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In order to improve the immunogenicity of peptide-based vaccines against group A Streptococcus (GAS), lipid moieties (C16 lipoamino acid and cholic acid) were conjugated with peptide antigen (P25-J8) and further modified with α-poly(glutamic acid) (α-PGA). Thus, positively charged lipopeptide vaccine candidates LCP-1 (P25-K(J8)-SS-C16-C16) and LCP-2 (P25-K(J8)-SS-K(cholic acid)) were synthesized. Negatively charged LCP-3 (P25-K(PGA-J8)-SS-K(cholic acid)) was also produced by attaching α-PGA to the J8 N-terminus of LCP-2. Polyelectrolyte complex (PEC) nanoparticles were formulated with heparin and/or trimethyl chitosan (TMC) for delivery of the lipopeptide vaccine candidates. The ability of the antigen-loaded nanoparticles to induce humoral immune responses was examined in outbred female Swiss mice following intranasal immunization. The antibodies produced were opsonic against all clinical GAS isolates tested., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Aim: To develop novel polymer-based nanoscale delivery system for lipopeptide-based vaccine against group A Streptococcus (GAS)., Materials & Methods: Four types of lipopeptide antigen-loaded polymeric nanoparticles (NP) were prepared. NP were accessed for their capacity to be taken up by dendritic cells; effect on dendritic cell maturation; ability to induce mucosal and systemic immunity; and capability to induce antibody responses that opsonize GAS bacteria., Results & Discussion: The combination of adjuvanting properties of lipopeptides and dextran/trimethyl chitosan-based NP had a synergistic effect on humoral immunity, and the produced antibodies showed high opsonic activity against clinical GAS isolates., Conclusion: Biocompatible NP-bearing trimethyl chitosan and dextran are efficient as mucosal adjuvants for the intranasal delivery of lipopeptide-based vaccines.

Unlabelled: Group A streptococcus (GAS), an exclusively human pathogen, causes a wide range of diseases ranging from trivial to life threatening. Treatment of infection is often ineffective following entry of bacteria into the bloodstream. To date, there is no vaccine available against GAS. In this study, cationic liposomes encapsulating lipopeptide-based vaccine candidates against GAS have been employed for intranasal vaccine delivery. Cationic liposomes were prepared with dimethyldioctadecylammonium bromide (DDAB) using the film hydration method. Female Swiss mice were immunized intranasally with the liposomes. In contrast to unmodified peptides, lipopeptides entrapped by liposomes induced both mucosal and systemic immunity, IgA and IgG (IgG1 and IgG2a) production in mice, respectively. High levels of antibody (IgA and IgG) titres were detected even five months post immunization. Thus, the combination of lipopeptides and liposomes generates a very promising delivery system for intranasal vaccines., Statement of Significance: Group A streptococcus, causing rheumatic heart diseases, kills approximately half a million people annually. There is no vaccine available against the infection. Mucosal immunity is vital in ensuring an individual is protected as this gram positive bacteria initially colonizes at the throat. Herein, we demonstrated that lipopeptides entrapped by liposomes induced both mucosal and systemic immunity. High levels of antibody (IgA and IgG) titres were detected even five months post immunization and lead vaccine candidate was able to induce humoral immune responses even after single immunization. Thus, the combination of lipopeptides and liposomes generates a very promising delivery system for intranasal vaccines., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Background: Group A streptococcus (GAS) is an exclusively human pathogenic bacteria. A delay in treatment of GAS infection often lead to severe diseases such as rheumatic heart disease which attributes to hundreds of thousands deaths annually. For the past few decades, the quest for a commercial GAS vaccine has been futile. Currently one of the most investigated strategies to develop vaccine against GAS includes the use of conserved epitopes from major virulent factor of GAS, M-protein., Methods: In this study, cationic liposomes of various sizes (70 nm to 1000 nm) were prepared with dimethyldioctadecylammonium bromide (DDAB) encapsulating lipopeptide bearing M-protein derived B-cell epitope (J14)., Results: Smaller liposomes induced higher antibody titres, though the differences between groups were not statistically significant., Conclusion: Nonetheless, all mice which were immunized with liposome-lipopeptide delivery system elicited high levels of systemic (IgG) and mucosal antibodies (IgA), which were discernably higher than those induced with the help of commercial adjuvant (cholera toxin B subunit)., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Peptides are of great interest to be used as vaccine antigens due to their safety, ease of manufacturing and specificity in generating immune response. There have been massive discoveries of peptide antigens over the past decade. However, peptides alone are poorly immunogenic, which demand co-administration with strong adjuvant to enhance their immunogenicity. Recently, fibril-forming peptides such as Q11 and lipoamino acid-based carrier have been identified to induce substantial immune responses when covalently linked to peptide epitope. In this study, we have incorporated either Q11 or lipoamino acids to a peptide epitope (J14) derived from M protein of group A streptococcus to develop self-adjuvanting vaccines. J14, Q11 and lipoamino acids were also conjugated together in a single vaccine construct in an attempt to evaluate the synergy effect of combining multiple adjuvants. Physicochemical characterization demonstrated that the vaccine constructs folded differently and self-assembled into nanoparticles. Significantly, only vaccine constructs containing double copies of lipoamino acids (regardless in conjugation with Q11 or not) were capable to induce significant dendritic cells uptake and subsequent J14-specific antibody responses in non-sizes dependent manners. Q11 had minimal impact in enhancing the immunogenicity of J14 even when it was used in combination with lipoamino acids. These findings highlight the impact of lipoamino acids moiety as a promising immunostimulant carrier and its number of attachment to peptide epitope was found to have a profound effect on the vaccine immunogenicity., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Infection with group A streptococcus (GAS) can result in a number of diseases, some of which are potentially life-threatening. The oral-nasal mucosa is a primary site of GAS infection, and a mucosally active vaccine candidate could form the basis of an antidisease and transmission-blocking GAS vaccine. In the present study, a peptide from the GAS M protein (J14) representing a B cell epitope was incorporated alongside a universal T cell helper epitope and a Toll-like receptor 2 targeting lipid moiety to form lipopeptide constructs. Through structure activity studies, we identified a vaccine candidate that induces J14-specific mucosal and systemic antibody responses when administered intranasally without additional adjuvants. The systemic antibodies elicited were capable of inhibiting the growth of GAS. In addition, J14-specific mucosal antibodies corresponded with reduced throat colonization after respiratory GAS challenge. These preclinical experiments show that this lipopeptide could form the basis of an optimal needle-free mucosal GAS vaccine.

Streptococcus pyogenes (group A streptococcus, GAS) is a Gram-positive bacterial pathogen responsible for a wide variety of diseases. To date, GAS vaccine development has focused primarily on the M-protein. The M-protein is highly variable at the amino (N)-terminus (determining serotype) but is conserved at the carboxyl (C)-terminus. Previously a 29 amino acid peptide (named J14) from the conserved region of the M-protein was identified as a potential vaccine candidate. J14 was capable of eliciting protective antibodies that recognized many GAS serotypes when co-administered with immuno-stimulants. This minimal epitope however showed no immunogenicity when administered alone. In an attempt overcome this immunological non-responsiveness, we developed a self-adjuvanting vaccine candidate composed of three components: the B-cell epitope (J14), a universal helper T-cell epitope (P25) and a lipid moiety consisting of lipoamino acids (Laas) which target Toll-like receptor 2 (TLR2). Immunological evaluation in B10.BR (H-2k) mice demonstrated that the epitope attachment to the point of lipid moiety, and the length of the Laa alkyl chain have a profound effect on vaccine immunogenicity after intranasal administration. It was demonstrated that a vaccine featuring C-terminal lipid moiety containing alkyl chains of 16 carbons, with P25 located at the N-terminus, and J14 attached to the side chain of a central lysine residue was capable of inducing optimal antibody response. These findings have considerable relevance to the development of a broad spectrum J14-based GAS vaccine and in particular provided a rational basis for peptide vaccine design based on this self-adjuvanting lipopeptide technology.

Immunological assessment of group A streptococcal (GAS) branched lipopeptides demonstrated the impact of spatial arrangement of vaccine components on both the quality and quantity of their immune responses. Each lipopeptide was composed of three components: a GAS B-cell epitope (J14), a universal CD4(+) T-cell helper epitope (P25), and an immunostimulant lipid moiety that differs only in its spatial arrangement. The best systemic immune responses were demonstrated by a lipopeptide featuring the lipid moiety at the lipopeptide C-terminus. However, this candidate did not achieve protection against bacterial challenge. The best protection (100%) was shown by a lipopeptide featuring a C-terminal J14, conjugated through a lysine residue to P25 at the N-terminus, and a lipid moiety on the lysine side chain. The former candidate features α-helical conformation required to produce protective J14-specific antibodies. Our results highlight the importance of epitope orientation and lipid position in the design of three-component synthetic vaccines.

Incorporation of lipoamino acids (LAAs) into peptide structures effectively imparts self-adjuvanting activity onto otherwise ineffective immunogens. Our fully synthetic lipopeptide vaccine candidates against group A streptococcus (GAS) were composed of J14 as a target GAS B-cell epitope alongside a universal helper T-cell epitope (P25) and a LAA-based lipid moiety. In the current study, we investigated the ability of our lipopeptides to activate nuclear factor-kappaB (NF-kappaB) in a toll-like receptor-2 (TLR2)-dependent manner as the possible mode of action and reported the structure-function requirements for novel TLR2 targeting lipopeptides based on LAAs. The NF-kappaB activation was dependent on the dose and the length of the alkyl chains of the incorporated lipid moieties with the hierarchy LAA 3 (16 carbons)>LAA 2 (14 carbons)>LAA 1 (12 carbons). The position of the lipid moiety (C-terminus vs. N(epsilon)-terminus of the central lysine residue) does not significantly affect NF-kappaB activation. Lipopeptides containing different copies of LAA 3 were synthesized and the di-lipidated analogue was the most effective in NFkappaB activation., (Copyright 2010 Elsevier Ltd. All rights reserved.)

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.