Final Report Summary - VLPSIRNA (Virus-like particles: the next step in gene therapy)
During the first two years in Prof. Finn's laboratory I was involved in two main projects that allowed me to explore the use of virus-like particles (VLPs) as carriers of poorly immunogenic epitopes for the development of vaccines. During the third year in Prof. Meijer laboratory the main focus of my research has been on the development of biocompatible hydrogels. These hydrogels have the potential to be used as implants for the slow release of drugs or vaccines, or as synthetic substitutes of the extracellular matrix.
VLPs: Multifunctional antigen carriers (Finn laboratory)
Carbohydrates are present on the surface of a variety of pathogens and malignant cells in a highly repetitive manner. This makes them ideal targets for vaccines. However, glycans are poorly immunogenic and carbohydrate-specific antibodies have low affinity compared with protein-specific antibodies. In able to prepare a glycan-based vaccine we aim to develop a strategy that uses a highly immunogenic delivery platform, synthetically pure carbohydrates, and an adjuvant designed to activate a specific part of the immune system.
We intend to increase the low immunogenicity of some glycans by using VLPs as multifunctional antigen carriers. VLPs have shown to be promising vaccine carriers due to their inherent immunogenicity, a consequence of their nanometre size, nucleoprotein content, and regular structure. VLPs can be obtained in large quantities, possess structures known to atomic resolution and their surfaces provide multiple functionalities that can be addressed in a rational manner both chemically and genetically. This provides a unique and robust platform on which to explore parameters for optimisation of antigen delivery.
(1) bacteriophage Q beta as platform for cell membrane sugars of Streptococcus Pneumoniae (pneumococcus), towards a VLP-based pneumococcal vaccine (in collaboration with Zinaida Polonskaya, a graduate student in the Finn laboratory, Prof. Luc Teyton at the Scripps Research Institute, and Prof. Paul Savage at Brigham Young University)
Pneumococcal infections cause a variety of illnesses in children and adults, and drug-resistant strains of the bacterium are a growing threat. The current vaccine (Prevnar) is a complex, expensive, and not very effective mixture of bacterial polysaccharides. Our goal is to investigate VLPs as glycan antigen carriers for the development of an improved pneumococcal conjugate vaccine.
To this end, the surface of Q beta was functionalised with sugar serotypes of pneumococcus prepared in Prof. Savage's group using azide-alkyne click chemistry. The conjugates were characterised by gel electrophoresis, mass spectrometry, and size exclusion chromatography, which allowed us to determine particle stability and sugar loading. To investigate the influence of the sugar loading on the antibody response and isotype, Q beta constructs carrying different sugar loadings were prepared. The immunogenicity of these constructs was evaluated in mice by analysing weekly samples of serum using enzyme-linked immunosorbent assay (ELISA) in order to analyse anti-glycan antibody production and determine the isotype. Furthermore, isolation of monoclonals was performed to determine affinity and sequence. The antibody response of two different mice strains was compared and the response of infected animals to the vaccine is research ongoing.
The resulting particles gave a much higher immune response and level of T-cell help than any other platform previously seen in other studies with synthetic carbohydrates. Using Q beta as glycan carrier induces the production of antibodies of high affinity and with a good secondary response. From ELISA studies, it became evident that the sugar density on the Q beta has an impact on the antibody response and on the kind of isotypes obtained. In addition these studies allowed us to gain insight in the antibody response derived from different chemical functional groups that were introduced using bioconjugation strategies.
This data shows promising and significant in vivo results on a new formulation of polysaccharide vaccines. Currently, the final stage using mice models takes place and the protective efficiency of the Qb-glycan constructs is investigated towards pneumococcus infection under both prophylactic and therapeutic settings. The results from these studies will provide the final results for the articles and patents derived from this research.
(2) VLPs as platforms for tumor associated carbohydrate antigens (TACAs) (in collaboration with Prof. Xuefei Huang at Michigan State University, and Prof. Lbachir BenMohamed at UC Irvine)
The main aim of this project is to investigate VLP based platforms as a multi-component construct for anti-cancer vaccines. We expect that the VLP constructs can induce powerful humoral and cellular immunity, which can provide protection against cancer. First studies carried out in the Finn group have shown positive results in the enhancement of immunogenicity of the Tn antigen when attached to the plant virus cowpea mosaic virus (CPMV), and other sugars when attached to the Q beta capsid.
In this case, two different VLP's were investigated: Q beta and the plant virus CPMV. Different cancer epitopes synthesised in the group of Prof. Xuefei Huang were conjugated to Q beta and CPMV by making use of either an NHS activated ester or applying click chemistry. These two strategies provided two chemically different spacers and provided different loading of antigens on the particles' surface. The immune response was investigated on mice by the collaborators and the formulation was optimised by varying parameters such as dosage and adjuvant formulation.
The first results obtained by this approach were recently published in ACS Chemical Biology.
Development of hydrogels based on the supramolecular self-assembly of ureido-pyrimidinone (UPy) for biomedical applications (Meijer laboratory)
Hydrogels based on the supramolecular self-assembly of UPy modified poly(ethylene glycols) (PEG) have been investigated for tissue engineering and drug delivery in the Meijer group. These hydrogels have shown to be biocompatible, highly dynamic and stable, and they offer a range of different mechanical properties that can be tuned to adapt to different applications. These properties make them potential candidates for implants for the slow release of the vaccines, but also as synthetic substitutes of the extracellular matrix for regenerative medicine. Since preliminary results towards the latter goal had already been obtained in the Meijer group, we decided to focus first our attention on the use of hydrogels as synthetic substitutes of the extracellular matrix.
Towards this goal we intend to develop a modular approach for the three-dimensional (3D) culture of cells and organoids in UPy-based supramolecular hydrogels. We aim to find a procedure that will provide ideal conditions in terms of adherence points, and biological and mechanical properties for each cell type. To this end, we collaborate with several research groups working on the field tissue engineering and use the hydrogels for the culture of two different cell types: myoblasts and chondrocytes. This allows us to investigate these polymers for two different tissues: muscle and cartilage. First studies have focused on cell viability, hydrogel stability and bioactivation.
Myoblasts culture experiments in UPy-based hydrogels have been carried out in collaboration with Dr Noortje Bax and Prof. Carlijn Boute from the Biomedical Engineering department at the TU/e. The resultant constructs showed a homogenous distribution of cells throughout the gel and good cell viability. For the bioactivation of the gels, collagen I was introduced in the gel resulting in the formation of attachment points for cells to adhere and spread, which is crucial for cell differentiation and tissue formation.
The culture of chondrocytes in UPy-based hydrogels is a project initiated with Dr Linda Kock and Dr Debby Gawlitta from the UMC Utrecht with the aim of starting a new approach to prepare hydrogels for cartilage regeneration. Preliminary results showed that the UP-PEG hydrogels were able to withstand the cell culture conditions and provided an environment for chondrocytes to survive and retain their natural phenotype. Ongoing experiments will investigate the formation of cartilage and matrix deposition by the cells.
VLPs: Multifunctional antigen carriers (Finn laboratory)
Carbohydrates are present on the surface of a variety of pathogens and malignant cells in a highly repetitive manner. This makes them ideal targets for vaccines. However, glycans are poorly immunogenic and carbohydrate-specific antibodies have low affinity compared with protein-specific antibodies. In able to prepare a glycan-based vaccine we aim to develop a strategy that uses a highly immunogenic delivery platform, synthetically pure carbohydrates, and an adjuvant designed to activate a specific part of the immune system.
We intend to increase the low immunogenicity of some glycans by using VLPs as multifunctional antigen carriers. VLPs have shown to be promising vaccine carriers due to their inherent immunogenicity, a consequence of their nanometre size, nucleoprotein content, and regular structure. VLPs can be obtained in large quantities, possess structures known to atomic resolution and their surfaces provide multiple functionalities that can be addressed in a rational manner both chemically and genetically. This provides a unique and robust platform on which to explore parameters for optimisation of antigen delivery.
(1) bacteriophage Q beta as platform for cell membrane sugars of Streptococcus Pneumoniae (pneumococcus), towards a VLP-based pneumococcal vaccine (in collaboration with Zinaida Polonskaya, a graduate student in the Finn laboratory, Prof. Luc Teyton at the Scripps Research Institute, and Prof. Paul Savage at Brigham Young University)
Pneumococcal infections cause a variety of illnesses in children and adults, and drug-resistant strains of the bacterium are a growing threat. The current vaccine (Prevnar) is a complex, expensive, and not very effective mixture of bacterial polysaccharides. Our goal is to investigate VLPs as glycan antigen carriers for the development of an improved pneumococcal conjugate vaccine.
To this end, the surface of Q beta was functionalised with sugar serotypes of pneumococcus prepared in Prof. Savage's group using azide-alkyne click chemistry. The conjugates were characterised by gel electrophoresis, mass spectrometry, and size exclusion chromatography, which allowed us to determine particle stability and sugar loading. To investigate the influence of the sugar loading on the antibody response and isotype, Q beta constructs carrying different sugar loadings were prepared. The immunogenicity of these constructs was evaluated in mice by analysing weekly samples of serum using enzyme-linked immunosorbent assay (ELISA) in order to analyse anti-glycan antibody production and determine the isotype. Furthermore, isolation of monoclonals was performed to determine affinity and sequence. The antibody response of two different mice strains was compared and the response of infected animals to the vaccine is research ongoing.
The resulting particles gave a much higher immune response and level of T-cell help than any other platform previously seen in other studies with synthetic carbohydrates. Using Q beta as glycan carrier induces the production of antibodies of high affinity and with a good secondary response. From ELISA studies, it became evident that the sugar density on the Q beta has an impact on the antibody response and on the kind of isotypes obtained. In addition these studies allowed us to gain insight in the antibody response derived from different chemical functional groups that were introduced using bioconjugation strategies.
This data shows promising and significant in vivo results on a new formulation of polysaccharide vaccines. Currently, the final stage using mice models takes place and the protective efficiency of the Qb-glycan constructs is investigated towards pneumococcus infection under both prophylactic and therapeutic settings. The results from these studies will provide the final results for the articles and patents derived from this research.
(2) VLPs as platforms for tumor associated carbohydrate antigens (TACAs) (in collaboration with Prof. Xuefei Huang at Michigan State University, and Prof. Lbachir BenMohamed at UC Irvine)
The main aim of this project is to investigate VLP based platforms as a multi-component construct for anti-cancer vaccines. We expect that the VLP constructs can induce powerful humoral and cellular immunity, which can provide protection against cancer. First studies carried out in the Finn group have shown positive results in the enhancement of immunogenicity of the Tn antigen when attached to the plant virus cowpea mosaic virus (CPMV), and other sugars when attached to the Q beta capsid.
In this case, two different VLP's were investigated: Q beta and the plant virus CPMV. Different cancer epitopes synthesised in the group of Prof. Xuefei Huang were conjugated to Q beta and CPMV by making use of either an NHS activated ester or applying click chemistry. These two strategies provided two chemically different spacers and provided different loading of antigens on the particles' surface. The immune response was investigated on mice by the collaborators and the formulation was optimised by varying parameters such as dosage and adjuvant formulation.
The first results obtained by this approach were recently published in ACS Chemical Biology.
Development of hydrogels based on the supramolecular self-assembly of ureido-pyrimidinone (UPy) for biomedical applications (Meijer laboratory)
Hydrogels based on the supramolecular self-assembly of UPy modified poly(ethylene glycols) (PEG) have been investigated for tissue engineering and drug delivery in the Meijer group. These hydrogels have shown to be biocompatible, highly dynamic and stable, and they offer a range of different mechanical properties that can be tuned to adapt to different applications. These properties make them potential candidates for implants for the slow release of the vaccines, but also as synthetic substitutes of the extracellular matrix for regenerative medicine. Since preliminary results towards the latter goal had already been obtained in the Meijer group, we decided to focus first our attention on the use of hydrogels as synthetic substitutes of the extracellular matrix.
Towards this goal we intend to develop a modular approach for the three-dimensional (3D) culture of cells and organoids in UPy-based supramolecular hydrogels. We aim to find a procedure that will provide ideal conditions in terms of adherence points, and biological and mechanical properties for each cell type. To this end, we collaborate with several research groups working on the field tissue engineering and use the hydrogels for the culture of two different cell types: myoblasts and chondrocytes. This allows us to investigate these polymers for two different tissues: muscle and cartilage. First studies have focused on cell viability, hydrogel stability and bioactivation.
Myoblasts culture experiments in UPy-based hydrogels have been carried out in collaboration with Dr Noortje Bax and Prof. Carlijn Boute from the Biomedical Engineering department at the TU/e. The resultant constructs showed a homogenous distribution of cells throughout the gel and good cell viability. For the bioactivation of the gels, collagen I was introduced in the gel resulting in the formation of attachment points for cells to adhere and spread, which is crucial for cell differentiation and tissue formation.
The culture of chondrocytes in UPy-based hydrogels is a project initiated with Dr Linda Kock and Dr Debby Gawlitta from the UMC Utrecht with the aim of starting a new approach to prepare hydrogels for cartilage regeneration. Preliminary results showed that the UP-PEG hydrogels were able to withstand the cell culture conditions and provided an environment for chondrocytes to survive and retain their natural phenotype. Ongoing experiments will investigate the formation of cartilage and matrix deposition by the cells.