Peptides Derived from the Tight Junction Protein CLDN1 Disrupt the Skin Barrier and Promote Responsiveness to an Epicutaneous Vaccine

Abstract

Keratinocytes express many pattern recognition receptors that enhance the skin's adaptive immune response to epicutaneous antigens. We have shown that these pattern recognition receptors are expressed below tight junctions (TJ), strongly implicating TJ disruption as a critical step in antigen responsiveness. To disrupt TJs, we designed peptides inspired by the first extracellular loop of the TJ transmembrane protein CLDN1. These peptides transiently disrupted TJs in the human lung epithelial cell line 16HBE and delayed TJ formation in primary human keratinocytes. Building on these observations, we tested whether vaccinating mice with an epicutaneous influenza patch containing TJ-disrupting peptides was an effective strategy to elicit an immunogenic response. Application of a TJ-disrupting peptide patch resulted in barrier disruption as measured by increased transepithelial water loss. We observed a significant increase in antigen-specific antibodies when we applied patches with TJ-disrupting peptide plus antigen (influenza hemagglutinin) in either a patch-prime or a patch-boost model. Collectively, these observations demonstrate that our designed peptides perturb TJs in human lung as well as human and murine skin epithelium, enabling epicutaneous vaccine delivery. We anticipate that this approach could obviate currently used needle-based vaccination methods that require administration by health care workers and biohazard waste removal.

Characterization of Human Keratinocyte Cell Lines for Barrier Studies

“…Reductions in CLDN1 have been observed in subjects with atopic dermatitis and CLDN1 knockout mice die shortly after birth due to cutaneous water loss, implicating its importance in barrier function (De Benedetto et al, 2011a, De Benedetto et al, 2011b, Furuse et al, 2002. Two days post-differentiation, all three cell types display circumferential OCLN immunoreactivity and the formation of the characteristic "honeycomb" pattern (Brewer et al, 2020). The only KC that showed distinct circumferential staining for J o u r n a l P r e -p r o o f OCLN at day two post-differentiation was PHFK, yet this pattern was still somewhat incomplete and was not seen across the entire field (Fig 1B contains a nuclear localization sequence and its localization to the nucleus has been previously reported (Hagen, 2017).…”

“…Immunofluorescent staining of TJ formation in PHFK, N/TERT2G, and HaCaT. PHFK, N/TERT2G, and HaCaT cells were plated onto sterilized glass coverslips and stained for immunofluorescent analysis of occludin, claudin-1 and DAPI as previously described (Brewer et al, 2020).…”

“…PHFKs, N/TERT2G cells, and HaCaT cells were plated onto sterilized glass coverslips and stained for immunofluorescent analysis for OCLN, CLDN1, and DAPI as previously described ( Brewer et al., 2020 ).…”

Skin Barriers in Dermal Drug Delivery: Which Barriers Have to Be Overcome and How Can We Measure Them?

“…Several approaches to enhance drug delivery via modification of TJ proteins, especially claudin-1, have been described. Application of a TJ-disrupting peptide patch addressing claudin-1 results in barrier disruption as measured by increased TEWL [ 79 ]. When using theses peptides in combination with an epicutaneous influenza vaccination patch, immune response was increased.…”

Emerging skin-targeted drug delivery strategies to engineer immunity: A focus on infectious diseases

“…Despite the attractive advantages of cutaneous vaccination over prevailing immunization routes, the skin has been an underutilized organ for clinical immunization largely due to the lack of effective drug delivery systems that can enable safe, reproducible, and patient-friendly deployment of antigens to skin microenvironments [64,[68][69][70][71]. Palpably, the stratum corneum, the outermost layer of the skin (Figure 1), consists of dead keratinocytes (corneocytes) and constitutes a formidable physical barrier to delivery of vaccine components to immunologically rich skin layers (viable epidermis and dermis, Figure 1), thereby necessitating sophisticated skin-targeted drug delivery strategies for effective intracutaneous immunization [72][73][74]. Topical drug delivery is an appealing method for patients due to its simplicity and non-invasiveness; however, topical administration of antigenic compounds and biological adjuvants, which are relatively complex biomolecules, is hampered by the formidable barrier function of the superficial cutaneous layers, which substantially reduces the bioavailability of vaccines, and in turn, the efficacy of immunization [75][76][77].…”