COVID-19 Infection, Vaccines, and Immunity—The Antibody Response Requires Detailed Analysis

Round 1

Reviewer 1 Report

This is a review on a topic of interest, however, its impact is diminished by its superficiality (very little introductory information is provide to understand some of the con cents discussed) and the fact that the argument for better evaluation of the antibody responses to COVID-19 is made solely based on data from Influenza infection or vaccination. Inclusion and discussion of more recent data from the literature on the analysis of the antibody responses to SARS-CoV-2 infection (E.g. E.g., Poh et al, Nature Communications volume 11,  (2020); Shrock et al, Science  27 Nov 2020:
Vol. 370, Issue 6520) could be used to further substantiate the benefits of a more careful analysis of COVID-19 antibody responses in the population. In addition suggestions of approaches for how this could be done in prospective studies would be welcome and make the review more comprehensive. 

The review would also benefit from language revision, some terms used are too colloquial and not appropriate (E.g "jab" instead of vaccination; "get-go" instead of start, etc.) There are truncated phrases and punctuation is also lacking.

Author Response

This is a review on a topic of interest, however, its impact is diminished by its superficiality (very little introductory information is provide to understand some of the con cents (njd says: should this read concepts?) discussed) and the fact that the argument for better evaluation of the antibody responses to COVID-19 is made solely based on data from Influenza infection or vaccination. Inclusion and discussion of more recent data from the literature on the analysis of the antibody responses to SARS-CoV-2 infection (E.g. E.g., Poh et al, Nature Communications volume 11, (2020); Shrock et al, Science 27 Nov 2020: Vol. 370, Issue 6520) could be used to further substantiate the benefits of a more careful analysis of COVID-19 antibody responses in the population. Reply: Thank you. I have included the references you suggest above and another by Zost et al. They nicely illustrate the level that antibody mapping has reached to date and at the same time how far there is to go. 2. Reviewer: In addition suggestions of approaches for how this could be done in prospective studies would be welcome and make the review more comprehensive. Reply: The reviewer puts his/her finger squarely on the problem – which I do underline in my article ‘’To determine person-to-person variation in the Covid-19 antibody response, it would be necessary to determine the epitope specificity of the antibodies stimulated in each individual. This is a problem that runs all through virology and radical new approaches are needed to solve it. Currently mapping of antibodies is pretty crude: they can be assigned to sub-regions of the S protein (Zost et al., 2020, or more specifically by binding to overlapping peptides (Poh et al., 2020; Shrock et al., 2020), but neutralization activity can only be inferred or shown by using the peptides to deplete the neutralizing activity of sera (Zost et al., 2020). A serious problem is that neutralization epitopes often comprise non-contiguous/conformational regions of the protein within the S protein monomer or at the interfaces in the trimer that cannot easily be mimicked by peptides.’’ If we had such methodology, there would not be a need for me to write the article in the first place. A major point of the article is to raise awareness of the pressing need to acquire data on the epitope specificity of Covid-19-specific antibodies, and the parallel need to devise a technology with to measure such epitope specificities. In fact, I have outlined in the article a methodology, based on our study and experience of influenza A virus, through which this could be achieved ‘’ An alternative approach would be to make a panel of multiple escape mutants by sequential exposure of virus to Covid-19-specific MAbs. These would then be utilised to analyse the epitope specificity of antibody samples, as used for influenza virus-specific sera (Lambkin et al., 1994; Lambkin and Dimmock, 1995, 1996.’’ However, such a methodology is not appropriate for high through-put use, and I am sure that with application others can solve this problem. The main difficulty, as I point out, is to replicate the conformational epitopes that form the majority of neutralizing epitopes. 3. Reviewer: The review would also benefit from language revision, some terms used are too colloquial and not appropriate (E.g "jab" instead of vaccination; "get-go" instead of start, etc.) There are truncated phrases and punctuation is also lacking. Reply: Language is always evolving, and I doubt there is anyone in the world these days who does not understand what a ‘jab’ is. Nonetheless if a word like ‘vaccination’ that is over 3-fold longer is preferred, so be it. I thought ‘get-go’ was a bit racy, but am happy for it to be changed if it grates with editorial policy. BTW English is my first language and I have written
over 200 research papers and several books, so I am an experienced author. Nonetheless I have checked the manuscript for infelicitous phrases and missing commas etc.

Reviewer 2 Report

Interesting analysis and hypothesis analysing the current and emerging problems and issues connected with the examination of the anti-SARS-CoV-2 antibodies and their clinical application. I have several comments and suggestions that could improve the manuscript:

• Despite the scientific interest in the "quality" and other characteristics of the examined antibodies, it has to be stated that the current clinical application of these examination is dominantly for infection diagnosis, not for the assessment of post-vaccination immunity. 
• There is no clear relationship between certain concentration of antibodies and the degree of achieved protection (after infection or vaccination). However, it is important to stress that the high concentration are more probably protective compared to low levels (logically).
• page 1, line 17 - S protein is spike protein, not surface protein
• It would be practical to name exactly all the antigens relevant in SARS-CoV-2 virion for immune attraction and production of antibodies. Also the differences of various antibodies in neutralization - anti-S versus anti-N etc.
• ADE effect has to be at least partially discussed and mentioned - since this could be the problem in case of low concentration of non-neutralizing antibodies (after infection). This effect was not described after vaccination.
• Page 1, line 36 - 9 isotypes... please, name all of them - especially for non-immunologists (I suppose that author though IgG1-4, IgA1-2, Inge, IgM, ID), or am I wrong?
• few words also about the cellular based assays (IGRA, T-spot) should be mentioned also, since this is another important issue of diagnostic approach
• Part 2 and 3 - sometimes it is hard to follow the ideas, confusing the facts about influenza and covid-19.
• for the lower spreading of virus in the society, it is important to have high concentration of neutralizing antibodies in the majority of the subjects (so-called herd immunity) and this is predominantly achieved by vaccination, since infection leads to variable immune response.
• Author has to conclude also the true clinical validity of the antibody examination according to the current knowledge.
• it would be interesting to add some comparison between vaccination and natural infection

Author Response

Interesting analysis and hypothesis analysing the current and emerging problems and issues connected with the examination of the anti-SARS-CoV-2 antibodies and their clinical application. I have several comments and suggestions that could improve the manuscript: 1. Reviewer: Despite the scientific interest in the "quality" and other characteristics of the examined antibodies, it has to be stated that the current clinical application of these examination is dominantly for infection diagnosis, not for the assessment of post-vaccination immunity.
Reply: Indeed, but I am making the point that knowledge of the epitope specificity of the antibodies produced would allow us to better understand and evaluate the immunity that results from infection, from vaccination – and from the different vaccines currently employed, and their longevity in the body. In addition we need this information to understand and evaluate the risk posed by virus variants. At the moment, we do not have this information and cannot make the necessary logical decisions. 2. Reviewer: There is no clear relationship between certain concentration of antibodies and the degree of achieved protection (after infection or vaccination). However, it is important to stress that the high concentration are more probably protective compared to low levels (logically).
Reply: I am not sure if the reviewer is referring to the overall covid-19-specific antibody concentration in serum or to the concentration of antibodies that react to a specific epitope. However, if the latter this could well be true, but we cannot make that judgement as we do are unable to identify antibodies specific for a particular epitope. 3. Reviewer: page 1, line 17 - S protein is spike protein, not surface protein
Reply: Thank you. S protein has been amended to spike protein in the text. 4. Reviewer: would it be practical to name exactly all the antigens relevant in SARS-CoV-2 virion for immune attraction and production of antibodies. Also the differences of various antibodies in neutralization - anti-S versus anti-N etc. Reply: It is highly likely that all SARS-CoV-2 proteins are immunogenic, but the major protein concerned with protection from infection is the S protein. Consequently I have focussed on the S protein in my article. 5. Reviewer: ADE effect has to be at least partially discussed and mentioned - since this could be the problem in case of low concentration of non-neutralizing antibodies (after infection). This effect was not described after vaccination.
Reply: ADE is an extremely interesting phenomenon, which is well evidenced in cell culture. However, its importance in establishing any infection in vivo is controversial. Therefore to deal with it here would distract from the main thrust of the article, which is not a general discussion of the interaction of SARS-CoV-2 and antibodies in humans but deals with the need to identify the epitope specificities of antibodies relevant to infection.
6. Reviewer: Page 1, line 36 - 9 isotypes... please, name all of them - especially for non-immunologists (I suppose that author though IgG1-4, IgA1-2, IgE, IgM, IgD), or am I wrong?
Reply: The reviewer is perfectly correct of course and has named all 9 human immunoglobulin isotypes. I am happy to have them named but I was thinking that this was too elementary to be included in an international virology journal. 7. Reviewer: few words also about the cellular based assays (IGRA, T-spot) should be mentioned also, since this is another important issue of diagnostic approach. Reply: The article is primarily concerned with the need to measure the epitope specificities of neutralizing antibodies, and I do not really think that the techniques mentioned are applicable in the circumstances. 8. Reviewer: Part 2 and 3 - sometimes it is hard to follow the ideas, confusing the facts about influenza and covid-19. Reply: I do apologise for not making myself clearer, but the relevant epitope mapping data were derived with influenza virus, and I have attempted (obviously not altogether successfully) to extrapolate from these data to the covid-19 situation. I will review the article and see how I can make the presentation clearer. 9. Reviewer: for the lower spreading of virus in the society, it is important to have high concentration of neutralizing antibodies in the majority of the subjects (so-called herd immunity) and this is predominantly achieved by vaccination, since infection leads to variable immune response. Reply: I do not in any way dispute the reviewer’s assertion of the importance of herd immunity, but my point in my article is that we need to understand what that immunity consists of in terms of the epitope-specificities of the antibodies that make up immunity in each individual. 10. Reviewer: Author has to conclude also the true clinical validity of the antibody examination according to the current knowledge. Reply: With respect, I am not addressing the current status of SARS-CoV-2 -specific antibody but have written a plea to the scientific community to make an all-out effort to analyse the epitope specific of such antibodies so that we can understand the nature of the ab response to infection and to vaccination. 11. Reviewer: it would be interesting to add some comparison between vaccination and natural infection. Reply: I absolutely agree that this comparison will be one of the most interesting to be made when it is possible to compare the constituent epitope specificities of the antibodies present in both cohorts.