, the bacterial culture was scraped from slants using tryptic soy broth (TSB) (1mL/slant) and collected and spread on TSA plates containing 0.7% yeast extract. After an incubation of 48 h, Brucella cultures were collected with 200 µL phosphate buffered saline (PBS)/plate using a sterile cell scraper. The bacterial suspension was centrifuged at 15,191 × g and supernatant was collected. The supernatant was filter sterilized twice using 0.22 µm filters and the sterility of the filtrate was tested by inoculating 50µl into 10mL TSB and incubating at 37°C for 48–72 h. Absence of any bacterial growth confirmed the sterility of the filtrate. To obtain the OMVs, the filtrate was ultracentrifuge at 176,508 × g for 2 h. The pellet was washed in 50 mL PBS and harvested by centrifugation as above. The washing step was repeated twice and the pellet containing OMVs was resuspended in 250 µL PBS and stored at −20°C.

Concentration of protein in OMVs was determined using Pierce BCA protein Assay kit (Thermo Scientific) according to the manufacturer’s protocol. For the assessment of structure of OMVs by electron microscopy, OMVs were mixed with 2% aqueous uranyl acetate solution for negative staining and placed onto 200-mesh formvar, carbon coated copper grids (Electron Microscopy Sciences). Excess liquid was soaked away using filter paper and the samples were viewed at 80,000× magnification on a Zeiss 10CA Transmission Electron Microscope (Virginia-Maryland Regional College of Veterinary Medicine). To determine the protein profile, first OMVs were mixed with different concentrations of P85, dissolved in phosphate buffered saline, using a double hub-syringe and then mixed with 2× Laemmli buffer (Bio-Rad) containing β-mercaptoethanol and heated for 5 min at 95°C. The samples were electrophoresed on 10% SDS-PAGE gels (Invitrogen) and stained with Coomassie blue to visualize protein bands.

To view the full paper, please see attached file.

Hoping this will be helpful,

Rafik