Antibodies to repeat-containing antigens in Plasmodium falciparum are exposure-dependent and short-lived in children in natural malaria infections

  1. Madhura Raghavan
  2. Katrina L Kalantar
  3. Elias Duarte
  4. Noam Teyssier
  5. Saki Takahashi
  6. Andrew F Kung
  7. Jayant V Rajan
  8. John Rek
  9. Kevin KA Tetteh
  10. Chris Drakeley
  11. Isaac Ssewanyana
  12. Isabel Rodriguez-Barraquer
  13. Bryan Greenhouse
  14. Joseph L DeRisi

  • Curated by eLife

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    This study describes the use of a new and valuable tool, namely phage display of Plasmodium falciparum proteome-wide peptides, for profiling of antibody targets. The study, conducted using plasma from Ugandan children and adults, represents an important aspect of naturally acquired antibodies with seroreactive responses to the intra-and inter-protein repeat regions. The results are, however, so far incomplete, and confirmatory data that antibodies to inter-protein repeat motifs do cross-react are needed.

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Abstract

Protection against Plasmodium falciparum , which is primarily antibody-mediated, requires recurrent exposure to develop. The study of both naturally acquired limited immunity and vaccine induced protection against malaria remains critical for ongoing eradication efforts. Towards this goal, we deployed a customized P. falciparum PhIP-seq T7 phage display library containing 238,068 tiled 62-amino acid peptides, covering all known coding regions, including antigenic variants, to systematically profile antibody targets in 198 Ugandan children and adults from high and moderate transmission settings. Repeat elements – short amino acid sequences repeated within a protein – were significantly enriched in antibody targets. While breadth of responses to repeat-containing peptides was twofold higher in children living in the high versus moderate exposure setting, no such differences were observed for peptides without repeats, suggesting that antibody responses to repeat-containing regions may be more exposure dependent and/or less durable in children than responses to regions without repeats. Additionally, short motifs associated with seroreactivity were extensively shared among hundreds of antigens, potentially representing cross-reactive epitopes. PfEMP1 shared motifs with the greatest number of other antigens, partly driven by the diversity of PfEMP1 sequences. These data suggest that the large number of repeat elements and potential cross-reactive epitopes found within antigenic regions of P. falciparum could contribute to the inefficient nature of malaria immunity.

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  1. Version published to 10.7554/elife.81401 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    DeRisi and colleagues used a new phage-display peptide platform, with 238,068 tiled 62-amino acid peptides covering all known P falciparum coding regions (and numerous other entities), to survey seroreactivity in 198 Ugandan children and adults from two cohorts. They find that the breadth of responses to repeat-containing peptides was twofold higher in children living in the high versus moderate exposure setting, while no such differences were observed for peptides without repeats. Additionally, short motifs associated with seroreactivity were extensively shared among hundreds of antigens, with much of this driven by motifs shared with PfEMP1 antigens.

    Malaria immunity is complex, and this new platform is a potentially valuable addition to the toolkit for understanding humoral responses. The two cohorts differed in fundamental ways: 1) high versus moderate exposure to infective bites; 2) samples drawn at the time of malaria for most donors in the high zone versus ~100 days after the last malaria episode in the moderate zone. The effect of acute malaria to boost short-term cross-reactive antibodies can confound the ability to draw inferences when comparing the two cohorts, and this should be further explored to understand its role in the patterns of seroreactivity observed.

    We thank the reviewer for this very insightful comment. In endemic areas, this potential confounder is a natural occurrence – in areas of higher transmission, people will on average be more likely to have an active or recent infection. The question is whether the differences seen in repeat-containing peptides are due to cumulative exposure or recency/active exposure. To address this point, we have added new analyses, as suggested, taking into account infection status in both exposure settings. In the moderate exposure setting, we find that the breadth of response in children to repeat containing peptides significantly narrows between the most recently exposed subjects, and those that have been infection free for >240 days, indicative of a short-lived response. This difference was not observed for peptides without repeats. (New figure: Figure 5, Supplement 4). We also observe an increase in breadth for repeat-containing peptides in high vs. moderate exposure settings, regardless of infection status (New figure: Figure 5, Supplement 3), a difference that was absent in non-repeat containing peptides. Overall, these data suggest that responses to repeats are not only more exposure-dependent, but also short-lived relative to non-repeats in children. We have included this new analysis (lines 409-435.)

    Reviewer #2 (Public Review):

    This work profiles naturally acquired antibodies against Plasmodium falciparum proteins in two Ugandan cohorts, at incredibly high resolution, using a comprehensive library of overlapping peptides. These findings highlight the ubiquity and importance of intra- and inter-protein repeat elements in the humoral immune response to malaria. The authors discuss evidence that repeat elements reside in more seroreactive proteins, and that the breadth of immunity to repeat-containing antigens is associated with transmission intensity in children.

    A key strength and value added to publicly available data are the breadth of proteome coverage and unprecedented resolution from using tiling peptides. The authors point out that a known limitation of PhIP-seq is that conformational and discontinuous-linear epitopes cannot be detected with short linear peptides. In addition, disulfide linkages and post-translational modifications would be absent in the T7 representations.

    Several significant conclusions drawn from the results in this study are based on the humoral response to repeat elements that are present in multiple locations, including different genes. If antibodies to these regions are cross-reactive as described, it is not clear how the assay can differentiate antibodies that were developed against one or many of these loci. This potential confounding could change the conclusions about inter-protein motifs.

    • We thank the reviewer for their comments on the study. We have added a note about post-translational modifications to the text (Line 675-676) as recommended.

    • With regards to interprotein motifs (Figure 6), we only suggest a potential for antibody cross-reactivity across these motifs based on sequence similarity alone. We do not claim direct evidence that they are indeed cross-reactive, especially given the complex polyclonal nature of the response we are measuring. We present this sequence analysis only as a landscape of potential cross-reactivity among linear epitopes in the proteome, derived from the pool of seroreactive peptides enriched in this cohort.

    • Regardless, we have included a new analysis following the suggestion of Reviewer #1 to determine whether reactivity to these shared motifs indeed correlates between peptides from different proteins sharing a motif within the same individual. While this analysis shows apparent cross reactivity within individuals, we point out that the data is derived from complex polyclonal repertoires inherent to each individual, and thus these observations must be taken in that context and do not definitively establish cross reactivity. Along with the new analysis (Line 495-503), we have sought to be clear on these limitations (Line 632-635).

    Reviewer #3 (Public Review):

    This work provides a new tool, a comprehensive PhIP-seq library, containing 238,068 individual 62-amino acids peptides tiled every 25-amino acid peptide covering all known 8,980 proteins of the deadliest malaria parasite, Plasmodium falciparum, to systematically profile antibody targets in high resolution. This phage display library has been screened by plasma samples obtained from 198 Ugandan children and adults in high and moderate malaria transmission settings and 86 US controls. This work identified that repeat elements were commonly targeted by antibodies. Furthermore, extensive sharing of motifs associated with seroreactivity indicated the potential for extensive cross-reactivity among antigens in P. falciparum. This paper provides a new proteome-wide high-throughput methodology to identify antibody targets that have been investigated by protein arrays and alpha screens to date. Importantly, only this methodology (PhIP-seq library) is able to investigate repeat-containing antigens and cross-reactive epitopes in high resolution (25-amino acid resolution).

    Strengths:

    1. Novel technology

    Firstly, the uniqueness of this study is the use of novel technology, the PhIP-seq library. This PhIP-seq library in this study contains >99.5% of the parasite proteome and is the highest coverage among existing proteome-wide tools for P. falciparum. Moreover, this library can identify antibody responses in high resolution (25 amino acids).

    Secondly, the PhIP-seq converts a proteomic assay (ie. protein array and alpha screen) into a genomic assay, leveraging the massive scale and low-cost nature of next-generation short-read sequencing.

    Thirdly, the phage display system is the ability to sequentially enrich and amplify the signal to noise. Finally, a high-quality strategic bioinformatic analysis of PhIP-seq data was applied.

    1. Novel findings

    The major findings of this study were obtained only by using this novel technology because of its full-proteome coverage and high resolution. Repeat elements were the common target of naturally acquired antibodies. Furthermore, extensive sharing of motifs associated with seroreactivity was observed among hundreds of parasite proteins, indicating the potential for extensive cross-reactivity among antigens in P. falciparum.

    1. Usefulness for the future research

    Importantly, plasma samples from longitudinal cohort studies will give the scientific community important insights into protective humoral immunity which will be important for the identification of vaccine and exposure-marker candidates in the near future.

    Weaknesses:

    Although the paper does have strengths in principle, the weaknesses of the paper are the insufficient description of the selected parasite proteins and seroreactivity ranking of the selected proteins such as TOP100 proteins.

    We thank the reviewer for their comments, corrections, and suggestions. We have made a number of changes and added new analyses, all of which have improved the work. These changes include the following:

    • Analysis of breadth of seroreactivity to repeat and non-repeat regions taking into account infection status in both exposure settings.

    • Analysis to test whether reactivity to peptides with interprotein motifs correlates within the same individual

    • A table listing top 100 proteins in terms of their seropositivity % in response to the reviewer’s comment (Supplementary table 2b).

  3. eLife

    eLife assessment

    This study describes the use of a new and valuable tool, namely phage display of Plasmodium falciparum proteome-wide peptides, for profiling of antibody targets. The study, conducted using plasma from Ugandan children and adults, represents an important aspect of naturally acquired antibodies with seroreactive responses to the intra-and inter-protein repeat regions. The results are, however, so far incomplete, and confirmatory data that antibodies to inter-protein repeat motifs do cross-react are needed.

  4. eLife

    Reviewer #1 (Public Review):

    DeRisi and colleagues used a new phage-display peptide platform, with 238,068 tiled 62-amino acid peptides covering all known P falciparum coding regions (and numerous other entities), to survey seroreactivity in 198 Ugandan children and adults from two cohorts. They find that breadth of responses to repeat-containing peptides was twofold higher in children living in the high versus moderate exposure setting, while no such differences were observed for peptides without repeats. Additionally, short motifs associated with seroreactivity were extensively shared among hundreds of antigens, with much of this driven by motifs shared with PfEMP1 antigens.

    Malaria immunity is complex, and this new platform is a potentially valuable addition to the toolkit for understanding humoral responses. The two cohorts differed in fundamental ways: 1) high versus moderate exposure to infective bites; 2) samples drawn at the time of malaria for most donors in the high zone versus ~100 days after the last malaria episode in the moderate zone. The effect of acute malaria to boost short-term cross-reactive antibodies can confound the ability to draw inferences when comparing the two cohorts, and this should be further explored to understand its role in the patterns of seroreactivity observed.

  5. eLife

    Reviewer #2 (Public Review):

    This work profiles naturally acquired antibodies against Plasmodium falciparum proteins in two Ugandan cohorts, at incredibly high resolution, using a comprehensive library of overlapping peptides. These findings highlight the ubiquity and importance of intra- and inter-protein repeat elements in the humoral immune response to malaria. The authors discuss evidence that repeat elements reside in more seroreactive proteins, and that the breadth of immunity to repeat-containing antigens is associated with transmission intensity in children.

    A key strength and value added to publicly available data are the breadth of proteome coverage and unprecedented resolution from using tiling peptides. The authors point out that a known limitation of PhIP-seq is that conformational and discontinuous-linear epitopes cannot be detected with short linear peptides. In addition, disulfide linkages and post-translational modifications would be absent in the T7 representations.

    Several significant conclusions drawn from the results in this study are based on the humoral response to repeat elements that are present in multiple locations, including different genes. If antibodies to these regions are cross-reactive as described, it is not clear how the assay can differentiate antibodies that were developed against one or many of these loci. This potential confounding could change the conclusions about inter-protein motifs.

  6. eLife

    Reviewer #3 (Public Review):

    This work provides a new tool, a comprehensive PhIP-seq library, containing 238,068 individual 62-amino acids peptides tiled every 25-amino acid peptide covering all known 8,980 proteins of the deadliest malaria parasite, Plasmodium falciparum, to systematically profile antibody targets in high resolution. This phage display library has been screened by plasma samples obtained from 198 Ugandan children and adults in high and moderate malaria transmission settings and 86 US controls. This work identified that repeat elements were commonly targeted by antibodies. Furthermore, extensive sharing of motifs associated with seroreactivity indicated the potential for extensive cross-reactivity among antigens in P. falciparum. This paper provides a new proteome-wide high-throughput methodology to identify antibody targets that have been investigated by protein arrays and alpha screens to date. Importantly, only this methodology (PhIP-seq library) is able to investigate repeat-containing antigens and cross-reactive epitopes in high resolution (25-amino acid resolution).

    Strengths:

    1. Novel technology
      Firstly, the uniqueness of this study is the use of novel technology, the PhIP-seq library. This PhIP-seq library in this study contains >99.5% of the parasite proteome and is the highest coverage among existing proteome-wide tools for P. falciparum. Moreover, this library can identify antibody responses in high resolution (25 amino acids).
      Secondly, the PhIP-seq converts a proteomic assay (ie. protein array and alpha screen) into a genomic assay, leveraging the massive scale and low-cost nature of next-generation short-read sequencing.
      Thirdly, the phage display system is the ability to sequentially enrich and amplify the signal to noise.
      Finally, a high-quality strategic bioinformatic analysis of PhIP-seq data was applied.

    2. Novel findings
      The major findings of this study were obtained only by using this novel technology because of its full-proteome coverage and high resolution. Repeat elements were the common target of naturally acquired antibodies. Furthermore, extensive sharing of motifs associated with seroreactivity was observed among hundreds of parasite proteins, indicating the potential for extensive cross-reactivity among antigens in P. falciparum.

    3. Usefulness for the future research
      Importantly, plasma samples from longitudinal cohort studies will give the scientific community important insights into protective humoral immunity which will be important for the identification of vaccine and exposure-marker candidates in the near future.

    Weaknesses:
    Although the paper does have strengths in principle, the weaknesses of the paper are the insufficient description of the selected parasite proteins and seroreactivity ranking of the selected proteins such as TOP100 proteins.

  7. Version published to 10.1101/2022.06.24.497532v1 on bioRxiv