Dendritic cell as therapeutic vaccines against tumors and its role in therapy for hepatocellular carcinoma

Dendritic cells （DCs） are the most potent professional antigen-presenting cells, and capable of stimulating naive T cells and driving primary immune responses. DCs are poised to capture antigen, migrate to draining lymphoid organs, and after a process of maturation, select antigen-specific lymphocytes to which they present the processed antigen, thereby inducing immune responses. The development of protocols for the ex vivo generation of DCs may provide a rationale for designing and developing DC-based vaccination for the treatment of tumors. There are now several strategies being applied to upload antigens to DCs and manipulate DC vaccines. DC vaccines are able to induce therapeutic and protective antitumor immunity. Numerous studies indicated that hepatocellular carcinoma （HCC） immunotherapies utilizing DC-presenting tumor-associated antigens could stimulate an antitumour T cell response leading to clinical benefit without any significant toxicity. DC-based tumor vaccines have become a novel immunoadjuvant therapy for HCC. Cellular ＆ Molecular Immunology. 2006;3（3）：197-203.     

Therapy of established tumour with a hybrid cellular vaccine generated by using granulocyte–macrophage colony-stimulating factor genetically modified dendritic cells

Dendritic cells (DCs) are the most powerful of all antigen-presenting cells and play a critical role in the induction of primary immune responses. DC-based vaccination represents a potentially powerful strategy for cancer immunotherapy. In this study, a new approach for a DC-based melanoma vaccine was described. Splenic DCs from C57BL/6 mice were fused with B16 melanoma cells, and the resultant B16/DC hybrid cells expressed major histocompatibility complex (MHC) molecules - B7 as well as the B16 tumour marker M562 - which were enriched by Ia-mediated positive selection with a MiniMACS column. The fusion rates were 12.7-26.8%. To generate hybrid tumour vaccines with potentially greater potent therapeutic efficacy, we genetically engineered DCs with granulocyte-macrophage colony-stimulating factor (GM-CSF) prior to cell fusion. Recombinant adenovirus vector was used to mediate gene transfer into DCs with high efficiency and DCs expressed GM-CSF at 96-138 ng/105 cells/ml 24 hr after GM-CSF gene transfer. GM-CSF gene-modified DCs (DC.GM) exhibited higher expression of B7 and co-stimulatory capacity in mixed lymphocyte reaction (MLR). Fusion of DC.GM with B16 cells generated B16/DC.GM hybrid cells secreting GM-CSF at 59-63 ng/105 cells/ml. Immunization of C57BL/6 mice with the B16/DC hybrid vaccine elicited a specific cytotoxic T-lymphocyte (CTL) response and protected the immunized mice from B16 tumour challenge, reduced pulmonary metastases and extended the survival of B16 tumour-bearing mice. The B16/DC.GM hybrid vaccine was able to induce a CTL response and protective immunity more potently and tended to be therapeutically more efficacious than the B16/DC vaccine. In vivo depletion of T-cell subsets demonstrated that both CD8+ and CD4+ T cells were essential for the therapeutic effects of B16/DC and B16/DC.GM hybrid vaccines. Additionally, other non-specific effector cells may also contribute to tumour rejection induced by the B16/DC.GM hybrid vaccine. These data indicate that a DC-based hybrid tumour vaccine may be an attractive strategy for cancer immunotherapy, and that GM-CSF gene-modified DCs may lead to the generation of hybrid vaccines with potentially increased therapeutic efficacy.     

Dendritic cells in cancer immunotherapy

Antigen presentation is a critical regulatory element for the induction of cellular immune responses. Thus, one of the principal current goals of tumor immunotherapy is to control and enhance tumor antigen presentation. In this respect, dendritic cells (DC) are now being widely investigated as immunotherapeutic agents for the treatment of disseminated malignancies. At present, numerous ways to employ DCs for tumor immunotherapy are being tested, ranging from direct in situ expansion and activation of DCs to adoptive transfer of ex vivo generated DCs, and numerous techniques have been designed to optimize DC activation, tumor antigen delivery to DCs, and induction of tumor-specific, as well as helper immune responses, in vivo. However, the results of recent preclinical studies and the diversity of the clinical phase I trials that are currently underway indicate that little is still known about the exact mechanisms by which DCs modulate tumor immunity and pose the concern that premature clinical trials might not yield the desired results and might be harmful to, rather than promote, the concept of DC-based tumor immunotherapy. This review summarizes some of the current approaches to induce tumor immunity by DC-based vaccination and discusses their advantages and concerns.     

Dendritic cells as vectors for immunotherapy of tumor and its application for gastric cancer therapy

Dendritic cells(DCs) are recognized as the most potent antigen-presenting cells(APCs) with the ability tostimulate naive resting T cells and initiate primary immune responses.DCs are poised to capture antigen(Ag),migrate to draining lymphoid organs,and,after a process of maturation,select Ag-specific lymphocytes towhich they present the processed Ag,thereby inducing immune responses.Numerous studies indicated thatimmunotherapies utilizing DC-presenting tumor-associated antigens can safely be administered to cancerpatients and induce significant immunologic and clinical responses.Moreover,it has been demonstrated thatDCs are related to clinical stage,invasion,metastasis and prognosis of gastric cancer.DC-based tumor vaccinesbecome a new effective immunoadjuvant therapy for gastric cancer.Cellular & Molecular Immunology.2004;1(5):351-356.     

抗肿瘤树突状细胞疫苗研究进展

近代肿瘤学的新发展主要在于其对抗原提呈过程的新认识 ,大多数抗原都需要经过抗原提呈细胞(ａｎｔｉｇｅｎｐｒｅｓｅｎｔｉｎｇｃｅｌｌｓ ,ＡＰＣｓ)的摄取、加工、内化 ,产生短链多肽并与主要组织相容性复合体 (ｍａｊｏｒｈｉｓｔｏ ｃｏｍｐａｔｉｂｉｌｉｔｙｃｏｍｐｌｅｘ ,ＭＨＣ)结合后表达于细胞膜表面才能被Ｔ细胞受体所识别 ;同时 ,ＡＰＣｓ与Ｔ细胞之间通过共刺激分子的结合发出第二信号 ,从而更加有效地促进Ｔ细胞的活化、增殖。因此 ,在抗肿瘤免疫过程中 ,ＡＰＣｓ的功能越来越受到人们的重视。近年来 ,作为抗原提呈能力最…     

Protective anti-tumour immune responses by murine dendritic cells pulsed with recombinant Tat-carcinoembryonic antigen derived from Escherichia coli

Carcinoembryonic antigen (CEA) is over-expressed on various human cancer cells and has been the target of immunotherapies using dendritic cells (DCs) pulsed with CEA-specific RNA or peptides, or transduced by CEA-expressing adenovirus or vaccinia virus. Because activated DCs do not phagocytose soluble protein antigens efficiently and pure immature DCs are not obtained easily ex vivo, an efficacious whole CEA protein-loaded DC vaccine has not been reported. To improve the antigen delivery into DCs, we utilized CEA conjugated to a protein-transduction domain, human immunodeficiency virus transactivating Tat. Furthermore, we purified the truncated non-glycosylated CEA from Escherichia coli to overcome the safety concerns and immunosuppressive functions associated with the native CEA protein. Using confocal microscopy and fluorescence activating cell sorter analysis, we demonstrated that the Tat-CEA protein entered the cytoplasm of DCs efficiently within 10 min of co-culture, compared with the negligible amount of CEA into DCs 30 min later. CEA-specific T cell proliferation and cytotoxic T cell responses were enhanced significantly in mice immunized with Tat-CEA-pulsed DCs [DC (Tat-CEA)] compared with those immunized with CEA-pulsed DCs [DC (CEA)]. T helper type 1 responses were more prominent in the DC (Tat-CEA) immunized mice whose splenocytes secreted more interferon-γ and less interleukin-4 than those from DC (CEA) immunized mice. In vivo, the DC (Tat-CEA) vaccine delayed tumour growth significantly and prolonged survival of tumour-bearing mice. These results suggest that protective epitopes are well preserved on bacteria-derived recombinant Tat-CEA. This strategy may provide a basic platform for DC-based anti-CEA vaccines that could be utilized in combination with advanced immune-enhancing therapeutics.     

Optimizing dendritic cell-based immunotherapy for cancer

Dendritic cells (DCs) are the most powerful professional antigen-presenting cells and are unique in their capability to initiate, maintain and regulate the intensity of primary immune responses, including specific antitumor responses. Development of practical procedures to prepare sufficient numbers of functional human DCs in culture from the peripheral blood precursors, paved the way for clinical trials to evaluate various DC-based strategies in patients with malignant diseases. However, no definite conclusions regarding the clinical and even immunological efficacy of DC vaccination can be stated, despite the fact that 12 years have passed since the first clinical trial utilizing DCs in cancer patients. Many unanswered questions hamper the development of DC-based vaccines, including the source of DC preparation and protocols for DC generation, activation and loading with tumor antigens, source of tumor antigens, route of vaccine administration and methods of immunomonitoring. Fortunately, in spite of the many obstacles, DC vaccines continue to hold promise for cancer therapy.     

体外培养树突状细胞成熟诱导物的研究进展

树突状细胞(DC)是体内功能最强的抗原提呈细胞(APC),参与多种自身免疫疾病、肿瘤、免疫缺陷病及某些炎症反应的病理过程.以成熟DC为基础制备的DC疫苗具有明显的诱发抗肿瘤免疫应答的能力,其显著的抗肿瘤效应已经得到广泛认可.目前体外培养DC的方法尚没有统一的标准,致使培养得到的DC在均一度和功能上有很大的差别,很大程度上限制了DC的基础研究和DC疫苗的临床应用工作的开展.以下综述了体外扩增DC所用成熟诱导物的特点,为相关研究者体外扩增DC提供了参考.     

Helper role of NK cells during the induction of anticancer responses by dendritic cells

Recent reports demonstrate that natural killer (NK) cells and dendritic cells (DC) support each other's activity in a positive feedback. We observed that activated NK cells induce the maturation of DCs into stable type-1 polarized DCs (DC1), characterized by up to 100-fold enhanced ability to produce IL-12p70 in response to subsequent interaction with Th cells. DC1 induction depends on NK cell-produced IFN-gamma and TNF-alpha, with a possible involvement of additional factors. DC1, induced by NK cells or by NK cell-related soluble factors, are stable, resistant to tumor-related suppressive factors, and show strongly enhanced ability to induce Th1 and CTL responses. In analogy to resting T cells, the induction of "helper" function of NK cells relies on a two-signal activation paradigm. While NKG2D-dependent tumor cell recognition is sufficient to induce the cytotoxic "effector" function of NK cells, the induction of "NK cell help" requires additional signals from type-1 IFNs, products of virally-infected cells, or from IL-2. Compared to non-polarized DCs currently-used in clinical trials, DC1s act as superior inducers of anti-cancer CTL responses during in vitro sensitization. The current data provides rationale for the clinical use of DC1s in cancer and chronic infections (such as HIV), as a new generation DC-based vaccines, uniquely combining fully mature DC status with an elevated, rather than "exhausted" ability to produce bioactive IL-12p70. We are currently implementing stage I/II clinical trials, testing the effectiveness of DC1s induced by NK cells or by NK cell-related factors, as therapeutic vaccines against melanoma.     

Immunogenicity of dendritic-tumor fusion hybrids and their utility in cancer immunotherapy

Cancer immunotherapy using fusion hybrid cells generated from dendritic cells (DCs) and tumor cells may be more effective than other DC-based vaccines. DC-tumor fusion potentially confers not only the DCs' antigen-presenting functionality but also a continuing source of endogenous tumor antigens for major-histocompatibility-complex-restricted T-cell sensitization. In animal models, many investigators demonstrated that vaccination with fusion hybrids was protective against tumor challenge and therapeutic, resulting in the regression of established tumors. In clinical trials for patients with a variety of metastatic diseases, fusion hybrid vaccines were well tolerated, but the overall objective response rate was only 10.9%. Careful scrutiny of a large number of publications revealed that, in most cases, no definitive evidence of heterokaryonic fusion cell formation was found. Further corroboration of this conclusion comes from reports that fusion hybrids generated from autologous (syngeneic) and allogeneic DCs displayed equivalent immunological function and therapeutic effects in vitro and in vivo. This puzzling finding suggests that effective fusion immunotherapy depends on tumor antigen scavenging and presentation by antigen-presenting cells (APCs) of host origin and is in violation of the basic tenet of the principle of DC function. We believe that conclusions drawn from reported clinical trials have not properly evaluated the efficacy of the DC-tumor hybrid vaccine, and therefore, they neither confirm nor disclaim the potential benefits that may be derived from this form of immunotherapy.     

Potent Dendritic Cell Vaccine Loaded with Latent Membrane Protein 2A （LMP2A）

Epstein-Barr virus （EBV）, a potential oncogenic herpesvirus, has been found to be associated with several malignancies. It＇s critical to elicit cellular immunity of the body to fight against EBV-associated tumor development. Using dendritic cells （DCs） loaded with latent membrane protein 2A （LMP2A） to elicit T cell response against tumor may be one of the most direct and safest immunotherapy approaches. The present study aimed to develop DCs-based cancer vaccine （DC loaded with LMP2A protein） and study its biological characteristics and immune functions. Purified LMP2A protein was extracted from a cell line L929/LMP2A stably expressing LMP2A. LMP2A could be loaded on DCs with no significant changes of the DC surface markers and cytomorphology. The percentage of DCs loaded with LMP2A was above 80%. LMP2A-loaded DCs markedly enhanced the proliferation of antigen-specific CD8^＋ T and CD4^＋ T cells by 3H-TdR incorporation assay. Besides, the specific cytotoxicity of the CTLs against LMP2A target cells was also significantly increased. These results indicated that DC-based vaccine loaded with virus antigen could elicit potent CTL response and provide a foundation for further study on the DC-based immunotherapy for nasopharygeal carcinoma and other EBV associated tumors. Cellular ＆ Molecular Immunology.     

Neem leaf glycoprotein overcomes indoleamine 2,3 dioxygenase mediated tolerance in dendritic cells by attenuating hyperactive regulatory T cells in cervical cancer stage IIIB patients

Tolerogenic dendritic cells (DCs) are a subset of DCs characterized by abundant indoleamine 2,3 dioxygenase (IDO) expressions. IDO may be co-operatively induced in DCs by regulatory T (Tregs) cells and various DC maturation agents. Tregs are markedly amplified in the physiological system of cancer patients, inducing over tolerance in DCs that leads to the hyper accumulation of immunosuppressive IDO in tumor microenvironment, thereby, hampering anti-tumor immunity. Consequently, a major focus of current immunotherapeutic strategies in cancer is to minimize IDO, which is possible by reducing Tregs and using various IDO inhibitors. Neem leaf glycoprotein (NLGP), a natural and nontoxic immunomodulator, demonstrated several unique immunoregulatory activities. Noteworthy activities of NLGP are to mature DCs and to inhibit Tregs. As Tregs are inducer of IDO in DCs and hyperactive Tregs is a hallmark of cancer, we anticipated that NLGP might abrogate IDO induction in DCs by inhibiting Tregs. Evidences are presented here that in a co-culture of DCs and Tregs isolated from cervical cancer stage IIIB (CaCx-IIIB) patients, NLGP does inhibit IDO induction in DCs by curtailing the over expression of Cytotoxic T-Lymphocyte Antigen 4 (CTLA4) on Tregs and concomitantly induces optimal DC maturation. In contrast, in the presence of LPS as maturation agent the DCs displays a tolerogenic profile. This finding suggests the reduction of tolerogenecity of DCs in CaCx-IIIB patients by reducing the IDO pool using NLGP. Accordingly, this study sheds more light on the diverse immunomodulatory repertoire of NLGP.     

Enhancement of anti-tumor immunity through local modulation of CTLA-4 and GITR by dendritic cells

Cancer vaccines have now demonstrated clinical efficacy, but immune modulatory mechanisms that prevent autoimmunity limit their effectiveness. Systemic administration of mAbs targeting the immune modulatory receptors CTLA-4 and glucocorticoid-induced TNFR-related protein (GITR) on Treg and effector T cells augments anti-tumor immunity both experimentally and clinically, but can induce life-threatening autoimmunity. We hypothesized that local delivery of anti-CTLA-4 and anti-GITR mAbs to the sites where T cells and tumor antigen-loaded DC vaccines interact would enhance the induction of anti-tumor immunity while avoiding autoimmunity. To achieve this goal, DCs transfected with mRNA encoding the H and L chains of anti-mouse CTLA-4 and GITR mAbs were co-administered with tumor antigen mRNA-transfected DCs. We observed enhanced induction of anti-tumor immunity and significantly improved survival in melanoma-bearing mice, without signs of autoimmunity. Using in vitro assays with human DCs, we demonstrated that DCs transfected with mRNA encoding a humanized anti-CTLA-4 mAb and mRNA encoding a soluble human GITR-L fusion protein enhance the induction of anti-tumor CTLs in response to DCs transfected with mRNAs encoding either melanoma or breast cancer antigens. Based on these results, this approach of using local delivery of immune modulators to enhance vaccine-induced immunity is currently being evaluated in a phase I clinical cancer immunotherapy trial.     

Co-culture of apoptotic breast cancer cells with immature dendritic cells: a novel approach for DC-based vaccination in breast cancer

A dendritic cell (DC)-based vaccine strategy could reduce the risk of recurrence and improve the survival of breast cancer patients. However, while therapy-induced apoptosis of hepatocellular and colorectal carcinoma cells can enhance maturation and antigen presentation of DCs, whether this effect occurs in breast cancer is currently unknown. In the present study, we investigated the effect of doxorubicin (ADM)-induced apoptotic MCF-7 breast cancer cells on the activation of DCs. ADM-induced apoptotic MCF-7 cells could effectively induce immature DC (iDC) maturation. The mean fluorescence intensity (MFI) of DC maturity marker CD83 was 23.3 in the ADM-induced apoptotic MCF-7 cell group compared with 8.5 in the MCF-7 cell group. The MFI of DC co-stimulatory marker CD86 and HLA-DR were also increased after iDCs were treated with ADM-induced apoptotic MCF-7 cells. Furthermore, the proliferating autologous T-lymphocytes increased from 14.2 to 40.3% after incubated with DCs induced by apoptotic MCF-7 cells. The secretion of interferon-γ by these T-lymphocytes was also increased. In addition, cell-cell interaction between apoptotic MCF-7 cells and iDCs, but not soluble factors released by apoptotic MCF-7 cells, was crucial for the maturation of iDCs. These findings constitute a novel in vitro DC-based vaccine strategy for the treatment of breast cancer by ADM-induced apoptotic MCF-7 cells.     

Superior efficacy of dendritic cell-tumor fusion vaccine compared with tumor lysate-pulsed dendritic cell vaccine in colon cancer

Dendritic cell (DC)-based tumor vaccine is a promising therapy for malignancies. Recent studies showed greater potency with DC/tumor fusion vaccines against acute myeloid leukemia and melanoma compared with lysate-pulsed DC vaccines. We compared these two vaccine strategies against murine colon cancer and investigated whether DC/tumor fusion cells continue to produce tumor antigens following fusion as a possible explanation for their increased potency. Using a mouse colon cancer model, CT26, we first showed that the DC/CT26 fusion vaccine is more effective in preventing tumor implantation than CT26 lysate-pulsed DC vaccine. Next, CT26 made to constitutively produce bioactive TGF-beta, a surrogate of tumor-derived products, was fused to DCs and found to produce bioactive TGF-beta 72 h after fusion. Our results suggest the DC/tumor fusion vaccine is more potent against colon cancer than the lysate-pulsed DC vaccine. These fusion cells have the distinct advantage of prolonged interaction with tumor antigens in vivo.     

Enhancement of dendritic cell-based vaccine potency by targeting antigen to endosomal/lysosomal compartments

Dendritic cells (DCs) are the central players in cancer immunotherapy because of their distinct ability to prime immune responses. In previous work with DNA vaccines, we described an intracellular targeting approach that routed a nuclear/cytoplasmic antigen, human papillomavirus (HPV) type 16 E7, into the endosomal and lysosomal compartments. It does so by linking E7 with the sorting signal of lysosome-associated membrane protein 1 (Sig/LAMP-1) to enhance the presentation of E7 antigen to MHC class I-restricted CD8(+) T cells, as well as to MHC class II-restricted CD4(+) T cells. To date, the Sig/LAMP-1 targeting strategy has not been tested in the context of DC-based vaccines. This study was designed to determine whether targeting HPV-16 E7 to the endosomal/lysosomal compartment can enhance the potency of DC vaccines. In immunological studies, DC-Sig/E7/LAMP-1 dramatically increased in vitro activation and in vivo expansion of E7-specific CD4(+) and CD8(+) T cells, compared with DC-E7 and DC-No insert. More importantly, in both tumor prevention and tumor treatment assays, DC-Sig/E7/LAMP-1 generated greater anti-tumor immunity against TC-1 than DC-E7. Our results demonstrate that linkage of the antigen gene to an endosomal/lysosomal targeting signal may greatly enhance the potency of DC-based vaccines.     

Potential applications of dendritic cells

Dendritic cells (DCs) are the professional antigen‐presenting cells of the immune system. Following infection or inflammation they undergo a complex process of maturation, and migrate to lymph nodes where they present antigens to T cells. Their decisive role in inducing immunity formed the rationale for DC immunotherapy: DCs loaded with tumor antigens are injected into cancer patients to stimulate T cells to eradicate tumors. Effective immune responses and favourable clinical outcomes have indeed been observed, but only in a minority of patients. For effective immunotherapy DCs need to traffic throughout the vascular and lymphatic system to reach the T‐cells located within lymph nodes. Though most immunotherapeutic agents are administered intravenously, DCs are predominantly administered intradermally. We observed that < 5% of intradermally administered mature DCs reach the draining lymph nodes amounting to inefficient homing. Despite this low number we could measure effective immune responses in some patients, but generally this may be too low. We demonstrated that DC maturation is a prerequisite to exert their immunostimulatory capacity in vivo. Immuno‐monitoring revealed a remarkable difference in immune responses. In patients vaccinated with immature DC the KLH responses as well as DTH reactivity against KLH and tumor‐peptides were weak and absent, respectively. In contrast, in patients vaccinated with mature DC a pronounced T cell as well a B cell response (IgG) against KLH were observed. Analysis of the response against the tumor peptides demonstrated little or no reactivity in blood. However, following intradermal administration of a delayed type hypersensitivity (DTH) challenge with gp100‐ and tyrosinase‐peptide loaded DC essentially all patients vaccinated with mature DC showed a positive induration. Moreover, we showed the predictive value of the presence or absence of antigen‐specific T cells in biopsies from DTH sites. In clinically responding patients, T cells specific for the antigen preferentially accumulated in the DTH site in accordance with the applied antigen in the DTH challenge.     

Phenotype and functional analysis of human monocyte-derived dendritic cells loaded with biodegradable poly(lactide-co-glycolide) microspheres for immunotherapy

Dendritic cells (DC) are increasingly explored as cellular vaccines for tumor immunotherapy. In most reported DC-based cancer vaccine trials, DC have been pulsed with soluble tumor antigen-derived peptide ligands of MHC molecules. Considering that the half-life of peptide/MHC complexes on the cell surface is relatively short and that soluble exogenous protein antigens cannot be efficiently processed via the MHC class I-processing pathway, the current vaccination procedure is not optimal for the induction of strong T cell responses aiming at tumor rejection. Recently, we have shown that antigen presentation can be prolonged when synthetic peptides were encapsulated in biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microspheres (MS) for uptake by DC. In the present study, we investigated the phenotypic and functional consequences of MS uptake by human monocyte-derived dendritic cells (MoDC) in vitro. We found that immature MoDC that were prepared in serum free media suitable for clinical application were able to phagocytose high numbers of MS, while matured MoDC showed a reduced capacity for phagocytosis of MS. The ingestion of MS did not change the cell surface expression of CD80, CD83, CD86 and HLA-DR of immature and mature DC, suggesting that MS uptake did not induce DC maturation but that maturation by cytokines or LPS was unaltered in the presence of MS. Furthermore, MS-loaded mature MoDC expressed normal levels of the chemokine receptor CCR7 and migrated as efficiently towards CCL19 or CCL21 as unloaded MoDC. DC viability and the secretion of TNF-alpha and IL-12 was not significantly changed by MS loading. Taken together, our data indicate that PLGA-MS loading has no negative effects on the pivotal properties of MoDC in vitro. It should therefore be feasible to further develop this antigen loading strategy for clinical use in immunotherapy against viral infections and tumors.     

Targeting DNA vaccines to myeloid cells using a small peptide

Targeting DNA vaccines to dendritic cells (DCs) greatly enhances immunity. Although several approaches have been used to target protein Ags to DCs, currently there is no method that targets DNA vaccines directly to DCs. Here, we show that a small peptide derived from the rabies virus glycoprotein fused to protamine residues (RVG‐P) can target DNA to myeloid cells, including DCs, which results in enhanced humoral and T‐cell responses. DCs targeted with a DNA vaccine encoding the immunodominant vaccinia B8R gene via RVG‐P were able to restimulate vaccinia‐specific memory T cells in vitro. Importantly, a single i.v. injection of B8R gene bound to RVG‐P was able to prime a vaccinia‐specific T‐cell response that was able to rapidly clear a subsequent vaccinia challenge in mice. Moreover, delivery of DNA in DCs was enough to induce DC maturation and efficient Ag presentation without the need for adjuvants. Finally, immunization of mice with a DNA‐vaccine encoding West Nile virus (WNV) prM and E proteins via RVG‐P elicited high titers of WNV‐neutralizing Abs that protected mice from lethal WNV challenge. Thus, RVG‐P provides a reagent to target DNA vaccines to myeloid cells and elicit robust T‐cell and humoral immune responses.