Peer review of: A cross-reactive human IgA monoclonal antibody blocks SARS-CoV-2 spike-ACE2 interaction (2020)

REVIEWER COMMENTS 

 

Reviewer #1 (Remarks to the Author): 

 

This manuscript describes a SARS/SARS2 cross-reactive antibody that was initially generated in a 
humanized mouse model a number of years ago during a SARS study. The authors show that the 
antibody likely binds to the ACE2 receptor binding site and blocks receptor engagement. The authors 
make the antibody as an IgG and IgA and observe better binding and neutralization in the IgA 
format, which they then suggest may have better in vivo properties for combatting SARS2 due to the 
preferential deposition in the mucosal tissue. The study is of moderate interest but there are a 
number of glaring and egregious mistakes and disconnected conclusions that give one serious pause 
as to the quality of the study. First and foremost is the “neutralization” assay that is used and the 
fact that they do not even report an IC50 value for comparison to other RBD antibodies. 

 

Major concerns: 

 

Fig. 1 the dynamic range of the association step is very small and it looks like the curves are appear 
not to be normalized to 0. 

 

It is difficult to understand why the IgA binds better than IgG to SARS-CoV-2 by Octet. Is it the 
dimeric version? If not then it should have the same variable regions and therefor bind similarly just 
like SARS-CoV. 

 

It would be good to show a sequence alignment between 80R and mAb362, both heavy and light 
chains. I assume only the heavy chain is 90% identical in framework. Also, are they predicted to be 
derived from the same germline genes? 

 

Additionally, just because the framework was 90% identical does not mean that the binding is similar 
in any way as the paratope is formed by the much more variable CDR loops. 

 

There are better programs to model antibody structures than Modeller, namely RosettaAntibody 
and RosettaAntibodyDesign. Also, given the level of modeling, prediction of specific contacts down 
to the level of Van der Waals and hydrogen bond is meaningless. 

 

While the mutagenesis studies suggest that they are in the right region of the RBD the modeling and 
docking based on 80R is not guaranteed to be very accurate. A crystal structure of the Fab alone or 
even better the Fab-RBD complex would be a significant addition to this paper. 

 

In the first paragraph of the results it would be nice to provide a short description of the 
immunization or infection and mouse model that was used to generate the panel of anti-SARS CoV 
mAbs that includes mAb362. Otherwise there is not appropriate context for the source of these 
antibodies even though the original study was properly referenced. 

 

IgG dimers are notoriously difficult to produce and are often heterogenous from monomers to 
oligomers when express with J chain. It is therefore important to run size exclusion chromatography 
to purify just the dimeric fraction. Otherwise there is likely a mixture of valencies, which confounds 
any binding studies. 

 

The ability of monomeric IgA to more potently neutralize than IgG is still somewhat controversial 
and there is only one other example in the HIV literature. Thus, it would have been nice if the 
authors made other SARS or SARS-CoV-2 control antibodies in both formats and tested binding and 
neutralization. I also find it curious that the authors did not test (or show) the dimeric IgA 
neutralization data for SARS in Fig. 4a. 

 

The emerging literature suggests that pseudovirus neutralization is a less good measure than live 
virus neutralization so the authors should consider redoing the neutralization assays. 

 

IC50 values for neutralization were either not calculated or not included for some reason, although 
the neutralization curves were shown in Fig. 4. Further, the abstract states that mAb362 neutralizes 
human epithelial cells expressing ACE2, which is very misleading. The authors instead used HEK 293 
cells transfected with ACE2 receptor. The E in HEK stands for “embryonic” not “epithelial”. Further, 
HEK cells were generated via a Adeno infection of an unknown cell type and are quite strange cells. 
Thus, while the ACE2 transfection is probably sufficient to measure blocking of infection by an 
antibody it is hardly representative of lung epithelium as suggested by the abstract. I am therefore 
lead to believe that this was an honest mistake out of ignorance or carefully crafted language to 
increase the impact of the study. 

 

 

 

 

Reviewer #2 (Remarks to the Author): 

 

The manuscript by Wang et al describes the discovery and characterisation human IgA monoclonal 
antibody, MAb363, which is cross-reactive for SARS-CoV and SARS-CoV-2. They demonstrate that 
Mab362 binds to the spike glycoprotein and competitively blocks the interaction with the human 
ACE2 receptor. Furthermore, they show that Mab362 can neutralise pseudotyped SARS-CoV and 
SARS-CoV-2 in human epithelial cells expressing hACE2, suggesting that IgA may play an important 
role in SARS-CoV-2 neutralization. However, the results, although important, lack robustness. Below 
are a number of experiments which could sufficiently strengthen the conclusions to warrant 
publication. 

 

Major concerns 

 

1) It is stated in the materials and methods section (lines 384-389) that recombinant spike 
ectodomains were produced, but the reported ELISA and bio-layer interferometry experiment were 
performed on the RBD only. Given that the proposed Mab363 epitope is only accessible in the open 
conformation of spike, binding experiments with RBD alone represent the most favourable, and less 
physiologically relevant, situation. Indeed, the SARS-CoV-2 spike protein is reported to sample the 
open conformation even less frequently than SARS-CoV 
(https://www.pnas.org/content/early/2020/05/05/2003138117). Therefore, additional binding 
experiments should be performed on the ectodomains (which were already produced by the 
authors). The authors should carefully check the quality of such ectodomains by negative-stain 
electron microscopy prior to using them for functional experiments to ensure that they display the 
expected prefusion conformation. 

 

2) No cryo-EM or crystallography data is reported but the authors go into great detail interpreting a 
predicted model of the Mab362-RBD complex (even going as far as to speculate about a water 
mediated interaction). Given the potential importance of SARS-CoV-2 neutralising antibodies, it does 
not seem appropriate to draw such firm conclusions about the binding interface without 
determining the structure experimentally. This entire section needs to be toned down. While it is not 
realistic to request that the authors obtain a structure, their predicted epitope could be further 
verified by introducing targeted SARS-CoV RBD residues into that of SARS-CoV-2 and demonstrating 
that this leads to increased binding of Mab362. If possible, it would also be nice to see an alignment 
of the Mab362 and 80R sequences, rather than taking the authors word for it. In addition, I would 
like to see the authors homology model so that I can compare it to the 80R-RBD complex (PDB-ID 
2GHW). 

 

3) Mab362 IgG is weakly neutralising but the IgA1, and its dimeric form, are able to potently 
neutralise pseudotype virus. The authors speculate that the increased flexibility of IgA may allow for 
multivalent interactions, hence explaining its increased potency. The dimeric form shows even 
greater neutralisation capacity, which I suspect may be due to crosslinking of virions. Given that 
increased avidity seems to be crucial for Mab362, it would be prudent to perform neutralisation 
experiments on SARS-CoV-2. The reason being that the pseudotype virus may not faithfully 
recapitulate the density of spikes which would be encountered on the native virion. 

 

4) I agree that IgA present an interesting therapeutic potential for mucosal protection against 
viruses. However, in contrast to monoclonal antibodies of the IgG isotype, their development as 
research tools or human therapeutics has been scarce. Given that the IgG Mab362 show relatively 
low potency, how feasible is it that the IgA isotype can be immediately translated into a prophylactic 
or treatment option? It is well known that the half-life of IgA is lower than that of IgG, but the 
authors do not discuss this. 

 

Minor points 

 

Line 86: Missing space “IgGor IgA1 isotypes”. 

 

Line 104: Remove the word “striking”. 

 

Line 105: Replace the word “outstanding” with “suitable”. 

 

Line 106: Please remove “build a highly accurate atomic”. You don’t have experimentally derived 
electron density or Coulomb potential so you had nothing to “build” into. “Highly accurate” implies 
that you verified your model with an actual structure. It would be sufficient to say “generate a 
homology model”. 

 

Line 110-131: The very detailed interpretation of the proposed model may be misleading to the 
untrained eye. Wherever possible, it needs to be made explicitly clear that this model is not derived 
experimentally. 

 

Line 132-133: As discussed above, gain of function would be more convincing. 

 

The labels in figure 2C-E and 2G-H are too small.