Biology subjects

Higgins, M. K.

Publications and source records attributed to Higgins, M. K..

At least 19 recordsLinked to original sources

bioRxiv2026

The helminth-derived effector protein HpBARI trimerises to selectively target membrane bound ST2 receptor

The ST2 receptor is activated by the alarmin cytokine IL-33, inducing and controlling immune responses. ST2-mediated signalling contributes to protection from parasitic infection, but is also involved in asthma, inflammatory bowel disease, fibrosis and allergic inflammation, making inhibitors of ST2 desirable. While signalling is mediated by membrane-associated ST2 on immune cells, extracellular fluids also contain a soluble form of ST2 which acts as a decoy, soaking up IL-33. An effective ST2-targeting therapeutic will selectively target membrane-associated ST2. The intestinal helminth H. polygyrus bakeri suppresses ST2-mediated signalling to reduce helminth clearance from the gut, with the effector HpBARI2 directly binding to and inhibiting ST2. Here, we present the structure of the ST2:HpBARI2 complex. HpBARI2 forms a stable, planar trimeric structure, with three vertices each binding a single ST2. HpBARI2 occludes the IL-33 binding site of ST2 and constraints the receptor in a conformation incompatible with recruitment of signalling co-receptor IL-1RAcP. The trimeric valency of HpBARI2 results in preferential binding to multiple membrane-associated ST2 molecules rather than the soluble decoy receptor. Therefore, HpBARI2 combines steric blockade, conformational restriction and multivalent binding to potently and selectively inhibit ST2 signalling in the context of soluble ST2. This insight will guide design of improved therapeutics which target ST2.

immunology↗

bioRxiv2026

Evolutionary adaptation of helminth immunomodulators to their hosts

Background: Parasitic nematodes have evolved secreted effector proteins which interact with host molecules to modulate the host immune response. These interactions can be highly specific and are under strong selective pressure. Here, we investigate the host-pathogen interaction between Heligmosomoides bakeri and its natural host Mus musculus, and between H. polygyrus and its natural host Apodemus sylvaticus. We focus on the Alarmin Release Inhibitor family (previously called HpARI) produced by heligmosomid parasites, which interact with IL-33 and heparan sulphate. Results: Using comparative genomics, we identified duplications and diversifications of the ARI family including multiple copies of ARI1 and conserved ARI3 homologues in both H. bakeri and H. polygyrus. We identify a new family member, HpolARI4, encoded by H. polygyrus, as well as a more distant homologue, HmixARI, encoded by Heligmosomum mixtum, a parasite of the bank vole (Myodes glareolus). Our functional assays identify that HpolARI4 preferentially blocks A. sylvaticus IL-33 but has little activity against M. musculus IL-33, whereas HbakARI2 is highly specific for M. musculus IL-33. We furthermore show differential interaction with host heparan sulphate, with HbakARI2 and HpolARI4 showing high affinity binding, whereas HbakARI3 shows no binding. We further test this family against non-host human IL-33 and show that functional suppression can be achieved by HbakARI3 in vitro and in vivo. Conclusion: By integrating genomic data, structural insights, and functional assays, we reveal how the expansion and diversification of the ARI family drive host-specific IL-33 modulation, providing a molecular model for nematode-host co-evolution.

immunology↗

bioRxiv2026

An African trypanosome surface protein inhibits amplification of the complement system

African trypanosomes replicate within the blood and tissue spaces of their mammalian hosts. As extracellular pathogens, they are constantly exposed to immune cells and molecules and are susceptible to killing by components of the complement system. In this study, we conducted a large-scale screen of a panel of human complement components against a set of putative T. brucei surface proteins and discovered a novel T. brucei receptor that binds to complement Factor B. Biochemical and structural characterisation of this protein revealed it to be a potent inhibitor of the C3bBb convertase, a central enzyme in amplification of the complement cascade. Structural studies show that this C3bBb receptor bridges C3b and Bb in a conformation which is incompatible with its catalytic activity, directly blocking C3bBb convertase function. This reveals a novel mechanism of C3bBb convertase regulation and deepens insight into how the African trypanosome cell surface has evolved to evade complement-mediated killing.

immunology↗

bioRxiv2026

A Trypanosoma brucei gambiense gene variant confers human serum resistance

Most species of African trypanosomes cannot infect humans due to the presence in our blood of trypanolytic factors (TLFs). These lipoprotein particles contain the apolipoprotein L1 (ApoLI) toxin which, when internalised by trypanosomes, forms pores and causes cell death. However, two subspecies of Trypanosoma brucei have evolved resistance to TLFs and cause Human African Trypanosomiasis (HAT). The mechanism of resistance of T. b. rhodesiense requires a single additional molecule, the serum resistance associated protein SRA. However, the mechanism of resistance of T. b. gambiense, which causes HAT in West Africa, has not been fully understood. Here we identify a single polymorphic variant of a PLAC8-domain containing protein which is required for human serum resistance and call this T. b. gambiense-specific resistance variant, TgsRV. African trypanosomes are coated with a dense layer of many copies of one member of the variant surface glycoprotein protein family (VSGs). For cells expressing some VSGs, TgsRV is sufficient for human serum resistance, while cells which express other VSGs require a second protein, TgsGP in addition to TgsRV. We therefore complete the identification of the molecular players required for human serum resistance by the African trypanosomes.

microbiology↗

bioRxiv2026

Immune receptor LILRB1 mediates cis-signalling which is targeted by RIFINs of the malaria parasite

The malaria parasite, Plasmodium falciparum, replicates within human erythrocytes, where it is susceptible to clearance by immune cells. It uses erythrocyte surface proteins, the RIFINs, to signal through immune receptors and to suppress immune cell function. Previous studies identified two groups of RIFINs which bind different sites on inhibitory immune receptor LILRB1. While some RIFINs bind an elongated LILRB1 conformation, triggering inhibitory immune signalling in trans, we show that other RIFINs stabilise a c-shaped LILRB1 conformation. This buckled LILRB1 binds MHC class I found on the same immune cell, which triggers inhibitory cis signalling. Therefore, LILRB1 exists in dynamic equilibrium, with an elongated conformation able to bind to ligands in trans on a target cell, while a buckled conformation signals through MHC class I in cis, setting the signalling threshold. Different clades of RIFINs exist to mediate inhibitory signalling through each of these LILRB1 conformations to prevent parasite destruction.

microbiology↗

bioRxiv2026

Dynamic hinge-motion of PfRIPR revealed by malaria invasion inhibitory antibodies

The PfPCRCR complex is essential for invasion of human erythrocytes by the deadliest malaria parasite, Plasmodium falciparum. Antibodies against each subunit of PfPCRCR prevent erythrocyte invasion and the PfRH5 component is currently the most advanced blood-stage malaria vaccine. Central within PfPCRCR is PfRIPR. This complex molecule contains a core and a flexible tail and allows PfPCRCR to bridge the parasite and erythrocyte during invasion. In this study, we generated a small panel of human monoclonal antibodies against PfRIPR. We structurally characterised four PfRIPR tail-binding antibodies in complex with PfRIPR fragments. We show that growth-inhibitory antibody RP.012 induces a kink in the PfRIPR tail while non-inhibitory antibodies do not. Furthermore, we show that these four antibodies modulate each other, either through antagonism or by acting synergistically. These studies have implications for the design of PfRIPR-based vaccine immunogens and indicate that the tail of PfRIPR undergoes essential conformational changes during erythrocyte invasion.

immunology↗

bioRxiv2025

HpBoRB, a helminth-derived CCP domain protein which binds RELMβ

Helminth infections persist by influencing host immunity through the release of immunomodulatory proteins which prevent immune ejection. The intestinal nematode Heligmosomoides polygyrus bakeri (Hpb) secretes multiple families of immunomodulatory proteins, many of which are composed of consecutive Complement Control Protein (CCP) domains. We hypothesized that further CCP domain proteins are secreted by the parasite to interact with the host. We identified an unusually large number of CCP domain-containing proteins in the genome of Hpb, and cloned a range of these for screening in an Avidity-based Extracellular Interaction Screening (AVEXIS) assay, focussing on interactions with host immune proteins. This screen confirmed the binding of known immunomodulators (HpBARI, TGM1) for their targets (ST2, TGFBR2) and identified a new interaction between a 2 CCP domain Hpb protein and mouse resistin-like molecule beta (RELM{beta}), a host protein demonstrated to have anti-helminth properties. This protein was named Binder of RELM{beta} (HpBoRB). This interaction was confirmed in ELISA, competition assays, size exclusion chromatography and surface plasmon resonance experiments, identifying a subnanomolar affinity interaction between HpBoRB and RELM{beta}. These data may indicate that Hpb interferes with the potent anti-helminth host protein RELM{beta} and adds to our knowledge of the host-parasite interactions mediated by Hpb secreted proteins.

immunology↗

bioRxiv2025

Cell surface localisation of GPI-anchored receptors in Trypanosoma brucei

Trypanosoma brucei, the causal agent of Human and Animal African trypanosomiasis proliferates in the extracellular milieu of mammals. It acquires host macromolecular nutrients, by receptor mediated endocytosis. The best characterised cell surface receptor is for transferrin (TfR) and it has been reported to be preferentially localised in the flagellar pocket domain of the plasma membrane, the sole site of endocytosis. In this location the TfR may be inaccessible to adaptive immune system effectors. The T. brucei genome encodes [~]15 TfR variants, and here we compared two, the first attached to the plasma membrane by a single glycosylphosphatidylinositol (GPI)-anchor and the other by two. Transferrin uptake kinetics were similar and rapid for both. Unexpectedly, initial binding of transferrin occurred over the whole cell surface suggesting the TfR was not localised solely in the flagellar pocket. This localisation was confirmed by immunofluorescence assays and was independent of the number of GPI-anchors. Two other GPI-anchored receptors were investigated to determine whether localisation to the whole cell surface was a general property of GPI-anchored receptors. Haptoglobin-haemoglobin uptake assays and immunofluorescence localisation of complement factor H receptor showed both were also whole cell surface localised. The mechanisms by which trypanosome receptors are protected from antibody-mediated attack are more complex than hiding in a pocket.

microbiology↗

bioRxiv2024

Scavenger receptor CD163 multimerises to allow uptake of diverse ligands

Scavenger receptors mediate cellular uptake of diverse ligands. CD163 is an archetypal scavenger receptor with a primary role in haemoglobin detoxification. Haemoglobin released from lysed erythrocytes causes oxidative damage. This is countered by formation of haemoglobin-haptoglobin complexes and CD163-mediated uptake into macrophages for degradation. Different haptoglobin isoforms form stoichiometrically diverse haptoglobin-haemoglobin complexes which must all be recognised by CD163. We show that CD163 forms multimeric complexes, with calcium-mediated interactions forming a trimeric base. Arms emerge from this base, moulding around the ligand to form a single promiscuous ligand-binding site. The arms can also bind to arms of adjacent protomers, blocking the ligand-binding surfaces in an autoinhibited state. This results in a scavenger receptor that can recognise diverse haemoglobin-containing complexes, allowing physiologically important detoxification.

biochemistry↗

bioRxiv2024

Structural basis for carbohydrate recognition by the Gal/GalNAc lectin of Entamoeba histolytica

Intestinal amoebiasis is caused by Entamoeba histolytica, one of the deadliest human-infective parasites. Central to its pathogenicity is its binding to mucosal carbohydrates, which precedes tissue damage by trogocytosis. Carbohydrate binding is mediated by a single adhesin, the galactose/N-acetylgalactosamine (Gal/GalNAc) lectin, which is the leading vaccine candidate for amoebiasis. We present the structure of the native heterodimeric lectin, revealing an ordered core containing the light chain and the N-terminal region of the heavy chain. Structures in the presence of ligand show that the Gal/GalNAc binding site is in the light chain. An elongated arm emerges from the heavy chain, which adopts multiple positions. This arm contains the epitope for a monoclonal antibody which blocks binding to target cells, located unexpectedly away from the Gal/GalNAc binding site. This study reveals the molecular basis for sugar binding by the Entamoeba histolytica Gal/GalNAc lectin and will guide future vaccine development against amoebiasis.

microbiology↗

bioRxiv2024

Structure-guided design of a Plasmodium vivax Duffy binding protein-based vaccine immunogen

Plasmodium vivax remains one of the major causative agents of human malaria and a vaccine is urgently required. It is an obligate intracellular parasites and replication within red blood cells is essential for development of disease and for transmission. The interaction between PvDBP on the parasite surface and the DARC receptor on human reticulocytes is essential for a Plasmodium vivax blood stage infection. Human vaccination with the RII region of PvDBP slowed parasite replication, showing that PvDBP is a promising vaccine candidate. However, it did not induce sterile protection, and further development is required to generate a vaccine which protects from clinical malaria. In this study, we develop a vaccine immunogen containing a region of PvDBP-RII, known as subdomain 3, which contains the epitope for a broadly-reactive growth-inhibitory antibody, DB9. We used structure-guided approaches to resurface subdomain 3 such that it folds as an isolated molecule. We show that this engineered subdomain 3 is more stable and more easily produced than PvDBP-RII and induces a more effective growth-inhibitory antibody response. We therefore present an improved PvDBP-based immunogen for use in blood stage vaccines to prevent malaria due to Plasmodium vivax. One sentence summaryStructure-guided design leads to a more effective Duffy-binding protein-based vaccine immunogen to prevent Plasmodium vivax.

immunology↗

bioRxiv2024

Heparan sulphate binding controls in vivo half-life of the HpARI protein family

The parasitic nematode Heligmosomoides polygyrus bakeri secretes the HpARI family, which bind to IL-33, either suppressing (HpARI1 and HpARI2) or enhancing (HpARI3) responses to the cytokine. We previously showed that HpARI2 also bound to DNA via its first Complement Control Protein (CCP1) domain. Here, we find that HpARI1 can also bind DNA, while HpARI3 cannot. Through the production of HpARI2/HpARI3 CCP1 domain-swapped chimeras, DNA-binding ability can be transferred, and correlates with in vivo half-life of administered proteins. We found that HpARI1 and HpARI2 (but not HpARI3) also binds to the extracellular matrix component heparan sulphate (HS), and structural modelling showed a basic charged patch in the CCP1 domain of HpARI1 and HpARI2 (but not HpARI3) which could facilitate these interactions. Finally, a mutant of HpARI2 was produced which lacked DNA and HS binding, and was also shown to have a short half-life in vivo. Therefore, we propose that during infection the suppressive HpARI1 and HpARI2 proteins have long-lasting effects at the site of deposition due to DNA and/or extracellular matrix interactions, while HpARI3 has a shorter half-life due to a lack of these interactions.

immunology↗

bioRxiv2024

RIFINs displayed on malaria-infected erythrocytes bind both KIR2DL1 and KIR2DS1

To discriminate between our own cells and foreign cells such as a pathogen, natural killer cells are armed with inhibitory and activating immune receptors. In some cases, such as the KIRs, these are found in pairs, with inhibitory and activating receptors containing nearly identical extracellular domains that are coupled to different intracellular signalling domains1. The balance in signalling mediated by these receptors determines whether an NK cell is activated to destroy a target cell. Previous studies have shown that RIFINs, displayed on surfaces of Plasmodium falciparum-infected erythrocytes, can bind to inhibitory immune receptors and dampen NK cell activation2,3, reducing parasite killing. Here, we identify a clade of RIFINs that bind to inhibitory immune receptor KIR2DL1 approximately ten-times more strongly than KIR2DL1 binds its host ligand, MHC class I. We show that this interaction mediates inhibitory signalling and reduces activation of KIR2DL1-expressing NK cells. We reveal the structural basis for KIR2DL1 binding and show that the RIFIN binding surface of KIR2DL1 is conserved in the activating immune receptor KIR2DS1. We find that KIR2DL1-binding RIFINs can also bind to KIR2DS1 and that these RIFINs cause activation of KIR2DS1-expressing NK cells. This highlights the evolutionary battle between pathogen and host, suggesting that activating KIRs may have evolved to allow detection of red blood cells infected with Plasmodium falciparum, helping the host to clear the parasite.

microbiology↗

bioRxiv2024

Rational structure-guided design of a blood stage malaria vaccine immunogen presenting a single epitope from PfRH5

Despite recent success in licencing of two malaria vaccines, there continues to be an urgent need for improved vaccine immunogens as the world aims for malaria eradication. The invasion of erythrocytes by Plasmodium falciparum is an essential step in the life cycle of the parasite, preceding symptoms of disease and parasite transmission. Antibodies which target the PfRH5 protein are highly effective at preventing erythrocyte invasion and the most effective growth-inhibitory antibodies bind to a single epitope. Here we used Rosetta-based protein design to produce a focused synthetic immunogen on which this epitope is presented on a small scaffold. Structural biology and biophysics were used to demonstrate that the immunogen is correctly folded and binds neutralising monoclonal antibodies with at least nanomolar affinity. In immunised rats, the immunogen induced PfRH5-targeting antibodies that inhibit parasite growth at a thousand-fold lower concentration that those induced through immunisation with PfRH5. Finally, we show that priming with the focused immunogen and boosting with PfRH5 achieves the best balance between antibody quality and quantity and induces the most effective growth-inhibitory response. This rationally designed vaccine immunogen is now available for use as part of future malaria vaccines, alone or in combination with other immunogens. One Sentence SummaryRational, structure-guided design has been used to produce a vaccine immunogen in which the epitope for the most growth-inhibitory monoclonal antibody targeting PfRH5 has been correctly presented on a synthetic scaffold for use in a vaccine to protect from blood-stage malaria.

immunology↗

bioRxiv2024

Development of an improved blood-stage malaria vaccine targeting the essential RH5-CyRPA-RIPR invasion complex

In recent years, reticulocyte-binding protein homologue 5 (RH5) has emerged as a leading blood-stage Plasmodium falciparum malaria vaccine antigen. The most advanced blood-stage vaccine candidate in a Phase 2b clinical trial, RH5.1/Matrix-M, is based on a full-length soluble protein-with-adjuvant formulation. RH5 interacts with cysteine-rich protective antigen (CyRPA) and RH5- interacting protein (RIPR) to form an essential heterotrimeric "RCR-complex". Here, we investigated whether a vaccine candidate based on the ternary RCR-complex could substantially improve upon the leading clinical candidate RH5.1/Matrix-M in preclinical studies. Using a panel of monoclonal antibodies (mAbs) we confirm that parasite growth-inhibitory epitopes on each antigen are exposed on the surface of the RCR-complex and that mAb pairs binding to different antigens can function additively or synergistically to mediate parasite growth inhibition activity (GIA) in vitro. However, immunisation of rats with the RCR-complex consistently fails to outperform RH5.1 alone. We show this is due to immuno-dominance of RIPR coupled with the inferior potency of anti-full length RIPR polyclonal IgG antibodies as compared to the anti-RH5 and anti-CyRPA response. To address this, we identified the growth-inhibitory antibody epitopes of RIPR are clustered within C-terminal EGF-like domains of RIPR. A fusion of these EGF domains to CyRPA, called "R78C", combined with RH5.1, provided a new vaccination strategy that improves upon the levels of in vitro GIA seen with RH5.1 alone. Superiority of the combination antigen vaccine candidate was achieved by the induction of a quantitatively higher, but qualitatively similar, polyclonal antibody response that demonstrated additive GIA across the three antigen targets. These preclinical data justified the advancement of the RH5.1+R78C/Matrix-M combination vaccine to a Phase 1 clinical trial.

immunology↗

bioRxiv2023

Natural malaria infection elicits rare but potent neutralizing antibodies to the blood-stage antigen RH5

Plasmodium falciparum RH5 is the most advanced blood-stage malaria vaccine candidate and is under evaluation for efficacy in endemic regions, emphasizing the need to study the underlying antibody response to RH5 during natural infection. Here, we found that RH5-reactive B cells were rare in malaria-exposed individuals despite repeated infections over multiple years. RH5-specific monoclonal antibodies isolated from these individuals were extensively mutated but mostly targeted non-neutralizing epitopes, in contrast to antibodies from RH5-vaccinated, malaria-naive individuals. However, infection-derived MAD8-151 and MAD8-502 were among the most potent neutralizers out of 186 antibodies isolated from both cohorts and target the same epitopes as the most effective vaccine-induced antibodies. Binding to basigin receptor-proximal epitopes was the primary factor governing the potency of RH5-specific antibodies from both natural infection and vaccination, followed by the strength of binding. These results indicate a clear strategy for the development of next-generation RH5 vaccines for use in malaria-endemic regions.

immunology↗

bioRxiv2023

Structural basis for IL-33 recognition and its antagonism by the helminth effector protein HpARI

Interleukin 33 (IL-33) plays a significant role in inflammation, allergy, and host defence against parasitic helminths. The model gastrointestinal nematode Heligmosomoides polygyrus bakeri (Hp) secretes the Alarmin Release Inhibitor (HpARI), a potent effector protein which suppresses protective immune responses and asthma in its host by inhibiting IL-33 signalling. Here we reveal the structure of HpARI bound to mouse IL-33. HpARI contains three CCP-like domains, and we show that it contacts IL-33 primarily through the second and third of these. A large loop which emerges from CCP3 directly contacts IL-33 and structural comparison shows that this overlaps with the binding site on IL-33 for its receptor, ST2, preventing formation of a signalling complex. Truncations of HpARI which lack the large loop from CCP3 are not able to block IL-33-mediated signalling in a cell-based assay and in an in vivo model of asthma. This shows that direct competition between HpARI and ST2 is responsible for suppression of IL-33-dependent responses. This first structure of IL-33 bound to a pathogen-based inhibitor will guide future approaches to design therapeutics blocking IL-33-mediated allergic and inflammatory conditions.

microbiology↗

bioRxiv2023

Molecular mechanism of complement inhibition by the trypanosome receptor ISG65

African trypanosomes replicate within infected mammals where they are exposed to the complement system. This system centres around complement C3, which is present in a soluble form in serum but becomes covalently deposited onto the surfaces of pathogens after proteolytic cleavage to C3b. Membrane-associated C3b triggers different complement-mediated effectors which promote pathogen clearance. To counter complement-mediated clearance, African trypanosomes have a cell surface receptor, ISG65, which binds to C3b and which decreases the rate of trypanosome clearance in an infection model. However, the mechanism by which ISG65 reduces C3b function has not been determined. We reveal through cryogenic electron microscopy that ISG65 has two distinct binding sites for C3b, only one of which is available in C3 and C3d. We show that ISG65 does not block the formation of C3b or the function of the C3 convertase which catalyses the surface deposition of C3b. However, we show that ISG65 forms a specific conjugate with C3b, perhaps acting as a decoy. ISG65 also occludes the binding sites for complement receptors 2 and 3, which may disrupt recruitment of immune cells, including B cells, phagocytes and granulocytes. This suggests that ISG65 protects trypanosomes by combining multiple approaches to dampen the complement cascade. Impact StatementA structure derived from cryogenic electron microscopy shows how ISG65, an African trypanosome receptor that aids virulence, binds C3b and suggests mechanisms through which ISG65 might aid complement resistance.

microbiology↗