Development of an innovative adenovirus-inspired self-assembling vaccine platform rapidly adaptable to coronaviruses and other emergent viruses

The COVID-19 pandemic clearly shows how emergent diseases can cause severe global health and economic problems. We must be prepared to react swiftly against new pathogenic agents and this requires the development of vaccines that are safe, efficient in the long-term and easily adaptable with a short revision time. To this end, the COVID-19 mRNA and adenoviral vector vaccines have been spectacular successes, permitting rapid vaccination across the world in an unprecedented manner. Here we report the design of a new adenovirus-derived vaccine technology based on non-infectious pseudo-viral nanoparticles from the serotype 3 human adenovirus. Each nanoparticle comprises sixty identical proteins that assemble to form a 30 nm diameter spherical particle. A sequence has been engineered into the surface of this protein that enables the display of a covalently-bound target antigens. To demonstrate the efficiency of this approach, we added the SARS-CoV 2 spike protein receptor binding domain (RBD), that interacts with host cell ACE2 receptors, to the surface of the nanoparticles. We first showed that the glycosylated RBD retained its ACE2-binding function when displayed on nanoparticles. We then measured the in vivo humoral response of our vaccine candidate in mice and observed a strong antibody response after the prime injection; further levels were achieved following a second booster injection. In mice preimmunized with underivatized adenoviral nanoparticles, we tested if adenovirus seroprevalence, as frequently observed in humans, was detrimental to the RBD-mediated protection provided by our vaccine candidate. Interestingly, a strong anti-coronaviral response was still observed suggesting that existing circulating anti-adenovirus antibodies are not deleterious to our vaccine platform. We then performed pseudo-CoV 2 neutralization assays and obtained higher ID50 values than observed with COVID-19 convalescent sera, thus showing the high potential efficacy of our vaccine platform. This new vaccine technology is a tool that is easily adaptable to future SARS-CoV 2 variants and, more generally, to future emergent viruses and pathogens

Développement et évaluation d'une plateforme vaccinale novatrice pour le traitement du mélanome

Virus like particles (VLPs) are versatile protein-based platforms which can be used as vaccine platform mainly in infectiology. In the present work we used adenoviruses dodecahedrons to display either short epitopes or a large tumor antigen. These non-infectious but immunogenic structures are formed during the adenovirus replication cycle, and have been modified to create a versatile platform named ADDomer (ADenovirus Dodecamer). Recently, it has been shown that ADDomers displaying a Chikungunya virus epitope successfully induced an anti-epitopic response in mice.Based on these results, this project aims at adapting the ADDomer platform to develop a cancer vaccine for melanoma. ADDomers displaying melanoma epitopes/antigens have successfully been produced and characterized. Epitopes can directly be genetically inserted inside the ADDomers’ exposed loops and are therefore exposed on the ADDomers’ surface. For antigens, the autocatalytic system SpyTag/SpyCatcher was adapted in order to covalently fix antigens on the ADDomers’ surface.Once the ADDomers were fully produced and characterized, we explored for the first time the immunogenicity of these ADDomers and their impact on human dendritic cell (DC) subsets’ features. We first demonstrated that A2L/MelA-ADDomers (ADDomers displaying human melanoma either epitope or antigen) displayed a strong immune-stimulating potential on human DC subsets (cDC2s, cDC1s, pDCs), which were able to internalize and cross-present tumor antigen, and subsequently cross-prime antigen-specific T-cell responses. To further limit off-target effects and enhance DC targeting, we engineered specific ligands to de-target untargeted cells (epithelial cells) and improve DCs’ addressing.Finally, we evaluated the ADDomers’ ability to control melanoma B16-OVA growth in mice. A set of adjuvants was screened showing that Poly I:C was well-suited to generate a homogenous cellular and humoral response against the desired epitopes. In a prophylactic setting, vaccination with the ADDomers displaying ovalbumin epitopes resulted in a total inhibition of tumor growth one month after vaccination. Remarkably, a therapeutic vaccination strategy showed a delay in the grafted tumor growth or its total rejection. Interestingly, if the ‘simple’ epitope display on the ADDomer was sufficient to prevent tumor growth, the improved engineered platform enabling the display of large antigen represents a tool to overcome the barrier of immune allele restriction, broadening the immune response, and paving the way to its potential use in humans as an off-the-shelf vaccine.Les particules pseudovirales constituent des plateformes protéiques polyvalentes qui peuvent être utilisées comme plateforme vaccinale, principalement en infectiologie. Dans ces travaux de thèse, nous avons utilisé des dodécaèdres d'adénovirus pour afficher soit des épitopes courts, soit un antigène tumoral de grande taille. Ces particules non infectieuses mais immunogènes sont formées pendant le cycle de réplication de l'adénovirus, et ont été modifiées pour créer une plateforme polyvalente appelée ADDomer (ADenovirus Dodecamer). Récemment, il a été démontré que des ADDomers affichant un épitope du virus Chikungunya induisaient avec succès une réponse anti-épitopique chez la souris.Sur la base de ces résultats, ce projet vise à adapter la plateforme ADDomer afin de développer un vaccin thérapeutique contre le mélanome. Des ADDomers affichant des épitopes/antigènes de mélanome ont été produits et caractérisés. Les épitopes peuvent être directement insérés génétiquement à l'intérieur des boucles de surface des ADDomers et seront donc affichés à leur surface. Pour l’affichage d’antigènes, le système autocatalytique SpyTag/SpyCatcher a été adapté afin de fixer de manière covalente les antigènes sur la surface des ADDomers.Une fois les ADDomers produits et caractérisés, nous avons exploré pour la première fois l'immunogénicité de ces ADDomers et leur impact sur des sous-types de cellules dendritiques (DC) humaines. Nous avons d'abord démontré que les ADDomers A2L et MelanA (épitope et antigène de mélanome humain) présentaient un fort potentiel de stimulation immunitaire des sous-types de cellules dendritiques humaines (cDC2s, cDC1s, pDCs), qui étaient capables d'internaliser et de cross présenter l'antigène tumoral, puis d'amorcer l’activation de réponses cellulaires T spécifiques de l'antigène. De plus, afin d’améliorer l’adressage spécifique des DCs, des ligands spécifiques ont été conçus.Enfin, nous avons évalué la capacité des ADDomers à contrôler la croissance du mélanome B16-OVA chez la souris. Plusieurs adjuvants ont été testés, montrant ainsi que le Poly I:C était bien adapté pour générer une réponse cellulaire et humorale homogène contre les épitopes souhaités. Dans un schéma prophylactique, la vaccination avec les ADDomers présentant des épitopes d'ovalbumine a entraîné une inhibition totale de la croissance tumorale. De plus, une stratégie de vaccination thérapeutique a montré un retard dans la croissance de la tumeur greffée ou son rejet total. Nous avons remarqué au cours de ces expériences que si l'affichage d'épitopes sur l'ADDomer était suffisant pour empêcher la croissance de la tumeur, l'affichage d'antigènes entiers présente le grand avantage de surmonter la barrière de la restriction allélique permettant d’obtenir une immunothérapie "prête à l'emploi" applicable à une large population

Développement et évaluation d'une plateforme vaccinale novatrice pour le traitement du mélanome

Virus like particles (VLPs) are versatile protein-based platforms which can be used as vaccine platform mainly in infectiology. In the present work we used adenoviruses dodecahedrons to display either short epitopes or a large tumor antigen. These non-infectious but immunogenic structures are formed during the adenovirus replication cycle, and have been modified to create a versatile platform named ADDomer (ADenovirus Dodecamer). Recently, it has been shown that ADDomers displaying a Chikungunya virus epitope successfully induced an anti-epitopic response in mice.Based on these results, this project aims at adapting the ADDomer platform to develop a cancer vaccine for melanoma. ADDomers displaying melanoma epitopes/antigens have successfully been produced and characterized. Epitopes can directly be genetically inserted inside the ADDomers’ exposed loops and are therefore exposed on the ADDomers’ surface. For antigens, the autocatalytic system SpyTag/SpyCatcher was adapted in order to covalently fix antigens on the ADDomers’ surface.Once the ADDomers were fully produced and characterized, we explored for the first time the immunogenicity of these ADDomers and their impact on human dendritic cell (DC) subsets’ features. We first demonstrated that A2L/MelA-ADDomers (ADDomers displaying human melanoma either epitope or antigen) displayed a strong immune-stimulating potential on human DC subsets (cDC2s, cDC1s, pDCs), which were able to internalize and cross-present tumor antigen, and subsequently cross-prime antigen-specific T-cell responses. To further limit off-target effects and enhance DC targeting, we engineered specific ligands to de-target untargeted cells (epithelial cells) and improve DCs’ addressing.Finally, we evaluated the ADDomers’ ability to control melanoma B16-OVA growth in mice. A set of adjuvants was screened showing that Poly I:C was well-suited to generate a homogenous cellular and humoral response against the desired epitopes. In a prophylactic setting, vaccination with the ADDomers displaying ovalbumin epitopes resulted in a total inhibition of tumor growth one month after vaccination. Remarkably, a therapeutic vaccination strategy showed a delay in the grafted tumor growth or its total rejection. Interestingly, if the ‘simple’ epitope display on the ADDomer was sufficient to prevent tumor growth, the improved engineered platform enabling the display of large antigen represents a tool to overcome the barrier of immune allele restriction, broadening the immune response, and paving the way to its potential use in humans as an off-the-shelf vaccine.Les particules pseudovirales constituent des plateformes protéiques polyvalentes qui peuvent être utilisées comme plateforme vaccinale, principalement en infectiologie. Dans ces travaux de thèse, nous avons utilisé des dodécaèdres d'adénovirus pour afficher soit des épitopes courts, soit un antigène tumoral de grande taille. Ces particules non infectieuses mais immunogènes sont formées pendant le cycle de réplication de l'adénovirus, et ont été modifiées pour créer une plateforme polyvalente appelée ADDomer (ADenovirus Dodecamer). Récemment, il a été démontré que des ADDomers affichant un épitope du virus Chikungunya induisaient avec succès une réponse anti-épitopique chez la souris.Sur la base de ces résultats, ce projet vise à adapter la plateforme ADDomer afin de développer un vaccin thérapeutique contre le mélanome. Des ADDomers affichant des épitopes/antigènes de mélanome ont été produits et caractérisés. Les épitopes peuvent être directement insérés génétiquement à l'intérieur des boucles de surface des ADDomers et seront donc affichés à leur surface. Pour l’affichage d’antigènes, le système autocatalytique SpyTag/SpyCatcher a été adapté afin de fixer de manière covalente les antigènes sur la surface des ADDomers.Une fois les ADDomers produits et caractérisés, nous avons exploré pour la première fois l'immunogénicité de ces ADDomers et leur impact sur des sous-types de cellules dendritiques (DC) humaines. Nous avons d'abord démontré que les ADDomers A2L et MelanA (épitope et antigène de mélanome humain) présentaient un fort potentiel de stimulation immunitaire des sous-types de cellules dendritiques humaines (cDC2s, cDC1s, pDCs), qui étaient capables d'internaliser et de cross présenter l'antigène tumoral, puis d'amorcer l’activation de réponses cellulaires T spécifiques de l'antigène. De plus, afin d’améliorer l’adressage spécifique des DCs, des ligands spécifiques ont été conçus.Enfin, nous avons évalué la capacité des ADDomers à contrôler la croissance du mélanome B16-OVA chez la souris. Plusieurs adjuvants ont été testés, montrant ainsi que le Poly I:C était bien adapté pour générer une réponse cellulaire et humorale homogène contre les épitopes souhaités. Dans un schéma prophylactique, la vaccination avec les ADDomers présentant des épitopes d'ovalbumine a entraîné une inhibition totale de la croissance tumorale. De plus, une stratégie de vaccination thérapeutique a montré un retard dans la croissance de la tumeur greffée ou son rejet total. Nous avons remarqué au cours de ces expériences que si l'affichage d'épitopes sur l'ADDomer était suffisant pour empêcher la croissance de la tumeur, l'affichage d'antigènes entiers présente le grand avantage de surmonter la barrière de la restriction allélique permettant d’obtenir une immunothérapie "prête à l'emploi" applicable à une large population

Développement et évaluation d'une plateforme vaccinale novatrice pour le traitement du mélanome

Virus like particles (VLPs) are versatile protein-based platforms which can be used as vaccine platform mainly in infectiology. In the present work we used adenoviruses dodecahedrons to display either short epitopes or a large tumor antigen. These non-infectious but immunogenic structures are formed during the adenovirus replication cycle, and have been modified to create a versatile platform named ADDomer (ADenovirus Dodecamer). Recently, it has been shown that ADDomers displaying a Chikungunya virus epitope successfully induced an anti-epitopic response in mice.Based on these results, this project aims at adapting the ADDomer platform to develop a cancer vaccine for melanoma. ADDomers displaying melanoma epitopes/antigens have successfully been produced and characterized. Epitopes can directly be genetically inserted inside the ADDomers’ exposed loops and are therefore exposed on the ADDomers’ surface. For antigens, the autocatalytic system SpyTag/SpyCatcher was adapted in order to covalently fix antigens on the ADDomers’ surface.Once the ADDomers were fully produced and characterized, we explored for the first time the immunogenicity of these ADDomers and their impact on human dendritic cell (DC) subsets’ features. We first demonstrated that A2L/MelA-ADDomers (ADDomers displaying human melanoma either epitope or antigen) displayed a strong immune-stimulating potential on human DC subsets (cDC2s, cDC1s, pDCs), which were able to internalize and cross-present tumor antigen, and subsequently cross-prime antigen-specific T-cell responses. To further limit off-target effects and enhance DC targeting, we engineered specific ligands to de-target untargeted cells (epithelial cells) and improve DCs’ addressing.Finally, we evaluated the ADDomers’ ability to control melanoma B16-OVA growth in mice. A set of adjuvants was screened showing that Poly I:C was well-suited to generate a homogenous cellular and humoral response against the desired epitopes. In a prophylactic setting, vaccination with the ADDomers displaying ovalbumin epitopes resulted in a total inhibition of tumor growth one month after vaccination. Remarkably, a therapeutic vaccination strategy showed a delay in the grafted tumor growth or its total rejection. Interestingly, if the ‘simple’ epitope display on the ADDomer was sufficient to prevent tumor growth, the improved engineered platform enabling the display of large antigen represents a tool to overcome the barrier of immune allele restriction, broadening the immune response, and paving the way to its potential use in humans as an off-the-shelf vaccine.Les particules pseudovirales constituent des plateformes protéiques polyvalentes qui peuvent être utilisées comme plateforme vaccinale, principalement en infectiologie. Dans ces travaux de thèse, nous avons utilisé des dodécaèdres d'adénovirus pour afficher soit des épitopes courts, soit un antigène tumoral de grande taille. Ces particules non infectieuses mais immunogènes sont formées pendant le cycle de réplication de l'adénovirus, et ont été modifiées pour créer une plateforme polyvalente appelée ADDomer (ADenovirus Dodecamer). Récemment, il a été démontré que des ADDomers affichant un épitope du virus Chikungunya induisaient avec succès une réponse anti-épitopique chez la souris.Sur la base de ces résultats, ce projet vise à adapter la plateforme ADDomer afin de développer un vaccin thérapeutique contre le mélanome. Des ADDomers affichant des épitopes/antigènes de mélanome ont été produits et caractérisés. Les épitopes peuvent être directement insérés génétiquement à l'intérieur des boucles de surface des ADDomers et seront donc affichés à leur surface. Pour l’affichage d’antigènes, le système autocatalytique SpyTag/SpyCatcher a été adapté afin de fixer de manière covalente les antigènes sur la surface des ADDomers.Une fois les ADDomers produits et caractérisés, nous avons exploré pour la première fois l'immunogénicité de ces ADDomers et leur impact sur des sous-types de cellules dendritiques (DC) humaines. Nous avons d'abord démontré que les ADDomers A2L et MelanA (épitope et antigène de mélanome humain) présentaient un fort potentiel de stimulation immunitaire des sous-types de cellules dendritiques humaines (cDC2s, cDC1s, pDCs), qui étaient capables d'internaliser et de cross présenter l'antigène tumoral, puis d'amorcer l’activation de réponses cellulaires T spécifiques de l'antigène. De plus, afin d’améliorer l’adressage spécifique des DCs, des ligands spécifiques ont été conçus.Enfin, nous avons évalué la capacité des ADDomers à contrôler la croissance du mélanome B16-OVA chez la souris. Plusieurs adjuvants ont été testés, montrant ainsi que le Poly I:C était bien adapté pour générer une réponse cellulaire et humorale homogène contre les épitopes souhaités. Dans un schéma prophylactique, la vaccination avec les ADDomers présentant des épitopes d'ovalbumine a entraîné une inhibition totale de la croissance tumorale. De plus, une stratégie de vaccination thérapeutique a montré un retard dans la croissance de la tumeur greffée ou son rejet total. Nous avons remarqué au cours de ces expériences que si l'affichage d'épitopes sur l'ADDomer était suffisant pour empêcher la croissance de la tumeur, l'affichage d'antigènes entiers présente le grand avantage de surmonter la barrière de la restriction allélique permettant d’obtenir une immunothérapie "prête à l'emploi" applicable à une large population

The use of magnetic susceptibility and viscosity measurements as a mapping tool for soil properties: DIGISOIL field results

International audienceThe clay (paramagnetic) and iron oxides (ferrimagnetic) contents carry the magnetic properties of soils. Changes in the local redox conditions influence the iron oxides fatum and, then, the magnetic properties of soils. Consequently, many processes (air circulation, microbial activities, heavy metal accumulation...) can influence the magnetic behaviour. The aim of this study is to evaluate which soil property(ies) (carbon content, bulk density, clay content...) can be mapped with the help of magnetic properties measurements (magnetic susceptibility, magnetic viscosity and there ratio). The survey has been achieved on the validation site defined in the DIGISOIL project and located in Luxembourg. This site is about 4 ha in area, it is cultivated and ploughed. We made 231 measurements with 5 devices (MS2D Bartington ltd, DECCO, TS6 Protovale, CS60 and VC100 prototype) in 7 configurations. 30 calibration cores were also collected to characterize soil properties (carbon content, clay content, stone content and water content) in the field. The results are used to analyse the relationship between soil and magnetic properties. When a relation appears, soil property maps derived from magnetic property ones are plotted and confronted with collected sample results

The Adenovirus Dodecahedron: Beyond the Platonic Story

International audienceMany geometric forms are found in nature, some of them adhering to mathematical laws or amazing aesthetic rules. One of the best-known examples in microbiology is the icosahedral shape of certain viruses with 20 triangular facets and 12 edges. What is less known, however, is that a complementary object displaying 12 faces and 20 edges called a 'dodecahedron' can be produced in huge amounts during certain adenovirus replication cycles. The decahedron was first described more than 50 years ago in the human adenovirus (HAdV3) viral cycle. Later on, the expression of this recombinant scaffold, combined with improvements in cryo-electron microscopy, made it possible to decipher the structural determinants underlying their architecture. Recently, this particle, which mimics viral entry, was used to fish the long elusive adenovirus receptor, desmoglein-2, which serves as a cellular docking for some adenovirus serotypes. This breakthrough enabled the understanding of the physiological role played by the dodecahedral particles, showing that icosahedral and dodecahedral particles live more than a simple platonic story. All these points are developed in this review, and the potential use of the dodecahedron in therapeutic development is discussed

Stimulation of the immune system by a tumor antigen-bearing adenovirus-inspired VLP allows control of melanoma growth

International audienceVirus-like particles (VLPs) are versatile protein-based platforms that can be used as a vaccine platform mainly in infectiology. In the present work, we compared a previously designed, non-infectious, adenovirus-inspired 60-mer dodecahedric VLP to display short epitopes or a large tumor model antigen. To validate these two kinds of platforms as a potential immuno-stimulating approach, we evaluated their ability to control melanoma B16-ovalbumin (OVA) growth in mice. A set of adjuvants was screened, showing that polyinosinic-polycytidylic acid (poly(I:C)) was well suited to generate a homogeneous cellular and humoral response against the desired epitopes. In a prophylactic setting, vaccination with the VLP displaying these epitopes resulted in total inhibition of tumor growth 1 month after vaccination. A therapeutic vaccination strategy showed a delay in grafted tumor growth or its total rejection. If the "simple" epitope display on the VLP is sufficient to prevent tumor growth, then an improved engineered platform enabling display of a large antigen is a tool to overcome the barrier of immune allele restriction, broadening the immune response, and paving the way for its potential utilization in humans as an off-the-shelf vaccine

Stimulation of the immune system by a tumor antigen-bearing adenovirus-inspired VLP allows control of melanoma growth

International audienceVirus-like particles (VLPs) are versatile protein-based platforms that can be used as a vaccine platform mainly in infectiology. In the present work, we compared a previously designed, non-infectious, adenovirus-inspired 60-mer dodecahedric VLP to display short epitopes or a large tumor model antigen. To validate these two kinds of platforms as a potential immuno-stimulating approach, we evaluated their ability to control melanoma B16-ovalbumin (OVA) growth in mice. A set of adjuvants was screened, showing that polyinosinic-polycytidylic acid (poly(I:C)) was well suited to generate a homogeneous cellular and humoral response against the desired epitopes. In a prophylactic setting, vaccination with the VLP displaying these epitopes resulted in total inhibition of tumor growth 1 month after vaccination. A therapeutic vaccination strategy showed a delay in grafted tumor growth or its total rejection. If the "simple" epitope display on the VLP is sufficient to prevent tumor growth, then an improved engineered platform enabling display of a large antigen is a tool to overcome the barrier of immune allele restriction, broadening the immune response, and paving the way for its potential utilization in humans as an off-the-shelf vaccine

Adenovirus-Inspired Virus-like-Particles Displaying Melanoma Tumor Antigen Specifically Target Human DC Subsets and Trigger Antigen-Specific Immune Responses

Virus-like particles constitute versatile vectors that can be used as vaccine platforms in many fields from infectiology and more recently to oncology. We previously designed non-infectious adenovirus-inspired 60-mer dodecahedric virus-like particles named ADDomers displaying on their surface either a short epitope or a large tumor/viral antigen. In this work, we explored for the first time the immunogenicity of ADDomers exhibiting melanoma-derived tumor antigen/epitope and their impact on the features of human dendritic cell (DC) subsets. We first demonstrated that ADDomers displaying tumor epitope/antigen elicit a strong immune-stimulating potential of human DC subsets (cDC2s, cDC1s, pDCs), which were able to internalize and cross-present tumor antigen, and subsequently cross-prime antigen-specific T-cell responses. To further limit off-target effects and enhance DC targeting, we engineered specific motifs to de-target epithelial cells and improve DCs’ addressing. The improved engineered platform making it possible to display large antigen represents a tool to overcome the barrier of immune allele restriction, broadening the immune response, and paving the way to its potential utilization in humans as an off-the-shelf vaccine

Elicitation of potent SARS-CoV-2 neutralizing antibody responses through immunization with a versatile adenovirus-inspired multimerization platform

International audienceVirus-like particles (VLPs) are highly suited platforms for protein-based vaccines. In the present work, we adapted a previously designed non-infectious adenovirus-inspired 60-mer dodecahedric VLP (ADDomer) to display a multimeric array of large antigens through a SpyTag/SpyCatcher system. To validate the platform as a potential COVID-19 vaccine approach, we decorated the newly designed VLP with the glycosylated receptor binding domain (RBD) of SARS-CoV-2. Cryoelectron microscopy structure revealed that up to 60 copies of this antigenic domain could be bound on a single ADDomer particle, with the symmetrical arrangements of a dodecahedron. Mouse immunization with the RBD decorated VLPs already showed a significant specific humoral response following prime vaccination, greatly reinforced by a single boost. Neutralization assays with SARS-CoV-2 spike pseudo-typed virus demonstrated the elicitation of strong neutralization titers, superior to those of COVID-19 convalescent patients. Notably, the presence of pre-existing immunity against the adenoviral-derived particles did not hamper the immune response against the antigen displayed on its surface. This plug and play vaccine platform represents a promising new highly versatile tool to combat emergent pathogens