ovalbumin and cytidylyl-3--5--guanosine

Codelivery nanovaccines of antigens and adjuvants have achieved positive therapy for cancer immunotherapy. The insufficient immunogenicity of these vaccines leads to the difficulty of eliciting robust immune effects for immune clearance due to the inadequate loading efficiency, complex preparation processes, low safety concerns, and weak immune responses. Herein, a visible codelivery nanovaccine of an antigen and adjuvant based on self-cross-linked antigen nanoparticles (ovalbumin nanoparticles (ONPs)) combined with the adjuvant (CpG) for cancer immunotherapy was prepared using antigens themselves as carriers. ONPs not only provide sufficient antigens for continuous simulation of the immune response with high antigen loading efficiency but also serve as natural carriers of CpG. In vitro and in vivo experiments proved that ONPs-CpG can elicit a robust immune response including DC maturity, T cell activation, and IFN-γ production. ONPs-CpG induced strong tumor-specific immunity and exhibited remarkable antitumor immunotherapy effects in vivo using mouse models of lymphoma. Furthermore, to perform the precise vaccine delivery, the dual fluorescent codelivery nanovaccine was monitored in real time in vivo by the visible imaging method. With regard to migration tracking, fluorescence imaging allowed for both high resolution and sensitivity of visible detection based on the fluorescence of ONPs and CpG. The multifunctional nanovaccine could function as a robust platform for cancer immunotherapy and a visible system for antigen-adjuvant tracking.

Topics: Adjuvants, Immunologic; Animals; Cancer Vaccines; Cells, Cultured; Dinucleoside Phosphates; Disease Models, Animal; Drug Delivery Systems; Fluorescent Dyes; Immunotherapy; Lymphoma; Mice; Nanoparticles; Optical Imaging; Ovalbumin; Particle Size

Memory T cells survive throughout the lifetime of an individual and are protective upon recall. It is not clear how memory T cells can live so long. Here, we demonstrate that at the resolution of a viral infection, low levels of antigen are captured by B cells and presented to specific CD4(+) memory T cells to render a state of unresponsiveness. We demonstrate in two systems that this process occurs naturally during the fall of antigen and is associated with a global gene expression program initiated with the clearance of antigen. Our study suggests that in the absence of antigen, a state of dormancy associated with low-energy utilization and proliferation can help memory CD4(+) T cells to survive nearly throughout the lifetime of mice. The dormant CD4(+) memory T cells become activated by stimulatory signals generated by a subsequent infection. We propose that quiescence might be a mechanism necessary to regulate long-term survival of CD4 memory T cells and to prevent cross-reactivity to self, hence autoimmunity.

Topics: Animals; Antigen Presentation; B-Lymphocytes; CD4-Positive T-Lymphocytes; Cell Survival; Dinucleoside Phosphates; Gene Expression Profiling; Homeostasis; Immunologic Memory; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Transgenic; Muramidase; Ovalbumin; T-Lymphocyte Subsets; Vaccinia virus

Immunostimulatory CpG-DNA targeting TLR9 is one of the most extensively evaluated vaccine adjuvants. Previously, we found that a particular form of natural phosphodiester bond CpG-DNA (PO-ODN) encapsulated in a phosphatidyl-Β-oleoyl- γ-palmitoyl ethanolamine (DOPE) : cholesterol hemisuccinate (CHEMS) (1 : 1 ratio) complex (Lipoplex(O)) is a potent adjuvant. Complexes containing peptide and Lipoplex(O) are extremely useful for B cell epitope screening and antibody production without carriers. Here, we showed that IL-12 production was increased in bone marrow derived dendritic cells in a CpG sequence-dependent manner when PO-ODN was encapsulated in Lipoplex(O), DOTAP or lipofectamine. However, the effects of Lipoplex(O) surpassed those of PO-ODN encapsulated in DOTAP or lipofectamine and also other various forms of liposome-encapsulated CpG-DNA in terms of potency for protein antigen-specific IgG production and Th1- associated IgG2a production. Therefore, Lipoplex(O) may have a unique potent immunoadjuvant activity which can be useful for various applications involving protein antigens as well as peptides.

Topics: Adjuvants, Immunologic; Animals; Cations; Dendritic Cells; Dinucleoside Phosphates; DNA; Esters; Immunoglobulin G; Interleukin-12; Liposomes; Male; Mice; Mice, Inbred BALB C; Muramidase; Oligodeoxyribonucleotides; Ovalbumin; Th1 Cells

Plasmacytoid dendritic cells (PDCs) are a unique leukocyte population capable of secreting high levels of type I interferon (IFN) in response to viruses and bacterial stimuli. In vitro experiments have shown that upon maturation, human and murine PDCs develop into potent immunostimulatory cells; however, their ability to prime an immune response in vivo remains to be addressed. We report that CpG-matured murine PDCs are capable of eliciting in naive mice antigen-specific CTLs against endogenous antigens as well as exogenous peptides, but not against an exogenous antigen. Type I IFN is not required for priming, as injection of CpG-matured PDCs into type I IFN receptor-deficient mice elicits functional CTL responses. Mature PDCs prime CTLs that secrete IFN-gamma and protect mice from a tumor challenge. In contrast, immature PDCs are unable to prime antigen-specific CTLs. However, mice injected with immature PDCs are fully responsive to secondary antigenic challenges, suggesting that PDCs have not induced long-lasting tolerance via anergic or regulatory T cells. Our results underline the heterogeneity and plasticity of different antigen-presenting cells, and reveal an important role of mature PDCs in priming CD8 responses to endogenous antigens, in addition to their previously reported ability to modulate antiviral responses via type I IFN.

Topics: Amino Acid Sequence; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 2; ATP-Binding Cassette Transporters; Autoantigens; CD8-Positive T-Lymphocytes; Dendritic Cells; Dinucleoside Phosphates; Isoantigens; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; Molecular Sequence Data; Ovalbumin; Peptide Fragments

Toll-like receptors (TLRs) control activation of adaptive immune responses by antigen-presenting cells (APCs). However, initiation of adaptive immune responses is also controlled by regulatory T cells (TR cells), which act to prevent activation of autoreactive T cells. Here we describe a second mechanism of immune induction by TLRs, which is independent of effects on costimulation. Microbial induction of the Toll pathway blocked the suppressive effect of CD4+CD25+ TR cells, allowing activation of pathogen-specific adaptive immune responses. This block of suppressor activity was dependent in part on interleukin-6, which was induced by TLRs upon recognition of microbial products.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antigens, CD; Antigens, Differentiation; Cells, Cultured; Culture Media, Conditioned; Dendritic Cells; Dinucleoside Phosphates; Drosophila Proteins; Immune Tolerance; Immunization; Interleukin-6; Lipopolysaccharides; Lymphocyte Activation; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Myeloid Differentiation Factor 88; Ovalbumin; Receptors, Cell Surface; Receptors, Immunologic; Self Tolerance; T-Lymphocytes; T-Lymphocytes, Regulatory; Toll-Like Receptors

Murine models of acute atopic asthma may be inadequate to study the effects of recurrent exposure to inhaled allergens, such as the epithelial changes seen in asthmatic patients. We developed a murine model in which chronic airway inflammation is maintained by repeated allergen [ovalbumin (OVA)] inhalation; using this model, we examined the response to mucosal administration of CpG DNA (oligonucleotides) and specific antigen immunotherapy. Mice repeatedly exposed to OVA developed significantly greater airway hyperresponsiveness and goblet cell hyperplasia, but not airway eosinophilia, compared with those exposed only twice. CpG-based immunotherapy significantly reversed both acute and chronic markers of inflammation as well as airway hyperresponsiveness. We further examined the effect of mucosal immunotherapy on the response to a second, unrelated antigen. Mice sensitized to both OVA and schistosome eggs, challenged with inhaled OVA, and then treated with OVA-directed immunotherapy demonstrated significant reduction of airway hyperresponsiveness and a moderate reduction in eosinophilia, after inhalation challenge with schistosome egg antigens. In this model, immunotherapy treatment reduced bronchoalveolar lavage (BAL) levels of Th2 cytokines (IL-4, IL-5, IL-13, and IL-10) without changing BAL IFN-gamma. Antigen recall responses of splenocytes from these mice demonstrated an antigen-specific (OVA) enhanced release of IL-10 from splenocytes of treated mice. These results suggest that CpG DNA may provide the basis for a novel form of immunotherapy of allergic asthma. Both antigen-specific and, to a lesser extent, antigen-nonspecific responses to mucosal administration of CpG DNA are seen.

Topics: Administration, Inhalation; Allergens; Animals; Asthma; Base Sequence; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Cytokines; Dinucleoside Phosphates; Disease Models, Animal; Female; Immunity, Mucosal; Immunotherapy; Mice; Mice, Inbred C57BL; Oligodeoxyribonucleotides; Ovalbumin

Receptors for conserved molecular patterns associated with microbial pathogens induce synthesis of co-stimulatory molecules and cytokines in immature dendritic cells (DC), as do antigen-reactive CD4 T helper cells via CD40 signaling. Once activated, antigen-presenting DC may activate CD8 T cell responses in a T helper cell-independent fashion. Using immunostimulatory CpG-oligonucleotides (ODN) mimicking bacterial CpG-DNA, we tested whether CpG-DNA bypasses the need for T helper cells in CTL responses towards proteins by directly activating antigen-presenting DC to transit into professional APC. We describe that immature DC in situ constitutively process soluble proteins and generate CD8 T cell determinants yet CD8 T cell responses remain abortive. Induction of primary antigen-specific CD8 cytotoxic T lymphocyte (CTL)-mediated responses becomes initiated in wild-type as well as T helper cell-deficient mice, provided soluble protein and CpG-ODN are draining into the same lymph node. Specifically we show that CpG-ODN trigger antigen-presenting immature DC within the draining lymph node to acutely up-regulate co-stimulatory molecules and produce IL-12. These results provide new insights for generating in vivo efficient CTL responses to soluble proteins which may influence vaccination strategies.

Topics: Animals; CD40 Antigens; CD40 Ligand; Dendritic Cells; Dinucleoside Phosphates; DNA, Bacterial; Female; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Ovalbumin; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Helper-Inducer

Foreign DNA has been shown to impinge on immune cell function by an as yet unidentified mechanism. We and others have demonstrated that single-stranded (ss) DNA containing the motif CpG flanked by two 5' purines and two 3' pyrimidines are mitogenic for B cells and activate macrophages to release tumor necrosis factor-alpha, interferon-gamma, interleukin (IL)-6 or IL-12. Because of these pro-inflammatory responses we investigated if ssDNA would serve as a potential vaccine adjuvant. Here we show that CpG-containing oligonucleotides represent a powerful adjuvant for both humoral and cellular immune responses. When ssDNA was incorporated into inocula, specific antibody titers of the IgG2 isotype were enhanced by greater than 100-fold. Primary cytotoxic T lymphocyte responses generated to either unprocessed protein antigen or major histocompatibility complex class I-restricted peptide were exceedingly strong. Evidence is also provided that oligomers directly influenced T cell receptor-triggered T cell proliferation. Thus ssDNA oligomers may serve as inexpensive and safe vaccine adjuvants and, in addition, differential effects due to sequence may allow for directed responses.

Topics: Adjuvants, Immunologic; Animals; B-Lymphocytes; Base Sequence; Cytotoxicity, Immunologic; Dinucleoside Phosphates; DNA, Single-Stranded; Lymph Nodes; Lymphocyte Activation; Macrophages; Mice; Mice, Inbred C57BL; Oligodeoxyribonucleotides; Ovalbumin; T-Lymphocytes