Targeting Of Dendritic Cells Research Articles

Nanovaccines silencing IL-10 production at priming phase for boosting immune responses to melanoma

Despite the significant efforts in developing cancer vaccines, there are still numerous challenges that need to be addressed to ensure their clinical efficacy. Herein, a lymphatic dendritic cell (DC)-targeted artificial nanovaccine mimicking tumor cell membrane (ATM-NV) is developed to boost effector immune response and control immunosuppression simultaneously. The NVs are formulated with lipids, tumor cell membrane proteins, imiquimod (IMQ), and IL-10 siRNA. IL-10 siRNA is incorporated to inhibit the secretion of IL-10, an immunosuppressive cytokine, of maturated DCs upon IMQ. To enhance the DC targeting ability, the nanovaccine surface was non-covalently conjugated with the anti-CD205 antibody. The IMQ and IL-10 siRNA co-loaded, CD205 receptor-targeted artificial tumor membrane NVs (IMQ/siR@ATM-NVs) efficiently migrate to the tumor-draining lymph node and target DCs. Furthermore, immunization with IMQ/siR@ATM-NVs reduces the production of IL-10 and increases Th1-driven antitumor immunity resulted in a great tumor inhibition efficacy. Our results suggest a potential strategy to promote the vaccination's antitumor efficacy by blocking the intrinsic negative regulators in DCs.

Abstract 1746: Designing a dendritic cell-targeted immunotherapy for the treatment of pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death. Low response rate, acquired resistance, severe side effects, highly desmoplastic and immunosuppressive microenvironment have limited the clinical outcomes of immunotherapy in PDAC. To overcome these hurdles, we set to design a novel immunotherapy that would be safe, efficacious, and selective to PDAC. The failure of immunotherapies to yield clinical response in PDAC is partially attributed to the inefficient antigen presentation by dendritic cells (DC) which results in insufficient T cells stimulation despite their expression of tumor-associated antigens (TAA). DC have an important role in the initiation and regulation of the innate and adaptive arms of the immune system. By efficient presentation of antigens, DC elicit an antigen-specific immune response. Trp53R172H is among the most common TP53 mutations in human PDAC, and is known as one of PDAC TAA. Despite the poor clinical outcomes, longer survival time was observed in PDAC patients with tumors that express high number of TAA combined with an abundant presentation of CD8+ T cell. To that end, we designed a DC-targeted nanoparticle (NP) containing PDAC Trp53R172H TAA and immune potentiators, with the DC targeting moiety, mannose, that was covalently conjugated to the NP, to enhance TAA presentation on DC. This approach led to specific activation of T cells against PDAC's TAA. The therapeutic activity of the NP was evaluated in vitro by our newly established 3D PDAC spheroids model, which is composed of freshly isolated murine stromal cells, therefore, better imitates the complex microenvironment found in PDAC. Along with an in vivo PDAC model, we evaluated the safety profile and therapeutic efficacy of our NP. The NP proposed herein successfully activated splenocytes and induced a specific cytotoxic reaction against PDAC cells ex vivo, which may result in improved survival rate. Citation Format: Ron Kleiner, Daniella Vaskovich, Sabina Pozzi, Anat Eldar-Boock, Dikla Ben-Shushan, Helena Florindo, Ronit Satchi-Fainaro. Designing a dendritic cell-targeted immunotherapy for the treatment of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1746.

Insights Into Dendritic Cells in Cancer Immunotherapy: From Bench to Clinical Applications.

Dendritic cells (DCs) are efficient antigen-presenting cells (APCs) and potent activators of naïve T cells. Therefore, they act as a connective ring between innate and adaptive immunity. DC subsets are heterogeneous in their ontogeny and functions. They have proven to potentially take up and process tumor-associated antigens (TAAs). In this regard, researchers have developed strategies such as genetically engineered or TAA-pulsed DC vaccines; these manipulated DCs have shown significant outcomes in clinical and preclinical models. Here, we review DC classification and address how DCs are skewed into an immunosuppressive phenotype in cancer patients. Additionally, we present the advancements in DCs as a platform for cancer immunotherapy, emphasizing the technologies used for in vivo targeting of endogenous DCs, ex vivo generated vaccines from peripheral blood monocytes, and induced pluripotent stem cell-derived DCs (iPSC-DCs) to boost antitumoral immunity.

Transdermal delivery of interleukin-12 gene targeting dendritic cells enhances the anti-tumour effect of programmed cell death protein 1 monoclonal antibody.

ABSTRACTRecent studies have suggested that the anti-tumour effect of the programmed cell death protein 1 monoclonal antibody (aPD-1) depends on the expression of interleukin-12 (IL-12) by dendritic cells (DCs). Since DCs are abundant in skin tissues, transdermal delivery of IL-12 targeting DCs may significantly improve the anti-tumour effect of aPD-1. In this study, a novel mannosylated chitosan (MC)-modified ethosome (Eth-MC) was obtained through electrostatic adsorption. The Eth-MC loaded with plasmid containing the IL-12 gene (pIL-12@Eth-MC) stimulated DCs to express mature-related molecular markers such as CD86, CD80, and major histocompatibility complex-II in a targeted manner. The pIL-12@Eth-MC was then mixed with polyvinyl pyrrolidone solution to make microspheres using the electrospray technique, and sprayed onto the surface of electrospun silk fibroin-polyvinyl alcohol nanofibres to obtain a PVP-pIL-12@Eth-MC/silk fibroin-polyvinyl alcohol composite nanofibrous patch (termed a transcutaneous immunization (TCI) patch). The TCI patch showed a good performance on transdermal drug release. Animal experiments on melanoma-bearing mice showed that topical application of the TCI patches promoted the expression of IL-12 and inhibited the growth of tumour. Furthermore, combined application of the TCI patch and aPD-1 showed a stronger anti-tumour effect than aPD-1 monotherapy. The combination therapy significantly promoted the expression of IL-12, interferon-γ and tumour necrosis factor-α, the infiltration of CD4+ and CD8+ T cells into tumour tissues, and thus promoted the apoptosis of tumour cells. The present study provides a convenient and non-invasive strategy for improving the efficacy of immune checkpoint inhibitor therapy. This study was approved by the Institutional Animal Care and Use Committee at Donghua University (approval No. DHUEC-NSFC-2020-11) on March 31, 2020.

A novel multifunctional vaccine platform with dendritic cell-targeting and pH-responsive for cancer immunotherapy: Antigen-directed biomimetic fabrication of a cabbage-like mannatide-zinc-antigen hybrid microparticles

Cancer vaccines, featuring with high-specificity and long-term immune memory effect, have received tremendous attention in the field of cancer immunotherapy. However, most current developed antigen-encapsulated multifunctional cancer vaccines are subjected to limited antigen loading capacity, low yield, complicated preparation process, inefficient antigen delivery, and potential systemic toxicity. Herein, a facile biomimetic strategy is reported to fabricate a multifunctional cancer vaccine with high antigen loading capacity, dendritic cells-targeting and pH-responsive. As a proof of strategy, we directly use model antigen ovalbumin (OVA) and dendritic cells-targeting molecule mannatide as organic templates, zinc (II) as inorganic component to construct antigen and mannatide co-loaded hybrid microparticles vaccine. These formed mannatide-zinc-ovalbumin hybrid microparticles (M-Zn-OVA HMPs) owns cabbage-like morphology with particle size of ~1.7 μm, high antigen loading capacity of 11.7%, high yield of 84.6%, good stability, and favorable biocompatibility. Taking advantage of high antigen loading, pH-responsive, and dendritic cells-targeting molecule, M−Zn−OVA HMPs can target deliver antigen to dendritic cells, and effectively maturation of dendritic cells to induce robust immune responses. As a result, such M−Zn−OVA HMPs shows outstanding prophylactic efficacy toward B16-OVA and E.G7-OVA tumors. Moreover, M−Zn−OVA HMPs demonstrates excellent therapeutic efficacy against established B16-OVA and E.G7-OVA tumors, and the therapeutic efficacy can be further enhanced when used in combination with anti-programmed death-1 (anti-PD-1) checkpoint blockade. Therefore, our work may present a facile and versatile strategy for large-scale fabrication of multifunctional vaccines for cancer immunotherapy.

Characterization of a Nanovaccine Platform Based on an α1,2-Mannobiose Derivative Shows Species-non-specific Targeting to Human, Bovine, Mouse, and Teleost Fish Dendritic Cells.

Dendritic cells serve as the main immune cells that trigger the immune response. We developed a simple and cost-effective nanovaccine platform based on the α1',2-mannobiose derivative for dendritic cell targeting. In previous work, we have formulated the α1,2-mannobiose-based nanovaccine platform with plasmid DNA and tested it in cattle against BoHV-1 infection. There, we have shown that the dendritic cell targeting using this nanovaccine platform in vivo can boost the immunogenicity, resulting in a long-lasting immunity. In this work, we aim to characterize the α1',2-mannobiose derivative, which is key in the nanovaccine platform. This DC-targeting strategy takes advantage of the specific receptor known as DC-SIGN and exploits its capacity to bind α1,2-mannobiose that is present at terminal ends of oligosaccharides in certain viruses, bacteria, and other pathogens. The oxidative conjugation of α1',2-mannobiose to NH2-PEG2kDa-DSPE allowed us to preserve the chemical structure of the non-reducing mannose of the disaccharide and the OH groups and the stereochemistry of all carbons of the reducing mannose involved in the binding to DC-SIGN. Here, we show specific targeting to DC-SIGN of decorated micelles incubated with the Raji/DC-SIGN cell line and uptake of targeted liposomes that took place in human, bovine, mouse, and teleost fish DCs in vitro, by flow cytometry. Specific targeting was found in all cultures, demonstrating a species-non-specific avidity for this ligand, which opens up the possibility of using this nanoplatform to develop new vaccines for various species, including humans.

Combining mannose receptor mediated nanovaccines and gene regulated PD-L1 blockade for boosting cancer immunotherapy

Tumor nanovaccines have potential applications in the prevention and treatment of malignant tumors. However, it remains a longstanding challenge in exploiting efficient nanocarriers for inducing potent specifically cellular immune responses. Toward this objective, we herein explore an intensive tumor immunotherapeutic strategy by combining mannosylated nanovaccines and gene regulated PD-L1 blockade for immune stimulation and killing activity. Here, we fabricate a mannose modified PLL-RT (Man-PLL-RT) mediated nanovaccines with dendritic cells (DCs) targeting capacity. Man-PLL-RT is capable of co-encapsulating with antigen (ovalbumin, OVA) and adjuvant (unmethylated cytosine-phosphate-guanine, CpG) by electrostatic interaction. This positively charged Man-PLL-RT/OVA/CpG nanovaccines can facilitate the endocytosis, maturation and cross presentation in DCs. However, the nanovaccines arouse limited inhibition of tumor growth, which is mainly due to the immunosuppressed microenvironment of tumors. Combining tumor nanovaccines with gene regulated PD-L1 blockade leads to an obvious tumor remission in B16F10 melanoma bearing mice. The collaborative strategy provides essential insights to boost the benefits of tumor vaccines by regulating the checkpoint blockade with gene therapy.

Dendritic cell targeting with Fc-enhanced CD40 antibody agonists induces durable antitumor immunity in humanized mouse models of bladder cancer.

Intravesical immunotherapy using Bacille Calmette-Guérin (BCG) attenuated bacteria delivered transurethrally to the bladder has been the standard of care for patients with high-risk non-muscle-invasive bladder cancer (NMIBC) for several decades. BCG therapy continues to be limited by high rates of disease recurrence and progression, and patients with BCG-unresponsive disease have few effective salvage therapy options besides radical cystectomy, highlighting a need for new therapies. We report that the immune-stimulatory receptor CD40 is highly expressed on dendritic cells (DCs) within the bladder tumor microenvironment of orthotopic bladder cancer mouse models, recapitulating CD40 expression by DCs found in human disease. We demonstrate that local CD40 agonism in mice with orthotopic bladder cancer through intravesical delivery of anti-CD40 agonist antibodies drives potent antitumor immunity and induces pharmacodynamic effects in the bladder tumor microenvironment, including a reduction in CD8+ T cells with an exhausted phenotype. We further show that type 1 conventional DCs (cDC1) and CD8+ T cells are required for both bladder cancer immune surveillance and anti-CD40 agonist antibody responses. Using orthotopic murine models humanized for CD40 and Fcγ receptors, we demonstrate that intravesical treatment with a fully human, Fc-enhanced anti-CD40 agonist antibody (2141-V11) induces robust antitumor activity in both treatment-naïve and treatment-refractory settings, driving long-term systemic antitumor immunity with no evidence of systemic toxicity. These findings support targeting CD40-expressing DCs in the bladder cancer microenvironment through an intravesical agonistic antibody approach for the treatment of NMIBC.

Development and Evaluation of an Ebola Virus Glycoprotein Mucin-Like Domain Replacement System as a New Dendritic Cell-Targeting Vaccine Approach against HIV-1.

ABSTRACTThe development of efficient vaccine approaches against HIV infection remains challenging in the vaccine field. Here, we developed an Ebola virus envelope glycoprotein (EboGP)-based chimeric fusion protein system and demonstrated that replacement of the mucin-like domain (MLD) of EboGP with HIV C2-V3-C3 (134 amino acids [aa]) or C2-V3-C3-V4-C4-V5-C5 (243 aa) polypeptides (EbGPΔM-V3 and EbGPΔM-V3-V5, respectively) still maintained the efficiency of EboGP-mediated viral entry into human macrophages and dendritic cells (DCs). Animal studies using mice revealed that immunization with virus-like particles (VLPs) containing the above chimeric proteins, especially EbGPΔM-V3, induced significantly more potent anti-HIV antibodies than HIV gp120 alone in mouse serum and vaginal fluid. Moreover, the splenocytes isolated from mice immunized with VLPs containing EbGPΔM-V3 produced significantly higher levels of gamma interferon (IFN-γ), interleukin 2 (IL-2), IL-4, IL-5, and macrophage inflammatory protein 1α (MIP-1α). Additionally, we demonstrated that coexpression of EbGPΔM-V3 and the HIV Env glycoprotein in a recombinant vesicular stomatitis virus (rVSV) vector elicited robust anti-HIV antibodies that may have specifically recognized epitopes outside or inside the C2-V3-C3 region of HIV-1 gp120 and cross-reacted with the gp120 from different HIV strains. Thus, this study has demonstrated the great potential of this DC-targeting vaccine platform as a new vaccine approach for improving immunogen delivery and increasing vaccine efficacy.IMPORTANCE Currently, there are more than 38.5 million reported cases of HIV globally. To date, there is no approved vaccine for HIV-1 infection. Thus, the development of an effective vaccine against HIV infection remains a global priority. This study revealed the efficacy of a novel dendritic cell (DC)-targeting vaccination approach against HIV-1. The results clearly show that the immunization of mice with virus-like particles (VLPs) and VSVs containing HIV Env and a fusion protein composed of a DC-targeting domain of Ebola virus GP with HIV C2-V3-C3 polypeptides (EbGPΔM-V3) could induce robust immune responses against HIV-1 Env and/or Gag in serum and vaginal mucosa. These findings provide a proof of concept of this novel and efficient DC-targeting vaccine approach in delivering various antigenic polypeptides of HIV-1 and/or other emergent infections to the host antigen-presenting cells to prevent HIV and other viral infections.

STINGing the Tumor Microenvironment to Promote Therapeutic Tertiary Lymphoid Structure Development

Tertiary lymphoid structures (TLS), also known as ectopic lymphoid structures (ELS) or tertiary lymphoid organs (TLO), represent a unique subset of lymphoid tissues noted for their architectural similarity to lymph nodes, but which conditionally form in peripheral tissues in a milieu of sustained inflammation. TLS serve as regional sites for induction and expansion of the host B and T cell repertoires via an operational paradigm involving mature dendritic cells (DC) and specialized endothelial cells (i.e. high endothelial venules; HEV) in a process directed by TLS-associated cytokines and chemokines. Recent clinical correlations have been reported for the presence of TLS within tumor biopsies with overall patient survival and responsiveness to interventional immunotherapy. Hence, therapeutic strategies to conditionally reinforce TLS formation within the tumor microenvironment (TME) via the targeting of DC, vascular endothelial cells (VEC) and local cytokine/chemokine profiles are actively being developed and tested in translational tumor models and early phase clinical trials. In this regard, a subset of agents that promote tumor vascular normalization (VN) have been observed to coordinately support the development of a pro-inflammatory TME, maturation of DC and VEC, local production of TLS-inducing cytokines and chemokines, and therapeutic TLS formation. This mini-review will focus on STING agonists, which were originally developed as anti-angiogenic agents, but which have recently been shown to be effective in promoting VN and TLS formation within the therapeutic TME. Future application of these drugs in combination immunotherapy approaches for greater therapeutic efficacy is further discussed.

Therapeutic Liposomal Vaccines for Dendritic Cell Activation or Tolerance.

Dendritic cells (DCs) are paramount in initiating and guiding immunity towards a state of activation or tolerance. This bidirectional capacity of DCs sets them at the center stage for treatment of cancer and autoimmune or allergic conditions. Accordingly, many clinical studies use ex vivo DC vaccination as a strategy to boost anti-tumor immunity or to suppress immunity by including vitamin D3, NF-κB inhibitors or retinoic acid to create tolerogenic DCs. As harvesting DCs from patients and differentiating these cells in vitro is a costly and cumbersome process, in vivo targeting of DCs has huge potential as nanoparticulate platforms equipped with activating or tolerogenic adjuvants can modulate DCs in their natural environment. There is a rapid expansion of the choices of nanoparticles and activation- or tolerance-promoting adjuvants for a therapeutic vaccine platform. In this review we highlight the most recent nanomedical approaches aimed at inducing immune activation or tolerance via targeting DCs, together with novel fundamental insights into the mechanisms inherent to fostering anti-tumor or tolerogenic immunity.

Dendritic Cell Tumor Vaccination via Fc Gamma Receptor Targeting: Lessons Learned from Pre-Clinical and Translational Studies.

Despite significant recent improvements in the field of immunotherapy, cancer remains a heavy burden on patients and healthcare systems. In recent years, immunotherapies have led to remarkable strides in treating certain cancers. However, despite the success of checkpoint inhibitors and the advent of cellular therapies, novel strategies need to be explored to (1) improve treatment in patients where these approaches fail and (2) make such treatments widely and financially accessible. Vaccines based on tumor antigens (Ag) have emerged as an innovative strategy with the potential to address these areas. Here, we review the fundamental aspects relevant for the development of cancer vaccines and the critical role of dendritic cells (DCs) in this process. We first offer a general overview of DC biology and routes of Ag presentation eliciting effective T cell-mediated immune responses. We then present new therapeutic avenues specifically targeting Fc gamma receptors (FcγR) as a means to deliver antigen selectively to DCs and its effects on T-cell activation. We present an overview of the mechanistic aspects of FcγR-mediated DC targeting, as well as potential tumor vaccination strategies based on preclinical and translational studies. In particular, we highlight recent developments in the field of recombinant immune complex-like large molecules and their potential for DC-mediated tumor vaccination in the clinic. These findings go beyond cancer research and may be of relevance for other disease areas that could benefit from FcγR-targeted antigen delivery, such as autoimmunity and infectious diseases.

Mannosylated gelatin nanoparticles enhanced inactivated PRRSV targeting dendritic cells and increased T cell immunity

The objective of the present work was to evaluate the efficacy of a novel antigen carrier using mannosylated gelatin nanoparticles with entrapped inactivated porcine reproductive and respiratory syndrome virus (PRRSV) in inducing T cell mediated immunity in vitro. Gelatin nanoparticles (GNP) were modified with mannose to form mannosylated gelatin nanoparticles (MnGNP), which can efficiently and specifically target monocyte derived dendritic cells (MoDCs). The inactivated PRRSV was encapsulated in the MnGNP and GNP, referred to as MnGNP-PRRSV and GNP-PRRSV, respectively. All these prepared nanometer particles were characterized for size, surface charge, drug encapsulation efficiency, and drug release. The efficacy of MnGNP in targeting MoDCs was investigated, as well as the subsequent MoDCs maturation and T cell mediated cytotoxicity. The developed MnGNP-PRRSV particle was characterized with a nanometric size of 302.67 ± 3.2 nm, surface charge of 23.81 ± 1.26 mV, and PRRSV encapsulation efficiency of 63.2 ± 1.85 %. The maximum uptake of MnGNP in MoDCs in vitro was 15.5 times higher than GNP with a shorter reaction time that peaked 4 h earlier. The uptake of MnGNP-PRRSV induced maturation of MoDCs and significantly enhanced expression of SWC-3a, CD80, CD1, SLA I, SLA II on MoDCs, compared to PRRSV (p < 0.001). The cytokine secretion of IL-1β, IL-6, IL-10, and IL-12 was also increased in MoDCs when treated with MnGNP-PRRSV, compared to PRRSV (p < 0.05). The matured MoDCs triggered T lymphocytes in autologous peripheral blood mononuclear cells (PBMCs) activation, proliferation, and differentiation into effector cytotoxic T lymphocyte, suggesting increased amount of activated T cells after MnGNP-PRRSV treatment. Additionally, the function of T cells to kill PRRSV infected cells was 83.98 ± 2.62 % when triggered by MnGNP-PRRSV, compared to 60 ± 4.7 % in PRRSV group (p < 0.001). These results indicate that MnGNP with entrapped inactivated PRRSV can effectively and specifically target dendritic cells for maturation and activation, and subsequently improve T cell activation, proliferation and function to kill PRRSV infected cells.

In Vivo Insulin Peptide Autoantigen Delivery by Mannosylated Sodium Alginate Nanoparticles Delayed but Could Not Prevent the Onset of Type 1 Diabetes in Nonobese Diabetic Mice.

Type 1 diabetes (T1D) is an autoimmune subtype of diabetes, mainly caused by the immune attack of self-insulin-producing cells. Immune modulation that delays the onset of T1D is able to reduce diabetic complications and mortality. We have previously reported that mannosylated sodium alginate nanoparticles (MAN-ALG) exhibited excellent dendritic cell targeting and in vivo antigen delivery efficacy. To investigate the role of MAN-ALG in an autoimmune context, we loaded the MAN-ALG with Ins29-23, a T1D autoantigen [MAN-ALG(PEP)], for T1D immune tolerance induction in nonobese diabetic (NOD) mice. We observed the delayed onset of T1D occurrence and some degree of blood glucose reduction accompanied by a larger islet area, attributable to augmented T-regulatory cell proportion in mice treated with MAN-ALG(PEP). However, MAN-ALG was also found to elicit lysosomal escape and cross-presentation of Ins29-23 in bone marrow-derived dendritic cells, leading to the immune activation of Ins29-23-recognizing T cells and destruction of Ins29-23-expressing islet cells. This dual impact resulted in delayed but a nonpreventive effect of MAN-ALG(PEP) on the T1D onset in NOD mice. Considering the potent immune stimulatory property of MAN-ALG, cautions should be implemented when using alginate-based biomaterials in an autoimmune context. Moreover, it is also noted that regarding the in vivo outcome of immune therapies, biomaterial-based delivery systems and their detailed role on immune regulation need to be examined.

Complement-Opsonized Nano-Carriers Are Bound by Dendritic Cells (DC) via Complement Receptor (CR)3, and by B Cell Subpopulations via CR-1/2, and Affect the Activation of DC and B-1 Cells.

The development of nanocarriers (NC) for biomedical applications has gained large interest due to their potential to co-deliver drugs in a cell-type-targeting manner. However, depending on their surface characteristics, NC accumulate serum factors, termed protein corona, which may affect their cellular binding. We have previously shown that NC coated with carbohydrates to enable biocompatibility triggered the lectin-dependent complement pathway, resulting in enhanced binding to B cells via complement receptor (CR)1/2. Here we show that such NC also engaged all types of splenic leukocytes known to express CR3 at a high rate when NC were pre-incubated with native mouse serum resulting in complement opsonization. By focusing on dendritic cells (DC) as an important antigen-presenting cell type, we show that CR3 was essential for binding/uptake of complement-opsonized NC, whereas CR4, which in mouse is specifically expressed by DC, played no role. Further, a minor B cell subpopulation (B-1), which is important for first-line pathogen responses, and co-expressed CR1/2 and CR3, in general, engaged NC to a much higher extent than normal B cells. Here, we identified CR-1/2 as necessary for binding of complement-opsonized NC, whereas CR3 was dispensable. Interestingly, the binding of complement-opsonized NC to both DC and B-1 cells affected the expression of activation markers. Our findings may have important implications for the design of nano-vaccines against infectious diseases, which codeliver pathogen-specific protein antigen and adjuvant, aimed to induce a broad adaptive cellular and humoral immune response by inducing cytotoxic T lymphocytes that kill infected cells and pathogen-neutralizing antibodies, respectively. Decoration of nano-vaccines either with carbohydrates to trigger complement activation in vivo or with active complement may result in concomitant targeting of DC and B cells and thereby may strongly enhance the extent of dual cellular/humoral immune responses.

Oral Immunization of Recombinant Lactococcus lactis and Enterococcus faecalis Expressing Dendritic Cell Targeting Peptide and Hexon Protein of Fowl Adenovirus 4 Induces Protective Immunity Against Homologous Infection

Hepatitis-hydropericardium syndrome (HPS) causes severe economic losses in the global poultry industry. The present study aims to explore oral immunization of recombinant Lactococcus lactis and Enterococcus faecalis expressing Hexon protein of fowl adenovirus 4 (FAdV-4). The bacteria L. lactis NZ9000 and E. faecalis MDXEF-1 were, respectively, modified as host strain to deliver truncated Hexon protein (ΔHexon) or ΔHexon protein fusing with dendritic cell (DC) targeting peptide (DC-ΔHexon) on the surface of bacteria. The expression of target protein in L. lactis NZ9000 and E. faecalis MDXEF-1 were detected by western blot. To evaluate the immune responses and protective efficacies provided by the live recombinant bacteria, chickens were immunized with the constructed ΔHexon-expressing bacteria three times at 2-week intervals, then experimentally challenged with hypervirulent FAdV-4/GX01. The results showed that oral immunizations with the four ΔHexon-expressing bacteria (NZ9000/ΔHexon-CWA, NZ9000/DC-ΔHexon-CWA, MDXEF-1/ΔHexon-CWA, and MDXEF-1/DC-ΔHexon-CWA), especially the two bacteria carrying DC-targeting peptide, stimulated higher levels of ΔHexon-specific sera IgG and secretory IgA (sIgA) in jejunal lavage fluid, higher proliferation of peripheral blood lymphocytes (PBLs) and higher levels of Th1/Th2-type cytokines, along with significantly decreased virus loads in liver and more offered protective efficacies against FAdV infection compared with PBS and empty vector control groups (p < 0.01). For chickens in the group MDXEF-1/DC-ΔHexon-CWA, the levels of aspartate transaminase (AST), alanine transaminase (ALT) and lactate dehydrogenase (LDH) in sera, and the virus loads in livers were significantly decreased vs. the other three ΔHexon-expressing bacteria (p < 0.01). The pathological changes in the hearts, livers, spleens and kidneys of chickens in MDXEF-1/DC-ΔHexon-CWA group were relatively slight compared to infection control group and other three ΔHexon-expressing bacteria groups. The rate of protection in MDXEF-1/DC-ΔHexon-CWA group was 90%. The present work demonstrated that cell surface-displayed target protein and immune enhancers in L. lactis and E. faecalis might be a promising approach to enhance immunity and immune efficacy against pathogen FAdV-4 infection.

Tolerogenic Immunotherapy: Targeting DC Surface Receptors to Induce Antigen-Specific Tolerance.

Dendritic cells (DCs) are well-established as major players in the regulation of immune responses. They either induce inflammatory or tolerogenic responses, depending on the DC-subtype and stimuli they receive from the local environment. This dual capacity of DCs has raised therapeutic interest for their use to modify immune-activation via the generation of tolerogenic DCs (tolDCs). Several compounds such as vitamin D3, retinoic acid, dexamethasone, or IL-10 and TGF-β have shown potency in the induction of tolDCs. However, an increasing interest exists in defining tolerance inducing receptors on DCs for new targeting strategies aimed to develop tolerance inducing immunotherapies, on which we focus particular in this review. Ligation of specific cell surface molecules on DCs can result in antigen presentation to T cells in the presence of inhibitory costimulatory molecules and tolerogenic cytokines, giving rise to regulatory T cells. The combination of factors such as antigen structure and conformation, delivery method, and receptor specificity is of paramount importance. During the last decades, research provided many tools that can specifically target various receptors on DCs to induce a tolerogenic phenotype. Based on advances in the knowledge of pathogen recognition receptor expression profiles in human DC subsets, the most promising cell surface receptors that are currently being explored as possible targets for the induction of tolerance in DCs will be discussed. We also review the different strategies that are being tested to target DC receptors such as antigen-carbohydrate conjugates, antibody-antigen fusion proteins and antigen-adjuvant conjugates.

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo.

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach invaccine design. Antigen is delivered to DC via antibodies specific for endocytosis receptors such as DEC-205 that induce uptake, processing, and MHC class I- and II-presentation. Efficient and reliable conjugation of the desired antigen to a suitable antibody is a critical step in DC targeting and among other factors depends on the format of the antigen. Chemical conjugation offull-length protein to purified antibodies is one possible strategy. In the past, we have successfully established cross-linking of the model antigen ovalbumin (OVA) and a DEC-205-specific IgG2a antibody (αDEC-205)for in vivo DC targeting studies in mice. The first step of the protocol is the purification of the antibody from the supernatant of the NLDC (non-lymphoid dendritic cells)-145 hybridoma by affinity chromatography. The purified antibody is activated for chemical conjugation by sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate) while at the same time the sulfhydryl-groups of the OVA protein are exposed through incubation with TCEP-HCl (tris (2-carboxyethyl) phosphine hydrochloride). Excess TCEP-HCl and sulfo-SMCC are removed and the antigen is mixed with the activated antibody for overnight coupling. The resulting αDEC-205/OVA conjugate is concentrated and freed from unbound OVA. Successful conjugation of OVA to αDEC-205 is verified by western blot analysis and enzyme-linked immunosorbent assay (ELISA). We have successfully used chemically crosslinked αDEC-205/OVA to induce cytotoxic T cell responses in the liver and to compare different adjuvants for their potential in inducing humoral and cellular immunity following in vivo targeting of DEC-205+ DC. Beyond that, such chemically coupled antibody/antigen conjugates offer valuable tools for the efficient induction of vaccine responses to tumor antigens and have been proven to be superior to classical immunization approaches regarding the prevention and therapy of various types of tumors.

Self-Amplifying Pestivirus Replicon RNA Encoding Influenza Virus Nucleoprotein and Hemagglutinin Promote Humoral and Cellular Immune Responses in Pigs

Self-amplifying replicon RNA (RepRNA) promotes expansion of mRNA templates encoding genes of interest through their replicative nature, thus providing increased antigen payloads. RepRNA derived from the non-cytopathogenic classical swine fever virus (CSFV) targets monocytes and dendritic cells (DCs), potentially promoting prolonged antigen expression in the DCs, contrasting with cytopathogenic RepRNA. We engineered pestivirus RepRNA constructs encoding influenza virus H5N1 (A/chicken/Yamaguchi/7/2004) nucleoprotein (Rep-NP) or hemagglutinin (Rep-HA). The inherent RNase-sensitivity of RepRNA had to be circumvented to ensure efficient delivery to DCs for intracellular release and RepRNA translation; we have reported how only particular synthetic delivery vehicle formulations are appropriate. The question remained concerning RepRNA packaged in virus replicon particles (VRPs); we have now compared an efficient polyethylenimine (PEI)-based formulation (polyplex) with VRP-delivery as well as naked RepRNA co-administered with the potent bis-(3’,5’)-cyclic dimeric adenosine monophosphate (c-di-AMP) adjuvant. All formulations contained a Rep-HA/Rep-NP mix, to assess the breadth of both humoral and cell-mediated defences against the influenza virus antigens. Assessment employed pigs for their close immunological relationship to humans, and as natural hosts for influenza virus. Animals receiving the VRPs, as well as PEI-delivered RepRNA, displayed strong humoral and cellular responses against both HA and NP, but with VRPs proving to be more efficacious. In contrast, naked RepRNA plus c-di-AMP could induce only low-level immune responses, in one out of five pigs. In conclusion, RepRNA encoding different influenza virus antigens are efficacious for inducing both humoral and cellular immune defences in pigs. Comparisons showed that packaging within VRP remains the most efficacious for delivery leading to induction of immune defences; however, this technology necessitates employment of expensive complementing cell cultures, and VRPs do not target human cells. Therefore, choosing the appropriate synthetic delivery vehicle still offers potential for rapid vaccine design, particularly in the context of the current coronavirus pandemic.

Environmental Factor-Mediated Transgenerational Inheritance of Igf2r Hypomethylation and Pulmonary Allergic Response via Targeting Dendritic Cells.

The developmental origin of allergic diseases has been suggested, but the molecular basis remains enigmatic. Exposure to environmental factors, such as di-(2-ethylhexyl) phthalate (DEHP; a common plasticizer), is suggested to be associated with increased childhood allergic asthma, but the causal relationship and its underlying mechanism remain unknown. This study explored the transgenerational mechanism of DEHP on allergic asthma and dendritic cell (DC) homeostasis through epigenetic modification. In a murine model, ancestral exposure of C57BL/6 mice to low-dose DEHP led to trans-generational promoter hypomethylation of the insulin-like growth factor 2 receptor (Igf2r), concomitant with enhanced Igf2r expression and increased apoptosis prominently in CD8α+ DCs upon ligand stimulation, with consequent reduction in their IL-12 secretion and subsequent T cell-derived IFN-γ, thereby promoting a default Th2-associated pulmonary allergic response. Increased apoptosis was also noted in circulating IGF2Rhigh human DCs. Further, in human placenta, the methylation level at the orthologous IGF2R promoter region was shown to be inversely correlated with the level of maternal DEHP intake. These results support the importance of ancestral phthalate exposure in conferring the trans-generational risk of allergic phenotypes, featuring hypo-methylation of the IGF2R gene and dysregulated DC homeostasis.