ovalbumin and maleic-acid

We aimed to highlight the utility of novel dissolving microneedle (MN)-based delivery systems for enhanced transdermal protein delivery. Vaccination remains the most accepted and effective approach in offering protection from infectious diseases. In recent years, much interest has focused on the possibility of using minimally invasive MN technologies to replace conventional hypodermic vaccine injections.. The focus of this study was exploitation of dissolving MN array devices fabricated from 20% w/w poly(methyl vinyl ether/maleic acid) using a micromoulding technique, for the facilitated delivery of a model antigen, ovalbumin (OVA).. A series of in-vitro and in-vivo experiments were designed to demonstrate that MN arrays loaded with OVA penetrated the stratum corneum and delivered their payload systemically. The latter was evidenced by the activation of both humoral and cellular inflammatory responses in mice, indicated by the production of immunoglobulins (IgG, IgG1, IgG2a) and inflammatory cytokines, specifically interferon-gamma and interleukin-4. Importantly, the structural integrity of the OVA following incorporation into the MN arrays was maintained.. While enhanced manufacturing strategies are required to improve delivery efficiency and reduce waste, dissolving MN are a promising candidate for 'reduced-risk' vaccination and protein delivery strategies.

Topics: Administration, Cutaneous; Animals; Antigens; Drug Delivery Systems; Interferon-gamma; Interleukin-4; Lasers; Maleates; Methyl Ethers; Mice; Microinjections; Needles; Ovalbumin; Polyvinyls; Skin; Skin Absorption; Solubility; Vaccination; Vaccines

Pathways for loading exogenous protein-derived peptides on MHC class I are thought to be present mainly in monocyte-lineage cells and to involve phagocytosis- or macropinocytosis-mediated antigenic leakage into either cytosol or extracellular milieu to give peptide access to MHC class I. We show that maleylation of OVA enhanced its presentation to an OVA-specific MHC class I-restricted T cell line by both macrophages and B cells. This enhanced presentation involved uptake through receptors of scavenger receptor (SR)-like ligand specificity, was TAP-1-independent, and was inhibited by low levels (2 mM) of ammonium chloride. No peptide loading of bystander APCs by maleylated (maleyl) OVA-pulsed macrophages was detected. Demaleylated maleyl-OVA showed enhanced MHC class I-restricted presentation through receptor-mediated uptake and remained highly sensitive to 2 mM ammonium chloride. However, if receptor binding of maleyl-OVA was inhibited by maleylated BSA, the residual presentation was relatively resistant to 2 mM ammonium chloride. Maleyl-OVA directly introduced into the cytosol via osmotic lysis of pinosomes was poorly presented, confirming that receptor-mediated presentation of exogenous maleyl-OVA was unlikely to involve a cytosolic pathway. Demaleylated maleyl-OVA was well presented as a cytosolic Ag, consistent with the dependence of cytosolic processing on protein ubiquitination. Thus, receptor-specific delivery of exogenous protein Ags to APCs can result in enhanced MHC class I-restricted presentation, suggesting that the exogenous pathway of peptide loading for MHC class I may be a constitutive property dependent mainly on the quantity of Ag taken up by APCs.

Topics: Ammonium Chloride; Animals; Antigen Presentation; ATP Binding Cassette Transporter, Subfamily B, Member 2; ATP-Binding Cassette Transporters; B-Lymphocytes; Cytosol; Endosomes; Histocompatibility Antigens Class I; Hydrogen-Ion Concentration; Ligands; Macrophages, Peritoneal; Maleates; Membrane Proteins; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Knockout; Ovalbumin; Protein Binding; Receptors, Immunologic; Receptors, Lipoprotein; Receptors, Scavenger; Scavenger Receptors, Class B; Serum Albumin, Bovine

We have maleylated proteins to target macrophage-specific scavenger receptors and have used this system to study changes in the epitopes and immunogenicity of such proteins. We show that maleylation of diphtheria toxoid (DT) induces targeting to macrophage scavenger receptors and enhances its immunogenicity. DT does not evoke detectable serum Ab responses upon injection as soluble protein. However, maleylated DT (mDT) does generate a significant Ab response. Furthermore, immunization with soluble mDT leads to a better T cell proliferative response in vitro than immunization with DT can generate, thereby demonstrating that maleylation leads to enhanced T cell immunogenicity in vivo. We also find that maleylation disrupts the native B cell epitopes of DT and creates new epitopes, because antisera to DT and mDT do not cross-react. At least some of the new epitopes generated are maleylation specific, because antisera against various maleylated proteins do cross-react. In contrast, maleylation does not significantly modify the repertoire of T cell epitopes generated from DT, because T cells generated by either DT or mDT immunization are cross-reactive, and both DT and mDT can stimulate T cells that are specific for single synthetic DT peptide. Maleylated proteins are better presented in vitro than are their native counterparts, and this enhancement of presentation is blocked by unrelated maleylated proteins. These results suggest that Ags targeted to scavenger receptors on macrophages by maleylation are better presented to T cells and are immunogenic in vivo without adjuvant.

Topics: Animals; Antigen-Presenting Cells; Antigens; Binding, Competitive; Cross Reactions; Diphtheria Toxoid; Endocytosis; Epitopes; Ligands; Lymphocyte Activation; Lymphocyte Cooperation; Macrophages; Maleates; Membrane Proteins; Mice; Mice, Inbred BALB C; Ovalbumin; Proteins; Receptors, Antigen, B-Cell; Receptors, Antigen, T-Cell; Receptors, Immunologic; Receptors, Lipoprotein; Receptors, Scavenger; Scavenger Receptors, Class B; Serum Albumin, Bovine; Tetanus Toxoid