Snakebite envenoming is a neglected tropical disease, with neurotoxic elapid venoms in particular a major therapeutic challenge due to the rapid action and inter-species diversity of α-neurotoxins. Consensus toxin approaches have emerged as a promising strategy for generating broadly cross-reactive antibodies, while virus-like particles (VLPs) have been proposed to overcome antigen immunogenicity challenges. Here, consensus short-chain (sc3FTx) and long-chain (lc3FTx) α-neurotoxins were evaluated as immunogens in sheep either alone or displayed on a VLP platform. Both toxin-only and VLP-displayed immunogens elicited subclass-specific antibody responses with broad geographical and taxonomic recognition of medically important elapids. A functional in vitro neurotoxicity assay successfully differentiated neutralising from non-neutralising binding responses and indicated superior efficacy of toxin-only antisera relative to VLP-toxin immunogens. However, despite promising in vitro neutralisation, translation to in vivo protection was limited with only modest improvements in murine survival observed following a 4× LD50 venom challenge. Increased concentrations of toxin-only experimental antivenom did, however, substantially improve protection relative to venom-only and VLP-toxin derived antivenom groups. In these experiments, VLP presentation did not enhance immunogenicity or protective efficacy under the conditions tested, although this may have been influenced by the substantially lower toxin antigen dose received by VLP-immunised sheep and should not be interpreted as definitive evidence against the VLP platform. Overall, these findings further demonstrate that consensus α-neurotoxin immunogens can generate broad subclass-specific antibody recognition, but that neutralisation remains difficult. This study highlights the importance of antigen presentation, immunisation strategy and functional epitope targeting in the development of next-generation recombinant antivenoms.

Malaria continues to exact a high disease burden worldwide, and improved vaccines and therapeutics are desperately needed. Both licensed vaccines target only the circumsporozoite protein (CSP) expressed during early stages of Plasmodium falciparum infection. Vaccines targeting the subsequent blood stage, in particular those based on RH5, are also showing clinical promise. Concrete data demonstrating the utility of a combination of antigens are lacking. Using a humanized liver mouse model, we show that anti-CSP monoclonal antibodies (mAbs) lose protection below ∼30 μg/mL serum concentration and plateau above ∼60 μg/mL. Yet breakthrough infections have multiple logs fewer parasites emerging from the liver. The addition of an anti-RH5 blood stage mAb to a partially protective anti-CSP mAb yields additional benefits by controlling the ensuing blood stage parasitemia to a transient and extremely low-level infection. These results provide the proof-of-concept evidence that multi-stage antibodies may be more protective compared to a single stage approach.

Immunotherapies hold great promise for cancer treatment, yet only a small fraction of patients respond to current approaches. We introduce a strategy that redirects pre-existing, vaccine-induced immunity to recognize and eliminate tumors. This method employs newly engineered phage-derived nanoparticles that achieve multilayered tumor specificity through ligand-mediated cell entry, transcriptional targeting, and the delivery of non-mammalian antigens absent from healthy tissues. By leveraging established immune memory, this platform enables highly specific and potent antitumor responses.We validated this concept using a malaria vaccine prototype for redirecting pathogen-specific immunity toward cancer. Specifically, we exploited the malaria epitope Pb9 (SYIPSAEKI), delivered by phage selectively to tumors in mice previously immunized with the Ad.ME-TRAP vaccine. In vitro, Pb9-expressing tumor cells were selectively recognized and destroyed by immune cells from immunized mice, accompanied by robust interferon-γ and tumor necrosis factor-α production. In vivo, systemic administration of the phage nanocarrier achieved highly selective Pb9 expression in tumors while sparing healthy organs. This tumor-restricted expression induced infiltration of antigen-specific cytotoxic T cells and natural killer cells, activation of pro-inflammatory pathways, and apoptosis within tumors. Interestingly, the combination of Ad.ME-TRAP immunization and phage-mediated Pb9 gene delivery led to complete tumor regression in a substantial proportion of animals, with durable long-term cures in over 40% of treated mice.These findings demonstrate a versatile immunotherapeutic strategy that redirects pre-existing vaccine-induced immune responses toward tumors using phage-derived, tumor-selective vectors. Beyond the malaria model, this platform offers a broadly applicable approach for repurposing preventive vaccines into safe and effective cancer immunotherapies.

Granulomatosis with polyangiitis is a life-threatening systemic vasculitis, characterised by anti-neutrophil cytoplasmic autoantibodies (ANCA) most commonly against proteinase 3 (PR3), a protease expressed intracellularly and on the surface of neutrophils. Most cell surface PR3 is bound to the receptor CD177; however, the molecular mechanism of the interactions is not well understood. Here, we present crystal structures of CD177 in complex with PR3 and unliganded CD177. We describe a mainly hydrophobic binding interface between PR3 and CD177, involving the first two Ly6/uPAR (LU) domains of CD177. These form a globular structure which is connected to downstream domains via a flexible linker. Using a panel of PR3-ANCA-positive patient samples, we show that a significant proportion of ANCAs target the CD177-binding site of PR3 in these samples. Structure-guided mutation of the CD177-binding site on PR3 is effective in reducing PR3-ANCA binding. The results demonstrate that the CD177-binding surface of PR3 harbours a major PR3-ANCA epitope, and that the extent of binding to this surface varies between different patients.

IntroductionThere is great interest in combining the licensed pre-erythrocytic malaria vaccine R21/Matrix-M® and the blood-stage candidate vaccine RH5.1/Matrix-M® to maximise efficacy against Plasmodium falciparum malaria. As protection is understood to be antibody-mediated for both vaccines, the success of a multistage vaccination strategy will be aided by understanding factors that impact the cellular drivers of humoral immunity, e.g. dosing regimen, exposure to malaria, and age. Prior analyses of adult vaccinees showed higher RH5-specific memory B cell responses with delayed fractional versus monthly booster dosing (50-50-10μg at 0-1-6months and 10-10-10μg at 0-1-2months, respectively), but the B cell impact of delayed booster regimens has not yet been evaluated in the target paediatric population. There have also not yet been NANP-specific B cell immunogenicity analyses with R21/Matrix-M.MethodsHere, pre- and post-vaccination PBMC from two Phase 1b clinical trials with R21/Matrix-M® (NCT03580824;Kenya) and RH5.1/Matrix-M® (NCT04318002; Tanzania) were analysed by flow cytometry for antigen-specific memory B cell and total (R21) or antigen-specific (RH5) circulating Tfh (cTfh) cell responses.ResultsFor R21, higher frequencies of NANP-specific activated memory IgG+ B cells were detected in infants versus adults, and also with a higher Matrix-M® dose (50μg versus 25μg). Both NANP-specific IgG+ memory B cells and total cTfh cells correlated with anti-NANP serum IgG. For RH5.1, higher previous malaria exposure was associated with increased RH5- specific cTfh and cTfh2 cell frequencies. RH5-specific IgG+ memory B cell responses were greatest with delayed booster dosing (10-10-10μg at 0-1-6months) compared to monthly or delayed fractional regimens, and correlated with both peak and late time point serum antibody (2-years after final vaccination). Interestingly, while late time point anti-RH5.1 serum IgG concentrations in delayed booster children vaccinees matched previous reports with RH5.1/AS01B delayed fractional dosing in adults (NCT02927145; UK), IgG durability and relationship to RH5-specific B cells differed between these two cohorts.DiscussionWhile our conclusions would be strengthened by further analyses in larger clinical trials, our data indicate that age, adjuvant dose, vaccine dose, and timing of final booster vaccination impact antigen-specific cellular responses to malaria vaccines and could thus inform the development of next-generation multi-stage malaria vaccination strategies.

Abstract Background Plasmodium vivax is an underappreciated cause of malaria disease burden. No reproducible and standardized full life-cycle controlled human malaria infection (CHMI) model to accelerate development of novel interventions is available. Methods This transmission-CHMI trial was conducted in Nijmegen, Netherlands. Healthy, malaria-naive adults were sequentially enrolled into three cohorts of four and inoculated with the asexual blood-stage isolate PvW1. Primary endpoint was proportion of oocyst-positive laboratory-reared Anopheles stephensi mosquitoes. The sequential design allowed for adaptations between cohorts. At parasitemia >10 parasites/µL or symptom onset, participants received oral gametocyte-sparing treatment (GST): mepacrine (Cohort 1 and 3; 100 mg at 0, 8 16 hours, then once daily for 3 days) or piperaquine (Cohort 3; 480 mg single-dose). Transmission was assessed by direct skin feeding (DSF) and membrane feeding assay (DMFA) with and without enrichment of gametocytes. End-of-study treatment was atovaquone-proguanil (1000/400 mg once daily for 3 days). The trial was registered: NL-OMON57011. Findings Participants were enrolled between September 17, 2024 and March 25, 2025, all (12/12) developed parasitemia and transmitted PvW1 to mosquitoes. No serious adverse events occurred. Most adverse reactions were related to malaria. Mepacrine and piperaquine reduced asexual parasitemia while preserving gametocytemia and transmission. Peak transmission occurred within 3 days after GST and depended on the parasite developmental cycle, with highest gametocyte-infectivity ∼48 h post ring-stage. In Cohort 3, mosquito infection reached 100% in all transmission assays. Median peak oocyst counts were 24 (IǪR: 14–31) for DSF, 17 (12–19) for DMFA, and 150 (116–199) for enriched DMFA. A two-fold increase in pre-GST maximal parasitemia was associated with 20 additional oocysts (95% CI 8·6–32) in enriched DMFA. Sporozoites were viable in primary human hepatocytes. Interpretation A PvW1 transmission-CHMI is reproducible and safe, enabling P. vivax sporozoite production, relapse models and evaluation of transmission-blocking interventions. Funding The OptiViVax project is supported by European Union Horizon Europe programme and UK Research and Innovation (UKRI); Swiss Government’s State Secretariat for Education, Research, and Innovation (SERI); National Institute for Health and Care Research Oxford Biomedical Research Centre (NIHR-BRC). Research in context Evidence before this study In its Malaria Vaccine Technology Roadmap, the World Health Organization prioritizes Plasmodium vivax research and vaccine development alongside Plasmodium falciparum . Controlled human malaria infections (CHMI) play an important role in the development of new interventions as they enable early evaluation of new drugs and vaccines in small groups of participants. Added value of this study To our knowledge this is the first study to achieve consistent transmission of a P. vivax clone to mosquitoes following blood-stage P. vivax CHMI. Reproducible high-level transmission was observed both in direct feeding assays and after gametocyte enrichment and parameters were identified that improve transmission success. It is also the first P. vivax CHMI trial that systematically assessed the use of piperaquine and mepacrine to attenuate asexual replication while preserving transmission-competent gametocytes. Implications of all the available evidence This study expands the repertoire of P. vivax CHMI models and builds on the success and knowledge of prior P. vivax and P. falciparum CHMIs. This optimised transmission model enables production of highly infected mosquitoes and sporozoites for downstream use with a genetically-defined clone, which is particularly important for P. vivax because it cannot be maintained in continuous culture. It also facilitates evaluation of transmission-blocking interventions.

Background Plasmodium vivax is the second leading cause of malaria worldwide. The lack of continuous in vitro culture of P. vivax hampers research efforts, including research on transmission to mosquitoes and sporozoite biology . Here, we present methodological improvements to achieve high mosquito infection rates. Methods We compared two gametocyte enrichment methods: magnetic cell sorting (MACS) and Percoll density gradient centrifugation (DGC) at 1500 g or 760 g. Blood samples before and after enrichment were offered to Anopheles stephensi mosquitoes by membrane feeding. Outcomes included the proportion of mosquitoes infected, oocyst density and sporozoite positivity. Protocols were first evaluated using in vitro cultured P. falciparum , before being further optimized using gametocyte-positive samples from eight healthy, malaria-naive adults infected with a P. vivax clone ‘PvW1’ during a controlled human malaria infection. Results DGC at a speed of 1500 g achieved the highest infection rates. Oocyst density increased approximately 86-fold (95% CI 14–525; p < 0.01) and the proportion of infected mosquitoes was on average 58% higher when compared to the non-enriched control (95% CI 42.6–74.0; p < 0.01). DGC at 760 g increased the oocyst density 11-fold (CI 1.8–69; p < 0.01) and the proportion of infected mosquitoes by 28% (CI 12.1–43.9; p < 0.01). MACS did not increase P. vivax mosquito infection compared to control. Infected mosquitoes became salivary gland positive, with the mean sporozoite load in infected mosquitoes increasing with oocyst density. Conclusions This study provides the first systematic comparison of MACS and DGC as enrichment techniques to increase P. vivax transmission and presents the DGC protocol that achieved the highest proportion of infected mosquitoes and oocyst densities. This protocol will facilitate P. vivax transmission studies and enable the generation of highly infected mosquitoes and sporozoites for downstream use.

Abstract Background Plasmodium vivax malaria relapses are a major source of morbidity and onward transmission of infection. The underlying mechanisms are poorly understood and current therapies sub-optimal. We examined the safety and feasibility of a controlled human malaria infection (CHMI) model for relapsing P. vivax . Methods We conducted an open-label, proof-of-concept, CHMI study of relapsing P. vivax . Healthy, malaria-naïve, Duffy-positive adults aged 18–45 years with extensive CYP2D6 metaboliser phenotype and normal blood glucose-6-phosphate dehydrogenase (G6PD) levels were recruited in Oxford, UK. Mosquito-bite CHMI was performed in Nijmegen, The Netherlands, using Anopheles stephensi mosquitoes infected with PvW1, a clonal isolate of P. vivax from Thailand. All follow-up visits were conducted in Oxford, UK. Primary P. vivax infections (qPCR > 500 genome copies/mL) were treated with artemether-lumefantrine (80mg/480mg at 8, 24, 36, 48 and 60 hours). From Day 28 following CHMI, participants attended a fortnightly clinic for clinical review and qPCR blood sampling, with additional assessments performed for any reported symptoms. P. vivax relapse infections (qPCR > 500 genome copies/mL) were treated with artemether-lumefantrine as per primary infection. Definitive anti-malarial treatment with atovaquone-proguanil (1000mg/400mg once daily for three days) and primaquine (0·5 mg/kg/day for 14 days) was administered six months following CHMI, regardless of parasitaemia or symptoms. The primary objective was to assess the safety, feasibility and frequency of relapsing P. vivax after CHMI. Remote follow-up (5 years) is ongoing. The study is registered with ISRCTN registry ( ISRCTN48625883 ). Findings 20 participants were screened for eligibility from 21 January 2025. Five participants (median age 22 years) underwent CHMI (five infected mosquitoes per participant) on 15 April 2025. All participants developed primary P. vivax infection and experienced at least one relapse infection. Two participants experienced a second relapse. Overall incidence rate was 3·6 relapse infections per person-year. Solicited adverse events were mild or moderate and there were no serious adverse events. Definitive anti-malarial treatment was administered to all participants. One participant experienced primaquine-induced methaemoglobinaemia, resolving with early discontinuation of treatment (total dose 5·3 mg/kg). To date, more than six months after primaquine treatment, no further relapses have been recorded. Interpretation CHMI of relapsing P. vivax is safe and feasible, allowing exploration of the mechanisms underlying relapse infections and providing a platform for future anti-relapse efficacy studies. Funding European Union Horizon Europe programme and UK Research and Innovation (UKRI) via OptiViVax consortium; UK National Institute for Health and Care Research Biomedical Research Centre: Oxford; and UK Medical Research Council. Research in Context Evidence before this study Relapse infections account for the majority of cases of Plasmodium vivax malaria. There are no licensed vaccines for P. vivax and current anti-relapse hypnozoiticidal therapies (primaquine and tafenoquine) have restrictive contraindications, side effects and variable efficacy. A controlled human malaria infection (CHMI) model for relapsing P. vivax , using a well-characterised clonal isolate of P. vivax administered by mosquito bite, would present an opportunity to improve understanding of hypnozoite immuno-biology and provide a platform for efficacy studies of novel anti-relapse interventions. We searched PubMed on 23 April 2026 for research articles using the terms (“P. vivax” OR “Plasmodium vivax” OR “vivax”) AND (“malaria challenge” OR “Controlled Human Malaria Infection” OR “CHMI” OR “sporozoite challenge”). No date or language filters were applied. The search identified seven published CHMI studies in which P. vivax was administered by mosquito bite. All these studies utilised mosquitoes infected from a source patient(s) with naturally-acquired infection, thereby limiting interstudy comparison and creating logistical challenges for reproducibility. Only one study reported relapse infections which occurred following primaquine treatment in two participants with poor and intermediate CYP2D6 metaboliser phenotype respectively. In addition to this PubMed search, historical records were identified which described the deliberate infection of humans with P. vivax i) in experiments conducted in the early 20 th Century, and ii) as part of malariotherapy for the treatment of “general paralysis of the insane” (neurosyphilis). Although some of these records used well-characterised strains of P. vivax (e.g. Madagascar strain) and included relapse infections, these pre-dated modern molecular and serological laboratory techniques. Having previously generated a cryopreserved inoculum of P. vivax -infected red blood cells (PvW1) with high-quality genome assembly, we set out to administer this clonal isolate in a CHMI study by mosquito bite, and characterise P. vivax relapse infections over a six-month period prior to the administration of primaquine treatment. Added value of this study This study reports the administration of PvW1 by mosquito bite to five healthy adult participants in the UK. All participants developed primary P. vivax infection and experienced at least one relapse infection in a six-month follow-up period. Two participants experienced a second relapse infection prior to the administration of definitive anti-malarial treatment with atovaquone-proguanil and primaquine. Solicited adverse events (foreseeable symptoms of malaria) were mild or moderate. Long-term remote follow-up is ongoing. However, to date, more than six months after primaquine treatment, no further relapse infections have been recorded. To our knowledge, this is the first CHMI study in the modern era to administer a well-characterised clonal isolate of P. vivax by mosquito bite. We demonstrate that a CHMI model for relapsing P. vivax is safe and feasible. Implications of all the available evidence The World Health Organisation (WHO) Technical Brief on the Control and Elimination of Plasmodium vivax Malaria calls for better understanding of the biology and epidemiology of P. vivax, and the development of new interventions and strategies. Specifically, it highlights a need for improved knowledge of the underlying mechanisms of P. vivax relapse infections; a single-dose hypnozoiticidal treatment that can be used for all population groups without significant side effects; and development of P. vivax vaccines. CHMI studies allow the study of host-pathogen interactions in detail and accelerate the development of vaccines and therapeutic drugs by providing evidence of efficacy early in clinical development. This proof-of-concept study presents an opportunity to explore the mechanisms behind hypnozoite formation and activation in humans, and provides a novel clinical platform for future drug and vaccine efficacy studies.

Plasmodium falciparum RH5-interacting protein (RIPR) is central to the essential PTRAMP-CSS-RIPR-CyRPA-RH5 (PCRCR) complex, a leading target of blood-stage malaria vaccines. However, mechanisms whereby anti-RIPR antibodies inhibit parasite invasion are poorly understood. We characterized 83 human IgG monoclonal antibodies (mAbs) from RIPR-vaccinated Kymouse platform mice. Single mAbs had minimal neutralizing activity; however, high-level synergistic inhibition was observed with pools of mAbs targeting the RIPR-tail region. Structural characterization and molecular dynamics simulations of RIPR-tail showed that mAbs targeting epidermal growth factor (EGF)-like domains 6-8 (RIPREGF (6-8)), but not RIPREGF (9-10) or the C-terminal domain (RIPRCTD), synergized to constrain the RIPR-tail conformation. The same antibodies dissociated PTRAMP-CSS from RIPR, thereby enabling anti-RIPREGF (9-10)-CTD mAbs or anti-CSS single-domain Abs to bind and potentiate anti-RIPREGF (6-8) IgG. Addition of these mAbs to IgG from humans immunized with the R78C (RIPREGF (7-8)-CyRPA) candidate vaccine enhanced malaria growth inhibition. These data provide a framework to guide next-generation blood-stage malaria vaccine design.

Abstract An efficacious blood-stage malaria vaccine would serve as a highly useful public health tool alongside licensed vaccines targeting the pre-erythrocytic life cycle stage of the Plasmodium falciparum parasite. RH5 is the leading blood-stage malaria vaccine candidate antigen due to its highly-conserved sequence and non-redundant role in merozoite invasion of red blood cells. Following encouraging immunogenicity data in UK and Tanzanian Phase Ia/b vaccine trials, RH5-based vaccines have progressed to Phase IIb evaluation in Burkina Faso in recent years. Here, we report a Phase Ia clinical trial in malaria-naïve UK adults to assess the safety and immunogenicity of the malaria vaccine candidate RH5.1 soluble protein with Matrix-M ® adjuvant using two different booster dosing regimens: 10-10-10 µg versus 50-50-10 µg RH5.1, both delivered in a 0-1-6-month schedule with 50µg Matrix-M ® adjuvant per dose ( ClinicalTrials.gov NCT06141057 ). A total of n=24 participants were recruited to this study, with n=23 completing all follow-up visits through to 1 year following final vaccination. The RH5.1/Matrix-M ® formulation was well-tolerated in this population, with injection site pain, myalgia and fatigue being the most commonly reported symptoms up to 7 days post-vaccination. There were no serious adverse events, adverse events of special interest, or suspected unexpected serious adverse reactions reported over the course of the trial. Both vaccination regimens were similarly immunogenic; no differences were observed in peak anti-RH5.1 serum IgG concentrations, in vitro functional anti-parasitic activity, avidity, or durability. Our findings build on other observations from clinical trials of adjuvanted RH5.1 indicating that humoral immunogenicity can be enhanced by delaying the final booster vaccination, but that there is limited impact of fractionation of the final dose. These insights can help to guide the next steps of multi-antigen, multi-stage malaria vaccine development in malaria-endemic settings.

mRNA-lipid nanoparticle (LNP) and adenoviral delivery of vaccines have proven effective during the COVID-19 pandemic. Herein, we explored whether antigen-presenting cell (APC) targeting of antigens could further enhance the immunogenicity of these delivery formats. Experiments were performed in mouse models for malaria (Plasmodium falciparum reticulocyte-binding protein homolog 5 [PfRH5] antigen) and influenza (hemagglutinin [HA] antigen). We used genetic constructs encoding bivalent fusion proteins that target antigens to MHC class II (MHCII) molecules on professional APCs. We demonstrate that MHCII-targeted fusion proteins bound to professional APCs and that such APC-targeting increased antibody and T cell responses as well as protection against the influenza virus. The results suggest that the injected mRNA-LNP and adenoviral vectors resulted in the secretion of fusion proteins that targeted APCs. Employing the APC-targeting principle could enhance the efficiency of adenoviral and mRNA-LNP vaccines against a variety of diseases.

Ebolavirus disease (EVD) is caused by multiple species of orthoebolavirus. Monoclonal antibodies (mAbs) against the virus glycoprotein (GP) are the only class of therapeutic approved for treatment of EVD caused by Orthoebolavirus zairense (Ebola virus, EBOV). Therefore, mAbs targeting multiple orthoebolavirus species may represent the next generation of EVD therapeutics. Broadly reactive anti-GP mAbs were produced; among these, mAbs 11886 and 11883 were broadly neutralizing in vitro. A 3.0 Å cryo-electron microscopy structure of EBOV GP bound to both mAbs shows that 11886 binds a novel epitope bridging the glycan cap (GC), 310 pocket and GP2 N-terminus, whereas 11883 binds the receptor binding region (RBR) and GC. In vitro, 11886 synergized with a range of mAbs with epitope specificities spanning the RBR/GC, including 11883. Notably, 11886 increased the breadth of neutralization by partner mAbs against different orthoebolavirus species. These data provide a strategic route to design improved mAb-based next-generation EVD therapeutics.

Background Malaria is the most common tropical infection in the UK. Current guidelines suggest that testing on 3 consecutive days is required following an initial negative result. This study aimed to see whether newer diagnostics (loop-mediated amplification assay [LAMP]) had sufficient sensitivity to support a change in diagnostic practice. Methods Blood samples from 11 participants who had undergone controlled human malaria infection (CHMI) with Plasmodium falciparum malaria were assessed from day 6 (C+6) for malaria positivity using the Carestart Malaria rapid diagnostic test (RDT) and from C+4 using the Alethia Malaria LAMP assay. Quantitative polymerase chain reaction had been performed twice daily during CHMI follow-up. A retrospective analysis of samples submitted to the Sheffield Teaching Hospitals for malaria testing over a 5-y period was conducted, evaluating the combination of the Carestart RDT alongside blood film analysis, as per UK guidelines. Results In CHMI samples, LAMP was positive for all parasitaemias >1000 parasites/ml, whereas RDTs were less reliable (59% positive for parasitaemias >1000 parasites/ml). The combination of RDT and blood films for clinical samples diagnosed most infections, but only a minority of negative samples had subsequent tests. Conclusions LAMP has higher sensitivity than current UK recommended methods, with a potential to review the requirement for additional days of testing in the majority of patients.

PfCyRPA and PfRIPR are promising next-generation malaria blood-stage vaccine candidate antigens that play an essential role in erythrocyte invasion of Plasmodium falciparum. CyRPA and RIPR orthologs are present in all human-infecting Plasmodium species, suggesting the potential for a cross-species vaccine. Using Growth Inhibition Assays (GIA), this study investigates seven anti-PfCyRPA and three anti-PfRIPR monoclonal antibodies targeting P. falciparum for their inhibitory activity against P. knowlesi, a non-falciparum species that contributes to a significant burden of zoonotic disease in South-East Asia, shares some biological features with Plasmodium vivax, and has a robust in vitro culture system. Despite their efficacy against P. falciparum and partially conserved epitopes, these antibodies exhibited minimal inhibition of P. knowlesi. Understanding the antigenic diversity and immune mechanisms across Plasmodium species is critical for advancing pan-species vaccine strategies.

An effective blood-stage vaccine is needed to protect against malaria pre-erythrocytic stage breakthrough. P. falciparum reticulocyte-binding protein homolog 5 (PfRH5) has emerged as a promising blood-stage vaccine antigen candidate, reducing parasite growth in humans during malaria challenge and showing field efficacy in children. Here, we characterize the human plasma IgG response to the RH5.1 vaccine candidate at monoclonal resolution, revealing that plasma repertoires are dominated by abundant, non-neutralizing antibodies. Using oligoclonal reconstitution experiments, in which defined pools of recombinant plasma mAbs are reassembled and functionally tested, we map how individual antibody interactions shape parasite growth inhibition activity. This approach allows us to discern which antibodies, within a polyclonal setting, act additively or synergistically, thereby revealing the emergent properties of anti-PfRH5 IgG. We further show that IgG lineages targeting linear epitopes lack neutralizing activity, while non-neutralizing IgG lineages that bind conformational epitopes can exhibit potent, interdependent synergy with each other and with neutralizing mAbs. These synergistic antibodies were identified in the plasma IgG compartments of five volunteers and highlight non-neutralizing PfRH5 epitopes that are critical for polyclonal-mediated growth inhibition. Our findings have broad implications for PfRH5 vaccine immunogen engineering and the role of non-neutralizing antibodies in infectious disease immunity. ### Competing Interest Statement KM, JRB and SJD are inventors on patent applications relating to RH5 malaria vaccines and/or antibodies. AMM and SJD have consulted to GSK on malaria vaccines. AMM has an immediate family member who is an inventor on patent applications relating to RH5 malaria vaccines and antibodies. All other authors have declared that no conflict of interest exists. United States Agency for International Development (USAID) Malaria Vaccine Development Program (MVDP), 7200AA20C00017