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

Synthetic self-assembling ADDomer platform for highly efficient vaccination by genetically encoded multiepitope display

International audienceSelf-assembling virus-like particles represent highly attractive tools for developing next-generation vaccines and protein therapeutics. We created ADDomer, an adenovirus-derived multimeric protein-based self-assembling nanoparticle scaffold engineered to facilitate plug-and-play display of multiple immunogenic epitopes from pathogens. We used cryo-electron microscopy at near-atomic resolution and implemented novel, cost-effective, high-performance cloud computing to reveal architectural features in unprecedented detail. We analyzed ADDomer interaction with components of the immune system and developed a promising first-in-kind ADDomer-based vaccine candidate to combat emerging Chikungunya infectious disease, exemplifying the potential of our approach

Intermediate-resolution crystal structure of the human adenovirus B serotype 3 fibre knob in complex with the EC2-EC3 fragment of desmoglein 2

International audienceThe cryo-electron microscopy (cryo-EM) structure of the complex between the trimeric human adenovirus B serotype 3 fibre knob and human desmoglein 2 fragments containing cadherin domains EC2 and EC3 has been published, showing 3:1 and 3:2 complexes. Here, the crystal structure determined at 4.5 Å resolution is presented with one EC2-EC3 desmoglein fragment bound per fibre knob monomer in the asymmetric unit, leading to an apparent 3:3 stoichiometry. However, in concentrated solution the 3:2 complex is predominant, as shown by small-angle X-ray scattering (SAXS), while cryo-EM at lower concentrations showed a majority of the 3:1 complex. Substitution of the calcium ions bound to the desmoglein domains by terbium ions allowed confirmation of the X-ray model using their anomalous scattering and shows that at least one binding site per cluster of calcium ions is intact and exchangeable and, combined with SAXS data, that the cadherin domains are folded even in the distal part that is invisible in the cryo-EM reconstruction

Human ficolin-2 recognition versatility extended: An update on the binding of ficolin-2 to sulfated/phosphated carbohydrates

International audienceFicolin-2 has been reported to bind to DNA and heparin, but the mechanism involved has not been thoroughly investigated. X-ray studies of the ficolin-2 fibrinogen-like domain in complex with several new ligands now show that sulfate and phosphate groups are prone to bind to the S3 binding site of the protein. Composed of Arg132, Asp133, Thr136 and Lys221, the S3 site was previously shown to mainly bind N-acetyl groups. Furthermore, DNA and heparin compete for binding to ficolin-2. Mutagenesis studies reveal that Arg132, and to a lesser extent Asp133, are important for this binding property. The versatility of the S3 site in binding N-acetyl, sulfate and phosphate groups is discussed through comparisons with homologous fibrinogen-like recognition proteins

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

Toward the understanding of DSG2 and CD46 interaction with HAdV-11 fiber, a super-complex analysis

International audienceABSTRACT Among human species B adenovirus, HAdV-11, has the particular feature of exploiting two primary receptors, desmoglein 2 (DSG2) and CD46, to mediate cell attachment. This serotype represents a particularly promising class of oncolytic viral vaccines. For instance, Enadenotucirev, a species B HAdV-11/HAdV-3 chimeric adenoviral vector, demonstrated preclinical tumor-selective cytotoxicity. Understanding the mechanism of HAdV-11 entry is a major challenge for the development of powerful vectors. Interactions between HAdV-11 and CD46 are well-characterized and clearly depend on the fiber protein, but the mechanism whereby HAdV-11 engages DSG2 has been still unresolved. In this study, we described a new cryo-EM structure of the HAdV-11 fiber in complex with DSG2. Kinetic interaction studies demonstrated that the binding stability of the HAdV-11 in complex with DSG2 was significantly lower than with CD46. Furthermore, competition assays with pseudotyped viruses suggested that the interaction of the HAdV-11 fiber with DSG2 was not required for infection but that DSG2 could serve as a receptor in the absence of CD46. Unexpectedly, we succeeded to isolate a HAdV-11 fiber/DSG2/CD46 “super complex” showing that these two receptors could simultaneously bind to the same trimeric HAdV-11K. Our work deciphers the use of two independent receptors by HAdV-11 from a biochemical and structural point of view, which can have an impact for future vectors design. IMPORTANCE The main limitation of oncolytic vectors is neutralization by blood components, which prevents intratumoral administration to patients. Enadenotucirev, a chimeric HAdV-11p/HAdV-3 adenovirus identified by bio-selection, is a low seroprevalence vector active against a broad range of human carcinoma cell lines. At this stage, there’s still some uncertainty about tropism and primary receptor utilization by HAdV-11. However, this information is very important, as it has a direct influence on the effectiveness of HAdV-11-based vectors. The aim of this work is to determine which of the two receptors, DSG2 and CD46, is involved in the attachment of the virus to the host, and what role they play in the early stages of infection

Differential Behaviours and Preferential Bindings of Influenza Nucleoproteins on Importins-α

International audienceInfluenza viruses are negative single-stranded RNA viruses with nuclear transcription and replication. They enter the nucleus by using the cellular importin-α/-β nuclear import machinery. Influenza nucleoproteins from influenza A, B, C and D viruses possess a nuclear localization signal (NLS) localized on an intrinsically disordered extremity (NPTAIL). In this paper, using size exclusion chromatography (SEC), SEC-multi-angle laser light scattering (SEC-MALLS) analysis, surface plasmon resonance (SPR) and fluorescence anisotropy, we provide the first comparative study designed to dissect the interaction between the four NPTAILs and four importins-α identified as partners. All interactions between NPTAILs and importins-α have high association and dissociation rates and present a distinct and specific behaviour. D/NPTAIL interacts strongly with all importins-α while B/NPTAIL shows weak affinity for importins-α. A/NPTAIL and C/NPTAIL present preferential importin-α partners. Mutations in B/NPTAIL and D/NPTAIL show a loss of importin-α binding, confirming key NLS residues. Taken together, our results provide essential highlights of this complex translocation mechanism

Binding Mechanism Elucidation of the Acute Respiratory Disease Causing Agent Adenovirus of Serotype 7 to Desmoglein-2

International audienceThe study of viruses causing acute respiratory distress syndromes (ARDS) is more essential than ever at a time when a virus can create a global pandemic in a matter of weeks. Among human adenoviruses, adenovirus of serotype 7 (HAdV7) is one of the most virulent serotypes. This virus regularly re-emerges in Asia and has just been the cause of several deaths in the United States. A critical step of the virus life cycle is the attachment of the knob domain of the fiber (HAd7K) to the cellular receptor desmoglein-2 (DSG2). Complexes between the fiber knob and two extracellular domains of DSG2 have been produced. Their characterization by biochemical and biophysical methods show that these two domains are sufficient for the interaction and that the trimeric HAd7K could accommodate up to three DSG2 receptor molecules. The cryo-electron microscopy (cryo-EM) structure of these complexes at 3.1 Å resolution confirmed the biochemical data, and allowed the identification of the critical amino acid residues for this interaction, which shows similarities with other DSG2 interacting adenoviruses, despite a low homology in the primary sequences

Glycosaminoglycans are interactants of Langerin: comparison with gp120 highlights an unexpected calcium-independent binding mode.

Langerin is a C-type lectin specifically expressed in Langerhans cells. As recently shown for HIV, Langerin is thought to capture pathogens and mediate their internalisation into Birbeck Granules for elimination. However, the precise functions of Langerin remain elusive, mostly because of the lack of information on its binding properties and physiological ligands. Based on recent reports that Langerin binds to sulfated sugars, we conducted here a comparative analysis of Langerin interaction with mannose-rich HIV glycoprotein gp120 and glycosaminoglycan (GAGs), a family of sulfated polysaccharides expressed at the surface of most mammalian cells. Our results first revealed that Langerin bound to these different glycans through very distinct mechanisms and led to the identification of a novel, GAG-specific binding mode within Langerin. In contrast to the canonical lectin domain, this new binding site showed no Ca(2+)-dependency, and could only be detected in entire, trimeric extracellular domains of Langerin. Interestingly binding to GAGs, did not simply rely on a net charge effect, but rather on more discrete saccharide features, such as 6-O-sulfation, or iduronic acid content. Using molecular modelling simulations, we proposed a model of Langerin/heparin complex, which located the GAG binding site at the interface of two of the three Carbohydrate-recognition domains of the protein, at the edge of the a-helix coiled-coil. To our knowledge, the binding properties that we have highlighted here for Langerin, have never been reported for C-type lectins before. These findings provide new insights towards the understanding of Langerin biological functions