Background:Malaria transmission in the Sahel persists during the dry season despite low microscopic parasite prevalence, maintaining reservoirs that hinder elimination efforts. Understanding serological markers of Plasmodium falciparum gametocyte exposure may help identify hidden transmission reservoirs during low‑transmission periods. Materials and Methods:We conducted a cross‑sectional survey in Kéniéroba, Mali, at three time points representing key seasonal periods: December 2024 (end of rainy season), May 2025 (dry season), and August 2025 (rainy season). A total of 356 participants were enrolled across three age groups (5-8, 9-17, and ≥18 yrs). Parasitaemia and gametocytaemia were assessed by microscopy. IgG and IgM responses to clinically isolated late-stage gametocytes were quantified by ELISA, and seropositivity thresholds defined using malaria‑naïve controls. Results:Microscopic P. falciparum infection prevalence remained stable across surveys (~13%), but gametocyte carriage declined from 5.7% in December to ~2% in May and August, whereas gametocyte density was higher in the latter months. IgG/IgM seropositivity exceeded 95% throughout, with IgM peaking in May, consistent with recent antigenic stimulation. Age‑stratified analyses revealed weak but statistically significant associations between antibody levels and gametocyte carriage restricted to children aged 5-8 yrs, while no associations were observed in older children or adults. Conclusions:In conclusion, serological analyses indicate widespread and seasonally variable exposure to P. falciparum in Kéniéroba, with consistently high IgG and IgM responses to gametocytes reflecting intense background malaria exposure and cross‑reactive immunity from asexual stages. Seasonal and age‑stratified patterns identify school‑aged children as an important reservoir sustaining transmission. Although overall correlations with gametocyte carriage were weak, age‑specific associations suggest an age‑dependent relationship between antibody responses and carriage.

Background/Objectives: Rapid development of vaccines against SARS-CoV-2 was pivotal to controlling the COVID-19 pandemic. The emergency also provided a rare opportunity to test novel vaccine platforms such as mRNA in large clinical trials. Most of the early vaccines used SARS-CoV-2 Spike protein as the target antigen. Nevertheless, subsequent studies have shown that Receptor Binding Domain (RBD) of Spike also can yield efficacious vaccines, and we previously demonstrated that chemical conjugation of RBD to a carrier protein, EcoCRM®, enhanced antibody responses and induced strong virus neutralization activity in mice. Methods: Here, we compared the immunogenicity of this conjugate to that of an approved mRNA vaccine from Pfizer/BioNTech in rhesus macaques over a period of nine months. Results: AS01-adjuvanted RBD conjugate induced a similar or better antibody response, receptor binding inhibition, and virus neutralization activity against different variants of SARS-CoV-2, compared to mRNA. IgG subclass profiles induced by conjugate and mRNA vaccines were initially dominated by IgG1 and IgG3 then switched to IgG2 and IgG4 dominant profiles during the subsequent six-month period. Polyclonal immune sera from the conjugate and mRNA had similar antibody avidity at multiple time points. Conclusions: In summary, antibody responses in rhesus macaques induced by the RBD-EcoCRM conjugate and the Spike mRNA vaccine are very similar. These results demonstrate the potential for the RBD-EcoCRM conjugate as a vaccine against SARS-CoV-2.

Importance:Pregnancy malaria (PM) is associated with adverse pregnancy outcomes such as stillbirth, early neonatal death, preterm delivery (PTD), and low birthweight. PM also increases the risk of malaria infection in infants. However, it is unknown whether PTD modifies the risk of malaria infection during early childhood. Objective:To investigate the association of PM and PTD with child susceptibility to malaria infection and disease. Design, Setting, and Participants:Pregnant women were enrolled between November 23, 2010, and December 9, 2014, into an observational longitudinal cohort study of mother-child pairs in Ouélessébougou, Mali, an area of high seasonal malaria transmission. Follow-up was completed through pregnancy. Children were enrolled at birth and followed up from January 21, 2011, to July 31, 2016, for as long as 5 years with monthly clinical visits during the malaria transmission season and every 2 months during the dry season. Data were analyzed from November 4, 2024, to July 15, 2025. Exposure:PM and PTD. Main Outcomes and Measures:Study end points included Plasmodium falciparum infection, clinical malaria, and severe malaria infections. Malaria diagnosis and clinical data were collected during scheduled examinations and unscheduled sick visits. Cox proportional hazards models were used to analyze whether hazards of first malaria infection and first clinical malaria infection were associated with PM and PTD. Associations between the incidence rate of parasitemia and risk factors (eg, maternal infection history, PTD) were estimated using negative binomial models. Cox proportional hazards and negative binomial models with an interaction term among PTD, pregnancy malaria, and gravidity were used to evaluate associations within strata of the 3 covariates. Results:In 1679 children included in adjusted models (848 female [50.5%] and 831 male [49.5%]), 760 (45.3%) were born during the malaria transmission season and 96 (5.7%) were born preterm. Children were followed up for a mean (SD) of 25.8 (16.1) months. PM was associated with an increased hazard of first malaria infection and first clinical malaria infection in children of women of all gravidities, while PTD (vs full-term delivery) was associated with increased hazard of first malaria infection (hazard ratio, 1.76; 95% CI, 1.05-2.95; P = .03) in offspring of multigravid women only. Further, the hazard ratio of first parasitemia for preterm compared with full-term offspring was 2.17 (95% CI, 1.25-3.75; P = .006) and 3.63 (95% CI, 1.90-5.93; P < .001) in offspring of uninfected secundigravida and multigravida women, respectively. The parasitemia infection incidence rate ratio for PTD was 2.74 (95% CI, 1.80-4.18) in offspring of uninfected multigravida women. Conclusions and Relevance:In this cohort study of young children, the association between PTD and the hazard of malaria varied based on maternal gravidity and maternal infection history during pregnancy. This information could be used to evaluate the health effects of active monitoring of P falciparum infection or adherence to malaria chemoprevention in children born preterm.

Dried blood spot (DBS) testing on confetti is one of the most common methods used to collect and extract DNA from field samples for molecular epidemiology of malaria studies. Here, we investigated whether ethanol or other solutions used to clean scissors are a source of sample contamination. DBS were prepared from 3 drops of blood from two Plasmodium falciparum cultures spotted on confetti, and 3 solutions (water, ethanol and DNase) were used to clean scissors between spots during DNA extraction. For each cleaning solution, two blank confetti were used as negative controls. Samples were analyzed by PCR-based genotyping of merozoite surface proteins 1 & 2 (msp-1 and msp-2), and P. falciparum chloroquine resistance transporter (Pfcrt). A total of 15 samples were analyzed. Based on msp-1 and msp-2 amplification, P. falciparum was detected on blank confetti when scissors were washed with ethanol. Based on Pfcrt amplification, DNA of P. falciparum was detected on blank confetti when scissors were cleaned with ethanol or water. No P. falciparum genes were detected on blank confetti when scissors were cleaned with DNase. Scissors cleaned with DNase prevented cross-contamination between samples during processing of dried blood spots, whereas ethanol (which is commonly used) fails to avert cross-contamination.

The population structure of the malaria parasite Plasmodium falciparum can reveal underlying demographic and adaptive evolutionary processes. Here, we analyse population structure in 4,376 P. falciparum genomes from 21 countries across Africa. We identified a strongly differentiated cluster of parasites, comprising ∼1.2% of samples analysed, geographically distributed over 13 countries across the continent. Members of this cluster, named AF1, carry a genetic background consisting of a large number of highly differentiated variants, rarely observed outside this cluster, at a multitude of genomic loci distributed across most chromosomes. At these loci, the AF1 haplotypes appear to have common ancestry, irrespective of the sampling location; outside the shared loci, however, AF1 members are genetically similar to their sympatric parasites. AF1 parasites sharing up to 23 genomic co-inherited regions were found in all major regions of Africa, at locations over 7,000 km apart. We coined the term cryptotype to describe a complex common background which is geographically widespread, but concealed by genomic regions of local origin. Most AF1 differentiated variants are functionally related, comprising structural variations and single nucleotide polymorphisms in components of the MSP1 complex and several other genes involved in interactions with red blood cells, including invasion and erythrocyte antigen export. We propose that AF1 parasites have adapted to some as yet unidentified evolutionary niche, by acquiring a complex compendium of interacting variants that rarely circulate separately in Africa. As the cryptotype spread across the continent, it appears to have been maintained mostly intact in spite of recombination events, suggesting a selective advantage. It is possible that other cryptotypes circulate in Africa, and new analysis methods may be needed to identify them.### Competing Interest StatementThe authors have declared no competing interest.

VAR2CSA is the Plasmodium falciparum variant surface antigen that mediates binding of infected erythrocytes to chondroitin sulfate A (CSA) and their sequestration in intervillous spaces of the placenta, leading to placental malaria (PM). Relatively high polymorphism in VAR2CSA sequences has hindered development of a vaccine that induces broadly neutralizing immunity. Recent research has highlighted that a broadly reactive human monoclonal antibody, called PAM1.4, binds to multiple conserved residues of different subfragments of VAR2CSA, forming a conformational epitope. In this short perspective, we describe evidence that residues located in the interdomain-1 fragment of VAR2CSA within the PAM1.4 binding epitope might be critical to broad reactivity of the antibody. Future investigation into broadly reactive anti-VAR2CSA antibodies may be important for the following: (1) identification of similar conformation epitopes targeted by broadly neutralizing antibodies; and (2) understanding different immune evasion mechanisms used by placenta-binding parasites through VAR2CSA polymorphism in critical epitopes.

BackgroundAnti-malarial drug resistance in Plasmodium falciparum is a major public health problem in malaria-endemic regions. Although various technical improvements in sequencing methods have been introduced to identify SNPs, the conventional approach with current tools does not discriminate mixed infections, and thus can be improved for more sensitive surveillance of anti-malarial resistance to better inform control strategies.MethodsWe developed a computational approach for deconvolution of chromatograms generated by standard Sanger sequencing of PCR amplicons in order to quantify molecular marker variants of anti-malarial drug resistance genes [Plasmodium falciparum dihydropteorate synthase (Pfdhps) and P. falciparum dihydrofolate reductase (Pfdhfr)]. We validated this computational approach using mixtures of V1/S and FCR3 at varying proportions between 0 and 100%, then applied it to field samples collected in Doneguebougou, Mali in 2018. We determined the mean fraction of resistance alleles in individual samples, as well as the prevalence of infections carrying resistant parasites.FindingsWe observed a highly significant correlation between the predicted and measured proportions of V1/S and FCR3 alleles in mixed laboratory samples (all p < 0.001). Among field samples, the mean fraction of resistant Pfdhps alleles was 4.7% 431V, 95.9% 436F/A, 49.9% 437G, 0.0% 540E, 1.2% 581G and 1.5% 613S/T; corresponding prevalences were 50.0%, 100%, 72.5%, 0.0%, 25.0%, and 12.5%, respectively. The mean fraction of resistant Pfdhfr alleles was 0.6% 16V, 11.1% 50R, 89.0% 51I, 98.3% 59R, 74.7% 108T/N, 8.6% 140L and 8.7% 164L; corresponding prevalences were 12.5%, 75.0%, 100%, 100%, 100%, 50.0%, and 28.6%, respectively. We identified two new point mutations on the Pfdhps gene at codons D484T and D545N.InterpretationComputational deconvolution of sequencing chromatograms can discriminate varying proportions of antimalarial drug-sensitive versus -resistant alleles. This cost-effective and quantitative variant-sequencing approach will be useful for population-based surveys that characterize mixed infections at the individual level to survey known and unknown mutations in P. falciparum drug-resistance genes.FundingThis work was supported by the Division of Intramural Research of the National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH). HM was supported by the African Postdoctoral Training Initiative (APTI) Fellowship program jointly managed by the US NIH, The African Academy of Sciences (AAS) and Bill & Melinda Gates Foundation (BMGF); Grant Reference Number: APTI-18-01.

BackgroundDespite decades of effort, Plasmodium falciparum malaria remains a leading killer of children. The absence of a highly effective vaccine and the emergence of parasites resistant to both diagnosis as well as treatment hamper effective public health interventions.Methods and resultsTo discover new vaccine candidates, we used our whole proteome differential screening method and identified PfGBP130 as a parasite protein uniquely recognized by antibodies from children who had developed resistance to P. falciparum infection but not from those who remained susceptible. We formulated PfGBP130 as lipid encapsulated mRNA, DNA plasmid, and recombinant protein-based immunogens and evaluated the efficacy of murine polyclonal anti-PfGBP130 antisera to inhibit parasite growth in vitro. Immunization of mice with PfGBP130-A (aa 111–374), the region identified in our differential screen, formulated as a DNA plasmid or lipid encapsulated mRNA, but not as a recombinant protein, induced antibodies that inhibited RBC invasion in vitro. mRNA encoding the full ectodomain of PfGBP130 (aa 89–824) also generated parasite growth-inhibitory antibodies.ConclusionWe are currently advancing PfGBP130-A formulated as a lipid-encapsulated mRNA for efficacy evaluation in non-human primates.

ABSTRACT The population structure of the malaria parasite Plasmodium falciparum can reveal underlying demographic and adaptive evolutionary processes. Here, we analyse population structure in 4,376 P. falciparum genomes from 21 countries across Africa. We identified a strongly differentiated cluster of parasites, comprising ∼1.2% of samples analysed, geographically distributed over 13 countries across the continent. Members of this cluster, named AF1, carry a genetic background consisting of a large number of highly differentiated variants, rarely observed outside this cluster, at a multitude of genomic loci distributed across most chromosomes. At these loci, the AF1 haplotypes appear to have common ancestry, irrespective of the sampling location; outside the shared loci, however, AF1 members are genetically similar to their sympatric parasites. AF1 parasites sharing up to 23 genomic co-inherited regions were found in all major regions of Africa, at locations over 7,000 km apart. We coined the term cryptotype to describe a complex common background which is geographically widespread, but concealed by genomic regions of local origin. Most AF1 differentiated variants are functionally related, comprising structural variations and single nucleotide polymorphisms in components of the MSP1 complex and several other genes involved in interactions with red blood cells, including invasion and erythrocyte antigen export. We propose that AF1 parasites have adapted to some as yet unidentified evolutionary niche, by acquiring a complex compendium of interacting variants that rarely circulate separately in Africa. As the cryptotype spread across the continent, it appears to have been maintained mostly intact in spite of recombination events, suggesting a selective advantage. It is possible that other cryptotypes circulate in Africa, and new analysis methods may be needed to identify them.

BackgroundPlasmodium falciparum malaria is still a leading cause of child mortality in sub-Saharan Africa. The clinical manifestations of malaria range from asymptomatic infection to severe disease. The variation in clinical presentation is partly attributed to host genetic factors with estimated narrow-sense heritability of 23%. Here, we investigate the associations between candidate gene polymorphisms and the likelihood of severe malaria (SM) in a cohort of Malian children.MethodsBased on our previous genome-wide association studies (GWAS) analysis, candidate genes were selected for in-depth analysis using several criteria including gene-level GWAS scores, functional overlap with malaria pathogenesis, and evidence of association with protection or susceptibility to other infectious or inflammatory diseases. Single Nucleotide Polymorphisms (SNPs) residing within these genes were selected mainly based on p-values from previous severe malaria susceptibility GWAS studies and minor allele frequency (MAF) in West African populations.ResultsOf 182 candidate genes reported in our previous study, 11 genes and 22 SNPs residing in these genes were selected. The selected SNPs were genotyped using KASP technology in 477 DNA samples (87 SM and 390 controls). Logistic regression analysis revealed that a common intron variant, rs13340578 in CUB and Sushi Multi Domain (CSMD1) gene, is associated with increased odds of SM in recessive mode of inheritance (MAF = 0.42, OR = 1.8, 95% CI = [1.78, 1.84], p = 0.029). The SNP is in linkage disequilibrium (LD) with multiple variants with regulatory features.ConclusionTaken together, the current study showed that an intron variant rs13340578, residing in CSMD1 gene, is associated with increased susceptibility to malaria. This finding suggests that modified regulation of complement may contribute to malaria disease severity. Further studies are needed to identify the causal variants and the underlying molecular mechanisms.

Malaria clinically affects over 250 million people each year through the bite of female Anopheles mosquitoes. Currently, there are two approved anti-infection vaccines and an investigational therapeutic human monoclonal antibody (huMAb) that target the Plasmodium falciparum circumsporozoite protein (CSP). The CSP is a disordered surface protein comprised of three regions: a charged amino-terminus, a central region composed of NPNA repeats that form a series of beta turns, and a carboxyl-terminus with a fucosylated thrombospondin repeat (TSR) like domain with a glycosylphosphatidylinositol anchor. Fucosylation is known to facilitate inter- and intra-protein interactions. Given that the anti-infection vaccines only include about half of the carboxyl-terminus and the huMAb recognizes a unique N-terminal epitope, we are furthering our investigation of the biology, subcellular localization, and structure of the CSP. We previously observed that native CSP may undergo structural changes on the surface of sporozoites. Efforts to crystallize a recombinant non-glycosylated CSP, identified as CSPM3, which mimics a processed form identified on infectious sporozoites failed to crystallize, while crystallization of a CSPM3 with O-linked mannosylation (CSPM3man) yielded crystals that did not diffract. We mapped the O-linked mannosylation on CSPM3man which were modeled on the TSR such that the mannosylation was within 10 angstroms of the native fucosylation site. To further understand the role of the glycosylation in CSPM3man, we performed atomic force microscopy which preliminarily has shown that this CSP favors complex assemblies and a collapsed phenotype which masks functional N- and C-terminal epitopes. Finally, we have developed a protective recombinant CSP repeat specific mAb with a human rhinovirus 3C proteolytic site for electron microscopy studies initially using PfCSPM3man and subsequently native CSP using fractionated sporozoites. An improved understanding of the CSP may support the clinical evaluation of a CSPM3 malaria vaccine.

The interpretation of a laboratory test result requires an appropriate reference range established in healthy subjects, and normal ranges may vary by factors such as geographic region, sex, and age. We examined hematological and clinical chemistry parameters in healthy residents at two rural vaccine trial sites: Bancoumana and Doneguebougou in Mali, West Africa. During screening of clinical studies in 2018 and 2019, peripheral blood samples from 1,192 apparently healthy individuals age 6 months to 82 years were analyzed at a laboratory accredited by the College of American Pathologists for a complete blood count, and creatinine and/or alanine aminotransferase levels. Based on manufacturers' reference range values, which are currently used in Malian clinical laboratories, abnormal values were common in this healthy population. In fact, 30.4% of adult participants had abnormal neutrophil levels and 19.8% had abnormal hemoglobin levels. Differences by sex were observed in those who were older, but not in those younger than 10 years, for several parameters, including hemoglobin, platelet, and absolute neutrophil counts in hematology, and creatinine in biochemistry. The site-specific reference intervals we report can be used in malaria vaccine clinical trials and other interventional studies, as well as in routine clinical care, to identify abnormalities in hematological and biochemical parameters among healthy Malian trial participants.

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BACKGROUND. Sanaria PfSPZ Vaccine, composed of attenuated Plasmodium falciparum (Pf) sporozoites (SPZ), protects against malaria. We conducted this clinical trial to assess the safety and efficacy of PfSPZ Vaccine in HIV-positive (HIV+) individuals, since the HIV-infection status of participants in mass vaccination programs may be unknown. METHODS. This randomized, double-blind, placebo-controlled trial enrolled 18- to 45-year-old HIV-negative (HIV-) and well-controlled HIV+ Tanzanians (HIV viral load <40 copies/mL, CD4 counts >500 cells/mu L). Participants received 5 doses of PfSPZ Vaccine or normal saline (NS) over 28 days, followed by controlled human malaria infection (CHMI) 3 weeks later. RESULTS. There were no solicited adverse events in the 9 HIV- and 12 HIV+ participants. After CHMI, 6 of 6 NS controls, 1 of 5 HIV- vaccinees, and 4 of 4 HIV+ vaccinees were Pf positive by quantitative PCR (qPCR). After immunization, anti-Pf circumsporozoite protein (anti-PfCSP) (isotype and IgG subclass) and anti-PfSPZ antibodies, anti-PfSPZ CD4(+) T cell responses, and V delta 2(+) gamma delta CD3(+) T cells were nonsignificantly higher in HIV- than in HIV+ vaccinees. Sera from HIV- vaccinees had significantly higher inhibition of PfSPZ invasion of hepatocytes in vitro and antibody-dependent complement deposition (ADCD) and Fc gamma 3B binding by anti-PfCSP and ADCD by anti-cell-traversal protein for ookinetes and SPZ (anti-PfCelTOS) antibodies. CONCLUSIONS. PfSPZ Vaccine was safe and well tolerated in HIV+ vaccinees, but not protective. Vaccine efficacy was 80% in HIV- vaccinees (P = 0.012), whose sera had significantly higher inhibition of PfSPZ invasion of hepatocytes and enrichment of multifunctional PfCSP antibodies. A more potent PfSPZ vaccine or regimen is needed to protect those living with HIV against Pf infection in Africa.

Most rare disease patients (75–50%) undergoing genomic sequencing remain unsolved, often due to lack of information about variants identified. Data review over time can leverage novel information regarding disease-causing variants and genes, increasing this diagnostic yield. However, time and resource constraints have limited reanalysis of genetic data in clinical laboratories setting. We developed RENEW, (REannotation of NEgative WES/WGS) an automated reannotation procedure that uses relevant new information in on-line genomic databases to enable rapid review of genomic findings. We tested RENEW in an unselected cohort of 1066 undiagnosed cases with a broad spectrum of phenotypes from the Mayo Clinic Center for Individualized Medicine using new information in ClinVar, HGMD and OMIM between the date of previous analysis/testing and April of 2022. 5741 variants prioritized by RENEW were rapidly reviewed by variant interpretation specialists. Mean analysis time was approximately 20 s per variant (32 h total time). Reviewed cases were classified as: 879 (93.0%) undiagnosed, 63 (6.6%) putatively diagnosed, and 4 (0.4%) definitively diagnosed. New strategies are needed to enable efficient review of genomic findings in unsolved cases. We report on a fast and practical approach to address this need and improve overall diagnostic success in patient testing through a recurrent reannotation process.