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Preparation and characterization of nanostructured ferric hydroxyphosphate adjuvants
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This article describes part of the results obtained during the development of a new generation of vaccine adjuvants based on nanostructured hydroxyphosphates of tunable composition and physicochemical characteristics. Colloidal gels of ferric hydroxyphosphates of various iron/phosphate ratios were prepared by precipitation techniques, sterilized by autoclaving and analyzed by transmission electron microscopy (TEM) and dark-field optical microscopy. The obtained materials were composed of a network of amorphous nanoparticles (<20 nm in size) that were aggregated into micron-sized structures in physiological saline. Preliminary adsorption experiments indicated the ability of the obtained materials to adsorb protein substances, which is an important prerequisite for their potential application as vaccine adjuvants and further optimization of the production process to achieve reproducibility of the physicochemical characteristics.
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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.
Use of highly pure antigens to improve vaccine safety has led to reduced vaccine immunogenicity and efficacy. This has led to the need to use adjuvants to improve vaccine immunogenicity. The ideal adjuvant should maximize vaccine immunogenicity without compromising tolerability or safety. Unfortunately, adjuvant research has lagged behind other vaccine areas such as antigen discovery, with the consequence that only a very limited number of adjuvants based on aluminium salts, monophosphoryl lipid A and oil emulsions are currently approved for human use. Recent strategic initiatives to support adjuvant development by the National Institutes of Health should translate into greater adjuvant choices in the future. Mechanistic studies have been valuable for better understanding of adjuvant action, but mechanisms of adjuvant toxicity are less well understood. The inflammatory or danger-signal model of adjuvant action implies that increased vaccine reactogenicity is the inevitable price for improved immunogenicity. Hence, adjuvant reactogenicity may be avoidable only if it is possible to separate inflammation from adjuvant action. The biggest remaining challenge in the adjuvant field is to decipher the potential relationship between adjuvants and rare vaccine adverse reactions, such as narcolepsy, macrophagic myofasciitis or Alzheimer's disease. While existing adjuvants based on aluminium salts have a strong safety record, there are ongoing needs for new adjuvants and more intensive research into adjuvants and their effects.
Aluminum compounds, including aluminum phosphate (AlPO4), aluminum hydroxide (Al(OH)3), and alum precipitated vaccines, historically referred to as protein aluminate, are currently the most commonly used adjuvants with human and veterinary vaccines (1–6). These adjuvants are often referred to as “alum” in the literature, which is misleading because (1) two most widely used adjuvants from this group, aluminum hydroxide and aluminum phosphate, have very different physical characteristics (7) and differ in their adjuvant properties (3,6); and (2) alum, chemically potassium aluminum sulfate (KAl(SO4)2.12H2O), has not been used as an adjuvant as such. Alum was originally used to partially purify protein antigens, mainly tetanus and diphtheria toxoids, by precipitating them in the presence of anions including phosphate, sulphate, and bicarbonate ions resulting in a mixture of compounds, mainly aluminum phosphate and aluminum hydroxide (4,8,9). The amounts of aluminum phosphate and aluminum hydroxide in the mixture depended upon the amount and nature of anions present in the reaction mixture and adjustment of pH of the final product with sodium hydroxide (3,4,6,10,11). Although alum-precipitated tetanus and diphtheria toxoids had been used for human immunization for many years, their use has declined considerably because of variability in production of alum precipitated toxoids (1,4,8,9,12).
Drug products containing iron oxide and hydroxide nanoparticles (INPs) are important for the treatment of iron deficiency anaemia. Pharmaceuticals prepared by the complexation of different kinds of INPs and carbohydrates have different physicochemical and biopharmaceutic characteristics. The increasing number of parenteral non-biological complex drugs (NBCD) containing iron requires physicochemical methods for characterization and enabling of cross comparisons. In this context the structure and the level of crystallinity of the iron phases may be connected to the in vitro and in vivo dissolution rates, which etiologically determine the therapeutic and toxic effects.
Research over the past several decades has provided insight into the mode of action of adjuvants. However, the main focus of attention has been the efficacy in the induction of protective immunogenicity, while less effort has been devoted to the study of toxicity mechanisms. Evidences suggest that several mechanisms that are responsible for the immunostimulating effects are, at the same time, responsible of the adverse effects. In this context, it is often very difficult to establish the boundaries between immunostimulation and immunotoxicity to reach the ideal balance of efficacy/safety. During decades, hundreds of adjuvants and adjuvant formulations have been proposed as immunostimulants for vaccines but very few have been used in human vaccines due to toxicity concerns. In this review, relevant aspects about immunotoxicology of adjuvants, based on clinical and experimental studies are discussed. Some effects are only observed under hyperstimulating regimens using non-approved adjuvants for human use, but these are nonetheless useful to understanding basic principles of adjuvant toxicity. The acute local and systemic reactions, during the first hours and those that can be observed after the third day of vaccination in the inoculation site and systemically are discussed.
We reviewed the three toxicokinetic reference studies commonly used to suggest that aluminum (Al)-based adjuvants are innocuous. A single experimental study was carried out using isotopic 26Al (Flarend et al., Vaccine, 1997). This study used aluminum salts resembling those used in vaccines but ignored adjuvant uptake by cells that was not fully documented at the time. It was conducted over a short period of time (28days) and used only two rabbits per adjuvant. At the endpoint, Al elimination in the urine accounted for 6% for Al hydroxide and 22% for Al phosphate, both results being incompatible with rapid elimination of vaccine-derived Al in urine. Two theoretical studies have evaluated the potential risk of vaccine Al in infants, by reference to an oral "minimal risk level" (MRL) extrapolated from animal studies. Keith et al. (Vaccine, 2002) used a high MRL (2mg/kg/d), an erroneous model of 100% immediate absorption of vaccine Al, and did not consider renal and blood-brain barrier immaturity. Mitkus et al. (Vaccine, 2011) only considered solubilized Al, with erroneous calculations of absorption duration. Systemic Al particle diffusion and neuro-inflammatory potential were omitted. The MRL they used was both inappropriate (oral Al vs. injected adjuvant) and still too high (1mg/kg/d) regarding recent animal studies. Both paucity and serious weaknesses of reference studies strongly suggest that novel experimental studies of Al adjuvants toxicokinetics should be performed on the long-term, including both neonatal and adult exposures, to ensure their safety and restore population confidence in Al-containing vaccines.
Adjuvants are substances that, when combined with an antigen in a vaccine formulation, potentiate the immune response against that antigen. Aluminium hydroxyphosphate is among the currently used adjuvants in vaccines, while studies on its iron analogue, ferric hydroxyphosphate, are quite limited. In this article we report on the preparation and physicochemical characterization of novel nanostructured iron and aluminium based mixed hydroxyphosphates as potential vaccine adjuvants, prepared by controlled co-precipitation from mixed solutions of Fe(III) and Al(III) chlorides with sodium phosphate and sterilized by autoclaving. The obtained hydroxyphosphates consisted of a network of platy nanoparticles (20–40 nm in size), which aggregated in aqueous medium to form secondary micron-sized particles. The obtained nanostructured hydroxyphosphates were characterized by energy dispersive X-ray spectroscopy for elemental composition, X-ray powder diffraction, infrared spectroscopy, zeta-potential measurements for determination of isoelectric point, protein adsorption, and dissolution rate in sodium citrate solution.
This chapter is concerned with the identification, characterization, and behavior of aluminum-containing adjuvants with proteins and anions similar to those occurring in vaccines and interstitial fluid. Aluminum-containing adjuvants referred to commercially as aluminum hydroxide have been identified as poorly crystalline aluminum oxyhydroxide with the structure of the mineral boehmite. Relevant properties of this material include its high surface area and its high pI, which provide the adjuvant with a high adsorptive capacity for positively charged proteins. Aluminum phosphate and alum-precipitated adjuvants may be classified as amorphous aluminum hydroxyphosphate with little or no specifically adsorbed sulfate. Variations in the molar PO4/A1 ratio of amorphous aluminum hydroxyphosphates result in PI values that range from 5 up to 7; the materials are negatively charged at a physiological pH of 7.4. The amorphous nature of these compounds gives them high surface area and high protein adsorptive capacity for positively charged proteins. Observations on the interactions of anions and charged proteins with charged adjuvant surfaces have provided a framework for predicting behavior of complex systems of vaccines and for designing specific combinations of adjuvants and antigens to optimize the stability and efficacy of vaccines.
Alum (or aluminum-containing) adjuvants are key components of many vaccines currently on the market. The immuno-potentiation effect of alum adjuvants is presumably due to their interaction with antigens, leading to adsorption on the alum particle surface. Understanding the mechanism of antigen adsorption/desorption and its influencing factors could provide guidance on formulation design and ensure proper in-vivo immuno-potentiation effect. In this paper, surface adsorption of several model proteins on two types of aluminum adjuvants (Alhydrogel(®) and Adjuphos(®)) are investigated to understand the underlying adsorption mechanisms, capacities, and potential influencing factors. It was found that electrostatic interactions are the major driving force for surface adsorption of all the model proteins except ovalbumin. Alhydrogel has a significantly higher adsorption capacity than Adjuphos. Several factors significantly change the adsorption capacity of both Alhydrogel and Adjuphos, including molecular weight of protein antigens, sodium chloride, phosphate buffer, denaturing agents, and size of aluminum particles. These important factors need to be carefully considered in the design of an effective protein antigen-based vaccine.
Aluminium-based vaccine adjuvants have been in use since the 1920s. Aluminium hydroxide (alum) that is the chemical basis of Alhydrogel, a widely used adjuvant, is a colloid that binds proteins to the particular surface for efficient presentation to the immune system during the vaccination process. Using conventional TEM and cryo-TEM we have shown that Alhydrogel can be finely dispersed by ultrasonication of the aqueous suspension. Clusters of ultrasonicated aluminium hydroxide micro-fibre crystals have been produced (∼ 10-100 nm), that are significantly smaller than those present the untreated Alhydrogel (∼ 2-12 μm). However, even prolonged ultrasonication did not produce a homogenous suspension of single aluminium hydroxide micro-fibres. The TEM images of unstained and negatively stained air-dried Alhydrogel are very similar to those obtained by cryo-electron microscopy. Visualization of protein on the surface of the finely dispersed Alhydrogel by TEM is facilitated by prior ultrasonication. Several examples are given, including some of medical relevance, using proteins of widely ranging molecular mass and oligomerization state. Even with the smaller mass proteins, their presence on the Alhydrogel surface can be readily defined by TEM. It has been found that low quantities of protein tend to cross-link and aggregate the small Alhydogel clusters, in a more pronounced manner than high protein concentrations. This indicates that complete saturation of the available Alhydrogel surface with protein may be achieved, with minimal cross-linkage, and future exploitation of this treatment of Alhydrogel is likely to be of immediate value for more efficient vaccine production.