Polysorbate 80 (PS80) is a commonly used surfactant in therapeutic protein formulations to mitigate adsorption and interface-induced protein aggregation. Several PS80 grades and qualities are... Show morePolysorbate 80 (PS80) is a commonly used surfactant in therapeutic protein formulations to mitigate adsorption and interface-induced protein aggregation. Several PS80 grades and qualities are available on the market for parenteral application. The role of PS80 grade on protein stability remains debatable, and the impact of (partially) degraded PS on protein aggregation is not yet well understood. In our study, a monoclonal antibody (IgG) was subjected to 3 different mechanical stress conditions in the presence of multicompendial (MC) and Chinese pharmacopeia (ChP) grade PS80. Furthermore, IgG formulations were spiked with (partly) hydrolyzed PS80 to investigate the effect of PS80 degradants on protein stability. PS80 functionality was assessed by measuring the extent of protein aggregation and particle formation induced during mechanical stress by using size-exclusion chromatography, dynamic light scattering, backgrounded membrane imaging, and flow imaging microscopy. No distinguishable differences in PS80 functionality between MC and ChP grade were observed in the 3 stress tests. However, with increasing degree of PS80 hydrolysis, higher counts of subvisible particles were measured after stress. Furthermore, higher levels of PS80 degradants at a constant PS80 concentration may destabilize the IgG. In conclusion, MC and ChP grade PS80 are equally protective, but PS80 degradants compromise IgG stability. Show less
Benne, N.; Duijn, J. van; Lozano Vigario, F.; Leboux, R.; Veelen, P. van; Jiskoot, W.; ... ; Slütter, B. 2019
In 2003, Crommelin et al. published an article titled: "Shifting paradigms: biopharmaceuticals versus low molecular weight drugs" (https://doi.org/10.1016/S0378-5173(03)00376-4). In the present... Show moreIn 2003, Crommelin et al. published an article titled: "Shifting paradigms: biopharmaceuticals versus low molecular weight drugs" (https://doi.org/10.1016/S0378-5173(03)00376-4). In the present commentary, 16 years later, we discuss pharmaceutically relevant aspects of the evolution of biologics since then. First, we discuss the increasing repertoire of biologics, in particular, the rapidly growing monoclonal antibody family and the advent of advanced therapy medicinal products. Next, we discuss trends in formulation and characterization as well as summarize our current insights into immunogenicity of biologics. We spend a separate section on new product(ion) paradigms for biologics, such as cell-free production systems, production of advanced therapy medicinal products, and downscaled production approaches. Furthermore, we share our views on issues related to reaching the patient, including routes and techniques of administration, alternative development models for affordable biologics, biosimilars, and handling of biologics. In the concluding section, we outline outstanding issues and make some suggestions for resolving those. Show less
Subunit vaccines often contain colloidal aluminum salt-based adjuvants to activate the innate immune system. These aluminum salts consist of micrometer-sized aggregates. It is well-known that... Show moreSubunit vaccines often contain colloidal aluminum salt-based adjuvants to activate the innate immune system. These aluminum salts consist of micrometer-sized aggregates. It is well-known that particle size affects the adjuvant effect of particulate adjuvants. In this study, the activation of human monocytes by hexagonal-shaped gibbsite (ø = 210 ± 40 nm) and rod-shaped boehmite (ø = 83 ± 827 nm) was compared with classical aluminum oxyhydroxide adjuvant (alum). To this end, human primary monocytes were cultured in the presence of alum, gibbsite, or boehmite. The transcriptome and proteome of the monocytes were investigated by using quantitative polymerase chain reaction and mass spectrometry. Human monocytic THP-1 cells were used to investigate the effect of the particles on cellular maturation, differentiation, activation, and cytokine secretion, as measured by flow cytometry and enzyme-linked immunosorbent assay. Each particle type resulted in a specific gene expression profile. IL-1ß and IL-6 secretion was significantly upregulated by boehmite and alum. Of the 7 surface markers investigated, only CD80 was significantly upregulated by alum and none by gibbsite or boehmite. Gibbsite hardly activated the monocytes. Boehmite activated human primary monocytes equally to alum, but induced a much milder stress-related response. Therefore, boehmite was identified as a promising adjuvant candidate. Show less
Over the past few years, loss of patent protection for blockbuster monoclonal antibody (mAb) drugs has caused a significant shift in the pharmaceutical industry towards the development of... Show moreOver the past few years, loss of patent protection for blockbuster monoclonal antibody (mAb) drugs has caused a significant shift in the pharmaceutical industry towards the development of biosimilar products. As a result, multiple biosimilar mAbs are becoming available for a single originator drug. As opposed to small-molecular drugs, protein biopharmaceuticals do not have fully defined and reproducible structures, making it impossible to create identical copies. Therefore, regulators demand biosimilar sponsors to demonstrate similarity with the reference product to prevent safety and efficacy issues with the proposed product. Protein glycosylation is considered a crucially important quality attribute, because of its major role in immunogenicity and clinical efficacy of therapeutic proteins. However, the intrinsic biological variability of glycan structures creates a significant challenge for the current analytical platforms. In this review, we discuss the importance of glycan characterization on therapeutic proteins, with a particular focus on the analytical techniques applied for glycan profiling of biosimilar mAb products. In addition, we present a case study on infliximab biosimilars to illustrate the potential clinical implications of differences in glycan profile between originator and biosimilar mAb products. Show less
Duijn, J. van; Elsas, M. van; Benne, N.; Depuydt, M.; Wezel, A.; Smeets, H.; ... ; Slutter, B. 2019
CD8+ T-cells can be atheroprotective in clinically relevant advanced stages of atherosclerosis, as their depletion results in less stable lesions with a more inflammatory phenotype. However, the... Show moreCD8+ T-cells can be atheroprotective in clinically relevant advanced stages of atherosclerosis, as their depletion results in less stable lesions with a more inflammatory phenotype. However, the phenotype and function of these cells in the lesional microenvironment remains to be determined. Here, we address how the atherosclerotic environment affects the functionality of CD8+ T-cells.We compared the cytokine production of CD8+ T-cells derived from spleens and aortas of apoE-/- mice with advanced atherosclerosis by flow cytometry.CD8+ T-cells isolated from atherosclerotic lesions produced lower amounts of IFN-γ and TNF-α than their splenic counterparts. The observed dysfunctional phenotype of the lesion-derived CD8+ T-cells was associated with an increased expression of the ectonucleotidase CD39, which converts inflammatory extracellular ATP into immunomodulatory adenosine. Indeed, pharmacological inhibition of CD39 in apoE-/- mice partly restored cytokine production by CD8+ T-cells. Using a bone-marrow transplantation approach, we showed that induction of CD39 was a consequence of antigen-specific CD8+ T-cell activation via T-cell receptor (TCR) signaling within the lesions. Importantly, analysis of human endarterectomy samples showed a clear microenvironment specific upregulation of CD39 on CD8+ T-cells in the plaques of human patients compared to matched CD8+ T-cells from the blood .Our results indicate that the continuous TCR signaling in the atherosclerotic plaque induces an immune regulatory CD8+ T-cell phenotype that is associated with decreased cytokine production through increased CD39 expression in both a murine atherosclerotic model and in atherosclerosis patients. This provides a new understanding of atheroprotective immune regulation by CD8+ T-cells. Show less
Jacobse, J.; Romeijn, S.G.; Tandon, A.; Voorde, W. ten; Grievink, W.; Rissmann, R.; ... ; Jiskoot, W. 2019
Aluminium phosphate is a commonly used adjuvant consisting of heterogeneously sized aggregates up to several micrometers. However, aluminium phosphate nanoparticles may exhibit an improved adjuvant... Show moreAluminium phosphate is a commonly used adjuvant consisting of heterogeneously sized aggregates up to several micrometers. However, aluminium phosphate nanoparticles may exhibit an improved adjuvant effect. In this study, nanoparticles were made by sonication of commercially available aluminium phosphate adjuvant, resulting in particles with a size (Z-average diameter) between 200-300 nm and a point of zero charge of 4.5. To prevent reaggregation, which occurred within 14 days, a screening of excipients was performed to identify stabilisers effective under physiological conditions (pH 7.4, 290 mOsm). The amino acids threonine, asparagine, and L-alanyl-L-1-aminoethylphosphonic acid (LAPA) stabilised sonicated aluminium phosphate. Particle sizes remained stable between 400-600 nm at 37 °C during 106 days. Contrarily, arginine induced strong reaggregation to a particle size larger than 1000 nm. The stability of aluminium phosphate nanoparticles was strongly affected by the pH. Aggregation mainly occurred below pH 7. The adsorption capacity, a potentially relevant parameter for adjuvants, was slightly reduced in the presence of asparagine, when using a model antigen (lysozyme). LAPA, arginine, threonine and aspartic acid reduced protein adsorption significantly. The adjuvant effect of aluminium phosphate nanoparticles was studied by immunisation of mice with diphtheria toxoid adjuvanted with the aluminium phosphate nanoparticles. The presence of LAPA, threonine, aspartic acid or asparagine did not alter diphtheria toxoid-specific antibody or toxin-neutralising antibody titres. Arginine increased diphtheria toxoid-specific antibody titres but not toxin-neutralising antibody titres. In conclusion, aluminium phosphate nanoparticles were stabilised by particular amino acids and induced an adjuvant effect comparable to that of aluminium phosphate microparticles. Show less
Maaden, K. van der; Schipper, P.; Jiskoot, W.; Bouwstra, J.A. 2019
For pharmaceutical, biological, and biomedical applications, the functionalization of gold surfaces with pH-sensitive groups has great potential. The aim of this work was to modify gold surfaces... Show moreFor pharmaceutical, biological, and biomedical applications, the functionalization of gold surfaces with pH-sensitive groups has great potential. The aim of this work was to modify gold surfaces with pH-sensitive groups and to determine the pKa of the modified gold surfaces using a fluorescent nanoparticle adhesion assay. To introduce pH-sensitive groups onto gold surfaces, we modified gold-coated silicon slides with four different bases: 4-mercaptopyridine (4-MP), 4-pyridylethylmercaptan (4-PEM), 4-aminothiophenol (4-ATP), and 2-mercaptoethylamine (2-MEA). To screen whether the modifications were successful, the binding of negatively charged fluorescently labeled nanoparticles to the positively charged surfaces was visualized by fluorescence microscopy and atomic force microscopy. Next, the pKa of the modified surfaces was determined by quantifying the pH-dependent adhesion of the fluorescently labeled nanoparticles with fluorescence spectroscopy. Fluorescence microscopy showed that the gold surfaces were successfully modified with the four different basic molecules. Moreover, fluorescence spectroscopy revealed that fluorescently labeled negatively charged nanoparticles bound onto gold surfaces that were modified with one of the four bases in a pH-dependent manner. By quantifying the adsorption of negatively charged fluorescently labeled nanoparticles onto the functionalized gold surfaces and using the Henderson–Hasselbalch equation, the pKa of these surfaces was determined to be 3.7 ± 0.1 (4-MP), 5.0 ± 0.1 (4-PEM), 5.4 ± 0.1 (4-ATP), and 7.4 ± 0.3 (2-MEA). We successfully functionalized gold surfaces with four different basic molecules, yielding modified surfaces with different pKa values, as determined with a fluorescent nanoparticle adhesion assay. Show less
The presence of protein aggregates is commonly believed to be an important risk factor for immunogenicity of therapeutic proteins. Among all types of aggregates, dimers are relatively abundant in... Show moreThe presence of protein aggregates is commonly believed to be an important risk factor for immunogenicity of therapeutic proteins. Among all types of aggregates, dimers are relatively abundant in most commercialized monoclonal antibody (mAb) products. The aim of this study was to investigate the immunogenicity of artificially created mAb dimers relative to that of unstressed and stressed mAb monomers. A monoclonal murine IgG1 (mIgG1) antibody was exposed to low pH, elevated temperature, or UV irradiation to induce dimerization. Dimers and monomers were purified via size-exclusion chromatography. Physicochemical analysis revealed that upon all stress conditions, new deamidation or oxidation or both of amino acids occurred. Nevertheless, the secondary and tertiary structures of all obtained dimers were similar to those of unstressed mIgG1. Isolated dimers were administered subcutaneously in Balb/c mice, and development of antidrug antibodies and accumulation of follicular T helper cells in draining lymph nodes and spleens were determined. None of the tested dimers or stressed monomers were found to be more immunogenic than the unstressed control in our mouse model. In conclusion, both dimers and monomers generated by using 3 different stress factors have a low immunogenicity similar to that of the unstressed monomers. Show less
Duijn, J. van; Elsas, M. van; Benne, N.; Depuydt, M.A.C.; Wezel, A.; Smeets, H.; ... ; Slütter, B.A. 2019
CD8+T-cells have been attributed both atherogenic and atheroprotective properties, butanalysis of CD8+T-cells has mostly been restricted to the circulation and secondary lymphoid organs.... Show moreCD8+T-cells have been attributed both atherogenic and atheroprotective properties, butanalysis of CD8+T-cells has mostly been restricted to the circulation and secondary lymphoid organs. Theatherosclerotic lesion, however, is a complex microenvironment containing a plethora of inflammatory signals,which may affect CD8+T-cell activation. Here, we address how this environment affects the functionality ofCD8+T-cells. We compared the cytokine production of CD8+T-cells derived from spleens and en-zymatically digested aortas ofapoE−/−mice with advanced atherosclerosis byflow cytometry. Aortic CD8+T-cells produced decreased amounts of IFN-γand TNF-αcompared to their systemic counterparts. The observeddysfunctional phenotype of the lesion-derived CD8+T-cells was not associated with classical exhaustion mar-kers, but with increased expression of the ectonucleotidase CD39. Indeed, pharmacological inhibition of CD39 inapoE−/−mice partly restored cytokine production by CD8+T-cells. Using a bone-marrow transplantation ap-proach, we show that TCR signaling is required to induce CD39 expression on CD8+T-cells in atheroscleroticlesions. Importantly, analysis of human endarterectomy samples showed a strong microenvironment specificupregulation of CD39 on CD8+T-cells in the plaques of human patients compared to matched blood samples. Our results suggest that the continuous TCR signaling in the atherosclerotic environment in thevessel wall induces an immune regulatory CD8+T-cell phenotype that is associated with decreased cytokineproduction through increased CD39 expression in both a murine atherosclerotic model and in atherosclerosispatients. This provides a new understanding of immune regulation by CD8+T-cells in atherosclerosis. Show less
Leone, M.; Priester, M.I.; Romeijn, S.; Nejadnik, M.R.; Mönkäre, J.; O'Mahony, C.; ... ; Bouwstra, J.A. 2019
The purpose of this study was to optimize the manufacturing of dissolving microneedles (dMNs) and to increase the antigen loading in dMNs to investigate the effect on their physicochemical... Show moreThe purpose of this study was to optimize the manufacturing of dissolving microneedles (dMNs) and to increase the antigen loading in dMNs to investigate the effect on their physicochemical properties. To achieve this, a novel single-array wells polydimethylsiloxane mold was designed, minimizing antigen wastage during fabrication and achieving homogeneous antigen distribution among the dMN arrays. Using this mold, hyaluronan (HA)-based dMNs were fabricated and tested for maximal ovalbumin (OVA) content. dMNs could be fabricated with an OVA:HA ratio as high as 1:1 (w/w), without compromising their properties such as shape and penetration into the ex vivo human skin, even after storage at high humidity and temperature. High antigen loading did not induce protein aggregation during dMN fabrication as demonstrated by complementary analytical methods. However, the dissolution rate in ex vivo human skin decreased with increasing antigen loading. About 2.7 µg OVA could be delivered in mice by using a single array with an OVA:HA ratio of 1:3 (w/w). Intradermal vaccination with dMNs induced an immune response similar as subcutaneous injection and faster than after hollow microneedle injection. In conclusion, results suggest that (i) the polydimethylsiloxane mold design has an impact on the manufacturing of dMNs, (ii) the increase in antigen loading in dMNs affects the microneedle dissolution and (iii) dMNs are a valid alternative for vaccine administration over conventional injection. Show less
Yu, M.; Silva, T.C.; Opstal, A. van; Romeijn, S.; Every, H.A.; Jiskoot, W.; ... ; Ottens, M. 2019
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... Show moreIn 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. Show less
Worldwide, enteric infections rank third among all causes of disease burdens, and vaccines able to induce a strong and long lasting intestinal immune responses are needed. Parenteral immunization... Show moreWorldwide, enteric infections rank third among all causes of disease burdens, and vaccines able to induce a strong and long lasting intestinal immune responses are needed. Parenteral immunization generally do not generate intestinal IgA. Recently however, injections of retinoic acid (RA) dissolved in oil, administered multiple times at the days before vaccination to precondition the vaccine-draining lymph nodes, enabled a parenteral vaccine strategy to induce intestinal IgA. As multiple injections of RA before vaccination is not an attractive strategy for clinical practice, we aimed to develop a 'one injection' vaccine formulation that upon parenteral administration induced intestinal IgA. Our vaccine formulation contained two liposomal delivery systems. One delivery system, based on 1,2-Distearoyl-sn-glycero-3-phosphocholine stabilized with PEG, was designed to exhibit fast drainage of RA to local lymph nodes to pre-condition these for a mucosal immune response before being subjected to the vaccine antigen. The other delivery system, based on the cationic liposomal adjuvant CAF01 stabilized with cholesterol, optimized for prolonged delivery of the antigen by migratory APCs to the pre-conditioned lymph node. Combined we call the adjuvant CAF23. We show that CAF23, administered by the subcutaneous route induces an antigen specific intestinal IgA response, making it a promising candidate adjuvant for vaccines against enteric diseases. Show less
Duijn, J. van; Elsas, M. van; Benne, N.; Depuydt, M.; Wezel, A.; Smeets, H.; ... ; Slütter, B. 2019
CD8+T-cells have been attributed both atherogenic and atheroprotective properties, butanalysis of CD8+T-cells has mostly been restricted to the circulation and secondary lymphoid organs.... Show moreCD8+T-cells have been attributed both atherogenic and atheroprotective properties, butanalysis of CD8+T-cells has mostly been restricted to the circulation and secondary lymphoid organs. Theatherosclerotic lesion, however, is a complex microenvironment containing a plethora of inflammatory signals,which may affect CD8+T-cell activation. Here, we address how this environment affects the functionality ofCD8+T-cells. We compared the cytokine production of CD8+T-cells derived from spleens and en-zymatically digested aortas ofapoE−/−mice with advanced atherosclerosis byflow cytometry. Aortic CD8+T-cells produced decreased amounts of IFN-γand TNF-αcompared to their systemic counterparts. The observeddysfunctional phenotype of the lesion-derived CD8+T-cells was not associated with classical exhaustion mar-kers, but with increased expression of the ectonucleotidase CD39. Indeed, pharmacological inhibition of CD39 inapoE−/−mice partly restored cytokine production by CD8+T-cells. Using a bone-marrow transplantation ap-proach, we show that TCR signaling is required to induce CD39 expression on CD8+T-cells in atheroscleroticlesions. Importantly, analysis of human endarterectomy samples showed a strong microenvironment specificupregulation of CD39 on CD8+T-cells in the plaques of human patients compared to matched blood samples. Our results suggest that the continuous TCR signaling in the atherosclerotic environment in thevessel wall induces an immune regulatory CD8+T-cell phenotype that is associated with decreased cytokineproduction through increased CD39 expression in both a murine atherosclerotic model and in atherosclerosispatients. This provides a new understanding of immune regulation by CD8+T-cells in atherosclerosis. Show less
Benne, N.; Leboux, R.J.T.; Glandrup, M.; Duijn, J. van; Lozano, Vigario F.; Neustrup, M.A.; ... ; Slütter, B. 2019
Regulatory T cells (Tregs) are vital for maintaining a balanced immune response and their dysfunction is oftenassociated with auto-immune disorders. We have previously shown that antigen-loaded... Show moreRegulatory T cells (Tregs) are vital for maintaining a balanced immune response and their dysfunction is oftenassociated with auto-immune disorders. We have previously shown that antigen-loaded anionic liposomescomposed of phosphatidylcholine (PC) and phosphatidylglycerol (PG) and cholesterol can induce strong antigenspecificTreg responses. We hypothesized that altering the rigidity of these liposomes while maintaining theirsize and surface charge would affect their capability of inducing Treg responses. The rigidity of liposomes isaffected in part by the length and saturation of carbon chains of the phospholipids in the bilayer, and in part bythe presence of cholesterol. We used atomic force microscopy (AFM) to measure the rigidity of anionic OVA323-containing liposomes composed of different types of PC and PG, with or without cholesterol, in a molar ratio of4:1(:2) distearoyl (DS)PC:DSPG (Young's modulus (YM) 3611 ± 1271 kPa), DSPC:DSPG:CHOL(1498 ± 531 kPa), DSPC:dipalmitoyl (DP)PG:CHOL (1208 ± 538), DPPC:DPPG:CHOL (1195 ± 348 kPa),DSPC:dioleoyl (DO)PG:CHOL (825 ± 307 kPa), DOPC:DOPG:CHOL (911 ± 447 kPa), and DOPC:DOPG(494 ± 365 kPa). Next, we assessed if rigidity affects the association of liposomes to bone marrow-deriveddendritic cells (BMDCs) in vitro. Aside from DOPC:DOPG liposomes, we observed a positive correlation betweenliposomal rigidity and cellular association. Finally, we show that rigidity positively correlates with Treg responsesin vitro in murine DCs and in vivo in mice. Our findings underline the suitability of AFM to measureliposome rigidity and the importance of this parameter when designing liposomes as a vaccine delivery system. Show less
Benne, N.; Leboux, R.J.T.; Glandrup, M.; Duijn, J. van; Lozano, Vigario F.; Neustrup, M.A.; ... ; Slütter, B. 2019
Regulatory T cells (Tregs) are vital for maintaining a balanced immune response and their dysfunction is oftenassociated with auto-immune disorders. We have previously shown that antigen-loaded... Show moreRegulatory T cells (Tregs) are vital for maintaining a balanced immune response and their dysfunction is oftenassociated with auto-immune disorders. We have previously shown that antigen-loaded anionic liposomescomposed of phosphatidylcholine (PC) and phosphatidylglycerol (PG) and cholesterol can induce strong antigenspecificTreg responses. We hypothesized that altering the rigidity of these liposomes while maintaining theirsize and surface charge would affect their capability of inducing Treg responses. The rigidity of liposomes isaffected in part by the length and saturation of carbon chains of the phospholipids in the bilayer, and in part bythe presence of cholesterol. We used atomic force microscopy (AFM) to measure the rigidity of anionic OVA323-containing liposomes composed of different types of PC and PG, with or without cholesterol, in a molar ratio of4:1(:2) distearoyl (DS)PC:DSPG (Young's modulus (YM) 3611 ± 1271 kPa), DSPC:DSPG:CHOL(1498 ± 531 kPa), DSPC:dipalmitoyl (DP)PG:CHOL (1208 ± 538), DPPC:DPPG:CHOL (1195 ± 348 kPa),DSPC:dioleoyl (DO)PG:CHOL (825 ± 307 kPa), DOPC:DOPG:CHOL (911 ± 447 kPa), and DOPC:DOPG(494 ± 365 kPa). Next, we assessed if rigidity affects the association of liposomes to bone marrow-deriveddendritic cells (BMDCs) in vitro. Aside from DOPC:DOPG liposomes, we observed a positive correlation betweenliposomal rigidity and cellular association. Finally, we show that rigidity positively correlates with Treg responsesin vitro in murine DCs and in vivo in mice. Our findings underline the suitability of AFM to measureliposome rigidity and the importance of this parameter when designing liposomes as a vaccine delivery system. Show less
Benne, N.; Leboux, R.J.T.; Glandrup, M.; Duijn, J. van; Lozano, Vigario F.; Neustrup, M.A.; ... ; Slütter, B. 2019
Regulatory T cells (Tregs) are vital for maintaining a balanced immune response and their dysfunction is often associated with auto-immune disorders. We have previously shown that antigen-loaded... Show moreRegulatory T cells (Tregs) are vital for maintaining a balanced immune response and their dysfunction is often associated with auto-immune disorders. We have previously shown that antigen-loaded anionic liposomes composed of phosphatidylcholine (PC) and phosphatidylglycerol (PG) and cholesterol can induce strong antigen-specific Treg responses. We hypothesized that altering the rigidity of these liposomes while maintaining their size and surface charge would affect their capability of inducing Treg responses. The rigidity of liposomes is affected in part by the length and saturation of carbon chains of the phospholipids in the bilayer, and in part by the presence of cholesterol. We used atomic force microscopy (AFM) to measure the rigidity of anionic OVA323-containing liposomes composed of different types of PC and PG, with or without cholesterol, in a molar ratio of 4:1(:2) distearoyl (DS)PC:DSPG (Young's modulus (YM) 3611 ± 1271 kPa), DSPC:DSPG:CHOL (1498 ± 531 kPa), DSPC:dipalmitoyl (DP)PG:CHOL (1208 ± 538), DPPC:DPPG:CHOL (1195 ± 348 kPa), DSPC:dioleoyl (DO)PG:CHOL (825 ± 307 kPa), DOPC:DOPG:CHOL (911 ± 447 kPa), and DOPC:DOPG (494 ± 365 kPa). Next, we assessed if rigidity affects the association of liposomes to bone marrow-derived dendritic cells (BMDCs) in vitro. Aside from DOPC:DOPG liposomes, we observed a positive correlation between liposomal rigidity and cellular association. Finally, we show that rigidity positively correlates with Treg responses in vitro in murine DCs and in vivo in mice. Our findings underline the suitability of AFM to measure liposome rigidity and the importance of this parameter when designing liposomes as a vaccine delivery system. Show less
Benne, N.; Duijn, J. van; Lozano Vigario, F.L.; Leboux, R.J.T.; Veelen, P. van; Kuiper, J.; ... ; Slütter, B. 2018
Atherosclerosis is the predominant underlying pathology of many types of cardiovascular disease and is one of the leading causes of death worldwide. It is characterized by the retention of oxidized... Show moreAtherosclerosis is the predominant underlying pathology of many types of cardiovascular disease and is one of the leading causes of death worldwide. It is characterized by the retention of oxidized low-density lipoprotein (ox-LDL) in lipid-rich macrophages (foam cells) in the intima of arteries. Autoantigens derived from oxLDL can be used to vaccinate against atherosclerosis. However, a major challenge is the induction of antigen-specific Tregs in a safe and effective way. Here we report that liposomes containing the anionic phospholipid 1,2distearoyl-sn-glycero-3-phosphoglycerol (DSPG) induce Tregs that are specific for the liposomes' cargo. Mechanistically, we show a crucial role for the protein corona that forms on the liposomes in the circulation, as uptake of DSPG-liposomes by antigen-presenting cells is mediated via complement component lq (Clq) and scavenger receptors (SRs). Vaccination of atherosclerotic mice on a western-type diet with DSPG-liposomes encapsulating an LDL-derived peptide antigen significantly reduced plaque formation by 50% and stabilized the plaques, and reduced serum cholesterol concentrations. These results indicate that DSPG-liposomes have potential as a delivery system in vaccination against atherosclerosis. Show less
Benne, N.; Duijn, J. van; Vigario, F.L.; Leboux, R.J.T.; Veelen, P. van; Kuiper, J.; ... ; Stutter, B. 2018
