High Potency of a Bivalent Human VH Domain in SARS-CoV-2 Animal Models

Novel COVID-19 therapeutics are urgently needed. We generated a phage-displayed human antibody VH domain library from which we identified a high-affinity VH binder ab8. Bivalent VH, VH-Fc ab8, bound with high avidity to membrane-associated S glycoprotein and to mutants found in patients. It potently neutralized mouse-adapted SARS-CoV-2 in wild-type mice at a dose as low as 2 mg/kg and exhibited high prophylactic and therapeutic efficacy in a hamster model of SARS-CoV-2 infection, possibly enhanced by its relatively small size. Electron microscopy combined with scanning mutagenesis identified ab8 interactions with all three S protomers and showed how ab8 neutralized the virus by directly interfering with ACE2 binding. VHFc ab8 did not aggregate and did not bind to 5,300 human membrane-associated proteins. The potent neutralization activity of VH-Fc ab8 combined with good developability properties and cross-reactivity to SARS-CoV-2 mutants provide a strong rationale for its evaluation as a COVID-19 therapeutic

Effects of N-Glycosylation Site Removal in Archaellins on the Assembly and Function of Archaella in Methanococcus maripaludis

In Methanococcus maripaludis S2, the swimming organelle, the archaellum, is composed of three archaellins, FlaB1S2, FlaB2S2 and FlaB3S2. All three are modified with an N-linked tetrasaccharide at multiple sites. Disruption of the N-linked glycosylation pathway is known to cause defects in archaella assembly or function. Here, we explored the potential requirement of N-glycosylation of archaellins on archaellation by investigating the effects of eliminating the 4 N-glycosylation sites in the wildtype FlaB2S2 protein in all possible combinations either by Asn to Glu (N to Q) substitution or Asn to Asp (N to D) substitutions of the N-glycosylation sequon asparagine. The ability of these mutant derivatives to complement a non-archaellated ΔflaB2S2 strain was examined by electron microscopy (for archaella assembly) and swarm plates (for analysis of swimming). Western blot results showed that all mutated FlaB2S2 proteins were expressed and of smaller apparent molecular mass compared to wildtype FlaB2S2, consistent with the loss of glycosylation sites. In the 8 single-site mutant complements, archaella were observed on the surface of Q2, D2 and D4 (numbers after N or Q refer to the 1st to 4th glycosylation site). Of the 6 double-site mutation complementations all were archaellated except D1,3. Of the 4 triple-site mutation complements, only D2,3,4 was archaellated. Elimination of all 4 N-glycosylation sites resulted in non-archaellated cells, indicating some minimum amount of archaellin glycosylation was necessary for their incorporation into stable archaella. All complementations that led to a return of archaella also resulted in motile cells with the exception of the D4 version. In addition, a series of FlaB2S2 scanning deletions each missing 10 amino acids was also generated and tested for their ability to complement the ΔflaB2S2 strain. While most variants were expressed, none of them restored archaellation, although FlaB2S2 harbouring a smaller 3-amino acid deletion was able to partially restore archaellation

Bypassing the Need for the Transcriptional Activator EarA through a Spontaneous Deletion in the BRE Portion of the fla Operon Promoter in Methanococcus maripaludis

In Methanococcus maripaludis, the euryarchaeal archaellum regulator A (EarA) is required for the transcription of the fla operon, which is comprised of a series of genes which encode most of the proteins needed for the formation of the archaeal swimming organelle, the archaellum. In mutants deleted for earA (ΔearA), there is almost undetectable transcription of the fla operon, Fla proteins are not synthesized and the cells are non-archaellated. In this study, we have isolated a spontaneous mutant of a ΔearA mutant in which the restoration of the transcription and translation of the fla operon (using flaB2, the second gene of the operon, as a reporter), archaella formation and swarming motility were all restored even in the absence of EarA. Analysis of the DNA sequence from the fla promoter of this spontaneous mutant revealed a deletion of three adenines within a string of seven adenines in the transcription factor B recognition element (BRE). When the three adenine deletion in the BRE was regenerated in a stock culture of the ΔearA mutant, very similar phenotypes to that of the spontaneous mutant were observed. Deletion of the three adenines in the fla promoter BRE resulted in the mutant BRE having high sequence identity to BREs from promoters that have strong basal transcription level in Mc. maripaludis and Methanocaldococcus jannaschii. These data suggest that EarA may help recruit transcription factor B to a weak BRE in the fla promoter of wild-type cells but is not required for transcription from the fla promoter with a strong BRE, as in the three adenine deletion version in the spontaneous mutant

Structural analysis of receptor engagement and antigenic drift within the BA.2 spike protein

Summary: The BA.2 sub-lineage of the Omicron (B.1.1.529) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant rapidly supplanted the original BA.1 sub-lineage in early 2022. Both lineages threatened the efficacy of vaccine-elicited antibodies and acquired increased binding to several mammalian ACE2 receptors. Cryoelectron microscopy (cryo-EM) analysis of the BA.2 spike (S) glycoprotein in complex with mouse ACE2 (mACE2) identifies BA.1- and BA.2-mutated residues Q493R, N501Y, and Y505H as complementing non-conserved residues between human and mouse ACE2, rationalizing the enhanced S protein-mACE2 interaction for Omicron variants. Cryo-EM structures of the BA.2 S-human ACE2 complex and of the extensively mutated BA.2 amino-terminal domain (NTD) reveal a dramatic reorganization of the highly antigenic N1 loop into a β-strand, providing an explanation for decreased binding of the BA.2 S protein to antibodies isolated from BA.1-convalescent patients. Our analysis reveals structural mechanisms underlying the antigenic drift in the rapidly evolving Omicron variant landscape

Complementation of an <i>aglB</i> Mutant of <i>Methanococcus maripaludis</i> with Heterologous Oligosaccharyltransferases

<div><p>The oligosaccharyltransferase is the signature enzyme for <i>N</i>-linked glycosylation in all domains of life. In Archaea, this enzyme termed AglB, is responsible for transferring lipid carrier-linked glycans to select asparagine residues in a variety of target proteins including archaellins, S-layer proteins and pilins. This study investigated the ability of a variety of AglBs to compensate for the oligosaccharyltransferase activity in <i>Methanococcus maripaludis</i> deleted for <i>aglB</i>, using archaellin FlaB2 as the reporter protein since all archaellins in <i>Mc</i>. <i>maripaludis</i> are modified at multiple sites by an <i>N</i>-linked tetrasaccharide and this modification is required for archaellation. In the <i>Mc</i>. <i>maripaludis</i> Δ<i>aglB</i> strain FlaB2 runs as at a smaller apparent molecular weight in western blots and is nonarchaellated. We demonstrate that AglBs from <i>Methanococcus voltae</i> and <i>Methanothermococcus thermolithotrophicus</i> functionally replaced the oligosaccharyltransferase activity missing in the <i>Mc</i>. <i>maripaludis</i> Δ<i>aglB</i> strain, both returning the apparent molecular weight of FlaB2 to wildtype size and restoring archaellation. This demonstrates that AglB from <i>Mc</i>. <i>voltae</i> has a relaxed specificity for the linking sugar of the transferred glycan since while the <i>N</i>-linked glycan present in <i>Mc</i>. <i>voltae</i> is similar to that of <i>Mc</i>. <i>maripaludis</i>, the <i>Mc</i>. <i>voltae</i> glycan uses <i>N</i>-acetylglucosamine as the linking sugar. In <i>Mc</i>. <i>maripaludis</i> that role is held by <i>N</i>-acetylgalactosamine. This study also identifies <i>aglB</i> from <i>Mtc</i>. <i>thermolithotrophicus</i> for the first time by its activity. Attempts to use AglB from <i>Methanocaldococcus jannaschii</i>, <i>Haloferax volcanii</i> or <i>Sulfolobus acidocaldarius</i> to functionally replace the oligosaccharyltransferase activity missing in the <i>Mc</i>. <i>maripaludis</i> Δ<i>aglB</i> strain were unsuccessful.</p></div

Primers used in this study.

<p>Primers used in this study.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

Comparison of <i>Mc</i>. <i>maripaludis</i> AglB to other AglBs used in this study (pairwise EMBOSS Needle).

<p>Comparison of <i>Mc</i>. <i>maripaludis</i> AglB to other AglBs used in this study (pairwise EMBOSS Needle).</p

Western blot analysis of FlaB2 in an <i>Mc</i>. <i>maripaludis</i> Δ<i>aglB</i>-<i>14-9</i> mutant complemented <i>in trans</i> with homologous <i>aglB</i>.

<p>The Δ<i>aglB-14-9</i> mutant was complemented with a plasmid borne version of <i>Mc</i>. <i>maripaludis aglB</i> under the control of the <i>nif</i> promoter. The complemented Δ<i>aglB-14-9</i> mutant was grown in nitrogen-free medium supplemented with alanine or NH₄Cl which results in transcription from the <i>nif</i> promoter being on (alanine) or off (NH₄Cl). Lane 1 is Mm900 (WT), lane 2 is Δ<i>aglB-14-9</i> mutant, lane 3 is Δ<i>aglB-14-9</i> mutant complemented cells grown in nitrogen-free medium supplemented with alanine, lane 4 is Δ<i>aglB-14-9</i> mutant complemented cells grown in nitrogen-free medium supplemented with NH₄Cl.</p