The Antibody Response to Plasmodium falciparum: Cues for Vaccine Design and the Discovery of Receptor-Based Antibodies.

Plasmodium falciparum remains a serious public health problem and a continuous challenge for the immune system due to the complexity and diversity of the pathogen. Recent advances from several laboratories in the characterization of the antibody response to the parasite have led to the identification of critical targets for protection and revealed a new mechanism of diversification based on the insertion of host receptors into immunoglobulin genes, leading to the production of receptor-based antibodies. These advances have opened new possibilities for vaccine design and passive antibody therapies to provide sterilizing immunity and control blood-stage parasites.

The RTS,S vaccine-a chance to regain the upper hand against malaria?

Malaria is estimated by the World Health Organization (WHO) to have killed 627,000 individuals worldwide in 2020, with nearly 80% of deaths in African children younger than five. The recent WHO approval of the RTS,S/AS01 vaccine, which targets Plasmodium falciparum pre-erythrocytic stages, provides hope that its use combined with other interventions can help reverse the current malaria resurgence.

Structural Basis for Blocking Sugar Uptake into the Malaria Parasite Plasmodium falciparum.

Plasmodium species, the causative agent of malaria, rely on glucose for energy supply during blood stage. Inhibition of glucose uptake thus represents a potential strategy for the development of antimalarial drugs. Here, we present the crystal structures of PfHT1, the sole hexose transporter in the genome of Plasmodium species, at resolutions of 2.6 Å in complex with D-glucose and 3.7 Å with a moderately selective inhibitor, C3361. Although both structures exhibit occluded conformations, binding of C3361 induces marked rearrangements that result in an additional pocket. This inhibitor-binding-induced pocket presents an opportunity for the rational design of PfHT1-specific inhibitors. Among our designed C3361 derivatives, several exhibited improved inhibition of PfHT1 and cellular potency against P. falciparum, with excellent selectivity to human GLUT1. These findings serve as a proof of concept for the development of the next-generation antimalarial chemotherapeutics by simultaneously targeting the orthosteric and allosteric sites of PfHT1.

SnapShot: Antimalarial Drugs.

Malaria is a prominent vector-borne illness caused by Plasmodium parasites. Therapeutic intervention remains a critical component for disease eradication efforts but is complicated by the emergence of drug resistance. This SnapShot summarizes the human-relevant stages of the P. falciparum life cycle and describes how licensed antimalarials, clinical candidates, and newly emerging compounds target each stage to prevent, treat, or block transmission of malaria. To view this SnapShot, open or download the PDF.

Human Antibodies that Slow Erythrocyte Invasion Potentiate Malaria-Neutralizing Antibodies.

The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is the leading target for next-generation vaccines against the disease-causing blood-stage of malaria. However, little is known about how human antibodies confer functional immunity against this antigen. We isolated a panel of human monoclonal antibodies (mAbs) against PfRH5 from peripheral blood B cells from vaccinees in the first clinical trial of a PfRH5-based vaccine. We identified a subset of mAbs with neutralizing activity that bind to three distinct sites and another subset of mAbs that are non-functional, or even antagonistic to neutralizing antibodies. We also identify the epitope of a novel group of non-neutralizing antibodies that significantly reduce the speed of red blood cell invasion by the merozoite, thereby potentiating the effect of all neutralizing PfRH5 antibodies as well as synergizing with antibodies targeting other malaria invasion proteins. Our results provide a roadmap for structure-guided vaccine development to maximize antibody efficacy against blood-stage malaria.

Steroid Hormone Function Controls Non-competitive Plasmodium Development in Anopheles.

Transmission of malaria parasites occurs when a female Anopheles mosquito feeds on an infected host to acquire nutrients for egg development. How parasites are affected by oogenetic processes, principally orchestrated by the steroid hormone 20-hydroxyecdysone (20E), remains largely unknown. Here we show that Plasmodium falciparum development is intimately but not competitively linked to processes shaping Anopheles gambiae reproduction. We unveil a 20E-mediated positive correlation between egg and oocyst numbers; impairing oogenesis by multiple 20E manipulations decreases parasite intensities. These manipulations, however, accelerate Plasmodium growth rates, allowing sporozoites to become infectious sooner. Parasites exploit mosquito lipids for faster growth, but they do so without further affecting egg development. These results suggest that P. falciparum has adopted a non-competitive evolutionary strategy of resource exploitation to optimize transmission while minimizing fitness costs to its mosquito vector. Our findings have profound implications for currently proposed control strategies aimed at suppressing mosquito populations.

Systems analysis and controlled malaria infection in Europeans and Africans elucidate naturally acquired immunity.

Controlled human infections provide opportunities to study the interaction between the immune system and malaria parasites, which is essential for vaccine development. Here, we compared immune signatures of malaria-naive Europeans and of Africans with lifelong malaria exposure using mass cytometry, RNA sequencing and data integration, before and 5 and 11 days after venous inoculation with Plasmodium falciparum sporozoites. We observed differences in immune cell populations, antigen-specific responses and gene expression profiles between Europeans and Africans and among Africans with differing degrees of immunity. Before inoculation, an activated/differentiated state of both innate and adaptive cells, including elevated CD161+CD4+ T cells and interferon-γ production, predicted Africans capable of controlling parasitemia. After inoculation, the rapidity of the transcriptional response and clusters of CD4+ T cells, plasmacytoid dendritic cells and innate T cells were among the features distinguishing Africans capable of controlling parasitemia from susceptible individuals. These findings can guide the development of a vaccine effective in malaria-endemic regions.

γδ T cells suppress Plasmodium falciparum blood-stage infection by direct killing and phagocytosis.

Activated Vγ9Vδ2 (γδ2) T lymphocytes that sense parasite-produced phosphoantigens are expanded in Plasmodium falciparum-infected patients. Although previous studies suggested that γδ2 T cells help control erythrocytic malaria, whether γδ2 T cells recognize infected red blood cells (iRBCs) was uncertain. Here we show that iRBCs stained for the phosphoantigen sensor butyrophilin 3A1 (BTN3A1). γδ2 T cells formed immune synapses and lysed iRBCs in a contact, phosphoantigen, BTN3A1 and degranulation-dependent manner, killing intracellular parasites. Granulysin released into the synapse lysed iRBCs and delivered death-inducing granzymes to the parasite. All intra-erythrocytic parasites were susceptible, but schizonts were most sensitive. A second protective γδ2 T cell mechanism was identified. In the presence of patient serum, γδ2 T cells phagocytosed and degraded opsonized iRBCs in a CD16-dependent manner, decreasing parasite multiplication. Thus, γδ2 T cells have two ways to control blood-stage malaria-γδ T cell antigen receptor (TCR)-mediated degranulation and phagocytosis of antibody-coated iRBCs.

Breadth of Fc-mediated effector function correlates with clinical immunity following human malaria challenge.

Malaria is a life-threatening disease of global health importance, particularly in sub-Saharan Africa. The growth inhibition assay (GIA) is routinely used to evaluate, prioritize, and quantify the efficacy of malaria blood-stage vaccine candidates but does not reliably predict either naturally acquired or vaccine-induced protection. Controlled human malaria challenge studies in semi-immune volunteers provide an unparalleled opportunity to robustly identify mechanistic correlates of protection. We leveraged this platform to undertake a head-to-head comparison of seven functional antibody assays that are relevant to immunity against the erythrocytic merozoite stage of Plasmodium falciparum. Fc-mediated effector functions were strongly associated with protection from clinical symptoms of malaria and exponential parasite multiplication, while the gold standard GIA was not. The breadth of Fc-mediated effector function discriminated clinical immunity following the challenge. These findings present a shift in the understanding of the mechanisms that underpin immunity to malaria and have important implications for vaccine development.

X marks the shot against malaria.

The development of a transmission-blocking vaccine (TBV) against malaria is hampered by poor understanding of functional antibody responses. In this issue of Immunity, Fabra-Garcia et al., Ivanochko et al., and Tang et al. isolate human monoclonal antibodies against the two most promising TBV candidates, Pfs48/45 and Pfs230, and map the epitopes responsible for potent transmission-reducing activity.

Potent transmission-blocking monoclonal antibodies from naturally exposed individuals target a conserved epitope on Plasmodium falciparum Pfs230.

Pfs230 is essential for Plasmodium falciparum transmission to mosquitoes and is the protein targeted by the most advanced malaria-transmission-blocking vaccine candidate. Prior understanding of functional epitopes on Pfs230 is based on two monoclonal antibodies (mAbs) with moderate transmission-reducing activity (TRA), elicited from subunit immunization. Here, we screened the B cell repertoire of two naturally exposed individuals possessing serum TRA and identified five potent mAbs from sixteen Pfs230 domain-1-specific mAbs. Structures of three potent and three low-activity antibodies bound to Pfs230 domain 1 revealed four distinct epitopes. Highly potent mAbs from natural infection recognized a common conformational epitope that is highly conserved across P. falciparum field isolates, while antibodies with negligible TRA derived from natural infection or immunization recognized three distinct sites. Our study provides molecular blueprints describing P. falciparum TRA, informed by contrasting potent and non-functional epitopes elicited by natural exposure and vaccination.

Highly potent, naturally acquired human monoclonal antibodies against Pfs48/45 block Plasmodium falciparum transmission to mosquitoes.

Malaria transmission-blocking vaccines (TBVs) aim to induce antibodies that interrupt malaria parasite development in the mosquito, thereby blocking onward transmission, and provide a much-needed tool for malaria control and elimination. The parasite surface protein Pfs48/45 is a leading TBV candidate. Here, we isolated and characterized a panel of 81 human Pfs48/45-specific monoclonal antibodies (mAbs) from donors naturally exposed to Plasmodium parasites. Genetically diverse mAbs against each of the three domains (D1-D3) of Pfs48/45 were identified. The most potent mAbs targeted D1 and D3 and achieved >80% transmission-reducing activity in standard membrane-feeding assays, at 10 and 2 µg/mL, respectively. Co-crystal structures of D3 in complex with four different mAbs delineated two conserved protective epitopes. Altogether, these Pfs48/45-specific human mAbs provide important insight into protective and non-protective epitopes that can further our understanding of transmission and inform the design of refined malaria transmission-blocking vaccine candidates.

Vaccination with a structure-based stabilized version of malarial antigen Pfs48/45 elicits ultra-potent transmission-blocking antibody responses.

Malaria transmission-blocking vaccines (TBVs) aim to elicit human antibodies that inhibit sporogonic development of Plasmodium falciparum in mosquitoes, thereby preventing onward transmission. Pfs48/45 is a leading clinical TBV candidate antigen and is recognized by the most potent transmission-blocking monoclonal antibody (mAb) yet described; still, clinical development of Pfs48/45 antigens has been hindered, largely by its poor biochemical characteristics. Here, we used structure-based computational approaches to design Pfs48/45 antigens stabilized in the conformation recognized by the most potently inhibitory mAb, achieving >25°C higher thermostability compared with the wild-type protein. Antibodies elicited in mice immunized with these engineered antigens displayed on liposome-based or protein nanoparticle-based vaccine platforms exhibited 1-2 orders of magnitude superior transmission-reducing activity, compared with immunogens bearing the wild-type antigen, driven by improved antibody quality. Our data provide the founding principles for using molecular stabilization solely from antibody structure-function information to drive improved immune responses against a parasitic vaccine target.

Malaria: Biology and Disease.

Malaria has been a major global health problem of humans through history and is a leading cause of death and disease across many tropical and subtropical countries. Over the last fifteen years renewed efforts at control have reduced the prevalence of malaria by over half, raising the prospect that elimination and perhaps eradication may be a long-term possibility. Achievement of this goal requires the development of new tools including novel antimalarial drugs and more efficacious vaccines as well as an increased understanding of the disease and biology of the parasite. This has catalyzed a major effort resulting in development and regulatory approval of the first vaccine against malaria (RTS,S/AS01) as well as identification of novel drug targets and antimalarial compounds, some of which are in human clinical trials.

A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes.

Apicomplexan parasites are leading causes of human and livestock diseases such as malaria and toxoplasmosis, yet most of their genes remain uncharacterized. Here, we present the first genome-wide genetic screen of an apicomplexan. We adapted CRISPR/Cas9 to assess the contribution of each gene from the parasite Toxoplasma gondii during infection of human fibroblasts. Our analysis defines ∼200 previously uncharacterized, fitness-conferring genes unique to the phylum, from which 16 were investigated, revealing essential functions during infection of human cells. Secondary screens identify as an invasion factor the claudin-like apicomplexan microneme protein (CLAMP), which resembles mammalian tight-junction proteins and localizes to secretory organelles, making it critical to the initiation of infection. CLAMP is present throughout sequenced apicomplexan genomes and is essential during the asexual stages of the malaria parasite Plasmodium falciparum. These results provide broad-based functional information on T. gondii genes and will facilitate future approaches to expand the horizon of antiparasitic interventions.

The PfRCR complex bridges malaria parasite and erythrocyte during invasion.

The symptoms of malaria occur during the blood stage of infection, when parasites invade and replicate within human erythrocytes. The PfPCRCR complex1, containing PfRH5 (refs. 2,3), PfCyRPA, PfRIPR, PfCSS and PfPTRAMP, is essential for erythrocyte invasion by the deadliest human malaria parasite, Plasmodium falciparum. Invasion can be prevented by antibodies3-6 or nanobodies1 against each of these conserved proteins, making them the leading blood-stage malaria vaccine candidates. However, little is known about how PfPCRCR functions during invasion. Here we present the structure of the PfRCR complex7,8, containing PfRH5, PfCyRPA and PfRIPR, determined by cryogenic-electron microscopy. We test the hypothesis that PfRH5 opens to insert into the membrane9, instead showing that a rigid, disulfide-locked PfRH5 can mediate efficient erythrocyte invasion. We show, through modelling and an erythrocyte-binding assay, that PfCyRPA-binding antibodies5 neutralize invasion through a steric mechanism. We determine the structure of PfRIPR, showing that it consists of an ordered, multidomain core flexibly linked to an elongated tail. We also show that the elongated tail of PfRIPR, which is the target of growth-neutralizing antibodies6, binds to the PfCSS-PfPTRAMP complex on the parasite membrane. A modular PfRIPR is therefore linked to the merozoite membrane through an elongated tail, and its structured core presents PfCyRPA and PfRH5 to interact with erythrocyte receptors. This provides fresh insight into the molecular mechanism of erythrocyte invasion and opens the way to new approaches in rational vaccine design.

Gut microbiota elicits a protective immune response against malaria transmission.

Glycosylation processes are under high natural selection pressure, presumably because these can modulate resistance to infection. Here, we asked whether inactivation of the UDP-galactose:ß-galactoside-α1-3-galactosyltransferase (α1,3GT) gene, which ablated the expression of the Galα1-3Galß1-4GlcNAc-R (α-gal) glycan and allowed for the production of anti-α-gal antibodies (Abs) in humans, confers protection against Plasmodium spp. infection, the causative agent of malaria and a major driving force in human evolution. We demonstrate that both Plasmodium spp. and the human gut pathobiont E. coli O86:B7 express α-gal and that anti-α-gal Abs are associated with protection against malaria transmission in humans as well as in α1,3GT-deficient mice, which produce protective anti-α-gal Abs when colonized by E. coli O86:B7. Anti-α-gal Abs target Plasmodium sporozoites for complement-mediated cytotoxicity in the skin, immediately after inoculation by Anopheles mosquitoes. Vaccination against α-gal confers sterile protection against malaria in mice, suggesting that a similar approach may reduce malaria transmission in humans.

Epigenetic Regulation and Chromatin Remodeling in Malaria Parasites.

Plasmodium falciparum, the human malaria parasite, infects two hosts and various cell types, inducing distinct morphological and physiological changes in the parasite in response to different environmental conditions. These variations required the parasite to adapt and develop elaborate molecular mechanisms to ensure its spread and transmission. Recent findings have significantly improved our understanding of the regulation of gene expression in P. falciparum. Here, we provide an up-to-date overview of technologies used to highlight the transcriptomic adjustments occurring in the parasite throughout its life cycle. We also emphasize the complementary and complex epigenetic mechanisms regulating gene expression in malaria parasites. This review concludes with an outlook on the chromatin architecture, the remodeling systems, and how this 3D genome organization is critical in various biological processes.

A Molecular Signature in Blood Reveals a Role for p53 in Regulating Malaria-Induced Inflammation.

Immunity that controls parasitemia and inflammation during Plasmodium falciparum (Pf) malaria can be acquired with repeated infections. A limited understanding of this complex immune response impedes the development of vaccines and adjunctive therapies. We conducted a prospective systems biology study of children who differed in their ability to control parasitemia and fever following Pf infection. By integrating whole-blood transcriptomics, flow-cytometric analysis, and plasma cytokine and antibody profiles, we demonstrate that a pre-infection signature of B cell enrichment, upregulation of T helper type 1 (Th1) and Th2 cell-associated pathways, including interferon responses, and p53 activation associated with control of malarial fever and coordinated with Pf-specific immunoglobulin G (IgG) and Fc receptor activation to control parasitemia. Our hypothesis-generating approach identified host molecules that may contribute to differential clinical outcomes during Pf infection. As a proof of concept, we have shown that enhanced p53 expression in monocytes attenuated Plasmodium-induced inflammation and predicted protection from fever.