In vitro production of infectious Plasmodium falciparum sporozoites.

An effective vaccine is needed for the prevention and elimination of malaria. The only immunogens that have been shown to have a protective efficacy of more than 90% against human malaria are Plasmodium falciparum (Pf) sporozoites (PfSPZ) manufactured in mosquitoes (mPfSPZ)1-7. The ability to produce PfSPZ in vitro (iPfSPZ) without mosquitoes would substantially enhance the production of PfSPZ vaccines and mosquito-stage malaria research, but this ability is lacking. Here we report the production of hundreds of millions of iPfSPZ. iPfSPZ invaded human hepatocytes in culture and developed to mature liver-stage schizonts expressing P. falciparum merozoite surface protein 1 (PfMSP1) in numbers comparable to mPfSPZ. When injected into FRGhuHep mice containing humanized livers, iPfSPZ invaded the human hepatocytes and developed to PfMSP1-expressing late liver stage parasites at 45% the quantity of cryopreserved mPfSPZ. Human blood from FRGhuHep mice infected with iPfSPZ produced asexual and sexual erythrocytic-stage parasites in culture, and gametocytes developed to PfSPZ when fed to mosquitoes, completing the P. falciparum life cycle from infectious gametocyte to infectious gametocyte without mosquitoes or primates.

Two chemoattenuated PfSPZ malaria vaccines induce sterile hepatic immunity.

The global decline in malaria has stalled1, emphasizing the need for vaccines that induce durable sterilizing immunity. Here we optimized regimens for chemoprophylaxis vaccination (CVac), for which aseptic, purified, cryopreserved, infectious Plasmodium falciparum sporozoites (PfSPZ) were inoculated under prophylactic cover with pyrimethamine (PYR) (Sanaria PfSPZ-CVac(PYR)) or chloroquine (CQ) (PfSPZ-CVac(CQ))-which kill liver-stage and blood-stage parasites, respectively-and we assessed vaccine efficacy against homologous (that is, the same strain as the vaccine) and heterologous (a different strain) controlled human malaria infection (CHMI) three months after immunization ( https://clinicaltrials.gov/ , NCT02511054 and NCT03083847). We report that a fourfold increase in the dose of PfSPZ-CVac(PYR) from 5.12 × 104 to 2 × 105 PfSPZs transformed a minimal vaccine efficacy (low dose, two out of nine (22.2%) participants protected against homologous CHMI), to a high-level vaccine efficacy with seven out of eight (87.5%) individuals protected against homologous and seven out of nine (77.8%) protected against heterologous CHMI. Increased protection was associated with Vδ2 γδ T cell and antibody responses. At the higher dose, PfSPZ-CVac(CQ) protected six out of six (100%) participants against heterologous CHMI three months after immunization. All homologous (four out of four) and heterologous (eight out of eight) infectivity control participants showed parasitaemia. PfSPZ-CVac(CQ) and PfSPZ-CVac(PYR) induced a durable, sterile vaccine efficacy against a heterologous South American strain of P. falciparum, which has a genome and predicted CD8 T cell immunome that differs more strongly from the African vaccine strain than other analysed African P. falciparum strains.

A protocol for directly accessing geminal C-4 diarylated pyrazol-5(4H)-ones via tandem C-H aryne insertion and their inceptive neurobiological evaluation.

Geminal C-4 diarylation of substituted pyrazol-5(4H)-ones with in situ generated arynes as the aryl source has been achieved in a one-flask operation. All the newly accessed C4-gem-diarylated pyrazolone entities were found to be non-cytotoxic with varying AChE enzyme inhibitory activities and BBB permeability attributes that augur well for further advancement towards CNS therapeutics for untreatable disorders.

Knockout of Anopheles stephensi immune gene LRIM1 by CRISPR-Cas9 reveals its unexpected role in reproduction and vector competence.

PfSPZ Vaccine against malaria is composed of Plasmodium falciparum (Pf) sporozoites (SPZ) manufactured using aseptically reared Anopheles stephensi mosquitoes. Immune response genes of Anopheles mosquitoes such as Leucin-Rich protein (LRIM1), inhibit Plasmodium SPZ development (sporogony) in mosquitoes by supporting melanization and phagocytosis of ookinetes. With the aim of increasing PfSPZ infection intensities, we generated an A. stephensi LRIM1 knockout line, Δaslrim1, by embryonic genome editing using CRISPR-Cas9. Δaslrim1 mosquitoes had a significantly increased midgut bacterial load and an altered microbiome composition, including elimination of commensal acetic acid bacteria. The alterations in the microbiome caused increased mosquito mortality and unexpectedly, significantly reduced sporogony. The survival rate of Δaslrim1 mosquitoes and their ability to support PfSPZ development, were partially restored by antibiotic treatment of the mosquitoes, and fully restored to baseline when Δaslrim1 mosquitoes were produced aseptically. Deletion of LRIM1 also affected reproductive capacity: oviposition, fecundity and male fertility were significantly compromised. Attenuation in fecundity was not associated with the altered microbiome. This work demonstrates that LRIM1's regulation of the microbiome has a major impact on vector competence and longevity of A. stephensi. Additionally, LRIM1 deletion identified an unexpected role for this gene in fecundity and reduction of sperm transfer by males.

PfSPZ-CVac efficacy against malaria increases from 0% to 75% when administered in the absence of erythrocyte stage parasitemia: A randomized, placebo-controlled trial with controlled human malaria infection.

PfSPZ-CVac combines 'PfSPZ Challenge', which consists of infectious Plasmodium falciparum sporozoites (PfSPZ), with concurrent antimalarial chemoprophylaxis. In a previously-published PfSPZ-CVac study, three doses of 5.12x104 PfSPZ-CVac given 28 days apart had 100% vaccine efficacy (VE) against controlled human malaria infection (CHMI) 10 weeks after the last immunization, while the same dose given as three injections five days apart had 63% VE. Here, we conducted a dose escalation trial of similarly condensed schedules. Of the groups proceeding to CHMI, the first study group received three direct venous inoculations (DVIs) of a dose of 5.12x104 PfSPZ-CVac seven days apart and the next full dose group received three DVIs of a higher dose of 1.024x105 PfSPZ-CVac five days apart. CHMI (3.2x103 PfSPZ Challenge) was performed by DVI 10 weeks after the last vaccination. In both CHMI groups, transient parasitemia occurred starting seven days after each vaccination. For the seven-day interval group, the second and third vaccinations were therefore administered coincident with parasitemia from the prior vaccination. Parasitemia was associated with systemic symptoms which were severe in 25% of subjects. VE in the seven-day group was 0% (7/7 infected) and in the higher-dose, five-day group was 75% (2/8 infected). Thus, the same dose of PfSPZ-CVac previously associated with 63% VE when given on a five-day schedule in the prior study had zero VE here when given on a seven-day schedule, while a double dose given on a five-day schedule here achieved 75% VE. The relative contributions of the five-day schedule and/or the higher dose to improved VE warrant further investigation. It is notable that administration of PfSPZ-CVac on a schedule where vaccine administration coincided with blood-stage parasitemia was associated with an absence of sterile protective immunity. Clinical trials registration: NCT02773979.

Differential Effects of Chronic Ethanol Use on Mouse Neuronal and Astroglial Metabolic Activity.

Chronic alcohol use disorder, a major risk factor for the development of neuropsychiatric disorders including addiction to other substances, is associated with several neuropathology including perturbed neuronal and glial activities in the brain. It affects carbon metabolism in specific brain regions, and perturbs neuro-metabolite homeostasis in neuronal and glial cells. Alcohol induced changes in the brain neurochemical profile accompany the negative emotional state associated with dysregulated reward and sensitized stress response to withdrawal. However, the underlying alterations in neuro-astroglial activities and neurochemical dysregulations in brain regions after chronic alcohol use are poorly understood. This study evaluates the impact of chronic ethanol use on the regional neuro-astroglial metabolic activity using 1H-[13C]-NMR spectroscopy in conjunction with infusion of [1,6-13C2]glucose and sodium [2-13C]acetate, respectively, after 48 h of abstinence. Besides establishing detailed 13C labeling of neuro-metabolites in a brain region-specific manner, our results show chronic ethanol induced-cognitive deficits along with a reduction in total glucose oxidation rates in the hippocampus and striatum. Furthermore, using [2-13C]acetate infusion, we showed an alcohol-induced increase in astroglial metabolic activity in the hippocampus and prefrontal cortex. Interestingly, increased astroglia activity in the hippocampus and prefrontal cortex was associated with a differential expression of monocarboxylic acid transporters that are regulating acetate uptake and metabolism in the brain.

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.

Iron mediated reductive cyclization/oxidation for the generation of chemically diverse scaffolds: An approach in drug discovery.

Chemically diverse scaffolds represent a main source of biologically important starting points in drug discovery. Herein, we report the development of such diverse scaffolds from nitroarene/ nitro(hetero)arenes using a key synthetic strategy. In a pilot-scale study, the synthesis of 10 diverse scaffolds was achieved. The 1,7-phenanthroline, thiazolo[5,4-f]quinoline, 2,3-dihydro-1H-pyrrolo[2,3-g]quinoline, pyrrolo[3,2-f]quinoline, 1H-[1,4]oxazino[3,2-g]quinolin-2(3H)-one, [1,2,5]oxadiazolo[3,4-h]quinoline, 7H-pyrido[2,3-c]carbazole, 3H-pyrazolo[4,3-f]quinoline, pyrido[3,2-f]quinoxaline were obtained from nitro hetero arenes in ethanol using iron-acetic acid treatment followed by reaction under oxygen atmosphere. This diverse library is compliant with the rule of five for drug-likeness. The mapping of chemical space represented by these scaffolds revealed a significant contribution to the underrepresented chemical diversity. Crucial to the development of this approach was the mapping of biological space covered by these scaffolds which revealed neurotropic and prophylactic anti-inflammatory activities. In vitro, neuro-biological assays revealed that compounds 14a and 15a showed excellent neurotropic potential and neurite growth compared to controls. Further, anti-inflammatory assays (in vitro and in vivo models) exhibited that Compound 16 showed significant anti-inflammatory activity by attenuating the LPS-induced TNF-α and CD68 levels by modulating the NFkB pathway. In addition, treatment with compound 16 significantly ameliorated the LPS-induced sepsis conditions, and pathological abnormalities (in lung and liver tissues) and improved the survival of the rats compared to LPS control. Owing to their chemical diversity along with bioactivities, it is envisaged that new quality pre-clinical candidates will be generated in the above therapeutic areas using identified leads.

Sterile protection against human malaria by chemoattenuated PfSPZ vaccine.

A highly protective malaria vaccine would greatly facilitate the prevention and elimination of malaria and containment of drug-resistant parasites. A high level (more than 90%) of protection against malaria in humans has previously been achieved only by immunization with radiation-attenuated Plasmodium falciparum (Pf) sporozoites (PfSPZ) inoculated by mosquitoes; by intravenous injection of aseptic, purified, radiation-attenuated, cryopreserved PfSPZ ('PfSPZ Vaccine'); or by infectious PfSPZ inoculated by mosquitoes to volunteers taking chloroquine or mefloquine (chemoprophylaxis with sporozoites). We assessed immunization by direct venous inoculation of aseptic, purified, cryopreserved, non-irradiated PfSPZ ('PfSPZ Challenge') to malaria-naive, healthy adult volunteers taking chloroquine for antimalarial chemoprophylaxis (vaccine approach denoted as PfSPZ-CVac). Three doses of 5.12 × 104 PfSPZ of PfSPZ Challenge at 28-day intervals were well tolerated and safe, and prevented infection in 9 out of 9 (100%) volunteers who underwent controlled human malaria infection ten weeks after the last dose (group III). Protective efficacy was dependent on dose and regimen. Immunization with 3.2 × 103 (group I) or 1.28 × 104 (group II) PfSPZ protected 3 out of 9 (33%) or 6 out of 9 (67%) volunteers, respectively. Three doses of 5.12 × 104 PfSPZ at five-day intervals protected 5 out of 8 (63%) volunteers. The frequency of Pf-specific polyfunctional CD4 memory T cells was associated with protection. On a 7,455 peptide Pf proteome array, immune sera from at least 5 out of 9 group III vaccinees recognized each of 22 proteins. PfSPZ-CVac is a highly efficacious vaccine candidate; when we are able to optimize the immunization regimen (dose, interval between doses, and drug partner), this vaccine could be used for combination mass drug administration and a mass vaccination program approach to eliminate malaria from geographically defined areas.

Efficient infection of non-human primates with purified, cryopreserved Plasmodium knowlesi sporozoites.

BACKGROUND: Plasmodium falciparum (Pf) sporozoite (SPZ) vaccines are the only candidate malaria vaccines that induce > 90% vaccine efficacy (VE) against controlled human malaria infection and the only malaria vaccines to have achieved reproducible VE against malaria in adults in Africa. The goal is to increase the impact and reduce the cost of PfSPZ vaccines by optimizing vaccine potency and manufacturing, which will benefit from identification of immunological responses contributing to protection in humans. Currently, there is no authentic animal challenge model for assessing P. falciparum malaria VE. Alternatively, Plasmodium knowlesi (Pk), which infects humans and non-human primates (NHPs) in nature, can be used to experimentally infect rhesus macaques (Macaca mulatta) to assess VE. METHODS: Sanaria has, therefore, produced purified, vialed, cryopreserved PkSPZ and conducted challenge studies in several naïve NHP cohorts. In the first cohort, groups of three rhesus macaques each received doses of 5 × 102, 2.5 × 103, 1.25 × 104 and 2.5 × 104 PkSPZ administered by direct venous inoculation. The infectivity of 1.5 × 103 PkSPZ cryopreserved with an altered method and of 1.5 × 103 PkSPZ cryopreserved for four years was tested in a second and third cohort of rhesus NHPs. The lastly, three pig-tailed macaques (Macaca nemestrina), a natural P. knowlesi host, were challenged with 2.5 × 103 PkSPZ cryopreserved six years earlier. RESULTS: In the first cohort, all 12 animals developed P. knowlesi parasitaemia by thick blood smear, and the time to positivity (prepatent period) followed a non-linear 4-parameter logistic sigmoidal model with a median of 11, 10, 8, and 7 days, respectively (r2 = 1). PkSPZ cryopreserved using a modified rapid-scalable method infected rhesus with a pre-patent period of 10 days, as did PkSPZ cryopreserved four years prior to infection, similar to the control group. Cryopreserved PkSPZ infected pig-tailed macaques with median time to positivity by thin smear, of 11 days. CONCLUSION: This study establishes the capacity to consistently infect NHPs with purified, vialed, cryopreserved PkSPZ, providing a foundation for future studies to probe protective immunological mechanisms elicited by PfSPZ vaccines that cannot be established in humans.

Multidose Priming and Delayed Boosting Improve Plasmodium falciparum Sporozoite Vaccine Efficacy Against Heterologous P. falciparum Controlled Human Malaria Infection.

BACKGROUND: A live-attenuated Plasmodium falciparum sporozoite (SPZ) vaccine (PfSPZ Vaccine) has shown up to 100% protection against controlled human malaria infection (CHMI) using homologous parasites (same P. falciparum strain as in the vaccine). Using a more stringent CHMI, with heterologous parasites (different P. falciparum strain), we assessed the impact of higher PfSPZ doses, a novel multi-dose prime regimen, and a delayed vaccine boost upon vaccine efficacy (VE). METHODS: We immunized 4 groups that each contained 15 healthy, malaria-naive adults. Group 1 received 5 doses of 4.5 x 105 PfSPZ (Days 1, 3, 5, and 7; Week 16). Groups 2, 3, and 4 received 3 doses (Weeks 0, 8, and 16), with Group 2 receiving 9.0 × 105/doses; Group 3 receiving 18.0 × 105/doses; and Group 4 receiving 27.0 × 105 for dose 1 and 9.0 × 105 for doses 2 and 3. VE was assessed by heterologous CHMI after 12 or 24 weeks. Volunteers not protected at 12 weeks were boosted prior to repeat CHMI at 24 weeks. RESULTS: At 12-week CHMI, 6/15 (40%) participants in Group 1 (P = .04) and 3/15 (20%) participants in Group 2 remained aparasitemic, as compared to 0/8 controls. At 24-week CHMI, 3/13 (23%) participants in Group 3 and 3/14 (21%) participants in Group 4 remained aparasitemic, versus 0/8 controls (Groups 2-4, VE not significant). Postboost, 9/14 (64%) participants versus 0/8 controls remained aparasitemic (3/6 in Group 1, P = .025; 6/8 in Group 2, P = .002). CONCLUSIONS: Administering 4 stacked priming injections (multi-dose priming) resulted in 40% VE against heterologous CHMI, while dose escalation of PfSPZ using single-dose priming was not significantly protective. Boosting unprotected subjects improved VE at 24 weeks, to 64%. CLINICAL TRIALS REGISTRATION: NCT02601716.

Transient knockdown of Anopheles stephensi LRIM1 using RNAi increases Plasmodium falciparum sporozoite salivary gland infections.

BACKGROUND: Plasmodium falciparum (Pf) sporozoites (PfSPZ) can be administered as a highly protective vaccine conferring the highest protection seen to date. Sanaria® PfSPZ vaccines are produced using aseptically reared Anopheles stephensi mosquitoes. The bionomics of sporogonic development of P. falciparum in A. stephensi to fully mature salivary gland PfSPZ is thought to be modulated by several components of the mosquito innate immune system. In order to increase salivary gland PfSPZ infections in A. stephensi and thereby increase vaccine production efficiency, a gene knock down approach was used to investigate the activity of the immune deficiency (IMD) signaling pathway downstream effector leucine-rich repeat immune molecule 1 (LRIM1), an antagonist to Plasmodium development. METHODS: Expression of LRIM1 in A. stephensi was reduced following injection of double stranded (ds) RNA into mosquitoes. By combining the Gal4/UAS bipartite system with in vivo expression of short hairpin (sh) RNA coding for LRIM1 reduced expression of LRIM1 was targeted in the midgut, fat body, and salivary glands. RT-qPCR was used to demonstrate fold-changes in gene expression in three transgenic crosses and the effects on P. falciparum infections determined in mosquitoes showing the greatest reduction in LRIM1 expression. RESULTS: LRIM1 expression could be reduced, but not completely silenced, by expression of LRIM1 dsRNA. Infections of P. falciparum oocysts and PfSPZ were consistently and significantly higher in transgenic mosquitoes than wild type controls, with increases in PfSPZ ranging from 2.5- to tenfold. CONCLUSIONS: Plasmodium falciparum infections in A. stephensi can be increased following reduced expression of LRIM1. These data provide the springboard for more precise knockout of LRIM1 for the eventual incorporation of immune-compromised A. stephensi into manufacturing of Sanaria's PfSPZ products.

Artemisinin resistance phenotypes and K13 inheritance in a Plasmodium falciparum cross and Aotus model.

Concerns about malaria parasite resistance to treatment with artemisinin drugs (ARTs) have grown with findings of prolonged parasite clearance t1/2s (>5 h) and their association with mutations in Plasmodium falciparum Kelch-propeller protein K13. Here, we describe a P. falciparum laboratory cross of K13 C580Y mutant with C580 wild-type parasites to investigate ART response phenotypes in vitro and in vivo. After genotyping >400 isolated progeny, we evaluated 20 recombinants in vitro: IC50 measurements of dihydroartemisinin were at similar low nanomolar levels for C580Y- and C580-type progeny (mean ratio, 1.00; 95% CI, 0.62-1.61), whereas, in a ring-stage survival assay, the C580Y-type progeny had 19.6-fold (95% CI, 9.76-39.2) higher average counts. In splenectomized Aotus monkeys treated with three daily doses of i.v. artesunate, t1/2 calculations by three different methods yielded mean differences of 0.01 h (95% CI, -3.66 to 3.67), 0.80 h (95% CI, -0.92 to 2.53), and 2.07 h (95% CI, 0.77-3.36) between C580Y and C580 infections. Incidences of recrudescence were 57% in C580Y (4 of 7) versus 70% in C580 (7 of 10) infections (-13% difference; 95% CI, -58% to 35%). Allelic substitution of C580 in a C580Y-containing progeny clone (76H10) yielded a transformant (76H10C580Rev) that, in an infected monkey, recrudesced regularly 13 times over 500 d. Frequent recrudescences of ART-treated P. falciparum infections occur with or without K13 mutations and emphasize the need for improved partner drugs to effectively eliminate the parasites that persist through the ART component of combination therapy.

A Mosquito Pick-and-Place System for PfSPZ-based Malaria Vaccine Production.

The treatment of malaria is a global health challenge that stands to benefit from the widespread introduction of a vaccine for the disease. A method has been developed to create a live organism vaccine using the sporozoites (SPZ) of the parasite Plasmodium falciparum (Pf), which are concentrated in the salivary glands of infected mosquitoes. Current manual dissection methods to obtain these PfSPZ are not optimally efficient for large-scale vaccine production. We propose an improved dissection procedure and a mechanical fixture that increases the rate of mosquito dissection and helps to deskill this stage of the production process. We further demonstrate the automation of a key step in this production process, the picking and placing of mosquitoes from a staging apparatus into a dissection assembly. This unit test of a robotic mosquito pick-and-place system is performed using a custom-designed micro-gripper attached to a four degree of freedom (4-DOF) robot under the guidance of a computer vision system. Mosquitoes are autonomously grasped and pulled to a pair of notched dissection blades to remove the head of the mosquito, allowing access to the salivary glands. Placement into these blades is adapted based on output from computer vision to accommodate for the unique anatomy and orientation of each grasped mosquito. In this pilot test of the system on 50 mosquitoes, we demonstrate a 100% grasping accuracy and a 90% accuracy in placing the mosquito with its neck within the blade notches such that the head can be removed. This is a promising result for this difficult and non-standard pick-and-place task. NOTE TO PRACTITIONERS­: Automated processes could help increase malaria vaccine production to global scale. Currently, production requires technicians to manually dissect mosquitoes, a process that is slow, tedious, and requires a lengthy training regimen. This paper presents an an improved manual fixture and procedure that reduces technician training time. Further, an approach to automate this dissection process is proposed and the critical step of robotic manipulation of the mosquito with the aid of computer vision is demonstrated. Our approach may serve as a useful example of system design and integration for practitioners that seek to perform new and challenging pick-and-place tasks with small, non-uniform, and highly deformable objects.

Increase of Dose Associated With Decrease in Protection Against Controlled Human Malaria Infection by PfSPZ Vaccine in Tanzanian Adults.

BACKGROUND: A vaccine would be an ideal tool for reducing malaria's impact. PfSPZ Vaccine (radiation attenuated, aseptic, purified, cryopreserved Plasmodium falciparum [Pf] sporozoites [SPZ]) has been well tolerated and safe in >1526 malaria-naive and experienced 6-month to 65-year-olds in the United States, Europe, and Africa. When vaccine efficacy (VE) of 5 doses of 2.7 × 105 PfSPZ of PfSPZ Vaccine was assessed in adults against controlled human malaria infection (CHMI) in the United States and Tanzania and intense field transmission of heterogeneous Pf in Mali, Tanzanians had the lowest VE (20%). METHODS: To increase VE in Tanzania, we increased PfSPZ/dose (9 × 105 or 1.8 × 106) and decreased numbers of doses to 3 at 8-week intervals in a double blind, placebo-controlled trial. RESULTS: All 22 CHMIs in controls resulted in parasitemia by quantitative polymerase chain reaction. For the 9 × 105 PfSPZ group, VE was 100% (5/5) at 3 or 11 weeks (P < .000l, Barnard test, 2-tailed). For 1.8 × 106 PfSPZ, VE was 33% (2/6) at 7.5 weeks (P = .028). VE of dosage groups (100% vs 33%) was significantly different (P = .022). Volunteers underwent repeat CHMI at 37-40 weeks after last dose. 6/6 and 5/6 volunteers developed parasitemia, but time to first parasitemia was significantly longer than controls in the 9 × 105 PfSPZ group (10.89 vs 7.80 days) (P = .039), indicating a significant reduction in parasites in the liver. Antibody and T-cell responses were higher in the 1.8 × 106 PfSPZ group. CONCLUSIONS: In Tanzania, increasing the dose from 2.7 × 105 to 9 × 105 PfSPZ increased VE from 20% to 100%, but increasing to 1.8 × 106 PfSPZ significantly reduced VE. CLINICAL TRIALS REGISTRATION: NCT02613520.

Potential Therapeutic Application of Zinc Oxide Nanoflowers in the Cerebral Ischemia Rat Model through Neuritogenic and Neuroprotective Properties.

Neuritogenesis, a complex process of the sprouting of neurites, plays a vital role in the structural and functional restoration of cerebral ischemia-injured neuronal tissue. Practically, there is no effective long-term treatment strategy for cerebral ischemia in clinical practice to date due to several limitations of conventional therapies, facilitating the urgency to develop new alternative therapeutic approaches. Herein, for the first time we report that pro-angiogenic nanomaterials, zinc oxide nanoflowers (ZONF), exhibit neuritogenic activity by elevating mRNA expression of different neurotrophins, following PI3K/Akt-MAPK/ERK signaling pathways. Further, ZONF administration to global cerebral ischemia-induced Fischer rats shows improved neurobehavior and enhanced synaptic plasticity of neurons via upregulation of Neurabin-2 and NT-3, revealing their neuroprotective activity. Altogether, this study offers the basis for exploitation of angio-neural cross talk of other pro-angiogenic nano/biomaterials for future advancement of alternative treatment strategies for cerebral ischemia, where neuritogenesis and neural repair are highly critical.

Increased stress and altered expression of histone modifying enzymes in brain are associated with aberrant behaviour in vitamin B12 deficient female mice.

A sub-optimal nutritional environment from early life can be envisaged as a stressor that translates into mental health problems in adulthood. After considering (a) the widespread prevalence of vitamin B12 deficiency especially amongst women in developing countries and (b) the importance of vitamin B12 in normal brain function, in this study we have elucidated the behavioural correlates of chronic severe and moderate vitamin B12 deficiency in C57BL/6 mice. Female weanling mice were assigned to three dietary groups: (a) control AIN-76A diet with cellulose as dietary fibre (b) vitamin B12 restricted AIN-76A diet with pectin as dietary fibre (severe deficiency group) and (c) vitamin B12 restricted AIN-76A diet with cellulose as dietary fibre (moderate deficiency group). The mice received these diets throughout pregnancy, lactation and thereafter. Nest-building, maternal care, anxiety and depressive behaviours were evaluated. Oxidative stress, activities of antioxidant enzymes and expression of various histone modifying enzymes in brain were investigated to unravel the probable underlying mechanisms. Our data suggests that both severe and moderate vitamin B12 deficiency induced anxiety and impaired maternal care. However, only severe vitamin B12 deficiency induced depression. Oxidative stress and poor antioxidant defense underlie the deleterious effects of both severe and moderate vitamin B12 deficiency. Altered expression of histone modifying enzymes in the brain of severely deficient mice is suggestive of epigenetic reprogramming. This study suggests that chronic vitamin B12 deficiency leads to behavioural anomalies in female C57BL/6 mice and the severity of these outcomes can be correlated to the level of deficiency.

Attenuated PfSPZ Vaccine induces strain-transcending T cells and durable protection against heterologous controlled human malaria infection.

A live-attenuated malaria vaccine, Plasmodium falciparum sporozoite vaccine (PfSPZ Vaccine), confers sterile protection against controlled human malaria infection (CHMI) with Plasmodium falciparum (Pf) parasites homologous to the vaccine strain up to 14 mo after final vaccination. No injectable malaria vaccine has demonstrated long-term protection against CHMI using Pf parasites heterologous to the vaccine strain. Here, we conducted an open-label trial with PfSPZ Vaccine at a dose of 9.0 × 105 PfSPZ administered i.v. three times at 8-wk intervals to 15 malaria-naive adults. After CHMI with homologous Pf parasites 19 wk after final immunization, nine (64%) of 14 (95% CI, 35-87%) vaccinated volunteers remained without parasitemia compared with none of six nonvaccinated controls (P = 0.012). Of the nine nonparasitemic subjects, six underwent repeat CHMI with heterologous Pf7G8 parasites 33 wk after final immunization. Five (83%) of six (95% CI, 36-99%) remained without parasitemia compared with none of six nonvaccinated controls. PfSPZ-specific T-cell and antibody responses were detected in all vaccine recipients. Cytokine production by T cells from vaccinated subjects after in vitro stimulation with homologous (NF54) or heterologous (7G8) PfSPZ were highly correlated. Interestingly, PfSPZ-specific T-cell responses in the blood peaked after the first immunization and were not enhanced by subsequent immunizations. Collectively, these data suggest durable protection against homologous and heterologous Pf parasites can be achieved with PfSPZ Vaccine. Ongoing studies will determine whether protective efficacy can be enhanced by additional alterations in the vaccine dose and number of immunizations.

Is Saglin a mosquito salivary gland receptor for Plasmodium falciparum?

BACKGROUND: Saglin, a 100 kDa protein composed of two 50 kDa homodimers, is present in the salivary glands of Anopheles gambiae and has been considered an essential receptor for sporozoites (SPZ) of Plasmodium berghei and Plasmodium falciparum (Pf), allowing SPZ to recognize, bind to, and infect mosquito salivary glands. Spatial and temporal patterns of Saglin expression reported here, however, suggest that this model does not fully describe the Saglin-SPZ interaction. RESULTS: Saglin protein was detected by indirect immunofluorescence microscopy only in the medial and proximal-lateral lobes, but not in the distal-lateral lobes, of the salivary glands of An. gambiae; the pattern of expression was independent of mosquito age or physiological state. These results were confirmed by steady-state Saglin transcript and protein expression using qRT-PCR and Western-blot analysis, respectively. Saglin was localized to the basal surface of the cells of the medial lobes and was undetectable elsewhere (intracellularly, on the lateral or apical membranes, the cells' secretory vacuoles, or in the salivary duct). In the cells of the proximal lateral lobes of the salivary glands, Saglin was distinctly intracellular and was not localized to any of the cell surfaces. Transgenic Anopheles stephensi were produced that expressed An. gambiae Saglin in the distal lateral lobes of the salivary gland. Additional Saglin expression did not enhance infection by PfSPZ compared to non-transgenic siblings fed on the same gametocyte-containing blood meal. CONCLUSIONS: The absence of Saglin in the distal lateral lobes of the salivary glands, a primary destination for SPZ, suggests Saglin is not an essential receptor for Plasmodium SPZ. The lack of any correlation between increased Saglin expression in the distal lateral lobes of the salivary glands of transgenic An. stephensi and PfSPZ infection is also consistent with Saglin not being an essential salivary gland receptor for Plasmodium SPZ.

Plasmodium 18S rRNA of intravenously administered sporozoites does not persist in peripheral blood.

BACKGROUND: Plasmodium 18S rRNA is a biomarker used to monitor blood-stage infections in malaria clinical trials. Plasmodium sporozoites also express this biomarker, and there is conflicting evidence about how long sporozoite-derived 18S rRNA persists in peripheral blood. If present in blood for an extended timeframe, sporozoite-derived 18S rRNA could complicate use as a blood-stage biomarker. METHODS: Blood samples from Plasmodium yoelii infected mice were tested for Plasmodium 18S rRNA and their coding genes (rDNA) using sensitive quantitative reverse transcription PCR and quantitative PCR assays, respectively. Blood and tissues from Plasmodium falciparum sporozoite (PfSPZ)-infected rhesus macaques were similarly tested. RESULTS: In mice, when P. yoelii sporozoite inoculation and blood collection were performed at the same site (tail vein), low level rDNA positivity persisted for 2 days post-infection. Compared to intact parasites with high rRNA-to-rDNA ratios, this low level positivity was accompanied by no increase in rRNA-to-rDNA, indicating detection of residual, non-viable parasite rDNA. When P. yoelii sporozoites were administered via the retro-orbital vein and blood sampled by cardiac puncture, neither P. yoelii 18S rRNA nor rDNA were detected 24 h post-infection. Similarly, there was no P. falciparum 18S rRNA detected in blood of rhesus macaques 3 days after intravenous injection with extremely high doses of PfSPZ. Plasmodium 18S rRNA in the rhesus livers increased by approximately 101-fold from 3 to 6 days post infection, indicating liver-stage proliferation. CONCLUSIONS: Beyond the first few hours after injection, sporozoite-derived Plasmodium 18S rRNA was not detected in peripheral blood. Diagnostics based on 18S rRNA are unlikely to be confounded by sporozoite inocula in human clinical trials.