Aluminum hydroxide exposure induces neurodevelopmental impairment in hESC-derived cerebral organoids.

Aluminum (Al) has been classified as a cumulative environmental pollutant that endangers human health. There is increasing evidence to suggest the toxic effects of Al, but the specific action on human brain development remains unclear. Al hydroxide (Al(OH)3), the most common vaccine adjuvant, is the major source of Al and poses risks to the environment and early childhood neurodevelopment. In this study, we explored the neurotoxic effect of 5 µg/ml or 25 µg/ml Al(OH)3 for six days on neurogenesis by utilizing human cerebral organoids from human embryonic stem cells (hESCs). We found that early Al(OH)3 exposure in organoids caused a reduction in the size, deficits in basal neural progenitor cell (NPC) proliferation, and premature neuron differentiation in a time and dose-dependent manner. Transcriptomes analysis revealed a markedly altered Hippo-YAP1 signaling pathway in Al(OH)3 exposed cerebral organoid, uncovering a novel mechanism for Al(OH)3-induced detrimental to neurogenesis during human cortical development. We further identified that Al(OH)3 exposure at day 90 mainly decreased the production of outer radial glia-like cells(oRGs) but promoted NPC toward astrocyte differentiation. Taken together, we established a tractable experimental model to facilitate a better understanding of the impact and mechanism of Al(OH)3 exposure on human brain development.

Neuroprotective properties of borax against aluminum hydroxide-induced neurotoxicity: Possible role of Nrf-2/BDNF/AChE pathways in fish brain.

The current study was designed to assess the possible neuroprotective effect of borax (BX) against the toxicity of aluminum hydroxide [AH, Al (OH)3] on brain of rainbow trout (Oncorhynchus mykiss) with multibiomarker approaches. For this purpose, the presence of the neuroprotective action by BX against the AH exposure was assessed by the activities of catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), myeloperoxidase (MPO), acetylcholinesterase (AChE). In addition, we evaluated glutathione (GSH), malondialdehyde (MDA), DNA damage (8-OHdG), apoptosis (caspase 3), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), nuclear factor erythroid-2 (Nrf-2), and brain-derived neurotrophic factor (BDNF) levels in 96 h semi-static treatment. In the 48th and 96th hour samplings, apoptosis induced by AH in the Nrf-2/BDNF/AChE pathways in rainbow trout brain tissue was revealed by DNA damage, enzyme inhibitions and lipid peroxidations. On the contrary applications of BX supported antioxidant capacity without leading apoptosis, lipid peroxidation, inflammatory response and DNA damage. BX also increased the BDNF levels and AChE activity. Moreover, BX exerted a neuroprotective effect against AH-induced neurotoxicity via down-regulating cytokine-related pathways, minimising DNA damage, apoptosis as well as up-regulating GSH, AChE, BDNF and antioxidant enzyme levels. It can be concluded that the combination of borax with AH modulated the toxic effects of AH.

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.

Inactivated whole hepatitis C virus vaccine employing a licensed adjuvant elicits cross-genotype neutralizing antibodies in mice.

BACKGROUND & AIMS: A prophylactic vaccine is required to eliminate HCV as a global public health threat. We developed whole virus inactivated HCV vaccine candidates employing a licensed adjuvant. Further, we investigated the effects of HCV envelope protein modifications (to increase neutralization epitope exposure) on immunogenicity. METHODS: Whole virus vaccine antigen was produced in Huh7.5 hepatoma cells, processed using a multistep protocol and formulated with adjuvant (MF-59 analogue AddaVax or aluminium hydroxide). We investigated the capacity of IgG purified from the serum of immunized BALB/c mice to neutralize genotype 1-6 HCV (by virus neutralization assays) and to bind homologous envelope proteins (by ELISA). Viruses used for immunizations were (i) HCV5aHi with strain SA13 envelope proteins and modification of an O-linked glycosylation site in E2 (T385P), (ii) HCV5aHi(T385) with reversion of T385P to T385, featuring the original E2 sequence determined in vivo and (iii) HCV5aHi(ΔHVR1) with deletion of HVR1. For these viruses, epitope exposure was investigated using human monoclonal (AR3A and AR4A) and polyclonal (C211 and H06) antibodies in neutralization assays. RESULTS: Processed HCV5aHi formulated with AddaVax induced antibodies that efficiently bound homologous envelope proteins and broadly neutralized cultured genotype 1-6 HCV, with half maximal inhibitory concentrations of between 14 and 192 µg/ml (mean of 36 µg/ml against the homologous virus). Vaccination with aluminium hydroxide was less immunogenic. Compared to HCV5aHi(T385) with the original E2 sequence, HCV5aHi with a modified glycosylation site and HCV5aHi(ΔHVR1) without HVR1 showed increased neutralization epitope exposure but similar immunogenicity. CONCLUSION: Using an adjuvant suitable for human use, we developed inactivated whole HCV vaccine candidates that induced broadly neutralizing antibodies, which warrant investigation in further pre-clinical studies. LAY SUMMARY: A vaccine against hepatitis C virus (HCV) is needed to prevent the estimated 2 million new infections and 400,000 deaths caused by this virus each year. We developed inactivated whole HCV vaccine candidates using adjuvants licensed for human use, which, following immunization of mice, induced antibodies that efficiently neutralized all HCV genotypes with recognized epidemiological importance. HCV variants with modified envelope proteins exhibited similar immunogenicity as the virus with the original envelope proteins.

Heterogeneous activation of hydrogen peroxide by cysteine intercalated layered double hydroxide for degradation of organic pollutants: Performance and mechanism.

A novel cysteine intercalated copper aluminum layered double hydroxide (CuAl-Cys-LDH) was synthesized and applied as heterogeneous catalyst for activating hydrogen peroxide (H2O2) to degrade rhodamine B (RhB) and 4-Nitrophenol (4-NP). The effects of initial pH, CuAl-Cys-LDH dosage, and H2O2 concentration on RhB and 4-NP removal were comprehensively investigated. The results indicated the intercalation of cysteine into the interlayer of LDH greatly enhanced its catalytic activity and stability. With 0.2 g/L CuAl-LDH and 50 mM H2O2, 93.7% of RhB and 80.2% of 4-NP could be removed in the CuAl-Cys-LDH activated H2O2 system. While the CuAl- LDH activated H2O2 system could only degrade 51.2% of RhB and 46.8% of 4-NP under the identical experimental conditions. Significantly, the CuAl-Cys-LDH catalyzed H2O2 system exhibited high degradation efficiency within a wide pH range from 4.0 to 10.0. Based on the electron paramagnetic resonance (EPR) tests and radical quenching experiments, it was inferred that •OH radical was the dominant species responsible for organic contaminants degradation. Mechanism study revealed that the intercalated cysteine in the interlayer of LDH strongly accelerated the rate-determining conversion of Cu(II) to Cu(I) by oxidation itself to cystine, thus enhanced the catalytic efficiency for H2O2 activation to produce •OH radicals. The findings of this work indicated that CuAl-Cys-LDH is a conveniently prepared and highly efficient and stable catalyst for the degradation of organic contaminants in environmental remediation.

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.

Non-linear dose-response of aluminium hydroxide adjuvant particles: Selective low dose neurotoxicity.

Aluminium (Al) oxyhydroxide (Alhydrogel®), the main adjuvant licensed for human and animal vaccines, consists of primary nanoparticles that spontaneously agglomerate. Concerns about its safety emerged following recognition of its unexpectedly long-lasting biopersistence within immune cells in some individuals, and reports of chronic fatigue syndrome, cognitive dysfunction, myalgia, dysautonomia and autoimmune/inflammatory features temporally linked to multiple Al-containing vaccine administrations. Mouse experiments have documented its capture and slow transportation by monocyte-lineage cells from the injected muscle to lymphoid organs and eventually the brain. The present study aimed at evaluating mouse brain function and Al concentration 180days after injection of various doses of Alhydrogel® (200, 400 and 800µg Al/kg of body weight) in the tibialis anterior muscle in adult female CD1 mice. Cognitive and motor performances were assessed by 8 validated tests, microglial activation by Iba-1 immunohistochemistry, and Al level by graphite furnace atomic absorption spectroscopy. An unusual neuro-toxicological pattern limited to a low dose of Alhydrogel® was observed. Neurobehavioural changes, including decreased activity levels and altered anxiety-like behaviour, were observed compared to controls in animals exposed to 200µg Al/kg but not at 400 and 800µg Al/kg. Consistently, microglial number appeared increased in the ventral forebrain of the 200µg Al/kg group. Cerebral Al levels were selectively increased in animals exposed to the lowest dose, while muscle granulomas had almost completely disappeared at 6 months in these animals. We conclude that Alhydrogel® injected at low dose in mouse muscle may selectively induce long-term Al cerebral accumulation and neurotoxic effects. To explain this unexpected result, an avenue that could be explored in the future relates to the adjuvant size since the injected suspensions corresponding to the lowest dose, but not to the highest doses, exclusively contained small agglomerates in the bacteria-size range known to favour capture and, presumably, transportation by monocyte-lineage cells. In any event, the view that Alhydrogel® neurotoxicity obeys "the dose makes the poison" rule of classical chemical toxicity appears overly simplistic.

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.

Glassy-state stabilization of a dominant negative inhibitor anthrax vaccine containing aluminum hydroxide and glycopyranoside lipid A adjuvants.

During transport and storage, vaccines may be exposed to temperatures outside of the range recommended for storage, potentially causing efficacy losses. To better understand and prevent such losses, dominant negative inhibitor (DNI), a recombinant protein antigen for a candidate vaccine against anthrax, was formulated as a liquid and as a glassy lyophilized powder with the adjuvants aluminum hydroxide and glycopyranoside lipid A (GLA). Freeze-thawing of the liquid vaccine caused the adjuvants to aggregate and decreased its immunogenicity in mice. Immunogenicity of liquid vaccines also decreased when stored at 40°C for 8 weeks, as measured by decreases in neutralizing antibody titers in vaccinated mice. Concomitant with efficacy losses at elevated temperatures, changes in DNI structure were detected by fluorescence spectroscopy and increased deamidation was observed by capillary isoelectric focusing (cIEF) after only 1 week of storage of the liquid formulation at 40°C. In contrast, upon lyophilization, no additional deamidation after 4 weeks at 40°C and no detectable changes in DNI structure or reduction in immunogenicity after 16 weeks at 40°C were observed. Vaccines containing aluminum hydroxide and GLA elicited higher immune responses than vaccines adjuvanted with only aluminum hydroxide, with more mice responding to a single dose.

Rapid deamidation of recombinant protective antigen when adsorbed on aluminum hydroxide gel correlates with reduced potency of vaccine.

Deamidation of the recombinant protective antigen (rPA) correlates with decreased effectiveness of the vaccine in protecting against infection by Bacillus anthracis. We present data demonstrating dramatic deamidation of amino acid positions 713 and 719 of rPA adsorbed onto aluminum hydroxide gel, an adjuvant, relative to rPA stored in solution without adjuvant. Although deamidation did not impact total levels of rPA-specific antibodies in a mouse model, it did correlate with a decrease in toxin-neutralizing antibodies. On the basis of these data, we hypothesize that interactions of rPA with aluminum hydroxide gel are destabilizing and are the direct cause of reduced vaccine efficacy.

Gene expression in primary cultured astrocytes affected by aluminum: alteration of chaperons involved in protein folding.

OBJECTIVES: Aluminum is notorious as a neurotoxic metal. The aim of our study was to determine whether endoplasmic reticulum (ER) stress is involved in aluminum-induced apoptosis in astrocytes. METHODS: Mitochondrial RNA (mRNA) was analyzed by reverse transcription (RT)-PCR following pulse exposure of aluminum glycinate to primary cultured astrocytes. Tunicamycin was used as a positive control. RESULTS: Gene expression analysis revealed that Ire1ß was up-regulated in astrocytes exposed to aluminum while Ire1α was up-regulated by tunicamycin. Exposure to aluminum glycinate, in contrast to tunicamycin, seemed to down-regulate mRNA expression of many genes, including the ER resident molecular chaperone BiP/Grp78 and Ca(2+)-binding chaperones (calnexin and calreticulin), as well as stanniocalcin 2 and OASIS. The down-regulation or non-activation of the molecular chaperons, whose expressions are known to be protective by increasing protein folding, may spell doom for the adaptive response. Exposure to aluminum did not have any significant effects on the expression of Bax and Bcl2 in astrocytes. CONCLUSIONS: The results of this study demonstrate that aluminum may induce apoptosis in astrocytes via ER stress by impairing the protein-folding machinery.

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.

Aluminum uptake and disease resistance in Nicotiana rustica leaves.

The comparative effectiveness of aluminum hydroxide and aluminum chloride has been studied in the development of bacterial wilt infection on leaves of Nicotiana rustica cv. Gansu yellow flower. We have analyzed the changes of foliar H(2)O(2) content, as well as of non-enzymatic and enzymatic antioxidants under aluminum stress. Pretreatment with aluminum hydroxide before pathogen challenge reduced the development of Ralstonia solanacearum infection and decreased the extent of leaf injury. The pretreatment also reduced the Al uptake in comparison to pretreatment with aluminum chloride. H(2)O(2) generation was significantly enhanced by pretreatment with aluminum hydroxide. Increased NADPH oxidase and superoxide dismutase activities were correlated with limited infection. Aluminum hydroxide pretreatment shifted the leaf redox homeostasis of AsA/DHA and GSH/GSSG toward oxidation, yielding higher oxidant levels than aluminum chloride before bacterial inoculation. The results support the idea that aluminum hydroxide induced H(2)O(2) accumulation through non-enzymatic and enzymatic regulation, ultimately resulting in resistance to tobacco wilt disease.

Long-term persistence of vaccine-derived aluminum hydroxide is associated with chronic cognitive dysfunction.

Macrophagic myofasciitis (MMF) is an emerging condition, characterized by specific muscle lesions assessing long-term persistence of aluminum hydroxide within macrophages at the site of previous immunization. Affected patients mainly complain of arthromyalgias, chronic fatigue, and cognitive difficulties. We designed a comprehensive battery of neuropsychological tests to prospectively delineate MMF-associated cognitive dysfunction (MACD). Compared to control patients with arthritis and chronic pain, MMF patients had pronounced and specific cognitive impairment. MACD mainly affected (i) both visual and verbal memory; (ii) executive functions, including attention, working memory, and planning; and (iii) left ear extinction at dichotic listening test. Cognitive deficits did not correlate with pain, fatigue, depression, or disease duration. Pathophysiological mechanisms underlying MACD remain to be determined. In conclusion, long-term persistence of vaccine-derived aluminum hydroxide within the body assessed by MMF is associated with cognitive dysfunction, not solely due to chronic pain, fatigue and depression.

[Microbial reduction and mobilization of adsorbed arsenate on ferric/aluminum hydroxides].

Although the mechanisms of arsenic release into groundwater remain poorly characterized, microbial reduction of As (V) adsorbed on the surface of iron oxides and the reductive dissolution of iron oxides are generally considered to play a key role in the mobilization of arsenic. We investigated the impact of bacterial reduction of adsorbed As (V) on a Al:Fe (1:0, 1:1, 0:1) hydroxides on arsenic mobilization using the mixed bacterial culture. After inoculation, the increase of dissolved As (III) concentration was observed, whereas As (V) was negligible in aqueous phase. Arenic release for the Al:Fe (1:0, 1:1, 0:1) hydroxides systems was 60 microg/L, 1.3 mg/L and 7.8 mg/L respectively. On the contrary, neither reduction nor release of arsenic was observed in the uninoculated groups. Furthermore, the introduction of aluminium may be responsible for the release of arsenic owing to its weaker affinity to As (III). In addition, our results showed that Fe reduction occurred far later than arsenic reduction and mobilization and obvious increase was not observed even after Fe reduction occurred. It suggested that in natural systems, the biotic reduction of As (V) adsorbed on ferric oxides or Fe (III) may not the major cause of arsenic release in sediment or groundwater system as previous works proposed. The reduction of As (V) bound to aluminum oxides or other minerals may play a key role.

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.