Browsing by Author "Collier, Joel H"

Vaccines have been a revolutionary intervention for infectious diseases over the past many decades. However, over 2 million people continue to die every year of vaccine-preventable causes, especially in low-resource areas. This is mainly due to inefficiencies in the vaccine supply chain which in entirety lead to loss of potency of vaccines worth over $250 million every year due to temperature fluctuations. Additionally, vaccines that actually reach users may lose effectiveness because of more challenges related to their direct delivery to the recipients; for example, contamination and injuries from misuse of needles. We sought to take a step in addressing these challenges by developing thermally stable vaccine tablets for the sublingual delivery of self-assembled peptide nanofibers. Tablets were engineered from a combination of supramolecular peptide nanofibers plus the excipients, dextran and mannitol. The tablet structure was characterized to assess the impact of tablet formation on the nanofiber structure, as well as for suitability of sublingual delivery. In vivo studies were then carried out in a mouse model to determine the capacity to raise antigen-specific immunogenic responses. Sublingual delivery of the tablet in the mouse model was achieved and an immunogenic response was raised in mice. This proof-of-concept study indicates a step towards improving vaccines with regard to addressing challenges of the vaccine supply chain and vaccine delivery, especially in low resource centers.

While promoting a highly targeted immune response, linear peptide epitopes generally have reduced immunogenicity and a lack of conformational specificity which has hampered the development of peptide-based vaccines. In contrast, supramolecular assembly is capable of considerably enhancing immunogenicity and promoting long-term immune responses to peptide epitopes. Immune responses generated with different vaccine platforms but targeted to the same peptide epitope, however, can vary greatly in magnitude and phenotype depending on the physicochemical cues, adjuvant, and density with which that immune epitope is displayed, influencing vaccine outcomes. Greater understanding of how design criteria such as adjuvant choice, epitope density, and multi-epitope immunization adjust the magnitude and phenotype of immune responses—in addition to how these changes in magnitude and phenotype affect therapeutic outcomes—would greatly benefit the development of peptide-based vaccines for treatment and prevention of disease. In this effort, the Collier lab has previously established that the density of T-cell epitope content within molecular self-assemblies can alter the cellular phenotype of antigen-specific T-cells in mice. Further modifications to nanofiber physical properties such as size and surface charge have also been recently shown to modulate the immunogenicity of peptide nanofiber constructs. It remains unclear, however, the extent to which combinations of B- and T-cell epitope content within nanofibers affects the antibody and T-cell responses. Further, the magnitude of antibody and T-cell responses can be magnified by the presence of adjuvants, but with adjuvant specific alterations to antibody subclass and T-cell phenotype. This work capitalizes on these advances in supramolecular nanofiber vaccines and extends them further in three separate contexts. Using peptide epitopes from inflammatory cytokines (Chapter 3), HIV protein mimics (Chapter 4), and influenza A virus (Chapter 5), we demonstrate how titrating epitope content controls adaptive immune responses and how these responses are influenced by the presence of different adjuvants. While previous work with adjuvants has identified general principles of phenotype, typically characterized as Th1 vs Th2 responses, this work seeks to understand and expand context-specific effects, e.g. optimized phenotypes for anti-cytokines immunizations compared to infectious disease. In Chapter 3, epitopes were predicted, tested, and optimized in the Q11 system to determine an immunogenic IL-17A epitope for the treatment of psoriasis. The epitopes were identified through a literature search and Kolaskar-Tongaonkar antigenicity prediction. High-scoring epitopes were tested in mice for immunogenicity and one was selected based on antibody titer. The formulation space combining the selected epitope with different combinations of T-cell epitope was also characterized, and combinations of B- and T-cell epitope content were determined to be predictive of antibody titer in this system. Responses were then enhanced with either a Th1 or a Th2 biasing adjuvant and tested for efficacy in a mouse model of psoriasis. Reduced disease symptoms were correlated with specific antibody subclasses in a first demonstration of the importance of antibody subclass for active immunotherapy. To further understand the quantitative importance of B cell signaling induced by supramolecular nanofibers, we developed a system of peptide-based activation of human B cells in vitro. In Chapter 4, we describe the first use, to our knowledge, of a peptide epitope for activation of the VRC01 Ramos B cell line. This cell line was described in 2017 and contains a B-cell Receptor (BCR) that is identical to the anti-HIV broadly-neutralizing monoclonal antibody VRC01. Other groups had previously published epitope mimicking peptide epitopes, termed mimotopes, which bind the VRC01 monoclonal antibody despite little to no sequence similarity to the native protein. We tested the highest performing mimotopes with the Q11 system and found an epitope-density-dependent effect for one of the mimotopes that was independent of nanofiber concentration. We then tested this epitope at a variety of epitope densities, with or without adjuvant. This study provides evidence that epitope density on supramolecular nanofibers has the potential to directly influence B-cell signaling, and how adjuvants can modulate this effect. Finally, we applied a Design-of-Experiments (DOE) approach to a multi-epitope system and combined it with the novel adjuvant KEYA20Q11. As attempts to make a universal influenza vaccine have become more refined, the specificity of peptide-based vaccines has had increased interest for their ability to avoid immunodominant epitopes in the native protein. Combining T-cell and B-cell responses has further shown promise in the field, but there remains a significant deficit in methods to enhance the T-cell response to a given epitope. In Chapter 5, we first optimize peptide nanofiber formulations in the context of adjuvant-free formulations. The addition of adjuvants does not alter the optimal response profile for antibody and T cell responses in this context, and we first use traditional adjuvants to enhance these responses before challenging with influenza virus. Unfortunately, this did not result in protection from viral challenge and a novel adjuvant based on the therapeutic polypeptide Glatiramer Acetate was used. This adjuvant provides universal T-cell help to enhance B-cell responses and increases the magnitude of co-assembled CD4+ and CD8+ responses. The protective efficacy of the peptide nanofiber formulation was enhanced by a combination of this novel adjuvant with the more traditional adjuvant of cyclic-di-AMP. While formulations with and without KEYA20Q11 both provided equal protection from influenza-induced weight loss, the enhanced T cell responses from the KEYA20Q11 group correlated with reduced viral titers. This work demonstrates how quantitative optimization in conjunction with adjuvant choice can lead to the enhancement of universal B- and T-cell responses for protection from influenza. Taken as a whole, this work represents a significant contribution to the understanding of how peptide epitope content and phenotype of adaptive immune responses influences their efficacy in the contexts of autoimmune and infectious disease. While not all materials-based vaccine platforms may follow the precise rules laid out through the use of the supramolecular peptide systems, the quantitative approach and analysis of immune cell phenotype should be broadly applicable. This work relies heavily on responses from in vivo models or from human cell responses which broadens the translatability of the approach. The completion of this work should facilitate the next generation of supramolecular nanofiber vaccines for autoimmunity and infectious disease.

Inflammatory bowel disease (IBD) is a chronic disorder characterized by persistent inflammation in the gastrointestinal tract. Current therapies for IBD, such as anti-inflammatory or immunosuppressant drugs and anti-cytokine biologics, only temporarily alleviate symptoms and vary widely in effectiveness among patients. Consequently, there exists a critical unmet need for a long-lasting and broadly effective IBD treatment. Natural antibodies against the small molecule epitope phosphorylcholine (PC) are an important component of innate immunity with diverse functions including the clearance of bacterial and autologous targets in a non-inflammatory manner. The cells that produce these antibodies, B1a cells, however, have been shown to be reduced in patients with IBD, with this decrease being associated with a more advanced disease state. It has also been demonstrated that the adoptive transfer of B1a cells in a murine model of IBD results in the increased production of anti-PC antibodies and lessens disease severity. Furthermore, active immunotherapies are an alternative to monoclonal antibody biologics and a promising approach for generating a long-lasting IBD therapy because they exploit the ability of a patient’s own immune system to produce antibodies against a therapeutic target. Building on these concepts, we strove to develop an active immunotherapy consisting of PC as an epitope displayed on self-assembling peptide nanofibers to produce a therapeutic anti-PC antibody response for the treatment of IBD. The first part of this dissertation (Chapter 3) describes the process of designing, determining the therapeutic efficacy of, and gaining mechanistic insights into a nanofiber-based anti-PC active immunotherapy. We began by developing conjugation strategies for attaching PC to Q11 self-assembling peptides to render PC immunogenic. In an effort to find a balance between immunogenicity, stability, and ease of synthesis, we compared phosphoramidite and phosphodiester linkages for PC and concluded that the phosphodiester linkage was critical for PC epitope integrity. We then investigated how altering the multivalency of PC on Q11 nanofibers could further augment the anti-PC antibody response by synthesizing two nanofiber and PC conjugates with either 1 or 1-4 PC copies per Q11 peptide termed PC-Q11 and PCM-Q11, respectively. Intraperitoneal (i.p.) immunization with PCM-Q11 was found to induce a significantly greater anti-PC antibody response than i.p. immunization with PC-Q11. Additionally, PCM-Q11 was more selectively taken up by and able to activate natural antibody-producing B1a cells compared to all other B cells than PC-Q11 or Q11 alone. Further, control over the immune phenotype elicited was achieved via the inclusion of a T-cell epitope and/or CpG adjuvant, with the addition of both greatly augmenting the immune response elicited. We then evaluated the efficacy of immunizations with PCM-Q11/T-cell epitope with or without CpG in several different dextran sodium sulfate (DSS)-induced murine colitis models. We first investigated the ability of these immunizations to prevent severe disease when administered prior to the induction of a 30-day chronic DSS colitis model. Interestingly, immunization with both formulations was protective, significantly improving weight loss, disease severity indices, and colon lengths over unimmunized controls. This efficacy was repeated in a 1 cycle (10-day) DSS colitis model in both male and female mice and not attributed to CpG administration alone. Immunizations against PC also lowered bacterial spread to the spleen due to colon damage, with this effect being more pronounced in female mice. Additionally, we determined the efficacy of PCM-Q11 immunizations in a therapeutic setting where mice received one cycle of DSS colitis followed by three immunizations and then one more cycle of DSS colitis. This showed that immunizations with PCM-Q11 have therapeutic efficacy by significantly improving weight loss, disease activity indices, colon lengths, and bacterial spread to the spleen in this model. Furthermore, we have conducted several other studies to gain mechanistic insight into the observed efficacy of PCM-Q11 immunizations. We found that anti-PC immunization decreases microbiome diversity. We were also able to use flow cytometry to detect IgG and IgM antibodies in serum from mice immunized with PCM-Q11 that bind apoptotic colon epithelial cells in vitro. Additionally, we have shown through passive transfer of PCM-Q11 immunized sera that induced anti-PC antibodies offer some protection against severe DSS-induced colitis. Moreover, through both histological examination of colon damage and immunofluorescence imaging of tight junction proteins, we determined that PCM-Q11 immunizations were not acting by improving barrier function in the colon. Finally, we observed reduced efficacy of PCM-Q11 immunizations in an Il10-/- murine model of colitis. Collectively, this data demonstrates that immunization with PCM-Q11 was both preventative and therapeutic in multiple DSS-induced models of colitis in mice, with considerable efficacy attributed to the induced anti-PC antibody response. In the second part of this dissertation (Chapter 4), peptide nanofibers were modified for use as oral vaccines. While oral delivery offers direct access for eliciting immune responses within the gastrointestinal tract, it poses substantial obstacles for vaccines to overcome including acidic and proteolytic environments, thick mucus barriers, and a limited window for absorption. We therefore focused on two main design improvements to peptide nanofibers: synthesis with protease-resistant D-amino acids and incorporation of muco-penetrative peptide sequences rich in proline, alanine, and serine (PAS). We showed that D-amino acid Q11 was not degraded in simulated gastrointestinal environments in contrast to its L-amino acid counterpart. Additionally, we determined that PASylation enhanced muco-penetration in vitro and accelerated nanofiber transport through the GI tract in vivo. Ultimately, however, we found that oral immunization with PASylated L-amino acid nanofibers with cholera toxin B subunit mucosal adjuvant was the optimal formulation for the generation of both local and systemic immune responses. The primary areas affected in IBD are the distal small intestines and the colon, so the induction of therapeutic antibodies in these tissues is paramount. Thus, we sought to translate the above design principles to the small molecule epitope, PC, to enable oral administration. Oral immunization with PASylated anti-PC formulations was able to generate both local and systemic anti-PC immune responses. We then illustrated that oral vaccination with PASylated PC-bearing nanofibers was effective in both therapeutic and prophylactic models of DSS-induced colitis, observing comparable reductions in disease severity to i.p. anti-PC immunizations, thus, increasing the translatability of our therapy by offering a needle-free formulation. Overall, this data indicates that PASylated supramolecular peptide nanofibers are a promising platform for oral immunization. This dissertation outlines an encouraging first example of an active immunotherapy engaging natural antibody responses against phosphorylcholine as a durable therapy for IBD. More broadly, the strategies developed offer a potentially versatile approach for engaging natural antibody therapies and oral nanofiber peptide vaccines towards a variety of inflammatory and infectious diseases.

Short peptides are poorly immunogenic when delivered sublingually – under the tongue. This challenge has prevented widespread investigation into sublingual peptide vaccines, leaving their considerable potential untapped. Sublingual immunization is logistically favorable due to its ease of administration and can raise both strong systemic immune responses and mucosal responses in tissues throughout the body. Peptide epitopes are highly specific, allowing for the generation of immune responses directed solely against precisely selected targets, without accompanying off-target antibody or T-cell production. To enable sublingual peptide immunization, we designed a strategy based on molecular self-assembly of epitopes within nanofibers. We then extract and expound several key implications from this finding, including insights into the mechanism of action, development of a translatable administration modality, and application to a currently unmet clinical need.

The design of our sublingual peptide immunization strategy is described in the first part of this thesis (Chapter 3). We sought to utilize nanomaterial delivery of peptides to enhance sublingual immunogenicity, a strategy that has proved successful in several other immunization routes. However, the salivary mucus layer is a significant barrier to nanomaterial delivery, particularly for supramolecular materials, due to its ability to entrap and clear these materials rapidly. We designed β-sheet nanofibers conjugated at a high-density to the mucus-inert polymer polyethylene glycol (PEG) to shield them from the mucus layer. Strikingly, sublingually delivered PEGylated peptide nanofibers (PEG-Q11) raised extremely durable antibody and T-cell responses against peptide epitopes when mixed with a mucosal adjuvant. We showed that PEG decreases nanofiber interactions with mucin in vitro, and extends the residence time of nanofibers at the sublingual space in vivo. Further, we showed that we could achieve similar results by adapting the use of PASylation (modification with peptide sequences rich in Pro, Ala, and Ser) to mucosal delivery.

In the second part of this thesis (Chapter 4) we designed a supramolecular strategy for enhancing sublingual nanofiber immunization. Mucosal adjuvants, such as cyclic-di-nucleotides (CDNs), can promote sublingual immune responses but must be co-delivered with the antigen to the epithelium for maximum effect. We designed peptide-polymer nanofibers displaying nona-arginine (R9) at a high density to promote complexation with CDNs via bidentate hydrogen-bonding with arginine side chains. We co-assembled PEG-Q11 and PEG-Q11R9 peptides to titrate the concentration of R9 within nanofibers. In vitro, PEG-Q11R9 fibers and cyclic-di-GMP or cyclic-di-AMP adjuvants had a synergistic effect on enhancing dendritic cell activation that was STING-dependent and increased monotonically with increasing R9 concentration. However, intermediate levels of R9 within sublingually-administered PEG-Q11 fibers were optimal for sublingual immunization, suggesting a balance between polyarginine’s ability to sequester CDNs along the nanofiber and its potentially detrimental mucoadhesive interactions. These findings reveal important design considerations for the continuing development of sublingual peptide nanofiber vaccines.

We sought to enhance the translational capacity of our immunization strategy by designing a highly accessible vaccine method (described in Chapter 5). Significant barriers exist to improving vaccine coverage in lower- and middle-income countries, including the costly requirements for cold-chain distribution and trained medical personnel to administer the vaccines. To address these barriers, we built upon our sublingual nanofiber platform to design a heat-stable and highly porous tablet vaccine that can be administered via simple dissolution under the tongue. We produced SIMPL (Supramolecular Immunization with Peptides SubLingually) tablet vaccines by freeze-drying a mixture of self-assembling peptide-polymer nanofibers, sugar excipients, and adjuvant. We showed that even after heating for 1 week at 45 °C, SIMPL tablets could raise antibody responses against a peptide epitope from M. tuberculosis, in contrast to a conventional carrier vaccine (KLH) which lost sublingual efficacy after heating. Our approach directly addresses the need for a heat-stable and easily deliverable vaccine to improve equity in global vaccine coverage.

To demonstrate the clinical usefulness of our technology, we designed a sublingual vaccine against uropathogenic E. coli (UPEC), the pathogen that causes most urinary tract infections (UTIs). Sublingual peptide immunization is uniquely advantageous for immunization against UTIs, as sublingual immunization raises antibodies in the blood and urinary tract, and peptide epitopes allow for targeting UPEC without perturbing commensals. We co-assembled PEG-Q11 nanofibers containing three different B-cell epitopes from UPEC along with a helper T-cell epitope, allowing us to raise simultaneous antibody responses against three targets systemically and in the urinary tract. These antibodies were highly specific for UPEC, exhibiting no binding to a non-pathogenic strain. Further, these antibodies demonstrated clinical potential by protecting mice from an intraperitoneal UPEC challenge. Finally, we showed the ability to use SIMPL tablets to raise anti-UPEC responses in rabbits, which contain an oral cavity with key similarities to humans.

This thesis demonstrates a critical enabling technology for sublingual peptide immunization and builds upon this technology to report findings with key implications for supramolecular biomaterial design, accessible vaccination, and treatment of UPEC-mediated diseases. This interdisciplinary research synthesizes and utilizes knowledge from materials science, immunology, vaccinology, pharmaceutical sciences, and pathology to present an important original contribution to the field of immune engineering.

Peptide-based vaccines have received growing interest due to their specificity and ability to limit off-target effects, and they are currently being explored toward a variety of infectious diseases and therapeutic targets. However, the efficacy and applicability of such epitope-based vaccines are currently limited by difficulties in predicting immunogenic epitopes in outbred populations and a reliance on carrier proteins and adjuvants that can cause pain and swelling. Current vaccine platforms are further limited in their ability to combine multiple different epitopes, making it difficult to adjust humoral and cellular responses systematically. A vaccine platform containing broadly reactive T-cell epitopes that boosts responses to co-delivered antigens with minimal inflammation could address these limitations. To that end, the focus of this dissertation was to create peptide epitopes that can be incorporated within a supramolecular nanomaterial platform, together acting as a nano-adjuvant, a term that we will use here to describe materials whose adjuvanting properties depend on their nanoscale structure. To achieve this, we took inspiration from a class of materials termed glatiramoids, which promote anti-inflammatory and TH2 immune responses. We created an immunomodulatory supramolecular nanomaterial system inspired by the randomized nature of glatiramoids termed KEYA-Q11. By creating a glatiramoid-like peptide library integrated within self-assembling Q11 nanofibers, numerous epitopes can be presented simultaneously along the nanofibers for maximum antigen presenting cell uptake and activation. The first half of this document (Chapters 3 and 4) describes how this nanomaterial increased immunogenicity of co-assembled epitopes while also creating a KEYA-specific non-inflammatory response to the randomized component. Additionally, capitalizing on the potential for KEYA-Q11 to amplify immune responses to co-assembled epitopes, this technology is applied in the second half of this document (Chapters 5 and 6) to an epitope-based influenza vaccine. Initially we designed and synthesized a self-assembling nanomaterial inspired by glatiramoids and evaluated its TH2 T-cell polarizing properties (Chapter 3). Glatiramoids raise strong, protective immune responses in patients and have been examined in a variety of contexts from Multiple Sclerosis to HIV. However, due to their randomized polysequence structure, it remains challenging to incorporate glatiramoids into other materials and strategies to optimize them for specific therapeutics. Therefore, we designed a polysequence peptide sequence and synthesized it onto the chemically defined, supramolecular Q11 nanofiber platform to straightforwardly titrate it into other nanomaterial formulations. This polysequence nanomaterial was termed KEYA-Q11 for the four amino acids, lysine, glutamic acid, tyrosine, and alanine, that comprise its structure. Due to the extensive number of possible KEYA sequences, multiple batches of KEYA-Q11 were first examined with an array of biophysical characterization techniques to confirm reproducible synthesis and assembly. The optimal number of polysequence amino acid additions was determined to be 20 amino acids as (KEYA)20Q11 could reliably be synthesized and raise strong Type 2/TH2/IL-4 immune responses. Moreover, by modulating the concentration of KEYA-Q11 in a Q11 immunization, the strength of KEYA-specific B-cell responses were similarly altered. KEYA modifications dramatically improved uptake of peptide nanofibers in vitro by antigen presenting cells and served as strong B-cell and T-cell epitopes in vivo, inducing a KEYA-specific Type 2/TH2/IL-4 phenotype. KEYA modifications also increased IL-4 production by T cells, extended the residence time of nanofibers, and decreased overall T cell expansion compared to unmodified nanofibers, further suggesting a TH2 T-cell response with minimal inflammation. Subsequently, we exploited the modularity of the self-assembling system to maximize application of KEYA-Q11 as a nanoscale adjuvant without inflammation (Chapter 4). Adjuvants are commonly required to raise strong immune responses to peptide therapeutics, but often induce swelling and pain at the injection site and typically drive immune phenotype. Relative to common adjuvants, KEYA-Q11 had no detectable injection site swelling and was more effective at raising humoral responses despite a genetically diverse in vivo population. Furthermore, when combined with peptide epitopes KEYA-Q11 augmented antibody production against co-assembled B-cell epitopes for cytokine TNF, D-chiral MMP cross linker, and a conserved segment of the M2 influenza protein, and increased T-cell stimulation specific to co-assembled T-cell epitopes PADRE and a conserved segment of the nucleoprotein of influenza. Likewise, when combined with the influenza surface protein hemagglutinin, KEYA modifications strengthened the resulting influenza-specific cellular immune responses. Augmented immune responses typically followed native epitope polarization, as in a co-assembly of KEYA-Q11 and the nucleoprotein epitope raised Type 2/TH2/IL4 producing KEYA-specific responses and magnified the Type 1/IFN producing nucleoprotein-specific responses that epitope would produce without an adjuvant, and thus using KEYA-Q11 as the adjuvant allowed for finer control over immune phenotype. Building on the success of KEYA-Q11 as a nano-scale adjuvant without inflammation, we utilized these properties to decrease the severity of influenza infection and provide broad protection via immunization with peptide epitopes (Chapters 5 and 6). Much of the current focus on influenza vaccines revolves around partial or whole proteins to induce broadly protective antibodies, while other have demonstrated cross-reactive T-cell responses are vital for heterologous protection. Conserved peptide epitopes have been discovered but typically are included with larger proteins and adjuvants to increase immunogenicity. Supramolecular assemblies based on the Q11 peptide system containing KEYA, a B-cell epitope from a conserved surface protein on influenza, and CD4+ and CD8+ T-cell epitopes from influenza nucleoprotein and polymerase acidic protein, respectively, raised strong immune responses against all three epitopes. Inclusion of the KEYA component in prophylactic immunizations with these materials significantly improved protection following a lethal influenza challenge. It has been established that while peptide-based immunotherapies can have finely directed specificity for chosen epitopes, they generally lack sufficient immunogenicity to provoke suitable immune responses. This new strategy for augmenting immune responses to peptide-based therapeutics, especially those employing nanomaterials, and especially for applications where non-inflammatory responses are prioritized, can be employed for a variety of potential applications in vaccine development, towards infectious diseases and towards non-infectious applications such as inflammatory autoimmune diseases, wound healing, or graft rejection. KEYA-Q11 is a unique fusion of two materials, a highly ordered system with a highly disordered system, and examination of this nanomaterial has provided valuable insight into both randomly polymerized structures and non-inflammatory nano-scale adjuvants.

The field of prostate cancer research suffers from a lack of suitable in vitro culture models, and primary human prostate cancer cells are notoriously difficult to culture reliably, especially in 3D. As an alternative to the ill-defined, costly, and highly variable Matrigel, self-assembled peptides have been developed, though they too suffer from the major drawback of low cell survival during encapsulation process, mostly due to its acidic environment. Here we designed a novel peptide, bQ13; it undergoes gelation at a comparatively neutral pH range. In this project, I will demonstrate its excellent cytocompatibility and ability to provide a chemically defined and controllable matrix for drug screening. My data suggest that bQ13 hydrogels indeed greatly enhanced the cell survival rate during the encapsulation process in comparison to PuraMatrix and Q11, previously developed self-assembling peptides. The increased cytocompatibility of bQ13 ultimately enabled to the formation of prostate cancer cell spheroids in long-term 3D culture. Drug screening results showed that bQ13 hydrogels also significantly shielded the spheroids from enzalutamide compared to the other hydrogel samples, indicating it is a system that more closely mimics the in vivo environment compared to Matrigel and other self-assembling peptides.

Peptide-based self-assembling biomaterials are a promising platform for biomedical applications such as immunomodulation, drug delivery, tissue repair and regeneration, cell delivery, and combinations thereof. However, many of these applications would benefit from the incorporation of folded proteins which have several advantages over their peptide counterparts. Despite their essential contribution to research progress over the years, bioactive peptides often fail to recapitulate the dynamic, high-affinity, and multifunctional nature of whole proteins. The ability to integrate and control the incorporation of protein components into self-assembling peptide materials would greatly broaden their applicability in biological contexts, particularly immune engineering and tissue engineering. In previous work, a strategy was established for inducing desired sets of expressed functional proteins to assemble directly into nanofibers or hydrogels through the use of a novel assembly tag known as the “βtail”. βtail proteins can be expressed and purified in a monomeric state, but they assemble in a modular fashion into compositionally defined nanofibers or gels when mixed with additional fibrillizing peptides. In this dissertation, we leveraged the novel βtail technology to elevate the function of self-assembling peptide materials for active immunotherapy and 3D cell culture applications. In concentrated form, peptide assemblies are well-studied matrices for the culture of many different cell types, but in more dilute formulations, they make nanofibers that are usefully immunostimulatory. The βtail system is useful for incorporating proteins in both contexts. The first half of this thesis (Chapters 3 and 4) describes the development of protein-bearing nanofibers for immunomodulation. We chose to focus on the protein C3dg, a late product of the complement cascade and key interface between innate and adaptive immunity. This protein has received considerable interest as a molecular adjuvant, but its utility in immunotherapies was yet to be fully realized. This was, in large part, due to an inability to assemble multiple copies of C3dg without utilizing chemistries that denature the protein or occlude its binding site. We overcame this issue with the βtail platform: by expressing a βtail-tagged version of C3dg and assembling multivalently into peptide nanofibers, we were able to enhance the humoral and cell-mediated immunogenic effects of C3dg. We initially investigated βtail-C3dg as a component in an active immunotherapy to mitigate TNF-mediated inflammation (Chapter 3). Active immunotherapies offer important advantages over existing biologics such as monoclonal antibodies (mAb), particularly towards chronic inflammatory diseases. Supramolecular assemblies based on a peptide system (Q11) containing βtail-tagged C3dg, B-cell epitopes from TNF, and the universal T-cell epitope PADRE raised strong antibody responses against both TNF and C3dg, and prophylactic immunization with these materials significantly improved protection in a lethal TNF-mediated inflammation model. Additionally, in a murine model of psoriasis induced by imiquimod, the C3dg-adjuvanted nanofiber vaccine performed as well as anti-TNF monoclonal antibodies. Nanofibers containing only βtail-C3dg and lacking the TNF B-cell epitope also showed improvements in both models, suggesting that supramolecular C3dg, by itself, played an important therapeutic role. We observed that immunization with βtail-C3dg caused the expansion of an autoreactive C3dg-specific T-cell population, which we believe acted to dampen the immune response, preventing excessive inflammation. These findings led us to believe that molecular assemblies displaying C3dg warrant further development as active immunotherapies. Due to its apparent anti-inflammatory characteristics, we sought to investigate the broad use of βtail-C3dg as a component in an active immunotherapy against another inflammatory molecule, complement component C5a (Chapter 4). There are no reported C5a B cell epitopes, so the epitopes investigated herein were predicted using the Kolaskar Tongaonkar Antigenicity Test and assembled with βtail-tagged C3dg and PADRE. Two out of the three selected epitopes raised IgG antibodies against C5a, and mice immunized with these formulations exhibited significantly reduced serum C5a concentrations. Interestingly, mice receiving prophylactic immunization with nanofiber formulations containing βtail-C3dg, C5a B-cell epitope, and PADRE exhibited reduced protection in a lethal sepsis model compared to formulations containing only the C5a B-cell epitope and PADRE. When we investigated the T-cell populations, we found that the combined C3dg/C5a immunizations elicited TH1-polarized autoreactive C3dg-specific T-cells. Because C5a plays a role in effector T-cell responses, we hypothesized that its combination with C3dg may have induced an inflammatory T-cell population against the C3dg component. Because formulations containing only the C5a B cell epitope and PADRE demonstrated efficacy, we proceeded to investigate formulations lacking the C3dg component. In a model of collagen antibody-induced arthritis, prophylactic immunization significantly improved the clinical severity of the disease. Despite the unexpectedly adverse contribution made by the βtail-C3dg component, this work represents a promising application of an active immunotherapy targeting complement C5a. The second half of this thesis (Chapters 5 and 6) focuses on the development of a tailored hydrogel matrix for prostate cancer cell growth in vitro using self-assembling peptides and proteins. One of the most significant challenges in establishing phenotypically accurate cultures of prostate cancer cells is constructing appropriate 3D culture environments. The utilization of matrices such as Matrigel has enabled the field to establish some prostate cancer organoid cultures, but Matrigel’s poor batch-to-batch consistency and “one size fits all” nature makes it difficult to customize for different cell types or different contexts. In response to these shortcomings, we designed a chemically defined nanofiber hydrogel (bQ13) that exhibits improved short- and long-term cytocompatibility for human prostate cancer cells compared to alternative commercially available 3D culture matrices. Building upon the success of bQ13, we sought to elevate the platform’s versatility by incorporating functional, structurally intricate proteins that interact directly with ligands and receptors to provoke a specific cellular response. Utilizing the βtail technology, we were able to assemble a range of concentrations of both green fluorescent protein (GFP) and the cell-binding domains of fibronectin (FN) into bQ13 nanofibers without altering the nanofiber structure or the mechanical properties of the hydrogel. The βtail did not interfere with the function of FN, allowing for the adhesion and spreading of cells in 2D and enhancing cell survival and proliferation in 3D. Additionally, the incorporation of an enzymatically cleavable sequence allowed for the controlled release of GFP from the bQ13 matrix, allowing for possible cell-monitoring applications. These results highlight the expanded adaptability of the bQ13 platform as a defined 3D matrix that can be tailored with folded proteins for various applications. This thesis outlines the first use of the βtail in specific biological applications and demonstrates its promising utility as a versatile biomaterial. The works discussed herein also propose significant contributions in the arenas of active immunotherapy and tissue engineering.

Human Immunodeficiency Virus (HIV) is a vaccine target that has remained elusive for decades. In 2015, 1.1 million people died of HIV-related causes and 2.1 million new infections occurred. Although an effective HIV vaccine has long been a major goal of the World Health Organization (WHO), HIV has been an extraordinarily challenging vaccine target. This challenge is due to several compounding factors including the evolution of the virus within individuals and across geographic regions. This evolution makes it difficult to develop a universal HIV vaccine that will neutralize all strains of the virus. Furthermore, the virus is exceptionally efficient at mutating to evade the immune systems of infected individuals. The outer surface of the virus is densely glycosylated, making it difficult for the immune system to make effective antibodies against such a shielded structure. Consequently, most antibodies generated against the virus are either non-neutralizing or only partially neutralizing and are ineffective at clearing HIV infection. Thus, alternative strategies to direct the immune system towards making neutralizing antibodies are required for an effective HIV vaccine. Attempts at creating an effective HIV vaccine have centered around stimulating high-affinity antibodies that effectively bind genetically diverse strains of the virus. Through protein engineering, variable dosing regimens, and creation of new antigens, HIV researchers have identified the key factors required for the human immune system to raise functional anti-HIV antibodies. Primarily, the immune system must be directed towards the highly conserved and functional antigenic regions of HIV surface proteins to make protective antibodies. To steer the immune system towards specific, neutralizing epitopes from HIV-1, the repertoire of B cells activated by HIV vaccines must be altered from those elicited by natural infection or traditional immunization approaches. Two ways to alter the B cell populations activated upon immunization are explored in this dissertation. Firstly, lowering the activation threshold of activated B cells by arraying antigens on materials allows for a shift of antibody repertoires toward more epitope specificities and potentially broader binding of antibodies to mutated viral strains. Secondly, the immune system can be focused toward specific epitopes using heterologous immunization regimens where antibodies are selected toward a certain specificity, then evolved to bind native antigens, such as those that would be displayed on HIV virions. Though both of these approaches have been explored in other systems, the unique impact of self-assembling peptides in this space has yet to be explored. Peptide biomaterials with fibrillar morphologies such as β-sheet peptides, worm-like micelles, and peptide amphiphiles have been explored towards numerous biomedical applications including scaffolds for tissue repair, immunotherapies for infectious diseases, cancer, or inflammatory conditions, and depots for sustained drug delivery. Although these materials have shown promise in preclinical applications, the immunological effects of their length and ligand valency are poorly understood. Because both of these features can be utilized to tune immune responses, optimizing them in the context of HIV immunization has the potential to improve the magnitude and quality of antibodies elicited by nanofiber immunogens. To examine the impact of antigen valency and nanofiber size in HIV immunization, structural tools to control these features were developed. The ability to control nanofiber length was achieved in this body of work be engineering a set of peptides we have designed and characterized to stabilize self-assembling interfaces. This newfound control over nanofiber length allows size-based targeting of materials which was not previously possible. In addition to studying size-dependent immunogenicity, the role of glycans in immune responses to nanoscale are a newfound area of interest amongst the biomaterials community. Though some glycans can be readily conjugated to peptide and polymer assemblies, many carbohydrates, such as sialic acids are extremely difficult to functionalize chemically. To overcome this, glycomimetic peptides which resemble diverse glycans have been designed, but are implemented in few therapeutic contexts. This system capitalizes upon the synthetic advantages of utilizing glycomimetic peptides in peptide-based immunogens and represents a broadly applicable strategy to impart lectin-binding properties to peptide materials. Although it is appreciated that multivalency and nanomaterial shape and size can influence immunogenicity, these aspects have yet to be fully exploited in the context of a specific disease. To meet these challenges, we have designed a self-assembling peptide system with control over the lengthwise assembly of nanofibers and have studied the effect of antigen valency on immune responses to these materials in the context of HIV. As immunogens, peptide nanofibers have a unique ability to activate low-affinity B cells, such as those which react to autologous targets, will likely be advantageous for HIV vaccination, where low-affinity B cells are precursors to the induction of broadly neutralizing antibody (bnAb) responses. To determine the utility of peptide nanofibers as platforms for HIV vaccination, We first constructed nanofibers that are covalently linked to the HIV envelope antigen gp120 which demonstrated their ability to raise antibodies with broad binding profiles. As an alternative approach for raising immune responses against HIV antigens, we have explored the use of peptide nanofibers displaying short, linear HIV epitopes as priming immunogens. This approach capitalizes on the ability of nanofibers to generate antibodies against short epitopes and tailors the accumulation of nanofibers in lymph nodes to prime epitope-focused antibodies against HIV virions. Taken together, the studies described here utilize supramolecular control over antigen valency and immunogen size to generate antibody responses to HIV with high affinity and high binding breadth. The supramolecular tools described here provide morphological controls for spontaneously assembling materials which have not yet been utilized for these types of platforms. This tight control over morphology allows us to ask questions with levels of immunological precision that are not common in biomaterials literature.

Tumor growth, survival, and metastasis depend upon interactions with the matrix and cells which compose the tumor microenvironment (TME). Tumor cell interactions with transformed extracellular matrix (ECM) and immune cells, such as tumor associated neutrophils (TANs) have been shown to affect tumor progression both clinically and in animal models. Unfortunately, while the complexity of the TME is difficult to recapitulate in standard cell culture, it is also difficult to analyze and to influence in vivo. Researchers have sought to circumvent these challenges by developing 3D models in naturally derived matrices like collagen and Matrigel, but these scaffolds often sacrifice biochemical tunability and fail to meet the stiffness regimes often seen in malignant ECM. Thus, there is a need for highly controlled 3D culture systems which mimic the biophysical properties and cell-cell interactions of the TME to better investigate how these interactions direct cancer development and progression. To meet this goal, researchers have employed synthetic hydrogel systems for 3D in vitro cell culture. Our lab has previously engineered a synthetic scaffold to serve as an ECM by incorporating a matrix metalloproteinase cleavable peptide into a biocompatible poly(ethylene glycol) (PEG) backbone and grafting in an integrin-binding peptide. This system allows independent tuning of adhesivity and matrix stiffness and supports tumor cell growth and spheroid formation. Here, we present two applications of this system to model cell-matrix interactions in the TME. First, we orthogonally tuned the adhesion ligand concentration and stiffness of our PEG-based hydrogels to investigate the individual and interactive impact of these matrix properties in a physiologically relevant regime. We assessed the tumor progression of a fibrosarcoma cell line (derived from mesenchymal cells) and a triple-negative breast carcinoma cell line (TNBC, derived from epithelial cells) cultured in and on these hydrogels. We observed that the cell proliferation, invasion, and focal complex formation in the fibrosarcoma cells responded to changes in matrix stiffness, while the same behaviors in the TNBC cell line occurred in response to changes in matrix adhesivity. to discern the differential behavior of these broad classes of tumors. We found no interactive effect between the two matrix properties within the conditions tested. These results helped to reiterate the importance of independently tunable systems in assessing cell response to specific matrix properties and established our system’s ability to discern differential tumorigenic behavior across diseases. Next, we incorporated neutrophil extracellular traps (NETs) in our system to study their impact on tumorigenesis in TNBC cells in a highly controlled environment. We observed that NETs helped to increase cell survival, proliferation, and pro-metastatic morphological phenotype. We also showed that the presence of NETs influenced the secretion of IL-8, a pro-NETosis factor, and that conditioned media from cells cultured in these gels influenced NETosis in an IL-8 dependent manner. The results observed in this system correlate with murine models and clinical studies in the literature and help to provide additional insight of the individual factors at play in the NET-mediated promotion of TNBC progression and metastasis. To expand upon current models of cell-matrix interactions within the TME, we applied an existing model to assess the differences in behavior between a fibrosarcoma and TNBC cell line and developed a new model for assessing NETs as a matrix biomolecule in TME models. These two reductionist models help to advance our understanding of the roles specific aspects of the TME play in tumor progression, ultimately helping to drive better rational design of TME-targeted therapies to improve clinical outcomes of cancer patients.