In two previously described donors, the extracellular domain of LAIR1, a collagen-binding inhibitory receptor encoded on chromosome 19 (ref. 1), was inserted between the V and DJ segments of an antibody. This insertion generated, through somatic mutations, broadly reactive antibodies against RIFINs, a type of variant antigen expressed on the surface of Plasmodium falciparum-infected erythrocytes2. To investigate how frequently such antibodies are produced in response to malaria infection, we screened plasma from two large cohorts of individuals living in malaria-endemic regions. Here we report that 5–10% of malaria-exposed individuals, but none of the European blood donors tested, have high levels of LAIR1-containing antibodies that dominate the response to infected erythrocytes without conferring enhanced protection against febrile malaria. By analysing the antibody-producing B cell clones at the protein, cDNA and gDNA levels, we characterized additional LAIR1 insertions between the V and DJ segments and discovered a second insertion modality whereby the LAIR1 exon encoding the extracellular domain and flanking intronic sequences are inserted into the switch region. By exon shuffling, this mechanism leads to the production of bispecific antibodies in which the LAIR1 domain is precisely positioned at the elbow between the VH and CH1 domains. Additionally, in one donor the genomic DNA encoding the VH and CH1 domains was deleted, leading to the production of a camel-like LAIR1-containing antibody. Sequencing of the switch regions of memory B cells from European blood donors revealed frequent templated inserts originating from transcribed genes that, in rare cases, comprised exons with orientations and frames compatible with expression. These results reveal different modalities of LAIR1 insertion that lead to public and dominant antibodies against infected erythrocytes and suggest that insertion of templated DNA represents an additional mechanism of antibody diversification that can be selected in the immune response against pathogens and exploited for B cell engineering.
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We thank M. Nussenzweig and H. Wardemann for reagents for antibody cloning and expression. This work was supported by the Swiss Vaccine Research Institute and by the Fondazione Aldo e Cele Daccò. The work on the healthy European blood donors was supported by the European Research Council (grant no. 670955 BROADimmune). The Mali study was funded by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health. A.L. and F.S. are supported by the Helmut Horten Foundation. This paper is published with the permission of the Director of KEMRI.
A.L. is the scientific founder and shareholder of Humabs BioMed. F.S. is a shareholder of Humabs BioMed.
Reviewer Information Nature thanks P. Preiser, M. Wahlgren and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Shown is one representative antibody from each donor. a, MGE9 (donor E); b, MGF21 (donor F); c, MGO3 (donor O); d, MGQ4 (donor Q).
Shown is one representative antibody for each donor; a, MMJ5 (donor J); b, MGM5 (donor M); c, MGB47 (donor B). The chromosome coordinates of the insertion sites are indicated in blue and green.
Extended Data Figure 3 Somatically mutated and conserved regions in the LAIR1 domains inserted in the VDJ or in the switch region.
a, Amino acid substitutions in antibodies isolated from different donors and mean R/S ratios at each residue. The mutational analysis takes into consideration the germline LAIR1 alleles found in each donor. In donor C, the P98L substitution is uncoloured because it may arise from polymorphism, since the donor is heterozygous at this position. The number of nucleotide mutations and amino acid substitutions are reported in brackets next to the antibody names. b, Graphic representation of mutational hot spots (red) and of most conserved regions (blue).
Extended Data Figure 4 Validation of switch region inserts combining Illumina and MinION technologies.
a, Illumina and MinION workflows. Switch regions of polyclonal naive or IgG/IgA switched B cells were amplified by PCR. For Illumina sequencing, PCR amplicons were fragmented, re-amplified during library preparation and sequenced using the 2 × 300 bp MiSeq system. The bioinformatic analysis included the assembly of contiguous, chimaeric reads. For insert confirmation, independently generated PCR-barcoded primary products were sequenced with MinION technology and analysed with a different bioinformatic approach for long, error-prone MinION reads. b, Multiple identical switch inserts for donor 6 were confirmed in biological replicate experiments with independent technical and analytical setups. Shown are the experimental designs, shared and unique reads in a Venn diagram and an alignment of Illumina and MinION sequences covering the switch insertion sites for two examples (LCP1, RAVER1). c, Shared and unique switch inserts in technical and biological replicate experiments of donor 5.
Shown is the scheme of the bioinformatics workflow used for the analysis of Illumina sequences.
Shown is the scheme of the bioinformatics workflow used for the analysis.
Shown is the original position of the inserts donated by PAX5 and EBF1 as well as a list of genes that donated two or more inserts.
Shown is the alignment of the contig sequence and the genomic region from which the insert was derived, as well as the potential amino acid sequence inserted between the VH and CH1.
Extended Data Figure 9 Relationship between LAIR1-IgG or LAIR1-IgM status and protection from febrile malaria.
Shown is the clinical status of 551 members of the Malian cohort, stratified by LAIR1-containing IgG (a) or LAIR1-containing IgM (b) status, over the years 2012 and 2013. Febrile malaria is defined as parasite density ≥ 2,500 asexual parasites per microlitre of blood and an axillary temperature of ≥ 37.5 °C.
This file contains Supplementary Figures 1-2 and Supplementary Tables 1-2.
This table contains a list of inserts in the switch region found by Illumina sequencing.
This table contains a list of inserts in the switch region found by MinION sequencing.
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Pieper, K., Tan, J., Piccoli, L. et al. Public antibodies to malaria antigens generated by two LAIR1 insertion modalities. Nature 548, 597–601 (2017). https://doi.org/10.1038/nature23670
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