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Malaria caused byPlasmodium vivaxis a pressing public health problem in tropical and subtropical areas.However, little progress has been made toward developing a P. vivaxvaccine, with only three candidates being tested in clinical studies. We previously reported that one chimeric recombinant protein (PvCSP-All epitopes) containing the conserved C-terminus of the P. vivax Circumsporozoite Protein (PvCSP), the three variant repeat domains, and aToll-like receptor-3 agonist,Poly(I:C), as an adjuvant (polyinosinic-polycytidylic acid, a dsRNA analog mimicking viral RNA), elicits strong antibody-mediated immune responses in mice to each of the three allelic forms of PvCSP. In the present study, a pre-clinical safety evaluation was performed to identify potential local and systemic toxic effects of the PvCSP-All epitopes combined with the Poly-ICLC (Poly I:C plus poly-L-lysine, Hiltonol®) or Poly-ICLC when subcutaneously injected into C57BL/6 mice and New Zealand White Rabbits followed by a 21-day recovery period. Overall, all observations were considered non-adverse and were consistent with the expected inflammatory response and immune stimulation following vaccine administration. High levels of vaccine-induced specific antibodies were detected both in mice and rabbits. Furthermore, mice that received the vaccine formulation were protected after the challenge with Plasmodium berghei sporozoites expressing CSP repeats from P. vivax sporozoites (Pb/Pv-VK210). In conclusion, in these non-clinical models, repeated dose administrations of the PvCSP-All epitopes vaccine adjuvanted with a Poly-ICLC were immunogenic, safe, and well tolerated., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: R.F.M., A.M.G., and I.S.S. are co-inventors of the potential P. vivax vaccine evaluated in this study. The patent is under evaluation process, application number BR102022005915-2. A.M.S is President and CEO of Oncovir, Inc., which is developing poly-ICLC (Hiltonol) for clinical trials. O.C. and A.S work for Orygen® which licensed Hiltonol from Oncovir for Latin America., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Membranolytic molecules constitute the first line of innate immune defense against pathogenic microorganisms. Plasmodium sporozoites are potentially exposed to these cytotoxic molecules in the hemolymph and salivary glands of mosquitoes, as well as in the skin, blood, and liver of the mammalian host. Here, we show that sporozoites are resistant to bacteriolytic concentration of cecropin B, a cationic amphipathic antimicrobial insect peptide. Intriguingly, anti-tumoral cell-penetrating peptides derived from the anti-apoptotic protein AAC11 killed P. berghei and P. falciparum sporozoites. Using dynamic imaging, we demonstrated that the most cytotoxic peptide, called RT39, did not significantly inhibit the sporozoite motility until the occurrence of a fast permeabilization of the parasite membrane by the peptide. Concomitantly, the cytosolic fluorescent protein constitutively expressed by sporozoites leaked from the treated parasite body while To-Pro 3 and FITC-labeled RT39 internalized, respectively, binding to the nucleic acids and membranes of sporozoites. This led to an increase in the parasite granularity as assessed by flow cytometry. Most permeabilization events started at the parasite's posterior end, resulting in the appearance of a fluorescent dot in the anterior part of sporozoites. Understanding and exploiting the susceptibility of sporozoites and other plasmodial stages to membranolytic molecules might foster strategies to eliminate the parasite and block its transmission.

The circumsporozoite protein (CSP) is the main surface antigen of the Plasmodium sporozoite (SPZ) and forms the basis of the currently only licensed anti-malarial vaccine (RTS,S/AS01). CSP uniformly coats the SPZ and plays a pivotal role in its immunobiology, in both the insect and the vertebrate hosts. Although CSP's N-terminal domain (CSP N ) has been reported to play an important role in multiple CSP functions, a thorough biophysical and structural characterization of CSP N is currently lacking. Here, we present an alternative method for the recombinant production and purification of CSP N from Plasmodium falciparum (PfCSP N ), which provides pure, high-quality protein preparations with high yields. Through an interdisciplinary approach combining in-solution experimental methods and in silico analyses, we provide strong evidence that PfCSP N is an intrinsically disordered region displaying some degree of compaction., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Vaccination strategies in mice inducing high numbers of memory CD8 + T cells specific to a single epitope are able to provide sterilizing protection against infection with Plasmodium sporozoites. We have recently found that Plasmodium-specific CD8 + T cells cluster around sporozoite-infected hepatocytes but whether such clusters are important in elimination of the parasite remains incompletely understood. Here, we used our previously generated data in which we employed intravital microscopy to longitudinally image 32 green fluorescent protein (GFP)-expressing Plasmodium yoelii parasites in livers of mice that had received activated Plasmodium-specific CD8 + T cells after sporozoite infection. We found significant heterogeneity in the dynamics of the normalized GFP signal from the parasites (termed 'vitality index' or VI) that was weakly correlated with the number of T cells near the parasite. We also found that a simple model assuming mass-action, additive killing by T cells well describes the VI dynamics for most parasites and predicts a highly variable killing efficacy by individual T cells. Given our estimated median per capita kill rate of k = 0.031/h we predict that a single T cell is typically incapable of killing a parasite within the 48 h lifespan of the liver stage in mice. Stochastic simulations of T cell clustering and killing of the liver stage also suggested that: (i) three or more T cells per infected hepatocyte are required to ensure sterilizing protection; (ii) both variability in killing efficacy of individual T cells and resistance to killing by individual parasites may contribute to the observed variability in VI decline, and (iii) the stable VI of some clustered parasites cannot be explained by measurement noise. Taken together, our analysis for the first time provides estimates of efficiency at which individual CD8 + T cells eliminate intracellular parasitic infection in vivo .

Human monoclonal antibodies (hmAbs) targeting the Plasmodium falciparum circumsporozoite protein (PfCSP) on the sporozoite surface are a promising tool for preventing malaria infection. However, their mechanisms of protection remain unclear. Here, using 13 distinctive PfCSP hmAbs, we provide a comprehensive view of how PfCSP hmAbs neutralize sporozoites in host tissues. Sporozoites are most vulnerable to hmAb-mediated neutralization in the skin. However, rare but potent hmAbs additionally neutralize sporozoites in the blood and liver. Efficient protection in tissues mainly associates with high-affinity and high-cytotoxicity hmAbs inducing rapid parasite loss-of-fitness in the absence of complement and host cells in vitro. A 3D-substrate assay greatly enhances hmAb cytotoxicity and mimics the skin-dependent protection, indicating that the physical stress imposed on motile sporozoites by the skin is crucial for unfolding the protective potential of hmAbs. This functional 3D cytotoxicity assay can thus be useful for downselecting potent anti-PfCSP hmAbs and vaccines., Competing Interests: Declaration of interests A.N. is a founder of Scians Ltd. R.A.S., A.H.I., and B.J.F. hold a patent describing antibody CIS43, issued on June 1, 2021; International Application No. PCT/US2018/017826. R.A.S., L.T.W., and J.R.F. hold a patent describing antibody L9, issued on May 4, 2020; International Application No. PCT/US2020/031345., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Plasmodium sporozoites actively migrate in the dermis and enter blood vessels to infect the liver. Despite their importance for malaria infection, little is known about these cutaneous processes. We combine intravital imaging in a rodent malaria model and statistical methods to unveil the parasite strategy to reach the bloodstream. We determine that sporozoites display a high-motility mode with a superdiffusive Lévy-like pattern known to optimize the location of scarce targets. When encountering blood vessels, sporozoites frequently switch to a subdiffusive low-motility behavior associated with probing for intravasation hotspots, marked by the presence of pericytes. Hence, sporozoites present anomalous diffusive motility, alternating between superdiffusive tissue exploration and subdiffusive local vessel exploitation, thus optimizing the sequential tasks of seeking blood vessels and pericyte-associated sites of privileged intravasation., (© 2023. The Author(s).)

Introduction: Plasmodium sporozoites (SPZ) inoculated by Anopheles mosquitoes into the skin of the mammalian host migrate to the liver before infecting hepatocytes. Previous work demonstrated that early production of IL-6 in the liver is detrimental for the parasite growth, contributing to the acquisition of a long-lasting immune protection after immunization with live attenuated parasites., Methods: Considering that IL-6 as a critical pro-inflammatory signal, we explored a novel approach whereby the parasite itself encodes for the murine IL-6 gene. We generated transgenic P. berghei parasites that express murine IL-6 during liver stage development., Results and Discussion: Though IL-6 transgenic SPZ developed into exo-erythrocytic forms in hepatocytes in vitro and in vivo , these parasites were not capable of inducing a blood stage infection in mice. Furthermore, immunization of mice with transgenic IL-6-expressing P. berghei SPZ elicited a long-lasting CD8 + T cell-mediated protective immunity against a subsequent infectious SPZ challenge. Collectively, this study demonstrates that parasite-encoded IL-6 attenuates parasite virulence with abortive liver stage of Plasmodium infection, forming the basis of a novel suicide vaccine strategy to elicit protective antimalarial immunity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Belhimeur, Briquet, Peronet, Pham, Commere, Formaglio, Amino, Scherf, Silvie and Mecheri.)

During transmission of malaria-causing parasites from mosquitoes to mammals, Plasmodium sporozoites migrate rapidly in the skin to search for a blood vessel. The high migratory speed and narrow passages taken by the parasites suggest considerable strain on the sporozoites to maintain their shape. Here, we show that the membrane-associated protein, concavin, is important for the maintenance of the Plasmodium sporozoite shape inside salivary glands of mosquitoes and during migration in the skin. Concavin-GFP localizes at the cytoplasmic periphery and concavin(-) sporozoites progressively round up upon entry of salivary glands. Rounded concavin(-) sporozoites fail to pass through the narrow salivary ducts and are rarely ejected by mosquitoes, while normally shaped concavin(-) sporozoites are transmitted. Strikingly, motile concavin(-) sporozoites disintegrate while migrating through the skin leading to parasite arrest or death and decreased transmission efficiency. Collectively, we suggest that concavin contributes to cell shape maintenance by riveting the plasma membrane to the subtending inner membrane complex. Interfering with cell shape maintenance pathways might hence provide a new strategy to prevent a malaria infection., (© 2022 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Malaria, an infection caused by apicomplexan parasites of the genus Plasmodium , continues to exact a significant toll on public health with over 200 million cases world-wide, and annual deaths in excess of 600,000. Considerable progress has been made to reduce malaria burden in endemic countries in the last two decades. However, parasite and mosquito resistance to frontline chemotherapies and insecticides, respectively, highlights the continuing need for the development of safe and effective vaccines. Here we describe the development of recombinant human antibodies to three target proteins from Plasmodium falciparum : reticulocyte binding protein homologue 5 (PfRH5), cysteine-rich protective antigen (Pf CyRPA), and circumsporozoite protein ( Pf CSP). All three proteins are key targets in the development of vaccines for blood-stage or pre-erythrocytic stage infections. We have developed potent anti- Pf RH5, Pf CyRPA and Pf CSP monoclonal antibodies that will prove useful tools for the standardisation of assays in preclinical research and the assessment of these antigens in clinical trials. We have generated some very potent anti- Pf RH5 and anti- Pf CyRPA antibodies with some clones >200 times more potent than the polyclonal anti-AMA-1 antibodies used for the evaluation of blood stage antigens. While the monoclonal and polyclonal antibodies are not directly comparable, the data provide evidence that these new antibodies are very good at blocking invasion. These antibodies will therefore provide a valuable resource and have potential as biological standards to help harmonise pre-clinical malaria research., Competing Interests: SJD is a named inventor on patent applications relating to PfRH5 and/or other malaria vaccines, mAbs, and immunisation regimes. UR and PH are employed by JPT Peptide Technologies GmbH. GK, RP, EJ, and AT are employed by Icosagen Cell Factory. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Nacer, Kivi, Pert, Juronen, Holenya, Aliprandini, Amino, Silvie, Quinkert, Le Duff, Hurley, Reimer, Tover, Draper, Gilbert, Ho and Bowyer.)

The uptake and digestion of host hemoglobin by malaria parasites during blood-stage growth leads to significant oxidative damage of membrane lipids. Repair of lipid peroxidation damage is crucial for parasite survival. Here, we demonstrate that Plasmodium falciparum imports a host antioxidant enzyme, peroxiredoxin 6 (PRDX6), during hemoglobin uptake from the red blood cell cytosol. PRDX6 is a lipid-peroxidation repair enzyme with phospholipase A 2 (PLA 2 ) activity. Inhibition of PRDX6 with a PLA 2 inhibitor, Darapladib, increases lipid-peroxidation damage in the parasite and disrupts transport of hemoglobin-containing vesicles to the food vacuole, causing parasite death. Furthermore, inhibition of PRDX6 synergistically reduces the survival of artemisinin-resistant parasites following co-treatment of parasite cultures with artemisinin and Darapladib. Thus, PRDX6 is a host-derived drug target for development of antimalarial drugs that could help overcome artemisinin resistance., Competing Interests: Declaration of interests A patent application has been filed by Institut Pasteur based on the results of this publication with M.P.W., C.E.C., L.T., O.G., C.H., P.F., and R.H. as inventors. A.B.F. and S.I.F. are shareholders in Peroxitech, Inc., a USA company that is developing a peptide-based PRDX6 inhibitor as a therapeutic agent., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The sole currently approved malaria vaccine targets the circumsporozoite protein-the protein that densely coats the surface of sporozoites, the parasite stage deposited in the skin of the mammalian host by infected mosquitoes. However, this vaccine only confers moderate protection against clinical diseases in children, impelling a continuous search for novel candidates. In this work, we studied the importance of the membrane-associated erythrocyte binding-like protein (MAEBL) for infection by Plasmodium sporozoites. Using transgenic parasites and live imaging in mice, we show that the absence of MAEBL reduces Plasmodium berghei hemolymph sporozoite infectivity to mice. Moreover, we found that maebl knockout ( maebl -) sporozoites display reduced adhesion, including to cultured hepatocytes, which could contribute to the defects in multiple biological processes, such as in gliding motility, hepatocyte wounding, and invasion. The maebl - defective phenotypes in mosquito salivary gland and liver infection were reverted by genetic complementation. Using a parasite line expressing a C-terminal myc-tagged MAEBL, we found that MAEBL levels peak in midgut and hemolymph parasites but drop after sporozoite entry into the salivary glands, where the labeling was found to be heterogeneous among sporozoites. MAEBL was found associated, not only with micronemes, but also with the surface of mature sporozoites. Overall, our data provide further insight into the role of MAEBL in sporozoite infectivity and may contribute to the design of future immune interventions.

Malaria is a major public health concern, as a highly effective human vaccine remains elusive. The efficacy of a subunit vaccine targeting the most abundant protein of the sporozoite surface, the circumsporozoite protein (CSP) has been hindered by difficulties in generating an effective humoral response in both quantity and quality. Using the rodent Plasmodium yoelii model we report here that immunization with CSP adjuvanted with 5'ppp-dsRNA, a RIG-I agonist, confers early and long-lasting sterile protection in mice against stringent sporozoite and mosquito bite challenges. The immunization induced high levels of antibodies, which were functional in targeting and killing the sporozoites and were sustained over time through the accumulation of long-lived plasma cells in the bone marrow. Moreover, 5'ppp-dsRNA-adjuvanted immunization with the CSP of P. falciparum was also significantly protective against challenges using a transgenic Pf CSP-expressing P. yoelii parasite. Conversely, using the TLR3 agonist poly(A:U) as adjuvant resulted in a formulation that despite inducing high antibody levels was unable to generate equally functional antibodies and was, consequently, less protective. In conclusion, we demonstrate that using 5'ppp-dsRNA as an adjuvant to vaccines targeting CSP induces effective anti- Plasmodium humoral immunity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a shared affiliation with one of the authors, SB, at time of review., (Copyright © 2022 Teixeira, Pérez-Cabezas, Costa, Sá, Golba, Sefiane-Djemaoune, Ribeiro, Kaneko, Iwanaga, Yuda, Tsuji, Boscardin, Amino, Cordeiro-da-Silva and Tavares.)

The calcium ion (Ca 2+ ) is a ubiquitous second messenger involved in key biological processes in prokaryotes and eukaryotes. In Plasmodium species, Ca 2+ signaling plays a central role in the parasite life cycle. It has been associated with parasite development, fertilization, locomotion, and host cell infection. Despite the lack of a canonical inositol-1,4,5-triphosphate receptor gene in the Plasmodium genome, pharmacological evidence indicates that inositol-1,4,5-triphosphate triggers Ca 2+ mobilization from the endoplasmic reticulum. Other structures such as acidocalcisomes, food vacuole and mitochondria are proposed to act as supplementary intracellular Ca 2+ reservoirs. Several Ca 2+ -binding proteins (CaBPs) trigger downstream signaling. Other proteins with no EF-hand motifs, but apparently involved with CaBPs, are depicted as playing an important role in the erythrocyte invasion and egress. It is also proposed that a cross-talk among kinases, which are not members of the family of Ca 2+ -dependent protein kinases, such as protein kinases G, A and B, play additional roles mediated indirectly by Ca 2+ regulation. This statement may be extended for proteins directly related to invasion or egress, such as SUB1, ERC, IMC1I, IMC1g, GAP45 and EBA175. In this review, we update our understanding of aspects of Ca 2+ -mediated signaling correlated to the developmental stages of the malaria parasite life cycle., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 de Oliveira, Alborghetti, Carneiro, Bastos, Amino, Grellier and Charneau.)

Transmission of malaria-causing parasites to and by the mosquito relies on active parasite migration and constitutes bottlenecks in the Plasmodium life cycle. Parasite adaption to the biochemically and physically different environments must hence be a key evolutionary driver for transmission efficiency. To probe how subtle but physiologically relevant changes in environmental elasticity impact parasite migration, we introduce 2D and 3D polyacrylamide gels to study ookinetes, the parasite forms emigrating from the mosquito blood meal and sporozoites, the forms transmitted to the vertebrate host. We show that ookinetes adapt their migratory path but not their speed to environmental elasticity and are motile for over 24 h on soft substrates. In contrast, sporozoites evolved more short-lived rapid gliding motility for rapidly crossing the skin. Strikingly, sporozoites are highly sensitive to substrate elasticity possibly to avoid adhesion to soft endothelial cells on their long way to the liver. Hence, the two migratory stages of Plasmodium evolved different strategies to overcome the physical challenges posed by the respective environments and barriers they encounter., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

In the malaria-causing parasite's life cycle, Plasmodium sporozoites must travel from the midgut of a mosquito to the salivary glands before they can infect a mammalian host. However, only a fraction of sporozoites complete the journey. Since salivary gland invasion is required for transmission of sporozoites, insights at the molecular level can contribute to strategies for malaria prevention. Recent advances in single-cell RNA sequencing provide an opportunity to assess sporozoite heterogeneity at a resolution unattainable by bulk RNA sequencing methods. In this study, we use a droplet-based single-cell RNA sequencing workflow to analyze the transcriptomes of over 8000 Plasmodium berghei sporozoites derived from the midguts and salivary glands of Anopheles stephensi mosquitoes. The detection of known marker genes confirms the successful capture and sequencing of samples composed of a mixed population of sporozoites. Using data integration, clustering, and trajectory analyses, we reveal differences in gene expression profiles of individual sporozoites, and identify both annotated and unannotated markers associated with sporozoite development. Our work highlights the utility of a high-throughput workflow for the transcriptomic profiling of Plasmodium sporozoites, and provides new insights into gene usage during the parasite's development in the mosquito.

Background: Different strategies for improvement of malaria control and elimination are based on the blockage of malaria parasite transmission to the mosquito vector. These strategies include the drugs that target the plasmodial sexual stages in humans and the early developmental stages inside mosquitoes., Objectives: Here we tested Malaria Box compounds in order to evaluate their activity against male and female gametocytes in Plasmodium berghei, mosquito infection in P. vivax and ookinete formation in both species., Methods/findings: The membrane feeding assay and the development of ookinetes by a 24 h ex vivo culture and the ookinete yield per 1000 erythrocytes were used to test transmission-blocking potential of the Malaria Box compounds in P. vivax. For P. berghei we used flow cytometry to evaluate male and female gametocyte time course and fluorescence microscopy to check the ookinete development. The two species used in this study showed similar results concerning the compounds' activity against gametocytes and ookinetes, which were different from those in P. falciparum. In addition, from the eight Malaria Box compounds tested in both species, compounds MMV665830, MMV665878 and MMV665941 were selected as a hit compounds due the high inhibition observed., Conclusion: Our results showed that P. berghei is suitable as an initial screening system to test compounds against P. vivax.

Deciphering the mechanisms by which Plasmodium parasites develop inside hepatocytes is an important step toward the understanding of malaria pathogenesis. We propose that the nature and the magnitude of the inflammatory response in the liver are key for the establishment of the infection. Here, we used mice deficient in the multidrug resistance-2 gene (Mdr2 -/- )-encoded phospholipid flippase leading to the development of liver inflammation. Infection of Mdr2 -/- mice with Plasmodium berghei ANKA ( Pb ANKA) sporozoites (SPZ) resulted in the blockade of hepatic exo-erythrocytic forms (EEFs) with no further development into blood stage parasites. Interestingly, cultured primary hepatocytes from mutant and wild-type mice are equally effective in supporting EEF development. The abortive infection resulted in a long-lasting immunity in Mdr2 -/- mice against infectious SPZ where neutrophils and IL-6 appear as key effector components along with CD8 + and CD4 + effector and central memory T cells. Inflammation-induced breakdown of liver tolerance promotes anti-parasite immunity and provides new approaches for the design of effective vaccines against malaria disease., (Copyright © 2020 Grand, Waqasi, Demarta-Gatsi, Wei, Peronet, Commere, Puig, Axelrod, Caldelari, Heussler, Amino and Mecheri.)

Discovering potent human monoclonal antibodies (mAbs) targeting the Plasmodium falciparum circumsporozoite protein (PfCSP) on sporozoites (SPZ) and elucidating their mechanisms of neutralization will facilitate translation for passive prophylaxis and aid next-generation vaccine development. Here, we isolated a neutralizing human mAb, L9 that preferentially bound NVDP minor repeats of PfCSP with high affinity while cross-reacting with NANP major repeats. L9 was more potent than six published neutralizing human PfCSP mAbs at mediating protection against mosquito bite challenge in mice. Isothermal titration calorimetry and multiphoton microscopy showed that L9 and the other most protective mAbs bound PfCSP with two binding events and mediated protection by killing SPZ in the liver and by preventing their egress from sinusoids and traversal of hepatocytes. This study defines the subdominant PfCSP minor repeats as neutralizing epitopes, identifies an in vitro biophysical correlate of SPZ neutralization, and demonstrates that the liver is an important site for antibodies to prevent malaria., Competing Interests: Declaration of Interests R.A.S., J.R.F., L.T.W., and R.V. have submitted US Provisional Patent Application 62/842,590, filed 3 May 2019, describing mAb L9. All other authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Infections with Plasmodium vivax are predominant in the Americas, representing 75% of malaria cases. Previously perceived as benign, malaria vivax is, in fact, a highly debilitating and economically important disease. Considering the high complexity of the malaria parasite life cycle, it has been hypothesized that an effective vaccine formulation against Plasmodium should contain multiple antigens expressed in different parasite stages. Based on that, we analyzed a recombinant P. vivax vaccine formulation mixing the apical membrane antigen 1 ectodomain (PvAMA-1) and a full-length circumsporozoite protein (PvCSP-All FL ) previously studied by our group, which elicits a potent antibody response in mice. Genetically distinct strains of mice (C57BL/6 and BALB/c) were immunized with the proteins, alone or in combination, in the presence of poly(I:C) adjuvant, a TLR3 agonist. In C57BL/6, high-antibody titers were induced against PvAMA-1 and the three PvCSP variants (VK210, VK247, and P. vivax -like). Meanwhile, mixing PvAMA-1 with PvCSP-All FL had no impact on total IgG antibody titers, which were long-lasting. Moreover, antibodies from immunized mice recognized VK210 sporozoites and blood-stage parasites by immunofluorescence assay. However, in the BALB/c model, the antibody response against PvCSP-All FL was relatively low. PvAMA-1-specific CD3 + CD4 + and CD3 + CD8 + T-cell responses were observed in C57BL/6 mice, and the cellular response was impaired by PvCSP-All FL combination. More relevant, the multistage vaccine formulation provided partial protection in mice challenged with a transgenic Plasmodium berghei sporozoite expressing the homologous PvCSP protein.

Plasmodium vivax is the most common species of human malaria parasite found outside Africa, with high endemicity in Asia, Central and South America, and Oceania. Although Plasmodium falciparum causes the majority of deaths, P. vivax can lead to severe malaria and result in significant morbidity and mortality. The development of a protective vaccine will be a major step toward malaria elimination. Recently, a formulation containing the three allelic variants of the P. vivax circumsporozoite protein (PvCSP-All epitopes) showed partial protection in mice after a challenge with the hybrid Plasmodium berghei (Pb) sporozoite, in which the PbCSP central repeats were replaced by the VK210 PvCSP repeats (Pb/Pv sporozoite). In the present study, the chimeric PvCSP allelic variants (VK210, VK247, and P. vivax -like) were fused with the mumps virus nucleocapsid protein in the absence (NLP-CSP R ) or presence of the conserved C-terminal (CT) domain of PvCSP (NLP-CSP CT ). To elicit stronger humoral and cellular responses, Pichia pastoris yeast was used to assemble them as nucleocapsid-like particles (NLPs). Mice were immunized with each recombinant protein adjuvanted with Poly (I:C) and presented a high frequency of antigen-specific antibody-secreting cells (ASCs) on days 5 and 30, respectively, in the spleen and bone marrow. Moreover, high IgG titers against all PvCSP variants were detected in the sera. Later, these immunized mice with NLP-CSP CT were challenged with Pb/Pv sporozoites. Sterile protection was observed in 30% of the challenged mice. Therefore, this vaccine formulation use has the potential to be a good candidate for the development of a universal vaccine against P. vivax malaria.

In comparing placental transfusion strategies, blood obtained from an umbilical cord that has been "milked" vs one in which clamping was simply delayed contains mesenchymal stromal cells in addition to solely hematopoietic stem cells, a composition more favorable for hematopoiesis, as suggested by its superior rescue of lethally irradiated bone marrow-depleted mice., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Following the RTS,S malaria vaccine, which showed only partial protection with short-term memory, there is strong support to develop second-generation malaria vaccines that yield higher efficacy with longer duration. The use of replicating viral vectors to deliver subunit vaccines is of great interest due to their capacity to induce efficient cellular immune responses and long-term memory. The measles vaccine virus offers an efficient and safe live viral vector that could easily be implemented in the field. Here, we produced recombinant measles viruses (rMV) expressing malaria "gold standard" circumsporozoïte antigen (CS) of Plasmodium berghei ( Pb ) and Plasmodium falciparum ( Pf ) to test proof of concept of this delivery strategy. Immunization with rMV expressing Pb CS or Pf CS induced high antibody responses in mice that did not decrease for at least 22 weeks post-prime, as well as rapid development of cellular immune responses. The observed long-term memory response is key for development of second-generation malaria vaccines. Sterile protection was achieved in 33% of immunized mice, as usually observed with the CS antigen, and all other immunized animals were clinically protected from severe and lethal Pb ANKA-induced cerebral malaria. Further rMV-vectored malaria vaccine candidates expressing additional pre-erythrocytic and blood-stage antigens in combination with rMV expressing Pf CS may provide a path to development of next generation malaria vaccines with higher efficacy., Competing Interests: The authors declare no competing interests.

Plasmodium sporozoites deposited in the skin following a mosquito bite must migrate and invade blood vessels to complete their development in the liver. Once in the bloodstream, sporozoites arrest in the liver sinusoids, but the molecular determinants that mediate this specific homing are not yet genetically defined. Here we investigate the involvement of the thrombospondin-related sporozoite protein (TRSP) in this process using knockout Plasmodium berghei parasites and in vivo bioluminescence imaging in mice. Resorting to a homing assay, trsp knockout sporozoites were found to arrest in the liver similar to control parasites. Moreover, we found no defects in the establishment of infection in mice following inoculation of trsp knockout sporozoites via intravenous and cutaneous injection or mosquito bite. Accordingly, mutant sporozoites were also able to successfully invade hepatocytes in vitro. Altogether, these results suggest TRSP may have a redundant role in the completion of the pre-erythrocytic phase of the malaria parasite. Nonetheless, identifying molecules with paramount roles in this phase could aid in the search for new antigens needed for the design of a protective vaccine against malaria.

Cerebral malaria (CM) is a multifactorial syndrome involving an exacerbated proinflammatory status, endothelial cell activation, coagulopathy, hypoxia, and accumulation of leukocytes and parasites in the brain microvasculature. Despite significant improvements in malaria control, 15% of mortality is still observed in CM cases, and 25% of survivors develop neurologic sequelae for life-even after appropriate antimalarial therapy. A treatment that ameliorates CM clinical signs, resulting in complete healing, is urgently needed. Previously, we showed a hyperbaric oxygen (HBO)-protective effect against experimental CM. Here, we provide molecular evidence that HBO targets brain endothelial cells by decreasing their activation and inhibits parasite and leukocyte accumulation, thus improving cerebral microcirculatory blood flow. HBO treatment increased the expression of aryl hydrocarbon receptor over hypoxia-inducible factor 1-α (HIF-1α), an oxygen-sensitive cytosolic receptor, along with decreased indoleamine 2,3-dioxygenase 1 expression and kynurenine levels. Moreover, ablation of HIF-1α expression in endothelial cells in mice conferred protection against CM and improved survival. We propose that HBO should be pursued as an adjunctive therapy in CM patients to prolong survival and diminish deleterious proinflammatory reaction. Furthermore, our data support the use of HBO in therapeutic strategies to improve outcomes of non-CM disorders affecting the brain.-Bastos, M. F., Kayano, A. C. A. V., Silva-Filho, J. L., Dos-Santos, J. C. K., Judice, C., Blanco, Y. C., Shryock, N., Sercundes, M. K., Ortolan, L. S., Francelin, C., Leite, J. A., Oliveira, R., Elias, R. M., Câmara, N. O. S., Lopes, S. C. P., Albrecht, L., Farias, A. S., Vicente, C. P., Werneck, C. C., Giorgio, S., Verinaud, L., Epiphanio, S., Marinho, C. R. F., Lalwani, P., Amino, R., Aliberti, J., Costa, F. T. M. Inhibition of hypoxia-associated response and kynurenine production in response to hyperbaric oxygen as mechanisms involved in protection against experimental cerebral malaria.

CD8 + T cells are the major effector cells that protect against malaria liver-stage infection, forming clusters around Plasmodium -infected hepatocytes and eliminating parasites after a prolonged interaction with these hepatocytes. We aimed to investigate the roles of specific and nonspecific CD8 + T cells in cluster formation and protective immunity. To this end, we used Plasmodium berghei ANKA expressing ovalbumin as well as CD8 + T cells from transgenic mice expressing a T cell receptor specific for ovalbumin (OT-I) and CD8 + T cells specific for an unrelated antigen, respectively. While antigen-specific CD8 + T cells were essential for cluster formation, both antigen-specific and nonspecific CD8 + T cells joined the clusters. However, nonspecific CD8 + T cells did not significantly contribute to protective immunity. In the livers of infected mice, specific CD8 + T cells expressed high levels of CD25, compatible with a local, activated effector phenotype. In vivo imaging of the liver revealed that specific CD8 + T cells interact with CD11c + cells around infected hepatocytes. The depletion of CD11c + cells virtually eliminated the clusters in the liver, leading to a significant decrease in protection. These experiments reveal an essential role of hepatic CD11c + dendritic cells and presumably macrophages in the formation of CD8 + T cell clusters around Plasmodium -infected hepatocytes. Once cluster formation is triggered by parasite-specific CD8 + T cells, specific and unrelated activated CD8 + T cells join the clusters in a chemokine- and dendritic cell-dependent manner. Nonspecific CD8 + T cells seem to play a limited role in protective immunity against Plasmodium parasites., (Copyright © 2018 American Society for Microbiology.)

Complete and long-lasting protective immunity against malaria can be achieved through vaccination with invasive live attenuated Plasmodium sporozoites, the motile stage inoculated in the host skin during a mosquito bite. Protective immunity relies primarily on effector CD8 + T cells targeting the parasite in the liver. Understanding the tissue-specific features of the immune response is emerging as a vital requirement for understanding protective immunity. The small parasite inoculum, the scarcity of infected cells and the tolerogenic properties of the liver represent hurdles for the establishment of protective immunity in endemic areas. In this review, we discuss recent advances on liver-specific features of immunity including innate recognition of malaria pre-erythrocytic stages, CD8 + T cell interactions with infected hepatocytes, antigen presentation for effective CD8 + T cell responses and generation of liver-resident memory CD8 + T cells. A better understanding of the factors involved in the induction and maintenance of effector CD8 + T cell immunity against malaria pre-erythrocytic stages is crucial for the development of an effective vaccine targeting the initial phase of malaria infection., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Plasmodium vivax is the most common species that cause malaria outside of the African continent. The development of an efficacious vaccine would contribute greatly to control malaria. Recently, using bacterial and adenoviral recombinant proteins based on the P. vivax circumsporozoite protein (CSP), we demonstrated the possibility of eliciting strong antibody-mediated immune responses to each of the three allelic forms of P. vivax CSP (PvCSP). In the present study, recombinant proteins representing the PvCSP alleles (VK210, VK247, and P. vivax -like), as well as a hybrid polypeptide, named PvCSP-All epitopes, were generated. This hybrid containing the conserved C-terminal of the PvCSP and the three variant repeat domains in tandem were successfully produced in the yeast Pichia pastoris . After purification and biochemical characterization, they were used for the experimental immunization of C57BL/6 mice in a vaccine formulation containing the adjuvant Poly(I:C). Immunization with a recombinant protein expressing all three different allelic forms in fusion elicited high IgG antibody titers reacting with all three different allelic variants of PvCSP. The antibodies targeted both the C-terminal and repeat domains of PvCSP and recognized the native protein on the surface of P. vivax sporozoites. More importantly, mice that received the vaccine formulation were protected after challenge with chimeric Plasmodium berghei sporozoites expressing CSP repeats of P. vivax sporozoites (Pb/PvVK210). Our results suggest that it is possible to elicit protective immunity against one of the most common PvCSP alleles using soluble recombinant proteins expressed by P. pastoris . These recombinant proteins are promising candidates for clinical trials aiming to develop a multiallele vaccine against P. vivax malaria.

Hematogenous dissemination followed by tissue tropism is a characteristic of the infectious process of many pathogens including those transmitted by blood-feeding vectors. After entering into the blood circulation, these pathogens must arrest in the target organ before they infect a specific tissue. Here, we describe a non-invasive method to visualize and quantify the homing of pathogens to the host tissues. By using in vivo bioluminescence imaging we quantify the accumulation of luciferase-expressing parasites in the host organs during the first minutes following their intravascular inoculation in mice. Using this technique we show that in the malarial infection, once in the blood circulation, most of bioluminescent Plasmodium berghei sporozoites, the parasite stage transmitted to the host skin by a mosquito bite, rapidly home to the liver where they invade and develop inside hepatocytes. This homing is specific to this developmental stage since blood stage parasites do not accumulate in the liver, as well as extracellular Trypanosoma brucei bloodstream forms and liver-infecting Leishmania infantum amastigotes. Finally, this method can be used to study the dynamics of tissue tropism of parasites, dissect the molecular and cellular basis of their increased arrest in organs and to evaluate immune interventions designed to block this targeted interaction., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Yeast cells represent an established bioreactor to produce recombinant proteins for subunit vaccine development. In addition, delivery of vaccine antigens directly within heat-inactivated yeast cells is attractive due to the adjuvancy provided by the yeast cell. In this study, Pichia pastoris yeast lysates carrying the nucleoprotein (N) from the measles vaccine virus were evaluated as a novel subunit vaccine platform to deliver the circumsporozoite surface antigen (CS) of Plasmodium. When expressed in Pichia pastoris yeast, the N protein auto-assembles into highly multimeric ribonucleoparticles (RNPs). The CS antigen from Plasmodium berghei (PbCS) was expressed in Pichia pastoris yeast in fusion with N, generating recombinant PbCS-carrying RNPs in the cytoplasm of yeast cells., Results: When evaluated in mice after 3-5 weekly subcutaneous injections, yeast lysates containing N-PbCS RNPs elicited strong anti-PbCS humoral responses, which were PbCS-dose dependent and reached a plateau by the pre-challenge time point. Protective efficacy of yeast lysates was dose-dependent, although anti-PbCS antibody titers were not predictive of protection. Multimerization of PbCS on RNPs was essential for providing benefit against infection, as immunization with monomeric PbCS delivered in yeast lysates was not protective. Three weekly injections with N-PbCS yeast lysates in combination with alum adjuvant produced sterile protection in two out of six mice, and significantly reduced parasitaemia in the other individuals from the same group. This parasitaemia decrease was of the same extent as in mice immunized with non-adjuvanted N-PbCS yeast lysates, providing evidence that the yeast lysate formulation did not require accessory adjuvants for eliciting efficient parasitaemia reduction., Conclusions: This study demonstrates that yeast lysates are an attractive auto-adjuvant and efficient platform for delivering multimeric PbCS on measles N-based RNPs. By combining yeast lysates that carry RNPs with a large panel of Plasmodium antigens, this technology could be applied to developing a multivalent vaccine against malaria.

Preventing malaria infection through vaccination requires preventing every sporozoite inoculated by mosquito bite: a major challenge for Plasmodium falciparum. Plasmodium vivax sporozoites consist of tachysporozoites causing primary infection and bradysporozoites leading to relapses. We hypothesise that a candidate P. vivax vaccine with low efficacy against primary infection may substantially reduce transmission by preventing relapses., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In vivo imaging has revealed new details about how the malaria parasite enters the bloodstream.

Malaria parasites undergo a complex life cycle between their hosts and vectors. During this cycle the parasites invade different types of cells, migrate across barriers, and transfer from one host to another. Recent literature hints at a misunderstanding of the difference between active, parasite-driven migration and passive, circulation-driven movement of the parasite or parasite-infected cells in the various bodily fluids of mosquito and mammalian hosts. Because both active migration and passive transport could be targeted in different ways to interfere with the parasite, a distinction between the two ways the parasite uses to get from one location to another is essential. We discuss the two types of motion needed for parasite dissemination and elaborate on how they could be targeted by future vaccines or drugs., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Plasmodium spp., which causes malaria, produces a histamine-releasing factor (HRF), an orthologue of mammalian HRF. Histamine-releasing factor produced by erythrocytic stages of the parasite is thought to play a role in the pathogenesis of severe malaria. Here, we show in a rodent model that HRF is not important during the erythrocytic but pre-erythrocytic phase of infection, which mainly consists in the transformation in the liver of the mosquito-injected parasite form into the erythrocyte-infecting form. Development of P. berghei ANKA cl15cy1 liver stages lacking HRF is impaired and associated with an early rise in systemic IL-6, a cytokine that strongly suppresses development of Plasmodium liver stages. The defect is rescued by injection of anti-IL-6 antibodies or infection in IL-6-deficient mice and parasite HRF is sufficient to decrease IL-6 synthesis, indicating a direct role of parasite HRF in reducing host IL-6. The target cells modulated by HRF for IL-6 production at early time points during liver infection are neutrophils. Parasite HRF is thus used to down-regulate a cytokine with anti-parasite activity. Our data also highlight the link between a prolonged transition from liver to blood-stage infection and reduced incidence of experimental cerebral malaria., (© 2014 John Wiley & Sons Ltd.)

Multiple immunizations using live irradiated sporozoites, the infectious plasmodial stage delivered into the host skin during a mosquito bite, can elicit sterile immunity to malaria. CD8(+) T cells seem to play an essential role in this protective immunity, since their depletion consistently abolishes sterilizing protection in several experimental models. So far, only a few parasite antigens are known to induce CD8(+) T cell-dependent protection, but none of them can reach the levels of protection afforded by live attenuated parasites. Systematic attempts to identify novel antigens associated with this efficient cellular protection were so far unsuccessful. In addition, the precise mechanisms involved in the recognition and elimination of parasitized hepatocytes in vivo by CD8(+) T cells still remain obscure. Recently, it has been shown that specific effector CD8(+) T cells, after recognition of parasitized hepatocytes, recruit specific and non-specific activated CD8(+) T cells to the site of infection, resulting in the formation of cellular clusters around and in the further elimination of intracellular parasites. The significance of this finding is discussed in the perspective of a general mechanism of antigen-dependent focalized inflammation and its consequences for the elimination of malaria liver stages.

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Anopheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.

Calcium is a key signalling molecule in apicomplexan parasites and plays an important role in diverse processes including gliding motility. Gliding is essential for the malaria parasite to migrate from the skin to the liver as well as to invade host tissues and cells. Here we investigated the dynamics of intracellular Ca(2+) in the motility of Plasmodium berghei sporozoites by live imaging and flow cytometry. We found that cytosolic levels of Ca(2+) increase when sporozoites are activated in suspension, which is sufficient to induce the secretion of integrin-like adhesins that are essential for gliding motility. By increasing intracellular Ca(2+) levels artificially with an ionophore, these adhesins are secreted onto the sporozoite surface, however, the parasite is not capable of gliding. A second level of Ca(2+) modulation was observed during attachment to and detachment from a solid substrate, leading to a further increase or a decrease in the cytoplasmic levels of Ca(2+) respectively. We also observed oscillations in the intracellular Ca(2+) level during gliding. Finally, an intracellular Ca(2+) chelator, an inhibitor of phosphoinositide-specific phospholipase C (PI-PLC), and an inhibitor of the inositol triphosphate (IP3) receptor blocked the rise in intracellular Ca(2+) , adhesin secretion, and motility of activated sporozoites, indicating that intracellular stores supply Ca(2+) during sporozoite gliding. Our study indicates that a rise in intracellular Ca(2+) is necessary but not sufficient to activate gliding, that Ca(2+) levels are modulated in several ways during motility, and that a PI-PLC/IP3 pathway regulates Ca(2+) release during the process of sporozoite locomotion., (© 2014 John Wiley & Sons Ltd.)

Plasmodium sporozoites are able to migrate through host cells by breaching their plasma membrane and gliding inside their cytoplasm. This migratory activity, called cell traversal (CT), was studied in vivo mainly using mutant sporozoites lacking the ability to wound host cells, and thus to perform CT. However, direct evidence of CT activity in host tissues by wild-type sporozoites remains scarce. Here, we describe a double-wounding assay to dynamically image CT activity in vivo and monitor cell membrane integrity over time. Based on the incorporation kinetics of a first live cell-impermeant dye, propidium iodide, we could determine whether traversed cells repair their wounded membranes or not. A second impermeant dye, SYTOX Green, was used to confirm the transient or the permanent loss of membrane integrity of traversed cells. This assay allowed, for the first time, the direct observation of sporozoites wounding and traversing host skin cells and showed that, while some traversed cells resealed their membrane, most became irreversibly permeable to these live cell-impermeant dyes. In combination with the study of CT-deficient sporozoites and the use of specific host cell markers, this intravital assay will provide the means to identify the nature of the cells traversed by sporozoites and will thus contribute to elucidating the role of CT by apicomplexan parasites in the vertebrate host., (© 2013.)