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

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. melitensis challenge. 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 nonspecific protection and enhances the efficacy of OMVs as a vaccine against B. melitensis.

Figure 1

Electron microscope image at 60000 × magnification showing the double membrane associated with the outer membrane vesicles (OMVs) obtained from B. melitensis 16M. Their sizes ranged between 50–100 nm.

Figure 2

Coomassie blue stained 10% SDS-GEL picture showing the protein profile of OMVs and OMVs mixed with different concentrations of P85. All samples contain 5 µg protein concentration of OMVs. Lane 1 and 6: Standard protein ladder, Lane 2: OMVs, Lane 3: OMVs + 0.003% P85, Lane 4: OMVs+ 0.03% P85, Lane 5: OMVs + 0.3% P85.

Figure 3

Concentration dependent cytotoxicity induced by P85 mixed with OMVs. J774A.1 cells were incubated with different concentrations of P85 and cell viability was determined using a MTS assay after 24 h of incubation. The cell viability was determined considering the viability of cells incubated with media only as 100%. Data presented here is the mean ± SEM of cell viability obtained from three independent experiments and asterisk (*) represents statistical significant difference at p ≤ 0.001.

Figure 4

Secretion of TNF-α by J774A.1 cells induced with OMVs alone or in combination with P85. To assess the stimulation of J774A.1 cells different concentrations of P85 mixed with OMVs or OMVs alone or P85 alone were added to the cells. After 24 h of incubation, levels of TNF-α were determined in culture supernatant using an indirect sandwich ELISA. Medium and E. coli LPS treated cell supernatants served as negative and positive controls respectively. Lower limit of detection was 15 pg mL⁻¹. Levels of TNF- α are shown as mean ± SEM of three independent experiments. One asterisk (*) represents the statistically significant difference at p ≤ 0.001 from medium and two asterisks (**) represent statistically significant difference at p ≤ 0.001 from medium as well as from OMVs alone.

Figure 5

Production of IFN-γ by splenocytes of BALB/c mice vaccinated with saline, P85, OMVs or OMVs + P85 after in vitro stimulation with OMVs. IFN-γ levels were determined in splenocyte culture supernatants 5 days after the stimulation using an indirect sandwich ELISA. Splenoctye supernatants from medium and ConA treated cells were negative and positive controls respectively. Higher and lower limits of detection of IFN-γ were 200 ng mL⁻¹ and 1.6 ng mL⁻¹ respectively. Data represent results from 3 mice per group and 3 independent samples per mouse. The graph shows the mean ± SEM of IFN-γ levels.

Figure 6

Antibody titers determined in the plasma of BALB/c mice vaccinated with OMVs or OMVs+P85. Levels of total IgG, IgG1 and IgG2a were determined using an indirect ELISA, at a 1:6400 dilution of plasma. Data represents results from 5 mice per group and each sample was tested in duplicates. The graph represents the mean ± SEM of absorbance at 450 nm of the color developed. Asterisk (*) Indicates the statistically significant difference (p ≤ 0.01) between OMVs and OMVs + P85 vaccinated mice.