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

Plasmodium falciparum reticulocyte-binding protein homolog 5 (RH5) is the most advanced blood-stage malaria vaccine candidate and is being evaluated for efficacy in endemic regions, emphasizing the need to study the underlying antibody response to RH5 during natural infection, which could augment or counteract responses to vaccination. Here, we found that RH5-reactive B cells were rare, and circulating immunoglobulin G (IgG) responses to RH5 were short-lived in malaria-exposed Malian individuals, despite repeated infections over multiple years. RH5-specific monoclonal antibodies isolated from eight malaria-exposed individuals mostly targeted non-neutralizing epitopes, in contrast to antibodies isolated from five RH5-vaccinated, malaria-naive UK individuals. However, MAD8-151 and MAD8-502, isolated from two malaria-exposed Malian individuals, were among the most potent neutralizers out of 186 antibodies from both cohorts and targeted the same epitopes as the most potent vaccine-induced antibodies. These results suggest that natural malaria infection may boost RH5-vaccine-induced responses and provide a clear strategy for the development of next-generation RH5 vaccines.

Analysis of the diverse antigenic landscape of the malaria protein RH5 identifies a potent vaccine-induced human public antibody clonotype.

The highly conserved and essential Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) has emerged as the leading target for vaccines against the disease-causing blood stage of malaria. However, the features of the human vaccine-induced antibody response that confer highly potent inhibition of malaria parasite invasion into red blood cells are not well defined. Here, we characterize 236 human IgG monoclonal antibodies, derived from 15 donors, induced by the most advanced PfRH5 vaccine. We define the antigenic landscape of this molecule and establish that epitope specificity, antibody association rate, and intra-PfRH5 antibody interactions are key determinants of functional anti-parasitic potency. In addition, we identify a germline IgG gene combination that results in an exceptionally potent class of antibody and demonstrate its prophylactic potential to protect against P. falciparum parasite challenge in vivo. This comprehensive dataset provides a framework to guide rational design of next-generation vaccines and prophylactic antibodies to protect against blood-stage malaria.

Infectivity of Plasmodium parasites to Aedes aegypti and Anopheles stephensi mosquitoes maintained on blood-free meals of SkitoSnack.

BACKGROUND: Aedes and Anopheles mosquitoes are responsible for tremendous global health burdens from their transmission of pathogens causing malaria, lymphatic filariasis, dengue, and yellow fever. Innovative vector control strategies will help to reduce the prevalence of these diseases. Mass rearing of mosquitoes for research and support of these strategies presently depends on meals of vertebrate blood, which is subject to acquisition, handling, and storage issues. Various blood-free replacements have been formulated for these mosquitoes, but none of these replacements are in wide use, and little is known about their potential impact on competence of the mosquitoes for Plasmodium infection. METHODS: Colonies of Aedes aegypti and Anopheles stephensi were continuously maintained on a blood-free replacement (SkitoSnack; SS) or bovine blood (BB) and monitored for engorgement and hatch rates. Infections of Ae. aegypti and An. stephensi were assessed with Plasmodium gallinaceum and P. falciparum, respectively. RESULTS: Replicate colonies of mosquitoes were maintained on BB or SS for 10 generations of Ae. aegypti and more than 63 generations of An. stephensi. The odds of engorgement by SS- relative to BB-maintained mosquitoes were higher for both Ae. aegypti (OR = 2.6, 95% CI 1.3-5.2) and An. stephensi (OR 2.7, 95% CI 1.4-5.5), while lower odds of hatching were found for eggs from the SS-maintained mosquitoes of both species (Ae. aegypti OR = 0.40, 95% CI 0.26-0.62; An. stephensi OR = 0.59, 95% CI 0.36-0.96). Oocyst counts were similar for P. gallinaceum infections of Ae. aegypti mosquitoes maintained on SS or BB (mean ratio = [mean on SS]/[mean on BB] = 1.11, 95% CI 0.85-1.49). Similar oocyst counts were also observed from the P. falciparum infections of SS- or BB-maintained An. stephensi (mean ratio = 0.76, 95% CI 0.44-1.37). The average counts of sporozoites/mosquito showed no evidence of reductions in the SS-maintained relative to BB-maintained mosquitoes of both species. CONCLUSIONS: Aedes aegypti and An. stephensi can be reliably maintained on SS over multiple generations and are as competent for Plasmodium infection as mosquitoes maintained on BB. Use of SS alleviates the need to acquire and preserve blood for mosquito husbandry and may support new initiatives in fundamental and applied research, including novel manipulations of midgut microbiota and factors important to the mosquito life cycle and pathogen susceptibility.

Preclinical development of a stabilized RH5 virus-like particle vaccine that induces improved antimalarial antibodies.

Plasmodium falciparum reticulocyte-binding protein homolog 5 (RH5) is a leading blood-stage malaria vaccine antigen target, currently in a phase 2b clinical trial as a full-length soluble protein/adjuvant vaccine candidate called RH5.1/Matrix-M. We identify that disordered regions of the full-length RH5 molecule induce non-growth inhibitory antibodies in human vaccinees and that a re-engineered and stabilized immunogen (including just the alpha-helical core of RH5) induces a qualitatively superior growth inhibitory antibody response in rats vaccinated with this protein formulated in Matrix-M adjuvant. In parallel, bioconjugation of this immunogen, termed "RH5.2," to hepatitis B surface antigen virus-like particles (VLPs) using the "plug-and-display" SpyTag-SpyCatcher platform technology also enables superior quantitative antibody immunogenicity over soluble protein/adjuvant in vaccinated mice and rats. These studies identify a blood-stage malaria vaccine candidate that may improve upon the current leading soluble protein vaccine candidate RH5.1/Matrix-M. The RH5.2-VLP/Matrix-M vaccine candidate is now under evaluation in phase 1a/b clinical trials.

Blood-stage malaria vaccine candidate RH5.1/Matrix-M in healthy Tanzanian adults and children; an open-label, non-randomised, first-in-human, single-centre, phase 1b trial.

BACKGROUND: A blood-stage Plasmodium falciparum malaria vaccine would provide a second line of defence to complement partially effective or waning immunity conferred by the approved pre-erythrocytic vaccines. RH5.1 is a soluble protein vaccine candidate for blood-stage P falciparum, formulated with Matrix-M adjuvant to assess safety and immunogenicity in a malaria-endemic adult and paediatric population for the first time. METHODS: We did a non-randomised, phase 1b, single-centre, dose-escalation, age de-escalation, first-in-human trial of RH5.1/Matrix-M in Bagamoyo, Tanzania. We recruited healthy adults (aged 18-45 years) and children (aged 5-17 months) to receive the RH5.1/Matrix-M vaccine candidate in the following three-dose regimens: 10 µg RH5.1 at 0, 1, and 2 months (Adults 10M), and the higher dose of 50 µg RH5.1 at 0 and 1 month and 10 µg RH5.1 at 6 months (delayed-fractional third dose regimen; Adults DFx). Children received either 10 µg RH5.1 at 0, 1, and 2 months (Children 10M) or 10 µg RH5.1 at 0, 1, and 6 months (delayed third dose regimen; Children 10D), and were recruited in parallel, followed by children who received the dose-escalation regimen (Children DFx) and children with higher malaria pre-exposure who also received the dose-escalation regimen (High Children DFx). All RH5.1 doses were formulated with 50 µg Matrix-M adjuvant. Primary outcomes for vaccine safety were solicited and unsolicited adverse events after each vaccination, along with any serious adverse events during the study period. The secondary outcome measures for immunogenicity were the concentration and avidity of anti-RH5.1 serum IgG antibodies and their percentage growth inhibition activity (GIA) in vitro, as well as cellular immunogenicity to RH5.1. All participants receiving at least one dose of vaccine were included in the primary analyses. This trial is registered at ClinicalTrials.gov, NCT04318002, and is now complete. FINDINGS: Between Jan 25, 2021, and April 15, 2021, we recruited 12 adults (six [50%] in the Adults 10M group and six [50%] in the Adults DFx group) and 48 children (12 each in the Children 10M, Children 10D, Children DFx, and High Children DFx groups). 57 (95%) of 60 participants completed the vaccination series and 55 (92%) completed 22 months of follow-up following the third vaccination. Vaccinations were well-tolerated across both age groups. There were five serious adverse events involving four child participants during the trial, none of which were deemed related to vaccination. RH5-specific T cell and serum IgG antibody responses were induced by vaccination and purified total IgG showed in vitro GIA against P falciparum. We found similar functional quality (ie, GIA per µg RH5-specific IgG) across all age groups and dosing regimens at 14 days after the final vaccination; the concentration of RH5.1-specific polyclonal IgG required to give 50% GIA was 14·3 µg/mL (95% CI 13·4-15·2). 11 children were vaccinated with the delayed third dose regimen and showed the highest median anti-RH5 serum IgG concentration 14 days following the third vaccination (723 µg/mL [IQR 511-1000]), resulting in all 11 who received the full series showing greater than 60% GIA following dilution of total IgG to 2·5 mg/mL (median 88% [IQR 81-94]). INTERPRETATION: The RH5.1/Matrix-M vaccine candidate shows an acceptable safety and reactogenicity profile in both adults and 5-17-month-old children residing in a malaria-endemic area, with all children in the delayed third dose regimen reaching a level of GIA previously associated with protective outcome against blood-stage P falciparum challenge in non-human primates. These data support onward efficacy assessment of this vaccine candidate against clinical malaria in young African children. FUNDING: The European and Developing Countries Clinical Trials Partnership; the UK Medical Research Council; the UK Department for International Development; the National Institute for Health and Care Research Oxford Biomedical Research Centre; the Division of Intramural Research, National Institute of Allergy and Infectious Diseases; the US Agency for International Development; and the Wellcome Trust.

Autoantibodies inhibit Plasmodium falciparum growth and are associated with protection from clinical malaria.

Many infections, including malaria, are associated with an increase in autoantibodies (AAbs). Prior studies have reported an association between genetic markers of susceptibility to autoimmune disease and resistance to malaria, but the underlying mechanisms are unclear. Here, we performed a longitudinal study of children and adults (n = 602) in Mali and found that high levels of plasma AAbs before the malaria season independently predicted a reduced risk of clinical malaria in children during the ensuing malaria season. Baseline AAb seroprevalence increased with age and asymptomatic Plasmodium falciparum infection. We found that AAbs purified from the plasma of protected individuals inhibit the growth of blood-stage parasites and bind P. falciparum proteins that mediate parasite invasion. Protected individuals had higher plasma immunoglobulin G (IgG) reactivity against 33 of the 123 antigens assessed in an autoantigen microarray. This study provides evidence in support of the hypothesis that a propensity toward autoimmunity offers a survival advantage against malaria.

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

Reticulocyte-binding protein homologue 5 (RH5), a leading blood-stage Plasmodium falciparum malaria vaccine target, interacts with cysteine-rich protective antigen (CyRPA) and RH5-interacting protein (RIPR) to form an essential heterotrimeric "RCR-complex". We investigate whether RCR-complex vaccination can improve upon RH5 alone. Using monoclonal antibodies (mAbs) we show that parasite growth-inhibitory epitopes on each antigen are surface-exposed on the RCR-complex and that mAb pairs targeting different antigens can function additively or synergistically. However, immunisation of female rats with the RCR-complex fails to outperform RH5 alone due to immuno-dominance of RIPR coupled with inferior potency of anti-RIPR polyclonal IgG. We identify that all growth-inhibitory antibody epitopes of RIPR cluster within the C-terminal EGF-like domains and that a fusion of these domains to CyRPA, called "R78C", combined with RH5, improves the level of in vitro parasite growth inhibition compared to RH5 alone. These preclinical data justify the advancement of the RH5.1 + R78C/Matrix-M™ vaccine candidate to Phase 1 clinical trial.

NAD activates olfactory receptor 1386 to regulate type I interferon responses in Plasmodium yoelii YM infection.

Olfactory receptors (Olfr) are G protein-coupled receptors that are normally expressed on olfactory sensory neurons to detect volatile chemicals or odorants. Interestingly, many Olfrs are also expressed in diverse tissues and function in cell-cell recognition, migration, and proliferation as well as immune responses and disease processes. Here, we showed that many Olfr genes were expressed in the mouse spleen, linked to Plasmodium yoelii genetic loci significantly, and/or had genome-wide patterns of LOD scores (GPLSs) similar to those of host Toll-like receptor genes. Expression of specific Olfr genes such as Olfr1386 in HEK293T cells significantly increased luciferase signals driven by IFN-ß and NF-κB promoters, with elevated levels of phosphorylated TBK1, IRF3, P38, and JNK. Mice without Olfr1386 were generated using the CRISPR/Cas9 method, and the Olfr1386-/- mice showed significantly lower IFN-α/ß levels and longer survival than wild-type (WT) littermates after infection with P. yoelii YM parasites. Inhibition of G protein signaling and P38 activity could affect cyclic AMP-responsive element promoter-driven luciferase signals and IFN-ß mRNA levels in HEK293T cells expressing the Olfr1386 gene, respectively. Screening of malaria parasite metabolites identified nicotinamide adenine dinucleotide (NAD) as a potential ligand for Olfr1386, and NAD could stimulate IFN-ß responses and phosphorylation of TBK1 and STAT1/2 in RAW264.7 cells. Additionally, parasite RNA (pRNA) could significantly increase Olfr1386 mRNA levels. This study links multiple Olfrs to host immune response pathways, identifies a candidate ligand for Olfr1386, and demonstrates the important roles of Olfr1386 in regulating type I interferon (IFN-I) responses during malaria parasite infections.

Evaluation of the precision of the Plasmodium knowlesi growth inhibition assay for Plasmodium vivax Duffy-binding protein-based malaria vaccine development.

Recent data indicate increasing disease burden and importance of Plasmodium vivax (Pv) malaria. A robust assay will be essential for blood-stage Pv vaccine development. Results of the in vitro growth inhibition assay (GIA) with transgenic P. knowlesi (Pk) parasites expressing the Pv Duffy-binding protein region II (PvDBPII) correlate with in vivo protection in the first PvDBPII controlled human malaria infection (CHMI) trials, making the PkGIA an ideal selection tool once the precision of the assay is defined. To determine the precision in percentage of inhibition in GIA (%GIA) and in GIA50 (antibody concentration that gave 50 %GIA), ten GIAs with transgenic Pk parasites were conducted with four different anti-PvDBPII human monoclonal antibodies (mAbs) at concentrations of 0.016 to 2 mg/mL, and three GIAs with eighty anti-PvDBPII human polyclonal antibodies (pAbs) at 10 mg/mL. A significant assay-to-assay variation was observed, and the analysis revealed a standard deviation (SD) of 13.1 in the mAb and 5.94 in the pAb dataset for %GIA, with a LogGIA50 SD of 0.299 (for mAbs). Moreover, the ninety-five percent confidence interval (95 %CI) for %GIA or GIA50 in repeat assays was calculated in this investigation. The error range determined in this study will help researchers to compare PkGIA results from different assays and studies appropriately, thus supporting the development of future blood-stage malaria vaccine candidates, specifically second-generation PvDBPII-based formulations.

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.

Vaccines and monoclonal antibodies: new tools for malaria control.

SUMMARYMalaria remains one of the biggest health problems in the world. While significant reductions in malaria morbidity and mortality had been achieved from 2000 to 2015, the favorable trend has stalled, rather significant increases in malaria cases are seen in multiple areas. In 2022, there were 249 million estimated cases, and 608,000 malaria-related deaths, mostly in infants and children aged under 5 years, globally. Therefore, in addition to the expansion of existing anti-malarial control measures, it is critical to develop new tools, such as vaccines and monoclonal antibodies (mAbs), to fight malaria. In the last 2 years, the first and second malaria vaccines, both targeting Plasmodium falciparum circumsporozoite proteins (PfCSP), have been recommended by the World Health Organization to prevent P. falciparum malaria in children living in moderate to high transmission areas. While the approval of the two malaria vaccines is a considerable milestone in vaccine development, they have much room for improvement in efficacy and durability. In addition to the two approved vaccines, recent clinical trials with mAbs against PfCSP, blood-stage vaccines against P. falciparum or P. vivax, and transmission-blocking vaccine or mAb against P. falciparum have shown promising results. This review summarizes the development of the anti-PfCSP vaccines and mAbs, and recent topics in the blood- and transmission-blocking-stage vaccine candidates and mAbs. We further discuss issues of the current vaccines and the directions for the development of next-generation vaccines.

Cross-reactivity of rPvs48/45, a recombinant Plasmodium vivax protein, with sera from Plasmodium falciparum endemic areas of Africa.

Background: Ps48/45, a Plasmodium gametocyte surface protein, is a promising candidate for malaria transmission-blocking (TB) vaccine. Due to its relevance for a multispecies vaccine, we explored the cross-reactivity and TB activity of a recombinant P. vivax Ps48/45 protein (rPvs48/45) with sera from P. falciparum-exposed African donors. Methods: rPvs48/45 was produced in Chinese hamster ovary cell lines and tested by ELISA for its cross-reactivity with sera from Burkina Faso, Tanzania, Mali, and Nigeria - In addition, BALB/c mice were immunized with the rPvs48/45 protein formulated in Montanide ISA-51 and inoculated with a crude extract of P. falciparum NF-54 gametocytes to evaluate the parasite-boosting effect on rPvs48/45 antibody titers. Specific anti-rPvs48/45 IgG purified from African sera was used to evaluate the ex vivo TB activity on P. falciparum, using standard mosquito membrane feeding assays (SMFA). Results: rPvs48/45 protein showed cross-reactivity with sera of individuals from all four African countries, in proportions ranging from 94% (Tanzania) to 40% (Nigeria). Also, the level of cross-reactive antibodies varied significantly between countries (p<0.0001), with a higher antibody level in Mali and the lowest in Nigeria. In addition, antibody levels were higher in adults (≥ 17 years) than young children (≤ 5 years) in both Mali and Tanzania, with a higher proportion of responders in adults (90%) than in children (61%) (p<0.0001) in Mali, where male (75%) and female (80%) displayed similar antibody responses. Furthermore, immunization of mice with P. falciparum gametocytes boosted anti-Pvs48/45 antibody responses, recognizing P. falciparum gametocytes in indirect immunofluorescence antibody test. Notably, rPvs48/45 affinity-purified African IgG exhibited a TB activity of 61% against P. falciparum in SMFA. Conclusion: African sera (exposed only to P. falciparum) cross-recognized the rPvs48/45 protein. This, together with the functional activity of IgG, warrants further studies for the potential development of a P. vivax and P. falciparum cross-protective TB vaccine.

A randomized first-in-human phase I trial of differentially adjuvanted Pfs48/45 malaria vaccines in Burkinabé adults.

BACKGROUNDMalaria transmission-blocking vaccines aim to interrupt the transmission of malaria from one person to another.METHODSThe candidates R0.6C and ProC6C share the 6C domain of the Plasmodium falciparum sexual-stage antigen Pfs48/45. R0.6C utilizes the glutamate-rich protein (GLURP) as a carrier, and ProC6C includes a second domain (Pfs230-Pro) and a short 36-amino acid circumsporozoite protein (CSP) sequence. Healthy adults (n = 125) from a malaria-endemic area of Burkina Faso were immunized with 3 intramuscular injections, 4 weeks apart, of 30 µg or 100 µg R0.6C or ProC6C each adsorbed to Alhydrogel (AlOH) adjuvant alone or in combination with Matrix-M (15 µg or 50 µg, respectively). The allocation was random and double-blind for this phase I trial.RESULTSThe vaccines were safe and well tolerated with no vaccine-related serious adverse events. A total of 7 adverse events, mild to moderate in intensity and considered possibly related to the study vaccines, were recorded. Vaccine-specific antibodies were highest in volunteers immunized with 100 µg ProC6C-AlOH with Matrix-M, and 13 of 20 (65%) individuals in the group showed greater than 80% transmission-reducing activity (TRA) when evaluated in the standard membrane feeding assay at 15 mg/mL IgG. In contrast, R0.6C induced sporadic TRA.CONCLUSIONAll formulations were safe and well tolerated in a malaria-endemic area of Africa in healthy adults. The ProC6C-AlOH/Matrix-M vaccine elicited the highest levels of functional antibodies, meriting further investigation.TRIAL REGISTRATIONPan-African Clinical Trials Registry (https://pactr.samrc.ac.za) PACTR202201848463189.FUNDINGThe study was funded by the European and Developing Countries Clinical Trials Partnership (grant RIA2018SV-2311).

Mesenchymal stem cells of the bone marrow raise infectivity of Plasmodium falciparum gametocytes.

Plasmodium falciparum is a parasite that causes the deadly human disease, malaria, and exhibits a complex life cycle in human and mosquito hosts. In the sexual stages of the parasite, gametocytes mature in the human body and propagate malaria when they are picked up by mosquitoes to infect new hosts. Previous research has shown that gametocytes home to the bone marrow of the host, where they complete their maturation and alter the behavior of resident mesenchymal stem cells (MSCs). In this study, we investigated the alternate side of this host-pathogen interaction, whether MSCs could alter the behavior of gametocytes. Gametocytes were co-cultured with MSCs until maturity and subsequently fed to mosquitoes to measure the oocysts produced. Here, we report, for the first time, that MSCs co-culture significantly elevated oocyst numbers in the infected mosquito compared to conventional culture medium. This enhancement appeared to be most effective during the early stages of gametocyte development and was not replicated by other cell types. MSC co-culture also increased the infectivity of field isolated P. falciparum parasites. This effect was partially mediated by soluble factor(s) as conditioned medium harvested from MSCs could also partially raise infectivity of gametocytes to nearly half compared to MSC co-culture. Together, this study reveals novel host-pathogen interactions, where the human MSCs are elevating the infectivity of malaria gametocytes. IMPORTANCE While prior research has established that Plasmodium gametocytes sequester in the bone marrow and can influence resident stem cells, the question of why they would choose this compartment and these cells remained a mystery. This study, for the first time, shows that being in the presence of mesenchymal stem cells (MSCs) alters the biology of the P. falciparum parasite and makes it more infectious to mosquitoes, hinting at novel mechanisms in its life cycle. This method also facilitates mosquito infections with field isolated parasites, affording research teams new infection models with parasites, which are challenging to infect into mosquitos using conventional culture methods. Finally, our findings that MSC-conditioned medium can also raise infectivity open avenues of investigation into mechanisms involved but can also serve as a practical tool for researchers hoping to increase oocyst yields.

Antibody gene features associated with binding and functional activity in vaccine-derived human mAbs targeting malaria parasites.

Adjuvants have been essential to malaria vaccine development, but their impact on the vaccine-induced antibody repertoire is poorly understood. Here, we used cDNA sequences from antigen-specific single memory B cells to express 132 recombinant human anti-Pfs230 monoclonal antibodies (mAbs). Alhydrogel®-induced mAbs demonstrated higher binding to Pfs230D1, although functional activity was similar between adjuvants. All Alhydrogel® mAbs using IGHV1-69 gene bound to recombinant Pfs230D1, but none blocked parasite transmission to mosquitoes; similarly, no AS01 mAb using IGHV1-69 blocked transmission. Functional mAbs from both Alhydrogel® and AS01 vaccines used IGHV3-21 and IGHV3-30 genes. Antibodies with the longest CDR3 sequences were associated with binding but not functional activity. This study assesses adjuvant effects on antibody clonotype diversity during malaria vaccination.

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.

Structure guided mimicry of an essential P. falciparum receptor-ligand complex enhances cross neutralizing antibodies.

Invasion of human erythrocytes by Plasmodium falciparum (Pf) merozoites relies on the interaction between two parasite proteins: apical membrane antigen 1 (AMA1) and rhoptry neck protein 2 (RON2). While antibodies to AMA1 provide limited protection against Pf in non-human primate malaria models, clinical trials using recombinant AMA1 alone (apoAMA1) yielded no protection due to insufficient functional antibodies. Immunization with AMA1 bound to RON2L, a 49-amino acid peptide from its ligand RON2, has shown superior protection by increasing the proportion of neutralizing antibodies. However, this approach relies on the formation of a complex in solution between the two vaccine components. To advance vaccine development, here we engineered chimeric antigens by replacing the AMA1 DII loop, displaced upon ligand binding, with RON2L. Structural analysis confirmed that the fusion chimera (Fusion-FD12) closely mimics the binary AMA1-RON2L complex. Immunization studies in female rats demonstrated that Fusion-FD12 immune sera, but not purified IgG, neutralized vaccine-type parasites more efficiently compared to apoAMA1, despite lower overall anti-AMA1 titers. Interestingly, Fusion-FD12 immunization enhanced antibodies targeting conserved epitopes on AMA1, leading to increased neutralization of non-vaccine type parasites. Identifying these cross-neutralizing antibody epitopes holds promise for developing an effective, strain-transcending malaria vaccine.

Dysfunction of CD169+ macrophages and blockage of erythrocyte maturation as a mechanism of anemia in Plasmodium yoelii infection.

Plasmodium parasites cause malaria with disease outcomes ranging from mild illness to deadly complications such as severe malarial anemia (SMA), pulmonary edema, acute renal failure, and cerebral malaria. In young children, SMA often requires blood transfusion and is a major cause of hospitalization. Malaria parasite infection leads to the destruction of infected and noninfected erythrocytes as well as dyserythropoiesis; however, the mechanism of dyserythropoiesis accompanied by splenomegaly is not completely understood. Using Plasmodium yoelii yoelii 17XNL as a model, we show that both a defect in erythroblastic island (EBI) macrophages in supporting red blood cell (RBC) maturation and the destruction of reticulocytes/RBCs by the parasites contribute to SMA and splenomegaly. After malaria parasite infection, the destruction of both infected and noninfected RBCs stimulates extramedullary erythropoiesis in mice. The continuous decline of RBCs stimulates active erythropoiesis and drives the expansion of EBIs in the spleen, contributing to splenomegaly. Phagocytosis of malaria parasites by macrophages in the bone marrow and spleen may alter their functional properties and abilities to support erythropoiesis, including reduced expression of the adherence molecule CD169 and inability to support erythroblast differentiation, particularly RBC maturation in vitro and in vivo. Therefore, macrophage dysfunction is a key mechanism contributing to SMA. Mitigating and/or alleviating the inhibition of RBC maturation may provide a treatment strategy for SMA.

Clinical needs assessment to inform development of a new assay to detect antimalarial drugs in patient samples: A case study.

Point-of-care assays have greatly increased access to diagnostic information and improved healthcare outcomes globally, especially in the case of tropical diseases in rural settings. Increased recognition of the impact of these tools and increased funding, along with advances in technology have led to a surge in development of new assays. However, many new tools fail to fulfill their intended purpose due to a lack of clinical impact, operational feasibility, and input from envisioned operators. To be successful, they must fit into existing clinical decision-making models and be designed in collaboration with end users. We describe a case study of the development of a new low-cost sensor for antimalarial drugs, from initial planning through collection and incorporation of design feedback to final assay design. The assay uses an aptamer-based sensor to detect antimalarial drugs from patient samples for tracking antimalarial use in Southeast Asia, a region with a long history of emerging antimalarial drug resistance. Design and use-case input was collected from malaria control experts, researchers, and healthcare workers to develop target product profiles. Data was collected via surveys and in-person interviews during assay development and ultimately informed a change in assay format. This aptamer sensor platform can be easily adapted to detect other small molecule and protein targets and the design process described here can serve as a model for the development of effective new assays to improve access to healthcare technology.

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.