Développement d'une plateforme vaccinale polyvalente basée sur l'utilisation des nanoparticules du virus mosaïque de la papaye (PapMV) et de la transpeptidase Sortase A de Staphylococcus aureus

La vaccination demeure à ce jour le moyen le plus efficace dans la prévention et le contrôle de maladies infectieuses. Les nanoparticules du PapMV ont été efficacement utilisées comme plateforme vaccinale permettant l’augmentation de l’immunogénicité d’antigènes. Bien que ces nanoparticules aient démontré un grand potentiel, la fusion d’antigène directement dans l’ORF de la protéine de capside (CP) peut nuire à sa capacité d’autoassemblage en nanoparticules. Nous avons développé une nouvelle méthode de modification du PapMV basé sur l’utilisation de la transpeptidase bactérienne sortase A (SrtA) permettant la conjugaison d’antigènes directement sur les nanoparticules post-assemblage. La SrtA a permis la conjugaison de longs antigènes (M2e et T20) sans affecter l’intégrité structurale et immunologique des nanoparticules. Ces nanoparticules ont induit de fortes réponses humorales spécifiques aux antigènes et ont induit une protection complète contre une infection à l’influenza chez les souris vaccinées avec PapMV-SrtA-M2e. La plateforme PapMV-SrtA permet l’ingénierie facile et rapide de nouveaux vaccins.Vaccination remains to date the most effective intervention in the prevention and control of infectious diseases. PapMV nanoparticles have shown to be an efficient vaccine platform to increase antigens immunogenicity. While they have shown great potential, the insertion of antigens in the open reading frame (ORF) of the coat protein (CP) can affect its capacity to assemble into nanoparticles. We developed a new method to modify PapMV nanoparticles based on the use of bacterial transpeptidase sortase A (SrtA) to attach antigens directly onto assembled nanoparticles. SrtA attached long antigenic peptides (M2e and T20) onto PapMV nanoparticles without affecting their structural of immunological integrity. These nanoparticles induced strong antigen specific antibodies and fully protected PapMV-SrtA-M2e vaccinated mice against an influenza challenge. The use of the PapMV-SrtA platform will enable the faster and easier development of new vaccines

Development of a versatile vaccination platform based on papaya mosaic virus (PapMV) nanoparticles

Over the past years, virus-like particles (VLPs) have shown great potential as highly immunogenic subunit vaccines. These non-infectious viral structures mimic the native pathogen’s organisation and conformation. VLPs contain highly repetitive and ordered viral epitopes leading to B cell activation through receptor cross-linking. By displaying heterologous epitopes on VLPs, one can mount an immune response against a different pathogen. These chimeric VLPs serve as presentation scaffold and can sometimes act as adjuvant to boost the immune response. However, VLP assembly can be affected by large epitope insertions altering intra or extra protein interactions impacting its conformation. Even if the insertion is successful, the epitopes have to be exposed at the particle surface to induce an immune response. To circumvent this problem, we have developed a new vaccine platform based on PapMV nanoparticules and sortase A (SrtA) transpeptidase. SrtA catalyzes the covalent conjugation of target antigenic epitopes to already assembled PapMV VLPs harbouring the SrtA recognition motif LPETG. Successful SrtA conjugations were achieved with peptides derived from Influenza (M2e) and HIV (T20). SrtA conjugated PapMV nanoparticles induce strong humoral responses in mice against both M2e and T20 peptides. PapMV-M2e vaccinated mice were protected against a lethal dose of Influenza H1N1 (A/WSN/33). Sera from PapMV-T20 vaccinated mice did not reduce in vitro HIV infection even with the high presence of specific antibodies. This new PapMV-SrtA platform eliminates the need for genetic fusion of the coat protein that can be difficult, time consuming and, sometime, unrealizable. The modification of PapMV VLP post-assembly facilitates its use in the rapid development of new vaccines by changing the nature of the target epitopes conjugated. This could be particularly useful when developing a pandemic vaccine or personalised vaccine for cancer therapy

A Type System for Privacy Properties (Technical Report)

Mature push button tools have emerged for checking trace properties (e.g. secrecy or authentication) of security protocols. The case of indistinguishability-based privacy properties (e.g. ballot privacy or anonymity) is more complex and constitutes an active research topic with several recent propositions of techniques and tools. We explore a novel approach based on type systems and provide a (sound) type system for proving equivalence of protocols, for a bounded or an unbounded number of sessions. The resulting prototype implementation has been tested on various protocols of the literature. It provides a significant speed-up (by orders of magnitude) compared to tools for a bounded number of sessions and complements in terms of expressiveness other state-of-the-art tools, such as ProVerif and Tamarin: e.g., we show that our analysis technique is the first one to handle a faithful encoding of the Helios e-voting protocol in the context of an untrusted ballot box

Omega-3 Polyunsaturated Fatty Acid: A Pharmaco-Nutraceutical Approach to Improve the Responsiveness to Ursodeoxycholic Acid

Ursodeoxycholic acid (UDCA) is the first line therapy for the treatment of cholestatic and autoimmune liver diseases. Its clinical use is currently limited by a significant proportion of non-responder patients. Polyunsaturated fatty acids (n-3 PUFAs) possess important anti-inflammatory properties and protect liver cells against bile acid (BA)-induced toxicity. The present study was designed to rapidly evaluate whether combining n-3 PUFAs (i.e., eicosapentaenoic [EPA] and docosahexaenoic [DHA] acids) to UDCA would provide additional benefits when compared to the drug alone. The parameters evaluated were (i) the expression of genes governing BA synthesis, transport, and metabolism; (ii) the prevention of BA-induced apoptosis and endoplasmic reticulum (ER)-stress; and (iii) the control of BA- and LPS-dependent inflammation. In the absence of n-3 PUFAs, most of the parameters investigated were unaffected by UDCA or were only altered by the higher dose (500 µM) of the drug. By contrast, in the presence of EPA/DHA (50/50 µM), all parameters showed a strongly improved response and the lowest UDCA dosage (50 µM) provided equal or better benefits than the highest dose used alone. For example, the combination EPA/DHA + UDCA 50 µM caused comparable down-regulation of the CYP7A1 gene expression and of the BA-induced caspase 3 activity as observed with UDCA 500 µM. In conclusion, these results suggest that the addition of n-3 PUFAs to UDCA may improve the response to the drug, and that such a pharmaco-nutraceutical approach could be used in clinic to open the narrow therapeutic dose of UDCA in cholestatic liver diseases

Increased Immunogenicity of Full-Length Protein Antigens through Sortase-Mediated Coupling on the PapMV Vaccine Platform

Background: Flexuous rod-shape nanoparticles—made of the coat protein of papaya mosaic virus (PapMV)—provide a promising vaccine platform for the presentation of viral antigens to immune cells. The PapMV nanoparticles can be combined with viral antigens or covalently linked to them. The coupling to PapMV was shown to improve the immune response triggered against peptide antigens (<39 amino acids) but it remains to be tested if large proteins can be coupled to this platform and if the coupling will lead to an immune response improvement. Methods: Two full-length recombinant viral proteins, the influenza nucleoprotein (NP) and the simian immunodeficiency virus group-specific protein antigen (GAG) were coupled to PapMV nanoparticles using sortase A. Mice were immunized with the nanoparticles coupled to the antigens and the immune response directed to the antigens were analyzed by ELISA and ELISPOT. Results: We showed the feasibility of coupling two different full-length proteins (GAG and NP) to the nanoparticle. We also showed that the coupling to PapMV nanoparticles improved significantly the humoral and the cytotoxic T lymphocyte (CTL) immune response to the antigens. Conclusion: This proof of concept demonstrates the versatility and the efficacy of the PapMV vaccine platform in the design of vaccines against viral diseases

A versatile papaya mosaic virus (PapMV) vaccine platform based on sortase-mediated antigen coupling

Abstract Background Flexuous rod-shaped nanoparticles made of the coat protein (CP) of papaya mosaic virus (PapMV) have been shown to trigger innate immunity through engagement of toll-like receptor 7 (TLR7). PapMV nanoparticles can also serve as a vaccine platform as they can increase the immune response to fused peptide antigens. Although this approach shows great potential, fusion of antigens directly to the CP open reading frame (ORF) is challenging because the fused peptides can alter the structure of the CP and its capacity to self assemble into nanoparticles—a property essential for triggering an efficient immune response to the peptide. This represents a serious limitation to the utility of this approach as fusion of small peptides only is tolerated. Results We have developed a novel approach in which peptides are fused directly to pre-formed PapMV nanoparticles. This approach is based on the use of a bacterial transpeptidase (sortase A; SrtA) that can attach the peptide directly to the nanoparticle. An engineered PapMV CP harbouring the SrtA recognition motif allows efficient coupling. To refine our engineering, and to predict the efficacy of coupling with SrtA, we modeled the PapMV structure based on the known structure of PapMV CP and on recent reports revealing the structure of two closely related potexviruses: pepino mosaic virus (PepMV) and bamboo mosaic virus (BaMV). We show that SrtA can allow the attachment of long peptides [Influenza M2e peptide (26 amino acids) and the HIV-1 T20 peptide (39 amino acids)] to PapMV nanoparticles. Consistent with our PapMV structural model, we show that around 30% of PapMV CP subunits in each nanoparticle can be fused to the peptide antigen. As predicted, engineered nanoparticles were capable of inducing a strong antibody response to the fused antigen. Finally, in a challenge study with influenza virus, we show that mice vaccinated with PapMV-M2e are protected from infection. Conclusions This technology will allow the development of vaccines harbouring long peptides containing several B and/or T cell epitopes that can contribute to a broad and robust protection from infection. The design can be fast, versatile and can be adapted to the development of vaccines for many infectious diseases as well as cancer vaccines