Identification of Human Norovirus GII.3 Blockade Antibody Epitopes
, Feifei Yin 1,2,* and Zhong Huang 3,*Round 1
Reviewer 1 Report
In this manuscript from Yi and colleagues, the authors developed anti-GII.3 monoclonal antibodies and mapped two blockade epitopes on the GII.3 norovirus capsid. Overall, the manuscript is straightforward and relatively easy to read. The authors appropriately utilized adequate experimental approaches to confirm the specificity of the newly developed mAbs. These antibodies provide valuable reagents for studying norovirus biology and development of diagnostic tools.
Author Response
Response: We thank this reviewer for recognizing the importance of the work.
Reviewer 2 Report
Identification of human norovirus GII.3 blockage epitopes
By Yufang Yi, Shuxia Wang, Xiaoli Wang et al (Corresponding authors: Chao Zhang, Feifei Yin, Zhong Huang)
Submitted to Viruses (Editorial No. viruses-1397953)
General Comments
This report describes the identification of norovirus GII.3 VP1 epitopes eliciting the formation of blockage antibodies. Monoclonal antibodies (mAbs) were produced from mice immunized with the virus-like particles (VLPs) of a GII.3 norovirus. Two mAbs (termed 8C7 and 8D1) were found to specifically bind GII.3 noroviruses and to block their reaction with a ligand histo-blood group antigen (HBGA). The epitopes to which these mAbs bind were identified by a clever series of chimeric VLPs and, by using a recently determined GII.3 P domain structure, found to either partially overlap with or being located close to the HBGA binding site, acting by direct blockage or steric hindrance of the interaction.
The topic has been introduced concisely. The methodology used is highly appropriate. The experimental data are novel and are very transparently presented. The discussion is short and to the point.
This reviewer enjoyed reading the manuscript very much and has only a few, relatively minor Specific Comments.
Specific Comments
Page
1 Consider slight change in title: … GII.3 blockage antibody epitopes
Abstract Line 3. Consider reading: … two distinct blockage antibody epitopes…
Line 3 from bottom. Should read: … to 10 (GI to GX) [3] ? Please consider.
2 Paragraph 3, line 8. … antibody epitopes. Although in silico analyses…
Bottom. Table 1 should be set on one page.
3 Table 1 Legend. Consider reading: … b According to Saito et al [33].
Section 2.2, end. Provide details about mAb purification.
Section 2.3, line 1. … incubated at 37 oC for 2 h…
4 Section 2.8. Please comment on using the P domain dimer structure of a strain which is not the strain used for the production of the mAbs.
6 Fig. 3. Read: Antiserum In Legend: Rabbit anti-GII.3 VLP antiserum…
9 Fig. 5. The mAb 8C7 epitope is also found in cluster 2 and 3 norovirus strains (KY767665, KJ145323, KF306203, etc; KF944111, JN565063). These strains should react with mAb 8C7. Please comment.
10 Line 6… despite the virus undergoing steady evolution.
11 Line 5. Consider phrasing: … within the P2 domain. The experimental definition of blockade antibody epitopes of GII.3 noroviruses is a novel observation.
Line 3 from bottom. See comment page 11 above.
13 Correct indentation of text of ref. 17.
15 Refs. 47, 52, 55, and 57 are incomplete.
Author Response
Dear reviewers,
We would like to thank you for your positive feedback. We have responded to the points made by the reviewers and marked up the revisions (“Track Changes”) in the revised text. We hope that revised manuscript is now suitable for publication.
Response to reviewer #2’ s comments:
Reviewer #2 (Comments to the Author):
This report describes the identification of norovirus GII.3 VP1 epitopes eliciting the formation of blockage antibodies. Monoclonal antibodies (mAbs) were produced from mice immunized with the virus-like particles (VLPs) of a GII.3 norovirus. Two mAbs (termed 8C7 and 8D1) were found to specifically bind GII.3 noroviruses and to block their reaction with a ligand histo-blood group antigen (HBGA). The epitopes to which these mAbs bind were identified by a clever series of chimeric VLPs and, by using a recently determined GII.3 P domain structure, found to either partially overlap with or being located close to the HBGA binding site, acting by direct blockage or steric hindrance of the interaction.
The topic has been introduced concisely. The methodology used is highly appropriate. The experimental data are novel and are very transparently presented. The discussion is short and to the point.
Response: We thank the reviewer for the positive comments. Below are our responses to the reviewer’s specific concerns.
Minor points:
Page 1 Consider slight change in title: GII.3 blockade antibody epitopes
Response: Thanks for the suggestion. We have added the word "antibody", to read “Identification of human norovirus GII.3 blockade antibody epitopes”. Besides, the term “blockade epitopes” has been changed to “blockade antibody epitopes” throughout the article.
Page 1 Abstract Line 3. Consider reading: two distinct blockade antibody epitopes
Response: Thanks. We have followed the suggestion to add the word "antibody", to read “two distinct blockade antibody epitopes”.
Page 1 Line 3 from bottom. Should read: to 10 (GI to GX) [3] ? Please consider.
Response: Thanks for pointing this out. We have cited reference #3.
Page 2 Paragraph 3, line 8. antibody epitopes. Although in silico analyses…
Response: Thanks. As suggested, we have modified the manuscript accordingly, to read “Although in silico analyses have predicted several sites…”.
Page 2 Bottom. Table 1 should be set on one page.
Response: Thanks for pointing this out. We have modified the manuscript accordingly.
Page 3 Table 1 Legend. Consider reading: b According to Saito et al [33].
Response: Thank you for the suggestion. We have modified the Table 1 Legend, to read “According to Saito et al [33]”.
Page 3 Section 2.2, end. Provide details about mAb purification.
Response: We have modified the manuscript accordingly, to read “Hybridoma cells were expanded and then injected intraperitoneally into female Balb/c mice previously irritated with liquid paraffin. Ascitic fluids were then obtained from the mice and clarified by centrifugation. The mAbs were purified from the supernatants using HiTrapTM Protein G affinity column (GE Healthcare, NJ, USA), according to the manufacturer’s instructions.”
Page 3 Section 2.3, line 1. incubated at 37 °C for 2 h…
Response: Thanks for pointing out the error. We have modified the manuscript accordingly, to read “incubated at 37 °C for 2 h”.
Page 4 Section 2.8. Please comment on using the P domain dimer structure of a strain which is not the strain used for the production of the mAbs.
Response: The P domain structure of GII.3 KJ strain, which was used for MAb production, has not been resolved. We have added the statement into the revised manuscript.
Page 6 Fig. 3. Read: Antiserum In Legend: Rabbit anti-GII.3 VLP antiserum…
Response: Thanks for pointing this out. As suggested, we have made modifications accordingly in our revised manuscript, to read “Rabbit anti-GII.3 VLP antiserum was used as a positive control.”.
Page 9 Fig. 5. The mAb 8C7 epitope is also found in cluster 2 and 3 norovirus strains (KY767665, KJ145323, KF306203, etc; KF944111, JN565063). These strains should react with mAb 8C7. Please comment.
Response: We agree with the reviewer’s comments. We have added the point into our revised manuscript, to read “It is worth noting that the 8C7 epitope is also found in several cluster 2 and 1 norovirus strains (KY767665, KJ145323, KF306213, KF944111, JN565063, etc) (Figure 5). It is reasonable to infer that these strains should react with mAb 8C7. Indeed, mAb 8C7 can react with KY767665 (KY) strain-derived VLP (Figure 3).”
Page 10 Line 6 despite the virus undergoing steady evolution.
Response: Thanks. As suggested, we have made modifications accordingly in our revised manuscript, to read “despite the virus undergoing steady evolution”.
Page 11 Line 5. Consider phrasing: within the P2 domain. The experimental definition of blockade antibody epitopes of GII.3 noroviruses is a novel observation.
Response: Thanks. As suggested, we have made modifications accordingly in our revised manuscript, to read “The experimental definition of blockade antibody epitopes of GII.3 norovirus is a novel observation”.
Page 11 Line 3 from bottom. See comment page 11 above.
Response: Thanks. As suggested, we have made modifications accordingly in our revised manuscript, to read “the present study developed a set of anti-GII.3 mAbs and experimentally defined blockade antibody epitopes of GII.3 norovirus”.
Page 13 Correct indentation of text of ref. 17.
Response: We have corrected this problem
Page 15 Refs. 47, 52, 55, and 57 are incomplete.
Response: We have used EndNote software to insert these references again. However, the same content as before was shown.
