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 Lö tvall, ∥ 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 immune- stimulating components as new vaccine candidates. Recently, extracellular vesicles, also known as exosomes and micro- vesicles 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 W ith increasing incidence of infectious diseases and accumulating resistance of existing pathogens to stand- ard 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 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 Received: September 12, 2014 Revised: November 28, 2014 Letter pubs.acs.org/NanoLett © XXXX American Chemical Society A dx.doi.org/10.1021/nl503508h | Nano Lett. XXXX, XXX, XXX−XXX