Felix Rueda

Controlled release of antigen and Toll-like receptor ligands from PLGA nanoparticles enhances immunogenicity

AIMnDendritic cells rapidly capture nanoparticles and induce a potent cellular immune response. It is yet unknown whether the immunological response induced by slow release of encapsulated versus soluble antigen and adjuvant is superior.nnnMATERIALS & METHODSnThe kinetics of poly(lactic-co-glycolic acid) PLGA nanoparticles antigen release was studied by the DQ-bovine serum albumin (BSA) self-quenching antigen model. The immunological response induced was evaluated by means of dendritic cell activation/maturation markers, cytokine production and their ability to drive antigen-specific T-cell proliferation.nnnRESULTS & CONCLUSIONnPLGA-encapsulated antigen and adjuvant showed an enhanced T-cell response when compared with soluble vaccine components by increasing antigenicity and adjuvanticity. Although the kinetic profile followed the same pattern, encapsulation increased strength and duration of the response.

Human dendritic cell activities are modulated by the omega-3 fatty acid, docosahexaenoic acid, mainly through PPARγ:RXR heterodimers: comparison with other polyunsaturated fatty acids

There is accumulating evidence that omega‐3 fatty acids may modulate immune responses. When monocytes were differentiated to dendritic cells (DCs) in the presence of docosahexaenoic acid (DHA), the expression of costimulatory and antigen presentation markers was altered in a concentration‐dependent way, positively or negatively, depending on the markers tested and the maturation stage of the DCs. Changes induced by eicosapentaenoic acid and linoleic acid were similar but less intense than those of DHA, whereas oleic acid had almost no effect. DHA‐treated, mature DCs showed inhibition of IL‐6 expression and IL‐10 and IL‐12 secretion, and their lymphoproliferative stimulation capacity was impaired. The phenotypic alterations of DCs induced by DHA were similar to those already reported for Rosiglitazone (Rosi), a peroxisome proliferator‐activated receptor γ (PPARγ) activator, and the retinoid 9‐cis‐retinoic acid (9cRA), a retinoid X receptor (RXR) activator. Moreover, DHA induced the expression of PPARγ target genes pyruvate dehydrogenase kinase‐4 and aP‐2 in immature DCs. The combination of DHA with Rosi or 9cRA produced additive effects. Furthermore, when DCs were cultured in the presence of a specific PPARγ inhibitor, all of the changes induced by DHA were blocked. Together, these results strongly suggest that the PPARγ:RXR heterodimer is the pathway component activated by DHA to induce its immunomodulatory effect on DCs.

Targeting nanoparticles to CD40, DEC-205 or CD11c molecules on dendritic cells for efficient CD8+ T cell response: A comparative study

Here we demonstrated the importance of targeting antigens (Ags) to dendritic cell (DC) receptors to achieve an efficient cytotoxic T cell response which was associated with a strong activation of DC. Pegylated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) were used to encapsulate ovalbumin (OVA) as a model Ag. This PLGA complex, together with Toll like receptor (TLR) 3 and 7 ligands, was then targeted to distinct DC cell-surface molecules. These cell-surface molecules, including CD40, a TNF-α family receptor, DEC-205, a C-type lectin receptor and CD11c, an integrin receptor, were targeted by means of specific monoclonal antibodies (mAbs) coupled to the NP. The efficiency of these different targeting strategies to activate DC and elicit a potent CD8(+) T cell response was studied. PLGA-(Ag/TLR3+7L) NP was more efficiently targeted to and internalized by DC in vitro compared to the control non-targeted NP. We observed a small but significantly improved internalization of CD40-targeted NP compared to DEC-205 or CD11c targeted NP. In contrast to non-targeted NP, all targeted NPs equally stimulated IL-12 production and expression of co-stimulatory molecules by DC, inducing strong proliferation and IFN-y production by T cells in vitro. Moreover, subcutaneous vaccination with CD40, DEC-205 and CD11c-targeted NP consistently showed higher efficacy than non-targeted NP in stimulating CD8+ T cell responses. However, all targeted NP vaccines showed an equal capacity to prime cytotoxic CD8+ T cells, which subsequently were able to induce targeted cell lysis. In conclusion, delivery of NP-vaccines to DC by targeting via cell-surface molecules leads to strong enhancement of vaccine potency and induction of T cell responses compared to non-specific delivery of NP to DC.

Targeting nanoparticles to dendritic cells for immunotherapy.

Dendritic cells (DCs) are key players in the initiation of adaptive immune responses and are currently exploited in immunotherapy for treatment of cancer and infectious diseases. Development of targeted nanodelivery systems carrying vaccine components, including antigens and adjuvants, to DCs in vivo represents a promising strategy to enhance immune responses. Delivering particulate vaccines specifically to DCs and preventing nonspecific uptake by other endocytotic cells are challenging. Size represents a critical parameter determining whether particulate vaccines can penetrate lymph nodes and reach resident DCs. Specific delivery is further enhanced by actively targeting DC-specific receptors. This chapter discusses the rationale for the use of particle-based vaccines and provides an overview of antigen-delivery vehicles currently under investigation. In addition, we discuss how vaccine delivery systems may be developed, focusing on liposomes, PLGA polymers, and gold nanoparticles, to obtain safe and efficacious vaccines.

Targeting nanosystems to human DCs via Fc receptor as an effective strategy to deliver antigen for immunotherapy.

Dendritic cells (DCs) are increasingly being explored as cellular vaccines for tumor immunotherapy, since they provide an effective system of antigen presentation both in vitro and in vivo. An additional advantage of this cell type is that it is possible to target specific antigens through the activation of receptors, such as FcR (the receptor for the IgG Fc fragment) and TLR (toll-like Receptor). Thus, the uptake capacity of DCs can be improved, thereby increasing antigen presentation. This, in turn, would lead to an enhanced immune response, and, in some instances, the tolerance/anergy of immune effector cells present in cancer patients could be reverted. Here we studied various nanotargeting systems, including liposomes and gold nanoparticles of a peptide-based immunotherapeutic vaccine for the treatment of androgen-responsive prostate cancer. Building blocks of the immunogenic peptide consisted of the luteinizing hormone-releasing hormone (LHRH), also known as gonadotropin-releasing hormone (GnRH) peptide (B- and T-cell epitope), in tandem with a T-helper epitope corresponding to the 830-844 region of tetanus toxoid. Three new peptides with several modifications at the N-terminal (palmitoyl, acetyl, and FITC) were synthesized. These peptides also contained a Cys as C-terminal residue to facilitate grafting onto gold nanoparticles. To target different antigen formulations to human DCs, the Fc was activated with a cross-linking spacer to generate a free thiol group and thus facilitate conjugation onto gold nanoparticles, liposomes, and peptide. Our results show that gold nanoparticles and liposomes targeted to FcRs of human DCs are effective antigen delivery carriers and induce a strong immune response with respect to nontargeted LHRH-TT-nanoparticle conjugates and a superior response to that of naked antigens. In addition, dual labeling using gold and FITC-peptide allowed DC tracking by flow cytometry as well as transmission electron microscopy. Nanoparticles were observed to show a homogeneous distribution throughout the cytoplasm. These results open up a new approach to the development of a novel strategy for cancer vaccines.

9-cis-Retinoic Acid (9cRA), a Retinoid X Receptor (RXR) Ligand, Exerts Immunosuppressive Effects on Dendritic Cells by RXR-Dependent Activation: Inhibition of Peroxisome Proliferator-Activated Receptor γ Blocks Some of the 9cRA Activities, and Precludes Them to Mature Phenotype Development

At nanomolar range, 9-cis-retinoic acid (9cRA) was able to interfere in the normal differentiation process from human monocyte to immature dendritic cell (DC) and produced a switch in mature DCs to a less stimulatory mode than untreated cells. 9cRA-treated mature DCs secreted high levels of IL-10 with an IL-12 reduced production. The phenotypic alterations unleashed by 9cRA were similar but not identical to other specific retinoid X receptor (RXR) agonists and to those already reported for rosiglitazone, a PPARγ activator, on DCs. The simultaneous addition of 9cRA and rosiglitazone on DCs displayed additive effects. Moreover, addition to cultures of GW9662, a specific inhibitor of PPARγ, or the RXR pan-antagonist HX603, blocked these changes. All these results suggest an activation of PPARγ-RXR and other RXR containing dimers by 9cRA in DCs. Finally, both GW9662 and HX603 by themselves altered the maturation process unleashed by TNFα, poly(I:C) or LPS on human DCs further suggesting that the heterodimer PPARγ-RXR must fulfill a significant role in the physiological maturation process of these cells in addition to the repressing effects reported till now for this nuclear receptor.

Combinatorial prospects of nano-targeted chemoimmunotherapy

Despite the significant increase in our knowledge on cancer initiation and progression, and the development of novel cancer treatments, overall patient survival rates have thus far only marginally improved. However, it can be expected that lasting tumor control will be attainable for an increasing number of cancer patients in the foreseeable future, which is likely to be achieved by combining cancer chemotherapy with anticancer immunotherapy. A plethora of new cancer chemotherapy reagents are expected to become accessible to the clinic in the coming years which can then be used for efficient tumor debulking and aid in antigen exposure to the immune system. Durable remission and the eradication of micrometastases are likely to be achieved with specialized monoclonal antibodies and therapeutic cancer vaccines that modulate the immune system to overcome immunosuppression and kill distant cancer cells. Moreover, the method of drug delivery to tumors, stromal and immune cells is expected to shift largely from conventional free drug molecules to encapsulated in targeted nano-vehicles, therapeutics often referred to or considered part of nanomedicine. Several biocompatible nano-vehicles, such as metal-nanoparticles, biodegradable-nanoparticles, liposomes or dendrimers are potential candidates for targeted drug delivery but may also serve additional purposes. A dexterous combination of nanomedicine, cancer immunotherapy and chemotherapeutic engineering are likely to become the basis for new hope in the form of targeted cancer therapies that could attack tumors early in their development. One can envision nano-vehicles that would selectively deliver effective doses of chemotherapeutic agents to cancer cells while leaving healthy cells untouched. Furthermore, given that after chemotherapeutic treatment there often remains a limited number of chemo-resistant tumor cells, which go on to drive tumor progression, nano-vehicles could also be engineered to provoke an appropriate immune response to destroy these cells. Here, we discuss the potential of the combinatorial role of cancer chemotherapy, cancer immunotherapy and the prospective of nanotechnology for the targeted delivery of chemoimmunotherapeutic agents.

Natural Docosahexaenoic Acid in the Triglyceride Form Attenuates In Vitro Microglial Activation and Ameliorates Autoimmune Encephalomyelitis in Mice

Many neurodegenerative diseases are associated, at least in part, to an inflammatory process in which microglia plays a major role. The effect of the triglyceride form of the omega-3 polyunsaturated fatty acid docosahexaenoic acid (TG-DHA) was assayed in vitro and in vivo to assess the protective and anti-inflammatory activity of this compound. In the in vitro study, BV-2 microglia cells were previously treated with TG-DHA and then activated with Lipopolysaccharide (LPS) and Interferon-gamma (IFN-γ). TG-DHA treatment protected BV-2 microglia cells from oxidative stress toxicity attenuating NO production and suppressing the induction of inflammatory cytokines. When compared with DHA in the ethyl-ester form, a significant difference in the ability to inhibit NO production in favor of TG-DHA was observed. TG-DHA inhibited significantly splenocyte proliferation but isolated CD4+ lymphocyte proliferation was unaffected. In a mice model of autoimmune encephalomyelitis (EAE), 250 mg/kg/day oral TG-DHA treatment was associated with a significant amelioration of the course and severity of the disease as compared to untreated animals. TG-DHA-treated EAE mice showed a better weight profile, which is a symptom related to a better course of encephalomyelitis. TG-DHA may be a promising therapeutic agent in neuroinflammatory processes and merit to be more extensively studied in human neurodegenerative disorders.

Enhancing immunogenicity and cross-reactivity of HIV-1 antigens by in vivo targeting to dendritic cells

Current retroviral treatments have reduced AIDS to a chronic disease for most patients. However, given drug-related side effects, the emergence of drug-resistant strains and the persistence of viral replication, the development of alternative treatments is a pressing need. This review focuses on recent developments in HIV immunotherapy treatments, with particular emphasis on current vaccination strategies for optimizing the induction of an effective immune response by the recruitment of dendritic cells. In addition to cell-based therapies, targeted strategies aiming to deliver synthetic HIV peptides to dendritic cell-specific receptors in vivo will be discussed.

Effect of TLR ligands co-encapsulated with multiepitopic antigen in nanoliposomes targeted to human DCs via Fc receptor for cancer vaccines

Nanoliposomes (NLs) hold promise as new highly specific nanomedicine for anti-tumor vaccines, since they could be targeted to specific receptors on dendritic cell (DC) to induce maturation and activation and increase the anti-tumor immune response. Here we studied a NLs formulation targeted or not to FcR (the receptor for the IgG Fc fragment) for the treatment of androgen-responsive prostate cancer. Luteinizing-hormone-releasing hormone (LHRH) peptide (B- and T-cell epitopes), in tandem with a tetanus toxoid T-helper epitope (830-844 region) and several TLR (Toll-Like Receptor) ligands as adjuvants were co-encapsulated. Specific uptake in vitro of LHRH-TT liposomes targeted to the FcRs of human DCs was enhanced. DC maturation/activation, cytokine production and lymphocyte activation were consistently higher in targeted than non-targeted liposomes. Similar increase was observed as more adjuvants were administrated. Targeting to specific receptor and co-encapsulation of several TLR adjuvants are essential factors for the immune response in peptide based liposome vaccine.