Therapeutic Immunization Research Articles

Diosgenin promotes antitumor immunity and PD-1 antibody efficacy against melanoma by regulating intestinal microbiota

Diosgenin, a natural steroidal saponin, can exert antitumor effect by regulating immune function and improving intestinal microbiota. The response to anti-PD-1 immunotherapy is associated with intestinal microbiota and effector T cells in tumor microenvironment. We hypothesize that the modulation of diosgenin on intestinal microbiota can facilitate antitumor immunity and the therapeutic efficacy of PD-1 antibody. In melanoma-bearing C57BL/6 mice, we observed that the anti-melanoma effect of diosgenin relied more on antitumor immunity than direct tumor inhibition activity evidenced by obvious CD4+/CD8+ T-cell infiltration and IFN-γ expression in tumor tissues, and it could improve the compositions of intestinal microbiota. Antibiotics impaired the therapeutic efficacy and immunity responses of diosgenin through disturbing intestinal microbiota, indicating the importance of intestinal microbiota in diosgenin’s in vivo antitumor activity. More importantly, the combined administration of PD-1 antibody with diosgenin aggravated the tumor necrosis and apoptosis by eliciting augmented T-cell responses. Taken together, diosgenin can be used as a microecological regulator to induce antitumor immunity and improve the efficacy of immune checkpoint antibody, making it more suitable for the treatment of malignant tumors.

Therapeutic Immunization against Glioblastoma

Glioblastoma is the most common form of brain cancer in adults that produces severe damage to the brain leading to a very poor survival prognosis. The standard of care for glioblastoma is usually surgery, as well as radiotherapy followed by systemic temozolomide chemotherapy, resulting in a median survival time of about 12 to 15 months. Despite these therapeutic efforts, the tumor returns in the vast majority of patients. When relapsing, statistics suggest an imminent death dependent on the size of the tumor, the Karnofsky Performance Status, and the tumor localization. Following the standard of care, the administration of Bevacizumab, inhibiting the growth of the tumor vasculature, is an approved medicinal treatment option approved in the United States, but not in the European Union, as well as the recently approved alternating electric fields (AEFs) generator NovoTTF/Optune. However, it is clear that regardless of the current treatment regimens, glioma patients continue to have dismal prognosis and novel treatments are urgently needed. Here, we describe different approaches of recently developed therapeutic glioma brain cancer vaccines, which stimulate the patient’s immune system to recognize tumor-associated antigens (TAA) on cancer cells, aiming to instruct the immune system to eventually attack and destroy the brain tumor cells, with minimal bystander damage to normal brain cells. These distinct immunotherapies may target particular glioma TAAs which are molecularly defined, but they may also target broad patient-derived tumor antigen preparations intentionally evoking a very broad polyclonal antitumor immune stimulation.

Immunotherapeutic strategies for treatment of hepatocellular carcinoma with antigen-loaded dendritic cells: in vivo study.

Hepatocellular carcinoma (HCC) is one of the major health problems in the world. DCs-based vaccines are a promising immunotherapeutic strategy that aims at the optimal for induction of a specific antitumor immune response and destruction of tumor cells. The present study was conducted to investigate the immunogenic characters of whole tumor lysate-pulsed DCs vaccine and its ability to induce a specific antitumor immune response in HCC mice model. We also evaluate the effectiveness of prophylactic and therapeutic immunization strategies against HCC in mice models. Mice-derived DCs were in vitro loaded with whole tumor lysate prepared from liver tissue of HCC mice and evaluated for expression of surface maturation markers CD83 and CD86. In vivo immunization of mice with whole tumor lysate-pulsed DCs was performed in two strategies; prophylactic (pre-exposure to HCC) and therapeutic (post-exposure to HCC). Effectiveness of both protocols was investigated in terms of histopathological examination of liver sections and measurement of serum levels of immune cytokines interferon-γ (IFN-γ) and interleukin-2 (IL-2). Loading of DCs with whole tumor cell lysate exhibited a significant increase in expression of CD83 and CD86. In vivo administration of prophylactic doses of whole tumor lysate-pulsed DCs in mice before induction of HCC evokes a strong antitumor immune response presented by absence of malignant cells in liver sections and the significant increase in IFN-γ and IL-2. Data herein indicated that prophylactic vaccination with whole tumor lysate-pulsed DCs exhibited an effective antitumor immune response against HCC more than therapeutic protocol.

Potent therapeutic efficacy of an alphavirus replicon DNA vaccine expressing human papilloma virus E6 and E7 antigens

ABSTRACTCervical cancer develops as a result of infection with high-risk human papillomavirus (HPV) through persistent expression of early proteins E6 and E7. Our group pioneered a recombinant viral vector system based on Semliki Forest virus (SFV) for vaccination against cervical cancer. The most striking benefit of this alphavirus vector-based vaccine platform is its high potency. DNA vaccines on the other hand, have a major advantage with respect to ease of production. In this study, the benefits associated with both SFV-based vaccines and DNA vaccines were combined with the development of a DNA-launched RNA replicon (DREP) vaccine targeting cervical cancer. Using intradermal delivery followed by electroporation, we demonstrated that DREP encoding for E6,7 (DREP-E6,7) induced effective, therapeutic antitumor immunity. While immunizations with a conventional DNA vaccine did not prevent tumor outgrowth, immunization with a 200-fold lower equimolar dose of DREP (0.05 µg of DREP) resulted in approximately 85% of tumor-free mice. To overcome the safety concern of potential malignant transformation at the vaccination site, we evaluated the anti-tumor effect of a DREP vaccine encoding a shuffled version of E7 (DREP-E7sh). DREP-E7sh delayed tumor growth yet not to the same extent as DREP-E6,7. In addition, inclusion of a helper cassette and an ER targeting signal (sigHelp) did not significantly further enhance the suppression of tumor outgrowth in the long term, albeit exhibiting better tumor control early after immunization. Collectively, this study points towards the clinical evaluation of DREP encoding HPV antigens as a potent immunotherapy for patients with HPV16 (pre)-malignancies.

Extracorporeal Photochemotherapy Drives Monocyte-to-Dendritic Cell Maturation to Induce Anticancer Immunity.

Extracorporeal photochemotherapy (ECP) is a cancer immunotherapy for cutaneous T-cell lymphoma (CTCL) operative in more than 350 centers worldwide. Although its efficacy and favorable safety profile have driven its widespread use, elucidation of its underlying mechanism has been difficult. In this study, we identify the principal contributors to the anticancer immunotherapeutic effects of ECP, with the goal of enhancing potency and broadening applicability to additional malignancies. First, we scaled down the clinical ECP leukocyte-processing device to mouse size. Second, we used that miniaturized device to produce a cellular vaccine that regularly initiated therapeutic antimelanoma immunity. Third, we individually subtracted key factors from either the immunizing inoculum or the treated animal to ascertain their contribution to the in vivo antimelanoma response. Platelet-signaled monocyte-to-dendritic cell (DC) differentiation followed by sorting/processing/presentation of tumor antigens derived from internalized apoptotic tumor cells were absolute requirements. As in clinical ECP, immunogenic cell death of tumor cells was finely titrated by DNA cross-linkage mediated by photoactivated 8-methoxypsoralen (8-MOPA). ECP-induced tumor-loaded DC were effective immunotherapeutic agents only if they were spared exposure to 8-MOPA, indicating that healthy DC are required for ECP. Infusion of responder T cells into naïve tumor-challenged mice established the protective role of stimulated T-cell antitumor immunity. Collectively, these results reveal that selective antitumor effects of ECP are initiated by tumor antigen-loaded, ECP-induced DC, which promote potent collaboration between CD4 and CD8 tumor-specific T cells. These mechanistic insights suggest potential therapeutic applicability of ECP to solid tumors in addition to CTCL.Significance: These findings identify principal cellular contributors to the anticancer immunotherapeutic impact of ECP and suggest this treatment may be applicable to a broad spectrum of immunogenic malignancies. Cancer Res; 78(14); 4045-58. ©2018 AACR.

Application of iNKT Cell-targeted Active Immunotherapy in Cancer Treatment.

In tumor immunity, invariant natural killer T (iNKT) cells play a pivotal role as a link between the innate and adaptive immune systems. With a precisely regulated activation mechanism, iNKT cells have the ability to respond quickly to antigenic stimulation and rapidly produce cytokines and chemokines, and subsequently an effective antitumor immune response. The development of iNKT cell-targeted active immunotherapy enables, not only an antitumor immune response through innate and acquired immunity, but also the conversion of an immunosuppressive into an immunogenic microenvironment. This review is focused on the activation mechanism and the role of iNKT cells after therapeutic active immunization. The therapeutic strategy targeting iNKT cells is expected to be applied to clinical practice in combination with surgery and chemotherapy.

Abstract 725: Targeting the skin microenvironment for effective cancer vaccines

Abstract Skin, the most accessible organ, harbors keratinocytes and immunocytes that contribute to both innate and adaptive immunity, rendering it an ideal anatomic target for immunization. Keratinocytes can drive skin inflammation by producing pro-inflammatory mediators that promote cutaneous immune responses. Thus, targeting accessible keratinocytes to express transgenic antigens and increase production of pro-inflammatory mediators is a salient and attractive approach to vaccination, but remains a critical challenge to achieve. Toward this goal, we evaluated the effects of transient overexpression of the stress response factor x-box binding protein 1 (XBP1) in keratinocytes on the skin microenvironment. In vitro, keratinocytes genetically engineered to overexpress XBP1 increased production of both co-transduced exogenous antigen and pro-inflammatory mediators. In vivo, overexpression of XBP1 and transgenic antigen in keratinocytes induced both protective and therapeutic antitumor immunity that was associated with enhanced cutaneous expression of pro-inflammatory mediators and co-delivered antigen, increased skin-infiltrating immunocytes including dendritic cells (DCs), and the induction of durable antigen-specific CD8+ T cells and skin-resident memory CD8+ T cells. Importantly, in human skin, correlative effects were observed: transient overexpression of XBP1 in skin triggered the expression of genes linked to a pro-immunogenic microenvironment, increased the expression of the co-delivered antigen, and promoted the activation and IFN-γ-producing T cell-stimulating function of skin DCs. These findings provide novel insights into the rational design of cancer vaccines with improved immunogenicity through strategic modulation of the skin microenvironment. Citation Format: Jack R. You, Rong Hao, Louis D. Falo, Guo Chen, Xingxing Hao, Cara Carey, Zhaoyang You, Louis D. Falo. Targeting the skin microenvironment for effective cancer vaccines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 725.

PO-413 Immune modulating properties of cyclophosphamide synergise with immunotherapy in preclinical models of neuroblastoma

IntroductionNeuroblastoma (NB) is one of the most common childhood cancers accounting for 15% of paediatric cancer deaths. Current therapies, which include chemotherapy, are highly toxic with significant treatment related mortality. There is little scope for further intensification therefore alternatives strategies such as immunotherapy, are a priority for improving patient outcomes. Classically, chemotherapy is regarded as immunosuppressive but recent work has highlighted that some may illicit an immunogenic form of cell death. This work sets out to identify the immunomodulating effects of cyclophosphamide (CPM) on tumour infiltrating immune cells and whether these properties can synergise with immunomodulatory antibodies in preclinical models of NB.Material and methodsThe effects of CPM and doxorubicin on tumour cells were investigated in vitro by analyses of immunogenic cell death (ICD) markers. Two different in vivo subcutaneous murine NB models (NXS2 +NB9464D), were treated i.p with different doses of CPM. Tissues were harvested and detailed immunophenotyping performed. Combination of CPM and anti-PD-1 therapy was investigated in both models using tumour growth and survival as end points. Combination therapy was also assessed in TH-MYCN mice which develop spontaneous neuroblastoma.Results and discussionsChemotherapy application in vitro led to an increase in expression of the ICD markers, ecto-calreticulin and Hsp-70. CPM was found to have numerous immune modulating activities in vivo, including the reduction of intratumoral Treg cells, even at low doses (20 mg/kg), in both NB models. Combination therapy was shown to increase CD8+ and CD4+ percentages within tumours, along with an increase CD4+ effector and memory cell proportions. Anti-PD-1 therapy synergised with CPM to improve median survival and slow tumour growth. Metronomic dosing of CPM and anti-PD-1 antibody led to tumour regression in TH-MYCN tumour bearing mice.ConclusionThis work supports combining low dose CPM to enhance immunomodulatory antibody therapy, to generate therapeutic anti-neuroblastoma immunity. Ongoing work is focused on elucidating the mechanism of action of the CPM and anti-PD-1 combination therapy, along with identifying the optimal chemotherapy and antibody dosing strategies.

Abstract 5722: Antigen identification for cancer immunotherapy by deep learning on tumor HLA peptides

Abstract A key goal in immuno-oncology is the identification of tumor antigens recognized by CD8 T cells. Immune modulators such as PD-1 inhibitors indirectly promote T-cell attack against tumor antigens, and may be augmented by antigen-directed therapeutic immunization or adoptive cell therapy to increase clinical benefit. Such personalized therapeutics require accurate antigen identification from patient samples, which remains elusive today. Methods: We generated the largest reported dataset of human tumor transcriptomes and HLA class I peptidomes (N=111) from specimens of multiple tumor types. We used these data to train a deep learning model of HLA peptide presentation for antigen prediction. Our model architecture addressed two key challenges: (1) learning HLA-allele-specific models from tumor data where each sample expressed up to 6 unique HLA class I alleles and (2) incorporating information about all aspects of HLA presentation, including gene expression, proteasomal processing and stable binding of peptides to HLA. We evaluated the performance of the model on two independent test datasets. First, we tested the model on HLA presented peptides from five held-out tumor mass spectrometry samples. Then, to establish that accurate prediction of HLA presentation translates to prediction of antigens recognized by T cells in vivo, we compiled from three published studies a dataset of >2,000 mutations in 16 patients, with 23 mutations (i.e. neoantigens) recognized by PD1+ PBMC or TIL CD8 T cells. Recognition of a peptide by TIL or activated peripheral T-cells implies not only tumor presentation of the peptide, but also its ability to prime T cell responses, and thus represents the most stringent test of tumor antigen prediction. Results: The model demonstrated a breakthrough in prediction accuracy. On the mass spectrometry test data, it achieved a >10-fold improvement in positive predictive value (PPV) vs standard HLA binding affinity (~50% PPV at 40% recall for the MS-based model vs ~5% PPV for binding affinity prediction). On the T-cell test dataset, the model ranked T-cell recognized neoantigens on average >4-fold higher than standard prediction (median rank ~7 for the MS-based model vs ~30 for binding affinity). When selecting candidate neoantigens for hypothetical 10-neoantigen personalized immunotherapy, the model prioritized at least 1 T-cell recognized neoantigen in the top 10 for 9/11 patients with neoantigen responses vs 3/11 for binding affinity. For a hypothetical 20-neoantigen immunotherapy, the model correctly selected the majority (16/23, 70%) of recognized neoantigens. Conclusion: We used the largest dataset of tumor transcriptomes and HLA peptidomes reported to-date to train a deep learning model of HLA epitope presentation. The new model significantly outperforms state of the art methods, and has sufficient predictive accuracy for in silico antigen selection for personalized cancer immunotherapy. Citation Format: Brendan Bulik-Sullivan, Jennifer Busby, Matthew Davis, Lauren Young, Tyler Murphy, Andrew Clark, Fujiko Duke, Michele Busby, Adnan Derti, Mojca Skoberne, Karin Jooss, Corinne E. Gustafson, Assunta De Rienzo, William G. Richards, Nhien T. Dao, Hyeong R. Kim, Jamie E. Anderson, Chang-Min Choi, Vincent De Montpreville, Se Jin Jang, Olaf Mercier, Raphael Bueno, Elie Fadel, Joshua Francis, Roman Yelensky. Antigen identification for cancer immunotherapy by deep learning on tumor HLA peptides [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5722.

Combined immunization against TGF-β1 enhances HPV16 E7-specific vaccine-elicited antitumour immunity in mice with grafted TC-1 tumours

Therapeutic vaccine appears to be a potential approach for the treatment of human papillomavirus (HPV)-associated tumours, but its efficacy can be dampened by immunosuppressive factors such as transforming growth factor (TGF)-β1. We sought to investigate whether active immunity against TGF-β1 enhances the anti-tumour immunity elicited by an HPV16 E7-specific vaccine that we developed previously. In this study, virus-like particles of hepatitis B virus core antigen were used as vaccine carriers to deliver either TGF-β1 B cell epitopes or E7 cytotoxic T-lymphocyte epitope. The combination of preventive immunization against TGF-β1 and therapeutic immunization with the E7 vaccine significantly reduced the growth of grafted TC-1 tumours in C57 mice, showing better efficacy than immunization with only one of the vaccines. The improved efficacy of combined immunization is evidenced by elevated IFN-γ and decreased IL-4 and TGF-β1 levels in cultured splenocytes, increased E7-specific IFN-γ-expressing splenocytes, and increased numbers of CD4+IFN-γ+ and CD8+IFN-γ+ cells and decreased numbers of Treg (CD4+Foxp3+) cells in the spleen and tumours. The results strongly indicate that targeting TGF-β1 through active immunization might be a potent approach to enhancing antigen-specific therapeutic vaccine-induced anti-tumour immune efficacy and providing a combined strategy for effective cancer immunotherapy.

Enhanced Anti-Mycobacterium tuberculosis Immunity over Time with Combined Drug and Immunotherapy Treatment

It is estimated that one third of the world’s population is infected with Mycobacterium tuberculosis (Mtb). This astounding statistic, in combination with costly and lengthy treatment regimens make the development of therapeutic vaccines paramount for controlling the global burden of tuberculosis. Unlike prophylactic vaccination, therapeutic immunization relies on the natural pulmonary infection with Mtb as the mucosal prime that directs boost responses back to the lung. The purpose of this work was to determine the protection and safety profile over time following therapeutic administration of our lead Mtb vaccine candidate, ID93 with a synthetic TLR4 agonist (glucopyranosyl lipid adjuvant in a stable emulsion (GLA-SE)), in combination with rifampicin, isoniazid, and pyrazinamide (RHZ) drug treatment. We assessed the host inflammatory immune responses and lung pathology 7–22 weeks post infection, and determined the therapeutic efficacy of combined treatment by enumeration of the bacterial load and survival in the SWR/J mouse model. We show that drug treatment alone, or with immunotherapy, tempered the inflammatory responses measured in brochoalveolar lavage fluid and plasma compared to untreated cohorts. RHZ combined with therapeutic immunizations significantly enhanced TH1-type cytokine responses in the lung over time, corresponding to decreased pulmonary pathology evidenced by a significant decrease in the percentage of lung lesions and destructive lung inflammation. These data suggest that bacterial burden assessment alone may miss important correlates of lung architecture that directly contribute to therapeutic vaccine efficacy in the preclinical mouse model. We also confirmed our previous finding that in combination with antibiotics therapeutic immunizations provide an additive survival advantage. Moreover, therapeutic immunizations with ID93/GLA-SE induced differential T cell immune responses over the course of infection that correlated with periods of enhanced bacterial control over that of drug treatment alone. Here we advance the immunotherapy model and investigate reliable correlates of protection and Mtb control.

Genetic characterization of the HIV-1 reservoir after Vacc-4x and romidepsin therapy in HIV-1-infected individuals.

Objective:Therapeutic HIV-1 immunization followed by latency reversal has been suggested as a strategy to eradicate HIV-1. Here we investigate the phylogenetic composition of the HIV-1 regions targeted by the therapeutic HIV-1 peptide vaccine Vacc-4x in participants in a clinical trial.Design:Seventeen participants on suppressive antiretroviral therapy were vaccinated with six doses of Vacc-4x followed by three doses of romidepsin. Seven study participants were selected for sequencing analysis. All participants underwent an analytical treatment interruption.Methods:Single-genome/proviral sequencing of the p24-RT region was performed to genetically characterize proviral DNA, cell-associated RNA and outgrowth viruses during therapy as well as plasma HIV-1 RNA during an analytical treatment interruption.Results:There were no changes in cell-associated HIV-1 RNA (P = 0.83) and DNA (P = 0.09) diversity over the course of the study and no difference between cell-associated HIV-1 RNA and DNA diversity (P = 0.32). Only one participant showed signs of potential vaccine-related selection in the rebounding plasma virus. In five of seven participants, we identified human leukocyte antigen-specific cytotoxic T lymphocytes (CTL) epitopes containing nonsilent mutations in 100% of the sequences.Conclusion:We detected no evidence of selective immune pressure reflected in proviral diversity or by occurrence of specific mutation in the vaccine-targeted epitopes. Preexisting CTL epitope mutations may affect the potency of this therapeutic vaccine. This highlights the challenges of developing effective HIV-1 therapeutic vaccines.

Hyperthermic treatment at 56 °C induces tumour-specific immune protection in a mouse model of prostate cancer in both prophylactic and therapeutic immunization regimens

Most active cancer immunotherapies able to induce a long-lasting protection against tumours are based on the activation of tumour-specific cytotoxic T lymphocytes (CTLs). Cell death by hyperthermia induces apoptosis followed by secondary necrosis, with the production of factors named “danger associated molecular pattern” (DAMP) molecules (DAMPs), that activate dendritic cells (DCs) to perform antigen uptake, processing and presentation, followed by CTLs cross priming. In many published studies, hyperthermia treatment of tumour cells is performed at 42–45 °C; these temperatures mainly promote cell surface expression of DAMPs. Treatment at 56 °C of tumour cells was shown to induce DAMPs secretion rather than their cell surface expression, improving DC activation and CTL cross priming in vitro.Thus we tested the relevance of this finding in vivo on the generation of a tumour-specific memory immune response, in the TRAMP-C2 mouse prostate carcinoma transplantable model. TRAMP-C2 tumour cells treated at 56 °C were able not only to activate DCs in vitro but also to trigger a tumour-specific CTL-dependent immune response in vivo. Prophylactic vaccination with 56 °C-treated TRAMP-C2 tumour cells alone provided protection against TRAMP-C2 tumour growth in vivo, whilst in the therapeutic regimen, control of tumour growth was achieved combining immunization with adjuvant chemotherapy.

Vaccinating with Stem Cells to Stop Cancer

In a recent study, Kooreman and colleagues identify a set of genes expressed in both induced pluripotent stem cells (iPSCs) and cancer cells. Vaccination of mice with iPSCs induces prophylactic and therapeutic anticancer immunity to shared antigens, opening a possible avenue towards rapid generation of iPSC-based, personalized cancer vaccines.

ARID1A deficiency promotes mutability and potentiates therapeutic antitumor immunity unleashed by immune checkpoint blockade.

ARID1A (the AT-rich interaction domain 1A, also known as BAF250a) is one of the most commonly mutated genes in cancer1,2. The majority of ARID1A mutations are inactivating mutations and lead to loss of ARID1A expression 3 , which makes ARID1A a poor therapeutic target. Therefore, it is of clinical importance to identify molecular consequences of ARID1A deficiency that create therapeutic vulnerabilities in ARID1A-mutant tumors. In a proteomic screen, we found that ARID1A interacts with mismatch repair (MMR) protein MSH2. ARID1A recruited MSH2 to chromatin during DNA replication and promoted MMR. Conversely, ARID1A inactivation compromised MMR and increased mutagenesis. ARID1A deficiency correlated with microsatellite instability genomic signature and a predominant C>T mutation pattern and increased mutation load across multiple human cancer types. Tumors formed by an ARID1A-deficient ovarian cancer cell line in syngeneic mice displayed increased mutation load, elevated numbers of tumor-infiltrating lymphocytes, and PD-L1 expression. Notably, treatment with anti-PD-L1 antibody reduced tumor burden and prolonged survival of mice bearing ARID1A-deficient but not ARID1A-wild-type ovarian tumors. Together, these results suggest ARID1A deficiency contributes to impaired MMR and mutator phenotype in cancer, and may cooperate with immune checkpoint blockade therapy.

Synergistic anti-tumor efficacy of combined TLR3 and CD40 neoantigen vaccine requires Batf3-dependent dendritic cells

Abstract Therapeutic cancer vaccine therapy targeting neoantigens has yielded encouraging results, yet overall clinical responses to the treatment have thus far limited. A better understanding of the mechanisms that regulate priming of neoantigen-specific T cells is required to improve vaccine strategy. To this end, we used murine MC38 colon adenocarcinoma cells that harbor a single-epitope mutation within the Adpgk protein with the neo-epitope presented in MHC-I H-2Db molecules. MC38 tumor-bearing mice were treated with Adpgk mutant epitope in combination with toll-like receptor (TLR) 3 agonist, anti-CD40 agonist, or both. Therapeutic vaccination with the Adpgk mutant peptide combined with TLR3 and anti-CD40 agonists (TLR3/CD40) significantly improved survival compared with either TLR3 or CD40 stimulation alone. This was associated with expansion and differentiation of CD8+ T cells in the periphery as well as in the tumor microenvironment. Significantly increased terminally differentiated neoantigen-specific CD8+ T cells in blood and spleen were identified using the tetramer staining assay. Interestingly, frequency of neoantigen-specific CD8+ T cells in blood was inversely correlated with the tumor volume, suggesting epitope spreading and broadening T-cell clonality induced by TLR3/CD40 stimulation. Importantly, therapeutic efficacy of neoantigen/TLR3/CD40 vaccination was abrogated in Batf3 knockout mouse, suggesting Batf3-dependent dendritic cells play a critical role in priming adaptive immunity. These studies provide the rational basis for the use of combined TLR3 and CD40 agonists together as essential adjuvants to optimize vaccines designed to elicit therapeutic immunity against cancer neoantigens.

Interferon γ and Its Important Roles in Promoting and Inhibiting Spontaneous and Therapeutic Cancer Immunity.

Originally identified in studies of cellular resistance to viral infection, interferon (IFN)-γ is now known to represent a distinct member of the IFN family and plays critical roles not only in orchestrating both innate and adaptive immune responses against viruses, bacteria, and tumors, but also in promoting pathologic inflammatory processes. IFN-γ production is largely restricted to T lymphocytes and natural killer (NK) cells and can ultimately lead to the generation of a polarized immune response composed of T helper (Th)1 CD4+ T cells and CD8+ cytolytic T cells. In contrast, the temporally distinct elaboration of IFN-γ in progressively growing tumors also promotes a state of adaptive resistance caused by the up-regulation of inhibitory molecules, such as programmed-death ligand 1 (PD-L1) on tumor cell targets, and additional host cells within the tumor microenvironment. This review focuses on the diverse positive and negative roles of IFN-γ in immune cell activation and differentiation leading to protective immune responses, as well as the paradoxical effects of IFN-γ within the tumor microenvironment that determine the ultimate fate of that tumor in a cancer-bearing individual.

Protective effects of chicken egg yolk immunoglobulins (IgY) against experimental Aeromonas hydrophila infection in blunt snout bream (Megalobrama amblycephala)

The emergence of multi antibiotic resistance by the pathogens and toxic impacts on host metabolism has opened new perspectives to rational novel vaccine techniques. Outbreaks of Aeromonas hydrophila in aquaculture caused high mortality throughout the world and resulted in the extensive economic loss in the aquaculture industry. In this study, we report the efficacy of anti-A. hydrophila IgY antibodies by passive vaccination and its prophylactic or therapeutic effects against A. hydrophila in blunt snout bream. Inactivated A. hydrophila immunized hens produced effective IgY antibodies that were stable at temperatures less than 60 °C or the pH value was >4. The specific IgY can be bound directly to A. hydrophila that efficiently agglutinated and inhibited the bacterial growth in a dose-dependent manner. The specific IgY had significantly enhanced the phagocytosis activity of macrophages and resulted in rapid bacterial clearance. Anti-A. hydrophila IgY antibodies significantly increased macrophage mediated respiratory burst, including nitric oxide and superoxide anion production and subsequently killed the pathogen. Histopathological studies of intestine and spleen from vaccinated blunt-snout bream challenged with A. hydrophila showed the structural integrity of the organs was maintained intact from the bacterial injury. In addition, the prophylactic and therapeutic immunization, protected the blunt snout bream and the survival is approximately about 60% and 50%, respectively. These data suggest that specific IgY has the potential for protecting blunt snout bream against A. hydrophila infection and show promise for the future development of harmless vaccines.

Tumor Ablation and Therapeutic Immunity Induction by an Injectable Peptide Hydrogel.

Immunosuppressive tumor microenvironments (TMEs) create tremendous obstacles for an effective cancer therapy. Herein, we developed a melittin-RADA32 hybrid peptide hydrogel loaded with doxorubicin (DOX) for a potent chemoimmunotherapy against melanoma through the active regulation of TMEs. The formed melittin-RADA32-DOX (MRD) hydrogel has an interweaving nanofiber structure and exhibits excellent biocompatibility, controlled drug release properties both in vitro and in vivo, and an enhanced killing effect to melanoma cells. A single-dose injection of MRD hydrogel retarded the growth of primary melanoma tumors by more than 95% due to loaded melittin and DOX, with concomitant recruitment of activated natural killer cells in the tumors. Furthermore, MRD hydrogel can activate dendritic cells of draining lymph nodes, specifically deplete M2-like tumor-associated macrophages (TAMs), and produce active, cytotoxic T cells to further defend the cells against remaining tumors, providing potent anticancer efficacy against subcutaneous and metastatic tumors in vivo. Multidose injection of MRD hydrogel eliminated 50% of the primary tumors and provided a strong immunological memory effect against tumor rechallenge after eradication of the initial tumors. Owing to its abilities to perform controlled drug release, regulate innate immune cells, deplete M2-like TAMs, direct anticancer and immune-stimulating capabilities, and reshape immunosuppressive TMEs, MRD hydrogel may serve as a powerful tool for anticancer applications.

Synergy effects of Polyinosinic-polycytidylic acid, CpG oligodeoxynucleotide, and cationic peptides to adjuvant HPV E7 epitope vaccine through preventive and therapeutic immunization in a TC-1 grafted mouse model

ABSTRACTCross-talk by pattern recognition receptors may facilitate the maturation of dendritic cells and fine tune the immune response. Thus, the inclusion of ligands agonistic to multiple receptors in a vaccine formula may be an effective strategy to elicit robust antitumor cellular immunity. We tested the adjuvant effects and possible synergy of CpG (CpG oligodeoxynucleotide), Poly I:C (polyinosinic-polycytidylic acid) and the cationic peptide Cramp (cathelicidin-related antimicrobial peptide) formulated in a DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) liposomal HPV E7 epitope vaccine on a TC-1 grafted mouse model. The vaccine formulations were administered both preventively and therapeutically. Based on our results, both CpG and Poly I:C-adjuvanted vaccines abolished tumor development in a preventive trial and significantly suppressed tumor growth in a therapeutic trial. Increased interferon (IFN)-γ expression and potent memory T cells in splenocytes as well as elevated CD8+IFN-γ+ cells in both spleen and tumor tissue indicated an elevated E744-62-specific cellular immune response. Although synergistic effects were detected between CpG and Poly I:C, their adjuvant effects were not enhanced further when combined with Cramp. Because the enhancement of tumor antigen-specific cellular immune responses is vital for the clearance of infected and cancerous cells, our results contribute a potential adjuvant combination for cancer vaccines.