Despite the tremendous potential of peptide-based cancer vaccines, their efficacy has been limited in humans. Recent innovations in tumor exome sequencing have signaled the new era of personalized immunotherapy with patient-specific neo-antigens, but a general methodology for stimulating strong CD8α+ cytotoxic T-lymphocyte (CTL) responses remains lacking. Here we demonstrate that high density lipoprotein-mimicking nanodiscs coupled with antigen (Ag) peptides and adjuvants can markedly improve Ag/adjuvant co-delivery to lymphoid organs and sustain Ag presentation on dendritic cells. Strikingly, nanodiscs elicited up to 47-fold greater frequencies of neoantigen-specific CTLs than soluble vaccines and even 31-fold greater than perhaps the strongest adjuvant in clinical trials (i.e. CpG in Montanide). Moreover, multi-epitope vaccination generated broad-spectrum T-cell responses that potently inhibited tumor growth. Nanodiscs eliminated established MC-38 and B16F10 tumors when combined with anti-PD-1 and anti-CTLA-4 therapy. These findings represent a new powerful approach for cancer immunotherapy and suggest a general strategy for personalized nanomedicine.

Infection of mice with sporozoites of Plasmodium berghei or Plasmodium yoelii has been used extensively to evaluate liver-stage protection by candidate preerythrocytic malaria vaccines. Unfortunately, repeated success of such vaccines in mice has not translated readily to effective malaria vaccines in humans. Thus, mice may be used better as models to dissect basic parameters required for immunity to Plasmodium-infection than as preclinical vaccine models. In turn, this basic information may aid in the rational design of malaria vaccines. Here, we describe a model of circumsporozoite-specific memory CD8 T cell generation that protects mice against multiple P. berghei sporozoite challenges for at least 19 months. Using this model we defined a threshold frequency of memory CD8 T cells in the blood that predicts long-term sterilizing immunity against liver-stage infection. Importantly, the number of Plasmodium-specific memory CD8 T cells required for immunity greatly exceeds the number required for resistance to other pathogens. In addition, this model allowed us to identify readily individual immunized mice that exceed or fall below the protective threshold before infection, information that should greatly facilitate studies to dissect basic mechanisms of protective CD8 T cell memory against liver-stage Plasmodium infection. Furthermore, the extremely large threshold in memory CD8 T cell frequencies required for long-term protection in mice may have important implications for development of effective malaria vaccines.

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The facultative intracellular bacterium Listeria monocytogenes is being developed as a cancer vaccine platform because of its ability to induce potent innate and adaptive immunity. For successful clinical application, it is essential to develop a Listeria platform strain that is safe yet retains the potency of vaccines based on wild-type bacteria. Here, we report the development of a recombinant live-attenuated vaccine platform strain that retains the potency of the fully virulent pathogen, combined with a >1,000-fold reduction in toxicity, as compared with wild-type Listeria. By selectively deleting two virulence factors, ActA (⌬actA) and Internalin B (⌬inlB), the immunopotency of Listeria was maintained and its toxicity was diminished in vivo, largely by blocking the direct internalin B-mediated infection of nonphagocytic cells, such as hepatocytes, and the indirect ActA-mediated infection by cellto-cell spread from adjacent phagocytic cells. In contrast, infection of phagocytic cells was not affected, leaving intact the ability of Listeria to stimulate innate immunity and to induce antigenspecific cellular responses. Listeria ⌬actA͞⌬inlB-based vaccines were rapidly cleared from mice after immunization and induced potent and durable effector and memory T-cell responses with no measurable liver toxicity. Therapeutic vaccination of BALB͞c mice bearing murine CT26 colon tumor lung metastases or palpable s.c. tumors (>100 mm 3 ) with recombinant Listeria ⌬actA͞⌬inlB expressing an endogenous tumor antigen resulted in breaking of self-tolerance and long-term survival. We propose that recombinant Listeria ⌬actA͞⌬inlB expressing human tumor-associated antigens represents an attractive therapeutic strategy for further development and testing in human clinical trials.C ancer immunotherapy represents a promising treatment strategy that has produced some tantalizing clinical responses for a variety of malignant diseases. Although promising, there continues to be a strong need for a practical immunization strategy that can be routinely adopted to specific malignancies and that consistently yields durable and robust therapeutic antitumor responses.Progress in molecular and cellular immunology, combined with increasing understanding of pathogen physiology and hostpathogen interaction has facilitated the design and use of attenuated bacteria as conventional vaccine vectors. However, the practical utility of live attenuated vaccines relies on achieving a proper balance between the virulence͞toxicity and immunogenicity of the vaccine. The potency of a pathogen to elicit adaptive immunity is related in part to its ability to stimulate significant innate immunity through recognition of microbial pathogen-associated molecular patterns by Toll-like receptors. Microbial encounter with professional antigen-presenting cells (APC), such as dendritic cells, results in activation and maturation (1) as well as secretion of high levels of T helper-1-type cytokines (2). This interaction initiates adaptive immune responses and therefore link...

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The anecdotal reports of promising results seen with immunotherapy and radiation in advanced malignancies have prompted several trials combining immunotherapy and radiation. However, the ideal timing of immunotherapy with radiation has not been clarified. Tumor bearing mice were treated with 20Gy radiation delivered only to the tumor combined with either anti-CTLA4 antibody or anti-OX40 agonist antibody. Immunotherapy was delivered at a single timepoint around radiation. Surprisingly, the optimal timing of these therapies varied. Anti-CTLA4 was most effective when given prior to radiation therapy, in part due to regulatory T cell depletion. Administration of anti-OX40 agonist antibody was optimal when delivered one day following radiation during the post-radiation window of increased antigen presentation. Combination treatment of anti-CTLA4, radiation, and anti-OX40 using the ideal timing in a transplanted spontaneous mammary tumor model demonstrated tumor cures. These data demonstrate that the combination of immunotherapy and radiation results in improved therapeutic efficacy, and that the ideal timing of administration with radiation is dependent on the mechanism of action of the immunotherapy utilized.

Cytotoxic therapies prime adaptive immune responses to cancer by stimulating the release of tumor-associated antigens. However, the tumor microenvironment into which these antigens are released is typically immunosuppressed, blunting the ability to initiate immune responses. Recently, activation of the DNA sensor molecule STING by cyclic dinucleotides was shown to stimulate infection-related inflammatory pathways in tumors. In this study, we report that the inflammatory pathways activated by STING ligands generate a powerful adjuvant activity for enhancing adaptive immune responses to tumor antigens released by radiation therapy. In a murine model of pancreatic cancer, we showed that combining CT-guided radiation therapy with a novel ligand of murine and human STING could synergize to control local and distant tumors. Mechanistic investigations revealed T cell-independent and TNFα-dependent hemorrhagic necrosis at early times followed by later CD8 T cell-dependent control of residual disease. Clinically, STING was found to be expressed extensively in human pancreatic tumor and stromal cells. Our findings suggest that this novel STING ligand could offer a potent adjuvant for leveraging radiotherapeutic management of pancreatic cancer.

Here, we describe a previously unrecognized pathway for activation of antigen-specific adaptive immune responses that was independent of Toll-Interleukin 1 Receptor signaling and directed toward detection of antigens expressed by apoptotic cells. This pathway is represented within Flt-3 Ligand-derived dendritic cells (DCs) that represent immature lymphoid DCs, but not within GM-CSF-treated bone marrow-derived dendritic cells. Exposure of these DCs to apoptotic cells resulted in production of type I interferon and favored the development of cytotoxic T cell responses. The N-Ethyl-N-Nitrosourea-induced germline mutation 3d (Unc3b1(3d/3d)) abolished both MHC class I and II responses elicited by this pathway, whereas a null allele of Cd36 selectively abolished class II responses. We propose that this mode of adaptive immune activation evolved to permit the sensitive detection of intracellular microbial infections, particularly viral infections, which frequently induce apoptotic cell death, but may also be important in transplantation, autoimmunity, and vaccine development.