Over the last decades, vaccine development has advanced significantly in pursuing higher safety with less side effects. However, this is often accompanied by a reduction in vaccine immunogenicity and an increased dependency on adjuvants to enhance vaccine potency. Especially for diseases like cancer, it is important that therapeutic vaccines contain adjuvants that promote strong T cell responses. An important mode of action for such adjuvants is to prolong antigen exposure to dendritic cells (DCs) and to induce their maturation. These mature DCs are extremely effective in the activation of antigen-specific T cells, which is a pre-requisite for induction of potent and long-lasting cellular immunity. For the activation of CD8+ cytotoxic T cell responses, however, the exogenous vaccine antigens need to gain access to the endogenous MHCI presentation pathway of DCs, a process referred to as antigen cross-presentation. In this review, we will focus on recent insights in clinically relevant vaccine adjuvants that impact DC cross-presentation efficiency, including aluminum-based nanoparticles, saponin-based adjuvants, and Toll-like receptor ligands. Furthermore, we will discuss the importance of adjuvant combinations and highlight new developments in cancer vaccines. Understanding the mode of action of adjuvants in general and on antigen cross-presentation in DCs in particular will be important for the design of novel adjuvants as part of vaccines able to induce strong cellular immunity.

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The recent decline in global malaria burden has stimulated efforts toward Plasmodium falciparum elimination. Understanding the biology of malaria transmission stages may provide opportunities to reduce or prevent onward transmission to mosquitoes. Immature P. falciparum transmission stages, termed stages I to IV gametocytes, sequester in human bone marrow before release into the circulation as mature stage V gametocytes. This process likely involves interactions between host receptors and potentially immunogenic adhesins on the infected red blood cell (iRBC) surface. Here, we developed a flow cytometry assay to examine immune recognition of live gametocytes of different developmental stages by naturally exposed Malawians. We identified strong antibody recognition of the earliest immature gametocyte-iRBCs (giRBCs) but not mature stage V giRBCs. Candidate surface antigens (n = 30), most of them shared between asexual- and gametocyte-iRBCs, were identified by mass spectrometry and mouse immunizations, as well as correlations between responses by protein microarray and flow cytometry. Naturally acquired responses to a subset of candidate antigens were associated with reduced asexual and gametocyte density, and plasma samples from malaria-infected individuals were able to induce immune clearance of giRBCs in vitro. Infected RBC surface expression of select candidate antigens was validated using specific antibodies, and genetic analysis revealed a subset with minimal variation across strains. Our data demonstrate that humoral immune responses to immature giRBCs and shared iRBC antigens are naturally acquired after malaria exposure. These humoral immune responses may have consequences for malaria transmission potential by clearing developing gametocytes, which could be leveraged for malaria intervention.

IntroductionIn situ tumor ablation releases a unique repertoire of antigens from a heterogeneous population of tumor cells. High-intensity focused ultrasound (HIFU) is a completely noninvasive ablation therapy that can be used to ablate tumors either by heating (thermal (T)-HIFU) or by mechanical disruption (mechanical (M)-HIFU). How different HIFU ablation techniques compare with respect to their antigen release profile, their activation of responder T cells, and their ability to synergize with immune stimuli remains to be elucidated.Methods and resultsHere, we compare the immunomodulatory effects of T-HIFU and M-HIFU ablation with or without the TLR9 agonist CpG in the ovalbumin-expressing lymphoma model EG7. M-HIFU ablation alone, but much less so T-HIFU, significantly increased dendritic cell (DC) activation in draining lymph nodes (LNs). Administration of CpG following T- or M-HIFU ablation increased DC activation in draining LNs to a similar extend. Interestingly, ex vivo co-cultures of draining LN suspensions from HIFU plus CpG treated mice with CD8+ OT-I T cells demonstrate that LN cells from M-HIFU treated mice most potently induced OT-I proliferation. To delineate the mechanism for the enhanced anti-tumor immune response induced by M-HIFU, we characterized the RNA, DNA and protein content of tumor debris generated by both HIFU methods. M-HIFU induced a uniquely altered RNA, DNA and protein profile, all showing clear signs of fragmentation, whereas T-HIFU did not. Moreover, western blot analysis showed decreased levels of the immunosuppressive cytokines IL-10 and TGF-β in M-HIFU generated tumor debris compared to untreated tumor tissue or T-HIFU.ConclusionCollectively, these results imply that M-HIFU induces a unique context of the ablated tumor material, enhancing DC-mediated T cell responses when combined with CpG.

BackgroundTumor ablation techniques, like cryoablation, are successfully used in the clinic to treat tumors. The tumor debris remaining in situ after ablation is a major antigen depot, including neoantigens, which are presented by dendritic cells (DCs) in the draining lymph nodes to induce tumor-specific CD8+T cells. We have previously shown that co-administration of adjuvants is essential to evoke strong in vivo antitumor immunity and the induction of long-term memory. However, which adjuvants most effectively combine with in situ tumor ablation remains unclear.Methods and resultsHere, we show that simultaneous administration of cytidyl guanosyl (CpG) with saponin-based adjuvants following cryoablation affects multifunctional T-cell numbers and interleukin (IL)-1 induced polymorphonuclear neutrophil recruitment in the tumor draining lymph nodes, relative to either adjuvant alone. The combination of CpG and saponin-based adjuvants induces potent DC maturation (mainly CpG-mediated), antigen cross-presentation (mainly saponin-based adjuvant mediated), while excretion of IL-1β by DCs in vitro depends on the presence of both adjuvants. Most strikingly, CpG/saponin-based adjuvant exposed DCs potentiate antigen-specific T-cell proliferation resulting in multipotent T cells with increased capacity to produce interferon (IFN)γ, IL-2 and tumor necrosis factor-α in vitro. Also in vivo the CpG/saponin-based adjuvant combination plus cryoablation increased the numbers of tumor-specific CD8+T cells showing enhanced IFNγ production as compared with single adjuvant treatments.ConclusionsCollectively, these data indicate that co-injection of CpG with saponin-based adjuvants after cryoablation induces an increased amount of tumor-specific multifunctional T cells. The combination of saponin-based adjuvants with toll-like receptor 9 adjuvant CpG in a cryoablative setting therefore represents a promising in situ vaccination strategy.