Peer review of: Development of A4 antibody for detection of neuraminidase I223R/H275Y-associated antiviral mu (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
In this work, the authors develop a new monoclonal antibody capable of specifically recognizing an 
influenza neuraminidase double-mutant which confers drug resistance. The goal stated by them is 
to create a point-of-care fast diagnosis method for drug-resistant strains. This is a real problem 
identified by the authors and they go in the direction of solving it. 
This is a very respectable work but, despite the paper’s merits, it cannot be framed as a protein 
engineering paper neither as diagnostic development paper since it lacks in both areas. I find that 
there are major issued that have to be addressed to substantiate their claims before it can be 
accepted in a publication such as Nature Communications. 
1. They show the antibody recognizing the double-mutant protein and not recognizing the wild-
type. However, they never use single-mutants in their study which also confers drug resistance 
and is more prevalent them the double mutants. It’s paramount to test the single mutants to show 
the diagnostic value of the antibody. 
2. They frame the paper as a diagnostic development work, but all the tests are done against 
recombinant protein or laboratory produced viruses. It’s paramount that their very interesting 
tools are tested on real human samples where it should be compared to traditional diagnostic 
methods such as DNA sequencing and/or qPCR to prove its ability to accurately differentiate the 
virus in real samples. Sensitivity and specificity numbers should be calculated after these tests to 
show the validity of the methods. 
3. They show computational studies for docking of the antibody-antigen complex without providing 
any methodological detail. Free energy number is discussed but it’s never explained how does the 
numbers were obtained. Different from protein structure modeling and despite its evolution in the 
past years, protein-protein docking methods are still only valid after experimental validation. Given 
two structures, a docking algorithm will always find many docking conformations even if the two 
proteins don’t actually bind. Without experimental validation of the docking structure, it’s 
impossible to assess its quality and validity. Without further evidence, there is no way to 
substantiate the claims made. Moreover, all the features pointed out for increased binding affinity 
consider hypothetical enthalpic changes, when in fact it could be entropic contributions. 
4. There are no considerations or references on phage display library specifications. This is very 
important for an antibody display paper. 
 
Those are the major points that I firmly believe should be addressed to make this paper, that has 
a lot of potentials, a very important piece of work that will push the current state-of-the-art on 
influenza diagnosis. Other minor points are described bellow 
 
 
• Affinities measured by ELISA are used for a rough estimation. Since the authors measured the 
affinity of the antibody by surface plasmon resonance, which is the state-of-art technique for such, 
the ELISA measurements should be kept as supplemental information or omitted since it does not 
add anything to the paper. Moreover, the methodology for measuring the affinity by ELISA was not 
described in the material and methods section. 
 
• There is no methodology for docking described in the paper and also there is no methodology for 
free energy calculation described in the paper. Please clarify this. 
 
• Different from protein structure modelling and despite its evolution in the past years, protein-
protein docking methods are still only valid after experimental validation. Given two structures, a 
docking algorithm will always find many docking conformations even if the two proteins don’t 
actually bind. Without experimental validation of the docking structure, it’s impossible to assess its 
quality and validity. Without further evidence there is no way to substantiate the claims made. 
Moreover, all the features pointed out for increased binding affinity consider hypothetical enthalpic 
changes, when in fact it could be entropic contributions. 

Line 34: It abbreviates neuraminidase to “NA” which has never been used in the text and so far. 
 
Line 121: please correct Delbecco to Dulbecco 
 
Line 130: why did you used insect expression system. Instead of mammalian expression system? 
 
Line 138: the centrifugation for 1h at 16,000g was to precipitate the protein or cell debris? I 
assume the protein was in the supernatant and that was used for affinity chromatography 
purification, but the text suggests that the “protein was protein was obtained by centrifugation” 
which implies that the protein was pelleted. 
 
Line 146: where the antibody library comes from? Is there a reference for this library? Is it 
human? Is it scFv os Fab format? What is the promoter? What is the helper phage used? 
 
Line 147: How many washes were performed in each round? It’s very important for the authors to 
give details of the method used to obtain the antibody so others can successfully follow. 
 
Line 156: How was the soluble Fab expressed? Was it fused to g3p? Is there an amber stop codon 
between the Fab gene and g3p so one can express soluble Fab when using a non-suppressor 
strain? 
 
Line 157: how was the Fab detected? Is there a tag so one can use a labeled anti-tag antibody? 
Please give more details. 
 
Line 160: Did the library only contain kappa light chains and no lambda light chains? 
 
Line 164: What polymerase was used? Please insert in the text. 
 
Line 165: Is recombinant PCR the same as overlap extension PCR? If so, I believe the later term is 
more widely used and well understood. 
 
Line 166: The fragments were not subcloned, but merely cloned. Subcloning is the technique 
where a fragment is directly excised with restriction enzyme from one plasmid and the ligated into 
another. In this case, the fragments were amplified by PCR, modified and then cloned. 
 
Line 176: What was the Bioanalyzer used for? It’s not clear. It looks like it was used for western 
blotting and SDS-PAGE. 
 
Line 221-222: It says Cys3-Protein G. I believe the authors meant Cy3, without the “s”. 
 
Line 303: I suggest giving the Kd in nanomolar, since it would more compliant to the standard 
using for such measurements. 
 
Line 315: The authors refer to Fig 4 when in fact, I’m led to believe, that they want to refer to 
figure 3. 
 
Line 320: correct the spelling of the word “later” 
 
 
 

Reviewer #2: 
Remarks to the Author: 
Guk et al. describe the development of an antibody for the detection of I223R/H275Y Influenza 
HA, which commonly renders the virus resistant to antiviral drugs, including oseltamivir. 
Furthermore, the authors describe the incorporation of this antibody within a lateral flow 
immunoassay. Even though the flow of experiments is logical, I do not believe that the manuscript 
at its current stage justifies the conclusions. Most importantly, all tests are performed with 
recombinant proteins or virus. In order to lay claim that this platform, in particular in combination 
with the LFI, would have any impact on clinical management of influenza virus infection, it would 
be absolutely essential that the authors evaluate performance of their assay directly in patient 
samples. 
Many diagnostic platforms have ultimately failed and a rigourous assessment using nasal swabs or 
broncheoalveolar lavage samples would be needed to assess the performance of this. 
Furthermore, the authors do not discuss the relevance of other drug resistance-associated variants 
in Influenza virus subtypes. Even though I223R/H275Y is an important resistence-associated 
variant, there are others playing an important role in other influenza virus subtypes. As far as I 
understand from the manuscript, the here-described antibody is specific to the H1N1 strain, even 
though it is not clear whether the authors tested other influenza virus subtypes. This would need 
to be included to ascertain the diagnostic validity of the assay. 
 
 
 
Reviewer #3: 
None