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Malaria presents a significant challenge to global public health, with around 247 million cases estimated to occur annually worldwide. The growing resistance of Plasmodium parasites to existing therapies underscores the urgent need for new and innovative antimalarial drugs. This study leveraged artificial intelligence (AI) to tackle this complex challenge. We developed multistage Machine Learning Quantitative Structure-Activity Relationship (ML-QSAR) models to effectively analyze large datasets and predict the efficacy of chemical compounds against multiple life cycle stages of Plasmodium parasites. We then selected 16 compounds for experimental evaluation, six of which showed at least dual-stage inhibitory activity and one inhibited all life cycle stages tested. Moreover, explainable AI (XAI) analysis provided insights into critical molecular features influencing model predictions, thereby enhancing our understanding of compound interactions. This study not only empowers the development of advanced predictive AI models but also accelerates the identification and optimization of potential antiplasmodial compounds., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Background: Malaria is an infectious disease caused by protozoan parasites belonging to the genus Plasmodium. Human-to-human transmission depends on a mosquito vector; thus, the interruption of parasite transmission from humans to mosquitoes is an important approach in the fight against malaria. The parasite stages infectious to mosquitoes are the gametocytes, sexual stages that are ingested by the vector during a blood meal and transform into male and female gametes in the midgut. Immunity against sexual stage antigens expressed by gametocytes, gametes, and the zygote formed after fertilisation can interrupt the parasite sexual cycle in the mosquito. This transmission blocking immunity is mediated by specific antibodies ingested during the mosquito blood feed, inhibiting the parasite development in the midgut. Merozoite thrombospondin related anonymous protein (MTRAP) is a merozoite and gametocyte surface protein essential for gamete egress from erythrocytes and for parasite transmission to mosquitoes., Objectives: Here, we evaluated the potential of the P. berghei MTRAP to elicit antibodies with the ability to inhibit gamete fertilisation in vitro., Methods: We expressed a soluble recombinant PbMTRAP and used it to immunise BALB/c mice. The transmission blocking activity of the anti-rPbMTRAP antibodies was tested through in vivo challenge experiments followed by in vitro conversion assays., Findings: Immunisations with the rPbMTRAP induced a strong antibody response and the antibodies recognised the native protein by Western Blot and IFA. Anti-rPbMTRAP present in the blood stream of immunised mice partially inhibited gamete conversion into ookinetes., Conclusion: Our results indicate that antibodies to PbMTRAP may reduce but are not sufficient to completely block transmission.

In tropical and subtropical areas, malaria stands as a profound public health challenge, causing an estimated 247 million cases worldwide annually. Given the absence of a viable vaccine, the timely and effective treatment of malaria remains a critical priority. However, the growing resistance of parasites to currently utilized drugs underscores the critical need for the identification of new antimalarial therapies. Here, we aimed to identify potential new drug candidates against Plasmodium falciparum , the main causative agent of malaria, by analyzing the transcriptomes of different life stages of the parasite and identifying highly expressed genes. We searched for genes that were expressed in all stages of the parasite's life cycle, including the asexual blood stage, gametocyte stage, liver stage, and sexual stages in the insect vector, using transcriptomics data from publicly available databases. From this analysis, we found 674 overlapping genes, including 409 essential ones. By searching through drug target databases, we discovered 70 potential drug targets and 75 associated bioactive compounds. We sought to expand this analysis to similar compounds to known drugs. So, we found a list of 1557 similar compounds, which we predicted as actives and inactives using previously developed machine learning models against five life stages of Plasmodium spp. From this analysis, two compounds were selected, and the reactions were experimentally evaluated. The compounds HSP-990 and silvestrol aglycone showed potent inhibitory activity at nanomolar concentrations against the P. falciparum 3D7 strain asexual blood stage. Moreover, silvestrol aglycone exhibited low cytotoxicity in mammalian cells, transmission-blocking potential, and inhibitory activity comparable to those of established antimalarials. These findings warrant further investigation of silvestrol aglycone as a potential dual-acting antimalarial and transmission-blocking candidate for malaria control., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Malaria is a global parasitic infection that leads to substantial illness and death. The most commonly-used drugs for treatment of malaria vivax are primaquine and chloroquine, but they have limitations, such as poor adherence due to frequent oral administration and gastrointestinal side effects. To overcome these limitations, we have developed nano-sized solid dispersion-based dissolving microarray patches (MAPs) for the intradermal delivery of these drugs. In vitro testing showed that these systems can deliver to skin and receiver compartment up to ≈60% of the payload for CQ-based dissolving MAPs and a total of ≈42% of drug loading for PQ-based dissolving MAPs. MAPs also displayed acceptable biocompatibility in cell tests. Pharmacokinetic studies in rats showed that dissolving MAPs could deliver sustained plasma levels of both PQ and CQ for over 7 days. Efficacy studies in a murine model for malaria showed that mice treated with PQ-MAPs and CQ-MAPs had reduced parasitaemia by up to 99.2%. This pharmaceutical approach may revolutionise malaria vivax treatment, especially in developing countries where the disease is endemic. The development of these dissolving MAPs may overcome issues associated with current pharmacotherapy and improve patient outcomes., Competing Interests: Declaration of Competing Interest The authors declare no competing interest., (Copyright © 2023. Published by Elsevier B.V.)

Safe and effective malaria transmission-blocking chemotherapeutics would allow a community-level approach to malaria control and eradication efforts by targeting the mosquito sexual stage of the parasite life cycle. However, only a single drug, primaquine, is currently approved for use in reducing transmission, and drug toxicity limits its widespread implementation. To address this limitation in antimalarial chemotherapeutics, we used a recently developed transgenic Plasmodium berghei line, Ookluc, to perform a series of high-throughput in vitro screens for compounds that inhibit parasite fertilization, the initial step of parasite development within the mosquito. Screens of antimalarial compounds, approved drug collections, and drug-like molecule libraries identified 185 compounds that inhibit parasite maturation to the zygote form. Seven compounds were further characterized to block gametocyte activation or to be cytotoxic to formed zygotes. These were further validated in mosquito membrane-feeding assays using Plasmodium falciparum and P. vivax. This work demonstrates that high-throughput screens using the Ookluc line can identify compounds that are active against the two most relevant human Plasmodium species and provides a list of compounds that can be explored for the development of new antimalarials to block transmission.

Adjuvants are essential for vaccine development, especially subunit-based vaccines such as those containing recombinant proteins. Increasing the knowledge of the immune response mechanisms generated by adjuvants should facilitate the formulation of vaccines in the future. The present work describes the immune phenotypes induced by Poly (I:C) and Montanide ISA 720 in the context of mice immunization with a recombinant protein based on the Plasmodium vivax  circumsporozoite protein (PvCSP) sequence. Mice immunized with the recombinant protein plus Montanide ISA 720 showed an overall more robust humoral response, inducing antibodies with greater avidity to the antigen. A general trend for mixed Th1/Th2 inflammatory cytokine profile was increased in Montanide-adjuvanted mice, while a balanced profile was observed in Poly (I:C)-adjuvanted mice. Montanide ISA 720 induced a gene signature in B lymphocytes characteristic of heme biosynthesis, suggesting increased differentiation to Plasma Cells. On the other hand, Poly (I:C) provoked more perturbations in T cell transcriptome. These results extend the understanding of the modulation of specific immune responses induced by different classes of adjuvants, and could support the optimization of subunit-based vaccines., 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 © 2022 Marques, de Melo, Novais, Soares, Bargieri and Gimenez.)

Competing Interests: We declare no competing interests.

Malaria is a serious public health problem that affects mostly the poorest countries in the world, killing more than 400,000 people per year, mainly children under 5 years old. Among the control and prevention strategies, the differential diagnosis of the Plasmodium -infecting species is an important factor for selecting a treatment and, consequently, for preventing the spread of the disease. One of the main difficulties for the detection of a specific Plasmodium sp is that most of the existing methods for malaria diagnosis focus on detecting P. falciparum . Thus, in many cases, the diagnostic methods neglect the other non-falciparum species and underestimate their prevalence and severity. Traditional methods for diagnosing malaria may present low specificity or sensitivity to non-falciparum spp. Therefore, there is high demand for new alternative methods able to differentiate Plasmodium species in a faster, cheaper and easier manner to execute. This review details the classical procedures and new perspectives of diagnostic methods for malaria non-falciparum differential detection and the possibilities of their application in different circumstances., 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 Gimenez, Marques, Regiart and Bargieri.)

Antimalarial drugs with novel modes of action and wide therapeutic potential are needed to pave the way for malaria eradication. Violacein is a natural compound known for its biological activity against cancer cells and several pathogens, including the malaria parasite, Plasmodium falciparum (Pf). Herein, using chemical genomic profiling (CGP), we found that violacein affects protein homeostasis. Mechanistically, violacein binds Pf chaperones, Pf Hsp90 and Pf Hsp70-1, compromising the latter's ATPase and chaperone activities. Additionally, violacein-treated parasites exhibited increased protein unfolding and proteasomal degradation. The uncoupling of the parasite stress response reflects the multistage growth inhibitory effect promoted by violacein. Despite evidence of proteotoxic stress, violacein did not inhibit global protein synthesis via UPR activation-a process that is highly dependent on chaperones, in agreement with the notion of a violacein-induced proteostasis collapse. Our data highlight the importance of a functioning chaperone-proteasome system for parasite development and differentiation. Thus, a violacein-like small molecule might provide a good scaffold for development of a novel probe for examining the molecular chaperone network and/or antiplasmodial drug design.

Increasing reports of multidrug-resistant malaria parasites urge the discovery of new effective drugs with different chemical scaffolds. Protein kinases play a key role in many cellular processes such as signal transduction and cell division, making them interesting targets in many diseases. Protein kinase 7 (PK7) is an orphan kinase from the Plasmodium genus, essential for the sporogonic cycle of these parasites. Here, we applied a robust and integrative artificial intelligence-assisted virtual-screening (VS) approach using shape-based and machine learning models to identify new potential PK7 inhibitors with in vitro antiplasmodial activity. Eight virtual hits were experimentally evaluated, and compound LabMol-167 inhibited ookinete conversion of Plasmodium berghei and blood stages of Plasmodium falciparum at nanomolar concentrations with low cytotoxicity in mammalian cells. As PK7 does not have an essential role in the Plasmodium blood stage and our virtual screening strategy aimed for both PK7 and blood-stage inhibition, we conducted an in silico target fishing approach and propose that this compound might also inhibit P. falciparum PK5, acting as a possible dual-target inhibitor. Finally, docking studies of LabMol-167 with P. falciparum PK7 and PK5 proteins highlighted key interactions for further hit-to lead optimization., (© 2020 Wiley-VCH GmbH.)

Drug resistance is a constant threat to malaria control efforts making it important to maintain a good pipeline of new drug candidates. Of particular need are compounds that also block transmission by targeting sexual stage parasites. Mature sexual stages are relatively resistant to all currently used antimalarials except the 8-aminoquinolines that are not commonly used due to potential side effects. Here, we synthesized a new Torin 2 derivative, NCATS-SM3710 with increased aqueous solubility and specificity for Plasmodium and demonstrate potent in vivo activity against all P. berghei life cycle stages. NCATS-SM3710 also has low nanomolar EC 50 s against in vitro cultured asexual P. falciparum parasites (0.38 ± 0.04 nM) and late stage gametocytes (5.77 ± 1 nM). Two independent NCATS-SM3710/Torin 2 resistant P. falciparum parasite lines produced by growth in sublethal Torin 2 concentrations both had genetic changes in PF3D7_0509800, annotated as a phosphatidylinositol 4 kinase ( Pf  PI4KIIIβ). One line had a point mutation in the putative active site (V1357G), and the other line had a duplication of a locus containing Pf  PI4KIIIβ. Both lines were also resistant to other Pf  PI4K inhibitors. In addition NCATS-SM3710 inhibited purified Pf  PI4KIIIβ with an IC 50 of 2.0 ± 0.30 nM. Together the results demonstrate that Pf  PI4KIIIβ is the target of Torin 2 and NCATS-SM3710 and provide new options for potent multistage drug development., Competing Interests: The authors declare no competing financial interest.

Widespread resistance against antimalarial drugs thwarts current efforts for controlling the disease and urges the discovery of new effective treatments. Drug repositioning is increasingly becoming an attractive strategy since it can reduce costs, risks, and time-to-market. Herein, we have used this strategy to identify novel antimalarial hits. We used a comparative in silico chemogenomics approach to select Plasmodium falciparum and Plasmodium vivax proteins as potential drug targets and analyzed them using a computer-assisted drug repositioning pipeline to identify approved drugs with potential antimalarial activity. Among the seven drugs identified as promising antimalarial candidates, the anthracycline epirubicin was selected for further experimental validation. Epirubicin was shown to be potent in vitro against sensitive and multidrug-resistant P. falciparum strains and P. vivax field isolates in the nanomolar range, as well as being effective against an in vivo murine model of Plasmodium yoelii Transmission-blocking activity was observed for epirubicin in vitro and in vivo Finally, using yeast-based haploinsufficiency chemical genomic profiling, we aimed to get insights into the mechanism of action of epirubicin. Beyond the target predicted in silico (a DNA gyrase in the apicoplast), functional assays suggested a GlcNac-1-P-transferase (GPT) enzyme as a potential target. Docking calculations predicted the binding mode of epirubicin with DNA gyrase and GPT proteins. Epirubicin is originally an antitumoral agent and presents associated toxicity. However, its antiplasmodial activity against not only P. falciparum but also P. vivax in different stages of the parasite life cycle supports the use of this drug as a scaffold for hit-to-lead optimization in malaria drug discovery., (Copyright © 2020 American Society for Microbiology.)

Two years ago, we held an exciting event entitled the São Paulo School of Advanced Sciences on Vaccines (SPSASV). Sixty-eight Ph.D. students, postdoctoral fellows and independent researchers from 37 different countries met at the Mendes Plaza Hotel located in the city of Santos, SP - Brazil to discuss the challenges and the new frontiers of vaccinology. The SPSASV provided a critical and comprehensive view of vaccine research from basics to the current state-of-the-art techniques performed worldwide. For 10 days, we discussed all the aspects of vaccine development in 36 lectures, 53 oral presentations and 2 poster sessions. At the end of the course, participants were further encouraged to present a model of a grant proposal related to vaccine development against individual pathogens. Among the targeted pathogens were viruses (Chikungunya, HIV, RSV, and Influenza), bacteria ( Mycobacterium tuberculosis and Streptococcus pyogenes ), parasites ( Plasmodium falciparum or Plasmodium vivax ), and the worm Strongyloides stercoralis . This report highlights some of the knowledge shared at the SPSASV., Competing Interests: Competing interests: The authors declare that they have no competing interests.

The lack of continuous in vitro cultures has been an obstacle delaying pre-clinical testing of Plasmodium vivax vaccine formulations based on known antigens. In this study, we generated a model to test available formulations based on the P. vivax MSP1 19 antigen. The Plasmodium berghei strains ANKA and NK65 were modified to express Pv MSP1 19 instead of the endogenous Pb MSP1 19 . The hybrid parasites were used to challenge C57BL/6 or BALB/c mice immunized with Pv MSP1 19 -based vaccine formulations. The Pv MSP1 19 was correctly expressed in the P. berghei hybrid mutant lines as confirmed by immunofluorescence using anti- Pv MSP1 19 monoclonal antibodies and by Western blot. Replacement of the Pb MSP1 19 by the Pv MSP1 19 had no impact on asexual growth in vivo . High titers of specific antibodies to Pv MSP1 19 were not sufficient to control initial parasitemia in the immunized mice, but late parasitemia control and a balanced inflammatory process protected these mice from dying, suggesting that an established immune response to Pv MSP1 19 in this model can help immunity mounted later during infection., (Copyright © 2020 Dobrescu, de Camargo, Gimenez, Murillo, Amorim, Marinho, Soares, Boscardin and Bargieri.)

Malaria is a tropical infectious disease that affects over 219 million people worldwide. Due to the constant emergence of parasitic resistance to the current antimalarial drugs, the discovery of new antimalarial drugs is a global health priority. Multi-target drug discovery is a promising and innovative strategy for drug discovery and it is currently regarded as one of the best strategies to face drug resistance. Aiming to identify new multi-target antimalarial drug candidates, we developed an integrative computational approach to select multi-kinase inhibitors for Plasmodium falciparum calcium-dependent protein kinases 1 and 4 (CDPK1 and CDPK4) and protein kinase 6 (PK6). For this purpose, we developed and validated shape-based and machine learning models to prioritize compounds for experimental evaluation. Then, we applied the best models for virtual screening of a large commercial database of drug-like molecules. Ten computational hits were experimentally evaluated against asexual blood stages of both sensitive and multi-drug resistant P. falciparum strains. Among them, LabMol-171, LabMol-172, and LabMol-181 showed potent antiplasmodial activity at nanomolar concentrations (EC 50 ≤ 700 nM) and selectivity indices >15 folds. In addition, LabMol-171 and LabMol-181 showed good in vitro inhibition of P. berghei ookinete formation and therefore represent promising transmission-blocking scaffolds. Finally, docking studies with protein kinases CDPK1, CDPK4, and PK6 showed structural insights for further hit-to-lead optimization studies., (Copyright © 2019 Lima, Cassiano, Tomaz, Silva, Sousa, Ferreira, Tavella, Calit, Bargieri, Neves, Costa and Andrade.)

Aim: Computer-aided drug design approaches were applied to identify chalcones with antiplasmodial activity. Methodology: The virtual screening was performed as follows: structural standardization of in-house database of chalcones; identification of potential Plasmodium falciparum protein targets for the chalcones; homology modeling of the predicted P. falciparum targets; molecular docking studies; and in vitro experimental validation. Results: Using these models, we prioritized 16 chalcones with potential antiplasmodial activity, for further experimental evaluation. Among them, LabMol-86 and LabMol-87 showed potent in vitro antiplasmodial activity against P. falciparum , while LabMol-63 and LabMol-73 were potent inhibitors of Plasmodium berghei progression into mosquito stages. Conclusion: Our results encourage the exploration of chalcones in hit-to-lead optimization studies for tackling malaria.

In recent work, Paton et al. (2019) report that mosquitoes exposed to atovaquone, a known antimalarial, are resistant to infection by the malaria parasite Plasmodium. Their results may be the foundation for the discovery of a new arsenal of antimalarials., (Published by Elsevier Inc.)

Malaria remains an important parasitic disease with a large morbidity and mortality burden. Plasmodium transmission-blocking (TB) compounds are essential for achieving malaria elimination efforts. Recent efforts to develop high-throughput screening (HTS) methods to identify compounds that inhibit or kill gametocytes, the Plasmodium sexual stage infectious to mosquitoes, have yielded insight into new TB compounds. However, the activities of these compounds against gametes, formed in the first minutes of mosquito infection, are typically not assessed, unless screened in a standard membrane feeding assay, a labor-intensive assay. We demonstrate here the generation of a Plasmodium model for drug screens against gametes and fertilization. The new P. berghei line, named Ookluc , was genetically and pharmacologically validated and scalable for HTS. Screening the Pathogen Box from the Medicines for Malaria Venture using the new model identified promising TB compounds. The use of Ookluc in different libraries of compounds may aid in the identification of transmission-blocking drugs not assessed in screens against asexual stages or gametocytes., (Copyright © 2018 American Society for Microbiology.)

Malaria is a life-threatening infectious disease caused by parasites of the genus Plasmodium , affecting more than 200 million people worldwide every year and leading to about a half million deaths. Malaria parasites of humans have evolved resistance to all current antimalarial drugs, urging for the discovery of new effective compounds. Given that the inhibition of deoxyuridine triphosphatase of Plasmodium falciparum ( Pf dUTPase) induces wrong insertions in plasmodial DNA and consequently leading the parasite to death, this enzyme is considered an attractive antimalarial drug target. Using a combi-QSAR (quantitative structure-activity relationship) approach followed by virtual screening and in vitro experimental evaluation, we report herein the discovery of novel chemical scaffolds with in vitro potency against asexual blood stages of both P. falciparum multidrug-resistant and sensitive strains and against sporogonic development of P. berghei . We developed 2D- and 3D-QSAR models using a series of nucleosides reported in the literature as Pf dUTPase inhibitors. The best models were combined in a consensus approach and used for virtual screening of the ChemBridge database, leading to the identification of five new virtual Pf dUTPase inhibitors. Further in vitro testing on P. falciparum multidrug-resistant (W2) and sensitive (3D7) parasites showed that compounds LabMol-144 and LabMol-146 demonstrated fair activity against both strains and presented good selectivity versus mammalian cells. In addition, LabMol-144 showed good in vitro inhibition of P. berghei ookinete formation, demonstrating that hit-to-lead optimization based on this compound may also lead to new antimalarials with transmission blocking activity.

Background: Technical limitations for culturing the human malaria parasite Plasmodium vivax have impaired the discovery of vaccine candidates, challenging the malaria eradication agenda. The immunogenicity of the M2 domain of the Merozoite Adhesive Erythrocytic Binding Protein (MAEBL) antigen cloned from the Plasmodium yoelii murine parasite, has been previously demonstrated., Results: Detailed epitope mapping of MAEBL through immunoinformatics identified several MHCI, MHCII and B cell epitopes throughout the peptide, with several of these lying in the M2 domain and being conserved between P. vivax, P. yoelii and Plasmodium falciparum, hinting that the M2-MAEBL is pan-reactive. This hypothesis was tested through functional assays, showing that P. yoelii M2-MAEBL antisera are able to recognize and inhibit erythrocyte invasion from both P. falciparum and P. vivax parasites isolated from Thai patients, in ex vivo assays. Moreover, the sequence of the M2-MAEBL is shown to be highly conserved between P. vivax isolates from the Amazon and Thailand, indicating that the MAEBL antigen may constitute a vaccine candidate outwitting strain-specific immunity., Conclusions: The MAEBL antigen is promising candidate towards the development of a malaria vaccine.

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.

Surface-associated TRAP (thrombospondin-related anonymous protein) family proteins are conserved across the phylum of apicomplexan parasites. TRAP proteins are thought to play an integral role in parasite motility and cell invasion by linking the extracellular environment with the parasite submembrane actomyosin motor. Blood stage forms of the malaria parasite Plasmodium express a TRAP family protein called merozoite-TRAP (MTRAP) that has been implicated in erythrocyte invasion. Using MTRAP-deficient mutants of the rodent-infecting P. berghei and human-infecting P. falciparum parasites, we show that MTRAP is dispensable for erythrocyte invasion. Instead, MTRAP is essential for gamete egress from erythrocytes, where it is necessary for the disruption of the gamete-containing parasitophorous vacuole membrane, and thus for parasite transmission to mosquitoes. This indicates that motor-binding TRAP family members function not just in parasite motility and cell invasion but also in membrane disruption and cell egress., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Malaria remains a world-threatening disease largely because of the lack of a long-lasting and fully effective vaccine. MAEBL is a type 1 transmembrane molecule with a chimeric cysteine-rich ectodomain homologous to regions of the Duffy binding-like erythrocyte binding protein and apical membrane antigen 1 (AMA1) antigens. Although MAEBL does not appear to be essential for the survival of blood-stage forms, ectodomains M1 and M2, homologous to AMA1, seem to be involved in parasite attachment to erythrocytes, especially M2. MAEBL is necessary for sporozoite infection of mosquito salivary glands and is expressed in liver stages. Here, the Plasmodium yoelii MAEBL-M2 domain was expressed in a prokaryotic vector. C57BL/6J mice were immunized with doses of P. yoelii recombinant protein rPyM2-MAEBL. High levels of antibodies, with balanced IgG1 and IgG2c subclasses, were achieved. rPyM2-MAEBL antisera were capable of recognizing the native antigen. Anti-MAEBL antibodies recognized different MAEBL fragments expressed in CHO cells, showing stronger IgM and IgG responses to the M2 domain and repeat region, respectively. After a challenge with P. yoelii YM (lethal strain)-infected erythrocytes (IE), up to 90% of the immunized animals survived and a reduction of parasitemia was observed. Moreover, splenocytes harvested from immunized animals proliferated in a dose-dependent manner in the presence of rPyM2-MAEBL. Protection was highly dependent on CD4(+), but not CD8(+), T cells toward Th1. rPyM2-MAEBL antisera were also able to significantly inhibit parasite development, as observed in ex vivo P. yoelii erythrocyte invasion assays. Collectively, these findings support the use of MAEBL as a vaccine candidate and open perspectives to understand the mechanisms involved in protection., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

A Plasmodium falciparum circumsporozoite protein (CSP)-based recombinant fusion vaccine is the first malaria vaccine to reach phase III clinical trials. Resistance to infection correlated with the production of antibodies to the immunodominant central repeat region of the CSP. In contrast to P. falciparum, vaccine development against the CSP of Plasmodium vivax malaria is far behind. Based on this gap in our knowledge, we generated a recombinant chimeric protein containing the immunodominant central repeat regions of the P. vivax CSP fused to Salmonella enterica serovar Typhimurium-derived flagellin (FliC) to activate the innate immune system. The recombinant proteins that were generated contained repeat regions derived from each of the 3 different allelic variants of the P. vivax CSP or a fusion of regions derived from each of the 3 allelic forms. Mice were subcutaneously immunized with the fusion proteins alone or in combination with the Toll-like receptor 3 (TLR-3) agonist poly(I·C), and the anti-CSP serum IgG response was measured. Immunization with a mixture of the 3 recombinant proteins, each containing immunodominant epitopes derived from a single allelic variant, rather than a single recombinant protein carrying a fusion of regions derived from each of 3 allelic forms elicited a stronger immune response. This response was independent of TLR-4 but required TLR-5/MyD88 activation. Antibody titers significantly increased when poly(I·C) was used as an adjuvant with a mixture of the 3 recombinant proteins. These recombinant fusion proteins are novel candidates for the development of an effective malaria vaccine against P. vivax.

Sporozoites, the stage of Plasmodium infectious to vertebrates when injected in the skin by a mosquito vector, are highly motile cells. Their unusual form of gliding motility is essential for infectivity, allowing the parasite to travel through both the mosquito and mammalian hosts, invading different cell types and escaping immune cell-mediated death. In this chapter, we describe techniques to study gliding motility of sporozoites in vitro and in vivo.

Apicomplexan parasites invade host cells by forming a ring-like junction with the cell surface and actively sliding through the junction inside an intracellular vacuole. Apical membrane antigen 1 is conserved in apicomplexans and a long-standing malaria vaccine candidate. It is considered to have multiple important roles during host cell penetration, primarily in structuring the junction by interacting with the rhoptry neck 2 protein and transducing the force generated by the parasite motor during internalization. Here, we generate Plasmodium sporozoites and merozoites and Toxoplasma tachyzoites lacking apical membrane antigen 1, and find that the latter two are impaired in host cell attachment but the three display normal host cell penetration through the junction. Therefore, apical membrane antigen 1, rather than an essential invasin, is a dispensable adhesin of apicomplexan zoites. These genetic data have implications on the use of apical membrane antigen 1 or the apical membrane antigen 1-rhoptry neck 2 interaction as targets of intervention strategies against malaria or other diseases caused by apicomplexans.

Transmission of Plasmodium species from a mammalian host to the mosquito vector requires the uptake, during an infected blood meal, of gametocytes, the precursor cells of the gametes. Relatively little is known about the molecular mechanisms involved in the developmental switch from asexual development to sexual differentiation or the maturation and survival of gametocytes. Here, we show that a gene coding for a novel putative transporter, NPT1, plays a crucial role in the development of Plasmodium berghei gametocytes. Parasites lacking NPT1 are severely compromised in the production of gametocytes and the rare gametocytes produced are unable to differentiate into fertile gametes. This is the earliest block in gametocytogenesis obtained by reverse genetics and the first to demonstrate the role of a protein with a putative transport function in sexual development. These results and the high degree of conservation of NPT1 in Plasmodium species suggest that this protein could be an attractive target for the development of novel drugs to block the spread of malaria., (© 2011 Blackwell Publishing Ltd.)

We describe here a highly efficient procedure for conditional mutagenesis in Plasmodium. The procedure uses the site-specific recombination FLP-FRT system of yeast and targets the pre-erythrocytic stages of the rodent Plasmodium parasite P. berghei, including the sporozoite stage and the subsequent liver stage. The technique consists of replacing the gene under study by an FRTed copy (i.e., flanked by FRT sites) in the erythrocytic stages of a parasite clone that expresses the flip (FLP) recombinase stage-specifically--called the 'deleter' clone. We present the available deleter clones, which express FLP at different times of the parasite life cycle, as well as the schemes and tools for constructing new deleter parasites. We also outline and discuss the various strategies for exchanging a wild-type gene with an FRTed copy and for generating conditional gene knockout or knockdown parasite clones. Finally, we detail the protocol for obtaining sporozoites that lack a protein of interest and for monitoring sporozoite-specific DNA excision and depletion of the target protein. The protocol should allow the functional analysis of any essential protein in the sporozoite, liver stage or hepatic merozoite stages of rodent Plasmodium parasites.

Recently, we described the improved immunogenicity of new malaria vaccine candidates based on the expression of fusion proteins containing immunodominant epitopes of merozoites and Salmonella enterica serovar Typhimurium flagellin (FliC) protein as an innate immune agonist. Here, we tested whether a similar strategy, based on an immunodominant B-cell epitope from malaria sporozoites, could also generate immunogenic fusion polypeptides. A recombinant His6-tagged FliC protein containing the C-terminal repeat regions of the VK210 variant of Plasmodium vivax circumsporozoite (CS) protein was constructed. This recombinant protein was successfully expressed in Escherichia coli as soluble protein and was purified by affinity to Ni-agarose beads followed by ion exchange chromatography. A monoclonal antibody specific for the CS protein of P. vivax sporozoites (VK210) was able to recognise the purified protein. C57BL/6 mice subcutaneously immunised with the recombinant fusion protein in the absence of any conventional adjuvant developed protein-specific systemic antibody responses. However, in mice genetically deficient in expression of TLR5, this immune response was extremely low. These results extend our previous observations concerning the immunogenicity of these recombinant fusion proteins and provide evidence that the main mechanism responsible for this immune activation involves interactions with TLR5, which has not previously been demonstrated for any recombinant FliC fusion protein.

In the past 25 years, the development of an effective malaria vaccine has become one of the biggest riddles in the biomedical sciences. Experimental data using animal infection models demonstrated that it is possible to induce protective immunity against different stages of malaria parasites. Nonetheless, the vast body of knowledge has generated disappointments when submitted to clinical conditions and presently a single antigen formulation has progressed to the point where it may be translated into a human vaccine. In parallel, new means to increase the protective effects of antigens in general have been pursued and depicted, such as the use of bacterial flagellins as carriers/adjuvants. Flagellins activate pathways in the innate immune system of both mice and humans. The recent report of the first Phase I clinical trial of a vaccine containing a Salmonella flagellin as carrier/adjuvant may fuel the use of these proteins in vaccine formulations. Herein, we review the studies on the use of recombinant flagellins as vaccine adjuvants with malarial antigens in the light of the current state of the art of malaria vaccine development. The available information indicates that bacterial flagellins should be seriously considered for malaria vaccine formulations to the development of effective human vaccines.

The Apical Membrane Antigen 1 (AMA-1) is considered a promising candidate for development of a malaria vaccine against asexual stages of Plasmodium. We recently identified domain II (DII) of Plasmodium vivax AMA-1 (PvAMA-1) as a highly immunogenic region recognised by IgG antibodies present in many individuals during patent infection with P. vivax. The present study was designed to evaluate the immunogenic properties of a bacterial recombinant protein containing PvAMA-1 DII. To accomplish this, the recombinant protein was administered to mice in the presence of each of the following six adjuvants: Complete/Incomplete Freund's Adjuvant (CFA/IFA), aluminium hydroxide (Alum), Quil A, QS21 saponin, CpG-ODN 1826 and TiterMax. We found that recombinant DII was highly immunogenic in BALB/c mice when administered in the presence of any of the tested adjuvants. Importantly, we show that DII-specific antibodies recognised the native AMA-1 protein expressed on the surface of P. vivax merozoites isolated from the blood of infected patients. These results demonstrate that a recombinant protein containing PvAMA-1 DII is immunogenic when administered in different adjuvant formulations, and indicate that this region of the AMA-1 protein should continue to be evaluated as part of a subunit vaccine against vivax malaria., ((c) 2010 Elsevier Ltd. All rights reserved.)

Background: Plasmodium falciparum and Plasmodium vivax are responsible for most of the global burden of malaria. Although the accentuated pathogenicity of P. falciparum occurs because of sequestration of the mature erythrocytic forms in the microvasculature, this phenomenon has not yet been noted in P. vivax. The increasing number of severe manifestations of P. vivax infections, similar to those observed for severe falciparum malaria, suggests that key pathogenic mechanisms (eg, cytoadherence) might be shared by the 2 parasites., Methods: Mature P. vivax-infected erythrocytes (Pv-iEs) were isolated from blood samples collected from 34 infected patients. Pv-iEs enriched on Percoll gradients were used in cytoadhesion assays with human lung endothelial cells, Saimiri brain endothelial cells, and placental cryosections., Results: Pv-iEs were able to cytoadhere under static and flow conditions to cells expressing endothelial receptors known to mediate the cytoadhesion of P. falciparum. Although Pv-iE cytoadhesion levels were 10-fold lower than those observed for P. falciparum-infected erythrocytes, the strength of the interaction was similar. Cytoadhesion of Pv-iEs was in part mediated by VIR proteins, encoded by P. vivax variant genes (vir), given that specific antisera inhibited the Pv-iE-endothelial cell interaction., Conclusions: These observations prompt a modification of the current paradigms of the pathogenesis of malaria and clear the way to investigate the pathophysiology of P. vivax infections.

In a recent study, we demonstrated the immunogenic properties of a new malaria vaccine polypeptide based on a 19 kDa C-terminal fragment of the merozoite surface protein-1 (MSP1(19)) from Plasmodium vivax and an innate immunity agonist, the Salmonella enterica serovar Typhimurium flagellin (FliC). Herein, we tested whether the same strategy, based on the MSP1(19) component of the deadly malaria parasite Plasmodium falciparum, could also generate a fusion polypeptide with enhanced immunogenicity. The His(6)FliC-MSP1(19) fusion protein was expressed from a recombinant Escherichia coli and showed preserved in vitro TLR5-binding activity. In contrast to animals injected with His(6)MSP1(19), mice subcutaneously immunised with the recombinant His(6)FliC-MSP1(19) developed strong MSP1(19)-specific systemic antibody responses with a prevailing IgG1 subclass. Incorporation of other adjuvants, such as CpG ODN 1826, complete and incomplete Freund's adjuvants or Quil-A, improved the IgG responses after the second, but not the third, immunising dose. It also resulted in a more balanced IgG subclass response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response, as determined by the detection of antigen-specific interferon-gamma secretion by immune spleen cells. MSP1(19)-specific antibodies recognised not only the recombinant protein, but also the native protein expressed on the surface of P. falciparum parasites. Finally, sera from rabbits immunised with the fusion protein alone inhibited the in vitro growth of three different P. falciparum strains. In summary, these results extend our previous observations and further demonstrate that fusion of the innate immunity agonist FliC to Plasmodium antigens is a promising alternative to improve their immunogenicity., ((c) 2010 Elsevier Ltd. All rights reserved.)

Salmonella flagellin, the flagellum structural subunit, has received particular interest as a vaccine adjuvant conferring enhanced immunogenity to soluble proteins or peptides, both for activation of antibody and cellular immune responses. In the present study, we evaluated the Salmonella enterica FliCd flagellin as a T cell vaccine adjuvant using as model the 9-mer (SYVPSAEQI) synthetic H2(d)-restricted CD8(+) T cell-specific epitope (CS(280-288)) derived from the Plasmodium yoelii circumsporozoite (CS) protein. The FliCd adjuvant effects were determined under two different conditions: (i) as recombinant flagella, expressed by orally delivered live S. Dublin vaccine strains expressing the target CS(280-288) peptide fused at the central hypervariable domain, and (ii) as purified protein in acellular vaccines in which flagellin was administered to mice either as a recombinant protein fused or admixed with the target CS(280-288) peptide. The results showed that CS(280-288)-specific cytotoxic CD8(+) T cells were primed when BALB/c mice were orally inoculated with the expressing the CS(280-288) epitope S. Dublin vaccine strain. In contrast, mice immunized with purified FliCd admixed with the CS(280-288) peptide and, to a lesser extent, fused with the target peptide developed specific cytotoxic CD8(+) T cell responses without the need of a heterologous booster immunization. The CD8(+) T cell adjuvant effects of flagellin, either fused or not with the target peptide, correlated with the in vivo activation of CD11c(+) dendritic cells. Taken together, the present results demonstrate that Salmonella flagellins are flexible adjuvant and induce adaptative immune responses when administered by different routes or vaccine formulations., ((c) 2009 Elsevier Ltd. All rights reserved.)

The present study evaluated the immunogenicity of new malaria vaccine formulations based on the 19kDa C-terminal fragment of Plasmodium vivax Merozoite Surface Protein-1 (MSP1(19)) and the Salmonella enterica serovar Typhimurium flagellin (FliC), a Toll-like receptor 5 (TLR5) agonist. FliC was used as an adjuvant either admixed or genetically linked to the P. vivax MSP1(19) and administered to C57BL/6 mice via parenteral (s.c.) or mucosal (i.n.) routes. The recombinant fusion protein preserved MSP1(19) epitopes recognized by sera collected from P. vivax infected humans and TLR5 agonist activity. Mice parenterally immunized with recombinant P. vivax MSP1(19) in the presence of FliC, either admixed or genetically linked, elicited strong and long-lasting MSP1(19)-specific systemic antibody responses with a prevailing IgG1 subclass response. Incorporation of another TLR agonist, CpG ODN 1826, resulted in a more balanced response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response measured by interferon-gamma secretion. Finally, we show that MSP1(19)-specific antibodies recognized the native protein expressed on the surface of P. vivax parasites harvested from infected humans. The present report proposes a new class of malaria vaccine formulation based on the use of malarial antigens and the innate immunity agonist FliC. It contains intrinsic adjuvant properties and enhanced ability to induce specific humoral and cellular immune responses when administered alone or in combination with other adjuvants.

The Apical Membrane Antigen-1 (AMA-1) of Plasmodium sp. has been suggested as a vaccine candidate against malaria. This protein seems to be involved in merozoite invasion and its extra-cellular portion contains three distinct domains: DI, DII, and DIII. Previously, we described that Plasmodium vivax AMA-1 (PvAMA-1) ectodomain is highly immunogenic in natural human infections. Here, we expressed each domain, separately or in combination (DI-II or DII-III), as bacterial recombinant proteins to map immunodominant epitopes within the PvAMA-1 ectodomain. IgG recognition was assessed by ELISA using sera of P. vivax-infected individuals collected from endemic regions of Brazil or antibodies raised in immunized mice. The frequencies of responders to recombinant proteins containing the DII were higher than the others and similar to the ones observed against the PvAMA-1 ectodomain. Moreover, ELISA inhibition assays using the PvAMA-1 ectodomain as substrate revealed the presence of many common epitopes within DI-II that are recognized by human immune antibodies. Finally, immunization of mice with the PvAMA-1 ectodomain induced high levels of antibodies predominantly to DI-II. Together, our results indicate that DII is particularly immunogenic during natural human infections, thus indicating that this region could be used as part of an experimental sub-unit vaccine to prevent vivax malaria.

Synthetic oligonucleotides (ODNs) containing immunostimulatory CpG motifs (CpG) are a new class of adjuvants suitable for the development of recombinant vaccines. Here we describe that endogenous interferon (IFN) was critical for the adjuvant activity of CpG ODN as genetically deficient mice developed significantly lower IgG antibody titers following immunization with recombinant proteins. In contrast, the absence of endogenous IL-12/IL-23 or IL-4 had little impact on the magnitude of the antibody response but instead caused a dramatic change in the pattern of IgG isotypes. The dependence on IFN-gamma was specific for CpG ODN and it was not observed with other adjuvants tested. IFN-gamma was produced by NK, dendritic cells, CD4+ and CD8+ T cells stimulated in vitro with CpG ODN. Adoptive transfer experiments confirmed that CD4+ or CD8+ T cells were in fact relevant sources of IFN-gamma in vivo. Following CpG ODN injection, splenic dendritic cells from IFN-gamma deficient mice did not up-regulate CD86 or CD40 expression, suggesting a role for these molecules. The importance of CD28 (CD86 ligand) was confirmed using CD28 deficient mice which presented severely impaired immune responses following CpG ODN-assisted immunization.

Recently, we generated two bacterial recombinant proteins expressing 89 amino acids of the C-terminal domain of the Plasmodium vivax merozoite surface protein-1 and the hexa-histidine tag (His6MSP1(19)). One of these recombinant proteins contained also the amino acid sequence of the universal pan allelic T-cell epitope (His6MSP1(19)-PADRE). In the present study, we evaluated the immunogenic properties of these antigens when administered via the intra-nasal route in the presence of distinct adjuvant formulations. We found that C57BL/6 mice immunized with either recombinant proteins in the presence of the adjuvants cholera toxin (CT) or the Escherichia coli heat labile toxin (LT) developed high and long lasting titers of specific serum antibodies. The induced immune responses reached maximum levels after three immunizing doses with a prevailing IgG1 subclass response. In contrast, mice immunized by intranasal route with His6MSP1(19)-PADRE in the presence of the synthetic oligonucleotides adjuvant CpG ODN 1826 developed lower antibody titers but when combined to CT, CpG addition resulted in enhanced IgG responses characterized by lower IgG1 levels. Considering the limitations of antigens formulations that can be used in humans, mucosal adjuvants can be a reliable alternative for the development of new strategies of immunization using recombinant proteins of P. vivax.

One of the most promising vaccine candidates against the erythrocytic forms of malaria is the 19 kDa C-terminal region of the merozoite surface protein 1 (MSP1(19)). As part of our studies aimed at the development of a Plasmodium vivax malaria vaccine, we characterized the immunogenic properties of a new bacterial recombinant protein containing the P. vivax MSP1(19) and two helper T-cell epitopes, the synthetic universal pan allelic DR epitope (PADRE) and a new internal MSP1 P. vivax epitope (DYDVVYLKPLAGMYK). We found that the recognition of His6MSP1(19)-DYDVVYLKPLAGMYK-PADRE was as good as the recognition of His6MSP1(19) indicating that the presence of the T-cell epitopes PADRE and DYDVVYLKPLAGMYK did not modify the MSP1(19) epitopes recognized by human IgG. The recombinant protein His6MSP1(19)-DYDVVYLKPLAGMYK-PADRE proved to be highly immunogenic in marmosets (Callithrix jacchus jacchus) when administered in incomplete Freund's adjuvant. However, when administered in other adjuvant formulations such as Quil A, CpG ODN 2006 or MPL/TDM, antibody titers to MSP1(19) were significantly lower. Among these three adjuvants, Quil A proved to be the most efficient one generating antibody titers significantly higher than the others. These results indicated that under the circumstances evaluated, adjuvants were key for the immunogenicity of the recombinant protein His6MSP1(19)-DYDVVYLKPLAGMYK-PADRE.