Abstract Host cell invasion by malaria parasites requires specific molecular interactions with host receptors. Plasmodium vivax merozoite invasion of reticulocytes is mediated by P. vivax Duffy binding protein (PvDBP) and its homolog, P. vivax erythrocyte binding protein (PvEBP). Here, we identify and characterize two novel P. vivax merozoite proteins, PvMP45 and PvMP36, which co- localize with PvDBP and PvEBP in the micronemes and bind reticulocyte receptors. PvMP45 and PvMP36 share high sequence identity with their P. knowlesi homologs, PkMP45 and PkMP36, which form a complex with other invasion related proteins. Field studies reveal that naturally acquired antibodies against PvMP36, PvEBP and PvDBP are associated with protection against clinical P. vivax malaria. We demonstrate that naturally acquired antibodies to PvEBP bind Fcy receptors and likely mediate protection by enabling opsonic phagocytosis. In addition, we show that combining antibodies against PvDBP and PvMP36 results in an additive invasion inhibitory effect against P. vivax blood stages. These results suggest that combining PvDBP, PvEBP and PvMP36 in a multivalent blood stage vaccine could elicit diverse immune mechanisms against P. vivax to achieve high efficacy. Importance All the clinical symptoms of malaria are attributed to the blood stage of malaria parasites during which merozoites invade and multiply within red blood cells. A clear understanding of the host- parasite interactions that enable invasion can open paths for development of novel methods to block parasite growth and prevent malaria. Here, we identify and characterize two novel invasion related proteins from P. vivax merozoites that form an invasion complex and bind host RBC receptors. We demonstrate that antibodies targeting these proteins can block RBC invasion by P. vivax and naturally acquired antibodies that develop following P. vivax infection against one of these proteins are associated with protection against P. vivax malaria. These studies not only expand our understanding of the molecular mechanisms that enable host cell invasion by P. vivax but open new avenues for development of vaccines to protect against P. vivax malaria.

Malaria parasite genomes have been generated predominantly using Illumina short-read sequencing that requires expensive equipment, is time-consuming with complex protocols, and does not adequately interrogate complex genomic regions that harbour important malaria virulence determinants. The portable Oxford Nanopore Technologies MinION platform generates long reads in real time and may overcome these limitations. We present compelling evidence that Nanopore sequencing delivers valuable additional information for malaria parasites with similar data fidelity for single nucleotide variant (SNV) calls compared to standard Illumina whole-genome sequencing. We demonstrate this through sequencing of pure Plasmodium falciparum DNA, mock infections and natural isolates from low-density, asymptomatic infections. Nanopore has low error rates for haploid SNV genotyping and identifies structural variants not detected with short reads. Nanopore genomes can be directly compared to publicly available genomes and produce high-quality end-to-end chromosome assemblies including complex, previously difficult-to-access regions. Nanopore sequencing could expedite whole-genome surveillance of malaria and provide new insights into parasite genome biology.

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.

IntroductionLong-term humoral immunity to Plasmodium vivax remains poorly understood, particularly the mechanisms sustaining persistent antibody responses to Duffy Binding Protein region II (DBPII), a key vaccine candidate. Here, a 15-year follow-up study of Amazonian malaria-exposed individuals investigated major cytokine pathways associated with sustained IgG responses to DBPII-related antigens, including DEKnull-2, an engineered immunogen targeting conserved DBPII epitopes.MethodsAs a proof-of-concept, this study was designed to compare two well-defined immunological phenotypes among P. vivax-exposed individuals: (i) Persistent Responders (PR, n = 11), who developed a long-lasting strain-transcending anti-DBPII immune response, defined here as sustained antibody responses to both DBPII and DEKnull-2, together with detectable binding-inhibitory antibody activity (BIAbs), throughout the follow-up period; and (ii) Non-Responders (NR; n = 14), who showed no detectable antibody response or BIAbs activity during follow-up. Cytokine responses were measured in supernatants of PBMC cultures stimulated with DBPII or DEKnull-2 by cytometric bead array (CBA), and IgG breadth was assessed using a panel of 16 P. vivax blood-stage antigens using Bio-Plex assay.ResultsRegardless of the antigen used for stimulation, P. vivax-exposed individuals (NR and PR) exhibited lower IL-6 and TNF levels than malaria-naïve non-exposed controls (NE, n = 14). Notably, the PR subgroup displayed a distinct profile characterized by a coordinated increase across inflammatory and anti-inflammatory cytokines. Even without antigen-specific stimulation, the PR group showed a distinct cytokine profile compared with NE controls, suggesting that host-intrinsic factors may influence DBPII-specific immune responses. Finally, PR (but not NR) displayed broader long-lasting antibody responses to multiple P. vivax blood-stage antigens, even in the absence of frequent reinfection-induced boosting.ConclusionOverall, these findings suggest that the PR profile is associated with a distinct and balanced cytokine profile. Our hypothesis-generating findings may improve our understanding of immune responses to P. vivax blood-stage antigens and inform future vaccine development.

BackgroundGlobal efforts to control and eventually eliminate malaria have been less effective for Plasmodium vivax relative to Plasmodium falciparum due to its unique biology, including dormant liver stages that cause later relapse, and earlier commitment to transmission stages. After the nationwide distribution of long-lasting insecticide treated nets (LLIN) in Papua New Guinea (PNG), P. vivax initially reduced to low prevalence, but again resurged to levels similar to those before LLIN distributions.MethodTo explore changes in P. vivax population structure and identify sources of resurgence over this period, we applied a previously validated genome-wide SNP barcode to genotype 336 P. vivax isolates obtained from serial cross-sectional surveys conducted over a decade in East Sepik (2005, 2012, 2016) and Madang Province (2006, 2010, 2014).ResultsPopulation genetic analyses of the resulting parasite genotypes revealed contrasting spatiotemporal patterns between the two provinces. In Madang, the complexity of infection, genetic diversity, and population structure varied with prevalence, with a possible population bottleneck and early clonal expansion at low transmission, and rapid recovery of the population with resurgence. In East Sepik, there was a less dramatic impact on the parasite population after prevalence decline, and ongoing transmission of multiple residual lineages throughout the study period. P. vivax decline was also accompanied by an increase in genetic differentiation between the two areas, which reduced with resurgence suggesting changes in parasite migration between areas associated with prevalence.ConclusionThe earlier implementation of LLIN in East Sepik, smaller rebound, heterogeneity in transmission and relative isolation, compared to Madang may have contributed to these differing patterns. The results demonstrate that long term sustained control efforts are essential to make a lasting impact on the P. vivax population, and that SNP barcodes can provide valuable insights into parasite transmission dynamics as a result of control efforts.

BACKGROUND:Plasmodium vivax presents a significant obstacle to malaria elimination due to its capacity to form dormant liver-stage hypnozoites that can cause relapses. Universal radical cure, which administers hypnozoite-targeting treatment to patients with P. falciparum malaria living in co-endemic areas, has potential to reduce P. vivax relapses. However, its implementation is hindered by the lack of a diagnostic tool for detecting hypnozoite-carriage. METHODS:P. vivax serological exposure markers (SEMs) were evaluated as a screening tool in P. falciparum patients for predicting risk of P. vivax microscopy-detected episodes over the following 63 days. Analysis was performed using samples from participants in the PRIMA study from Ethiopia, Indonesia, and Bangladesh (NCT03916003). An existing random forest serological classification algorithm was used and evaluated for sensitivity and specificity, then further optimized by re-training a study-specific model. RESULTS:IgG antibodies were measured in 244 P. falciparum participant samples, of which 22 had a vivax microscopy-detected episode during follow-up. Serological exposure markers showed high sensitivity (82%) but low specificity (27%), likely due to sustained antibody responses in moderate-to-high transmission areas or undetected submicroscopic P. vivax infections during follow-up. A study-specific algorithm increased specificity to 68%, but with a corresponding drop in sensitivity to 68%. CONCLUSIONS:Although P. vivax SEMs showed limited suitability for guiding radical cure in P. falciparum patients in this study (where the outcome was microscopy-detected vivax episodes), they could play a valuable role in building community trust and acceptance of universal radical cure when supported by strong communication and implementation strategies.

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 remains a major public health challenge in Pakistan. Radical cure is required to eliminate liver stages of the parasite and prevent relapses, however the optimal primaquine regimen in Pakistan is unknown. Clinical outcomes following treatment with the national standard regimen of chloroquine plus 14-day low-dose (3·5mg/kg total dose) primaquine were evaluated in a prospective cohort of patients with P. vivax malaria. Methods: Data from a prospective clinical cohort study in Thatta district, Sindh Province, Pakistan, were analysed to evaluate treatment outcomes. Adult patients with microscopy-confirmed P. vivax monoinfection and glucose-6-phosphate dehydrogenase (G6PD) activity ≥70% of the adjusted male median were treated, according to national guidelines, with chloroquine (25 mg/kg over 3 days) plus primaquine (0·25 mg/kg/day for 14 days; total dose 3·5 mg/kg). Treatment was provided with adherence support through phone calls and participants were followed for nine months. The cumulative risk of recurrent P. vivax parasitaemia at 9-months post treatment was estimated using Kaplan–Meier analysis. Safety outcomes included assessment of symptoms on day 14 and changes in haemoglobin concentrations during follow-up. Findings: Between August 2023 and April 2024, 80 participants were enrolled, three of whom had P. vivax recurrences on days 61, 158 and 189 after treatment. The cumulative incidence of recurrence at 9 months was 3·9% (95% confidence interval 1·3 – 11·6). Treatment was well tolerated, with no serious adverse events. None of the patients had clinically significant haemolysis and haematological recovery following acute malaria was rapid. Interpretation: In G6PD-normal adults with P. vivax malaria in southern Pakistan, there was a low risk of P. vivax recurrence within 9 months following 14-day low-dose primaquine.

Understanding the genetic relatedness of Plasmodium vivax recurrences is essential for distinguishing between relapse, reinfection, and recrudescence-a distinction critical for evaluating treatment efficacy and transmission dynamics. We developed P. vivax AmpSeq (PvAmpSeq), an amplicon sequencing assay targeting 11 single-nucleotide polymorphism (SNP)-rich genomic regions. PvAmpSeq was applied to field isolates from a clinical trial in the Solomon Islands and a longitudinal cohort in Peru, and statistical models were applied for the genetic classification of recurrences. In the Solomon Islands trial, where participants received antimalarials at baseline, half of the recurrent infections showed >50% identity-by-descent relatedness to baseline parasites, allowing statistical classification as probable relapses and recrudescences, although with wide uncertainty. In the Peruvian cohort, 68% of the recurrences exhibited <25% relatedness. PvAmpSeq provides high-resolution genotyping to characterize P. vivax recurrences, offering insights into transmission and treatment outcomes. We also discuss the nuances and limitations of available statistical methods for the classification of P. vivax genotyping data.

Background Plasmodium vivax (P. vivax) has emerged as the primary cause of malaria in Cambodia. Achieving malaria elimination and securing malaria-free certification requires a focused effort on addressing P. vivax malaria. This is essential because the elimination of P. vivax often lags behind that of Plasmodium falciparum, making it a critical component in the overall strategy. This study assesses the feasibility of the Mass Drug Administration (MDA) and P. vivax Serological Testing and Treatment (PvSeroTAT) integrated with Reactive Case Detection (RACD) in two of the highest malaria burden operational districts of Cambodia and examines the potential for integrating these two approaches with existing malaria elimination efforts. Methods This study employs an observational, prospective cohort design. MDA with chloroquine (CQ) will be conducted in Stung Treng through four monthly rounds, while RACD with PvSeroTAT will be implemented in Sen Monorom, targeting households near confirmed P. vivax cases. Data on coverage, compliance, cost, and stakeholder perceptions will be collected through surveys, interviews, and malaria case monitoring. A Composite Feasibility Index will integrate quantitative and qualitative indicators. Cost and budget impact analyses will assess scalability for malaria-endemic districts. Discussion Innovative and targeted public health approaches and tools are necessary to ensure the elimination of the malaria parasite reservoir, including the hidden hypnozoites. While MDA with CQ clears active blood-stage infections leading to immediate reductions in malaria prevalence, PvSeroTAT can detect past exposure to P. vivax by using serological markers allowing for targeted treatment of individuals at risk of developing relapsing infections with an 8-aminoquinoline. This helps reduce the parasite reservoir more efficiently. This study will provide insight into operational feasibility, implementation costs, community acceptance, and long-term sustainability. The findings will guide Cambodia’s malaria elimination efforts through improved surveillance and targeted interventions. Trial Registration OSF Preregistration: https://doi.org/10.17605/OSF.IO/5KZH7, retrospectively registered 15 October 2025. ### Competing Interest Statement TD and EJ are employed by MMV Medicines for Malaria Venture. MS, TH, and CAL are consultants engaged by MMV Medicines for Malaria Venture. STK, SS, JP, IM and LR declare no competing interests. ### Clinical Protocols ### Funding Statement Yes ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Before the commencement of this study, ethical approval was obtained from the National Ethics Committee for Health Research (No. 085 NECHR). In addition, written informed consent will be obtained from all participants in Khmer, ensuring full understanding. Verbal assent will be taken from children <18 years along with a signed consent from their guardians. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes No datasets were generated or analysed during the current study. All relevant data from this study will be made available upon study completion.

The key ligand involved in Plasmodium vivax reticulocyte invasion is the Duffy Binding Protein (PvDBP) which binds the Duffy receptor on reticulocytes. Anti-PvDBP human monoclonal antibodies can inhibit PvDBP-Duffy receptor binding and neutralize reticulocytes invasion in vitro. However, parasites with multiple copies of the pvdbp gene are protected in vitro against neutralization. Here, we evaluated whether this gene amplification also protects parasites in vivo.We hypothesized that: (i) multi-pvdbp copy parasites are more frequent in areas with a high P. vivax prevalence, (ii) individuals with naturally acquired binding inhibitory anti-PvDBP Abs (BIAbs) are predominantly infected over time by multi-copy parasites and (iii) multi-copy parasites infect asymptomatic carriers more frequently than symptomatic individuals. We analyzed samples from a 2019-2020 longitudinal cohort of individuals living in nine villages in Eastern Cambodia with low (~5%) to high (~30%) P. vivax prevalence. Using a PCR assay targeting the boundaries of the pvdbp duplication, we estimated the frequency of multi-copy parasites over time. Then, using a flow cytometry assay, we determined the presence of naturally acquired BIAbs in 657 participants' plasma. Finally, we compared parasite gene copy number over the 21-month follow-up in cohort participants according to the presence of BIAbs at the start of the study. In parallel, we determined the frequency of pvdbpduplication in samples collected among symptomatic treatment-seeking patients in the same area over the same period. We compared the frequency of infection with multi-copy parasites between asymptomatic cohort members and symptomatic patients. We found a significant association between P. vivax prevalence and the proportion of multi-copy parasites, which ranged from 35% in low prevalence villages to 47% in high prevalence villages (p=0.0246). We also observed that the more inhibitory the Abs in the hosts' plasma, the higher the proportion of multi-copy parasites: 87% (40/46) from individuals with high BIAbs while 38% (193/514) from individuals without any BIAbs (p<0.0001). This association between immunity and infection by multi-copy parasites remained consistent over the 21-month longitudinal follow-up. Finally, we found that the frequency of multi-copy parasites was higher in asymptomatic carriers than in symptomatic individuals. Overall, these results indicate that pvdbp duplication helps the parasites to avoid the hosts' anti-PvDBP immunity in vivo. It warrants further investigations to determine if immunization with a PvDBP vaccine could overcome this immune evasion mechanism.

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.

Objective To investigate whether IgG to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens could reveal undetected SARS-CoV-2 exposure in a cohort of Malawian pregnant women participating in the REVAMP clinical trial.Design A secondary analysis of serological samples from a randomised controlled trial of iron supplementation in pregnancy, which began recruiting women in November 2018 and had its last visit occurring in September 2021.Setting Resource-limited setting in Zomba and Blantyre, Southern Malawi.Participants Pregnant women with ultrasound-confirmed singleton pregnancies in the second trimester, with haemoglobin <100 g/L and randomised at enrolment to receive either intravenous ferric carboxymaltose or standard oral iron, where no coronavirus disease 2019 (COVID-19) clinical cases or SARS-CoV-2 positive tests were reported during the pandemic (April 2020–September 2021).Primary and secondary outcomes Primary outcome was the levels of antibodies and seropositivity to SARS-CoV-2 antigens in the cohort of women across the duration of the trial. Secondary outcomes were the impact of IgG levels and seropositivity to SARS-CoV-2 on pregnancy outcomes.Results At delivery, IgG levels to SARS-CoV-2 antigens increased sharply by 18.5%–29.7% every 30 days during COVID-19 waves 2 and 3. Overall seropositivity reached 39.3% during the pandemic; however, 14.7% pre-pandemic seropositivity demonstrates cross-reactive antibody responses. Pandemic pregnancies showed improved outcomes with longer gestations (mean difference: 0.6 weeks (95% CI 0.2 to 0.9)) and higher birth weights (mean difference: 169.3 g (65.9–272.6)). SARS-CoV-2 IgG levels were not associated with pregnancy outcomes.Conclusion Serological testing was able to detect exposure to SARS-CoV-2 in a population without clinical indications of the disease, suggesting that serosurveillance is more sensitive than relying on clinical data to monitor pathogen exposure in the community. Additionally, this highlights pregnancy cohorts as valuable sentinel populations for infectious disease surveillance in resource-limited settings.Trial registration number This trial was prospectively registered at ANZCTR: ACTRN12618001268235.

Background Asymptomatic Plasmodium vivax infections undermine malaria control by sustaining transmission. This study aimed to validate an eight-marker serological panel (selected from 14 antigens) of P. vivax exposure to detect individuals likely harbouring hypnozoites in rural Cambodia, and to compare it with molecular surveillance for identifying at-risk populations in low-transmission settings. Methods Two studies were conducted in Mondulkiri, Cambodia. In a longitudinal cohort (N = 471), participants were tested monthly for one year for P. vivax using qPCR. At 12 months, IgG responses to 14 antigens were assessed, and Random Forest models (‘Cambodia-specific’ and ‘global’) were used to classify previous infection, with PCR as the reference standard. In a cross-sectional survey (N = 3538), risk factors for sero- and PCR positivity were assessed using generalized linear mixed-effects models. Findings PCR detected P. vivax in 29.9% (141/471) of participants over the 12-month follow-ups, while 31.8% (150/471) were classified as seropositive at month 12. Of 141 PCR-confirmed cases, 95 were seropositive (67.4%) and 46 seronegative (32.6%) under the 8-antigen panel. The ‘global’ model achieved an AUC of 0.81, with 83.3% specificity and 69.5% sensitivity for infections within nine months, increasing to 82% sensitivity for last-month infections. In the cross-sectional study, serology identified more positives than PCR, reflecting detection of exposure not captured by PCR. Interpretation This study provides the first validation of serological markers of P. vivax exposure in Cambodia, showing that such tools can identify recent and asymptomatic infections, though sensitivity was below target. Serology-based surveillance and strategies such as PvSeroTAT could accelerate malaria elimination. Funding NIH/NIAID, ICEMR Asia-Pacific U19AI129392.

BACKGROUND:Autoantibodies have been implicated as key players in COVID-19 pathogenesis, with evidence of impacting disease severity and poor outcomes. METHODS:We analyzed autoantibody profiles in 435 PCR-confirmed COVID-19 patients in Manaus, Brazil, comparing hospitalized (263) and nonhospitalized (172) individuals, and 37 healthy controls. Twenty autoantibodies and SARS-CoV-2-specific IgA/IgG antibodies were measured using multiplex Luminex® assays from plasma samples collected on days 1, 7, 14, and 28. RESULTS:Hospitalized patients had significantly higher levels of autoantibodies targeting ACE2, MPO, IFNΩ, IFNα1, and IFNα2 at baseline, with 127 hospitalized patients positive for IFNα1, 161 for IFNα2 and 68 for IFNΩ. Longitudinal analysis revealed progressively increasing levels of anti-ACE2, B2G1, C1q, IFNα1, IFNα2, PADI4, PF4, and PR3 autoantibodies in hospitalized patients; however, the neutralizing capacity of IFNs were not investigated. Survival analysis showed that elevated type I IFN autoantibodies correlated with reduced survival (P = .023). Stronger correlations between autoantibodies and SARS-CoV-2 IgA/IgG were found in severe cases. CONCLUSIONS:In this cohort, autoantibodies were linked to COVID-19 severity and mortality, indicating their potential as biomarkers for patient risk stratification and therapeutic targets. Further research is needed to explore their impact in long-term immune dysregulation and postacute sequelae in COVID-19 survivors. LAY SUMMARY:COVID-19 affects people differently. Some recover quickly, while others become very sick or die. Scientists have found that some COVID-19 patients develop autoantibodies, or "self-attacking" antibodies. How and which autoantibodies were relevant in COVID-19 remains to be described. This study conducted in Manaus, Brazil, aimed to understand if these autoantibodies contributed to severe COVID-19 and poor survival outcomes. Manaus was one of the hardest-hit regions by the pandemic. We studied 435 COVID-19 patients (263 hospitalized, 172 nonhospitalized), and 37 healthy individuals. Blood samples were collected over 28 days and tested for 20 different autoantibodies and antibodies against the coronavirus using advanced laboratory assays. Hospitalized patients had notably higher levels of 2 specific autoantibodies. Those that block the body's natural antiviral proteins (type I interferons) and ACE2 (helps regulate systemic and local inflammation, and fluid homeostasis). These autoantibodies were strongly linked with severe illness and a higher risk of death, and increased over time. Patients with strong autoantibody responses also had higher levels of antibodies against the virus, suggesting a link between infection and immune self-attack. Autoantibodies were strongly linked to severe COVID-19 and death. Screening for them could help doctors identify high-risk patients early for effective treatment strategies.

Asymptomatic Plasmodium vivax infections sustain transmission and derail elimination efforts, yet these hidden reservoirs evade current diagnostics. Here, we integrated computational immunology with empirical serology to discover high-performance B-cell epitopes. From a library of 142 P. vivax proteins, we prioritized six antigens (AMA1, CSP, DBP, MSP1, MSP9, and P12), synthesized 64 epitopes, and screened them against serum from P. vivax-infected cases from the China-Myanmar border (CMB) via ELISA, with multiplex immunoassay validation across Brazil, the Solomon Islands, and Papua New Guinea samples. Rigorous assessment of specificity, sensitivity, and asymptomatic discrimination, along with antigenic conservation analyses in CMB clinical isolates and global homologues, yielded ten immunodominant epitopes. Four exhibited outstanding performance, namely, MSP9_603-609 (90.4% sensitivity, AUC = 0.980), AMA1_457-466 (AUC = 0.910), MSP9_458-464(AUC = 0.802), and MSP9_425-433 (AUC = 0.898), all with absolute specificity against P. falciparum (P < 0.0001). Crucially, the MSP9_384-389 epitope distinguished asymptomatic infections (AUC = 0.756, P < 0.0005). Despite variable detection rates across regions (2.2-22.2%), with the highest rates in Brazil, all the epitopes remained 99-100% conserved globally, reflecting strong functional immunological constraints. These epitopes enable unprecedented detection of both symptomatic and asymptomatic P. vivax infections - addressing a critical surveillance gap - while their scalability and species specificity make them ideal for elimination campaigns, particularly in low-resource settings. This work lays the foundation for next-generation serological tools to accelerate vivax malaria eradication.

[This corrects the article DOI: 10.3389/fgene.2025.1621920.].

Inoculation with Plasmodium vivax malaria parasites can lead to blood-stage infections from the primary infection and relapses from liver-stage parasites or non-circulating merozoites. Understanding the risk of clinical illness following primary infection and relapse would inform surveillance and intervention strategies, but the probabilities are uncertain in people living in endemic areas. A major difficulty lies in the inability to distinguish primary infections and relapses. In this study, we estimate the probabilities of clinical illness using the different seasonal patterns of primary infection and relapse. Children aged one to three years in Ilaita, Papua New Guinea, were followed up over 16 months for illness (fever with ≥500 parasites/µl) with fortnightly active and passive case detection, and for blood-stage infection every two months. Estimates of the number of primary infections and relapses for each two-month time-period, age-group, village and ITN use category were derived from previous analyses using genotyping data. In this study, we use a Bayesian statistical model to relate the number of observed P. vivax clinical cases in each covariate category to the expected numbers of primary infections and relapses. We include the cumulative number of primary infections experienced since birth as a proxy for acquired immunity. To reflect uncertainty, we use varying assumptions about whether relapses can cause illness in different circumstances. The probability of illness decayed exponentially with increasing cumulative numbers of primary infections experienced. The estimated probability of illness following relapses was lower than that for primary infection, how much lower depended on how they were defined. Later relapses within the same brood tended to have lower probabilities than earlier ones. Varying seasonally, relapses were estimated to contribute half of P. vivax illness in this cohort despite accounting for 80% of the force of blood-stage infection. The results can inform estimates of the burden of P. vivax and provide building blocks for mathematical models for predicting the impact of interventions. Interventions triggered by clinical cases would focus on more recent infections and age-groups with less acquired immunity.

BACKGROUND:Plasmodium vivax forms dormant liver stages (hypnozoites) that can reactivate weeks to months after primary infection. Radical cure requires a combination of antimalarial drugs to kill both the blood-stage and liver-stage parasites. Hypnozoiticidal efficacy of the liver-stage drugs primaquine and tafenoquine cannot be estimated directly because hypnozoites are undetectable. We aimed to estimate hypnozoiticidal efficacy from clinical trial data, and quantify the community-level impact of implementing case management with radical cure. METHODS:We calibrated a novel P vivax Recurrence Model to publicly available data from prospective clinical trials to estimate the hypnozoiticidal efficacy of different supervised primaquine (3·5 mg/kg or 7 mg/kg over 7 or 14 days) and tafenoquine (5 mg/kg or 7·5 mg/kg single dose) regimens in patients with normal glucose-6-phosphate dehydrogenase (G6PD) activity. We used an existing P vivax Individual-Based Model to quantify the 5-year impact of case management with unsupervised primaquine or tafenoquine regimens across various transmission settings. FINDINGS:We estimated median hypnozoiticidal efficacies of 99·1% (95% credible interval 96·0-100) for primaquine 7 mg/kg over 14 days; 96·3% (90·8-99·7) for primaquine 7 mg/kg over 7 days; 72·3% (68·1-76·3) for primaquine 3·5 mg/kg over 7 or 14 days; 62·4% (49·1-76·3) for tafenoquine 5 mg/kg single dose; and 87·5% (62·1-99·3) for tafenoquine 7·5 mg/kg single dose. 5 years of community-level tafenoquine case management was estimated to reduce P vivax transmission by 74-79% where pre-intervention prevalence as measured by PCR was low (<2%) and by 17-20% where prevalence as measured by PCR was high (around 35%). Similar 5-year reductions were estimated with primaquine case management only when adherence to the primaquine regimen was above 50%. INTERPRETATION:Substantial reductions in prevalence as measured by PCR were predicted with primaquine and tafenoquine regimens if these could be implemented with high coverage and adherence. The benefits of preventing P vivax relapses need to be balanced against the risks of inducing severe haemolysis in patients with G6PD deficiency. FUNDING:Bill & Melinda Gates Foundation and Horizon Europe.

Plasmodium falciparum Pfs230 and Pfs48/45, part of a core fertilization complex, are leading malaria transmission-blocking vaccine candidates. However, how the two proteins interact is unknown. Here we report a 3.36 Å resolution cryo-electron microscopy structure of the endogenous Pfs230-Pfs48/45 complex. We show that Pfs48/45 interacts with Pfs230 domains 13 and 14, domains that are not included in current Pfs230 vaccine immunogens. Using a transgenic parasite line with a domain 13 to 14 deletion, we show that these domains are essential for Pfs230 localization on the gamete surface. Nanobodies against domains 13 and 14 inhibit Pfs230-Pfs48/45 complex formation, reduce transmission and structural analyses reveal their binding epitopes. Furthermore, domains 13 and 14 are targets of naturally acquired immunity and when delivered as mRNA-LNP vaccines induce potent immune responses. Our comprehensive structural insights on a core P. falciparum fertilization complex will guide the design of novel transmission-blocking vaccine candidates against malaria. ### Competing Interest Statement The authors have declared no competing interest.