Periodic Reporting for period 1 - FEBRIS (In vitro brain microvascular model to tackle fever in cerebral malaria)
Berichtszeitraum: 2022-09-01 bis 2024-08-31
Malaria remains a significant global health challenge and economic burden in 85 countries, with Africa bearing the heaviest impact. In 2022, there were 249 million new cases and 608,000 deaths reported worldwide. Infection is primarily caused by Plasmodium falciparum, which accounts for over 90% of cases and deaths, particularly affecting children under five. These children are especially vulnerable to cerebral malaria, one of the most fatal outcomes of P. falciparum infection. Currently, no vaccine or treatment specifically targets this condition, highlighting the urgent need for innovative tools and interventions.
Cerebral malaria (CM) is characterised by the accumulation of malaria-infected red blood cells in the brain vasculature which lead to vascular blockage, blood flow impairment, brain-blood-barrier inflammation and disfunction leading to brain haemorrhages and swelling.
A major challenge in studying cerebral malaria (CM) is the brain's inaccessibility during active infection. Current knowledge is based largely on autopsies, which do not reflect disease onset or progression. This gap highlights the need for models that capture the complex pathogenesis of CM. Existing in vitro models, such as 2D cultures and flow chambers, lack flow or fail to replicate the vascular tree's multicellular complexity. While brain organoids are promising as 3D models, their heterogeneous architectures and lack of perfusable vasculature limit their use. Furthermore, animal models show minimal intracerebral accumulation, a key feature of human malaria infection.
To overcome these limitations, during my Marie Skłodowska-Curie postdoctoral fellowship in the Bernabeu Lab at EMBL Barcelona, I developed a bioengineered 3D model of human brain microvessels to study the pathogenesis of cerebral malaria (CM). I investigated the binding mechanism between malaria-infected red blood cells and endothelial proteins in the brain microvessels. Specifically, I:
1. Identified antibodies from individuals exposed to malaria that inhibit a specific parasite-host interaction, representing a common mechanism of acquired immunity to CM.
2. Demonstrated that febrile temperatures during malaria infection increase parasite accumulation in the brain microvessels.
3. Designed and fabricated prototypes of a 3D in vitro microvasculatures, serving as preliminary models for developing brain-region-specific microvasculature chips for white matter, grey matter, and the basal ganglia.
Cerebral malaria (CM) is characterised by the accumulation of malaria-infected red blood cells in the brain vasculature which lead to vascular blockage, blood flow impairment, brain-blood-barrier inflammation and disfunction leading to brain haemorrhages and swelling.
A major challenge in studying cerebral malaria (CM) is the brain's inaccessibility during active infection. Current knowledge is based largely on autopsies, which do not reflect disease onset or progression. This gap highlights the need for models that capture the complex pathogenesis of CM. Existing in vitro models, such as 2D cultures and flow chambers, lack flow or fail to replicate the vascular tree's multicellular complexity. While brain organoids are promising as 3D models, their heterogeneous architectures and lack of perfusable vasculature limit their use. Furthermore, animal models show minimal intracerebral accumulation, a key feature of human malaria infection.
To overcome these limitations, during my Marie Skłodowska-Curie postdoctoral fellowship in the Bernabeu Lab at EMBL Barcelona, I developed a bioengineered 3D model of human brain microvessels to study the pathogenesis of cerebral malaria (CM). I investigated the binding mechanism between malaria-infected red blood cells and endothelial proteins in the brain microvessels. Specifically, I:
1. Identified antibodies from individuals exposed to malaria that inhibit a specific parasite-host interaction, representing a common mechanism of acquired immunity to CM.
2. Demonstrated that febrile temperatures during malaria infection increase parasite accumulation in the brain microvessels.
3. Designed and fabricated prototypes of a 3D in vitro microvasculatures, serving as preliminary models for developing brain-region-specific microvasculature chips for white matter, grey matter, and the basal ganglia.
I fabricated 3D brain endothelial microvessels to test monoclonal antibodies that inhibit malaria interaction with endothelial receptors associated with severe disease outcomes. Additionally, I studied the effect of fever on parasite binding in these microvessels. Malaria binding experiments were conducted on both a monolayer of endothelial cells (2D) and within 3D microvessels. As proof of concept, I fabricated narrow microchannels by the multiphoton ablation technique available at the Centre for Genomic Regulation in Barcelona.
To dissect the mechanisms behind increased binding under hyperthermia, I used immunofluorescence, ELISA, cytokine antibody arrays, and proteomics in collaboration with the Proteomics Facility at EMBL Heidelberg, while endothelial barrier breakdown was measured using the XCELLigence system. Imaging techniques included confocal and light-sheet microscopy at the Mesoscopic Imaging Facility at EMBL Barcelona. Ongoing experiments by a Master’s student that I am supervising, are optimising the first protocol for expansion microscopy of our microvessel network.
To recreate sections of complex brain microvasculature, I simulated flow hydrodynamics in prototype designs and shifted focus to retina microvasculature due to its similarity to cerebral malaria. I fabricated preliminary designs of retina microvasculature for both children and adults using soft-lithography and am currently testing these models.
Results exploitation, and dissemination. We first published our results as a preprint on bioRxiv, followed by an open-access publication in Nature including raw data. Together with my host PI, we disseminated the findings through social media (Twitter, ResearchGate) and presented them at international conferences (including GRC in 2023, and BioMalPar, MAM, TERMIS in 2024). We also communicated our results to scientific and lay communities via an article in EMBLetc. Magazine and an EMBL press release.
To dissect the mechanisms behind increased binding under hyperthermia, I used immunofluorescence, ELISA, cytokine antibody arrays, and proteomics in collaboration with the Proteomics Facility at EMBL Heidelberg, while endothelial barrier breakdown was measured using the XCELLigence system. Imaging techniques included confocal and light-sheet microscopy at the Mesoscopic Imaging Facility at EMBL Barcelona. Ongoing experiments by a Master’s student that I am supervising, are optimising the first protocol for expansion microscopy of our microvessel network.
To recreate sections of complex brain microvasculature, I simulated flow hydrodynamics in prototype designs and shifted focus to retina microvasculature due to its similarity to cerebral malaria. I fabricated preliminary designs of retina microvasculature for both children and adults using soft-lithography and am currently testing these models.
Results exploitation, and dissemination. We first published our results as a preprint on bioRxiv, followed by an open-access publication in Nature including raw data. Together with my host PI, we disseminated the findings through social media (Twitter, ResearchGate) and presented them at international conferences (including GRC in 2023, and BioMalPar, MAM, TERMIS in 2024). We also communicated our results to scientific and lay communities via an article in EMBLetc. Magazine and an EMBL press release.
Progress beyond the state of the art. I have constructed a 3D model of brain endothelial microvessels embedded in a collagen hydrogel, perfused with malaria-infected erythrocytes. This model is the most physiologically relevant platform for understanding binding kinetics between infected erythrocytes and endothelial cells. As part of a worldwide collaboration, I tested human antibodies from Ugandan adults exposed to malaria, identifying two promising antibodies that inhibit the binding of the virulence protein PfEMP1 to the endothelial EPCR receptor.
My results also indicate that febrile temperatures increase parasite binding to brain microvessels at 40°C in a receptor and flow-dependent manner, suggesting surface molecular rearrangements and shedding of endothelial glycocalyx.
Expected results.
• Publication: R.A. Reyes$, S.S.R. Raghavan$, N.K. Hurlburt$, V. Introini$ et al., (2024). "Broadly inhibitory antibodies against severe malaria virulence proteins", Preprint bioRxiv, DOI: 10.1101/2024.01.25.577124. Accepted in Nature.
• Publication on febrile temperature effects on cerebral malaria pathogenesis.
• Functioning microfluidic prototypes for 3D brain and retina microvasculature-on-a-chip.
• Organizer of the Malaria Gordon Research Conference and Seminar 2025.
• Presenting my MSCA work at international conferences.
• Supervised two Master’s students.
• Secured independent funding from EMBL Infection Biology: £5,000 for bulk RNA sequencing and £25,000 Transition to Independence Award.
• Received an offer for a Group Leader position at a leading research institute in Europe.
Potential impacts. Evaluating the effect of fever on malaria cytoadhesion enhances understanding CM and supports informed therapeutic decisions in malaria-endemic countries. This knowledge can lead to the development of treatments that prevent or cure CM pathology. Insights from FEBRIS will empower healthcare professionals, improving patient outcomes and potentially reducing healthcare costs.
The interdisciplinary approach of the FEBRIS project addresses critical knowledge gaps while promoting educational outreach on malaria. Sharing findings at international conferences, enhanced public support for malaria research. My commitment to mentoring and career development underscores my dedication to fostering future researchers. FEBRIS project advances our understanding of cerebral malaria and positions me to significantly contribute to healthcare challenges and long-term public health outcomes.
My results also indicate that febrile temperatures increase parasite binding to brain microvessels at 40°C in a receptor and flow-dependent manner, suggesting surface molecular rearrangements and shedding of endothelial glycocalyx.
Expected results.
• Publication: R.A. Reyes$, S.S.R. Raghavan$, N.K. Hurlburt$, V. Introini$ et al., (2024). "Broadly inhibitory antibodies against severe malaria virulence proteins", Preprint bioRxiv, DOI: 10.1101/2024.01.25.577124. Accepted in Nature.
• Publication on febrile temperature effects on cerebral malaria pathogenesis.
• Functioning microfluidic prototypes for 3D brain and retina microvasculature-on-a-chip.
• Organizer of the Malaria Gordon Research Conference and Seminar 2025.
• Presenting my MSCA work at international conferences.
• Supervised two Master’s students.
• Secured independent funding from EMBL Infection Biology: £5,000 for bulk RNA sequencing and £25,000 Transition to Independence Award.
• Received an offer for a Group Leader position at a leading research institute in Europe.
Potential impacts. Evaluating the effect of fever on malaria cytoadhesion enhances understanding CM and supports informed therapeutic decisions in malaria-endemic countries. This knowledge can lead to the development of treatments that prevent or cure CM pathology. Insights from FEBRIS will empower healthcare professionals, improving patient outcomes and potentially reducing healthcare costs.
The interdisciplinary approach of the FEBRIS project addresses critical knowledge gaps while promoting educational outreach on malaria. Sharing findings at international conferences, enhanced public support for malaria research. My commitment to mentoring and career development underscores my dedication to fostering future researchers. FEBRIS project advances our understanding of cerebral malaria and positions me to significantly contribute to healthcare challenges and long-term public health outcomes.