Plasmodium knowlesi in pig-tailed macaques: a potential new model for malaria vaccine research.

BACKGROUND: Plasmodium knowlesi is an established experimental model for basic and pre-clinical malaria vaccine research. Historically, rhesus macaques have been the most common host for malaria vaccine studies with P. knowlesi parasites. However, rhesus are not natural hosts for P. knowlesi, and there is interest in identifying alternative hosts for vaccine research. The study team previously reported that pig-tailed macaques (PTM), a natural host for P. knowlesi, could be challenged with cryopreserved P. knowlesi sporozoites (PkSPZ), with time to blood stage infection equivalent to in rhesus. Here, additional exploratory studies were performed to evaluate PTM as potential hosts for malaria vaccine studies. The aim was to further characterize the parasitological and veterinary health outcomes after PkSPZ challenge in this macaque species. METHODS: Malaria-naïve PTM were intravenously challenged with 2.5 × 103 PkSPZ and monitored for blood stage infection by Plasmodium 18S rRNA RT-PCR and thin blood smears. Disease signs were evaluated by daily observations, complete blood counts, serum chemistry tests, and veterinary examinations. After anti-malarial drug treatment, a subset of animals was re-challenged and monitored as above. Whole blood gene expression analysis was performed on selected animals to assess host response to infection. RESULTS: In naïve animals, the kinetics of P. knowlesi blood stage replication was reproducible, with parasite burden rising linearly during an initial acute phase of infection from 6 to 11 days post-challenge, before plateauing and transitioning into a chronic low-grade infection. After re-challenge, infections were again reproducible, but with lower blood stage parasite densities. Clinical signs of disease were absent or mild and anti-malarial treatment was not needed until the pre-defined study day. Whole blood gene expression analysis identified immunological changes associated with acute and chronic phases of infection, and further differences between initial challenge versus re-challenge. CONCLUSIONS: The ability to challenge PTM with PkSPZ and achieve reliable blood stage infections indicate this model has significant potential for malaria vaccine studies. Blood stage P. knowlesi infection in PTM is characterized by low parasite burdens and a benign disease course, in contrast with the virulent P. knowlesi disease course commonly reported in rhesus macaques. These findings identify new opportunities for malaria vaccine research using this natural host-parasite combination.

Screening for lipid nanoparticles that modulate the immune activity of helper T cells towards enhanced antitumour activity.

Lipid nanoparticles (LNPs) can be designed to potentiate cancer immunotherapy by promoting their uptake by antigen-presenting cells, stimulating the maturation of these cells and modulating the activity of adjuvants. Here we report an LNP-screening method for the optimization of the type of helper lipid and of lipid-component ratios to enhance the delivery of tumour-antigen-encoding mRNA to dendritic cells and their immune-activation profile towards enhanced antitumour activity. The method involves screening for LNPs that enhance the maturation of bone-marrow-derived dendritic cells and antigen presentation in vitro, followed by assessing immune activation and tumour-growth suppression in a mouse model of melanoma after subcutaneous or intramuscular delivery of the LNPs. We found that the most potent antitumour activity, especially when combined with immune checkpoint inhibitors, resulted from a coordinated attack by T cells and NK cells, triggered by LNPs that elicited strong immune activity in both type-1 and type-2 T helper cells. Our findings highlight the importance of optimizing the LNP composition of mRNA-based cancer vaccines to tailor antigen-specific immune-activation profiles.

More time to kill: A longer liver stage increases T cell-mediated protection against pre-erythrocytic malaria.

Liver stage (LS) Plasmodia mature in 2-2.5 days in rodents compared to 5-6 days in humans. Plasmodium-specific CD8+ T cell expansion differs across these varied timespans. To mimic the kinetics of CD8+ T cells of human Plasmodium infection, a two-dose challenge mouse model that achieved 4-5 days of LS antigen exposure was developed. In this model, mice were inoculated with a non-protective, low dose of late-arresting, genetically attenuated sporozoites to initiate T cell activation and then re-inoculated 2-3 days later with wild-type sporozoites. Vaccines that partially protected against traditional challenge completely protected against two-dose challenge. During the challenge period, CD8+ T cell frequencies increased in the livers of two-dose challenged mice but not in traditionally challenged mice, further suggesting that this model better recapitulates kinetics of CD8+ T cell expansion in humans during the P. falciparum LS. Vaccine development and antigen discovery efforts may be aided by using the two-dose challenge strategy.

Multi-step screening of DNA/lipid nanoparticles and co-delivery with siRNA to enhance and prolong gene expression.

Lipid nanoparticles hold great potential as an effective non-viral vector for nucleic acid-based gene therapy. Plasmid DNA delivery can result in extended transgene expression compared to mRNA-based technologies, yet there is a lack of systematic investigation into lipid nanoparticle compositions for plasmid DNA delivery. Here, we report a multi-step screening platform to identify optimized plasmid DNA lipid nanoparticles for liver-targeted transgene expression. To achieve this, we analyze the role of different helper lipids and component ratios in plasmid DNA lipid nanoparticle-mediated gene delivery in vitro and in vivo. Compared to mRNA LNPs and in vivo-jetPEI/DNA nanoparticles, the identified plasmid DNA lipid nanoparticles successfully deliver transgenes and mediate prolonged expression in the liver following intravenous administration in mice. By addressing different physiological barriers in a stepwise manner, this screening platform can efficiently down select effective lipid nanoparticle candidates from a lipid nanoparticle library of over 1000 formulations. In addition, we substantially extend the duration of plasmid DNA nanoparticle-mediated transgene expression using a DNA/siRNA co-delivery approach that targets transcription factors regulating inflammatory response pathways. This lipid nanoparticle-based co-delivery strategy further highlights the unique advantages of an extended transgene expression profile using plasmid DNA delivery and offers new opportunities for DNA-based gene medicine applications.

Additional Feeding Reveals Differences in Immune Recognition and Growth of Plasmodium Parasites in the Mosquito Host.

Mosquitoes may feed multiple times during their life span in addition to those times needed to acquire and transmit malaria. To determine the impact of subsequent blood feeding on parasite development in Anopheles gambiae, we examined Plasmodium parasite infection with or without an additional noninfected blood meal. We found that an additional blood meal significantly reduced Plasmodium berghei immature oocyst numbers, yet had no effect on the human parasite Plasmodium falciparum These observations were reproduced when mosquitoes were fed an artificial protein meal, suggesting that parasite losses are independent of blood ingestion. We found that feeding with either a blood or protein meal compromises midgut basal lamina integrity as a result of the physical distention of the midgut, enabling the recognition and lysis of immature P. berghei oocysts by mosquito complement. Moreover, we demonstrate that additional feeding promotes P. falciparum oocyst growth, suggesting that human malaria parasites exploit host resources provided with blood feeding to accelerate their growth. This is in contrast to experiments with P. berghei, where the size of surviving oocysts is independent of an additional blood meal. Together, these data demonstrate distinct differences in Plasmodium species in evading immune detection and utilizing host resources at the oocyst stage, representing an additional, yet unexplored component of vectorial capacity that has important implications for the transmission of malaria.IMPORTANCE Mosquitoes must blood feed multiple times to acquire and transmit malaria. However, the impact of an additional mosquito blood meal following malaria parasite infection has not been closely examined. Here, we demonstrate that additional feeding affects mosquito vector competence; namely, additional feeding significantly limits Plasmodium berghei infection, yet has no effect on infection of the human parasite P. falciparum Our experiments support that these killing responses are mediated by the physical distension of the midgut and by temporary damage to the midgut basal lamina that exposes immature P. berghei oocysts to mosquito complement, while human malaria parasites are able to evade these killing mechanisms. In addition, we provide evidence that additional feeding promotes P. falciparum oocyst growth. This is in contrast to P. berghei, where oocyst size is independent of an additional blood meal. This suggests that human malaria parasites are able to exploit host resources provided by an additional feeding to accelerate their growth. In summary, our data highlight distinct differences in malaria parasite species in evading immune recognition and adapting to mosquito blood feeding. These observations have important, yet previously unexplored, implications for the impact of multiple blood meals on the transmission of malaria.

The 20-hydroxyecdysone agonist, halofenozide, promotes anti-Plasmodium immunity in Anopheles gambiae via the ecdysone receptor.

Mosquito physiology and immunity are integral determinants of malaria vector competence. This includes the principal role of hormonal signaling in Anopheles gambiae initiated shortly after blood-feeding, which stimulates immune induction and promotes vitellogenesis through the function of 20-hydroxyecdysone (20E). Previous studies demonstrated that manipulating 20E signaling through the direct injection of 20E or the application of a 20E agonist can significantly impact Plasmodium infection outcomes, reducing oocyst numbers and the potential for malaria transmission. In support of these findings, we demonstrate that a 20E agonist, halofenozide, is able to induce anti-Plasmodium immune responses that limit Plasmodium ookinetes. We demonstrate that halofenozide requires the function of ultraspiracle (USP), a component of the canonical heterodimeric ecdysone receptor, to induce malaria parasite killing responses. Additional experiments suggest that the effects of halofenozide treatment are temporal, such that its application only limits malaria parasites when applied prior to infection. Unlike 20E, halofenozide does not influence cellular immune function or AMP production. Together, our results further demonstrate the potential of targeting 20E signaling pathways to reduce malaria parasite infection in the mosquito vector and provide new insight into the mechanisms of halofenozide-mediated immune activation that differ from 20E.

20-Hydroxyecdysone Primes Innate Immune Responses That Limit Bacterial and Malarial Parasite Survival in Anopheles gambiae.

Blood feeding is an integral behavior of mosquitoes to acquire nutritional resources needed for reproduction. This requirement also enables mosquitoes to serve as efficient vectors to acquire and potentially transmit a multitude of mosquito-borne diseases, most notably malaria. Recent studies suggest that mosquito immunity is stimulated following a blood meal, independent of infection status. Since blood feeding promotes production of the hormone 20-hydroxyecdysone (20E), we hypothesized that 20E plays an important role in priming the immune response for pathogen challenge. Here, we examine the immunological effects of priming Anopheles gambiae with 20E prior to pathogen infection, demonstrating a significant reduction in bacteria and Plasmodium berghei survival in the mosquito host. Transcriptome sequencing (RNA-seq) analysis following 20E treatment identifies several known 20E-regulated genes, as well as several immune genes with previously reported function in antipathogen defense. Together, these data demonstrate that 20E influences cellular immune function and antipathogen immunity following mosquito blood feeding, arguing the importance of hormones in the regulation of mosquito innate immune function.IMPORTANCE Blood feeding is required to provide nutrients for mosquito egg production and serves as a mechanism to acquire and transmit pathogens. Shortly after a blood meal is taken, there is a peak in the production of 20-hydroxyecdysone (20E), a mosquito hormone that initiates physiological changes, including yolk protein production and mating refractoriness. Here, we examine additional roles of 20E in the regulation of mosquito immunity, demonstrating that priming the immune system with 20E increases mosquito resistance to pathogens. We identify differentially expressed genes in response to 20E treatment, including several involved in innate immune function as well as lipid metabolism and transport. Together, these data argue that 20E stimulates mosquito cellular immune function and innate immunity shortly after blood feeding.