Peptide-Based Vaccines in Diseases
An innovative approach is represented by peptide-based prototypes (Skwarczynski and Toth, 2016; Malonis et al., 2020) that are often able to overcome disadvantages encountered by other strategies as a more safe profile since for easiness of purification (Marasco et al., 2008; Marasco and Scognamiglio, 2015). Their chemical synthesis renders them suitable for large scale production with low costs and high reproducibility (Tizzano et al., 2005). Normally, they are also soluble in water and more stable in storage conditions (Li et al., 2014) but they can be unstable in the body and easily degraded by proteases before eliciting an efficient immune response. Furthermore, they are usually weak immunogens and need adjuvants (additional immune stimulants) to stimulate B- and T-lymphocytes and to induce an effective response. Peptide-based vaccines are usually made of synthetic B- or T- cells epitopes (class I or class II) that can also be combined. T-cells recognize peptide sequences complexed with major histocompatibility complex (MHC) class I or II molecules on the surface of APCs. CD8+ T-cells (also called cytotoxic T lymphocytes (CTL)), kill infected cells, while CD4+ T helper cells, that recognize MHC II-epitope complex, interact with CTL to reinforce their activity and with B-cells to activate the production of specific antibodies against the pathogen (Sanchez-Trincado et al., 2017). Epitope mapping represents the identification of the binding site, i.e. “epitope”, of an antibody on its target antigen (Gershoni et al., 2007). The most practical strategy relies on the employment of in silico computational methods (Hager-Braun and Tomer, 2005) (as schematically reported in Figure 2, applied to SARS-CoV-2); once identified, peptide epitopes are usually modified in order to optimize their functional properties, to enhance immune recognition and to elicit significant immune responses. Ala-scan or combinatorial approaches could be performed to investigate the role of each amino acid residue in antigen/antibody recognition and hence linear flank-modified or cyclic epitopes can be obtained also through stapling insertions (Lau et al., 2015; Godel et al., 2012). Such epitopes, once improved in their antigenic properties, can be included in specific delivery carriers to protect them from degradation (Allahyari and Mohit, 2016): they include VLPs, liposomes, polymeric micro-/nano-particles and dendrimeric systems that are schematically described in Figure 2 (Neek et al., 2019; Singh et al., 2007). In VLPs, (Figure 2A), the antigen can be immobilized on the surface through biochemical tools or covalent links (Sapsford et al., 2013; Qian et al., 2020; Koho et al., 2015; Syomin and Ilyin, 2019). The advantages of VLPs include similar size and antigenicity of the corresponding viruses and lack of viral genome and, as consequence, of danger. A VLP modification is constituted by VLPs assembled from the coat protein of bacteriophages (Bao et al., 2019; Manoutcharian, 2005; Hess and Jewell, 2020). In liposomes, antigen can be entrapped inside them (soluble antigens) or incorporated into lipophilic bilayer (lipophilic antigens) (Figure 2B) (De Serrano and Burkhart, 2017). Also, polymeric micro and nanoparticles can contain encapsulated and/or surface-engrafted antigens with shape and size similar to the virus (Figure 2C). On the basis of the percentage of biopolymers, among others chitosan and poly(lactic-co-glycolic acid) (PLGA) (Duran et al., 2019), it is possible to obtain controlled release from micro/nanoparticles able to extend antigen interaction with the immune cells to induce an effective immune response (Sadler and Tam, 2002). Dendrimers, as polyamidoamine (PAMAM), polypropyleneimine (PPI) and multiple antigenic peptides (MAPs) (Figure 2D) are hyper-branched polymers used for the delivery of different therapeutics (Sadler and Tam, 2002). Due to their well-defined, multifunctional and stable construct, the use of dendrimers has a dual purpose: 1) multi-copies of antigenic peptides usually enhance immunogenic response and 2) their unnatural structure is stable to proteases degradation and can be used for delivery (Joshi et al., 2013). They are conceived in two forms: 1) homotropic that contains multimers of the same peptide or 2) heterotropic, obtained by the combination of different epitopes, used for example for vaccine development of classical swine fever virus (CSFV), a typical B4T-type vaccine containing one T-cell epitope from the NS2-3 protein linked to four copies of B-cell epitopes from the E2 protein (Monso et al., 2011; Joshi et al., 2013).
Peptide-Based Vaccines in Clinical Trials for Wide-Impact Diseases
Peptide-based vaccines have been under investigation for several decades. The earliest peptide vaccination study was reported by Anderer and co-workers in 1963. They observed that a peptide of six amino acids corresponding to the linear sequence of tobacco mosaic virus could elicit virus-neutralizing antibody. However, only in 1980 the immune response to peptides has been investigated in detail (Arnon, 1972; Brown, 1994). Several researchers observed that degradation of proteins into small peptides was crucial for their presentation to T-cells and was necessary for the antigen-processing events. Moreover, it was observed that peptides could stimulate protective immune responses against some viruses, such as hepatitis B (Li et al., 2014). These results prompted researchers to study if it would be possible to apply this knowledge to make vaccines. Since 1990, over 100 synthetic peptide vaccines have entered phase I and II clinical trials but none of them is currently available on the market. These failures are due to several problems that characterized these vaccines, such as insufficient immunogenicity, chemical instability due to degradation and conformational instability (Chen et al., 2009). The continuous research has however allowed to overcome many of these obstacles. For example, the problem of low immunogenicity is now overcome thanks to several multiple antigenic peptides, moreover, different innovative administration routes (e.g., intradermal, oral, intranasal) (Wang et al., 2015; Waghule et al., 2019) and formulations (e.g., liposomes, nanoparticles) are now able to impair peptide degradation (Singh et al., 2020). Many efforts have yet to be made to obtain a valid peptide-based vaccine, but currently different peptide-based vaccines are under investigation in clinical trials for several diseases as reported in Table 2 and seven of them are actually in phase III clinical trials.
A combinatorial epitope approach was also used in the development of the Ii-Key peptide vaccine, and implies the use of a vaccine technology based on hybrid peptides, as proposed by EpiVax, Inc. company in collaboration with Generex Biotechnology Corp (Kallinteris et al., 2006; Wu, 2020). The Ii-Key peptide technology, already adopted for cancer vaccine development, is used to overcome the weakness of presentation of MHC class II epitope vaccine peptides (Kissick et al., 2014). This method assumes that “hybrid molecules” can enhance CD4+ T cell response: the hybrid is composed by N-terminus of MHC II epitopes covalently linked, through a simple polymethylene spacer (or other chemical linkers), to the C terminus of the Ii-Key peptide (four amino acids: LRMK). In vitro, this chimeric peptide exhibited an enhanced presentation of the antigens of about 200 times in comparison with the epitope alone. In vivo, the T- helper cell response is enhanced up to 8 times (measured with ELISPOT test) (Kallinteris et al., 2006); by combining Ii-Key peptide technology with the computational tool for vaccine design, iVAX (De Groot et al., 2020), the resulting vaccine will blend short peptide sequences of the virus with Ii-Key peptides. The aim is to provide broad T cell-mediated protection against SARS-CoV-2 avoiding, at the same time, antibody dependent enhancement (ADE) (Tirado and Yoon, 2003).