Biology subjects

Levashina, E. A.

Publications and source records attributed to Levashina, E. A..

16 recordsLinked to original sources

bioRxiv2026

B Lymphocyte Protein Factories produced by Hematopoietic Stem Cell Gene Editing

Long-term in vivo production of therapeutic proteins and development of vaccines that elicit protective levels of broadly neutralizing antibodies (bNAbs) against major pathogens face challenges. Here we report on an alternative gene-editing approach using small numbers of hematopoietic stem and progenitor cells (HSPCs) to direct long-term, high-level expression of antibodies or cargo proteins. Edited B lymphocyte offspring can be activated by cognate antigen to undergo clonal expansion and develop into specific antibody or cargo protein-synthesizing plasma cells. These cells produce long-lasting, therapeutic levels of serum antibody against HIV-1 or malaria and an anti-influenza virus bNAb that mediated universal protection from heterologous lethal challenge. Our data provide a paradigm for cell therapy approaches to prevent or treat disease using self-amplifying B cell protein factories.

immunology↗

bioRxiv2025

The major role of the REL2/NF-κB pathway in the regulation of midgut bacterial homeostasis in the malaria vector Anopheles gambiae

Multicellular organisms harbor diverse microbial communities that play essential roles in host physiology. While often beneficial, these interactions require tight regulation to prevent dysbiosis and disease. This study examines the tissue-specific immune responses of mosquitoes to blood feeding and Plasmodium falciparum infection in Anopheles females. We demonstrate that REL2 signaling regulates antimicrobial peptide (AMP) expression and shapes midgut bacterial composition post-blood meal. Loss of REL2 leads to midgut dysbiosis, characterized by the overgrowth of Serratia spp., and mosquito lethality within a day of feeding. Interestingly, Serratia-induced dysbiosis also reduces P. falciparum prevalence in surviving mosquitoes. Our findings highlight the critical role of immune system in maintaining midgut bacterial homeostasis and uncover complex interactions between mosquito immunity, gut microbiota, and malaria parasites. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=147 HEIGHT=200 SRC="FIGDIR/small/643338v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@29ddfeorg.highwire.dtl.DTLVardef@ad6037org.highwire.dtl.DTLVardef@87f198org.highwire.dtl.DTLVardef@10640ca_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

Somatic hypermutation-mediated paratope flexibility improves the cross-reactivity of human malaria antibodies

The protective capacity of antibodies targeting circumsporozoite protein on sporozoites of the malaria parasite Plasmodium falciparum (PfCSP) is linked to high affinity and cross-reactivity with the PfCSP central repeat domain and N-terminal junction. However, the role of somatic hypermutation (SHM) in the development of such antibodies remains unclear. Here we define the contributions of SHM to the high affinity and strong repeat and N-junction cross-reactivity of the potent anti-PfCSP monoclonal antibody (mAb) 4493 and of similar antibodies with shared SHM and affinity maturation trajectories. Molecular dynamics simulations reveal that SHM reduces the flexibility of the unbound mAb 4493 but increases the flexibility of the antigen-bound complex, thereby lowering the entropic cost for antigen binding. Furthermore, we identify an inverse relation between antibody affinity and serum stability, which limits the protective capacity of these antibodies. Our study provides molecular level evidence for the different roles that the SHM process plays in increasing VH3-49+V{kappa}3-20 antibody affinity and cross-reactivity and demonstrates how antibody affinity maturation can negatively impact antibody stability and thereby function.

immunology↗

bioRxiv2023

Larval thermosensitivity shapes adult population dynamics in Anopheles mosquitoes

Mosquitoes are vectors of human life-threatening pathogens, posing a significant global health threat. While the influence of temperature on mosquito life-history traits has been extensively studied in laboratory settings, the ecological factors shaping mosquito development and population dynamics in natural environments remain poorly understood. Here, we used a multi-disciplinary approach, integrating field data from Mali, laboratory experiments, and mathematical modeling, to investigate the causal relationships between climate variables and the abundance of Anopheles gambiae s.l. mosquitoes. Using convergent-cross mapping analyses an adult abundance in the Nanguilabou village, we observed that the dynamics of adult mosquito populations was driven by larval thermosensitivity. To elucidate the underlying mechanisms, we conducted experimental studies that revealed a density-dependent larval thermal response. Through mathematical modeling, we quantified the complex interplay between temperature and larval density, demonstrating that temperature and density have independent, non-synergistic effects on larval developmental speed, mortality, and pupation rates. Our findings provide a mechanistic understanding of how larval development shapes adult mosquito populations, highlighting the significance of multidisciplinary approaches in studying climate-driven mosquito population dynamics.

ecology↗

bioRxiv2023

Generation of a Plasmodium falciparum reporter line for studies of parasite biology throughout the life cycle

Fluorescence reporter strains of human malaria parasites are powerful tools to study the interaction of the parasites with both human and mosquito hosts. However, low fluorescence intensity in transmission-relevant parasite stages and the choice of insertion loci that cause parasite developmental defects in the mosquito largely limits usefulness of currently available lines. To overcome these limitations, we used a CRISPR-Cas9-mediated approach to generate PfOBC13GFP, a novel selection marker-free reporter parasite in the background of the African NF54 Plasmodium falciparum line. As docking site, we selected the OBC13 locus that is dispensable for asexual and sexual development in vitro. PfOBC13GFP parasites encode GFP flanked by hsp70 UTRs that drive strong fluorescence reporter expression throughout blood and mosquito stages, enabling parasite detection by such high throughput methods as flow cytometry. When compared to the parental line, PfOBC13GFP parasites showed normal development during blood and mosquito stages, and they efficiently infected the main African vector Anopheles coluzzii, overcoming one of the limitations of the previously developed fluorescent reporter lines based on the Pfs47 locus. PfOBC13GFP constitutes a potent tool enabling host-pathogen studies throughout Plasmodium life cycle. ImportanceFluorescence reporter strains have been very useful in malaria research, however, up to date they had limitations in mosquito infectivity and fluorescence intensity. Here we report the generation of PfOBC13GFP, a new fluorescent parasite strain of the human malaria parasite P. falciparum. PfOBC13GFP parasites are highly fluorescent throughout the life cycle, making them an ideal tool for the study the parasite progression through blood and mosquito stages. They efficiently infect the African mosquito vector A. coluzzii, allowing the study of this African parasite in its biological background. Moreover, strong parasite fluorescence enables flow cytometry and live microscopy characterization of all parasite stages, especially those involved in transmission.

microbiology↗

bioRxiv2023

Comparative assessment of mosquito size and receptiveness to P. falciparum infection of two rearing procedures for Anopheles mosquitoes

Malaria is transmitted when Anopheline mosquitoes ingest Plasmodium parasites during blood-feeding. Artificial feeding assays allow mosquitoes to take up blood from membrane feeders, and are widely used to study malaria transmission. These assays require large quantities of mosquitoes; insectaries optimize their rearing procedures to generate high yields of permissive, homogeneous mosquito populations. Rearing of Anopheline stephensi mosquitoes was protocolized at the Radboudumc in the 1980s, yet infection outcomes remain heterogeneous. This study explores possible improvements in mosquito rearing to improve homogeneity of the resulting mosquito populations. It compares the current mass-rearing standard with an adapted alternative approach from another institute that optimizes larval density per tray and applies a diet that has previously been reported. Differences between procedures were assessed by measuring mosquito size by proxy of wing-length and imbibed blood meal volume. To assess receptiveness to P. falciparum infection mosquitoes were fed cultured gametocytes. We observed a slight decrease in mosquito size when applying the alternative rearing procedure, but generated equally parasite-receptive P. falciparum mosquitoes compared to the standard procedure. We conclude that both rearing protocols can be used to generate susceptible mosquitoes for conducting malaria research.

systems biology↗

bioRxiv2023

Molecular and functional properties of human Plasmodium falciparum CSP C-terminus antibodies

Human monoclonal antibodies (mAbs) against the central repeat and junction domain of Plasmodium falciparum circumsporozoite protein (PfCSP) have been studied extensively to guide malaria vaccine design compared to antibodies against the PfCSP C terminus. Here, we describe the molecular characteristics and protective potential of a panel of 73 germline and mutated human mAbs against the highly immunogenic PfCSP C-terminal domain. Two mAbs recognized linear epitopes in the C-terminal linker with sequence similarity to repeat and junction motifs, whereas all others targeted conformational epitopes in the -thrombospondin repeat (-TSR) domain. Specificity for the polymorphic Th2R/Th3R but not the conserved RII+ region in the -TSR was associated with IGHV3-21/IGVL3-21 or IGLV3-1 gene usage. Although the C terminus specific mAbs showed signs of more efficient affinity maturation and class-switching compared to anti-repeat mAbs, parasite inhibitory activity was limited to a single C-linker reactive mAb with cross-reactivity to the central repeat and junction. The data provide novel insights in the human anti-C-linker and anti--TSR antibody response that support exclusion of the PfCSP C terminus from malaria vaccine designs.

immunology↗

bioRxiv2022

Mosquito metabolism shapes life-cycle strategies of Plasmodium parasites

The life-history of multicellular organisms is a collection of traits determining fitness described by growth, survival, and reproduction. Within-host survival and between-host transmission are key life-history traits of single-celled malaria parasites. Therefore, understanding the evolutionary forces that shape these components is crucial to predict malaria epidemiology, drug resistance, and virulence. The evolutionary strategies of Plasmodium parasites have been largely investigated in the vertebrate host. In contrast, very little is known about their adaptation strategies in the mosquito vector, possibly due to the experimental challenges encountered while studying vector-parasite interactions. Mathematical models offer a unique tool to study such complex biological systems, and have been extensively employed in malaria epidemiology. However, all models developed so far do not consider mosquito physiology. Here, we examine the life-history evolution of Plasmodium parasites with a novel individual-based model of malaria transmission that includes mosquito metabolism. Specifically, we model the metabolic cascade of resource allocation induced by blood-feeding, as well as the influence of multiple blood meals on parasite development. Our model shows that successful vector-to-human transmission events are rare, and are caused by long-lived mosquitoes. Interestingly, we observe that the life-history strategies of malaria parasites depend on the mosquito metabolic status. In our model, additional resources provided by multiple blood meals benefit selection for parasites with slow or intermediate developmental time. These results challenge the current concept that evolution selects for fast developing parasites to maximize their chances to complete their within-mosquito life cycle. We propose that the long sporogonic cycle observed for Plasmodium is not a constraint but rather an adaptation to increase transmission potential.

evolutionary biology↗

bioRxiv2020

Kinetics of Plasmodium midgut invasion in Anopheles mosquitoes

Malaria-causing Plasmodium parasites traverse the mosquito midgut cells to establish infection at the basal side of the midgut. This dynamic process is a determinant of mosquito vector competence, yet the kinetics of the parasite migration is not well understood. Here we used transgenic mosquitoes of two Anopheles species and a Plasmodium berghei fluorescence reporter line to track parasite passage through the mosquito tissues at high spatial resolution. We provide new quantitative insight into malaria parasite invasion in African and Indian Anopheles species and demonstrate that species-specific kinetics of Plasmodium invasion is shaped by the mosquito complement-like system. Author SummaryThe traversal of the mosquito midgut cells is one of the critical stages in the life cycle of malaria parasites. Motile parasite forms, called ookinetes, traverse the midgut epithelium in a dynamic process which is not fully understood. Here, we harnessed transgenic reporters to track invasion of Plasmodium parasites in African and Indian mosquito species. We found important differences in parasite dynamics between the two anopheline species and demonstrated an unexpected role of mosquito complement-like system in regulation of parasite invasion.

immunology↗

bioRxiv2020

A high-affinity antibody against the CSP N-terminal domain lacks Plasmodium falciparum inhibitory activity

Malaria is a global health concern and research efforts are ongoing to develop a superior vaccine to RTS,S/AS01. To guide immunogen design, we seek a comprehensive understanding of the protective humoral response against Plasmodium falciparum circumsporozoite protein (PfCSP). In contrast to the well-studied responses to the repeat region and the C-terminus, the antibody response against the N-terminal domain of PfCSP (N-CSP) remains obscure. Here, we characterized the molecular recognition and functional efficacy of the N-CSP-specific monoclonal antibody 5D5. The crystal structure at 1.85 [A] resolution revealed that 5D5 binds an -helical epitope in N-CSP with high affinity through extensive shape and charge complementarity, and the unusual utilization of an N-linked glycan. Nevertheless, functional studies indicated low 5D5 binding to live Pf sporozoites, and lack of sporozoite inhibition in vitro and in mosquitoes. Overall, our data on low recognition and inhibition of sporozoites do not support the inclusion of the 5D5 epitope into the next generation of CSP-based vaccines. Summary StatementThe Plasmodium falciparum sporozoite surface protein, PfCSP, is an attractive vaccine target, but the antibody response against the CSP N-terminal domain has remained understudied. Here, to guide immunogen design, Thai et al. provide insights into the binding motif and functional efficacy of the N-terminal domain-specific monoclonal antibody, 5D5.

immunology↗

bioRxiv2019

Evolution of protective human antibodies against Plasmodium falciparum circumsporozoite protein repeat motifs

Circumsporozoite protein of the human malaria parasite Plasmodium falciparum (PfCSP) is the main target of antibodies that prevent the infection and disease. Protective antibodies recognize the central PfCSP domain, but our understanding of how parasite inhibition is associated with recognition of this domain and with the evolution of potent antibodies remains scattered. Here, we characterized the epitope specificity of 200 human monoclonal PfCSP antibodies. We show that the majority of PfCSP antibodies bind to NANP and NANP-like motifs with different preferences and define the molecular basis for recognition. Epitope cross-reactivity evolved with increasing antibody affinity around a conserved (N/D)P-NANP-N(V/A) core. High affinity to this motif, but not binding to NANP-like motifs, was associated with parasite inhibition and protection. Thus, NANP drives the development of potent PfCSP antibodies independently of their cross-reactivity profile, a finding with direct implications for the design of a second-generation PfCSP-based malaria vaccine.\n\nHIGHLIGHTSO_LIThe majority of human PfCSP antibodies recognize multiple epitopes\nC_LIO_LINANP affinity maturation drives the evolution of cross-reactive PfCSP antibodies\nC_LIO_LIPreferential PfCSP antibody binding to a conserved core motif\nC_LIO_LIHigh affinity not epitope specificity is associated with PfCSP antibody potency\nC_LI

immunology↗

bioRxiv2019

Anopheles gambiae TEP1 forms a complex with the coiled-coil domain of LRIM1/APL1C following a conformational change in the thioester domain

The complement-like protein thioester-containing protein 1 (TEP1) is a key factor in the immune response of the malaria vector Anopheles gambiae to pathogens. Multiple allelic variants of TEP1 have been identified in laboratory strains and in the field, and are correlated with distinct immunophenotypes. TEP1 is tightly regulated by conformational changes induced by cleavage in a protease-sensitive region. Cleaved TEP1 forms a soluble complex with a heterodimer of two leucine-rich repeat proteins, LRIM1 and APL1C, and precipitates in the absence of this complex. The molecular structure and oligomeric state of the TEP1/LRIM1/APL1C complex is unclear. We have analyzed the stability of the cleaved form of four TEP1 alleles. Soluble TEP1 forms exhibit significant variation in stability from hours to days at room temperature. Stability is correlated with allelic variation within two specific loops in direct proximity to the thioester bond. The variable loops are part of an interface between the TED and MG8 domains TEP1 that protect the thioester from hydrolysis. Engineering specific disulfide bonds to prevent separation of the TED-MG8 interface stabilizes the cleaved form of TEP1 for months at room temperature. The C-terminal coiled-coil domain of the LRIM1/APL1C complex is sufficient to stabilize the cleaved form of TEP1 in solution but cleaved forms of disulfide-stabilized TEP1 do not interact with LRIM1/APL1C. This implies that formation of the TEP1cut/LRIM1/APL1C complex is dependent on the same conformational change that induces the precipitation of cleaved TEP1. Author SummaryThe mosquito Anopheles gambiae is the principal vector for malaria in Sub-Saharan Africa. A mosquitos own immune system affects how readily it transmits disease. A protein in A. gambiae called TEP1 is responsible for targeting malaria parasites that traverse the mosquitos midgut. TEP1 has multiple alleles and some are associated with a stronger immune response to malaria than others. How genetic variability in TEP1 is linked to phenotypic diversity is not understood. We show that the variation between TEP1 alleles affects the stability of the protein in solution. We also show that the different TEP1 alleles have a wide range in stability of the protein, from hours to days. Engineering disulfide bonds into TEP1 can increase this stability to months. TEP1 activity in vivo is maintained by a complex of two leucine-rich proteins called LRIM1 and APL1C, which binds TEP1 through its C-terminal coiled-coil domain. We found that LRIM1/APL1C does not bind disulfide-stabilized TEP1, suggesting that LRIM1/APL1C binds to activated TEP1. This research advances our molecular understanding of a key immune response that affects the capacity of A. gambiae mosquitoes to transmit malaria.

immunology↗

bioRxiv2019

Metabolic balancing by miR-276 shapes the mosquito reproductive cycle and Plasmodium falciparum development

Anopheles mosquitoes are obligate vectors of the human malaria parasite Plasmodium falciparum. The blood-feeding behavior of Anopheles females delivers essential nutrients for egg development and drives transmission from one human host to another. Plasmodium growth is adapted to the vector reproductive cycle, but how changes in the reproductive cycle impact parasite development is poorly understood. Here, we show that the blood meal-induced miR-276-5p fine-tunes the duration of the mosquito reproductive cycle. Silencing of miR-276 prolonged amino acid catabolism and increased female fertility rates, suggesting that timely termination of the reproductive cycle restricts mosquito investment into reproduction. Prolongation of the reproductive period in P. falciparum-infected females compromised the development of the transmissible parasite form called sporozoite. Our results suggest that Plasmodium sporogony exploits surplus resources after mosquito reproductive investment and demonstrate the crucial role of the mosquito amino acid metabolism in parasite within-vector proliferation and malaria transmission.

molecular biology↗

bioRxiv2017

Single-cell RNA-sequencing redefines blood cell type classification in mosquitoes

Mosquito blood cells are ancestral immune cells that help control infection by vector-borne pathogens. Despite their importance, little is known about mosquito blood cell biology beyond the ambiguous morphological and functional criteria used for their classification. Here we combined the power of single-cell RNA-sequencing, imaging flow cytometry and single-molecule RNA hybridization to analyze blood cells of the malaria mosquito Anopheles gambiae. By demonstrating that blood cells express nearly half of the mosquito transcriptome, our dataset represents an unprecedented view into their transcriptional machinery. Analyses of differentially expressed genes identified transcriptional signatures of two distinct cell types that challenge the current morphology-based classification of these cells. We further demonstrated an active transfer of a cellular marker between blood cells that confounds their identity. We propose that cell-to-cell exchange is broadly relevant for cell type classification and may account for the remarkable cellular diversity observed in nature.

immunology↗

bioRxiv2017

microRNA tissue atlas of the malaria mosquito Anopheles gambiae

Anopheles gambiae mosquitoes transmit the human malaria parasite Plasmodium falciparum, which causes the majority of fatal malaria cases worldwide. The hematophagous life style defines the mosquito reproductive biology and is exploited by P. falciparum for its own sexual reproduction and transmission. The two main phases of the mosquito reproductive cycle, pre-vitellogenic (PV) and post-blood meal (PBM) shape its capacity to transmit malaria. Transition between these phases is tightly coordinated to ensure homeostasis between mosquito tissues and successful reproduction. One layer of control is provided by microRNAs, well known regulators of blood meal digestion and egg development in Aedes mosquitoes. Here, we report a global overview of tissue-specific miRNA expression during the PV and PBM phases and identify miRNAs regulated during PV to PBM transition. The observed coordinated changes in the expression levels of a set of miRNAs in the energy-storing tissues suggest a role in the regulation of blood meal-induced metabolic changes.

genomics↗

bioRxiv2017

Mosquito lipids regulate Plasmodium sporogony and infectivity to the mammalian host

Malaria is a fatal human parasitic disease transmitted by a mosquito vector. The evolution of within-host malaria virulence has been the focus of many empirical and theoretical studies. However, the vectors contribution to virulence evolution is not well understood. Here we explored how within-vector resource exploitation impacts evolutionary trajectories of within-host Plasmodium virulence. We developed a nested model of within-vector dynamics and malaria epidemiology, which predicted that non-competitive resource exploitation within-vector restricts within-host parasite virulence. To validate our model, we experimentally manipulated mosquito lipid trafficking and gauged within-vector parasite development, within-host infectivity and virulence. We found that mosquito-derived lipids determine within-host parasite virulence by shaping development and metabolic activity of transmissible sporozoites. Our findings uncover the role of within-vector environment in regulating within-host Plasmodium virulence and identify Plasmodium metabolic traits that may contribute to the evolution of malaria virulence.

microbiology↗