One of the greatest pharmaceutical challenges in vaccinology is the delivery of antigens to the cytosol of antigen-presenting cells (APCs) in order to allow for the stimulation of major histocompatibility complex (MHC) class I-restricted CD8(+) T-cell responses, which may act on intracellular infections or cancer. Recently, we described a novel method for cytotoxic T-lymphocyte (CTL) vaccination by combining antigens with a photosensitizer and light for cytosolic antigen delivery. The goal of the current project was to test this immunization method with particle-based formulations. Liposomes were prepared from dipalmitoylphosphatidylcholine and cholesterol, and the antigen ovalbumin (OVA) or the photosensitizer tetraphenyl chlorine disulfonate (TPCS2a) was separately encapsulated. C57BL/6 mice were immunized intradermally with OVA liposomes or a combination of OVA and TPCS2a liposomes, and light was applied the next day for activation of the photosensitizer resulting in cytosolic release of antigen from phagosomes. Immune responses were tested both after a prime only regime and after a prime-boost scheme with a repeat immunization 2 weeks post priming. Antigen-specific CD8(+) T-cell responses and antibody responses were analyzed ex vivo by flow cytometry and ELISA methods. The physicochemical stability of liposomes upon storage and light exposure was analyzed in vitro. Immunization with both TPCS2a- and OVA-containing liposomes greatly improved CD8(+) T-cell responses as compared to immunization without TPCS2a and as measured by proliferation in vivo and cytokine secretion ex vivo. In contrast, OVA-specific antibody responses (IgG1 and IgG2c) were reduced after immunization with TPCS2a-containing liposomes. The liposomal formulation protected the photosensitizer from light-induced inactivation during storage. In conclusion, the photosensitizer TPCS2a was successfully formulated in liposomes and enabled a shift from MHC class II to MHC class I antigen processing and presentation for stimulation of strong CD8(+) T-cell responses. Therefore, photosensitive particulate vaccines may have the potential to add to current vaccine practice a new method of vaccination that, as opposed to current vaccines, can stimulate strong CD8(+) T-cell responses.

Allergen-specific subcutaneous (SCIT) as well as sublingual (SLIT) immunotherapy both confer long-term protection against the highly prevalent IgE-mediated allergies, such as allergic rhino-conjunctivitis and asthma. As both SCIT and SLIT are both very laborious and time-consuming treatments, we and others have investigated whether the immunotherapy could be enhanced by so-called intralymphatic immunotherapy (ILIT), which aims at improving treatment efficacy and safety and to shorten treatment duration, thereby making allergen immunotherapy (AIT) more patient friendly. In the original studies made by us [1, 2] and by Cardell et al. [3], ILIT was administered three times with four-week intervals. In 2013, Witten et al. questioned the efficacy of ILIT with grass pollen, based on the results of a clinical study of their in 38 adult hayfever patients [4]. While promising immunological changes such as increased IgG4 and Treg responses were observed, they concluded that the clinical outcome was at odds with that measured by us in 165 hay-fever and 20 cat-dander allergic patients as well as in Cardell's study with 28 hay-fever patients. The protocols in these studies differed in one major point. While our and Cardell's studies used a dosing schedule of 4 weeks, Witten's study utilized a shorter dosing schedule of 2 weeks. We chose the longer time intervals based on general vaccine recommendations as they allow buildup of successive waves of allergen-specific immune responses with efficient memory B-cell formation and affinity maturation without antibody interference. Moreover, CD4 T cells compete for antigen, and highaffinity T cells have a competitive advantage [5]. Of note, no FDA-approved childhood vaccine has a shorter time interval than 4 weeks, and the official guidelines consider a vaccination 'invalid' if the time interval is shortened to less than the recommended minimum.Comparably less is known about the role of time intervals in AIT, and especially in newer methods such as ILIT. To address this question experimentally, a preclinical test in mice was run to investigate the effects of varying ILIT dosing intervals on certain characteristics of the antibody response. Groups of five female BALB/c mice were sensitized with 1 lg ovalbumin (OVA) adsorbed to aluminium hydroxide by 4 weekly subcutaneous (s.c.) injections. Three weeks later, the mice received ILIT injections with 10 lg OVA into the inguinal lymph nodes [6]. The ILIT was repeated for a total of three injections, the time interval between each injection being one, two or four weeks (Fig. 1a). ILIT preparations with OVA were made with aluminium hydroxide [6,7] as this was the adjuvant also used in the clinical ILIT trials [1-4]. Blood was collected from tail veins at different time points during the study. OVA-specific IgG 1 , IgG 2a and IgE in serum as well as the OVA-specific affinity of IgG were measured at different time points by means of ELISA.Sensitization caused only a weak induction of anti-OVA IgG 1 a barely measurable induction of IgG 2a , but ...

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