ResearchPad - sporozoites https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Novel malaria antigen <i>Plasmodium yoelii</i> E140 induces antibody-mediated sterile protection in mice against malaria challenge]]> https://www.researchpad.co/article/elastic_article_14592 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.

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<![CDATA[A probabilistic model of pre-erythrocytic malaria vaccine combination in mice]]> https://www.researchpad.co/article/5c3fa5a2d5eed0c484ca6b39

Malaria remains one the world’s most deadly infectious diseases, with almost half a million deaths and over 150 million clinical cases each year. An effective vaccine would contribute enormously to malaria control and will almost certainly be required for eventual eradication of the disease. However, the leading malaria vaccine candidate, RTS,S, shows only 30–50% efficacy under field conditions, making it less cost-effective than long-lasting insecticide treated bed nets. Other subunit malaria vaccine candidates, including TRAP-based vaccines, show no better protective efficacy. This has led to increased interest in combining subunit malaria vaccines as a means of enhancing protective efficacy. Mathematical models of the effect of combining such vaccines on protective efficacy can help inform optimal vaccine strategies and decision-making at all stages of the clinical process. So far, however, no such model has been developed for pre-clinical murine studies, the stage at which all candidate antigens and combinations begin evaluation. To address this gap, this paper develops a mathematical model of vaccine combination adapted to murine malaria studies. The model is based on simple probabilistic assumptions which put the model on a firmer theoretical footing than previous clinical models, which rather than deriving a relationship between immune responses and protective efficacy posit the relationship to be either exponential or Hill curves. Data from pre-clinical murine malaria studies are used to derive values for unknowns in the model which in turn allows simulations of vaccine combination efficacy and suggests optimal strategies to pursue. Finally, the ability of the model to shed light on fundamental biological variables of murine malaria such as the blood stage growth rate and sporozoite infectivity is explored.

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<![CDATA[Inter-subunit interactions drive divergent dynamics in mammalian and Plasmodium actin filaments]]> https://www.researchpad.co/article/5b6006f7463d7e39c55261ba

Cell motility is essential for protozoan and metazoan organisms and typically relies on the dynamic turnover of actin filaments. In metazoans, monomeric actin polymerises into usually long and stable filaments, while some protozoans form only short and highly dynamic actin filaments. These different dynamics are partly due to the different sets of actin regulatory proteins and partly due to the sequence of actin itself. Here we probe the interactions of actin subunits within divergent actin filaments using a comparative dynamic molecular model and explore their functions using Plasmodium, the protozoan causing malaria, and mouse melanoma derived B16-F1 cells as model systems. Parasite actin tagged to a fluorescent protein (FP) did not incorporate into mammalian actin filaments, and rabbit actin-FP did not incorporate into parasite actin filaments. However, exchanging the most divergent region of actin subdomain 3 allowed such reciprocal incorporation. The exchange of a single amino acid residue in subdomain 2 (N41H) of Plasmodium actin markedly improved incorporation into mammalian filaments. In the parasite, modification of most subunit–subunit interaction sites was lethal, whereas changes in actin subdomains 1 and 4 reduced efficient parasite motility and hence mosquito organ penetration. The strong penetration defects could be rescued by overexpression of the actin filament regulator coronin. Through these comparative approaches we identified an essential and common contributor, subdomain 3, which drives the differential dynamic behaviour of two highly divergent eukaryotic actins in motile cells.

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<![CDATA[Interventions that effectively target Anopheles funestus mosquitoes could significantly improve control of persistent malaria transmission in south–eastern Tanzania]]> https://www.researchpad.co/article/5989db5cab0ee8fa60bdfee7

Malaria is transmitted by many Anopheles species whose proportionate contributions vary across settings. We re-assessed the roles of Anopheles arabiensis and Anopheles funestus, and examined potential benefits of species-specific interventions in an area in south-eastern Tanzania, where malaria transmission persists, four years after mass distribution of long-lasting insecticide-treated nets (LLINs). Monthly mosquito sampling was done in randomly selected households in three villages using CDC light traps and back-pack aspirators, between January-2015 and January-2016, four years after the last mass distribution of LLINs in 2011. Multiplex polymerase chain reaction (PCR) was used to identify members of An. funestus and Anopheles gambiae complexes. Enzyme-linked immunosorbent assay (ELISA) was used to detect Plasmodium sporozoites in mosquito salivary glands, and to identify sources of mosquito blood meals. WHO susceptibility assays were done on wild caught female An. funestus s.l, and physiological ages approximated by examining mosquito ovaries for parity. A total of 20,135 An. arabiensis and 4,759 An. funestus were collected. The An. funestus group consisted of 76.6% An. funestus s.s, 2.9% An. rivulorum, 7.1% An. leesoni, and 13.4% unamplified samples. Of all mosquitoes positive for Plasmodium, 82.6% were An. funestus s.s, 14.0% were An. arabiensis and 3.4% were An. rivulorum. An. funestus and An. arabiensis contributed 86.21% and 13.79% respectively, of annual entomological inoculation rate (EIR). An. arabiensis fed on humans (73.4%), cattle (22.0%), dogs (3.1%) and chicken (1.5%), but An. funestus fed exclusively on humans. The An. funestus populations were 100% susceptible to organophosphates, pirimiphos methyl and malathion, but resistant to permethrin (10.5% mortality), deltamethrin (18.7%), lambda-cyhalothrin (18.7%) and DDT (26.2%), and had reduced susceptibility to bendiocarb (95%) and propoxur (90.1%). Parity rate was higher in An. funestus (65.8%) than An. arabiensis (44.1%). Though An. arabiensis is still the most abundant vector species here, the remaining malaria transmission is predominantly mediated by An. funestus, possibly due to high insecticide resistance and high survival probabilities. Interventions that effectively target An. funestus mosquitoes could therefore significantly improve control of persistent malaria transmission in south–eastern Tanzania.

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<![CDATA[Evaluation of Insecticides Susceptibility and Malaria Vector Potential of Anopheles annularis s.l. and Anopheles vagus in Assam, India]]> https://www.researchpad.co/article/5989d9e3ab0ee8fa60b6a2bc

During the recent past, development of DDT resistance and reduction to pyrethroid susceptibility among the malaria vectors has posed a serious challenge in many Southeast Asian countries including India. Current study presents the insecticide susceptibility and knock-down data of field collected Anopheles annularis sensu lato and An. vagus mosquito species from endemic areas of Assam in northeast India. Anopheles annularis s.l. and An. vagus adult females were collected from four randomly selected sentinel sites in Orang primary health centre (OPHC) and Balipara primary health centre (BPHC) areas, and used for testing susceptibility to DDT, malathion, deltamethrin and lambda-cyhalothrin. After insecticide susceptibility tests, mosquitoes were subjected to VectorTest assay kits to detect the presence of malaria sporozoite in the mosquitoes. An. annularis s.l. was completely susceptible to deltamethrin, lambda-cyhalothrin and malathion in both the study areas. An. vagus was highly susceptible to deltamethrin in both the areas, but exhibited reduced susceptibility to lambda-cyhalothrin in BPHC. Both the species were resistant to DDT and showed very high KDT50 and KDT99 values for DDT. Probit model used to calculate the KDT50 and KDT99 values did not display normal distribution of percent knock-down with time for malathion in both the mosquito species in OPHC (p<0.05) and An. vagus in BPHC (χ2 = 25.3; p = 0.0), and also for deltamethrin to An. vagus in BPHC area (χ2 = 15.4; p = 0.004). Minimum infection rate (MIR) of Plasmodium sporozoite for An. vagus was 0.56 in OPHC and 0.13 in BPHC, while for An. annularis MIR was found to be 0.22 in OPHC. Resistance management strategies should be identified to delay the expansion of resistance. Testing of field caught Anopheles vectors from different endemic areas for the presence of malaria sporozoite may be useful to ensure their role in malaria transmission.

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<![CDATA[A unique profilin-actin interface is important for malaria parasite motility]]> https://www.researchpad.co/article/5989db5cab0ee8fa60be0243

Profilin is an actin monomer binding protein that provides ATP-actin for incorporation into actin filaments. In contrast to higher eukaryotic cells with their large filamentous actin structures, apicomplexan parasites typically contain only short and highly dynamic microfilaments. In apicomplexans, profilin appears to be the main monomer-sequestering protein. Compared to classical profilins, apicomplexan profilins contain an additional arm-like β-hairpin motif, which we show here to be critically involved in actin binding. Through comparative analysis using two profilin mutants, we reveal this motif to be implicated in gliding motility of Plasmodium berghei sporozoites, the rapidly migrating forms of a rodent malaria parasite transmitted by mosquitoes. Force measurements on migrating sporozoites and molecular dynamics simulations indicate that the interaction between actin and profilin fine-tunes gliding motility. Our data suggest that evolutionary pressure to achieve efficient high-speed gliding has resulted in a unique profilin-actin interface in these parasites.

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<![CDATA[Molecular Markers of Radiation Induced Attenuation in Intrahepatic Plasmodium falciparum Parasites]]> https://www.researchpad.co/article/5989da0eab0ee8fa60b789a7

Experimental immunization with radiation attenuated sporozoites (RAS) and genetically attenuated sporozoites has proved to be a promising approach for malaria vaccine development. However, parasite biomarkers of growth attenuation and enhanced immune protection in response to radiation remain poorly understood. Here, we report on the effect of an attenuating dose of γ-irradiation (15 krad) on the Plasmodium falciparum sporozoite (PfSPZ) ultrastructure by electron microscopy, growth rate of liver stage P. falciparum in liver cell cultures, and genome-wide transcriptional profile of liver stage parasites by microarray. We find that γ-irradiation treated PfSPZ retained a normal cellular structure except that they were vacuous with a partially disrupted plasma membrane and inner membrane complex. A similar infection rate was observed by γ-irradiation-treated and untreated PfSPZ in human HCO-4 liver cells (0.47% versus 0.49%, respectively) on day 3 post-infection. In the microarray studies, cumulatively, 180 liver stage parasite genes were significantly transcriptionally altered on day 3 and/or 6 post-infection. Among the transcriptionally altered biomarkers, we identified a signature of seven candidate parasite genes that associated with functionally diverse pathways that may regulate radiation induced cell cycle arrest of the parasite within the hepatocyte. A repertoire of 14 genes associated with protein translation is transcriptionally overexpressed within the parasite by radiation. Additionally, 37 genes encode proteins expressed on the cell surface or exported into the host cell, 4 encode membrane associated transporters, and 10 encode proteins related to misfolding and stress-related protein processing. These results have significantly increased the repertoire of novel targets for 1) biomarkers of safety to define proper attenuation, 2) generating genetically attenuated parasite vaccine candidates, and 3) subunit candidate vaccines against liver stage malaria.

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<![CDATA[Molecular Characterization and Functional Analysis of a Novel Calcium-Dependent Protein Kinase 4 from Eimeria tenella]]> https://www.researchpad.co/article/5989d9f8ab0ee8fa60b70f98

Eimeria tenella is an obligate intracellular parasite that actively invades cecal epithelial cells of chickens. The basis of cell invasion is not completely understood, but some key molecules of host cell invasion have been discovered. This paper investigated the characteristics of calcium-dependent protein kinase 4 (EtCDPK4), a critical molecule in E. tenella invasion of host cells. A full-length EtCDPK4 cDNA was identified from E. tenella using rapid amplification of cDNA ends. EtCDPK4 had an open reading frame of 1803 bp encoding a protein of 600 amino acids. Quantitative real-time PCR and western blotting were used to explore differences in EtCDPK4 transcription and translation in four developmental stages of E. tenella. EtCDPK4 was expressed at higher levels in sporozoites, but translation was higher in second-generation merozoites. In vitro invasion inhibition assays explored whether EtCDPK4 was involved in invasion of DF-1 cells by E. tenella sporozoites. Polyclonal antibodies against recombinant EtCDPK4 (rEtCDPK4) inhibited parasite invasion, decreasing it by approximately 52%. Indirect immunofluorescence assays explored EtCDPK4 distribution during parasite development after E. tenella sporozoite invasion of DF-1 cells in vitro. The results showed that EtCDPK4 might be important in sporozoite invasion and development. To analyze EtCDPK4 functional domains according to the structural characteristics of EtCDPK4 and study the kinase activity of rEtCDPK4, an in vitro phosphorylation system was established. We verified that rEtCDPK4 was a protein kinase that was completely dependent on Ca2+ for enzyme activity. Specific inhibitors of rEtCDPK4 activity were screened by kinase activity in vitro. Some specific inhibitors were applied to assays of DF-1 cell invasion by E. tenella sporozoites to confirm that the inhibitors functioned in vitro. W-7, H-7, H-89, and myristoylated peptide inhibited DF-1 invasion by E. tenella sporozoites. The experimental results showed that EtCDPK4 may be involved in E. tenella invasion of chicken cecal epithelial cells.

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<![CDATA[Indoor residual spraying with micro-encapsulated pirimiphos-methyl (Actellic® 300CS) against malaria vectors in the Lake Victoria basin, Tanzania]]> https://www.researchpad.co/article/5989db5aab0ee8fa60bdf6d6

Background

The indoor residual spraying programme for malaria vectors control was implemented in four districts of the Lake Victoria basin of Tanzania namely Ukerewe, Sengerema, Rorya andSerengeti. Entomological monitoring activities were implemented in one sentinel village in each district to evaluate the efficacy of pirimiphos-methyl 300 CS sprayed on different wall surfaces and its impact against malaria vectors post-IRS intervention.

Methods

The residual decay rate of p-methyl 300 CS applied at a target dosage of 1g a.i./m2 on thesprayed wall surfaces was monitored for a period of 43 weeks post-IRSusing the WHO cone wall bioassay method. The bioassays were performed by exposing 2–5 days old unfed susceptible female Anopheles gambiae s.s. (Kisumu strain) to sprayed wall surfaces for a period of 30 minutes. In each sentinel village, mosquito collection was carried out by trained community mosquito collectors. Monthly mosquito collections were carried out from 6.00pm to 6.00am using CDC light traps and clay pot methods for indoors host seekingand outdoors resting mosquitoes respectively. Six traps (2 CDC light traps and 4 clay pots) were set per sentinel village per night for28 consecutive days in a moon. PCR and ELISA were used for mosquito species identification and sporozoite detection, respectively.

Results

Based on the WHOPES recommendation, insecticides should have a minimum efficacy of ≥ 80% mosquito mortality at 24 hours post exposure on the sprayed wall surfaces to be considered effective. In this study, p-methyl 300 CS was demonstrated to have a long residual efficacy of 21–43 weeks post-IRS on mud, cement, painted and wood wall surfaces. Numberof anopheline mosquitoes decreased post-IRS interventions in all sentinel villages. The highest numbers ofanopheline mosquitoes were collected in November-December, 38–43 weeks post-IRS. A total of 270 female anopheline mosquitoes were analyzed by PCR; out of which 236 (87.4%) were An. gambiae s.l. and 34 (12.6%) were An. funestus group. Of the 236 An. gambiae s.l.identified 12.6% (n = 34) were An. gambiae s.s. and 68.6% (n = 162) were An. arabiensis. Ofthe 34 An. funestus group indentified 91.2% (n = 31) were An. parensis and 8.8% (n = 3) were An. rivulorum. The overall Plasmodium falciparum sporozoite rate was 0.7% (n = 2,098).

Conclusions

Pirimiphos-methyl 300 CS was found to be effective for IRS in the Lake Victoria basin,Tanzania. P-methyl 300 CShas a long residual efficacy on sprayed wall surfaces and therefore it is effective in controlling principal malaria vectors of An. gambiae s.l and An. funestus which rest on wall surfaces after and before feeding.

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<![CDATA[Translational Control of UIS4 Protein of the Host-Parasite Interface Is Mediated by the RNA Binding Protein Puf2 in Plasmodium berghei Sporozoites]]> https://www.researchpad.co/article/5989da80ab0ee8fa60b9a54a

UIS4 is a key protein component of the host-parasite interface in the liver stage of the rodent malaria parasite Plasmodium berghei and required for parasite survival after invasion. In the infectious sporozoite, UIS4 protein has variably been shown to be translated but also been reported to be translationally repressed. Here we show that uis4 mRNA translation is regulated by the P. berghei RNA binding protein Pumilio-2 (PbPuf2 or Puf2 from here on forward) in infectious salivary gland sporozoites in the mosquito vector. Using RNA immunoprecipitation we show that uis4 mRNA is bound by Puf2 in salivary gland sporozoites. In the absence of Puf2, uis4 mRNA translation is de-regulated and UIS4 protein expression upregulated in salivary gland sporozoites. Here, using RNA immunoprecipitation, we reveal the first Puf2-regulated mRNA in this parasite.

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<![CDATA[Probability of Transmission of Malaria from Mosquito to Human Is Regulated by Mosquito Parasite Density in Naïve and Vaccinated Hosts]]> https://www.researchpad.co/article/5989db54ab0ee8fa60bdcfc1

Over a century since Ronald Ross discovered that malaria is caused by the bite of an infectious mosquito it is still unclear how the number of parasites injected influences disease transmission. Currently it is assumed that all mosquitoes with salivary gland sporozoites are equally infectious irrespective of the number of parasites they harbour, though this has never been rigorously tested. Here we analyse >1000 experimental infections of humans and mice and demonstrate a dose-dependency for probability of infection and the length of the host pre-patent period. Mosquitoes with a higher numbers of sporozoites in their salivary glands following blood-feeding are more likely to have caused infection (and have done so quicker) than mosquitoes with fewer parasites. A similar dose response for the probability of infection was seen for humans given a pre-erythrocytic vaccine candidate targeting circumsporozoite protein (CSP), and in mice with and without transfusion of anti-CSP antibodies. These interventions prevented infection more efficiently from bites made by mosquitoes with fewer parasites. The importance of parasite number has widespread implications across malariology, ranging from our basic understanding of the parasite, how vaccines are evaluated and the way in which transmission should be measured in the field. It also provides direct evidence for why the only registered malaria vaccine RTS,S was partially effective in recent clinical trials.

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<![CDATA[Simulating within-vector generation of the malaria parasite diversity]]> https://www.researchpad.co/article/5989db5cab0ee8fa60bdff4a

Plasmodium falciparum, the most virulent human malaria parasite, undergoes asexual reproduction within the human host, but reproduces sexually within its vector host, the Anopheles mosquito. Consequently, the mosquito stage of the parasite life cycle provides an opportunity to create genetically novel parasites in multiply-infected mosquitoes, potentially increasing parasite population diversity. Despite the important implications for disease transmission and malaria control, a quantitative mapping of how parasite diversity entering a mosquito relates to diversity of the parasite exiting, has not been undertaken. To examine the role that vector biology plays in modulating parasite diversity, we develop a two-part model framework that estimates the diversity as a consequence of different bottlenecks and expansion events occurring during the vector-stage of the parasite life cycle. For the underlying framework, we develop the first stochastic model of within-vector P. falciparum parasite dynamics and go on to simulate the dynamics of two parasite subpopulations, emulating multiply infected mosquitoes. We show that incorporating stochasticity is essential to capture the extensive variation in parasite dynamics, particularly in the presence of multiple parasites. In particular, unlike deterministic models, which always predict the most fit parasites to produce the most sporozoites, we find that occasionally only parasites with lower fitness survive to the sporozoite stage. This has important implications for onward transmission. The second part of our framework includes a model of sequence diversity generation resulting from recombination and reassortment between parasites within a mosquito. Our two-part model framework shows that bottlenecks entering the oocyst stage decrease parasite diversity from what is present in the initial gametocyte population in a mosquito’s blood meal. However, diversity increases with the possibility for recombination and proliferation in the formation of sporozoites. Furthermore, when we begin with two parasite subpopulations in the initial gametocyte population, the probability of transmitting more than two unique parasites from mosquito to human is over 50% for a wide range of initial gametocyte densities.

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<![CDATA[Targeted Deletion of a Plasmodium Site-2 Protease Impairs Life Cycle Progression in the Mammalian Host]]> https://www.researchpad.co/article/5989db53ab0ee8fa60bdcdb2

Site-2 proteases (S2P) belong to the M50 family of metalloproteases, which typically perform essential roles by mediating activation of membrane–bound transcription factors through regulated intramembrane proteolysis (RIP). Protease-dependent liberation of dormant transcription factors triggers diverse cellular responses, such as sterol regulation, Notch signalling and the unfolded protein response. Plasmodium parasites rely on regulated proteolysis for controlling essential pathways throughout the life cycle. In this study we examine the Plasmodium-encoded S2P in a murine malaria model and show that it is expressed in all stages of Plasmodium development. Localisation studies by endogenous gene tagging revealed that in all invasive stages the protein is in close proximity to the nucleus. Ablation of PbS2P by reverse genetics leads to reduced growth rates during liver and blood infection and, hence, virulence attenuation. Strikingly, absence of PbS2P was compatible with parasite life cycle progression in the mosquito and mammalian hosts under physiological conditions, suggesting redundant or dispensable roles in vivo.

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<![CDATA[A Putative Small Solute Transporter Is Responsible for the Secretion of G377 and TRAP-Containing Secretory Vesicles during Plasmodium Gamete Egress and Sporozoite Motility]]> https://www.researchpad.co/article/5989da5eab0ee8fa60b9085a

Regulated protein secretion is required for malaria parasite life cycle progression and transmission between the mammalian host and mosquito vector. During transmission from the host to the vector, exocytosis of highly specialised secretory vesicles, such as osmiophilic bodies, is key to the dissolution of the red blood cell and parasitophorous vacuole membranes enabling gamete egress. The positioning of adhesins from the TRAP family, from micronemes to the sporozoite surface, is essential for gliding motility of the parasite and transmission from mosquito to mammalian host. Here we identify a conserved role for the putative pantothenate transporter PAT in Plasmodium berghei in vesicle fusion of two distinct classes of vesicles in gametocytes and sporozoites. PAT is a membrane component of osmiophilic bodies in gametocytes and micronemes in sporozoites. Despite normal formation and trafficking of osmiophilic bodies to the cell surface upon activation, PAT-deficient gametes fail to discharge their contents, remain intraerythrocytic and unavailable for fertilisation and further development in the mosquito. Sporozoites lacking PAT fail to secrete TRAP, are immotile and thus unable to infect the subsequent rodent host. Thus, P. berghei PAT appears to regulate exocytosis in two distinct populations of vesicles in two different life cycle forms rather than acting as pantothenic transporter during parasite transmission.

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<![CDATA[Complex Minigene Library Vaccination for Discovery of Pre-Erythrocytic Plasmodium T Cell Antigens]]> https://www.researchpad.co/article/5989da75ab0ee8fa60b9674f

Development of a subunit vaccine targeting liver-stage Plasmodium parasites requires the identification of antigens capable of inducing protective T cell responses. However, traditional methods of antigen identification are incapable of evaluating T cell responses against large numbers of proteins expressed by these parasites. This bottleneck has limited development of subunit vaccines against Plasmodium and other complex intracellular pathogens. To address this bottleneck, we are developing a synthetic minigene technology for multi-antigen DNA vaccines. In an initial test of this approach, pools of long (150 bp) antigen-encoding oligonucleotides were synthesized and recombined into vectors by ligation-independent cloning to produce two DNA minigene library vaccines. Each vaccine encoded peptides derived from 36 (vaccine 1) and 53 (vaccine 2) secreted or transmembrane pre-erythrocytic P. yoelii proteins. BALB/cj mice were vaccinated three times with a single vaccine by biolistic particle delivery (gene gun) and screened for interferon-γ-producing T cell responses by ELISPOT. Library vaccination induced responses against four novel antigens. Naïve mice exposed to radiation-attenuated sporozoites mounted a response against only one of the four novel targets (PyMDH, malate dehydrogenase). The response to PyMDH could not be recalled by additional homologous sporozoite immunizations but could be partially recalled by heterologous cross-species sporozoite exposure. Vaccination against the dominant PyMDH epitope by DNA priming and recombinant Listeria boosting did not protect against sporozoite challenge. Improvements in library design and delivery, combined with methods promoting an increase in screening sensitivity, may enable complex minigene screening to serve as a high-throughput system for discovery of novel T cell antigens.

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<![CDATA[An in vitro model of intestinal infection reveals a developmentally regulated transcriptome of Toxoplasma sporozoites and a NF-κB-like signature in infected host cells]]> https://www.researchpad.co/article/5989db50ab0ee8fa60bdc1be

Toxoplasmosis is a zoonotic infection affecting approximately 30% of the world’s human population. After sexual reproduction in the definitive feline host, Toxoplasma oocysts, each containing 8 sporozoites, are shed into the environment where they can go on to infect humans and other warm-blooded intermediate hosts. Here, we use an in vitro model to assess host transcriptomic changes that occur in the earliest stages of such infections. We show that infection of rat intestinal epithelial cells with mature sporozoites primarily results in higher expression of genes associated with Tumor Necrosis Factor alpha (TNFα) signaling via NF-κB. Furthermore, we find that, consistent with their biology, these mature, invaded sporozoites display a transcriptome intermediate between the previously reported day 10 oocysts and that of their tachyzoite counterparts. Thus, this study uncovers novel host and pathogen factors that may be critical for the establishment of a successful intracellular niche following sporozoite-initiated infection.

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<![CDATA[Seroprevalence of Antibodies against Plasmodium falciparum Sporozoite Antigens as Predictive Disease Transmission Markers in an Area of Ghana with Seasonal Malaria Transmission]]> https://www.researchpad.co/article/5989d9d3ab0ee8fa60b64be6

Introduction

As an increasing number of malaria-endemic countries approach the disease elimination phase, sustenance of control efforts and effective monitoring are necessary to ensure success. Mathematical models that estimate anti-parasite antibody seroconversion rates are gaining relevance as more sensitive transmission intensity estimation tools. Models however estimate yearly seroconversion and seroreversion rates and usually predict long term changes in transmission, occurring years before the time of sampling. Another challenge is the identification of appropriate antigen targets since specific antibody levels must directly reflect changes in transmission patterns. We therefore investigated the potential of antibodies to sporozoite and blood stage antigens for detecting short term differences in malaria transmission in two communities in Northern Ghana with marked, seasonal transmission.

Methods

Cross-sectional surveys were conducted during the rainy and dry seasons in two communities, one in close proximity to an irrigation dam and the other at least 20 Km away from the dam. Antibodies against the sporozoite-specific antigens circumsporozoite protein (CSP) and Cell traversal for ookinetes and sporozoites (CelTOS) and the classical blood stage antigen apical membrane antigen 1 (AMA1) were measured by indirect ELISA. Antibody levels and seroprevalence were compared between surveys and between study communities. Antibody seroprevalence data were fitted to a modified reversible catalytic model to estimate the seroconversion and seroreversion rates.

Results

Changes in sporozoite-specific antibody levels and seroprevalence directly reflected differences in parasite prevalence between the rainy and dry seasons and hence the extent of malaria transmission. Seroconversion rate estimates from modelled seroprevalence data did not however support the above observation.

Conclusions

The data confirms the potential utility of sporozoite-specific antigens as useful markers for monitoring short term/seasonal changes in malaria transmission. It may however be essential to update models to allow for assessment of seasonal changes in malaria transmission, which usually occur within four to six months.

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<![CDATA[A Full-Length Plasmodium falciparum Recombinant Circumsporozoite Protein Expressed by Pseudomonas fluorescens Platform as a Malaria Vaccine Candidate]]> https://www.researchpad.co/article/5989d9dbab0ee8fa60b67b14

The circumsporozoite protein (CSP) of Plasmodium falciparum is a major surface protein, which forms a dense coat on the sporozoite's surface. Preclinical research on CSP and clinical evaluation of a CSP fragment-based RTS, S/AS01 vaccine have demonstrated a modest degree of protection against P. falciparum, mediated in part by humoral immunity and in part by cell-mediated immunity. Given the partial protective efficacy of the RTS, S/AS01 vaccine in a recent Phase 3 trial, further improvement of CSP-based vaccines is crucial. In this report, we describe the preclinical development of a full-length, recombinant CSP (rCSP)-based vaccine candidate against P. falciparum malaria suitable for current Good Manufacturing Practice (cGMP) production. Utilizing a novel high-throughput Pseudomonas fluorescens expression platform, we demonstrated greater efficacy of full-length rCSP as compared to N-terminally truncated versions, rapidly down-selected a promising lead vaccine candidate, and developed a high-yield purification process to express immunologically active, intact antigen for clinical trial material production. The rCSP, when formulated with various adjuvants, induced antigen-specific antibody responses as measured by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA), as well as CD4+ T-cell responses as determined by ELISpot. The adjuvanted rCSP vaccine conferred protection in mice when challenged with transgenic P. berghei sporozoites containing the P. falciparum repeat region of CSP. Furthermore, heterologous prime/boost regimens with adjuvanted rCSP and an adenovirus type 35-vectored CSP (Ad35CS) showed modest improvements in eliciting CSP-specific T-cell responses and anti-malarial protection, depending on the order of vaccine delivery. Collectively, these data support the importance of further clinical development of adjuvanted rCSP, either as a stand-alone product or as one of the components in a heterologous prime/boost strategy, ultimately acting as an effective vaccine candidate for the mitigation of P. falciparum-induced malaria.

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<![CDATA[Sporozoite Immunization of Human Volunteers under Mefloquine Prophylaxis Is Safe, Immunogenic and Protective: A Double-Blind Randomized Controlled Clinical Trial]]> https://www.researchpad.co/article/5989daa9ab0ee8fa60ba8926

Immunization of healthy volunteers with chloroquine ChemoProphylaxis and Sporozoites (CPS-CQ) efficiently and reproducibly induces dose-dependent and long-lasting protection against homologous Plasmodium falciparum challenge. Here, we studied whether chloroquine can be replaced by mefloquine, which is the only other licensed anti-malarial chemoprophylactic drug that does not affect pre-erythrocytic stages, exposure to which is considered essential for induction of protection by CPS immunization. In a double blind randomized controlled clinical trial, volunteers under either chloroquine prophylaxis (CPS-CQ, n = 5) or mefloquine prophylaxis (CPS-MQ, n = 10) received three sub-optimal CPS immunizations by bites from eight P. falciparum infected mosquitoes each, at monthly intervals. Four control volunteers received mefloquine prophylaxis and bites from uninfected mosquitoes. CPS-MQ immunization is safe and equally potent compared to CPS-CQ inducing protection in 7/10 (70%) versus 3/5 (60%) volunteers, respectively. Furthermore, specific antibody levels and cellular immune memory responses were comparable between both groups. We therefore conclude that mefloquine and chloroquine are equally effective in CPS-induced immune responses and protection.

Trial Registration

ClinicalTrials.gov NCT01422954

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<![CDATA[Sterile Immunity to Malaria after DNA Prime/Adenovirus Boost Immunization Is Associated with Effector Memory CD8+T Cells Targeting AMA1 Class I Epitopes]]> https://www.researchpad.co/article/5989da73ab0ee8fa60b957bd

Background

Fifteen volunteers were immunized with three doses of plasmid DNA encoding P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1) and boosted with human adenovirus-5 (Ad) expressing the same antigens (DNA/Ad). Four volunteers (27%) demonstrated sterile immunity to controlled human malaria infection and, overall, protection was statistically significantly associated with ELISpot and CD8+ T cell IFN-γ activities to AMA1 but not CSP. DNA priming was required for protection, as 18 additional subjects immunized with Ad alone (AdCA) did not develop sterile protection.

Methodology/Principal Findings

We sought to identify correlates of protection, recognizing that DNA-priming may induce different responses than AdCA alone. Among protected volunteers, two and three had higher ELISpot and CD8+ T cell IFN-γ responses to CSP and AMA1, respectively, than non-protected volunteers. Unexpectedly, non-protected volunteers in the AdCA trial showed ELISpot and CD8+ T cell IFN-γ responses to AMA1 equal to or higher than the protected volunteers. T cell functionality assessed by intracellular cytokine staining for IFN-γ, TNF-α and IL-2 likewise did not distinguish protected from non-protected volunteers across both trials. However, three of the four protected volunteers showed higher effector to central memory CD8+ T cell ratios to AMA1, and one of these to CSP, than non-protected volunteers for both antigens. These responses were focused on discrete regions of CSP and AMA1. Class I epitopes restricted by A*03 or B*58 supertypes within these regions of AMA1 strongly recalled responses in three of four protected volunteers. We hypothesize that vaccine-induced effector memory CD8+ T cells recognizing a single class I epitope can confer sterile immunity to P. falciparum in humans.

Conclusions/Significance

We suggest that better understanding of which epitopes within malaria antigens can confer sterile immunity and design of vaccine approaches that elicit responses to these epitopes will increase the potency of next generation gene-based vaccines.

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