Review Reports - Immunological Strategies for Enhancing Viral Neutralization and Protection in Antibody-Guided Vaccine Design

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review is about antibody guided vaccine design focussing on viral neutralization (title).

Major comments

I do not see a clear distinction between in vivo neutralization and in vitro neutralization of viruses by antibodies while both mechanisms vary considerably. In vivo neutralization also encompasses opsonophagocytosis, which is relevant for viral diseases as well. A major novel paper is (Liu et al., 2023), who exploit this mechanism by making bispecific recombinant antibodies against influenza and endogenous polyclonal antibodies (light chain), and in that manner indirectly recruit effector functions. I think the first publication about this indirect recruitment of opsonophagocytosis by bispecific antibodies was from (Holliger et al., 1997). The importance of opsonophagocytosis for protection against foot-and-mouth disease (FMD) is also known from the work of Ken McCullough, e.g. (McCullough et al., 1988). In more recent research (Li et al., 2024), where the mechanism of antibody mediated neutralization of FMD virus (FMDV) is elucidated, such opsonophagocytosis is mostly ignored. A thorough discussion on the relevance of opsonophagocytosis and other cellular immune mechanisms where antibodies mediate protection by being recognized through their Fc by immune cells (macrophages, killer T cells) would make the review more into depth. I think that in diseases where opsonophagocytosis is important for antibody mediated protection, the focus on sites that confer in vitro neutralization is not needed. In vitro neutralization often relies on mechanisms such as blocking of virus-receptor interaction and thus cell entry, or destabilization of viruses. It requires recognition of specific sites. However, opsonophagocytosis can occur with antibodies irrespective of the site recognized. It would be interesting to see if a monoclonal antibody that does not confer in vitro neutralization does confer in vivo neutralization. I did not read Liu et al 2023 well enough to check this. Lessons learned from such studies on opsonophagocytosis for vaccine design would then be that one should not focus on specific epitopes that are relevant for in vitro neutralization for vaccine design, but should focus on the overall integrity (3D structure!) of the viral antigen for making good vaccines. I think Li et al 2024 is a good example of showing this. This discussion could also be broadened with ‘antigenic competition’ (line 55). I personally think that antigenic competition (an immune response against a part of a pathogen that does not contribute to protection) is only occurring with parts of pathogens that are shedded or secreted, but not likely with proteins that are integral part of the virus. One may dispute this also by stating that especially the HA of influenza and spikes of coronavirus are immunogenic, which mutate rapidly and are therefore not ideal vaccine candidates.

Although the writing is essentially good, I find it generally has too many paragraphs. I think this fits with the lack of coherence in writing. It is just summarizing findings in one or two paper without placing it in a broader context.

At many places quite general statements are made about how antibodies work, which is simply textbook knowledge, which does not make the review interesting. Sometimes this is needed, but these parts should be kept brief.

I do not understand how antibody engineering can help vaccine design? It is mentioned in the abstract and at other places. Antibody engineering can perfectly help in improving antibody therapy, but what has it got to do with vaccine design? Antibodies can help identify sites of pathogens relevant for protection, but one does need to engineer the antibody for that.

Lines 312 to 327 describe the antigenic drift and shift of influenza HA and NA, but do not that influenza is a segmented virus, not that HA and NA are on different segments, not that there are 16 HA and 10 NA serotypes (or perhaps even more nowadays) and that reassortment of those segments causes novel serotype combinations. Therefore, the discussion is not into depth. Furthermore, the relevance for antibody guided vaccine design is absent.

I find that conclusions of papers are simply stated, without explaining to the reader what was done. Examples are the “goldilocks immunity” and “immuno wave” model (lines 104-105), the PME method (line 406).

Furthermore, the link with antibody guided vaccine design is missing at many places. Lines 103-109 (ref 22), Section 8.2 about personalized vaccines based on immunogenetics, Section 8.1 about next generation vaccine platforms, 7.2 computational modelling and machine learning. These paragraphs describe important novel research fields but do not have a link with vaccine DESIGN, or with antibody -guided vaccine design.

When describing the influenza (268-273) I find a lot of literature missing on this topic. Here, many years ago, when I followed influenza literature well, there was a great effort to develop monoclonal antibody therapy for influenza focussing in HA. Indeed it was found that there is a conserved site on the HA that has a particular conformation, which can be fixed by antibody binding and thereby prevent the infection of cells. This region is called the fusion peptide if I remember well. Since the globular head encodes the 16 serotypes (is highly variable) this fusion peptide was preferred for developing antibodies. There are many Nature and Science papers on this topic, which describe the mechanisms of antibody mediated protection and the parts of HA fusion peptide that are conserved. Initially researchers found two antibodies covering two clades of the HA molecules, that together could neutralize all 16 HA types. Later antibodies were found that could neutralize even all 16 HA’s. Later people also thought about designing improved vaccines that better induce antibodies against the stalk instead of the head. I did not follow this literature well anymore. I think a lot more interesting could be said here that is precisely about the topic of this review. Here antibody studies indeed identified the relevant epitope (fusionpeptide) that was later used in vaccine design. Here are some refs (not exhaustive and old, focussing on antibody part for epitope identification) (Beukenhorst et al., 2024; Ekiert et al., 2009; Ekiert et al., 2011; Friesen et al., 2010; Friesen et al., 2014)

A topic that is mentioned in the Table, but not thoroughly discussed in the review is identification of (T- and ) B-cell epitopes, for making vaccines. This is again fitting well with the topic of the review, and here much more can be said. There are now many studies where people have made concatemers of T and B-cell epitopes as vaccine candidates. One could discuss these in more detail. Often the problem is that only linear epitopes can be easily produced, but most epitopes are conformational, making these vaccines not very effective. See e.g. the Li et al 2024 paper on FMDV, which shows the importance of even small conformational changes already on FMDV vaccine efficacy.

Lines 302-310 are about a bacterial agent, while I thought the review was about viruses.

Minor comments

Line 47: HIV and RSV are abbreviations that should be defined

Line 79 says: Protection is primarily mediated by pathogen-specific antibodies produced 79 by plasma cells, a subset of terminally differentiated B lymphocytes. Here cellular immunity is completely ignored…..

Beukenhorst, A.L., Frallicciardi, J., Rice, K.L., Koldijk, M.H., Moreira de Mello, J.C., Klap, J.M., Hadjichrysanthou, C., Koch, C.M., da Costa, K.A.S., Temperton, N., de Jong, B.A., Vietsch, H., Ziere, B., Julg, B., Koudstaal, W., Goudsmit, J., 2024. A pan-influenza monoclonal antibody neutralizes H5 strains and prophylactically protects through intranasal administration. Scientific reports 14, 3818, https://doi.org/10.1038/s41598-024-53049-5.

Ekiert, D.C., Bhabha, G., Elsliger, M.A., Friesen, R.H., Jongeneelen, M., Throsby, M., Goudsmit, J., Wilson, I.A., 2009. Antibody recognition of a highly conserved influenza virus epitope. Science 324, 246-251, https://doi.org/10.1126/science.1171491.

Ekiert, D.C., Friesen, R.H., Bhabha, G., Kwaks, T., Jongeneelen, M., Yu, W., Ophorst, C., Cox, F., Korse, H.J., Brandenburg, B., Vogels, R., Brakenhoff, J.P., Kompier, R., Koldijk, M.H., Cornelissen, L.A., Poon, L.L., Peiris, M., Koudstaal, W., Wilson, I.A., Goudsmit, J., 2011. A highly conserved neutralizing epitope on group 2 influenza A viruses. Science 333, 843-850, https://doi.org/10.1126/science.1204839.

Friesen, R.H., Koudstaal, W., Koldijk, M.H., Weverling, G.J., Brakenhoff, J.P., Lenting, P.J., Stittelaar, K.J., Osterhaus, A.D., Kompier, R., Goudsmit, J., 2010. New class of monoclonal antibodies against severe influenza: prophylactic and therapeutic efficacy in ferrets. PLoS ONE 5, e9106, https://doi.org/10.1371/journal.pone.0009106.

Friesen, R.H., Lee, P.S., Stoop, E.J., Hoffman, R.M., Ekiert, D.C., Bhabha, G., Yu, W., Juraszek, J., Koudstaal, W., Jongeneelen, M., Korse, H.J., Ophorst, C., Brinkman-van der Linden, E.C., Throsby, M., Kwakkenbos, M.J., Bakker, A.Q., Beaumont, T., Spits, H., Kwaks, T., Vogels, R., Ward, A.B., Goudsmit, J., Wilson, I.A., 2014. A common solution to group 2 influenza virus neutralization. Proc. Natl. Acad. Sci. 111, 445-450, https://doi.org/10.1073/pnas.1319058110.

Holliger, P., Wing, M., Pound, J.D., Bohlen, H., Winter, G., 1997. Retargeting serum immunoglobulin with bispecific diabodies. Nature Biotechnology 15, 632-636, https://doi.org/10.1038/nbt0797-632.

Li, H., Liu, P., Dong, H., Dekker, A., Harmsen, M.M., Guo, H., Wang, X., Sun, S., 2024. Foot-and-mouth disease virus antigenic landscape and reduced immunogenicity elucidated in atomic detail. Nat Commun 15, 8774, https://doi.org/10.1038/s41467-024-53027-5.

Liu, X., Balligand, T., Carpenet, C., Ploegh, H.L., 2023. An armed anti-immunoglobulin light chain nanobody protects mice against influenza A and B infections. Sci Immunol 8, eadg9459, https://doi.org/10.1126/sciimmunol.adg9459.

McCullough, K.C., Parkinson, D., Crowther, J.R., 1988. Opsonization-enhanced phagocytosis of foot-and-mouth disease virus. Immunology 65, 187-191.

Author Response

This review is about antibody guided vaccine design focussing on viral neutralization (title).

Authors` reply: We sincerely appreciate the time and effort you have invested in reviewing our manuscript. Your insightful comments have been instrumental in enhancing the depth and coherence of our work.

Major comments

I find the review well written with good quality of English. However, it lacks an in depth analysis of modern literature on this topic and only addresses some already well known old stories. Furthermore, it lacks some coherence and focus. Assuming that the focus of the review is indeed on what the title suggests then I have the following criticism.I do not see a clear distinction between in vivo neutralization and in vitro neutralization of viruses by antibodies while both mechanisms vary considerably. In vivo neutralization also encompasses opsonophagocytosis, which is relevant for viral diseases as well. A major novel paper is (Liu et al., 2023), who exploit this mechanism by making bispecific recombinant antibodies against influenza and endogenous polyclonal antibodies (light chain), and in that manner indirectly recruit effector functions. I think the first publication about this indirect recruitment of opsonophagocytosis by bispecific antibodies was from (Holliger et al., 1997).

Authors' reply: We acknowledge the need for a clearer distinction between in vivo and in vitro neutralization mechanisms. In response, we have elaborated on the differences, emphasizing that in vivo neutralization encompasses processes such as opsonophagocytosis, where antibodies facilitate the engulfment and destruction of viruses by immune cells. We have incorporated discussions on recent advancements, including the work by Liu et al. (2023), which explores the use of bispecific recombinant antibodies against influenza to recruit effector functions indirectly. Additionally, we have referenced the foundational study by Holliger et al. (1997) on bispecific antibodies and their role in immune response modulation. These additions provide a comprehensive understanding of the multifaceted roles antibodies play in viral neutralization.

The importance of opsonophagocytosis for protection against foot-and-mouth disease (FMD) is also known from the work of Ken McCullough, e.g. (McCullough et al., 1988). In more recent research (Li et al., 2024), where the mechanism of antibody mediated neutralization of FMD virus (FMDV) is elucidated, such opsonophagocytosis is mostly ignored. A thorough discussion on the relevance of opsonophagocytosis and other cellular immune mechanisms where antibodies mediate protection by being recognized through their Fc by immune cells (macrophages, killer T cells) would make the review more into depth. I think that in diseases where opsonophagocytosis is important for antibody mediated protection, the focus on sites that confer in vitro neutralization is not needed. In vitro neutralization often relies on mechanisms such as blocking of virus-receptor interaction and thus cell entry, or destabilization of viruses. It requires recognition of specific sites. However, opsonophagocytosis can occur with antibodies irrespective of the site recognized. It would be interesting to see if a monoclonal antibody that does not confer in vitro neutralization does confer in vivo neutralization. I did not read Liu et al 2023 well enough to check this. Lessons learned from such studies on opsonophagocytosis for vaccine design would then be that one should not focus on specific epitopes that are relevant for in vitro neutralization for vaccine design, but should focus on the overall integrity (3D structure!) of the viral antigen for making good vaccines. I think Li et al 2024 is a good example of showing this.

Authors' reply: We appreciate your suggestion to delve deeper into the role of opsonophagocytosis and other antibody-mediated cellular immune mechanisms. Accordingly, we have expanded our discussion to include the significance of Fc-mediated interactions, where antibodies, through their Fc regions, engage with immune cells such as macrophages and killer T cells to mediate protection. We have also highlighted studies demonstrating the importance of these mechanisms in diseases like foot-and-mouth disease, referencing the work of McCullough et al. (1988). This comprehensive analysis underscores the importance of considering these pathways in vaccine design and therapeutic interventions.

This discussion could also be broadened with ‘antigenic competition’ (line 55). I personally think that antigenic competition (an immune response against a part of a pathogen that does not contribute to protection) is only occurring with parts of pathogens that are shedded or secreted, but not likely with proteins that are integral part of the virus. One may dispute this also by stating that especially the HA of influenza and spikes of coronavirus are immunogenic, which mutate rapidly and are therefore not ideal vaccine candidates.

Authors' reply: We have addressed the concept of antigenic competition, particularly in the context of vaccine design, we completely agree with the reviewer. Our discussion now includes a paragraph based on your valuable suggestions and recommendations.

Authors' reply: We have revised the manuscript to improve coherence and focus.

Authors' reply: We acknowledge that certain sections of our manuscript contained general statements, we have condensed these sections, ensuring they provide necessary background without overshadowing novel insights.

Authors' reply: We appreciate your critical observation regarding the role of antibody engineering in vaccine design. Upon reflection, we recognize that while antibody engineering is pivotal in therapeutic applications, its direct influence on vaccine design is less pronounced. Consequently, we have revised the manuscript and add a paragraph on clarifying this issue.

Authors' reply: We have expanded our discussion on influenza virus antigenic drift and shift, emphasizing the segmented nature of the influenza genome and the reassortment events leading to novel serotype combinations. We have detailed that hemagglutinin (HA) and neuraminidase (NA) are on separate gene segments, with 18 HA and 11 NA subtypes identified to date.

Authors' reply: We have provided detailed explanations of concepts such as "goldilocks immunity," "immuno wave" model, thank you for the critical point.

Authors' reply: We agree that some paragraphs of our review do not directly cover the topic, however, we believe that these passages are significant part of the topic. Therefore, we have strengthened the connection between the discussed topics and antibody-guided vaccine design throughout the manuscript. Sections on personalized vaccines, next-generation vaccine platforms, and computational modeling now explicitly address how these innovations contribute to the rational design of vaccines that elicit effective antibody responses.

Authors' reply: We are very thankful for the have enriched our discussion on influenza by incorporating seminal studies that identified conserved epitopes in the HA stem region, particularly the fusion peptide. These studies demonstrate how targeting conserved regions can lead to the development of broadly neutralizing antibodies, informing universal influenza vaccine design. References to key works, such as proposed by the referee, have been included to substantiate this discussion.

Authors' reply: We have introduced a comprehensive section on the identification of B-cell and T-cell epitopes for vaccine development. This includes discussions on the challenges of eliciting responses to conformational versus linear epitopes and the implications for vaccine efficacy. We have highlighted recent advancements in epitope mapping and vaccine design strategies that address these challenges.

Lines 302-310 are about a bacterial agent, while I thought the review was about viruses.

Authors' reply: We mentioned bacterial serotypes only to emphasize the importance of antigenic variation.

Minor comments

Line 47: HIV and RSV are abbreviations that should be defined

Authors' reply: We have defined abbreviations such as HIV (Human Immunodeficiency Virus) and RSV (Respiratory Syncytial Virus) upon their first occurrence to ensure clarity.

Authors' reply: We have revised the statement regarding antibody-mediated protection to acknowledge the integral role of cellular immunity, including T-cell responses, in conjunction with pathogen-specific antibodies produced by plasma cells. This provides a more balanced perspective on the immune mechanisms involved in protection.

We are confident that these revisions have addressed your valuable comments and have significantly strengthened our manuscript. Thank you once again for your critical insights and constructive feedback, and also for the valuable references suggested.

Reviewer 2 Report

Comments and Suggestions for Authors

Minor comments:

Lines 162-171: Authors mention that nanoparticle display is novel approach and that this method shows promising results in preclinical studies. The referenced papers are old (2010 and 2020, respectively). Novavax’s COVID-19 nanoparticle vaccines has been approved in humans. There are several other approved vaccines based on nanoparticle platform like VLPs (example see reference article below - https://doi.org/10.1038/s41541-021-00330-7 )

Line 237 : Typo – should be ‘Novavax’

Line 281-283: Please include appropriate reference

Table 1: ‘Advances and limitations in immunological strategies for antibody-guided vaccine design’. Please include reference for all strategies.

Line 139-140: “To achieve that, techniques such as..” This sentence seems incomplete. Please review and revise.

Table 1: Viral vector-based vaccines: The authors give examples of ‘Johnson & Johnson, Astrazeneca vaccines’. These are names of vaccine manufacturers. Please revise to reflect examples of vaccines name and targeted disease.

Major comments:

The title of section 4 says ‘advances in antibody engineering for vaccine design’. In my opinion, it should say ‘advances in antibody-guided vaccine design’. Discussion of antibody engineering is not within the scope of this review article.

Lines 126-127 ‘The field of vaccine development has advanced significantly with the integration of antibody engineering especially in designing vaccines that elicit specific immune responses to effectively neutralize viral threats’. The authors mention that integration of antibody engineering has significantly advanced vaccine development. Can authors elaborate on this ? Do they mean to say protein engineering (antigen) instead of antibody engineering?

In section 4, the authors lay out various antibody-guided vaccine design strategies including structure-based, epitope-focused, nanoparticle display, and scaffold-based. They begin by discussing rational antigen design based on structural insights into antigen and antibodies and provide example of pre-fusion stabilized spike protein in COVID 19 mRNA vaccine. Authors then discuss epitope-focused design to direct the immune response to certain preferred epitopes critical to pathogen neutralization with an example of influenza vaccine design. Following this, nanoparticle design for multimetric antigen display is described, followed by a parallel strategy to design vaccines to mimic neutralizing antibody epitopes to guide the immune system to direct the response to these epitopes (e.g., pre-fusion spike stabilization in mRNA COVID 19 vaccine). Finally, scaffold-based design is highlighted.

I suggest authors to divide this section into multiple sub-sections with each sub-section focusing on different design aspect with relevant specific supporting examples (including last 5 years) from the literature. In its current form, there is scope to improve the flow of this section as well as reference to more recently published research work. For example, the parallel strategy of mimicking neutralizing epitopes to steer immune response can be combined with structure-based design.

Lines 195-202: The authors mention scaffold-based design as a strategy to design effective vaccines by promoting epitope presentation to B-cells. In my opinion, this is not a very well-known strategy and is most advanced specifically for HIV. Therefore, suggest authors to, in a few lines, describe what this approach entails with specifics on how it has shown promise for HIV vaccine development efforts.

Line 22-24: In the abstract, the authors claim that they discuss antibody engineering and novel adjuvant strategies to improve vaccine efficacy. In my opinion, antibody engineering is not within the scope of this review. Can authors clarify this. Furthermore, there is a very brief mention of use of adjuvants to enhance immune response with specific example of Matrix M adjuvant in Novavax’s COVID 19 vaccine. No other novel adjuvant has been discussed. A more detailed discussion on novel adjuvants and their use as immunomodulators is warranted.

Table 1: The authors mention immune checkpoint inhibition as a strategy for antibody-guided vaccine design. No reference or additional context has been provided. Suggest authors to elaborate on this in the main text and include supporting evidence from literature. Same comment for some other strategies listed in this table.

In this review, the authors have described various antibody-guided vaccine design strategies and highlighted few successful advances in case of SARS-CoV-2, HIV and influenza vaccines. The authors have done a good job commenting on emerging techniques and future directions as well as challenges of antigen drift/ shift that hinder optimal vaccine design. Overall, minor revision as highlighted in my comments is needed. English is well-written but additional references are required as several places in the manuscript.

Author Response

We sincerely appreciate the time and effort you have invested in reviewing our manuscript. Your insightful and detailed comments have been instrumental in enhancing the depth, coherence, and focus of our work.

Minor comments:

Lines 162-171: Authors mention that nanoparticle display is novel approach and that this method shows promising results in preclinical studies. The referenced papers are old (2010 and 2020, respectively). Novavax’s COVID-19 nanoparticle vaccines has been approved in humans. There are several other approved vaccines based on nanoparticle platform like VLPs (example see reference article below - https://doi.org/10.1038/s41541-021-00330-7 )

Authors' reply: We acknowledge that nanoparticle vaccine platforms have advanced significantly since the references cited. We have updated the references and data in the manuscript to reflect these developments and included more recent references to provide a current perspective on nanoparticle-based vaccines.

Line 237 : Typo – should be ‘Novavax’

Authors' reply: We have corrected the typographical error on line 237 to 'Novavax'.

Line 281-283: Please include appropriate reference

Authors' reply: An appropriate reference has been added to lines 281-283 to substantiate the information presented.

Table 1: ‘Advances and limitations in immunological strategies for antibody-guided vaccine design’. Please include reference for all strategies.

Authors' reply: References have been included for all strategies listed in Table 1, ensuring proper attribution and allowing readers to consult the original sources for more detailed information.

Line 139-140: “To achieve that, techniques such as..” This sentence seems incomplete. Please review and revise.

Authors' reply: The sentence on lines 139-140 has been reviewed and revised for clarity and completeness.

Table 1: Viral vector-based vaccines: The authors give examples of ‘Johnson & Johnson, Astrazeneca vaccines’. These are names of vaccine manufacturers. Please revise to reflect examples of vaccines name and targeted disease.

Authors' reply: In Table 1, we have revised the examples of viral vector-based vaccines to specify the vaccine names and the diseases they target, providing clearer and more precise information.

Major comments:

The title of section 4 says ‘advances in antibody engineering for vaccine design’. In my opinion, it should say ‘advances in antibody-guided vaccine design’. Discussion of antibody engineering is not within the scope of this review article.

Authors' reply: We agree with your observation and have revised the title of Section 4 to 'Advances in Antibody-Guided Vaccine Design' to accurately reflect the content discussed.

Lines 126-127 ‘The field of vaccine development has advanced significantly with the integration of antibody engineering especially in designing vaccines that elicit specific immune responses to effectively neutralize viral threats’. The authors mention that integration of antibody engineering has significantly advanced vaccine development. Can authors elaborate on this ? Do they mean to say protein engineering (antigen) instead of antibody engineering?

Authors' reply: We recognize the ambiguity in our original statement. Our intention was to highlight how structural insights into antigen-antibody interactions have informed vaccine design. We have revised the manuscript to clarify that structural biology and protein engineering, rather than antibody engineering per se, have significantly advanced vaccine development by identifying key epitopes and informing the design of immunogens that elicit desired immune responses.

In section 4, the authors lay out various antibody-guided vaccine design strategies including structure-based, epitope-focused, nanoparticle display, and scaffold-based. They begin by discussing rational antigen design based on structural insights into antigen and antibodies and provide example of pre-fusion stabilized spike protein in COVID 19 mRNA vaccine. Authors then discuss epitope-focused design to direct the immune response to certain preferred epitopes critical to pathogen neutralization with an example of influenza vaccine design. Following this, nanoparticle design for multimetric antigen display is described, followed by a parallel strategy to design vaccines to mimic neutralizing antibody epitopes to guide the immune system to direct the response to these epitopes (e.g., pre-fusion spike stabilization in mRNA COVID 19 vaccine). Finally, scaffold-based design is highlighted.

Authors' reply: We have restructured Section 4 into multiple sub-sections, each focusing on a distinct design strategy, such as structure-based design, epitope-focused design, nanoparticle display, and scaffold-based design. Each sub-section now includes recent examples from the literature, particularly from the last five years, to provide up-to-date and relevant information. This reorganization enhances the flow and coherence of the section.

Lines 195-202: The authors mention scaffold-based design as a strategy to design effective vaccines by promoting epitope presentation to B-cells. In my opinion, this is not a very well-known strategy and is most advanced specifically for HIV. Therefore, suggest authors to, in a few lines, describe what this approach entails with specifics on how it has shown promise for HIV vaccine development efforts.

Authors' reply: We have expanded the discussion on scaffold-based design to provide a more detailed explanation of this approach, including its application in HIV vaccine development. We have included specific examples and references to illustrate how scaffold-based design promotes epitope presentation to B-cells and its potential advantages in eliciting robust immune responses.

Line 22-24: In the abstract, the authors claim that they discuss antibody engineering and novel adjuvant strategies to improve vaccine efficacy. In my opinion, antibody engineering is not within the scope of this review. Can authors clarify this. Furthermore, there is a very brief mention of use of adjuvants to enhance immune response with specific example of Matrix M adjuvant in Novavax’s COVID 19 vaccine. No other novel adjuvant has been discussed. A more detailed discussion on novel adjuvants and their use as immunomodulators is warranted.

Authors' reply: We acknowledge that the discussion on antibody engineering was beyond the intended scope of this review. We have revised the manuscript and added a few paragraphs on this topic, not to delve in the details but to mention. Regarding novel adjuvants, we have expanded the manuscript to include a more detailed discussion on recent advancements in adjuvant development, and their roles as immunomodulators in enhancing vaccine efficacy.

Table 1: The authors mention immune checkpoint inhibition as a strategy for antibody-guided vaccine design. No reference or additional context has been provided. Suggest authors to elaborate on this in the main text and include supporting evidence from literature. Same comment for some other strategies listed in this table.

Authors' reply: We have elaborated on the strategy of immune checkpoint inhibition listed in Table 1, providing context and supporting evidence from the literature..

We sincerely appreciate your positive feedback on our manuscript, we have conducted a thorough review of recent literature and incorporated pertinent citations throughout the manuscript. These additions aim to provide a more comprehensive and up-to-date perspective on the topics discussed. We appreciate your positive feedback regarding the quality of English in our manuscript. To further enhance clarity and readability, we have meticulously reviewed the text and made necessary improvements.

Reviewer 3 Report

Comments and Suggestions for Authors

The title of the paper suggests that it will describe how neutralising or other antibodies have been used to develop better vaccines. The article mostly presents a narrative on vaccines in general and does not address the subject of the title in an analytical way or accurately present the conclusions of others. It does provide some references of interest.

The focus on covid points out that the vaccines work by presenting the spike protein which contains the receptor binding domain. This could be broadly considered to antibody guided but no mention is made of the neutralising antibodies that bind to the N-terminal domain of the spike protein and how they might work or be useful.

The broadly neutralising antibodies for HIV are of considerable interest especially since they can mediate protection in primate models. They are not well discussed. Some consideration could be given to their further development especially when there are several well defined epitopes. The investigators found that it was important to understand the ontogeny of the development of the antibodies (that occur late in infection) and that B-cell clones that did not produce HIV-binding antibody had to be first expanded and further stimulated for favourable mutations to develop. This is a very important concept.

Author Response

We appreciate your thorough review and insightful comments, which have significantly contributed to enhancing the depth and focus of our manuscript. We acknowledge your observation regarding the alignment between the title and the manuscript's content. To address this, we have revised the manuscript to provide a more analytical discussion on how neutralizing and other antibodies have been utilized in the development of improved vaccines.

We appreciate your suggestion to include information on neutralizing antibodies targeting the NTD of the SARS-CoV-2 spike protein. In response, we have expanded the relevant section to discuss the role of NTD-specific neutralizing antibodies. Notably, studies have identified potent neutralizing antibodies that bind to the NTD, contributing to viral neutralization and offering potential avenues for vaccine design

We concur with your assessment of the significance of broadly neutralizing antibodies in HIV research. To enhance our discussion, we have included an in-depth commenting of bNAbs, highlighting their ability to neutralize diverse HIV strains and their protective efficacy in primate models. Furthermore, understanding the ontogeny of bNAbs has revealed that their development involves the expansion and maturation of specific B-cell clones, a process that occurs later in infection.
We are confident that these revisions address your concerns and enhance the manuscript's relevance and analytical depth. Thank you once again for your valuable feedback.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors did a good job to improve the manuscript based on my suggestions. I can see they worked hard and tried to improve the manuscript accordingly. However, I find the paper still needs improvements. Therefore I give another review report with detailed suggestions to improve the paper. Please be ambitious and motivate yourself for writing a well-cited review! I disliked that the authors did not use track changes modus in the revised version. Although the yellow highlighting helped, I prefer track changes. This helps me to look the next time for what is changed and limits my time spent.

Line 62-63 says: The mechanisms of virus neutralization are not fully understood and are much debated. The reason is that it is possible to inhibit events after virus entry.

The first sentence is quite general and ‘empty’, obvious. But the link with ‘the reason’ really surprises me. There are many things known about virus neutralization and inhibition of events (infection?) after virus entry (into cells?) is not the most important, widely occurring issue. It is possible that this occurs, but then please give a good description of the process and a good reference. Furthermore, I dislike the link between these sentences. I would not mind deleting these sentences.

Line 64-66 says: Sometimes neutralization occurs by Fab fragments (fragment antigen-binding region) almost as well as by whole antibody molecules, indicating that the viral envelope or the spikes of the envelope are not always required.

I do not understand how equal neutralization by Fab and whole antibody indicate that the viral envelope or the spikes of the envelope are not always needed. I think this is nonsense. I think that equal neutralization by Fab and IgG indicate that the Fc is not needed for in vivo (!) neutralization, and thus indicate that effector mechanisms like ADCC (antibody dependent cellular cytotoxicity) and ADCP (Antibody dependent cellular phagocytosis) are not relevant in vivo. I furthermore would like to note that the reference 19 (Pinto et al) given for the above statement did not compare Fab and IgG in vivo neutralization and furthermore have shown that ADCC and ADCP are important for in vivo neutralization of SARS-COV by mAbs. This flaws the above statement. I did not check the other 2 references given. Please check refs and rephrase the last part of the sentence into something about role of Fc encoded effector functions. I would have liked to see an explanation earlier in the introduction stating that Fc part of IgG is important for recruiting many effector functions such as ADCC and ADCP by binding to cellular receptors.

Line 69 says: describe the use of bispecific antibody fragments (diabodies) to….

Here it is not described where these bispecific antibody fragments bind to (model antigen that could be viral antigen and host (mouse) immunoglobulin) and that the diabodies themselves lack Fc and thus do not recruit effector functions. However, the diabodies crosslink antigen to host Fc and thereby recruit effector functions. This is essential to know for readers to understand why this is relevant. Please rephrase. Also move this part later to lines 273-275.

Lines 78-81 says: In 2024, Li et al. ignored opsonophagocytosis and elucidated the mechanism of antibody-mediated neutralization of FMD virus [27]. Their study shows how surface proteins and immunogenic characteristics will help and guide the structure-based design of new broad-spectrum and stable vaccines for protection against FMD.

Please rephrase into:

In 2024, Li et al. demonstrated that the correct 3D conformation of the viral surface proteins in vaccines is crucial for antibody-mediated neutralization of FMD virus [27]. Their study will help and guide the structure-based design of new broad-spectrum and stable vaccines for protection against FMD. I also prefer that this text is moved to later in the review where this is (again) discussed: lines 348-354. See also my comment there.

Line 87-88 says:

The human immunodeficiency virus (HIV) is even more variable, and there is still no vaccine that induces an immune response [32].

This is wrongly phrased since most vaccines against HIV induce an immune response, but this response is apparently insufficient for protection. Please rephrase.

Lines 102 to 114 are well written!

Line 138: please replace ‘primarily’ by ‘often’ since cellular immunity sometimes is more important than humoral immunity. This is the case for example for PRRSV.

Line 142: rephrase “eliminate infected cells and helper T cells that support B-cell activation and antibody production.” Into “eliminate infected cells. Furthermore, helper T cells support B-cell activation and antibody production. This does not classify helper T-cells as cellular immunity that is not part of humoral immunity. I would consider Helper T cells part of humoral immunity.

Line 142-143: please rephrase ‘This coordinated interplay’ into ‘A coordinated interplay’.

Line 182: should ‘different reactions’ not be ‘aspecific reactions’?

Line 217: replace ‘antibodies’ by ‘epitopes’ since the structure of the antibodies is not relevant, but the structure of the epitopes recognized, and especially neutralizing antibody epitopes, is relevant.

Line 242: replace ‘we agree that’ by ‘since’ and in line 243 replace ‘and we have clarified’ by ‘we have only clarified’. It appeared that you were talking to a reviewer by stating ‘we agree that’…….

Lines 273-275 says: Therefore, as initial studies revealed a couple antibodies that could neutralize all 16 HA serotypes, the subsequent advancements identified antibodies capable of neutralizing all 16 types [72].

Here the point is missed that this paper describes the use of bispecific recombinant antibodies against host IgG to retarget antigens (influenza) to host antibodies containing an Fc domain for obtaining opsonophagocytosis (cellular immunity). Please rephrase this sentence to make this point. I would like to see the Holliger et al story in lines 68-71 also moved to this place, since it’s the same approach.

Lines 294-298 discuss antigenic drift and shift of influenza, but this is rather late, since influenza is already discussed in 4.1 above. I prefer moving this part up, after ‘For the influenza virus e.g. HA and neuraminidase (NA) are promising targets for protective activity [65,66].’ in line 255.

Lines 353-354 says: ‘This aspect of vaccine development aligns well with the broader themes of the review and warrants further discussion [27].’ I prefer to remove this sentence and move reference [27] to the sentence ending in line 350. I also prefer that the text at lines 78-81 is moved to this place and properly merged. It is now a duplication.

Line 396-397 says ‘They showed that wеrе polyfunctional antibodies induction and a reduced risk of acquiring infection.’ This sentence is grammatically not good. I think the word ‘were’ should be out, but also ‘antibodies induction’ is not good. Please check and rewrite.

Section 5.2 (lines 447 – 481) is about HIV and influenza again and contains a lot of repetition of what is already said earlier in section 4. I prefer that this section 5.2 is deleted as a special section and that the information contained is merged at several appropriate places in section 4. I find the text in lines 482-487 not informative and propose to delete it. Then section 5.1 is the only section in section 5. I propose to delete section 5 as a separate section and place section 5.1 under section 4, as section 4.5.

Lines 547 to 555 discuss ADE. I prefer that the authors specifically mention that ADE is only described with specific viruses. It is well described for dengue virus, but also appears to occur with SARS-COV. However, for many other viruses it does not occur.

Line 564: remove ‘antibody-guided’ since checkpoint inhibition has nothing to do with antibody guided vaccine design. In general I think that the review is not sufficiently focussing on the antibody-guided part of vaccine design, but talking about a lot of other aspects of vaccination strategies. These aspects are interesting, but do not fall under what the title says. I therefore propose to change the title instead into:

Immunological Strategies in Enhancing Viral Neutralization and Protection and Antibody-Guided Vaccine Design

Antibody-Guided Vaccine Design and further Immunological Strategies to enhance Viral Neutralization and Protection

In this manner it is clear that other aspects of vaccine design are mentioned, but that antibody-guided vaccine design is an important aspect.

I give the following additional papers focusing on antibody guided vaccine design for FMD by identifying broadly neutralizing antibodies and using these to find epitopes that are conserved across multiple FMD strains within a serotypes and thus useful for vaccine design:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8610593/pdf/jvi.01308-21.pdf

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8513477/pdf/jvi.00881-21.pdf

These could be added to parts where FMD is described as another example of antibody-guided vaccine design.

Further interesting findings related to antibody-guiding are the following (I tried to phrase it ready for copy-paste…). Antibody-guiding also helped improve vaccine design by analysing the effect of oil emulgation on conformation and thus antigenicity and immunogenicity of protein antigens. It appears that oil-emulsification can have a denaturing effect that drastically affects antigenicity and immunogenicity. This was shown for the model protein beta-lactamase (http://www.ncbi.nlm.nih.gov/pubmed/16754878) and FMD (http://www.ncbi.nlm.nih.gov/pubmed/25843267). In the latter work methods to improve vaccine formulation, by adding sucrose and BSA as excipients, were also developed using antibody-based assays to evaluate antigen integrity. This can be considered antibody-guided vaccine formulation design. I think these papers also fit nicely with the main topic (from its title) of this review. Since one of the papers is about FMD (again) they could perhaps be added somewhere with other FMD papers.

I like section 7.2! This section is really about the focus point of the paper (antibody-guided….), but from a different viewpoint.

In section 8.1 I propose to add more specifically that the current trend to develop recombinant vaccines using various platforms rather than inactivated authentic viruses offers much opportunity for antibody-guided vaccine design.

Line 674: I dislike ‘antibody engineering’ since this is modification of antibodies. I think this is not meant here. Perhaps rephrase to ‘antigenicity analysis’

Line 696: the same applies here as in 674. Note that I already earlier criticized ‘antibody engineering’ in my first review. The authors replied that they corrected this, but this correction was not thorough.

Lines 690 to 698: I think the review is not about antibodies to be used for passive vaccination, but antibodies to be used for design of antigens for active immunization.

Therefore in line 693-694 change ‘By improving the specificity and potency of antibodies, these vaccines can specifically target and neutralize pathogens’ into ‘Antibody-guiding can improve vaccine design for increased specificity and potency of antibodies that neutralize pathogens’

Minor comments:

Why are not all authors given for refs 19, 21, 27, 35, 48, 49, 84, 88, 92, 93, 95 and many more thereafter (find et al in refs)?

Line 169: remove ‘etc.’ (it already says e.g. earlier….)

Line 170: again remove ‘etc.’ (it already says e.g. earlier….), and make Cholera, Influenza, Rotavirus lowercase.

Line 193: remove ‘for’ (before ‘diabetes type 1’).

Line 228: remove ‘etc.’, and do this throughout the document. Its an emptly word that can be added to any list of items. It is not an informative word. Either extend the list with more items or keep it short if it is clear. Adding e.g. in front of the list makes more sense to me.

Line 383: remove the word ‘of’ at two places.

Line 597: please remove ‘is’.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

Improvements made

Author Response

Thank you very much for the valuable help in improving the manuscript.