Introduction: Active immunotherapies are versatile biological tools that can be utilized to induce protective or therapeutic immune responses against specific targets, yet to date their application remains limited to select infectious diseases. Broadening the utility of active immunotherapies to new diseases -even those beyond infectious diseases- requires delivery strategies where targets can be reached with precision to stringently control the immune phenotype.
Lung inflammatory diseases such as chronic obstructive pulmonary disorders, idiopathic pulmonary fibrosis, and cystic fibrosis have limited therapeutic treatment options but could potentially be treated with an active immunotherapy approach. To do so, it is necessary to engineer materials that can access the lymphoid tissues of the lower respiratory tract (Woodward et al., 2024). In this study, we investigated the delivery of peptide nanofibers, an interesting class of biomaterials that can serve as an engineerable active immunotherapy platform. Specifically, C29 is an - helical peptide that self-assembles into a nanofiber structure and can be delivered via multiple routes (subcutaneous, sublingual, intranasal) (Wu et al., 2017). These nanofibers can further be modified with B-cell epitopes of inflammatory cytokines including IL-1 and TNF at the N-terminus to be presented by antigen-presenting cells (APCs) and elicit anti-inflammatory antibody responses (Shores et al., 2017). This study investigated the extent to which intranasally delivered peptide nanofibers can engage pulmonary immune cells, towards a therapy for chronic inflammatory lung conditions.
Materials and
Methods: C29 peptides appended with B-cell epitopes from IL-1 and TNF were produced using solid phase peptide synthesis, confirmed with MALDI, and purified via HPLC. Self-assembly of the synthesized peptides into nanofibers was accomplished with previously published methods. Assembly and complexation with the mucosal adjuvant cyclic-di-AMP was measured using TEM-energy dispersive X-ray spectroscopy (EDS).
To measure lung delivery in vivo, mice were intranasally immunized with nanofibers modified with B-cell epitopes at varying doses and adjuvanting conditions and compared to subcutaneously immunized control groups. Epitope-specific humoral responses in sera and lung parenchyma were measured by ELISA, and lung-specific immune cells were assessed with flow cytometry. Immune cell infiltrates to the lung were assessed histologically, and functional differences in respiration were quantified using whole-body plethysmography.
Results, Conclusions, and Discussions: Following intranasal delivery of the nanofibers, anti-cytokine IgG responses were detected in the lungs up to 14 days following the last immunization. Neither the choice of delivery route nor dose had a significant impact on the strength of the IgG antibody response. In contrast, IgA responses against TNF were dependent on adjuvanting conditions. Mice immunized intranasally with 50 L of the dual-cytokine epitope nanofibers with adjuvant significantly raised IgA responses greater than the non-adjuvanting and subcutaneous delivery groups. The co-delivery of cyclic-di-AMP could have contributed to this heighted antibody response, because the small molecule is a mucosal adjuvant which amplifies the activity of APCs in presenting available antigens in mucosal sites such as the lungs.
Comparison of systemic IgG responses against both IL-1 and TNF by ELISA analysis indicated that antibody responses were epitope-dependent. The lowest dose of intranasally delivered nanofibers was able to engage responses comparable to that of a 2-fold greater dose against IL-1. The addition of adjuvant also significantly contributed to sustained IgG responses against IL-1 up to 14 days following the last immunization. Both results contributed to dose-sparing implications for later active immunotherapy formulations. In contrast, neither dose nor adjuvanting conditions impacted IgG serum responses against the TNF epitope. This could result from preferential immunodominance of one cytokine epitope over the other upon co-delivery.
Whole body plethysmography of mice treated with the C29 peptides were compared against a naïve group, a diseased-LPS treatment group, and an alternative peptide fiber (Q11) treatment group. Breath frequency, tidal volume ventilation, and peak inspiratory/expiratory flow measurements revealed that administration of C29 with the anti-inflammatory cytokine epitopes intranasally were comparable to that of the naïve group and almost 2x greater than that of the diseased and Q11 treatment groups. This suggests that the intranasal administration of C29 peptide nanofibers does not deleterious affect lung function.
In sum, peptide nanofibers can raise systemic and lung mucosal antibody responses after intranasal immunization, and we identified nanofiber formulations that can accomplish this without affecting lung functionality. These results indicate a future path for lung delivery of peptide nanofibers for active immunotherapies.
Acknowledgements (Optional): This work was supported by Duke University and by NIH Grant R01AI67300.
