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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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Neutralisation of VLP cell entry by sera from vaccinated animals.(A) Huh7 cells (5×105 cells) were incubated with FITC labelled VLPs (200 ng) in the presence of a 1∶40 dilution of anti-CD81 antibody or serum from naïve mice. Cells were then washed and analysed for fluorescence by flow cytometry. (B) Neutralisation of VLP entry was determined by pre-incubating FITC labelled VLPs (200 ng) with a 1∶5 dilution of immune sera from mice inoculated with VLPs in saline, Alhydrogel or E8Pam2Cys. Supernatants were clarified by centrifugation, incubated with Huh7 cells (5×105) in a total volume of (500 µl) for 1 hour. Cells were then harvested and cellular fluorescence levels analysed by flow cytometry. All bar graphs represent the percentage reduction in VLP entry relative to baseline levels obtained using serum from naïve mice.
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pone-0047492-g005: Neutralisation of VLP cell entry by sera from vaccinated animals.(A) Huh7 cells (5×105 cells) were incubated with FITC labelled VLPs (200 ng) in the presence of a 1∶40 dilution of anti-CD81 antibody or serum from naïve mice. Cells were then washed and analysed for fluorescence by flow cytometry. (B) Neutralisation of VLP entry was determined by pre-incubating FITC labelled VLPs (200 ng) with a 1∶5 dilution of immune sera from mice inoculated with VLPs in saline, Alhydrogel or E8Pam2Cys. Supernatants were clarified by centrifugation, incubated with Huh7 cells (5×105) in a total volume of (500 µl) for 1 hour. Cells were then harvested and cellular fluorescence levels analysed by flow cytometry. All bar graphs represent the percentage reduction in VLP entry relative to baseline levels obtained using serum from naïve mice.

Mentions: To assess the neutralising activity of antibodies induced by vaccination, we first set out to investigate if VLP entry into human hepatocyte cell line Huh7 could be inhibited. Pre-incubation of FITC-labelled VLPs (VLP-FITC) with PBS or naïve serum resulted in minimal inhibition of VLP entry (Figure 5A). However, the presence of an antibody against CD81, a cell surface molecule implicated in HCV entry into hepatocytes [30], was able to prevent VLP entry into these cells by >90% confirming that these VLPs also utilise this molecule to facilitate cell entry. We next analysed the ability of sera obtained from mice inoculated with VLPs to inhibit the binding and entry of VLPs into Huh 7 cells (Figure 5B). Neutralisation of binding of VLPs to Huh7 cells was significantly greater in sera obtained from mice inoculated with VLPs in E8Pam2Cys (∼50%) compared to sera obtained from mice inoculated with VLPs administered in Alhydrogel (∼30%) or in saline (∼20%).


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)

Neutralisation of VLP cell entry by sera from vaccinated animals.(A) Huh7 cells (5×105 cells) were incubated with FITC labelled VLPs (200 ng) in the presence of a 1∶40 dilution of anti-CD81 antibody or serum from naïve mice. Cells were then washed and analysed for fluorescence by flow cytometry. (B) Neutralisation of VLP entry was determined by pre-incubating FITC labelled VLPs (200 ng) with a 1∶5 dilution of immune sera from mice inoculated with VLPs in saline, Alhydrogel or E8Pam2Cys. Supernatants were clarified by centrifugation, incubated with Huh7 cells (5×105) in a total volume of (500 µl) for 1 hour. Cells were then harvested and cellular fluorescence levels analysed by flow cytometry. All bar graphs represent the percentage reduction in VLP entry relative to baseline levels obtained using serum from naïve mice.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3472981&req=5

pone-0047492-g005: Neutralisation of VLP cell entry by sera from vaccinated animals.(A) Huh7 cells (5×105 cells) were incubated with FITC labelled VLPs (200 ng) in the presence of a 1∶40 dilution of anti-CD81 antibody or serum from naïve mice. Cells were then washed and analysed for fluorescence by flow cytometry. (B) Neutralisation of VLP entry was determined by pre-incubating FITC labelled VLPs (200 ng) with a 1∶5 dilution of immune sera from mice inoculated with VLPs in saline, Alhydrogel or E8Pam2Cys. Supernatants were clarified by centrifugation, incubated with Huh7 cells (5×105) in a total volume of (500 µl) for 1 hour. Cells were then harvested and cellular fluorescence levels analysed by flow cytometry. All bar graphs represent the percentage reduction in VLP entry relative to baseline levels obtained using serum from naïve mice.
Mentions: To assess the neutralising activity of antibodies induced by vaccination, we first set out to investigate if VLP entry into human hepatocyte cell line Huh7 could be inhibited. Pre-incubation of FITC-labelled VLPs (VLP-FITC) with PBS or naïve serum resulted in minimal inhibition of VLP entry (Figure 5A). However, the presence of an antibody against CD81, a cell surface molecule implicated in HCV entry into hepatocytes [30], was able to prevent VLP entry into these cells by >90% confirming that these VLPs also utilise this molecule to facilitate cell entry. We next analysed the ability of sera obtained from mice inoculated with VLPs to inhibit the binding and entry of VLPs into Huh 7 cells (Figure 5B). Neutralisation of binding of VLPs to Huh7 cells was significantly greater in sera obtained from mice inoculated with VLPs in E8Pam2Cys (∼50%) compared to sera obtained from mice inoculated with VLPs administered in Alhydrogel (∼30%) or in saline (∼20%).

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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