Bispecific IgG neutralizes SARS-CoV-2 variants and prevents escape in mice.

Neutralizing antibodies that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein are among the most promising approaches against COVID-191,2. A bispecific IgG1-like molecule (CoV-X2) has been developed on the basis of C121 and C135, two antibodies derived from donors who had recovered from COVID-193. Here we show that CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable spike binding to the cellular receptor of the virus, angiotensin-converting enzyme 2 (ACE2). Furthermore, CoV-X2 neutralizes wild-type SARS-CoV-2 and its variants of concern, as well as escape mutants generated by the parental monoclonal antibodies. We also found that in a mouse model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, the simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, and combines the advantages of antibody cocktails with those of single-molecule approaches.

Stable and selective permeable hydrogel microcapsules for high-throughput cell cultivation and enzymatic analysis.

BACKGROUND: Miniaturization of biochemical reaction volumes within artificial microcompartments has been the key driver for directed evolution of several catalysts in the past two decades. Typically, single cells are co-compartmentalized within water-in-oil emulsion droplets with a fluorogenic substrate whose conversion allows identification of catalysts with improved performance. However, emulsion droplet-based technologies prevent cell proliferation to high density and preclude the feasibility of biochemical reactions that require the exchange of small molecule substrates. Here, we report on the development of a high-throughput screening method that addresses these shortcomings and that relies on a novel selective permeable polymer hydrogel microcapsule. RESULTS: Hollow-core polyelectrolyte-coated chitosan alginate microcapsules (HC-PCAMs) with selective permeability were successfully constructed by jet break-up and layer-by-layer (LBL) technology. We showed that HC-PCAMs serve as miniaturized vessels for single cell encapsulation, enabling cell growth to high density and cell lysis to generate monoclonal cell lysate compartments suitable for high-throughput analysis using a large particle sorter (COPAS). The feasibility of using HC-PCAMs as reaction compartments which exchange small molecule substrates was demonstrated using the transpeptidation reaction catalyzed by the bond-forming enzyme sortase F from P. acnes. The polyelectrolyte shell surrounding microcapsules allowed a fluorescently labelled peptide substrate to enter the microcapsule and take part in the transpeptidation reaction catalyzed by the intracellularly expressed sortase enzyme retained within the capsule upon cell lysis. The specific retention of fluorescent transpeptidation products inside microcapsules enabled the sortase activity to be linked with a fluorescent readout and allowed clear separation of microcapsules expressing the wild type SrtF from those expressing the inactive variant. CONCLUSION: A novel polymer hydrogel microcapsule-based method, which allows for high-throughput analysis based on encapsulation of single cells has been developed. The method has been validated for the transpeptidation activity of sortase enzymes and represents a powerful tool for screening of libraries of sortases, other bond-forming enzymes, as well as of binding affinities in directed evolution experiments. Moreover, selective permeable microcapsules encapsulating microcolonies provide a new and efficient means for preparing novel caged biocatalyst and biosensor agents.

Characterization of the housekeeping sortase from the human pathogen Propionibacterium acnes: first investigation of a class F sortase.

Sortase enzymes play an important role in Gram-positive bacteria. They are responsible for the covalent attachment of proteins to the surface of the bacteria and perform this task via a highly sequence-specific transpeptidation reaction. Since these immobilized proteins are often involved in pathogenicity of Gram-positive bacteria, characterization of this type of enzyme is also of medical relevance. Different classes of sortases (A-F) have been found, which recognize characteristic recognition sequences present in substrate proteins. Up to date, sortase A from Staphylococcus aureus, a housekeeping class A sortase, is the most thoroughly studied representative of the sortase family of enzymes. Here we report the in-depth characterization of the class F sortase from Propionibacterium acnes, a class of sortases that has not been investigated before. As Sortase F is the only transpeptidase found in the P. acnes genome, it is the housekeeping sortase of this organism. Sortase F from P. acnes shows a behavior similar to sortases from class A in terms of pH dependence, recognition sequence and catalytic activity; furthermore, its activity is independent of bivalent ions, which contrasts to sortase A from S. aureus We demonstrate that sortase F is useful for protein engineering applications, by producing a site-specifically conjugated homogenous antibody-drug conjugate with a potency similar to that of a conjugate prepared with sortase A. Thus, the detailed characterization presented here will not only enable the development of anti-virulence agents targeting P. acnes but also provides a powerful alternative to sortase A for protein engineering applications.

Elucidation of the Recognition Sequence of Sortase B from Bacillus anthracis by Using a Newly Developed Liquid Chromatography-Mass Spectrometry-Based Method.

Sortases are enzymes that are responsible for the attachment of secreted proteins to the cell wall of Gram-positive bacteria. Hereby, the sortases recognize short, five-residue amino acid sequences present in the target proteins and fuse them to the peptidoglycan layer via a transpeptidation reaction, creating a new peptide bond between the C-terminus of the recognition sequence and the cell wall. The transpeptidation activity of sortases is widely used in protein engineering for modification of target proteins. The majority of protocols rely on the high activity of the well-characterized Staphylococcus aureus SrtA and variants thereof, while sortases from other classes are not used for this purpose. This can be attributed to the lower activity of other sortases and to the limited sequence specificity data available for the different sortases. We set out to determine the sequence specificity of Bacillus anthracis SrtB. To this end, we developed a new method for sequence specificity determination of sortases or other bond-forming enzymes that recognize an amino acid sequence. Using mixtures of recognition peptides of limited complexity, which are reacted with biotinylated substrates, the biotinylated transpeptidation products are isolated with magnetic streptavidin beads and analyzed via liquid chromatography and mass spectrometry. With this, peptide sequences that are recognized by the sortase and function as substrates can be determined and quantified. The method, developed with the highly active evolved SrtA from S. aureus, allowed for the first time unbiased in-depth analysis of the sequence specificity for SrtB from B. anthracis, which is 104-fold less active than SrtA from S. aureus.

Fiber sensors multiplexing using vectorial wave mixing in a photorefractive crystal.

We propose a novel multiplexing system for sensing of dynamic strains excited in different multimode optical fibers. Multiplexing of the sensors is implemented by using vectorial wave mixing technique in the reflection geometry of hologram formation in a photorefractive crystal of CdTe:V. We analyzed different mechanisms of the crosstalk between measuring channels and showed that system performance is strongly affected by residual stresses of the photorefractive crystal.

Sensing of multimode-fiber strain by a dynamic photorefractive hologram.

We present a strain sensor in which a multimode fiber is used as a sensitive element. High sensitivity to dynamic strains is achieved by means of vectorial wave mixing in a photorefractive CdTe:V crystal. It was found that the largest source of noise in our sensor is related to the instability of the polarization state of speckles emerging from the fiber. This noise is significantly diminished in fiber with a core of large diameter (550 microm).

Fast adaptive interferometer on dynamic reflection hologram in CdTe:V.

We present an adaptive interferometer based on the reflection dynamic hologram recorded in photorefractive CdTe:V crystal with no external electric field. Linear phase-to-intensity transformation is achieved by vectorial mixing of two waves with different polarization states (linear and elliptical) in the anisotropic diffraction geometry. Comparison of reflection and transmission geometries considering both sensitivity and adaptability is carried out. It is shown that the reflection geometry is characterized by better combination of these parameters provided that the crystal possesses high enough concentration of photorefractive centers.