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1.  Disparate adjuvant properties among three formulations of “alum” 
Vaccine  2012;31(4):653-660.
Aluminum adjuvants, commonly referred to as “alum,” are the most widespread immunostimulants in human vaccines. Although the mechanisms that promote humoral responses to alum-adsorbed antigens are still enigmatic, alum is thought to form antigen depots and induce inflammatory signals that, in turn, promote antibody production. It was recently noted that Imject® alum, a commercial aluminum-containing adjuvant commonly used in animal studies, is not the physicochemical equivalent of aluminum adjuvant present in human vaccines. This difference raises concerns about the use of Imject® alum in animal research as a model for approved aluminum adjuvants. Here, we compared the capacity of Imject® alum, Alhydrogel®, and a traditional alum-antigen precipitate to induce humoral responses in mice to the hapten-carrier antigen, NP-CGG [(4-hydroxy-3-nitrophenyl)acetyl-chicken γ-globulin]. The magnitude of humoral responses elicited by Alhydrogel® and precipitated alum was significantly greater than that induced by Imject® alum. The strength of the humoral responses elicited by different alum formulations was correlated with the quantity of pro-inflammatory cytokines induced and the numbers of inflammatory cells at the site of immunization. Moreover, Imject® exhibited a severely reduced capacity to adsorb protein antigens compared to Alhydrogel® and precipitated alum. These findings reveal substantial differences in the immunostimulatory properties of distinct alum preparations, an important point of consideration for the evaluation of novel adjuvants, the assessment of new alum-based vaccines, and in mechanistic studies of adjuvanticity.
PMCID: PMC3541451  PMID: 23200935
alum; adjuvant; antibody; inflammation
2.  Chitin Particles Are Multifaceted Immune Adjuvants 
Rationale: Chitin is a ubiquitous polysaccharide in fungi, insects, allergens, and parasites that is released at sites of infection. Its role in the generation of tissue inflammation, however, is not fully understood.
Objectives: We hypothesized that chitin is an important adjuvant for adaptive immunity.
Methods: Mice were injected with a solution of ovalbumin and chitin.
Measurements and Main Results: We used in vivo and ex vivo/in vitro approaches to characterize the ability of chitin fragments to foster adaptive immune responses against ovalbumin and compared these responses to those induced by aluminum hydroxide (alum). In vivo, ovalbumin challenge caused an eosinophil-rich pulmonary inflammatory response, Th2 cytokine elaboration, IgE induction, and mucus metaplasia in mice that had been sensitized with ovalbumin plus chitin or ovalbumin plus alum. Toll-like receptor-2, MyD88, and IL-17A played critical roles in the chitin-induced responses, and MyD88 and IL-17A played critical roles in the alum-induced responses. In vitro, CD4+ T cells from mice sensitized with ovalbumin plus chitin were incubated with ovalbumin-stimulated bone marrow–derived dendritic cells. In these experiments, CD4+ T-cell proliferation, IL-5, IL-13, IFN-γ, and IL-17A production were appreciated. Toll-like receptor-2, MyD88, and IL-17A played critical roles in these in vitro adjuvant properties of chitin. TLR-2 was required for cell proliferation, whereas IL-17 and TLR-2 were required for cytokine elaboration. IL-17A also inhibited the generation of adaptive Th1 responses.
Conclusions: These studies demonstrate that chitin is a potent multifaceted adjuvant that induces adaptive Th2, Th1, and Th17 immune responses. They also demonstrate that the adjuvant properties of chitin are mediated by a pathway(s) that involves and is regulated by TLR-2, MyD88, and IL-17A.
PMCID: PMC3029935  PMID: 20656945
chitin; adjuvant; ovalbumin; aluminum hydroxide; alum
3.  Biopersistence and Brain Translocation of Aluminum Adjuvants of Vaccines 
Aluminum oxyhydroxide (alum) is a crystalline compound widely used as an immunological adjuvant of vaccines. Concerns linked to the use of alum particles emerged following recognition of their causative role in the so-called macrophagic myofasciitis (MMF) lesion detected in patients with myalgic encephalomyelitis/chronic fatigue/syndrome. MMF revealed an unexpectedly long-lasting biopersistence of alum within immune cells in presumably susceptible individuals, stressing the previous fundamental misconception of its biodisposition. We previously showed that poorly biodegradable aluminum-coated particles injected into muscle are promptly phagocytosed in muscle and the draining lymph nodes, and can disseminate within phagocytic cells throughout the body and slowly accumulate in brain. This strongly suggests that long-term adjuvant biopersistence within phagocytic cells is a prerequisite for slow brain translocation and delayed neurotoxicity. The understanding of basic mechanisms of particle biopersistence and brain translocation represents a major health challenge, since it could help to define susceptibility factors to develop chronic neurotoxic damage. Biopersistence of alum may be linked to its lysosome-destabilizing effect, which is likely due to direct crystal-induced rupture of phagolysosomal membranes. Macrophages that continuously perceive foreign particles in their cytosol will likely reiterate, with variable interindividual efficiency, a dedicated form of autophagy (xenophagy) until they dispose of alien materials. Successful compartmentalization of particles within double membrane autophagosomes and subsequent fusion with repaired and re-acidified lysosomes will expose alum to lysosomal acidic pH, the sole factor that can solubilize alum particles. Brain translocation of alum particles is linked to a Trojan horse mechanism previously described for infectious particles (HIV, HCV), that obeys to CCL2, signaling the major inflammatory monocyte chemoattractant.
PMCID: PMC4318414
alum; vaccine adjuvants; macrophagic myofasciitis; neurotoxicity; genetics; monocytes; CCL2; MCP1
4.  Slow CCL2-dependent translocation of biopersistent particles from muscle to brain 
BMC Medicine  2013;11:99.
Long-term biodistribution of nanomaterials used in medicine is largely unknown. This is the case for alum, the most widely used vaccine adjuvant, which is a nanocrystalline compound spontaneously forming micron/submicron-sized agglomerates. Although generally well tolerated, alum is occasionally detected within monocyte-lineage cells long after immunization in presumably susceptible individuals with systemic/neurologic manifestations or autoimmune (inflammatory) syndrome induced by adjuvants (ASIA).
On the grounds of preliminary investigations in 252 patients with alum-associated ASIA showing both a selective increase of circulating CCL2, the major monocyte chemoattractant, and a variation in the CCL2 gene, we designed mouse experiments to assess biodistribution of vaccine-derived aluminum and of alum-particle fluorescent surrogates injected in muscle. Aluminum was detected in tissues by Morin stain and particle induced X-ray emission) (PIXE) Both 500 nm fluorescent latex beads and vaccine alum agglomerates-sized nanohybrids (Al-Rho) were used.
Intramuscular injection of alum-containing vaccine was associated with the appearance of aluminum deposits in distant organs, such as spleen and brain where they were still detected one year after injection. Both fluorescent materials injected into muscle translocated to draining lymph nodes (DLNs) and thereafter were detected associated with phagocytes in blood and spleen. Particles linearly accumulated in the brain up to the six-month endpoint; they were first found in perivascular CD11b+ cells and then in microglia and other neural cells. DLN ablation dramatically reduced the biodistribution. Cerebral translocation was not observed after direct intravenous injection, but significantly increased in mice with chronically altered blood-brain-barrier. Loss/gain-of-function experiments consistently implicated CCL2 in systemic diffusion of Al-Rho particles captured by monocyte-lineage cells and in their subsequent neurodelivery. Stereotactic particle injection pointed out brain retention as a factor of progressive particle accumulation.
Nanomaterials can be transported by monocyte-lineage cells to DLNs, blood and spleen, and, similarly to HIV, may use CCL2-dependent mechanisms to penetrate the brain. This occurs at a very low rate in normal conditions explaining good overall tolerance of alum despite its strong neurotoxic potential. However, continuously escalating doses of this poorly biodegradable adjuvant in the population may become insidiously unsafe, especially in the case of overimmunization or immature/altered blood brain barrier or high constitutive CCL-2 production.
PMCID: PMC3616851  PMID: 23557144
Alum; Vaccine adverse effect; Vaccine adjuvant; Nanomaterial biodistribution; Nanomaterial neurodelivery; Macrophages; Macrophagic myofasciitis; CCL-2; Single nucleotide polymorphisms (SNPs)
5.  The NLRP3 Inflammasome is Critical for Alum-Mediated IL-1β Secretion but Dispensable for Adjuvant Activity 
European journal of immunology  2008;38(8):2085-2089.
Alum (aluminiun hydroxide) is the most widely used adjuvant in human vaccines, but the immune mechanisms that are activated by alum remain poorly understood. Alum has been recently shown to promote caspase-1 activation and IL-1β secretion but the cellular pathways involved remain elusive. Here we report that the release of IL-1β triggered by alum is abrogated in macrophages deficient in Nlrp3 and Asc but not Nlrc4. The requirement of the Nlrp3 inflammasome was specific for IL-1β in that secretion of TNF-α was independent of Nlrp3 or Asc. Consistently, processing of pro-caspase-1 induced by alum was abolished in macrophages lacking Nlrp3 or Asc. Unlike caspase-1 processing and IL-1β secretion triggered by LPS, alum-mediated activation of the inflammasome did not require exogenous ATP. Importantly, induction of IgG production against human serum albumin by alum was unimpaired in mice deficient in Nlrp3. These results indicate that alum induces IL-1β via the Nlrp3 inflammasome but this activity is dispensable for alum-mediated adjuvant activity.
PMCID: PMC2759997  PMID: 18624356
Adjuvant; Alum; caspase-1; NLR
6.  106 Neisseria Meningitidis Derived Proteoliposome as an Adjuvant for Allergen Vaccines 
In recent years one important trend of Allergen-specific immunotherapy is to investigate new adjuvants with immunomodulatory properties. The outer membrane vesicle or proteoliposome (PL) from Neisseria meningitidis serogroup B has been reported as a potent adjuvant inducing a Th1-skewed response. The aim of this work was to assess the immunogenicity of a novel anti-allergic vaccine candidate based on purified allergens from Dermatophagoides siboney mite and PL as adjuvant, both components adsorbed onto Aluminum hydroxide.
In a preventative experimental setting BALB/c mice were administered with 3 doses containing 5 μg of Der s 1 allergen at one week intervals by subcutaneous route. Further, mice were subjected to allergen challenge by aerosol inhalation. In another experiment, mice were administered first with 2 doses of PL + Alum and later with the whole vaccines formulation, including the allergen. The allergen-specific antibody response was assessed determining serum levels of IgE, IgG1, and IgG2a by ELISA. The local allergic inflammatory response was evaluated by measuring cytokine levels (IL-4, IL-5, IFNg and IL-10) in broncho-alveolar lavage (BAL) by ELISA.
The formulation consistently induced IgG2a, as well as IgG1 antibodies with a potential anti-IgE blocking effect. The induction of IgG2a was clearly PL dependent while IgG1 was dependent mostly of Alum. Prior administration of the proteoliposome with alum without allergen showed to enhance this allergen-specific immunogenic effect. The vaccine prevented the development of systemic (IgE) and local allergic response in mice subjected to allergen exposure by inhalant route. Vaccinated mice showed lower levels of serum IgE, Th2 cytokines (IL-4, IL-5) in BAL and lower eosinophil counting in blood as compared to controls. Histological examination of lungs showed also a diminished allergic inflammatory response in vaccinated mice in contrast with mice which were administered with the conventional formulation of Alum-adsorbed allergen.
The antiallergic protective effect was proven in a preventative setting, showing to decrease the inflammatory response in the lungs of mice exposed to allergen aerosol, as well as, a Th2-antagonistic immune response with few injections.
PMCID: PMC3512719
7.  Tailoring Subunit Vaccine Immunity with Adjuvant Combinations and Delivery Routes Using the Middle East Respiratory Coronavirus (MERS-CoV) Receptor-Binding Domain as an Antigen 
PLoS ONE  2014;9(11):e112602.
The development of an effective vaccine is critical for prevention of a Middle East respiratory syndrome coronavirus (MERS-CoV) pandemic. Some studies have indicated the receptor-binding domain (RBD) protein of MERS-CoV spike (S) is a good candidate antigen for a MERS-CoV subunit vaccine. However, highly purified proteins are typically not inherently immunogenic. We hypothesised that humoral and cell-mediated immunity would be improved with a modification of the vaccination regimen. Therefore, the immunogenicity of a novel MERS-CoV RBD-based subunit vaccine was tested in mice using different adjuvant formulations and delivery routes. Different vaccination regimens were compared in BALB/c mice immunized 3 times intramuscularly (i.m.) with a vaccine containing 10 µg of recombinant MERS-CoV RBD in combination with either aluminium hydroxide (alum) alone, alum and polyriboinosinic acid (poly I:C) or alum and cysteine-phosphate-guanine (CpG) oligodeoxynucleotides (ODN). The immune responses of mice vaccinated with RBD, incomplete Freund’s adjuvant (IFA) and CpG ODN by a subcutaneous (s.c.) route were also investigated. We evaluated the induction of RBD-specific humoral immunity (total IgG and neutralizing antibodies) and cellular immunity (ELISpot assay for IFN-γ spot-forming cells and splenocyte cytokine production). Our findings indicated that the combination of alum and CpG ODN optimized the development of RBD-specific humoral and cellular immunity following subunit vaccination. Interestingly, robust RBD-specific antibody and T-cell responses were induced in mice immunized with the rRBD protein in combination with IFA and CpG ODN, but low level of neutralizing antibodies were elicited. Our data suggest that murine immunity following subunit vaccination can be tailored using adjuvant combinations and delivery routes. The vaccination regimen used in this study is promising and could improve the protection offered by the MERS-CoV subunit vaccine by eliciting effective humoral and cellular immune responses.
PMCID: PMC4236105  PMID: 25405618
8.  Novel human polysaccharide adjuvants with dual Th1 and Th2 potentiating activity 
Vaccine  2006;24(Suppl 2):S2-26-9.
Pure soluble, recombinant and synthetic antigens, despite their better tolerability, are unfortunately often much less immunogenic than live or killed whole organism vaccines. Thus, the move towards the development of safer subunit vaccines has created a major need for more potent adjuvants. In particular, there is an urgent need for adjuvants capable of boosting cellular (Th1) immunity but without unacceptable toxicity. The adjuvant activity of aluminium compounds (aluminium phosphate or hydroxide) was first described by Glenny and colleagues in 1926. Surprisingly, despite the description of over one hundred adjuvants in the scientific literature, alum remains the only adjuvant approved for human use in the USA. Unfortunately, alum has no effect on cellular immunity and is faced with increasing concerns regarding potential for cumulative aluminium toxicity. Why then has alum not been replaced in human vaccines? Despite the enormous number of candidates, potency has invariably been associated with increased toxicity, and this more than anything else has precluded their use, particularly in prophylactic vaccines where safety issues are paramount. Hence, there is a major unmet need for a safe efficacious adjuvant capable of boosting cellular plus humoral immunity. The extensive data on inulin-based adjuvants indicate that these are excellent candidates to replace alum as the adjuvant of choice for many vaccines. Particular advantages offered by inulin-based adjuvants is that they induce cellular in addition to humoral immunity and offer excellent safety, tolerability, ease of manufacture and formulation. Thus, adjuvants based on inulin have enormous potential for use in vaccines against both pathogens and cancer.
PMCID: PMC3101117  PMID: 16823913
Adjuvant; Vaccine; Inulin; Complement; Cellular; Immune; Th1; Th2
9.  Immunization with the Chlamydia trachomatis Mouse Pneumonitis Major Outer Membrane Protein by Use of CpG Oligodeoxynucleotides as an Adjuvant Induces a Protective Immune Response against an Intranasal Chlamydial Challenge  
Infection and Immunity  2002;70(9):4812-4817.
Recently, we have shown that a vaccine consisting of a purified preparation of the Chlamydia trachomatis mouse pneumonitis (MoPn) major outer membrane protein (MOMP) and Freund's adjuvant can protect mice against a genital challenge. Here, we wanted to determine if CpG motifs could be used as an immune modulator to the MOMP to induce protection in mice against an intranasal (i.n.) challenge. One-week-old BALB/c mice were immunized intramuscularly and subcutaneously either once or three times at 2-week intervals with MOMP and CpG suspended in aluminum hydroxide (alum). Negative controls received ovalbumin, CpG, and alum. Positive controls were immunized i.n. with C. trachomatis MoPn elementary bodies (EB). Six weeks after the last immunization, mice were challenged i.n. with 104 inclusion-forming units (IFU) of the C. trachomatis MoPn serovar. Mice that received MOMP, CpG, and alum had a strong immune response, as shown by a high titer of serum antibodies to Chlamydia and significant lymphoproliferation of T-cells following stimulation with C. trachomatis EB. After the i.n. challenge mice immunized with MOMP, CpG, and alum showed significantly less body weight loss than the corresponding control mice immunized with ovalbumin, CpG, and alum. Ten days after the challenge the animals were euthanized, their lungs were weighed, and the numbers of IFU in the lungs were determined. The average weight of the lungs of the mice immunized with MOMP, CpG, and alum was significantly less than average weight of the lungs of the mice immunized with ovalbumin, CpG, and alum. Also, the average number of IFU recovered per mouse immunized with MOMP, CpG, and alum was significantly less than the average number of IFU per mouse detected in the mice inoculated with ovalbumin, CpG, and alum. In conclusion, our data show that CpG sequences can be used as an effective adjuvant with the C. trachomatis MoPn MOMP to elicit a protective immune response in mice against a chlamydial respiratory challenge.
PMCID: PMC128273  PMID: 12183524
10.  Macrophagic myofasciitis: characterization and pathophysiology 
Lupus  2012;21(2):184-189.
Aluminium oxyhydroxide (alum), a nano-crystaline compound forming agglomerates, has been introduced in vaccine for its immunologic adjuvant effect in 1927. Alum is the most commonly used adjuvant in human and veterinary vaccines but mechanisms by which it stimulates immune responses remains incompletely understood. Although generally well tolerated, alum may occasionally cause disabling health problems in presumably susceptible individuals. A small proportion of vaccinated people present with delayed onset of diffuse myalgia, chronic fatigue and cognitive dysfunction, and exhibit very long-term persistence of alum-loaded macrophages at site of previous intra-muscular (i.m.) immunization, forming a granulomatous lesion called macrophagic myofasciitis (MMF). Clinical symptoms associated with MMF are paradigmatic of the recently delineated “autoimmune/inflammatory syndrome induced by adjuvants” (ASIA). The stereotyped cognitive dysfunction is reminiscent of cognitive deficits described in foundry workers exposed to inhaled Al particles. Alum safety concerns will largely depend on whether the compound remains localized at site of injection or may diffuse and accumulate in distant organs. Animal experiments indicate that biopersistent nanomaterials taken-up by monocytes-lineage cells in tissues, e.g. fluorescent alum surrogates, can first translocate to draining lymph nodes, and thereafter circulate in blood within phagocytes and reach the spleen, and, eventually, slowly accumulate in brain.
PMCID: PMC3623725  PMID: 22235051
Adjuvants, Immunologic; adverse effects; Alum Compounds; adverse effects; Animals; Fasciitis; chemically induced; immunology; pathology; physiopathology; Humans; Myositis; chemically induced; immunology; pathology; physiopathology; Nanostructures; Phagocytes; metabolism; Syndrome
11.  Periodontal Bacterial DNA Suppresses the Immune Response to Mutans Streptococcal Glucosyltransferase▿  
Infection and Immunity  2007;75(8):4088-4096.
Certain CpG motifs found in bacterial DNA enhance immune responses through Toll-like receptor 9 (TLR-9) and may also demonstrate adjuvant properties. Our objective was to determine if DNA from bacteria associated with periodontal disease could affect the immune response to other bacterial antigens in the oral cavity. Streptococcus sobrinus glucosyltransferase (GTF), an enzyme involved in dental caries pathogenesis, was used as a test antigen. Rowett rats were injected with aluminum hydroxide (alum) with buffer, alum-GTF, or alum-GTF together with either Escherichia coli DNA, Fusobacterium nucleatum DNA, or Porphyromonas gingivalis DNA. Contrary to expectation, animals receiving alum-GTF plus bacterial DNA (P. gingivalis in particular) demonstrated significantly reduced serum immunoglobulin G (IgG) antibody, salivary IgA antibody, and T-cell proliferation to GTF compared to animals immunized with alum-GTF alone. A diminished antibody response was also observed after administration of alum-GTF with the P. gingivalis DNA either together or separately, indicating that physical complexing of antigen and DNA was not responsible for the reduction in antibody. Since TLR triggering by DNA induces synthesis of prospective suppressive factors (e.g., suppressor of cytokine signaling [SOCS]), the effects of P. gingivalis DNA and GTF exposure on rat splenocyte production of SOCS family molecules and inflammatory cytokines were investigated in vitro. P. gingivalis DNA significantly up-regulated SOCS1 and SOCS5 expression and down-regulated interleukin-10 expression by cultured splenocytes. These results suggested that DNA from periodontal disease-associated bacteria did not enhance, but in fact suppressed, the immune response to a protein antigen from cariogenic streptococci, potentially through suppressive SOCS components triggered by innate mechanisms.
PMCID: PMC1952018  PMID: 17517867
12.  Molecular and Cellular Response Profiles Induced by the TLR4 Agonist-Based Adjuvant Glucopyranosyl Lipid A 
PLoS ONE  2012;7(12):e51618.
Toll-like receptor (TLR)4 agonists are known potent immunostimulatory compounds. These compounds can be formulated as part of novel adjuvants to enhance vaccine medicated immune responses. However, the contribution of the formulation to the innate in vivo activity of TLR4 agonist compounds is not well understood.
Methodology and Principal Findings
We evaluated synthetic TLR4 agonist Glucopyranosyl Lipid A (GLA) for its effects on molecular and cellular innate immune responses in the murine model. Microarray techniques were used to compare the responses to GLA in an aqueous formulation or in an oil-in-water Stable Emulsion formulation (GLA-SE) versus either SE alone or the mineral salt aluminum hydroxide (alum) at the muscle injection site over multiple timepoints. In contrast to the minimal gene upregulation induced by SE and alum, both GLA and GLA-SE triggered MyD88- and TRIF-dependent gene expression. Genes for chemokines, cytokine receptors, signaling molecules, complement, and antigen presentation were also strongly upregulated by GLA and GLA-SE. These included chemokines for TH1-type T cells (CXCL9 and CXCL10) and mononuclear leukocytes (CCL2, CCL3) among others. GLA-SE induced stronger and more sustained gene upregulation than GLA in the muscle; GLA-SE induced genes were also detected in local draining lymph nodes and at lower levels in peripheral blood. Both GLA and GLA-SE resulted in increased cellular trafficking to the draining lymph nodes and upregulated MHC molecules and ICAM1 on local dendritic cells. GLA and GLA-SE transiently upregulated circulating MCP-1, TNFα, IFNγ and IP-10 in blood.
While GLA and GLA-SE activate a large number of shared innate genes and proteins, GLA-SE induces a quantitatively and qualitatively stronger response than GLA, SE or alum. The genes and proteins upregulated could be used to facilitate selection of appropriate adjuvant doses in vaccine formulations.
PMCID: PMC3532059  PMID: 23284726
13.  Antigen-Based Therapy with Glutamic Acid Decarboxylase (GAD) Vaccine in Patients with Recent-Onset Type 1 Diabetes: A Randomised Double-Masked Controlled Trial 
Lancet  2011;378(9788):319-327.
Type 1 diabetes (T1DM) is an autoimmune disease leading to destruction of insulin producing beta cells and life-long requirement for insulin therapy. Glutamic acid decarboxylase (GAD) is a major target of this immune response. Studies in animal models of autoimmunity have shown that treatment with a target antigen can modulate aggressive autoimmunity. We evaluated immunization with GAD formulated in aluminum hydroxide (alum) as an adjuvant in recent onset T1DM.
In this multicentre, double-masked, randomised controlled trial, 145 subjects (ages 3-45) with T1DM for less than 3 months received 3 injections of 20 μg GAD-alum (48 subjects), 2 injections of GAD-alum and one of alum alone (49 subjects) or 3 injections of alum (48 subjects) subcutaneously at baseline, 4 weeks later and 8 weeks after the second injection. Primary outcome was baseline-adjusted geometric mean 2-hour area under the curve (AUC) serum C-peptide following a mixed meal tolerance test at one year. Secondary outcomes included changes in HbA1c and insulin dose, and safety. This trial is registered in (NCT00529399).
The ratio (experimental to control) of the adjusted population mean of C-peptide for the GAD-alum ×3 and GAD-alum ×2/alum ×1 groups is 0.998 (95% CI: [0.779, 1.22], p = 0.98) and 0.926 (95% CI: [0.720, 1.13], p = 0.50), respectively. HbA1c and insulin use did not differ between groups. There was no difference in rate or severity of adverse events between groups.
Antigen-based immunotherapy therapy using GAD-alum given subcutaneously in two or three doses over 4 to 12 weeks does not alter the course of loss of insulin secretion over one year in subjects with recently diagnosed T1DM. While antigen-based therapy is a highly desireable treatment and is effective in animal models, translation to human autoimmune disease remains a challenge.
National Institutes of Health.
PMCID: PMC3580128  PMID: 21714999
glutamic acid decarboxylase; type 1 diabetes; antigen specific therapy; immune modulation; children
14.  ATP release and purinergic signaling: a common pathway for particle-mediated inflammasome activation 
Cell Death & Disease  2012;3(10):e403-.
Deposition of uric acid crystals in joints causes the acute and chronic inflammatory disease known as gout and prolonged airway exposure to silica crystals leads to the development of silicosis, an irreversible fibrotic pulmonary disease. Aluminum salt (Alum) crystals are frequently used as vaccine adjuvant. The mechanisms by which crystals activate innate immunity through the Nlrp3 inflammasome are not well understood. Here, we show that uric acid, silica and Alum crystals trigger the extracellular delivery of endogenous ATP, which just precedes the secretion of mature interleukin-1β (IL-1β) by macrophages, both events depending on purinergic receptors and connexin/pannexin channels. Interestingly, not only ATP but also ADP and UTP are involved in IL-1β production upon these Nlrp3 inflammasome activators through multiple purinergic receptor signaling. These findings support a pivotal role for nucleotides as danger signals and provide a new molecular mechanism to explain how chemically and structurally diverse stimuli can activate the Nlrp3 inflammasome.
PMCID: PMC3481132  PMID: 23059822
ATP; danger signal; inflammasome; P2R; NLR
15.  Robust Stimulation of Humoral and Cellular Immune Responses following Vaccination with Antigen-Loaded β-Glucan Particles 
mBio  2010;1(3):e00164-10.
β-Glucan particles (GPs) are purified Saccharomyces cerevisiae cell walls treated so that they are primarily β1,3-d-glucans and free of mannans and proteins. GPs are phagocytosed by dendritic cells (DCs) via the Dectin-1 receptor, and this interaction stimulates proinflammatory cytokine secretion by DCs. As the hollow, porous GP structure allows for high antigen loading, we hypothesized that antigen-loaded GPs could be exploited as a receptor-targeted vaccine delivery system. Ovalbumin (OVA) was electrostatically complexed inside the hollow GP shells (GP-OVA). Incubation of C57BL/6J mouse bone marrow-derived DCs with GP-OVA resulted in phagocytosis, upregulation of maturation markers, and rapid proteolysis of OVA. Compared with free OVA, GP-OVA was >100-fold more potent at stimulating the proliferation of OVA-reactive transgenic CD8+ OT-I and CD4+ OT-II T cells, as measured by in vitro [3H]thymidine incorporation using DCs as antigen-presenting cells. Next, immune responses in C57BL/6J mice following subcutaneous immunizations with GP-OVA were compared with those in C57BL/6J mice following subcutaneous immunizations with OVA absorbed onto the adjuvant alum (Alum/OVA). Vaccination with GP-OVA stimulated substantially higher antigen-specific CD4+ T-cell lymphoproliferative and enzyme-linked immunospot (ELISPOT) responses than that with Alum/OVA. Moreover, the T-cell responses induced by GP-OVA were Th1 biased (determined by gamma interferon [IFN-γ] ELISPOT assay) and Th17 biased (determined by interleukin-17a [IL-17a] ELISPOT assay). Finally, both the GP-OVA and Alum/OVA formulations induced strong secretions of IgG1 subclass anti-OVA antibodies, although only GP-OVA induced secretion of Th1-associated IgG2c antibodies. Thus, the GP-based vaccine platform combines adjuvanticity and antigen delivery to induce strong humoral and Th1- and Th17-biased CD4+ T-cell responses.
Most licensed vaccines work by promoting protective antibody responses. However, for many infectious diseases, antibody-mediated protection appears to play a relatively minor role, and vaccination has met with limited success. While live-attenuated organisms generally elicit T-cell responses, their use in vaccines is limited by the potential for causing disease. Thus, there is an urgent need for new vaccine platforms that deliver antigens in such a manner as to promote strong T-cell-mediated responses. Here we designed a novel vaccine platform consisting of yeast-derived β-glucan particles (GPs) that combines antigen delivery and adjuvant activity. GPs loaded with the model antigen ovalbumin (OVA) stimulated robust humoral and T-cell responses in mice. In addition, the cellular response was Th1 and Th17 biased. This work has implications for the design of vaccines that stimulate biased T-cell responses as well as for understanding how immunity to fungal pathogens develops.
PMCID: PMC2925077  PMID: 20802824
16.  Improved Immunogenicity and Efficacy of the Recombinant 19-Kilodalton Merozoite Surface Protein 1 by the Addition of Oligodeoxynucleotide and Aluminum Hydroxide Gel in a Murine Malaria Vaccine Model  
Infection and Immunity  2002;70(2):692-701.
Vaccination of mice with yeast-secreted Plasmodium yoelii-derived 19-kilodalton merozoite surface protein 1 (yMSP119) has been shown to afford protection from challenge with a lethal strain of P. yoelii. Sterile immunity can be achieved when MSP119 is emulsified in Freund adjuvant but not when it is adsorbed to aluminum hydroxide gel (alum). Because complete Freund adjuvant is not an acceptable adjuvant for use in humans, alternative adjuvants must be identified for formulating MSP119 as a vaccine for use in humans. To determine whether oligodeoxynucleotides with CpG motifs (ODN), reported to be a powerful new class of adjuvants, could enhance the immunogenicity of yMSP119, C57BL/6 mice were vaccinated either with yMSP119 formulated with Freund adjuvant, with alum, or with ODN plus alum and challenged intravenously with P. yoelii 17XL asexual blood-stage parasites. Adsorption of immunogen and adjuvant to alum was optimized by adjusting buffer (phosphate versus acetate) and pH. We found that the adjuvant combination of ODN plus alum with yMSP119, injected intraperitoneally (i.p.), increased immunoglobulin G (IgG) yMSP119-specific antibody production 12-fold over Freund adjuvant given i.p., 3-fold over Freund adjuvant given subcutaneously (s.c.), 300-fold over alum given i.p., and 48-fold over alum given s.c. The predominant antibody isotype in the group receiving alum-ODN-yMSP119 was IgG1. Increased antibody levels correlated to protection from a challenge with P. yoelii 17XL. Supernatant cytokine levels of gamma interferon in yMSP119-stimulated splenocytes were dramatically elevated in the alum-ODN-yMSP119 group. Interleukin-10 (IL-10) levels were also elevated; however, no IL-5 was detected. The cytokine profile, as well as the predominant IgG1 antibody isotype, suggests the protective immune response was a mixed Th1/Th2 response.
PMCID: PMC127724  PMID: 11796601
17.  Evaluation of recombinant Onchocerca volvulus activation associated protein-1 (ASP-1) as a potent Th1-biased adjuvant with a panel of protein or peptide-based antigens and commercial inactivated vaccines 
Vaccine  2008;26(39):5022-5029.
Alum, the only adjuvant approved for clinical applications, can induce strong humoral (Th2) but weak cellular (Th1) immune responses. It is necessary to develop safe and effective adjuvants capable of inducing both humoral and cellular immune responses. We previously showed that activation-associated protein-1 (ASP-1) derived from Onchocerca volvulus has potent adjuvant activity. In this study, we have further evaluated the adjuvanticity of recombinant ASP-1 using a panel of recombinant proteins or synthetic peptide-based antigens, including ovalbumin (OVA), synthetic HIV peptide (HIV-p), recombinant HIV gp41 (rgp41) and HBV HBsAg, as well as three commercially available inactivated vaccines against haemorrhagic fever with renal syndrome (HFRS), Influenza and Rabies. Our results indicate that ASP-1 induced significantly higher IgG1 (Th2-associated) and IgG2a (Th1-associated) responses than alum adjuvant against OVA antigen, HIV-p, and rgp41. Consistently, it induced similar level of IgG1 responses as alum but higher level of IgG2a and IFN-γ-producing T cell responses than alum adjuvant against HBsAg. Further, ASP-1 improved both IgG1 and IgG2a responses to three commercial inactivated vaccines when used separately or in combination. In conclusion, the recombinant ASP-1, unlike alum adjuvant, is able to induce both Th1 and Th2-associated humoral responses and Th1 cellular responses, suggesting that it can be further developed as a promising adjuvant for subunit-based and inactivated vaccines.
PMCID: PMC2597511  PMID: 18675867
Adjuvant; Onchocerca volvulus; Activation-associated protein-1; Vaccine
18.  Engineering an Effective Immune Adjuvant by Designed Control of Shape and Crystallinity of Aluminum Oxyhydroxide Nanoparticles 
ACS nano  2013;7(12):10834-10849.
Adjuvants based on aluminum salts (Alum) are commonly used in vaccines to boost the immune response against infectious agents. However, the detailed mechanism of how Alum enhances adaptive immunity and exerts its adjuvant immune effect remains unclear. Other than being comprised of micron-sized aggregates that include nanoscale particulates, Alum lacks specific physicochemical properties to explain activation of the innate immune system, including the mechanism by which aluminum-based adjuvants engage the NLRP3 inflammasome and IL-1β production. This is putatively one of the major mechanisms required for an adjuvant effect. Because we know that long aspect ratio nanomaterials trigger the NLRP3 inflammasome, we synthesized a library of aluminum oxyhydroxide (AlOOH) nanorods to determine whether control of the material shape and crystalline properties could be used to quantitatively assess NLRP3 inflammasome activation and linkage of the cellular response to the material’s adjuvant activities in vivo. Using comparison to commercial Alum, we demonstrate that the crystallinity and surface hydroxyl group display of AlOOH nanoparticles quantitatively impact the activation of the NLRP3 inflammasome in human THP-1 myeloid cells or murine bone marrow-derived dendritic cells (BMDCs). Moreover, these in vitro effects were correlated with the immunopotentiation capabilities of the AlOOH nanorods in a murine OVA immunization model. These results demonstrate that shape, crystallinity and hydroxyl content play an important role in NLRP3 inflammasome activation and are therefore useful for quantitative boosting of antigen-specific immune responses. These results show that the engineered design of aluminum-based adjuvants in combination with dendritic cell property-activity analysis can be used to design more potent aluminum-based adjuvants.
PMCID: PMC3899397  PMID: 24261790
NLRP3 Inflammasome; IL-1β; Aluminum Oxyhydroxide; Oxidative Stress; Vaccine Adjuvant; Humoral and Cellular Immune Responses; Alum
19.  Poly(I:C)/Alum Mixed Adjuvant Priming Enhances HBV Subunit Vaccine-Induced Immunity in Mice When Combined with Recombinant Adenoviral-Based HBV Vaccine Boosting 
PLoS ONE  2013;8(1):e54126.
Virus-specific cellular immune responses play a critical role in virus clearance during acute or chronic HBV infection. Currently, the commercially available HBV vaccine is combined with alum adjuvant, which stimulates mainly Th2 immune responses. Therefore, development of new therapeutic HBV vaccine adjuvants and immune strategies that also promote Th1 and CTL responses is urgently needed.
Methodology/Principal findings
To improve the immunity induced by the novel HBSS1 HBV vaccine, we evaluated the ability of adjuvants, including alum, CpG and polyriboinosinic polyribocytidylic acid [poly(I:C)], to enhance the response when boosted with the recombinant adenoviral vector vaccine rAdSS1. The immune responses to different adjuvant combinations were assessed in C57BL/6 mice by enzyme-linked immunosorbent assay (ELISA), ELISpot and cytokine release assays. Among the combinations tested, a HBV protein particle vaccine with CpG/alum and poly(I:C)/alum priming combinations accelerated specific seroconversion and produced high antibody (anti-PreS1, anti-S antibody) titres with a Th1 bias. After boosting with recombinant adenoviral vector vaccine rAdSS1, both groups produced a strong multi-antigen (S and PreS1)-specific cellular immune response. HBSS1 immunisation with poly(I:C)/alum priming also generated high-level CD4+ and CD8+ T cell responses in terms of Th1 cytokines (IFN-γand IL-2).
The protein-vaccine HBSS1 with mixed poly(I:C)/alum adjuvant priming, followed by a rAdSS1 vaccine boost, maximises specific antibody and Th1-biased cellular immune responses. This regime might prove useful in the development of HBV therapeutic vaccines. Furthermore, this promising strategy might be applied to vaccines against other persistent infections, such as human immunodeficiency virus and tuberculosis.
PMCID: PMC3545998  PMID: 23335993
20.  Vaccination with Novel Immunostimulatory Adjuvants against Blood-Stage Malaria in Mice  
Infection and Immunity  2003;71(9):5178-5187.
An important aspect of malaria vaccine development is the identification of an appropriate adjuvant which is both capable of stimulating a protective immune response and safe for use by humans. Here, we investigated the feasibility of using novel immunostimulatory molecules as adjuvants combined with a crude antigen preparation and coadsorbed to aluminum hydroxide (alum) as a vaccine against blood-stage Plasmodium chabaudi AS malaria. Prior to challenge infection, immunization of genetically susceptible A/J mice with the combination of malaria antigen plus recombinant interleukin-12 (IL-12) in alum induced a Th1 immune response with production of high levels of gamma interferon (IFN-γ) and diminished IL-4 levels by spleen cells stimulated in vitro with parasite antigen compared to mice immunized with antigen alone, antigen in alum, or antigen plus IL-12. Mice immunized with malaria antigen plus recombinant IL-12 in alum had high levels of total malaria-specific antibody and immunoglobulin G2a. Compared to unimmunized mice, immunization with antigen plus IL-12 in alum induced the highest level of protective immunity against challenge infection with P. chabaudi AS, which was evident as a significantly decreased peak parasitemia level and 100% survival. Protective immunity was dependent on CD4+ T cells, IFN-γ, and B cells and was long-lasting. Replacement of IL-12 as an adjuvant by synthetic oligodeoxynucleotides (ODN) containing CpG motifs induced a similar level of vaccine-induced protection against challenge infection with P. chabaudi AS. These results illustrate that it is possible to enhance the potency of a crude malaria antigen preparation delivered in alum by inclusion of immunostimulatory molecules, such as IL-12 or CpG-ODN.
PMCID: PMC187300  PMID: 12933862
21.  Prototype Alzheimer’s disease epitope vaccine induced strong Th2-type anti-Aβ antibody response with Alum to Quil A adjuvant switch 
Vaccine  2005;24(13):2275-2282.
β-Amyloid (Aβ) peptide has been proposed to be a causal factor in Alzheimer’s disease (AD). Currently being investigated, active and passive Aβ-immunotherapy significantly reduce Aβ plaque deposition, neuritic dystrophy, and astrogliosis in the brains of APP transgenic (APP/Tg) mice. Immunization with Aβ42 formulated in the Th1-type adjuvant QS21 was beneficial for AD patients with significant titers of anti-Aβ antibodies, however, 6% of participants developed meningoencephalitis, likely due to anti-Aβ-specific autoimmune Th1 cells. Thus, successful Aβ vaccination requires the development of strong antibody responses without Th1-type cellular immunity. In this study, we compared the induction of humoral immune responses with Th1-type (Quil A) and Th2-type (Alum) adjuvants singly and in combination, using our novel epitope vaccine composed of self B cell epitope Aβ1–15 and foreign T cell epitope PADRE (PADRE-Aβ1–15-MAP). Formulated in Quil A, this vaccine resulted in significantly higher anti-Aβ antibody responses in both BALB/c (H-2d) and C57BL/6 (H-2b) mice, compared with Alum. Anti-Aβ antibodies induced by Alum were predominantly IgG1 type accompanied by lower levels of IgG2a and IgG2b. Quil A induced robust and almost equal titers of anti-Aβ antibodies of IgG1 and IgG2a isotypes and slightly lower levels of IgG2b. Switching adjuvants from Alum to Quil A induced higher concentrations of antibodies than injections with Alum only, however slightly lower than Quil A only. Switching both adjuvants did not change the profile of antibody responses generated by the initial adjuvant injected. These results suggest that switching from Alum to Quil A would be beneficial for AD patients because anti-Aβ antibody production was enhanced without changing the initially generated and likely beneficial Th2-type humoral response.
PMCID: PMC2081151  PMID: 16368167
Alzheimer’s disease (AD); Epitope vaccine immunization; Th1 and Th2 adjuvants
22.  Silica Nanoparticles as the Adjuvant for the Immunisation of Mice Using Hepatitis B Core Virus-Like Particles 
PLoS ONE  2014;9(12):e114006.
Advances in nanotechnology and nanomaterials have facilitated the development of silicon dioxide, or Silica, particles as a promising immunological adjuvant for the generation of novel prophylactic and therapeutic vaccines. In the present study, we have compared the adjuvanting potential of commercially available Silica nanoparticles (initial particles size of 10–20 nm) with that of aluminium hydroxide, or Alum, as well as that of complete and incomplete Freund's adjuvants for the immunisation of BALB/c mice with virus-like particles (VLPs) formed by recombinant full-length Hepatitis B virus core (HBc) protein. The induction of B-cell and T-cell responses was studied after immunisation. Silica nanoparticles were able to adsorb maximally 40% of the added HBc, whereas the adsorption capacity of Alum exceeded 90% at the same VLPs/adjuvant ratio. Both Silica and Alum formed large complexes with HBc VLPs that sedimented rapidly after formulation, as detected by dynamic light scattering, spectrophotometry, and electron microscopy. Both Silica and Alum augmented the humoral response against HBc VLPs to the high anti-HBc level in the case of intraperitoneal immunisation, whereas in subcutaneous immunisation, the Silica-adjuvanted anti-HBc level even exceeded the level adjuvanted by Alum. The adjuvanting of HBc VLPs by Silica resulted in the same typical IgG2a/IgG1 ratios as in the case of the adjuvanting by Alum. The combination of Silica with monophosphoryl lipid A (MPL) led to the same enhancement of the HBc-specific T-cell induction as in the case of the Alum and MPL combination. These findings demonstrate that Silica is not a weaker putative adjuvant than Alum for induction of B-cell and T-cell responses against recombinant HBc VLPs. This finding may have an essential impact on the development of the set of Silica-adjuvanted vaccines based on a long list of HBc-derived virus-like particles as the biological component.
PMCID: PMC4250084  PMID: 25436773
23.  A Novel Vaccine Adjuvant for Recombinant Flu Antigens 
Many new vaccines under development consist of rationally designed recombinant proteins that are relatively poor immunogens unless combined with potent adjuvants. There is only one adjuvant in common use in the U.S., aluminum phosphate or hydroxide (e.g. alum). This adjuvant, however, has significant limitations, particularly regarding the generation of strong cell-mediated (T cell) immune responses. A novel adjuvant, JVRS-100, composed of cationic liposome-DNA complexes (CLDC) has been evaluated for immune enhancing activity. The JVRS-100 adjuvant has been shown to elicit robust immune responses compared to CpG oligonucleotides, alum, and MPL adjuvants, and efficiently enhances both humoral and cellular immune responses. Safety has been evaluated in preclinical studies, and the adjuvant is now in early-stage clinical development. One application of this novel adjuvant is to augment the immune responses to recombinant subunit antigens, which are often poorly immunogenic. The JVRS-100 adjuvant, when combined with a recombinant influenza hemagglutinin (H1), elicited increased specific antibody and T-cell responses in mice. Single-dose vaccination and prime/boost vaccinations with JVRS-100-H1 were both shown to be protective (i.e., survival, reduced weight loss) following H1N1 (PR/8/34) virus challenge. Enhanced immunological responses could be critically important for improved efficacy and dose-sparing of a recombinant influenza vaccine.
PMCID: PMC2693274  PMID: 19285425
Adjuvant; vaccine; influenza
24.  Alum increases antigen uptake, reduces antigen degradation and sustains antigen presentation by DCs in vitro 
Immunology Letters  2012;147(1-2):55-62.
► The EαGFP/YAe system can be used to study the impact of alum on antigen uptake and presentation by DCs. ► Alum acts as an antigen delivery system. ► Alum slows down protein degradation in DCs. ► Alum eventually enhances magnitude and duration of expression of peptide/MHC complexes on the DC surface.
Aluminium adjuvants (alum) have been the only widely approved adjuvants for use in human vaccines since the 1920s, however, the mechanism of action of these adjuvants remains elusive. Due to increasing demand for novel adjuvants, a clearer understanding of the mechanisms that allow these important agents to affect adaptive immune responses will make a significant contribution to the rational design of future vaccines.
Using a novel approach to tracking antigen and antigen presentation, we demonstrate that alum induces higher antigen accumulation and increased antigen presentation by dendritic cells (DCs) in vitro. Antigen accumulation was 100-fold higher and antigen presentation 10-fold higher following alum treatment when compared with soluble protein alone. We also observed that alum causes an initial reduction in presentation compared with soluble antigen, but eventually increases the magnitude and duration of antigen presentation. This was associated with reduced protein degradation in DCs following alum treatment. These studies demonstrate the dynamic alterations in antigen processing and presentation induced by alum that underlie enhanced DC function in response to this adjuvant.
PMCID: PMC3477319  PMID: 22732235
BMDC, bone marrow-derived dendritic cell; EαGFP, Ealpha green fluorescence protein; Alum; Antigen uptake; Processing; Presentation; YAe system
25.  Enhancement of humoral immunity in mice by coupling pUCpGs10 and aluminium to the HCV recombinant immunogen 
Virology Journal  2011;8:507.
To investigate the enhancement of humoral immunity when CpG ODN (cytidine phosphate guanosine oligodeoxynucleotides) and aluminium adjuvants are complexed with the HCV (Hepatitis C virus) recombinant immunogen in mice.
After immunizing Balb/c mice with the recombination HCV antigen adjuvanted with pUCpGs10 and/or aluminium(antigen+CpG+alum, antigen+CpG, antigen+alum, antigen+PBS), enzyme-linked immunosorbent assay (ELISA) was used to measure the specific serum antibody titers of IgG, to determine the neutralization response to various peptide genotypes, and to determine the concentration of IL-6 and IL-10 in supernatants of in vitro cultured splenic lymphocytes. Enzyme-linked immunospot assay (ELISPOT) was used to quantify the non-specific and specific splenic antibody-secreting cells (ASCs), and flow cytometry (FCM) determined the ratio of different splenic lymphocytes. The serum of rabbits immunized with the recombinant pBVGST/HVR1 antigen immunoprecipitated the HCV isolated from 12 patients' serum.
The sera antibody titers were 1:51200, 1:9051, 1:18102, 1:6400 respectively after the final immunization and demonstrated good neutralization responses to the six gene peptide containing 1a, 1b, 2a, 3a, 4a and 6a. The aluminum adjuvant increased the population of both specific ASCs (P < 0.01) and total ASCs(P < 0.05), with a proportional rise in concentrations of CD19+CD27+ (P < 0.05), as well as levels of IL-6, IL-10 (P < 0.05) in splenic lymphocytes. The results clearly indicated a significantly higher number of CD19+CD38+ splenic lymphocytes with the aluminum and pUCpGs10 adjuvant present compared to the control group(P < 0.05). Anti-HVR1 antibody in induced mice can cross-reactively capture HCV particles (10/12).
1. The aluminum adjuvant induces a potent Th2-biased immune response by increasing both the populations of specific and total ASCs and the ratio of CD19+CD27+ cells. 2. The pUCpGs10 complexed with the aluminum adjuvant boosts the population of plasma cells and increase the efficiency of the immune response. 3. The two adjuvants have synergistic effects on humoral immunity. 4. The recombinant HVR1 protein has the possibility of generating broadly reactive anti-HVR1 antibody.
PMCID: PMC3261835  PMID: 22054420
HCV; humoral immunity; adjuvant; ELISPOT; FCM

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