Peer review of: An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
Hanke et al. present a fine study on nanobody (Ty1) isolated from the alpaca Tyson against 
the SARS-CoV2 spike protein specifically the RBD of the spike (immunisations with spike 
trimer and later only RBD). Using a pseudotyped lentivirus model expressing SARS-CoV2 
spike protein for infection of HEK293 expressing ACE2 they find that Ty1 blocks infection. 
Lentivirus expressing VSV-G is not functional and a nanobody against influenza 
nucleoprotein A shows no effect. The characterise the binding by BLI and ITC to find 
consistently a 5-10 nM binding affinity, and they characterise the complex using cryo-EM 
determining the structure at overall 2.9 Å resolution. These are fine and consistent results, 
and they present a good report to also highlight the implications and requirements for 
clinical application of their findings. 
It would be nice to compare to recent results for glycosylation dynamics/shielding of the 
spike protein (see e.g. https://www.biorxiv.org/content/10.1101/2020.05.20.103325v1 and 
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7217288/). Furthermore, to recent studies 
of human antibody binding sites https://www.cell.com/cell/pdf/S0092-8674(20)30757-1.pdf 
- this could go to the discussion section in a minor revision. 
 
minor point 
The front page states an equal contribution note, but I dont see it applied to anyone? 
 
 
 
Reviewer #2: 
Remarks to the Author: 
The consortium identified an affinity-matured nanobody against RBD of SARS-Cov2, 
retrieved form an immune alpaca Nb library. The selection was performed after only two 
rounds of phage display pannings, and enrichments were checked by NGS. The highest 
enriched clone, named Ty1 was further analysed in a virus neutralisation capacity, in 
staining S protein generating cells. The affinity of binding was measured by bio 
interferometry and ITC. Cryo-EM was employed to identify the binding site. Interestingly, 
this nanobody binds simultaneously to the ACE2 accessible and in the two non-accessible 
conformers. 
 
This reviewer is impressed by the quality and quantity of work that has been achieved in a 
short period of time. The experiments are well performed and the resulting data are highly 
interesting and important. Nevertheless, I would like to suggest a few minor amendments 
that could be taken into account. 
 
Line 99 (and also later) the authors are using NP-VHH1 as a non-RBD binding control. 
However, I missed the specificity of this nanobody. 
Line 202 and later, human FC is probably human Fc (from IgG1?) 
 
Line 250: the sequence of the Ty1 nanobody should start with QVQL, the MA is presumably 

a leftover of the pel B leader signal. 
It is surprising that it is nowhere mentioned that the sequence of the VHH in FR2 
corresponds to the VH germline sequences. V at pos 37 (Kabat) and PEW at pos 45-47. 
However, the R instead of W at pos 103 proofs that it is a VHH (part of a heavy chain 
antibody) as explained in Muyldermans (2013) Ann Rev Biochem. 82. 
 
It is also surprising that the authors missed the paper by Justin D Walter (Market Seeger 
lab) submitted to BioRxiv doi.org/10.1101/2020.04.16.045419; while they refer to the 
paper by Chi, X in BioRxiv ref 21) This paper Walter et al. should be added.