Self-Assembling Peptides for Vaccine Development and Antibody Production
Self-assembling peptides have shown great potential for drug delivery, cancer cell inhibition, and regenerative medicine. Recently studies indicate that they are also promising for subunit vaccine delivery. We summarize in this tutorial review two strategies to deliver subunit vaccines, one by covalently conjugating and the other one by physically mixing. By the former strategy, protein and peptide antigens are covalently connected with self-assembling peptides, and the resulting peptides can self-assemble into nanofibers by themselves or by mixing with the original self-assembling peptides. For the latter one, antigens including DNA, proteins, and attenuated cells physically interact with nanofibers of self-assembling peptides via charge interaction, hydrogen bonding, hydrophobic interaction, etc. Both strategies can prolong the lifetime of subunit vaccines at injection sites, assist antigen uptake by antigen-presenting cells (APCs), facilitate transportation of antigens from injection sites to lymph nodes, and stimulate downstream immune responses. Vaccines based on self-assembling peptides can raise stronger antibody productions, which is useful for protective vaccine development and antibody production. Besides, several vaccines capable of eliciting strong CD8+ T-cell response are also introduced in this paper, and they are promising for the development of vaccines to treat important diseases such as cancers and HIV. Challenges remained are also discussed in the last section of the paper. Overall, self-assembling peptides are very useful for antibody production and the development of novel vaccines to treat important diseases.
Vaccines based on live attenuated pathogens have been widely applied to efficiently protect against former epidemics and significantly prolong human life [1, 2, 3]. However, the direct use of live attenuated pathogens in human beings may cause side effects and possible risks. In modern vaccine industry, subunit vaccines have been extensively studied and developed due to their well-defined molecular structure, good safety, specificity, and ease of production and storage. However, they are usually needed to be formulated with adjuvants because of their weak immunogenicity . Many nanomaterials have shown promising vaccine adjuvant potency, which are capable of prolonging the lifetime at injection sites, enhancing cellular uptake by antigen-presenting cells (APCs), promoting lymph node accumulation, and facilitating downstream immune responses of subunit vaccines [5, 6, 7, 8].
Through non-covalent interactions such as hydrophobic interaction, hydrogen bonding, aromatic interaction, and charge interaction, self-assembling peptides and peptide derivatives can self-assemble into nanofibers and hydrogels [9, 10, 11, 12, 13, 14, 15]. These nanofibers and hydrogels have been widely applied for the delivery of therapeutic agents including small molecular pharmaceutics and growth factors [16, 17, 18, 19]. Recent studies also demonstrate that they are promising vaccine adjuvants because of the good biocompatibility, ease of integration of subunit vaccines, and well-defined molecular structures . There are two strategies to apply self-assembling peptides for antigen delivery, one by covalently connecting peptide or protein antigens with self-assembling peptides and the other one by physically interacting antigens with self-assembling peptides. In this tutorial review, we summarize recent progresses in using self-assembling peptides for vaccine development and antibody production.
Vaccines Based on Self-Assembling Peptides Covalently Conjugated with Peptide and Protein Antigens
With the development of vaccines, synthetic peptide ligands or epitopes as well as recombinant proteins have been widely used as subunit vaccines. However, the low immunogenicity and fast degradation property of subunit vaccines hinder their clinical applications. Making antigens into larger aggregates beyond single molecules will prolong their in vivo lifetimes and enhance immune response against these antigens . Therefore, nanofibers of self-assembling peptides provide an ideal platform for the delivery of subunit vaccines. Peptide epitopes and protein antigens can be covalently connected with self-assembling peptides, and the resulting conjugates can self-assemble by themselves or co-assemble with self-assembling peptides to form nanofibers. The antigens are displayed at the surface of resulting nanofibers, which will not affect the activity of subunit vaccines. The formation of nanomaterials enhances the lifetime of subunit vaccines. More importantly, strong antibody responses against peptide epitopes or proteins can be achieved specifically after administrating these epitope-bearing nanofibers without additional adjuvants. These self-adjuvanting nanomaterials will be promising vaccine candidates for disease prevention and immunotherapy.
Conjugating Self-Assembling Peptides with Peptide Epitopes to Raise Antibody Production
Surprisingly, after mice being immunized subcutaneously with different peptides, they found a high IgG production elicited by O-Q11 without additional adjuvants in mice due to fibrillization. As shown in Fig. 1h, fibrillized Q11 alone was not immunogenic whether delivered with PBS or CFA. On the contrary, fibrillized O-Q11 without additional adjuvants exhibited similar IgG titers to OVA delivered in CFA. What’s more, O-Q11 delivered in CFA acquired even higher IgG titers (Fig. 1i). The similar production of antibody isotypes between OVA in CFA and fibrillized O-Q11 demonstrated that Q11 peptide itself acted as a powerful adjuvant. O-Q11 antiserum also reacted to OVA-coated plates, indicating the availability of the epitopes on the fibers (Fig. 1j). Yet OVA-specific antibody responses were not elicited after eliminating covalent bonding between Q11 self-assembling domain and OVA epitope domain, demonstrating the self-adjuvant properties of Q11 were entirely dependent on its covalent conjugation to the epitope peptide (Fig. 1k). In general, antibody production resulted from T cell help by producing cytokines or antigen presentation. This study not only demonstrated for the first time peptide epitope-bonding self-assembling peptide could elicited high specific antibody titers but also provided a novel idea for the development of vaccines and clinical application of self-assembling peptides [22, 23].
Adjustable Immune Responses to Self-Assembling Peptides
Co-assembly of Multiple Proteins into Nanofibers to Produce Multiple Protein Antigen Vaccines
Enhanced Protective Immune Responses In Vivo by Using a Self-Assembling Peptide Amphiphile (PA)
To evaluate the potency of diC16-OVA micelles acting as antigen delivery vehicles in vivo, a preventive immune assay was performed. After three subcutaneous immunizations with phosphate buffer saline (PBS), diC16-OVA in PBS, and OVA peptide in incomplete Freund’s adjuvant (IFA), female C57BL/6 mice were inoculated with cancer cells expressing ovalbumin. Slower tumor growth and longer survival rate were received in the diC16-OVA group compared to PBS group and OVA in IFA group (Fig. 4d, e). The tumor prevention in diC16-OVA group was mediated by OVA-specific cytotoxic T cells. As shown in Fig. 4f, there was more splenocytes stained with fluorescently labeled MHC-I pentamer loaded with the SIINFEKL peptide obtained from the diC16-OVA group compared to those obtained from PBS and OVA in IFA groups. As an effective antigenic peptide delivery system, self-assembling PAs containing T-cell epitope will be doubtlessly an excellent cancer vaccine candidate.
Physical Encapsulation of Antigens in Peptide-Based Hydrogels
Until now, there are only two FDA-approved vaccine adjuvants, aluminum salts (alum) and the recently approved monophosphoryl lipid A (MPL). MPL is an amphiphilic molecule derived from lipopolysaccharide (LPS). It can form vesicles, and antigens should be encapsulated into the formed vesicles for functions. Alum is gel-like material that can provide porous cavities for antigen adsorption by simple mixing. Nanomaterials with the size of 20–200 nm carrying antigens can move from draining lymphatic capillary to lymph nodes to stimulate immune responses. Therefore, many nanomaterials including MPL and abovementioned ones are very useful vaccine adjuvants especially for the development of vaccines to treat cancers, HIV, etc. Though alum cannot elicit strong cellular immune response and it can only be applied for the development of prophylactic vaccines, it attracts extensive research interests to develop adjuvants similar to alum because of the ease of formulation in large scales. Hydrogels are promising materials for this goal . For example, a whole-cell vaccine can be obtained by physically mixing attenuated cancer cells with injectable cryogel for cancer therapy . In the following section, we introduce using supramolecular nanofibers/hydrogels formed by self-assembling peptides to physically interact with and deliver antigens.
Enhanced Immunostimulatory Effects of DNA-Encapsulated Peptide Hydrogels
DNA that encodes tumor-specific antigens represents potential immunostimulatory agents. However, rapid enzymatic degradation and mechanical fragmentation from high shear stresses during injection restricted its development. In general, DNA vaccines must be injected with a delivery system to enhance their immune responses. Therefore, developing a high-efficient DNA delivery system was regarded as an urgent requirement. Self-assembling peptides became a popular supramolecular material for biomedical applications in the last two decades not only because of its security but also its ease of design and modification. Self-assembling peptides rich in lysine, arginine, and histidine behave positive charge and are able to catch the negative charged DNA in hydrogels. Besides the sustained release and protective effect of DNA in hydrogels, the nanostructures in hydrogels (mostly nanofibers) facilitate the cell internalization of DNA via endocytosis, thus enhancing DNA delivery efficacy and facilitating immune responses.
A Peptide-Based Nanofibrous Hydrogel as a Promising DNA Nanovector for Optimizing the Efficacy of HIV Vaccine
Enzyme-Catalyzed Formation of Co-assembled Nanofibers in Hydrogels for Protein Vaccine Delivery
C57BL/6J mice were then immunized subcutaneously with different hydrogel-based vaccines, and soluble OVA in PBS was used as a control while the alum-adjuvant-containing OVA as a positive control. Compared with soluble OVA group, alum evoked 163-fold of IgG antibody, while L-gel-2 and D-gel-2 stimulated higher IgG antibody production than alum (209- and 622-fold, respectively, Fig. 7d). Both gels can enhance antigen uptake (Fig. 7e) and promote and prolong accumulation of antigen in lymph nodes (Fig. 7f), as well as evoke germinal center formation. They also demonstrated the capability of hydrogels to promote DC maturation. BMDCs treated with both gels showed significantly enhanced expression of CD80, which were the co-stimulatory molecules of DCs (Fig. 7g). The D-gel significantly promoted the expression of CD40 on BMDCs, while L-gel only slightly increased its expression (Fig. 7h). In addition, both gels significantly induced TNF-α production (Fig. 7i), while D-gel but not L-gel significantly promoted IL-6 production (Fig. 7j). Furthermore, both gels moderately induced IL-12 secretion by BMDCs (Fig. 7k). In a therapeutic tumor inhibition assay, D-gel-2 prevented EG-7-OVA tumor growth more significantly than its L-counterpart. These observations suggested that D-gel was more powerful to stimulate CD8+ T-cell response. The good biocompatibility of the gels and more powerful cellular immune response eliciting property of D-gel suggested its potential in the development of protein vaccines to treat cancers.
A Powerful CD8+ T-Cell Stimulating D-Tetra-peptide Hydrogel
The cellular immune response, especially the CD8+ T-cell immune response, is important for immunotherapy because the CD8+ T-cell immune response is an immune process that can directly kill bacteria, viruses, and tumor cells . Jiang, Yang, and co-workers have demonstrated that self-assembling peptides could form co-assembled nanofibers for vaccine delivery to evoke the cellular immune response [33, 34]. However, it is difficult to obtain the pro-gelator (Nap-GFFpY-NMe or Nap-GFFpY-OMe) in large scales (e.g., grams). Besides, recent studies have indicated that the pathway to prepare supramolecular hydrogels is very important to the property of resulting gels . Therefore, the kinetics of enzyme reaction may affect the vaccine adjuvant property of resulting gels. It is therefore worth investigating the vaccine adjuvant property of hydrogels formed by the general heating–cooling process. Therefore, Yang and co-workers recently tested the vaccine adjuvant property of a serial of hydrogels formed by heating–cooling process .
L-Rhamnose-Containing Supramolecular Nanofibrils as Potential Immunosuppressive Materials
The pioneering works introduced in this tutorial review have demonstrated that self-assembling peptides are powerful nanomaterials for immune modulation, which are very useful for vaccine development and antibody production. However, challenges still remained as formidable tasks. Peptides and proteins are needed to be folded into correct conformation to stimulate specific antibody . While nanomaterials formed by self-assembling peptides are metastable materials [41, 42], their property will be significantly affected by the pathway of preparation [36, 43, 44]. In order to assist peptide folding, the kinetics of peptide self-assembly, pathway to prepare self-assembled nanomaterials, and presence of additives are needed to be screened and optimized. For antibody production, complete Freund’s adjuvant is very powerful but is still unable to enhance the proportion of specialized antibody for phosphorylated and acetylated proteins. Self-assembling peptides may prevent the dephosphorylation and deacetylation of the antigens and therefore specifically evoke antibody production for these proteins. Besides, a perfect mixed of multiple adjuvants may further increase the efficiency of antibody production . Combination therapy will generally lead to better therapeutic effects for cancer treatment. The effect of combination of cancer vaccine with chemotherapy, cell therapy, or other immunotherapy on cancer treatment is worth investigating. Though many challenges remained, we image a brilliant future of self-assembling peptides in the development of novel vaccines and peptide-based therapeutics.
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