ovalbumin and resiquimod

Previously, high-aspect- ratio ribbon-like microconfetti (MC) composed of acetalated dextran (Ace-DEX) have been shown to form a subcutaneous depot for sustained drug release. In this study, MC were explored as an injectable vaccine platform. Production of MC by electrospinning followed by high-shear homogenization allowed for precise control over MC fabrication. Three distinct sizes of MC, small (0.67 × 10.2 μm

Topics: Adjuvants, Immunologic; Animals; Biopolymers; Cytokines; Dendritic Cells; Dextrans; Drug Carriers; Imidazoles; Immunity, Humoral; Mice; Mice, Inbred C57BL; Ovalbumin; Particle Size; RAW 264.7 Cells; Vaccines, Synthetic

Growing evidence indicates that the Toll-like receptor7/8(TLR7/8) agonist resiquimod (R848) is a potential inhibitor of type-2 immunity. However, the mechanisms mediating its therapeutic effects are not fully understood. This study investigated the effects of R848 on OVA-induced allergic rhinitis(AR) mice and the expression of IL-25, IL-33, TSLP, T-cell immunoglobulin mucin1 (TIM1) and T-cell immunoglobulin mucin3 (TIM3). BALB/c mice were intranasally sensitized and challenged with ovalbumin (OVA), and R848 was intraperitoneally injected into AR mice. Histological changes in the nasal mucosa were evaluated by hematoxylin and eosin (H & E) and Periodic Acid-Schiff (PAS) staining; cytokine levels in serum were measured with enzyme-linked immunosorbent assays (ELISAs);the mRNA expression levels of IFN-γ, IL-17 and Foxp3 in the spleen determined by quantitative real-time RT-PCR (qRT-PCR); the proportions of Th1, Th2, Th17, Treg and TIM3 + IFN-γ + Th1 cells in the spleen were assessed with flow cytometry; TIM1, TIM3 and IL-33 expression levels in the nasal mucosa were evaluated with immunofluorescence staining(IF).R848 alleviated the nasal allergic symptoms; reduced eosinophil cell infiltration, goblet cell hyperplasia in the nasal mucosa; reduced IL-13, IL-17, IL-25 and IL-33 levels in serum; upregulated the relative mRNA expression of IFN-γ and Foxp3, and downregulated the relative mRNA expression of IL-17 in the spleen; decreased Th2, Th17 and TIM3 + IFN-γ + Th1 cells ratios, increased the proportion of Th1 and Treg cells in the spleen; suppressed TIM1 and TIM3,but increased IL-33 expression in the nasal mucosa in OVA-induced AR mice. R848 suppresses IL-25, IL-33 released and TIM1, TIM3 expression, which may contribute to its anti-allergic effects.

Topics: Animals; Anti-Allergic Agents; Antigens; Cytokines; Disease Models, Animal; Female; Hepatitis A Virus Cellular Receptor 1; Hepatitis A Virus Cellular Receptor 2; Imidazoles; Mice, Inbred BALB C; Nasal Mucosa; Ovalbumin; Rhinitis, Allergic; Spleen

Therapeutic vaccination is and remains a major challenge, particularly in cancer treatment. In this process, the effective activation of dendritic cells by a combination of distinctly acting adjuvants and an antigen is crucial for success. While most common vaccine formulations lack the efficiency to trigger sufficient T cell responses in a therapeutic tumor treatment, nanovaccines offer unique properties to tackle that challenge. Here, we report the stepwise development of a nanocapsule for vaccination approaches, comprising a shell consisting of antigen and loaded with a superadditive adjuvant combination. In a first initial step, we identified the combination of resiquimod (R848) and muramyl dipeptide (MDP) to have a superadditive stimulatory potential. Particulated in Spermine-modified dextran-nanoparticles, the dual-adjuvant maintains its superadditive character and stimulates murine dendritic cells (DC) stronger than the soluble equivalents. The second step was to evaluate a protein-based nanocapsule as suitable antigen source for the induction of antigen-specific T cell responses. Therefore, the DC-mediated antigen-specific T cell proliferation upon treatment with nanocapsules, whose shell consists of ovalbumin (OVA), was assessed. At least, the superadditive adjuvant combination was encapsulated into OVA-nanocapsules to create the final nanovaccine. Its immunostimulatory potential for DC was extensively tested by measuring the expression of co-stimulatory surface markers, the secretion of pro-inflammatory cytokines and the capability to mediate OVA-specific T cell responses. The developed nanovaccine triggers strong superadditive dendritic cell stimulation and potent antigen-specific CD4

Topics: Acetylmuramyl-Alanyl-Isoglutamine; Adjuvants, Pharmaceutic; Animals; Antigens; Cancer Vaccines; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Line; Cell Proliferation; Cell Survival; Cytokines; Dendritic Cells; Dextrans; Humans; Imidazoles; Mice, Inbred C57BL; Mice, Transgenic; Nanocapsules; Ovalbumin; Spermine; T-Lymphocytes

Objective To compare and characterize the Th1 immune responses induced by the most commonly used commercial Toll-like receptor (TLR) agonists through in vivo and ex vivo experiments. Methods The concentrations of IL-12 were tested by ELISA after mouse dendritic cells (DCs) in vitro were stimulated by one of tested TLR agonists, including poly(I:C), monophosphoryl lipid A (MPLA), resiquimod (R848), cytosine polyguanine-C (CpG-C). The changes in percentage and phenotype of DCs, NK cells and effector T cells in the draining lymph nodes were analyzed by flow cytometry after BALB/c mice were immunized with ovalbumin (OVA) mixed with selected TLR agonist. The serum concentrations of specific anti-OVA IgG2a in the immunized mice were determined by ELISA. Results Only CpG-C and R848 could significantly induce the production of IL-12 from bone marrow-derived DCs in vitro. Among the tested TLR agonists, R848 was the most effective adjuvant in recruiting DCs and NK cells into lymph nodes, inducing the proliferation of CD4

Topics: Adjuvants, Immunologic; Animals; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cytokines; Dendritic Cells; Imidazoles; Interleukin-12; Killer Cells, Natural; Lymph Nodes; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Ovalbumin; Toll-Like Receptors

Caspase-1 is required for nephritis and robust autoantibody development in the pristane model of murine lupus. The objective of this study was to evaluate the immune response and to study the splenic B and T cell populations in wild-type (WT) and caspase-1-/- mice following pristane injection in order to develop an understanding of why absence of caspase-1 is protective in pristane-induced lupus.. Immunization responses to NP-Ficoll and NP-ovalbumin were assessed in WT and caspase-1-/- mice. In vitro IgM and IgG responses to R848 were measured by ELISA. Serum IgM anti-dsDNA and IL-1β were also measured by ELISA. B and T cell populations 2 weeks and 6 months following pristane injection were measured by flow cytometry in WT and caspase-1-/- mice.. Caspase-1-/- mice generate equivalent IgG responses to NP-Ficoll and NP-ova antigens when compared to wild-type mice. Additionally, they secrete IgM and IgG in response to TLR7 activation. Pristane injected WT and caspase-1-/- mice generate robust IgM anti-dsDNA responses. Caspase-1-/- mice have a significant reduction in marginal zone B cell populations compared to WT 6 months after pristane exposure whereas T cell responses are intact in these mice.. Caspase-1-/- mice have intact immune responses but do not develop an expanded marginal zone B cell population in response to pristane-induced lupus. This may be one explanation for reduced IgG autoantibody production in these mice.

Topics: Animals; Antibodies, Antinuclear; B-Lymphocytes; Caspase 1; Cells, Cultured; Disease Models, Animal; Ficoll; Genetic Predisposition to Disease; Imidazoles; Immunization; Immunoglobulin G; Immunoglobulin M; Lupus Erythematosus, Systemic; Mice, Inbred BALB C; Mice, Knockout; Nitrophenols; Ovalbumin; Phenotype; Phenylacetates; Spleen; T-Lymphocytes; Terpenes; Time Factors

Cross-presentation of cellular antigens is crucial for priming CD8(+) T cells, and generating immunity to intracellular pathogens--particularly viruses. It is unclear which intestinal phagocytes perform this function in vivo. To address this, we examined dendritic cells (DCs) from the intestinal lymph of IFABP-tOVA 232-4 mice, which express ovalbumin in small intestinal epithelial cells (IECs). Among lymph DCs (LDCs) only CD103(+) CD11b(-) CD8α(+) DCs cross-present IEC-derived ovalbumin to CD8(+) OT-I T cells. Similarly, in the mesenteric lymph nodes (MLNs), cross-presentation of IEC-ovalbumin was limited to the CD11c(+) MHCII(hi) CD8α(+) migratory DCs, but absent from all other subsets, including the resident CD8α(hi) DCs. Crucially, delivery of purified CD8α(+) LDCs, but not other LDC subsets, into the MLN subcapsular lymphatic sinus induced proliferation of ovalbumin-specific, gut-tropic CD8(+) T cells in vivo. Finally, in 232-4 mice treated with R848, CD8α(+) LDCs were uniquely able to cross-prime interferon γ-producing CD8(+) T cells and drive their migration to the intestine. Our results clearly demonstrate that migrating CD8α(+) intestinal DCs are indispensable for cross-presentation of cellular antigens and, in conditions of inflammation, for the initial differentiation of effector CD8(+) T cells. They may therefore represent an important target for the development of antiviral vaccinations.

Topics: Animals; Antigens; CD8 Antigens; CD8-Positive T-Lymphocytes; Cell Movement; Cell Proliferation; Cells, Cultured; Cross-Priming; Dendritic Cells; Imidazoles; Interferon-gamma; Intestinal Mucosa; Lymph; Lymphocyte Activation; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Ovalbumin; Toll-Like Receptor 7

Delivering vaccine antigens to mucosal surfaces is potentially very attractive, especially as protection from mucosal infections may be mediated by local immune responses. However, to date mucosal immunization has had limited successes, with issues of both safety and poor immunogenicity. One approach to improve immunogenicity is to develop adjuvants that are effective and safe at mucosal surfaces. Differences in immune responses between mice and men have overstated the value of some experimental adjuvants which have subsequently performed poorly in the clinic. Due to their closer similarity, non-human primates can provide a more accurate picture of adjuvant performance. In this study we immunised rhesus macaques (Macaca mulatta) using a unique matrix experimental design that maximised the number of adjuvants screened while reducing the animal usage. Macaques were immunised by the intranasal, sublingual and intrarectal routes with the model protein antigens keyhole limpet haemocyanin (KLH), β-galactosidase (β-Gal) and ovalbumin (OVA) in combination with the experimental adjuvants Poly(I:C), Pam3CSK4, chitosan, Thymic Stromal Lymphopoietin (TSLP), MPLA and R848 (Resiquimod). Of the routes used, only intranasal immunization with KLH and R848 induced a detectable antibody response. When compared to intramuscular immunization, intranasal administration gave slightly lower levels of antigen specific antibody in the plasma, but enhanced local responses. Following intranasal delivery of R848, we observed a mildly inflammatory response, but no difference to the control. From this we conclude that R848 is able to boost antibody responses to mucosally delivered antigen, without causing excess local inflammation.

Topics: Adjuvants, Immunologic; Administration, Intranasal; Animals; Antibodies; Antibody Formation; beta-Galactosidase; Chitosan; Cytokines; Female; Hemocyanins; Imidazoles; Immunity, Mucosal; Immunization; Immunoglobulin A; Immunoglobulin G; Inflammation; Injections, Intramuscular; Lipopeptides; Macaca mulatta; Mucous Membrane; Ovalbumin; Poly I-C; Thymic Stromal Lymphopoietin

The trafficking of effector T cells is tightly regulated by the expression of site-specific sets of homing molecules. In contrast, naive T cells are generally assumed to express a uniform pattern of homing molecules and to follow a random distribution within the blood and secondary lymphoid organs. In this study, we demonstrate that systemic infection fundamentally modifies the trafficking of circulating naive CD8(+) T cells. We show that on naive CD8(+) T cells, the constitutive expression of the integrin α4β7 that effects their entry into GALT is downregulated following infection of mice with Salmonella typhimurium. We further show that this downregulation is dependent on TLR signaling, and that the TLR-activated naive CD8(+) T cells are blocked from entering GALT. This contrasts strongly with Ag-experienced effector T cells, for which TLR costimulation in the GALT potently upregulates α4β7 and enhances trafficking to intestinal tissues. Thus, TLR activation leads to opposite effects on migration of naive and effector CD8(+) T cells. Our data identify a mechanism that excludes noncognate CD8(+) T cells from selected immune compartments during TLR-induced systemic inflammation.

Topics: Animals; CD8-Positive T-Lymphocytes; Cell Movement; Cell Proliferation; Dendritic Cells; Down-Regulation; Female; Imidazoles; Integrins; Interleukin-12 Subunit p40; Interleukin-6; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; Ovalbumin; Salmonella Infections; Salmonella typhimurium; Signal Transduction; T-Lymphocyte Subsets; Toll-Like Receptors

Epidemiological studies suggest that viral infections during childhood are a risk factor for the development of asthma. However, the role of virus-specific pattern recognition receptors in this process is not well defined. In the current study, we compare the effects of the inhaled viral TLR ligands polyinosinic-polycytidylic acid (TLR3) and resiquimod (TLR7/8) on sensitization to a model allergen (OVA) in a murine model. Both compounds enhance the migration, activation, and Ag-processing of myeloid dendritic cells from the lung to the draining lymph nodes comparable to the effects of LPS. Application of polyinosinic-polycytidylic acid [poly(I:C)] or LPS induces production of allergen-specific IgE and IgG1, whereas resiquimod (R848) had no effect. In addition, rechallenge of mice with OVA resulted in airway inflammation and mucus production in animals that received either poly(I:C) or LPS but not after application of R848. In summary, these results show that activation of TLR3 in combination with inhaled allergen results in induction of dendritic cell activation and migration similar to the effects of LPS. This leads to the development of allergic airway disease after allergen rechallenge, whereas mice treated with R848 did not develop allergic airway disease. These findings give further insight into the effects of stimulation of different TLRs on the development of asthma.

Topics: Administration, Inhalation; Allergens; Animals; Dendritic Cells; Disease Models, Animal; Hypersensitivity; Imidazoles; Ligands; Lipopolysaccharides; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Ovalbumin; Poly I-C; Toll-Like Receptor 3; Toll-Like Receptor 7; Toll-Like Receptor 8

It has been documented that TLR7 stimulation triggers not only antiviral responses, but also alleviates experimental asthma. Considering the implication of invariant NKT (iNKT) cells in both situations, we postulated that they might contribute to the anti-inflammatory effect of TLR7 ligands. We show in this study that spleen cells activated by the TLR7 agonist resiquimod (R848) attenuate allergic inflammation upon adoptive transfer when they are recovered from wild-type, but not from iNKT cell-deficient Jα18(-/-) mice, which proves the specific involvement of this regulatory population. Furthermore, we provide evidence that IFN-γ is critical for the protective effect, which is lost when transferred iNKT cells are sorted from IFN-γ-deficient mice. In support of a direct activation of iNKT cells through TLR7 signaling in vivo, we observed a prompt increase of serum IFN-γ levels, associated with upregulation of CD69 expression on iNKT cells. Moreover, we demonstrate that iNKT cells effectively express TLR7 and respond to R848 in vitro by producing high levels of IFN-γ in the presence of IL-12, consistent with the conclusion that their contribution to the alleviation of allergic inflammation upon treatment with TLR7 ligands is mediated through IFN-γ.

Topics: Adoptive Transfer; Animals; Anti-Allergic Agents; Asthma; Drug Delivery Systems; Imidazoles; Inflammation Mediators; Interferon-gamma; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Natural Killer T-Cells; Ovalbumin; Toll-Like Receptor 7

The evolution of allergic asthma is tightly controlled by effector and regulatory cells, as well as cytokines such as IL-10 and/or TGF-β, and it is widely acknowledged that environmental exposure to allergens and infectious agents can influence these processes. In this context, the recognition of pathogen-associated motifs, which trigger TLR activation pathways, plays a critical role with important consequences for disease progression and outcome. We addressed the question whether the TLR7 ligand resiquimod (R848), which has been shown to be protective in several experimental allergic asthma protocols, can also suppress typical asthma symptoms once the disease is established. To this end, we used an OVA-induced experimental model of murine allergic asthma in which R848 was injected after a series of challenges with aerosolized OVA. We found that the treatment attenuated allergic symptoms through a mechanism that required Tregs, as assessed by the expansion of this population in the lungs of mice having received R848, and the loss of R848-mediated suppression of allergic responses after in vivo Treg depletion. IL-10 provided only a minor contribution to this suppressive effect that was largely mediated through a TGF-β-dependent pathway, a finding that opens new therapeutic opportunities for the pharmacological targeting of Tregs.

Topics: Animals; Asthma; Disease Models, Animal; Imidazoles; Interleukin-10; Lung; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Ovalbumin; T-Lymphocytes, Regulatory; Toll-Like Receptor 7; Transforming Growth Factor beta

TLR agonists have been suggested to directly impact Tregs, thereby enhancing or reversing their suppressive function. Here, in order to select TLR agonists leading to potent effector T-cell responses, while minimizing Treg inhibitory function, we used a model antigen, covalently linked to an inert delivery system, combined with a large panel of TLR agonists, for the immunization of mice with an attenuated/depleted or intact Treg subset. We observed that the negative modulation of effector CD4(+) T cells exerted by Tregs cannot be circumvented, whatever the TLR agonist used as adjuvant. To better understand the impact of TLR agonists on Tregs, we investigated (i) the TLR expression profile of highly purified CD4(+) Foxp3(+) Tregs, at steady state or subsequent to in vivo activation by TLR agonists and (ii) the Treg phenotype after in vivo and in vitro activation by TLR agonists. Our results demonstrate that TLR agonists, as single signal inducers, are not able to directly activate Tregs. The phenotypic Treg activation observed in vivo, following TLR administration, does not result from cross-talk with conventional T cells but is rather a consequence of the interaction with other immune cell type(s).

Topics: Adjuvants, Immunologic; Amino Acid Sequence; Animals; CD4-Positive T-Lymphocytes; Female; Flow Cytometry; Forkhead Transcription Factors; Gene Expression Profiling; Green Fluorescent Proteins; Imidazoles; Lipopolysaccharides; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Molecular Sequence Data; Oligodeoxyribonucleotides; Ovalbumin; Peptide Fragments; Poly I-C; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes, Regulatory; Toll-Like Receptors; Zymosan

Resiquimod is a compound belonging to the imidazoquinoline family of compounds known to signal through Toll-like receptor 7. Resiquimod treatment has been demonstrated to inhibit the development of allergen induced asthma in experimental models. The aim of the present study was to elucidate the molecular processes that were altered following resiquimod treatment and allergen challenge in a mouse model of allergic asthma. Employing microarray analysis, we have characterized the "asthmatic" transcriptome of the lungs of A/J and C57BL/6 mice and determined that it includes genes involved in the control of cell cycle progression, the complement and coagulation cascades, and chemokine signaling. Our results demonstrated that resiquimod treatment resulted in the normalization of the expression of genes involved with airway remodeling, and generally, chemokine signaling. Resiquimod treatment also altered the expression of cell adhesion molecules, and molecules involved in natural killer (NK) cell-mediated cytotoxicity. Furthermore, we have demonstrated that systemic resiquimod administration resulted in the recruitment of NK cells to the lungs and livers of the mice, although no causal relationship between NK cell recruitment and treatment efficacy was found. Overall, our findings identified several genes, important in the development of asthma pathology, that were normalized following resiquimod treatment, thus improving our understanding of the molecular consequences of resiquimod treatment in the lung milieu. The recruitment of NK cells to the lungs may also have application in the treatment of virally induced asthma exacerbations.

Topics: Animals; Asthma; Bronchoalveolar Lavage Fluid; CD8 Antigens; Flow Cytometry; Gene Expression Profiling; Gene Expression Regulation; Imidazoles; Killer Cells, Natural; Mice; Mice, Inbred C57BL; Mice, Knockout; Ovalbumin; Reverse Transcriptase Polymerase Chain Reaction

Based on epidemiological data, the hygiene hypothesis associates poor hygienic living conditions during childhood with a lower risk for the development of allergic diseases such as bronchial asthma. The role of viral infections, and especially of viral TLR ligands, within this context remains to be clarified. Viral TLR ligands involve dsRNA and ssRNA which are recognized by TLR-3 or TLR-7, respectively. In this study, we evaluated the impact of TLR-3 or TLR-7 activation on experimental asthma in mice. Systemic application of the synthetic TLR-3 or TLR-7 ligands polycytidylic-polyinosinic acid (p(I:C)) or R-848, respectively, during the sensitization phase prevented the production of OVA-specific IgE and IgG1 Abs and subsequently abolished all features of experimental asthma including airway hyperresponsiveness and allergic airway inflammation. Furthermore, administration of p(I:C) or R-848 to animals with already established primary allergic responses revealed a markedly reduced secondary response following allergen aerosol rechallenges. In contrast to wild-type animals, application of p(I:C) or R-848 to IL-12p35(-/-) mice had no effect on airway inflammation, goblet cell hyperplasia, and airway hyperresponsiveness. However, in the absence of IL-12, the numbers of eosinophils and lymphocytes in bronchoalveolar lavage fluids were still significantly reduced. These partial effects could also be abolished by neutralizing anti-IL-10 Abs in IL-12p35(-/-) mice. These data indicate that TLR-3 or TLR-7 activation by viral TLR ligands has both preventive as well as suppressive effects on experimental asthma which is mediated by the additive effects of IL-12 and IL-10.

Topics: Animals; Antibodies; Antiviral Agents; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Cytokines; Female; Imidazoles; Immunoglobulin E; Immunoglobulin G; Immunosuppression Therapy; Interleukin-10; Interleukin-12; Interleukin-12 Subunit p35; Ligands; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mice, Mutant Strains; Ovalbumin; Poly I-C; Toll-Like Receptor 3; Toll-Like Receptor 7

Complexing TLR9 agonists such as plasmid DNA to cationic liposomes markedly potentiates their ability to activate innate immunity. We therefore reasoned that liposomes complexed with DNA or other TLR agonists could be used as effective vaccine adjuvants. To test this hypothesis, the vaccine adjuvant effects of liposomes complexed to TLR agonists were assessed in mice. We found that liposomes complexed to nucleic acids (liposome-Ag-nucleic acid complexes; LANAC) were particularly effective adjuvants for eliciting CD4(+) and CD8(+) T cell responses against peptide and protein Ags. Notably, LANAC containing TLR3 or TLR9 agonists effectively cross-primed CD8(+) T cell responses against even low doses of protein Ags, and this effect was independent of CD4(+) T cell help. Ag-specific CD8(+) T cells elicited by LANAC adjuvants were functionally active and persisted for long periods of time in tissues. In a therapeutic tumor vaccine model, immunization with the melanoma peptide trp2 and LANAC adjuvant controlled the growth of established B16 melanoma tumors. In a prophylactic vaccine model, immunization with the Mycobacterium tuberculosis protein ESAT-6 with LANAC adjuvant elicited significant protective immunity against aerosol challenge with virulent M. tuberculosis. These results suggest that certain TLR agonists can be combined with cationic liposomes to produce uniquely effective vaccine adjuvants capable of eliciting strong T cell responses against protein and peptide Ags.

Topics: Adaptor Proteins, Signal Transducing; Adjuvants, Immunologic; Animals; Cancer Vaccines; Cations; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cross-Priming; Female; Imidazoles; Lipopolysaccharides; Liposomes; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Knockout; Mycobacterium tuberculosis; Myeloid Differentiation Factor 88; Oligodeoxyribonucleotides; Ovalbumin; Poly I-C; Signal Transduction; Toll-Like Receptor 3; Toll-Like Receptor 9; Zymosan

Imiquimod, an immune response modifier and inducer of cytokines in vitro and in vivo, has been shown to have potent antiviral and antitumour activity and to act as an adjuvant for protein vaccination. We have undertaken studies in mice to investigate the potential of imiquimod and resiquimod to adjuvant DNA vaccination. These imidazoquinolines were administered by subcutaneous injection at the vaccination site immediately after particle-mediated immunotherapeutic delivery of plasmid DNA using a gene gun. Imiquimod was found to increase the number and maturation status of dendritic cells in draining lymph nodes, and to enhance antigen-specific CD4(+) and CD8(+) T cell responses, as assessed by analyses of clonal expansion, and the quantity and kinetics of cytokine production from these cells in lymph nodes and spleens collected after vaccination. A more substantial increase in IFN-gamma-producing, compared with IL-4-producing CD4(+) T cells suggested that imiquimod biased the immune response towards a predominance of Th1 cells. The analogue resiquimod was found to be to produce a similar Th1 biased immune response with a 10-fold reduced dose compared with imiquimod. Collectively, these studies suggest that both imiquimod and resiquimod may be suitable adjuvants for therapeutic DNA vaccines requiring induction of potent cytotoxic T cell responses.

Topics: Adjuvants, Immunologic; Aminoquinolines; Animals; Biolistics; CD11 Antigens; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cytokines; Imidazoles; Imiquimod; Immunization; Immunotherapy; Injections, Subcutaneous; Interferon-gamma; Lymph Nodes; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Microspheres; Ovalbumin; Plasmids; Th1 Cells; Vaccines, DNA

Allergen-induced sensitization and airway disease are the results of adverse immune reactions against environmental antigens that may be prevented or inhibited by immune modifying strategies.. To investigate the effects of the novel immune response modifier resiquimod (R-848), from the family of imidazol-derivates, in a murine model of allergen-mediated Th2-immune responses and concomitant airway inflammation and airway hyper-reactivity.. BALB/c mice were systemically sensitized with ovalbumin (OVA) on days 1 and 14 and challenged with OVA aerosol on days 28 and 29. R-848 was applied intranasally to sensitized animals once prior to the first allergen airway challenge, on day 27.. A single application of R-848 significantly reduced numbers of eosinophils and lymphocytes in bronchoalveolar lavage fluid and inhibited mucus gland hyperplasia, compared with sensitized and challenged controls. Associated with the decrease in airway inflammation, single intranasal treatment with R-848 abolished the development of airway hyper-reactivity after allergen sensitization and airway challenges. Additionally, Th2-cytokine production in lung tissues from sensitized and R-848-treated animals was reduced, whereas IL-12 and IFN-gamma production was increased, compared with non-treated sensitized mice.. These data indicate that R-848 effectively inhibits allergen-induced airway inflammation and hyper-reactivity by modulation of increased Th2-immune responses.

Topics: Allergens; Animals; Asthma; Bronchi; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Female; Imidazoles; Immunologic Factors; Interferon-gamma; Interleukin-12; Interleukin-4; Interleukin-5; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Ovalbumin; Th2 Cells

Imiquimod and R-848 are members of a family of immune response modifiers that stimulate cytokine production in monocyte/macrophages and dendritic cell cultures. This study evaluated the effects of the imidazoquinolines, imiquimod and R-848, on B lymphocyte activation. Both agents induced proliferation of murine T-cell-depleted and highly purified splenic B cell preparations as well as purified human B cells. Resting and activated B cells responded to these agents, with activated cells responding more efficiently. B cells from the LPS-hyporesponsive C3H/HeJ mice and guanosine-hyporesponsive SJL mice proliferated in response to imiquimod and R-848, indicating a different mechanism of action than lipopolysaccharide and guanine nucleosides. B cells were also stimulated by imiquimod and R-848 to produce increased immunoglobulin levels. Increased expression of a number of B cell activation markers were seen following imiquimod or R-848 stimulation. Finally, R-848 was shown to act as a vaccine adjuvant enhancing OVA-specific IgG2a levels while suppressing total IgE. These results indicate that R-848 and imiquimod are potent activators of B lymphocytes and are capable of augmenting antigen-specific immunoglobulin production.

Topics: Adjuvants, Immunologic; Aminoquinolines; Animals; Antibody Specificity; Antigens, CD; B-Lymphocytes; B7-2 Antigen; Cells, Cultured; Guanosine; Humans; Imidazoles; Imiquimod; Immunoglobulin Class Switching; Immunoglobulin E; Immunoglobulin G; Lipopolysaccharides; Lymphocyte Activation; Membrane Glycoproteins; Mice; Mice, Inbred Strains; Ovalbumin; Spleen

R-848 and imiquimod belong to a class of immune response modifiers that are potent inducers of cytokines, including IFN-alpha, TNF-alpha, IL-12, and IFN-gamma. Many of these cytokines can affect the acquired immune response. This study examines the effects of R-848 on aspects of acquired immunity, including immunoglobulin secretion, in vivo cytokine production, and Ag-specific T cell cytokine production. Results are compared with those of Th1 CpG ODN. R-848 and CpG ODN are effective at skewing immunity in the presence of Alum toward a Th1 Ab response (IgG2a) and away from a Th2 Ab response (IgE). R-848 and CpG ODN are also capable of initiating an immune response in the absence of additional adjuvant by specifically enhancing IgG2a levels. Both R-848 and imiquimod showed activity when given subcutaneously or orally, indicating that the compound mechanism was not through generation of a depot effect. Although CpG ODN behaves similarly to R-848, CpG ODN has a distinct cytokine profile, is more effective than R-848 when given with Alum in the priming dose, and is active only when given by the same route as the Ag. The mechanism of R-848's adjuvant activity is linked to cytokine production, where increases in IgG2a levels are associated with IFN-alpha, TNF-alpha, IL-12, and IFN-gamma induction, and decreases in IgE levels are associated with IFN-alpha and TNF-alpha. Imiquimod also enhances IgG2a production when given with Ag. The above results suggest that the imidazoquinolines R-848 and imiquimod may be attractive compounds for use as vaccine adjuvants and in inhibiting pathological responses mediated by Th2 cytokines.

Topics: Adjuvants, Immunologic; Administration, Oral; Animals; Cell Separation; Cytokines; Female; Imidazoles; Immunization, Secondary; Immunoglobulin E; Immunoglobulin G; Interferon-gamma; Mice; Mice, Inbred BALB C; Oligodeoxyribonucleotides; Ovalbumin; Spleen; Th1 Cells; Th2 Cells; Vaccination