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Liposomal co-entrapment of CD40mAb induces enhanced IgG responses against bacterial polysaccharide and protein.

Hatzifoti C, Bacon A, Marriott H, Laing P, Heath AW - PLoS ONE (2008)

Bottom Line: Antibody against CD40 is effective in enhancing immune responses to vaccines when chemically conjugated to the vaccine antigen.Unfortunately the requirement for chemical conjugation presents some difficulties in vaccine production and quality control which are compounded when multivalent vaccines are required.After subcutaneous immunization of BALB/c female mice, anti-polysaccharide and DT/TT responses were measured by ELISA.

View Article: PubMed Central - PubMed

Affiliation: Adjuvantix Ltd, Sheffield, United Kingdom.

ABSTRACT

Background: Antibody against CD40 is effective in enhancing immune responses to vaccines when chemically conjugated to the vaccine antigen. Unfortunately the requirement for chemical conjugation presents some difficulties in vaccine production and quality control which are compounded when multivalent vaccines are required. We explore here an alternative to chemical conjugation, involving the co-encapsulation of CD40 antibody and antigens in liposomal vehicles.

Methodology/principal findings: Anti-mouse CD40 mAb or isotype control mAb were co-entrapped individually in cationic liposomal vehicles with pneumococcal polysaccharides or diphtheria and tetanus toxoids. Retention of CD40 binding activity upon liposomal entrapment was assessed by ELISA and flow cytometry. After subcutaneous immunization of BALB/c female mice, anti-polysaccharide and DT/TT responses were measured by ELISA. Simple co-encapsulation of CD40 antibody allowed for the retention of CD40 binding on the liposome surface, and also produced vaccines with enhanced imunogenicity. Antibody responses against both co-entrapped protein in the form of tetanus toxoid, and Streptococcus pneumoniae capsular polysaccharide, were enhanced by co-encapsulation with CD40 antibody. Surprisingly, liposomal encapsulation also appeared to decrease the toxicity of high doses of CD40 antibody as assessed by the degree of splenomegaly induced.

Conclusions/significance: Liposomal co-encapsulation with CD40 antibody may represent a practical means of producing more immunogenic multivalent vaccines and inducing IgG responses against polysaccharides without the need for conjugation.

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Related in: MedlinePlus

Assessment of liposome CD40 binding activity.Fig 1a) ELISA plates were coated with anti-human IgG, blocked with 3% BSA, and then incubated with recombinant murine CD40-human IgG1. After washing, various liposomal preparations containing entrapped pneumococcal type 3 polysaccharide (PS) and/or CD40 mAb at varying concentrations (6 or 20 µg per 0.5 ml of liposomal preparation) were added to the plate in two-fold dilutions. Liposomal binding to CD40-was detected using HRP labelled goat anti-rat IgG. 1b) To assess liposomal binding to cell surface CD40, CD154 transfected (filled histograms) or CD40 transfected (open histograms) L929 cells were stained with liposomal preparations at a 1/10 dilution in FACS buffer. Binding was detected using FITC labelled anti-rat IgG.
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pone-0002368-g001: Assessment of liposome CD40 binding activity.Fig 1a) ELISA plates were coated with anti-human IgG, blocked with 3% BSA, and then incubated with recombinant murine CD40-human IgG1. After washing, various liposomal preparations containing entrapped pneumococcal type 3 polysaccharide (PS) and/or CD40 mAb at varying concentrations (6 or 20 µg per 0.5 ml of liposomal preparation) were added to the plate in two-fold dilutions. Liposomal binding to CD40-was detected using HRP labelled goat anti-rat IgG. 1b) To assess liposomal binding to cell surface CD40, CD154 transfected (filled histograms) or CD40 transfected (open histograms) L929 cells were stained with liposomal preparations at a 1/10 dilution in FACS buffer. Binding was detected using FITC labelled anti-rat IgG.

Mentions: Because CD40 antibody conjugates are thought to bind directly to antigen specific B cells [8], and because it was unknown whether simple liposomal formulation of antibody would allow it to bind CD40, initial experiments were performed to assess the effectiveness of simple liposomal formulations in binding to CD40. Assessment of CD40 binding by the liposomes was performed by Flow cytometric analysis on CD40 transfected or normal L929 cells, and by ELISA assay using plates coated with recombinant murine CD40-Fc, and with detection in both cases by anti-rat antibody conjugates (the monoclonal antibody is rat anti-mouse CD40). The two assays were consistent in that the CD40mAb containing liposomes clearly were able to bind to both recombinant CD40-Fc (Figure 1a), and to cell expressed CD40 (Fig 1b). While the binding found in the ELISA assay may have been attributable, at least in part, to leakage of the liposomal contents in the presence of Tween, the Flow cytometric staining was done in the absence of detergent. The liposomes are stable even at room temperature in the absence of Tween. In a separate experiment less than 1% of entrapped carboxyfluorescein was released over a 20 minute incubation even at room temperature (not shown). These observations, taken together, are consistent with the ability of liposomal formulations to entrap proteins both within the aqueous core and in the lipid membrane of the liposome.


Liposomal co-entrapment of CD40mAb induces enhanced IgG responses against bacterial polysaccharide and protein.

Hatzifoti C, Bacon A, Marriott H, Laing P, Heath AW - PLoS ONE (2008)

Assessment of liposome CD40 binding activity.Fig 1a) ELISA plates were coated with anti-human IgG, blocked with 3% BSA, and then incubated with recombinant murine CD40-human IgG1. After washing, various liposomal preparations containing entrapped pneumococcal type 3 polysaccharide (PS) and/or CD40 mAb at varying concentrations (6 or 20 µg per 0.5 ml of liposomal preparation) were added to the plate in two-fold dilutions. Liposomal binding to CD40-was detected using HRP labelled goat anti-rat IgG. 1b) To assess liposomal binding to cell surface CD40, CD154 transfected (filled histograms) or CD40 transfected (open histograms) L929 cells were stained with liposomal preparations at a 1/10 dilution in FACS buffer. Binding was detected using FITC labelled anti-rat IgG.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2387064&req=5

pone-0002368-g001: Assessment of liposome CD40 binding activity.Fig 1a) ELISA plates were coated with anti-human IgG, blocked with 3% BSA, and then incubated with recombinant murine CD40-human IgG1. After washing, various liposomal preparations containing entrapped pneumococcal type 3 polysaccharide (PS) and/or CD40 mAb at varying concentrations (6 or 20 µg per 0.5 ml of liposomal preparation) were added to the plate in two-fold dilutions. Liposomal binding to CD40-was detected using HRP labelled goat anti-rat IgG. 1b) To assess liposomal binding to cell surface CD40, CD154 transfected (filled histograms) or CD40 transfected (open histograms) L929 cells were stained with liposomal preparations at a 1/10 dilution in FACS buffer. Binding was detected using FITC labelled anti-rat IgG.
Mentions: Because CD40 antibody conjugates are thought to bind directly to antigen specific B cells [8], and because it was unknown whether simple liposomal formulation of antibody would allow it to bind CD40, initial experiments were performed to assess the effectiveness of simple liposomal formulations in binding to CD40. Assessment of CD40 binding by the liposomes was performed by Flow cytometric analysis on CD40 transfected or normal L929 cells, and by ELISA assay using plates coated with recombinant murine CD40-Fc, and with detection in both cases by anti-rat antibody conjugates (the monoclonal antibody is rat anti-mouse CD40). The two assays were consistent in that the CD40mAb containing liposomes clearly were able to bind to both recombinant CD40-Fc (Figure 1a), and to cell expressed CD40 (Fig 1b). While the binding found in the ELISA assay may have been attributable, at least in part, to leakage of the liposomal contents in the presence of Tween, the Flow cytometric staining was done in the absence of detergent. The liposomes are stable even at room temperature in the absence of Tween. In a separate experiment less than 1% of entrapped carboxyfluorescein was released over a 20 minute incubation even at room temperature (not shown). These observations, taken together, are consistent with the ability of liposomal formulations to entrap proteins both within the aqueous core and in the lipid membrane of the liposome.

Bottom Line: Antibody against CD40 is effective in enhancing immune responses to vaccines when chemically conjugated to the vaccine antigen.Unfortunately the requirement for chemical conjugation presents some difficulties in vaccine production and quality control which are compounded when multivalent vaccines are required.After subcutaneous immunization of BALB/c female mice, anti-polysaccharide and DT/TT responses were measured by ELISA.

View Article: PubMed Central - PubMed

Affiliation: Adjuvantix Ltd, Sheffield, United Kingdom.

ABSTRACT

Background: Antibody against CD40 is effective in enhancing immune responses to vaccines when chemically conjugated to the vaccine antigen. Unfortunately the requirement for chemical conjugation presents some difficulties in vaccine production and quality control which are compounded when multivalent vaccines are required. We explore here an alternative to chemical conjugation, involving the co-encapsulation of CD40 antibody and antigens in liposomal vehicles.

Methodology/principal findings: Anti-mouse CD40 mAb or isotype control mAb were co-entrapped individually in cationic liposomal vehicles with pneumococcal polysaccharides or diphtheria and tetanus toxoids. Retention of CD40 binding activity upon liposomal entrapment was assessed by ELISA and flow cytometry. After subcutaneous immunization of BALB/c female mice, anti-polysaccharide and DT/TT responses were measured by ELISA. Simple co-encapsulation of CD40 antibody allowed for the retention of CD40 binding on the liposome surface, and also produced vaccines with enhanced imunogenicity. Antibody responses against both co-entrapped protein in the form of tetanus toxoid, and Streptococcus pneumoniae capsular polysaccharide, were enhanced by co-encapsulation with CD40 antibody. Surprisingly, liposomal encapsulation also appeared to decrease the toxicity of high doses of CD40 antibody as assessed by the degree of splenomegaly induced.

Conclusions/significance: Liposomal co-encapsulation with CD40 antibody may represent a practical means of producing more immunogenic multivalent vaccines and inducing IgG responses against polysaccharides without the need for conjugation.

Show MeSH
Related in: MedlinePlus