Bacterial Protoplast-Derived Nanovesicles as Vaccine Delivery

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Letter pubs.acs.org/NanoLett

Bacterial Protoplast-Derived Nanovesicles as Vaccine Delivery System against Bacterial Infection Oh Youn Kim,† Seng Jin Choi,† Su Chul Jang,† Kyong-Su Park,† Sae Rom Kim,† Jun Pyo Choi,† Ji Hwan Lim,† Seung-Woo Lee,‡ Jaesung Park,§ Dolores Di Vizio,⊥ Jan Lötvall,∥ Yoon-Keun Kim,*,∇ and Yong Song Gho*,† †

Department of Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea § Department of Mechanical Engineering, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea ⊥ Division of Cancer Biology and Therapeutics, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, California 90048, United States ∥ Krefting Research Centre, Department of Internal Medicine, Institute of Medicine, The Sahlgrenska Academy, University of Gothenburg, Gothenburg 405 30, Sweden ∇ Ewha Womans University Medical Center, Seoul 120-808, Republic of Korea ‡

S Supporting Information *

ABSTRACT: The notion that widespread infectious diseases could be best managed by developing potent, adjuvant-free vaccines has resulted in the use of various biological immunestimulating 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. KEYWORDS: protoplast, extracellular vesicles, vaccination, outer membrane vesicles, exosome-mimetics

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Extracellular vesicles are spherical bilayered proteolipids of 20− 1000 nm in diameter produced ubiquitously by all living cells.11−16 These vesicles are very attractive candidates to develop novel diagnostic tools, targeted drug delivery systems, and vaccines because they harbor specific subsets of proteins, DNAs, RNAs, and lipids, which play diverse physiological and pathological functions.8−10,16−18 Extracellular vesicles are highly biocompatible nanosized structures and represent an appealing vaccine delivery system that encloses immune modulating components capable of stimulating antigen-specific immune response.8−10,16

ith increasing incidence of infectious diseases and accumulating resistance of existing pathogens to standard interventions, the development of an effective and safe vaccine platform is crucial for overcoming the burden of infectious diseases. The use of nanosized vehicles made of lipids, polymers, gold, and silica is the burgeoning area of vaccine delivery system today as they can easily travel through the blood and lymphatic vessels for effective vaccine delivery.1−6 However, there still remain challenges to be solved, including the difficulty in loading the desired antigen to the vehicle and effectiveness of the adjuvant, in order to develop a delivery system that is both safe and effective for broad range of vaccines.7 Recently, extracellular vesicles, also known as exosomes and microvesicles in mammalian cells and outer membrane vesicles (OMVs) in Gram-negative bacteria, have gained attention for the next generation vaccine.8−10 © XXXX American Chemical Society

Received: September 12, 2014 Revised: November 28, 2014

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Scheme 1. Schematic Diagram of PDNVs and Antigen-Loaded PDNVs Preparation

(Scheme 1). These purified nanovesicles did not contain any live bacteria: no bacterial colonies were observed in PDNVs (20 μg in total protein)-inoculated lysogeny broth agar plate. The size of PDNVs ranged from 68 to 220 nm in diameter with the average size being 114 ± 9.45 nm (n = 5) as determined by dynamic light scattering analysis (Figure 1A). Examination of the purified PDNVs using scanning electron microscopy (Supporting Information Figure S1), transmission electron microscopy (Supporting Information Figure S2), and cryotransmission electron microscopy (Figure 1B and Supporting Information Figure S3) revealed that they were intact nanosized vesicles with a lipid bilayer devoid of contaminants like cellular debris and protein aggregates. Purified PDNVs were depleted of outer membrane components including outer membrane protein A (OmpA) and lipid A (a core component of LPS), while they harbored FtsZ, a bacterial cytoplasmic protein (Figure 1C and Supporting Information Figure S4). To test the immunogenicity of PDNVs, we prepared antigenloaded PDNVs using genetically engineered E. coli. Green fluorescence protein (GFP), OmpA and Staphylococcus aureus specific coagulase (SAcoagulase) loaded PDNVs, designated as GFP PDNVs, OmpAPDNVs, and SAcoaPDNVs, respectively, were chosen as bacterial antigen cargo to test the preventive vaccination effect of PDNVs. The presence of each antigen in antigen-loaded PDNVs was confirmed by Western blotting analyses (Figure 1D). Dynamic light scattering analyses revealed no changes in size between antigen-loaded and control PDNVs (Figure 1E). The total protein measured in PDNVs and antigen-loaded PDNVs was 200 times more abundant than in naturally produced OMVs (Figure 1F), indicating a significantly higher yield for PDNVs. We next tested the potential toxicity of PDNVs examining the inflammatory response to PDNVs in vitro and in vivo. PDNVs did not increase the expression of pro-inflammatory cytokine IL-6 by mouse macrophage cell line Raw 264.7 in vitro (Figure 1G). In addition, systemic inflammatory indexes like the level of serum IL-6 or number of leukocytes and platelets in the blood remained unchanged 6 h after the intraperitoneal injection of PDNVs (5 μg in total protein) in vivo (Figure 1H and Supporting Information Figure S5). In contrast, bacteriafree E. coli OMVs (5 μg in total protein), used as positive control, significantly induced expression of the inflammatory

Gram-negative bacteria OMVs, enriched with multiple virulence factors and immune stimulating molecules, such as bacterial endotoxin lipopolyshaccharide (LPS) and outer membrane proteins,13,16 have recently gained interests as acellular, adjuvant-free vaccines.17−24 In fact, OMVs are being used as vaccines against meningococcal diseases in children in several countries such as Norway, New Zealand, and Cuba.24,25 However, the commercialization of OMVs as vaccines in humans is concerning for several reasons. First, although most of the OMVs considered safe for use as vaccine or drug delivery are LPS-free,24−26 increasing evidence suggests that LPS is not the only virulent factor in OMVs and that other components of the OMVs, such as the outer membrane proteins and lipoproteins are also important for provoking systemic inflammatory response syndrome leading to lethality in mice.27 In addition, bacteria release relatively low quantities of OMVs, indicating that their cost-effective mass production may become inconvenient. In this study, in an attempt to optimizing the current vaccine delivery system, we designed bioinspired bacterial protoplastderived nanovesicles (PDNVs), which are depleted of toxic outer membrane components to generate a universal adjuvantfree vaccine delivery system. These PDNVs exhibited significantly higher yield 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 a new avenue for the creation of next generation, adjuvant-free, less toxic vaccines, to be used to prevent infectious diseases. PDNVs and antigen-loaded PDNVs were prepared from Escherichia coli following the protocol illustrated in Scheme 1. Briefly, E. coli or genetically engineered E. coli expressing the antigen was treated with lysozyme to remove the outer membrane and periplasmic components. These resulting protoplasts were extruded serially through polycarbonate membranes having pore sizes of 10 μm, 5 μm, and finally 1 μm to obtain nanosized vesicles, a procedure that has been used to generate exosome-mimetic nanovesicles.28 We then purified PDNVs in a two-step OptiPrep ultracentrifugation gradient from the interface layer between 10% and 50% iodixanol B

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Figure 1. Production and characterization of bacteria protoplast-derived nanovesicles (PDNVs). (A) Size distribution of PDNVs measured by dynamic light scattering analysis (n = 5). (B) Cryotransmission electron micrograph images of PDNVs. Scale bars, 100 nm. (C) Western blotting analysis of outer membrane components (OmpA and lipid A) of PDNVs and E. coli whole cell lysate (WCL). Same amount of protein of PDNVs and E. coli WCL (10 μg) were loaded. Bacterial cytoplasmic protein, FtsZ was used as internal control. (D) GFPPDNVs, OmpAPDNVs, and SAcoa PDNVs protein lysates (500 ng) were blotted with the indicated antibodies. For positive controls, 300 ng of OmpA and 100 ng of GFP and SAcoagulase recombinant proteins were loaded. (E) Size distribution of GFPPDNVs, OmpAPDNVs, and SAcoaPDNVs measured by dynamic light scattering analysis (n = 5). (F) The yields of PDNVs, GFPPDNVs, OmpAPDNVs, SAcoaPDNVs, and OMVs measured as the total protein amount in five independent preparations. CFU, colony forming units. (G) Levels of in vitro pro-inflammatory cytokine IL-6 production from mice macrophage cell line Raw 264.7 after the treatment with PDNVs or OMVs for 16 h (n = 3). (H) In vivo serum level of IL-6 after 6 h of intraperitoneal injection of PDNVs (5 μg) or OMVs (5 μg) in mice (n = 5). (I) Survival rate of mice intraperitoneally injected with PDNVs (n = 10), GFPPDNVs (n = 5), OmpA PDNVs (n = 5), or OMVs (n = 10). n.s., **, and *** indicate not significant, P < 0.01, and P < 0.001, respectively.

increase of local IL-6 level in peritoneal fluid though significantly lower than the level increased by E. coli OMVs

cytokine in vitro and systemic inflammation in vivo.27 We observed that intraperitoneally injected PDNVs induced slight C

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Figure 2. PDNVs are taken up by bone marrow-derived DCs and can activate immunostimulatory responses. (A, B) Uptake of PDNVs (red fluorescent signal) by bone marrow-derived DCs (green fluorescent signal) was examined using the super-resolution structured illumination microscopy. Representative two-dimensional (A) and three-dimensional (B) fluorescence images. Scale bars, 20 μm. (C) The production of IL-6, TNF-α, and IL-12p40 measured from the cell supernatant of DCs after the treatment with PDNVs for 12 h (n = 3). OMVs (100 ng/mL) were used as positive control. ** and *** indicate P < 0.01 and P < 0.001, respectively.

Figure 3. PDNVs immunogenicity evaluation using GFP model protein. (A) Study protocol for the immunization and evaluations. Mice were intraperitoneally immunized with empty PDNVs (5 μg in total protein), free GFP protein (2.5 μg), GFPPDNVs (5 μg in total protein), free GFP protein (2.5 μg) mixed with adjuvant aluminum hydroxide, and GFPLiposome. Each injected treatment harbored 2.5 μg of GFP except for PDNVs control. (B) Levels of GFP reactive IgG titer following immunization with the indicated vehicles. (C) GFP specific production of IFN-γ, IL-17, and IL-10 from splenic T cells. Results (n = 5 per group) are representative of two independent experiments performed (total n = 10). ** and *** indicate P < 0.01 and P < 0.001, respectively.

(Supporting Information Figure S6). Moreover, no hepatic injury was observed following the injection of OmpAPDNVs (100 μg), whereas signs of hepatic injury was clearly shown

(Supporting Information Figure S5). In addition, we observed that intraperitoneal injection of OmpAPDNVs (100 μg) still did not cause increase in systemic inflammatory response D

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Figure 4. Vaccination effect of bacterial antigens OmpA-loaded OmpAPDNVs against E. coli-induced bacterial sepsis. Mice were immunized by intraperitoneal injection with OmpAPDNVs (5 μg of total protein) three times with 1 week intervals and challenged with lethal dose E. coli (1.0 × 109 CFU) 10 days after the final immunization. (A) OmpA protein-reactive IgG levels in serum 1 week after each injection. (B) OmpA protein specific T cell responses from isolated splenic T cells a week after the final immunization. (C) Survival rates of mice after the lethal dose E. coli challenge. Results (n = 10) are representative of three independent experiments (total n = 30) for (A) to (C). (D) Blood serum levels of OmpA specific IgG after the final immunizations with OmpAPDNVs in wild type and TLR4-deficient mice (n = 4). (E) OmpA specific T cell response of splenic T cells isolated from wild type and TLR4-deficient mice after OmpAPDNVs immunization (n = 4). (F) Survival rates of wild type and TLR4-deficient mice after the E. coli challenge. Results (n = 10) are representative of two independent experiments (total n = 20). n.s., **, and *** indicate not significant, P < 0.01, and P < 0.001, respectively.

these results indicate that PDNVs can serve as antigen delivery vehicles that can be produced in large quantities and are less toxic. Dendritic cells (DCs) are the first line of cells that initiate the antigen specific immune response. When bone marrow− derived DCs were treated with labeled PDNVs, we observed strong fluorescent signal against PDNVs in the cytoplasm of DCs, indicating DCs uptake PDNVs (Figure 2A,B). Furthermore, treatment of PDNVs to DCs induced production of

after the injection of CCl4 (40% in corn oil), a well-known liver toxic agent (Supporting Information Table S1).29 Finally, to further test the toxicity of PDNVs, mice were injected with a high dose (1 mg) of PDNVs, GFPPDNVs, and OmpAPDNVs, and monitored at given intervals for 5 days. As a control group, mice were injected with 25 μg of OMVs. Our results show that all mice treated with 1 mg of empty or antigen-loaded PDNVs survived, whereas 80% of the mice treated with OMVs died within 48 h from the injection (Figure 1I).27,30 Collectively, E

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Figure 5. Vaccination effect of Gram-positive bacterial antigen SAcoagulase-loaded SAcoaPDNVs against S. aureus-induced lethal pneumonia and antigen specificity of PDNV-mediated immunization. Mice were intraperitoneally injected with 5 μg protein of SAcoaPDNVs, OmpAPDNVs, or phosphate-buffered saline (PBS) three times with 1 week intervals and challenged after 10 days from the final immunization. (A) SAcoagulase specific IgG titer after immunization. (B) SAcoagulase specific production of IFN-γ and IL-17 from splenic T cells isolated from immunized mice 1 week after the final immunization. (C, D) Survival rates of mice challenged with S. aureus (5.0 × 109 CFU) intratracheally (C) or E. coli (1.0 × 109 CFU) intraperitoneally (D) 10 days after the last immunization. ** and *** indicates P < 0.01 and P < 0.001, respectively. Results (n = 10) are representative of two independent experiments (total n = 20).

in hospital populations.27 Therefore, we next investigated whether the Gram-negative bacterial antigen delivered by PDNVs can actually prevent bacterial sepsis. OmpA, one of the major outer membrane proteins, is highly conserved among the Gram-negative bacteria family and is known to be involved in bacterial virulence and growth.31−33 Therefore, we used OmpA as the model antigen protein to investigate the PDNV-mediated preventive vaccination effect against Gram-negative bacteria sepsis. Mice were immunized intraperitoneally with OmpA PDNVs for 3 weeks at 1 week intervals. All OmpAPDNVimmunized mice developed high levels of serum OmpA-specific IgG antibody (Figure 4A). OmpA specific induction of Th1 and Th17 cytokines IFN-γ and IL-17, respectively, was also observed in OmpAPDNV-immunized mice, whereas the level of IL-4, a representative Th2 cytokine, was not (Figure 4B). This induction of T cell-mediated immunity is an important indicator of effective vaccination as Th1 and Th17 cellular responses are the key host immunity effectors against intracellular bacteria infections.21,34 On the other hand, Th2 cellular response, usually involved in asthma and extracellular parasite infections, was not triggered by OmpA PDNVs immunization.34 When the mice were challenged with lethal doses of E. coli 10 days after the final immunization, all OmpA PDNV-immunized mice survived, whereas the shamimmunized mice died within 24 h (Figure 4C). After the bacterial challenge, sham-immunized mice had developed serious systemic inflammatory symptoms including eye exudates, piloerection, and hypothermia, whereas OmpAPDNVimmunized mice did not (data not shown). Moreover, this preventive vaccination effect was independent of the PDNV administration routes as the vaccination effect was also

immune modulating cytokines, IL-6, TNF-α, and IL-12p40 in a dose-dependent manner (Figure 2C). Taken together, these results suggest that PDNVs are effectively taken up by DCs and activate these antigen presenting cells to induce cytokines polarizing specific immune response. We next investigated whether PDNVs have the ability as adjuvant and induce antigen specific immunity effectively. We used GFP as the model antigen cargo because GFP itself is poorly immunogenic.20 When the antigen specific B cell antibody and T cell responses were examined, mice immunized with GFPPDNVs developed high levels of GFP specific antibody titers and GFP specific T cell responses, whereas immunized groups with PDNVs and free GFP did not (Figure 3A−C). We further compared the antigen specific immunogenicity of GFP PDNVs with the commercially available adjuvant aluminum hydroxide to evaluate the adjuvant efficacy and with GFPloaded liposomes (GFPLiposome) to find out if the strong antigen specific adaptive responses were triggered by the nanosized delivery vehicle effect of PDNVs. Although the production of GFP specific antibody and T cell responses were all observed from the mice immunized with GFP + aluminum hydroxide and GFPLiposomes, GFPPDNV-immunized mice group showed the highest increase in both B and T cell responses, despite the use of same dose of GFP in all cases (Figure 3B,C). These results collectively suggest that GFP PDNVs can activate both B cell and T cell antigen specific responses effectively without the aid of additional adjuvants. When our immune system breaks down, Gram-negative bacteria such as E. coli, which constitutes the human micro flora could induce strong inflammatory response that could eventually provoke sepsis, one of the leading causes of death F

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Figure 6. Long-term vaccination effect of PDNVs. (A) Study protocol scheme for long-term vaccination. Mice were injected intraperitoneally three times with OmpAPDNVs (5 μg in protein amount), and challenged 6 weeks after the final immunization (n = 10 per group). (B) Serum OmpA specific IgG titer after immunization. (C) OmpA specific T cell response of splenic T cells isolated from OmpAPDNV-immunized mice 6 weeks after the final immunization. (D) Survival rate of mice after E. coli (5.0 × 109 CFU) challenge after 6 weeks of final immunization. ** and *** indicates P < 0.01 and P < 0.001, respectively.

effect against Gram-negative E. coli infection (Figure 5D). Note that OmpA is a Gram-negative bacterial specific antigen while SAcoagulase is S. aureus specific antigen. Taken together, these results suggest that the PDNV-mediated vaccination effect is antigen specific, and that the immunization efficiency is retained when using different types of antigens. We finally examined the long-term vaccination effect of PDNVs immunization. High levels of antigen specific antibody titer and antigen specific T cell responses were maintained for 6 weeks in mice immunized with OmpAPDNVs (Figure 6A−C). We also observed a long-term preventive effect on Gramnegative bacteria-induced lethality by OmpAPDNVs vaccination (Figure 6D). All the OmpAPDNV-immunized mice survived the lethal dose E. coli challenge after 6 weeks of the last immunization except for one mouse that died almost right after the challenge. Together, the results imply that the protective effect of OmpAPDNVs is maintained in long-term. In this study, we report a series of experiments to develop nanosized bacterial protoplast-derived vesicles depleted of outer membrane components, as a safe and effective, adjuvant-free vaccine delivery system. By fabricating protoplasts of genetically engineered antigen-expressing E. coli, antigen-loaded PDNVs are easily produced with significantly higher yield and these PDNVs are less toxic compared with E. coli OMVs. Moreover, immunization with antigen-loaded PDNVs induces potent antigen specific humoral and cellular immune responses and confers effective protection against both Gram-negative and Gram-positive bacterial sepsis in murine models. Because the preventive effect of vaccine is specific to the specific antigen,20 utilization of PDNVs loaded with single or multiple bacterial antigens may enhance future vaccination efficacy against most virulent bacterial infections.40,41 Our findings open up a new era in the next-generation acellular vaccine delivery field.

observed in mice administrated with intramuscular immunization route (Supporting Information Figure S7). These results demonstrate that immunization with OmpAPDNVs effectively prevents E. coli-induced lethality via activation of both B cell and T cell antigen specific responses. To exclude the possibility that LPS contamination was responsible for the observed PDNVs vaccination effects, we immunized, with OmpAPDNVs, mice deficient for the LPS receptor TLR4.35,36 Both TLR4deficient and wild type immunized mice produced similar levels of OmpA protein specific antibody titers and IFN-γ and survived to the lethal bacteria challenge (Figure 4D−F), indicating that LPS is not involved in the vaccination effect of PDNVs. To evaluate the extent of antigen specificity of the PDNVmediated immunization, and to test whether it could have a broad clinical usefulness, we carried out vaccination experiments using PDNVs loaded with antigens from different types of bacteria. For this purpose, we used Gram-positive bacteria S. aureus specific Sacoagulase to examine the protective effect of PDNVs immunization against S. aureus-induced pneumonia.37 S. aureus is one of the major causes of life-threatening natural infections in hospital populations but the development of staphylococcal vaccines has failed despite the increasing antibiotics resistance of S. aureus.38,39 High serum levels of SAcoagulase specific antibody titer were detected in mice immunized with SAcoaPDNVs, but not in sham- and OmpAPDNV -immunized mice (Figure 5A). Additionally, SAcoagulasespecific T cell responses were induced in mice immunized with SAcoaPDNVs (Figure 5B). Furthermore, when sham-, OmpA PDNV-, and SAcoaPDNV-immunized mice were challenged with S. aureus to induce lethal pneumonia, only the mice immunized with SAcoaPDNVs survived (Figure 5C). In addition, immunization with SAcoaPDNVs did not confer any protective G

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(13) Lee, E. Y.; Choi, D. Y.; Kim, D. K.; Kim, J. W.; Park, J. O.; Kim, S.; Kim, S. H.; Desiderio, D. M.; Kim, Y. K.; Kim, K. P.; Gho, Y. S. Proteomics 2009, 9, 5425−5436. (14) Marcilla, A.; Trelis, M.; Cortes, A.; Sotillo, J.; Cantalapiedra, F.; Minguez, M. T.; Valero, M. L.; Sanchez del Pino, M. M.; MunozAntoli, C.; Toledo, R.; Bernal, D. PLoS One 2012, 7, e45974. (15) Martin-Jaular, L.; Nakayasu, E. S.; Ferrer, M.; Almeida, I. C.; Del Portillo, H. A. PLoS One 2011, 6, e26588. (16) Lee, E. Y.; Choi, D. S.; Kim, K. P.; Gho, Y. S. Mass Spectrom. Rev. 2008, 27, 535−555. (17) Ohno, S.; Takanashi, M.; Sudo, K.; Ueda, S.; Ishikawa, A.; Matsuyama, N.; Fujita, K.; Mizutani, T.; Ohgi, T.; Ochiya, T.; Gotoh, N.; Kuroda, M. Mol. Ther. 2013, 21, 185−191. (18) Valadi, H.; Ekstrom, K.; Bossios, A.; Sjostrand, M.; Lee, J. J.; Lotvall, J. O. Nat. Cell Biol. 2007, 9, 654−659. (19) Oster, P.; O’Hallahan, J.; Aaberge, I.; Tilman, S.; Ypma, E.; Martin, D. Vaccine 2007, 25, 3075−3079. (20) Chen, D. J.; Osterrieder, N.; Metzger, S. M.; Buckles, E.; Doody, A. M.; DeLisa, M. P.; Putnam, D. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 3099−3104. (21) Kim, O. Y.; Hong, B. S.; Park, K. S.; Yoon, Y. J.; Choi, S. J.; Lee, W. H.; Roh, T. Y.; Lotvall, J.; Kim, Y. K.; Gho, Y. S. J. Immunol. 2013, 190, 4092−4102. (22) Kulp, A.; Kuehn, M. J. Annu. Rev. Microbiol. 2010, 64, 163−184. (23) Keenan, J.; Day, T.; Neal, S.; Cook, B.; Perez-Perez, G.; Allardyce, R.; Bagshaw, P. FEMS Microbiol. Lett. 2000, 182, 259−264. (24) Ferrari, G.; Garaguso, I.; Adu-Bobie, J.; Doro, F.; Taddei, A. R.; Biolchi, A.; Brunelli, B.; Giuliani, M. M.; Pizza, M.; Norais, N.; Grandi, G. Proteomics 2006, 6, 1856−1866. (25) Granoff, D. M. Clin. Infect. Dis. 2010, 50 (Suppl 2), S54−S65. (26) Gujrati, V.; Kim, S.; Kim, S. H.; Min, J. J.; Choy, H. E.; Kim, S. C.; Jon, S. ACS Nano 2014, 8, 1525−1537. (27) Park, K. S.; Choi, K. H.; Kim, Y. S.; Hong, B. S.; Kim, O. Y.; Kim, J. H.; Yoon, C. M.; Koh, G. Y.; Kim, Y. K.; Gho, Y. S. PLoS One 2010, 5, e11334. (28) Jang, S. C.; Kim, O. Y.; Yoon, C. M.; Choi, D. S.; Roh, T. Y.; Park, J.; Nilsson, J.; Lotvall, J.; Kim, Y. K.; Gho, Y. S. ACS Nano 2013, 7, 7698−7710. (29) Yoon, Y. J.; Chang, S.; Kim, O. Y.; Kang, B. K.; Park, J.; Lim, J. H.; Huang, J. Y.; Kim, Y. K.; Byun, J. H.; Gho, Y. S. PLoS One 2013, 8, e68600. (30) Shah, B.; Sullivan, C. J.; Lonergan, N. E.; Stanley, S.; Soult, M. C.; Britt, L. D. Shock 2012, 37, 621−628. (31) Smith, S. G.; Mahon, V.; Lambert, M. A.; Fagan, R. P. FEMS Microbiol. Lett. 2007, 273, 1−11. (32) Kurupati, P.; Ramachandran, N. P.; Poh, C. L. Clin Vaccine Immunol. 2011, 18, 82−88. (33) Yan, W.; Faisal, S. M.; McDonough, S. P.; Chang, C. F.; Pan, M. J.; Akey, B.; Chang, Y. F. Vaccine 2010, 28, 2277−2283. (34) Zhu, J.; Paul, W. E. Blood 2008, 112, 1557−1569. (35) Takeuchi, O.; Akira, S. Cell 2010, 140, 805−820. (36) Mata-Haro, V.; Cekic, C.; Martin, M.; Chilton, P. M.; Casella, C. R.; Mitchell, T. C. Science 2007, 316, 1628−1632. (37) Cheng, A. G.; McAdow, M.; Kim, H. K.; Bae, T.; Missiakas, D. M.; Schneewind, O. PLoS Pathog. 2010, 6, e1001036. (38) Gordon, L. A. Clin. Infect. Dis. 1998, 26, 1179−1181. (39) Kollef, M. H.; Micek, S. T. Curr. Opin. Infect. Dis. 2006, 2, 161− 168. (40) Moriel, D. G.; Bertoldi, I.; Spagnuolo, A.; Marchi, S.; Rosini, R.; Nesta, B.; Pastorello, I.; Corea, V. A.; Torricelli, G.; Cartocci, E.; Savino, S.; Scarselli, M.; Dobrindt, U.; Hacker, J.; Tettelin, H.; Tallon, L. J.; Sullivan, S.; Wieler, L. H.; Ewers, C.; Pickard, D.; Dougan, G.; Fontana, M. R.; Rappuoli, R.; Pizza, M.; Serino, L. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 9072−9077. (41) Pizza, M.; Scarlato, V.; Masignani, V.; Giuliani, M. M.; Aricò, B.; Comanducci, M.; Jennings, G. T.; Baldi, L.; Bartolini, E.; Capecchi, B.; Galeotti, C. L.; Luzzi, E.; Manetti, R.; Marchetti, E.; Mora, M.; Nuti, S.; Ratti, G.; Santini, L.; Savino, S.; Scarselli, M.; Storni, E.; Zuo, P.; Broeker, M.; Hundt, E.; Knapp, B.; Blair, E.; Mason, T.; Tettelin, H.;

Nanosized bacterial protoplast-derived vesicles represent a versatile system that could accommodate also viral or bacterial antigens for future applications as a novel universal vaccine to prevent the various infectious diseases of today. However, the overall safety of PDNV-based vaccination, and the elucidation of the key component and detailed mechanism of how PDNVs triggers antigen-specific protective immune response, are required in future studies.

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ASSOCIATED CONTENT

S Supporting Information *

Materials and methods and additional figures and table as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Y.S.G.). *E-mail: [email protected] (Y.-K.K). Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare the following competing financial interest(s): O.Y.K., S.C.J., Y.-K.K., and Y.S.G. are the inventors of a patent regarding the use of bacterial protoplast nanovesicles as delivery vehicles (International Application No.: PCT/KR2011/004820; Publication Date: 30.06.2011). All rights have been assigned to Aeon Medix.

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ACKNOWLEDGMENTS This work was supported by the Midcareer Researcher Program through an NRF grant funded by the MEST (2014023004 and 20110028845) and National Junior Research Fellowship (2014009828).

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dx.doi.org/10.1021/nl503508h | Nano Lett. XXXX, XXX, XXX−XXX

Nano Letters

Letter

Hood, D. W.; Jeffries, A. C.; Saunders, N. J.; Granoff, D. M.; Venter, J. C.; Moxon, E. R.; Grandi, G.; Rappuoli, R. Science 2000, 287, 1816− 1820.

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dx.doi.org/10.1021/nl503508h | Nano Lett. XXXX, XXX, XXX−XXX