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Neutrophils and macrophages confine pathogens by entrapping them in extracellular traps (ETs) through activating TLR9 function. However, plasmodial parasites secreted TatD-like DNases (TatD) to counteract ETs-mediated immune clearance. We found that TLR9 mutant mice increased susceptibility to rodent malaria, suggesting TLR9 is a key protein for host defense. We found that the proportion of neutrophils and macrophages in response to plasmodial parasite infection in the TLR9 mutant mice was significantly reduced compared to that of the WT mice. Importantly, PbTatD can directly bind to the surface TLR9 (sTLR9) on macrophages, which blocking the phosphorylation of mitogen-activated protein kinase and nuclear factor-κB, negatively regulated the signaling of ETs formation by both macrophages and neutrophils. Such, P. berghei TatD is a parasite virulence factor that can inhibit the proliferation of macrophages and neutrophils through directly binding to TLR9 receptors on the cell surface, thereby blocking the activation of the downstream MyD88-NF-kB pathways., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Chinese1 is the predominant Toxoplasma gondii lineage in China, and significant phenotypic differences are observed within the lineage. WH3 and WH6 are two representative strains of Chinese 1, which exhibit divergent virulence and pathogenicity in mice. However, virulence determinants and their modulating mechanisms remain elusive. A global genome expression analysis of the WH3 and WH6 transcriptional profiles identified microneme secretory protein 6 (MIC6), which may be associated with the phenotypic difference observed in WH3. In the present study, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome-editing technique was used to generate a T. gondii microneme secretory protein (TgMIC6) knockout in WH3. Wild-type mice and different mouse and human cell lines were infected with the WH3, WH3-Δmic6, and WH6 strains. The survival rate of mice, related cytokine levels in serum, and the proliferation of parasites were observed. These results suggested that TgMIC6 is an important effector molecule that determines the differential virulence of WH3 in vivo and in vitro. Furthermore, MIC6 may enhance WH3 virulence via inhibition of host cell autophagy and activation of key molecules in the epidermal growth factor receptor (EGFR)-Akt-mammalian target of rapamycin (mTOR) classical autophagy pathway. CD40L was cleared in vivo by i.p injection of CD40L monoclonal antibody, and it was found that the virulence of WH3-Δmic6 to mice was restored to a certain extent in the absence of CD40L. This study elucidates the virulence determinants and immune escape strategies of Toxoplasma gondii in China. Moreover, these data will aid the development of effective strategies for the prevention and control of toxoplasmosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Toxoplasma gondii is an obligate intracellular parasite that can infect a variety of mammals including humans and causes toxoplasmosis. Unfortunately, a protective and safe vaccine against toxoplasmosis hasn't been developed yet. In this study, we developed a DNA vaccine encoding the SRS13 protein and immunized BALB/c mice thrice with pVAX1-SRS13 through the intramuscular route (IM) or intradermally using an electroporation device (ID + EP). The immunogenicity of pVAX1-SRS13 was analyzed by ELISA, Western blot, cytokine ELISA, and flow cytometry. The protective efficacy of the pVAX1-SRS13 was investigated by challenging mice orally with T. gondii PRU strain tissue cysts. The results revealed that pVAX1-SRS13 administered through IM or ID + EP routes induced high level of anti-SRS13 IgG antibody responses (P = 0.0037 and P < 0.0001). The IFN-γ level elicited by the pVAX1-SRS13 (ID + EP) was significantly higher compared to the control group (P = 0.00159). In mice administered with pVAX1-SRS13 (ID + EP), CD8 + cells secreting IFN-γ was significantly higher compared to pVAX1-SRS13 (IM) (P = 0.0035) and the control group (P = 0.0068). Mice vaccinated with the SRS13 DNA vaccine did not induce significant IL-4 level. Moreover, a significant reduction in the number of tissue cysts and the load of T. gondii DNA was detected in brains of mice administered with pVAX1-SRS13 through ID + EP and IM routes compared to controls. In conclusion, the SRS13 DNA vaccine was found to be highly immunogenic and confers strong protection against chronic toxoplasmosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Establishing an intact intracellular parasitophorous vacuole (PV) that enables efficient nutrient uptake and protein trafficking is essential for the survival and proliferation of Toxoplasma gondii. Although the PV membrane (PVM)-localized dense granule protein 17 (GRA17) and GRA23 mediate the permeability of the PVM to small molecules, including nutrient uptake and excretion of metabolic by-products, the molecular mechanism by which T. gondii acquires nutrients remains unclear. In this study, we showed that the secreted protein GRA47 contributed to normal PV morphology, PVM permeability to small molecules, growth, and virulence in T. gondii. Co-immunoprecipitation analysis demonstrated potential interaction of GRA47 with GRA72, and the loss of GRA72 affected PV morphology, parasite growth and infectivity. To investigate the biological relationship among GRA47, GRA72, GRA17 and GRA23, attempts were made to construct strains with double gene deletion and overexpressing strains. Only Δgra23Δgra72 was successfully constructed. This strain exhibited a significant increase in the proportion of aberrant PVs compared with the Δgra23 strain. Overexpressing one of the three related GRAs partially rescued PVs with aberrant morphology in Δgra47, Δgra72 and Δgra17, while the expression of the Plasmodium falciparum PVM protein PfExp2, an ortholog of GRA17 and GRA23, fully rescued the PV morphological defect in all three Δgra strains. These results suggest that these GRA proteins may not be functionally redundant but rather work in different ways to regulate nutrient acquisition. These findings highlight the versatility of the nutrient uptake mechanisms in T. gondii, which may contribute to the parasite's remarkable ability to grow in different cellular niches in a very broad range of hosts., (Copyright © 2024 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Myanmar aims to eliminate malaria by 2030. However, recent increase of malaria incidence is a great challenge to archive that goal. Increasing prevalence of Plasmodium vivax also hinders this endeavor. Monitoring genetic structure of the parasite is necessary to understand genetic nature and evolutionary aspect of P. vivax population in Myanmar. Partial fragment flanking blocks I and II of merozoite surface protein-3 alpha of P. vivax (pvmsp-3α) was amplified from P. vivax isolates collected in Pyin Oo Lwin, Mandalay Region, Myanmar in 2013-2015. Sequence analysis of pvmsp-3α was performed to determine genetic diversity and natural selection of this gene. Spatio-temporal genetic changes of pvmsp-3α in Myanmar P. vivax population were also investigated via comparative analysis of gene sequences obtained in this study and previously reported Myanmar pvmsp-3α sequences. Genetic diversity of Myanmar pvmsp-3α was detected in P. vivax isolates analyzed. Size polymorphisms in block I and amino acid changes and recombination events in block II were main factors contributing to the genetic diversity of pvmsp-3α. Comparative spatio-temporal analysis with previously reported Myanmar pvmsp-3α populations revealed the presence of genetic differences by population with moderate genetic differentiation between populations. Similar pattern of natural selection was also detected in Myanmar pvmsp-3α populations. These suggested that enough size of the P. vivax population sufficient to generate or maintain the genetic diversity remains in the population. Thus, continuous molecular surveillance of genetic structure of Myanmar P. vivax is necessary., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Malaria remains one of the most important infectious diseases in the world, with the greatest burden in sub-Saharan Africa, primarily from Plasmodium falciparum infection. The treatment and control of malaria is challenged by resistance to most available drugs, but partial resistance to artemisinins (ART-R), the most important class for the treatment of malaria, was until recently confined to southeast Asia. This situation has changed, with the emergence of ART-R in multiple countries in eastern Africa. ART-R is mediated primarily by single point mutations in the P falciparum kelch13 protein, with several mutations present in African parasites that are now validated resistance mediators based on clinical and laboratory criteria. Major priorities at present are the expansion of genomic surveillance for ART-R mutations across the continent, more frequent testing of the efficacies of artemisinin-based regimens against uncomplicated and severe malaria in trials, more regular assessment of ex-vivo antimalarial drug susceptibilities, consideration of changes in treatment policy to deter the spread of ART-R, and accelerated development of new antimalarial regimens to overcome the impacts of ART-R. The emergence of ART-R in Africa is an urgent concern, and it is essential that we increase efforts to characterise its spread and mitigate its impact., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2024 World Health Organization. Published by Elsevier Ltd.. All rights reserved.)

In malaria parasites, the erythrocyte binding-like proteins (EBL) are a family of invasion proteins that are attractive vaccine targets. In the case of Plasmodium vivax, the widespread malaria parasite, blood-stage vaccines have been largely focused on a single EBL candidate, the Duffy binding-like domain (DBL) of the Duffy binding protein (DBPII), due to its well-characterized role in the reticulocyte invasion. A novel P. vivax EBL family member, the Erythrocyte binding protein (EBP2, also named EBP or DBP2), binds preferentially to reticulocytes and may mediate an alternative P. vivax invasion pathway. To gain insight into the natural genetic diversity of the DBL domain of EBP2 (region II; EBP2-II), we analyzed ebp2-II gene sequences of 71 P. vivax isolates collected in different endemic settings of the Brazilian Amazon rainforest, where P. vivax is the predominant malaria-associated species. Although most of the substitutions in the ebp2-II gene were non-synonymous and suggested positive selection, the results showed that the DBL domain of the EBP2 was much less polymorphic than that of DBPII. The predominant EBP2 haplotype in the Amazon region corresponded to the C127 reference sequence first described in Cambodia (25% C127-like haplotype). An overview of ebp2-II gene sequences available at GenBank (n = 352) from seven countries (Cambodia, Madagascar, Myanmar, PNG, South Korea, Thailand, Vietnam) confirmed the C127-like haplotype as highly prevalent worldwide. Two out of 43 haplotypes (5 to 20 inferred per country) showed a global frequency of 60%. The results presented here open new avenues of research pursuit while suggesting that a vaccine based on the DBL domain of EBP2 should target a few haplotypes for broad coverage., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Toxoplasmosis is a common parasitic zoonosis that can be life-threatening in immunocompromised patients. About one-third of the human population is infected with Toxoplasma gondii. Primary infection triggers an innate immune response wherein IFN-γ-induced host cell GTPases, namely IRG and GBP proteins, serve as a vital component for host cell resistance. In the past decades, interest in elucidating the function of these GTPase families in controlling various intracellular pathogens has emerged. Numerous T. gondii effectors were identified to inactivate particular IRG proteins. T. gondii is re-optimizing its effectors to combat IRG function and in this way secures transmission. We discuss the IRG-specific effectors employed by the parasite in murine infections, contributing to a better understanding of T. gondii virulence., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Transmission-blocking vaccines interrupting malaria transmission within mosquitoes represent an ideal public health tool to eliminate malaria at the population level. Plasmodium falciparum and P. vivax account for more than 90% of the global malaria burden, co-endemic in many regions of the world. P25 and P48/45 are two leading candidates for both species and have shown promising transmission-blocking activity in preclinical and clinical studies. However, neither of these target antigens as individual vaccines has induced complete transmission inhibition in mosquitoes. In this study, we assessed immunogenicity of combination vaccines based on P25 and P48/45 using a DNA vaccine platform to broaden vaccine specificity against P. falciparum and P. vivax. Individual DNA vaccines encoding Pvs25, Pfs25, Pvs48/45 and Pfs48/45, as well as various combinations including (Pvs25 + Pvs48/45), (Pfs25 + Pfs48/45), (Pvs25 + Pfs25), and (Pvs48/45 + Pfs48/45), were evaluated in mice using in vivo electroporation. Potent antibody responses were induced in mice immunized with individual and combination DNA vaccines, and specific antibody responses were not compromised when combinations of DNA vaccines were evaluated against individual DNA vaccines. The anti-Pvs25 IgG from individual and combination groups revealed concentration-dependent transmission-reducing activity (TRA) in direct membrane feeding assays (DMFA) using blood from P. vivax-infected donors in Brazil and independently in ex vivo MFA using Pvs25-transgenic P. berghei. Similarly, anti-Pfs25 and anti-Pfs48/45 IgGs from mice immunized with Pfs25 and Pfs48/45 DNA vaccines individually and in various combinations revealed antibody dose-dependent TRA in standard membrane feeding assays (SMFA) using culture-derived P. falciparum gametocytes. However, antibodies induced by immunization with Pvs48/45 DNA vaccines were ineffective in DMFA and require further vaccine construct optimization, considering the possibility of induction of both transmission-blocking and transmission-enhancing antibodies revealed by competition ELISA. These studies provide a rationale for combining multiple antigens to simultaneously target transmission of malaria caused by P. falciparum and P. vivax., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

LeishGEM is a genome-wide functional annotation community resource for Leishmania mexicana, where deletion mutant growth in vitro and in vivo is measured and protein localisation is determined by endogenous tagging and LOPIT-DC (localisation of organelle proteins by isotope tagging with differential centrifugation) spatial proteomics. Data are being made available pre-publication via http://leishgem.org which allows data-driven identification of the mechanisms for Leishmania parasitism., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Cryptosporidiosis is an infectious enteric disease caused by species (some of them zoonotic) of the genus Cryptosporidium that in many countries are under surveillance. Typing assays critical to the surveillance of cryptosporidiosis typically involve characterization of Cryptosporidium glycoprotein 60 genes (gp60). Here, we characterized the gp60 of Cryptosporidium suis from two samples-a human and a porcine faecal sample-based on which a preliminary typing scheme was developed. A conspicuous feature of the C. suis gp60 was a novel type of tandem repeats located in the 5' end of the gene and that took up 777/1635 bp (48%) of the gene. The C. suis gp60 lacked the classical poly-serine repeats (TCA/TCG/TCT), which is usually subject to major genetic variation, and the length of the tandem repeat made a typing assay incorporating this region based on Sanger sequencing practically unfeasible. We therefore designed a typing assay based on the post-repeat region only and applied it to C. suis-positive samples from suid hosts from Norway, Denmark, and Spain. We were able to distinguish three different subtypes; XXVa-1, XXVa-2, and XXVa-3. Subtype XXVa-1 had a wider geographic distribution than the other subtypes and was also observed in the human sample. We think that the present data will inform future strategies to develop a C. suis typing assay that could be even more informative by including a greater part of the gene, including the tandem repeat region, e.g., by the use of long-read next-generation sequencing., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

The protozoan parasite Plasmodium falciparum, responsible for the deadliest form of human malaria, employs antigenic variation via monoallelic expression as a key survival strategy. The selective activation of one out of the 60-member var gene family is key to understanding the parasite's ability to cause severe disease and evade the host immune response. var gene activation is initiated by its relocation to a specialized expression site. While the perinuclear expression site (PES) plays a crucial role in enabling the expression of a single allele, the characteristics of this PES remain largely obscure. Recent breakthroughs in genome editing tools and the discovery of regulatory noncoding RNAs have shed light on this intriguing biological feature, offering significant insights into the mechanisms of pathogen virulence., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Plasmodium vivax Merozoite Surface Protein 8 (PvMSP8) is a promising candidate target for the development of multi-component vaccines. Therefore, determining the genetic variation pattern of Pvmsp8 is essential in providing a reference for the rational design of the P. vivax malaria vaccines. This study delves into the genetic characteristics of the Pvmsp8 gene, specifically focusing on samples from the China-Myanmar border (CMB) region, and contrasts these findings with broader global patterns. The study uncovers that Pvmsp8 exhibits a notable level of conservation across different populations, with limited polymorphisms and relatively low nucleotide diversity (0.00023-0.00120). This conservation contrasts starkly with the high polymorphisms found in other P. vivax antigens such as Pvmsp1. A total of 25 haplotypes and 14 amino acid mutation sites were identified in the global populations, and all mutation sites were confined to non-functional regions. The study also notes that most CMB Pvmsp8 haplotypes are shared among Burmese, Cambodian, Thai, and Vietnamese populations, indicating less geographical variance, but differ notably from those found in Pacific island regions or the Panama. The findings underscore the importance of considering regional genetic diversity in P. vivax when developing targeted malaria vaccines. Non departure from neutral evolution were found by Tajima's D test, however, statistically significant differences were observed between the kn/ks rates. The study's findings are crucial in understanding the evolution and population structure of the Pvmsp8 gene, particularly during regional malaria elimination efforts. The highly conserved nature of Pvmsp8, combined with the lack of mutations in its functional domain, presents it as a promising candidate for developing a broad and effective P. vivax vaccine. This research thus lays a foundation for the rational development of multivalent malaria vaccines targeting this genetically stable antigen., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Gamete development is a precisely programmed process in Cryptosporidium parvum, a leading cause of diarrheal disease worldwide. Nava et al. recently described the developmentally regulated expression of CDPK5 during male gametogenesis. Here we discuss their main findings, posing this protein kinase as a promising target for antiparasitic interventions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Recent data indicate increasing disease burden and importance of Plasmodium vivax (Pv) malaria. A robust assay will be essential for blood-stage Pv vaccine development. Results of the in vitro growth inhibition assay (GIA) with transgenic P. knowlesi (Pk) parasites expressing the Pv Duffy-binding protein region II (PvDBPII) correlate with in vivo protection in the first PvDBPII controlled human malaria infection (CHMI) trials, making the PkGIA an ideal selection tool once the precision of the assay is defined. To determine the precision in percentage of inhibition in GIA (%GIA) and in GIA 50 (antibody concentration that gave 50 %GIA), ten GIAs with transgenic Pk parasites were conducted with four different anti-PvDBPII human monoclonal antibodies (mAbs) at concentrations of 0.016 to 2 mg/mL, and three GIAs with eighty anti-PvDBPII human polyclonal antibodies (pAbs) at 10 mg/mL. A significant assay-to-assay variation was observed, and the analysis revealed a standard deviation (SD) of 13.1 in the mAb and 5.94 in the pAb dataset for %GIA, with a LogGIA 50 SD of 0.299 (for mAbs). Moreover, the ninety-five percent confidence interval (95 %CI) for %GIA or GIA 50 in repeat assays was calculated in this investigation. The error range determined in this study will help researchers to compare PkGIA results from different assays and studies appropriately, thus supporting the development of future blood-stage malaria vaccine candidates, specifically second-generation PvDBPII-based formulations., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Chetan E. Chitnis is an inventor on patents that relate to binding domains of erythrocyte-binding proteins of Plasmodium parasites including PvDBP (patent no. 6962987; binding domains from Plasmodium vivax and Plasmodium falciparum erythrocyte binding proteins)., (Copyright © 2024. Published by Elsevier Ltd.)

Microtubules (MTs) play a vital role as key components of the eukaryotic cytoskeleton. The phylum Apicomplexa comprises eukaryotic unicellular parasitic organisms defined by the presence of an apical complex which consists of specialized secretory organelles and tubulin-based cytoskeletal elements. One apicomplexan parasite, Toxoplasma gondii, is an omnipresent opportunistic pathogen with significant medical and veterinary implications. To ensure successful infection and widespread dissemination, T. gondii heavily relies on the tubulin structures present in the apical complex. Recent advances in high-resolution imaging, coupled with reverse genetics, have offered deeper insights into the composition, functionality, and dynamics of these tubulin-based structures. The apicomplexan tubulins differ from those of their mammalian hosts, endowing them with unique attributes and susceptibility to specific classes of inhibitory compounds., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Significant variations in the abundance of mitochondrial RNA processing proteins and their target RNAs across trypanosome life stages present an opportunity to explore the regulatory mechanisms that drive these changes. Utilizing omics approaches can uncover unconventional targets, aiding our understanding of the parasites' adaptation and enabling targeted interventions for differentiation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The micropore, a mysterious structure found in apicomplexan species, was recently shown to be essential for nutrient acquisition in Plasmodium falciparum and Toxoplasma gondii. However, the differences between the micropores of these two parasites questions the nature of a general apicomplexan micropore structure and whether the formation process model from Plasmodium can be applied to other apicomplexans. We analyzed the literature on different apicomplexan micropores and found that T. gondii probably harbors a more representative micropore type than the more widely studied ones in Plasmodium. Using recent knowledge of the Kelch 13 (K13) protein interactome and gene depletion phenotypes in the T. gondii micropore, we propose a model of micropore formation, thus enriching our wider understanding of micropore protein function., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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.)

Plasmodium vivax malaria is increasingly recognized as a major global health problem and the socio-economic impact of P.vivax-induced burden is huge. Vaccine development against P. vivax malaria has been hampered by the lack of an in vitro culture system and poor access to P. vivax sporozoites. The recent generation of Plasmodium falciparum parasites that express a functional P. vivax AMA1 molecule has provided a platform for in vitro evaluation of PvAMA1 as a potential blood stage vaccine. Three so-called PvAMA1 Diversity Covering (DiCo) proteins were designed to assess their potential to induce a functional and broad humoral immune response to the polymorphic PvAMA1 molecule. Rabbits were immunized with the mixture of three, Pichia-produced, PvAMA1 DiCo proteins, as well as with 2 naturally occurring PvAMA1 alleles. For these three groups, the experimental adjuvant raffinose fatty acid sulfate ester (RFASE) was used, while in a fourth group the purified main mono-esterified constituent (RSL10) of this adjuvant was used. Animals immunized with the mixture of the three PvAMA1 DiCo proteins in RFASE showed high anti-PvAMA1 antibody titers against three naturally occurring PvAMA1variants while also high growth-inhibitory capacity was observed against P. falciparum parasites expressing PvAMA1. This supports further clinical development of the PvAMA1 DiCo mixture as a potential malaria vaccine. However, as the single allele PvAMA1 SalI-group showed similar characteristics in antibody titer and inhibition levels as the PvAMA1 DiCo mixture-group, this raises the question whether a mixture is really necessary to overcome the polymorphism in the vaccine candidate. RFASE induced strong humoral responses, as did the animals immunized with the purified component, RSL10. This suggests that RSL10 is the active ingredient. However, one of the RSL10-immunized animal showed a delayed response, necessitating further research into the clinical development of RSL10., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Toxoplasma gondii (T. gondii) is one of the most common pathogenic protozoa in the world, and causes toxoplasmosis, which in varying degrees causes significant economic losses and poses a serious public health challenge globally. To date, the development of an effective vaccine for human toxoplasmosis remains a challenge. Given that T.gondii calcium-dependent protein kinase 3 (CDPK3), dense granule protein 35 (GRA35) and rhoptry organelle protein 46 (ROP46) play key roles during Toxoplasma gondii invasion of host cells, we developed a protein vaccine cocktail including these proteins and validated its protective efficacy. The specific protective effects of vaccine on mice were analyzed by measuring serum antibodies, cytokines, splenocyte proliferation, the percentage of CD 4+ and CD 8+ T-lymphocytes, survival rate, and parasite cyst burden. The results showed that mice vaccinated with a three-protein cocktail produced the highest levels of immune protein antibodies to IgG, and high levels of IFN-γ, IL-2, IL-4, and IL-10 compared to other mice vaccinated with two proteins. In addition, CD 4+ and CD 8+ T cell percentages were significantly elevated. Compared to the control groups, mice vaccinated with the three-protein cocktail survived significantly longer after acute infection with T. gondii and had significantly fewer cysts after chronic infection. These results demonstrated that a cocktail vaccine of TgCDPK3, TgGRA35, and TgROP46 can effectively induce cellular and humoral immune responses with good protective effects in mice, indicating its potential as vaccine candidates for toxoplasmosis., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ran An, Jian Du reports financial support was provided by National Natural Science Foundation of China., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Pathogenicity of the zoonotic pathogen Toxoplasma gondii largely depends on the secretion of effector proteins into the extracellular milieu and host cell cytosol, including the dense granule proteins (GRAs). The protein-encoding gene TGME49&#95;299780 was previously identified as a contributor to parasite fitness. However, its involvement in parasite growth, virulence and infectivity in vitro and in vivo remains unknown. Here, we comprehensively examined the role of this new protein, termed GRA76, in parasite pathogenicity. Subcellular localization revealed high expression of GRA76 in tachyzoites inside the parasitophorous vacuole (PV). However, its expression was significantly decreased in bradyzoites. A CRISPR-Cas9 approach was used to knock out the gra76 gene in the T. gondii type I RH strain and type II Pru strain. The in vitro plaque assays and intracellular replication showed the involvement of GRA76 in replication of RH and Pru strains. Deletion of the gra76 gene significantly decreased parasite virulence, and reduced the brain cyst burden in mice. Using RNA sequencing, we detected a significant increase in the expression of bradyzoite-associated genes such as BAG1 and LDH2 in the PruΔgra76 strain compared with the wild-type Pru strain. Using an in vitro bradyzoite differentiation assay, we showed that loss of GRA76 significantly increased the propensity for parasites to form bradyzoites. Immunization with PruΔgra76 conferred partial protection against acute and chronic infection in mice. These findings show the important role of GRA76 in the pathogenesis of T. gondii and highlight the potential of PruΔgra76 as a candidate for a live-attenuated vaccine., (Copyright © 2023 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Background: Plasmodium falciparum is an apicomplexan parasite responsible for lethal cases of malaria. According to WHO recommendations, P falciparum cases are treated with artemisinin-based combination therapy including dihydroartemisinin-piperaquine. However, the emergence of resistant parasites against dihydroartemisinin-piperaquine was reported in southeast Asia in 2008 and, a few years later, suspected in South America., Methods: To characterise resistance emergence, a treatment efficacy study was performed on the reported patients infected with P falciparum and treated with dihydroartemisinin-piperaquine in French Guiana (n=6, 2016-18). Contemporary isolates collected in French Guiana were genotyped for P falciparum chloroquine resistance transporter (pfCRT; n=845) and pfpm2 and pfpm3 copy number (n=231), phenotyped using the in vitro piperaquine survival assay (n=86), and analysed through genomic studies (n=50). Additional samples from five Amazonian countries and one outside the region were genotyped (n=1440)., Findings: In field isolates, 40 (47%) of 86 (95% CI 35·9-57·1) were resistant to piperaquine in vitro; these phenotypes were more associated with pfCRT C350R (ie, Cys350Arg) and pfpm2 and pfpm3 amplifications (Dunn test, p<0·001). Those markers were also associated with dihydroartemisinin-piperaquine treatment failure (n=3 [50%] of 6). A high prevalence of piperaquine resistance markers was observed in Suriname in 19 (83%) of 35 isolates and in Guyana in 579 (73%) of 791 isolates. The pfCRT C350R mutation emerged before pfpm2 and pfpm3 amplification in a temporal sequence different from southeast Asia, and in the absence of artemisinin partial resistance, suggesting a geographically distinctive epistatic relationship between these genetic markers., Interpretation: The high prevalence of piperaquine resistance markers in parasite populations of the Guianas, and the risk of associated therapeutic failures calls for caution on dihydroartemisinin-piperaquine use in the region. Furthermore, greater attention should be given to potential differences in genotype to phenotype mapping across genetically distinct parasite populations from different continents., Funding: Pan American Health Organization and WHO, French Ministry for Research, European Commission, Santé publique France, Agence Nationale de la Recherche, Fundação de Amparo à Pesquisa do Estado do Amazonas, Ministry of Health of Brazil, Oswaldo Cruz Foundation, and National Institutes of Health., Translations: For the French and Portuguese translations of the abstract see Supplementary Materials section., Competing Interests: Declaration of interests M-PA and PR are staff members of WHO. The authors alone are responsible for the views expressed in this publication, which do not necessarily represent the decisions, policies, or views of WHO. All other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Mitochondrial uridine insertion/deletion RNA editing, catalyzed by a multiprotein complex (editosome), is essential for gene expression in trypanosomes and Leishmania parasites. As this process is absent in the human host, a drug targeting this mechanism promises high selectivity and reduced toxicity. Here, we successfully miniaturized our FRET-based full-round RNA editing assay, which replicates the complete RNA editing process, adapting it into a 1536-well format. Leveraging this assay, we screened over 100,000 compounds against purified editosomes derived from Trypanosoma brucei, identifying seven confirmed primary hits. We sourced and evaluated various analogs to enhance the inhibitory and parasiticidal effects of these primary hits. In combination with secondary assays, our compounds marked inhibition of essential catalytic activities, including the RNA editing ligase and interactions of editosome proteins. Although the primary hits did not exhibit any growth inhibitory effect on parasites, we describe eight analog compounds capable of effectively killing T. brucei and/or Leishmania donovani parasites within a low micromolar concentration. Whether parasite killing is - at least in part - due to inhibition of RNA editing in vivo remains to be assessed. Our findings introduce novel molecular scaffolds with the potential for broad antitrypanosomal effects., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Emerging resistance against artemisinin (ART) poses a major challenge in controlling malaria. Parasites with mutations in PfKelch13, the major marker for ART resistance, are known to reduce hemoglobin endocytosis, induce unfolded protein response (UPR), elevate phosphatidylinositol-3-phosphate (PI3P) levels, and stimulate autophagy. Nonetheless, PfKelch13-independent resistance is also reported, indicating extensive complementation by reconfiguration in the parasite metabolome and transcriptome. These findings implicate that there may not be a single 'universal identifier' of ART resistance. This review sheds light on the molecular, transcriptional, and metabolic pathways associated with ART resistance, while also highlighting the interplay between cellular heterogeneity, environmental stress, and ART sensitivity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The ability to accurately measure the intensity of malaria transmission in areas with low transmission is extremely important to guide elimination efforts. Plasmodium falciparum Cell-traversal protein for ookinetes and sporozoites (PfCelTOS) is an important conserved sporozoite antigen reported as one of the promising malaria vaccine candidates, and could be used to estimate malaria transmission intensity. This study aimed at determining whether the diversity of PfCelTOS gene reflects the changes in malaria transmission that occurred between 2007 and 2014 in Dielmo, a Senegalese village, before and after the implementation of insecticide treated bed nets (ITNs). Of the 109 samples positive for PfCelTOS PCR, 96 (88%) were successfully sequenced and analysed for polymorphisms and population diversity. The number of segregating sites was higher during the pre-intervention period (13) and the malaria resurgences (11) than during the intervention period (5). Similarly, the number and diversity of haplotypes were higher during the pre-intervention period (16 and 0.914, respectively) and the malaria resurgences (6 and 0.821, respectively) than during the intervention period (4 and 0.758, respectively). Moreover, the average number of nucleotide differences was higher during the pre-intervention (3.792) and during malaria resurgences (3.467) than during the intervention period (2.189). The 3D7 KSSFNEP haplotype was only observed during the intervention period. Only two haplotypes were shared in both the pre-intervention and intervention periods while four haplotypes were shared between the pre-intervention and the malaria resurgences. The Fst values indicate moderate differentiation between pre-intervention and intervention periods (0.17433), and between intervention and malaria resurgences period (0.19198) as well as between pre-intervention and malaria resurgences periods (0.06607). PfCelTOS genetic diversity reflected changes of malaria transmission, with higher polymorphisms recorded before the large-scale implementation of ITNs and during the malaria resurgences. PfCelTOS is also a candidate vaccine; mapping its diversity across multiple endemic environments will facilitate the design and optimisation of a broad and efficacious vaccine., Competing Interests: Declaration of Competing Interest The author(s) declare that they have no competing interests., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Malaria affects ∼ ¼ billion people globally and requires the development of additional tools to aid in elimination efforts. The recently approved RTS,S/AS01 vaccine represents a positive step, however, the moderate efficacy necessitates the development of more efficacious vaccines. PfCSP is a key target antigen for pre-erythrocytic vaccines aimed at preventing Plasmodium falciparum malaria infections. Epitopes within the central repeat region and at the junction of the repeat and N-terminal domain are well documented as major protective B cell epitopes. On the other hand, a majority of antibodies against the epitopes in the C-terminal domain, have been shown to be non-protective against sporozoite challenge. The C-terminal domain, however, contains CD4 +  and CD8 +  T cell epitopes previously shown to be important for regulating immune responses. The present study was designed to further explore the immunomodulatory potential of the C-terminal domain using DNA vaccines encoding PfCSP with sequential C-terminal truncations following known T cell epitopes. Five DNA vaccines encoding different truncations of PfCSP within the C-terminal domain were administered via intramuscular route and in vivo electroporation for effective immunogenicity. Protection in mice was evaluated by challenge with transgenic P. berghei expressing PfCSP. In Balb/c mice, antibody responses and protective efficacy were both affected progressively with sequential deletion of C-terminal amino acid residues. Similar studies in C57Bl/6 mice revealed that immunizations with plasmids encoding truncated PfCSP showed partial protection from sporozoite challenge with no significant differences in antibody titers observed compared to full-length PfCSP DNA immunized mice. Further analysis revealed murine strain-specific differences in the recognition of specific epitopes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Plasmodium knowlesi is the leading cause of malaria in Malaysia. Serine Repeat Antigens (SERAs) have an essential role in the parasite life cycle. However, genetic characterization on P. knowlesi SERA3 Ag2 (PkSERA3 Ag2) is lacking. In the present study, nucleotide diversity, natural selection, and haplotypes of PkSERA3 Ag2 in clinical samples from Peninsular Malaysia and Malaysian Borneo were investigated. A total of 50 P. knowlesi clinical samples were collected from Peninsular Malaysia and Malaysian Borneo. The PkSERA3 Ag2 gene was amplified using PCR, and subsequently cloned and sequenced. Genetic diversity, haplotype, natural selection as well as genetic structure and differentiation of PkSERA3 Ag2 were analysed. In addition, in silico analyses were performed to identify repeat motifs, B-cell epitopes, and antigenicity indices of the protein. Analysis of 114 PkSERA3 Ag2 sequences revealed high nucleotide diversity of the gene in Malaysia. A codon-based Z-test indicated that the gene underwent purifying selection. Haplotype and population structure analyses identified two distinct PkSERA3 Ag2 clusters (K = 2, ΔK = 721.14) but no clear genetic distinction between PkSERA3 Ag2 from Peninsular Malaysia and Malaysian Borneo. F ST index indicated moderate differentiation of the gene. In silico analyses revealed unique repeat motifs among PkSERA3 Ag2 isolates. Moreover, the amino acid sequence of PkSERA3 Ag2 exhibited potential B-cell epitopes and possessed high antigenicity indices. These findings enhance the understanding of PkSERA3 Ag2 gene as well as its antigenic properties. Further validation is necessary to ascertain the utility of PkSERA3 Ag2 as a serological marker for P. knowlesi infection., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Trypanosoma brucei and related kinetoplastid parasites possess unique RNA processing pathways, including in their mitochondria, that regulate metabolism and development. Altering RNA composition or conformation through nucleotide modifications is one such pathway, and modifications including pseudouridine regulate RNA fate and function in many organisms. We surveyed pseudouridine synthase (PUS) orthologs in trypanosomatids, with a particular interest in mitochondrial enzymes due to their potential importance for mitochondrial function and metabolism. Trypanosoma brucei mitochondrial (mt)-LAF3 is an ortholog of human and yeast mitochondrial PUS enzymes, and a mitoribosome assembly factor, but structural studies differ in their conclusion as to whether it has PUS catalytic activity. Here, we generated T. brucei cells that are conditionally null (CN) for mt-LAF3 expression and showed that mt-LAF3 loss is lethal and disrupts mitochondrial membrane potential (ΔΨm). Addition of a mutant gamma ATP synthase allele to the CN cells permitted ΔΨm maintenance and cell survival, allowing us to assess primary effects on mitochondrial RNAs. As expected, these studies showed that loss of mt-LAF3 dramatically decreases levels of mitochondrial 12S and 9S rRNAs. Notably, we also observed decreases in mitochondrial mRNA levels, including differential effects on edited vs. pre-edited mRNAs, indicating that mt-LAF3 is required for mitochondrial rRNA and mRNA processing, including of edited transcripts. To assess the importance of PUS catalytic activity in mt-LAF3 we mutated a conserved aspartate that is necessary for catalysis in other PUS enzymes and showed it is not essential for cell growth, or maintenance of ΔΨm and mitochondrial RNA levels. Together, these results indicate that mt-LAF3 is required for normal expression of mitochondrial mRNAs in addition to rRNAs, but that PUS catalytic activity is not required for these functions. Instead, our work, combined with previous structural studies, suggests that T. brucei mt-LAF3 acts as a mitochondrial RNA-stabilizing scaffold., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The local diversity and population structure of malaria parasites vary across different regions of the world, reflecting variations in transmission intensity, host immunity, and vector species. This study aimed to use amplicon sequencing to investigate the genotypic patterns and population structure of P. vivax isolates from a highly endemic province of Thailand in recent years. Amplicon deep sequencing was performed on 70 samples for the 42-kDa region of pvmsp1 and domain II of pvdbp. Unique haplotypes were identified and a network constructed to illustrate genetic relatedness in northwestern Thailand. Based on this dataset of 70 samples collected between 2015 and 2021, 16 and 40 unique haplotypes were identified in pvdbp II and pvmsp1 42kDa , respectively. Nucleotide diversity was higher in pvmsp1 42kDa than in pvdbp II (π = 0.027 and 0.012), as was haplotype diversity (Hd = 0.962 and 0.849). pvmsp1 42kDa also showed a higher recombination rate and higher levels of genetic differentiation (F st ) in northwestern Thailand versus other regions (0.2761-0.4881). These data together suggested that the genetic diversity of P. vivax in northwestern Thailand at these two studied loci evolved under a balancing selection, most likely host immunity. The lower genetic diversity of pvdbp II may reflect its stronger functional constrain. In addition, despite the balancing selection, a decrease in genetic diversity was observed. Hd of pvdbp II decreased from 0.874 in 2015-2016 to 0.778 in 2018-2021; π of pvmsp1 42kDa decreased from 0.030 to 0.022 over the same period. Thus, the control activities must have had a strong impact on the parasite population size. The findings from this study provide an understanding of P. vivax population structure and the evolutionary force on vaccine candidates. They also established a new baseline for tracking future changes in P. vivax diversity in the most malarious area of Thailand., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Development of next-generation vaccines against Plasmodium falciparum (Pf) is a priority. Many malaria vaccines target the pre-erythrocytic sporozoite (SPZ) and liver stages. These include subunit vaccines based on the Pf circumsporozoite protein (CSP) and attenuated PfSPZ vaccines. However, these strategies require 3-4 doses and have not achieved optimal efficacy against field-transmitted malaria. Prime-and-trap is a recently developed two-step heterologous vaccine strategy that combines priming with DNA encoding CSP followed by a single dose of attenuated SPZ. This strategy aims to induce CD8 + T cells that can eliminate parasites in the liver. Prior data has demonstrated that prime-and-trap with P. yoelii CSP and PySPZ was immunogenic and protective in mice. Here we report preliminary data on the immunogenicity of PfCSP prime and PfSPZ trap vaccine in rhesus macaques. This vaccine induced PfCSP-specific antibodies and T cell responses in all animals. However, response magnitude differed between individuals, suggesting further study is required., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: S. C. M. filed a patent application on selected aspects of the prime-and-trap concept through the University of Washington. S. C. M. has equity in a startup company (Sound Vaccines, Inc.) that is negotiating with the University of Washington for rights to this intellectual property. The relationship between the authors and Sound Vaccines, Inc., has been reviewed by the University of Washington and complies with all University and State of Washington policies on such activities. N.K.C., B. K. L. S., and S. L. H. are paid employees of Sanaria Inc., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Malaria still poses a major burden on human health around the world, especially in endemic areas. Plasmodium resistance to several antimalarial drugs has been one of the major hindrances in control of malaria. Thus, the World Health Organization recommended artemisinin-based combination therapy (ACT) as a front-line treatment for malaria. The emergence of parasites resistant to artemisinin, along with resistant to ACT partner drugs, has led to ACT treatment failure. The artemisinin resistance is mostly related to the mutations in the propeller domain of the kelch13 (k13) gene that encodes protein Kelch13 (K13). The K13 protein has an important role in parasite reaction to oxidative stress. The most widely spread mutation in K13, with the highest degree of resistance, is a C580Y mutation. Other mutations, which are already identified as markers of artemisinin resistance, are R539T, I543T, and Y493H. The objective of this review is to provide current molecular insights into artemisinin resistance in Plasmodium falciparum. The trending use of artemisinin beyond its antimalarial effect is described. Immediate challenges and future research directions are discussed. Better understanding of the molecular mechanisms underlying artemisinin resistance will accelerate implementation of scientific findings to solve problems with malarial infection., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2023. Published by Elsevier B.V.)

A key morphological feature of kinetoplastid parasites is the position and length of flagellum attachment to the cell body. This lateral attachment is mediated by the flagellum attachment zone (FAZ), a large complex cytoskeletal structure, which is essential for parasite morphogenesis and pathogenicity. Despite the complexity of the FAZ only two transmembrane proteins, FLA1 and FLA1BP, are known to interact and connect the flagellum to the cell body. Across the different kinetoplastid species, each only has a single FLA/FLABP pair, except in Trypanosoma brucei and Trypanosoma congolense where there has been an expansion of these genes. Here, we focus on the selection pressure behind the evolution of the FLA/FLABP proteins and the likely impact this will have on host-parasite interactions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Malaria outbreaks are important public health concerns that can cause resurgence in endemic regions approaching elimination. We investigated a Plasmodium falciparum outbreak in Attapeu Province, Laos, during the 2020-21 malaria season, using genomic epidemiology methods to elucidate parasite population dynamics and identify its causes., Methods: In this genetic analysis, 2164 P falciparum dried blood spot samples were collected from southern Laos between Jan 1, 2017, and April 1, 2021, which included 249 collected during the Attapeu outbreak between April 1, 2020, and April 1, 2021, by routine surveillance. Genetic barcodes obtained from these samples were used to investigate epidemiological changes underpinning the outbreak, estimate population diversity, and analyse population structure. Whole-genome sequencing data from additional historical samples were used to reconstruct the ancestry of outbreak strains using identity-by-descent analyses., Findings: The outbreak parasite populations were characterised by unprecedented loss of genetic diversity, primarily caused by rapid clonal expansion of a multidrug-resistant strain (LAA1) carrying the kelch13 Arg539Thr (R539T) mutation. LAA1 replaced kelch13 Cys580Tyr (C580Y) mutants resistant to dihydroartemisinin-piperaquine (KEL1/PLA1) as the dominant strain. LAA1 inherited 58·8% of its genome from a strain circulating in Cambodia in 2008. A secondary outbreak strain (LAA2) carried the kelch13 C580Y allele, and a genome that is essentially identical to a Cambodian parasite from 2009. A third, low-frequency strain (LAA7) was a recombinant of KEL1/PLA1 with a kelch13 R539T mutant., Interpretation: These results strongly suggest that the outbreak was driven by a selective sweep, possibly associated with multidrug-resistant phenotypes of the outbreak strains. Established resistant populations can circulate at low frequencies for years before suddenly overwhelming dominant strains when the conditions for selection become favourable-eg, when front-line therapies change. Genetic surveillance can support elimination by characterising key properties of outbreaks such as population diversity, drug resistance marker prevalence, and the origins of outbreak strains., Funding: Bill & Melinda Gates Foundation; The Global Fund to Fight AIDS, Tuberculosis and Malaria; Wellcome Trust., Translation: For the Lao translation of the abstract see Supplementary Materials section., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

TrypTag was a 4-year project to tag the N- and C-termini of almost all Trypanosoma brucei proteins with a fluorescent protein and record the subcellular localisation through images and manual annotation. We highlight the new routes to cell biological discovery this transformative resource is enabling for parasitologists and cell biologists., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

DNA damage inducible 1 protein (DDI1) is involved in a variety of cellular processes including proteasomal degradation of specific proteins. All DDI1 proteins contain a ubiquitin-like (UBL) domain and a retroviral protease (RVP) domain. Some DDI1 proteins also contain a ubiquitin-associated (UBA) domain. The three domains confer distinct activities to DDI1 proteins. The presence of a RVP domain makes DDI1 a potential target of HIV protease inhibitors, which also block the development of malaria parasites. Hence, we investigated the DDI1 of malaria parasites to identify its roles during parasite development and potential as a therapeutic target. DDI1 proteins of Plasmodium and other apicomplexan parasites share the UBL-RVP domain architecture, and some also contain the UBA domain. Plasmodium DDI1 is expressed across all the major life cycle stages and is important for parasite survival, as conditional depletion of DDI1 protein in the mouse malaria parasite Plasmodium berghei and the human malaria parasite Plasmodium falciparum compromised parasite development. Infection of mice with DDI1 knock-down P. berghei was self-limiting and protected the recovered mice from subsequent infection with homologous as well as heterologous parasites, indicating the potential of DDI1 knock-down parasites as a whole organism vaccine. Plasmodium falciparum DDI1 (PfDDI1) is associated with chromatin and DNA-protein crosslinks. PfDDI1-depleted parasites accumulated DNA-protein crosslinks and showed enhanced susceptibility to DNA-damaging chemicals, indicating a role of PfDDI1 in removal of DNA-protein crosslinks. Knock-down of PfDDI1 increased susceptibility to the retroviral protease inhibitor lopinavir and antimalarial artemisinin, which suggests that simultaneous inhibition of DDI1 could potentiate antimalarial activity of these drugs. As DDI1 knock-down parasites confer protective immunity and it could be a target of HIV protease inhibitors, Plasmodium DDI1 is a potential therapeutic target for malaria control., (Copyright © 2023 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

The host-parasite evolutionary arms race is a fundamental process with medical implications. During this process, the host develops parasite resistance, and the parasite develops host immune evasion strategies. Thus, this process accelerates relevant protein evolution. This study test hypothesizes that proteins subject to sequence evolution structural constraints play a crucial role and that these constraints hinder the modification of such proteins in this process. These hypotheses were tested using Plasmodium falciparum model and evaluated protein structures predicted for the entire proteome by the AlphaFold method. Based on dN/dS test results and P. falciparum and P. reichenowi comparisons, the presented approach identified proteins subject to purifying selection acting on the whole sequence and buried residues (dN < dS) and positive selection on nonburied residues. Of the 26 proteins, some known antigens (ring-exported protein 3, RAP protein, erythrocyte binding antigen-140, and protein P47) targeted by the host immune system are promising vaccine candidates. The set also contained 11 enzymes, including FIKK kinase, which modifies host proteins. This set was compared with genes for which the dN/dS test suggested that positive selection acts on the whole gene (i.e., dN > dS). The present study found that such genes encode enzymes and antigenic vaccine candidates less frequently than genes for which evolution is not subject to selection constraints and positive selection acts on only exposed residues. The analysis was repeated comparing P. falciparum with P. alderi, which is more distantly related. The study discusses the potential implications of the presented methodology for rational vaccine design and the parasitology and evolutionary biology fields., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author. Published by Elsevier B.V. All rights reserved.)

Background: The genetic diversity of malaria parasites traces the origin and spread of new variants and can be used to evaluate the effectiveness of malaria control measures. Therefore, this study aims to improve the understanding of the molecular epidemiology of Plasmodium vivax malaria at the China-Myanmar border by genotyping the PvMSP-3α and PvMSP-3β genes., Methods: Blood samples were collected from P. vivax malaria patients along the China-Myanmar border. The PvMSP-3α and PvMSP-3β genes were amplified by polymerase chain reaction (PCR) and the genetic polymorphism and haplotype of the two genes were analyzed., Results: A total of 422 blood samples were used for this study, of which 224 were analyzed at PvMSP-3α and 126 at PvMSP-3β. Samples mainly were from young adults aged 18-45 years, although local patients were significantly younger than migrant laborers crossing the border at Tengchong (P < 0.0001). Molecular evolutionary analysis revealed that PvMSP-3α and PvMSP-3β underwent diversifying natural selection, and intragenic recombination contributed to the diversity of the isolates. Based on the length of the genes, we identified three types of PvMSP-3α [1.9-2.0 kb (Type-A), 1.4-1.5 kb (Type-B), and 1.1-1.3 kb (Type-C)] and two types of PvMSP-3β [1.7-2.2 kb (Type-A) and 1.4-1.5 kb (Type-B)]. Migrant laborers returning to China through Tengchong bore P. vivax infections displaying significantly higher genetic diversity than local residents., Conclusions: Both PvMSP-3 paralogs were subjected to diversifying selection in each sample population. Clustering of alleles supports ephemeral endemic differentiation of alleles, but the broader phylogeny suggests that alleles transit the globe, perhaps accelerated by movements of migrants such as those transiting Tengchong., Competing Interests: Declaration of Competing Interest 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 The Authors. Published by Elsevier B.V. All rights reserved.)

Immunity to Plasmodium falciparum is non-sterilising, thus individuals residing in malaria-endemic areas are at risk of infection throughout their lifetime. Here we seek to find a genomic epidemiological explanation for why residents of all ages harbour blood stage infections despite lifelong exposure to P. falciparum in areas of high transmission. We do this by exploring, for the first known time, the age-specific patterns of diversity of variant antigen encoding (var) genes in the reservoir of infection. Microscopic and submicroscopic P. falciparum infections were analysed at the end of the wet and dry seasons in 2012-2013 for a cohort of 1541 residents aged from 1 to 91 years in an area characterised by high seasonal malaria transmission in Ghana. By sequencing the near ubiquitous Duffy-binding-like alpha domain (DBLα) that encodes immunogenic domains, we defined var gene diversity in an estimated 1096 genomes detected in sequential wet and dry season sampling of this cohort. Unprecedented var (DBLα) diversity was observed in all ages with 42,399 unique var types detected. There was a high degree of maintenance of types between seasons (>40% seen more than once), with many of the same types, especially upsA, appearing multiple times in isolates from different individuals. Children and adolescents were found to be significant reservoirs of var DBLα diversity compared with adults. Var repertoires within individuals were highly variable, with children having more related var repertoires compared to adolescents and adults. Individuals of all ages harboured multiple genomes with var repertoires unrelated to those infecting other hosts. High turnover of parasites with diverse isolate var repertoires was also observed in all ages. These age-specific patterns are best explained by variant-specific immune selection. The observed level of var diversity for the population was then used to simulate the development of variant-specific immunity to the diverse var types under conservative assumptions. Simulations showed that the extent of observed var diversity with limited repertoire relatedness was sufficient to explain why adolescents and adults in this community remain susceptible to blood stage infection, even with multiple genomes., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Despite the development of prophylactic anti-malarial drugs and practices to prevent infection, malaria remains a health concern. Preclinical testing of novel malaria vaccine strategies achieved through rational antigen selection and novel particle-based delivery platforms is yielding encouraging results. One such platform, self-assembling virus-like particles (VLP) is safer than attenuated live viruses, and has been approved as a vaccination tool by the FDA. We explore the use of Norovirus sub-viral particles lacking the natural shell (S) domain forming the interior shell but that retain the protruding (P) structures of the native virus as a vaccine vector. Epitope selection and their surface display has the potential to focus antigen specific immune responses to crucial epitopes. Recombinant P-particles displaying epitopes from two malaria antigens, Plasmodium falciparum (Pf) CelTOS and Plasmodium falciparum (Pf) CSP, were evaluated for immunogenicity and their ability to confer protection in a murine challenge model. Immune responses induced in mice resulted either in sterile protection (displaying PfCelTOS epitopes) or in antibodies with functional activity against sporozoites (displaying PfCSP epitopes) in an in vitro liver-stage development assay (ILSDA). These results are encouraging and support further evaluation of this platform as a vaccine delivery system., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

Background: Molecular markers for monitoring resistance could help improve malaria treatment policies. Delayed clearance of Plasmodium falciparum by artemisinin-based combination therapies (ACTs) has been reported in several countries. In addition to PfKelch13 (pfk13), new drug resistance genes, P. falciparum ubiquitin-specific protease 1 (pfubp1) and the eadaptor protein complex 2 mu subunit (pfap2mu), have been identified as being linked to ACTs. This study investigated the prevalence of single-nucleotide polymorphisms (SNPs) in clinical P. falciparum isolates pfubp1 and pfap2mu imported from Africa and Southeast Asia (SEA) to Wuhan, China, to provide baseline data for antimalarial resistance monitoring in this region., Methods: Peripheral venous blood samples were collected in Wuhan, China, from August 2011 to December 2019. The Pfubp1 and pfap2mu SNPs of P. falciparum were determined by nested PCR and Sanger sequencing., Results: In total, 296 samples were collected. Subsequently, 92.23% (273/296) were successfully amplified and sequenced for Pfubp1. There were 60.07% (164/273) wild-type strains and 39.93% (109/273) mutant strains. The pfap2mu gene was divided into three fragments for amplification, and 82.77% (245/296), 90.20% (267/296) and 94.59% (280/296) were sequenced successfully. Genotypes reportedly associated with ACTs resistance detected in this study included pfubp1 D1525E as well as E1528D and pfap2mu S160N. The mutation prevalence rates were 10.99% (30/273), 13.19% (36/273) and 11.24% (30/267), respectively. These are all focused on Congo, Nigeria and Angola. Known delayed-clearance parasite mutations have also been found in SEA., Conclusions: The existence of mutation sites of known clearance genes detected in the isolates in this study, including D1525E and E1528D in the pfubp1 gene and S160N in the pfap2mu gene, further proved the risk of ACTs resistance. Constant vigilance is therefore needed to protect the effectiveness of ACTs and to prevent the spread of drug-resistant P. falciparum. Further studies in malaria-endemic countries are needed to further validate potential genetic markers for monitoring parasite populations in Africa and SEA., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Merosome development is a very important part of the Plasmodium life cycle. It constitutes the last step of liver stage development after which merozoites egress to the bloodstream and invade red blood cells. Many parasite proteins have been shown to play key roles in this process. Phospholipases are known to be actively involved in membrane formation and remodeling. At least one phospholipase A2, localized to the parasitophorous vacuolar membrane, is known to be directly important for parasitophorous vacuolar membrane (PVM) rupture and subsequent release of merozoites. Here, we characterize a Plasmodium berghei phosphatidic acid (PA) preferring phospholipase A1 (PbPla1) homolog. C-terminal 3XHA-mCherry tagging revealed that PbPla1 is expressed in all life cycle stages except sporozoites and is present in the parasite's cytoplasm. Targeted disruption of PbPla1 revealed its non-essentiality for the blood and mosquito stages. Pla1 - sporozoites were found to be late in their ability to establish successful blood stage infection in mice. While exoerythrocytic form development was found to be normal in vitro, a decrease in the number of merosomes was observed. PVM rupture in late exo-erythrocytic forms (EEFs) was quantified by counting the number of parasites with intact PVMs, and was found to be significantly higher in Pla1 - . Our findings indicate the role of PbPla1 in merosome release., (Copyright © 2022 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

The protozoan parasite Giardia lamblia acquires cholesterol from the environment since it is unable to synthesise cholesterol de novo and this is vital for trophozoite growth. Conversely, the lack of cholesterol was described as an essential event to trigger encystation, the differentiation of trophozoites to mature cysts. During the G. lamblia cell cycle, cholesterol is acquired as a free molecule as well as through receptor-mediated endocytosis (RME) of lipoproteins. In this work, we describe the involvement of RME in the cell differentiation process of G. lamblia. We found that a reduction in the expression of the medium subunit (Glµ2) of the giardial adaptin protein GlAP2 impaired RME, triggering the process of encystation in growing cells. Contrary to expectations, decreasing Glµ2 expression produced a cohort of trophozoites that yielded significantly less mature cysts when cells were induced to encyst. Analysis of the subcellular localization of Glµ2 and the cyst wall protein 1 (CWP1) during encystation was later performed, to dissect the process. Our results showed, on one hand, that blocking RME by inhibiting Glµ2 expression, and probably cholesterol entry, is sufficient to induce cell differentiation but not to complete the process of encystation. On the other hand, we observed that GlAP2 is necessary to accomplish the final steps of encystation by sorting CWP1 to the plasma membrane for cyst wall formation. The understanding of the mechanisms involved in cyst formation should provide novel insights into the control of giardiasis, an endemic worldwide neglected disease., (Copyright © 2022 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

A critical step in malaria blood-stage infections is the invasion of red blood cells (RBCs) by merozoite forms of the Plasmodium parasite. Much progress has been made in defining the parasite ligands and host receptors that mediate this critical step. However, less well understood are the RBC biophysical determinants that influence parasite invasion. In this review we explore how Plasmodium falciparum merozoites interact with the RBC membrane during invasion to modulate RBC deformability and facilitate invasion. We further highlight RBC biomechanics-related polymorphisms that might have been selected for in human populations due to their ability to reduce parasite invasion. Such an understanding will reveal the translational potential of targeting host pathways affecting RBC biomechanical properties for the treatment of malaria., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Recent studies have documented Pfhrp2/3 gene deletion globally as one of the biological threats in the fight against malaria. For malaria diagnosis, PfHRP2 based RDTs are most widely used in India, and performance of these RDTs are affected by deleted Pfhrp2/3 gene in Plasmodium falciparum. This study was planned to confirm Pfhrp2/3 gene deletion incidences and genetic variation in PfHRP2-based RDT positive with P.falciparum malaria cases from India., Methodology: Confirmed positive samples by PfHRP2-based RDTs as P. falciparum (n = 240) from six different endemic regions of India were validated by PCR to assure the actual infection. Two hundred forty samples qualified for DNA intactness by single-copy genes were subjected to amplification for the Pfhrp2/3 gene and its neighbouring gene (downstream and upstream) by PCR genotyping. Genetic variation in samples was analysed post-sequencing using Mega X software. Statistical analysis was performed to validate the genetic variation using Mann-Whitney Test., Results: RDT target region of Pfhrp2 gene (exon2) was found deleted in a single sample with presence of the Pfhrp3 exon2. Complete gene deletion of 4.2% was observed in the Pfhrp3 gene. Partial gene deletion was recorded for both pfhrp2 gene (exon2-0.4%, upstream 25.8% and downstream -9.1%) and Pfhrp3 gene (exon2-18.75%, upstream - 22.08% and downstream 13.3%). Eleven new unique types of amino acid repeat sequence and earlier reported amino acid repeat type was found in the Pfhrp2 gene, prompting high genetic variation., Conclusions: This study suggests that parasites lacking Pfhrp2/3 gene and its neighbouring gene (downstream and upstream) are present in malaria endemic areas of India, resulting in false positive results by RDT. Systematic countrywide monitoring for malaria control and elimination of malaria is warranted in this regard., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Early transcribed membrane proteins form a unique protein family in malaria parasites. These molecules are expressed during Plasmodium intracellular phases and inserted at the parasite parasitophorus vacuole membrane, which constitutes the host-parasite interface. Upregulated in infectious sporozoites 4 (UIS4) is an essential early transcribed membrane protein of liver stages of the murine malaria model parasite Plasmodium berghei. Despite its relevance for liver stage maturation, the molecular functions of UIS4 remain elusive, and UIS4 orthologs in human malaria parasites have not yet been identified. In order to characterise functional domains of UIS4, we generated P. berghei parasites carrying a carboxy-terminally truncated version of UIS4. We observed that uis4Δc parasites are severely impaired in liver stage development, similar to uis4(-) parasites, indicating an important role of the C-terminal domain for UIS4 function. To test whether members of the P. falciparum early transcribed membrane protein family are potential UIS4 orthologs, we selected candidates based on structural homology and parasitophorous vacuole membrane localization. We generated transgenic P. berghei parasites where UIS4 was replaced by Plasmodium falciparum ETRAMP8 or ETRAMP10.3. Both early transcribed membrane proteins were expressed in transgenic parasite lines, but liver stage maturation was impaired, indicating that the selected early transcribed membrane proteins failed to substitute the function of UIS4. As a control, we included the UIS4 ortholog from the murine parasite Plasmodium chaubaudi. We observed that PcUIS4 successfully restores UIS4 function in P. berghei. Together, these results suggest that Plasmodium parasites express tailor-made parasitophorous vacuole membrane proteins that might at least partially explain the narrow host range of malaria parasites., (Copyright © 2021 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

North Central Nigeria is one region in Nigeria with a significant incidence of malaria caused majorly by Plasmodium falciparum. This study utilizes the msp1 and msp2 genes of P. falciparum to examine its diversity and multiplicity of infection (MOI). Blood samples were collected from 247 children across selected healthcare facilities in Minna, from infants and children aged 6 months to 17 years. Of the total collection, 143 (58%) of the children were infected with P. falciparum with parasite density ≥ 1000 μl, and from which fifty (50) samples was randomly selected and presented for PCR for the characterization of msp1 and msp2 gene using nested-PCR method. Overall, 57 msp1 genotypes, including K1, MAD20 and RO33 were identified, ranging from (250-1000 bp), (100-500 bp) and (400-500 bp), respectively. In addition, 54 different msp2 genotypes of FC27 and 3D7 alleles ranging from (100-900 bp) and (100-800 bp), respectively were selected. A monoclonal infection of 39% and a polyclonal infection of 61% was recorded, however, a particularity about this study is the polyclonal nature of RO33. Determination of gene diversity revealed MAD20 as the predominant allele for msp1 with a mean MOI of 1.35 and FC27 for msp2 with 1.72 MOI. The overall MOI recorded for the study was 1.60. There was, however, no statistical significance difference between MOI and age of the child (P > 0.05). Meanwhile, findings from this study revealed P. falciparum populations were not genetically diverse with Heterozygosity (He) index of 0.0636. However, a significant level gene diversity within the antigenic markers of msp1 and msp2 was observed with He index of 0.714 and 0.830, respectively. This study has demonstrated the potential of gene diversity and MOI of P. falciparum, as important markers for assessing differences in malaria transmission intensity. Continuous malaria genetic surveillance is therefore recommended as a fundamental tool for monitoring changes in gene types and for intervention programs' effectiveness., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Plasmodium vivax-induced malaria is among the leading causes of morbidity and mortality in sub-tropical and tropical regions and infect 2.85 billion people globally. The continual rise and propagation of resistance against anti-malarial drugs is a prerequisite to develop a potent vaccine candidate for Plasmodium vivax (P. vivax). Circumsporozoite protein (CSP) is an important immunogen of malaria parasite that has the conserved CSP structure as an immune dominant B-cell epitope. In current study, we focused on designing multi-epitope vaccines (MEVs) using various immunoinformatics tools against Pakistani based allelic variants VK210 and VK247 of P. vivax CSP (PvCSP) gene. Antigenicity, allergic potential and physicochemical parameters of both PvCSP variants were assessed for the designed MEVs and they were within acceptable range suitable for post experimental investigations. The three-dimensional structures of both MEVs have been predicted ab initio, optimized, and validated by using different online servers. The both MEVs candidates were stable and free from aggregation-prone regions. The stability of both MEVs had been improved by a disulfide engineering approach. To estimate the binding energy and stability of the MEVs, molecular docking simulation and binding free energy calculations with TLR-4 immune receptor have been conducted. The docking score of PvCSP210 and PvCSP247 for TLR-4 was -6.34 kJ/mol and - 2.3 kJ/mol, respectively. For PvCSP210-TLR4 system, mean RMSD was 4.96 Å while PvCSP247-TLR4 system, average RMSD was 4.49 Å. The binding free energy of PvCSP210-TLR4 complex and PvCSP247-TLR4 complex was -50.49/-117.15 kcal/mol (MMGBSA/MMPSA) and -52.94/-96.26 kcal/mol (MMGBSA/MMPSA), respectively. The expression of both MEVs produced in Escherichia coli K12 expression system by in silico cloning was significant. Immune simulation revealed that the proposed MEVs induce strong humoral and cellular immunological responses, in addition to significant production of interleukins and cytokines. In conclusions, we believed that the MEVs proposed in current research, using combine approach of immunoinformatics, structural biology and biophysical approaches, could induce protective and effective immune responses against P. vivax and the experimental validation of our findings could contribute to the development of potential malaria vaccine., (Copyright © 2021. Published by Elsevier B.V.)

The ability of malaria parasites to develop resistance to antimalarial drugs has made it necessary to continuously survey malaria parasite populations for resistance markers. Mutations in specific malaria parasite genes confer resistance to antimalarial drugs. The study compared mutations in Pfcrt and Pfmdr1 genes of P. falciparum from two ecologically different areas of Nigeria. Plasmodium falciparum dried blood spots collected from New Bussa (Northcentral Nigeria) and Ijede (Southwest Nigeria) were analysed by PCR-RFLP for Pfcrt, K76 T, Pfmdr1, N86Y and Y184F mutations. Pfmdr1 copy number was determined by quantitative-PCR. A total of 145 blood spots [Ijede = 55; New Bussa = 90 blood spots] were analysed, but Pfcrt gene was successfully amplified in 144 samples while Pfmdr1 was amplified in 132 samples. Overall, prevalence of mutant forms of Pfcrt 76 T,Pfmdr1 86Y and 184F were 74.3% (95% CI: 66.4-81.2%), 18.2% (95% CI: 12.0-25.8%) and 35.6% (95% CI: 27.5-44.4%). The frequency of Pfcrt 76 T was similar in both study sites [Ijede: 81.8% (95%CI: 69.1-90.9%); New Bussa: 69.7% (95%CI: 59.0-79.0), p = 0.105]. However, the frequencies of Pfmdr1 86Y and 184F were significantly higher in Ijede (28.3% and 62.3%) than in New Bussa (11.4% and 17.7%), respectively (P < 0.05). Eight parasite genotypes based on three codons of the two genes were identified. The most frequent genotype was TNY 53(40.5%) while the least was KYF 1 (0.8%). The most frequent genotype in Ijede and New Bussa were TNF 18(34.0%) and TNY 40 (51.3%) respectively. The frequency of wild strain KNF in Ijede and New Bussa were 3 (5.7%) and 18 (23.1%), respectively. The distribution of the genotypes differed significantly by location. The genotypes with more than two or more mutations were more in Ijede 32 (60.4%) than in New Bussa 16 (20.5%) (p < 0.001). Amplification of Pfmdr1 copy number was not observed in the two study sites. The prevalence of Pfcrt 76 T was similar in both locations while Pfmdr1 86Y and 184F differed in both locations. Single nucleotide polymorphisms in the three codons assessed were more in Ijede than in New Bussa., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background The major variant surface antigen (VSA) in Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) encoded by var gene family has an important role in cytoadhesion/sequestration and rosetting by adhesion of uninfected erythrocytes to infected erythrocytes leading to disease severity. DBL1α domain in the PfEMP-1, protein is crucial in the cytoadhesion phenomena in P. falciparum infections and this review aims to analyse the genetic diversity of DBL1α domain sequences in PfEMP-1 from different geographical regions globally. Methods All available DBL1α sequence data was reviewed by using the electronic database PubMed, ResearchGate, Google, Google scholar, MEDLINE with the following Keywords-Plasmodium falciparum", "var gene", "DBL1α", "field isolate", "diversity", "polymorphism", "Africa", "America", "Asia" and "Caribbean" from different geographical regions across the world. Results A total of 240 studies were identified initially but only 20 studies qualified for this systematic review. The overall ratio of distinct sequences DBL1α domain was 24.62/1167 the highest in African region (33.59/766 isolates) and lowest in South America (5.6/215 isolates). In the 18 included studies, the presence of distinct DBL1α sequences was the highest in Oceania 55.32% (1186/2144) followed by Africa (38.43%), Asia (22.45%) and South America (16.48%), though the sample size in Oceania was comparatively smaller to that of Africa and South America. Conclusion This review highlights the ratio and percentage of distinct sequences of DBL1α domain of var gene in different geographical regions giving an idea of the existing diversity prevalent in this potential vaccine target gene which may contribute to designing the preventive measures towards disease severity., (Copyright © 2021 Elsevier B.V. All rights reserved.)