Broad neutralization of SARS-related viruses by human monoclonal antibodies

Broadly protective vaccines against known and preemergent human coronaviruses (HCoVs) are urgently needed. To gain a deeper understanding of cross-neutralizing antibody responses, we mined the memory B cell repertoire of a convalescent severe acute respiratory syndrome (SARS) donor and identified 200 SARS coronavirus 2 (SARS-CoV-2) binding antibodies that target multiple conserved sites on the spike (S) protein. A large proportion of the non-neutralizing antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of preexisting memory B cells elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a target for the rational design of pan-sarbecovirus vaccines

Parkin structure and activation

Parkinson’s disease (PD) is the second most common neurodegenerative disease. Around 10% of PD cases are familial and have genetic causes. The most frequent form of autosomal recessive PD is caused by mutations in the PARK2 gene. This gene encodes an E3 ubiquitin ligase, named parkin, which participates in the ubiquitin proteasome system and in mitophagy. In its resting state parkin is auto-inhibited by intra-molecular contacts occluding the catalytic cysteine and the putative binding site of the E2 enzyme and needs to be activated in order to ubiquitinate substrates. Recently, phosphorylation of parkin and ubiquitin have been found to be essential for parkin activation; however, not much is known about the structural effects of these post-translational modifications on parkin. Previously available crystal structures do not show the molecular interactions between the RING1 domain and the ubiquitin-like domain (UbLD) of parkin. These interactions play an important role in controlling parkin’s activation. The research presented in this thesis includes the crystallization and the solution of the structure of a parkin linker-deletion mutant at 2.54 Å. The structure exposes the molecular interactions underlying binding of the UbLD to the RING1 domain of parkin. Small-angle X-ray scattering and isothermal titration calorimetry experiments show that parkin phosphorylation causes release of the UbLD and the repressor element of parkin from their respective binding sites. The results also show that phospho-ubiquitin binds parkin through residue histidine 302, but this binding in itself is not enough to activate parkin. These results underscore the important role that phosphorylation plays in the parkin pathway and contribute to our understanding of the molecular events underlying parkin activation.La maladie de Parkinson est la deuxième maladie neuro-dégénérative la plus répandue. Près de 10% des cas de cette maladie ont des causes génétiques. La forme la plus répandue de maladie de Parkinson autosomale récessive est causée par des mutations dans le gène PARK2. Ce gène encode une ubiquitine-ligase E3 appelée parkin qui joue un rôle important dans le système ubiquitine-protéasome ainsi que dans la mitophagie. À l’état de repos, parkin adopte une conformation auto-inhibée due à des interactions intra-moléculaires qui bloquent la cystéine catalytique et le site putatif de liaison de l’enzyme E2. Ainsi, parkin doit être activée avant de pouvoir ubiquitiner des substrats. Récemment, des recherches ont démontré que la phosphorylation de parkin ainsi que de l’ubiquitine sont essentielles à l’activation de parkin; toutefois, il y a peu de données permettant de comprendre les effets de ces phosphorylations sur la structure de parkin. Les structures cristallographiques de parkin disponibles jusqu’à maintenant ne donnent aucune information sur les interactions moléculaires entre le domaine RING1 et le domaine semblable-à-l’ubiquitine de parkin (UbLD). Ces interactions jouent un rôle important dans le contrôle de l’activation de parkin. Les résultats de recherche présentés dans ce mémoire incluent la cristallisation et la solution de la structure d’un mutant de parkin, qui contient une délétion dans une région charnière flexible, à une résolution de 2,54 Å. Cette structure démontre les interactions moléculaires entre les domaines UbLD et RING1 de parkin. Des expériences de diffusion des rayons X aux petits angles et de titration calorimétrique isotherme démontrent que la phosphorylation de parkin cause le relâchement du domaine UbLD et de l’élément répresseur de parkin de leurs sites de liaison respectifs. Ces résultats démontrent aussi que la phospho-ubiquitine lie parkin par l’intermédiaire de l’histidine 302, mais que cette liaison n’est pas suffisante pour activer parkin. Ces résultats soulignent le rôle très important que la phosphorylation joue dans la régulation de parkin et contribuent à faire évoluer notre compréhension des évènements moléculaires qui permettent l’activation de parkin

Assessing the binding properties of the anti-PD-1 antibody landscape using label-free biosensors.

Here we describe an industry-wide collaboration aimed at assessing the binding properties of a comprehensive panel of monoclonal antibodies (mAbs) against programmed cell death protein 1 (PD-1), an important checkpoint protein in cancer immunotherapy and validated therapeutic target, with well over thirty unique mAbs either in clinical development or market-approved in the United States, the European Union or China. The binding kinetics of the PD-1/mAb interactions were measured by surface plasmon resonance (SPR) using a Carterra LSA instrument and the results were compared to data collected on a Biacore 8K. The effect of chip type on the SPR-derived binding rate constants and affinities were explored and the results compared with solution affinities from Meso Scale Discovery (MSD) and Kinetic Exclusion Assay (KinExA) experiments. When using flat chip types, the LSA and 8K platforms yielded near-identical kinetic rate and affinity constants that matched solution phase values more closely than those produced on 3D-hydrogels. Of the anti-PD-1 mAbs tested, which included a portion of those known to be in clinical development or approved, the affinities spanned from single digit picomolar to nearly 425 nM, challenging the dynamic range of our methods. The LSA instrument was also used to perform epitope binning and ligand competition studies which revealed over ten unique competitive binding profiles within this group of mAbs

Longitudinal dynamics of the human B cell response to the yellow fever 17D vaccine

A comprehensive understanding of the development and evolution of human B cell responses duced by pathogen exposure will facilitate the design of next-generation vaccines. Here, we utilized a gh-throughput single B cell cloning technology to longitudinally track the human B cell response to the llow fever virus 17D (YFV-17D) vaccine. The earlymemory B cell (MBC) response was mediated by both assical immunoglobulin M (IgM) (IgM(+)CD27(+)) and switched immunoglobulin (swIg(+)) MBC pulations; however, classical IgM MBCs waned rapidly, whereas swIg(+) and atypical IgM(+) and IgD(+) MBCs were stable over time. Affinity maturation continued for 6 to 9 mo following vaccination, providing evidence for the persistence of germinal center activity long after the period of active viral replication in peripheral blood. Finally, a substantial fraction of the neutralizing antibody response was mediated by public clones that recognize a fusion loop-proximal antigenic site within domain II of the viral envelope glycoprotein. Overall, our findings provide a framework for understanding the dynamics and complexity of human B cell responses elicited by infection and vaccination