**3. Peptide vaccines**

*Peptide Synthesis*

with such a combination of chromatography and spectroscopy are useful for the complete characterization of synthetic peptides. While chromatographic analyses allow us to have knowledge about the peptides' purity, the molecular weight of the peptides is determined by MS analyses. It is also found in some systems that determine the amino acid sequence of the peptide molecule like amino acid analyzer and LC-MS-MS. Information on structural properties of peptides can also be obtained by NMR and FTIR methods. With NMR and FTIR, the structures of the peptides are further elucidated via specific binding and functional groups [12, 35]. Moreover circular dichroism (CD) spectroscopy gives information about the conformation and secondary structures of polypeptides. This technique can be used to distinguish

between random coil, alpha-helix, or beta-sheet structures [38, 39].

**8**

**Figure 1.**

*Some of the fundamental linkers used on the Fmoc-based SPSS [22].*

The increase in studies on peptide vaccines in recent years shows that peptide vaccines will be an important part of new-generation vaccine systems. Vaccines are indispensable in protecting human and community health in terms of reducing infectious diseases, disability, and deaths and, most importantly, eliminating and eradicating the long-term disease. The development of the vaccine, which began with the observations of Edward Jenner at the end of the eighteenth century, has significantly reduced the number of infections and diseases until now [40–42]. Traditional vaccines (live-attenuated or inactivated vaccines) have been widely used over the last hundred years to develop effective vaccines against many diseases. However, the use of this technology is a source of concern because of the problems encountered in the preparation and use of such vaccines. The disadvantages of classical vaccines such as the increased need for safety, high cost in the production of large-scale vaccine preparations, high genetic variability of pathogens, side effects, and risk of leak of the disease agent have necessitated research to develop new technological vaccines [43, 44]. The basis for new vaccine technologies is the production of high-purity subunit vaccines that contain a small part of the pathogen necessary to generate an immune response. Subunit vaccines can be composed of polysaccharides and proteins or their peptide fragments. Synthetic peptide vaccines consist of 20–30 amino acids containing the specific epitope of a corresponding antigen against various diseases. There is no limitation from cancer diseases to allergies in diseases targeted by peptide vaccines. The advantages of peptide vaccines have enabled peptides to be preferred in vaccine technology [45–48]. Although many peptide vaccine studies continue, there is no approved peptide vaccine for human use. According to the database of clinical studies maintained on ClinicalTrials.gov, in the search on the topic of peptide vaccine until mid-December 2018, 374 clinical trials in phase I, 272 clinical trials in phase II, 14 clinical trials in phase III, and 2 clinical trials in phase IV have been found [47, 49]. Furthermore, it is reported in the literature that a veterinary peptide vaccine against canine parvovirus, which is targeted against animal diseases, is effective [50]. Advantages involved in the choice of peptide-based vaccine can be summarized as follows: Peptide-based vaccines are more reliable than classical vaccines because they are produced using chemical synthetic approaches almost exclusively. It does not produce any unwanted immune response as it does not contain biological material. Also, this situation eliminates the risk of biological contamination. The rapid, easy, inexpensive, and high-purity synthesis of peptides by microwave-assisted solid-phase peptide synthesis methods is an important advantage for the use of peptides in vaccine systems. These synthesized peptide antigens can be fully characterized by analytical methods. "Cold chain" is not required generally during transport and storage of peptide vaccines. Peptide vaccines have high stability and are typically water-soluble. Allergic and/ or reactogenic effects are avoided by the usage of peptide vaccines. Peptide vaccines can be customized using the peptide epitope of one or more antigens [46, 51, 52].

Despite all its advantages, there are some limitations of peptide-based vaccines. The peptides alone are generally weakly immunogenic, have poor release properties and are easily undergo enzymatic degradation. These disadvantages can be overcome using adjuvants and delivery systems. Adjuvants, derived from the Latin word "adjuvare," which means "to help," play a crucial role to the elicitation of a strong immune response to an antigen [49, 53–56]. The first step in the development of peptide vaccines is the identification of the structure of peptide epitopes which will provide effective and long-term immunity. After the synthesis and characterization of specific antigens to be determined, the appropriate adjuvant and delivery system selection is carried out [43, 46]. Currently, instead of classical adjuvants, the use of

new generation of more effective adjuvants is being studied. It is aimed to overcome the disadvantages of the traditional adjuvants with the development of modern adjuvants and delivery systems. Alum (insoluble salts of aluminum), which is a classical adjuvant, is a weak immune stimulant; its mechanisms of action are not fully elucidated and have a possibility of side effects. Emulsion adjuvants such as incomplete Freund's adjuvant (IFA), complete Freund's adjuvant (CFA), and lipid A are used; however, it is important to develop safer adjuvants due to their toxicity [46, 49, 57, 58]. Protein and synthetic polymer carriers play an important role in new-generation vaccine systems. The immunogenicity of peptide vaccines based on conjugation to protein and polymeric carriers is enhanced. Bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), human serum albumin (HSA), and ovalbumin (OVA) are the most preferred proteins in peptide-protein conjugates [51, 59, 60]. In recent years, peptide-polymer conjugations using synthetic polymeric carriers have gained importance. Polymers to be selected in peptide-polymer conjugates, should be biocompatible, be relatively easy to synthesize and be modifiable according to the recommended use. This situation is pointed out that such polymers are important carrier candidates for peptide vaccines [61–63].

Nanoparticles are investigated for their potential use as vaccine delivery systems. The use of nanoparticles in peptide-based vaccines may be useful in elimination of limitations with small peptide antigens. It can play a critical role in increasing the size of the antigen molecule, protecting the peptides from enzymatic degradation, and the delivery of the peptides to the targeted cells. Inorganic NPs, lipid-based NPs, polymeric NPs, and carbohydrates are common classes of nanoparticles. Especially polymeric NPs are of great interest for vaccination due to their biocompatibility and predictable degradation. In addition to natural polymers such as chitosan, dextran, and albumin, synthetic polymers such as polyesters, polyanhydrides, and polyamides can be mentioned. Mostly studied poly(lactic-coglycolic acid) (PLGA) is a synthetic copolymer composed of lactide and glycolide and approved by the European Medicine Agency (EMA) and US Food and Drug Administration (FDA) [64]. This polymer, which is biodegradable and biocompatible, is suitable for vaccine and drug delivery systems in humans. In the usage of polymeric nanoparticles as delivery systems for peptide vaccines, the peptide may be covalently/non-covalently attached to the surface of the particles through functional groups, or the peptide may be loaded into the nanoparticles [65–69].

Peptide vaccines are considered to be critical in the production of effective, safe, inexpensive, and easy to produce vaccines by the abovementioned advantages and the development of new adjuvants.
