Protective anti-prion antibodies in human immunoglobulin repertoires

  1. Assunta Senatore
  2. Karl Frontzek
  3. Marc Emmenegger
  4. Andra Chincisan
  5. Marco Losa
  6. Regina Reimann
  7. Geraldine Horny
  8. Jingjing Guo
  9. Sylvie Fels
  10. Silvia Sorce
  11. Caihong Zhu
  12. Nathalie George
  13. Stefan Ewert
  14. Thomas Pietzonka
  15. Simone Hornemann
  16. Adriano Aguzzi

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Abstract

Prion immunotherapy may hold great potential, but antibodies against certain PrP epitopes can be neurotoxic. Here we identified >6000 PrP-binding antibodies in a synthetic human Fab phage display library, 49 of which we characterized in detail. Antibodies directed against the flexible tail of PrP conferred neuroprotection against infectious prions. We then mined published repertoires of circulating B cells from healthy humans and found antibodies similar to the protective phage-derived antibodies. When expressed recombinantly, these antibodies exhibited anti-PrP reactivity. Furthermore, we surveyed 48’718 samples from 37’894 hospital patients for the presence of anti-PrP IgGs, and found 21 high-titer individuals. The clinical files of these individuals did not reveal any enrichment of specific pathologies, suggesting that anti-PrP autoimmunity is innocuous. The existence of protective anti-prion antibodies in unbiased human immunological repertoires, combined with the reported lack of such antibodies in carriers of disease-associated PRNP mutations, suggests a link to the low incidence of spontaneous prion diseases in human populations.

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  1. Review Commons

    Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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    Reply to the reviewers

    Dear dr. Monaco,

    We thank you and the reviewers for the positive and encouraging reviews on our manuscript entitled “Protective anti-prion antibodies in human immunoglobulin repertoires” and are glad to address the reviewer’s suggestions

    In the following you will find a point-by-point response to the referees' critique.

    __Reviewer #1 (Evidence, reproducibility and clarity (Required)): __

    __Comment 1: __Abtract: Although it is clear and direct, the last sentence where it refers to "a link to the low incidence of spontaneous prion diseases in human populations", is not easy to understand without a detailed explanation as given in the Discussion. I suggest a re-wording.

    __Response 1: __We have reworded the sentence in the abstract and given more explanation in the discussion (see also Reviewer 2, Comment 2).

    __Comment 2: __Results: It is clear how these Fabs act in preventing prion-induced neurotoxicity as shown in the COCS model. In addition to this effect, they also inhibit prion spreading, although this appears to be a lesser effect than inhibition of neurotoxicity. Thus, it would be interesting to discuss the possible effect of a Fab therapy, which provide a fully inhibition of the neurotoxicity but only partially inhibition of the prion propagation.

    • __Response 2: __As suggested by the reviewer, we have added appropriate text to the discussion to comment on the option of a potential Fab therapy with a fully inhibition of neurotoxicity and partially inhibition of prion propagation. __Comment 3: __The therapeutic effect of the Fabs in the cell model was performed by adding the Fabs to the medium 1 h after infection and during splitting. Is there any study that evaluates the effect of Fabs added to the medium before inoculation or at later times?

    __Response 3: __The goal of these experiments was to investigate whether the antibodies in question would counteract prion infections in principle, rather than performing a precise range-finding of the optimal therapeutic window. We have opted to not add the Fabs before inoculation, because past experience (and many papers) show that the “prophylactic” treatment rarely correlated with post-exposure efficacy. We also have not treated the cells after prion infection at later time points, because the data at later time points may be less pronounced and more variable. As for the treatment of cells with anti-PrP antibodies prior to exposure to prions, a study has been conducted in N2a cells (Pankievicz J et al., 2006, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1779824/). There, preincubation of N2a cells with mouse monoclonal anti-PrP antibodies (Mabs) before prion infection (22L) and preincubation of the inoculum with Mabs before infection of the cells led to a significant reduction in PrPSc levels as assessed by proteinase-K Western blot. This paper is now discussed in our manuscript.

    Comment 4: Discussion: The authors repeatedly refer to the toxicity that antibodies against GD might have. Related to this, there is currently a therapy (experimental medicine) in humans using an antibody against this region. Perhaps it would be interesting to make a comment on this.

    __Response 4: __Our findings (Sonati et al, Nature 2013, and several following papers) are fundamentally incompatible with those of the London lab on the toxicity of anti-GD antibodies, and elsewhere I have warned loudly against the use of such antibodies in humans. However, this discussion is peripheral to the findings presented here. We have added some text to the discussion but we would rather not expand on this specific issue.

    __Comment 5: __Page 15. I have found the speculative comment: "Accordingly, clinically silent prion generation may occasionally occur in healthy individuals. PrPSc aggregates arising de novo may result in exposure of neoepitopes and/or epitopes occluded in cell-borne PrPC." interesting. However, some of the auto-antibodies found in healthy humans are against a region believed to be structurally unaltered in PrPSc, which it doesn't fit with the theory of exposure to neo-epitopes.

    • __Response 5: __I still believe that my hypothesis is viable, but of course I concede that – thus far – I have no supporting data. We have therefore modified the text to alleviate this comment.

    __Reviewer #2: __

    __Comment 1: __This is a technically advanced and carefully executed study that clearly demonstrate the presence of natural autoantibodies to PrP, some of which show protective properties, in an unselected human population. Although this finding is interesting on its own right, its impact on issues such as incidence of sporadic prion diseases is unclear given that apparently only 0.06% of the nearly 38,000 subjects examined carried these antibodies "in high titer".

    Response 1: We agree with the reviewer and have modified the statement as follows: “The frequency of high-titer anti-PrP antibody carriers (0.06%) is much lower than the occurrence of Fab71-like HCDR3 sequences in published human repertoires. This discrepancy could mean that most anti-PrP specificities exist in a dormant state, or are expressed as B-cell receptors, but do not produce circulating antibodies. It will be interesting to discover the triggers that may ignite antibody production and, possibly, afford protection against prions”. The discrepancy between the frequency of anti-PrP antibodies found in the plasma screen and by analysis of the antibody repertoires in the NGS datasets could stem from the fact that most anti-PrP specificities exist in a dormant state, or are expressed as B-cell receptors, but do not produce circulating antibodies (Joseena Iype et al., J Immunol 2019; now also included in the manuscript).

    __Comment 2: __Furthermore, this reviewer could not locate the base of the pivotal statement made in the Abstract that these autoantibodies lack in carriers of disease-associated PRNP mutations. These two points need to be clarified.

    __Response 2: __The statement refers to the study by Frontzek et al. (citation #48: Frontzek, K.* et al.* Autoantibodies against the prion protein in individuals with PRNP mutations Neurologyhttps://n.neurology.org/content/early/2020/02/25/WNL.0000000000009183?rss=1). Although listed in the references, the citation got lost in the discussion. We have inserted the reference again.

    Comment 3: The manuscript suffers for the excessive amount of data that are crammed in the five figures. Combined these figures display a total of 33 panels some of which are quite complicated. The authors should be more selective and roll over some of the nonessential information i.e. that related to methodology, to the Supplement.

    • __Response 3: __We agree with the reviewer and have moved several panels to the Supplement.

    __Comment 4: __The use of acronyms is excessive and should be reduced (see for example COCS).

    • Response 4: We have attempted to reduce the number of acronyms. We have however introduced the term COCS in Falsig et al., Nature Neuroscience 2007, and have used it regularly in more than a dozen follow-up papers. Comment 5: The legends need to be carefully checked for clarity, especially figure 4

    • __Response 5: __We have revised the legends to improve their clairity.

    __Reviewer #3: __

    __Comment 1: __On page 10, the authors state that Fab71 (Figure 3e) and Fab100 (Extended data Figure 7) substantially lowered PrPSc levels in prion-infected cells. However, in both cases, only about half of the cultures tested showed less PrPSc than either the control samples or samples treated with other Fabs. This variability undercuts the conclusion that what they are observing is a substantial, reproducible effect. The authors should consider moderating their conclusion somewhat to better fit the data.

    __Response 1: __We agree with the reviewer. The effect of Fab71 and Fab100 in reducing PrPSc levels in cells as compared to control samples and samples treated with the other Fabs is only partially present and variable among the replicates, but still statistically significant (One-way ANOVA; p

    Comment 2: In figure 2, the legend to panel a does not match the figure. Fab3 and Fab71 are represented by the blue lines, not the red lines as stated in the legend.

    • __Response 2: __We thank the referee for pointing this out. We have now corrected it.

    __Comment 3: __In the legend to Extended data figure S4, please give the epitopes to Fab10 and Fab53.

    Response 3: We have included the epitopes of these two Fabs (OR51-91 for Fab10 and CC2-HC92-120 for Fab53) in the Figure legend.

    Comment 4: In Figure 3c, the lines indicating the significant groups are not well-aligned. In the left side of the panel, the lines should connect the dark gray control group squares with the Fab25 pink diamonds. Likewise, in the right side of the panel, the lower set of lines should connect the dark gray control group squares with the Fab83 dark blue triangles.

    • Response 4: We have corrected this issue.

    __Comment 5: __I agree with the comments of both reviewers. The suggestion of reviewer #2 to move methodology-related panels in the main figures to supplemental data would make it much easier for the reader to focus on the critical experimental data.

    • __Response 5: __See response to comment 2 of reviewer 2. With all issues addressed, we hope that our revised manuscript will now be found suitable for proceeding to the next steps.

    Best regards,

    Adriano Aguzzi

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    The manuscript by Senatore et al. is large scale study looking for natural human antibodies directed against prion protein (PrP). Using a synthetic human Fab phage display library, they found and characterized multiple human anti-PrP Fabs most of which recognized epitopes to a region of PrP from amino acid residues 92-120. Based on this information, they searched for and found low affinity, long-lived anti-PrP antibodies in both a repertoire of human antibodies and in 27 of almost 38,000 human clinical samples. They speculate that anti-PrP antibodies may help to protect against sporadic forms of prion disease and conclude that they may represent a source of potential immunotherapeutics against human prion infection.

    Minor comments:

    1. On page 10, the authors state that Fab71 (Figure 3e) and Fab100 (Extended data Figure 7) substantially lowered PrPSc levels in prion-infected cells. However, in both cases, only about half of the cultures tested showed less PrPSc than either the control samples or samples treated with other Fabs. This variability undercuts the conclusion that what they are observing is a substantial, reproducible effect. The authors should consider moderating their conclusion somewhat to better fit the data.

    2. In figure 2, the legend to panel a does not match the figure. Fab3 and Fab71 are represented by the blue lines, not the red lines as stated in the legend.

    3. In the legend to Extended data figure S4, please give the epitopes to Fab10 and Fab53.

    4. In Figure 3c, the lines indicating the significant groups are not well-aligned. In the left side of the panel, the lines should connect the dark gray control group squares with the Fab25 pink diamonds. Likewise, in the right side of the panel, the lower set of lines should connect the dark gray control group squares with the Fab83 dark blue triangles.

    Significance

    This is an extensive, well-written study which provides significant data suggesting that humans can make anti-PrP antibodies. This is a novel finding that raises important questions about how the body may respond to spontaneous formation of infectious prions. Technically, the study is sound with appropriately interpreted data. Overall the study and the antibodies it characterizes, some of which are novel, will be of interest to prion researchers.

    Referees cross commenting

    I agree with the comments of both reviewers. The suggestion of reviewer #2 to move methodology-related panels in the main figures to supplemental data would make it much easier for the reader to focus on the critical experimental data.

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    The authors extensively and rigorously characterized a subset of antibodies to PrP identified in a human Fab phage display library. These selected antibodies were compared and found to be similar to repertoires of naturally occurring human antibodies present in circulating B cells. Profiling of antibodies harvested from an unbiased 38,000 patient population uncovered the presence of high titer anti-PrP autoantibodies in 21 individuals sharing no specific pathologies. This finding demonstrates the presence of apparently innocuous immunity to prion in an unselected population. Based also on "the reported lack of such antibodies in carriers of disease-associated PRNP mutations" the authors propose that the low incidence of "spontaneous" prion diseases may be linked to the presence of these protective antibodies in the general population.

    Major comments:

    This is a technically advanced and carefully executed study that clearly demonstrate the presence of natural autoantibodies to PrP, some of which show protective properties, in an unselected human population. Although this finding is interesting on its own right, its impact on issues such as incidence of sporadic prion diseases is unclear given that apparently only 0.06% of the nearly 38,000 subjects examined carried these antibodies "in high titer". Furthermore, this reviewer could not locate the base of the pivotal statement made in the Abstract that these autoantibodies lack in carriers of disease-associated PRNP mutations. These two points need to be clarified. The manuscript suffers for the excessive amount of data that are crammed in the five figures. Combined these figures display a total of 33 panels some of which are quite complicated. The authors should be more selective and roll over some of the nonessential information i.e. that related to methodology, to the Supplement.

    Minor comments:

    The use of acronyms is excessive and should be reduced (see for example COCS). The legends need to be carefully checked for clarity, especially figure 4

    Significance

    Significance

    See above

    Referees Cross Commenting

    I agree with most of the comments by Reviewers 1 and 3. However, my queries remain.

  4. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    This is a very interesting article with important implications in the prion field. It is extremely well detailed and exquisitely well written. The objective of the article is very clear and the results obtained are not only interesting but also have very important implications for understanding prion diseases.

    I have some comments and a few minor concerns.

    Abtract:

    Although it is clear and direct, the last sentence where it refers to "a link to the low incidence of spontaneous prion diseases in human populations", is not easy to understand without a detailed explanation as given in the Discussion. I suggest a re-wording.

    Results:

    It is clear how these Fabs act in preventing prion-induced neurotoxicity as shown in the COCS model. In addition to this effect, they also inhibit prion spreading, although this appears to be a lesser effect than inhibition of neurotoxicity. Thus, it would be interesting to discuss the possible effect of a Fab therapy, which provide a fully inhibition of the neurotoxicity but only partially inhibition of the prion propagation.

    The therapeutic effect of the Fabs in the cell model was performed by adding the Fabs to the medium 1 h after infection and during splitting. Is there any study that evaluates the effect of Fabs added to the medium before inoculation or at later times?

    Discussion:

    The authors repeatedly refer to the toxicity that antibodies against GD might have. Related to this, there is currently a therapy (experimental medicine) in humans using an antibody against this region. Perhaps it would be interesting to make a comment on this.

    Page 15. I have found the speculative comment: "Accordingly, clinically silent prion generation may occasionally occur in healthy individuals. PrPSc aggregates arising de novo may result in exposure of neoepitopes and/or epitopes occluded in cell-borne PrPC." interesting. However, some of the auto-antibodies found in healthy humans are against a region believed to be structurally unaltered in PrPSc, which it doesn't fit with the theory of exposure to neo-epitopes.

    Significance

    The advance is highly significance for two reasons: 1) the tools that the authors have generated are really useful for the community and 2) The fact the healthy humans can generate anti-PrP antibodies is completely new and open new ways to understand the prion diseases mechanisms.

    The audience is principally for those working on prion and prion-like diseases.

    My expertise is in prion and prion-like diseases.

  5. Version published to 10.1101/2020.02.05.933721 on bioRxiv