In vitro and in vivo functions of SARS-CoV-2 infection-enhancing and neutralizing antibodies

SARS-CoV-2 neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) and the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-g (FcgR)-mediated enhancement of virus infection in vitro, while five non-neutralizing NTD antibodies mediated FcgR-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Three of 46 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo, increased lung inflammation can rarely occur in SARS-CoV-2 antibody-infused macaques

Chimeric β-Lactamases: Global Conservation of Parental Function and Fast Time-Scale Dynamics with Increased Slow Motions

<div><p>Enzyme engineering has been facilitated by recombination of close homologues, followed by functional screening. In one such effort, chimeras of two class-A β-lactamases – TEM-1 and PSE-4 – were created according to structure-guided protein recombination and selected for their capacity to promote bacterial proliferation in the presence of ampicillin (Voigt <em>et al</em>., Nat. Struct. Biol. 2002 9:553). To provide a more detailed assessment of the effects of protein recombination on the structure and function of the resulting chimeric enzymes, we characterized a series of functional TEM-1/PSE-4 chimeras possessing between 17 and 92 substitutions relative to TEM-1 β-lactamase. Circular dichroism and thermal scanning fluorimetry revealed that the chimeras were generally well folded. Despite harbouring important sequence variation relative to either of the two ‘parental’ β-lactamases, the chimeric β-lactamases displayed substrate recognition spectra and reactivity similar to their most closely-related parent. To gain further insight into the changes induced by chimerization, the chimera with 17 substitutions was investigated by NMR spin relaxation. While high order was conserved on the ps-ns timescale, a hallmark of class A β-lactamases, evidence of additional slow motions on the µs-ms timescale was extracted from model-free calculations. This is consistent with the greater number of resonances that could not be assigned in this chimera relative to the parental β-lactamases, and is consistent with this well-folded and functional chimeric β-lactamase displaying increased slow time-scale motions.</p> </div

Thermal denaturation of parental and chimeric β-lactamases monitored by CD spectroscopy and thermal scanning fluorimetry.

<p>A) Far-UV CD spectra at 25°C of TEM-1 (blue), PSE-4 (red), cTEM-17m (gold), cTEM-67m (green) and cTEM-92m (black). B) Mean molar residual ellipticity (MRW) measured at 222 nm during thermal denaturation at a rate of 20°C/hour for TEM-1 (blue), PSE-4 (red) and the chimeras cTEM-17m (gold), cTEM-67m (green) and cTEM-92m (black). C, D) First derivative analysis of representative thermal melting curves observed by fluorescence of SYPRO Orange. Melting curves are shown for a ratio of 3.33 × SYPRO Orange to 1 µM (dashed line) or 2 µM (full line) protein, for C) TEM-1 (blue), PSE-4 (red) and D) the chimeras cTEM-17m (gold), cTEM-67m (green) and cTEM-92m (black).</p

T_m values obtained upon thermal denaturation monitored by circular dichroism (CD) at 222nm and thermal scanning fluorimetry for TEM-1, the chimeric β-lactamases and PSE-4.

<p>T_m values obtained upon thermal denaturation monitored by circular dichroism (CD) at 222nm and thermal scanning fluorimetry for TEM-1, the chimeric β-lactamases and PSE-4.</p

Average backbone ¹⁵N spin relaxation parameters for TEM-1, cTEM-17m and PSE-4.

a<p>Values taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052283#pone.0052283-Savard1" target="_blank">[23]</a>.</p>b<p>Spin relaxation statistics for residues with data at two magnetic fields (N = 167).</p>c<p>Values taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052283#pone.0052283-Morin1" target="_blank">[24]</a>.</p

Model-free parameters for TEM-1, cTEM-17m and PSE-4.

a<p>Values taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052283#pone.0052283-Savard1" target="_blank">[23]</a>.</p>b<p>Minimized using an ellipsoid diffusion tensor.</p>c<p>Values taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052283#pone.0052283-Morin1" target="_blank">[24]</a>.</p>d<p>Values are given as the average +/− standard deviation from the mean.</p

Comparison of slow motions for the parental and chimeric enzymes.

<p>Residues for which the conformational exchange term R_ex was extracted from model-free models <i>m3</i>, <i>m4</i>, <i>m7</i>, <i>m8</i>, or <i>m9</i>, indicative of dynamics on the µs-ms timescale, were scaled to the same field (600 MHz) and mapped as spheres for TEM-1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052283#pone.0052283-Savard1" target="_blank">[23]</a>, PSE-4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052283#pone.0052283-Morin1" target="_blank">[24]</a> and cTEM-17m. The spheres are colored according to the magnitude of R_ex, as defined in the scale below. Residues for which backbone NMR assignments are missing, potentially indicating the presence of µs-ms motions, are in blue. The active-site serine is in sticks representation.</p

Kinetic constants for hydrolysis of the chromogenic substrate CENTA by TEM-1, the chimeric β-lactamases and PSE-4 ^a.

a<p>Values are given as the average +/− standard deviation from the mean.</p

Kinetic constants for the hydrolysis of penicillins and cephalosporins by TEM-1, the chimeric β-lactamases and PSE-4 ^a.

a<p>Values are given as the average +/− standard deviation from the mean.</p>b<p>ND: Not determined, the activity being too low or undetectable.</p

Sequence blocks exchanged between parental TEM-1 and PSE-4 β-lactamases in the selected, functional chimeras.

<p>A) Numbering of the sequence blocks originating from TEM-1 (blue) and PSE-4 (red) in cTEM-17m, cTEM-67m and cTEM-92m, according to the Ambler numbering <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052283#pone.0052283-Ambler1" target="_blank">[64]</a>. The nomenclature of the chimeras is as follows: ‘c’ indicates chimeragenesis, and the number refers to the number of substitutions (or mutations: ‘m’) in each chimera relative to the TEM-1 parental sequence. The catalytic nucleophile, Ser70, and the catalytically-relevant Ω-loop, are indicated to highlight their parental origin. B) Structural representation of the sequence blocks exchanged during chimeragenesis, coloured according to parental origin as in panel A. The TEM-1 coordinates (PDB 1ZG4) were used for the representation. C) The sequence identity to each parental sequence is given. TEM-1 and PSE-4 differ by 150 residues (for a sequence identity of 40%) between residues 26 (the <i>N</i>-terminus following cleavage of the leader sequence of TEM-1) to 290 (<i>C</i>-terminus of TEM-1). The deletion at position 58 in PSE-4 is considered as a mutation relative to TEM-1. Thus, the additional four residues at the <i>N</i>-terminus and five residues at the <i>C</i>-terminus of PSE-4 are not included in the comparison to TEM-1.</p