[Show abstract][Hide abstract] ABSTRACT: H1N1 influenza viruses mutate rapidly, rendering vaccines developed in any given year relatively ineffective in subsequent years. Thus it is necessary to generate new vaccines every year, but this is time-consuming and resource-intensive. Should a highly virulent influenza strain capable of human-to-human transmission emerge, these factors will severely limit the number of people that can be effectively immunised against that strain in time to prevent a pandemic. An adjuvant and mode of administration capable of rendering ordinarily unprotective vaccine doses protective would thus be highly advantageous.
The carbohydrate mannan was conjugated to whole inactivated H1N1 influenza virus at a range of ratios, and mixed with it at a range of ratios, and various doses of the resulting preparations were administered to mice via the intranasal (IN) route. Serum immunity was assessed via antigen-specific IgG ELISA and the haemagglutination-inhibition (HI) assay, and mucosal immunity was assessed via IgA ELISA of bronchio-alveolar lavages.
IN-administered inactivated H1N1 mixed with mannan induced higher serum IgG and respiratory-tract IgA than inactivated H1N1 conjugated to mannan, and HIN1 alone. Adjuvantation was mannan-dose-dependent, with 100 μg of mannan adjuvanting 1 μg of H1N1 more effectively than 10 or 50 μg of mannan. Serum samples from mice immunised with 1 μg H1N1 adjuvanted with 10 μg mannan did not inhibit agglutination of red blood cells (RBCs) at a dilution factor of 10 in the HI assay, but samples resulting from adjuvantation with 50 and 100 μg mannan inhibited agglutination at dilution factors of ≥ 40. Both serum IgG1 and IgG2a were induced by IN mannan-adjuvanted H1N1 vaccination, suggesting the induction of humoral and cellular immunity.
Mixing 100 μg of mannan with 1 μg of inactivated H1N1 adjuvanted the vaccine in mice, such that IN immunisation induced higher serum IgG and respiratory tract IgA than immunisation with virus alone. The serum from mice thus immunised inhibited H1N1-mediated RBC agglutination strongly in vitro. If mannan similarly adjuvants low doses of influenza vaccine in humans, it could potentially be used for vaccine 'dose-sparing' in the event that a vaccine shortage arises from an epidemic involving a highly virulent human-to-human transmissable influenza strain.
[Show abstract][Hide abstract] ABSTRACT: Cell penetrating peptides (CPP), including the TAT peptide from the human immunodeficiency virus transactivator of transcription (HIV-TAT) protein and penetratin from Drosophila Antennapedia homeodomain protein, translocate various cargos including peptides and proteins across cellular barriers. This mode of delivery has been harnessed by our group and others to deliver antigenic proteins or peptides into the cytoplasm of antigen processing cells (APC) such as monocyte-derived dendritic cells (MoDC). Antigens or T cell epitopes delivered by CPP into APC in vivo generate antigen-specific cytotoxic T cell and helper T cell responses in mice. Furthermore, mice immunised with these peptides or proteins are protected from a tumour challenge. The functional properties of CPP are dependent on the various cargos being delivered and the target cell type. Despite several studies demonstrating superior immunogenicity of TAT and Antp-based immunogens, none has compared the immunogenicity of antigens delivered by TAT and Antp CPP. In the current study we demonstrate that a cytotoxic T cell epitope from the mucin 1 (MUC1) tumour associated antigen, when delivered by TAT or Antp, generates identical immune responses in mice resulting in specific MUC1 T cell responses as measured by in vivo CTL assays, IFNγ ELISpot assays and prophylactic tumour protection.
[Show abstract][Hide abstract] ABSTRACT: The interaction of Abs with their specific FcRs is of primary importance in host immune effector systems involved in infection and inflammation, and are the target for immune evasion by pathogens. FcγRIIa is a unique and the most widespread activating FcR in humans that through avid binding of immune complexes potently triggers inflammation. Polymorphisms of FcγRIIa (high responder/low responder [HR/LR]) are linked to susceptibility to infections, autoimmune diseases, and the efficacy of therapeutic Abs. In this article, we define the three-dimensional structure of the complex between the HR (arginine, R134) allele of FcγRIIa (FcγRIIa-HR) and the Fc region of a humanized IgG1 Ab, hu3S193. The structure suggests how the HR/LR polymorphism may influence FcγRIIa interactions with different IgG subclasses and glycoforms. In addition, mutagenesis defined the basis of the epitopes detected by FcR blocking mAbs specific for FcγRIIa (IV.3), FcγRIIb (X63-21), and a pan FcγRII Ab (8.7). The epitopes detected by these Abs are distinct, but all overlap with residues defined by crystallography to contact IgG. Finally, crystal structures of LR (histidine, H134) allele of FcγRIIa and FcγRIIa-HR reveal two distinct receptor dimers that may represent quaternary states on the cell surface. A model is presented whereby a dimer of FcγRIIa-HR binds Ag-Ab complexes in an arrangement that possibly occurs on the cell membrane as part of a larger signaling assembly.
The Journal of Immunology 08/2011; 187(6):3208-17. DOI:10.4049/jimmunol.1101467 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cytoplasmic delivery and cross-presentation of proteins and peptides is necessary for processing and presentation of antigens for the generation of cytotoxic T cells. We previously described the use of the 16 amino acid peptide penetratin from the Drosophila Antennapedia homeodomain (penetratin, Antp) to transport cytotoxic T lymphocyte epitopes derived from ovalbumin (OVA) or the Mucin-1 tumor-associated antigen into cells. We have now shown that penetratin covalently conjugated to OVA protein and linked in tandem to CD4(+) and/or CD8(+) T-cell epitopes from OVA-stimulated T cells in vitro (B3Z T-cell hybridoma and OT-I and OT-II T cells). The induction of these responses was directly mediated by the penetratin peptide as linking a nonspecific 16-mer peptide to OVA or mixing did not induce CD8(+) or CD4(+) T-cell responses in vitro. Furthermore, interferon (IFN)-γ-secreting CD4(+) and CD8(+) T cells were induced which suppressed B16.OVA tumor growth in C57BL/6 mice. Tumor protection was mediated by a CD8(+) T-cell-dependent mechanism and did not require CD4(+) help to protect mice 7 days after a boost immunization. Alternatively, 40 days after a boost immunization, the presence of CD4(+) help enhanced antigen-specific IFN-γ-secreting CD8(+) T cells and tumor protection in mice challenged with B16.OVA. Long-term CD8 responses were equally enhanced by antigen-specific and universal CD4 help. In addition, immunization with AntpOVA significantly delayed growth of B16.OVA tumors in mice in a tumor therapy model.
[Show abstract][Hide abstract] ABSTRACT: Receptor mediated gene delivery is an attractive non-viral method for targeting genetic material to specific cell types. We have previously utilized oxidized (OMPLL) and reduced mannan poly-L-lysine (RMPLL) to target DNA vaccines to antigen presenting cells and demonstrated that it could induce far stronger immune responses in mice compared to naked DNA immunization. In this study, we describe the immune enhancing attributes of mannan-PLL mediated DNA vaccination at the molecular level. Several attributes observed in similar gene delivery conjugates, such as entry via the endocytic pathway, low toxicity, protection from nucleases and compaction of particle size, were also evident here. In addition, OMPLL and RMPLL conjugates had profound effects on the antigen presentation functions of dendritic cells and macrophages, through the stimulation of cytokine production and maturation of dendritic cells. Interestingly, we demonstrate that OMPLL-DNA and RMPLL-DNA are able to mediate dendritic cell activation via toll-like receptor 2 as opposed to mannan alone which mediates via toll-like receptor 4. Overall, this report leads to greater understanding of how oxidized and reduced mannan mediated gene delivery could augment immune responses to DNA vaccination and provide insights into ways of further improving its immunogenicity.
[Show abstract][Hide abstract] ABSTRACT: The interaction of immune complexes with the human Fc receptor, FcgammaRIIa, initiates the release of inflammatory mediators and is implicated in the pathogenesis of human autoimmune diseases, including rheumatoid arthritis and systemic lupus erythematosus, so this FcR is a potential target for therapy. We have used the three-dimensional structure of an FcgammaRIIa dimer to design small molecule inhibitors, modeled on a distinct groove and pocket created by receptor dimerization, adjacent to the ligand-binding sites. These small chemical entities (SCEs) blocked immune complex-induced platelet activation and aggregation and tumor necrosis factor secretion from macrophages in a human cell line and transgenic mouse macrophages. The SCE appeared specific for FcgammaRIIa, as they inhibited only immune complex-induced responses and had no effect on responses to stimuli unrelated to FcR, for example platelet stimulation with arachidonic acid. In vivo testing of the SCE in FcgammaRIIa transgenic mice showed that they inhibited the development and stopped the progression of collagen-induced arthritis (CIA). The SCEs were more potent than methotrexate and anti-CD3 in sustained suppression of CIA. Thus, in vitro and in vivo activity of these SCE FcgammaRIIa receptor antagonists demonstrated their potential as anti-inflammatory agents for autoimmune diseases involving immune complexes.
[Show abstract][Hide abstract] ABSTRACT: DNA immunization is an attractive form of vaccination, which has shown promising results only in small animal models. There is a need to develop efficient gene delivery systems. We previously demonstrated that oxidized (OM) and reduced mannan (RM) complexed to ovalbumin DNA via poly-l-lysine (PLL), were able to generate potent immune responses in mice. Herein, we further investigated the suitability of OMPLL and RMPLL as carriers for mucin 1 (MUC1) DNA vaccination for cancer immunotherapy. Studies presented here showed that immune responses in C57BL/6 mice induced by OMPLL-MUC1 DNA and RMPLL-MUC1 DNA immunization were more immunogenic compared to MUC1 DNA alone. Moreover, tumor protection was evident at a dose as low as 0.5 microg. In addition, strong T cell responses were induced in HLA-A2 transgenic and human MUC1 transgenic mice. These results demonstrate the potential of OM and RM as efficient non-viral gene delivery carriers for DNA vaccines for use in cancer immunotherapy.
[Show abstract][Hide abstract] ABSTRACT: 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 cross-present 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.
European Journal of Immunology 03/2008; 38(2):424-36. DOI:10.1002/eji.200737578 · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Perturbation of the homeostatic balance between activation and inhibition via signals transmitted after cross linking of cell surface receptors such as FcR can result in autoimmune disease due to activation of T cells, B cells and initiation of the inflammatory cytokine cascade. FcgammaRIIa, the most widespread activating FcR in humans, with alleles associated with autoimmunity, is not normally found in mice. We have previously shown that mice carrying a huFcgammaRIIa transgene on a mixed background (SJL/B6) are susceptible to both induced and spontaneous autoimmune disease. Here we show that mice with this transgene on a non-susceptible C57BL/6 background can develop both induced and passively transferred immune complex mediated arthritis, but no spontaneous disease. Thus the transgene acting in concert with other susceptibility genes increased disease development. The transgene is expressed on mature bone marrow derived DC, neutrophils, platelets and macrophages and increased activation of these cells may promote susceptibility to disease in transgenic animals. Disease can be treated with anti-T or B cell agents, or specifically by blocking the FcgammaRIIa with anti-FcgammaRIIa fragments or small chemical entities designed to bind to the FcR dimer interface. The presence of enhanced passive and induced autoimmunity in two transgenic mice strains supports the hypothesis that huFcgammaRIIa is an important factor in the pathogenesis of autoimmune inflammation and a valid target for therapeutic intervention.