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Vaccination with nanoparticles combined with micro-adjuvants protects against cancer

Vaccination with nanoparticles combined with micro-adjuvants protects against cancer Background: Induction of strong T cell responses, in particular cytotoxic T cells, is a key for the generation of efficacious therapeutic cancer vaccines which yet, remains a major challenge for the vaccine developing world. Here we demonstrate that it is possible to harness the physiological properties of the lymphatic system to optimize the induction of a protective T cell response. Indeed, the lymphatic system sharply distinguishes between nanoscale and microscale particles. The former reaches the fenestrated lymphatic system via diffusion, while the latter either need to be transported by dendritic cells or form a local depot. Methods: Our previously developed cucumber-mosaic virus-derived nanoparticles termed (CuMV -VLPs) incorporating TT a universal Tetanus toxoid epitope TT830–843 were assessed for their draining kinetics using stereomicroscopic imaging. A nano-vaccine has been generated by coupling p33 epitope as a model antigen to CuMV -VLPs using bio-orthogonal TT Cu-free click chemistry. The CuMV -p33 nano-sized vaccine has been next formulated with the micron-sized TT microcrystalline tyrosine (MCT) adjuvant and the formed depot effect was studied using confocal microscopy and trafficking experiments. The immunogenicity of the nanoparticles combined with the micron-sized adjuvant was next assessed in an aggressive transplanted murine melanoma model. The obtained results were compared to other commonly used adjuvants such as B type CpGs and Alum. Results: Our results showed that CuMV -VLPs can efficiently and rapidly drain into the lymphatic system due to their TT nano-size of ~ 30 nm. However, formulating the nanoparticles with the micron-sized MCT adjuvant of ~ 5 μMresultedin a local depot for the nanoparticles and a longer exposure time for the immune system. The preclinical nano-vaccine CuMV -p33 formulated with the micron-sized MCT adjuvant has enhanced the specific T cell response in the stringent TT B16F10p33 murine melanoma model. Furthermore, the micron-sized MCT adjuvant was as potent as B type CpGs and clearly superior to the commonly used Alum adjuvant when total CD8 , specific p33 T cell response or tumour protection were assessed. Conclusion: The combination of nano- and micro-particles may optimally harness the physiological properties of the lymphatic system. Since the nanoparticles are well defined virus-like particles and the micron-sized adjuvant MCT has been used for decades in allergen-specific desensitization, this approach may readily be translated to the clinic. Keywords: Cucumber-mosaic virus CuMV, Virus-like particle VLP, Microcrystalline tyrosine MCT, Nano-vaccine * Correspondence: Mona.mohsen@dbmr.unibe.ch; Monamona20@icloud.com Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK Department of BioMedical Research, Immunology RIA, Inselspital, University of Bern, Bern, Switzerland Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mohsen et al. Journal for ImmunoTherapy of Cancer (2019) 7:114 Page 2 of 12 Introduction T 1-biased immunological profile compared with the Nanoparticles, specifically virus-like particles (VLPs), have widely used Alum adjuvant [12, 15]. This is also fur- succeeded as prophylactic vaccines and are now widely ther indicated where immunological synergy is ob- used. However, mounting an efficient immune response served when MCT is combined with Monophosphoryl by therapeutic cancer vaccines is still a challenging area, Lipid A®, since it was shown that a combination of highlighting the need for improved vaccine formulations. MCT and MPL was synergistic in enhancing murine Three main parameters are pivotal for the development of antigen specific IgG Ab responses without increasing an effective cancer vaccine: a cancer antigen, a delivery antigen specific IgE responses [15]. A short-course platform and an adjuvant. A large number of different for- allergy vaccine using this adjuvant combination has mulations have been studied extensively in the past years; recently completed a successful Phase II study in however head-to-head comparative data are still scarce Europe and has been marketed as a named patient and remain untested in humans [1]. Furthermore, vaccine product for a number of years [16]. Further evidence formulations have not been studied or optimized with re- has now emerged on MCT’s more specific immuno- spect to the size of delivery platforms versus adjuvants. modulatory functions. MCT activates the inflamma- Cucumber-mosaic virus (CuMV) coat protein may be some in vitro and has been demonstrated to induce expressed as a recombinant plant nano-sized virus-like par- sustained and robust innate responses, including, spe- ticle (VLP) and has been studied as a promising candidate cific adaptive T cell responses in a variety of immune- vaccine platform by displaying relevant epitopes for the in- applications [11, 13, 14]. Consequently, proof of duction of immune responses [2]. CuMV-VLPs are icosahe- concept studies using MCT across a broader vaccine dral nanoparticles capable of inducing both humoral and scope is being pursued more rigorously and has since cellular immune responses by generating neutralizing anti- highlighted its adaptable nature within various formu- + + bodies (Abs), CD4 T cells and CD8 cytotoxic T lympho- lation designs with other adjuvants / delivery systems cytes (CTLs) [3, 4]. Previously, we have developed an in optimizing immune responses [17]. engineered nano-sized cucumber-mosaic virus-derived VLP Here we harnessed the influence of particle’ssizeon by incorporating a universal Tetanus toxoid epitope TT830– draining properties and their efficacy in producing effective 843 which we termed CuMV -VLPs [5]. The incorporation CTLs response against an aggressive B16F10 tumour model TT of the universal T cell epitope has been shown to be a by combining a CuMV nano-vaccine with the micron- TT powerful enhancer of the immune response in Tetanus tox- size MCT adjuvant. We compared the response to other oid immunized mice. Using the engineered nano-sized commonly used adjuvants such as the well-established CuMV -VLPs as a vaccine platform in humans is also ex- Alum and the potent B type immunostimulatory CpGs. We TT pected to enhance their immune responses, since this epi- show that the micron-sized MCT is a powerful adjuvant for tope is recognized in essentially all humans who all have CuMV -p33 nano-vaccine displaying p33 epitope derived TT memory CD4 T cells specific for the epitope due to vaccin- from LCMV, rivaling CpGs and clearly performing better ation against tetanus [5]. We have shown in several studies than Alum in inducing CTLs and tumour protection. Thus, that CuMV -VLPs nano-vaccine induce protective and this combination may be an optimal way to formulate TT therapeutic Ab responses in mice, horses and dogs [5–7]. cancer nano-vaccines with micron-sized adjuvants thereby Depot-forming adjuvants can prolong antigen presenta- taking advantage of the physiological properties of the tion time to antigen-presenting cells (APCs), protect anti- lymphatic system. gen from degradation and are optimal for T cell enhancement and clonal expansion [8, 9]. Microcrystalline Materials and methods tyrosine (MCT) is a classical adjuvant used in the niche Expression and production of CuMV -VLPs TT area of allergy immunotherapy, some of which include CuMV -VLPs expression and production was performed TT products that are licensed or are currently in late stage clin- as described in detail in [5]. ical development [10]. MCT forms crystals of natural L-Tyrosine and due to their micron-size, they cannot read- Electron microscopy ily enter the lymphatics and remain at the injection site, Physical stability and integrity of CuMV -VLPs were TT forming a depot and local inflammation [11]. The introduc- visualized by transmission electron microscopy using the tion of MCT as a depot was based on its favorable bio- Philips CM12 EM. For imaging, sample-grids were glow degradable properties where a 48 h half-life at the injection discharged and 5 μl of VLP solution was added for 30 s. site has been previously reported, making it suitable to The grids were then washed 3x with ddH O and nega- adapt within formulations designed for weekly-dose tivly stained with 5 μl of 5% uranyl acetate for 30 s. Fi- administration as is also often used for cancer vac- nally, excess uranyl acetate was removed by pipetting cines [11–14]. Moreover, early immunological studies and the grids were air dried for 10 min. Images were in various animal models showed a more favorable taken with 84,000x and 110,000x magnification. Mohsen et al. Journal for ImmunoTherapy of Cancer (2019) 7:114 Page 3 of 12 Mice days at 37 °C and scanned at Leica SP8 with 10x lense, Wild type C57BL/6 mice were purchased from Harlan. 1024 × 1024 resolution, tile scan was performed in LNs −/− RAG2 mice on a C57BL/6 background were provided that were not fitting in the field of view of the 10x lense. by Ochsenbein’ lab and were bred in our pathogen-free Images were analyzed and segmented the individual animal facility. All in vivo experiments used 8–12-week-old CMV particles using Imaris Software v9.2.1 (Bitplane). female. All animal procedures were performed in accord- ance with the Swiss Animals Act (455.109.1) (September Depot effect with trafficking experiment 2008, 5th) of University of Bern. 10 μgof AF488 CuMV -p33 nano-vaccine formulated or TT not formulated with 50 μl of 4% MCT adjuvant was Stereomicroscopic imaging injected in WT C57BL/6 mice footpads (8–12 weeks; Har- WT C57BL/6 mice (8–12 weeks; Harlan) were anesthe- lan) using isoflurane anesthesia. Popliteal LNs were col- tized and prepared for imaging by shaving their right leg. lected 3 h, 24 h, 48 h, 96 h and 216 h after injection and Skin and adipose tissues were removed to expose the pop- treated with collagenase D (Roch) in 10%FSC containing liteal lymph node (LN) as described in detail in [18]. The DMEM for 25 min at 37 °C. Cells were stained with live/ anesthetized mice were then stabilized on a customized dead dye (eBioscience) and analyzed for total number of platform for imaging. The popliteal LN was located by FITC (CuMV -p33). Naïve mice were used as a control. TT bright field illumination imaging. A dose of 10 μgof CuMV -VLPs vaccine was labelled with Alexa Fluor 488 Measuring p33 specific CD8 T cell response in the spleen TT (AF488) according to the manufacturer’sinstructions Six groups of WT C57BL/6 mice (8–12 weeks old; Harlan) (Thermo Fisher SCIENTIFIC) and injected subcutane- were vaccinated s.c. with a single dose of: 1st group 70 μg ously (s.c.) into the mouse footpad to study the draining of CuMV -VLPs, 2nd group 70 μgofCuMV -Actin TT TT kinetics of CuMV -VLPs. Fluorescent light illumination nano-vaccine, 3rd group 70 μgofCuMV p33 nano-vac- TT TT- with a CCD Nikon camera was used for imaging. cine 4th group 70 μgofCuMV p33 nano-vaccine , TT- admixed with 15 nmol of B type CpGs 5′′-TCC ATG ACG Development of CuMV -p33 nano-vaccine using bio- TTC CTG ATG CT-3′′) (20 mer) (Invivogen), 5th group TT orthogonal cu-free click chemistry 70 μgofCuMV p33 nano-vaccine formulated with 4% TT- CuMV -VLPs were derivatized using 10-fold molar ex- MCT adjuvant (40 mg/ ml) (Allergy Therapeutics Ltd. TT cess of DBCO cross-linker (Dibenzocyclooctyne-N-hy- Worthing, UK) and the 6th group 70 μgofCuMV p33 TT- droxysuccinimidyl ester) (Sigma-Aldrich) in 2 mM nano-vaccine formulated with 100 μl of Alhydrogel EDTA and 20 mM NaP, pH 7.5 for 30 min at RT in a adjuvant 2% (InvivoGen). Formulating CuMV -p33 with TT shaker at 400 rpm. Excess uncoupled DBCO was re- MCT or Alum requires prolonged mixing of both moved by diafiltration steps. Modified p33 peptide components for 1 h at RT in shaker at 400 rpm to ensure H-KAVYNFATMGGCK(N3)-NH2 was purchased from adequate adsorption of the VLPs on MCT or Alum surface. (Pepscan PRESTO) and reconstituted using DMSO. Seven days later, spleens were collected and staining was 10-fold molar excess of the modified peptide was then performed using Fc-block, live/dead, anti-CD8 (eBioscience) added to the derivatized CuMV -VLPs. 5-fold molar and p33 (KAVYNFATM) tetramer designed using H-2Db TT excess of TCEP was added to liberate cysteine residues allele and PE fluorochrome (TCMetrix). at the C-terminus on the VLPs. The coupling was performed for 1 h at RT in a shaker at 400 rpm. Excess Intra-cellular cytokine (ICS) staining for IFN-γ and TNF-α peptide was removed using 100 kDa MWCO amicon Intra-cellular cytokine staining was performed on centrifuge tubes (Sigma Aldrich). The efficiency of the spleens and TILs of vaccinated WT C57BL/6 mice for coupling was tested by SDS-PAGE (Bio-RAD) and measuring of IFN-γ and TNF-α cytokines as described assessed by densitometric analysis of SDS-PAGE of Cu in detail in [20]. MV -VLP monomer bands compared to CuMV -VLP TT TT monomer plus p33 after coupling. Tumour experiments 1×10 cells of B16F10p33 melanoma cell line (From −/− Depot effect with confocal microscopy Ochsenbein lab) was injected into the flank of RAG2 10 μg of AF488 CuMV -p33 nano-vaccine formulated C57BL/6 mice (From Ochsenbein lab). Twelve days later TT or not formulated with 50 μl of 4% MCT adjuvant was the growing tumours were collected and processed into injected in WT C57BL/6 mice footpads (8–12 weeks; ~2mm fragments for transplantation into the flank of Harlan) using isoflurane anesthesia. Popliteal LNs were WT C57BL/6 mice (8–12 weeks old; Harlan) under full collected 3 h, 24 h, 48 h, 96 h and 216 h after injection. anesthesia. The transplanted WT C57BL/6 mice were Lymph nodes were fixed in PFA 2% for 3 h at RT. The treated 3 times over 14 days (mice in the control group LNs were transferred to CUBIC 1 medium [19] for 8 reached the humane end-point at day 14). In the first Mohsen et al. Journal for ImmunoTherapy of Cancer (2019) 7:114 Page 4 of 12 tumor experiment mice were vaccinated s.c. as follows: VLP monomer show efficient coupling of p33 peptide to 1st group 70 μg of CuMV -VLPs, 2nd group 70 μgof CuMV -VLP monomers (Fig. 1d). TT TT CuMV -p33 nano-vaccine and 3rd group 70 μgof TT CuMV -p33 nano-vaccine formulated with 4% MCT The micron-sized MCT adjuvant displays depot effect TT adjuvant (40 mg/ ml) (Allergy Therapeutics Ltd. Wor- when combined with CuMV -p33 nano-vaccine TT thing, UK). In the second tumour experiment mice were Microcrystalline tyrosine (MCT) is considered to be a vaccinated s.c. as follows: 1st group 70 μg of CuM depot-forming adjuvant facilitating the slow but prolonged V -VLPs, 2nd group 70 μg of CuMV -p33 nano-vac- release of antigens. Formulating the nano-vaccine TT TT cine admixed with 15 nmol of B type CpGs 5′′-TCC CuMV -p33 with the micron-sized MCT adjuvant may TT ATG ACG TTC CTG ATG CT-3′′) (20 mer) (Invivo- therefore enhance the slow release of the nanoparticles dis- gen), 3rd group 70 μg of CuMV -p33 nano-vaccine playing the target epitope and extend their exposure to the TT formulated with 4% MCT adjuvant (40 mg/ ml) (Allergy immune system. To test that, we have first formulated the Therapeutics Ltd. Worthing, UK) and 4th group 70 μg AF488 CuMV -p33 nano-vaccine with MCT in vitro to TT of CuMV -p33 nano-vaccine formulated with 100 μlof visualize the binding of the nanoparticles to the TT Alhydrogel adjuvant 2% (InvivoGen). Tumour growth micron-sized MCT adjuvant by confocal microscopy. The was followed daily and measured using calipers. Tu- results showed that the labelled nanoparticles bind and dec- mours were collected and measured on day 14. TILs orate the surface of the micron-sized crystals (Fig. 2a). To were isolated by treating the tumours with collagenase D further study this hypothesis in vivo, we injected the AF488 (Roch) in 10%FSC containing DMEM for 25 min at 37 ° CuMV -p33 nano-vaccine (alone or formulated with TT C. Cells were passed through a cell strainer of 100 μm MCT adjuvant) into the footpad of WT C57BL/6 mice as il- (Corning) and TILs were separated using Ficoll (Sig- lustrated in Fig. 2b and collected the popliteal LNs at differ- ma-Aldrich). TILs were stained with Fc block, live/dead, ent time-points 3 h, 24 h, 48 h, 96 h and 216 h to assess the anti-CD8 (eBioscience) and p33 tetramers (TCMetrix). persistence of the labelled nanoparticles by flow cytometry. The results demonstrate that CuMV -p33 injected in free TT form disappears from the popliteal LN in ~ 4 days while Statistics formulating the nano-vaccine with the micron-sized MCT Tumour growth curves were compared by calculating adjuvant causes slower but prolonged release of the nano- the area-under curve (AUC) and analyzed by One-Way particles over 9 days (Fig. 2c). These findings were also ANOVA (Turkey’s Multiple Comparison Test). Other supported when imaging the popliteal LNs by confocal data has been analyzed and presented using Unpaired microscopy (Fig. 2d). Student’s t test. GraphPad Prism7 or 8 software was used for the analysis. Formulating CuMV -p33 nano-vaccine with the micron- TT sized MCT adjuvant induces significant p33 specific T cell Results response and enhances cytokines secretion CuMV -VLPs demonstrate fast kinetics and constitute an We then tested whether formulating CuMV -p33 TT TT efficient vaccine platform for displaying target peptides/ nano-vaccine with MCT would enhance the specific T epitopes cell response in vivo. Therefore, six vaccines and formu- In a first step, we produced the engineered CuMV -VLPs lations were prepared as outlined in Fig. 3a. CpG 1668 TT and confirmed their morphology, integrity and nano-size and Alum were independently formulated with by electron microscopy (Fig. 1a). Next, we studied the CuMV -p33 nano-vaccine to benchmark the potency TT draining-kinetics of CuMV -VLPs utilizing stereomiscro- of the micron-sized MCT adjuvant. Actin coupled to TT scopic imaging. To this end, CuMV -VLPs were labelled CuMV -VLPs was used as a non-specific control TT TT with the fluorescent dye AF488 and injected s.c. in the peptide to show that the obtained response is specific to footpad of WT C57BL/6 mice. Our results show that the p33 peptide. The different vaccine formulations were labelled nanoparticles accumulate in the popliteal LN in injected once s.c. in WT C57BL/6 mice and spleens were less than 1 min, demonstrating fast and efficient draining collected seven days later for tetramer and intra-cellular of free 30 nm CuMV -VLPs (Fig. 1b). To study the effi- cytokine staining. The H2-D allele p33 (KAVYNFATM) TT cacy of CuMV -VLPs as a cytotoxic T cell based nano- tetramers have been used to enable direct visualization TT vaccine, we have used the H-2D restricted p33 peptide and quantification of p33 specific T cells. The results derived from LCMV as a model antigen. The model pep- showed that admixing CuMV -p33 nano-vaccine with TT tide was coupled to CuMV -VLPs using our developed CpGs 1668 or formulating it with the micron-sized TT method based on “biorthogonal Cu-free click chemistry” MCT adjuvant induced the highest percentage of p33 as illustrated in Fig. 1c. The efficiency of the coupling was specific T cells upon single injection (Fig. 3b and f). We tested using SDS-PAGE. The additional bands above the then assessed the cytokine secretion in each group, Mohsen et al. Journal for ImmunoTherapy of Cancer (2019) 7:114 Page 5 of 12 Fig. 1 CuMV -VLPs demonstrate fast kinetics and constitute an efficient vaccine platform for displaying target peptides/epitopes. a Electron TT microscopy imaging of CuMV -VLPs, (3.5 mg/ml) adsorped on carbon grids and negatively stained with uranyl acetate solution, scale bar 200 nm, TT CuMV -VLPs sized ∼30 nm. b Stereomicroscopy images of mice popliteal LN following s.c. injection of AF488 CuMV -VLPs in mice footpad. [1]bright TT TT field of the popliteal LN (identified by the arrowhead) [2] fluorescent image prior to injection of AF488 CuMV -VLPs, [3–5] 1 min, 5 min and 10 min TT post injection of AF488 CuMV -VLPs taken with the appropriate fluorescent filters. c A sketch illustrating the coupling of p33 epitope to CuMV -VLPs TT TT using Cu-free click chemistry (Dibenzocyclooctyne-N-hydroxysuccinimidyl ester (DBCO) cross-linker). DBCO cross-linker reacts with Lys residues on CuMV -VLPs and incorporates a cyclooctyne moiety. The formed dibenzocyclooctyne will then react with azide-labelled p33 peptide forming a stable TT triazole linkage without Cu catalyst. d SDS-PAGE of CuMV -p33 nano-vaccine using DBCO “Dibenzocyclooctyne-N-hydroxysuccinimidyl ester” cross- TT linker; arrows indicate CuMV -VLP monomers and dimers (formed by DBCO cross-linking of 2 monomers) with coupled p33 peptide TT mainly IFN-γ and TNF-α. The secretion of both cyto- melanoma model, we have adapted a challenging melan- kines was enhanced when combining the nano-vaccine oma murine model based on transplanting ~2mm of with CpGs 1668 or MCT adjuvant (Fig. 3c and d). Formu- B16F10p33 tumour fragment into the flank of WT lating CuMV -p33 nano-vaccine with Alum did not en- C57BL/6 mice. The tumour was allowed to grow for 5 TT hance the production of p33 specific T cells nor the days more following transplantation before the vaccin- secretion of IFN-γ or TNF-α.Furthermore,whenanalyzing ation regimen started (Fig. 4a). Three groups were pre- the dual secretion of IFN-γ and TNF-α in polyfunctional T pared as illustrated in Fig. 4b, CuMV -VLPs as a TT cells, a large percentage of the cytokine-producing T cells control, CuMV -p33 nano-vaccine alone and CuM TT was found to be polyfunctional in the groups admixed with V -p33 nano-vaccine formulated with MCT. Tumours TT CpGs 1668 or MCT (Fig. 3eand g). were collected for analysis 14 days after tumour trans- plantation as the control group reached the ethically Formulating CuMV -p33 nano-vaccine with the micron- allowed maximal size of ~1000mm . The obtained TT sized MCT adjuvant delays tumour growth and enhances results revealed that formulating CuMV -p33 nano- TT CD8 and p33 specific CTL infiltration into B16F10p33 vaccine with the micron-sized MCT could significantly tumours hinder B16F10p33 tumour progression when compared In order to test the immunogenicity and efficacy of com- to the control group (p < 0.0001) or to the group vacci- bining nanoparticles with micron-sized adjuvants in a nated with CuMV -p33 nano-vaccine alone (p 0.0055) TT Mohsen et al. Journal for ImmunoTherapy of Cancer (2019) 7:114 Page 6 of 12 Fig. 2 The micron-sized MCT adjuvant displays depot effect when combined with CuMV -p33 nano-vaccine. a Confocal microscopy imaging of TT AF488 CuMV -p33 nano-vaccine following formulation with the micron-sized MCT adjuvant, 1) GFP signal of AF488 CuMV -p33 nano-vaccine 2) TT TT bright light field 3) an overlay 4–6 3D images with bright light field 4) an MCT crystal decorated with CuMV -p33 nano-vaccine particles. b A TT sketch illustrates the two prepared nano-vaccines, the 1st group consists of AF488 CuMV -p33 nano-vaccine and the 2nd consists of AF488 TT CuMV -p33 nano-vaccine formulated with MCT adjuvant. d Total number of AF488 CuMV -p33 nanoparticles in the popliteal LNs collected 3 h, TT TT 24 h, 48 h, 96 h and 216 h post-injection of the two prepared nano-vaccine groups in mice footpad. Statistical analysis by unpaired Student’s t test. c Confocal microscopy images of popliteal LNs 24 h and 216 h post-injection of the two prepared nano-vaccine groups in mice footpad, GFP signal was detected in LNs, whole mount view of z-stacks was acquired. One representative experiment of 3 similar experiments is shown (Fig. 4c and d). Tumour-infiltrating lymphocytes (TILs) then compared to the immune-stimulatory B type CpGs represent a prognostic factor for effective immune responses and the widely used adjuvant Alum using the same ag- especially in melanoma [21, 22]. Therefore, we measured the gressive B16F10p33 tumour model. Four groups were total number of the infiltrated CD8 T cells and p33 specific prepared as shown in Fig. 5a. The results again revealed CTLs (Fig. 4e) in TILs and calculated the density of these that formulating CuMV -p33 nano-vaccine with CpGs TT cells in each vaccinated group (number of cells divided by 1668 or MCT would significantly (p 0.0072, 0.0129 re- tumour volume). Formulating CuMV -p33 nano-vaccine spectively) hinder B16F10p33 tumour progression, TT with MCT significantly increased the density of total CD8 which was not the case when formulating CuMV -p33 TT T cells (p. 0.0024) (Fig. 4f) as well as the density of p33 spe- with Alum (p 0.4188) (Fig. 5b). In a next step, we mea- cific CTL measured by tetramers (p. 0.0093) (Fig. 4g) in sured the total number of infiltrated CD8 T cells (Fig. comparison to mice vaccinated with CuMV -p33 nano- 5c) and p33 specific CTLs (Fig. 5d) in the tumour and TT vaccine alone. IFN-γ production was also enhanced when calculated the density. There was a general increase in formulating CuMV -p33 with MCT (Fig. 4h). Thus, for- the groups mixed with CpGs 1668 or formulated with TT mulating the a nano-vaccine in MCT enhanced infiltration MCT or Alum (Fig. 5e and f). Formulating by specific T cells as well as anti-tumor protection. CuMV -p33 nano-vaccine with the micron-sized MCT TT adjuvant showed comparable results to CpGs 1668, the The micron-sized MCT adjuvant shows comparable gold standard adjuvant in mice. Furthermore, MCT ad- activity to B-type CpGs and is superior to alum in driving juvant was more potent at increasing the infiltration of protection against B16F10p33 melanoma total CD8 and p33-specific T cells into the tumour The immunogenicity of the CuMV -p33 nano-vaccine microenvironment when compared to formulating the TT formulated with the micron-sized MCT adjuvant was vaccine with Alum. Mohsen et al. Journal for ImmunoTherapy of Cancer (2019) 7:114 Page 7 of 12 Fig. 3 Formulating CuMV -p33 nano-vaccine with the micron-sized MCT adjuvant induces significant p33 specific T cell response and enhances TT cytokines secretion. a Vaccination scheme for six vaccine groups, CuMV -VLPs, CuMV -Actin, CuMV -p33, CuMV -p33 admixed with CpG 1668, TT TT TT TT + + CuMV -p33 formulated with MCT and CuMV -p33 formulated with Alum. b Percentage of CD8 Tetramer CTLs (means ± SEM) in the spleen in TT TT + + each vaccinated group. c Percentage of CD8 IFN-γ secreting cells (means ± SEM) in the spleen in each vaccinated group. d Percentage of + + + + + CD8 TNF-α cells (means ± SEM) in the spleen in each vaccinated group. e Percentage of CD8 IFN-γ /TNF-α secreting cells (means ± SEM) in the spleen in each vaccinated group. Statistical analysis by Oneway ANOVA (Turkey’s Multiple Comparison Test). f Representative flow cytometry + + dot plots showing the percentage of CD8 Tetramer CTLs in each vaccinated group. g Representative flow cytometry dot plots showing the + + + percentage of CD8 IFN-γ /TNF-α secreting cells in each vaccinated group. (3 mice per group), one representative of 3 similar experiments is shown The micron-sized MCT adjuvant shows comparable universal T cell epitope of Tetanus toxin (CuM production of cytokines to B type CpGs and is superior to V -VLPs). These nanoparticles constitute a promis- TT alum in B16F10p33 melanoma ing vaccine platform as the incorporated Tetanus Production of IFN-γ and TNF-a cytokines by TILs was toxin epitope can enhance their immunogenicity and also assessed upon immunization with CuMV -p33 the production of robust Ab and CTL responses espe- TT admixed with B type CpGs or formulated with MCT or cially in aging populations [5, 6]. Displaying epitopes Alum. Production of IFN-γ (Fig. 6a and b) and TNF-a on CuMV -VLP’s exterior surface can be achieved TT (Fig. 6c and d) was assessed separately or as dual cytokine by simple chemical techniques such as the SMPH production in polyfunational T cells (Fig. 6e and f). There heterobifunctional cross-linker. Such techniques have was no significant difference in the production of single shown efficacy and good results in many different IFN-γ, TNF-a or dual IFN-γ/TNF cytokines between the clinical settings [23–25]. However, we have lately en- groups admixed with CpGs 1668 or formulated with hanced the coupling efficacy of epitopes to bacterio- MCT adjuvant (p 0.3986, 0.3433 and 0.4120 respectively). phage Qβ-VLP using the biorthogonal Cu-free click However, there was a significant difference when compar- chemistry (Mohsen et al., submitted). Here we have ing the group formulated with MCT to the one formu- shown that such method can also be efficiently used lated with Alum (p 0.0179, 0.0187 and 0.006, respectively). to couple peptides/epitopes to CuMV -VLPs. Gener- TT ally, Cu-free click chemistry is a safe, non-toxic coup- Discussion ling method as the azide moiety attached to the In this study, we have developed cucumber mosaic target epitope does not react with any of the body’s virus-derived nanoparticles genetically fused to the natural molecules [26–28]. Mohsen et al. Journal for ImmunoTherapy of Cancer (2019) 7:114 Page 8 of 12 Fig. 4 Formulating CuMV -p33 nano-vaccine with the micron-sized MCT adjuvant causes tumour regression and enhances CD8 and p33 TT specific CTL infiltration into B16F10p33 tumours. a A diagram illustrating the adapted tumour experimental method based on injecting ~ 1 × 10 −/− B16F10p33 melanoma cell line into the flank of RAG2 deficient C57BL/6 mice. Twelve to thirteen days later the growing tumours were collected and processed in ~2mm for transplantation into the flank of WT C57BL/6. b Vaccination scheme for three vaccine groups, CuMV - TT VLPs, CuMV -p33 and CuMV -p33 formulated with MCT. c Tumour growth curve of subcutaneous B16F10p33 melanomas in each vaccinated TT TT 3 3 group, mice were euthanized when the tumour reached ~1000mm , arrows indicate start of treatment. d Tumour volume mm (mean ± SEM) measured at day 14 post tumour collection in each vaccinated group, each dot represents a tumour. e Representative flow cytometry dot plots + + + showing the total number of CD8 Tetramer CTLs in each vaccinated group. f Density of CD8 T cells (means ± SEM) in each vaccinated group, + ” + + “measured by dividing the total number of CD8 cells in TILs by the tumour volume . g Density of CD8 Tetramer CTLs (means ± SEM) in each + + ” + + vaccinated group, “measured by dividing the total number of CD8 Tetramer CTL by tumour volume . h Percentage of CD8 IFN-γ secreting cells (means ± SEM) in each vaccinated group. Statistical analysis by Student’s t test. (5 mice per group), one representative of 3 similar experiments is shown Immunostimulatory adjuvants such as synthetic mostly in mice and more rarely in humans [37]. In mice, CpG-oligonucleotides are TLR-agonists and have shown TLR-9 is expressed by all DCs while in human it is promising therapeutic potential by activating both the mainly expressed by pDCs in lymphoid organs but not innate and adaptive immune system [29–31]. CpGs have by conventional DCs [38]. pDCs, however, respond also been successfully used clinically to adjuvant cancer much more efficiently to A type CpGs rather than B vaccines [32, 33]. However, CpGs have some drawbacks type CpGs as only the former induce strong production including their unfavourable pharmacokinetics and their of type I IFN [39]. Hence, it may be difficult to directly propensity to cause splenomegaly, at least in mice [34, translate findings with B type CpGs from mice to 35]. It has been shown previously that packaging CpGs humans. With this respect, it is interesting to note that into VLPs such as Qβ or HBcAg can improve the CuMV VLPs naturally package RNA from the E. coli TT pharmacokinetics and dynamics of the DNA oligomers expression strain, a ligand for TLR7/8 which is [36]. Nevertheless, it is not always feasible to package expressed in all human DCs. VLPs with CpGs as some nanoparticles are unstable and Several preclinical and clinical studies have indicated packaging with reassembly processes may be time con- that some of the adjuvants used in licensed products are suming when targeting translational approaches. Fur- not optimal for developing effective cancer vaccines. Ex- thermore, TLR-9 agonists have been widely used with amples are the commonly used Montanide (Incomplete VLP-based vaccines to enhance T and B cell responses Freund’s adjuvant) and Alum [40–43]. Such limitations Mohsen et al. Journal for ImmunoTherapy of Cancer (2019) 7:114 Page 9 of 12 Fig. 5 The micron-sized MCT adjuvant shows comparable activity to B type CpGs and is superior to Alum in driving protection against B16F10p33 melanoma. a vaccination scheme for four vaccine groups, CuMV -VLPs, CuMV -p33 admixed with CpG 1668, CuMV -p33 formulated TT TT TT with MCT and CuMV -p33 formulated with Alum. b Tumour growth curve of subcutaneous B16F10p33 melanomas in each vaccinated group, TT mice were euthanized when the tumour reached ~1000mm , arrows indicate start of treatment. c Representative flow cytometry dot plots showing the total number of CD8 T cells in each vaccinated group, gated on TILs. d Representative flow cytometry dot plots showing the total + + + + number of CD8 Tetramer CTLs in each vaccinated group, gated on CD8 T cells. e Density of CD8 T cells (means ± SEM) in each vaccinated + ” + + group, “measured by dividing the total number of CD8 cells in TILs by the tumour volume . f Density of CD8 Tetramer CTLs (means ± SEM) in + ” each vaccinated group, “measured by dividing the total number of p33 tetramer CTLs by tumour volume . Statistical analysis by Student’s t test. (4 mice per group), one representative of 3 similar experiments is shown may include the inadequate ability of these adjuvants to immunotherapy. Protection against IgE-mediated allergic induce CTLs [44]. Nevertheless, use of B-type CpGs and response was achieved in mouse models, independently Alum in mice is useful where direct comparative adju- of inflammasome and TLR signaling in vivo. As has been vant studies are concerned. Since they are well charac- seen for Alum, the adjuvant activity of MCT was inde- terized in mice, both adjuvants are a good benchmark pendent of the inflammasome in vivo despite its ability when comparing other new or newly used adjuvants to activate the inflammasome in vitro [11]. In malaria, such as MCT. MCT has been shown to consistently enhance protective MCT is a micron-sized adjuvant that forms crystals of IgG responses and more protective IgG subclasses about ~ 5 μM that readily adsorb proteins including resulting in enhanced protection against malaria. Similar protein-based nanoparticles such as VLPs. MCT is well observations were made for influenza vaccine candidates known in the world of specific allergy immunotherapy, [13, 14, 17]. as it is categorized as a depot excipient in registered sub- Here, we have studied the draining kinetics of cutaneous immunotherapy products for the treatment of CuMV -p33 vaccine alone or formulated with adjuvant TT allergies. However, the knowledge about its mechanism MCT. Our results indicate that the free AF488 of action and its potential in different fields of vaccinol- CuMV -p33 can drain rapidly into the draining LN but TT ogy is only expanding now. In a recent study, MCT was fades away after ~ 4 days. In contrast, the release of shown to be an effective adjuvant in allergen-specific AF488 CuMV -p33 nano-vaccine formulated with TT Mohsen et al. Journal for ImmunoTherapy of Cancer (2019) 7:114 Page 10 of 12 Fig. 6 The micron-sized MCT adjuvant shows comparable production of cytokines to B type CpGs and is superior to Alum in B16F10p33 + + melanoma. a Percentage of CD8 IFN-γ secreting cells (means ± SEM) in each vaccinated group. b Representative flow cytometry dot plots + + + + showing the frequency of CD8 IFN-γ secreting cells in each vaccinated group. c Percentage of CD8 TNF- secreting cells (means ± SEM) in + + each vaccinated group. d Representative flow cytometry dot plots showing the frequency of CD8 TNF- secreting cells in each vaccinated + + + group. e Percentage of dual IFN-γ and TNF- secreting cells (means ± SEM) in each vaccinated tumour, gated on CD8 cells in TILs. f + + + Representative flow cytometry dot plots showing the frequency of dual IFN-γ and TNF- secreting cells in each group, gated on CD8 cells in TILs. Statistical analysis by Student’s t test. (4 mice per group), one representative of 3 similar experiments is shown MCT was delayed and more consistent over a longer The transplantation of solid tumor fragments allows period of time. Previous studies have mostly supported studying tumor development in the context of already the importance of the depot-forming adjuvants in T cell established tumor stroma which is more reflective of the based vaccine development as they delay the clearance physiological situation. When transplanted as solid frag- of the vaccine that results in enhancing the generation ments, even very immunogenic tumors grow in im- of effective antigen-specific CTL responses [45, 46]. munocompetent hosts and the vascularized tumours To study the immunogenicity of the developed rapidly grow to lethal size (47–50). The results show that CuMV -p33 nano-vaccine formulated with MCT, the formulating CuMV -p33 nano-vaccine with MCT was TT TT protective capacity of the induced CTL-responses was more potent in blocking tumour growth than using the assessed in an aggressive murine melanoma model con- nano-vaccine alone. The protective capacity of the sisting of B16F10 cells transfected with H-2D restricted CuMV -p33 with MCT is therefore strong, as the TT p33 epitope derived from LCMV. To generate tumors model used is very challenging. Previous studies have in- with maximal physiological properties, we have used a dicated that melanoma tumours exhibiting increased challenging tumour model based on transplanting solid numbers of tumour infiltrating CD8 T cells have better tumour fragments rather than single cell suspension. prognosis. TILs have led to a better understating of the Mohsen et al. Journal for ImmunoTherapy of Cancer (2019) 7:114 Page 11 of 12 interaction between hosts and tumours, mainly because Additional file 3: Movie S3. showing the popliteal LN 24 h after their study allowed better characterization of effective injecting CuMV -p33 nano-vaccine formulated with MCT adjuvant in TT mice footpad. (MP4 12060 kb) therapeutic responses [22, 47]. TILs isolated from the Additional file 4: Movie S4 showing the popliteal LN 216 h after vaccinated groups have been assessed for the presence injecting CuMV -p33 nano-vaccine in mice footpad. (MP4 30385 kb) TT of p33 specific CTLs by means of tetramers and intracel- Additional file 5: Movie S5 showing the popliteal LN 216 h after lular cytokine staining. CuMV -p33 vaccine formulated TT injecting CuMV -p33 nano-vaccine formulated with MCT adjuvant in TT with MCT adjuvant enhanced the infiltration of CD8 mice footpad. (MP4 34519 kb) and p33 specific CTLs into the tumour and the produc- tion of IFN-γ. These results indicate that MCT may be a Acknowledgments None. promising cancer adjuvant. When comparing MCT adju- vant to the potent B type CpGs or the widely used Alum, Funding the overall adjuvants activity of MCT was comparable to This work was supported by Bencard Adjuvant Systems, Dominion Way, UK, the Swiss Cancer League (KFS-4132-02-2017) and Qatar Foundation. CpGs and superior to Alum. It has been previously seen that Alum may increase over- Availability of data and materials all IgG responses at least as good as MCT. In contrast to Data are available in the main text and supplementary materials, raw data available upon request by the correspondent author. Alum, however, MCT induced superior IgG2a responses, which is usually associated with T 1 responses and/or TLR Authors’ contributions activity [14]. This may be compatible with the observed Design of experiments, acquisition of data, interpretation of data, analysis of data: MOM, MDH, GCM, MV, CR, DS, ER, DS and MFB; VLPs expression and ability of MCT to enhance CTL responses, which Alum production: CL, AZ; Confocal microscopy imaging and stereomicroscopic failed to do. Further work will be required to elucidate the imaging: MS, ER, PE and JVS. Writing, revise and revision of manuscript: mechanism of this difference, as both Alum and MCT form MOM, TMK, AK, DS, MV, MAS, MFK and MFB. Technical and material support: MOM, MDH, DS, LZ, AK and MFB. Study supervision: MFB. All authors read a depot and may activate the inflammasome pathway. An and approved the final manuscript. obvious hypothesis is that Alum induces a T 2driving pathway in addition to the inflammasome or vice versa, Ethics approval All procedures on animals were conducted at University of Bern in MCT may activate a T 1 driving pathway. The distinctions accordance with the Swiss Animals Act (455.109.1) (September 2008, 5th) portrayed in their respective immunological profiles are University of Bern. also likely to be be partly governed by their inherent formu- Consent for publication lation/structural characteristics (i.e. particle size, morph- Not applicable. ology, antigen adsorption etc). In addition to this, it is known that tryptophan or arginine serve as direct Competing interests The authors declare that they have no competing interests. Martin immune-modulators, a possibility that has not been exten- Bachmann and Mona Mohsen own shares of DeepVax GmbH involved in sively studied for tyrosine [48, 49]. developing virus-like particles based vaccines for cancer, other authors de- Taken together, this study shows that MCT is a potent clare that they have no competing interests. enhancer of CTL responses and may be viewed as a multi-purpose adjuvant with novel indications. As such, Publisher’sNote its effectiveness and compatibility in a mix- and match Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. adjuvant systems approach should be further tested in preclinical and human immunotherapy trials. Combin- Author details ation of MCT with nanoparticles appears particularly at- Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK. Department of BioMedical Research, Immunology RIA, tractive, as the micron-sized adjuvants will form a local Inselspital, University of Bern, Bern, Switzerland. Bencard Adjuvant Systems, depot at the injection site with concomitant activation of Dominion Way, Worthing, UK. Latvian Biomedical Research & Study Centre, skin-resident antigen-presenting cells. Nanoparticles will Riga, Latvia. Institute of anatomy, University of Bern, Bern, Switzerland. 6 7 Theodor Kocher Institute, University of Bern, Bern, Switzerland. Department be released over time, draining to local LNs for extended of Medical Oncology, Bern University Hospital, University of Bern, Bern, time-periods, causing an optimal immune reaction. Switzerland. International Immunology Center, Anhui Agricultural University, Thus, the combination of nanoparticles with micron- Hefei, Anhui, China. Department of dermatology, University of Zurich, Bern, Switzerland. Department of Oncology, University of Lausanne, Bern, sized adjuvants may optimally harness the properties of Switzerland. National Center for Cancer Care & Research (NCCCR), Doha, the lymphatic system. State of Qatar. Received: 13 November 2018 Accepted: 2 April 2019 Additional files References Additional file 1: Movie S1. showing AF488 CuMV -VLPs decorating the TT 1. Hu Z, Ott PA, Wu CJ. Towards personalized, tumour-specific, therapeutic surface of microcrystalline tyrosine crystals MCT adjuvant. (MP4 13481 kb) vaccines for cancer. Nat Rev Immunol. 2017;18(3):168–182. 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Abstract

Background: Induction of strong T cell responses, in particular cytotoxic T cells, is a key for the generation of efficacious therapeutic cancer vaccines which yet, remains a major challenge for the vaccine developing world. Here we demonstrate that it is possible to harness the physiological properties of the lymphatic system to optimize the induction of a protective T cell response. Indeed, the lymphatic system sharply distinguishes between nanoscale and microscale particles. The former reaches the fenestrated lymphatic system via diffusion, while the latter either need to be transported by dendritic cells or form a local depot. Methods: Our previously developed cucumber-mosaic virus-derived nanoparticles termed (CuMV -VLPs) incorporating TT a universal Tetanus toxoid epitope TT830–843 were assessed for their draining kinetics using stereomicroscopic imaging. A nano-vaccine has been generated by coupling p33 epitope as a model antigen to CuMV -VLPs using bio-orthogonal TT Cu-free click chemistry. The CuMV -p33 nano-sized vaccine has been next formulated with the micron-sized TT microcrystalline tyrosine (MCT) adjuvant and the formed depot effect was studied using confocal microscopy and trafficking experiments. The immunogenicity of the nanoparticles combined with the micron-sized adjuvant was next assessed in an aggressive transplanted murine melanoma model. The obtained results were compared to other commonly used adjuvants such as B type CpGs and Alum. Results: Our results showed that CuMV -VLPs can efficiently and rapidly drain into the lymphatic system due to their TT nano-size of ~ 30 nm. However, formulating the nanoparticles with the micron-sized MCT adjuvant of ~ 5 μMresultedin a local depot for the nanoparticles and a longer exposure time for the immune system. The preclinical nano-vaccine CuMV -p33 formulated with the micron-sized MCT adjuvant has enhanced the specific T cell response in the stringent TT B16F10p33 murine melanoma model. Furthermore, the micron-sized MCT adjuvant was as potent as B type CpGs and clearly superior to the commonly used Alum adjuvant when total CD8 , specific p33 T cell response or tumour protection were assessed. Conclusion: The combination of nano- and micro-particles may optimally harness the physiological properties of the lymphatic system. Since the nanoparticles are well defined virus-like particles and the micron-sized adjuvant MCT has been used for decades in allergen-specific desensitization, this approach may readily be translated to the clinic. Keywords: Cucumber-mosaic virus CuMV, Virus-like particle VLP, Microcrystalline tyrosine MCT, Nano-vaccine * Correspondence: Mona.mohsen@dbmr.unibe.ch; Monamona20@icloud.com Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK Department of BioMedical Research, Immunology RIA, Inselspital, University of Bern, Bern, Switzerland Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mohsen et al. Journal for ImmunoTherapy of Cancer (2019) 7:114 Page 2 of 12 Introduction T 1-biased immunological profile compared with the Nanoparticles, specifically virus-like particles (VLPs), have widely used Alum adjuvant [12, 15]. This is also fur- succeeded as prophylactic vaccines and are now widely ther indicated where immunological synergy is ob- used. However, mounting an efficient immune response served when MCT is combined with Monophosphoryl by therapeutic cancer vaccines is still a challenging area, Lipid A®, since it was shown that a combination of highlighting the need for improved vaccine formulations. MCT and MPL was synergistic in enhancing murine Three main parameters are pivotal for the development of antigen specific IgG Ab responses without increasing an effective cancer vaccine: a cancer antigen, a delivery antigen specific IgE responses [15]. A short-course platform and an adjuvant. A large number of different for- allergy vaccine using this adjuvant combination has mulations have been studied extensively in the past years; recently completed a successful Phase II study in however head-to-head comparative data are still scarce Europe and has been marketed as a named patient and remain untested in humans [1]. Furthermore, vaccine product for a number of years [16]. Further evidence formulations have not been studied or optimized with re- has now emerged on MCT’s more specific immuno- spect to the size of delivery platforms versus adjuvants. modulatory functions. MCT activates the inflamma- Cucumber-mosaic virus (CuMV) coat protein may be some in vitro and has been demonstrated to induce expressed as a recombinant plant nano-sized virus-like par- sustained and robust innate responses, including, spe- ticle (VLP) and has been studied as a promising candidate cific adaptive T cell responses in a variety of immune- vaccine platform by displaying relevant epitopes for the in- applications [11, 13, 14]. Consequently, proof of duction of immune responses [2]. CuMV-VLPs are icosahe- concept studies using MCT across a broader vaccine dral nanoparticles capable of inducing both humoral and scope is being pursued more rigorously and has since cellular immune responses by generating neutralizing anti- highlighted its adaptable nature within various formu- + + bodies (Abs), CD4 T cells and CD8 cytotoxic T lympho- lation designs with other adjuvants / delivery systems cytes (CTLs) [3, 4]. Previously, we have developed an in optimizing immune responses [17]. engineered nano-sized cucumber-mosaic virus-derived VLP Here we harnessed the influence of particle’ssizeon by incorporating a universal Tetanus toxoid epitope TT830– draining properties and their efficacy in producing effective 843 which we termed CuMV -VLPs [5]. The incorporation CTLs response against an aggressive B16F10 tumour model TT of the universal T cell epitope has been shown to be a by combining a CuMV nano-vaccine with the micron- TT powerful enhancer of the immune response in Tetanus tox- size MCT adjuvant. We compared the response to other oid immunized mice. Using the engineered nano-sized commonly used adjuvants such as the well-established CuMV -VLPs as a vaccine platform in humans is also ex- Alum and the potent B type immunostimulatory CpGs. We TT pected to enhance their immune responses, since this epi- show that the micron-sized MCT is a powerful adjuvant for tope is recognized in essentially all humans who all have CuMV -p33 nano-vaccine displaying p33 epitope derived TT memory CD4 T cells specific for the epitope due to vaccin- from LCMV, rivaling CpGs and clearly performing better ation against tetanus [5]. We have shown in several studies than Alum in inducing CTLs and tumour protection. Thus, that CuMV -VLPs nano-vaccine induce protective and this combination may be an optimal way to formulate TT therapeutic Ab responses in mice, horses and dogs [5–7]. cancer nano-vaccines with micron-sized adjuvants thereby Depot-forming adjuvants can prolong antigen presenta- taking advantage of the physiological properties of the tion time to antigen-presenting cells (APCs), protect anti- lymphatic system. gen from degradation and are optimal for T cell enhancement and clonal expansion [8, 9]. Microcrystalline Materials and methods tyrosine (MCT) is a classical adjuvant used in the niche Expression and production of CuMV -VLPs TT area of allergy immunotherapy, some of which include CuMV -VLPs expression and production was performed TT products that are licensed or are currently in late stage clin- as described in detail in [5]. ical development [10]. MCT forms crystals of natural L-Tyrosine and due to their micron-size, they cannot read- Electron microscopy ily enter the lymphatics and remain at the injection site, Physical stability and integrity of CuMV -VLPs were TT forming a depot and local inflammation [11]. The introduc- visualized by transmission electron microscopy using the tion of MCT as a depot was based on its favorable bio- Philips CM12 EM. For imaging, sample-grids were glow degradable properties where a 48 h half-life at the injection discharged and 5 μl of VLP solution was added for 30 s. site has been previously reported, making it suitable to The grids were then washed 3x with ddH O and nega- adapt within formulations designed for weekly-dose tivly stained with 5 μl of 5% uranyl acetate for 30 s. Fi- administration as is also often used for cancer vac- nally, excess uranyl acetate was removed by pipetting cines [11–14]. Moreover, early immunological studies and the grids were air dried for 10 min. Images were in various animal models showed a more favorable taken with 84,000x and 110,000x magnification. Mohsen et al. Journal for ImmunoTherapy of Cancer (2019) 7:114 Page 3 of 12 Mice days at 37 °C and scanned at Leica SP8 with 10x lense, Wild type C57BL/6 mice were purchased from Harlan. 1024 × 1024 resolution, tile scan was performed in LNs −/− RAG2 mice on a C57BL/6 background were provided that were not fitting in the field of view of the 10x lense. by Ochsenbein’ lab and were bred in our pathogen-free Images were analyzed and segmented the individual animal facility. All in vivo experiments used 8–12-week-old CMV particles using Imaris Software v9.2.1 (Bitplane). female. All animal procedures were performed in accord- ance with the Swiss Animals Act (455.109.1) (September Depot effect with trafficking experiment 2008, 5th) of University of Bern. 10 μgof AF488 CuMV -p33 nano-vaccine formulated or TT not formulated with 50 μl of 4% MCT adjuvant was Stereomicroscopic imaging injected in WT C57BL/6 mice footpads (8–12 weeks; Har- WT C57BL/6 mice (8–12 weeks; Harlan) were anesthe- lan) using isoflurane anesthesia. Popliteal LNs were col- tized and prepared for imaging by shaving their right leg. lected 3 h, 24 h, 48 h, 96 h and 216 h after injection and Skin and adipose tissues were removed to expose the pop- treated with collagenase D (Roch) in 10%FSC containing liteal lymph node (LN) as described in detail in [18]. The DMEM for 25 min at 37 °C. Cells were stained with live/ anesthetized mice were then stabilized on a customized dead dye (eBioscience) and analyzed for total number of platform for imaging. The popliteal LN was located by FITC (CuMV -p33). Naïve mice were used as a control. TT bright field illumination imaging. A dose of 10 μgof CuMV -VLPs vaccine was labelled with Alexa Fluor 488 Measuring p33 specific CD8 T cell response in the spleen TT (AF488) according to the manufacturer’sinstructions Six groups of WT C57BL/6 mice (8–12 weeks old; Harlan) (Thermo Fisher SCIENTIFIC) and injected subcutane- were vaccinated s.c. with a single dose of: 1st group 70 μg ously (s.c.) into the mouse footpad to study the draining of CuMV -VLPs, 2nd group 70 μgofCuMV -Actin TT TT kinetics of CuMV -VLPs. Fluorescent light illumination nano-vaccine, 3rd group 70 μgofCuMV p33 nano-vac- TT TT- with a CCD Nikon camera was used for imaging. cine 4th group 70 μgofCuMV p33 nano-vaccine , TT- admixed with 15 nmol of B type CpGs 5′′-TCC ATG ACG Development of CuMV -p33 nano-vaccine using bio- TTC CTG ATG CT-3′′) (20 mer) (Invivogen), 5th group TT orthogonal cu-free click chemistry 70 μgofCuMV p33 nano-vaccine formulated with 4% TT- CuMV -VLPs were derivatized using 10-fold molar ex- MCT adjuvant (40 mg/ ml) (Allergy Therapeutics Ltd. TT cess of DBCO cross-linker (Dibenzocyclooctyne-N-hy- Worthing, UK) and the 6th group 70 μgofCuMV p33 TT- droxysuccinimidyl ester) (Sigma-Aldrich) in 2 mM nano-vaccine formulated with 100 μl of Alhydrogel EDTA and 20 mM NaP, pH 7.5 for 30 min at RT in a adjuvant 2% (InvivoGen). Formulating CuMV -p33 with TT shaker at 400 rpm. Excess uncoupled DBCO was re- MCT or Alum requires prolonged mixing of both moved by diafiltration steps. Modified p33 peptide components for 1 h at RT in shaker at 400 rpm to ensure H-KAVYNFATMGGCK(N3)-NH2 was purchased from adequate adsorption of the VLPs on MCT or Alum surface. (Pepscan PRESTO) and reconstituted using DMSO. Seven days later, spleens were collected and staining was 10-fold molar excess of the modified peptide was then performed using Fc-block, live/dead, anti-CD8 (eBioscience) added to the derivatized CuMV -VLPs. 5-fold molar and p33 (KAVYNFATM) tetramer designed using H-2Db TT excess of TCEP was added to liberate cysteine residues allele and PE fluorochrome (TCMetrix). at the C-terminus on the VLPs. The coupling was performed for 1 h at RT in a shaker at 400 rpm. Excess Intra-cellular cytokine (ICS) staining for IFN-γ and TNF-α peptide was removed using 100 kDa MWCO amicon Intra-cellular cytokine staining was performed on centrifuge tubes (Sigma Aldrich). The efficiency of the spleens and TILs of vaccinated WT C57BL/6 mice for coupling was tested by SDS-PAGE (Bio-RAD) and measuring of IFN-γ and TNF-α cytokines as described assessed by densitometric analysis of SDS-PAGE of Cu in detail in [20]. MV -VLP monomer bands compared to CuMV -VLP TT TT monomer plus p33 after coupling. Tumour experiments 1×10 cells of B16F10p33 melanoma cell line (From −/− Depot effect with confocal microscopy Ochsenbein lab) was injected into the flank of RAG2 10 μg of AF488 CuMV -p33 nano-vaccine formulated C57BL/6 mice (From Ochsenbein lab). Twelve days later TT or not formulated with 50 μl of 4% MCT adjuvant was the growing tumours were collected and processed into injected in WT C57BL/6 mice footpads (8–12 weeks; ~2mm fragments for transplantation into the flank of Harlan) using isoflurane anesthesia. Popliteal LNs were WT C57BL/6 mice (8–12 weeks old; Harlan) under full collected 3 h, 24 h, 48 h, 96 h and 216 h after injection. anesthesia. The transplanted WT C57BL/6 mice were Lymph nodes were fixed in PFA 2% for 3 h at RT. The treated 3 times over 14 days (mice in the control group LNs were transferred to CUBIC 1 medium [19] for 8 reached the humane end-point at day 14). In the first Mohsen et al. Journal for ImmunoTherapy of Cancer (2019) 7:114 Page 4 of 12 tumor experiment mice were vaccinated s.c. as follows: VLP monomer show efficient coupling of p33 peptide to 1st group 70 μg of CuMV -VLPs, 2nd group 70 μgof CuMV -VLP monomers (Fig. 1d). TT TT CuMV -p33 nano-vaccine and 3rd group 70 μgof TT CuMV -p33 nano-vaccine formulated with 4% MCT The micron-sized MCT adjuvant displays depot effect TT adjuvant (40 mg/ ml) (Allergy Therapeutics Ltd. Wor- when combined with CuMV -p33 nano-vaccine TT thing, UK). In the second tumour experiment mice were Microcrystalline tyrosine (MCT) is considered to be a vaccinated s.c. as follows: 1st group 70 μg of CuM depot-forming adjuvant facilitating the slow but prolonged V -VLPs, 2nd group 70 μg of CuMV -p33 nano-vac- release of antigens. Formulating the nano-vaccine TT TT cine admixed with 15 nmol of B type CpGs 5′′-TCC CuMV -p33 with the micron-sized MCT adjuvant may TT ATG ACG TTC CTG ATG CT-3′′) (20 mer) (Invivo- therefore enhance the slow release of the nanoparticles dis- gen), 3rd group 70 μg of CuMV -p33 nano-vaccine playing the target epitope and extend their exposure to the TT formulated with 4% MCT adjuvant (40 mg/ ml) (Allergy immune system. To test that, we have first formulated the Therapeutics Ltd. Worthing, UK) and 4th group 70 μg AF488 CuMV -p33 nano-vaccine with MCT in vitro to TT of CuMV -p33 nano-vaccine formulated with 100 μlof visualize the binding of the nanoparticles to the TT Alhydrogel adjuvant 2% (InvivoGen). Tumour growth micron-sized MCT adjuvant by confocal microscopy. The was followed daily and measured using calipers. Tu- results showed that the labelled nanoparticles bind and dec- mours were collected and measured on day 14. TILs orate the surface of the micron-sized crystals (Fig. 2a). To were isolated by treating the tumours with collagenase D further study this hypothesis in vivo, we injected the AF488 (Roch) in 10%FSC containing DMEM for 25 min at 37 ° CuMV -p33 nano-vaccine (alone or formulated with TT C. Cells were passed through a cell strainer of 100 μm MCT adjuvant) into the footpad of WT C57BL/6 mice as il- (Corning) and TILs were separated using Ficoll (Sig- lustrated in Fig. 2b and collected the popliteal LNs at differ- ma-Aldrich). TILs were stained with Fc block, live/dead, ent time-points 3 h, 24 h, 48 h, 96 h and 216 h to assess the anti-CD8 (eBioscience) and p33 tetramers (TCMetrix). persistence of the labelled nanoparticles by flow cytometry. The results demonstrate that CuMV -p33 injected in free TT form disappears from the popliteal LN in ~ 4 days while Statistics formulating the nano-vaccine with the micron-sized MCT Tumour growth curves were compared by calculating adjuvant causes slower but prolonged release of the nano- the area-under curve (AUC) and analyzed by One-Way particles over 9 days (Fig. 2c). These findings were also ANOVA (Turkey’s Multiple Comparison Test). Other supported when imaging the popliteal LNs by confocal data has been analyzed and presented using Unpaired microscopy (Fig. 2d). Student’s t test. GraphPad Prism7 or 8 software was used for the analysis. Formulating CuMV -p33 nano-vaccine with the micron- TT sized MCT adjuvant induces significant p33 specific T cell Results response and enhances cytokines secretion CuMV -VLPs demonstrate fast kinetics and constitute an We then tested whether formulating CuMV -p33 TT TT efficient vaccine platform for displaying target peptides/ nano-vaccine with MCT would enhance the specific T epitopes cell response in vivo. Therefore, six vaccines and formu- In a first step, we produced the engineered CuMV -VLPs lations were prepared as outlined in Fig. 3a. CpG 1668 TT and confirmed their morphology, integrity and nano-size and Alum were independently formulated with by electron microscopy (Fig. 1a). Next, we studied the CuMV -p33 nano-vaccine to benchmark the potency TT draining-kinetics of CuMV -VLPs utilizing stereomiscro- of the micron-sized MCT adjuvant. Actin coupled to TT scopic imaging. To this end, CuMV -VLPs were labelled CuMV -VLPs was used as a non-specific control TT TT with the fluorescent dye AF488 and injected s.c. in the peptide to show that the obtained response is specific to footpad of WT C57BL/6 mice. Our results show that the p33 peptide. The different vaccine formulations were labelled nanoparticles accumulate in the popliteal LN in injected once s.c. in WT C57BL/6 mice and spleens were less than 1 min, demonstrating fast and efficient draining collected seven days later for tetramer and intra-cellular of free 30 nm CuMV -VLPs (Fig. 1b). To study the effi- cytokine staining. The H2-D allele p33 (KAVYNFATM) TT cacy of CuMV -VLPs as a cytotoxic T cell based nano- tetramers have been used to enable direct visualization TT vaccine, we have used the H-2D restricted p33 peptide and quantification of p33 specific T cells. The results derived from LCMV as a model antigen. The model pep- showed that admixing CuMV -p33 nano-vaccine with TT tide was coupled to CuMV -VLPs using our developed CpGs 1668 or formulating it with the micron-sized TT method based on “biorthogonal Cu-free click chemistry” MCT adjuvant induced the highest percentage of p33 as illustrated in Fig. 1c. The efficiency of the coupling was specific T cells upon single injection (Fig. 3b and f). We tested using SDS-PAGE. The additional bands above the then assessed the cytokine secretion in each group, Mohsen et al. Journal for ImmunoTherapy of Cancer (2019) 7:114 Page 5 of 12 Fig. 1 CuMV -VLPs demonstrate fast kinetics and constitute an efficient vaccine platform for displaying target peptides/epitopes. a Electron TT microscopy imaging of CuMV -VLPs, (3.5 mg/ml) adsorped on carbon grids and negatively stained with uranyl acetate solution, scale bar 200 nm, TT CuMV -VLPs sized ∼30 nm. b Stereomicroscopy images of mice popliteal LN following s.c. injection of AF488 CuMV -VLPs in mice footpad. [1]bright TT TT field of the popliteal LN (identified by the arrowhead) [2] fluorescent image prior to injection of AF488 CuMV -VLPs, [3–5] 1 min, 5 min and 10 min TT post injection of AF488 CuMV -VLPs taken with the appropriate fluorescent filters. c A sketch illustrating the coupling of p33 epitope to CuMV -VLPs TT TT using Cu-free click chemistry (Dibenzocyclooctyne-N-hydroxysuccinimidyl ester (DBCO) cross-linker). DBCO cross-linker reacts with Lys residues on CuMV -VLPs and incorporates a cyclooctyne moiety. The formed dibenzocyclooctyne will then react with azide-labelled p33 peptide forming a stable TT triazole linkage without Cu catalyst. d SDS-PAGE of CuMV -p33 nano-vaccine using DBCO “Dibenzocyclooctyne-N-hydroxysuccinimidyl ester” cross- TT linker; arrows indicate CuMV -VLP monomers and dimers (formed by DBCO cross-linking of 2 monomers) with coupled p33 peptide TT mainly IFN-γ and TNF-α. The secretion of both cyto- melanoma model, we have adapted a challenging melan- kines was enhanced when combining the nano-vaccine oma murine model based on transplanting ~2mm of with CpGs 1668 or MCT adjuvant (Fig. 3c and d). Formu- B16F10p33 tumour fragment into the flank of WT lating CuMV -p33 nano-vaccine with Alum did not en- C57BL/6 mice. The tumour was allowed to grow for 5 TT hance the production of p33 specific T cells nor the days more following transplantation before the vaccin- secretion of IFN-γ or TNF-α.Furthermore,whenanalyzing ation regimen started (Fig. 4a). Three groups were pre- the dual secretion of IFN-γ and TNF-α in polyfunctional T pared as illustrated in Fig. 4b, CuMV -VLPs as a TT cells, a large percentage of the cytokine-producing T cells control, CuMV -p33 nano-vaccine alone and CuM TT was found to be polyfunctional in the groups admixed with V -p33 nano-vaccine formulated with MCT. Tumours TT CpGs 1668 or MCT (Fig. 3eand g). were collected for analysis 14 days after tumour trans- plantation as the control group reached the ethically Formulating CuMV -p33 nano-vaccine with the micron- allowed maximal size of ~1000mm . The obtained TT sized MCT adjuvant delays tumour growth and enhances results revealed that formulating CuMV -p33 nano- TT CD8 and p33 specific CTL infiltration into B16F10p33 vaccine with the micron-sized MCT could significantly tumours hinder B16F10p33 tumour progression when compared In order to test the immunogenicity and efficacy of com- to the control group (p < 0.0001) or to the group vacci- bining nanoparticles with micron-sized adjuvants in a nated with CuMV -p33 nano-vaccine alone (p 0.0055) TT Mohsen et al. Journal for ImmunoTherapy of Cancer (2019) 7:114 Page 6 of 12 Fig. 2 The micron-sized MCT adjuvant displays depot effect when combined with CuMV -p33 nano-vaccine. a Confocal microscopy imaging of TT AF488 CuMV -p33 nano-vaccine following formulation with the micron-sized MCT adjuvant, 1) GFP signal of AF488 CuMV -p33 nano-vaccine 2) TT TT bright light field 3) an overlay 4–6 3D images with bright light field 4) an MCT crystal decorated with CuMV -p33 nano-vaccine particles. b A TT sketch illustrates the two prepared nano-vaccines, the 1st group consists of AF488 CuMV -p33 nano-vaccine and the 2nd consists of AF488 TT CuMV -p33 nano-vaccine formulated with MCT adjuvant. d Total number of AF488 CuMV -p33 nanoparticles in the popliteal LNs collected 3 h, TT TT 24 h, 48 h, 96 h and 216 h post-injection of the two prepared nano-vaccine groups in mice footpad. Statistical analysis by unpaired Student’s t test. c Confocal microscopy images of popliteal LNs 24 h and 216 h post-injection of the two prepared nano-vaccine groups in mice footpad, GFP signal was detected in LNs, whole mount view of z-stacks was acquired. One representative experiment of 3 similar experiments is shown (Fig. 4c and d). Tumour-infiltrating lymphocytes (TILs) then compared to the immune-stimulatory B type CpGs represent a prognostic factor for effective immune responses and the widely used adjuvant Alum using the same ag- especially in melanoma [21, 22]. Therefore, we measured the gressive B16F10p33 tumour model. Four groups were total number of the infiltrated CD8 T cells and p33 specific prepared as shown in Fig. 5a. The results again revealed CTLs (Fig. 4e) in TILs and calculated the density of these that formulating CuMV -p33 nano-vaccine with CpGs TT cells in each vaccinated group (number of cells divided by 1668 or MCT would significantly (p 0.0072, 0.0129 re- tumour volume). Formulating CuMV -p33 nano-vaccine spectively) hinder B16F10p33 tumour progression, TT with MCT significantly increased the density of total CD8 which was not the case when formulating CuMV -p33 TT T cells (p. 0.0024) (Fig. 4f) as well as the density of p33 spe- with Alum (p 0.4188) (Fig. 5b). In a next step, we mea- cific CTL measured by tetramers (p. 0.0093) (Fig. 4g) in sured the total number of infiltrated CD8 T cells (Fig. comparison to mice vaccinated with CuMV -p33 nano- 5c) and p33 specific CTLs (Fig. 5d) in the tumour and TT vaccine alone. IFN-γ production was also enhanced when calculated the density. There was a general increase in formulating CuMV -p33 with MCT (Fig. 4h). Thus, for- the groups mixed with CpGs 1668 or formulated with TT mulating the a nano-vaccine in MCT enhanced infiltration MCT or Alum (Fig. 5e and f). Formulating by specific T cells as well as anti-tumor protection. CuMV -p33 nano-vaccine with the micron-sized MCT TT adjuvant showed comparable results to CpGs 1668, the The micron-sized MCT adjuvant shows comparable gold standard adjuvant in mice. Furthermore, MCT ad- activity to B-type CpGs and is superior to alum in driving juvant was more potent at increasing the infiltration of protection against B16F10p33 melanoma total CD8 and p33-specific T cells into the tumour The immunogenicity of the CuMV -p33 nano-vaccine microenvironment when compared to formulating the TT formulated with the micron-sized MCT adjuvant was vaccine with Alum. Mohsen et al. Journal for ImmunoTherapy of Cancer (2019) 7:114 Page 7 of 12 Fig. 3 Formulating CuMV -p33 nano-vaccine with the micron-sized MCT adjuvant induces significant p33 specific T cell response and enhances TT cytokines secretion. a Vaccination scheme for six vaccine groups, CuMV -VLPs, CuMV -Actin, CuMV -p33, CuMV -p33 admixed with CpG 1668, TT TT TT TT + + CuMV -p33 formulated with MCT and CuMV -p33 formulated with Alum. b Percentage of CD8 Tetramer CTLs (means ± SEM) in the spleen in TT TT + + each vaccinated group. c Percentage of CD8 IFN-γ secreting cells (means ± SEM) in the spleen in each vaccinated group. d Percentage of + + + + + CD8 TNF-α cells (means ± SEM) in the spleen in each vaccinated group. e Percentage of CD8 IFN-γ /TNF-α secreting cells (means ± SEM) in the spleen in each vaccinated group. Statistical analysis by Oneway ANOVA (Turkey’s Multiple Comparison Test). f Representative flow cytometry + + dot plots showing the percentage of CD8 Tetramer CTLs in each vaccinated group. g Representative flow cytometry dot plots showing the + + + percentage of CD8 IFN-γ /TNF-α secreting cells in each vaccinated group. (3 mice per group), one representative of 3 similar experiments is shown The micron-sized MCT adjuvant shows comparable universal T cell epitope of Tetanus toxin (CuM production of cytokines to B type CpGs and is superior to V -VLPs). These nanoparticles constitute a promis- TT alum in B16F10p33 melanoma ing vaccine platform as the incorporated Tetanus Production of IFN-γ and TNF-a cytokines by TILs was toxin epitope can enhance their immunogenicity and also assessed upon immunization with CuMV -p33 the production of robust Ab and CTL responses espe- TT admixed with B type CpGs or formulated with MCT or cially in aging populations [5, 6]. Displaying epitopes Alum. Production of IFN-γ (Fig. 6a and b) and TNF-a on CuMV -VLP’s exterior surface can be achieved TT (Fig. 6c and d) was assessed separately or as dual cytokine by simple chemical techniques such as the SMPH production in polyfunational T cells (Fig. 6e and f). There heterobifunctional cross-linker. Such techniques have was no significant difference in the production of single shown efficacy and good results in many different IFN-γ, TNF-a or dual IFN-γ/TNF cytokines between the clinical settings [23–25]. However, we have lately en- groups admixed with CpGs 1668 or formulated with hanced the coupling efficacy of epitopes to bacterio- MCT adjuvant (p 0.3986, 0.3433 and 0.4120 respectively). phage Qβ-VLP using the biorthogonal Cu-free click However, there was a significant difference when compar- chemistry (Mohsen et al., submitted). Here we have ing the group formulated with MCT to the one formu- shown that such method can also be efficiently used lated with Alum (p 0.0179, 0.0187 and 0.006, respectively). to couple peptides/epitopes to CuMV -VLPs. Gener- TT ally, Cu-free click chemistry is a safe, non-toxic coup- Discussion ling method as the azide moiety attached to the In this study, we have developed cucumber mosaic target epitope does not react with any of the body’s virus-derived nanoparticles genetically fused to the natural molecules [26–28]. Mohsen et al. Journal for ImmunoTherapy of Cancer (2019) 7:114 Page 8 of 12 Fig. 4 Formulating CuMV -p33 nano-vaccine with the micron-sized MCT adjuvant causes tumour regression and enhances CD8 and p33 TT specific CTL infiltration into B16F10p33 tumours. a A diagram illustrating the adapted tumour experimental method based on injecting ~ 1 × 10 −/− B16F10p33 melanoma cell line into the flank of RAG2 deficient C57BL/6 mice. Twelve to thirteen days later the growing tumours were collected and processed in ~2mm for transplantation into the flank of WT C57BL/6. b Vaccination scheme for three vaccine groups, CuMV - TT VLPs, CuMV -p33 and CuMV -p33 formulated with MCT. c Tumour growth curve of subcutaneous B16F10p33 melanomas in each vaccinated TT TT 3 3 group, mice were euthanized when the tumour reached ~1000mm , arrows indicate start of treatment. d Tumour volume mm (mean ± SEM) measured at day 14 post tumour collection in each vaccinated group, each dot represents a tumour. e Representative flow cytometry dot plots + + + showing the total number of CD8 Tetramer CTLs in each vaccinated group. f Density of CD8 T cells (means ± SEM) in each vaccinated group, + ” + + “measured by dividing the total number of CD8 cells in TILs by the tumour volume . g Density of CD8 Tetramer CTLs (means ± SEM) in each + + ” + + vaccinated group, “measured by dividing the total number of CD8 Tetramer CTL by tumour volume . h Percentage of CD8 IFN-γ secreting cells (means ± SEM) in each vaccinated group. Statistical analysis by Student’s t test. (5 mice per group), one representative of 3 similar experiments is shown Immunostimulatory adjuvants such as synthetic mostly in mice and more rarely in humans [37]. In mice, CpG-oligonucleotides are TLR-agonists and have shown TLR-9 is expressed by all DCs while in human it is promising therapeutic potential by activating both the mainly expressed by pDCs in lymphoid organs but not innate and adaptive immune system [29–31]. CpGs have by conventional DCs [38]. pDCs, however, respond also been successfully used clinically to adjuvant cancer much more efficiently to A type CpGs rather than B vaccines [32, 33]. However, CpGs have some drawbacks type CpGs as only the former induce strong production including their unfavourable pharmacokinetics and their of type I IFN [39]. Hence, it may be difficult to directly propensity to cause splenomegaly, at least in mice [34, translate findings with B type CpGs from mice to 35]. It has been shown previously that packaging CpGs humans. With this respect, it is interesting to note that into VLPs such as Qβ or HBcAg can improve the CuMV VLPs naturally package RNA from the E. coli TT pharmacokinetics and dynamics of the DNA oligomers expression strain, a ligand for TLR7/8 which is [36]. Nevertheless, it is not always feasible to package expressed in all human DCs. VLPs with CpGs as some nanoparticles are unstable and Several preclinical and clinical studies have indicated packaging with reassembly processes may be time con- that some of the adjuvants used in licensed products are suming when targeting translational approaches. Fur- not optimal for developing effective cancer vaccines. Ex- thermore, TLR-9 agonists have been widely used with amples are the commonly used Montanide (Incomplete VLP-based vaccines to enhance T and B cell responses Freund’s adjuvant) and Alum [40–43]. Such limitations Mohsen et al. Journal for ImmunoTherapy of Cancer (2019) 7:114 Page 9 of 12 Fig. 5 The micron-sized MCT adjuvant shows comparable activity to B type CpGs and is superior to Alum in driving protection against B16F10p33 melanoma. a vaccination scheme for four vaccine groups, CuMV -VLPs, CuMV -p33 admixed with CpG 1668, CuMV -p33 formulated TT TT TT with MCT and CuMV -p33 formulated with Alum. b Tumour growth curve of subcutaneous B16F10p33 melanomas in each vaccinated group, TT mice were euthanized when the tumour reached ~1000mm , arrows indicate start of treatment. c Representative flow cytometry dot plots showing the total number of CD8 T cells in each vaccinated group, gated on TILs. d Representative flow cytometry dot plots showing the total + + + + number of CD8 Tetramer CTLs in each vaccinated group, gated on CD8 T cells. e Density of CD8 T cells (means ± SEM) in each vaccinated + ” + + group, “measured by dividing the total number of CD8 cells in TILs by the tumour volume . f Density of CD8 Tetramer CTLs (means ± SEM) in + ” each vaccinated group, “measured by dividing the total number of p33 tetramer CTLs by tumour volume . Statistical analysis by Student’s t test. (4 mice per group), one representative of 3 similar experiments is shown may include the inadequate ability of these adjuvants to immunotherapy. Protection against IgE-mediated allergic induce CTLs [44]. Nevertheless, use of B-type CpGs and response was achieved in mouse models, independently Alum in mice is useful where direct comparative adju- of inflammasome and TLR signaling in vivo. As has been vant studies are concerned. Since they are well charac- seen for Alum, the adjuvant activity of MCT was inde- terized in mice, both adjuvants are a good benchmark pendent of the inflammasome in vivo despite its ability when comparing other new or newly used adjuvants to activate the inflammasome in vitro [11]. In malaria, such as MCT. MCT has been shown to consistently enhance protective MCT is a micron-sized adjuvant that forms crystals of IgG responses and more protective IgG subclasses about ~ 5 μM that readily adsorb proteins including resulting in enhanced protection against malaria. Similar protein-based nanoparticles such as VLPs. MCT is well observations were made for influenza vaccine candidates known in the world of specific allergy immunotherapy, [13, 14, 17]. as it is categorized as a depot excipient in registered sub- Here, we have studied the draining kinetics of cutaneous immunotherapy products for the treatment of CuMV -p33 vaccine alone or formulated with adjuvant TT allergies. However, the knowledge about its mechanism MCT. Our results indicate that the free AF488 of action and its potential in different fields of vaccinol- CuMV -p33 can drain rapidly into the draining LN but TT ogy is only expanding now. In a recent study, MCT was fades away after ~ 4 days. In contrast, the release of shown to be an effective adjuvant in allergen-specific AF488 CuMV -p33 nano-vaccine formulated with TT Mohsen et al. Journal for ImmunoTherapy of Cancer (2019) 7:114 Page 10 of 12 Fig. 6 The micron-sized MCT adjuvant shows comparable production of cytokines to B type CpGs and is superior to Alum in B16F10p33 + + melanoma. a Percentage of CD8 IFN-γ secreting cells (means ± SEM) in each vaccinated group. b Representative flow cytometry dot plots + + + + showing the frequency of CD8 IFN-γ secreting cells in each vaccinated group. c Percentage of CD8 TNF- secreting cells (means ± SEM) in + + each vaccinated group. d Representative flow cytometry dot plots showing the frequency of CD8 TNF- secreting cells in each vaccinated + + + group. e Percentage of dual IFN-γ and TNF- secreting cells (means ± SEM) in each vaccinated tumour, gated on CD8 cells in TILs. f + + + Representative flow cytometry dot plots showing the frequency of dual IFN-γ and TNF- secreting cells in each group, gated on CD8 cells in TILs. Statistical analysis by Student’s t test. (4 mice per group), one representative of 3 similar experiments is shown MCT was delayed and more consistent over a longer The transplantation of solid tumor fragments allows period of time. Previous studies have mostly supported studying tumor development in the context of already the importance of the depot-forming adjuvants in T cell established tumor stroma which is more reflective of the based vaccine development as they delay the clearance physiological situation. When transplanted as solid frag- of the vaccine that results in enhancing the generation ments, even very immunogenic tumors grow in im- of effective antigen-specific CTL responses [45, 46]. munocompetent hosts and the vascularized tumours To study the immunogenicity of the developed rapidly grow to lethal size (47–50). The results show that CuMV -p33 nano-vaccine formulated with MCT, the formulating CuMV -p33 nano-vaccine with MCT was TT TT protective capacity of the induced CTL-responses was more potent in blocking tumour growth than using the assessed in an aggressive murine melanoma model con- nano-vaccine alone. The protective capacity of the sisting of B16F10 cells transfected with H-2D restricted CuMV -p33 with MCT is therefore strong, as the TT p33 epitope derived from LCMV. To generate tumors model used is very challenging. Previous studies have in- with maximal physiological properties, we have used a dicated that melanoma tumours exhibiting increased challenging tumour model based on transplanting solid numbers of tumour infiltrating CD8 T cells have better tumour fragments rather than single cell suspension. prognosis. TILs have led to a better understating of the Mohsen et al. Journal for ImmunoTherapy of Cancer (2019) 7:114 Page 11 of 12 interaction between hosts and tumours, mainly because Additional file 3: Movie S3. showing the popliteal LN 24 h after their study allowed better characterization of effective injecting CuMV -p33 nano-vaccine formulated with MCT adjuvant in TT mice footpad. (MP4 12060 kb) therapeutic responses [22, 47]. TILs isolated from the Additional file 4: Movie S4 showing the popliteal LN 216 h after vaccinated groups have been assessed for the presence injecting CuMV -p33 nano-vaccine in mice footpad. (MP4 30385 kb) TT of p33 specific CTLs by means of tetramers and intracel- Additional file 5: Movie S5 showing the popliteal LN 216 h after lular cytokine staining. CuMV -p33 vaccine formulated TT injecting CuMV -p33 nano-vaccine formulated with MCT adjuvant in TT with MCT adjuvant enhanced the infiltration of CD8 mice footpad. (MP4 34519 kb) and p33 specific CTLs into the tumour and the produc- tion of IFN-γ. These results indicate that MCT may be a Acknowledgments None. promising cancer adjuvant. When comparing MCT adju- vant to the potent B type CpGs or the widely used Alum, Funding the overall adjuvants activity of MCT was comparable to This work was supported by Bencard Adjuvant Systems, Dominion Way, UK, the Swiss Cancer League (KFS-4132-02-2017) and Qatar Foundation. CpGs and superior to Alum. It has been previously seen that Alum may increase over- Availability of data and materials all IgG responses at least as good as MCT. In contrast to Data are available in the main text and supplementary materials, raw data available upon request by the correspondent author. Alum, however, MCT induced superior IgG2a responses, which is usually associated with T 1 responses and/or TLR Authors’ contributions activity [14]. This may be compatible with the observed Design of experiments, acquisition of data, interpretation of data, analysis of data: MOM, MDH, GCM, MV, CR, DS, ER, DS and MFB; VLPs expression and ability of MCT to enhance CTL responses, which Alum production: CL, AZ; Confocal microscopy imaging and stereomicroscopic failed to do. Further work will be required to elucidate the imaging: MS, ER, PE and JVS. Writing, revise and revision of manuscript: mechanism of this difference, as both Alum and MCT form MOM, TMK, AK, DS, MV, MAS, MFK and MFB. Technical and material support: MOM, MDH, DS, LZ, AK and MFB. Study supervision: MFB. All authors read a depot and may activate the inflammasome pathway. An and approved the final manuscript. obvious hypothesis is that Alum induces a T 2driving pathway in addition to the inflammasome or vice versa, Ethics approval All procedures on animals were conducted at University of Bern in MCT may activate a T 1 driving pathway. The distinctions accordance with the Swiss Animals Act (455.109.1) (September 2008, 5th) portrayed in their respective immunological profiles are University of Bern. also likely to be be partly governed by their inherent formu- Consent for publication lation/structural characteristics (i.e. particle size, morph- Not applicable. ology, antigen adsorption etc). In addition to this, it is known that tryptophan or arginine serve as direct Competing interests The authors declare that they have no competing interests. Martin immune-modulators, a possibility that has not been exten- Bachmann and Mona Mohsen own shares of DeepVax GmbH involved in sively studied for tyrosine [48, 49]. developing virus-like particles based vaccines for cancer, other authors de- Taken together, this study shows that MCT is a potent clare that they have no competing interests. enhancer of CTL responses and may be viewed as a multi-purpose adjuvant with novel indications. As such, Publisher’sNote its effectiveness and compatibility in a mix- and match Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. adjuvant systems approach should be further tested in preclinical and human immunotherapy trials. Combin- Author details ation of MCT with nanoparticles appears particularly at- Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK. Department of BioMedical Research, Immunology RIA, tractive, as the micron-sized adjuvants will form a local Inselspital, University of Bern, Bern, Switzerland. Bencard Adjuvant Systems, depot at the injection site with concomitant activation of Dominion Way, Worthing, UK. Latvian Biomedical Research & Study Centre, skin-resident antigen-presenting cells. Nanoparticles will Riga, Latvia. Institute of anatomy, University of Bern, Bern, Switzerland. 6 7 Theodor Kocher Institute, University of Bern, Bern, Switzerland. Department be released over time, draining to local LNs for extended of Medical Oncology, Bern University Hospital, University of Bern, Bern, time-periods, causing an optimal immune reaction. Switzerland. International Immunology Center, Anhui Agricultural University, Thus, the combination of nanoparticles with micron- Hefei, Anhui, China. Department of dermatology, University of Zurich, Bern, Switzerland. Department of Oncology, University of Lausanne, Bern, sized adjuvants may optimally harness the properties of Switzerland. National Center for Cancer Care & Research (NCCCR), Doha, the lymphatic system. State of Qatar. Received: 13 November 2018 Accepted: 2 April 2019 Additional files References Additional file 1: Movie S1. showing AF488 CuMV -VLPs decorating the TT 1. Hu Z, Ott PA, Wu CJ. Towards personalized, tumour-specific, therapeutic surface of microcrystalline tyrosine crystals MCT adjuvant. (MP4 13481 kb) vaccines for cancer. Nat Rev Immunol. 2017;18(3):168–182. 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Journal for ImmunoTherapy of CancerSpringer Journals

Published: Apr 26, 2019

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