RESUMO
Subunit vaccines would benefit from a safe particle-based adjuvant. Elastin-like polypeptide (ELP)-based micelles are interesting candidate adjuvants due to their well-defined size and easy modification with protein-based cargo. Coiled coils can facilitate noncovalent modifications, while potentially enhancing antigen delivery through interaction with cell membranes. ELP micelles comprise ELP diblock copolymers that self-assemble above a critical micelle temperature. In this study, an amphiphilic ELP was conjugated to peptide "K", which forms a heterodimeric coiled-coil complex with peptide "E". Self-assembled "covalent" micelles containing ELP-OVA323 (i.e., model antigen OVA323 conjugated to ELP), "coiled-coil" micelles containing ELP-K/E-OVA323 and "hybrid" micelles containing ELP-K and ELP-OVA323 were shown to be monodisperse and spherical. Dendritic cells (DCs) were exposed to all micelle compositions, and T-cell proliferation was investigated. The presence of ELP-K enhanced micelle uptake and subsequent DC maturation, resulting in enhanced CD4+ T-cell proliferation, which makes ELPs with coiled coil-associated antigens a promising vaccine platform.
Assuntos
Polipeptídeos Semelhantes à Elastina , Micelas , Elastina/química , Peptídeos/química , Antígenos , Ativação LinfocitáriaRESUMO
Synthetic vaccines, based on antigenic peptides that comprise MHC-I and MHC-II T-cell epitopes expressed by tumors, show great promise for the immunotherapy of cancer. For optimal immunogenicity, the synthetic peptides (SPs) should be adjuvanted with suitable immunostimulatory additives. Previously, we have shown that improved immunogenicity inâ vivo is obtained with vaccine modalities in which an SP is covalently connected to an adjuvanting moiety, typically a ligand to Toll-like receptor 2 (TLR2). SPs were covalently attached to UPam, which is a derivative of the classic TLR2 ligand Pam3 CysSK4 . A disadvantage of the triply palmitoylated UPam is its high lipophilicity, which precludes universal adoption of this adjuvant for covalent modification of various antigenic peptides as it renders the synthetic vaccine insoluble in several cases. Here, we report a novel conjugatable TLR2 ligand, mini-UPam, which contains only one palmitoyl chain, rather than three, and therefore has less impact on the solubility and other physicochemical properties of a synthetic peptide. In this study, we used SPs that contain the clinically relevant neoepitopes identified in a melanoma patient who completely recovered after T-cell therapy. Homogeneous mini-UPam-SP conjugates have been prepared in good yields by stepwise solid-phase synthesis that employed a mini-UPam building block pre-prepared in solution and the standard set of Fmoc-amino acids. The immunogenicity of the novel mini-UPam-SP conjugates was demonstrated by using the cancer patient's T-cells.
Assuntos
Antígenos de Neoplasias/química , Vacinas Anticâncer/imunologia , Ligantes , Receptor 2 Toll-Like/química , Vacinas Sintéticas/imunologia , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/química , Linhagem Celular , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Desenho de Fármacos , Humanos , Interleucina-8/metabolismo , Lipopeptídeos/síntese química , Lipopeptídeos/química , Lipopeptídeos/imunologia , Lipoilação , Ativação Linfocitária , Receptor 2 Toll-Like/metabolismo , Vacinas Sintéticas/químicaRESUMO
AIMS: To evaluate feasibility of intradermal (i.d.) adalimumab administration using hollow microneedles, and to compare a single i.d. dose of adalimumab using a hollow microneedle with a single subcutaneous (s.c.) dose using a conventional needle. METHODS: In this single-centre double-blind, placebo-controlled, double-dummy clinical trial in 24 healthy adults we compared 40 mg adalimumab (0.4 mL) administered i.d. using a hollow microneedle with a s.c. dose using a conventional needle. Primary parameters were pain, acceptability and local tolerability; secondary parameters safety, pharmacokinetics and immunogenicity. We explored usability of optical coherence tomography, clinical photography, thermal imaging, and laser speckle contrast imaging to evaluate skin reaction after i.d. injections. In vitro protein analysis was performed to assess compatibility of adalimumab with the hollow microneedle device. RESULTS: While feasible and safe, injection pain of i.d. adalimumab was higher compared to s.c. adalimumab (35.4 vs. 7.9 on a 100-point visual analogue scale). Initial absorption rate and relative bioavailability were higher after i.d. adalimumab (time to maximum plasma concentration = 95 h [47-120]; Frel = 129% [6.46%]) compared to s.c. adalimumab (time to maximum plasma concentration = 120 h [96-221]). Anti-adalimumab antibodies were detected in 50% and 83% of the subjects after i.d. and s.c. adalimumab, respectively. We observed statistically significantly more erythema and skin perfusion after i.d. adalimumab, compared to s.c. adalimumab and placebo injections (P < .0001). Cytokine secretion after whole blood lipopolysaccharide challenge was comparable between administration routes. CONCLUSIONS: Intradermal injection of adalimumab using hollowing microneedles was perceived as more painful and less accepted than s.c. administration, but yields a higher relative bioavailability with similar safety and pharmacodynamic effects.
Assuntos
Agulhas , Pele , Adalimumab , Adulto , Humanos , Injeções Intradérmicas , Injeções Subcutâneas , Medição da DorRESUMO
Clearance of nanoparticles (NPs) after intravenous injection - mainly by the liver - is a critical barrier for the clinical translation of nanomaterials. Physicochemical properties of NPs are known to influence their distribution through cell-specific interactions; however, the molecular mechanisms responsible for liver cellular NP uptake are poorly understood. Liver sinusoidal endothelial cells and Kupffer cells are critical participants in this clearance process. Here we use a zebrafish model for liver-NP interaction to identify the endothelial scavenger receptor Stabilin-1 as a non-redundant receptor for the clearance of small anionic NPs. Furthermore, we show that physiologically, Stabilin-1 is required for the removal of bacterial lipopolysaccharide (LPS/endotoxin) from circulation and that Stabilin-1 cooperates with its homolog Stabilin-2 in the clearance of larger (~100 nm) anionic NPs. Our findings allow optimization of anionic nanomedicine biodistribution and targeting therapies that use Stabilin-1 and -2 for liver endothelium-specific delivery.
Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Endotélio/metabolismo , Nanopartículas , Proteínas de Peixe-Zebra/fisiologia , Animais , Ânions , Proteínas de Ligação ao Cálcio/genética , Técnicas de Silenciamento de Genes , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genéticaRESUMO
Formaldehyde-inactivated toxoid vaccines have been in use for almost a century. Despite formaldehyde's deceptively simple structure, its reactions with proteins are complex. Treatment of immunogenic proteins with aqueous formaldehyde results in heterogenous mixtures due to a variety of adducts and cross-links. In this study, we aimed to further elucidate the reaction products of formaldehyde reaction with proteins and report unique modifications in formaldehyde-treated cytochrome c and corresponding synthetic peptides. Synthetic peptides (Ac-GDVEKGAK and Ac-GDVEKGKK) were treated with isotopically labeled formaldehyde (13CH2O or CD2O) followed by purification of the two main reaction products. This allowed for their structural elucidation by (2D)-nuclear magnetic resonance and nanoscale liquid chromatography-coupled mass spectrometry analysis. We observed modifications resulting from (i) formaldehyde-induced deamination and formation of α,ß-unsaturated aldehydes and methylation on two adjacent lysine residues and (ii) formaldehyde-induced methylation and formylation of two adjacent lysine residues. These products react further to form intramolecular cross-links between the two lysine residues. At higher peptide concentrations, these two main reaction products were also found to subsequently cross-link to lysine residues in other peptides, forming dimers and trimers. The accurate identification and quantification of formaldehyde-induced modifications improves our knowledge of formaldehyde-inactivated vaccine products, potentially aiding the development and registration of new vaccines.
Assuntos
Citocromos c/química , Formaldeído/farmacologia , Lisina/química , Peptídeos/química , Aldeídos/química , Cromatografia Líquida de Alta Pressão/métodos , Reagentes de Ligações Cruzadas/química , Desaminação/efeitos dos fármacos , Cinética , Espectroscopia de Ressonância Magnética/métodos , Espectrometria de Massas/métodos , Metilação/efeitos dos fármacos , Estrutura Molecular , Vacinas de Produtos Inativados/químicaRESUMO
In this study, we developed a microfluidics method, using a so-called H-cell microfluidics device, for the determination of protein diffusion coefficients at different concentrations, pHs, ionic strengths, and solvent viscosities. Protein transfer takes place in the H-cell channels between two laminarly flowing streams with each containing a different initial protein concentration. The protein diffusion coefficients are calculated based on the measured protein mass transfer, the channel dimensions, and the contact time between the two streams. The diffusion rates of lysozyme, cytochrome c, myoglobin, ovalbumin, bovine serum albumin, and etanercept were investigated. The accuracy of the presented methodology was demonstrated by comparing the measured diffusion coefficients with literature values measured under similar solvent conditions using other techniques. At low pH and ionic strength, the measured lysozyme diffusion coefficient increased with the protein concentration gradient, suggesting stronger and more frequent intermolecular interactions. At comparable concentration gradients, the measured lysozyme diffusion coefficient decreased drastically as a function of increasing ionic strength (from zero onwards) and increasing medium viscosity. Additionally, a particle tracing numerical simulation was performed to achieve a better understanding of the macromolecular displacement in the H-cell microchannels. It was found that particle transfer between the two channels tends to speed up at low ionic strength and high concentration gradient. This confirms the corresponding experimental observation of protein diffusion measured via the H-cell microfluidics.
Assuntos
Dispositivos Lab-On-A-Chip , Proteínas/química , Animais , Difusão , Concentração de Íons de Hidrogênio , Muramidase/química , Concentração Osmolar , Solventes/química , ViscosidadeRESUMO
OBJECTIVES: Therapeutic antibodies can provoke an antidrug antibody (ADA) response, which can form soluble immune complexes with the drug in potentially high amounts. Nevertheless, ADA-associated adverse events are usually rare, although with notable exceptions including infliximab. The immune activating effects and the eventual fate of these 'anti-idiotype' complexes are poorly studied, hampering assessment of ADA-associated risk of adverse events. We investigated the in vitro formation and biological activities of ADA-drug anti-idiotype immune complexes using patient-derived monoclonal anti-infliximab antibodies. METHODS: Size distribution and conformation of ADA-drug complexes were characterised by size-exclusion chromatography and electron microscopy. Internalisation of and immune activation by complexes of defined size was visualised with flow imaging, whole blood cell assay and C4b/c ELISA. RESULTS: Size and conformation of immune complexes depended on the concentrations and ratio of drug and ADA; large complexes (>6 IgGs) formed only with high ADA titres. Macrophages efficiently internalised tetrameric and bigger complexes in vitro, but not dimers. Corroborating these results, ex vivo analysis of patient sera demonstrated only dimeric complexes in circulation.No activation of immune cells by anti-idiotype complexes was observed, and only very large complexes activated complement. Unlike Fc-linked hexamers, anti-idiotype hexamers did not activate complement, demonstrating that besides size, conformation governs immune complex potential for triggering effector functions. CONCLUSIONS: Anti-idiotype ADA-drug complexes generally have restricted immune activation capacity. Large, irregularly shaped complexes only form at high concentrations of both drug and ADA, as may be achieved during intravenous infusion of infliximab, explaining the rarity of serious ADA-associated adverse events.
Assuntos
Anticorpos/imunologia , Formação de Anticorpos/efeitos dos fármacos , Complexo Antígeno-Anticorpo/imunologia , Antirreumáticos/imunologia , Infliximab/imunologia , Cromatografia em Gel , Ensaio de Imunoadsorção Enzimática , Humanos , Soro/imunologiaRESUMO
PURPOSE: To investigate the potential of two flow imaging microscopy (FIM) techniques (Micro-Flow Imaging (MFI) and FlowCAM) to determine total cell concentration and cell viability. METHODS: B-lineage acute lymphoblastic leukemia (B-ALL) cells of 2 different donors were exposed to ambient conditions. Samples were taken at different days and measured with MFI, FlowCAM, hemocytometry and automated cell counting. Dead and live cells from a fresh B-ALL cell suspension were fractionated by flow cytometry in order to derive software filters based on morphological parameters of separate cell populations with MFI and FlowCAM. The filter sets were used to assess cell viability in the measured samples. RESULTS: All techniques gave fairly similar cell concentration values over the whole incubation period. MFI showed to be superior with respect to precision, whereas FlowCAM provided particle images with a higher resolution. Moreover, both FIM methods were able to provide similar results for cell viability as the conventional methods (hemocytometry and automated cell counting). CONCLUSION: FIM-based methods may be advantageous over conventional cell methods for determining total cell concentration and cell viability, as FIM measures much larger sample volumes, does not require labeling, is less laborious and provides images of individual cells.
Assuntos
Contagem de Células/instrumentação , Sobrevivência Celular , Imagem Óptica/instrumentação , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Microscopia/instrumentação , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologiaRESUMO
PURPOSE: Filgrastim, a recombinant human granulocyte-colony stimulating factor, is widely used to treat congenital and acquired neutropenia. Following patent expiration of the innovator filgrastim product, biosimilar filgrastim products have been approved in the EU and shown to be comparable with the innovator with respect to quality, safety and efficacy. In less regulated markets, copy filgrastim products are available but data about their quality are scarce. In the present study, we provide a head-to-head comparative study on the quality of biosimilar and copy filgrastim products. METHODS: Innovator filgrastim product, Neupogen®, two EU-licensed biosimilars, Zarzio® and Tevagrastim®, and two copy filgrastim products, Biocilin® and PDgrastim®, were subjected to peptide mapping, circular dichroism spectroscopy, fluorescence spectroscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis, high performance size-exclusion chromatography, reversed-phase ultra-performance liquid chromatography, endotoxin test, flow imaging microscopy and in vitro potency assay. RESULTS: Zarzio® and Tevagrastim® have comparable quality to Neupogen®, while Biocilin® showed a significantly lower and PDgrastim® a higher specific activity. Moreover, PDgrastim® showed a higher level of impurities and a lower thermo stability than the other products. CONCLUSIONS: Except for the deviating specific activities of the two copy filgrastim products, we found no substantial differences in product quality between the filgrastim products studied.
Assuntos
Medicamentos Biossimilares/química , Filgrastim/química , Fármacos Hematológicos/química , Medicamentos Biossimilares/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Contaminação de Medicamentos , Estabilidade de Medicamentos , Filgrastim/farmacologia , Fármacos Hematológicos/farmacologia , Humanos , Estabilidade ProteicaRESUMO
PURPOSE: Personalized peptide-based cancer vaccines will be composed of multiple patient specific synthetic long peptides (SLPs) which may have various physicochemical properties. To formulate such SLPs, a flexible vaccine delivery system is required. We studied whether cationic liposomes are suitable for this purpose. METHODS: Fifteen SIINFEKL T cell epitope-containing SLPs, widely differing in hydrophobicity and isoelectric point, were separately loaded in cationic liposomes via the dehydration-rehydration method. Particle size and polydispersity index (PDI) were measured via dynamic light scattering (DLS), and zeta potential with laser Doppler electrophoresis. Peptide loading was fluorescently determined and the immunogenicity of the formulated peptides was assessed in co-cultures of dendritic cells (DCs) and CD8+ T-cells in vitro. RESULTS: All SLPs were loaded in cationic liposomes by using three different loading method variants, depending on the SLP characteristics. The fifteen liposomal formulations had a comparable size (< 200 nm), PDI (< 0.3) and zeta potential (22-30 mV). Cationic liposomes efficiently delivered the SLPs to DCs that subsequently activated SIINFEKL-specific CD8+ T-cells, indicating improved immunological activity of the SLPs. CONCLUSION: Cationic liposomes can accommodate a wide range of different SLPs and are therefore a potential delivery platform for personalized cancer vaccines.
Assuntos
Vacinas Anticâncer/administração & dosagem , Portadores de Fármacos/química , Epitopos de Linfócito T , Lipossomos/química , Oligopeptídeos/administração & dosagem , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/química , Vacinas Anticâncer/imunologia , Cátions , Composição de Medicamentos , Liberação Controlada de Fármacos , Corantes Fluorescentes/química , Humanos , Ativação Linfocitária , Oligopeptídeos/química , Oligopeptídeos/imunologia , Ovalbumina/química , Tamanho da Partícula , Fragmentos de Peptídeos/química , Biblioteca de Peptídeos , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/química , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
PURPOSE: To examine the immunogenicity of diphtheria toxoid (DT) loaded mesoporous silica nanoparticles (MSNs) after coated and hollow microneedle-mediated intradermal immunization in mice. METHODS: DT was loaded into MSNs and the nanoparticle surface was coated with a lipid bilayer (LB-MSN-DT). To prepare coated microneedles, alternating layers of negatively charged LB-MSN-DT and positively charged N-trimethyl chitosan (TMC) were coated onto pH-sensitive microneedle arrays via a layer-by-layer approach. Microneedle arrays coated with 5 or 3 layers of LB-MSN-DT were used to immunize mice and the elicited antibody responses were compared with those induced by hollow microneedle-injected liquid formulation of LB-MSN-DT. Liquid DT formulation with and without TMC (DT/TMC) injected by a hollow microneedle were used as controls. RESULTS: LB-MSN-DT had an average size of about 670 nm and a zeta potential of -35 mV. The encapsulation efficiency of DT in the nanoparticles was 77%. The amount of nano-encapsulated DT coated onto the microneedle array increased linearly with increasing number of the coating layers. Nano-encapsulated DT induced stronger immune responses than DT solution when delivered intradermally via hollow microneedles, but not when delivered via coated microneedles. CONCLUSION: Both the nano-encapsulation of DT and the type of microneedles affect the immunogenicity of the antigen.
Assuntos
Toxoide Diftérico/administração & dosagem , Nanopartículas/química , Dióxido de Silício/química , Animais , Toxoide Diftérico/química , Toxoide Diftérico/imunologia , Composição de Medicamentos , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Imunização , Imunogenicidade da Vacina , Injeções Intradérmicas , Camundongos , Camundongos Endogâmicos BALB C , Tamanho da Partícula , Porosidade , Propriedades de SuperfícieRESUMO
OBJECTIVES: Anti-citrullinated protein antibodies (ACPA) are highly specific for rheumatoid arthritis (RA). Here, we studied binding of ACPA-IgG immune complexes (IC) to individual Fc gamma receptors (FcγR) to identify potential effector mechanisms by which ACPA could contribute to RA pathogenesis. METHODS: ACPA-IgG1 and control IgG1(IgG1 depleted of ACPA-IgG1) were isolated from plasma and synovial fluid (SF) of RA patients by affinity chromatography using CCP2 peptides. Subsequently, IC were generated using fluorescently labelled F(ab')2 fragments against the F(ab')2 region of IgG, or by using citrullinated fibrinogen. IC were incubated with FcγR-transfected CHO cell lines or neutrophils from healthy donors. FcγR binding of IC was analysed by flow cytometry in the presence or absence of specific blocking antibodies. RESULTS: ACPA-IgG1 IC predominantly bound to FcγRI and FcγRIIIA on FcγR-transfected CHO cell lines, while much lower binding was observed to FcγRIIA and FcγRIIB. ACPA-IgG1 IC showed reduced binding to FcγRIIIA compared to control IgG1 IC, in line with enhanced ACPA-IgG1 Fc core-fucosylation. Neutrophils activated in vitro to induce de novo expression of FcγRI showed binding of ACPA-IgG IC, and blocking studies revealed that almost 30% of ACPA-IgG IC binding to activated neutrophils was mediated by FcγRI. CONCLUSIONS: Our studies show that ACPA-IgG1 IC bind predominately to activating FcγRI and FcγRIIIA, and highlight FcγRI expressed by activated neutrophils as relevant receptor for these IC. As neutrophils isolated from SF exhibit an activated state and express FcγRI in the synovial compartment, this IC-binding could contribute to driving disease pathogenesis in RA.
Assuntos
Anticorpos Antiproteína Citrulinada/metabolismo , Complexo Antígeno-Anticorpo/metabolismo , Artrite Reumatoide/etiologia , Receptores de IgG/metabolismo , Idoso , Artrite Reumatoide/imunologia , Feminino , Glicosilação , Humanos , Masculino , Pessoa de Meia-Idade , Neutrófilos/fisiologia , Receptores de IgG/fisiologia , Sinovite/etiologiaRESUMO
Bone morphogenetic proteins (BMPs) are responsible for bone formation during embryogenesis and bone regeneration and remodeling. The osteoinductive action of BMPs, especially BMP-2 and BMP-7, has led to their use in a range of insurmountable treatments where intervention is required for effective bone regeneration. Introduction of BMP products to the market, however, was not without reports of multiple complications and side effects. Aiming for optimization of the therapeutic efficacy and safety, efforts have been focused on improving the delivery of BMPs to lower the administered dose, localize the protein, and prolong its retention time at the site of action. A major challenge with these efforts is that the protein stability should be maintained. With this review we attempt to shed light on how the stability of BMPs can be affected in the formulation and delivery processes. We first provide a short overview of the current standing of the complications experienced with BMP products. We then discuss the different delivery parameters studied in association with BMPs, and their influence on the efficacy and safety of BMP treatments. In particular, the literature addressing the stability of BMPs and their possible interactions with components of the delivery system as well as their sensitivity to conditions of the formulation process is reviewed. In summary, recent developments in the fields of bioengineering and biopharmaceuticals suggest that a good understanding of the relationship between the formulation/delivery conditions and the stability of growth factors such as BMPs is a prerequisite for a safe and effective treatment.
Assuntos
Proteínas Morfogenéticas Ósseas/administração & dosagem , Regeneração Óssea/efeitos dos fármacos , Composição de Medicamentos/métodos , Sistemas de Liberação de Medicamentos/métodos , Animais , Liberação Controlada de Fármacos , Estabilidade de Medicamentos , Humanos , Polímeros/química , Transdução de Sinais , Engenharia Tecidual , Alicerces Teciduais/químicaRESUMO
PURPOSE: To investigate the effect of nanoparticulate impurities (NPIs) isolated from pharmaceutical-grade sucrose, on the stability of monoclonal antibodies (mAbs). METHODS: NPIs were purified from pharmaceutical-grade sucrose and spiked into trastuzumab, rituximab, infliximab, and cetuximab formulations. The stability of the mAbs as a function of storage time, temperature, and NPI concentration was assessed by visual inspection, flow-imaging microscopy, nanoparticle tracking analysis, size-exclusion chromatography, capillary isoelectric focusing, and intrinsic differential scanning fluorimetry. RESULTS: NPIs negatively affected the stability of all mAbs, albeit it to different extents. After spiking with NPIs, trastuzumab formulations showed high numbers of µm-sized particles and turbidity, rituximab and cetuximab formulations contained high numbers of nm-sized particles, while infliximab formed nm- and µm-sized particles, and showed turbidity. Low-molecular-weight species were observed for rituximab and infliximab, whereas high-molecular-weight species were detected for cetuximab. Whereas the stability of trastuzumab and infliximab formulations was affected directly after spiking NPIs, degradation of rituximab and cetuximab was observed only after 14 weeks at elevated temperatures. Moreover, the stability of rituximab and infliximab was affected by NPI concentrations that are potentially present in final drug products. CONCLUSIONS: The presence of sucrose-derived NPIs in (bio-)pharmaceutical formulations may pose a threat to the stability of mAbs.
Assuntos
Anticorpos Monoclonais/química , Excipientes/química , Nanopartículas/química , Sacarose/química , Composição de Medicamentos , Estabilidade de Medicamentos , Estabilidade Proteica , ProteóliseRESUMO
PURPOSE: To physicochemically characterize and compare monoclonal antibody (mAb) solutions containing aggregates generated via metal catalyzed oxidation (MCO). METHODS: Two monoclonal IgG2s (mAb1 and mAb2) and one monoclonal IgG1 (rituximab) were exposed to MCO with the copper/ascorbic acid oxidative system, by using several different methods. The products obtained were characterized by complementary techniques for aggregate and particle analysis (from oligomers to micron sized species), and mass spectrometry methods to determine the residual copper content and chemical modifications of the proteins. RESULTS: The particle size distribution and the morphology of the protein aggregates generated were similar for all mAbs, independent of the MCO method used. There were differences in both residual copper content and in chemical modification of specific residues, which appear to be dependent on both the protein sequence and the protocol used. All products showed a significant increase in the levels of oxidized His, Trp, and Met residues, with differences in extent of modification and specific amino acid residues modified. CONCLUSION: The extent of total oxidation and the amino acid residues with the greatest oxidation rate depend on a combination of the MCO method used and the protein sequence.
Assuntos
Anticorpos Monoclonais/química , Antineoplásicos Imunológicos/química , Cobre/química , Imunoglobulina G/química , Agregados Proteicos , Rituximab/química , Ácido Ascórbico/química , Ácido Ascórbico/farmacologia , Catálise , Humanos , Modelos Moleculares , Oxirredução/efeitos dos fármacos , Agregados Proteicos/efeitos dos fármacos , SoluçõesRESUMO
PURPOSE: To develop a new intradermal antigen delivery system by coating microneedle arrays with lipid bilayer-coated, antigen-loaded mesoporous silica nanoparticles (LB-MSN-OVA). METHODS: Synthesis of MSNs with 10-nm pores was performed and the nanoparticles were loaded with the model antigen ovalbumin (OVA), and coated with a lipid bilayer (LB-MSN-OVA). The uptake of LB-MSN-OVA by bone marrow-derived dendritic cells (BDMCs) was studied by flow cytometry. The designed LB-MSN-OVA were coated onto pH-sensitive pyridine-modified microneedle arrays and the delivery of LB-MSN-OVA into ex vivo human skin was studied. RESULTS: The synthesized MSNs demonstrated efficient loading of OVA with a maximum loading capacity of about 34% and the lipid bilayer enhanced the colloidal stability of the MSNs. Uptake of OVA loaded in LB-MSN-OVA by BMDCs was higher than that of free OVA, suggesting effective targeting of LB-MSN-OVA to antigen-presenting cells. Microneedles were readily coated with LB-MSN-OVA at pH 5.8, yielding 1.5 µg of encapsulated OVA per microneedle array. Finally, as a result of the pyridine modification, LB-MSN-OVA were effectively released from the microneedles upon piercing the skin. CONCLUSION: Microneedle arrays coated with LB-MSN-OVA were successfully developed and shown to be suitable for intradermal delivery of the encapsulated protein antigen.
Assuntos
Antígenos/administração & dosagem , Nanopartículas/química , Agulhas , Ovalbumina/administração & dosagem , Dióxido de Silício/química , Células Apresentadoras de Antígenos/metabolismo , Portadores de Fármacos , Liberação Controlada de Fármacos , Humanos , Concentração de Íons de Hidrogênio , Injeções Intradérmicas , Bicamadas Lipídicas , Macrófagos/metabolismo , Tamanho da Partícula , Porosidade , Pele , Propriedades de SuperfícieRESUMO
PURPOSE: The aim of this study was to investigate the depth-dependent intradermal immunogenicity of inactivated polio vaccine (IPV) delivered by depth-controlled microinjections via hollow microneedles (HMN) and to investigate antibody response enhancing effects of IPV immunization adjuvanted with CpG oligodeoxynucleotide 1826 (CpG) or cholera toxin (CT). METHODS: A novel applicator for HMN was designed to permit depth- and volume-controlled microinjections. The applicator was used to immunize rats intradermally with monovalent IPV serotype 1 (IPV1) at injection depths ranging from 50 to 550 µm, or at 400 µm for CpG and CT adjuvanted immunization, which were compared to intramuscular immunization. RESULTS: The applicator allowed accurate microinjections into rat skin at predetermined injection depths (50-900 µm), -volumes (1-100 µL) and -rates (up to 60 µL/min) with minimal volume loss (±1-2%). HMN-mediated intradermal immunization resulted in similar IgG and virus-neutralizing antibody titers as conventional intramuscular immunization. No differences in IgG titers were observed as function of injection depth, however IgG titers were significantly increased in the CpG and CT adjuvanted groups (7-fold). CONCLUSION: Intradermal immunogenicity of IPV1 was not affected by injection depth. CpG and CT were potent adjuvants for both intradermal and intramuscular immunization, allowing effective vaccination upon a minimally-invasive single intradermal microinjection by HMN.
Assuntos
Adjuvantes Imunológicos/farmacologia , Formação de Anticorpos/imunologia , Vacina Antipólio de Vírus Inativado/imunologia , Adjuvantes Farmacêuticos/farmacologia , Animais , Anticorpos Antivirais/imunologia , Feminino , Imunoglobulina G/imunologia , Injeções Intradérmicas/métodos , Injeções Intramusculares/métodos , Microinjeções/métodos , Oligodesoxirribonucleotídeos/imunologia , Ratos , Ratos Wistar , Vacinação/métodosRESUMO
Detection of cerebral ß-amyloid (Aß) by targeted contrast agents is of great interest for in vivo diagnosis of Alzheimer's disease (AD). Partly because of their planar structure several bis-styrylbenzenes have been previously reported as potential Aß imaging agents. However, these compounds are relatively hydrophobic, which likely limits their in vivo potential. Based on their structures, we hypothesized that less hydrophobic bis-pyridylethenylbenzenes may also label amyloid. We synthesized several bis-pyridylethenylbenzenes and tested whether these compounds indeed display improved solubility and lower LogP values, and studied their fluorescent properties and Aß binding characteristics. Bis-pyridylethenylbenzenes showed a clear affinity for Aß plaques on both human and murine AD brain sections. Competitive binding experiments suggested a different binding site than Chrysamine G, a well-known stain for amyloid. With a LogP value between 3 and 5, most bis-pyridylethenylbenzenes were able to enter the brain and label murine amyloid in vivo with the bis(4-pyridylethenyl)benzenes showing the most favorable characteristics. In conclusion, the presented results suggest that bis-pyridylethenylbenzene may serve as a novel backbone for amyloid imaging agents.
Assuntos
Peptídeos beta-Amiloides/química , Meios de Contraste/química , Corantes Fluorescentes/química , Placa Amiloide/diagnóstico por imagem , Piridinas/química , Estirenos/química , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Meios de Contraste/síntese química , Corantes Fluorescentes/síntese química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Camundongos Transgênicos , Microscopia de Fluorescência , Imagem Molecular , Ligação Proteica , Piridinas/síntese química , Solubilidade , Estilbenos/química , Estirenos/síntese químicaRESUMO
The current resurgence of whooping cough is alarming, and improved pertussis vaccines are thought to offer a solution. Outer membrane vesicle vaccines (omvPV) are potential vaccine candidates, but omvPV-induced humoral responses have not yet been characterized in detail. The purpose of this study was to determine the antigen composition of omvPV and to elucidate the immunogenicity of the individual antigens. Quantitative proteome analysis revealed the complex composition of omvPV. The omvPV immunogenicity profile in mice was compared to those of classic whole cell vaccine (wPV), acellular vaccine (aPV), and pertussis infection. Pertussis-specific antibody levels, antibody isotypes, IgG subclasses, and antigen specificity were determined after vaccination or infection by using a combination of multiplex immunoassays, two-dimensional immunoblotting, and mass spectrometry. The vaccines and infection raised strong antibody responses, but large quantitative and qualitative differences were measured. The highest antibody levels were obtained by omvPV. All IgG subclasses (IgG1/IgG2a/IgG2b/IgG3) were elicited by omvPV and in a lower magnitude by wPV, but not by aPV (IgG1) or infection (IgG2a/b). The majority of omvPV-induced antibodies were directed against Vag8, BrkA, and LPS. The broad and balanced humoral response makes omvPV a promising pertussis vaccine candidate.
Assuntos
Anticorpos Antibacterianos/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Vacinas Bacterianas/imunologia , Bordetella pertussis/imunologia , Proteoma , Animais , Anticorpos Antibacterianos/biossíntese , Proteínas da Membrana Bacteriana Externa/imunologia , Cromatografia Líquida , Eletroforese em Gel Bidimensional , Camundongos , Espectrometria de Massas em Tandem , Coqueluche/prevenção & controleRESUMO
The aim of this work was to coat pH-sensitive microneedle arrays with inactivated polio vaccine (IPV) particles and N-trimethyl chitosan chloride (TMC) via electrostatic interactions, and assess the immunogenicity of the vaccine after topical application of the coated microneedles in rats. The surface of 200 µm long microneedles was first chemically modified with pH-sensitive (pyridine) groups and then coated with negatively charged IPV and a positively charged polymer (TMC). To obtain a sufficient high antigen dose, 10 layers of IPV were alternately coated with TMC. The binding of IPV and TMC onto pH-sensitive microneedles was quantified and visualized by using fluorescently labeled TMC and IPV. The release of IPV and TMC from the microneedles was evaluated in ex vivo human skin by fluorescence and the immunogenicity of (unlabeled) IPV was assessed after topical application of the coated microneedles in rats. pH-sensitive microneedles were homogeneously coated with 10 layers of both IPV and TMC, resulting in 45 D antigen units IPV and 700 ng TMC per microneedle array. Fluorescence microscopy imaging revealed that both IPV and TMC were released into ex vivo human skin upon application of the coated microneedles. Finally, in vivo application of IPV-TMC-coated pH-sensitive microneedles in rats led to the induction of IPV specific antibody responses, illustrating that they are practically applicable. Topical administration of pH-sensitive microneedles coated with polyelectrolyte multinanolayers of antigens and oppositely charged polymers may be a useful approach for microneedle-based vaccination.