Functionalization of a self-assembling peptide with a TLR7 agonist promotes robust humoral and cellular immune responses

Introduction: Each year, 12.7 million deaths occur because we lack potent vaccines against key pathogens, such as malaria, tuberculosis, and HIV. Developing antigens that confer long-lasting, sterilizing immunity represents a major challenge; therefore, adjuvants have been employed to enhance the immune response. These generally function by stimulating the innate immune system which, in turn, helps increase the magnitude of the adaptive immune response to the antigen of interest. Alum, which greatly enhances the humoral (Th2) immune response, has been the gold standard vaccine adjuvant for nearly a century; however, its efficacy in boosting cellular (Th1) immunity is relatively limited. This is a key shortcoming that has motivated the development of new vaccine adjuvants that are capable of enhancing cellular immunity, which may be especially important for providing protection against intracellular bacteria, helminths, and cancer. Therefore, we set out to develop an adjuvant that would enhance both cellular and humoral immunity by attaching 1V209, a small-molecule toll-like receptor 7 (TLR7) agonist, to a positively-charged multidomain peptide, K2, which is known to self-assemble into hydrogels. We hypothesized that conjugating these molecules would improve the otherwise very poor water solubility of 1V209, improve its ability to access TLR7 on the endosome of antigen-presenting cells, and spatially and temporally co-localize the adjuvant with antigen entrapped within the peptide’s hydrogel network.

Materials and

Methods: Solid-phase peptide synthesis was used to produce K2 with 1V209 attached directly or in series with a variety of linkers and purified using high-performance liquid chromatography and mass spectroscopy. The TLR7 agonist activity of 1V209-K2 was then measured using a cell-based reporter system (HEK-Blue mTLR7). Next, a cell uptake study was performed by co-incubating fluorescently labeled K2 with HEK293 cells and quantifying internalization using flow cytometry. FTIR and rheology were then used to characterize peptide self-assembly and the resulting hydrogel’s mechanical properties, respectively. In vivo experiments were then performed to assess antigen release from the 1V209-functionalized hydrogels, measure the rate of hydrogel clearance, and determine the immunological benefits of co-delivering the hydrogel with the antigen. Briefly, fluorescent ovalbumin and fluorescently labeled 1V209-K2 were co-injected subcutaneously into SKH1-Elite mice and imaged longitudinally using an In Vivo Imaging System (IVIS) to assess antigen release. Blood and tissues from vaccinated mice were also collected to assess antibody titers via enzyme-linked immunosorbent assay (ELISA), immune cell type distribution and antigen specificity in the spleen using flow cytometry and enzyme-linked immunospot (ELISpot), and the local inflammatory response via histology and immunohistochemistry.

Results, Conclusions, and Discussions: Attaching 1V209 to K2 via its carboxylic acid improved the TLR7 agonist’s solubility by more than 200-fold and effectively increased its bioactivity (Figure 1A), which we show is likely attributable to the increase in drug uptake that occurs because of 1V209-K2’s positive charge (Figure 1B). Employing linkers between 1V209 and K2 did not meaningfully affect bioactivity, so all subsequent studies used 1V209 directly attached to the N-terminus of K2 (Figure 1A). Attaching 1V209 to K2 did not inhibit K2’s ability to self-assemble into beta sheets or form hydrogels (Figure 1C,D); however, the mechanical properties of the resulting hydrogel were poor relative to K2, and therefore blends of 1V209-K2 and the stronger hydrogel-forming K2 were used. IVIS studies revealed that 1V209-K2 hydrogels remained at the injection site for at least 3 months while antigen was 75% released within 1 week (Figure 1E,F). Interestingly, the kinetics of ovalbumin release from 1V209-K2 hydrogels was very similar to release from alum-adsorbed ovalbumin. Immunologically, 1V209-K2 promoted the infiltration of cells (Figure 1G), including CD11c dendritic cells. Immunohistochemical staining revealed increased levels of TNF-alpha in 1V209-K2 hydrogels relative to the alum depot. Larger numbers of F4/80 macrophages were also observed surrounding the 1V209-K2 hydrogel compared to the alum and K2 injection sites. Antibody titers achieved when ovalbumin was delivered in 1V209-K2 were slightly higher than titers achieved with alum, though the difference was not significant (Figure 1H). Antibody subclasses suggested a more balanced immune response in mice treated with 1V209-K2 compared to alum with a significantly lower IgG1:IgG2a ratio of 1.4 ± 1.1 for 1V209-K2 compared to 9 ± 4.2 for alum on a log2 scale. ELISpot results also revealed a significant increase in the number of interferon-gamma- and TNF-alpha-producing splenocytes following re-stimulation with ovalbumin compared to mice receiving alum-adjuvanted formulations (Figure 1I,J), again suggestive of enhanced cellular immunity. Taken together, these results suggest that 1V209-K2 enhances the humoral immune response, like alum, and also meaningfully enhances the cellular immune response, unlike alum, making it a promising tool for improving vaccines against a number of infectious diseases and cancer.

Acknowledgements (Optional): This work was supported by the National Institute of Health (R01 DE030140, K22AI146215, R35GM143101), the Welch Foundation (C-2141), and the Cancer Prevention and Research Institute of Texas (RR190056).