BACKGROUND:Malaria transmission-blocking vaccines (TBVs) targeting parasite transmission by mosquitoes represent a valuable public health tool for malaria control and elimination through herd immunity in the community. Pfs230, a surface protein expressed in gametocytes and gametes of Plasmodium falciparum, plays a critical role in gamete fertilization and further development within mosquitoes. Prior studies have advanced the N-terminal pro-domain (Pro) and domain 1 (D1) of Pfs230 as a putative TBV target. However, whether other Pfs230 domains function as TBV candidates needs further study. METHODS:DNA vaccines encoding Pfs230 domains D7 to D10 (D7D10) were developed and evaluated in BALB/c mice. Antigen-specific antibody responses were assessed via enzyme-linked immunosorbent assay. Transmission-reducing activity (TRA) of antibodies induced by D7D10 DNA and protein vaccines were evaluated in standard membrane feeding assays (SMFAs) using in vitro-cultured P falciparum gametocytes. In addition, we investigated functional activity of antibodies induced by DNA vaccines encoding smaller subdomains D7 and D7D8. RESULTS:Potent D7D10-specific antibody responses were elicited in mice immunized with DNA vaccines. Anti-D7D10 IgGs purified from mice revealed strong dose-dependent TRA against P falciparum in SMFA, irrespective of the presence or absence of active complement. Western blot analysis using various Pfs230 fragments (D5-D12) suggested that antibodies elicited by D7D10 vaccines predominantly target D7. Furthermore, DNA vaccines encoding D7 and D7D8 similarly induced antibodies with strong TRA in SMFAs. CONCLUSIONS:Our studies identify Pfs230-D7D10, particularly D7, as a novel and promising P falciparum TBV candidate.

Background:Malaria remains a major global health burden, with children under five years in sub-Saharan Africa disproportionately affected. While immunity develops with repeated exposure, the specific correlates of protection in early childhood are not well defined. To address this, we assessed IgG and IgM antibody responses to Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) domains in plasma samples from 80 Burkinabe children (aged 0-60 months) collected before (pre-malaria season) and during peak malaria transmission. Methods:Using AlphaScreen, we measured antibody responses to 271 PfEMP1 (3D7) domains expressed via wheat germ cell-free synthesis. Results:Pre-season analysis showed that 96% of domains were IgG-reactive and IgG breadth increased with age, whereas 71% were IgM-reactive, but IgM breadth showed no age trend. IgG responses to 132 domains (49%) remained significantly associated with reduced risk of clinical malaria after false discovery rate (FDR) correction, including many non-adhesion domains. Five (5) IgM responses were nominally protective, but none remained significant after FDR correction. Fc-dependent opsonic phagocytosis assay using a subset of the top-ranked domains revealed that opsonic phagocytosis activity correlated with protection for only one antigen, suggesting that PfEMP1-specific antibodies may mediate protection through mechanisms beyond phagocytosis in children under 5 years old. Conclusions:This study provides the first comprehensive characterization of IgG and IgM responses to a large panel of PfEMP1 domains in young children, revealing broad antibody recognition and selective opsonic phagocytosis activity. Findings from the study deepen our understanding of early-life immunity to malaria and help identify PfEMP1 regions of interest for future vaccine development.

After invasion and replication, intracellular pathogens must egress from infected host cells. Toxoplasma gondii facilitates this process by permeabilizing host cells through induced secretion of perforin-like protein 1 (PLP1). However, the precise mechanism of host cell permeabilization remains enigmatic. Here, we identify the secretory microneme protein MIC11 as a key factor for membrane disruption. A CRISPR-based in vivo screen identifies MIC11 as the top in vivo fitness-conferring gene. Deletion of MIC11 results in severe defects in membrane rupture and egress. Scanning mutagenesis identifies functional motifs in MIC11, and mechanistic analyses support an association between MIC11 and PLP1, suggesting that MIC11 is involved in PLP1-dependent membrane disruption. Moreover, the merozoite-specific paralogue MIC22 functionally complements MIC11 deletion, suggesting a conserved mechanism of egress in the feline-restricted stages of T. gondii. Collectively, the discovery of MIC11 advances our understanding of how parasites disrupt host cells to facilitate rapid egress and successful dissemination.

Protein-protein interaction analysis requires not only the detection of a single protein but also multiple interacting proteins. The proximity-dependent biotin identification (BioID) method using biotin proximity-labelled enzymes is a protein-protein interaction method used in cells and in vivo; however, the detection tags are limited because even the lysine residues of the amino acids in the detection tag are labelled by the enzyme. Herein, we demonstrated a new affinity tag system without lysine residues suitable for use in the BioID method. After immunisation of rabbits with Plasmodium falciparum PfRipr5 protein, 22 anti-PfRipr5 monoclonal antibodies were isolated by ImmunoSpot Array Assay on a Chip, and their binding and specificity were analysed. The results showed that No. 6 anti-PfRipr5 monoclonal antibody had the highest sensitivity and specificity. The epitope was identified to contain one lysine residue; therefore, the mutated epitope, which was replaced with an arginine residue, showed similar binding properties. It was named REC tag for further analysis, and the REC tag was compatible with IB, AlphaScreen, and immunostaining and showed resistance to biotinylation using TurboID in vitro. These results indicate that a new detection tag system could be developed, in which REC tags could be used to detect BioID methods.

Plasmodium vivax is the most widespread cause of malaria with a high burden of disease. Progress in reducing the global malaria burden has stalled with no vaccines available partly due to a limited knowledge of targets and mechanisms of protective immunity. We developed a platform to quantify antibody functions to multiple P. vivax antigens and dissect immunity in a longitudinal cohort of children from Papua New Guinea at risk of P. vivax malaria. We identified antigens targeted by multiple functional antibodies, including interactions with Fcγ receptors, which mediate different cellular effector functions, and complement fixation, advancing our understanding of P. vivax immunity. We identified specific antigens targeted by antibodies associated with protection from P. vivax malaria. Evaluating thousands of possible combinations, we identified subsets of antigens in the most protective combinations providing leads for developing highly protective multi-antigen P. vivax vaccines eliciting multi-functional antibody responses to achieve and sustain elimination.

Antibody-mediated immunity directed against Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is an important immune mechanism for protection against clinical malaria and control of parasitemia. Current studies on PfEMP1-based immunity are constrained by the protein’s high molecular weight, complex multidomain architecture, and extensive polymorphism. Controlled human malaria infection (CHMI) studies uniquely enable the assessment of strain-specific PfEMP1 immunity prior to exposure and its correlation with infection outcomes using the same parasite strain. In this study, different serological assays targeting both full-length native PfEMP1 and individual domains were applied to plasma samples obtained prior to challenge infection at study baseline from 25 individuals enrolled in a CHMI study of PfSPZ Challenge (NF54), that included malaria-naïve Europeans and lifelong malaria-exposed Africans with varying degrees of immunity. All assays showed strong predictive value of both PfEMP1 antibody levels and breadth at study baseline for CHMI outcome. A random forest machine learning analysis of antibody recognition profiles, measured by AlphaScreen nearly covering all extracellular PfEMP1 domains of the NF54-CHMI parasite strain, suggested that a broad antibody repertoire, including antibodies against the most diversified group B PfEMP1s, represents a discriminative feature distinguishing volunteers with high versus low susceptibility to CHMI. In parasites re-isolated from the volunteers, dominant var transcripts encoded PfEMP1 variants are not recognized by the individuals’ pre-existing antibodies at study baseline. Given that the CHMI strain NF54 predominantly expresses B-type var genes at the onset of blood-stage infection, effective control of CHMI was associated with a broad antibody repertoire, particularly including antibodies targeting B-type PfEMP1. In line with the “hole-in-the-wall” theory, this likely reflects an antibody-mediated restriction of the parasite population emerging from the liver to parasites expressing PfEMP1 variants not previously encountered by the host immune system.

Toxoplasma gondii, which can virtually infect all warm-blooded cells, secretes various virulence factors to evade interferon-gamma (IFN-γ)-dependent host immunity. While these secreted proteins are widely characterized, the molecular mechanisms important for virulence within the parasite are unclear. In this study, we aimed to investigate the roles of non-secretory proteins of T. gondii in immunosuppression. Deletion of deubiquitinase TgJosephin resulted in attenuated virulence in wild-type mice but not in mice lacking the interferon-gamma receptor (IFNγR). Moreover, TgJosephin expression was maintained by TgRad23, a protein involved in DNA repair and protein shuttling. Notably, TgJosephin depletion increased ubiquitination of subpellicular microtubule protein 1 (SPM1), a stabilizing component of the parasite microtubules, and mutating its ubiquitination sites restored virulence in the absence of TgJosephin. We propose TgJosephin as a novel virulence factor maintained by TgRad23 and a virulence pathway involving SPM1 metabolism.IMPORTANCEToxoplasma gondii is an obligate parasite whose infection can be detrimental when combined with pregnancy or immunodeficiency. Studies on T. gondii virulence have revealed various secretory proteins that inhibit the host interferon-gamma (IFN-γ) immune response. However, much of the broader virulence landscape remains unclear. To explore the unknown molecular pathways of T. gondii virulence in mice, we searched for immunosuppressive functions in genes encoding non-secretory proteins, associated with fundamental cellular processes of the virulent type I strain. Here, we found that TgJosephin, a highly conserved deubiquitinase, was important for virulence in wild-type mice but not mice lacking the IFN-γ receptor (IFNγR). In addition, TgJosephin expression was dependent on TgRad23, and loss of TgJosephin led to increased ubiquitination of a microtubule protein SPM1. Our results suggest a novel anti-IFN-γ pathway of T. gondii mediated by TgJosephin and SPM1 deubiquitination.

Prioritising malaria vaccine targets requires understanding immunity to genetically and structurally diverse parasite antigens, influencing antibody measurements and durability. We measured total IgG levels to 25 Plasmodium falciparum antigens and assessed their association with protection and antigen features. Antibodies were quantified in two longitudinal cohorts of Papua New Guinean children (5-14 years; n=647) experiencing high or moderate transmission. Associations between antibody levels and time to first clinical malaria episode were evaluated using Cox regression and Bayesian antibody-kinetics models, incorporating antigen genetic diversity and structural properties. In high-transmission settings, antibody levels were elevated and stable, with the strongest protection observed for conserved, low-diversity antigens dominated by the 3D7-reference-matching haplotypes and enriched for intrinsically disordered and alpha-helical regions. In moderate transmission, antibody levels were variable, decayed over time, and reflected recent exposure. These findings identify antigen diversity as a key modifier of malaria immunity and underscore the importance of antigen features.

Background Plasmodium vivax poses a major obstacle to malaria elimination because this parasite can lie dormant in the liver for weeks to months before reactivating and causing a relapse of infection. These dormant forms (hypnozoites) cannot be detected using standard diagnostics, but P vivax exposure in the previous 9 months and, by proxy, hypnozoite carriage, can be inferred using serological markers. In this study, we aimed to examine how genetic variation in P vivax affects the utility of these markers and whether redesigned antigens could improve performance. Methods In this observational diagnostic accuracy study, we analysed global P vivax genetic data to assess variation in leading serological markers (n=14). Accordingly, we expressed new haplotypes that better reflect global sequence diversity for eight antigens, compared with the commonly used reference strain (Sal-1). Antibody responses against these were tested using samples from cohorts in Brazil and Thailand, with magnitude assessed in relation to how recently participants had a qPCR-detectable blood-stage P vivax infection. We compared the ability of the haplotypes versus the reference to correctly identify individuals infected within the previous 9 months. Findings Extensive global genetic diversity was identified in two P vivax antigens, MSP5 (π=14·8 × 10-3) and DBPII (π=7·7 × 10-3). Several antigens had large numbers of circulating haplotypes, with the percentage with similar sequence identity to the reference Sal-1 ranging from 0·4% (MSP5) to 99% (S16). Samples for immune analysis were previously collected between April 2013 and June 2014, with 774 and 923 participants included in the current analysis from Thailand and Brazil, respectively. Two antigens showed strong differences in immunogenicity by region and construct (RBP2a and DBPII). However, for most proteins (five of eight: MSP5, RiPR, PTEX150, Pv-fam-a, and RBP2b), these differences had no significant effect on the accuracy of identifying recent exposure. Affected performance (eg, RBP2a) was overcome by adding multiple antigens into the classification model. Interpretation Even highly diverse antigens can be effective serological markers. Our findings highlight the importance of testing the effect of genetic diversity and suggest practical strategies to ensure consistent performance across regions. Funding Australian National Health and Medical Research Council.

Background:The Plasmodium falciparum Rh5-interacting protein (PfRipr) is a key component of the pentameric PTRAMP-CSS-PfRipr-CyRPA-RH5 (PCRCR) complex, which is essential for erythrocyte invasion. Antibodies against PfRipr can inhibit parasite growth, but the full-length protein is structurally complex and challenging to produce as a recombinant antigen. We previously found that a specific PfRipr fragment, PfRipr5, was the most potent antigen; however, identifying minimal functional regions within PfRipr5 is critical for improving the vaccine design. Methods:We investigated PfRipr5, a truncated fragment of PfRipr consisting of EGF-like domains 5-9, and identified the epitope recognized by the growth-inhibitory monoclonal antibody 29B11. Epitope characterization was conducted using Western blotting with cysteine-substituted mutants and surface plasmon resonance (SPR) analysis with a single-site kinetics model. Results and conclusion:The identified 20-amino-acid region represents a cysteine-associated epitope recognized by the growth-inhibitory monoclonal antibody 29B11. This study defines a growth-inhibitory epitope within PfRipr5 whose recognition is associated with cysteine integrity. These findings provide a tractable molecular entry point for dissecting PfRipr function and support epitope-focused strategies for rational design of subunit vaccines against blood-stage malaria.

BACKGROUND:Malaria transmission-blocking vaccines induce functional immunity, impeding Plasmodium parasite development in mosquitoes and thereby reducing malaria transmission. We aim to identify target antigens of naturally acquired transmission-blocking antibodies against Plasmodium vivax. METHODS:Thirty plasma samples obtained from Thai P. vivax patients were evaluated for the presence of IgM and IgG antibodies against a library of 659 P. vivax recombinant proteins expressed by the wheat germ cell-free protein expression system using AlphaScreen. Spearman's rank tests were used to compare the antibody levels and transmission-reducing activity (TRA) of the plasma samples, measured by direct membrane feeding assay. RESULTS:Most proteins were immunoreactive to IgM (85.4%) and IgG (97.9%). TRA is significantly correlated with the level of IgM and IgG to 89 and 8 antigens (p < 0.05), respectively. Notably, IgM responses against the N-terminal Pvs230 was identified. After false discovery rate adjustment, significant correlations of TRA and IgM responses to 11 antigens remained. Some of those TRA-associated antigens exhibited elevated IgM levels in individuals with significant TRA compared to those with non-significant TRA. CONCLUSION:Our findings demonstrate the association of IgM with naturally acquired transmission-blocking immunity and highlight potential targets of P. vivax transmission-reducing antibodies. Further investigations are needed to validate the targets, elucidate the underlying mechanisms and facilitate the research on transmission-blocking vaccines.

The Pfs230:Pfs48/45 complex forms the basis for leading malaria transmission-blocking vaccine candidates, yet little is known about its molecular assembly. Here, we used cryo-electron microscopy to elucidate the structure of the endogenous Pfs230:Pfs48/45 complex bound to six transmission-blocking antibodies. Our structure revealed that Pfs230 consists of multiple domain clusters rigidified by interactions mediated through insertion domains. Membrane-anchored Pfs48/45 formed a disk-like structure, interacting with a short C-terminal peptide on Pfs230 that was critical for Pfs230 membrane-retention in vivo. Membrane retention through this interaction was not essential for transmission to mosquitoes, suggesting that complex disruption is not a mode of action for transmission-blocking antibodies. Analyses of Pfs48/45- and Pfs230-targeted antibodies identified conserved epitopes on the Pfs230:Pfs48/45 complex and provided a structural paradigm for complement-dependent activity of Pfs230-targeting antibodies. Altogether, the antibody-bound Pfs230:Pfs48/45 structure improves our molecular understanding of this biological complex, informing the development of next-generation Plasmodium falciparum transmission-blocking interventions.

INTRODUCTION:The World Health Organization has recommended two pre-erythrocytic malaria vaccines targeting Plasmodium falciparum. However, there is currently no vaccine available for Plasmodium vivax, the second leading cause of malaria. To eliminate malaria, transmission-blocking vaccines (TBVs) that can prevent infection of mosquitoes from humans would be helpful. AREAS COVERED:This review summarizes the identification of targets, progress, and prospects in developing malaria TBVs. We searched PubMed for studies published up to 11 April 2025, using the terms ['Pfs25' OR 'Pfs230' OR 'Pfs48/45' OR 'Pvs25' OR 'Pvs230' OR 'Pvs48/45' OR 'AnAPN1'] AND ['malaria transmission-blocking vaccine']. EXPERT OPINION:After over 30 years of research and development, Pfs230 for P. falciparum and Pvs25 for P. vivax are the most advanced candidates for transmission-blocking vaccines.

Understanding the red blood cell (RBC) invasion mechanism by Plasmodium falciparum, the causative agent of human malaria, is critical for developing blood-stage vaccines. Most research on the roles of parasite molecules during invasion into the RBC has focused on the pre-invasion and invasion phases, particularly on merozoite proteins in these early steps. Consequently, well-established blood-stage vaccine candidates, such as MSP1, AMA1, EBA175, Rh5, CyRPA, and Ripr, have been discovered by research targeting the early invasion phases. Recently, we identified that Liver Stage Antigen 3 (LSA3) is a merozoite dense granule protein and a novel blood-stage vaccine candidate, given that antigen-specific antibodies inhibit parasite growth by 24%. However, the precise role of LSA3 in RBC invasion remains unclear. In this study, we investigated the function of LSA3 during merozoite invasion of RBCs. In the late-invasion phase, LSA3 localized to the posterior end of invading wild-type merozoites accessible to anti-LSA3 antibodies in the culture medium. In vitro cultured lsa3 knockout parasites (ΔLSA3) exhibited slowed parasite growth. Time-lapse imaging revealed that knockout of lsa3 resulted in a 65% reduction in normal invasion completion relative to the wild-type. Furthermore, ΔLSA3 parasites displayed a high frequency of accolé-form: aberrant morphology, with a protruding convex-shaped ring-stage just beneath the RBC membrane, following successful internalization into RBCs. This study provides the first evidence that a merozoite dense granule protein potentially plays an important role in the late-invasion phase of the P. falciparum erythrocytic cycle.

Malaria transmission-blocking vaccines (TBV) target sexual stage parasites that are transmitted to mosquitoes and are critical for spread of the pathogen. The clinically most advanced TBV candidate contains part of the Pro-domain (Pro) and Domain 1 (D1) of Plasmodium falciparum surface protein Pfs230. Subunit vaccines that contain other domains of Pfs230 have so far failed to induce functional antibodies. Here, we produced eight single-domain fragments of Pfs230 in Drosophila melanogaster S2 cells and assessed their immunogenicity in mouse immunizations. In addition to D1-specific antibodies, antibodies raised against D12 showed strong functional transmission-reducing activity in membrane feeding assays with cultured parasites, an activity that was complement dependent. Murine D12-specific antibodies further reduced mosquito transmission of parasites acquired from naturally infected parasite carriers. The D12 antigen was recognized by sera from an all-age cohort of individuals who had been naturally exposed to P. falciparum with antibody levels increasing with age. In conclusion, we identified Pfs230D12 as a promising TBV candidate.

Naturally acquired immunity (NAI) against malaria is developed through repeated exposure of humans to malaria parasites. Efforts to decrease malaria incidence can potentially alter the development of immunity. This work aimed to monitor total IgG titres against PfEBA-175RIII-V during the implementation of a quarterly mass testing, treatment, and tracking (MTTT) strategy in a high malaria transmission setting in Ghana. In total, 314 individuals aged 6 months–90 years participating in four quarterly MTTT studies were selected for this study. Finger-pricked blood was collected from each participant and used to prepare dried blood spots, which were used for molecular diagnosis of Plasmodium falciparum infection and elution of antibodies for serological evaluation of PfEBA-175RIII-V specific antibodies. The infection prevalence by PCR was reduced from 56.2

Background Pregnant women in malaria-endemic regions exhibit heightened susceptibility to Plasmodium falciparum infection due to accumulation of infected red blood cells (iRBCs) in the placenta. This is aided by the variant antigen 2 chondroitin sulfate A (VAR2CSA) protein which remains a target for pregnancy-associated malaria (PAM) vaccine studies. However, polymorphism in this antigen presents a significant challenge in developing broadly efficacious vaccines. This study explored PfCSA-L, a protein co-expressed and co-localized with VAR2CSA, as a potential alternative vaccine target due to its hypothesized role in PAM pathogenesis. Methods Sera and dried blood spots were collected from pregnant women attending antenatal care clinic at Webuye County Hospital, Western Kenya. P. falciparum infection status was confirmed by PCR. Recombinant PfCSA-L, expressed using a eukaryotic wheat germ cell-free system (WGCFS), was used to assess IgG antibody responses via ELISA. Results We observed a statistically significant increase in IgG levels as gestation advanced, suggesting potential exposure-driven antibody maturation against PfCSA-L. Primigravida women exhibited a trend toward higher anti-PfCSA-L antibody levels compared to multigravida during the second visit, possibly reflecting a more vigorous immune response during the first pregnancy. Genetic analysis of field parasite isolates revealed a high conservation of PfCSA-L at both DNA and protein levels. B-cell epitope prediction identified potential targets on the PfCSA-L surface within the conserved region. Conclusion These findings, coupled with the observed IgG response, further positions PfCSA-L as a promising vaccine candidate for PAM. However, further investigations are warranted to elucidate the functional role of anti-PfCSA-L antibodies and definitively validate PfCSA-L and/or the identified epitopes as potential PAM vaccine targets.

[This corrects the article DOI: 10.3389/fimmu.2019.00730.].