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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 maturation.(A) D1 dendritic cells (2×105 cells) were incubated with VLPs (5 µg) alone or formulated with E8Pam2Cys (0.01 nmoles/ml) or Alhydrogel (5 µg) in a total volume of 1 ml. For comparative purposes within all experiments, cells were also either left untreated, exposed to LPS (5 µg/ml) or to similar amounts of each adjuvant alone. Cell surface MHC class II expression was determined after 16 hours using a PE-conjugated anti-IA/IE antibody. Cells expressing low levels of MHC Class II molecules were deemed to be immature whilst those expressing high levels were considered to be mature. Shown are representative histograms depicting cell surface MHC class II expression from one of three experiments conducted separately. MHC Class IIhigh expressing cells are shaded in grey. For dosing experiments, cells were also incubated with increasing amounts of (B) VLPs, (C) E8Pam2Cys, (D) Alhydrogel or VLPs (5 µg) formulated with increasing amounts of (E) E8Pam2Cys, or (F) Alhydrogel.
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pone-0047492-g002: Dendritic cell maturation.(A) D1 dendritic cells (2×105 cells) were incubated with VLPs (5 µg) alone or formulated with E8Pam2Cys (0.01 nmoles/ml) or Alhydrogel (5 µg) in a total volume of 1 ml. For comparative purposes within all experiments, cells were also either left untreated, exposed to LPS (5 µg/ml) or to similar amounts of each adjuvant alone. Cell surface MHC class II expression was determined after 16 hours using a PE-conjugated anti-IA/IE antibody. Cells expressing low levels of MHC Class II molecules were deemed to be immature whilst those expressing high levels were considered to be mature. Shown are representative histograms depicting cell surface MHC class II expression from one of three experiments conducted separately. MHC Class IIhigh expressing cells are shaded in grey. For dosing experiments, cells were also incubated with increasing amounts of (B) VLPs, (C) E8Pam2Cys, (D) Alhydrogel or VLPs (5 µg) formulated with increasing amounts of (E) E8Pam2Cys, or (F) Alhydrogel.

Mentions: To investigate the ability of HCV VLPs and E8Pam2Cys to cause activation of DCs, we measured the expression of surface MHC class II molecules following incubation with the various antigens. The results (Figure 2A) indicate that untreated DCs contained two populations of cells which were MHC class IIlow and MHC class IIhigh, the latter comprising ∼24% of the population analysed. While the distribution of these populations was not affected by exposure to HCV VLPs alone, incubation with HCV VLPs mixed with E8Pam2Cys caused a dramatic shift in the distribution of MHC class II expressing cells such that 83% of cells were MHC class IIhigh. The upregulation of MHC class II expression on these cells were comparable to those cultured in the presence of LPS which is a potent DC maturation stimulus.


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 maturation.(A) D1 dendritic cells (2×105 cells) were incubated with VLPs (5 µg) alone or formulated with E8Pam2Cys (0.01 nmoles/ml) or Alhydrogel (5 µg) in a total volume of 1 ml. For comparative purposes within all experiments, cells were also either left untreated, exposed to LPS (5 µg/ml) or to similar amounts of each adjuvant alone. Cell surface MHC class II expression was determined after 16 hours using a PE-conjugated anti-IA/IE antibody. Cells expressing low levels of MHC Class II molecules were deemed to be immature whilst those expressing high levels were considered to be mature. Shown are representative histograms depicting cell surface MHC class II expression from one of three experiments conducted separately. MHC Class IIhigh expressing cells are shaded in grey. For dosing experiments, cells were also incubated with increasing amounts of (B) VLPs, (C) E8Pam2Cys, (D) Alhydrogel or VLPs (5 µg) formulated with increasing amounts of (E) E8Pam2Cys, or (F) Alhydrogel.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3472981&req=5

pone-0047492-g002: Dendritic cell maturation.(A) D1 dendritic cells (2×105 cells) were incubated with VLPs (5 µg) alone or formulated with E8Pam2Cys (0.01 nmoles/ml) or Alhydrogel (5 µg) in a total volume of 1 ml. For comparative purposes within all experiments, cells were also either left untreated, exposed to LPS (5 µg/ml) or to similar amounts of each adjuvant alone. Cell surface MHC class II expression was determined after 16 hours using a PE-conjugated anti-IA/IE antibody. Cells expressing low levels of MHC Class II molecules were deemed to be immature whilst those expressing high levels were considered to be mature. Shown are representative histograms depicting cell surface MHC class II expression from one of three experiments conducted separately. MHC Class IIhigh expressing cells are shaded in grey. For dosing experiments, cells were also incubated with increasing amounts of (B) VLPs, (C) E8Pam2Cys, (D) Alhydrogel or VLPs (5 µg) formulated with increasing amounts of (E) E8Pam2Cys, or (F) Alhydrogel.
Mentions: To investigate the ability of HCV VLPs and E8Pam2Cys to cause activation of DCs, we measured the expression of surface MHC class II molecules following incubation with the various antigens. The results (Figure 2A) indicate that untreated DCs contained two populations of cells which were MHC class IIlow and MHC class IIhigh, the latter comprising ∼24% of the population analysed. While the distribution of these populations was not affected by exposure to HCV VLPs alone, incubation with HCV VLPs mixed with E8Pam2Cys caused a dramatic shift in the distribution of MHC class II expressing cells such that 83% of cells were MHC class IIhigh. The upregulation of MHC class II expression on these cells were comparable to those cultured in the presence of LPS which is a potent DC maturation stimulus.

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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