Hollow Aluminum Hydroxide Modified Silica Nanoadjuvants with Amplified Immunotherapy Effects through Immunogenic Cell Death Induction and Antigen Release.

In spite of the widespread application of vaccine adjuvants in various preventive vaccines at present, the existing adjuvants are still hindered by weak cellular immunity responses in therapeutic cancer vaccines. Herein, a hollow silica nanoadjuvant containing aluminum hydroxide spikes on the surface (SiAl) is synthesized for the co-loading of chemotherapeutic drug doxorubicin (Dox) and tumor fragment (TF) as tumor antigens (SiAl@Dox@TF). The obtained nanovaccines show significantly elevated anti-tumor immunity responses thanks to silica and aluminum-based composite nanoadjuvant-mediated tumor antigen release and Dox-induced immunogenic cell death (ICD). In addition, the highest frequencies of dendritic cells (DCs), CD4+ T cells, CD8+ T cells, and memory T cells as well as the best mice breast cancer (4T1) tumor growth inhibitory are also observed in SiAl@Dox@TF group, indicating favorable potential of SiAl nanoadjuvants for further applications. This work is believed to provide inspiration for the design of new-style nanoadjuvants and adjuvant-based cancer vaccines.

A comparison between adjuvant and delivering functions of calcium phosphate, aluminum hydroxide and chitosan nanoparticles, using a model protein of Brucella melitensis Omp31.

Vaccination is the most efficient and economic approach used to hinder infection and intense consequences caused by viruses, bacteria, or other pathogenic organisms. Since the intrinsic immunogenicity of recombinant antigens is usually low, safe and potent vaccine adjuvants are needed to ensure the success of those recombinant vaccines. Nanoparticles (NPs) have attracted much interest as adjuvants and delivery systems. Previous studies have shown that calcium phosphate (CP), aluminum hydroxide (AH) and chitosan (CS) NPs are promising delivery systems for immunization. In addition, it has been determined that Omp31 is a good candidate for inducing protection against Brucella (B) melitensis and B. ovis. Our aim in the present study was to compare the functions of CP, AH and CS NPs for stimulation of the immune response and protection against B. melitensis by using omp31 as a model protein. Based on the cytokine profile and subclasses of the antibody, vaccination with Omp31 load CP (CP/Omp31) and Omp31 load AH (AH/Omp31) NPs induced T helper type 1 (Th1)-T helper type 2 (Th2) immune response, whereas immunization by Omp31 load CS (CS/Omp31) NPs induced Th1 immune response. CP/Omp31 NPs elicited protection toward B. melitensis challenge equivalent to the vaccine strain B. melitensis Rev.1. Compared to CS/Omp31 NPs, CP/Omp31 NPs elicited a low increase in protection level against B. melitensis 16 M. In conclusion, the obtained results indicated that CP NPs were potent antigen delivery systems to immunize brucellosis.

Quantitative Analysis of Vaccine Antigen Adsorption to Aluminum Adjuvant Using an Automated High-Throughput Method.

Aluminum-containing adjuvants have been widely used in vaccine formulations to safely and effectively potentiate the immune response. The examination of the extent of antigen adsorption to aluminum adjuvant is always evaluated during the development of aluminum adjuvant containing vaccines. A rapid, automated, high-throughput assay was developed to measure antigen adsorption in a 96-well plate format using a TECAN Freedom EVO® (TECAN). The antigen adsorption levels at a constant adjuvant concentration for each sample were accurately measured at 12 antigen/adjuvant (w/w) formulation ratios. These measurements were done at aluminum adjuvant concentrations similar to normal vaccine formulations, unlike previous non-automated and automated adjuvant adsorption studies. Two high-sensitivity analytical methods were used to detect the non-absorbed antigens. The antigen-to-adjuvant adsorption curves were fit to a simple Langmuir adsorption model for quantitatively analyzing the antigen to the adjuvant adsorption level and strength. The interaction of two model antigens, bovine serum albumin and lysozyme, with three types of aluminum adjuvant, were quantitatively analyzed in this report. Automated, high-throughput methodologies combined with sensitive analytical methods are useful for accelerating practical vaccine formulation development.LAY ABSTRACT: Vaccines are probably the most effective public health method to prevent epidemics of many infectious diseases. Many of the most effective vaccines contain aluminum adjuvant. This report describes novel technology that can be used to better optimize the efficacy and stability of aluminum adjuvant-containing vaccines.

Competition between bacteria and phosphate for adsorption sites on gibbsite: An in-situ ATR-FTIR spectroscopic and macroscopic study.

Sorption and desorption of phosphate (P) on Fe and Al (hydr)oxides may be affected by bacteria in soils because their ubiquitous and strong interactions. The role of Bacillus subtilis and Pseudomonas fluorescens in adsorption of P on gibbsite (γ-AlOOH) was systematically investigated under a wide range of conditions by combining in-situ attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy with batch macroscopic experiments. In-situ ATR-FTIR observations of the ternary systems (bacteria, P, and gibbsite) showed simultaneous desorption of P from, and adhesion of the bacteria to, gibbsite, indicating a competition between the two for surface sites. Batch desorption experiments showed that bacteria could mobilize the P from gibbsite into solution, and macroscopic adsorption data showed that the amount of P adsorbed on the bacteria-gibbsite complex was less than that on gibbsite alone over durations from 0h to 26h, concentrations of P from 0.1mM to 2.0mM, pH from 5 to 8, and ionic strength from 0M to 0.5M, suggesting that bacteria inhibit the adsorption of P on gibbsite. The degree of inhibition increased with the number of bacteria in the system and was significantly but non-linearly correlated with the decline in the positive charge on gibbsite induced by the bacteria. Therefore, competition for suitable sites on the surface of gibbsite between P and the bacteria and reduction in the positive charge on the surface of gibbsite induced by bacteria are proposed as two important mechanisms that inhibit P adsorption. These findings highlight the role of bacteria in regulating the availability of P to plants and its mobility in natural environments.

Chemical mimicking of bio-assisted aluminium extraction by Aspergillus niger's exometabolites.

Presence of microorganisms in soils strongly affects mobility of metals. This fact is often excluded when mobile metal fraction in soil is studied using extraction procedures. Thus, the first objective of this paper was to evaluate strain Aspergillus niger's exometabolites contribution on aluminium mobilization. Fungal exudates collected in various time intervals during cultivation were analyzed and used for two-step bio-assisted extraction of alumina and gibbsite. Oxalic, citric and gluconic acids were identified in collected culture media with concentrations up to 68.4, 2.0 and 16.5 mmol L-1, respectively. These exometabolites proved to be the most efficient agents in mobile aluminium fraction extraction with aluminium extraction efficiency reaching almost 2.2%. However, fungal cultivation is time demanding process. Therefore, the second objective was to simplify acquisition of equally efficient extracting agent by chemically mimicking composition of main organic acid components of fungal exudates. This was successfully achieved with organic acids mixture prepared according to medium composition collected on the 12th day of Aspergillus niger cultivation. This mixture extracted similar amounts of aluminium from alumina compared to culture medium. The aluminium extraction efficiency from gibbsite by organic acids mixture was lesser than 0.09% which is most likely because of more rigid mineral structure of gibbsite compared to alumina. The prepared organic acid mixture was then successfully applied for aluminium extraction from soil samples and compared to standard single step extraction techniques. This showed there is at least 2.9 times higher content of mobile aluminium fraction in soils than it was previously considered, if contribution of microbial metabolites is considered in extraction procedures. Thus, our contribution highlights the significance of fungal metabolites in aluminium extraction from environmental samples, but it also simplifies the extraction procedure inspired by bio-assisted extraction of aluminium by common soil fungus A. niger.

Mechanisms of Antigen Adsorption Onto an Aluminum-Hydroxide Adjuvant Evaluated by High-Throughput Screening.

The adsorption mechanism of antigen on aluminum adjuvant can affect antigen elution at the injection site and hence the immune response. Our aim was to evaluate adsorption onto aluminum hydroxide (AH) by ligand exchange and electrostatic interactions of model proteins and antigens, bovine serum albumin (BSA), ß-casein, ovalbumin (OVA), hepatitis B surface antigen, and tetanus toxin (TT). A high-throughput screening platform was developed to measure adsorption isotherms in the presence of electrolytes and ligand exchange by a fluorescence-spectroscopy method that detects the catalysis of 6,8-difluoro-4-methylumbelliferyl phosphate by free hydroxyl groups on AH. BSA adsorption depended on predominant electrostatic interactions. Ligand exchange contributes to the adsorption of ß-casein, OVA, hepatitis B surface antigen, and TT onto AH. Based on relative surface phosphophilicity and adsorption isotherms in the presence of phosphate and fluoride, the capacities of the proteins to interact with AH by ligand exchange followed the trend: OVA < ß-casein < BSA < TT. This could be explained by both the content of ligands available in the protein structure for ligand exchange and the antigen's molecular weight. The high-throughput screening platform can be used to better understand the contributions of ligand exchange and electrostatic attractions governing the interactions between an antigen adsorbed onto aluminum-containing adjuvant.

Biodistribution and toxicity of spherical aluminum oxide nanoparticles.

With the rapid development of the nano-industry, concerns about their potential adverse health effects have been raised. Thus, ranking accurately their toxicity and prioritizing for in vivo testing through in vitro toxicity test is needed. In this study, we used three types of synthesized aluminum oxide nanoparticles (AlONPs): γ-aluminum oxide hydroxide nanoparticles (γ-AlOHNPs), γ- and α-AlONPs. All three AlONPs were spherical, and the surface area was the greatest for γ-AlONPs, followed by the α-AlONPs and γ-AlOHNPs. In mice, γ-AlOHNPs accumulated the most 24 h after a single oral dose. Additionally, the decreased number of white blood cells (WBC), the increased ratio of neutrophils and the enhanced secretion of interleukin (IL)-8 were observed in the blood of mice dosed with γ-AlOHNPs (10 mg kg(-1)). We also compared their toxicity using four different in vitro test methods using six cell lines, which were derived from their potential target organs, BEAS-2B (lung), Chang (liver), HACAT (skin), H9C2 (heart), T98G (brain) and HEK-293 (kidney). The results showed γ-AlOHNPs induced the greatest toxicity. Moreover, separation of particles was observed in a transmission electron microscope (TEM) image of cells treated with γ-AlOHNPs, but not γ-AlONPs or α-AlONPs. In conclusion, our results suggest that the accumulation and toxicity of AlONPs are stronger in γ-AlOHNPs compared with γ-AlONPs and α-AlONPs owing their low stability within biological system, and the presence of hydroxyl group may be an important factor in determining the distribution and toxicity of spherical AlONPs.

Unequivocal identification of intracellular aluminium adjuvant in a monocytic THP-1 cell line.

Aluminium-based adjuvants (ABA) are the predominant adjuvants used in human vaccinations. While a consensus is yet to be reached on the aetiology of the biological activities of ABA several studies have identified shape, crystallinity and size as critical factors affecting their adjuvanticity. In spite of recent advances, the fate of ABA following their administration remains unclear. Few if any studies have demonstrated the unequivocal presence of intracellular ABA. Herein we demonstrate for the first time the unequivocal identification of ABA within a monocytic T helper 1 (THP-1) cell line, using lumogallion as a fluorescent molecular probe for aluminium. Use of these new methods revealed that particulate ABA was only found in the cell cytoplasm. Transmission electron microscopy revealed that ABA were contained within vesicle-like structures of approximately 0.5-1 µm in diameter.

Increasing the potency of an alhydrogel-formulated anthrax vaccine by minimizing antigen-adjuvant interactions.

Aluminum salts are the most widely used vaccine adjuvants, and phosphate is known to modulate antigen-adjuvant interactions. Here we report an unexpected role for phosphate buffer in an anthrax vaccine (SparVax) containing recombinant protective antigen (rPA) and aluminum oxyhydroxide (AlOH) adjuvant (Alhydrogel). Phosphate ions bind to AlOH to produce an aluminum phosphate surface with a reduced rPA adsorption coefficient and binding capacity. However, these effects continued to increase as the free phosphate concentration increased, and the binding of rPA changed from endothermic to exothermic. Crucially, phosphate restored the thermostability of bound rPA so that it resembled the soluble form, even though it remained tightly bound to the surface. Batches of vaccine with either 0.25 mM (subsaturated) or 4 mM (saturated) phosphate were tested in a disease model at batch release, which showed that the latter was significantly more potent. Both formulations retained their potency for 3 years. The strongest aluminum adjuvant effects are thus likely to be via weakly attached or easily released native-state antigen proteins.

The immunobiology of aluminium adjuvants: how do they really work?

Aluminium adjuvants potentiate the immune response, thereby ensuring the potency and efficacy of typically sparingly available antigen. Their concomitant critical importance in mass vaccination programmes may have prompted recent intense interest in understanding how they work and their safety. Progress in these areas is stymied, however, by a lack of accessible knowledge pertaining to the bioinorganic chemistry of aluminium adjuvants, and, consequently, the inappropriate application and interpretation of experimental models of their mode of action. The objective herein is, therefore, to identify the many ways that aluminium chemistry contributes to the wide and versatile armoury of its adjuvants, such that future research might be guided towards a fuller understanding of their role in human vaccinations.

Bioaccumulation of heavy metals by Phragmites australis cultivated in synthesized substrates.

Accumulation of heavy metals from various oxides with adsorbed cadmium by wetland plant Phragmites australis was studied to evaluate the fate of heavy metals in the sediment of constructed wetlands. Hoagland solution was used as nutrition supply, and single metal oxide with adsorbed cadmium was applied as contaminant to study the accumulation characteristics of cadmium and the substrate metals by P. australis. After 45-d treatment, the bioaccumulation degree in root followed the order: Al(OH)3 > Al2O3 > Fe3O4 > MnO2 > FeOOH. Heavy metals absorbed by P. australis were largely immobilized by the roots with little translocation to aboveground parts.

Effect of the strength of adsorption of hepatitis B surface antigen to aluminum hydroxide adjuvant on the immune response.

Hepatitis B surface antigen (HBsAg) is known to adsorb to aluminum hydroxide adjuvant (AH) by ligand exchange between its accessible phosphate groups and surface hydroxyl groups of the adjuvant. To study the effect of the binding strength, five vaccines were prepared with AH or four samples of AH that were modified by pretreatment with different concentrations of potassium dihydrogen phosphate. The adsorptive coefficients ranged from 3660 to 250mL/mg based on the Langmuir adsorption isotherm and degrees of elution ranged from 1 to 31% when the vaccines were exposed to interstitial fluid in vitro. When tested in mice the four vaccines containing phosphate-treated AH (PTAH) induced significantly greater antibody responses than the vaccine containing AH, which had the highest adsorptive coefficient and the smallest degree of elution of HBsAg. The results indicated that antibody production is reduced when the antigen is adsorbed too strongly. Thus, the strength of adsorption of the antigen to an aluminum-containing adjuvant can affect the immunogenicity of the vaccine and should be optimized during vaccine formulation.

Transesterification of triglycerides in high and low quality oil feeds over an HT2 hydrotalcite catalyst.

The use of a heterogeneous catalyst, in the transesterification reaction of refined and acidic cottonseed oil for the production of methyl-esters (biodiesel) has been studied. The basic Mg-Al-CO3 hydrotalcite catalyst used showed a high activity for methanolysis and esterification reactions in a refined and an acidic cottonseed oil as well as in a representative high water content animal fat feed. The experiments were performed in a temperature range between 180 and 210 degrees C, in a batch reactor. The methanol to vegetable oil molar ratio was 6 to 1, while the catalyst concentration was fixed at 1 wt.% of the oil mass. Non-calcined and calcined forms of the catalyst were tested. The activity of the calcined catalyst was lower than the initial activity of the non-calcined catalytic system but it appeared the same with the reused non-calcined system.

Relationship between physical and chemical properties of aluminum-containing adjuvants and immunopotentiation.

Aluminum-containing adjuvants are an important component of many vaccines because they safely potentiate the immune response. The structure and properties of aluminum hydroxide adjuvant, aluminum phosphate adjuvant and alum-precipitated adjuvants are presented in this review. The major antigen adsorption mechanisms, electrostatic attraction and ligand exchange, are related to the adjuvant structure. The manner by which aluminum-containing adjuvants potentiate the immune response is related to the structure, properties of the adjuvant and adsorption mechanism. Immunopotentiation occurs through the following sequential steps: inflammation and recruitment of antigen-presenting cells, retention of antigen at the injection site, uptake of antigen, dendritic cell maturation, T-cell activation and T-cell differentiation.

Aluminum control of phosphorus sorption by lake sediments.

Release of reactive (phosphate-like) phosphorus (P) from freshwater sediments represents a significant internal P source for many lakes. Hypolimnetic P release occurs under reducing conditions that cause reductive dissolution of ferric hydroxide [Fe(OH)3]. This hypolimnetic P release may be naturally low or artificially reduced by sediment with naturally high or artificially elevated concentrations of aluminum hydroxide [Al(OH)3]. We presentfield and laboratory data for a common extraction analysis of sediments from 43 lakes differing in trophic status, pH regime, climate, and P loading. The results indicate that a simple sequential extraction of sediment may be a useful predictor of sediment's ability to release P. Sequential extractions of sediment P, Al, and Fe by water (H2O), bicarbonate-dithionite (BD), and NaOH (at 25 degrees C) showed that negligible amounts of P would be released from lake sediments during hypolimnetic anoxia if either (1) the molar Al(NaOH-25):Fe(BD) ratio is > 3 or (2) the molar Al(NaOH-25):P(H2O+BD) ratio is > 25. These ratios can be used as operational targets for estimation of sediment P release potential and Al dosing of P-rich sediment to prevent hypolimnetic P release under anoxic conditions.

Detoxification of endotoxin by aluminum hydroxide adjuvant.

Langmuir adsorption isotherms of endotoxin and aluminum-containing adjuvants at pH 7.4 and 25 degrees C revealed that aluminum hydroxide adjuvant has a greater adsorption capacity (283 microg/mg Al) and adsorption coefficient (1.3x10(4) ml/miccrog) than aluminum phosphate adjuvant (3.0 microg/mg Al, 0.20 ml/microg). The difference in endotoxin adsorption was related to two adsorption mechanisms: electrostatic attraction and covalent bonding. The isoelectric point (iep) of endotoxin is approximately 2. An electrostatic attractive force will be present with aluminum hydroxide adjuvant (iep=11.4), and an electrostatic repulsive force will operate with aluminum phosphate adjuvant (iep=4.6). Endotoxin contains two phosphate groups in the lipid A portion. Covalent bonding occurs with surface aluminum in aluminum hydroxide adjuvant but is inhibited by surface phosphate in aluminum phosphate adjuvant. In-vitro desorption experiments using components of interstitial fluid showed that endotoxin adsorbed by aluminum hydroxide adjuvant was not desorbed by interstitial anions (5 mM phosphate or 2.7 mM citrate) or interstitial proteins (25 mg albumin/ml). The effect of aluminum-containing adjuvants on the systemic response of Sprague-Dawley rats to a 15 microg/kg subcutaneous dose of endotoxin was determined by measuring the serum concentration of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). TNF-alpha and IL-6 were observed in the group which received an endotoxin solution or endotoxin and aluminum phosphate adjuvant. No TNF-alpha or IL-6 was detected in the group that received endotoxin and aluminum hydroxide adjuvant. Aluminum hydroxide adjuvant detoxifies endotoxin by adsorbing it in the vaccine and then not releasing it in interstitial fluid upon administration.

Adjuvanticity of aluminium hydroxide and calcium phosphate in diphtheria-tetanus vaccines--I.

The potencies of two diphtheria-tetanus vaccines (DT) adsorbed to either aluminium hydroxide or calcium phosphate were compared in mice and guinea pigs. The vaccines were made from the same batches of purified toxoids and contained the same amounts of antigens. Immunizations were done once or twice with different doses of vaccine injected undiluted, diluted in saline or diluted in the corresponding adjuvant. The various potency assays showed that the adjuvanticity of calcium phosphate was lower than or equal to aluminium hydroxide. Despite the range of potency assays done, none of the methods reflected the efficacy of these vaccines in revaccination of humans. A simplified potency assay is suggested for release of final vaccine formulations to reduce the number of animals in quality control.

Phosphate-binding properties and electrolyte content of aluminum hydroxide antacids.

The phosphate-binding capacities of 19 liquid and solid aluminum hydroxide gel antacids were determined in vitro under varying pH conditions. The resulting data provide a basis explaining the phosphate-binding characteristics observed when patients are treated with long-term aluminum hydroxide therapy. No antacid, liquid or solid, showed significant binding at pH 1.0. Maximum phosphate binding (expressed as phosphorus; P) was observed at pH 2.0 and 3.0 for most antacids and decreased markedly at alkaline pH. The liquid antacids showed a significantly greater phosphate-binding capacity than did tablets or capsules (p less than 0.01). At pH 2.0, the liquid antacids bound a mean of 22.3 mg P/5 ml. At pH 8.0 binding was reduced to a mean of 7.3 mg P/5 ml. Significant interbrand differences were observed. At pH 2.0, the solid antacids bound a mean of 15.3 mg P/tablet or capsule. At pH 8.0, binding was reduced to a mean of 5.8 mg P/tablet or capsule. Interbrand differences, while substantial, were less than those observed among the liquid antacids. Variations in sodium and potassium content were clinically insignificant for most of the antacids in this study, while the differences in phosphate-binding properties were sufficient to warrant attention in the patient with renal failure.

Aluminum deposition in the central nervous system. Preferential accumulation in the hippocampus in weanling rats.

Simultaneous administration of 1,25-dihydroxyvitamin-D3, citrate, and aluminum-containing phosphate binders is frequently used in patients with chronic renal failure. In order to investigate whether citrate may represent a risk factor of aluminum intoxication, 16 Sprague-Dawley weanling rats were randomly assigned to four groups: 1,25-dihydroxyvitamin-D3 at 16 ng/kg/day was given to all groups except the control; in addition, two groups received either aluminum hydroxide at 160 mg elemental aluminum/kg/day, or aluminum citrate at 160 mg elemental aluminum/kg/day, respectively. The control group received only the vehicle. Extremely high aluminum concentrations were detected in the hippocampus of rats receiving aluminum compounds. This content of aluminum (microgram/g dry weight) was far higher than that found in other brain areas of the same animals (146.40 +/- 51.23 versus 4.49 +/- 0.62, P less than 0.001) as well as that detected in the hippocampus of the control animals (2.73 +/- 0.40). Thus, in non-uremic, weanling rats supplemented with 1,25-dihydroxyvitamin-D3, the administration of aluminum favors selective accumulation in the hippocampus. No differences between aluminum hydroxide and aluminum citrate administration were observed.

A comparison between pyridinic and non-pyridinic extraction of allergenic materials.

The effect of addition of pyridine in the extracting solvent of allergenic materials was studied in the following substances. Dermatophagoides farinae, Chenopodium album, Parietaria judaica, Phleum pratense, Poa pratensis, and Secale cereale. Pyridinated extracts differed significantly in protein content, isoelectric focusing profile, and allergenic activity from the non-pyridinated extracts. On the other hand, no difference was detected between both extraction systems when their adsorption capacity to aluminium gels was compared.