Toll-Like Receptor and Cytokine Responses to Infection with Endogenous and Exogenous Koala Retrovirus, and Vaccination as a Control Strategy

Round 1

Reviewer 1 Report

In this review, the authors summarize the current knowledge on the innate immune response to koala retrovirus (KRV) infection, and the progress in vaccine development as the more realistic option of a control strategy for this life-threatening infection in Koala populations. In general, the review is very interesting and is a good update on the recent advances on this topic. However, there are some points that should be reviewed by the authors.

The title does not accurately reflect the content of the review. It should include the innate immune response and vaccination, since the description of the alternative control strategies are not very relevant in the total of the review. The review of the innate immune response to KoRV infection is the central part of the review, with interesting aspects about TLR and cytokines, and that is why it should be mentioned in the title.

Specific comments:

Line 36: LTRs are present only in the provirus DNA, not in the RNA genome of the virus. The authors should correct this error.

Line 47- 50-The authors should explain more clearly the existence of endogenous and exogenous forms of KoRV and in which Koala populations they can be found.

Line 130-142 and figure 1:  It is difficult to interpret these results if there are not data from KoRV-negative and endogenous KoRV-A only infected koalas. How many koalas have been analyzed to characterize the TLRs expression patterns? The authors should add to the legend of Figure 1 that upregulations were observed compared to KoRV-A only infected koalas.

Line 166-167 and figure 2. Again, cytokines response it is difficult to interpret without data from KoRV-negative and endogenous KoRV-A only infected koalas. The authors should add to the legend of Figure 2 that response were observed compared to KoRV-A only infected koalas.

Sections 3.3, 3.4 and 3.5 should not be included in section 3 of the innate immune response, as they describe control strategies and vaccines. Section 3.3 should be section 4, and change 3.4 to 4.1 and 3.5 to 4.2.

Figure 3 is very difficult to interpret. It would be convenient to indicate the vaccine used in each study and write it in the legend of the figure. Where do the data on the gradual or linear increase in IgG in ages older or younger than 7 years come from? What exactly do they mean?

Author Response

In this review, the authors summarize the current knowledge on the innate immune response to koala retrovirus (KRV) infection, and the progress in vaccine development as the more realistic option of a control strategy for this life-threatening infection in Koala populations. In general, the review is very interesting and is a good update on the recent advances on this topic. However, there are some points that should be reviewed by the authors.

The title does not accurately reflect the content of the review. It should include the innate immune response and vaccination, since the description of the alternative control strategies are not very relevant in the total of the review. The review of the innate immune response to KoRV infection is the central part of the review, with interesting aspects about TLR and cytokines, and that is why it should be mentioned in the title.

Response: Thanks for the reviewer sincere comment. In line with reviewer comments, we have modified the title as ‘Toll-like receptor and cytokine responses to infection with endogenous and exogenous koala retrovirus and vaccination as a control strategy’.

 

Specific comments:

Line 36: LTRs are present only in the provirus DNA, not in the RNA genome of the virus. The authors should correct this error.

Response: According to the reviewer comment we have corrected the error (page 1, line 37-38).

Line 47- 50-The authors should explain more clearly the existence of endogenous and exogenous forms of KoRV and in which Koala populations they can be found.

Response: Thanks for the reviewer comments. We have updated the text in line with reviewer suggestion (page 2, line 64-68).

Line 130-142 and figure 1:  It is difficult to interpret these results if there are not data from KoRV-negative and endogenous KoRV-A only infected koalas. How many koalas have been analyzed to characterize the TLRs expression patterns? The authors should add to the legend of Figure 1 that upregulations were observed compared to KoRV-A only infected koalas.

Response: Thanks for the reviewer comments. In line 144-145 we have indicated the limitations of that study, as reviewer highlighted. A total of 11 koalas were used for TLRs expression patterns analysis. In accordance with reviewer comments, we have added a legend in Figure 1 (page 4, line 175-178).

Line 166-167 and figure 2. Again, cytokines response it is difficult to interpret without data from KoRV-negative and endogenous KoRV-A only infected koalas. The authors should add to the legend of Figure 2 that response were observed compared to KoRV-A only infected koalas.

Response: Thanks for the reviewer comments. In line 175-176 we have discussed the limitations of that study, as reviewer highlighted. According to reviewer comments, we have added a legend in Figure 2 (page 5, line 205).

Sections 3.3, 3.4 and 3.5 should not be included in section 3 of the innate immune response, as they describe control strategies and vaccines. Section 3.3 should be section 4, and change 3.4 to 4.1 and 3.5 to 4.2.

Response: Thanks for the reviewer comments. According to reviewer comments, we have updated the section numbers.

Figure 3 is very difficult to interpret. It would be convenient to indicate the vaccine used in each study and write it in the legend of the figure. Where do the data on the gradual or linear increase in IgG in ages older or younger than 7 years come from? What exactly do they mean?

Response: Thanks for the reviewer comments. We have indicated each study in the figure for easy following, as reviewer commented. Also, we have made update on the figure legends. In addition, we have discussed an association of anti-IgG levels and koala age (page 6, line 249-253).

Reviewer 2 Report

The authors have written a review article about the immune response of koalas to KoRV and the routes to control KoRV in the koala population. I am not an expert on immune response myself and cannot comment on the science of it.

1) It might be important to discuss the existence of germline insertions and cite relevant papers. 

2) It would be beneficial to have a small part on evolutionary control and control of the endogenous KORV by piRNAs - which is just mentioned briefly.

3) the vaccination part is largely based on one publication and should be more concise and add a view of the authors on the direction in the field would benefit.

Author Response

The authors have written a review article about the immune response of koalas to KoRV and the routes to control KoRV in the koala population. I am not an expert on immune response myself and cannot comment on the science of it.

1) It might be important to discuss the existence of germline insertions and cite relevant papers. 

Response: In line with reviewer comments, we have updated the text with relevant discussion (page 1, line 47-48).

2) It would be beneficial to have a small part on evolutionary control and control of the endogenous KORV by piRNAs - which is just mentioned briefly.

Response: According to reviewer comments we have updated the text discussing control of KoRV by piRNAs (page 7, line 315-331, page 8, line 332-340).

3) the vaccination part is largely based on one publication and should be more concise and add a view of the authors on the direction in the field would benefit.

Response: In line with reviewer comments, we have concised and updated the text (page 7, line 292-294).

Round 2

Reviewer 1 Report

The authors have followed my recommendations and made the necessary changes to the manuscript. The title is much more appropriate for the content of the manuscript. And it is much easier to interpret the text and figures. But there are still minor observations.

- Line 36: LTRs are present only in the provirus DNA, not in the RNA genome of the virus. The authors should correct this error. Response: According to the reviewer comment we have corrected the error (page 1, line 37-38).

Ok, but the sentence must said “an integrated KoRV provirus additionally contains long terminal repeats at both ends”

- Line 130-142 and figure 1:  It is difficult to interpret these results if there are not data from KoRV-negative and endogenous KoRV-A only infected koalas. How many koalas have been analyzed to characterize the TLRs expression patterns? The authors should add to the legend of Figure 1 that upregulations were observed compared to KoRV-A only infected koalas.

Response: Thanks for the reviewer comments. In line 144-145 we have indicated the limitations of that study, as reviewer highlighted. A total of 11 koalas were used for TLRs expression patterns analysis (it would be interesting to include the number of animals analysed in the text itself). 

- Line 166-167 and figure 2. Again, cytokines response it is difficult to interpret without data from KoRV-negative and endogenous KoRV-A only infected koalas. The authors should add to the legend of Figure 2 that response were observed compared to KoRV-A only infected koalas.

Response: Thanks for the reviewer comments. In line 175-176 we have discussed the limitations of that study, as reviewer highlighted (I have not found the limitations of this study in version 2 of the text. Could the authors review it?). 

Author Response

Please see the attachment

Author Response File: Author Response.pdf