Continuous propagation of Babesia duncani in vitro in human erythrocytes and the availability of a mouse model of B. duncani lethal infection make this parasite an ideal model to study Babesia biology and pathogenesis. Two culture media, HL-1 and Claycomb, with proprietary formulations are the only culture media known to support the parasite growth in human erythrocytes; however, the HL-1 medium has been discontinued and the Claycomb medium is often unavailable leading to major interruptions in the study of this pathogen. To identify alternative media conditions, we evaluated the growth of B. duncani in various culture media with well-defined compositions. We report that the DMEM-F12 culture medium supports the continuous growth of the parasite in human erythrocytes to levels equal to those achieved in the HL-1 and Claycomb media. We generated new clones of B. duncani from the parental WA-1 clinical isolate after three consecutive subcloning events in this medium. All clones showed a multiplication rate in vitro similar to that of the WA-1 parental isolate and cause fatal infection in C3H/HeJ mice. The culture medium, which can be readily reconstituted from its individual components, and the tools and resources developed here will facilitate the study of B. duncani.

We report the discovery of MED6-189, an analog of the kalihinol family of isocyanoterpene natural products that is effective against drug-sensitive and drug-resistant Plasmodium falciparum strains, blocking both asexual replication and sexual differentiation. In vivo studies using a humanized mouse model of malaria confirm strong efficacy of the compound in animals with no apparent hemolytic activity or toxicity. Complementary chemical, molecular, and genomics analyses revealed that MED6-189 targets the parasite apicoplast and acts by inhibiting lipid biogenesis and cellular trafficking. Genetic analyses revealed that a mutation in PfSec13 , which encodes a component of the parasite secretory machinery, reduced susceptibility to the drug. Its high potency, excellent therapeutic profile, and distinctive mode of action make MED6-189 an excellent addition to the antimalarial drug pipeline.

Virus-like vesicles (VLV) are hybrid vectors based on an evolved Semliki Forest virus (SFV) RNA replicon and the envelope glycoprotein (G) from vesicular stomatitis virus (VSV). Previously, we showed that VLV can be used to express protein antigens and generate protective antigen-specific CD8⁺ T cells. This report describes VLV vectors designed for enhanced protein expression and immunogenicity. Expressing hepatitis B virus (HBV) middle S antigen (MHBs) from VLV using a dual subgenomic promoter significantly increased MHBs-specific CD8⁺ T cell and antibody production in mice. Furthermore, envelope glycoprotein switch from VSV Indiana to the glycoprotein of Chandipura virus enabled prime-boost immunization and further increased responses to MHBs. Therapeutic efficacy was evaluated in a mouse model of chronic HBV infection initiated by HBV delivery with adeno-associated virus. Mice with lower or intermediate HBV antigen levels demonstrated a significant and sustained reduction of HBV replication following VLV prime-boost immunization. However, mice with higher HBV antigen levels showed no changes in HBV replication, emphasizing the importance of HBV antigenemia for implementing immunotherapies. This report highlights the potential of VLV dual promoter vectors to induce effective antigen-specific immune responses and informs the further development and evaluation of hybrid viral vaccine platforms for preventative and therapeutic purposes.

The significant rise in the number of tick-borne diseases represents a major threat to public health worldwide. One such emerging disease is human babesiosis, which is caused by several protozoan parasites of the Babesia genus of which B. microti is responsible for most clinical cases reported to date. Recent studies have shown that during its intraerythrocytic life cycle, B. microti exports several antigens into the mammalian host using a novel vesicular-mediated secretion mechanism. One of these secreted proteins is the immunodominant antigen BmGPI12, which has been demonstrated to be a reliable biomarker of active B. microti infection. The major immunogenic determinants of this antigen remain unknown. Here we provide a comprehensive molecular and serological characterization of a set of eighteen monoclonal antibodies developed against BmGPI12 and a detailed profile of their binding specificity and suitability in the detection of active B. microti infection. Serological profiling and competition assays using synthetic peptides identified five unique epitopes on the surface of BmGPI12 which are recognized by a set of eight monoclonal antibodies. ELISA-based antigen detection assays identified five antibody combinations that specifically detect the secreted form of BmGPI12 in plasma samples from B. microti-infected mice and humans but not from other Babesia species or P. falciparum.

Human PANK1, PANK2, and PANK3 genes encode several pantothenate kinase isoforms that catalyze the phosphorylation of vitamin B5 (pantothenic acid) to phosphopantothenate, a critical step in the biosynthesis of the major cellular cofactor, Coenzyme A (CoA). Mutations in the PANK2 gene, which encodes the mitochondrial pantothenate kinase (PanK) isoform, have been linked to pantothenate-kinase associated neurodegeneration (PKAN), a debilitating and often fatal progressive neurodegeneration of children and young adults. While the biochemical properties of these enzymes have been well-characterized in vitro, their expression in a model organism such as yeast in order to probe their function under cellular conditions have never been achieved. Here we used three yeast mutants carrying missense mutations in the yeast PanK gene, CAB1, which are associated with defective growth at high temperature and iron, mitochondrial dysfunction, increased iron content, and oxidative stress, to assess the cellular function of human PANK genes and functional conservation of the CoA-controlled processes between humans and yeast. Overexpression of human PANK1 and PANK3 in these mutants restored normal cellular activity whereas complementation with PANK2 was partial and could only be achieved with an isoform, PanK2^mtmΔ, lacking the mitochondrial transit peptide. These data, which demonstrate functional conservation of PanK activity between humans and yeast, set the stage for the use of yeast as a model system to investigate the impact of PKAN-associated mutations on the metabolic pathways altered in this disease.

Human babesiosis is an emerging and potentially fatal tick-borne disease caused by intraerythrocytic parasites of the Babesia genus. Among these, Babesia duncani is particularly notable for causing severe and life-threatening illness in humans. Accurate diagnosis and effective disease management hinge on the detection of active B. duncani infections. While molecular assays are available to detect the parasite in blood, a reliable method for identifying biomarkers of active infection remains elusive. We developed the first B. duncani antigen capture assays, targeting two immunodominant antigens, BdV234 and BdV38. These assays were validated using established in vitro and in vivo B. duncani infection models, and following drug treatment. The assays demonstrated no cross-reactivity with other species such as B. microti, B. divergens, Babesia MO1, or Plasmodium falciparum, and can detect as few as 115 infected erythrocytes/µl of blood. Screening of 1731 blood samples from various biorepositories, including samples previously identified as Lyme and/or B. microti-positive, as well as new specimens from wild mice, revealed no evidence of B. duncani infection or cross-reactivity. These assays hold significant promise for various applications, including point-of-care testing for the early detection of B. duncani in patients, field tests for screening reservoir hosts, and high-throughput screening of blood samples intended for transfusion.