Plasmodium Berghei Sporozoites Research Articles

Late sporogonic stages of Plasmodium parasites are susceptible to the melanization response in Anopheles gambiae mosquitoes.

The malaria-causing parasites have to complete a complex infection cycle in the mosquito vector that also involves attack by the insect's innate immune system, especially at the early stages of midgut infection. However, Anopheles immunity to the late Plasmodium sporogonic stages, such as oocysts, has received little attention as they are considered to be concealed from immune factors due to their location under the midgut basal lamina and for harboring an elaborate cell wall comprising an external layer derived from the basal lamina that confers self-properties to an otherwise foreign structure. Here, we investigated whether Plasmodium berghei oocysts and sporozoites are susceptible to melanization-based immunity in Anopheles gambiae. Silencing of the negative regulator of melanization response, CLIPA14, increased melanization prevalence without significantly increasing the numbers of melanized oocysts, while co-silencing CLIPA14 with CLIPA2, a second negative regulator of melanization, resulted in a significant increase in melanized oocysts and melanization prevalence. Only late-stage oocysts were found to be melanized, suggesting that oocyst rupture was a prerequisite for melanization-based immune attack, presumably due to the loss of the immune-evasive features of their wall. We also found melanized sporozoites inside oocysts and in the hemocoel, suggesting that sporozoites at different maturation stages are susceptible to melanization. Silencing the melanization promoting factors TEP1 and CLIPA28 rescued oocyst melanization in CLIPA2/CLIPA14 co-silenced mosquitoes. Interestingly, silencing of CTL4, that protects early stage ookinetes from melanization, had no effect on oocysts and sporozoites, indicating differential regulation of immunity to early and late sporogonic stages. Similar to previous studies addressing ookinete stage melanization, the melanization of Plasmodium falciparum oocysts was significantly lower than that observed for P. berghei. In summary, our results provide conclusive evidence that late sporogonic malaria parasite stages are susceptible to melanization, and we reveal distinct regulatory mechanisms for ookinete and oocyst melanization.

A two-dose viral-vectored Plasmodium vivax multistage vaccine confers durable protection and transmission-blockade in a pre-clinical study.

Among Plasmodium spp. responsible for human malaria, Plasmodium vivax ranks as the second most prevalent and has the widest geographical range; however, vaccine development has lagged behind that of Plasmodium falciparum, the deadliest Plasmodium species. Recently, we developed a multistage vaccine for P. falciparum based on a heterologous prime-boost immunization regimen utilizing the attenuated vaccinia virus strain LC16m8Δ (m8Δ)-prime and adeno-associated virus type 1 (AAV1)-boost, and demonstrated 100% protection and more than 95% transmission-blocking (TB) activity in the mouse model. In this study, we report the feasibility and versatility of this vaccine platform as a P. vivax multistage vaccine, which can provide 100% sterile protection against sporozoite challenge and >95% TB efficacy in the mouse model. Our vaccine comprises m8Δ and AAV1 viral vectors, both harboring the gene encoding two P. vivax circumsporozoite (PvCSP) protein alleles (VK210; PvCSP-Sal and VK247; -PNG) and P25 (Pvs25) expressed as a Pvs25-PvCSP fusion protein. For protective efficacy, the heterologous m8Δ-prime/AAV1-boost immunization regimen showed 100% (short-term; Day 28) and 60% (long-term; Day 242) protection against PvCSP VK210 transgenic Plasmodium berghei sporozoites. For TB efficacy, mouse sera immunized with the vaccine formulation showed >75% TB activity and >95% transmission reduction activity by a direct membrane feeding assay using P. vivax isolates in blood from an infected patient from the Brazilian Amazon region. These findings provide proof-of-concept that the m8Δ/AAV1 vaccine platform is sufficiently versatile for P. vivax vaccine development. Future studies are needed to evaluate the safety, immunogenicity, vaccine efficacy, and synergistic effects on protection and transmission blockade in a non-human primate model for Phase I trials.

Non-clinical toxicity and immunogenicity evaluation of a Plasmodium vivax malaria vaccine using Poly-ICLC (Hiltonol®) as adjuvant

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.

Protein O-Fucosyltransferase Is Required for the Efficient Invasion of Hepatocytes by Plasmodium berghei Sporozoites.

The O-fucosylation of the thrombospondin type I repeat (TSR) domain is important for TSR-containing proteins' optimal folding and stability. However, the importance of Plasmodium O-fucosyltransferase 2 (POFut2) remains unclear due to two different reports. Here, we disrupted the POFut2 gene in Plasmodium berghei and demonstrated that POFut2 KO parasites develop normally in blood and mosquito stages but show reduced infectivity in mice. We found that the reduced infectivity of POFut2 KO sporozoites was due to a diminished level of TRAP that affected the parasite gliding motility and hepatocyte infectivity. Using all-atom MD simulation, we also hypothesize that O-fucosylation impacts the TSR domain's stability more than its heparin binding capacity.

Establishing RTS,S/AS01 as a benchmark for comparison to next-generation malaria vaccines in a mouse model

New strategies are needed to reduce the incidence of malaria, and promising approaches include vaccines targeting the circumsporozoite protein (CSP). To improve upon the malaria vaccine, RTS,S/AS01, it is essential to standardize preclinical assays to measure the potency of next-generation vaccines against this benchmark. We focus on RTS,S/AS01-induced antibody responses and functional activity in conjunction with robust statistical analyses. Transgenic Plasmodium berghei sporozoites containing full-length P. falciparum CSP (tgPb-PfCSP) allow two assessments of efficacy: quantitative reduction in liver infection following intravenous challenge, and sterile protection from mosquito bite challenge. Two or three doses of RTS,S/AS01 were given intramuscularly at 3-week intervals, with challenge 2-weeks after the last vaccination. Minimal inter- and intra-assay variability indicates the reproducibility of the methods. Importantly, the range of this model is suitable for screening more potent vaccines. Levels of induced anti-CSP antibody 2A10 equivalency were also associated with activity: 105 μg/mL (95% CI: 68.8, 141) reduced liver infection by 50%, whereas 285 μg/mL (95% CI: 166, 404) is required for 50% sterile protection from mosquito bite challenge. Additionally, the liver burden model was able to differentiate between protected and non-protected human plasma samples from a controlled human malaria infection study, supporting these models’ relevance and predictive capability. Comparison in animal models of CSP-based vaccine candidates to RTS,S/AS01 is now possible under well controlled conditions. Assessment of the quality of induced antibodies, likely a determinant of durability of protection in humans, should be possible using these methods.

Late sporogonic stages of Plasmodium parasites are susceptible to the melanization response in Anopheles gambiae mosquitoes.

The malaria-causing parasites have to complete a complex infection cycle in the mosquito vector that also involves attack by the insect's innate immune system, especially at the early stages of midgut infection. However, Anopheles immunity to the late Plasmodium sporogonic stages, such as oocysts, has received little attention as they are considered to be concealed from immune factors due to their location under the midgut basal lamina and for harboring an elaborate cell wall comprising an external layer derived from the basal lamina that confers self-properties to an otherwise foreign structure. Here, we investigated whether Plasmodium berghei oocysts and sporozoites are susceptible to melanization-based immunity in Anopheles gambiae. Silencing of the negative regulator of melanization response, CLIPA14, increased melanization prevalence without significantly increasing the numbers of melanized oocysts, while co-silencing CLIPA14 with CLIPA2, a second negative regulator of melanization, resulted in a significant increase in melanized oocysts and melanization prevalence. Only late-stage oocysts were found to be melanized, suggesting that oocyst rupture was a prerequisite for melanization-based immune attack, presumably due to the loss of the immune-evasive features of their wall. We also found melanized sporozoites inside oocysts and in the hemocoel, suggesting that sporozoites at different maturation stages are susceptible to melanization. Silencing the melanization promoting factors TEP1 and CLIPA28 rescued oocyst melanization in CLIPA2/CLIPA14 co-silenced mosquitoes. Interestingly, silencing of CTL4, that protects early stage ookinetes from melanization, had no effect on oocysts and sporozoites, indicating differential regulation of immunity to early and late sporogonic stages. Similar to previous studies addressing ookinete stage melanization, the melanization of Plasmodium falciparum oocysts was significantly lower than that observed for P. berghei. In summary, our results provide conclusive evidence that late sporogonic malaria parasite stages are susceptible to melanization, and we reveal distinct regulatory mechanisms for ookinete and oocyst melanization.

Protective antibody threshold of RTS,S/AS01 malaria vaccine correlates antigen and adjuvant dose in mouse model

Mouse models are useful for the early down-selection of malaria vaccine candidates. The Walter Reed Army Institute of Research has optimized a transgenic Plasmodium berghei sporozoite challenge model to compare the efficacy of Plasmodium falciparum circumsporozoite protein (CSP) vaccines. GSK’s RTS,S vaccine formulated in the adjuvant AS01 can protect malaria-naïve individuals against malaria. We report that the RTS,S/AS01 vaccine induces high level sterile protection in our mouse model. Down titration of the antigen at a constant AS01 dose revealed a potent antigen dose-sparing effect and the superiority of RTS,S/AS01 over a soluble CSP antigen. RTS,S-mediated protective immunity was associated with a threshold of major repeat antibody titer. Combined titration of the antigen and adjuvant showed that reducing the adjuvant could improve antibody boosting post-3rd vaccination and reduce the threshold antibody concentration required for protection. Mouse models can provide a pathway for preclinical assessment of strategies to improve CSP vaccines against malaria.

MRNA vaccine against malaria tailored for liver-resident memory T cells.

Malaria is caused by Plasmodium species transmitted by Anopheles mosquitoes. Following a mosquito bite, Plasmodium sporozoites migrate from skin to liver, where extensive replication occurs, emerging later as merozoites that can infect red blood cells and cause symptoms of disease. As liver tissue-resident memory T cells (Trm cells) have recently been shown to control liver-stage infections, we embarked on a messenger RNA (mRNA)-based vaccine strategy to induce liver Trm cells to prevent malaria. Although a standard mRNA vaccine was unable to generate liver Trm or protect against challenge with Plasmodium berghei sporozoites in mice, addition of an agonist that recruits T cell help from type I natural killer T cells under mRNA-vaccination conditions resulted in significant generation of liver Trm cells and effective protection. Moreover, whereas previous exposure of mice to blood-stage infection impaired traditional vaccines based on attenuated sporozoites, mRNA vaccination was unaffected, underlining the potential for such a rational mRNA-based strategy in malaria-endemic regions.

Generating Genetically Modified Plasmodium berghei Sporozoites.

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of Plasmodium deposited by mosquitoes in the skin of vertebrate hosts undergoes a phase of mandatory development in the liver before initiating clinical malaria. We know little about the biology of Plasmodium development in the liver; access to the sporozoite stage and the ability to genetically modify such sporozoites are critical tools for studying the nature of Plasmodium infection and the resulting immune response in the liver. Here, we present a comprehensive protocol for the generation of transgenic Plasmodium berghei sporozoites. We genetically modify blood-stage P. berghei and use this form to infect Anopheles mosquitoes when they take a blood meal. After the transgenic parasites undergo development in the mosquitoes, we isolate the sporozoite stage of the parasite from the mosquito salivary glands for in vivo and in vitro experimentation. We demonstrate the validity of the protocol by generating sporozoites of a novel strain of P. berghei expressing the green fluorescent protein (GFP) subunit 11 (GFP11), and show how it could be used to investigate the biology of liver-stage malaria.

Preliminary Assessment of Intramuscular Depot of Lipid-Based Decoquinate Formulation for Long-Term Chemoprophylaxis of Malaria.

Sustained-release formulations of decoquinate were evaluated for the long-term prophylaxis of malaria. In the initial experiment, mice were protected from liver-stage Plasmodium infection by intramuscular administration of a lipids-based formulation at a dose of decoquinate 200 mg/kg. The mice that were inoculated with Plasmodium berghei sporozoites 34 days after the administration of a one-time drug dose were continuously monitored for 60 days and shown to be free of Plasmodium parasites. The optimized formulation for the sustained release of decoquinate was prepared by hot melt extrusion, constructed by lipids including cholesterol and mono or diglycerides, and had a drug load of 20 to 40% and particle size of 30 to 50 μm. Decoquinate of the lipids-based formulation was slowly released in vitro at a constant rate for the duration of two months, and was examined and continuously exposed at a therapeutic level in the blood for as long as 4 to 6 months. Further evaluation showed that the lipids-based formulation at doses of decoquinate 100 to 150 mg/kg could protect mice from Plasmodium infection for a period of 120 days. It is the first time that cholesterol has been used for a controlled drug delivery system of decoquinate. The results may provide useful information, not only for preparing a formulation of long-acting decoquinate but also in general for developing a controlled drug release system. The one-time administration of pharmaceutical agents in such a slow-release system may serve patients with no concerns about compliance.

Interactions with Asialo-Glycoprotein Receptors and Platelets Are Dispensable for CD8+ T Cell Localization in the Murine Liver.

Liver-resident CD8+ T cells can play critical roles in the control of pathogens, including Plasmodium and hepatitis B virus. Paradoxically, it has also been proposed that the liver may act as the main place for the elimination of CD8+ T cells at the resolution of immune responses. We hypothesized that different adhesion processes may drive residence versus elimination of T cells in the liver. Specifically, we investigated whether the expression of asialo-glycoproteins (ASGPs) drives the localization and elimination of effector CD8+ T cells in the liver, while interactions with platelets facilitate liver residence and protective function. Using murine CD8+ T cells activated in vitro, or in vivo by immunization with Plasmodium berghei sporozoites, we found that, unexpectedly, inhibition of ASGP receptors did not inhibit the accumulation of effector cells in the liver, but instead prevented these cells from accumulating in the spleen. In addition, enforced expression of ASGP on effector CD8+ T cells using St3GalI-deficient cells lead to their loss from the spleen. We also found, using different mouse models of thrombocytopenia, that severe reduction in platelet concentration in circulation did not strongly influence the residence and protective function of CD8+ T cells in the liver. These data suggest that platelets play a marginal role in CD8+ T cell function in the liver. Furthermore, ASGP-expressing effector CD8+ T cells accumulate in the spleen, not the liver, prior to their destruction.

Imaging Infection by Vector-Borne Protozoan Parasites Using Whole-Mouse Bioluminescence.

Vector-borne protozoan parasites such as Plasmodium spp. Leishmaniaspp. and Trypanosoma brucei are responsible for several serious diseases. Significant advances in parasitology have been made using rodent models combined with live imaging techniques, including whole-mouse bioluminescence imaging (BLI). This technique has been applied to investigate parasite dissemination, infectivity, and growth. It has also been used in drug and vaccine testing. This chapter focuses on the methods that utilize whole-mouse BLI to (i) evaluate the homing and infectivity of Plasmodium berghei sporozoites; (ii) conductin vivo testing of promising chemical entities against Leishmania infantum infection; and (iii) study molecular mechanisms of host susceptibility to Trypanosoma brucei brucei infection.

Immunomodulation by Mosquito Salivary Protein AgSAP Contributes to Early Host Infection by Plasmodium.

ABSTRACTMalaria is caused when Plasmodium sporozoites are injected along with saliva by an anopheline mosquito into the dermis of a vertebrate host. Arthropod saliva has pleiotropic effects that can influence local host responses, pathogen transmission, and exacerbation of the disease. A mass spectrometry screen identified mosquito salivary proteins that are associated with Plasmodium sporozoites during saliva secretions. In this study, we demonstrate that one of these salivary antigens, Anopheles gambiae sporozoite-associated protein (AgSAP), interacts directly with Plasmodium falciparum and Plasmodium berghei sporozoites. AgSAP binds to heparan sulfate and inhibits local inflammatory responses in the skin. The silencing of AgSAP in mosquitoes reduces their ability to effectively transmit sporozoites to mice. Moreover, immunization with AgSAP decreases the Plasmodium burden in mice that are bitten by Plasmodium-infected mosquitoes. These data suggest that AgSAP facilitates early Plasmodium infection in the vertebrate host and serves as a target for the prevention of malaria.

Dissection-independent production of Plasmodium sporozoites from whole mosquitoes.

Progress towards a protective vaccine against malaria remains slow. To date, only limited protection has been routinely achieved following immunisation with either whole-parasite (sporozoite) or subunit-based vaccines. One major roadblock to vaccine progress, and to pre-erythrocytic parasite biology in general, is the continued reliance on manual salivary gland dissection for sporozoite isolation from infected mosquitoes. Here, we report development of a multi-step method, based on batch processing of homogenised whole mosquitoes, slurry, and density-gradient filtration, which combined with free-flow electrophoresis rapidly produces a pure, infective sporozoite inoculum. Human-infective Plasmodium falciparum and rodent-infective Plasmodium berghei sporozoites produced in this way are two- to threefold more infective than salivary gland dissection sporozoites in in vitro hepatocyte infection assays. In an in vivo rodent malaria model, the same P. berghei sporozoites confer sterile protection from mosquito-bite challenge when immunisation is delivered intravenously or 60-70% protection when delivered intramuscularly. By improving purity, infectivity, and immunogenicity, this method represents a key advancement in capacity to produce research-grade sporozoites, which should impact delivery of a whole-parasite based malaria vaccine at scale in the future.

Clustering and Erratic Movement Patterns of Syringe-Injected versus Mosquito-Inoculated Malaria Sporozoites Underlie Decreased Infectivity.

ABSTRACTMalaria vaccine candidates based on live, attenuated sporozoites have led to high levels of protection. However, their efficacy critically depends on the sporozoites’ ability to reach and infect the host liver. Administration via mosquito inoculation is by far the most potent method for inducing immunity but highly impractical. Here, we observed that intradermal syringe-injected Plasmodium berghei sporozoites (syrSPZ) were 3-fold less efficient in migrating to and infecting mouse liver than mosquito-inoculated sporozoites (msqSPZ). This was related to a clustered dermal distribution (2-fold-decreased median distance between syrSPZ and msqSPZ) and, more importantly, a 1.4-fold (significantly)-slower and more erratic movement pattern. These erratic movement patterns were likely caused by alteration of dermal tissue morphology (>15-μm intercellular gaps) due to injection of fluid and may critically decrease sporozoite infectivity. These results suggest that novel microvolume-based administration technologies hold promise for replicating the success of mosquito-inoculated live, attenuated sporozoite vaccines.IMPORTANCE Malaria still causes a major burden on global health and the economy. The efficacy of live, attenuated malaria sporozoites as vaccine candidates critically depends on their ability to migrate to and infect the host liver. This work sheds light on the effect of different administration routes on sporozoite migration. We show that the delivery of sporozoites via mosquito inoculation is more efficient than syringe injection; however, this route of administration is highly impractical for vaccine purposes. Using confocal microscopy and automated imaging software, we demonstrate that syringe-injected sporozoites do cluster, move more slowly, and display more erratic movement due to alterations in tissue morphology. These findings indicate that microneedle-based engineering solutions hold promise for replicating the success of mosquito-inoculated live, attenuated sporozoite vaccines.

Transcriptional heterogeneity and tightly regulated changes in gene expression during Plasmodium berghei sporozoite development

Despite the critical role of Plasmodium sporozoites in malaria transmission, we still know little about the mechanisms underlying their development in mosquitoes. Here, we use single-cell RNA sequencing to characterize the gene expression profiles of 16,038 Plasmodium berghei sporozoites isolated throughout their development from midgut oocysts to salivary glands, and from forced salivation experiments. Our results reveal a succession of tightly regulated changes in gene expression occurring during the maturation of sporozoites and highlight candidate genes that could play important roles in oocyst egress, sporozoite motility, and the mechanisms underlying the invasion of mosquito salivary glands and mammalian hepatocytes. In addition, the single-cell data reveal extensive transcriptional heterogeneity among parasites isolated from the same anatomical site, suggesting that Plasmodium development in mosquitoes is asynchronous and regulated by intrinsic as well as environmental factors. Finally, our analyses show a decrease in transcriptional activity preceding the translational repression observed in mature sporozoites and associated with their quiescent state in salivary glands, followed by a rapid reactivation of the transcriptional machinery immediately upon salivation.

Single-cell RNA sequencing reveals developmental heterogeneity among Plasmodium berghei sporozoites

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

A Multistage Formulation Based on Full-Length CSP and AMA-1 Ectodomain of Plasmodium vivax Induces High Antibody Titers and T-cells and Partially Protects Mice Challenged with a Transgenic Plasmodium berghei Parasite

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

Protective Malaria Vaccine in Mice Based on the Plasmodium vivax Circumsporozoite Protein Fused with the Mumps Nucleocapsid Protein.

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

An in vitro DNA Sensor-based Assay to Measure Receptor-specific Adhesion Forces of Eukaryotic Cells and Pathogens.

Motility of eukaryotic cells or pathogens within tissues is mediated by the turnover of specific interactions with other cells or with the extracellular matrix. Biophysical characterization of these ligand-receptor adhesions helps to unravel the molecular mechanisms driving migration. Traction force microscopy or optical tweezers are typically used to measure the cellular forces exerted by cells on a substrate. However, the spatial resolution of traction force microscopy is limited to ~2 µm and performing experiments with optical traps is very time-consuming. Here we present the production of biomimetic surfaces that enable specific cell adhesion via synthetic ligands and at the same time monitor the transmitted forces by using molecular tension sensors. The ligands were coupled to double-stranded DNA probes with defined force thresholds for DNA unzipping. Receptor-mediated forces in the pN range are thereby semi-quantitatively converted into fluorescence signals, which can be detected by standard fluorescence microscopy at the resolution limit (~0.2 µm). The modular design of the assay allows to vary the presented ligands and the mechanical strength of the DNA probes, which provides a number of possibilities to probe the adhesion of different eukaryotic cell types and pathogens and is exemplified here with osteosarcoma cells and Plasmodium berghei Sporozoites.