Review Reports - Multi-Influenza HA Subtype Protection of Ferrets Vaccinated with an N1 COBRA-Based Neuraminidase

Round 1

Reviewer 1 Report

Influenza A virus (IAV) is an important human respiratory pathogen and represents a considerable threat to global public health. Current vaccination strategies to prevent IAV infections are only partially effective. Therefore, the development of novel prophylactic approaches, including universal vaccines, are required for the efficient control of IAV infections. In this manuscript "Multi-influenza HA subtype protection of ferrets vaccinated with an N1 COBRA-based neuraminidase" by Zhang et al., authors have evaluated the potential of a new NA-based IAV vaccine (N1-I COBRA NA) against several subtypes of IAV. For that, authors have used ferrets, which is a well-established animal model. The data provided in this manuscript show interesting results, the manuscript is nicely written and experiments are logical. I have some minor comments and suggestions that would improve the manuscript.

Specific comments

· Authors should provide evidences regarding the quality of the NA purification for each of the NA proteins used in this study. Eg, Coomassie staining gel from the purification.

· Please, include in the manuscript the sequence of N1-I COBRA NA. In addition, authors should include an alignment using N1-I COBRA NA and the others NAs or viruses used in the manuscript (including Sing/86).

· It could be worthy indicate in the alignment the region involved in Neuraminidase activity. This can help to explain or discuss data from figure 7, showing differences between the ability of antibodies to specifically inhibit one or multiple types of NA.

· Related with figure 7, authors should perform an ELISA to determine the total amount of antibodies (IGs) against the different NAs. In figure 7, it is inferred only the antibodies with anti-neuraminidase activity.

· Page 11, Line 436: “There were no viral nasal wash titers detected at any time point post-infection”. Since some animals lost weight and even some of them died (Mock and CA/09), it is weird that virus is not detected in the nasal wash. Could the authors explain or discuss this finding?

Author Response

December 25, 2022

Dear Editor: We are re-submitting the Manuscript #viruses 2108206 for Viruses entitled “Multi-influenza HA subtype protection of ferrets vaccinated with an N1 COBRA-based neuraminidase”. We have responded to the referee comments and revised the manuscript for resubmission. We look forward to receiving your comments. We thank the referee for the helpful comments and suggestions.

Specific Reviewer comments:

Response Letter

Referee #1

Influenza A virus (IAV) is an important human respiratory pathogen and represents a considerable threat to global public health. Current vaccination strategies to prevent IAV infections are only partially effective. Therefore, the development of novel prophylactic approaches, including universal vaccines, are required for the efficient control of IAV infections. In this manuscript "Multi-influenza HA subtype protection of ferrets vaccinated with an N1 COBRA-based neuraminidase" by Zhang et al., authors have evaluated the potential of a new NA-based IAV vaccine (N1-I COBRA NA) against several subtypes of IAV. For that, authors have used ferrets, which is a well-established animal model. The data provided in this manuscript show interesting results, the manuscript is nicely written and experiments are logical. I have some minor comments and suggestions that would improve the manuscript.

Response: Thank you for reviewing our manuscript.

Specific comments

Authors should provide evidence regarding the quality of the NA purification for each of the NA proteins used in this study. Eg, Coomassie staining gel from the purification.

Response: Thank you. The Coomassie staining for the purified NA proteins are showing below:

Supplemental Figure 1. Coomassie staining for the purified NA proteins.

Please, include in the manuscript the sequence of N1-I COBRA NA. In addition, authors should include an alignment using N1-I COBRA NA and the others NAs or viruses used in the manuscript (includingSing/86).

Response: Thank you for this great comment. Studies have shown that the NA catalytic site (in red boxes) consists of eight highly conserved residues (N2 numbering): Arg118, Asp151, Arg152, Arg224, Glu276, Arg292, Arg371 and Tyr406. There are another 10 framework residues (in blue boxes): Gle119, Arg156, Trp178, Ser179, Asp198, Ile222, Glu227, Glu277, Asn294, and Glu425. These residues stabilize the geometry of the catalytic site. We marked all these residues in the alignment.

Supplemental Figure 2. NA Protein sequences alignment. The NA catalytic site residues are in red boxes, while the framework residues are in blue boxes.

It could be worthy indicate in the alignment the region involved in Neuraminidase activity. This can help to explain or discuss data from figure 7, showing differences between the ability of antibodies to specifically inhibit one or multiple types of NA.

Response: Thank you for this great comment. As showing in Supplemental Figure 2, the NA catalytic site residues are highly conserved among all the NA subtypes of influenza A and B viruses, thus this information might not be helpful to understand the data in Figure 7. It has been shown that, conserved epitopes on NA proteins that are responsible for eliciting broadly reactive antibodies have been identified using monoclonal antibodies. Furthermore, the universal monoclonal antibodies usually target the lateral region of NA head domain instead of the catalytic site. For example, there was a single conserved epitope on the H1N1 and H5N1 NA proteins that elicited cross-reactive antibodies against both H1N1 seasonal, H1N1pdm, and H5N1 NA proteins. And the N1-I COBRA NA immunogen retains this conserved epitope (amino acid residues 273, 338, and 339). And our data indicated that the N1-I COBRA NA vaccine potentially elicits antibodies specific to this conserved NA epitope.

Related with figure 7, authors should perform an ELISA to determine the total amount of antibodies (IGs) against the different NAs. In figure 7, it is inferred only the antibodies with anti-neuraminidase activity.

Response: Thank you for the suggestion. We didn’t perform ELISA for the serum samples collected in this study. But we do have ELISA data from our previously published study done in mice. In both studies, the same antigens were used and robust antibody responses were seen as determined by ELISA or ELLA. The anti-NA antibodies determined by ELISA can bind multiple epitopes on the NA protein but not necessary to have the ability to inhibit the NA activity. For example, in our previous study, the Bris/07 NA-specific elicited antibodies bound to the NA protein of Viet/04, but they did not inhibit the NA enzymatic activity of the Viet/04 (H5N1) virus. Therefore, we only used ELLA to exam the antibody responses after vaccination because we want to evaluate the level of NA-inhibiting antibodies which correlates to the protection against influenza viral infection.

Page 11, Line 436: “There were no viral nasal wash titers detected at any time point post-infection”. Since some animals lost weight and even some of them died (Mock and CA/09), it is weird that virus is not detected in the nasal wash. Could the authors explain or discuss this finding?

Response: Thank you for pointing this out. Studies have shown that ferrets that challenged with A/Vietnam/1203/2004 (VN/04) virus at a higher dose (10^6 PFU/animal) usually shed small amount of virus in the nasal wash samples. In this study, a lower dose (10^5 PFU/animal) was used to challenge the ferrets, and the virus preferentially replicates in the lower respiratory tract (lung) where the α2,3-linked sialosides (α2,3SAL) receptors abound. As a result, the virus replication could be restricted in the lung tissue and the spreading of virus to the upper respiratory tract (nasal turbinate and trachea) could be reduced, thus limiting the shedding of virus in the nasal cavity.

Best regards,

Ted M. Ross, Ph.D.

GRA Eminent Scholar in Infectious Diseases

Director - Center for Vaccines and Immunology

Email: tedross@uga.edu

Author Response File: Author Response.pdf

Reviewer 2 Report

The study by Zhang et al addresses an important subject of improving immunity against influenza via vaccination. A preclinical ferret model is used to evaluate the effectiveness of a novel neuraminidase vaccine in naive and pre immune animals. The study is thorough and well executed.

Concerns regarding the manuscript in its current form are as follows:

1. The manuscript would benefit from extensive English editing. Several sections have unclear sentences and unconventional phraseology.

2. The abstract, as written, does not clearly reflect the study goals and findings.

3. Several terms require earlier definition or are not defined. For example, COBRA is not introduced until the end of introduction and BF is undefined.

4. Figure 1 would benefit from re-working as it the experimental plan is not clearly portrayed and is almost impossible to interpret.

5. How the unchallenged animals in Figure 3D have increased nasal inflammatory responses requires explanation. It is not clear from the methods whether these animals were anesthetized for the infection and received diluent or not. These essential details need to be added to the methods.

6. Does collecting nasal washes from animals impact the ability of the animals to transmit virus? Virus was transmitted but was transmission/titer of the virus in the nares greatly reduced by this procedure? Were the animals anesthetized for this procedure as well?

Author Response

December 25, 2022

Specific Reviewer comments:

Response Letter

Referee #2

Response: Thank you for reviewing our manuscript.

Concerns regarding the manuscript in its current form are as follows:

The manuscript would benefit from extensive English editing. Several sections have unclear sentences and unconventional phraseology.

Response: Thank you for the comment. We have had our manuscript checked by a native English-speaking colleague.

The abstract, as written, does not clearly reflect the study goals and findings.

Response: Thank you for the comment. We have re-written the abstract (Line 14-21).

Several terms require earlier definition or are not defined. For example, COBRA is not introduced until the end of introduction and BF is undefined.

Response: Thank you for the comment. We have addressed this in the manuscript. The COBRA is defined in the abstract (Line 14-15) and the “BF” refers to “neutral-buffered, 10% formalin fixative solution” (see Line 173 for details).

Figure 1 would benefit from re-working as it the experimental plan is not clearly portrayed and is almost impossible to interpret.

Response: Thank you for the comment. Figure 1 only shows the timeline for each experiment: protection or transmission. In order to avoid any confusion, we showed the group information in the figures of challenge result.

How the unchallenged animals in Figure 3D have increased nasal inflammatory responses requires explanation. It is not clear from the methods whether these animals were anesthetized for the infection and received diluent or not. These essential details need to be added to the methods.

Response: Thank you for the comment. About Figure 3D, the unchallenged group had a minimal group histopathological score detected between the levels 3 and 9 of the nose, with the highest score of 2 at level 5-7. Within these levels, the degree of inflammation was minimal and was considered non-significant, background inflammation. We have defined the "Group Histopathological Score" and how it was calculated in the section of "Materials and Methods". (Line: 193-194)

Does collecting nasal washes from animals impact the ability of the animals to transmit virus? Virus was transmitted but was transmission/titer of the virus in the nares greatly reduced by this procedure? Were the animals anesthetized for this procedure as well?

Response: Thank you for the comment.

(1) No, this procedure will not affect the ability of the animals to transmit virus. Firstly, only small amount (3 mL) of PBS was used to collect the shed virus in the nostril of ferrets. Secondly, this procedure was done every two days which is not often. There is plenty of time for infected ferrets re-produce, shed virus and then transmit virus to their cage mates.

(2) It’s okay if the amount of virus in the nares was reduced by the procedure as those ferrets were co-housed all the time. And the infected animals are shedding virus all the time whether the nasal wash procedure was performed or not. There will be virus everywhere in the cage and the cage mate will contact with it all the time. Thus, even if the nasal wash procedure has taken small amount of the virus in the nostril of ferrets, the ferrets still have a big chance to be infected.

(3) Yes, ferrets were anesthetized with vaporized isoflurane before bleeds, vaccination, infection, nasal washes, and euthanasia. This states in the materials and methods section.