Human coronavirus OC43 nanobody neutralizes virus and protects mice from infection.

Human coronavirus (hCoV) OC43 is endemic to global populations and usually causes asymptomatic or mild upper respiratory tract illness. Here, we demonstrate the neutralization efficacy of isolated nanobodies from alpacas immunized with the S1B and S1C domain of the hCoV-OC43 spike glycoprotein. A total of 40 nanobodies bound to recombinant OC43 protein with affinities ranging from 1 to 149 nM. Two nanobodies WNb 293 and WNb 294 neutralized virus at 0.21 and 1.79 nM, respectively. Intranasal and intraperitoneal delivery of WNb 293 fused to an Fc domain significantly reduced nasal viral load in a mouse model of hCoV-OC43 infection. Using X-ray crystallography, we observed that WNb 293 bound to an epitope on the OC43 S1B domain, distal from the sialoglycan-binding site involved in host cell entry. This result suggests that neutralization mechanism of this nanobody does not involve disruption of glycan binding. Our work provides characterization of nanobodies against hCoV-OC43 that blocks virus entry and reduces viral loads in vivo and may contribute to future nanobody-based therapies for hCoV-OC43 infections. IMPORTANCE: The pandemic potential presented by coronaviruses has been demonstrated by the ongoing COVID-19 pandemic and previous epidemics caused by severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus. Outside of these major pathogenic coronaviruses, there are four endemic coronaviruses that infect humans: hCoV-OC43, hCoV-229E, hCoV-HKU1, and hCoV-NL63. We identified a collection of nanobodies against human coronavirus OC43 (hCoV-OC43) and found that two high-affinity nanobodies potently neutralized hCoV-OC43 at low nanomolar concentrations. Prophylactic administration of one neutralizing nanobody reduced viral loads in mice infected with hCoV-OC43, showing the potential for nanobody-based therapies for hCoV-OC43 infections.

Structure of Plasmodium falciparum Rh5-CyRPA-Ripr invasion complex.

Plasmodium falciparum causes the severe form of malaria that has high levels of mortality in humans. Blood-stage merozoites of P. falciparum invade erythrocytes, and this requires interactions between multiple ligands from the parasite and receptors in hosts. These interactions include the binding of the Rh5-CyRPA-Ripr complex with the erythrocyte receptor basigin1,2, which is an essential step for entry into human erythrocytes. Here we show that the Rh5-CyRPA-Ripr complex binds the erythrocyte cell line JK-1 significantly better than does Rh5 alone, and that this binding occurs through the insertion of Rh5 and Ripr into host membranes as a complex with high molecular weight. We report a cryo-electron microscopy structure of the Rh5-CyRPA-Ripr complex at subnanometre resolution, which reveals the organization of this essential invasion complex and the mode of interactions between members of the complex, and shows that CyRPA is a critical mediator of complex assembly. Our structure identifies blades 4-6 of the ß-propeller of CyRPA as contact sites for Rh5 and Ripr. The limited contacts between Rh5-CyRPA and CyRPA-Ripr are consistent with the dissociation of Rh5 and Ripr from CyRPA for membrane insertion. A comparision of the crystal structure of Rh5-basigin with the cryo-electron microscopy structure of Rh5-CyRPA-Ripr suggests that Rh5 and Ripr are positioned parallel to the erythrocyte membrane before membrane insertion. This provides information on the function of this complex, and thereby provides insights into invasion by P. falciparum.

Cryo-EM structure of an essential Plasmodium vivax invasion complex.

Plasmodium vivax is the most widely distributed malaria parasite that infects humans1. P. vivax invades reticulocytes exclusively, and successful entry depends on specific interactions between the P. vivax reticulocyte-binding protein 2b (PvRBP2b) and transferrin receptor 1 (TfR1)2. TfR1-deficient erythroid cells are refractory to invasion by P. vivax, and anti-PvRBP2b monoclonal antibodies inhibit reticulocyte binding and block P. vivax invasion in field isolates2. Here we report a high-resolution cryo-electron microscopy structure of a ternary complex of PvRBP2b bound to human TfR1 and transferrin, at 3.7 Å resolution. Mutational analyses show that PvRBP2b residues involved in complex formation are conserved; this suggests that antigens could be designed that act across P. vivax strains. Functional analyses of TfR1 highlight how P. vivax hijacks TfR1, an essential housekeeping protein, by binding to sites that govern host specificity, without affecting its cellular function of transporting iron. Crystal and solution structures of PvRBP2b in complex with antibody fragments characterize the inhibitory epitopes. Our results establish a structural framework for understanding how P. vivax reticulocyte-binding protein engages its receptor and the molecular mechanism of inhibitory monoclonal antibodies, providing important information for the design of novel vaccine candidates.

Nanobody cocktails potently neutralize SARS-CoV-2 D614G N501Y variant and protect mice.

Neutralizing antibodies are important for immunity against SARS-CoV-2 and as therapeutics for the prevention and treatment of COVID-19. Here, we identified high-affinity nanobodies from alpacas immunized with coronavirus spike and receptor-binding domains (RBD) that disrupted RBD engagement with the human receptor angiotensin-converting enzyme 2 (ACE2) and potently neutralized SARS-CoV-2. Epitope mapping, X-ray crystallography, and cryo-electron microscopy revealed two distinct antigenic sites and showed two neutralizing nanobodies from different epitope classes bound simultaneously to the spike trimer. Nanobody-Fc fusions of the four most potent nanobodies blocked ACE2 engagement with RBD variants present in human populations and potently neutralized both wild-type SARS-CoV-2 and the N501Y D614G variant at concentrations as low as 0.1 nM. Prophylactic administration of either single nanobody-Fc or as mixtures reduced viral loads by up to 104-fold in mice infected with the N501Y D614G SARS-CoV-2 virus. These results suggest a role for nanobody-Fc fusions as prophylactic agents against SARS-CoV-2.

Complement in malaria immunity and vaccines.

Developing efficacious vaccines for human malaria caused by Plasmodium falciparum is a major global health priority, although this has proven to be immensely challenging over the decades. One major hindrance is the incomplete understanding of specific immune responses that confer protection against disease and/or infection. While antibodies to play a crucial role in malaria immunity, the functional mechanisms of these antibodies remain unclear as most research has primarily focused on the direct inhibitory or neutralizing activity of antibodies. Recently, there is a growing body of evidence that antibodies can also mediate effector functions through activating the complement system against multiple developmental stages of the parasite life cycle. These antibody-complement interactions can have detrimental consequences to parasite function and viability, and have been significantly associated with protection against clinical malaria in naturally acquired immunity, and emerging findings suggest these mechanisms could contribute to vaccine-induced immunity. In order to develop highly efficacious vaccines, strategies are needed that prioritize the induction of antibodies with enhanced functional activity, including the ability to activate complement. Here we review the role of complement in acquired immunity to malaria, and provide insights into how this knowledge could be used to harness complement in malaria vaccine development.

Antibody Fc-binding profiles and ACE2 affinity to SARS-CoV-2 RBD variants.

Emerging SARS-CoV-2 variants, notably Omicron, continue to remain a formidable challenge to worldwide public health. The SARS-CoV-2 receptor-binding domain (RBD) is a hotspot for mutations, reflecting its critical role at the ACE2 interface during viral entry. Here, we comprehensively investigated the impact of RBD mutations, including 5 variants of concern (VOC) or interest-including Omicron (BA.2)-and 33 common point mutations, both on IgG recognition and ACE2-binding inhibition, as well as FcγRIIa- and FcγRIIIa-binding antibodies, in plasma from two-dose BNT162b2-vaccine recipients and mild-COVID-19 convalescent subjects obtained during the first wave using a custom-designed bead-based 39-plex array. IgG-recognition and FcγR-binding antibodies were decreased against the RBD of Beta and Omicron, as well as point mutation G446S, found in several Omicron sub-variants as compared to wild type. Notably, while there was a profound decrease in ACE2 inhibition against Omicron, FcγR-binding antibodies were less affected, suggesting that Fc functional antibody responses may be better retained against the RBD of Omicron in comparison to neutralization. Furthermore, while measurement of RBD-ACE2-binding affinity via biolayer interferometry showed that all VOC RBDs have enhanced affinity to human ACE2, we demonstrate that human ACE2 polymorphisms, E35K (rs1348114695) has reduced affinity to VOCs, while K26R (rs4646116) and S19P (rs73635825) have increased binding kinetics to the RBD of VOCs, potentially affecting virus-host interaction and, thereby, host susceptibility. Collectively, our findings provide in-depth coverage of the impact of RBD mutations on key facets of host-virus interactions.

Nanobodies against Pfs230 block Plasmodium falciparum transmission.

Transmission blocking interventions can stop malaria parasite transmission from mosquito to human by inhibiting parasite infection in mosquitos. One of the most advanced candidates for a malaria transmission blocking vaccine is Pfs230. Pfs230 is the largest member of the 6-cysteine protein family with 14 consecutive 6-cysteine domains and is expressed on the surface of gametocytes and gametes. Here, we present the crystal structure of the first two 6-cysteine domains of Pfs230. We identified high affinity Pfs230-specific nanobodies that recognized gametocytes and bind to distinct sites on Pfs230, which were isolated from immunized alpacas. Using two non-overlapping Pfs230 nanobodies, we show that these nanobodies significantly blocked P. falciparum transmission and reduced the formation of exflagellation centers. Crystal structures of the transmission blocking nanobodies with the first 6-cysteine domain of Pfs230 confirm that they bind to different epitopes. In addition, these nanobodies bind to Pfs230 in the absence of the prodomain, in contrast with the binding of known Pfs230 transmission blocking antibodies. These results provide additional structural insight into Pfs230 domains and elucidate a mechanism of action of transmission blocking Pfs230 nanobodies.

Naturally acquired antibody kinetics against Plasmodium vivax antigens in people from a low malaria transmission region in western Thailand.

BACKGROUND: Plasmodium vivax (P. vivax) is the dominant Plasmodium spp. causing the disease malaria in low-transmission regions outside of Africa. These regions often feature high proportions of asymptomatic patients with sub-microscopic parasitaemia and relapses. Naturally acquired antibody responses are induced after Plasmodium infection, providing partial protection against high parasitaemia and clinical episodes. However, previous work has failed to address the presence and maintenance of such antibody responses to P. vivax particularly in low-transmission regions. METHODS: We followed 34 patients in western Thailand after symptomatic P. vivax infections to monitor antibody kinetics over 9 months, during which no recurrent infections occurred. We assessed total IgG, IgG subclass and IgM levels to up to 52 P. vivax proteins every 2-4 weeks using a multiplexed Luminex® assay and identified protein-specific variation in antibody longevity. Mathematical modelling was used to generate the estimated half-life of antibodies, long-, and short-lived antibody-secreting cells. RESULTS: Generally, an increase in antibody level was observed within 1-week post symptomatic infection, followed by an exponential decay of different rates. We observed mostly IgG1 dominance and IgG3 sub-dominance in this population. IgM responses followed similar kinetic patterns to IgG, with some proteins unexpectedly inducing long-lived IgM responses. We also monitored antibody responses against 27 IgG-immunogenic antigens in 30 asymptomatic individuals from a similar region. Our results demonstrate that most antigens induced robust and long-lived total IgG responses following asymptomatic infections in the absence of (detected) boosting infections. CONCLUSIONS: Our work provides new insights into the development and maintenance of naturally acquired immunity to P. vivax and will guide the potential use of serology to indicate immune status and/or identify populations at risk.

Surface area-to-volume ratio, not cellular viscoelasticity, is the major determinant of red blood cell traversal through small channels.

The remarkable deformability of red blood cells (RBCs) depends on the viscoelasticity of the plasma membrane and cell contents and the surface area to volume (SA:V) ratio; however, it remains unclear which of these factors is the key determinant for passage through small capillaries. We used a microfluidic device to examine the traversal of normal, stiffened, swollen, parasitised and immature RBCs. We show that dramatic stiffening of RBCs had no measurable effect on their ability to traverse small channels. By contrast, a moderate decrease in the SA:V ratio had a marked effect on the equivalent cylinder diameter that is traversable by RBCs of similar cellular viscoelasticity. We developed a finite element model that provides a coherent rationale for the experimental observations, based on the nonlinear mechanical behaviour of the RBC membrane skeleton. We conclude that the SA:V ratio should be given more prominence in studies of RBC pathologies.

Comparison of total immunoglobulin G antibody responses to different protein fragments of Plasmodium vivax Reticulocyte binding protein 2b.

BACKGROUND: Plasmodium vivax is emerging as the dominant and prevalent species causing malaria in near-elimination settings outside of Africa. Hypnozoites, the dormant liver stage parasite of P. vivax, are undetectable to any currently available diagnostic test, yet are a major reservoir for transmission. Advances have been made to harness the naturally acquired immune response to identify recent exposure to P. vivax blood-stage parasites and, therefore, infer the presence of hypnozoites. This in-development diagnostic is currently able to detect infections within the last 9-months with 80% sensitivity and 80% specificity. Further work is required to optimize protein expression and protein constructs used for antibody detection. METHODS: The antibody response against the top performing predictor of recent infection, P. vivax reticulocyte binding protein 2b (PvRBP2b), was tested against multiple fragments of different sizes and from different expression systems. The IgG induced against the recombinant PvRBP2b fragments in P. vivax infected individuals was measured at the time of infection and in a year-long observational cohort; both conducted in Thailand. RESULTS: The antibody responses to some but not all different sized fragments of PvRBP2b protein are highly correlated with each other, significantly higher 1-week post-P. vivax infection, and show potential for use as predictors of recent P. vivax infection. CONCLUSIONS: To achieve P. vivax elimination goals, novel diagnostics are required to aid in detection of hidden parasite reservoirs. PvRBP2b was previously shown to be the top candidate for single-antigen classification of recent P. vivax exposure and here, it is concluded that several alternative recombinant PvRBP2b fragments can achieve equal sensitivity and specificity at predicting recent P. vivax exposure.

Nanobody generation and structural characterization of Plasmodium falciparum 6-cysteine protein Pf12p.

Surface-associated proteins play critical roles in the Plasmodium parasite life cycle and are major targets for vaccine development. The 6-cysteine (6-cys) protein family is expressed in a stage-specific manner throughout Plasmodium falciparum life cycle and characterized by the presence of 6-cys domains, which are ß-sandwich domains with conserved sets of disulfide bonds. Although several 6-cys family members have been implicated to play a role in sexual stages, mosquito transmission, evasion of the host immune response and host cell invasion, the precise function of many family members is still unknown and structural information is only available for four 6-cys proteins. Here, we present to the best of our knowledge, the first crystal structure of the 6-cys protein Pf12p determined at 2.8 Šresolution. The monomeric molecule folds into two domains, D1 and D2, both of which adopt the canonical 6-cys domain fold. Although the structural fold is similar to that of Pf12, its paralog in P. falciparum, we show that Pf12p does not complex with Pf41, which is a known interaction partner of Pf12. We generated 10 distinct Pf12p-specific nanobodies which map into two separate epitope groups; one group which binds within the D2 domain, while several members of the second group bind at the interface of the D1 and D2 domain of Pf12p. Characterization of the structural features of the 6-cys family and their associated nanobodies provide a framework for generating new tools to study the diverse functions of the 6-cys protein family in the Plasmodium life cycle.

Plasmodium vivax Reticulocyte Binding Proteins for invasion into reticulocytes.

Plasmodium vivax is responsible for most of the malaria infections outside Africa and is currently the predominant malaria parasite in countries under elimination programs. P. vivax preferentially enters young red cells called reticulocytes. Advances in understanding the molecular and cellular mechanisms of entry are hampered by the inability to grow large numbers of P. vivax parasites in a long-term in vitro culture. Recent progress in understanding the biology of the P. vivax Reticulocyte Binding Protein (PvRBPs) family of invasion ligands has led to the identification of a new invasion pathway into reticulocytes, an understanding of their structural architecture and PvRBPs as targets of the protective immune response to P. vivax infection. This review summarises current knowledge on the role of reticulocytes in P. vivax infection, the function of the PvRBP family of proteins in generating an immune response in human populations, and the characterization of anti-PvRBP antibodies in blocking parasite invasion.

Evolutionary history of human Plasmodium vivax revealed by genome-wide analyses of related ape parasites.

Wild-living African apes are endemically infected with parasites that are closely related to human Plasmodium vivax, a leading cause of malaria outside Africa. This finding suggests that the origin of P. vivax was in Africa, even though the parasite is now rare in humans there. To elucidate the emergence of human P. vivax and its relationship to the ape parasites, we analyzed genome sequence data of P. vivax strains infecting six chimpanzees and one gorilla from Cameroon, Gabon, and Côte d'Ivoire. We found that ape and human parasites share nearly identical core genomes, differing by only 2% of coding sequences. However, compared with the ape parasites, human strains of P. vivax exhibit about 10-fold less diversity and have a relative excess of nonsynonymous nucleotide polymorphisms, with site-frequency spectra suggesting they are subject to greatly relaxed purifying selection. These data suggest that human P. vivax has undergone an extreme bottleneck, followed by rapid population expansion. Investigating potential host-specificity determinants, we found that ape P. vivax parasites encode intact orthologs of three reticulocyte-binding protein genes (rbp2d, rbp2e, and rbp3), which are pseudogenes in all human P. vivax strains. However, binding studies of recombinant RBP2e and RBP3 proteins to human, chimpanzee, and gorilla erythrocytes revealed no evidence of host-specific barriers to red blood cell invasion. These data suggest that, from an ancient stock of P. vivax parasites capable of infecting both humans and apes, a severely bottlenecked lineage emerged out of Africa and underwent rapid population growth as it spread globally.

Neutralising antibodies block the function of Rh5/Ripr/CyRPA complex during invasion of Plasmodium falciparum into human erythrocytes.

An effective vaccine is a priority for malaria control and elimination. The leading candidate in the Plasmodium falciparum blood stage is PfRh5. PfRh5 assembles into trimeric complex with PfRipr and PfCyRPA in the parasite, and this complex is essential for erythrocyte invasion. In this study, we show that antibodies specific for PfRh5 and PfCyRPA prevent trimeric complex formation. We identify the EGF-7 domain on PfRipr as a neutralising epitope and demonstrate that antibodies against this region act downstream of complex formation to prevent merozoite invasion. Antibodies against the C-terminal region of PfRipr were more inhibitory than those against either PfRh5 or PfCyRPA alone, and a combination of antibodies against PfCyRPA and PfRipr acted synergistically to reduce invasion. This study supports prioritisation of PfRipr for development as part of a next-generation antimalarial vaccine.

Evaluation of 4-Amino 2-Anilinoquinazolines against Plasmodium and Other Apicomplexan Parasites In Vitro and in a P. falciparum Humanized NOD-scid IL2Rγnull Mouse Model of Malaria.

A series of 4-amino 2-anilinoquinazolines optimized for activity against the most lethal malaria parasite of humans, Plasmodium falciparum, was evaluated for activity against other human Plasmodium parasites and related apicomplexans that infect humans and animals. Four of the most promising compounds from the 4-amino 2-anilinoquinazoline series were equally as effective against the asexual blood stages of the zoonotic P. knowlesi, suggesting that they could also be effective against the closely related P. vivax, another important human pathogen. The 2-anilinoquinazoline compounds were also potent against an array of P. falciparum parasites resistant to clinically available antimalarial compounds, although slightly less so than against the drug-sensitive 3D7 parasite line. The apicomplexan parasites Toxoplasma gondii, Babesia bovis, and Cryptosporidium parvum were less sensitive to the 2-anilinoquinazoline series with a 50% effective concentration generally in the low micromolar range, suggesting that the yet to be discovered target of these compounds is absent or highly divergent in non-Plasmodium parasites. The 2-anilinoquinazoline compounds act as rapidly as chloroquine in vitro and when tested in rodents displayed a half-life that contributed to the compound's capacity to clear P. falciparum blood stages in a humanized mouse model. At a dose of 50 mg/kg of body weight, adverse effects to the humanized mice were noted, and evaluation against a panel of experimental high-risk off targets indicated some potential off-target activity. Further optimization of the 2-anilinoquinazoline antimalarial class will concentrate on improving in vivo efficacy and addressing adverse risk.

Recruitment of Human C1 Esterase Inhibitor Controls Complement Activation on Blood Stage Plasmodium falciparum Merozoites.

The complement system is a front-line defense system that opsonizes and lyses invading pathogens. To survive, microbes exposed to serum must evade the complement response. To achieve this, many pathogens recruit soluble human complement regulators to their surfaces and hijack their regulatory function for protection from complement activation. C1 esterase inhibitor (C1-INH) is a soluble regulator of complement activation that negatively regulates the classical and lectin pathways of complement to protect human tissue from aberrant activation. In this article, we show that Plasmodium falciparum merozoites, the invasive form of blood stage malaria parasites, actively recruit C1-INH to their surfaces when exposed to human serum. We identified PfMSP3.1, a member of the merozoite surface protein 3 family of merozoite surface proteins, as the direct interaction partner. When bound to the merozoite surface, C1-INH retains its ability to complex with and inhibit C1s, MASP1, and MASP2, the activating proteases of the complement cascade. P. falciparum merozoites that lack PfMSP3.1 showed a marked reduction in C1-INH recruitment and increased C3b deposition on their surfaces. However, these ΔPfMSP3.1 merozoites exhibit enhanced invasion of RBCs in the presence of active complement. This study characterizes an immune-evasion strategy used by malaria parasites and highlights the complex relationship between merozoites and the complement system.

Structurally conserved erythrocyte-binding domain in Plasmodium provides a versatile scaffold for alternate receptor engagement.

Understanding how malaria parasites gain entry into human red blood cells is essential for developing strategies to stop blood stage infection. Plasmodium vivax preferentially invades reticulocytes, which are immature red blood cells. The organism has two erythrocyte-binding protein families: namely, the Duffy-binding protein (PvDBP) and the reticulocyte-binding protein (PvRBP) families. Several members of the PvRBP family bind reticulocytes, specifically suggesting a role in mediating host cell selectivity of P. vivax. Here, we present, to our knowledge, the first high-resolution crystal structure of an erythrocyte-binding domain from PvRBP2a, solved at 2.12 Å resolution. The monomeric molecule consists of 10 α-helices and one short ß-hairpin, and, although the structural fold is similar to that of PfRh5--the essential invasion ligand in Plasmodium falciparum--its surface properties are distinct and provide a possible mechanism for recognition of alternate receptors. Sequence alignments of the crystallized fragment of PvRBP2a with other PvRBPs highlight the conserved placement of disulfide bonds. PvRBP2a binds mature red blood cells through recognition of an erythrocyte receptor that is neuraminidase- and chymotrypsin-resistant but trypsin-sensitive. By examining the patterns of sequence diversity within field isolates, we have identified and mapped polymorphic residues to the PvRBP2a structure. Using mutagenesis, we have also defined the critical residues required for erythrocyte binding. Characterization of the structural features that govern functional erythrocyte binding for the PvRBP family provides a framework for generating new tools that block P. vivax blood stage infection.

Recruitment of Factor H as a Novel Complement Evasion Strategy for Blood-Stage Plasmodium falciparum Infection.

The human complement system is the frontline defense mechanism against invading pathogens. The coexistence of humans and microbes throughout evolution has produced ingenious molecular mechanisms by which microorganisms escape complement attack. A common evasion strategy used by diverse pathogens is the hijacking of soluble human complement regulators to their surfaces to afford protection from complement activation. One such host regulator is factor H (FH), which acts as a negative regulator of complement to protect host tissues from aberrant complement activation. In this report, we show that Plasmodium falciparum merozoites, the invasive form of the malaria parasites, actively recruit FH and its alternative spliced form FH-like protein 1 when exposed to human serum. We have mapped the binding site in FH that recognizes merozoites and identified Pf92, a member of the six-cysteine family of Plasmodium surface proteins, as its direct interaction partner. When bound to merozoites, FH retains cofactor activity, a key function that allows it to downregulate the alternative pathway of complement. In P. falciparum parasites that lack Pf92, we observed changes in the pattern of C3b cleavage that are consistent with decreased regulation of complement activation. These results also show that recruitment of FH affords P. falciparum merozoites protection from complement-mediated lysis. Our study provides new insights on mechanisms of immune evasion of malaria parasites and highlights the important function of surface coat proteins in the interplay between complement regulation and successful infection of the host.

Plasmodium falciparum Adhesins Play an Essential Role in Signalling and Activation of Invasion into Human Erythrocytes.

The most severe form of malaria in humans is caused by the protozoan parasite Plasmodium falciparum. The invasive form of malaria parasites is termed a merozoite and it employs an array of parasite proteins that bind to the host cell to mediate invasion. In Plasmodium falciparum, the erythrocyte binding-like (EBL) and reticulocyte binding-like (Rh) protein families are responsible for binding to specific erythrocyte receptors for invasion and mediating signalling events that initiate active entry of the malaria parasite. Here we have addressed the role of the cytoplasmic tails of these proteins in activating merozoite invasion after receptor engagement. We show that the cytoplasmic domains of these type 1 membrane proteins are phosphorylated in vitro. Depletion of PfCK2, a kinase implicated to phosphorylate these cytoplasmic tails, blocks P. falciparum invasion of red blood cells. We identify the crucial residues within the PfRh4 cytoplasmic domain that are required for successful parasite invasion. Live cell imaging of merozoites from these transgenic mutants show they attach but do not penetrate erythrocytes implying the PfRh4 cytoplasmic tail conveys signals important for the successful completion of the invasion process.

Revealing the sequence and resulting cellular morphology of receptor-ligand interactions during Plasmodium falciparum invasion of erythrocytes.

During blood stage Plasmodium falciparum infection, merozoites invade uninfected erythrocytes via a complex, multistep process involving a series of distinct receptor-ligand binding events. Understanding each element in this process increases the potential to block the parasite's life cycle via drugs or vaccines. To investigate specific receptor-ligand interactions, they were systematically blocked using a combination of genetic deletion, enzymatic receptor cleavage and inhibition of binding via antibodies, peptides and small molecules, and the resulting temporal changes in invasion and morphological effects on erythrocytes were filmed using live cell imaging. Analysis of the videos have shown receptor-ligand interactions occur in the following sequence with the following cellular morphologies; 1) an early heparin-blockable interaction which weakly deforms the erythrocyte, 2) EBA and PfRh ligands which strongly deform the erythrocyte, a process dependant on the merozoite's actin-myosin motor, 3) a PfRh5-basigin binding step which results in a pore or opening between parasite and host through which it appears small molecules and possibly invasion components can flow and 4) an AMA1-RON2 interaction that mediates tight junction formation, which acts as an anchor point for internalization. In addition to enhancing general knowledge of apicomplexan biology, this work provides a rational basis to combine sequentially acting merozoite vaccine candidates in a single multi-receptor-blocking vaccine.