Liposomal Co-Entrapment of CD40mAb Induces Enhanced IgG Responses against Bacterial Polysaccharide and Protein

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

Construction, evaluation and immune responses elicited by a helicobacter pylori urease B DNA construct via different routes of vaccine delivery

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Immune responses against Tetanus toxoid.

<p>Immune responses against Tetanus toxoid induced by TT/DT co-encapsulated in liposomes (3 µg DT, 1 µg TT) along with either 10 µg or 1mg of CD40mAb (solid lines) or isotype control mAb (dashed lines). On the right hand side the effect of 1mg of free antibody mixed with 1 µg TT are shown. All plots are ELISA assays on individual sera. All results day 14 post-injection except for the bottom graph, which is day 28.</p

Antibody titres induced against Tetanus toxoid.

<p>Antibody titres induced against Tetanus toxoid.</p

Immune responses against pneumococcal capsular polysaccharides.

<p>a) IgG responses at day 30 to PS3 in mice immunized once with liposomes incorporating PS3 (10 µg), PS14 (10 µg) and CD40mAb (10 µg) (solid lines), or PS14, PS3 and control mAb (all 10 µg) (broken lines). CD40mAb/liposome immunised group produced significantly higher titres against PS3 than the control mAb liposome group (IgG GMTs 105 and 11 respectively) p = 0.007, Student's t test. b) IgG responses at day 30 to PS3 in mice immunized once with liposomes incorporating PS3 (10 µg), PS14 (10 µg) and either CD40mAb (10 µg) (solid lines), or control mAb (all 10 µg) (broken lines) incorporated into separate liposomes. Geometric mean titers against PS3 were 11.5 for the control mAb group, and 38 for the CD40mAb group. c) IgG responses 14 days post-boost against PS14 in mice immunized as above. Only two of the 5 mice from the CD40mAb group, and none of the mice from the control mAb group, produced a detectable IgG response against PS14. Overall there was no significant enhancement of responses.</p

Assessment of liposome CD40 binding activity.

<p>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.</p