Choose your models wisely: how different murine bone marrow-derived dendritic cell protocols influence the success of nanoparticulate vaccines in vitro

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Anti-tumor immunity induced by a novel nanoparticulate vaccine : a mechanistic approach

Tese de doutoramento, Farmácia (Tecnologia Farmacêutica), Universidade de Lisboa, Faculdade de Farmácia, 2016Biodegradable polymeric nanoparticles (NP) are promising tools for tumor eradication. Different nanovaccines based on the aliphatic-polyester (poly(lactic-co-glycolic acid) (PLGA) have been developed and optimized to investigate how different methods for antigen association to nanoparticulate carriers affect antigen uptake by antigen presenting cells (APC) and the generation and nature of antigen-specific immune responses. The different experimental parameters have been tested in order to predict the effect of the nature of protein association (adsorbed vs. entrapped) and polymers/surfactant concentrations, on NP average mean diameter, polydispersity index, surface charge, encapsulation efficiency, protein integrity and surfactant residual amount. This development procedure allowed for the rational identification of particle composition and experimental conditions that led to the antigenassociated nanocarrier presenting the ideal product specifications previously identified for optimal immune cell modulation, having in consideration literature evidences and our previous data. We used PLGA and pegylated (PEG)-PLGA as a matrix polymer. When preparing NP with double-emulsion solvent-evaporation technique, we used α-lactalbumin (LALBA) as an antigen, glycol chitosan (CS) to increase the viscosity of the internal aqueous phase (IP) and polyvinyl alcohol (PVA) or Pluronic F127 (PF127) to stabilization the interphase of double emulsion. Various concentrations of surfactants has been used (2, 4, 5, 8, 10, 12 % v/v) to best meet the criteria of the most optimal nanoparticulate delivery system. We aimed for NP below 200 nm, polydispersity index (PDI) under 0.2 and ζ potential close to neutrality. The formulations with 10 % v/v PVA solution in IP and 0.3 % v/v PVA or PF127 in external aqueous phase (EP) presented stable and repetitive NP formulations with desired physiochemical properties. The highest encapsulation efficacy (EE) values were obtained for the formulation prepared with PVA (EntrapLALBA(PVA)), reaching 80 % of the integrated protein, while NP with PF127 (EntrapLALBA(PF127)) presented around 70 % EE. As regards the protein adsorbed, NP formulations prepared with PVA (AdsLALBA(PVA)) or PF127 (AdsLALBA(PF127), reached the values of 40 % and 50 %, respectively. Dynamic light scattering (DLS) and laser doppler velocimetry (LDV) were used to determine average particle size, polydispersity (PDI) and ζ potential, respectively. EntrapLALBA(PVA) presented the average size of 171 nm with PDI around 0.15 and ζ potential 0.51 mV. EntrapLALBA(PF127) had 185 nm with PDI 0.17 and ζ potential 0.37 mV. On the other side AdsLALBA(PVA) and AdsLALBA(PF127) presented slightly biggersize 188 nm and 195 nm, respectively, with more negative ζ potential, -1,89 and -1,42 mV, respectively. NP size, PDI and geometry were also confirmed by atomic force microscopy (AFM). Protein integrity was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fourier transform infrared spectroscopy (FTIR) was further used to study the interaction of protein adsorption onto surfaces of polymeric NP. The presence of bands at the wavelengths 1662 cm-1 and 1562 cm-1, that are specific for primary amides and amines, were detected in formulations AdsLALBA(PVA) and AdsLALBA(PF127), where LALBA was adsorbed onto the surface of the NP. While in NP EntrapLALBA(PVA) and EntrapLALBA(PF127) this bands were absent indicating the protein incorporation into the polymeric matrix. Differential scanning calorimetry (DSC) showed that the formulation process did not alter the structure of the polymers used in the preparation of NP. The ability of the optimal formulations for systemic delivery and activation of DC was further evaluated in vitro and in vivo on bone marrow derived dendritic cells (BMDC). Similar activation and maturation profile of APC were obtained after the internalization of NP with antigen entrapped (EntrapNP) or adsorbed onto NP surface (AdsNP) evaluated at 3, 6, 16 and 24 h (ImageStreamTM and flow cytometry). The DC-NP interaction increased with the incubation time, presenting internalization values between 50-60% and 30-40%, in vitro and in vivo, respectively. Interestingly, MHCII was upregulated after the immunization with AdsNP and MHCI after the vaccination with EntrapNP, suggesting more efficient crosspresentation. To evaluate the activation of antigen-specific immune response, we replaced LALBA for ovalbumin (OVA). It is a highly immunogenic model antigen for which, in immunology, we have well-established animal models that can help to study the effect of the antigen on immune system without any unspecific immune response. Phenotype, frequency and efficacy of immune cell stimulation induced by NP loaded with OVA (EntrapNP or AdsNP) and with/without TLR ligands (CpG and Monophosphoryl Lipid A (MPLA)) were characterized and their specificity clarified by the engrafted OT-I (CD8+) and OT-II (CD4+) T cells. EntrapNP with OVA and adjuvants presented the strongest antigen-specific cytotoxic immune response, following NP with AdsOVA-Ajds. Moreover, long-lasting memory of cytotoxic T lymphocytes (CTL), was evaluated 8 weeks after a single immunization in response to the different ways of antigen delivery, PBS, OVA in solution, OVA & Adjs in solution and NP AdsOVA, AdsOVA-Adjs, EntrapOVA, or EntrapOVA-Adjs. Immunizations where we used antigen alone did not induce efficient CTL reactivation. Overall, Entrap OVA-Adj NP presented the most robust immune response in inducing the memory, followed by AdsOVAAdjs NP and OVA & Adjs in solution. To explore whether the immunization with formulated NP results in efficient crosspriming, an in vivo killing assay in steady-state conditions was performed. 24 h prior to immunization, animals were engrafted with CD8+ (OT-I) cells. 5 days p.i., mice were injected with an equal mix of targeted and untargeted CD45.1 splenocytes. Targeted cells were pulsed with OVA peptide, SIINFEKL, and labeled with higher dose of CFSE (CFSEhi) while untargeted control cells were labeled with a lower dose of CFSE (CSFElow). The in vivo clearance of both type of grafted cells was evaluated 16 h later in LN and spleens by flow cytometry. All NPs, with or without adjuvants, generated the most efficient in vivo killing activity towards the SIINFEKL-pulsed cells, as shown by the disappearance of the CFSEhi peak of cells. Indicating the successful induction of cross-priming and activation of antigenspecific CTL that could potentially lead to a broad and effective immune response pivotal in case of tumor rejections. To test the efficacy of the therapeutic vaccine and to have a good read-out of the antigen-specific T cells, we used B16.MO5 melanoma-bearing mice using different vaccination schedules, single and three time immunization with OVA and adjuvants- loaded NP. The groups immunized with PBS and Empty NP served as a control and presented similar tumor growth. All treated groups showed a significant reduction in tumor growth. EntrapOVA-Adjs NP (3x immunization) presented the slowest tumor growth (more than 12x smaller final tumor volume), followed by the group immunized with AdsOVA-Adjs NP (3x immunization) (more than 9x smaller final tumor volume) and single vaccination with EntrapOVA-Adjs NP (more than 8x smaller final tumor volume). The highest amount of tumor infiltrating lymphocytes (TILs) at the tumor site was detected for EntrapOVA-Adjs NP (3x immunization), suggesting the best remission and survival prognosis of the treated group. Two inflammatory cytokines (TNF-α and IFN-γ) produced by TILs were quantified in tumor microenvironment. 4-time high levels of TNF-α upon 3-time immunizations were detected in tumor microenvironment which kept the tumor growth under control. On the other side in the spleens, repeated vaccination prevented upregulation of both inflammatory markers, TNF-α and IFN-γ, indicating absence of systemic inflammation. Overall, the present work shows that the design of safe but efficacious nanoparticulate cancer vaccines requires a deep understanding of NP biological effect on immune cells, both under steady-state and cancerous environment. A detailed characterization of the imune cell-related pathways modulated by the optimal antigen-loaded NP and their correlation with the nature and anti-tumor efficacy of the induced immune response was performed in vivo in healthy and pathological conditions, showing that the nature of antigen delivery is crucial for a specific T cell activation and targeted cytotoxic effect

Development and evaluation of novel biodegradable nanoparticles for vaccine delivery

The development and evaluation of a chitosan-dextran sulfate nanoparticulate vaccine delivery system, with incorporation of Immunoglobulin-A as an immunological adjuvant with M-cell targeting potential, are described in this thesis. This research project highlights the importance of nanoparticulate systems and novel adjuvants in the development of efficient and safe vaccines with an emphasis on defining a correlation between pharmaceutical formulation factors and in-vivo immunological responses

Oral and nasal vaccination: current prospects, challenges, and impact of nanotechnology-based delivery systems

Currently, mucosal vaccine administration has stood out as an easier and non-invasive application method. It can also be used to induce local and systemic immune responses. In the COVID-19 pandemic context, nasal and oral vaccines have been developed based on different technological platforms. This review addressed relevant aspects of mucosal vaccine administration, with emphasis on oral and nasal vaccinations, in addition to the importance of using nanotechnology-based delivery systems to enable these strategies

Dendritic Cell Endocytosis Essential for Viruses and Vaccines

Protective immune defences are dependent upon critical roles played by dendritic cells (DCs), rendering them important targets for both vaccine delivery and virus infection. Studies in these areas led to successful development of targeted vaccine delivery, including synthetic virus-like particle (SVLP) and nanoparticulate RNA vaccines. A major consideration is DC endocytosis, whereby the different endocytic routes influencing the outcome. Rapid clathrin-mediated endocytosis likely favours degradative pathways. Slower processes such as macropinocytosis, caveolar endocytosis and retrograde transport to endoplasmic reticulum relate more to the processing rates leading to antigen presentation by DCs. These pathways are also influential in promoting the initiation of virus replication following infection. DC endocytosis of RNA viruses and RNA vaccines must lead to cytosolic translocation of the RNA for translation, relating to the process of antigen cross-presentation. One can learn from observations on both virus infections and cross-presentation for delivering RNA vaccines. Accordingly, recent advances in nanoparticulate delivery have been applied with self-amplifying replicon RNA (RepRNA), providing efficient delivery to DCs and promoting replicon-encoded antigen translation. Through realising the important relationships between DC endocytic pathways and induction of immune responses, delivery of SVLP and RepRNA vaccines to DCs offers high value for the development of future synthetic vaccine platforms

Self-assembled hydrogel nanoparticles for drug delivery applications

Hydrogel nanoparticles—also referred to as polymeric nanogels or macromolecular micelles—are emerging as promising drug carriers for therapeutic applications. These nanostructures hold versatility and properties suitable for the delivery of bioactive molecules, namely of biopharmaceuticals. This article reviews the latest developments in the use of self-assembled polymeric nanogels for drug delivery applications, including small molecular weight drugs, proteins, peptides, oligosaccharides, vaccines and nucleic acids. The materials and techniques used in the development of self-assembling nanogels are also described

Development Of A Nanoparticulate Drug Delivery Vehicle For Retinoic Acid

Retinoic acid (RA) is a small molecule capable of shunting developing T cells away from the Th17 lineage and towards the Treg phenotype, making it a potentially useful therapeutic for autoimmune and inflammatory diseases. However, therapy can be complicated by systemic toxicity and unpredictable bioavailability, making a targeted drug delivery vehicle for local therapy desirable. A promising approach is the use of nanoparticles, which have been demonstrated to increase potency and decrease toxicity of therapies in a variety of disease models including Th17 mediated diseases. We therefore constructed a nanoparticulate drug delivery platform from poly(lactic-co-glycolic acid) (PLGA) capable of encapsulating and releasing RA. Here we report the fabrication, characterization, and in vitro bioactivity of this platform. We demonstrate that RA containing PLGA nanoparticles suppress IL-17 and IFN-gamma production and ROR-gamma(t) expression in T cells polarized towards the Th17 phenotype in vitro with similar potency to that of free drug. Furthermore, we show that these particles enhance TGF-beta dependent Foxp3 expression and IL-10 production of T cells in vitro with similar potency to free RA. Finally, we demonstrate that T cells polarized towards the Th17 phenotype in the presence of free RA and nanoparticulate RA have similarly suppressed ability to induce IL-6 production by fibroblasts. Our findings demonstrate the feasibility of RA delivery via biodegradable nanoparticles and represent an exciting technology for the treatment of autoimmune and inflammatory diseases

A comparison of nanoparticullate CpG immunotherapy with and without allergens in spontaneously equine asthma-affected horses, an animal model

Introduction: New therapeutic strategies to modulate the immune response of human and equine allergic asthma are still under extensive investigation. Immunomodulating agents stimulating T-regulatory cells offer new treatment options beyond conventional symptomatic treatment or specific immunotherapy for human and equine allergic airway diseases, with the goal of a homoeostatic T-helper cell balance. The aim of this study was to evaluate the effects of a nebulized gelatin nanoparticle-CpG formulation (CpG-GNP) with and without specific allergens for the treatment of spontaneous allergic equine asthma as a model for human asthma. Methods: Twenty equine asthma-affected horses were treated either with CpG-GNP alone or CpG-GNP with allergens. Two specific allergens were selected for each horse based on history and an in-vitro test. Each horse received seven administrations of the respective nebulized composition and was examined before treatment, immediately after and 6 weeks after the treatment course. Results: Clinical parameters such as breathing rate, indirect interpleural measurement, arterial blood gases, amount of tracheal mucus and percentage of neutrophils and cytokines in tracheal washes and serum samples were evaluated. Treatment with CpG-GNP alone as well as in combinations with relevant allergens resulted in clinical improvement of nasal discharge, breathing rate, amount of secretion and viscosity, neutrophil percentage and partial oxygen pressure directly after and 6 weeks after treatment. There were no significant differences between the two treatments in clinical parameters or local cytokine profiles in the tracheal wash fluid (IL-10, IFN-g, and IL-17). IL-4 concentrations decreased significantly in both groups. Conclusion: Nonspecific CpG-GNP-based immunotherapy shows potential as a treatment for equine and possibly also human allergic asthma

Synthetic Nanoparticles for Vaccines and Immunotherapy

The immune system plays a critical role in our health. No other component of human physiology plays a decisive role in as diverse an array of maladies, from deadly diseases with which we are all familiar to equally terrible esoteric conditions: HIV, malaria, pneumococcal and influenza infections; cancer; atherosclerosis; autoimmune diseases such as lupus, diabetes, and multiple sclerosis. The importance of understanding the function of the immune system and learning how to modulate immunity to protect against or treat disease thus cannot be overstated. Fortunately, we are entering an exciting era where the science of immunology is defining pathways for the rational manipulation of the immune system at the cellular and molecular level, and this understanding is leading to dramatic advances in the clinic that are transforming the future of medicine.1,2 These initial advances are being made primarily through biologic drugs– recombinant proteins (especially antibodies) or patient-derived cell therapies– but exciting data from preclinical studies suggest that a marriage of approaches based in biotechnology with the materials science and chemistry of nanomaterials, especially nanoparticles, could enable more effective and safer immune engineering strategies. This review will examine these nanoparticle-based strategies to immune modulation in detail, and discuss the promise and outstanding challenges facing the field of immune engineering from a chemical biology/materials engineering perspectiveNational Institutes of Health (U.S.) (Grants AI111860, CA174795, CA172164, AI091693, and AI095109)United States. Department of Defense (W911NF-13-D-0001 and Awards W911NF-07-D-0004