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Only a small fraction of the antigens expressed by malaria parasites have been evaluated as vaccine candidates. A successful malaria subunit vaccine will likely require multiple antigenic targets to achieve broad protection with high protective efficacy. Here we describe protective efficacy of a novel antigen, Plasmodium yoelii (Py) E140 (PyE140), evaluated against P. yoelii challenge of mice. Vaccines targeting PyE140 reproducibly induced up to 100% sterile protection in both inbred and outbred murine challenge models. Although PyE140 immunization induced high frequency and multifunctional CD8+ T cell responses, as well as CD4+ T cell responses, protection was mediated by PyE140 antibodies acting against blood stage parasites. Protection in mice was long-lasting with up to 100% sterile protection at twelve weeks post-immunization and durable high titer anti-PyE140 antibodies. The E140 antigen is expressed in all Plasmodium species, is highly conserved in both P. falciparum lab-adapted strains and endemic circulating parasites, and is thus a promising lead vaccine candidate for future evaluation against human malaria parasite species., Competing Interests: The authors have read the journal’s policy and have the following conflicts: ECS, KJL, and JCA are named inventors on a patent application US PCT/US18/25510 covering methods and compositions for vaccinating against malaria. JCA is a named inventor on US patent number 10, 195, 260, US Patent Application No. 16/230,512, and related foreign applications covering the PfE140 antigen. NR, KO, JW, and JCA are/were employed by CAMRIS International, which is a commercial company that owns the rights to the PfE140 antigen under patent number 10, 195, 260. This does not alter our adherence to PLOS ONE policies on sharing data and materials. ERPi Inc., Meso Scale Diagnostics, Sanaria, and General Dynamics Information Technology Inc. are current affiliations for some authors who previously contributed to the study. Neither ERPi Inc., Meso Scale Diagnostics, Sanaria, nor General Dynamics Information Technology Inc. made any contribution to the study.

Malaria is a devastating disease resulting in significant morbidity and mortality, especially in the developing world. Previously, we showed that the gut microbiome modulates severity of malaria in mice, though the exact mechanism was unknown. One well-studied mechanism by which the intestinal microbiota exerts an effect on host health is by synthesis of short-chain fatty acids (SCFAs). SCFAs have pleiotropic effects on the host, including modulating the immune system and altering susceptibility to pathogens. The objective of the current work was to explore if gut microbiota-mediated resistance and susceptibility to malaria in mice is through differential production of SCFAs. Of the eight detected SCFAs, only propionic acid (C3) was different between two groups of resistant and two groups of susceptible mice, with higher levels in feces of susceptible mice compared to resistant mice. Nevertheless, subsequent analysis revealed no robust correlation between malaria severity and levels of fecal propionic acid. In spite of the broad effect of SCFAs on host physiology, including host immunity, this study shows that gut microbiota-mediated modulation of malaria severity in mice is independent of fecal SCFA levels. Additionally, our data indicates that intestinal SCFAs do not function as biomarkers for prediction of malaria disease severity., Competing Interests: The authors have declared that no competing interests exist.

The emergence of malaria pathogens having resistance against antimalarials implies the necessity for the development of new drugs. Recently, we have demonstrated a resistance against malaria infection of α-tocopherol transfer protein knockout mice showing undetectable plasma levels of α-tocopherol, a lipid-soluble antioxidant. However, dietary restriction induced α-tocopherol deficiency is difficult to be applied as a clinical antimalarial therapy. Here, we report on a new strategy to potentially treat malaria by using probucol, a drug that can reduce the plasma α-tocopherol concentration. Probucol pre-treatment for 2 weeks and treatment throughout the infection rescued from death of mice infected with Plasmodium yoelii XL-17 or P. berghei ANKA. In addition, survival was extended when the treatment started immediately after parasite inoculation. The ratio of lipid peroxidation products to parent lipids increased in plasma after 2 weeks treatment of probucol. This indicates that the protective effect of probucol might be mediated by the oxidative stressful environment induced by α-tocopherol deficiency. Probucol in combination with dihydroartemisin suppressed the proliferation of P. yoelii XL-17. These results indicated that probucol might be a candidate for a drug against malaria infection by inducing α-tocopherol deficiency without dietary α-tocopherol restriction.

The pre-erythrocytic stages of Plasmodium spp. are increasingly recognised as ideal targets for prophylactic vaccines and drug treatments. Intense research efforts in the last decade have been focused on in vitro culture and in vivo detection and quantification of liver stage parasites to assess the effects of candidate vaccines or drugs. Typically, the onset of blood stage parasitaemia is used as a surrogate endpoint to estimate the efficacy of vaccines and drugs targeting pre-erythrocytic parasite stages in animal models. However, this provides no information on the parasite burden in the liver after vaccination or treatment and therefore does not detect partial efficacy of any vaccine or drug candidates. Herein, we describe a quantitative RT-PCR method adapted to detect and quantitate Plasmodium yoelii liver stages in mice with increased sensitivity even after challenge with as few as 50 cryopreserved sporozoites (corresponding to approximately 5-10 freshly isolated sporozoites). We have validated our quantitative RT-PCR assay according to the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines and established high reproducibility and accuracy. Our assay provides a rapid and reproducible assessment of liver stage parasite burden in rodent malaria models, thereby facilitating the evaluation of the efficacy of anti-malarial drugs or prophylactic vaccines with high precision and efficacy.

Plasmodium falciparum malaria remains one of the most serious health problems globally and a protective malaria vaccine is desperately needed. Vaccination with attenuated parasites elicits multiple cellular effector mechanisms that lead to Plasmodium liver stage elimination. While granule-mediated cytotoxicity requires contact between CD8+ effector T cells and infected hepatocytes, cytokine secretion should allow parasite killing over longer distances. To better understand the mechanism of parasite elimination in vivo, we monitored the dynamics of CD8+ T cells in the livers of naïve, immunized and sporozoite-infected mice by intravital microscopy. We found that immunization of BALB/c mice with attenuated P. yoelii 17XNL sporozoites significantly increases the velocity of CD8+ T cells patrolling the hepatic microvasculature from 2.69±0.34 μm/min in naïve mice to 5.74±0.66 μm/min, 9.26±0.92 μm/min, and 7.11±0.73 μm/min in mice immunized with irradiated, early genetically attenuated (Pyuis4-deficient), and late genetically attenuated (Pyfabb/f-deficient) parasites, respectively. Sporozoite infection of immunized mice revealed a 97% and 63% reduction in liver stage density and volume, respectively, compared to naïve controls. To examine cellular mechanisms of immunity in situ, naïve mice were passively immunized with hepatic or splenic CD8+ T cells. Unexpectedly, adoptive transfer rendered the motile CD8+ T cells from immunized mice immotile in the liver of P. yoelii infected mice. Similarly, when mice were simultaneously inoculated with viable sporozoites and CD8+ T cells, velocities 18 h later were also significantly reduced to 0.68±0.10 μm/min, 1.53±0.22 μm/min, and 1.06±0.26 μm/min for CD8+ T cells from mice immunized with irradiated wild type sporozoites, Pyfabb/f-deficient parasites, and P. yoelii CS280-288 peptide, respectively. Because immobilized CD8+ T cells are unable to make contact with infected hepatocytes, soluble mediators could potentially play a key role in parasite elimination under these experimental conditions.

Anopheline mosquitoes are the major vectors of human malaria. Parasite-mosquito interactions are a critical aspect of disease transmission and a potential target for malaria control. Current investigations into parasite-mosquito interactions frequently assume that genetically resistant and susceptible mosquitoes exist in nature. Therefore, comparisons between the Plasmodium susceptibility profiles of different mosquito species may contribute to a better understanding of vectorial capacity. Anopheles stephensi is an important malaria vector in central and southern Asia and is widely used as a laboratory model of parasite transmission due to its high susceptibility to Plasmodium infection. In the present study, we identified a rodent malaria-refractory strain of A. stephensi mysorensis (Ehime) by comparative study of infection susceptibility. A very low number of oocysts develop in Ehime mosquitoes infected with P. berghei and P. yoelii, as determined by evaluation of developed oocysts on the basal lamina. A stage-specific study revealed that this reduced susceptibility was due to the impaired formation of ookinetes of both Plasmodium species in the midgut lumen and incomplete crossing of the midgut epithelium. There were no apparent abnormalities in the exflagellation of male parasites in the ingested blood or the maturation of oocysts after the rounding up of the ookinetes. Overall, these results suggest that invasive-stage parasites are eliminated in both the midgut lumen and epithelium in Ehime mosquitoes by strain-specific factors that remain unknown. The refractory strain newly identified in this report would be an excellent study system for investigations into novel parasite-mosquito interactions in the mosquito midgut.

In order to propagate within the mammalian host, malaria parasites must invade red blood cells (RBCs). This process offers a window of opportunity in which to target the parasite with drugs or vaccines. However, most of the studies relating to RBC invasion have analyzed the molecular interactions of parasite proteins with host cells under static conditions, and the dynamics of these interactions remain largely unstudied. Time-lapse imaging of RBC invasion is a powerful technique to investigate cell invasion and has been reported for Plasmodium knowlesi and Plasmodium falciparum. However, experimental modification of genetic loci is laborious and time consuming for these species. We have established a system of time-lapse imaging for the rodent malaria parasite Plasmodium yoelii, for which modification of genetic loci is quicker and simpler. We compared the kinetics of RBC invasion by P. yoelii with that of P. falciparum and found that the overall kinetics during invasion were similar, with some exceptions. The most striking of these differences is that, following egress from the RBC, the shape of P. yoelii merozoites gradually changes from flat elongated ovals to spherical bodies, a process taking about 60 sec. During this period merozoites were able to attach to and deform the RBC membrane, but were not able to reorient and invade. We propose that this morphological change of P. yoelii merozoites may be related to the secretion or activation of invasion-related proteins. Thus the P. yoelii merozoite appears to be an excellent model to analyze the molecular dynamics of RBC invasion, particularly during the morphological transition phase, which could serve as an expanded window that cannot be observed in P. falciparum.

It was recently reported that when mosquitoes infected with P. berghei sporozoites feed on mice, they deposit approximately 100-300 sporozoites in the dermis. When we inoculate P. yoelii (Py) sporozoites intravenously (IV) into BALB/c mice, the 50% infectious dose (ID(50)) is often less than 3 sporozoites, indicating that essentially all Py sporozoites in salivary glands are infectious. Thus, it should only take the bite of one infected mosquito to infect 100% of mice. In human subjects, it takes the bite of at least 5 P. falciparum-infected mosquitoes to achieve 100% blood stage infection. Exposure to 1-2 infected mosquitoes only leads to blood stage infection in approximately 50% of subjects. If mosquitoes carrying Py sporozoites inoculate 100-300 sporozoites per bite, and 1 to 2 mosquito bites achieve 50% blood stage infection rates, then this would suggest that the majority of sporozoites inoculated by mosquitoes into the dermis are not responsible for a productive infection, or that a significant number of sporozoite-infected mosquitoes do not inoculate any sporozoites. The objective of this study was to determine if this is the case. We therefore studied the infectivity to mice of the bites of 1, 2, 4, or 5-8 Py-infected mosquitoes. The bite of one Py sporozoite-infected mosquito caused blood stage infection in 41.4% (12/29) of mice, two bites infected 66.7% (22/33), four bites infected 75% (18/24), and five to eight bites infected 100% (21/21). These findings demonstrate that inoculation of sporozoites by mosquito bite is much less efficient than IV inoculation of Py sporozoites by needle and syringe. Such data may have implications for determining the best route and dose of administration to humans of our attenuated P. falciparum sporozoite vaccine, the scientific basis of which is immunity by bites from irradiated infected mosquitoes, and suggest that the challenge is to develop a method of administration that approximates IV inoculation, not one that mimics mosquito bite.