Transport of ferrihydrite nanoparticles in saturated porous media: role of ionic strength and flow rate

The use of nanoscale ferrihydrite particles, which are known to effectively enhance microbial degradation of a wide range of contaminants, represents a promising technology for in situ remediation of contaminated aquifers. Thanks to their small size, ferrihydrite nanoparticles can be dispersed in water and directly injected into the subsurface to create reactive zones where contaminant biodegradation is promoted. Field applications would require a detailed knowledge of ferrihydrite transport mechanisms in the subsurface, but such studies are lacking in the literature. The present study is intended to fill this gap, focusing in particular on the influence of flow rate and ionic strength on particle mobility. Column tests were performed under constant or transient ionic strength, including injection of ferrihydrite colloidal dispersions, followed by flushing with particle-free electrolyte solutions. Particle mobility was greatly affected by the salt concentration, and particle retention was almost irreversible under typical salt content in groundwater. Experimental results indicate that, for usual ionic strength in European aquifers (2 to 5 mM), under natural flow condition ferrihydrite nanoparticles are likely to be transported for 5 to 30 m. For higher ionic strength, corresponding to contaminated aquifers, (e.g., 10 mM) the travel distance decreases to few meters. A simple relationship is proposed for the estimation of travel distance with changing flow rate and ionic strength. For future applications to aquifer remediation, ionic strength and injection rate can be used as tuning parameters to control ferrihydrite mobility in the subsurface and therefore the radius of influence during field injection

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

Insight into the cellular fate and toxicity of aluminium adjuvants used in clinically approved human vaccinations

Aluminium adjuvants remain the most widely used and effective adjuvants in vaccination and immunotherapy. Herein, the particle size distribution (PSD) of aluminium oxyhydroxide and aluminium hydroxyphosphate adjuvants was elucidated in attempt to correlate these properties with the biological responses observed post vaccination. Heightened solubility and potentially the generation of Al3+ in the lysosomal environment were positively correlated with an increase in cell mortality in vitro, potentially generating a greater inflammatory response at the site of simulated injection. The cellular uptake of aluminium based adjuvants (ABAs) used in clinically approved vaccinations are compared to a commonly used experimental ABA, in an in vitro THP-1 cell model. Using lumogallion as a direct-fluorescent molecular probe for aluminium, complemented with transmission electron microscopy provides further insight into the morphology of internalised particulates, driven by the physicochemical variations of the ABAs investigated. We demonstrate that not all aluminium adjuvants are equal neither in terms of their physical properties nor their biological reactivity and potential toxicities both at the injection site and beyond. High loading of aluminium oxyhydroxide in the cytoplasm of THP-1 cells without immediate cytotoxicity might predispose this form of aluminium adjuvant to its subsequent transport throughout the body including access to the brain

Nanoparticle-Based Adjuvants and Delivery Systems for Modern Vaccines

Ever since the development of the first vaccine, vaccination has had the great impact on global health, leading to the decrease in the burden of numerous infectious diseases. However, there is a constant need to improve existing vaccines and develop new vaccination strategies and vaccine platforms that induce a broader immune response compared to traditional vaccines. Modern vaccines tend to rely on certain nanotechnology platforms but are still expected to be readily available and easy for large-scale manufacturing and to induce a durable immune response. In this review, we present an overview of the most promising nanoadjuvants and nanoparticulate delivery systems and discuss their benefits from tehchnological and immunological standpoints as well as their objective drawbacks and possible side effects. The presented nano alums, silica and clay nanoparticles, nanoemulsions, adenoviral-vectored systems, adeno-associated viral vectors, vesicular stomatitis viral vectors, lentiviral vectors, virus-like particles (including bacteriophage-based ones) and virosomes indicate that vaccine developers can now choose different adjuvants and/or delivery systems as per the requirement, specific to combatting different infectious diseases

Unraveling the enigma: elucidating the relationship between the physicochemical properties of aluminium-based adjuvants and their immunological mechanisms of action

Aluminium salts are by far the most commonly used adjuvants in vaccines. There are only two aluminium salts which are used in clinically-approved vaccines, Alhydrogel® and AdjuPhos®, while the novel aluminium adjuvant used in Gardasil® is a sulphated version of the latter. We have investigated the physicochemical properties of these two aluminium adjuvants and specifically in milieus approximating to both vaccine vehicles and the composition of injection sites. Additionally we have used a monocytic cell line to establish the relationship between their physicochemical properties and their internalisation and cytotoxicity. We emphasise that aluminium adjuvants used in clinically approved vaccines are chemically and biologically dissimilar with concomitantly potentially distinct roles in vaccine-related adverse events

Nano toolbox in immune modulation and nanovaccines.

Despite the great success of vaccines over two centuries, the conventional strategy is based on attenuated/altered microorganisms. However, this is not effective for all microbes and often fails to elicit a protective immune response, and sometimes poses unexpected safety risks. The expanding nano toolbox may overcome some of the roadblocks in vaccine development given the plethora of unique nanoparticle (NP)-based platforms that can successfully induce specific immune responses leading to exciting and novel solutions. Nanovaccines necessitate a thorough understanding of the immunostimulatory effect of these nanotools. We present a comprehensive description of strategies in which nanotools have been used to elicit an immune response and provide a perspective on how nanotechnology can lead to future personalized nanovaccines

Development of novel vaccine carriers

In this work, combined interdisciplinary research in the fields of Pharmaceutical Technology, (Bio)Materials Science, and Biology is presented to understand and promote lyotropic liquid crystalline dispersions as novel vaccine carriers. The work was initiated with a detailed development of lyotropic mesophases based on phytantriol. The aim was to gain knowledge on the phase behavior of the system and also on the structural impact of additives. Therefore, formulations with increasing degree of complexity were sequentially characterized. The study started establishing a phase diagram, in which bulk phases of phytantriol in excess of highly purified water were characterized, upon temperature increase, applying polarization microscopy and small angle X-ray scattering (SAXS). Subsequently, pursuing the development of a system with immunostimulatory properties, the impact of mannide monooleate on phytantriol-based mesophases was investigated. Mannide monooleate is an emulsifier widely used in adjuvant formulations and found to promote immune responses in emulsion form. Thus, progressively higher amounts of mannide monooleate were incorporated into the system and the structural modifications were monitored upon temperature increase. The phase diagram allowed for a deep understanding of the mesophase behavior and enabled the selection of a feasible phytantriol/mannide monooleate ratio for the subsequent investigations. In this way, a stable formulation, featuring inverse hexagonal structure was identified. Next, to enable dispersion of the characterized bulk and to produce a nanoparticulate system, different steric stabilizers were evaluated. Here, the selection criteria were based on the dispersion efficiency at low concentrations and on the impact on the internal structure of the particles. The last part of the formulation development was concerning the application and customization of the formed hexosomes. Thus, the particle loading with the model antigens ovalbumin and lysozyme was studied, as well as strategies to introduce new features to the particulate system in a controlled way, preserving the inverse hexagonal structure. Here, negatively and positively charged hexosomes could be prepared through the incorporation of charged phospholipids into the formulation. Additionally, a formulation based on phytantriol and mannide monooleate, which typically formed an inverse hexagonal structure was shown to self-assemble with bicontinuous cubic double diamond structure upon addition of octyl-β-D-glucopyranoside. The aim of the second part of this work was the verification of one of the most important hypothesis with respect to the biological performance of non-lamellar lyotropic liquid crystalline dispersions. More specifically, it is known that non-lamellar structures are involved in fusogenic processes of the cell membrane. For this reason, it was speculated that particles internally structured with non-lamellar phases, such as hexosomes, could have fusogenic properties. Thus, to verify this hypothesis and elucidate the cellular uptake mechanism of hexosomes, we investigated the interactions of these particles with cells and with models of the cell membrane. For this purpose, the established hexosomes, based on phytantriol/mannide monooleate, were fluorescently labeled and their toxicity in cultures of HeLa cells was determined. Series of uptake experiments were carried out after suppression of the major endocytic pathways using highly specific approaches (e.g. single and double knockdown of regulatory proteins), and also inhibition strategies of broader effect (e.g. temperature reduction to 4°C, hypotonic treatment, cytochalasin D treatment). Additionally, experiments using models of the cell membrane (e.g. phospholipid monolayers and bilayers) were performed. All the analyses were carried out with hexosomes and liposomes in parallel to enable the comparison between particles featuring non-lamellar and lamellar structures, respectively. The results revealed a steeper toxicity curve and faster internalization kinetics for hexosomes in comparison to liposomes. Interestingly, against the expectations, indications of fusogenic properties were not observed. However, strong evidences that hexosomes have an alternative cell entry pathway that bypasses standard endocytosis were identified. In contrast, liposomes appeared to enter the cells through well-known endocytic pathways, such as caveolae-mediated and clathrin- mediated endocytosis. The final part of this work consisted of the evaluation of the biological performance of hexosomes within the vaccination context. The experiments were performed in vitro and in vivo, in parallel with the already established and promising cationic liposomal formulation CAF04, which is based on dimethyldioctadecylammonium (DDA) and on a synthetic analogue of monomycoloyl glycerol (MMG-1). Since MMG-1 and the positive charge of DDA are considered the core of the success of this formulation, differently charged hexosomes containing MMG-1 were developed and included in the study. The development process was driven by the lessons learned in the first phase of the project with phytantriol/mannide monooleate systems. In this way, to create particles internally structured with inverse hexagonal phase, MMG-1 was combined to phytantriol. After the establishment of the formulations, the immunogenicity of plain particles (without antigen) was investigated in vitro in cultures of dendritic cells derived from human peripheral blood mononuclear cells. Through the evaluation of the expression of several surface markers, it was found that, while CAF04 liposomes induced the upregulation of the homing receptor CCR7, all hexosomal formulations tested did not provoke any sign of adjuvanticity. Considering that the cultures of dendritic cells represent a dramatic simplification of the immune system, further experiments were performed in vivo. Thus, mice were immunized with the formulations loaded with Chlamydia trachomatis major outer membrane protein (MOMP) antigen. The immunological output was analyzed through several parameters (e.g. T-cell activation, cytokine expression and release, polyfunctionality, specific serum IgG) and all of them revealed dramatic differences between the immunizations performed with the unadjuvanted antigen and antigen adjuvanted with either liposomes or hexosomes. In comparison to the unadjuvanted systems, liposomes and hexosomes were both able to increase the magnitude of the immune responses. However, the immune responses elicited by CAF04 liposomes and by hexosomes were very distinct, the first eliciting mainly cellular mediated responses and the second humoral responses. Throughout this work, it was shown that stable and robust lipid-based lyotropic liquid formulations, that allow customization without impairment of the internal structure, can be rationally designed. In addition, keeping track of the structural modifications induced by each additive (separately) was shown to be a key strategy to enable further optimization in advanced stages of the formulation development. Regarding the performance of hexosomes in the biological environment, we have obtained accurate results demonstrating, in vitro and in vivo, drastic differences in comparison to liposomes. This intriguing outcome was observed for different formulations and in completely independent experiments. Overall, the insights presented in this thesis show hexosomes as promising delivery systems with uncommon properties and an applicability potential that goes beyond the vaccination context

Strategies to improve the immunogenicity of subunit vaccine candidates

Subunit vaccines contain highly defined macromolecular components of a pathogen that are capable of eliciting protective immunity. They possess several advantages over other vaccine types (e.g. live attenuated and inactivated) such as improved safety profiles, highly defined nature, ease of production, and potential for lower cost of goods. One critical limitation of subunit vaccines, however, is their weak immunogenicity owing to their inability to replicate, monovalent structures, and the absence of other immunostimulatory components. Common approaches to enhance the immunogenicity of subunit vaccines include polyvalent antigen display strategies, the use of adjuvants, etc. The polyvalent antigen display strategy requires the use of a scaffold, which can be protein-based or some other materials. Chapter 2 focuses on the biophysical properties of a potential scaffold for polyvalent antigen display-Bacillus anthracis lumazine synthase (LS), an icosahedral homo-oligomeric protein. LS in PBS buffer showed a minor thermal transition around 50 ᵒC, and a major one at 95 ᵒC. The minor transition arose from the dissociation of the LS/phosphate complex, which formed in PBS buffer at room temperature. The major transition corresponded to the dissociation of LS oligomers, thermal unfolding, and aggregation. In chapter 3, I describe an attempt to develop ricin vaccine candidates in which LS was used as a scaffold to achieve polyvalent display of a linear neutralizing epitope (designated PB10) from ricin. PB10 was genetically inserted onto the C terminus of LS, and the fusion protein (designated LS_PB10) was expressed in an E.coli system. LS_PB10 self-assembled into spherical particles. Fusion of the PB10 peptide did not affect the structure and stability of LS. LS_PB10 showed tight binding to a mAb targeting the PB10 epitope. Immunization of LS_PB10 in mice elicited a moderate level of anti-ricin serum titers, which, however, failed to offer protection during a challenge study using a 10x lethal dose of ricin. Such an unsatisfactory end result may be attributable to 1) limited efficacies of using the PB10 epitope alone, 2) loss of secondary structure of PB10 on LS; 3) an epitope suppression effect induced by the highly immunogenic nature of the LS scaffold. Studying antigen/adjuvant compatibility is critical for the development of adjuvanted vaccine formulations. Chapter 4 discusses the utilization of biophysical tools to understand effects of two emulsion-based adjuvants (designated as ME.0 and SE) on the structure and thermal stability of alpha-toxin (AT), a potential vaccine candidate for Staphylococcus aureus infection. Both adjuvants are oil-in-water (O/W) emulsions using squalene as the oil phase. DSC analysis showed the ME.0 emulsion thermally destabilized AT, probably because of changes in the buffer composition of AT upon mixing. The SE emulsion caused increased alpha-helix and decreased beta-sheet content in AT, and a blue shift in Trp fluorescence emission spectra of AT. DSC analysis showed SE exerted a dramatic thermal stabilization effect on AT, probably attributable to an interaction between AT and SE. Size exclusion chromatography showed a complete loss in the recovery of AT when mixed with SE, but not ME.0, indicating a high degree of interaction with SE. The goal of protein formulation development is to identify optimal conditions for long-term storage. Certain commercial conditions (e.g., high protein concentration or turbid adjuvanted samples) impart additional challenges to biophysical characterization. Formulation screening studies for such conditions are usually performed using a simplified format in which the target protein is studied at a low concentration in a clear solution. The failure of study conditions to model the actual formulation environment may cause a loss of ability to identify the optimal conditions for target proteins in their final commercial formulations. In chapter 5, we utilized a steady-state/lifetime fluorescence-based high-throughput platform to develop a general workflow for direct formulation optimization under analytically challenging but commercially relevant conditions. A high-concentration monoclonal antibody and an Alhydrogel-adjuvanted antigen were investigated. A large discrepancy in screening results was observed for both proteins under these two different conditions (simplified versus commercially relevant). This study demonstrates the feasibility of using a steady-state/lifetime fluorescence plate reader for direct optimization of challenging formulation conditions and highlights the importance of performing formulation optimization under commercially relevant conditions

Improved Delivery of a Broadly Active Influenza Subunit Vaccine

Influenza is a major burden every season causing up to 9,000,000 illnesses in the United States. Vaccines are available; however, their efficacy is limited. Antigenic drift/shift of the virus means that the vaccine must be redesigned every year to incorporate new circulating strains, and predictions for what the major circulating strains will be are not always correct leading to incomplete protection. To subvert the mutating virus, computationally optimized broadly reactive antigen (COBRA) hemagglutinin (HA) immunogens have been created that induce a broadly neutralizing antibody response; however, the immunogenicity of a COBRA subunit vaccine is limited but can be improved by delivery strategies that can enhance targeting to immune cells and act as adjuvants to activate the immune system. The work presented herein aims to improve the efficacy of a COBRA HA vaccine using drug delivery platforms. First, polymeric microparticles (MPs) composed of acetalated dextran (Ace-DEX) encapsulating COBRA HA and a STING agonist are investigated. Mice vaccinated with these MPs showed a broadly active humoral response, an improved cellular response when compared to an FDA-approved vaccine adjuvant, protection after lethal influenza challenge, and storage stability outside the cold chain. The efficacy of this platform was also assessed in immunocompromised mouse models such as obesity, aging, and chemotherapy-induced immunosuppression. Next, a coordination polymer is used to deliver COBRA HA along with aTLR agonist. Again, this platform shows efficacy in vivo as evidenced by a strong cellular and humoral response, protection after a lethal influenza challenge, and stability outside the cold chain. Finally, the efficacy of lipid nanoparticles as a carrier for mRNA encoding COBRA was evaluated, and an in vivo efficacy study showed that the adjuvant containing MPs decreased the humoral response to the COBRA HA LNPs. Overall, in the development of a broadly active influenza vaccine it is important to consider multiple delivery platforms. The work described herein shows that each platform has its own set of advantages and disadvantages. Together this work will aid in the selection of the most efficacious platform for a broadly active influenza vaccine that will help to reduce the major yearly burden of influenza.Doctor of Philosoph

Rational design of adjuvants for subunit vaccines : the format of cationic adjuvants affects the induction of antigen-specific antibody responses

A range of cationic delivery systems have been investigated as vaccine adjuvants, though few direct comparisons exist. To investigate the impact of the delivery platform, we prepared four cationic systems (emulsions, liposomes, polymeric nanoparticles and solid lipid nanoparticles) all containing equal concentrations of the cationic lipid dimethyldioctadecylammonium bromide in combination with the Neisseria adhesin A variant 3 subunit antigen. The formulations were physicochemically characterized and their ability to associate with cells and promote antigen processing (based on degradation of DQ-OVA, a substrate for proteases which upon hydrolysis is fluorescent) was compared in vitro and their vaccine efficacy (antigen-specific antibody responses and IFN-γ production) and biodistribution (antigen and adjuvant) were evaluated in vivo. Due to their cationic nature, all delivery systems gave high antigen loading (> 85%) with liposomes, lipid nanoparticles and emulsions being <200 nm, whilst polymeric nanoparticles were larger (~350 nm). In vitro, the particulate systems tended to promote cell uptake and antigen processing, whilst emulsions were less effective. Similarly, whilst the particulate delivery systems induced a depot (of both delivery system and antigen) at the injection site, the cationic emulsions did not. However, out of the systems tested the cationic emulsions induced the highest antibody responses. These results demonstrate that while cationic lipids can have strong adjuvant activity, their formulation platform influences their immunogenicity