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


RR2
 Reviewers' Comments:
 Reviewer #1:
 Remarks to the Author:
 Dear Editors & Authors;
 I welcome the possibility of reviewing the paper for the 2nd time. In the first version the authors
 developed a new antibody aiming to improve Influenza diagnosis, however, many things remained
 to be clarified.
 In this 2nd revision, the authors have addressed the major concerns I had previously:
 - They have tested the antibody against the single mutant;
 - They tested the antibody against donor samples;
 - They provided some experimental validation on the antibody-antigen binding mechanism;
 - The methodology section has been significantly improved, providing details regarding the phage
 display methodology and the docking methodology.
 I believe the paper has scientific merit and recommend it to be published at Nature
 Communications.
 Best wishes,
 André
 Reviewer #4:
 Remarks to the Author:
 Review of Author’s response to reviewer #2 questions.
 Reviewer #2
 Question #1.
 Reviewer#2 raised a very valid concern on the application of the LFA in a clinical setting without
 vigorous validation of the assay performance using real clinical material. The authors responded by
 including a small study (n=14) using NP/Op swabs spiked with 103 pfu pH1N1 viruses with
 I223R/H275Y markers. A few issues remain:
 1). Sensitivity is often an issue for LFA assays. the authors did not demonstrate a dose effect with
 the detection of the I223R/H275Y in NP/OP swabs. 2). Matrix effect from different NP/OP
 swabs/broncheoalveolar lavage samples may also impact the sensitivity of the LFA detection, a
 larger number of the actual clinical samples collected from different patients are needed in order to
 evaluate the performance of this assay. 3). A more vigorous study using actual clinical specimen
 that are conducted side-by-side with sequencing are indeed needed to truly demonstrate the
 sensitivity and specificity of the assay and its true value in the actual clinical setting.
 Question 2
 "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
 resistanceassociated
 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 #2 raised a good point. The authors did not sufficiently addressed this question.
 I223R/H275Y is a recognized marker for A(H1N1) oseltamivir/zanamivir resistance, but not
 necessarily for other subtypes. Furthermore, there are several other genetic markers that were
 identified to be associated for drug resistance, and not all drug resistance are based on
 neuraminidase. The authors should consider modifying the language in manuscript to address this

 limitation, including in the title to avoid overstatement of the study findings, for example the title
 can be modified to:
 “Development of novel A4 antibody for detection of neuraminidase I223R/H275Y associated
 antiviral multidrug-resistant influenza viruse”