Development of lipid-core-peptide (LCP) based vaccines for the prevention of the group A streptococcal (GAS) infection

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.

Synthesis of a highly pure lipid core peptide based self-adjuvanting triepitopic group A Streptococcal vaccine, and subsequent immunological evaluation

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.

Engineering stable peptide toxins by means of backbone cyclization: Stabilization of the alpha-conotoxin MII

Conotoxins (CTXs), with their exquisite specificity and potency, have recently created much excitement as drug leads. However, like most peptides, their beneficial activities may potentially be undermined by susceptibility to proteolysis in vivo. By cyclizing the alpha-CTX MII by using a range of linkers, we have engineered peptides that preserve their full activity but have greatly improved resistance to proteolytic degradation. The cyclic MII analogue containing a seven-residue linker joining the N and C termini was as active and selective as the native peptide for native and recombinant neuronal nicotinic acetylcholine receptor subtypes present in bovine chromaffin cells and expressed in Xerl oocytes, respectively. Furthermore, its resistance to proteolysis against a specific protease and in human plasma was significantly improved. More generally, to our knowledge, this report is the first on the cyclization of disulfide-rich toxins. Cyclization strategies represent an approach for stabilizing bioactive peptides while keeping their full potencies and should boost applications of peptide-based drugs in human medicine.

Physical enhancement of transdermal drug application: is delivery technology keeping up with pharmaceutical development?

Advances in molecular biology have given us a wide range of protein and peptide-based drugs that are unsuitable for oral delivery because of their high degree of first-pass metabolism. Though parenteral delivery is the obvious answer, for the successful development of commercial chronic and self-administration usage formulations it is not the ideal choice. Transdermal delivery is emerging as the biggest application target for these agents, however, the skin is extremely efficient at keeping out such large molecular weight compounds and therapeutic levels are never going to be realistically achieved by passive absorption. Physical enhancement mechanisms including: iontophoresis, electroporation, ultrasound, photomechanical waves, microneedles and jet-propelled particles are emerging as solutions to this topical delivery dilemma. Adding proteins and peptides to the list of other large molecular weight drugs with insufficient passive transdermal fluxes to be therapeutically useful, we have a collection of pharmacological agents waiting for efficient delivery methods to be introduced. This article reviews the current state of physical transdermal delivery technology, assesses the pros and cons of each technique and summarises the evidence-base of their drug delivery capabilities.

Group A streptococcal vaccine delivery by immunization with a self-adjuvanting M protein-based lipid core peptide construct

Background & objectives: To develop a broad strain coverage GAS vaccine, several strategies have been investigated which included multi-epitope approaches as well as targeting the M protein conserved C-region. These approaches, however, have relied on the use of adjuvants that are toxic for human application. The development of safe and effective adjuvants for human use is a key issue in the development of effective vaccines. In this study, we investigated the lipid polylysine core peptide (LCP) system as a self-adjuvanting GAS vaccine delivery approach. Methods: An LCP-GAS construct was synthesised incorporating multiple copies of a protective peptide epitope (J8) from the conserved carboxy terminal C-repeat region of the M protein. B10.BR mice were immunized parenterally with the LCP-J8 construct, with or without conventional adjuvant, prior to the assessment of immunogenicity and the induction of serum opsonic antibodies. Results: Our data demonstrated immunogenicity of LCP-J8 when coadministered in complete Freund's adjuvant (CFA), or administered in the absence of conventional adjuvant. In both cases, immunization led to the induction of high-titre J8 peptide-specific serum IgG antibody responses, and the induction of heterologous opsonic antibodies that did not cross-react with human heart tissue proteins. Interpretation & conclusion: These data indicated the potential of a novel self-adjuvanting LCP vaccine delivery system incorporating a synthetic GAS M protein C-region peptide immunogen in the induction of broadly protective immune responses, and pointed to the potential application of this system in human vaccine development against infectious diseases.

Synthesis and immunological evaluation of M protein targeted tetra-valent and tri-valent group A streptococcal vaccine candidates based on the lipid-core peptide system

Group A streptococcus (GAS) is responsible for causing many clinical complications including the relatively benign streptococcal pharyngitis and impetigo. However. if left untreated. these conditions may lead to more severe diseases such as rheumatic fever (RF) and rheumatic heart disease (RHD). These diseases exhibit high morbidity and mortality, Particularly in developing countries and in indigenous populations of affluent countries. Only ever occur following GAS infection, a vaccine offers Promise for their Prevention. As stich, we have investigated the Use of the lipid-core peptide (LCP) system for the development of multi-valent Prophylactic GAS vaccines. The current study has investigated the capacity of this system to adjuvant LIP to four different GAS peptide epitopes. Presented are the synthesis and immunological assessment of tetra-valent and tri-valent GAS LCP systems. We demonstrated their capacity to elicit systemic IgG antibody responses in B10.BR mice to all GAS peptide epitopes. The data also showed that the LCP systems Were self-adjuvanting. These findings are particularly encouraging for the development of multi-valent LCP-based GAS vaccines.

Lipid and carbohydrate based adjuvant/carriers in immunology

This review discusses various issues regarding vaccines:what are they and how they work, safety aspects, the role of adjuvants and carriers in vaccination, synthetic peptides as immunogens, and new technologies for vaccine development and delivery including the identification of novel adjuvants for mucosal vaccine delivery. There has been a recent increase of interest, in the use of lipids and carbohydrates as adjuvants, and so a particular emphasis is placed on adjuvants derived from lipids or carbohydrates, or from both. Copyright (C) 2003 European Peptide Society and John Wiley Sons, Ltd.

Method for the synthesis of highly pure vaccines using the lipid core peptide system

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. Copyright (c) 2006 European Peptide Society and John Wiley & Sons, Ltd.

Population-based detection of systolic and diastolic dysfunction with amino-terminal pro-B-type natriuretic peptide

Background There is limited information regarding the clinical utility of amino-terminal pro-B-type natriuretic pepticle (NT-proBNP) for the detection of left ventricular (LV) dysfunction in the community. We evaluated predictors of circulating NT-proBNP levels and determined the utility of NT-proBNP to detect systolic and diastolic LV dysfunction in older adults. Methods. A population-based sample of 1229 older adults (mean age 69.4 years, 50.1% women) underwent echocardiographic assessment of cardiac structure and function and measurement of circulating NT-proBNP levels. Results Predictors of NT-proBNP included age, female sex, body mass index, and cardiorenal parameters (diastolic dysfunction [DID] severity; LV mass and left atrial volume; right ventricular overload; decreasing ejection fraction [EF] and creatinine clearance). The performance of NT-proBNP to detect any degree of LV dysfunction, including mild DID, was poor (area under the curve 0.56-0.66). In contrast, the performance of NT-proBNP for the detection of EF 0.90 regardless of age and sex; history of hypertension, diabetes, coronary artery disease; or body mass category. The ability of NT-proBNP to detect EF