Nanofiber-Based Active Immunotherapy: A Multimodal Approach Targeting Chronic Inflammation

Abstract

Chronic inflammatory diseases represent a significant global health challenge, with current therapies limited by high costs, frequent dosing requirements, and systemic side effects. This dissertation explores the development of innovative nanofiber-based active immunotherapies targeting pro-inflammatory cytokines to address these limitations. Leveraging the modularity of Q11 and C29 supramolecular peptide platforms, we designed self-assembling nanofibers incorporating B-cell epitopes from inflammatory mediators alongside exogenous T-cell epitopes to elicit durable neutralizing antibody responses without adjuvants. The dissertation is structured around three main parts, each addressing a distinct aspect of inflammatory disease and immune dysfunction.In the first part, we demonstrate the efficacy of IL-1β-targeted nanofibers in reducing epidermal thickening in a murine psoriasis model. These nanofibers achieved results comparable to gold-standard monoclonal antibody treatments, highlighting their potential as a novel therapeutic approach for chronic skin inflammation. Specifically, IL-1β-Q11 nanofibers raised robust anti-IL-1β titers without adjuvant and significantly reduced inflammatory IL-1β levels in an acute inflammation model. In a chronic psoriasis-like model histological analysis revealed a marked reduction in epidermal thickness, comparable to that observed with anti-IL-1β monoclonal antibody treatment. Furthermore, the nanofiber treatment led to a significant decrease in the expression of psoriasis-associated pro-inflammatory genes in the skin such as TNF and IL-6 as measured by RT-qPCR. We also observed an increase in IgG1/Th2-biased responses and CD62L+ regulatory T-cells in the treatment groups, suggesting the modulation of the adaptive immune system as a key mechanism of action for the nanofibers. The second part extends this approach to develop needle-free dual-target formulations against IL-1β and TNF for pulmonary inflammation. Intranasal delivery of these nanofibers enhanced both systemic and mucosal antibody responses while significantly reducing airway resistance and neutrophil infiltration in an acute lung injury model. This work demonstrates the versatility of the platform in addressing complex inflammatory cascades in pulmonary diseases. Notably, dual-target nanofibers outperformed plain nanofiber formulations in reducing pro-inflammatory cytokines and preserving lung function in an LPS-induced acute lung injury model. Specifically, mice treated with dual-target nanofibers showed a significant improvement in airway resistance as measured by flexiVent over untreated LPS controls and marked reduction in BAL fluid levels of TNF and IL-6 compared to plain nanofiber controls. Differential cell count analysis of BAL fluid revealed a significant increase in neutrophil infiltration across all LPS-challenge groups but a significant decrease in myeloperoxidase activity in the lungs of the dual-target nanofiber mice compared to untreated mice, indicating a strong local anti-inflammatory response in the lungs of treated animals. The third part investigates the potential of unadjuvanted anti-IL-1β nanofibers to counteract immunosenescence in aged mice. By reducing baseline inflammation, this approach improved antibody responses to influenza vaccination, highlighting its potential for enhancing vaccine efficacy in elderly populations. Aged mice (18-22 months old) treated with IL1β-Q11 nanofibers prior to influenza vaccination showed a 2.5-fold increase in neutralizing antibody titers compared to untreated aged controls. Remarkably, flow cytometry analysis of lymph nodes revealed IL1β-Q11 nanofiber treatment in aged mice restored germinal center B-cell activity and reduced T-cell exhaustion markers to levels comparable with young mice, indicating improved adaptive immune function. This study provides insights into the broader applications of nanofiber-based immunotherapies in addressing age-related immune dysfunction. Throughout this work, we demonstrate the versatility of nanofiber-based immunotherapies across diverse disease contexts, from localized skin inflammation to systemic age-related immune dysfunction. The modularity of the Q11 and C29 platforms enables precise control over epitope composition and immune response phenotypes, offering advantages over traditional protein-based or virus-like particle vaccines. Key findings include robust antibody production against target cytokines without the need for adjuvants with both platforms. Enhanced mucosal immunity was achieved through intranasal and sublingual delivery routes with C29, with IgA levels in BAL fluid increased 5-fold compared to subcutaneous administration. Additionally, intranasally or sublingually treated animals exhibited balanced Th1/Th2 responses, as measured by IFN-γ and IL-4 production in restimulated splenocytes, and improved isotype distribution, with a more balanced IgG1/IgG2a,b ratio compared to subcutaneous routes. Finally, The Q11 nanofiber platform demonstrated the ability to restore age-related immune dysfunction through targeted cytokine blockade, as evidenced by the 2.5-fold increase in vaccine responsiveness in aged mice. This dissertation establishes proof-of-concept for supramolecular peptide nanofibers as a transformative platform for active immunotherapy against inflammatory cytokines. By providing durable immune modulation without the need for frequent dosing or adjuvants, this approach addresses critical gaps in current treatment paradigms for chronic inflammatory diseases. The findings not only advance our understanding of cytokine blockade therapy but also enable the design of biomaterial-based strategies for treating a wide range of inflammatory conditions with unmet clinical needs. The potential applications of this technology extend beyond the specific diseases studied here, offering a versatile tool for addressing complex inflammatory disorders and age-related immune dysfunction in a targeted and personalized manner.