PPATHOGENS-D-26-00578

Predictive value of PfEMP1 antibody profiles for the course of controlled human malaria infections

PLOS Pathogens

Dear Dr. Bachmann,

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Academic Editor

PLOS Pathogens

Tracey Lamb

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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1) Please ensure that the CRediT author contributions listed for every co-author are completed accurately and in full.

At this stage, the following Authors/Authors require contributions: Yannick D. Höppner, Ralf Krumkamp, Hikaru Nagaoka, Charlotte Wapler, Hannah Honner, Louise Turner, Jean Claude Dejon-Agobé, Yabo J. Honkpehedji, Jeannot Fréjus Zinsou, Jana Held, Meral Esen, Heidrun von Thien, Iris Bruchhaus, B. Kim Lee Sim, Stephen L. Hoffman, Peter G. Kremsner, Tim-Wolf Gilberger, Bertrand Lell, Takafumi Tsuboi, Thomas Lavstsen, Eizo Takashima, Benjamin Mordmüller, and Anna Bachmann. Please ensure that the full contributions of each author are acknowledged in the "Add/Edit/Remove Authors" section of our submission form.

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Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: Dr Bachmann and colleagues have performed an in-depth study of antibody responses to PfEMP1 in 20 Gabonese and 5 malaria-naïve adults who participated in a CHMI (controlled human malaria infection) study. The authors have previous published a study (Bachmann et al PLos Pathogens 2019) which uses the same samples to perform a much less detailed serological study, showing that individuals who controlled or cleared infections had higher levels of antibody to a range of PfEMP1 domains (and to some merozoite and other antigens) than those who were malaria naïve and those who failed to control their infections. The present study extends this by (1) including a 3D7 specific multiplex array (2) measuring antibody against almost all 3D7/NF54 PfEMP1 domains by alpha screen and (3) assessing antibody to IEs using two parasite lines with fixed PfEMP1 expression and a control with no surface PfEMP1. This is a very comprehensive assessment of the PfEMP1 antibody response.

The overall conclusions from the current study are broadly similar to the first study, but they extend this to show more clearly the importance of antiPfEMP1 antibody as a probable determinant of the types of PfEMP1 expression in parasites that emerge after CHMI challenge.

Reviewer #2: Study summary

This study investigated the role of antibodies against PfEMP1, either expressed on the surface of infected erythrocytes or represented by recombinant extracellular domains, in regulating in vivo parasite growth and var gene expression within a controlled human malaria infection (CHMI) model. The cohort comprised five malaria-naïve European adults and twenty malaria-exposed Gabonese adults.

All participants were infected with Plasmodium falciparum sporozoites via direct venous inoculation. Based on the prepatent period, participants were stratified into four groups:

• Naïve (European volunteers)

• Non-controllers (prepatent period 14–18 days)

• Controllers (17–25 days)

• Clearers (remained blood-smear negative up to day 28)

For downstream analyses, naïve individuals and non-controllers were grouped as the high-susceptibility group, whereas controllers and clearers were classified as the low-susceptibility group.

Main findings

• The low-susceptibility group had significantly higher levels of pre-existing, naturally acquired antibodies to:

1. Native PfEMP1 expressed on the surface of infected erythrocytes

2. Recombinant proteins representing specific extracellular domains of PfEMP1

• Individuals with antibodies recognizing a broader repertoire of PfEMP1 variants, including group B PfEMP1, demonstrated improved control of parasite growth.

• Parasites from controllers exhibited a more restricted var gene expression profile compared to those from non-controllers and the var diversity was negatively correlated with the breadth of pre-existing naturally acquired antibodies to PfEMP1 variants.

• General assessment

The manuscript is well written, and the data are clearly presented. The findings are largely consistent with prior studies, reinforcing existing knowledge. However, the study applies a more exhaustive experimental approach (e.g. AlphaScreen) and novel analytical frameworks, which add value.

Overall comments

• The concept of naturally acquired immunity should be more explicitly emphasized, as the baseline antibody measurements reflect immune exposure acquired through natural infection and are central to interpreting CHMI outcomes

The AlphaScreen assay is introduced as though it is widely familiar. A brief definition and explanation would improve accessibility for readers.

Specific comments:

Abstract

• The term “AlphaScreen data” is not intuitive and may be difficult for readers to interpret. Consider rephrasing including paraphrasing to capture the assay

• Lines 46–48: “All assays showed strong predictive value…”

This statement is vague. It would be more informative to explicitly state whether recognition of homologous or heterologous PfEMP1 domains showed stronger predictive performance.

Introduction

• Lines 92–94: The concept of mutually exclusive var gene expression has been challenged by recent work (PMID: 40379932). This should be incorporated to reflect current understanding.

• Lines 133–136: “In contrast uncomplicated malaria...”. The is not unclear. uncomplicated malaria is associated with less diverse var expression, but this statement seems to suggest otherwise. Clarify the intended distinction.

Results

• Line 189: Define TBS (thick blood smear) at first mention.

• Line 276: Clarify that baseline antibody levels represent “naturally acquired immunity”.

Figures

• Figure 1e–f: Specify the statistical test used to calculate p-values. Given the time-to-event nature of the data, Cox regression would be appropriate, but it is not clear whether this was applied.

• Figure 2: Panels d and e are difficult to interpret. In the key, replace “low” and “high” with “low-susceptibility” and “high-susceptibility” for clarity. Or make it more explicit in the figure legend as done in Figure 3 legend.

Reviewer #3: This is a novel and conceptually strong manuscript suggesting that pre-existing immunity may influence early parasite antigenic expression, also is highly interesting and well-designed study addressing an important question in malaria immunity. The integration of clinical phenotyping, serology, and parasite gene expression is a major strength. However, the manuscript currently: overinterprets correlative data as mechanistic, relies on small sample sizes in critical analyses, lacks validation for predictive models

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: I do not recommend additional experiments

Reviewer #2: none

Reviewer #3: The main concern with the conclusions is the overinterpret correlative findings as evidence of protection and mechanism. While the data show an association between broader PfEMP1 specific antibody responses and improved control of parasitemia, there is no direct functional evidence demonstrating that these antibodies mediate parasite clearance, making the use of terms like “protection” and “antiparasite immunity” too strong. In addition, the conclusion emphasizes antibody breadth, but the results also point to the importance of specific domains.

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: MAJOR COMMENTS:

1. Whilst it is clear that the presence of higher levels of PfEMP1 antibody correlate with lower breadth of PfEMP1 expression (and one can infer a causative link here), it is not as clear that the PfEMP1 antibodies are what drives the outcomes of challenge in the study participants. Certainly increasing breadth and strength of PfEMP1 response appears to correlate with outcomes, but isn’t it equally possible that the relationship between PfEMP1 antibody and outcome could be mediated through greater immunity to merozoite antigens, limiting parasite replication? The authors imply causation, and they are possible correct (e.g. lines 281-3), but they need to be open to other explanations.

2. In Figure 4 the authors refer to their profiling as “systems serology”. This is a misuse of the term, which refers to functional and/or biophysical profiling of antibody responses, of the sort the authors refer to at the end of the discussion.

3. In terms of relative importance of each PfEMP1 group to immunity, it seems from the Random Forest that of the seven most important features, their break down by group (A, B,C) is not unexpected or favouring any one group- two A three B and two C; B are about half the 3D7 PfEMP1s. Most of the 83 differently detected proteins (Figure 5e) are making pretty tiny contributions to the accuracy of classification.

4. In the text (lines 267-269) the authors state that representation differs from what would be expected by chance. It is not clear- is this “0” if all domains are equally represented? Some aid to interpretation is needed for the reader.

5. Given the relatively small sample size (24 individuals, after censoring) the robustness of the analysis needs to be raised as a potential shortcoming.

Minor comments

1. My understanding and my experience is that the IT4VAR19 parasite line binds EPCR but NOT ICAM-1, it has a CIDRalpha 1.1 domain and is a DC8 type variant.

2. Line 255-257: I do not understand this terminology e.g. 98% DBLalpha 0/1/2, how is this lower than the 89% CIDRalpha?

3. In Figure 3 e and f and Figure 4 it is not clear what the numbers in brackets ref to e.g. (n=1) or (n=14/56)

Reviewer #2: Since var expression is dependent on antibody levels, I suggest antibody levels be on the x-axis and var expression on the y-axis.

Reviewer #3: The manuscript is generally well presented, but several minor issues could improve clarity. The definition of serorecognition based on a naïve mean + 2 SD cutoff is somewhat arbitrary and may influence breadth estimates; a sensitivity analysis or presentation of continuous data would strengthen confidence in the results.

Introduction: Line 155-157. It is more appropriate to suggest that this pattern may reflect early recognition of parasites expressing B-type var genes upon emergence from the liver. This interpretation aligns with the broader concept of PfEMP1 diversity, where parasites expressing novel variants can evade pre-existing immunity, potentially delaying parasitemia.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Dear Dr. Bachmann,

We are pleased to inform you that your manuscript 'Predictive value of PfEMP1 antibody profiles for the course of controlled human malaria infections' has been provisionally accepted for publication in PLOS Pathogens.

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Best regards,

Marcelo U. Ferreira, MD, PhD

Academic Editor

PLOS Pathogens

Tracey Lamb

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The authors have revised their manuscript and taken all my suggestions into account.

Reviewer #2: as provided in my initial review report

Reviewer #3: This is a well-conducted and informative study that provides novel insights into PfEMP1-specific antibody responses and parasite control during controlled human malaria infection. The manuscript is well written, the analyses are appropriate, and the conclusions are generally supported by the data.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: No experiments required

Reviewer #2: all previous concerns have been adequately addressed

Reviewer #3: Thank you for addressing my previous concern regarding the interpretation of the antibody findings. The revised text now more appropriately frames the results as associations rather than direct evidence of protective mechanisms. I also appreciate the inclusion of specific PfEMP1 domains alongside the discussion of antibody breadth, which provides a more balanced interpretation of the findings. I have no further major concerns regarding this point.

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: no further issues

Reviewer #2: none

Reviewer #3: I would mark my previous comments as resolved.

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If you choose “no”, your identity will remain anonymous but your review may still be made public.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No