Recently, we characterized the novel immunization method DIVA based on the unique combination of the two adjuvants IMQ and dithranol, suitable for the induction of antigen-specific memory T cell responses (9). DIVA induces memory T cells solely by non-invasive application on native skin. In continuation of this work, we wanted to examine whether the application sequence of the adjuvants and peptides and their dose could be optimized further boosting T cell activation.

Firstly, we asked whether the order of application for the adjuvants is relevant. Therefore, we treated the ears of C57BL/6 mice first with dithranol and afterwards with IMQ (as in DIVA) or vice versa and observed the induced ear swelling as well as the frequency of antigen-specific T cells in the primary immune response. Interestingly, one week after treatment ear swelling was significantly increased after the reversed application order (IMQ-dithranol) compared to the application of dithranol followed by IMQ ( Figure 1B ) indicating an augmented local inflammatory reaction. In contrast, the median frequency of antigen-specific T cells was reduced (0.8%) after IMQ-dithranol sequence and lower compared to dithranol-IMQ application (1.7%) ( Figure 1C ). While the CTL frequency displayed no statistically significant difference, IFN-ɤ production was strongly enhanced in mice immunized with dithranol-IMQ in comparison to IMQ-dithranol ( Figure 1D ), thus demonstrating that the application of dithranol prior to IMQ is important for the induction of a functionally relevant CTL response upon DIVA along with alleviating local skin irritation.

To further reduce skin irritation, it was investigated whether both adjuvants need to be colocalized at the same skin site. Therefore, we compared the application of DIVA as described before with single application of the two adjuvants on two distinct skin sites. Mice were treated with dithranol on the left ear followed by IMQ the next day on the right ear and analyzed for primary T cell responses ( Figure 1A ). As shown in Figures 1E, F , performing DIVA on only one ear, and thus decreasing the treated skin area by 50% resulted lower specific CTL frequencies along with a 50% reduction in specific target cell lysis in vivo. This suggests that the treated skin area directly correlates with the specific cytolytic immune response upon DIVA. Beyond this, when dithranol and IMQ were applied on separated skin areas, no specific CTL response was detectable ( Figures 1E, F ), indicating that both adjuvants crucially require local concurrence of both compounds for the induction of a potent immune response upon DIVA.

To further define the required dosages for the synergistic effect of dithranol and IMQ in DIVA, we used titrated amounts IMQ or dithranol in DIVA, respectively, and subsequently analyzed the primary immune response. As depicted in Figure 2 , the administration of IMQ at 2.5 mg per ear was sufficient to induce a median frequency of antigen-specific T cells at about 2% with a reduction decreasing the amounts of IMQ ( Figure 2A ). This was paralleled for the specific cytolytic activity defining an amount of about 600 µg IMQ per ear as minimal dose for the induction of a detectable CTL response upon DIVA ( Figure 2B ) and demonstrates that the amount of IMQ correlates with the frequency and activity of antigen-specific T cells. For dithranol, we found that 16 µg or 8 µg per ear were necessary for the optimal induction of a primary immune response characterized by a significantly increased frequency of antigen-specific T cells ( Figure 2C ).

Finally, we also sought to define the optimal amount of antigen required for the induction of T cell responses upon DIVA and titrated the peptide amounts. As we hypothesized that the antigen dose during the priming will impact not only the primary immune response, but also memory formation (discussed in detail in (11)), we assessed the CTL frequency not only at 7- or 14-days post DIVA, but also after 35 days by ex vivo ELISpot assay without prior in vitro expansion. For the primary CTL response, we were able to detect antigen-specific T cells in the peripheral blood applying as little as 1 mg peptide onto the skin ( Figure 2D ), indicating that only minute amounts of peptide must pass the skin barrier to prime a specific CTL response at the peak to expansion 14 days post DIVA. However, to induce a persistent memory response peptide amounts of at least 30 µg were required ( Figure 2D ). Investigating functionality of the memory T cells by IFN-ɤ ELISpot assay, we used OVA_257-264 or OVA_323-337 peptides to detect specific CD8- or CD4-T cell activities in the memory phase. Here, the highest IFN-ɤ production by T cells was observed after immunization with 100 µg OVA_257-264 peptide ( Figure 2E ) or 100 µg OVA_323-337 peptide ( Figure 2F ). Different from our previous publication (9) the IFN-ɤ production is apparently different, most likely due to technical reasons.

In summary, for an optimal immune response, the efficacy of DIVA relies on dithranol and IMQ in a dose dependent manner. While both adjuvants as well as peptide antigen can be effective already at minute amounts to induce a primary specific immune reaction and concurrently reduce local skin irritation, at least 30 µg peptide antigens are required for the generation of effector memory T cells.

IMQ formulated in IMI-Sol is a solid nano-emulsion of crystalline IMQ of crumble consistency ( Figure 3A ) that becomes viscous upon exposure to shear force to release IMQ cargo (8, 12). However, this formulation has limited shelf life as the lipophilic components tend to oxidize and degrade, becoming hygroscopic at room temperature, thus requiring below –20°C for storage (13). Due to the shear-thinning behavior, IMI-Sol cannot be filled, stored and applied in standard aluminum tubes, which are otherwise frequently used for topical products. To increase shelf life and applicability in the light of our aim to translate DIVA for application in humans, we modified IMI-Sol to a spreadable semi-solid oleogel formulation termed IMI-Sol+ ( Figure 3B ). This was achieved by adding an oleogel consisting of aerosil, isopropyl-myristate and medium chain trigylcerides and evaluated whether this alteration would affect the efficacy of DIVA. Mice were treated with DIVA using either IMI-Sol+ or IMI-Sol together with OVA_257-264. The generation of OVA_257-264-specific T cells was determined in the peripheral blood as before after one week and followed up until memory phase. In the primary immune response, both formulations induced potent frequencies of antigen-specific CD8⁺ T cells ( Figure 3C ) with comparable effector functions assessed by in vivo cytolytic activity ( Figure 3D ). In the memory phase on day 35, a significant population of OVA_257-264-specific T cells was detectable in both groups ( Figure 3E ) with comparable IFN-ɤ production ( Figure 3F ). DIVA-induced side effects, characterized by ear swelling, were comparable between the two IMQ formulations ( Figure 3G ). Thus, the formulation IMI-Sol+ facilitates the standardized administration of IMQ with equivalent activity in DIVA compared to IMI-Sol and improved shelf life and storage properties.

DIVA induces a potent primary T cell response in a frequency of up to 10% of circulating CD8 T cells after 14 days protective against a lethal vaccinia virus challenge (9). To assess whether DIVA-induced T cell responses are limited to single application, we investigated whether it is possible to further boost the generated cellular immune response by a repeated immunization. Since we defined a minimal dose of dithranol required for the induction of a primary immune response ( Figure 2C ), we reasoned that for repeated application lower doses of dithranol or prolonged intervals between treatments might be helpful to enhance vaccination efficacy and ameliorate local skin reactions. Therefore, modulations of DIVA application scheme included the dose of dithranol (ranging from 2 to 8 µg dithranol/ear) and up to three treatment cycles as detailed in Supplementary Figure 1A . While in case of repeated application of DIVA more than 8 µg dithranol caused severe skin irritations (not shown), we observed a massive increase in the frequency of specific CD8⁺ T cells in the peripheral blood upon repeated application on day 14 compared to single treatment also using lower doses of dithranol ( Supplementary Figure 1B ). Interestingly, monitoring the CTL frequency over time, we found the most pronounced CTL expansion up to 60% using DIVA twice with 8 µg Dithranol (DIVA²) ( Supplementary Figures 1C, D ). Notably, lower doses of dithranol or an additional treatment cycle yielded lower CTL frequencies. Moreover, this strong CTL response was accompanied with transiently aggravated local skin reactions ( Supplementary Figure 1E ) indicating that repeated DIVA treatment is limited by adverse local skin reaction as well as in terms of immune cell stimulation favoring one additional vaccination for conversion to persistent memory T-cell responses.

Next, we asked for the optimal interval between treatments for DIVA. Therefore, we compared DIVA as single treatment with an additional treatment cycle after 7 or 14 days. As shown in Figure 4 , we observed that an additional treatment cycle resulted in a major increase in specific CTL frequency compared to single application. Although, single application of DIVA resulted in a considerable primary and memory CTL response, a second treatment cycle massively boosted the antigen-specific CTL response at both time points (at 7 or 14 days post priming). However, the peak expansion up to 40% peptide specific CTLs occurred upon secondary treatment after 7 days ( Figure 4B ) compared to only 20% specific CTLs after 14 days ( Figure 4C ). Notably, also IFN-ɤ production ex vivo and effector phenotype persisted until day 49 indicative of a durable effector memory CTL response. We also revealed the impact of the application area needed for immunization. In this context we reduced the immunization area for DIVA² by treating only one ear which corresponds to an area of about 1.5 cm² with different antigen doses. In this context, antigen-specific CTL response is not significantly affected by reducing the skin area by half. Additionally, we analyzed the boosting effect of DIVA² when the second treatment was performed on a different skin area. In detail, mice were treated on one ear in the first immunization and boosted after 7 days on the other ear. In this context, antigen-specific CTL frequencies were not affected after high dose immunization or addition of MHC class II restricted OVA_323-337 peptides compared to boosting the same skin side twice ( Supplementary Figure 3 ). Overall, this allows the conclusion that DIVA² with a secondary boost after 7 days results in superior CTL expansion and memory formation.

After establishing the optimized vaccination scheme DIVA² we evaluated the biological efficacy of the induced specific T cell responses in a tumor model using MC38 colon carcinoma cells transfected to express OVA as a target neoantigen (MC38mOVA). C57BL/6 mice were immunized once or twice and subsequently challenged by subcutaneous injection of 5x10⁴ MC38mOVA tumor cells ( Figure 5A ). The vaccination efficiency of DIVA was assessed on day 0 by the frequencies of antigen-specific CD8⁺ T cells in the peripheral blood. As demonstrated before, repeated vaccination by DIVA² yielded a high frequency of antigen-specific T cells and an approximately 30-fold increase compared to single administration of DIVA ( Figure 5B ). Although, single DIVA induced specific CTL frequencies up to 5%, we observed tumor outgrowth in 17% tumor challenged mice (2 of 12 mice injected with MC38mOVA). In contrast, in DIVA²-treated mice no tumors were detectable, thus 100% tumor protection was achieved ( Figures 5C, D ), highlighting the relevance of sufficient numbers of activated CTLs for full tumor clearance.

Taken together, these data indicate that DIVA² boosts specific CD8⁺ T cell generation that converts to effective primary and memory responses that are highly functional in clearing MC38mOVA tumor cells in vivo in a prophylactic tumor vaccination setting.

C57BL/6 mice - purchased from the Envigo Laboratory (Envigo, Indianapolis, USA) or were obtained from the local animal facility of the Johannes Gutenberg-University of Mainz (TARC) - were used at the age of 8-10 weeks. All animal studies were conducted according to the national guidelines and were reviewed and confirmed by an institutional review board/ethics committee headed by the local animal welfare officer of the University Medical Center (Mainz, Germany). The responsible national authority (National Investigation Office Rhineland-Palatine, Koblenz, Germany) gave approval of the animal experiments (Approval ID: AZ 23 177-07/G18-1-096).

The IMQ-containing formulation IMI-Sol was formulated as previously described (8, 12). IMI-Sol+ is produced by dispersing IMI-Sol into an oleogel formulation consisting of 7% aerosol and 10% isopropyl-myristate (IPM) in medium chain triglyceride-base (MCT). Unification of the two formulations was performed in a melanin bowl in a ratio of 1:1 and filled into aponorm aluminum tubes. The formulation can be stored at 4°C.

Immunizations were performed under isoflurane/oxygen anesthesia. For single DIVA (9), mice were treated on both ears with 25 mg dithranol-petrolatum (0.6 µg/mg - 0.08 µg/mg), corresponding to a total amount of dithranol per ear of 16 µg - 2 µg. Dithranol formulations were manufactured by the Pharmacy of the UMC Mainz according to European Pharmacopeia (Ph. Eur.) standards. On the following day, mice were treated with 50 mg-125 mg of IMI-Sol formulation or IMI-Sol+ containing 5% IMQ manufactured by the group of Prof. Peter Langguth, JGU Mainz, Germany) (corresponding to a total amount of 2.5 mg - 0.6 mg IMQ per ear). Ovalbumin peptides OVA_257-264 (SIINFEKL) and OVA_323-337 (OTII) (concentrations as indicated in the range of 1-100 µg each, from peptides & elephants, Henningsdorf, Germany) were solved in DMSO and applied stirred in hydrophobic DAC basic cream immediately after IMQ treatment. For boost immunization, treatment was repeated after 7 or 14 days.

Prior to tumor cell inoculation, mice were immunized two times with DIVA as indicated above. For the inoculation of MC38mOVA tumor cells mice were anesthetized. 5x10⁴ tumor cells diluted in 100 μl PBS per mouse were implanted subcutaneously (s.c.) on the shaved right flank. After 6 days tumors were palpable. Tumor size was measured three times per week with a digital caliper and the tumor volume was calculated. The survival of the mice was monitored. The Tumor experiment was stopped when the tumor volume of a mouse exceeded 600 mm³ or when ulceration of a tumor was observed.

At the end of an immunization experiment, mice were sacrificed, spleens were removed and homogenized over a 70 µm cell strainer with a syringe plunger. Erythrocytes were lysed in a hypotonic lysis buffer followed by resuspension in PBS. For ELISpot analysis, cells were counted using animal blood counter Scil Vet ABC™ (Scil animal care company GmbH, Viernheim, Germany) and resuspended in Iscove`s modified Dulbecco’s medium (IMDM, Gibco, Carlsbad, USA) supplemented with 10% FCS, 1% glutamine an 1% sodium pyruvate at a concentration of 2x10⁷ cells/ml). For flow cytometric analysis of the in vivo cytotoxicity, cells were resuspended in PBS supplemented with 0.5% BSA and 5mM EDTA (FACS buffer).

For flow cytometric analysis of DIVA-induced specific T cell responses blood samples were obtained through a puncture of the tail. Erythrocytes were lysed via hypotonic lysis with ACK buffer for 10 minutes at RT. Cells were resuspended in FACS buffer. Samples were incubated for 30 min at 4°C with fluorescently labeled antibodies against CD8 (Pacific Blue-conjugated, clone 53-6.7, BioLegend, Koblenz, Germany), CD44 (APC-conjugated, clone IM7, BioLegend, San Diego, USA) and CD62L (FITC-conjugated, clone MEL-14, BioLegend, San Diego, USA). T cells specific for -H2K^b-OVA_-257-264 were detected by H2-K^b tetramer (PE-conjugated, in-house production, Institute for Immunology, Johannes Gutenberg University Medical Center, Mainz, Germany). All analyses were performed with a LSRII Flow Cytometer and FACSDiva software (BD Pharmingen, Hamburg, Germany).

The effector function of the antigen-specific CD8⁺ T cells was assessed by transfer of 2 x 10⁷ target splenocytes labelled with either 4 mM (CFSEhigh) or 0.4 mM (CFSElow) carboxyfluorescein diacetate succinimidyl ester. The CFSElow cells were additionally loaded with SIINFEKL-peptide (OVA_257-264) (2 mM) for 1h at 37°C. Both populations were transferred i. v. in a 1:1 ratio. 20 h after in vivo cell transfer, blood samples or spleen cell suspensions of immunized and naïve control mice were obtained and the specific lysis of the peptide-pulsed target cells was determined by flow cytometry. All analyses were performed with a LSRII Flow Cytometer and FACSDiva software (BD Pharmingen, Hamburg, Germany).

Peptide-specific release of IFN-ɤ was assessed as previously described (9). Briefly, 96 Well MultiScreenHTS IP plates (0.45 mm, Merck Millipore, Darmstadt, Germany) were coated over night at 4°C with murine anti-IFN-ɤ antibody (clone AN18, Mabtech, Nacka Strand, Sweden (10 μg/ml)). After a washing step with PBS, the membrane was blocked with IMDM (Gibco, Carlsbad, USA) supplemented with 10% FCS, 1% glutamine and 1% sodium pyruvate for at least 60 min at 37°C. Spleen single cell suspensions were prepared as described before. 5x10⁵ splenocytes were incubated in the absence or presence of OVA_257-264 or OVA_323-337 peptides (each 1 μM). After 20 h of incubation at 37° C and 5% CO₂ the plate was washed six times with PBS including 0.05% Tween and the produced IFN-ɤ was detected with a biotinylated anti-IFN-ɤ antibody (clone R4-6A2, Mabtech, Nacka Strand, Sweden (2μg/ml)). After an additional washing step as described above, the enzyme-substrate reaction was performed to visualize the occurred binding using the Vectastain ABC Kit (Vector Laboratories, Burlingame, USA) together with AEC (Sigma-Aldrich, Taufkirchen, Germany) as described by the manufacturer. The reaction was stopped with H₂O. After drying, spot forming units (SFU) were measured with the automated image analysis system AID iSpot ELISpot reader (AID Autoimmun Diagnostika, Straßberg, Germany).

The statistical analysis was performed using GraphPad Prism (version 9.4.1 for Mac OS, GraphPad Software, San Diego California, USA). Multiple comparisons between more than two groups were performed with Kruskal-Wallis test and Dunn’s post-test or with two-way ANOVA and Bonferroni’s post-test as appropriate. Comparisons of survival curves were performed by Log-rank (Mantel-Cox) test. The significance level was determined as a p value </= 0.05.

Gating strategies for flow cytometry Figure S2 .