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Hepatitis C VLPs delivered to dendritic cells by a TLR2 targeting lipopeptide results in enhanced antibody and cell-mediated responses.

Chua BY, Johnson D, Tan A, Earnest-Silveira L, Sekiya T, Chin R, Torresi J, Jackson DC - PLoS ONE (2012)

Bottom Line: Although many studies provide strong evidence supporting the development of HCV virus-like particle (VLP)-based vaccines, the fact that heterologous viral vectors and/or multiple dosing regimes are required to induce protective immunity indicates that it is necessary to improve their immunogenicity.While co-formulation of this lipopeptide with VLPs only resulted in marginal improvements in dendritic cell (DC) uptake, its ability to concomitantly induce DC maturation at very small doses is a feature not observed using VLPs alone or in the presence of an aluminium hydroxide-based adjuvant (Alum).These results suggest overall that the immunogenicity of HCV VLPs can be significantly improved by the addition of this novel adjuvant by targeting their delivery to DCs and could therefore constitute a viable vaccine strategy for the treatment of HCV.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, The University of Melbourne, Parkville, Victoria, Australia.

ABSTRACT
Although many studies provide strong evidence supporting the development of HCV virus-like particle (VLP)-based vaccines, the fact that heterologous viral vectors and/or multiple dosing regimes are required to induce protective immunity indicates that it is necessary to improve their immunogenicity. In this study, we have evaluated the use of an anionic self-adjuvanting lipopeptide containing the TLR2 agonist Pam(2)Cys (E(8)Pam(2)Cys) to enhance the immunogenicity of VLPs containing the HCV structural proteins (core, E1 and E2) of genotype 1a. While co-formulation of this lipopeptide with VLPs only resulted in marginal improvements in dendritic cell (DC) uptake, its ability to concomitantly induce DC maturation at very small doses is a feature not observed using VLPs alone or in the presence of an aluminium hydroxide-based adjuvant (Alum). Dramatically improved VLP and E2-specific antibody responses were observed in VLP+E(8)Pam(2)Cys vaccinated mice where up to 3 doses of non-adjuvanted or traditionally alum-adjuvanted VLPs was required to match the antibody titres obtained with a single dose of VLPs formulated with this lipopeptide. This result also correlated with significantly higher numbers of specific antibody secreting cells that was detected in the spleens of VLP+E(8)Pam(2)Cys vaccinated mice and greater ability of sera from these mice to neutralise the binding and uptake of VLPs by Huh7 cells. Moreover, vaccination of HLA-A2 transgenic mice with this formulation also induced better VLP-specific IFN-γ-mediated responses compared to non-adjuvanted VLPs but comparable levels to that achieved when coadministered with complete freund's adjuvant. These results suggest overall that the immunogenicity of HCV VLPs can be significantly improved by the addition of this novel adjuvant by targeting their delivery to DCs and could therefore constitute a viable vaccine strategy for the treatment of HCV.

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Dendritic cell uptake of fluorescenated VLPs.CD11c+ MHC Class II+ splenocyte-derived cultured D1 dendritic cells (2×105 cells) were cultured in the presence of 5 µg FITC-labelled VLPs alone or in association with E8Pam2Cys (0.01 nmoles/ml). Cells were harvested after 24 hours, washed, fixed in 1% paraformaldehyde and analysed for green fluorescence by flow cytometry. Representative histograms show both surface (A) and intracellular (B) associated fluorescence derived from the analysis of a minimum of 1×104 cells in each sample. To determine the level of fluorescence emanating from the cell interior, extracellular fluorescence was quenched by exposing cells to trypan blue for 1 minute prior to analysis. Bar graphs show the percentage of cells deemed to be positive for (C) whole cell or (D) intracellular associated fluorescence based on a marker that was set using untreated cells and are representative of results from one of three experiments conducted separately.
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pone-0047492-g001: Dendritic cell uptake of fluorescenated VLPs.CD11c+ MHC Class II+ splenocyte-derived cultured D1 dendritic cells (2×105 cells) were cultured in the presence of 5 µg FITC-labelled VLPs alone or in association with E8Pam2Cys (0.01 nmoles/ml). Cells were harvested after 24 hours, washed, fixed in 1% paraformaldehyde and analysed for green fluorescence by flow cytometry. Representative histograms show both surface (A) and intracellular (B) associated fluorescence derived from the analysis of a minimum of 1×104 cells in each sample. To determine the level of fluorescence emanating from the cell interior, extracellular fluorescence was quenched by exposing cells to trypan blue for 1 minute prior to analysis. Bar graphs show the percentage of cells deemed to be positive for (C) whole cell or (D) intracellular associated fluorescence based on a marker that was set using untreated cells and are representative of results from one of three experiments conducted separately.

Mentions: The syntheses of the branched anionic peptide construct containing eight N-terminal glutamic acid residues (E8) using traditional Fmoc chemistry has been described previously [23]. Briefly, synthesis was carried out manually using PEG-S RAM solid support (Rapp Polymere, Tübingen, Germany; substitution factor 0.27 mmol/g). Fmoc-lysine(Mtt)-OH (Novabiochem, Läufelfingen, Switzerland) was first coupled to the support and the Fmoc protecting group present on the α-amino group then removed and Fmoc-lysine(Fmoc)-OH was then coupled to the exposed N-terminal amino group. Subsequent de-protection and acylation of another two rounds of Fmoc-lysine(Fmoc)-OH yielded eight branch points to which glutamic acid residues were coupled. The primary amino groups of the glutamic acid residues were then acetylated using a 20-fold excess of acetic anhydride and a 40-fold excess of diisopropylethylamine (DIPEA; Sigma, Australia) to generate E8 which has an overall charge of −8. Lipidation of E8 was then carried out by removing the Mtt protective group present on the ε-amino group of the C-terminal lysine followed by acylation of the exposed ε-amino group with two serially added serine residues. The Pam2Cys lipid moiety was then coupled according to Zeng et al [25] to generate E8(Pam2Cys) (Figure 1A).


Hepatitis C VLPs delivered to dendritic cells by a TLR2 targeting lipopeptide results in enhanced antibody and cell-mediated responses.

Chua BY, Johnson D, Tan A, Earnest-Silveira L, Sekiya T, Chin R, Torresi J, Jackson DC - PLoS ONE (2012)

Dendritic cell uptake of fluorescenated VLPs.CD11c+ MHC Class II+ splenocyte-derived cultured D1 dendritic cells (2×105 cells) were cultured in the presence of 5 µg FITC-labelled VLPs alone or in association with E8Pam2Cys (0.01 nmoles/ml). Cells were harvested after 24 hours, washed, fixed in 1% paraformaldehyde and analysed for green fluorescence by flow cytometry. Representative histograms show both surface (A) and intracellular (B) associated fluorescence derived from the analysis of a minimum of 1×104 cells in each sample. To determine the level of fluorescence emanating from the cell interior, extracellular fluorescence was quenched by exposing cells to trypan blue for 1 minute prior to analysis. Bar graphs show the percentage of cells deemed to be positive for (C) whole cell or (D) intracellular associated fluorescence based on a marker that was set using untreated cells and are representative of results from one of three experiments conducted separately.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3472981&req=5

pone-0047492-g001: Dendritic cell uptake of fluorescenated VLPs.CD11c+ MHC Class II+ splenocyte-derived cultured D1 dendritic cells (2×105 cells) were cultured in the presence of 5 µg FITC-labelled VLPs alone or in association with E8Pam2Cys (0.01 nmoles/ml). Cells were harvested after 24 hours, washed, fixed in 1% paraformaldehyde and analysed for green fluorescence by flow cytometry. Representative histograms show both surface (A) and intracellular (B) associated fluorescence derived from the analysis of a minimum of 1×104 cells in each sample. To determine the level of fluorescence emanating from the cell interior, extracellular fluorescence was quenched by exposing cells to trypan blue for 1 minute prior to analysis. Bar graphs show the percentage of cells deemed to be positive for (C) whole cell or (D) intracellular associated fluorescence based on a marker that was set using untreated cells and are representative of results from one of three experiments conducted separately.
Mentions: The syntheses of the branched anionic peptide construct containing eight N-terminal glutamic acid residues (E8) using traditional Fmoc chemistry has been described previously [23]. Briefly, synthesis was carried out manually using PEG-S RAM solid support (Rapp Polymere, Tübingen, Germany; substitution factor 0.27 mmol/g). Fmoc-lysine(Mtt)-OH (Novabiochem, Läufelfingen, Switzerland) was first coupled to the support and the Fmoc protecting group present on the α-amino group then removed and Fmoc-lysine(Fmoc)-OH was then coupled to the exposed N-terminal amino group. Subsequent de-protection and acylation of another two rounds of Fmoc-lysine(Fmoc)-OH yielded eight branch points to which glutamic acid residues were coupled. The primary amino groups of the glutamic acid residues were then acetylated using a 20-fold excess of acetic anhydride and a 40-fold excess of diisopropylethylamine (DIPEA; Sigma, Australia) to generate E8 which has an overall charge of −8. Lipidation of E8 was then carried out by removing the Mtt protective group present on the ε-amino group of the C-terminal lysine followed by acylation of the exposed ε-amino group with two serially added serine residues. The Pam2Cys lipid moiety was then coupled according to Zeng et al [25] to generate E8(Pam2Cys) (Figure 1A).

Bottom Line: Although many studies provide strong evidence supporting the development of HCV virus-like particle (VLP)-based vaccines, the fact that heterologous viral vectors and/or multiple dosing regimes are required to induce protective immunity indicates that it is necessary to improve their immunogenicity.While co-formulation of this lipopeptide with VLPs only resulted in marginal improvements in dendritic cell (DC) uptake, its ability to concomitantly induce DC maturation at very small doses is a feature not observed using VLPs alone or in the presence of an aluminium hydroxide-based adjuvant (Alum).These results suggest overall that the immunogenicity of HCV VLPs can be significantly improved by the addition of this novel adjuvant by targeting their delivery to DCs and could therefore constitute a viable vaccine strategy for the treatment of HCV.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, The University of Melbourne, Parkville, Victoria, Australia.

ABSTRACT
Although many studies provide strong evidence supporting the development of HCV virus-like particle (VLP)-based vaccines, the fact that heterologous viral vectors and/or multiple dosing regimes are required to induce protective immunity indicates that it is necessary to improve their immunogenicity. In this study, we have evaluated the use of an anionic self-adjuvanting lipopeptide containing the TLR2 agonist Pam(2)Cys (E(8)Pam(2)Cys) to enhance the immunogenicity of VLPs containing the HCV structural proteins (core, E1 and E2) of genotype 1a. While co-formulation of this lipopeptide with VLPs only resulted in marginal improvements in dendritic cell (DC) uptake, its ability to concomitantly induce DC maturation at very small doses is a feature not observed using VLPs alone or in the presence of an aluminium hydroxide-based adjuvant (Alum). Dramatically improved VLP and E2-specific antibody responses were observed in VLP+E(8)Pam(2)Cys vaccinated mice where up to 3 doses of non-adjuvanted or traditionally alum-adjuvanted VLPs was required to match the antibody titres obtained with a single dose of VLPs formulated with this lipopeptide. This result also correlated with significantly higher numbers of specific antibody secreting cells that was detected in the spleens of VLP+E(8)Pam(2)Cys vaccinated mice and greater ability of sera from these mice to neutralise the binding and uptake of VLPs by Huh7 cells. Moreover, vaccination of HLA-A2 transgenic mice with this formulation also induced better VLP-specific IFN-γ-mediated responses compared to non-adjuvanted VLPs but comparable levels to that achieved when coadministered with complete freund's adjuvant. These results suggest overall that the immunogenicity of HCV VLPs can be significantly improved by the addition of this novel adjuvant by targeting their delivery to DCs and could therefore constitute a viable vaccine strategy for the treatment of HCV.

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