A human antibody epitope map of Pfs230D1 derived from analysis of individuals vaccinated with a malaria transmission-blocking vaccine.

Pfs230 domain 1 (Pfs230D1) is an advanced malaria transmission-blocking vaccine antigen demonstrating high functional activity in clinical trials. However, the structural and functional correlates of transmission-blocking activity are not defined. Here, we characterized a panel of human monoclonal antibodies (hmAbs) elicited in vaccinees immunized with Pfs230D1. These hmAbs exhibited diverse transmission-reducing activity, yet all bound to Pfs230D1 with nanomolar affinity. We compiled epitope-binning data for seventeen hmAbs and structures of nine hmAbs complexes to construct a high-resolution epitope map and revealed that potent transmission-reducing hmAbs bound to one face of Pfs230D1, while non-potent hmAbs bound to the opposing side. The structure of Pfs230D1D2 revealed that non-potent transmission-reducing epitopes were occluded by the second domain. The hmAb epitope map delineated binary hmAb combinations that synergized for extremely high-potency, transmission-reducing activity. This work provides a high-resolution guide for structure-based design of enhanced immunogens and informs diagnostics that measure the transmission-reducing response.

Structure-guided design of VAR2CSA-based immunogens and a cocktail strategy for a placental malaria vaccine.

Placental accumulation of Plasmodium falciparum infected erythrocytes results in maternal anemia, low birth weight, and pregnancy loss. The parasite protein VAR2CSA facilitates the accumulation of infected erythrocytes in the placenta through interaction with the host receptor chondroitin sulfate A (CSA). Antibodies that prevent the VAR2CSA-CSA interaction correlate with protection from placental malaria, and VAR2CSA is a high-priority placental malaria vaccine antigen. Here, structure-guided design leveraging the full-length structures of VAR2CSA produced a stable immunogen that retains the critical conserved functional elements of VAR2CSA. The design expressed with a six-fold greater yield than the full-length protein and elicited antibodies that prevent adhesion of infected erythrocytes to CSA. The reduced size and adaptability of the designed immunogen enable efficient production of multiple variants of VAR2CSA for use in a cocktail vaccination strategy to increase the breadth of protection. These designs form strong foundations for the development of potent broadly protective placental malaria vaccines.

Plasmodium falciparum exploits CD44 as a coreceptor for erythrocyte invasion.

The malaria parasite Plasmodium falciparum invades and replicates asexually within human erythrocytes. CD44 expressed on erythrocytes was previously identified as an important host factor for P falciparum infection through a forward genetic screen, but little is known about its regulation or function in these cells, nor how it may be used by the parasite. We found that CD44 can be efficiently deleted from primary human hematopoietic stem cells using CRISPR/Cas9 genome editing, and that the efficiency of ex vivo erythropoiesis to enucleated cultured red blood cells (cRBCs) is not affected by lack of CD44. However, the rate of P falciparum invasion was reduced in CD44-null cRBCs relative to isogenic wild-type control cells, validating CD44 as an important host factor for this parasite. We identified 2 P falciparum invasion ligands as binding partners for CD44, erythrocyte binding antigen 175 (EBA-175) and EBA-140 and demonstrated that their ability to bind to human erythrocytes relies primarily on their canonical receptors, glycophorin A and glycophorin C, respectively. We further show that EBA-175 induces phosphorylation of erythrocyte cytoskeletal proteins in a CD44-dependent manner. Our findings support a model in which P falciparum exploits CD44 as a coreceptor during invasion of human erythrocytes, stimulating CD44-dependent phosphorylation of host cytoskeletal proteins that alter host cell deformability and facilitate parasite entry.

Implications of conformational flexibility, lipid binding, and regulatory domains in cell-traversal protein CelTOS for apicomplexan migration.

Malaria and other apicomplexan-caused diseases affect millions of humans, agricultural animals, and pets. Cell traversal is a common feature used by multiple apicomplexan parasites to migrate through host cells and can be exploited to develop therapeutics against these deadly parasites. Here, we provide insights into the mechanism of the Cell-traversal protein for ookinetes and sporozoites (CelTOS), a conserved cell-traversal protein in apicomplexan parasites and malaria vaccine candidate. CelTOS has previously been shown to form pores in cell membranes to enable traversal of parasites through cells. We establish roles for the distinct protein regions of Plasmodium vivax CelTOS and examine the mechanism of pore formation. We further demonstrate that CelTOS dimer dissociation is required for pore formation, as disulfide bridging between monomers inhibits pore formation, and this inhibition is rescued by disulfide-bridge reduction. We also show that a helix-destabilizing amino acid, Pro127, allows CelTOS to undergo significant conformational changes to assemble into pores. The flexible C terminus of CelTOS is a negative regulator that limits pore formation. Finally, we highlight that lipid binding is a prerequisite for pore assembly as mutation of a phospholipids-binding site in CelTOS resulted in loss of lipid binding and abrogated pore formation. These findings identify critical regions in CelTOS and will aid in understanding the egress mechanism of malaria and other apicomplexan parasites as well as have implications for studying the function of other essential pore-forming proteins.

The structure of Acinetobacter-secreted protease CpaA complexed with its chaperone CpaB reveals a novel mode of a T2SS chaperone-substrate interaction.

Members of the Acinetobacter baumannii-calcoaceticus complex are nosocomial pathogens frequently causing multidrug-resistant infections that are increasing at alarming rates. A. baumannii has become the Gram-negative bacterium with the highest rate of multidrug resistance. As such, it is categorized by the World Health Organization as a critical priority for the research and development of new antimicrobial therapies. The zinc-dependent metalloendopeptidase CpaA is a predominant substrate of the type II secretion system (T2SS). CpaA is also a virulence factor of medically relevant Acinetobacter strains that specifically degrade the human glycoprotein coagulation factor XII and not its deglycosylated form, but the mechanism for this specificity is unknown. CpaB is a membrane-anchored T2SS chaperone that interacts with CpaA and is required for its stability and secretion. Here, we report the crystal structure of the CpaAB complex at 2.6-Å resolution, revealing four glycan-binding domains in CpaA that were not predicted from its primary sequence and may explain CpaA's glycoprotein-targeting activity. The structure of the complex identified a novel mode for chaperone-protease interactions in which the protease surrounds the chaperone. The CpaAB organization was akin to zymogen inactivation, with CpaB serving as a prodomain that inhibits catalytically active CpaA. CpaB contains a C-terminal tail that appears to block access to the CpaA catalytic site, and functional experiments with truncated variants indicated that this tail is dispensable for CpaA expression and secretion. Our results provide new insight into the mechanism of CpaA secretion and may inform the future development of therapeutic strategies for managing Acinetobacter infections.

Plasmodium falciparum erythrocyte-binding antigen 175 triggers a biophysical change in the red blood cell that facilitates invasion.

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.

Moderately Neutralizing Epitopes in Nonfunctional Regions Dominate the Antibody Response to Plasmodium falciparum EBA-140.

Plasmodium falciparum erythrocyte-binding antigen 140 (EBA-140) plays a role in tight junction formation during parasite invasion of red blood cells and is a potential vaccine candidate for malaria. Individuals in areas where malaria is endemic possess EBA-140-specific antibodies, and individuals with high antibody titers to this protein have a lower rate of reinfection by parasites. The red blood cell binding segment of EBA-140 is comprised of two Duffy-binding-like domains, called F1 and F2, that together create region II. The sialic acid-binding pocket of F1 is essential for binding, whereas the sialic acid-binding pocket in F2 appears dispensable. Here, we show that immunization of mice with the complete region II results in poorly neutralizing antibodies. In contrast, immunization of mice with the functionally relevant F1 domain of region II results in antibodies that confer a 2-fold increase in parasite neutralization compared to that of the F2 domain. Epitope mapping of diverse F1 and F2 monoclonal antibodies revealed that the functionally relevant F1 sialic acid-binding pocket is a privileged site inaccessible to antibodies, that the F2 sialic acid-binding pocket contains a nonneutralizing epitope, and that two additional epitopes reside in F1 on the opposite face from the sialic acid-binding pocket. These studies indicate that focusing the immune response to the functionally important F1 sialic acid binding pocket improves the protective immune response of EBA-140. These results have implications for improving future vaccine designs and emphasize the importance of structural vaccinology for malaria.

DARC extracellular domain remodeling in maturating reticulocytes explains Plasmodium vivax tropism.

Plasmodium vivax is the most prevalent parasite species that causes malaria in humans and exclusively infects reticulocytes. Reticulocyte infection is facilitated by P vivax Duffy binding protein (DBP), which utilizes DARC (Duffy antigen receptor for chemokines) as an entry point. However, the selective tropism of P vivax for transferrin receptor (CD71)-positive reticulocytes remained unexplained, given the constitutive expression of DARC during reticulocyte maturation. CD71/RNA double staining of reticulocytes enriched from adult peripheral blood reveals 4 distinct reticulocyte populations: CD71high/RNAhigh (∼0.016%), CD71low/RNAhigh (∼0.059%), CD71neg/RNAhigh (∼0.37%), CD71neg/RNAlow (∼0.55%), and erythrocytes CD71neg/RNAneg (∼99%). We hypothesized that selective association of DBP with a small population of immature reticulocytes could explain the preference of P vivax for reticulocytes. Binding of specific monoclonal anti-DARC antibodies and recombinant DBP to CD71high/RNAhigh reticulocytes was significantly higher compared with other reticulocyte populations and erythrocytes. Interestingly, the total DARC protein throughout reticulocyte maturation was constant. The data suggest that selective exposure of the DBP binding site within DARC is key to the preferential binding of DBP to immature reticulocytes, which is the potential mechanism underlying the preferential infection of a reticulocyte subset by P vivax.

Broadly neutralizing epitopes in the Plasmodium vivax vaccine candidate Duffy Binding Protein.

Plasmodium vivax Duffy Binding Protein (PvDBP) is the most promising vaccine candidate for P. vivax malaria. The polymorphic nature of PvDBP induces strain-specific immune responses, however, and the epitopes of broadly neutralizing antibodies are unknown. These features hamper the rational design of potent DBP-based vaccines and necessitate the identification of globally conserved epitopes. Using X-ray crystallography, small-angle X-ray scattering, hydrogen-deuterium exchange mass spectrometry, and mutational mapping, we have defined epitopes for three inhibitory mAbs (mAbs 2D10, 2H2, and 2C6) and one noninhibitory mAb (3D10) that engage DBP. These studies expand the currently known inhibitory epitope repertoire by establishing protective motifs in subdomain three outside the receptor-binding and dimerization residues of DBP, and introduce globally conserved protective targets. All of the epitopes are highly conserved among DBP alleles. The identification of broadly conserved epitopes of inhibitory antibodies provides critical motifs that should be retained in the next generation of potent vaccines for P. vivax malaria.

Defining the interaction of the protease CpaA with its type II secretion chaperone CpaB and its contribution to virulence in Acinetobacter species.

Acinetobacter baumannii, Acinetobacter nosocomialis, and Acinetobacter pittii are a frequent cause of multidrug-resistant, healthcare-associated infections. Our previous work demonstrated that A. nosocomialis M2 possesses a functional type II secretion system (T2SS) that is required for full virulence. Further, we identified the metallo-endopeptidase CpaA, which has been shown previously to cleave human Factor V and deregulate blood coagulation, as the most abundant type II secreted effector protein. We also demonstrated that its secretion is dependent on CpaB, a membrane-bound chaperone. In this study, we show that CpaA expression and secretion are conserved across several medically relevant Acinetobacter species. Additionally, we demonstrate that deletion of cpaA results in attenuation of A. nosocomialis M2 virulence in moth and mouse models. The virulence defects resulting from the deletion of cpaA were comparable with those observed upon abrogation of T2SS activity. The virulence defects resulting from the deletion of cpaA are comparable with those observed upon abrogation of T2SS activity. We also show that CpaA and CpaB strongly interact, forming a complex in a 1:1 ratio. Interestingly, deletion of the N-terminal transmembrane domain of CpaB results in robust secretion of CpaA and CpaB, indicating that the transmembrane domain is dispensable for CpaA secretion and likely functions to retain CpaB inside the cell. Limited proteolysis of spheroplasts revealed that the C-terminal domain of CpaB is exposed to the periplasm, suggesting that this is the site where CpaA and CpaB interact in vivo Last, we show that CpaB does not abolish the proteolytic activity of CpaA against human Factor V. We conclude that CpaA is, to the best of our knowledge, the first characterized, bona fide virulence factor secreted by Acinetobacter species.

Red cell receptors as access points for malaria infection.

PURPOSE OF REVIEW: Red cell receptors provide unique entry points for Plasmodium parasites to initiate blood-stage malaria infection. Parasites encode distinct ligands that bind specifically to both highly abundant and low-copy receptors. Recent advances in the understanding of molecular and structural mechanisms of these interactions provide fundamental insights into receptor-ligand biology and molecular targets for intervention. RECENT FINDINGS: The review focuses on the requirements for known interactions, insight derived from complex structures, and mechanisms of receptor/ligand engagement. Further, novel roles for established red cell membrane proteins, parasite ligands and associated interacting partners have recently been established in red cell invasion. SUMMARY: The new knowledge underlines the intricacies involved in invasion by a eukaryotic parasite into a eukaryotic host cell demonstrated by expanded parasite ligand families, redundancy in red cell receptor engagement, multitiered temporal binding, and the breadth of receptors engaged.

Comparison of Type 1 D-3-phosphoglycerate dehydrogenases reveals unique regulation in pathogenic Mycobacteria.

D-3-phosphoglycerate dehydrogenases (PGDH) from all organisms catalyze the conversion of D-3-phosphoglycerate to phosphohydroxypyruvate as the first step in the biosynthesis of l-serine. This investigation compares the properties of Type 1 PGDHs from seven different species and demonstrates that conserved residues in the ACT and ASB domains of some allow l-serine to act as a feedback inhibitor at low micromolar concentrations. In addition, the serine sensitivity is dependent on the presence of phosphate ions. These residues are most highly conserved among PGDHs from the actinomycetales family, but only certain pathogenic mycobacteria appear to have the full complement of residues required for high sensitivity to serine. These basic residues are also responsible for the presence of dual pH optima in the acidic region that is also phosphate dependent. Analytical ultracentrifugation analysis demonstrates that the dual pH optima do not require changes in oligomeric state. This study also demonstrates that substrate inhibition is a common feature of Type 1 PGDHs and that it is suppressed by phosphate, indicating that phosphate likely interacts at both the catalytic and regulatory sites. The unique features resulting from the complement of basic residues conserved in pathogenic mycobacteria may impart important metabolic advantages to these organisms.

A quantitative assay for binding and inhibition of Plasmodium falciparum Erythrocyte Binding Antigen 175 reveals high affinity binding depends on both DBL domains.

Plasmodium falciparum Erythrocyte Binding Antigen 175 (PfEBA-175) engages Glycophorin A (GpA) on erythrocytes during malaria infection. The two Duffy binding like domains (F1 and F2) of PfEBA-175 that form region II (RII) are necessary for binding GpA, and are the target of neutralizing antibodies. Recombinant production of RII in Pichia pastoris and baculovirus has required mutations to prevent aberrant glycosylation or deglycosylation resulting in modifications to the protein surface that may affect antibody recognition and binding. In this study, we developed a recombinant system in Escherichia coli to obtain RII and F2 without mutations or glycosylation through oxidative refolding. The system produced refolded protein with high yields and purity, and without the need for mutations or deglycosylation. Biophysical characterization indicated both proteins are well behaved and correctly folded. We also demonstrate the recombinant proteins are functional, and develop a quantitative functional flow cytometry binding assay for erythrocyte binding ideally suited to measure inhibition by antibodies and inhibitors. This assay showed far greater binding of RII to erythrocytes over F2 and that binding of RII is inhibited by a neutralizing antibody and sialyllactose, while galactose had no effect on binding. These studies form the framework to measure inhibition by antibodies and small molecules that target PfEBA-175 in a rapid and quantitative manner using RII that is unmodified or mutated. This approach has significant advantages over current methods for examining receptor-ligand interactions and is applicable to other erythrocyte binding proteins used by the parasite.

A Self-Assembling Pfs230D1-Ferritin Nanoparticle Vaccine Has Potent and Durable Malaria Transmission-Reducing Activity.

Malaria is caused by eukaryotic protozoan parasites of the genus Plasmodium. There are 249 million new cases and 608,000 deaths annually, and new interventions are desperately needed. Malaria vaccines can be divided into three categories: liver stage, blood stage, or transmission-blocking vaccines. Transmission-blocking vaccines prevent the transmission of disease by the mosquito vector from one human to another. Pfs230 is one of the leading transmission-blocking vaccine antigens for malaria. Here, we describe the development of a 24-copy self-assembling nanoparticle vaccine comprising domain 1 of Pfs230 genetically fused to H. pylori ferritin. The single-component Pfs230D1-ferritin construct forms a stable and homogenous 24-copy nanoparticle with good production yields. The nanoparticle is highly immunogenic, as two low-dose vaccinations of New Zealand White rabbits elicited a potent and durable antibody response with high transmission-reducing activity when formulated in two distinct adjuvants suitable for translation to human use. This single-component 24-copy Pfs230D1-ferritin nanoparticle vaccine has the potential to improve production pipelines and the cost of manufacturing a potent and durable transmission-blocking vaccine for malaria control.

Naturally acquired antibodies against Plasmodium vivax pre-erythrocytic stage vaccine antigens inhibit sporozoite invasion of human hepatocytes in vitro.

In Plasmodium vivax, the most studied vaccine antigens are aimed at blocking merozoite invasion of erythrocytes and disease development. Very few studies have evaluated pre-erythrocytic (PE) stage antigens. The P. vivax circumsporozoite protein (CSP), is considered the leading PE vaccine candidate, but immunity to CSP is short-lived and variant specific. Thus, there is a need to identify other potential candidates to partner with CSP in a multivalent vaccine to protect against infection and disease. We hypothesize that sporozoite antigens important for host cell infection are considered potential targets. In this study, we evaluated the magnitude and quality of naturally acquired antibody responses to four P. vivax PE antigens: sporozoite surface protein 3 (SSP3), sporozoite protein essential for traversal 1 (SPECT1), cell traversal protein of ookinetes and sporozoites (CelTOS) and CSP in plasma of P. vivax infected patients from Thailand. Naturally acquired antibodies to these antigens were prevalent in the study subjects, but with significant differences in magnitude of IgG antibody responses. About 80% of study participants had antibodies to all four antigens and only 2% did not have antibodies to any of the antigens. Most importantly, these antibodies inhibited sporozoite infection of hepatocytes in vitro. Significant variations in magnitude of antigen-specific inhibitory antibody responses were observed with individual samples. The highest inhibitory responses were observed with anti-CelTOS antibodies, followed by anti-SPECT1, SSP3 and CSP antibodies respectively. These data highlight the vaccine potential of these antigens in protecting against hepatocyte infection and the need for a multi-valent pre-erythrocytic vaccine to prevent liver stage development of P. vivax sporozoites.

RBD design increases the functional antibody titers elicited by SARS-CoV-2 spike vaccination.

Most COVID-19 vaccines contain the SARS-CoV-2 spike protein as an antigen, but they lose efficacy as neutralizing antibody titers wane and escape variants emerge. Modifying the spike antigen to increase neutralizing antibody titers would help counteract this decrease in titer. We previously used a structure-based computational design method to identify nine amino acid changes in the receptor-binding domain (RBD) of spike that stabilize the RBD and increase the neutralizing antibody titers elicited by vaccination. Here, we introduce those enhancing amino acid changes into a full-length spike (FL-S-2P) ectodomain representative of most approved vaccine antigens. These amino acid changes can be incorporated into the FL-S-2P protein without negatively effecting expression or stability. Furthermore, the amino acid changes improved functional antibody titers in both mice and monkeys following vaccination. These amino acid changes could increase the duration of protection conferred by most COVID-19 vaccines.

A potent and durable malaria transmission-blocking vaccine designed from a single-component 60-copy Pfs230D1 nanoparticle.

Malaria transmission-blocking vaccines (TBVs) reduce disease transmission by breaking the continuous cycle of infection between the human host and the mosquito vector. Domain 1 (D1) of Pfs230 is a leading TBV candidate and comprises the majority of transmission-reducing activity (TRA) elicited by Pfs230. Here we show that the fusion of Pfs230D1 to a 60-copy multimer of the catalytic domain of dihydrolipoyl acetyltransferase protein (E2p) results in a single-component nanoparticle composed of 60 copies of the fusion protein with high stability, homogeneity, and production yields. The nanoparticle presents a potent human transmission-blocking epitope within Pfs230D1, indicating the antigen is correctly oriented on the surface of the nanoparticle. Two vaccinations of New Zealand White rabbits with the Pfs230D1 nanoparticle elicited a potent and durable antibody response with high TRA when formulated in two distinct adjuvants suitable for translation to human use. This single-component nanoparticle vaccine may play a key role in malaria control and has the potential to improve production pipelines and the cost of manufacturing of a potent and durable TBV.

Plasmodium falciparum exploits CD44 as a co-receptor for erythrocyte invasion.

The malaria parasite Plasmodium falciparum invades and replicates asexually within human erythrocytes. CD44 expressed on erythrocytes was previously identified as an important host factor for P. falciparum infection through a forward genetic screen, but little is known about its regulation or function in these cells, nor how it may be utilized by the parasite. We found that CD44 can be efficiently deleted from primary human hematopoietic stem cells using CRISPR/Cas9 genome editing, and that the efficiency of ex-vivo erythropoiesis to enucleated cultured red blood cells (cRBCs) is not impacted by lack of CD44. However, the rate of P. falciparum invasion was substantially reduced in CD44-null cRBCs relative to isogenic wild-type (WT) control cells, validating CD44 as an important host factor for this parasite. We identified two P. falciparum invasion ligands as binding partners for CD44, Erythrocyte Binding Antigen-175 (EBA-175) and EBA-140, and demonstrated that their ability to bind to human erythrocytes relies primarily on their canonical receptors-glycophorin A and glycophorin C, respectively. We further show that EBA-175 induces phosphorylation of erythrocyte cytoskeletal proteins in a CD44-dependent manner. Our findings support a model where P. falciparum exploits CD44 as a co-receptor during invasion of human erythrocytes, stimulating CD44-dependent phosphorylation of host cytoskeletal proteins that alter host cell deformability and facilitate parasite entry.

Design of a stabilized non-glycosylated Pfs48/45 antigen enables a potent malaria transmission-blocking nanoparticle vaccine.

A malaria vaccine that blocks parasite transmission from human to mosquito would be a powerful method of disrupting the parasite lifecycle and reducing the incidence of disease in humans. Pfs48/45 is a promising antigen in development as a transmission blocking vaccine (TBV) against the deadliest malaria parasite Plasmodium falciparum. The third domain of Pfs48/45 (D3) is an established TBV candidate, but production challenges have hampered development. For example, to date, a non-native N-glycan is required to stabilize the domain when produced in eukaryotic systems. Here, we implement a SPEEDesign computational design and in vitro screening pipeline that retains the potent transmission blocking epitope in Pfs48/45 while creating a stabilized non-glycosylated Pfs48/45 D3 antigen with improved characteristics for vaccine manufacture. This antigen can be genetically fused to a self-assembling single-component nanoparticle, resulting in a vaccine that elicits potent transmission-reducing activity in rodents at low doses. The enhanced Pfs48/45 antigen enables many new and powerful approaches to TBV development, and this antigen design method can be broadly applied towards the design of other vaccine antigens and therapeutics without interfering glycans.

Structure-based design of a strain transcending AMA1-RON2L malaria vaccine.

Apical membrane antigen 1 (AMA1) is a key malaria vaccine candidate and target of neutralizing antibodies. AMA1 binds to a loop in rhoptry neck protein 2 (RON2L) to form the moving junction during parasite invasion of host cells, and this complex is conserved among apicomplexan parasites. AMA1-RON2L complex immunization achieves higher growth inhibitory activity than AMA1 alone and protects mice against Plasmodium yoelii challenge. Here, three single-component AMA1-RON2L immunogens were designed that retain the structure of the two-component AMA1-RON2L complex: one structure-based design (SBD1) and two insertion fusions. All immunogens elicited high antibody titers with potent growth inhibitory activity, yet these antibodies did not block RON2L binding to AMA1. The SBD1 immunogen induced significantly more potent strain-transcending neutralizing antibody responses against diverse strains of Plasmodium falciparum than AMA1 or AMA1-RON2L complex vaccination. This indicates that SBD1 directs neutralizing antibody responses to strain-transcending epitopes in AMA1 that are independent of RON2L binding. This work underscores the importance of neutralization mechanisms that are distinct from RON2 blockade. The stable single-component SBD1 immunogen elicits potent strain-transcending protection that may drive the development of next-generation vaccines for improved malaria and apicomplexan parasite control.