Progress 10/01/07 to 09/30/12
Outputs
OUTPUTS: The research conducted over the past five years has contributed to an improved understanding of the mechanisms by which aluminum adjuvants enhance the immune response, and has enhanced our knowledge of formulation conditions that optimize the immune response to aluminum-adjuvanted vaccines. The research findings have been disseminated through publications in peer-reviewed journals and presentations at international meetings and at private and public research institutions. PARTICIPANTS: The project involved collaborators in the College of Pharmacy at Purdue University. The project provided training for graduate students and post-docs. TARGET AUDIENCES: The knowledge generated in this project is important for scientists in the pharmaceutical and animal health industry and in academia. The project provides information on the formulation of vaccines with aluminum adjuvants and this knowledge is critical to developing an effective vaccine. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Aluminum adjuvants are added to veterinary and human vaccines to enhance the antibody response to vaccine antigens. We have demonstrated that the adsorption of vaccine antigens onto aluminum adjuvants can be modified by pretreatment of adjuvants with phosphate buffer and by controlled phosphorylation of antigen using newly designed linker molecules. The adsorption can be measured in terms of adsorptive capacity and adsorptive strength (affinity). Thus, we have provided methods to control and assess the formulation of aluminum adjuvanted vaccines. Adsorption of vaccine antigens slows the diffusion of antigens from the injection site and is required to enhance the immune response at low antigen concentrations. On the other hand, a high adsorptive strength can interfere with the antibody response. The two main aluminum adjuvants used, aluminum hydroxide adjuvant (AH) and aluminum phosphate adjuvant (AP) differ in their adsorptive characteristics and can also induce qualitatively different immune responses. The latter was demonstrated with a killed whole cell streptococcal vaccine in which formulation with AH induced an IL-17 response, whereas formulation with AP failed to do so. The mechanisms involved in the adsorption of antigens to aluminum adjuvants also regulate the interactions between other immunomodulatory compounds and aluminum adjuvants. For example, lipopolysaccharide with two terminal phosphate groups is tightly adsorbed to AH resulting in neutralization of its activity. In contrast, monophosphoryl lipid A (MPLA) with one phosphate group has a lower affinity for AH and can enhance the adjuvant activity of AH. This information is important in the development of combination adjuvants.
Publications
- Hansen B, Belfast M, Soung G, Song L, Egan PM, Capen R, HogenEsch H, Mancinelli R, Hem SL (2009) Effect of the strength of adsorption of hepatitis B surface antigen to aluminum hydroxide adjuvant on the immune response. Vaccine, 27:888-892.
- HogenEsch H, Noe S, Green M, Hem S (2010) Lack of correlation between retention of antigen at the injection site and the immune response to aluminium adjuvant-containing vaccines. 4th Global Vaccine Congress. Vienna, Austria. (abstract).
- Noe S, Green MA, HogenEsch H, Hem SL (2010) Mechanism of immunopotentiation by aluminum-containing adjuvants elucidated by the relationship between antigen retention at the inoculation site and the immune response. Vaccine, 28:3588-3594.
- Hem SL, HogenEsch H, Middaugh CR, Volkin DB (2010) Preformulation studies--The next advance in aluminum adjuvant-containing vaccines. Vaccine, 28:4868-4870.
- Hansen B, Malyala P, Singh M, Sun Y, Srivastava I, HogenEsch H, Hem SL (2011) Effect of the strength of adsorption of hiv 1 sf162dv2gp140 to aluminum-containing adjuvants on the immune response. J Pharm Sci, 100:3245-3252.
- HogenEsch H, Dunham A, Hansen B, Anderson K, Maisonneuve JF, Hem SL (2011) Formulation of a killed whole cell pneumococcus vaccine. Effect of aluminum adjuvants on the antibody and IL-17 response. J Immune Based Ther Vaccines, 9:5.
- Hem SL, HogenEsch H (2007) Aluminum-containing vaccine adjuvants (Invited review). Expert Rev Vaccines, 6:685-698
- Hem SL and HogenEsch H (2007). Aluminum-containing adjuvants: Properties, formulation and use. In: Vaccine Adjuvants and Delivery Systems, M. Singh, Ed., J. Wiley & Sons, Publ., pp. 81-114.
- Sokolovska A, HogenEsch H (2008) Effect of combination adjuvants composed of aluminium salts and TLR4 agonists on the immune response. 2nd Vaccine Conference, Boston, MA, 2008. Vaccine, Suppl. (abstract).
- Anderson KA, HogenEsch H (2008) Effect of the dose of aluminum adjuvants on the immune response. 2nd Vaccine Conference, Boston, MA, 2008. Vaccine, Suppl. (abstract).
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: Aluminum-containing adjuvants are widely used in veterinary vaccines, but the mechanism by which they enhance the immune response is poorly understood. Experiments were conducted to determine the optimal formulation of killed whole cell bacteria with aluminum adjuvants. Two commercial aluminum adjuvants, aluminum hydroxide (AH) and aluminum phosphate (AP) adjuvant were used. Both adjuvants enhanced the antibody response to the bacterial antigens in C57BL/6 mice. However, at a low antigen dose, a stronger antibody response was observed with AP, whereas AH induced a stronger antibody response at a high antigen dose. Preliminary evaluation of antibody specificity by Western blots suggested differences in the antibody repertoire induced by AH and AP adjuvants. As a measure of cell-mediated immune responses, the secretion of IL-17 was assessed ex vivo by peripheral blood cells from immunized mice. Only vaccines formulated with AH induced an IL-17 response, whereas no response was observed with vaccines without antigen or formulated with AP. PARTICIPANTS: Collaborators: Stanley Hem - Department of Industrial and Physical Pharmacy, Purdue University, West Lafayette, IN Manmohan Singh - Novartis Vaccines and Diagnostics (Cambridge, MA) Jean-Francois Maisonneuve - PATH (Seattle, WA) TARGET AUDIENCES: Investigators working on vaccines in academia, government and the private sector. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
This work enhances our understanding of the mechanisms by which aluminum-containing adjuvants work and helps in correctly formulating aluminum-adjuvanted vaccines. Formulation is a key issue in vaccine development and requires detailed knowledge of the antigens and adjuvants. Aluminum-containing adjuvants probably work by inducing a local immune response with recruitment of antigen-presenting cells. However, the exact mechanism by which these adjuvants induce inflammation that leads to an enhanced immune response remains unknown. Our work indicates that investigators should pay close attention to the choice of aluminum adjuvants used in vaccines as this may affect the quality and quantity of the immune response.
Publications
- Hansen B, Malyala P, Singh M, Sun Y, Srivastava I, HogenEsch H, Hem SL (2011) Effect of the strength of adsorption of hiv 1 sf162dv2gp140 to aluminum-containing adjuvants on the immune response. J Pharm Sci, 100:3245-3252.
- HogenEsch H, Dunham A, Hansen B, Anderson K, Maisonneuve JF, Hem SL (2011) Formulation of a killed whole cell pneumococcus vaccine. Effect of aluminum adjuvants on the antibody and IL-17 response. J Immune Based Ther Vaccines, 9:5.
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: Aluminum-containing adjuvants have traditionally been thought to work by forming depots of adsorbed antigen from which the antigen is slowly released. The relation between depot formation and the immune response was studied by formulating vaccines with labeled antigen mixed with adjuvant either adsorbed or not adsorbed. These experiments demonstrated that there was no difference in the rate of diffusion of antigen from the injection site between soluble antigen without aluminum-containing adjuvant and non-adsorbed antigen mixed with aluminum adjuvant. As expected, the rate of diffusion from the injection site was significantly slower for adsorbed antigens. However, the antibody response to the vaccine formulated with non-adsorbed antigen was much higher than the immune response to soluble antigen. This work demonstrates that the aluminum adjuvant needs to be present with antigen to enhance the immune response, but adsorption of the antigen is not necessary. Experiments were conducted to determine the optimal formulation of killed whole cell bacteria with aluminum adjuvants. Adsorption of the bacteria to aluminum adjuvants and the strength of binding were determined. The adsorption was affected by phosphate treatment of the aluminum adjuvants. The effect of the formulation on the immune response to the bacteria is being studied. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: The target audience is scientists in academic institutions and industry who are developing vaccines. Formulation is a key, but often underappreciated, aspect of vaccine development. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This work enhances our understanding of the mechanisms by which aluminum-containing adjuvants work and helps in correctly formulating aluminum-adjuvanted vaccines. Formulation is a key issue in vaccine development and requires detailed knowledge of the antigens and adjuvants. Aluminum-containing adjuvants probably work by inducing a local immune response with recruitment of antigen-presenting cells. However, the exact mechanism by which these adjuvants induce inflammation that leads to a biased Th2 immune response remains unknown. This knowledge is necessary to design new and safe adjuvants that enhance a broader immune response.
Publications
- Noe S, Green MA, HogenEsch H, Hem SL (2010) Mechanism of immunopotentiation by aluminum-containing adjuvants elucidated by the relationship between antigen retention at the inoculation site and the immune response. Vaccine, 28:3588-3594.
- Hem SL, HogenEsch H, Middaugh CR, Volkin DB (2010) Preformulation studies - The next advance in aluminum adjuvant-containing vaccines. Vaccine, 28:4868-4870.
- HogenEsch H (2010) Aluminum-containing adjuvants: Formulation and mechanism of immunopotentiation. Conference on Infectious Diseases. Novel strategies for design and development of drugs and vaccines. Mumbai, India, S7.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Aluminum-containing adjuvants have long been thought to work by forming depots of adsorbed antigen from which the antigen is slowly released. To address the relation between depot formation and the immune response, model vaccines were formulated in which the antigen was mixed with adjuvant either adsorbed or not adsorbed. The vaccine antigen was labeled with a tag to allow visualization of the antigen following injection in rats. These experiments demonstrated that there was no difference in the rate of diffusion of antigen from the injection site between soluble antigen without aluminum-containing adjuvant and non-adsorbed antigen mixed with aluminum adjuvant. As expected, the rate of diffusion from the injection site was significantly slower for adsorbed antigens. However, the antibody response to the vaccine formulated with non-adsorbed antigen was much higher than the immune response to soluble antigen. This work demonstrates that the aluminum adjuvant needs to be present with antigen to enhance the immune response, but adsorption of the antigen is not necessary. Indeed, as previously reported, strong adsorption may be detrimental to the immune response induced by injection of aluminum-adsorbed antigens. PARTICIPANTS: The research provides an opportunity to train graduate students and post-docs. TARGET AUDIENCES: The target audience is primarly comprised of scientists working with aluminum-containing adjuvants in vaccine formulation in academia, animal health industry and the pharmaceutical inductry. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This work enhances our understanding of the mechanisms by which aluminum-containing adjuvants work and will help in correctly formulating aluminum-adjuvanted vaccines. Aluminum-containing adjuvants probably work by inducing a local immune response with recruitment of antigen-presenting cells. However, the exact mechanism by which these adjuvants induce inflammation that leads to a biased Th2 immune response remains unknown. This knowledge is necessary to design new and safe adjuvants that enhance a broader immune response.
Publications
- Anderson KA, HogenEsch H (2008) Effect of the dose of aluminum adjuvants on the immune response. 2nd Vaccine Conference, Boston, MA, December 2008. Vaccine, Suppl. S.
- Hansen B, Belfast M, Soung G, Song L, Egan PM, Capen R, HogenEsch H, Mancinelli R, Hem SL (2009) Effect of the strength of adsorption of hepatitis B surface antigen to aluminum hydroxide adjuvant on the immune response. Vaccine, 27:888-892.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: The combination of immunomodulatory molecules may allow the synthesis of novel vaccine adjuvants in which the desirable properties of the compounds are combined. The interaction between the Toll-like receptor 4 agonists lipopolysaccharide (LPS) and its derivative, monophosphoryl lipid A (MPLA), and aluminum adjuvants was determined. The secretion of IL-12 induced by LPS and MPLA was inhibited by mixing with aluminum hydroxide adjuvant, whereas aluminum phosphate adjuvant had a minimal effect. The combination of LPS and MPLA with aluminum phosphate adjuvant enhanced the secretion of IL-1. The combination adjuvant also induced a stronger IgG response to vaccine antigens. In another series of experiments, the effect of increasing the dose of aluminum adjuvants was examined. Higher doses induced a greater antibody response and the induced antibodies had a higher affinity than vaccines with lower doses of aluminum adjuvants. PARTICIPANTS: Collaborator: Stan Hem - Professor of Industrial and Physical Pharmacy, Purdue University. The project provided training for a post-doc (Anna Sokolovska) and an undergraduate student (Fanjia Lu). TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The research has implications for our understanding of the mechanism by which aluminum-containing adjuvants enhance the immune response and for vaccine formulations. It emphasizes the complexities of aluminum adjuvants and the need to carefully choose the right aluminum adjuvant, aluminum hydroxide vs. aluminum phosphate, to induce the desired immune response. In addition, the dose of adjuvant should be maximized to induce an optimal antibody response.
Publications
- Hem SL, HogenEsch H (2007) Aluminum-containing vaccine adjuvants (Invited review). Expert Rev Vaccines, 6:685-698
- Sokolovska A, HogenEsch H (2008) Evaluation of the immunopotentiation by TLR4 agonists combined with aluminum-containing adjuvants. 11th Annual Conference on Vaccine Research, Baltimore, p 54 (Abstract)
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: The findings are reported through publications and presentations at scientific meetings. The research has demonstrated that aluminum-containing adjuvants directly activate dendritic cells and elucidated a mechanism for the selective stimulation of humoral immune responses by these adjuvants.
TARGET AUDIENCES: The target audience includes anyone involved in the development, formulation and production of veterinary and human vaccines.
Impacts
The research findings are leading to a better understanding of the mechanism by which aluminum adjuvants work and to improved vaccine formulations.
Publications
- Romero-Mendez IZ, Shi Y, HogenEsch H, Hem SL (2007) Potentiation of the immune response to non-adsorbed antigens by aluminum-containing adjuvants. Vaccine, 25:825-833.
- Sokolovska A, Hem SL, HogenEsch H (2007) Activation of dendritic cells and induction of CD4+ T cell differentiation by aluminum-containing adjuvants. Vaccine, 25:4575-4585.
- Hansen B, Sokolovska A, HogenEsch H, Hem SL (2007) Relationship between the strength of antigen adsorption to an aluminum-containing adjuvant and the immune response. Vaccine, 25:6618-24.
- Sokolovska A, HogenEsch H (2007) Activation of dendritic cells and induction of CD4+ T cell differentiation by aluminum-containing adjuvants. 10th Annual Conference on Vaccine Research. Baltimore, P25.
- Hem SL and HogenEsch H (2007). Aluminum-containing adjuvants: Properties, formulation and use. In: Vaccine Adjuvants and Delivery Systems, M. Singh, Ed., J. Wiley & Sons, Publ., pp. 81-114
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Progress 10/01/05 to 09/30/06
Outputs
Aluminum-containing adjuvants adsorb antigens via 3 mechanisms: Electrostatic adsorption, ligand exchange and hydrophobic interaction. We have investigated the effect of the adsorption mechanism on the elution of the antigen in simulated interstitial fluid. These studies led to the conclusion that antigens that adsorb to aluminum-containing adjuvants by electrostatic attraction are more likely to elute upon intramuscular or subcutaneous administration than antigens that adsorb by ligand exchange. In related studies, the role of adsorption on the immune response to antigens was determined. Using three different model systems, the data indicate that aluminum-containing adjuvants enhance the immune response to antigens even when the antigens are not adsorbed to the adjuvant particles.
Impacts
The research enhances our understanding of the mechanism(s) by which aluminum-containing adjuvants enhance the immune response and may lead to the development of more effective vaccines. An understanding of the role of adsorption is important for vaccine formulations.
Publications
- Jiang D, Morefield GL, HogenEsch H, Hem SL (2006) Relationship of adsorption mechanism of antigens by aluminum-containing adjuvants to in vitro elution in interstitial fluid. Vaccine, 24:1665-1669.
- Sokolovska A, HogenEsch H (2006) Effect of aluminum-containing adjuvants on TH1/TH2 differentiation. J Immunol 176, S86.
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Progress 10/01/04 to 09/30/05
Outputs
Previous studies demonstrated that aluminum-containing adjuvants directly activate dendritic cells. The activated dendritic cells express and secrete IL-1. We have investigated the molecular pathways that lead to IL-1 secretion and found that the aluminum-containing adjuvant induced IL-1 secretion is inhibited by inhibitors of caspase 1. Further studies have shown that aluminum-containing adjuvants induce the secretion of IL-18. The dendritic cells activated by aluminum adjuvants induced secretion of IL-4, IL-5 and IFN-gamma in an antigen-dependent manner by naive T cells. The secretion of IL-4 and IL-5 was in part dependent on IL-1 and IL-18. The effect of adsorption of antigens on aluminum-containing adjuvants was determined. Fluorochrome-labeled antigen in solution or adsorbed to aluminum phosphate or aluminum hydroxide adjuvant was incubated with dendritic cells, and antigen uptake was assessed by flow cytometry and confocal microscopy. Aluminum adjuvants enhanced
the uptake of antigen. Smaller aluminum-containing particles were more efficient than larger particles.
Impacts
Our studies have revealed potential mechanisms by which aluminum adjuvants enhance the immune response, in particular the humoral immune response. These studies may lead to the development of modified vaccine adjuvants that combine the safety of aluminum-containing adjuvants with increased efficacy.
Publications
- Morefield G, Jiang D, Romero-Mendez IZ, Geahlen RL, HogenEsch H, Hem SL (2005) Effect of phosphorylation of ovalbumin on adsorption by aluminum-containing adjuvants and elution upon exposure to interstitial fluid. Vaccine, 23:1502-1506.
- Morefield GL, Sokolovska A, Jiang D, HogenEsch H, Robinson JP, Hem SL (2005) Role of aluminum-containing adjuvants in antigen internalization by dendritic cells in vitro. Vaccine, 23: 1588-95.
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Progress 10/01/03 to 09/29/04
Outputs
1. Previous studies demonstrated that aluminum-containing adjuvants induce the expression and secretion of IL-1 by dendritic cells. This allows us to determine the pathways involved in activation of dendritic cells and led to the identification of NF-kB as one of the signaling pathways involved. Blockage of IL-1 with neutralizing antibodies did not inhibit the ability of dendritic cells to activate antigen-specific T cells suggesting that IL-1 has no direct role in the immune stimulating effect of aluminum adjuvants 2. Studies of the mechanisms of antigen adsorption on aluminum adjuvants have identified ligand exchange and electrostatic mechanisms. Ligand exhange requires an accessible phosphate group on the antigen and results in high affinity adsorption. Although adsorption helps to keep the antigen localized to the site of injection, high affinity adsorption results in a reduced immune response, probably because of failure to process and present the antigen
appropriately.
Impacts
Our studies are the first to demonstrate that aluminum adjuvants have a direct effect on dendritic cells. This opens new avenues to delineate the molecular mechanisms by which aluminum adjuvants work and may lead to the development of new adjuvants that are safe and effective. Modulation of the surface of aluminum adjuvants with phosphate ions alters the adsorption and elution characteristics of antigens and may influence the magnitude of the immune response.
Publications
- Iyer S, HogenEsch H, Hem SL (2003) Effect of the degree of phosphate substitution in aluminum hydroxide adjuvant on the adsorption of phosphorylated proteins. Pharm Dev Tech, 8:81-86.
- Iyer S, Robinett RSR, HogenEsch H, Hem SL (2004) Mechanism of adsorption of hepatitis B surface antigen by aluminum hydroxide adjuvant. Vaccine, 22:1475-1479.
- Morefield GL, HogenEsch H, Robinson JP, Hem SL (2004) Distribution of adsorbed antigen in mono-valent and combination vaccines. Vaccine, 22:1973-1984.
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Progress 10/01/02 to 09/30/03
Outputs
Dendritic cells play a critical role in the initiation of the immune response. They take up and process antigen, present antigenic peptides to T cells, activate T cells and influence their differentiation. This research focuses on the interaction between dendritic cells and aluminum adjuvants. Our hypothesis is that aluminum compounds enhance the immune response in part by selective activation of dendritic cells. To determine if aluminum adjuvants enhance the ability of dendritic cells to activate T cells, dendritic cells were cultured with soluble antigen (ovalbumin) or ovalbumin adsorbed to aluminum phosphate or aluminum hydroxide adjuvants. Dendritic cells activated T cells much more efficiently when incubated with the adsorbed ovalbumin. This could be due to increased uptake of ovalbumin, activation of dendritic cells or a combination. Indeed, we have shown that aluminum adjuvants enhance the uptake of ovalbumin. Activation of dendritic cells may lead to increased
expression of cell surface molecules and secretion of cytokines. Aluminum adjuvants with or without ovalbumin only modestly increased the expression of MHC II, CD40, CD80, and CD86 in comparison with lipopolysaccharide (LPS), a known activator of dendritic cells. Aluminum adjuvants did not induce the secretion of IL-4, IL-6, IL-10 or IL-12, but they induced marked secretion of IL-1 comparable to the secretion induced by LPS. The secretion of IL-1 was inhibited by caffeic acid phenethyl ester (CAPE), an inhibitor of the NF- B signaling pathway. Experiments were conducted to determine changes in global gene expression in dendritic cells incubated with aluminum hydroxide or aluminum phosphate in the absence or presence of ovalbumin. RNA collected after 4 hours of incubation was assayed using microarrays (Clontech). Preliminary analysis revealed changes in large sets of genes. Some of the changes were conserved between aluminum phosphate and aluminum hydroxide, whereas other changes were
unique to each adjuvant.
Impacts
Our studies are the first to demonstrate that aluminum adjuvants have a direct effect on dendritic cells. This opens new avenues to delineate the molecular mechanisms by which aluminum adjuvants work and may lead to the development of new adjuvants that are safe and effective.
Publications
- Iyer S, HogenEsch H, Hem SL (2003) Relationship between the degree of antigen adsorption to aluminum hydroxide adjuvant in interstitial fluid and antibody production. Vaccine, 21:1219-1223.
- Sokolovska A, HogenEsch H (2003) Impact of aluminum adjuvants on IL-1 beta production by dendritic cells. FASEB J 17:C200.
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