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ABSTRACT: A model cytotoxic T-cell epitope is linked to a synthetic lipid tail, forming a peptide amphiphile that self-assembles into cylindrical micelles. The micelles are capable of inducing a cytotoxic T-cell response in mice that slows the growth of tumors expressing the tumor antigen.
Advanced Materials 05/2012; 24(28):3845-9. · 13.88 Impact Factor
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ABSTRACT: Traditional vaccines consisting of whole attenuated micro-organisms, or microbial components administered with adjuvant, have
been demonstrated as one of the most cost-effective and successful public health interventions. Their use in large scale immunisation
programs has lead to the eradication of smallpox, reduced morbidity and mortality from many once common diseases, and reduced
strain on health services. However, problems associated with these vaccines including risk of infection, adverse effects,
and the requirement for refrigerated transport and storage have led to the investigation of alternative vaccine technologies.
Peptide vaccines, consisting of either whole proteins or individual peptide epitopes, have attracted much interest, as they
may be synthesised to high purity and induce highly specific immune responses. However, problems including difficulties stimulating
long lasting immunity, and population MHC diversity necessitating multiepitopic vaccines and/or HLA tissue typing of patients
complicate their development. Furthermore, toxic adjuvants are necessary to render them immunogenic, and as such non-toxic
human-compatible adjuvants need to be developed. Lipidation has been demonstrated as a human compatible adjuvant for peptide
vaccines. The lipid-core-peptide (LCP) system, incorporating lipid adjuvant, carrier, and peptide epitopes, exhibits promise
as a lipid-based peptide vaccine adjuvant. The studies reviewed herein investigate the use of the LCP system for developing
vaccines to protect against group A streptococcal (GAS) infection. The studies demonstrate that LCP-based GAS vaccines are
capable of inducing high-titres of antigen specific IgG antibodies. Furthermore, mice immunised with an LCP-based GAS vaccine
were protected against challenge with 8830 strain GAS.
Letters in Peptide Science 04/2012; 10(5):605-613.
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ABSTRACT: Virus-like particles (VLPs) based on the small envelope protein of hepatitis B virus (HBsAg-S) are immunogenic at the B- and T-cell level. In this study, we inserted overlapping sequences encoding the carboxy terminus of the Helicobacter pylori katA gene product into HBsAg-S. The HBsAg-S-KatA fusion proteins were able to assemble into secretion-competent VLPs (VLP-KatA). The VLP-KatA proteins were able to induce KatA-specific antibodies in immunized mice. The mean total IgG antibody titers 41 days post-primary immunization with VLP-KatA (2.3 × 10(3)) were significantly greater (P < 0.05) than those observed for vaccination with VLP alone (5.2 × 10(2)). Measurement of IgG isotypes revealed responses to both IgG1 and IgG2a (mean titers, 9.0 × 10(4) and 2.6 × 10(4), respectively), with the IgG2a response to vaccination with VLP-KatA being significantly higher than that for mice immunized with KatA alone (P < 0.05). Following challenge of mice with H. pylori, a significantly reduced bacterial load in the gastric mucosa was observed (P < 0.05). This is the first report describing the use of VLPs as a delivery vehicle for H. pylori antigens.
Clinical and vaccine immunology: CVI 12/2011; 19(2):268-76. · 2.37 Impact Factor
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ABSTRACT: Considerable success has been made with many peptide antigen formulations and it appears that peptide-based vaccines are emerging as the next generation of prophylactic and remedial immunotherapy. However, peptides are typically poorly immunogenic and rely on delivery with potent immunostimulatory adjuvants that activate the innate and adaptive arms of the immune system. Our research aims to develop novel peptide antigen delivery systems that incorporate multiple pattern-recognition receptor (PRR) agonists and is focused on those designed to stimulate Toll-like receptors (TLRs) on dendritic cells (DCs). The cytokine (IL-4, IL-6, IL-10, IL-12 and IL-23) profiles of DCs induced by individual TLR agonists have been evaluated. From this data we predicted which TLR agonists may influence a particular T helper cell (Th) response. Using purified DCs that were derived from precursor cells in murine bone marrow and then stimulated simultaneously with multiple TLR agonists, we have shown synergy between various TLR agonist pairs leading to enhanced cytokine production. Using various mitogen-activated protein kinase (MAPK) inhibitors (c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38 MAPK) we have demonstrated the importance of p38 MAPK and ERK signaling pathways in IL-12p70 and IL-12p40 production in DCs induced by TLR stimulation, whereas the JNK pathway appeared to have a negative regulatory role on cytokine production in DCs stimulated with certain TLR agonists. An important role for nuclear factor-kappa B and phosphoinositol-3-kinase as positive regulators of TLR signaling in DCs leading to cytokine production was also demonstrated. The significance of this research lies not only in improving potency, but by understanding the immunological mechanisms of adjuvanticity, in being able to tailor peptide vaccines to generate specific types of Th responses required for immunity against various types of pathogens.
Human vaccines 01/2011; 7 Suppl:85-93. · 3.58 Impact Factor
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ABSTRACT: Triggering Toll-like receptors (TLRs) on dendritic cells (DCs) induces inflammatory cytokine production necessary for T helper type 1 immunity. The present study investigated whether simultaneous stimulation of two TLRs that signal through the same or different pathway(s) enhances cytokine production in DCs.
Fms-like tyrosine kinase-3 ligand-generated murine DCs were used in stimulation assays with TLR agonists with or without pharmacological inhibitors of cell signaling pathways. Cytokine levels were evaluated by enzyme-linked immunosorbent assay or cytometric bead array.
There was synergistic enhancement of interleukin (IL)-6 and IL-12, which were significantly inhibited by inhibitors of nuclear factor-kappaB and phosphatidylinositol 3-kinase. IL-12p40 was significantly inhibited by both p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase inhibitors, whereas IL-12p70 was inhibited by p38 MAPK inhibitor alone. IL-6 was significantly inhibited by extracellular signal-regulated kinase and, variably, by p38 MAPK and c-Jun N-terminal kinase inhibitors.
Production of cytokines in DCs after simultaneous stimulation of TLRs that signal through the same or different pathway(s) showed differential use of MAPK signaling pathways, yet both nuclear factor-kappaB and the phosphatidylinositol 3-kinase pathway as a positive regulator of TLR signaling were important. Our data suggest an important role for MyD88-dependent signaling pathways in TLR-mediated synergistic enhancement of inflammatory cytokine production in DCs.
The Journal of Infectious Diseases 07/2010; 202(2):318-29. · 6.41 Impact Factor
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ABSTRACT: Production of chemokines in dendritic cells (DCs) may be crucial in modulating immune responses generated through Toll-like receptor (TLR)-mediated recognition of microbial products. We evaluated chemokine production in DCs induced by TLR agonists and investigated the role of signaling pathways. DCs were generated from mouse bone marrow cells cultured with Fms-like tyrosine kinase-3 ligand and stimulated with a wide array of individual TLR agonists or simultaneously with pairs of combinations. Production of monocyte chemoattractant protein-1 (MCP-1/CCL2), macrophage inflammatory protein-1 (MIP-1/CCL3) and regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), were determined in cell culture supernatants by ELISA or cytokine cytometric bead array. Pharmacological inhibitors of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), nuclear factor-kappaB (NF-kB) and phosphatidylinositol 3-kinase (PI3K), were used to investigate the role of signaling pathways. TLR agonists induced significantly elevated MCP-1, RANTES, and MIP-1. Production of RANTES and MIP-1 was particularly prominent after stimulation of DCs with TLR3 (Poly(I:C)), and TLR7/8 (R848) or TLR9 (CpG ODN) agonists, respectively. However, down-modulation of chemokine production was observed in simultaneously TLR-stimulated DCs. A positive role was identified for NF-kB, PI3K and ERK, whereas JNK had a negative regulatory effect on chemokine production in DCs. Positive and negative regulatory roles for the p38 MAPK pathway were observed. Thus, chemokine levels differed and most notably there was down-modulation of chemokines in DCs stimulated with combined TLR agonists. Furthermore, analysis of signaling pathways revealed a role for MAPKs in positive and negative regulation of chemokine production in DCs. The chemokine response of DCs induced by TLR agonists appears complex and could have important implications for vaccine design.
Molecular Immunology 07/2010; 47(11-12):2065-73. · 2.90 Impact Factor
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ABSTRACT: Considerable success has been made with many peptide antigen formulations, and peptide-based vaccines are emerging as the next generation of prophylactic and remedial immunotherapy. However, finding an optimal platform balancing all of the requirements for an effective, specific and safe immune response remains a major challenge for many infectious and chronic diseases. This review outlines how peptide immunogenicity is influenced by the way in which peptides are presented to the immune system, underscoring the need for multifunctional delivery systems that couple antigen and adjuvant into a single construct. Particular attention is given to the ability of Toll-like receptor agonists to act as adjuvants. A survey of recent approaches to developing peptide antigen delivery systems is given, many of which incorporate Toll-like receptor agonists into the design.
Expert Review of Vaccines 02/2010; 9(2):157-73. · 4.25 Impact Factor
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ABSTRACT: Although most commercial vaccines are delivered by injection, there is an increasing interest in needle-free vaccine delivery for reasons including the ability to elicit immune responses at mucosal surfaces, ease of administration, and the ability to administer vaccines without the need for trained medical professionals. This review summarizes strategies and technologies that are being used to improve oral vaccine absorption. Peptides and proteins, which comprise important vaccine components, exhibit unfavorable physicochemical properties including degradation in the gastrointestinal tract, and poor transport across the intestinal wall, which hinder oral vaccine development. Approaches to overcome these obstacles aim to provide new vaccines and delivery systems that are capable of eliciting protective immune responses, and are making an impact on current vaccine development.
Current Drug Delivery 09/2009; 6(4):347-58.
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ABSTRACT: The etiology of rheumatic fever and rheumatic heart disease (RF/RHD) is believed to be autoimmune, involving immune responses initiated between streptococcal and host tissue proteins through a molecular mimicry mechanism(s). We sought to investigate the humoral and cellular responses elicited in a Lewis rat model of group A streptococcus M-protein- or peptide-induced experimental valvulitis/carditis, a recently developed animal model which may, in part, represent human rheumatic carditis. Recombinant streptococcal M5 protein elicited opsonic antibodies in Lewis rats, and anti-M5 antisera recognized epitopes within the B- and C-repeat regions of M5. One peptide from the streptococcal M5 protein B-repeat region (M5-B.6, amino acids 161 to 180) induced lymphocytes that responded to both recombinant M5 and cardiac myosin. Rats immunized with streptococcal M5 protein developed valvular lesions, distinguished by infiltration of CD3(+), CD4(+), and CD68(+) cells into valve tissue, consistent with human studies that suggest that RF/RHD are mediated by inflammatory CD4(+) T cells and CD68(+) macrophages. The current study provides additional information that supports the use of the rat autoimmune valvulitis model for investigating RF/RHD.
Infection and immunity 04/2009; 77(5):2177-83. · 4.21 Impact Factor
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ABSTRACT: Bone marrow (BM)-FMS-like tyrosine kinase 3 (Flt3) ligand-murine dendritic cells (DC) in response to group A streptococcal (GAS) lipopeptide vaccines containing an analogue of a Toll-like receptor (TLR) 2 agonist showed significant up-regulation in expression of DC maturation markers CD40 and CD80 when compared to unstimulated controls. There were significant increases in MHC class II, CD40, CD80 and CD86 expression on classical DC and plasmacytoid DC after in vivo administration or in vitro stimulation of BM-granulocyte macrophage colony stimulating factor (GMCSF)-DC with lipopolysaccharide but not lipopeptide GAS vaccines. Our results indicate that an LCP-GAS vaccine induced phenotypic maturation of BM-Flt3-DC but not BM-GMCSF-DC.
Vaccine 03/2009; 27(25-26):3313-8. · 3.77 Impact Factor
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Advances in experimental medicine and biology 02/2009; 611:347-9. · 1.09 Impact Factor
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Advances in experimental medicine and biology 02/2009; 611:345-6. · 1.09 Impact Factor
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ABSTRACT: Group A streptococcus (GAS) is associated with many human diseases, ranging in severity from benign to life-threatening. A promising strategy for developing vaccines against GAS involves the use of carbohydrates as carriers for peptide antigens. This study describes the optimized synthesis of d-glucose and d-galactose derived carriers, bearing an adipate linker and four tert-butoxycarbonyl protected aminopropyl groups. Prophylactic GAS vaccine candidates were synthesized by conjugating multiple copies of a single GAS M protein derived peptide antigen (either J8 or J14) onto the carbohydrate carriers. These antigens contain peptide sequences, which are highly conserved and offer the potential to prevent infections caused by up to 70% of GAS strains. Lipophilic amino acids were also conjugated to the d-glucose anomeric carbon to produce a self-adjuvanting liposaccharide vaccine. High serum IgG antibody titers against each of the incorporated peptide epitopes were detected following subcutaneous immunization of B10.BR (H-2 (k)) mice with the liposaccharide vaccine candidates.
Journal of Medicinal Chemistry 04/2008; 51(5):1447-52. · 5.25 Impact Factor
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ABSTRACT: Four lipid-core peptide systems were synthesized using stepwise solid-phase peptide synthesis, incorporating a sequence from the human papillomavirus type-16 (HPV-16) E7 protein (E744-62), for the purpose of developing vaccines against HPV-16 associated cervical cancer. d-Mannose was conjugated to the vaccine in order to investigate whether the targeting of dendritic cell mannose receptors would improve vaccine efficacy. The ability of the vaccines to clear or reduce the size of HPV-16 associated tumors was assessed in C57BL/6 (H-2b) mice using the TC-1 HPV-16 tumor model. Overall, significant reductions in the size of TC-1 tumors were observed in the mouse model, with the conjugation of mannose to these vaccines demonstrated to clear or reduce the size of TC-1 tumors to a greater extent than non-mannose-containing vaccines (37 out of 40 versus 21 out of 30 tumors cleared, respectively).
Journal of Medicinal Chemistry 10/2007; 50(19):4721-7. · 5.25 Impact Factor
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Colleen Olive
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ABSTRACT: Infection with the human bacterial pathogen group A Streptococcus (GAS) is estimated to cause over 500,000 deaths per year, the majority of which are related to rheumatic fever (RF) and rheumatic heart disease (RHD). While GAS is an important cause of morbidity and mortality globally, the burden of GAS-associated diseases is greater in less developed countries and in indigenous populations of developed countries. The antiphagocytic bacterial surface M protein is a major candidate antigen in the development of a vaccine to prevent GAS infection and RF/RHD. A major obstacle, however, in the development of an M-protein-based vaccine is the widespread diversity of circulating GAS strains and M protein types. Added to this is the possibility of inducing autoimmunity following vaccination as a result of molecular mimicry between the M protein and host tissue proteins. Research has been aimed at the development of a safe GAS vaccine that is able to induce broad-coverage protective immunity. The development of subunit vaccine approaches targeting the M protein using various vaccine delivery technologies is the focus of this review.
Current opinion in molecular therapeutics 03/2007; 9(1):25-34. · 3.68 Impact Factor
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ABSTRACT: Traditional vaccines consisting of whole attenuated microorganisms, killed microorganisms, or microbial components, administered with an adjuvant (e.g. alum), have been proved to be extremely successful. However, to develop new vaccines, or to improve upon current vaccines, new vaccine development techniques are required. Peptide vaccines offer the capacity to administer only the minimal microbial components necessary to elicit appropriate immune responses, minimizing the risk of vaccination associated adverse effects, and focusing the immune response toward important antigens. Peptide vaccines, however, are generally poorly immunogenic, necessitating administration with powerful, and potentially toxic adjuvants. The attachment of lipids to peptide antigens has been demonstrated as a potentially safe method for adjuvanting peptide epitopes. The lipid core peptide (LCP) system, which incorporates a lipidic adjuvant, carrier, and peptide epitopes into a single molecular entity, has been demonstrated to boost immunogenicity of attached peptide epitopes without the need for additional adjuvants. The synthesis of LCP systems normally yields a product that cannot be purified to homogeneity. The current study describes the development of methods for the synthesis of highly pure LCP analogs using native chemical ligation. Because of the highly lipophilic nature of the LCP lipid adjuvant, difficulties (e.g. poor solubility) were experienced with the ligation reactions. The addition of organic solvents to the ligation buffer solubilized lipidic species, but did not result in successful ligation reactions. In comparison, the addition of approximately 1% (w/v) sodium dodecyl sulfate (SDS) proved successful, enabling the synthesis of two highly pure, tri-epitopic Streptococcus pyogenes LCP analogs. Subcutaneous immunization of B10.BR (H-2(k)) mice with one of these vaccines, without the addition of any adjuvant, elicited high levels of systemic IgG antibodies against each of the incorporated peptides.
Journal of Peptide Science 01/2007; 12(12):800-7. · 1.80 Impact Factor
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ABSTRACT: We have developed a highly pure, self-adjuvanting, triepitopic Group A Streptococcal vaccine based on the lipid core peptide system, a vaccine delivery system incorporating lipidic adjuvant, carrier, and peptide epitopes into a single molecular entity. Vaccine synthesis was performed using native chemical ligation. Due to the attachment of a highly lipophilic adjuvant, addition of 1% (w/v) sodium dodecyl sulfate was necessary to enhance peptide solubility in order to enable ligation. The vaccine was synthesized in three steps to yield a highly pure product (97.7% purity) with an excellent overall yield. Subcutaneous immunization of B10.BR (H-2(k)) mice with the synthesized vaccine, with or without the addition of complete Freund's adjuvant, elicited high serum IgG antibody titers against each of the incorporated peptide epitopes.
Journal of Medicinal Chemistry 11/2006; 49(21):6364-70. · 5.25 Impact Factor
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ABSTRACT: The aim of this study was to investigate methods for the synthesis of highly pure, well-characterized analogues of the lipid core peptide (LCP) system. Difficulties synthesizing and purifying conventional LCP systems have led to the requirement for a technique to produce highly pure, LCP-based vaccines for potential use in human clinical trials. The current study describes methods for the attachment of lipophilic adjuvants onto multi-epitopic peptide vaccines. Described is the synthesis, using native chemical ligation, of a highly pure, tri-epitopic, group A streptococcal (GAS) lipopeptide vaccine candidate. Intranasal immunization of the described tri-epitopic GAS lipopeptide with the mucosal adjuvant cholera toxin B subunit induced high serum IgG antibody titers specific for each of the incorporated peptide epitopes.
The Journal of Organic Chemistry 10/2006; 71(18):6846-50. · 4.45 Impact Factor
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ABSTRACT: We investigated the lipid core peptide (LCP) system for mucosal vaccine delivery against infection with group A streptococcus (GAS)--the causative pathogen of rheumatic fever and rheumatic heart disease.
An LCP vaccine formulation containing 2 different peptide epitopes of the antiphagocytic M protein of GAS--a conformational epitope from the carboxyterminal conserved C-repeat region and an aminoterminal serotypic epitope--was intranasally administered to mice with cholera toxin B subunit or without additional adjuvant.
Our data demonstrate that the LCP vaccine formulation induced the elicitation of antigen-specific systemic immunoglobulin G responses when administered with or without cholera toxin B subunit, whereas cholera toxin B subunit was required for the induction of antigen-specific mucosal immunoglobulin A responses. Immune serum samples from vaccinated mice were capable of opsonization of a homologous GAS strain, as well as opsonization of a heterologous GAS strain. Furthermore, mice were protected from GAS challenge following immunization with the LCP vaccine formulation, even in the absence of additional adjuvant.
These data support the potential of the LCP system in the development of a self-adjuvanting, synthetic, peptide-based mucosal GAS vaccine for the prevention of diseases caused by GAS.
The Journal of Infectious Diseases 09/2006; 194(3):316-24. · 6.41 Impact Factor
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ABSTRACT: The development of a vaccine to prevent infection with group A streptococcus (GAS) is hampered by the widespread diversity of circulating GAS strains and M protein types, and it is widely believed that a multivalent vaccine would provide better protective immunity.
We investigated the efficacy of incorporating 3 M protein serotypic amino-terminal epitopes from GAS isolates that are common in Australian Aboriginal communities and a conformational epitope from the conserved carboxy-terminal C-repeat region into a single synthetic lipid core peptide (LCP) vaccine construct in inducing broadly protective immune responses against GAS after parenteral delivery to mice.
Immunization with the tetraepitopic LCP vaccine construct led to high titers of systemic, antigen-specific IgG responses and the induction of broadly protective immune responses, as was demonstrated by the ability of immune serum to opsonize multiple GAS strains. Systemic challenge of mice with a lethal dose of GAS given 60 or 300 days after primary immunization showed that, compared with the control mice, the vaccinated mice were significantly protected against GAS infection, demonstrating that the vaccination stimulated long-lasting protective immunity.
These data support the efficacy of the LCP vaccine delivery system in the development of a synthetic, multiepitopic vaccine for the prevention of GAS infection.
The Journal of Infectious Diseases 07/2006; 193(12):1666-76. · 6.41 Impact Factor
