Potential of lipid core peptide technology as a novel self-adjuvanting vaccine delivery system for multiple different synthetic peptide immunogens

This study demonstrates the effectiveness of a novel self-adjuvanting vaccine delivery system for multiple different synthetic peptide immunogens by use of lipid core peptide (LCP) technology. An LCP formulation incorporating two different protective epitopes of the surface antiphagocytic M protein of group A streptococci (GAS)-the causative agents of rheumatic fever and subsequent rheumatic heart disease-was tested in a murine parenteral immunization and GAS challenge model. Mice were immunized with the LCP-GAS formulation, which contains an M protein amino-terminal type-specific peptide sequence (8830) in combination with a conserved non-host-cross-reactive carboxy-terminal C-region peptide sequence (J8) of the M protein. Our data demonstrated immunogenicity of the LCP-8830-J8 formulation in B10.BR mice when coadministered in complete Freund's adjuvant and in the absence of a conventional adjuvant. In both cases, immunization led to induction of high-titer GAS peptide-specific serum immunoglobulin G antibody responses and induction of highly opsonic antibodies that did not cross-react with human heart tissue proteins. Moreover, mice were completely protected from GAS infection when immunized with LCP-8830-J8 in the presence or absence of a conventional adjuvant. Mice were not protected, however, following immunization with an LCP formulation containing a control peptide from a Schistosoma sp. These data support the potential of LCP technology in the development of novel self-adjuvanting multi-antigen component vaccines and point to the potential application of this system in the development of human vaccines against infectious diseases.

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.

theta-defensins prevent HIV-1 Env-mediated fusion by binding gp41 and blocking 6-helix bundle formation

Retrocyclin-1, a 0-defensin, protects target cells from human immunodeficiency virus, type 1 (HIV-1) by preventing viral entry. To delineate its mechanism, we conducted fusion assays between susceptible target cells and effector cells that expressed HIV-1 Env. Retrocyclin-1 (4 mu M) completely blocked fusion mediated by HIV-1 Envs that used CXCR4 or CCR5 but had little effect on cell fusion mediated by HIV-2 and simian immunodeficiency virus Envs. Retrocyclin-1 inhibited HIV-1 Env-mediated fusion without impairing the lateral mobility of CD4, and it inhibited the fusion of CD4-deficient cells with cells bearing CD4-independent HIV-1 Env. Thus, it could act without cross-linking membrane proteins or inhibiting gp120-CD4 interactions. Retrocyclin-1 acted late in the HIV-1 Env fusion cascade but prior to 6-helix bundle formation. Surface plasmon resonance experiments revealed that retrocyclin bound the ectodomain of gp41 with high affinity in a glycan-independent manner and that it bound selectively to the gp41 C-terminal heptad repeat. Native-PAGE, enzyme-linked immunosorbent assay, and CD spectroscopic analyses all revealed that retrocyclin-1 prevented 6-helix bundle formation. This mode of action, although novel for an innate effector molecule, resembles the mechanism of peptidic entry inhibitors based on portions of the gp41 sequence.

Inhibition of in vitro VEGF expression and choroidal neovascularization by synthetic dendrimer peptide mediated delivery of a sense oligonucleotide

Ocular neovascularisation is the leading cause of blindness in developed countries and the most potent angiogenic factor associated with neovascularisation is vascular endothelial growth factor (VEGF). We have previously described a sense oligonucleotide (ODN-1) that possesses anti-human and rat VEGF activity. This paper describes the synthesis of lipid-lysine dendrimers and their subsequent ability to delivery ODN-1 to its target and mediate a reduction in VEGF concentration both in vitro and in vivo. Positively charged dendrimers were used to deliver ODN-1 into the nucleus of cultured D407 cells. The effects on VEGF mRNA transcription and protein expression were analysed using RT-PCR and ELISA, respectively. The most effective dendrimers in vitro were further investigated in vivo using an animal model of choroidal neovascularisation (CNV). All dendrimer/ODN-1 complexes mediated in a significant reduction in VEGF expression during an initial 24 hr period (40-60%). Several complexes maintained this level of VEGF reduction during a subsequent, second 24 hr period, which indicated protection of ODN-1 from the effects of endogenous nucleases. In addition, the transfection efficiency of dendrimers that possessed 8 positive charges (chi = 81(.)51%) was significantly better (P = 0(.)0036) than those that possessed 4 positive charges (chi = 56(.)8%). RT-PCR revealed a correlation between levels of VEGF protein mRNA. These results indicated that the most effective structural combination was three branched chains of intermediate length with 8 positive charges such as that found for dendrimer 4. Dendrimer 4 and 7/ODN-1 complexes were subsequently chosen for in vivo analysis. Fluorescein angiography demonstrated that both dendrimers significantly (P < 0(.)0001) reduced the severity of laser mediated CNV for up to two months post-injection. This study demonstrated that lipophilic, charged dendrimer mediated delivery of ODN-1 resulted in the down-regulation of in vitro VEGF expression. In addition, in vivo delivery of ODN-1 by two of the dendrimers resulted in significant inhibition of CNV in an inducible rat model. Time course studies showed that the dendrimer/ODN-1 complexes remained active for up to two months indicating the dendrimer compounds provided protection against the effects of nucleases. (C) 2004 Elsevier Ltd. All rights reserved.

Cyclization of pyrrhocoricin retains structural elements crucial for the antimircobial activity of the native peptide

Pyrrhacoricin is a naturally occurring antimicrobial peptide from the European fire bug Pyrrhocoris apterus. It has submicromolar activity against a range of Gram-negative bacterial strains and has created recent interest as a lead for the development of novel antibiotic compounds. In this study, we have used NMR spectroscopy to determine the solution structures of pyrrhocoricin and a synthetic macrocyclic derivative that has improved in vivo pharmaceutical properties. Native pyrrhocoricin is largely disordered in solution, but there is evidence of a subpopulation with ordered turn regions over residues 2-5, 4-7, and 16-19. The macrocyclic derivative incorporates a nine amino acid linker joining the N- and C-termini, which does not adversely affect the antimicrobial potency but leads to a broader spectrum of activity. The NMR data suggest that the turn conformations in the cyclic derivative are similar to those in the native form, thus implicating them in the biological function. (C) 2004 Wiley Periodicals, Inc.

Toward the development of a synthetic group A streptococcal vaccine of high purity and broad protective coverage

Using native chemical ligation, we synthesized a group A streptococcal. (GAS) vaccine that contained three different GAS M protein peptide epitopes in a chemically well-characterized construct in high purity. Two of the peptide epitopes represented variable amino terminal serotype determinants, and the third represented a carboxyl terminal conserved region determinant of the GAS M protein. We also synthesized a lipid core peptide (LCP) construct containing the same three peptides. Upon immunization of mice, the non-LCP construct only elicited antibody responses to all three epitopes with the use of adjuvant. The LCP construct, however, elicited excellent antibody responses to all three epitopes without the need for any additional adjuvant or carrier. We have synthesized the LCP synthetic vaccine system with good reproducibility.

A versatile synthetic approach to peptidyl privileged structures using a "safety-catch" linker

Peptidyl privileged structures have been widely used by many groups to discover biologically active molecules. In this context, privileged substructures are used as hydrophobic anchors, to which peptide functionality is appended to gain specificity. Utilization of this concept has led to the discovery of many different active compounds at a wide range of biological receptors. A synthetic approach to these compounds has been developed on a safety-catch linker that allows rapid preparation of large libraries of these molecules. Importantly, amide bond formation/cleavage through treatment with amines is the final step; it is a linker strategy that allows significant diversification to be easily incorporated, and it only requires the inclusion of an amide bond. In addition, chemistry has been developed that permits the urea moiety to be inserted at the N-terminus of the peptide, allowing the same set of amines (either privileged substructures or amino acid analogues) to be used at both the N- and C-termini of the molecule. To show the robustness of this approach, a small library of peptidyl privileged structures were synthesized, illustrating that large combinatorial libraries can be synthesized using these technologies.

Solution structure and characterization of the LGR8 receptor binding surface of insulin-like peptide 3

Insulin-like peptide 3 (INSL3), a member of the relaxin peptide family, is produced in testicular Leydig cells and ovarian thecal cells. Gene knock-out experiments have identified a key biological role in initiating testes descent during fetal development. Additionally, INSL3 has an important function in mediating male and female germ cell function. These actions are elicited via its recently identified receptor, LGR8, a member of the leucine-rich repeat-containing G-protein- coupled receptor family. To identify the structural features that are responsible for the interaction of INSL3 with its receptor, its solution structure was determined by NMR spectroscopy together with in vitro assays of a series of B-chain alanine-substituted analogs. Synthetic human INSL3 was found to adopt a characteristic relaxin/ insulin-like fold in solution but is a highly dynamic molecule. The four termini of this two-chain peptide are disordered, and additional conformational exchange is evident in the molecular core. Alanine-substituted analogs were used to identify the key residues of INSL3 that are responsible for the interaction with the ectodomain of LGR8. These include Arg(B16) and Val(B19), with His(B12) and Arg(B20) playing a secondary role, as evident from the synergistic effect on the activity in double and triple mutants involving these residues. Together, these amino acids combine with the previously identified critical residue, Trp(B27), to form the receptor binding surface. The current results provide clear direction for the design of novel specific agonists and antagonists of this receptor.