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.

CD40-mediated enhancement of immune responses against three forms of influenza vaccine.

There is potential for influenza A infections to cause massive morbidity and mortality. Vaccination may be the primary defence against pandemic influenza, and potential pandemic'flu vaccines may be produced conventionally, in embryonated eggs, or as recombinant protein or synthetic peptide vaccines. However the vaccines are produced, the supply may be limiting, and it will be important to enhance the immunogenicity of the vaccines as much as possible. We have shown that conjugation to CD40 binding antibody is a very efficient way of enhancing immune responses against model antigens, but were interested in assessing the effectiveness of this system using influenza vaccines. We produced conjugates of CD40 monoclonal antibody (mAb) and isotype control with three potential influenza vaccines: a peptide-based vaccine containing T- and B-cell epitopes from virus haemagglutinin; a whole, killed virus vaccine; and a commercially produced split virus vaccine. CD40 mAb conjugates in each case were more immunogenic, but the adjuvant effect of CD40 conjugation was greatest with the split vaccine, where antibody responses were enhanced by several hundred-fold after a single immunization, and lymphocyte proliferation in response to antigen in vitro was also strongly enhanced.

Antibodies against cell surface antigens as very potent immunological adjuvants.

We describe here two very potent adjuvant systems which are thought to work directly on antigen specific lymphocytes, thus by-passing the normal route for adjuvants, which is to activate antigen presenting cells (APCs) inducing release of inflammatory cytokines with resultant side effects of local and systemic reactogenicity. CD40 and CD28 based adjuvants are extremely potent and should avoid the inflammatory side effects induced by most adjuvants.

Mucosal immunization with a urease B DNA vaccine induces innate and cellular immune responses against Helicobacter pylori.

BACKGROUND: Helicobacter pylori is recognized as a major risk factor for recurrent gastroduodenal inflammatory diseases and gastric adenocarcinoma. The high prevalence of H. pylori infection worldwide, the risks of side-effects from antibiotic therapy, and increasing resistance to antibiotics are the main primers for the development of improved H. pylori vaccines. The antigenic potential of its urease enzyme, a critical virulence factor required for colonization of the gastric mucosa, has been demonstrated in animal and human studies. An important but controversial issue in H. pylori vaccine studies is the type of immune response required to control infection. A new approach in H. pylori vaccinology is the administration of DNA vaccines, which has included heat-shock protein and catalase DNA vaccines. MATERIALS AND METHODS: The H. pylori urease subunit B construct or vector alone was administered to mice via the intranasal route. Spleens and stomachs were examined on day 0 and weeks 3, 6, and 12 after immunization. Proliferation of spleen cells was assessed using the carboxyfluorescein diacetate succinimidyl ester-based flow cytometry assay and cytokine secretion from cultured spleen cells was detected by ELISA, after stimulation with the urease subunit B recombinant antigen. Total RNA was isolated from stomach and spleen tissue and the expression of beta-defensin and cytokine genes was monitored by reverse transcription followed by polymerase chain reaction (RT-PCR). Immunized mice were challenged with H. pylori and bacterial DNA quantified by TaqMan PCR. RESULTS: The urease B subunit DNA vaccine increased INF-gamma secretion and splenocyte proliferation without inducing adverse effects in the spleen. Increase in gastric beta-defensin 1 and marked induction in local IL-10 : IFN-gamma ratio up to 12 weeks post-immunization suggest a potential role for local innate immune responses in protection at the site of infection. Although significant bacterial reduction in the stomachs of urease B subunit DNA-immunized mice was observed, intermediate reduction was also noted in the vector group. Increased defensin expression and adjuvant effects of the cytosine preceding guanosine motifs may contribute to this phenomenon. Our data confirm that cytosine preceding guanosine motifs, even without coadministration with antigen, can reduce extracellular bacterial load. CONCLUSIONS: In this study, a DNA construct encoding the urease B subunit was assessed for its immune profile and its ability to reduce bacterial colonization in the murine stomach. Our studies suggest that local innate immune responses may play a greater role than previously supposed in limiting H. pylori colonization in the gastric mucosa.

A plasmid immunization construct encoding urease B of Helicobacter pylori induces an antigen-specific antibody response and upregulates the expression of beta-defensins and IL-10 in the stomachs of immunized mice.

The objectives of this study were to investigate the efficacy of a prototype DNA immunization construct encoding the urease B subunit enzyme of Helicobacter pylori (H. pylori) for inducing adaptive and innate immune responses in mice immunized via intramuscular or subcutaneous routes and to further explore the adjuvant effects of the CpG motifs in the vector. Antibody, cytokine, and beta-defensin profiles were assessed in the stomachs of immunized animals: experiments were terminated 3 months after immunization because there was a significant increase in the anti-H. pylori urease B antibody response at Week 6 in mice immunized with the urease B construct. A long lasting expression of IL-10 mRNA was noted. Furthermore, a marked and sustained increase in the mRNA expression of beta-defensins was also observed, particularly beta1. This study demonstrates that an H. pylori urease B DNA construct can induce innate as well as adaptive immune responses in the stomachs of immunized mice. Upregulation of beta-defensin gene expression followed immunization and we believe that this is the first report of a DNA vaccine inducing innate anti-microbial responses. Such complex molecular interactions that modulate both innate and adaptive immune responses may be of critical importance in the control of mucosal pathogens, such as H. pylori.