Transduction Of Dendritic Cells Research Articles

A pseudotyped adenovirus serotype 5 vector with serotype 49 fiber knob is an effective vector for vaccine and gene therapy applications

Adenoviruses (Ad) have demonstrated significant success as replication-deficient (RD) viral vectored vaccines, as well as broad potential across gene therapy and cancer therapy. Ad vectors transduce human cells via direct interactions between the viral fiber knob and cell surface receptors, with secondary cellular integrin interactions. Ad receptor usage is diverse across the extensive phylogeny. Commonly studied human Ad serotype 5 (Ad5), and chimpanzee Ad-derived vector “ChAdOx1” in licensed ChAdOx1 nCoV-19 vaccine, both form primary interactions with the Coxsackie and Adenovirus Receptor (CAR), which is expressed on human epithelial cells and erythrocytes. CAR usage is suboptimal for targeted gene delivery to cells with low/negative CAR expression, including human dendritic cells (DCs) and vascular smooth muscle cells (VSMCs). We evaluated the performance of a RD Ad5 vector pseudotyped with the fiber knob of human Ad serotype 49, termed Ad5/49K vector. Ad5/49K demonstrated superior transduction of murine and human DCs over Ad5, which translated into significantly increased T cell immunogenicity when evaluated in a mouse cancer vaccine model using 5T4 tumour-associated antigen. Additionally, Ad5/49K exhibited enhanced transduction of primary human VSMCs. These data highlight the potential of Ad5/49K vector for both vascular gene therapy applications and as a potent vaccine vector.

Checkpoint inhibitor-expressing lentiviral vaccine suppresses tumor growth in preclinical cancer models

BackgroundWhile immunotherapy has been highly successful for the treatment of some cancers, for others, the immune response to tumor antigens is weak leading to treatment failure. The resistance of tumors...

TFDP3 as E2F Unique Partner, Has Crucial Roles in Cancer Cells and Testis.

Transcription factor DP family member 3 (TFDP3) is a cancer-testis antigen, mainly expressed in normal testis and multiple cancers. TFDP3 gene (Gene ID: 51270) is located on the chromosome X and shares a high degree of sequence homology with TFDP1 and TFDP2, which can form heterodimers with E2F family members and enhance DNA-binding activity of E2Fs. In contrast to TFDP1 and TFDP2, TFDP3 downregulates E2F-mediated transcriptional activation. During DNA damage response in cancer cells, TFDP3 is induced and can inhibit E2F1-mediated apoptosis. Moreover, TFDP3 is involved in cell autophagy and epithelial-mesenchymal transition. Regarding cancer therapy opportunity, the transduction of dendritic cells with recombinant adenovirus-encoding TFDP3 can activate autologous cytotoxic T lymphocytes to target hepatoma cells. Here, we review the characterization of TFDP3, with an emphasis on the biological function and molecular mechanism. A better understanding of TFDP3 will provide new insights into the pathological mechanisms and therapeutic strategies for cancers.

Lentiviral vector induces high-quality memory T cells via dendritic cells transduction

We report a lentiviral vector harboring the human β2-microglobulin promoter, with predominant expression in immune cells and minimal proximal enhancers to improve vector safety. This lentiviral vector efficiently transduces major dendritic cell subsets in vivo. With a mycobacterial immunogen, we observed distinct functional signatures and memory phenotype in lentiviral vector- or Adenovirus type 5 (Ad5)-immunized mice, despite comparable antigen-specific CD8+ T cell magnitudes. Compared to Ad5, lentiviral vector immunization resulted in higher multifunctional and IL-2-producing CD8+ T cells. Furthermore, lentiviral vector immunization primed CD8+ T cells towards central memory phenotype, while Ad5 immunization favored effector memory phenotype. Studies using HIV antigens in outbred rats demonstrated additional clear-cut evidence for an immunogenic advantage of lentiviral vector over Ad5. Additionally, lentiviral vector provided enhance therapeutic anti-tumor protection than Ad5. In conclusion, coupling lentiviral vector with β2-microglobulin promoter represents a promising approach to produce long-lasting, high-quality cellular immunity for vaccinal purposes.

Adenovirus-Antibody Complexes Contributed to Lethal Systemic Inflammation in a Gene Therapy Trial

Intra-arterial administration of an adenovirus serotype 5 (Ad5) vector in a gene therapy trial caused lethal, systemic inflammation in subject 019 with ornithine transcarbamylase deficiency. This unanticipated inflammatory response was absent in another subject receiving the same vector dose and in 16 subjects receiving lower vector doses. We hypothesized that an immune memory to a previous natural adenovirus infection enhanced the immune response to high-dose systemic Ad5 vector,causing the exaggerated immune response in subject 019. To investigate this, we found that rabbit polyclonal sera to Ad5 and pooled human immunoglobulin (Ig) inhibited Ad5 vector transduction of non-immune cells invitro, but enhanced transduction and activation of human dendritic cells(DCs). Sera from approximately 7% of normal human subjects and 50% of patients treated topically with Ad5 vectors enhanced Ad5 transduction and activation of DCs, apparently from formation of Ig-Ad5 immune complexes and binding to DCs through FcγR. Subject 019's blood substantially increased Ad5-vector activation of human DC primary cultures at levels exceeding those from normal subjects. Although this study is based on one event in a single subject, the results implicatea pre-existing humoral immune response to Ad5 in the lethal systemic inflammatory response that occurred in subject019.

Vector uncoating limits adeno-associated viral vector-mediated transduction of human dendritic cells and vector immunogenicity

AAV vectors poorly transduce Dendritic cells (DC), a feature invoked to explain AAV’s low immunogenicity. However, the reason for this non-permissiveness remained elusive. Here, we performed an in-depth analysis using human monocyte-derived immature DC (iDC) as model. iDC internalized AAV vectors of various serotypes, but even the most efficient serotype failed to transduce iDC above background. Since AAV vectors reached the cell nucleus, we hypothesized that AAV’s intracellular processing occurs suboptimal. On this basis, we screened an AAV peptide display library for capsid variants more suitable for DC transduction and identified the I/VSS family which transduced DC with efficiencies of up to 38%. This property correlated with an improved vector uncoating. To determine the consequence of this novel feature for AAV’s in vivo performance, we engineered one of the lead candidates to express a cytoplasmic form of ovalbumin, a highly immunogenic model antigen, and assayed transduction efficiency as well as immunogenicity. The capsid variant clearly outperformed the parental serotype in muscle transduction and in inducing antigen-specific humoral and T cell responses as well as anti-capsid CD8+ T cells. Hence, vector uncoating represents a major barrier hampering AAV vector-mediated transduction of DC and impacts on its use as vaccine platform.

Decreased expression of A20 is associated with ocular Behcet’s disease (BD) but not with Vogt-Koyanagi-Harada (VKH) disease

PurposeA20 is a ubiquitously expressed and inducible cytosolic protein, which plays an important role in the negative regulation of inflammation and immunity. In this study, we investigated the role of...

First-in-Human Treatment With a Dendritic Cell-targeting Lentiviral Vector-expressing NY-ESO-1, LV305, Induces Deep, Durable Response in Refractory Metastatic Synovial Sarcoma Patient

Effective induction of antitumor T cells is a pivotal goal of cancer immunotherapy. To this end, lentiviral vectors (LV) are uniquely poised to directly prime CD8 T-cell responses via transduction of dendritic cells in vivo and have shown promise as active cancer therapeutics in preclinical tumor models. However, until now, significant barriers related to production and regulation have prevented their widespread use in the clinic. We developed LV305, a dendritic cell-targeting, integration-deficient, replication incompetent LV from the ZVex platform, encoding the full-length cancer-testis antigen NY-ESO-1. LV305 is currently being evaluated in phase 1 and 2 trials in metastatic recurrent cancer patients with NY-ESO-1 positive solid tumors as a single agent and in combination with anti-PD-L1. Here we report on the first patient treated with LV305, a young woman with metastatic, recurrent, therapy-refractive NY-ESO-1 synovial sarcoma. The patient developed a robust NY-ESO-1-specific CD4 and CD8 T-cell response after 3 intradermal injections with LV305, and subsequently over 85% disease regression that is continuing for >2.5 years posttherapy. No adverse events >grade 2 occurred. This case demonstrates that LV305 can be safely administered and has the potential to induce a significant clinical benefit and immunologic response in a patient with advanced stage cancer.

Abstract 5092: ZVex® lentiviral vector strongly activates pro-inflammatory, antigen processing, and anti-viral defense response pathways in monocyte-derived dendritic cells

Abstract Background: Dendritic cells (DCs) are professional antigen presenting cells that effectively bridge the innate and adaptive immune responses and require activation for successful priming of naïve T-cells. We have developed ZVex, an integration deficient, hybrid lentiviral vector engineered to target DC-SIGN expressed on immature myeloid DCs for genetic immunization against tumor antigens. As lentiviruses normally don't efficiently infect DCs, little is known about the functional effect of LV transduction of conventional DCs. Here, the effect of DC transduction with ZVex vectors was studied by gene expression profiling. Material and Methods: Human moDCs from multiple donors were prepared by 5-day stimulation with IL-4 and GM-CSF and transduced with ZVex-GFP or control vectors, such as a ZVex with nonfunctional reverse transcriptase (RT-dead), ZVex particles generated to contain no vector genome (empty ZVex), and a heat-inactivated ZVex preparation. Cellular RNA was isolated at different time points from transduced DC cultures and used for gene expression profiling with Nanostring’s human pan cancer immune panel (770 genes). Results: DCs transduced with ZVex vectors displayed statistically significant up-regulation of genes involved in pro-inflammatory, antigen processing, and anti-viral defense pathways. Noteworthy among the up-regulated genes were classical anti-viral response genes like OAS3, MX1, and interferon stimulated genes like ISG15 and ISG20. Incubation with the RT-dead mutant vector also led to up-regulation of these genes, albeit to a much lower extent, suggesting that LV-RNA itself can result in the activation of these pathways in DCs, though reverse transcription appears to further potentiate the response. Of note, cells transduced with empty ZVex, which consists of an intact virion particle but lacks encapsulated LV RNA, also mediated a low magnitude and breadth of DC transcriptional activation, possibly via signaling through DC-SIGN. Conclusions: ZVex incorporates several elements capable of inducing a potent innate immune activation in DCs. DCs are relatively insensitive to lentiviral infection, but the use of accessory proteins in ZVex makes DCs conducive to ZVex transduction and results in the induction of a Type-1 IFN response that seems to be largely dependent on reverse transcription. In addition, other engineered vector particle components such as non-reverse transcribed viral RNA, viral structural proteins, and/or engagement of the DC-SIGN receptor, also contribute to DC activation. Citation Format: Anshika Bajaj, Lisa Y. Ngo, Peter Berglund, Jan ter Meulen. ZVex® lentiviral vector strongly activates pro-inflammatory, antigen processing, and anti-viral defense response pathways in monocyte-derived dendritic cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5092. doi:10.1158/1538-7445.AM2017-5092

Abstract 4561: Non-integrative lentiviral particles for immunotherapy: RNA delivery to drive tumoral antigen presentation in a safe and efficient way

Abstract Safe and efficient cancer therapies using adoptive transfer of engineered cells are very promising but very challenging approaches. Opportunities to improve gene transfer into primary cells involve a better design of the vectors used. Such improvements must lead to an increase of the efficiency of gene expression, the cell phenotype preservation and the increase of the gene number delivered. The use of lentiviral vectors has largely increased in clinical protocols over the past few years but safety concerns have slowed down this progression. Actually, the permanent genetic modification remains a focus of significant regulatory oversight. On another side, mRNA delivery is a versatile, flexible, and safe mean for protein therapies, but existing techniques, such as chemical or electroporation-based transfection protocols, are known to induce cell toxicity and phenotype modifications of the target cells. Here, we present an innovative tool, named LentiFlash, allowing RNA delivery into target cells without any genomic scar. This tool combines RNA delivery, providing the best expression system to get efficient and transient expression, and lentiviral delivery which exhibits the best efficiency of entry into primary and stem cells. The RNA encapsidation is mediated via an RNA/protein interaction using properties of the MS2 bacteriophage. This new vector breaks with all existing systems. The resulting lentiviral particle is able to efficiently deliver non-viral coding or non-coding RNA into the cytoplasm of any cell type. These LentiFlash particles, are able to transduce delicate primary cells, such as dendritic cells, T cells and hematopoietic stem cells. They show an efficient, fast and transient expression of protein and RNA, such as antigenic peptides or genome editing tools, with no cell phenotype modification. Here we show a proof of concept on primary murine dendritic cells transduction with LentiFlash particles, successfully used to deliver several tumoral antigens. As a result, these dendritic cells are able to present the antigens to the immune system which in turn can efficiently fight a tumor development into wild type mice. This new delivery system gives the opportunity for multiple antigens co-expression to enhance immune responses, avoiding tumor relapse. Such multiple co-expressions into immune cells are expected to mimic the innate and adaptive immune responses. This transient RNA delivery mediated by LentiFlash is a powerful tool to induce an efficient gene delivery. The possibility to express multiple genes at once in the target cells is an attractive therapeutic perspective. The absence of any viral sequence avoids any integration event, an important safety consideration for human use. Finally, another advantage of the LentiFlash system is its ability to utilize lentiviral production platforms already validated in clinical settings. Citation Format: Pascale Bouillé, Christine Duthoit, Nicolas Martin, Lucille Lamouroux, Jean-Christophe Pages. Non-integrative lentiviral particles for immunotherapy: RNA delivery to drive tumoral antigen presentation in a safe and efficient way [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4561. doi:10.1158/1538-7445.AM2017-4561

Dendritic Cells Cross-Present Immunogenic Lentivector-Encoded Antigen from Transduced Cells to Prime Functional T Cell Immunity

Recombinant lentiviral vectors (LVs) are highly effective vaccination vehicles that elicit protective T cell immunity in disease models. Dendritic cells (DCs) acquire antigen at sites of vaccination and migrate to draining lymph nodes, where they prime vaccine-specific T cells. The potency with which LVs activate CD8+ T cell immunity has been attributed to the transduction of DCs at the immunization site and durable presentation of LV-encoded antigens. However, it is not known how LV-encoded antigens continue to be presented to T cells once directly transduced DCs have turned over. Here, we report that LV-encoded antigen is efficiently cross-presented by DCs in vitro. We have further exploited the temporal depletion of DCs in the murine CD11c.DTR (diphtheria toxin receptor) model to demonstrate that repopulating DCs that were absent at the time of immunization cross-present LV-encoded antigen to T cells in vivo. Indirect presentation of antigen from transduced cells by DCs is sufficient to prime functional effector T cells that control tumor growth. These data suggest that DCs cross-present immunogenic antigen from LV-transduced cells, thereby facilitating prolonged activation of T cells in the absence of circulating LV particles. These are findings that may impact on the future design of LV vaccination strategies.

Improvement of the cytotoxic T lymphocyte response against hepatocellular carcinoma by transduction of cancer cells with an adeno-associated virus carrying the interferon-γ gene.

Dendritic cell (DC)-based antigen-targeted immunotherapy may offer effective adjuvant therapy for hepatocellular carcinoma (HCC), in which cytotoxic T lymphocytes (CTLs) are key. However, in a number of cases, the activity of CTLs is completely inhibited due to the downregulated expression of major human leukocyte antigen (HLA) class I molecules by HCC cells. The aim of the present study was to overcome this issue. Hep3B cells were transduced by HCC‑specific recombinant adeno‑associated virus (rAAV) carrying human α‑fetoprotein promoter (AFPp) and the interferon‑γ (IFN‑γ) gene (rAAV/AFPp‑IFN‑γ). rAAV carrying the cytomegalovirus promoter (CMVp) and human α‑fetoprotein (AFP) gene (rAAV/CMVp‑AFP) was used to transduce professional antigen‑presenting DCs for the purpose of stimulating a CTL response. It was observed that transduction of DCs with rAAV/CMVp‑AFP resulted in: (i) AFP and interleukin‑12 expression; (ii) high expression levels of cluster of differentiation (CD)80, CD83, CD86, CD40, HLA‑death receptor and CD1a; (iii) T cell populations with marked IFN‑γ expression; (iv) a high percentage of CD69+/CD8+ T cells; and (v) the activity of CTLs against HLA‑A2‑expressing Hep3B cells. The transduction of Hep3B cells with rAAV/AFPp‑IFN‑γ resulted in: (i) IFN‑γ expression; (ii) upregulated expression of HLA‑A2; and (iii) an improved CTL response against HLA‑A2‑deficient Hep3B cells. rAAV/CMVp‑AFP‑transduced DCs elicited an AFP‑specific and HLA‑class I‑restricted CTL response against Hep3B cells. In conclusion, it was shown that the transduction of Hep3B with rAAV/AFPp-IFN-γ upregulated the expression of HLA-A2 and improved the sensitivity to CTL response.

Functional modification of dendritic cells with recombinant adenovirus encoding interleukin 10 for the treatment of sepsis.

Control of dendritic cell (DC) function is critical for strategies to modulate innate and acquired immune responses. We examined whether transduction of murine DCs with adenoviral vectors (Adv) expressing interleukin (IL)-10 could alter their phenotype and T cell stimulatory function. Murine bone marrow-derived DCs were transduced with AdV encoding human IL-10 or green fluorescent protein (GFP). Whereas transduction of immature DCs with AdV/GFP resulted in dose-dependent maturation, DCs transduced with Adv/IL-10 maintained an immature state with low major histocompatibility complex (MHC) class II, CD86, and IL-12 expression. The Adv/IL-10 transduced DCs were phenotypically unique, characterized by suppression of IL-12 expression, failure to stimulate Th1 or Th2 cytokine responses, and retained capacity to endocytose antigen. Importantly, Adv/IL-10-transduced DCs were biologically active in vivo, in that administration of these DCs into mice before a generalized peritonitis significantly improved survival. We conclude that Adv/IL-10 transduction of DCs provides an efficient means to modulate DC function. The capacity to modify DCs by adenoviral expression of IL-10 may provide a novel ex vivo or in vivo approach to mitigate acute and chronic inflammatory diseases like sepsis.

Abstract A25: Immunotherapy using LOAd700 armed with CD40 ligand controls experimental pancreatic cancer and activates immune responses

Abstract The aim of this study was to evaluate the efficacy of LOAd immunotherapy to treat pancreatic cancer in a xenograft model as well as to determine the capacity of LOAds to stimulate the immune system in in vitro models. LOAd viruses are oncolytic adenoviruses (5/35) armed with immunostimulatory genes in order to shift the tumor milieu towards immune activation at the same time providing release and spread of tumor antigens due to oncolysis. In this manner, LOAd virus is an antigen-independent immunotherapy for various solid tumors. LOAd armed with CD40 ligand (LOAd700) was constructed from the ICOVIR system by changing the virus shaft and knob to that of serotype 35 to broaden virus binding and entry into cells (pending patent EP14163704). Oncolysis in OCOVIRs is restricted to cells with a disrupted Rb pathway. Viruses were produced using A549 cells. The pancreatic cancer cell lines BxPC3, Panc01, MiaPaCa2 and PaCa3 were used for in vitro evaluation and Panc01 was used in the xenograft model in Nu/Nu mice. Human dendritic cells (DCs) were obtained by differentiation of CD14+ monocytes with GM-CSF and IL4 for 7 days. LOAd viruses infected both the panel of pancreatic tumor cell lines and human DCs with high efficacy and both the cell lines and the DCs expressed the CMV driven transgene/s post transduction. The oncolytic LOAd virus did not kill DCs nor healthy pancreatic cells present in donor islet cell isolation surplus material. In contrast, oncolysis was restricted to the tumor cell lines. The LOAd-transduced DCs increased markers of maturation such as MHC II, CD86, CD70 and CD83 and produced high levels of IL12. LOAd stimulated DCs were able to activate and expand antigen-specific T cells and NK cells as demonstrated in a CMV in vitro system. LOAd viruses could control tumor growth in a xenograft immunodeficient model due to oncolysis alone. In conclusion, LOAd700 can kill pancreatic tumor cells in vitro and control growing tumor in mice due to oncolysis alone. Further, upon DC transduction, LOAd viruses mature DCs to efficient antigen presenters and stimulators of Th1 effector cells such as T cells and NK cells. LOAd is an interesting new immunotherapy for solid malignancies including pancreatic cancer. Citation Format: Emma Svensson, Rafael Moreno, Ioanna Milenova, Lisa Christiansson, Ramon Alemany, Angelica Loskog. Immunotherapy using LOAd700 armed with CD40 ligand controls experimental pancreatic cancer and activates immune responses. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A25.

Vpx-Independent Lentiviral Transduction and shRNA-Mediated Protein Knock-Down in Monocyte-Derived Dendritic Cells.

The function of dendritic cells (DCs) in the immune system is based on their ability to sense and present foreign antigens. Powerful tools to research DC function and to apply in cell-based immunotherapy are either silencing or overexpression of genes achieved by lentiviral transduction. To date, efficient lentiviral transduction of DCs or their monocyte derived counterparts (MDDCs) required high multiplicity of infection (MOI) or the exposure to the HIV-2/SIV protein Vpx to degrade viral restriction factor SAM domain and HD domain-containing protein 1 (SAMHD1). Here we present a Vpx-independent method for efficient (>95%) transduction of MDDCs at lower MOI. The protocol can be used both for ectopic gene expression and knock-down. Introducing shRNA targeting viral entry receptor CD4 and restriction factor SAMHD1 into MDDCs resulted in down-regulation of targeted proteins and, consequently, expected impact on HIV infection. This protocol for MDDCs transduction is robust and free of the potential risk arising from the use of Vpx which creates a virus infection-prone environment, potentially dangerous in clinical setting.

Vpx-containing dendritic cell vaccine induces CTLs and reactivates latent HIV-1 in vitro.

Eradication of HIV-1 from an infected individual requires a means of inducing production of virus from latently infected cells and stimulating an immune response against the infected cells. We report the development of lentiviral vectors that transduce dendritic cells (DCs) to both induce production of virus from latently infected cells and stimulate antigen-specific CTLs. The vectors package Vpx, a lentiviral accessory protein that counteracts the SAMHD1-mediated block to DC transduction, allowing for long-term expression of vector-encoded proteins. The vectors encode influenza or HIV-1-derived epitopes fused via a self-cleaving peptide to CD40L that releases the peptide into the endoplasmic reticulum for entry into the antigen presentation pathway. Expression of CD40L caused transduced DCs to mature and produce Th1-skewing cytokines. The DCs presented antigen to CD8 T cells, enhancing antigen-specific CTLs. Coculture of the transduced DCs with latently infected cells induced high level virus production, an effect that was mediated by TNF-α. The ability of a DC vaccine to reactivate latent HIV-1 and stimulate an adaptive immune response provides a means to reduce the size of the latent reservoir in patients. This strategy can also be applied to develop DC vaccines for other diseases.

Early interaction of adeno-associated virus serotype 8 vector with the host immune system following intramuscular delivery results in weak but detectable lymphocyte and dendritic cell transduction.

Following in vivo recombinant adeno-associated virus (rAAV)-based gene transfer, adaptive immune responses specific to the vector or the transgene product have emerged as a potential roadblock to successful clinical translation. The occurrence of such responses depends on several parameters, including the route of vector administration as well as the viral serotype and the genome configuration, either self-complementary (sc) or single-stranded (ss). These parameters influence rAAV vector-associated immunity by modulating the crosstalk between the vector and the host immune system, including vector ability to interact or even transduce lymphoid tissues in general and antigen-presenting cells (APCs) in particular. Little is known about immune cell populations that are targeted in vivo by rAAV vectors. Moreover, the transduction of dendritic cells is still controversial and not directly demonstrated. Here, we show that intramuscular administration of an sc rAAV8 vector in the mouse leads to a rapid distribution of viral genomes in the lymphoid tissues that is associated with transgene expression. Transduced cells were detected in follicular areas of the spleen and the draining lymph nodes. In addition to B and T lymphocytes, transduced professional APCs were detected although at very low frequency. In addition, viral genomes and transgene transcripts were also detected in these cell populations after ss rAAV8 vector administration. Although the functional significance of those observations needs further explorations, our results highlight an early and intricate interaction between the rAAV vector upon its in vivo delivery and the host immune system.

Clinical testing of a dendritic cell targeted therapeutic vaccine in patients with chronic hepatitis C virus infection

The lack of antiviral cellular immune responses in patients with chronic hepatitis C virus (HCV) infection suggests that T-cell vaccines may provide therapeutic benefit. Due to the central role that dendritic cells (DC) play in the activation of T-cell responses, our aim was to carry out a therapeutic vaccination clinical trial in HCV patients using DC. Five patients with chronic HCV infection were vaccinated with three doses of 5 × 106 or 107 autologous DC transduced with a recombinant adenovirus encoding NS3 using the adapter protein CFh40L, which facilitates DC transduction and maturation. No significant adverse effects were recorded after vaccination. Treatment caused no changes in serum liver enzymes nor in viral load. Vaccination induced weak but consistent expansion of T-cell responses against NS3 and adenoviral antigens. Patients’ DC, as opposed to murine DC or DC from healthy subjects, secreted high IL-10 levels after transduction, inducing the activation of IL-10–producing T cells. IL-10 blockade during vaccine preparation restored its ability to stimulate anti-NS3 Th1 responses. Thus, vaccination with adenovirus-transduced DC is safe and induces weak antiviral immune responses. IL-10 associated with vaccine preparation may be partly responsible for these effects, suggesting that future vaccines should consider concomitant inhibition of this cytokine.

Abstract 702: DCVex(TM): A novel integration-deficient lentivector technology that incorporates genetic and post-translational elements to target dendritic cells

Abstract With the goal of creating antigen-directed immunotherapeutics that can be safely administered directly to patients, Immune Design has developed a platform of novel integration-deficient lentiviral vectors that target and deliver antigen-encoding nucleic acids to dendritic cells (DCs) in order to promote the activation of antigen-specific effector CD8 T cells. This platform, termed DCVex(TM), utilizes a novel genetic variant of a Sindbis virus envelope glycoprotein with post-translational carbohydrate modifications in combination with Vpx, a SIVmac viral accessory protein, to achieve efficient targeting and transduction of DCs. In addition, DCVex(TM) incorporates safety features in its design that include redundant mechanisms to render DCVex(TM) integration-deficient, as well as genetic modifications that eliminate psi-gag recombination between split genome components. The characteristics that allow DCVex(TM) to specifically transduce human DCs and the advances that DCVex(TM) brings to conventional third-generation lentiviral vector design demonstrate its potential as a vaccine designed to utilize DCs for cancer immunotherapy. Citation Format: Semih U. Tareen, Brenna Kelley-Clarke, Christopher J. Nicolai, Megan M. Slough, Chintan D. Vin, Neal Van Hoeven, Scott H. Robbins, Jan H. ter Meulen, Peter Berglund. DCVex(TM): A novel integration-deficient lentivector technology that incorporates genetic and post-translational elements to target dendritic cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 702. doi:10.1158/1538-7445.AM2014-702

Astragalus mongholicus regulate the Toll-like-receptor 4 meditated signal transduction of dendritic cells to restrain stomach cancer cells.

Backgroud : According to the traditional view, we depend on three methods to treat tumors; surgery, chemotherapy and radiotherapy. However, these methods have its own limitations in application. Traditional Chinese Medicine (TCM) is one of the oldest healing systems. Astragalus mongholicus (AMs) that is the common herbal medicine, the biggest part of TCM, have been proved to be effective in treating cancers from lots of clinical cases. However, we have not fully understood the anti-tumor mechanism of AMs, and this has lead to some doubt for some Western-Medicine scholars and restricts its wide use. The main objective of this research is to discuss the effect and mechanism of AMs to human stomach cancer. Materials and Methods : To observe the effect and mechanism of tumor treatment by AMs, we have done the research from three major aspects, the influence of DCs, the inhibition of tumor in vitro as well as the animal studies in vivo after treatment. First, we culture the mouse dendritic cells (DCs) from bone marrow of mouse hind legs according to the method using Interleukin-4(IL-4) and Granulocyte-macrophage colony stimulating factor (GM-CSF), which refer to the way established by Inaba (Inaba K, 1992). And then we investigate the growth-rate of the DCs co-cultured with AMs injection. We analyze the expression of the Toll-like-receptor 4(TLR4), with SYBR-Green I Real-time PCR and the I-kappa-B-alpha (IκB-α) with Western-Blot, the main regulatory protein to control nuclear factor NFκB-p65 nuclear translocation. Second, we choose the human gastric cancer cell lines MKN 45 as the target cell, which was co-cultured with DCs,T cells from spleen of mouse and AMs injection, and use MTT assay to judge the amount of cell lines and Immnunoflurescene to analyze the expression of anti-active caspase 3 pAb anti-PARP P85 fragment pAb, the mark of apoptosis of cells. Third, we have conducted the animal studies beside the basic experiment in vitro . The nude mouse developed stomach cancer, due to intra-preritoneal injection with MKN45 have been divided into two groups: the treatment group challenged with AMs injection and the control group with saline injection. We took the average of the diameter of each group as the y axis and the days after administered with AMs as x axis. After 40 days, all animals were killed by detruncation, and the tumor were removed and measured. We compare the diameter ( 40 days) of the tumor as well as the survival days between different groups to investigate the effect of inhibition of cancer. Results : All results show that AMs is effective in treating human stomach cancer and the mechanism might be regulated by TLR4 mediated signal transduction of DCs. The results are briefly introduced as follows:First, we succeed in culturing the DCs induced by IL-4 and GM-CSF and find the positive rate of CD11c expression, the mark of DCs,is beyond 90% (Fig-1). We detect AMs can precipitate DCs maturation by upregulating TLR4 in SYBR-Green I Real-time PCR (Fig-2) and suppressing IκB-αby Western-Blot (Fig-3). Second, after the MKN45 co-cultured with DCs,T cells and AMs injection, the result show that AMs can great reduce the amount of cell lines by MTT assay (Fig-4) and induce apoptosis with Immunofluorescence (Fig-5). Finally, we have conducted animal studies beside the experiment in vitro , and the result in vivo show that AMs can delay tumor development from the diameter and weight of the tumor (Fig-6, Fig-7), prolong life-span and improve life-quality. Conclusion : Ams Can play a great role in treating human stomach cancers as a good Chinese herbal medicine by precipitating DCs maturation, which is probably due to its effects by regulating the TLR4 mediated signal transduction. Key words : Astragalus mongholicus ; huaman stomach cancer; Traditional Chinese Medicine (TCM); Chinese herbal medicine; Toll-like-receptors 4; NFκB-p65; IκB-α