Supramolecular Self-Assembled Peptide-Based Vaccines: Current State and Future Perspectives

Despite the undeniable success of vaccination programs in preventing diseases, effective vaccines against several life-threatening infectious pathogens such as human immunodeficiency virus are still unavailable. Vaccines are designed to boost the body's natural ability to protect itself against foreign pathogens. To enhance vaccine-based immunotherapies to combat infections, cancer, and other conditions, biomaterials have been harnessed to improve vaccine safety and efficacy. Recently, peptides engineered to self-assemble into specific nanoarchitectures have shown great potential as advanced biomaterials for vaccine development. These supramolecular nanostructures (i.e., composed of many peptides) can be programmed to organize into various forms, including nanofibers, nanotubes, nanoribbons, and hydrogels. Additionally, they have been designed to be responsive upon exposure to various external stimuli, providing new innovations in the development of smart materials for vaccine delivery and immunostimulation. Specifically, self-assembled peptides can provide cell adhesion sites, epitope recognition, and antigen presentation, depending on their biochemical and structural characteristics. Furthermore, they have been tailored to form exquisite nanostructures that provide improved enzymatic stability and biocompatibility, in addition to the controlled release and targeted delivery of immunomodulatory factors (e.g., adjuvants). In this mini review, we first describe the different types of self-assembled peptides and resulting nanostructures that have recently been investigated. Then, we discuss the recent progress and development trends of self-assembled peptide-based vaccines, their challenges, and clinical translatability, as well as their future perspectives.


INTRODUCTION
Vaccination has been considered as one of the crowning achievements of humankind, and gained remarkable triumph in treating many life-threatening and epidemic diseases, such as influenza, tuberculosis, and smallpox (Malonis et al., 2019;Piot et al., 2019). Vaccines can greatly reduce the burden of preventable infectious diseases by working with the body's natural defenses to safely develop immunity to diseases (Piot et al., 2019;Parvizpour et al., 2020). For instance, to respond to the new and unprecedented coronavirus disease 2019  crisis, vaccination is considered to be the best strategy to end this pandemic (Graham, 2020;Lurie et al., 2020). Conventional vaccination methods are based on pathogens (e.g., attenuated, inactivated, toxins, subunits) that can provoke immunity against antigens and provide long-lasting protection against the diseases (Chiang et al., 2010;Cockburn and Seder, 2018). Despite these advantages, this approach is suffering from a number of drawbacks, including low immunogenicity, high cost, manufacturing challenges, vaccine instability in the cold chain, and potential contaminations during manufacturing which could alter vaccine efficacy and cause a strong allergic shock (Skwarczynski and Toth, 2016).
Although great advances have been made in the development of vaccines and immunotherapies, there is an increasing demand for enhanced control over the immune responses induced against infectious diseases and cancer. Biomaterials can be leveraged for modulating the immune system and subsequently controlling immune responses (Bookstaver et al., 2018). For instance, recent immunization strategies have centered on biomaterial-based vaccines in which specific cellular components were used as antigens to stimulate the immune response against cancer (Bencherif et al., 2015;Malonis et al., 2019). Within these biomaterial-based vaccines, full-length proteins, and peptides have been extensively studied as antigens, but they can also be used as structural biomaterials (Skwarczynski and Toth, 2016). Peptides are biomolecules that generally consist of sequences of 2-50 amino acids and that have a molecular structure that is generally much simpler than that of proteins (Malonis et al., 2019). Using peptides has become a widespread trend in vaccine development since they are easily processed and presented by antigen-presenting cells, leading to potent T-cell-mediated immune responses (Purcell et al., 2007). Furthermore, current challenges associated with cell-based or protein-based vaccination, such as manufacturing complexity, biological contamination, off-target effects, and autoimmunity, can be prevented with peptide-based vaccines (Purcell et al., 2007;Skwarczynski and Toth, 2016). Additionally, peptides are capable of self-assembly into ordered supramolecular structures, making them excellent building blocks to form nanofibers, nanovesicles, nanotubes, nanomicelles, and hydrogels (Habibi et al., 2016;Qi et al., 2018). These peptide assemblies can have a multivalent character and present peptides in their native 3D conformation, which is essential for B-cell stimulation. Furthermore, they allow mixing of multiple components with precise stoichiometry (Wen and Collier, 2015). Ultimately, immunogenic, long-lasting, stable, and self-adjuvanted vaccines can be engineered by combining epitopes, antigens, and immunomodulatory moieties within the self-assembled peptide structures (Wen and Collier, 2015).
Besides being exploited as immunostimulatory materials in vaccine development, self-assembled supramolecular peptides are also excellent candidates to serve as carriers for the delivery of immunological factors (Sis and Webber, 2019;Xiao et al., 2019b). The macrostructural features of supramolecular peptide assemblies can be fine-tuned by altering the amino acid sequences. Additionally, bioactive segments can be introduced into the peptides during their self-assembly to design stimuliresponsive and cell-or organ-targeted vaccine delivery vehicles (Sis and Webber, 2019;Xiao et al., 2019a;Lampel, 2020). Moreover, combining immunogenic peptide epitopes with nonimmunogenic peptides that form delivery carriers could enhance vaccine efficacy while reducing unwanted side effects (Eskandari et al., 2017).
In this mini review, we first describe the various strategies employed to create self-assembled supramolecular peptides with sophisticated hierarchical nanostructures. Next, their application in subunit vaccine design and delivery for cellular and humoral immunity is highlighted. Finally, we discuss the challenges and clinical translatability of self-assembled peptide-based vaccines, as well as their future perspectives.

DESIGN OF SUPRAMOLECULAR SELF-ASSEMBLED PEPTIDES
Molecular self-assembly is a bottom-up approach for achieving highly ordered and stable nanoscale structures or patterns. This technique is based on the spontaneous assembly of small molecules or nanosized building blocks under thermodynamic equilibrium conditions (Qi et al., 2018). Intermolecular and intramolecular interactions, such as hydrogen bonding, amphiphilic interactions, and aromatic stacking, have been used for supramolecular peptide assembly, allowing the construction of complexes with stratified nanostructures (Lampel, 2020). Specifically, peptides can be designed to exhibit distinctive secondary structures, such as β-sheets, β-hairpins, and α-helices, and these natural motifs can be leveraged to drive a complex hierarchical architecture (Rad-Malekshahi et al., 2015). For instance, β-sheets consisting of alternating hydrophobic and hydrophilic amino acids have been extensively applied in driving self-assembly of peptides into extended fibrillar nanostructures (Moore et al., 2018;Sis and Webber, 2019). Several groups have designed selfassembled nanofibrils from the synthetic amphiphilic peptide RADA16 (RADARADARADARADA) through the β-sheet motifs ( Figure 1A), in which alternating regions of hydrophobic alanine and hydrophilic arginine/aspartate residues can yield a stable β-sheet-rich structure (Cormier et al., 2013;Lu et al., 2019). Furthermore, the antiparallel orientation of doublelayered β-sheets, such as β-hairpins, have shown to generate another secondary structure, which promotes the formation of peptide-based nanofibrous hydrogels under physiological conditions (Worthington et al., 2017). Smith et al. developed a multiphase transitioning, injectable hydrogel through the molecular self-assembly of a peptide-based β-hairpin. Their anastomosis photocage 1 (APC1) peptide, which contains seven lysine residues, was folded into a β-hairpin and rapidly self-assembled into a cross-linked fibrillar hydrogel (Smith et al., 2016). Alternatively, α-helices could also be used to form a highly ordered structure through helical self-assembly, which closely resembles the sophisticated hierarchical coiled coil-type structures of native proteins, such as collagen (Lampel, FIGURE 1 | Strategies to induce supramolecular self-assembly of peptides and peptide-based vaccines. (A) Formation of β-strands from self-assembling peptide RADA16-I (top) and their arrangement into nanofibers with different symmetry classes arranged into stacks of two β-sheets (bottom). β-sheets are formed by alternating segments of hydrophobic alanine (green) and hydrophilic arginine/aspartate (blue/red) regions (reproduced with permission from Cormier et al., 2013). (B) Symmetry (top) and sequence (bottom) of coiled-coils (CCs) made of peptides containing a heptad repeat of hydrophobic (h) and polar (p) residues in a hpphppp pattern. Helical wheels for classical Type-N, and Type-1, Type-2, and Type-3 interfaces are depicted. All are viewed along the α-helices from the N to C termini, labels are for the canonical a-g nomenclature and the teardrop shapes indicate the direction of Cα-Cβ bonds (reproduced with permission from Rhys et al., 2018). (C) Effect of different divalent transition metal ions on the morphological transformation of self-assembled PAs, consisting of hexahistidine as a hydrophilic head and oligostyrene as a hydrophobic tail (reproduced from Knight et al., 2018). (D) Schematic illustration of immune stimulation by self-assembled peptide-based vaccines. Peptides undergo self-assembly upon intra-muscular or subcutaneous vaccination, after which they can induce humoral and/or cellular immune responses to protect individuals against pathogens. Humoral immune responses arise once B-cells encounter antigens in secondary lymphoid organs, inducing their activation. Mechanistically, B-cell activation occurs upon binding of antigens to the B-cell receptor (BCR), resulting in the internalization of antigens via receptor-mediated endocytosis. These antigens are then processed, and antigenic peptide-loaded MHC II is displayed on the cell surface, allowing CD4+ helper T-cell binding. CD4+ helper T-cells further stimulate B-cells to undergo proliferation, immunoglobulin (Ig) class-switching, and differentiation into antigen-specific antibody-secreting plasma cells and memory B-cells. To induce CTL-mediated cellular immune responses, antigens need to be processed and presented by antigen-presenting cells (APCs), such as dendritic cells and macrophages. APCs take up the antigen by pinocytosis, phagocytosis, and receptor-mediated phagocytosis, cleave the antigen into smaller peptide fragments, and present these peptides on MHC. In the lymph nodes, extracellular antigenic peptides are presented on MHC II to CD4+ T-cells, whereas intracellular proteins, including viral and tumor-associated antigens, are presented on MHC I to CD8+ T-cells. A subset of APCs can also present extracellular antigenic peptides on MHC I in a process known as cross-presentation. Once activated, naïve CD8+ T-cells proliferate into CTLs to mediate antigen-specific cytotoxicity. A fraction of CD8+ T-cells become memory CD8+ T-cells to ensure long-term protection. Upon activation, naïve CD4+ T-cells differentiate into helper T-cells which secrete cytokines to support CTL expansion and survival along with B-cell proliferation and differentiation. Memory B-and T-cells persist in the body for a long time and mount an immediate and efficient antigen-specific immune response upon reinfection with the same pathogen. 2020). Thomson et al. designed supercoiled coil α-helical barrels by self-assembling 5-7 α-helices twisted around each other with cyclic or dihedral symmetry ( Figure 1B). The peptides used in their study have a canonical hpphppp heptad repeat sequence, in which h and p represent hydrophobic and polar amino acids, respectively (Thomson et al., 2014;Rhys et al., 2018). Di-phenylalanine peptide (FF) is another interesting building block to trigger peptide self-assembly into desirable nanostructures such as nanotubes, nanospheres, and nanoribbons (Habibi et al., 2016). While self-assembly of the FF motif is usually achieved in the form of amyloid-like sheets (Lampel, 2020), Bera et al. interestingly generated a helical architecture by simply adding one more amino acid (proline) to the FF peptide, forming proline-phenylalanine-phenylalanine (PFF) (Bera et al., 2019).
Additionally, non-peptidic compounds such as alkyls, amino acids, and metal ions have been used to form self-assembled peptide complexes with a wide variety of structures and functionalities (Sis and Webber, 2019). Alkyls, for instance, have been used as a hydrophobic tail to produce peptide amphiphiles (PAs) that undergo self-assembly via hydrophobic shielding (Sis and Webber, 2019). As a result, the nanostructure and other properties of self-assembled PAs can be controlled by switching their hydrophobic components (Qi et al., 2018). Choi et al. proved that the nanostructure of self-assembled amphiphilic Janus peptide dendrimers (JPDs) can be induced via a simple chemical bifurcation. Various JPDs were synthesized by varying the length of their hydrophilic and hydrophobic regions, and their self-assembly resulted in the formation of spherical/cylindrical micelles or bi-layered vesicles (Choi et al., 2019). PAs can also be made by conjugating peptides with functional polymers (Habibi et al., 2016). Chin et al. designed a muscle-inspired anisotropic actuator in the form of a hollow fibrous peptide-based hydrogel, in which VVVAAAEEE peptides were functionalized with a lysine-coupled bromoisobutyryl moiety to enable the grafting of thermo-responsive polymers via atom transfer radical polymerization (Chin et al., 2018). Recent studies also revealed that the nanostructure of selfassembled peptides can be reshaped in a controllable manner by incorporating metal ions (Sis and Webber, 2019). Knight et al. studied the effect of different metal ions on the morphological transformation of self-assembled PAs consisting of hexahistidine as a hydrophilic head and oligostyrene as a hydrophobic tail ( Figure 1C). Interestingly, the presence of manganese(II) promoted the formation of multilamellar vesicles, while nickel(II) and cadmium(II) gave rise to micelle nanoparticles. In contrast, zinc(II), copper(II), and cobalt(II) led to agglomerated micelles (Knight et al., 2018).
Finally, there has been a growing interest in designing smart nanomaterials that initiate the assembly of molecular building blocks upon application of external stimuli such as enzymes, pH, heat, and light. This strategy can provide additional spatiotemporal control over the formation and structure of supramolecular peptides (Lampel, 2020). For instance, enzymeinstructed self-assembly (EISA) of peptides has recently been applied to allow assembly in complex biological systems . Li et al. synthesized several dipeptidic precursors, consisting of FF with various stereoisomers, in which N-and C-terminal peptide sequences are linked to 2-(naphthalen-2-yl)acetic acid and 2-(4-(2-aminoethoxy)-4oxobutanamido)ethane-1-sulfonic acid, respectively. Molecular self-assembly (i.e., EISA) of these peptides is induced by carboxylesterase, which is present inside the cells and in the extracellular space of tissues. Using this approach, intracellular and intercellular peptide self-assembly could be achieved at different rates . Additionally, some stimuliresponsive peptides, such as elastin-like polypeptides (ELPs), exhibit reversible transition behavior upon exposure to specific stimuli (Zhang et al., 2015;Saha et al., 2020). Dreher et al. prepared several ELP block copolymers (with varying molecular weights and block ratios) in a linear AB diblock architecture by homogeneously fusing an N-terminal hydrophilic ELP block to a C-terminal hydrophobic ELP block. They observed that copolymers with suitable diblock ratios are highly water-soluble at normal body temperature (37 • C) and can self-assemble into spherical micelles at the tumor temperature (42 • C). When the temperature was lowered again, the copolymers showed the inverse transition behavior (Dreher et al., 2008). Hassouneh et al. developed a theoretical model to explain the mechanism for this special reversible transition behavior of the copolymers (Hassouneh et al., 2015).

APPLICATION OF SELF-ASSEMBLED PEPTIDES IN VACCINE DESIGN AND DELIVERY
A wide variety of vaccines have been designed to stimulate the immune system to combat pathogens (e.g., bacteria, viruses) or tumors. In particular, vaccines aim to induce an adaptive immune response that leads to immunological memory, as illustrated in Figure 1D. There are two subdivisions of the adaptive immune system: the cell-mediated immune response, which is executed by cytotoxic T lymphocytes (CTLs) that can kill infected or cancerous cells, and the humoral immune response, which is mediated by activated B-cells that produce antibodies to neutralize extracellular pathogens (Molnar and Gair, 2015). Peptide-based vaccines generally require three major components-an antigen, an adjuvant, and a delivery vehicle-to generate efficient adaptive immune responses. When peptides are used as antigens, the conformation of the specific regions recognized by the immune system, known as epitopes, is particularly important for inducing humoral immunity. Specifically, B-cells need to bind epitopes in their native conformation to allow antibody binding. B-cell epitopes usually have α-helical, loop, and β-strand conformations that are generally integrated into vaccines as longer peptides to allow them to adopt their native conformation (Skwarczynski and Toth, 2016;Malonis et al., 2019). When designing vaccines, self-assembly of peptides can be utilized to ensure correct folding of antigenic epitopes. In vaccine applications where antibody affinity and titer are essential, self-assembling peptides that also have inherent CD4+ T-cell epitopes, such as those included in the Coil29 (QARILEADAEILRAYARILEAHAEILRAD) peptide, can be incorporated to induce strong follicular helper T-cell engagement that further promotes B-cell responses (Wu et al., 2020). Similar to CD4+ T-cell stimulation, the peptide sequence is more critical than the epitope conformation itself to induce CTL-mediated cellular immunity. Therefore, shorter peptides can be used to induce T-cell responses, as CD4+ T-cells recognize 12-16 amino acid long peptides presented by MHC II on antigen-presenting cells (APCs), while CD8+ T-cells bind to slightly shorter 8-10 amino acid long peptides displayed by MHC I (Skwarczynski and Toth, 2016;Malonis et al., 2019). Unlike attenuated pathogen-based vaccines, peptidebased vaccines generally incorporate adjuvants to boost the overall immune response to antigens and mimic the natural "danger signals" which follow infections. The choice of adjuvants depends on several factors such as immunogenicity and toxicity. Several studies have established that the adjuvanticity of selfassembled peptides is mediated via antigen presentation in an ordered and repetitive array that resembles pathogenassociated molecular patterns (PAMPs), resulting in strong immune responses mediated through Toll-like receptor (TLR)inflammasome signaling pathways via TLR2 and TLR4 activation (Azmi et al., 2014;Negahdaripour et al., 2017;Tandon et al., 2018;Zottig et al., 2020). Additionally, self-assembling peptides may act as adjuvants themselves by forming an antigen depot, directing vaccines to APCs, and ultimately enhancing immune-cell priming (Grenfell et al., 2015;Acar et al., 2017;Negahdaripour et al., 2017).

Self-Assembled Peptide-Based Vaccines for Cellular Immunity
Supramolecular self-assembling peptides can form excellent structures to induce cytotoxic immune responses, which is particularly important for cancer immunotherapy. For this purpose, peptide assemblies can function as a platform for safe and controlled delivery of antigens, adjuvants, immune cells and/or drugs ( Table 1). For example, Wang et al. utilized a tumor-penetrating peptide Fmoc-KCRGDK-based hydrogel formulation to encapsulate a BRD4 inhibitor, a photothermal agent (indocyanine green), and autologous tumor cells. Upon laser irradiation, the personalized cancer vaccine released tumorassociated antigens. This process promoted DC maturation, T-cell infiltration, the formation of memory immune cells to prevent tumor relapse, and inhibited distant tumors (Wang T. et al., 2018). Interestingly, Xu et al. observed that the configuration and the number of lysine residues in the peptide are critical to enhance CTL response. They described a supramolecular NF-κB-activating nano-adjuvant hydrogel, synthesized by pH-triggered self-assembly of Ada-GFFYGKKK-NH2 peptide, for cancer immunotherapy. According to their findings, nano-adjuvants containing D-configured peptides and 3 lysine residues encapsulated antigens more efficiently through charge-charge interaction than 2 lysine residues, and as a result, generated more robust adaptive and innate immune responses than peptides with L-configuration . Yang et al. showed that a nanofibrous RADA16 peptidebased hydrogel scaffold encapsulating bone-marrow derived DCs, model antigen ovalbumin (OVA), and anti-PD-1 antibody recruited and stimulated endogenous and exogenous DCs. This process increased DC migration to the lymph nodes, driving a more robust antigen-specific immune response against EG7-OVA tumors . Furthermore, Wang et al. developed glutathione-responsive nanocomposites by coassembling a positively charged cell-penetrating CWWRCRCRC peptide with a negatively charged protein such as ovalbumin (OVA) via electrostatic interactions. After being internalized by APCs, intracellular glutathione degraded the disulfide bonds of the peptide, inducing rapid release of the antigen into the cytoplasm, which was then cross-presented to induce potent CD8+ T-cell responses. When compared to free OVA, the peptide-based nanocomposites improved antigen uptake by DCs, promoted DC activation and maturation, and enhanced cellular as well as humoral immune responses (Wang K. et al., 2018). Overall, the use of self-assembled peptides as a delivery vehicle provides multiple benefits, including efficient cell and antigen loading, minimal loss of active components, as well as controlled and targeted release in the lymphoid organs (Zhang, 2017;Lee et al., 2019).
Apart from acting as a delivery vehicle, self-assembling peptides can also serve as an antigen source themselves to induce cellular immunity. For example, Black et al. developed cancer vaccines consisting of self-assembling tumor antigen peptides with enhanced immunogenicity. Conjugating a synthetic lipid tail with two palmitic residues to an OVA-derived peptide containing a CTL epitope initiated the self-assembly of PAs into cylindrical micelles, resulting in multivalent epitope presentation. These micelles were internalized by APCs, likely due to the fusion of their hydrophobic tails with the cell membrane, leading to CTL activation in absence of additional adjuvants. Additionally, these cylindrical micelles act as antigen depots, thereby protecting the peptides from degradation and prolonging antigen exposure to the APCs. Improved in vivo protection was observed against OVA-expressing tumor cells after immunization with the diC16-OVA micelles compared to free OVA peptide formulated with incomplete Freund's adjuvant (Black et al., 2012). Xing et al. developed injectable, peptidebased supramolecular hydrogels by co-assembling poly-L-lysine (PLL) and FF dipeptide via an electrostatic coupling. Fmoc-FF/PLL-SH hydrogels have a nanofibrous structure with α-helical conformation which resembles natural fimbrial antigens, thereby acting as an adjuvant. When injected around a tumor, the hydrogels activated T-cell responses and efficiently suppressed tumor growth without the addition of other adjuvants or antigens. In this case, the tumor cells themselves act as the source of antigen to stimulate the immune response (Xing et al., 2017). Self-assembly can also be used to incorporate longer peptides containing multiple epitopes to ensure optimal APC stimulation, increase the magnitude of CTL response, and simultaneously promote helper T-cell activity (Lynn et al., 2020). Overall, peptide-based self-assembly can induce robust cellular immune responses by serving as a durable source of antigen that allows easy uptake and processing by APCs, providing multiple epitopes, and conferring self-adjuvanticity to the vaccine.

Self-Assembled Peptide-Based Vaccines for Humoral Immunity
Robust humoral immune responses are essential to treat infectious diseases. For this purpose, self-assembly designs have included approaches such as flanking the antigen with a selfassembling peptide sequence to display the secondary structure of the antigen, presenting antigens in a highly ordered and repetitive form, and optimizing the distance between repeated epitopes for optimal B-cell receptor (BCR) engagement (Raman et al., 2006;Black et al., 2010;Babapoor et al., 2011;Trent et al., 2015;Skwarczynski et al., 2020). Self-assembling supramolecular Frontiers in Chemistry | www.frontiersin.org β-sheet-rich nanofibers P. falciparum vaccine for malaria Rudra et al., 2012 peptides can serve as a delivery scaffold to present antigenic peptides, and they can simultaneously possess self-adjuvanting characteristics to induce B-cell responses. For instance, Grenfell et al. exploited RADA4 peptide-based hydrogels that selfassembled into hydrated nanofibers after injection into the tissue to form a gel matrix depot, to deliver a recombinant antigen for the hepatitis B virus. This system elicited enhanced adjuvant-free humoral and cellular responses when compared to the antigen delivered with aluminum hydroxide (alum) and complete Freund's adjuvant. The authors credited the slow release of antigen from the depot to have improved the activation of APCs and prolonged immunostimulation (Grenfell et al., 2015). In a different study, Tian et al. utilized a supramolecular hydrogel-based nanovector comprised of Nap-GFFY-NMe (naphthylacetic acid-modified tetrapeptide GFFY with C-terminal methyl amide group), using an EISA approach to encapsulate a DNA sequence encoding gp145, an envelope glycoprotein of the human immunodeficiency virus (HIV). Alkaline phosphatase triggered self-assembly of the peptide, leading to formation of nanofibrous hydrogels. The strong cellular and humoral immune responses were attributed to the ability of left-handed structure of nanofibers to effectively condense DNA and prevent it from degradation, thus enhancing DNA transfection and gene expression in cells (Tian et al., 2014). Self-assembling peptides can also be an antigen source to induce humoral immunity. For instance, Kaba et al. utilized a self-assembling peptide-based nanoparticle platform to present repeated immunodominant B-cell circumsporozoite peptide epitope (DPPPPNPN) 2 D of the malarial parasite. The polypeptide consisted of two oligomerization domains-a de novo trimeric coiled-coil domain and a pentameric coiledcoil domain-which were fused together with flexible diglycine residues. The self-assembled nanoparticles, containing multiple coiled-coil domains, displayed the B-cell peptide epitope in a highly ordered and repetitive array, thereby triggering robust helper T-cell-dependent and long-lasting antibody responses with higher avidity and titer (Kaba et al., 2009). Pimentel et al. utilized a similar polypeptide-based self-assembling nanoparticle platform to display the C-terminal heptad repeat region (HRC) of the SARS-CoV S-protein in its native α-helical trimeric coiledcoil conformation. These nanoparticles not only maintained this conformational integrity, but also provided repetitive presentation of the B-cell epitope and displayed icosahedral symmetry that resembled viral protein capsids. The SARS-CoV vaccine evoked conformation-specific neutralizing antibodies against the B-cell epitope without additional adjuvants (Pimentel et al., 2009). A potent peptide-based vaccine against Streptococcus pneumoniae was proposed by Dorosti et al., who incorporated CTL epitopes such as pneumococcal surface protein A (PspA) and choline-binding protein A (CbpA), helper T-cell epitopes such as pneumococcal histidine triad protein D (PhtD) and a lipoprotein from pneumococcal iron ABC transporter (PiuA) and universal helper T-cell epitopes like diphtheria toxoids on a coiled-coil self-assembling interface. The designed vaccine was predicted to exhibit stronger immunogenic responses compared to an analogous epitope-based vaccine (Dorosti et al., 2019). In addition, Rudra et al. developed a peptide-based vaccine to prevent malaria by combining self-assembling β-sheet peptides with epitopes of protozoan parasite P. falciparum (Rudra et al., 2012). In another example, Trent et al. designed a selfadjuvanting peptide-based vaccine for group A streptococcus (GAS). When the J8 antigenic peptide of the GAS-M protein is taken out of its native protein environment, its α-helical structure is lost, and as a result, it adopts a random conformation in the solution. The addition of two C16 alkyl chains at the N terminus of the J8 peptide triggered its self-assembly into cylindrical micelles, and reinforced α-helicity of the antigen, which subsequently induced a strong B-cell response in mice. Since the hierarchical micellar structure kept thousands of peptides in close proximity, it conferred potent adjuvanticity while maintaining the native antigen conformation at the injection site. In addition, the micellar structure increased the local antigen concentration available to immune cells in comparison to free peptide, which quickly diffused away from the injection site (Trent et al., 2015). Taken together, these studies have demonstrated that self-assembled peptides can efficiently induce humoral immune responses through the display of secondary structures, thereby maintaining the conformational integrity of epitopes. Furthermore, these selfassembly platforms offer a highly ordered and multivalent display of relevant antigens, resulting in efficient stimulation and proliferation of B-cells, and their subsequent differentiation into antibody-secreting plasma cells. Inclusion of helper T-cell epitopes further enhances vaccine efficacy by imparting longlasting B-cell immunity.

CHALLENGES AND FUTURE PERSPECTIVES
Despite the great potential of self-assembled supramolecular peptides for vaccine design and engineering, many challenges still persist. Compared to conventional vaccines containing live-attenuated or inactivated pathogens (viruses, bacteria, etc.), most peptide-based vaccines induce a weak immune response (Malonis et al., 2019). The use of adjuvants can overcome this obstacle, and deepen our understanding of their mechanism of action and their safety (Skwarczynski and Toth, 2016). Additionally, maintaining the stability and efficacy of peptidebased vaccines in complex biological environments remain challenging. For instance, their rapid enzymatic degradation and structural integrity in the body is a major limiting factor . Self-assembled peptides must exhibit improved stability when interfacing with biological barriers (e.g., pH, enzymes) that are encountered upon administration in tissues (Eskandari et al., 2017). Rational systematic design of the supramolecular peptide structure to include covalent cross-linking-or via improved intermolecular interactions such as hydrogen bonding, π-π stacking, and hydrophobic interactions-may augment their resilience and mechanical stability (Khalily et al., 2015;Li et al., 2019). The development of supramolecular self-assembled peptide-based vaccines is hindered by our limited understanding of the interface between self-assembled nanostructures and immune cells. In-depth investigation of the interactions between the self-assembled structures and the receptors on human immune cells, their uptake by APCs, as well as their effect on DC maturation, B-cell and T-cell priming, and cytokine profile, is needed . Challenges exist in translating the ease in design and smallscale synthesis of supramolecular peptides to an industrial scale. Furthermore, advanced engineering of supramolecular assembly (e.g., cell-penetrating peptides) may target intracellular vaccine delivery more precisely, ultimately enhancing antigen uptake, promoting endosomal escape and cross-presentation by APCs, which are critical steps in inducing a robust cellular immune response . Existing technologies such as machine learning, bioinformatics, and computational modeling can potentially be leveraged for the macromolecular engineering of immunostimulatory peptide assemblies with improved vaccine immunogenicity, efficacy, and safety . Moving forward, biologically inspired supramolecular peptides, an excellent and sparsely explored class of materials, could be further exploited in designing the next generation of vaccines that are effective, safe, affordable, and accessible to everyone. Despite their importance and great potential, self-assembled peptidebased vaccines do require further investigation and validation prior to regulatory approval and clinical use.

AUTHOR CONTRIBUTIONS
The manuscript was written and edited with contribution from all authors. All authors have given approval to the final version of the manuscript.

FUNDING
SB acknowledges the support from the NSF CAREER award (DMR 1847843). This project was partially funded by the Science and Technology Unit-King Abdulaziz University-Kingdom of Saudi Arabia, award number UE-41-106.