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Immunization with attenuated whole Plasmodium sporozoites constitutes a promising vaccination strategy. Compared to replication-deficient parasites, immunization with replication-competent parasites confers better protection and also induces a type I IFN (IFN-1) response, but whether this IFN-1 response has beneficial or adverse effects on vaccine-induced adaptive immunity is not known. Here, we show that IFN-1 signaling-deficient mice immunized with replication-competent sporozoites exhibit superior protection against infection. This correlates with superior CD8 T cell memory including reduced expression of the exhaustion markers PD-1 and LAG-3 on these cells and increased numbers of memory CD8 T cells in the liver. Moreover, the adoptive transfer of memory CD8 T cells from the livers of previously immunized IFN-1 signaling-deficient mice confers greater protection against liver stage parasites. However, the detrimental role of IFN-1 signaling is not CD8 T cell intrinsic. Together, our data demonstrate that liver stage-engendered IFN-1 signaling impairs hepatic CD8 T cell memory via a CD8 T cell-extrinsic mechanism. Here, Minkah et al . show that, while immunization with replication-competent Plasmodium parasites can confer sterile protection against infection, it also induces a type I interferon response that adversely affects anti-malaria immunity by affecting numbers of protective hepatic CD8 T cells and CD8 T cell function.

Background The in vitro cultivation of individual stages of the Plasmodium falciparum mosquito life cycle is notably challenging. The main difficulty is replicating the intricate nutrient and metabolite exchanges necessary for oocyst development and sporozoite (SPZ) formation in the three-dimensional environment of the mosquito midgut. Replicating these conditions is essential for understanding the biological interactions between mosquito and parasite, as well as advancing malaria vaccine development. Methods An in vitro three-dimensional system was developed that closely mimics the mosquito midgut epithelium, basal lamina, and haemolymph, facilitating the production of substantial quantities of haemolymph-like Pf SPZ. Results Use of an extracellular matrix-coated Alvetex ® Strata scaffold, combined with optimized culture medium, supports efficient oocyst attachment and provides the necessary nutrients for robust production of haemolymph-like SPZ. This system enables full maturation of oocysts, as shown by immunofluorescence assays (IFA), and allows timely release of in vitro SPZ (IVS) between days 11 and 15, comparable to the in vivo mosquito timeline. Haemolymph-like SPZ generated were found to be infectious, as evidenced by successful HC04 infection in in vitro and in vivo studies using FRG-huHep mice. Similar outcomes were observed across different P. falciparum strains. Conclusions Implementation of the Alvetex Strata scaffold, optimized medium, and improved ookinete production consistently enables in vitro generation of large quantities of haemolymph-like SPZ.

Background Plasmodium vivax represents the most geographically widespread human malaria parasite affecting civilian and military populations in endemic areas. Targeting the pre-erythrocytic (PE) stage of the parasite life cycle is especially appealing for developing P. vivax vaccines as it would prevent disease and transmission. Here, naturally acquired immunity to a panel of P. vivax PE antigens was explored, which may facilitate vaccine development and lead to a better understanding of naturally acquired PE immunity. Methods Twelve P. vivax PE antigens orthologous to a panel of P. falciparum antigens previously identified as highly immunogenic in protected subjects after immunization with radiation attenuated sporozoites (RAS) were used for evaluation of humoral and cellular immunity by ELISA and IFN-γ ELISpot. Samples from P. vivax infected individuals (n = 76) from a low endemic malaria region in the Peruvian Amazon Basin were used. Results In those clinical samples, all PE antigens evaluated showed positive IgG antibody reactivity with a variable prevalence of 58–99% in recently P. vivax diagnosed patients. The magnitude of the IgG antibody response against PE antigens was lower compared with blood stage antigens MSP1 and DBP-II, although antibody levels persisted better for PE antigens (average decrease of 6% for PE antigens and 43% for MSP1, p < 0.05). Higher IgG antibodies was associated with one or more previous malaria episodes only for blood stage antigens (p < 0.001). High IgG responders across PE and blood stage antigens showed significantly lower parasitaemia compared to low IgG responders (median 1,921 vs 4,663 par/µl, p < 0.05). In a subgroup of volunteers (n = 17),positive IFN-γ T cell response by ELISPOT was observed in 35% vs 9–35% against blood stage MSP1 and PE antigens, respectively, but no correlation with IgG responses . Conclusions These results demonstrate clear humoral and T cell responses against P. vivax PE antigens in individuals naturally infected with P. vivax. These data identify novel attractive PE antigens suitable for use in the potential development and selection of new malaria vaccine candidates which can be used as a part of malaria prevention strategies in civilian and military populations living in P. vivax endemic areas.

Targeted protein degradation (TPD) is a novel strategy for developing therapeutics against pathogens. Prior to causing malaria, Plasmodium parasites replicate within hepatocytes as liver stages, surrounded by a parasitophorous vacuole membrane (PVM). We hypothesized that TPD can be employed to trigger host-driven degradation of essential liver stage PVM proteins and lead to parasite death. To explore this, we took advantage of the proteolysis-targeting-chimera HaloPROTAC3, a molecule that recruits the host von Hippel-Lindau (VHL) E3 ligase to the HaloTag (HT). Parasites expressing HT fused to the host cytosol-exposed domain of the PVM protein UIS4 (UIS4-HT) were generated in Plasmodium berghei and Plasmodium cynomolgi , but only P. berghei UIS4-HT enabled productive liver stage infection experiments in vitro. Although HaloPROTAC3 triggered the degradation of HT proteins in host cells, it had no impact on the survival of P. berghei UIS4-HT liver stages. Furthermore, HaloPROTAC3 bound to P. berghei UIS4-HT but did not recruit VHL or trigger ubiquitination of the PVM. Overall, although this study did not establish whether host-driven TPD can degrade Plasmodium PVM proteins, it highlights the challenges of developing TPD approaches against novel targets and offers insights for advancing this therapeutic strategy against pathogens.

Following their inoculation by the bite of an infected Anopheles mosquito, the malaria parasite sporozoite forms travel from the bite site in the skin into the bloodstream, which transports them to the liver. The thrombospondin-related anonymous protein (TRAP) is a type 1 transmembrane protein that is released from secretory organelles and relocalized on the sporozoite plasma membrane. TRAP is required for sporozoite motility and host infection, and its extracellular portion contains adhesive domains that are predicted to engage host receptors. Here, we identified the human platelet-derived growth factor receptor β (hPDGFRβ) as one such protein receptor. Deletion constructs showed that the von Willebrand factor type A and thrombospondin repeat domains of TRAP are both required for optimal binding to hPDGFRβ-expressing cells. We also demonstrate that this interaction is conserved in the human-infective parasite Plasmodium vivax , but not the rodent-infective parasite Plasmodium yoelii . We observed expression of hPDGFRβ mainly in cells associated with the vasculature suggesting that TRAP:hPDGFRβ interaction may play a role in the recognition of blood vessels by invading sporozoites.

Background The potential benefits of long-acting injectable chemoprotection (LAI-C) against malaria have been recently recognized, prompting a call for suitable candidate drugs to help meet this need. On the basis of its known pharmacodynamic and pharmacokinetic profiles after oral dosing, ELQ-331, a prodrug of the parasite mitochondrial electron transport inhibitor ELQ-300, was selected for study of pharmacokinetics and efficacy as LAI-C in mice. Methods Four trials were conducted in which mice were injected with a single intramuscular dose of ELQ-331 or other ELQ-300 prodrugs in sesame oil with 1.2% benzyl alcohol; the ELQ-300 content of the doses ranged from 2.5 to 30 mg/kg. Initial blood stage challenges with Plasmodium yoelii were used to establish the model, but the definitive study measure of efficacy was outcome after sporozoite challenge with a luciferase-expressing P. yoelii , assessed by whole-body live animal imaging. Snapshot determinations of plasma ELQ-300 concentration ([ELQ-300]) were made after all prodrug injections; after the highest dose of ELQ-331 (equivalent to 30 mg/kg ELQ-300), both [ELQ-331] and [ELQ-300] were measured at a series of timepoints from 6 h to 5½ months after injection. Results A single intramuscular injection of ELQ-331 outperformed four other ELQ-300 prodrugs and, at a dose equivalent to 30 mg/kg ELQ-300, protected mice against challenge with P. yoelii sporozoites for at least 4½ months. Pharmacokinetic evaluation revealed rapid and essentially complete conversion of ELQ-331 to ELQ-300, a rapidly achieved (< 6 h) and sustained (4–5 months) effective plasma ELQ-300 concentration, maximum ELQ-300 concentrations far below the estimated threshold for toxicity, and a distinctive ELQ-300 concentration versus time profile. Pharmacokinetic modeling indicates a high-capacity, slow-exchange tissue compartment which serves to accumulate and then slowly redistribute ELQ-300 into blood, and this property facilitates an extremely long period during which ELQ-300 concentration is sustained above a minimum fully-protective threshold (60–80 nM). Conclusions Extrapolation of these results to humans predicts that ELQ-331 should be capable of meeting and far-exceeding currently published duration-of-effect goals for anti-malarial LAI-C. Furthermore, the distinctive pharmacokinetic profile of ELQ-300 after treatment with ELQ-331 may facilitate durable protection and enable protection for far longer than 3 months. These findings suggest that ELQ-331 warrants consideration as a leading prototype for LAI-C.

Background/Objectives: Malaria, caused by infection with Plasmodium parasites, exacts a heavy toll worldwide. There are two licensed vaccines for malaria as well as two monoclonal antibodies that have shown promising efficacy in field trials. The vaccines and monoclonal antibodies target the major surface protein (circumsporozoite protein, CSP) of Plasmodium falciparum. Yet P. falciparum is only one of the four major species of Plasmodium that infect humans. Plasmodium vivax is the second leading cause of malaria, but the P. vivax vaccine and monoclonal development lags far behind that for P. falciparum owing to the lack of basic preclinical tools such as in vitro culture or mouse models that replicate the key biological features of P. vivax. Notably among these features is the ability to form dormant liver stages (hypnozoites) that reactivate and drive the majority of the P. vivax malaria burden. Plasmodium cynomolgi is a simian parasite which is genotypically very close and phenotypically similar to P. vivax; it can infect non-human primates commonly used in research and replicates many features of P. vivax, including relapsing hypnozoites. Methods: Recently, a strain of P. cynomolgi has been adapted to in vitro cultures allowing parasite transgenesis. Here, we created a transgenic P. cynomolgi parasite in which the endogenous P. cynomolgi CSP has been replaced with P. vivax CSP, with the goal of enabling the preclinical study of anti-P. vivax CSP interventions to protect against primary and relapse infections. Results: We show that the in vitro-generated transgenic Pcy[PvCSP] parasite expresses both serotypes of P. vivax CSP and retains full functionality in vivo, including the ability to transmit to laboratory-reared Anopheles mosquitoes and cause relapsing infections in rhesus macaques. To our knowledge, this is the first gene replacement in a relapsing Plasmodium species. Conclusions: This work can directly enable the in vivo development of anti-P. vivax CSP interventions and provide a blueprint for the study of relapsing malaria through reverse genetics.

Chimeric rodent malaria parasites with the endogenous circumsporozoite protein ( csp ) gene replaced with csp from the human parasites Plasmodium falciparum ( Pf ) and P. vivax ( Pv ) are used in preclinical evaluation of CSP vaccines. Chimeric rodent parasites expressing Pf CSP have also been assessed as whole sporozoite (WSP) vaccines. Comparable chimeric P. falciparum parasites expressing CSP of P. vivax could be used both for clinical evaluation of vaccines targeting Pv CSP in controlled human P. falciparum infections and in WSP vaccines targeting P. vivax and P. falciparum . We generated chimeric P. falciparum parasites expressing both Pf CSP and Pv CSP. These Pf - Pv CSP parasites produced sporozoite comparable to wild type P. falciparum parasites and expressed Pf CSP and Pv CSP on the sporozoite surface. Pf - Pv CSP sporozoites infected human hepatocytes and induced antibodies to the repeats of both Pf CSP and Pv CSP after immunization of mice. These results support the use of Pf - Pv CSP sporozoites in studies optimizing vaccines targeting Pv CSP.

Vaccine-induced sterilizing protection from infection by Plasmodium parasites, the pathogens that cause malaria, will be essential in the fight against malaria as it would prevent both malaria-related disease and transmission. Stopping the relatively small number of parasites injected by the mosquito before they can migrate from the skin to the liver is an attractive means to this goal. Antibody-eliciting vaccines have been used to pursue this objective by targeting the major parasite surface protein present during this stage, the circumsporozoite protein (CSP). While CSP-based vaccines have recently had encouraging success in disease reduction, this was only achieved with extremely high antibody titers and appeared less effective for a complete block of infection (i.e., sterile protection). While such disease reduction is important, these and other results indicate that strategies focusing on CSP alone may not achieve the high levels of sterile protection needed for malaria eradication. Here, we show that monoclonal antibodies (mAbs) recognizing another sporozoite protein, TRAP/SSP2, exhibit a range of inhibitory activity and that these mAbs may augment CSP-based protection despite conferring no sterile protection on their own. Therefore, pursuing a multivalent subunit vaccine immunization is a promising strategy for improving infection-blocking malaria vaccines.

New therapeutics are necessary for preventing Plasmodium vivax malaria due to easy transmissibility and dormancy in the liver that increases the clinical burden due to recurrent relapse. In this manuscript we characterize 12 Pv Apical Membrane Antigen 1 (PvAMA1) specific human monoclonal antibodies from Peripheral Blood Mononuclear Cells of a Pv-exposed individual. PvAMA1 is essential for sporozoite and merozoite invasion, making it a unique therapeutic target. We show that humAb 826827 blocks the invasion of human reticulocytes using Pv clinical isolates and inhibits sporozoite invasion of human hepatocytes in vitro (IC 50 of 0.3 – 3.7 µg/mL). Inoculation of human liver transgenic (FRG-humHep) female mice with humAb 826827 significantly reduces liver infection in vivo. The crystal structure of rPvAMA1 bound to 826827 shows that 826827 partially occupies the highly conserved hydrophobic groove in PvAMA1 that binds its known receptor, RON2. We have isolated a potent humAb that is isolate-transcendent, blocks both pre-erythrocytic and blood stage infection, and could be a potential therapy for Pv. Here the authors isolate monoclonal antibodies specific for Plasmodium vivax apical membrane antigen 1, and characterize the epitope of one antibody that inhibits invasion of reticulocytes and hepatocytes, and reduces liver infection in a mouse model.