Infection can protect against subsequent disease by induction of both humoral and cellular immunity, but inert protein-based vaccines are not as effective. In this study, we present a new vaccine design, with Ag covalently conjugated to solid core nano-beads of narrowly defined size (0.04–0.05 μm) that localize to dendritic cells (DEC205+ CD40+, CD86+) in draining lymph nodes, inducing high levels of IFN-γ production (CD8 T cells: precursor frequencies 1/5000 to 1/1000) and high Ab titers in mice. Conjugation of Ag to these nano-beads induced responses that were significantly higher (2- to 10-fold) than those elicited by other bead sizes, and higher than a range of currently used adjuvants (alum, QuilA, monophosphoryl lipid A). Responses were comparable to CFA/IFA immunization for Abs and ex vivo peptide-pulsed dendritic cell immunization for CD8 T cells. A single dose of Ag-conjugated beads protected mice from tumors in two different model challenges and caused rapid clearance of established tumors in mice. Thus, a range of Ags conjugated to nano-beads was effective as immunogens in both therapeutic and prophylactic scenarios.

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The induction of CD8+ cytotoxic T lymphocytes (CTLs) is desirable for immunization against many diseases, and recombinant-synthetic peptide antigens are now favored agents to use. However, a major problem is how to induce CTLs, which requires a Ti-type response to such synthetic antigens. We (refs. 4 and 5; unpublished data). The unusual finding is that if the conjugation is done under oxidizing conditions, then cell-mediated immunity is selectively stimulated, compared with using reducing conditions for conjugation when antibody responses occur.A successful vaccine for cancer immunotherapy requires a suitable target antigen and production of cytotoxic T lymphocyte (CTL) responses (6). In adenocarcinomas, it appears that mucins, particularly MUC1, may provide a suitable target as there is a 10-fold increase in mucin expression on the cell surface, and altered glycosylation leads to exposure of normally hidden peptide sequences (e.g., APDTR). These changes in cancer generate a potential new target for immunotherapy that is apparently absent in normal mucin (7-10). The APDTR sequence [from the variable number of tandem repeats (VNTR) region] is immunogenic in mice, leading to antibody formation, whether the antigen is administered as MUC1+ cancer cells, purified mucin (HMFG), or as peptides (11,12). Such studies of immunogenicity in mice would be of little relevance to humans were it not for the findings that tumorspecific CTL precursors (CTLp) exist in the lymph nodes of patients with cancer of breast, ovary, or pancreas, or in multiple myeloma patients (13-16). The CTLp can be stimulated by antigen and interleukin 2 (IL-2) in vitro to become functional CD8+ CTLs, the target antigen being the APDTR sequence of MUC1 (13-16 MATERIALS AND METHODSChemical Studies. MUC1 FP containing 5 VNTR of human MUC1 and peptide (Cpl3-32) were produced as described (2). FP was conjugated to mannan in two ways. (i) Ox-M-FP: Mannan (Sigma), at 14 mg/ml in 0.1 M phosphate buffer (pH 6.0), was oxidized with sodium periodate (0.01 M) for 60 min at 40C. Ethanediol (10 ,ul) was added and incubated for a further 30 min at 4°C, and the mixture was passed through a Sephadex-G25 column equilibrated in bicarbonate buffer (pH 6.0-9.0). The oxidized mannan that eluted in the void volume (2 ml) was mixed with 900 gg of MUC1 FP, incubated overnight at room temperature, and used without further purification. (ii) Reduced (Red)-M-FP: The ox-M-FP mixture was treated with sodium borohydride (1 mg/ml) for 3 hr and used without further purification.Immunizations 10128The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Antigen mannosylation has been shown to be an effective approach to potentiate antigen immunogenicity, due to the enhanced antigen uptake and presentation by APC. To overcome disadvantages associated with conventional methods used to mannosylate antigens, we have developed a novel mannose-based antigen delivery system that utilizes a polyamidoamine (PAMAM) dendrimer. It is demonstrated that mannosylated dendrimer ovalbumin (MDO) is a potent immune inducer. With a strong binding avidity to DC, MDO potently induced OVA-specific T cell response in vitro. It was found that the immunogenicity of MDO was due not only to enhanced antigen presentation, but also to induction of DC maturation. Mice immunized with MDO generated strong OVA-specific CD4 + /CD8 + T cell and antibody responses. MDO also targeted lymph node DC to crosspresent OVA, leading to OTI CD8 + T cell proliferation. Moreover, upon challenge with B16-OVA tumor cells, tumors in mice pre-immunized with MDO either did not grow or displayed a much more delayed onset, and had slower kinetics of growth than those of OVA-immunized mice. This mannose-based antigen delivery system was applied here for the first time to the immunization study. With several advantages and exceptional adjuvanticity, we propose mannosylated dendrimer as a potential vaccine carrier.

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SummaryMannan, a polysaccharide isolated from yeast binds to C-type lectins of the mannose receptor family, expressed by antigen-presenting cells (APCs) including dendritic cells (DCs) and macrophages. As these receptors mediate endocytosis, they have been targeted with ligands to deliver antigens into APCs to initiate immune responses. Immunization with tumour antigen MUC1 conjugated to oxidized mannan (OM) or reduced mannan (RM) induced differential immune responses in mice, and only mice immunized with OM-MUC1 elicited strong MUC1-specific cytotoxic T lymphocyte responses and protected mice from a MUC1 tumour challenge. In this study, the adjuvant effect of mannan and its derivatives including OM and RM, in comparison to lipopolysaccharide, on DCs were investigated. Mannan, OM and RM were capable of stimulating mouse bone marrow-derived DC in vitro, eliciting enhanced allogeneic T-cell proliferation and enhancing OTI/OTII peptide-specific T-cell responses. Injection of mice with mannan, OM and RM induced a mature phenotype of lymph node and splenic DCs. Analysis by reverse transcription-polymerase chain reaction indicated that Manna, OM and RM also stimulated up-regulation of inflammatory cytokines including interleukin-1b and tumour necrosis factor-a, and differential T helper 1 (Th1)/Th2 cytokines. Subsequent experiments demonstrated that activation of DCs was Toll-like receptor-4-dependent. The data presented here, together with evidence reported previously on OM and RM in induction of immune responses in vivo, suggest that OM and RM exert a dual capacity to target antigen to APCs as well as mature DCs.

Antigens such as MUC1 coupled to oxidized mannan lead to rapid and efficient MHC class I presentation to CD8+ cells and a preferential T1 response; after reduction there is class II presentation and a T2 immune response. We now show that the selective advantage of the oxidized mannan‐MUC1 is due to the presence of aldehydes and not Schiff bases, and that oxidized mannan‐MUC1 binds to the mannose and not scavenger receptors and is internalized and presented by MHC class I molecules 1000 times more efficiently than when reduced. After internalization there is rapid access to the class I pathway via endosomes but not lysosomes, proteasomal processing and transport to the endoplasmic reticulum, Golgi apparatus and cell surface. Aldehydes cause rapid entry into the class I pathway, and can therefore direct the subsequent immune response.