resPAINT: Accelerating Volumetric Super-Resolution Localisation Microscopy by Active Control of Probe Emission.

Points for accumulation in nanoscale topography (PAINT) allows practically unlimited measurements in localisation microscopy but is limited by background fluorescence at high probe concentrations, especially in volumetric imaging. We present reservoir-PAINT (resPAINT), which combines PAINT and active control of probe photophysics. In resPAINT, an activatable probe "reservoir" accumulates on target, enabling a 50-fold increase in localisation rate versus conventional PAINT, without compromising contrast. By combining resPAINT with large depth-of-field microscopy, we demonstrate super-resolution imaging of entire cell surfaces. We generalise the approach by implementing various switching strategies and 3D imaging techniques. Finally, we use resPAINT with a Fab to image membrane proteins, extending the operating regime of PAINT to include a wider range of biological interactions.

Heterotypic interactions drive antibody synergy against a malaria vaccine candidate.

Understanding mechanisms of antibody synergy is important for vaccine design and antibody cocktail development. Examples of synergy between antibodies are well-documented, but the mechanisms underlying these relationships often remain poorly understood. The leading blood-stage malaria vaccine candidate, CyRPA, is essential for invasion of Plasmodium falciparum into human erythrocytes. Here we present a panel of anti-CyRPA monoclonal antibodies that strongly inhibit parasite growth in in vitro assays. Structural studies show that growth-inhibitory antibodies bind epitopes on a single face of CyRPA. We also show that pairs of non-competing inhibitory antibodies have strongly synergistic growth-inhibitory activity. These antibodies bind to neighbouring epitopes on CyRPA and form lateral, heterotypic interactions which slow antibody dissociation. We predict that such heterotypic interactions will be a feature of many immune responses. Immunogens which elicit such synergistic antibody mixtures could increase the potency of vaccine-elicited responses to provide robust and long-lived immunity against challenging disease targets.

resPAINT: Accelerating Volumetric Super-Resolution Localisation Microscopy by Active Control of Probe Emission.

Points for accumulation in nanoscale topography (PAINT) allows practically unlimited measurements in localisation microscopy but is limited by background fluorescence at high probe concentrations, especially in volumetric imaging. We present reservoir-PAINT (resPAINT), which combines PAINT and active control of probe photophysics. In resPAINT, an activatable probe "reservoir" accumulates on target, enabling a 50-fold increase in localisation rate versus conventional PAINT, without compromising contrast. By combining resPAINT with large depth-of-field microscopy, we demonstrate super-resolution imaging of entire cell surfaces. We generalise the approach by implementing various switching strategies and 3D imaging techniques. Finally, we use resPAINT with a Fab to image membrane proteins, extending the operating regime of PAINT to include a wider range of biological interactions.

Structural basis for the neutralization of SARS-CoV-2 by an antibody from a convalescent patient.

The COVID-19 pandemic has had an unprecedented health and economic impact and there are currently no approved therapies. We have isolated an antibody, EY6A, from an individual convalescing from COVID-19 and have shown that it neutralizes SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds the receptor binding domain (RBD) of the viral spike glycoprotein tightly (KD of 2 nM), and a 2.6-Å-resolution crystal structure of an RBD-EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues within this footprint are key to stabilizing the pre-fusion spike. Cryo-EM analyses of the pre-fusion spike incubated with EY6A Fab reveal a complex of the intact spike trimer with three Fabs bound and two further multimeric forms comprising the destabilized spike attached to Fab. EY6A binds what is probably a major neutralizing epitope, making it a candidate therapeutic for COVID-19.