Elena A. Levashina

Flow cytometry is widely used to identify and sort rare cells, but conventional approaches rely on fluorescent labels. Label-free discrimination of rare targets in complex, heterogeneous samples remains challenging. Here, we introduce a label-free strategy that combines multi-angle pulse shape flow cytometry (MAPS-FC), which exploits the full temporal structure of the signal pulses during cell or particles transit through the laser spot, with a deep learning–based analysis. As a biologically relevant test case, we focus on Plasmodium falciparum sporozoites, the infectious stage of the malaria parasite, which need to be isolated from mosquito salivary glands and are embedded in a heterogeneous mosquito-derived background. Using a GFP-expressing reporter line for validation, we trained a long short-term memory (LSTM) neural network to classify sporozoites solely from their scattering pulse shapes captured through MAPS-FC. Using training data from multiple days and setting a threshold on the classification confidence, the network can distinguish sporozoites from mosquito debris without fluorescent labels. This work establishes MAPS-FC with deep learning as a platform for label-free identification of rare parasites in complex biological samples.

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. In this study, 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. In mice, edited B lymphocytes derived from transplanted HSPCs were activated by cognate antigen, underwent clonal expansion, and developed into specific antibody-synthesizing or cargo protein–synthesizing plasma cells. These cells produced long-lasting, therapeutic levels of serum antibody against HIV-1, malaria, or 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.

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.

Summary: 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

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+Vκ3-20 antibody affinity and cross-reactivity and demonstrates how antibody affinity maturation can negatively impact antibody stability and thereby function.

Within-host survival and between-host transmission are key life-history traits of single-celled malaria parasites. Understanding the evolutionary forces that shape these traits is crucial to predict malaria epidemiology, drug resistance, and virulence. However, very little is known about how Plasmodium parasites adapt to their mosquito vectors. Here, we examine the evolution of the time Plasmodium parasites require to develop within the vector (extrinsic incubation period) with an 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 predicts that successful vector-to-human transmission events are rare, and are caused by long-lived mosquitoes. Importantly, our results show that the life-history strategies of malaria parasites depend on the mosquito’s metabolic status. In our model, additional resources provided by multiple blood meals lead to selection for parasites with slow or intermediate developmental time. These results challenge the current assumption that evolution favors 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.

Round table discussion on challenges and opportunities in malaria research with Elena Levashina, Dominique Soldati-Favre, Andrew Waters, Friedrich Frischknecht, and Julian Rayner.

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.