Biology subjects

Paesen, G. C.

Publications and source records attributed to Paesen, G. C..

6 recordsLinked to original sources

bioRxiv2026

Conserved dimerization architecture in C-type lectins from virus-vector mosquitoes

C-type lectins (CTLs) play key roles in innate immunity and microbial carbohydrate recognition. In the disease vector mosquito Aedes aegypti, the CTLD-S family comprises 34 soluble CTLs whose members are implicated in flavivirus dissemination and microbial homeostasis, yet their structure and organization remain uncharacterized. Here, we combine X-ray crystallography, small-angle X-ray scattering (SAXS), molecular dynamics, and machine learning-based structure prediction to characterize CTLs in Aedes aegypti. We determined the crystal structures of four representative CTLD-S proteins: mosGCTL-1, -3, -6, and -20. All crystals featured mosGCTL proteins in an identical homodimer arrangement, positioning both carbohydrate-binding sites on the same molecular face. Dimerization was confirmed in solution and AlphaFold predictions across the entire CTLD-S family indicated that dimer formation may be a unifying feature of mosquito CTLD-S proteins. For one mosGCTL structure, paucimannose glycans bound at a Ca2+-dependent site, demonstrating bi-dentate glycan-binding through one dimer. Finally, machine learning based predictions indicated hundreds of possible CTLD-S heterodimers may be viable, with wide-ranging implications for preferred glycan binding through one dimer. Our findings reveal a conserved dimeric arrangement among mosquito lectins that may underpin carbohydrate recognition relevant to vector-pathogen interactions.

biochemistry↗

bioRxiv2024

Loop Grafting Improves Neuraminidase Protein Vaccine Production

Influenza virus neuraminidase is a crucial target for protective antibodies, yet the development of recombinant neuraminidase protein as a vaccine has been held back by instability and variable expression. We have taken a pragmatic approach to improving expression and stability of neuraminidase by grafting antigenic surface loops from low-expressing neuraminidase proteins onto the scaffold of high-expressing counterparts. The resulting hybrid proteins retained the antigenic properties of the loop donor while benefiting from the high-yield expression, stability, and tetrameric structure of the loop recipient. These hybrid proteins were recognised by a broad set of human monoclonal antibodies elicited by influenza infection or vaccination, with X-ray structures validating the accurate structural conformation of the grafted loops and the enzymatic cavity. Immunisation of mice with neuraminidase hybrids induced inhibitory antibodies to the loop donor and protected against lethal influenza challenge. This pragmatic technique offers a robust solution for improving the expression and stability of influenza neuraminidase proteins for vaccine development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/617814v3_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@14d4b4eorg.highwire.dtl.DTLVardef@18af09aorg.highwire.dtl.DTLVardef@ba1982org.highwire.dtl.DTLVardef@1e0d143_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

Structure and stabilization of the antigenic glycoprotein building blocks of the New World mammarenavirus spike complex

The spillover of New World (NW) arenaviruses from rodent reservoirs into human populations poses a continued risk to human health. NW arenaviruses present a glycoprotein (GP) complex on the envelope surface of the virion, which orchestrates host-cell entry and is a key target of the immune response arising from infection and immunization. Each protomer of the trimeric GP is composed of a stable signal peptide (SSP), a GP1 attachment glycoprotein, and a GP2 fusion glycoprotein. To glean insights into the architecture of this key therapeutic target, we determined the crystal structures of NW GP1-GP2 heterodimeric complexes from Junin virus (JUNV) and Machupo virus (MACV). Due to the metastability of the interaction between GP1 and GP2, structural elucidation required the introduction of a disulfide bond at the GP1-GP2 complex interface, but no other stabilizing modifications were required. While the overall assembly of NW GP1-GP2 is conserved with that presented by Old World (OW) arenaviruses, including Lassa virus (LASV) and lymphocytic choriomeningitis virus (LCMV), NW GP1-GP2 complexes are structurally distinct. Indeed, we note that when compared to the OW GP1-GP2 complex, the globular portion of NW GP1 undergoes limited structural alterations upon detachment from its cognate GP2. We further demonstrate that our engineered GP1-GP2 heterodimers are antigenically relevant and recognized by neutralizing antibodies. These data provide insights into the distinct assemblies presented by NW and OW arenaviruses, as well as provide molecular-level blueprints that may guide vaccine development. ImportanceAlthough the emergence of New World (NW) hemorrhagic fever mammarenaviruses poses an unceasing threat to human health, there is a paucity of reagents capable of protecting against the transmission of these pathogens from their natural rodent reservoirs. This is, in part, attributed to our limited understanding of structure and function of the NW glycoprotein spike complex presented on the NW arenavirus surface. Here, we provide a detailed molecular-level description of how the two major components of this key therapeutic target assemble to form a key building block of the NW arenaviral spike complex. The insights gleaned from this work provide a framework for guiding the structure-based development of NW arenaviral vaccines.

biochemistry↗

bioRxiv2022

VelcroVax: a bolt-on vaccine platform technology improves antibody titres against a viral glycoprotein in mice

Having varied approaches to the design and manufacture of vaccines is critical in being able to respond to worldwide needs and to newly emerging pathogens. Virus-like particle (VLP) vaccines form the basis of two of the most successful licensed vaccines (against hepatitis B virus (HBV) and human papillomavirus). They are produced by recombinant expression of viral structural proteins, which self-assemble into immunogenic nanoparticles. VLPs can also be modified to present unrelated antigens, and here we describe a universal bolt-on vaccine platform (termed VelcroVax) where the capturing VLP and the target antigen (hapten) are produced separately. We utilise a modified HBV core (HBcAg) VLP, with surface expression of a high-affinity binding sequence (Affimer) directed against a SUMO tag and use this to capture SUMO-tagged gp1 glycoprotein from the arenavirus, Junin virus (JUNV). Using this model system, we have solved high-resolution structures of VelcroVax VLPs, and shown that the VelcroVax-JUNV gp1 complex induces superior humoral immune responses compared to the non-complexed viral protein. We propose that this system could be modified to present a range of haptens and therefore form the foundation of future rapid-response vaccination strategies.

immunology↗

bioRxiv2021

Antibody evasion by the Brazilian P.1 strain of SARS-CoV-2

Terminating the SARS-CoV-2 pandemic relies upon pan-global vaccination. Current vaccines elicit neutralizing antibody responses to the virus spike derived from early isolates. However, new strains have emerged with multiple mutations: P.1 from Brazil, B.1.351 from South Africa and B.1.1.7 from the UK (12, 10 and 9 changes in the spike respectively). All have mutations in the ACE2 binding site with P.1 and B.1.351 having a virtually identical triplet: E484K, K417N/T and N501Y, which we show confer similar increased affinity for ACE2. We show that, surprisingly, P.1 is significantly less resistant to naturally acquired or vaccine induced antibody responses than B.1.351 suggesting that changes outside the RBD impact neutralisation. Monoclonal antibody 222 neutralises all three variants despite interacting with two of the ACE2 binding site mutations, we explain this through structural analysis and use the 222 light chain to largely restore neutralization potency to a major class of public antibodies.

microbiology↗

bioRxiv2020

Molecular rationale for hantavirus neutralization by a reservoir host-derived monoclonal antibody

The intricate lattice of Gn and Gc glycoprotein spike complexes at the surface of hantaviruses facilitates host-cell entry and is the primary target of the neutralizing antibody-mediated immune response. Here, through study of a neutralizing monoclonal antibody (mAb 4G2) generated in a bank vole reservoir host following infection with Puumala virus (PUUV), we provide molecular-level insights into how antibody-mediated targeting of the hantaviral glycoprotein lattice effectively neutralizes the virus. Crystallographic analysis reveals that mAb 4G2 binds to a multi-domain site on Gc in the pre-fusion state, and that Fab binding is incompatible with the conformational changes of the Gc that are required for host cell entry. Cryo-electron microscopy of PUUV-like particles treated with Fab 4G2 demonstrates that the antibody binds to monomeric Gc at breaks in the Gn-Gc lattice, highlighting the immunological accessibility of Gc monomers on the mature hantavirus surface and the plastic nature of the higher-order lattice assembly. This work provides a structure-based blueprint for rationalizing antibody-mediated targeting of hantaviruses.

microbiology↗