Paragraph: Peptide based-vaccines are becoming one of the most widely investigated prophylactic and therapeutic health care interventions against a variety of diseases, including cancer. However, the lack of a safe and highly efficient adjuvant (immune stimulant) is regarded as the biggest obstacle to vaccine development. The incorporation of a peptide antigen in a nanostructure-based delivery system was recently shown to overcome this obstacle. Nanostructures are often formed from antigens conjugated to molecules such as polymers, lipids, and peptide, with the help of selfassembly phenomenon. This review describes the application of self-assembly process for the production of peptide-based vaccine candidates and the ability of these nanostructures to stimulate humoral and cellular immune responses. T stimulate a cytotoxic T lymphocyte response to eliminate cells that are already infected [8] [9] [10] . Therapeutic vaccines are a growing pharmaceutical sector. Sipuleucel-T (Provenge®) was approved by Food and Drug Administration (FDA) in 2010 for prostate cancer [11, 12] and talimogene laherparepvec (T-VEC) (Imlygic™) was introduced against metastatic melanoma in 2015 [13] [14] [15] . Despite their advantages, vaccines also have some drawbacks. Traditional vaccines based on attenuated and inactivated pathogens may revert to virulent form [16, 17] , cause autoimmunity or strong allergic responses [18, 19] . Additionally, the production and stability of this type of vaccine is often challenging. Subunit vaccines contain a small fragment of the whole pathogenic organism, for instance a recombinant protein, peptide, carbohydrate [20] [21] [22] [23] . Recombinant protein-based vaccines cause fewer adverse effects, but the difficulty with protein extraction and possible contamination during production are significant disadvantages. Recombinant vaccine can also trigger undesired immune responses. To avoid these obstacles minimal fragment of protein caring immunological information (peptide epitope) can be used for vaccine design [3] . However, peptide-based vaccines still have their own Achilles' heel. Peptides themselves are usually not immunogenic [3, 24] ; therefore, the use of immunostimulating agents (adjuvants) are essential for the success of this vaccine formulation [25] . Adjuvants are compounds that are able to evoke or enhance the immune response against co-delivered antigens [26] [27] [28] . A number of adjuvants have already been developed, for example Freund's adjuvant, lipid A, cholera toxin, aluminum salts, cytokines, liposomes, saponins, and CpG oligodeoxynucleotides [29] [30] [31] . However, the immunostimulating properties of these adjuvants are often poor or their toxicity is significant, therefore most of them are not suitable for human treatment. Thus, safer and more efficient adjuvants are urgently required [27] . Nanotechnology is one of the most quickly growing fields of science and engineering. It has been widely applied in materials, electronics, manufacturing, agricultural science, chemistry and bioscience [32] [33] [34] [35] [36] [37] . In bioscience, nanotechnology is used to create sensors, medical imaging and diagnostic tools, and for drug delivery. The most widely used nanostructures in this field are nanoparticles. Nanoparticles are often defined as particles with diameters ranging from 1 to 100 nm [24, 38] , while particles with diameter within the range of 100 to 1000 nm are considered sub-micro particles. However, following common understanding of "nano-size" range, nanostructures in this article are defined as any nanometer-sized object ranging from 1 to 1000 nm [39, 40] . Nanoparticles are especially useful in vaccine design. Many pathogens are within the nanometer range, nanoparticles are usually taken up efficiently by antigen-presenting cells (APCs) [41] . APCs are the key element of the primary innate immune system [42] and are responsible for triggering further adaptive immune responses. Therefore nanostructures often have immunostimulating properties and help to trigger immune reactions against the antigens they delivered (Fig. 1) [24, 43, 44] . Moreover, the surface of nanostructures can be modified with a specific targeting moiety for better APC recognition and uptake [45] [46] [47] . There are two main approaches to creating nanostructures: (1) mechanically breaking a large material into nanostructures, or (2) building up nanostructures from basic segments, often via a self-assembly process [48] . The first approach uses external forces like grinding, milling, or high pressure to break down the original material [49] [50] [51] . This process adds additional complexity to producing a nanostructure and the harsh conditions used to break down the initial component may damage the antigen. Nanostructures produced by this method typically have a low loading capacity and are often not biodegradable and thus may accumulate in the human tissues. Meanwhile, self-assembly-based strategies for production of nanoparticle vaccines can overcome these problems. Here, we highlight how the use of carrier-antigen conjugates forming self-assembling nanostructures can be effectively combined with the rational vaccine design. The formation of self-assembled nanoparticles from carrier-antigen (peptide epitopes) conjugates, and their ability to generate humoral/cellular immune responses are discussed (Fig. 1) . Extensive reviews on non-conjugated selfassembled antigen carrier, such as liposomes, can be found in the recent literature [52] [53] [54] [55] . Similarly, virus-like particles (VLPs), which are formed by self-assembly processes, have been reviewed extensively in the recent literature [56-58] and therefore are Fig. 1. Schematic representation of the self-assembled peptide subunit vaccines and the immune responses induced upon immunization with these nanostructures. G. Zhao et al. 
 Title: Feature Article -Review The application of self-assembled nanostructures in peptide-based subunit vaccine development
Output: True