Cyclodextrin microneedles for the delivery of a nanoparticle-based peptide antigen vaccine.

In recent years, microneedles (MNs) have gained considerable interest in drug formulation due to their non-invasive and patient-friendly nature. Dissolving MNs have emerged as a promising approach to enhance drug delivery across the skin in a painless manner without generating sharp waste and providing the possibility for self-administration. Cyclodextrins, a group of cyclic oligosaccharides, are well-established in pharmaceutical products due to their safety and unique ability to form inclusion complexes with various drug molecules. In this manuscript, we report the development and characterization of dissolving MNs composed of cyclodextrins for intradermal delivery of a cyclodextrin-based nanoparticulate vaccine. Different cyclodextrins were tested and the most promising candidates were fabricated into MNs by micromolding. The MNs' piercing effectiveness and drug permeation across the skin were tested ex vivo. Furthermore, in vivo studies were carried out to assess the skin's tolerance to cyclodextrin-based MNs, and to evaluate the immune response using a model peptide antigen in a mouse model. The data revealed that the MNs were well-tolerated and effective, even leading to dose-sparing effects. This study highlights the potential of cyclodextrin-based dissolving MNs as a versatile platform for intradermal vaccine delivery, providing a compatible matrix for nanoparticulate formulations to enhance immune responses.

Photochemically-Mediated Inflammation and Cross-Presentation of Mycobacterium bovis BCG Proteins Stimulates Strong CD4 and CD8 T-Cell Responses in Mice.

Conventional vaccines are very efficient in the prevention of bacterial infections caused by extracellular pathogens due to effective stimulation of pathogen-specific antibodies. In contrast, considering that intracellular surveillance by antibodies is not possible, they are typically less effective in preventing or treating infections caused by intracellular pathogens such as Mycobacterium tuberculosis. The objective of the current study was to use so-called photochemical internalization (PCI) to deliver a live bacterial vaccine to the cytosol of antigen-presenting cells (APCs) for the purpose of stimulating major histocompatibility complex (MHC) I-restricted CD8 T-cell responses. For this purpose, Mycobacterium bovis BCG (BCG) was combined with the photosensitiser tetraphenyl chlorine disulfonate (TPCS2a) and injected intradermally into mice. TPCS2a was then activated by illumination of the injection site with light of defined energy. Antigen-specific CD4 and CD8 T-cell responses were monitored in blood, spleen, and lymph nodes at different time points thereafter using flow cytometry, ELISA and ELISPOT. Finally, APCs were infected and PCI-treated in vitro for analysis of their activation of T cells in vitro or in vivo after autologous vaccination of mice. Combination of BCG with PCI induced stronger BCG-specific CD4 and CD8 T-cell responses than treatment with BCG only or with BCG and TPCS2a without light. The overall T-cell responses were multifunctional as characterized by the production of IFN-γ, TNF-α, IL-2 and IL-17. Importantly, PCI induced cross-presentation of BCG proteins for stimulation of antigen-specific CD8 T-cells that were particularly producing IFN-γ and TNF-α. PCI further facilitated antigen presentation by causing up-regulation of MHC and co-stimulatory proteins on the surface of APCs as well as their production of TNF-α and IL-1ß in vivo. Furthermore, PCI-based vaccination also caused local inflammation at the site of vaccination, showing strong infiltration of immune cells, which could contribute to the stimulation of antigen-specific immune responses. This study is the first to demonstrate that a live microbial vaccine can be combined with a photochemical compound and light for cross presentation of antigens to CD8 T cells. Moreover, the results revealed that PCI treatment strongly improved the immunogenicity of M. bovis BCG.

Cyclosporine A, in Contrast to Rapamycin, Affects the Ability of Dendritic Cells to Induce Immune Tolerance Mechanisms.

Following organ transplantation, it is essential that immune tolerance is induced in the graft recipient to reduce the risk of rejection and avoid complications associated with the long-term use of immunosuppressive drugs. Immature dendritic cells (DCs) are considered to promote transplant tolerance and may minimize the risk of graft rejection. The aim of the study was to evaluate the effects of immunosuppressive agents: rapamycin (Rapa) and cyclosporine A (CsA) on generation of human tolerogenic DCs (tolDCs) and also to evaluate the ability of these cells to induce mechanisms of immune tolerance. tolDCs were generated in the environment of Rapa or CsA. Next, we evaluated the effects of these agents on surface phenotypes (CD11c, MHC II, CD40, CD80, CD83, CD86, CCR7, TLR2, TLR4), cytokine production (IL-4, IL-6, IL-10, IL-12p70, TGF-ß), phagocytic capacity and resistant to lipopolysaccharide activation of these DCs. Moreover, we assessed ability of such tolDCs to induce T cell activation and apoptosis, Treg differentiation and production of Th1- and Th2-characteristic cytokine profile. Data obtained in this study demonstrate that rapamycin is effective at generating maturation-resistant tolDCs, however, does not change the ability of these cells to induce mechanisms of immune tolerance. In contrast, CsA affects the ability of these cells to induce mechanisms of immune tolerance, but is not efficient at generating maturation-resistant tolDCs.

Photochemical internalization (PCI)-mediated activation of CD8 T cells involves antigen uptake and CCR7-mediated transport by migratory dendritic cells to draining lymph nodes.

Antigen cross-presentation to cytotoxic CD8+ T cells is crucial for the induction of anti-tumor and anti-viral immune responses. Recently, co-encapsulation of photosensitizers and antigens into microspheres and subsequent photochemical internalization (PCI) of antigens in antigen presenting cells has emerged as a promising new strategy for inducing antigen-specific CD8+ T cell responses in vitro and in vivo. However, the exact cellular mechanisms have hardly been investigated in vivo, i.e., which cell types take up antigen-loaded microspheres at the site of injection, or in which secondary lymphoid organ does T cell priming occur? We used spray-dried poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with ovalbumin and the photosensitizer tetraphenyl chlorine disulfonate (TPCS2a) to investigate these processes in vivo. Intravital microscopy and flow cytometric analysis of the murine ear skin revealed that dendritic cells (DCs) take up PLGA microspheres in peripheral tissues. Illumination then caused photoactivation of TPCS2a and induced local tissue inflammation that enhanced CCR7-dependent migration of microsphere-containing DCs to tissue-draining lymph nodes (LNs), i.e., the site of CD8+ T cell priming. The results contribute to a better understanding of the functional mechanism of PCI-mediated vaccination and highlight the importance of an active transport of vaccine microspheres by antigen presenting cells to draining LNs.

Photochemical Internalization: Light Paves Way for New Cancer Chemotherapies and Vaccines.

Photochemical internalization (PCI) is a further development of photodynamic therapy (PDT). In this report, we describe PCI as a potential tool for cellular internalization of chemotherapeutic agents or antigens and systematically review the ongoing research. Eighteen published papers described the pre-clinical and clinical developments of PCI-mediated delivery of chemotherapeutic agents or antigens. The studies were screened against pre-defined eligibility criteria. Pre-clinical studies suggest that PCI can be effectively used to deliver chemotherapeutic agents to the cytosol of tumor cells and, thereby, improve treatment efficacy. One Phase-I clinical trial has been conducted, and it demonstrated that PCI-mediated bleomycin treatment was safe and identified tolerable doses of the photosensitizer disulfonated tetraphenyl chlorin (TPCS2a). Likewise, PCI was pre-clinically shown to mediate major histocompatibility complex (MHC) class I antigen presentation and generation of tumor-specific cytotoxic CD8+ T-lymphocytes (CTL) and cancer remission. A first clinical Phase I trial with the photosensitizer TPCS2a combined with human papilloma virus antigen (HPV) was recently completed and results are expected in 2020. Hence, photosensitizers and light can be used to mediate cytosolic delivery of endocytosed chemotherapeutics or antigens. While the therapeutic potential in cancer has been clearly demonstrated pre-clinically, further clinical trials are needed to reveal the true translational potential of PCI in humans.