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Structural variants are mapped that are correlated with a reduced risk of severe malaria. Large-scale deletions and duplications of genes, referred to as structural variants (SVs), are common within the human genome and have been linked to disease. Examining a genomic region that appears to confer a selective benefit, Leffler et al. used fine mapping to identify a specific SV that reduces the risk of severe malaria by an estimated 40% (see the Perspective by Winzeler). Data from African individuals revealed that populations harbor different SVs in this region. Furthermore, by dissecting a highly complex genomic region, the authors identified the likely causal element. This element encodes hybrid genes that affect glycophorin proteins, which are used by the malarial parasite in infection and are associated with resistance to severe disease. Science , this issue p. eaam6393 ; see also p. 1122 The malaria parasite Plasmodium falciparum invades human red blood cells by a series of interactions between host and parasite surface proteins. By analyzing genome sequence data from human populations, including 1269 individuals from sub-Saharan Africa, we identify a diverse array of large copy-number variants affecting the host invasion receptor genes GYPA and GYPB . We find that a nearby association with severe malaria is explained by a complex structural rearrangement involving the loss of GYPB and gain of two GYPB-A hybrid genes, which encode a serologically distinct blood group antigen known as Dantu. This variant reduces the risk of severe malaria by 40% and has recently increased in frequency in parts of Kenya, yet it appears to be absent from west Africa. These findings link structural variation of red blood cell invasion receptors with natural resistance to severe malaria.

A short half-life in the circulation limits the application of therapeutics such as single-domain antibodies (VHHs). We utilize red blood cells to prolong the circulatory half-life of VHHs. Here we present VHHs against botulinum neurotoxin A (BoNT/A) on the surface of red blood cells by expressing chimeric proteins of VHHs with Glycophorin A or Kell. Mice whose red blood cells carry the chimeric proteins exhibit resistance to 10,000 times the lethal dose (LD 50 ) of BoNT/A, and transfusion of these red blood cells into naive mice affords protection for up to 28 days. We further utilize an improved CD34+ culture system to engineer human red blood cells that express these chimeric proteins. Mice transfused with these red blood cells are resistant to highly lethal doses of BoNT/A. We demonstrate that engineered red blood cells expressing VHHs can provide prolonged prophylactic protection against bacterial toxins without inducing inhibitory immune responses and illustrates the potentially broad translatability of our strategy for therapeutic applications. The therapeutic use of single-chain antibodies (VHHs) is limited by their short half-life in the circulation. Here the authors engineer mouse and human red blood cells to express VHHs against botulinum neurotoxin A (BoNT/A) on their surface and show that an infusion of these cells into mice confers long lasting protection against a high dose of BoNT/A.

Glycophorins are transmembrane proteins of red blood cells (RBCs), heavily glycosylated on their external-facing surface. In humans, there are four glycophorin proteins, glycophorins A, B, C and D. Glycophorins A and B are encoded by two similar genes GYPA and GYPB, and glycophorin C and glycophorin D are encoded by a single gene, GYPC. The exact function of glycophorins remains unclear. However, given their abundance on the surface of RBCs, it is likely that they serve as a substrate for glycosylation, giving the RBC a negatively charged, complex glycan “coat”. GYPB and GYPE (a closely related pseudogene) were generated from GYPA by two duplication events involving a 120-kb genomic segment between 10 and 15 million years ago. Non-allelic homologous recombination between these 120-kb repeats generates a variety of duplication alleles and deletion alleles, which have been systematically catalogued from genomic sequence data. One allele, called DUP4, encodes the Dantu NE blood type and is strongly protective against malaria as it alters the surface tension of the RBC membrane. Glycophorins interact with other infectious pathogens, including viruses, as well as the malarial parasite Plasmodium falciparum, but the role of glycophorin variation in mediating the effects of these pathogens remains underexplored.

Invasion of the red blood cell (RBC) by the Plasmodium parasite defines the start of malaria disease pathogenesis. To date, experimental investigations into invasion have focused predominantly on the role of parasite adhesins or signaling pathways and the identity of binding receptors on the red cell surface. A potential role for signaling pathways within the erythrocyte, which might alter red cell biophysical properties to facilitate invasion, has largely been ignored. The parasite erythrocyte-binding antigen 175 (EBA175), a protein required for entry in most parasite strains, plays a key role by binding to glycophorin A (GPA) on the red cell surface, although the function of this binding interaction is unknown. Here, using real-time deformability cytometry and flicker spectroscopy to define biophysical properties of the erythrocyte, we show that EBA175 binding to GPA leads to an increase in the cytoskeletal tension of the red cell and a reduction in the bending modulus of the cell’s membrane. We isolate the changes in the cytoskeleton and membrane and show that reduction in the bending modulus is directly correlated with parasite invasion efficiency. These data strongly imply that the malaria parasite primes the erythrocyte surface through its binding antigens, altering the biophysical nature of the target cell and thus reducing a critical energy barrier to invasion. This finding would constitute a major change in our concept of malaria parasite invasion, suggesting it is, in fact, a balance between parasite and host cell physical forces working together to facilitate entry.

A multi-centre genome-wide association study of severe malaria in African children uncovers a new resistance locus close to a cluster of genes encoding glycophorins, which are receptors used by the malaria-causing parasite to invade red blood cells. Gene variants associated with resistance to malaria This multi-centre genome-wide association study of severe malaria in more than 11,000 African children uncovers a new variant associated with resistance to malaria that is close to a cluster of genes encoding glycophorins — membrane receptors used by the Plasmodium falciparum parasite to invade red blood cells. Interestingly, the variant is linked to polymorphisms previously found to have been maintained for millions of years, based on genome analysis of humans and chimpanzees. The high prevalence of sickle haemoglobin in Africa shows that malaria has been a major force for human evolutionary selection, but surprisingly few other polymorphisms have been proven to confer resistance to malaria in large epidemiological studies 1 , 2 , 3 . To address this problem, we conducted a multi-centre genome-wide association study (GWAS) of life-threatening Plasmodium falciparum infection (severe malaria) in over 11,000 African children, with replication data in a further 14,000 individuals. Here we report a novel malaria resistance locus close to a cluster of genes encoding glycophorins that are receptors for erythrocyte invasion by P. falciparum . We identify a haplotype at this locus that provides 33% protection against severe malaria (odds ratio = 0.67, 95% confidence interval = 0.60–0.76, P value = 9.5 × 10 −11 ) and is linked to polymorphisms that have previously been shown to have features of ancient balancing selection, on the basis of haplotype sharing between humans and chimpanzees 4 . Taken together with previous observations on the malaria-protective role of blood group O 1 , 2 , 3 , 5 , these data reveal that two of the strongest GWAS signals for severe malaria lie in or close to genes encoding the glycosylated surface coat of the erythrocyte cell membrane, both within regions of the genome where it appears that evolution has maintained diversity for millions of years. These findings provide new insights into the host–parasite interactions that are critical in determining the outcome of malaria infection.

Serological typing of MNS polymorphic antigens - M, N, S and s - remains a fundamental technique in transfusion medicine and prenatal care, providing essential information for matching blood donors and recipients and managing haemolytic disease. Although this method is well proven and routinely used, it is not a comprehensive solution, as it has several weaknesses. Alternatively, multiplex polymerase chain reaction (PCR) is a commonly used genotyping tool due to its potency and ability to amplify several DNA targets simultaneously in a single reaction. In this work, we aimed to develop multiplex PCR and evaluate its performance for GYPA*M, GYPA*N, GYPB*S, and GYPB*s allele identification using serological and DNA sequencing methods. We also aimed to investigate the correlation between these alleles and Mi -associated hybrid glycophorins (GPs). Remarkably, multiplex PCR was well optimised, and the results aligned with serological phenotyping and DNA sequencing data with maximum accuracy and reliability; this confirmed our findings on its validity in predicting MNSs phenotypes. In addition, this work strongly demonstrates, for the first time, a moderate correlation between the GYPA*M/M and GYPB*s/s genotypes and Mi -associated hybrid GPs among Thai donors. Individuals with the GYPA*M/M and GYPB*s/s genotypes, predicted M + N - S- s + phenotypes, will thus most likely to express the Mi(a+) antigen. Nevertheless, further studies are required to validate these results and elucidate the underlying correlations.

Disrupting erythrocyte invasion by Plasmodium falciparum is an attractive approach to combat malaria. P. falciparum EBA-175 (PfEBA-175) engages the host receptor Glycophorin A (GpA) during invasion and is a leading vaccine candidate. Antibodies that recognize PfEBA-175 can prevent parasite growth, although not all antibodies are inhibitory. Here, using x-ray crystallography, small-angle x-ray scattering and functional studies, we report the structural basis and mechanism for inhibition by two PfEBA-175 antibodies. Structures of each antibody in complex with the PfEBA-175 receptor binding domain reveal that the most potent inhibitory antibody, R217, engages critical GpA binding residues and the proposed dimer interface of PfEBA-175. A second weakly inhibitory antibody, R218, binds to an asparagine-rich surface loop. We show that the epitopes identified by structural studies are critical for antibody binding. Together, the structural and mapping studies reveal distinct mechanisms of action, with R217 directly preventing receptor binding while R218 allows for receptor binding. Using a direct receptor binding assay we show R217 directly blocks GpA engagement while R218 does not. Our studies elaborate on the complex interaction between PfEBA-175 and GpA and highlight new approaches to targeting the molecular mechanism of P. falciparum invasion of erythrocytes. The results suggest studies aiming to improve the efficacy of blood-stage vaccines, either by selecting single or combining multiple parasite antigens, should assess the antibody response to defined inhibitory epitopes as well as the response to the whole protein antigen. Finally, this work demonstrates the importance of identifying inhibitory-epitopes and avoiding decoy-epitopes in antibody-based therapies, vaccines and diagnostics.

Plasmodium falciparum, the cause of almost all human malaria mortality, is a member of the Laverania subgenus which infects African great apes. Interestingly, Laverania parasites exhibit strict host specificity in their natural environment: P. reichenowi, P. billcollinsi, and P. gaboni infect only chimpanzees; P. praefalciparum, P. blacklocki, and P. adleri are restricted to gorillas, and P. falciparum is pandemic in humans. The molecular mechanism(s) responsible for these host restrictions are not understood, although the interaction between the parasite blood-stage invasion ligand EBA175 and the host erythrocyte receptor Glycophorin-A (GYPA) has been implicated previously. We reexamined the role of the EBA175—GYPA interaction in host tropism using recombinant proteins and biophysical assays and found that EBA175 orthologs from the chimpanzee-restricted parasites P. reichenowi and P. billcollinsi both bound to human GYPA with affinities similar to that of P. falciparum, suggesting that the EBA175—GYPA interaction is unlikely to be the sole determinant of Laverania host specificity. We next investigated the contribution of the recently discovered Reticulocyte-binding protein Homolog 5 (RH5)—Basigin (BSG) interaction in host-species selectivity and found that P. falciparum RH5 bound chimpanzee BSG with a significantly lower affinity than human BSG and did not bind gorilla BSG, mirroring the known host tropism of P. falciparum. Using site-directed mutagenesis, we identified residues in BSG that are responsible for the species specificity of PfRH5 binding. Consistent with the essential role of the PfRH5—BSG interaction in erythrocyte invasion, we conclude that species-specific differences in the BSG receptor provide a molecular explanation for the restriction of P. falciparum to its human host.

Background Approximately 5% of the human genome shows common structural variation, which is enriched for genes involved in the immune response and cell-cell interactions. A well-established region of extensive structural variation is the glycophorin gene cluster, comprising three tandemly-repeated regions about 120 kb in length and carrying the highly homologous genes GYPA , GYPB and GYPE . Glycophorin A (encoded by GYPA ) and glycophorin B (encoded by GYPB ) are glycoproteins present at high levels on the surface of erythrocytes, and they have been suggested to act as decoy receptors for viral pathogens. They are receptors for the invasion of the protist parasite Plasmodium falciparum, a causative agent of malaria. A particular complex structural variant, called DUP4, creates a GYPB-GYPA fusion gene known to confer resistance to malaria. Many other structural variants exist across the glycophorin gene cluster, and they remain poorly characterised. Results Here, we analyse sequences from 3234 diploid genomes from across the world for structural variation at the glycophorin locus, confirming 15 variants in the 1000 Genomes project cohort, discovering 9 new variants, and characterising a selection of these variants using fibre-FISH and breakpoint mapping at the sequence level. We identify variants predicted to create novel fusion genes and a common inversion duplication variant at appreciable frequencies in West Africans. We show that almost all variants can be explained by non-allelic homologous recombination and by comparing the structural variant breakpoints with recombination hotspot maps, confirm the importance of a particular meiotic recombination hotspot on structural variant formation in this region. Conclusions We identify and validate large structural variants in the human glycophorin A-B-E gene cluster which may be associated with different clinical aspects of malaria.

In the war against Plasmodium, humans have evolved to eliminate or modify proteins on the erythrocyte surface that serve as receptors for parasite invasion, such as the Duffy blood group, a receptor for Plasmodium vivax, and the Gerbich-negative modification of glycophorin C for Plasmodium falciparum. In turn, the parasite counters with expansion and diversification of ligand families. The high degree of polymorphism in glycophorin B found in malaria-endemic regions suggests that it also may be a receptor for Plasmodium, but, to date, none has been identified. We provide evidence from erythrocyte-binding that glycophorin B is a receptor for the P. falciparum protein EBL-1, a member of the Duffy-binding-like erythrocyte-binding protein (DBL-EBP) receptor family. The erythrocyte-binding domain, region 2 of EBL-1, expressed on CHO-K1 cells, bound glycophorin B⁺ but not glycophorin B-null erythrocytes. In addition, glycophorin B⁺ but not glycophorin B-null erythrocytes adsorbed native EBL-1 from the P. falciparum culture supernatants. Interestingly, the Efe pygmies of the Ituri forest in the Democratic Republic of the Congo have the highest gene frequency of glycophorin B-null in the world, raising the possibility that the DBL-EBP family may have expanded in response to the high frequency of glycophorin B-null in the population.