<i>In vitro</i> immunogenicity of <i>S</i>. <i>aureus</i>-derived EVs (SEVs).

<p>(A) Uptake of SEVs by bone marrow-derived dendritic cells (BMDCs). BMDCs were treated with SEVs (10 μg/ml) for 24 h. BMDCs cytoplasm were stained with CellTracker Green CMFDA (5-chloromethylfluorescein diacetate, green), nuclei with Hoechst (blue), and SEVs with DiI (red). The quantification of SEV-florescence in no-treatment and SEV-treatment group (n = 20, each group). (B) The expression of co-stimulatory molecules in BMDCs. The expression of CD80 and CD86 in BMDCs were measured 24 h after treatment with SEVs (10 μg/ml) or PBS. (C) Production of pro-inflammatory cytokines from BMDCs 24 h after SEVs treatment. BMDCs were treated with various concentrations of SEVs, and the levels of TNF-ɑ, IL-6, and IL-12 in the cell supernatants were measured by ELISA. *** indicates p< 0.001.</p

Efficacy of <i>S</i>. <i>aureus</i> EVs (SEVs) vaccination on protection against lethality induced by <i>S</i>. <i>aureus</i> lung infection.

<p>(A) Determination of lethal and sub-lethal doses of <i>S</i>. <i>aureus</i> in mouse pneumonia model. Survival rates in mice were evaluated after oropharyngeal application with different doses (1 × 10⁸, 2 × 10⁸, 3 × 10⁸ and 4 × 10⁸ CFU) of <i>S</i>. <i>aureus</i>. Survival was monitored every 12 h for 3 days (n = 10, each group). (B) Histologic image of mouse lung after oropharyngeal application of <i>S</i>. <i>aureus</i> (1 × 10⁸ CFU) 24 h post-infection. (C) Study protocol for SEV-immunization and challenge of the lethal dose (4 × 10⁸ CFU<i>)</i> of <i>S</i>. <i>aureus</i>. SEVs and sham (PBS) were injected intramuscularly at weekly intervals for 3 weeks, and then <i>S</i>. <i>aureus</i> was applied via oropharyngeal route one week after the last immunization. (D) Efficacy of different doses (1, 5, and 10 μg) of SEV vaccination. Survival was monitored every 12 h for 3 days (n = 10, each group). (E) Efficacy of SEV vaccination according to immunization frequency. Survival rates were monitored every 12 h for 3 days in mice immunized with SEVs (5 μg) once, twice, or three times (n = 10, each group).</p

Antibody and T cell responses after SEV vaccination.

<p>For (A) and (B), SEVs (5 μg) and sham (PBS) were injected intramuscularly to mice at weekly intervals for 3 weeks (n = 10, each group). (A) The levels of SEV-reactive IgG in serum. Sera were obtained from SEV- and sham-immunized mice 7 days after each immunization and serum levels of SEV-reactive IgG were measured by ELISA. (B) SEV-specific production of IFN-γ, IL-17, and IL-4 from splenic T cells. Splenic T cells were isolated from spleens of SEV- and sham-immunized mice, and then stimulated with anti- CD3/CD28 for 72 h. The levels of IFN-γ, IL-17, and IL-4 in the cell supernatants were measured by ELISA. For (C) and (D) SEVs (5 μg) and sham (PBS) were applied intraperitoneally (IP), subcutaneously (SC), or intramuscularly (IM) at weekly intervals for 3 weeks (n = 10, each group). (C) The levels of SEV-reactive IgG in serum. Sera were obtained from SEV- and sham (PBS)-immunized mice 7 days after the last immunization. (D) SEV-specific production of IFN-γ, IL-17, and IL-4 from splenic T cells. Splenic T cells were isolated from spleen of SEV- and sham (PBS)-immunized mice, and then stimulated with anti-CD3/CD28 for 72 h. The levels of IFN-γ, IL-17 and IL-4 in the cell supernatants were measured by ELISA. * indicates p< 0.05 vs. PBS and ** indicates p< 0.01 vs. the other groups.</p

Efficacy of SEV vaccination on protection against pneumonia induced by sub-lethal dose of <i>S</i>. <i>aureus</i>.

<p>For all figures, SEVs (5 μg) and sham (PBS) were injected intramuscularly to mice at weekly intervals for 3 weeks, and then sub-lethal dose (1 × 10⁸ CFU<i>)</i> of <i>S</i>. <i>aureus</i> was applied via the oropharyngeal route one week after the last immunization. Normal: PBS-immunized and PBS-challenged mice; PBS: PBS-immunized and <i>S</i>. <i>aureus</i>-challenged mice; SEV: SEV-immunized and <i>S</i>. <i>aureus</i>-challenged mice. (A) Colony forming unit (CFU) counts from lung of SEV- and sham (PBS)-immunized mice 24 h after the <i>S</i>. <i>aureus</i> challenge (n = 10, each group). (B) Histology (left panel) and gross image (right panel) of lung from SEV- and sham (PBS)-immunized mice after the sub-lethal dose of <i>S</i>. <i>aureus</i> challenge. (C) Distribution of <i>S</i>. <i>aureus</i> before and after SEV-immunization. Cy7-labeled <i>S</i>. <i>aureus</i> was applied via the oropharyngeal route to SEV- and sham (PBS)-immunized mice. Cy7 fluorescence of whole mouse (upper panel) or lung (lower panel) was acquired by IVIS spectrum 24 h after the <i>S</i>. <i>aureus</i> challenge. (D) Bioluminescence signal in the lung tissue after Cy7-labeled <i>S</i>. <i>aureus</i> administration. The amount of the bioluminescence signal (photons/s) in the lung tissue was measured by IVIS spectrum 24h after <i>S</i>. <i>aureus</i> challenge (n = 5, each group). (E) The levels of IL-β and IL-6 in serum of SEV- and sham (PBS)-immunized mice 24 h after the <i>S</i>. <i>aureus</i> challenge (n = 10, each group). * indicates p< 0.05 vs. PBS.</p

Long-term effect of SEV vaccination on the protection against lethality induced by <i>S</i>. <i>aureus</i> infection.

<p>(A) Study protocol for SEV vaccination and bacterial challenge. SEVs (5 μg) and sham (PBS) were injected intramuscularly at weekly intervals for 3 weeks, and then <i>S</i>. <i>aureus</i> (4 × 10⁸ CFU) was challenged by oropharyngeal application 40 days after the last immunization. (B) Survival rates of SEV- and sham-immunized mice challenged with <i>S</i>. <i>aureus</i> (n = 10, each group). Survival was monitored every 12 h for 40 days.</p

Toxicity of SEV vaccine.

<p>Mice were intramuscularly administered with 5 μg or 50 μg of SEV and monitored for 14 days. (n = 10) (A) The survival rate, body temperature, and body weight of mice measured at indicated times. (B) The levels of IL-1β, IL-6 and TNF-α in serum of SEV- and sham (PBS)-immunized mice 24 h after the SEV administration. n.s. indicates not significant.</p

Bacterial Protoplast-Derived Nanovesicles as Vaccine Delivery System against Bacterial Infection

The notion that widespread infectious diseases could be best managed by developing potent, adjuvant-free vaccines has resulted in the use of various biological immune-stimulating components as new vaccine candidates. Recently, extracellular vesicles, also known as exosomes and microvesicles in mammalian cells and outer membrane vesicles in Gram-negative bacteria, have gained attention for the next generation vaccine. However, the more invasive and effective the vaccine is in delivery, the more risk it holds for severe immune toxicity. Here, in optimizing the current vaccine delivery system, we designed bacterial protoplast-derived nanovesicles (PDNVs), depleted of toxic outer membrane components to generate a universal adjuvant-free vaccine delivery system. These PDNVs exhibited significantly higher productivity and safety than the currently used vaccine delivery vehicles and induced strong antigen-specific humoral and cellular immune responses. Moreover, immunization with PDNVs loaded with bacterial antigens conferred effective protection against bacterial sepsis in mice. These nonliving nanovesicles derived from bacterial protoplast open up a new avenue for the creation of next generation, adjuvant-free, less toxic vaccines to be used to prevent infectious diseases