Studying the effect of chloroquine on sporozoite-induced protection and immune responses in Plasmodium berghei malaria

BACKGROUND Sporozoite immunization of animals and humans under a chemo-prophylactic cover of chloroquine (CPS-CQ) efficiently induces sterile protection against malaria. In humans, CPS-CQ is strikingly more efficient than immunization with radiation attenuated sporozoites (RAS), raising the hypothesis that this might be partially due to CQ. Chloroquine, an established anti-malarial drug, is also well known for its immune modulating properties including improvement of cross-presentation. The aim of this study was to investigate whether co-administration of CQ during sporozoite immunization improves cellular responses and protective efficacy in Plasmodium berghei models. METHODS A number of experiments in selected complimentary P. berghei murine models in Balb/cByJ and C57BL/6j mice was performed. First, the effect of CQ administration on the induction of protection and immune responses by RAS immunization was studied. Next, the effect of CQ on the induction of circumsporozoite (CS) protein-specific CD8(+) T cells by immunization with P. berghei parasites expressing a mutant CS protein was investigated. Finally, a direct comparison of CPS-CQ to CPS with mefloquine (MQ), an anti-malarial with little known immune modulating effects, was performed. RESULTS When CQ was co-administered during immunization with graded numbers of RAS, this did not lead to an increase in frequencies of total memory CD8(+) T cells or CS protein-specific CD8(+) T cells. Also parasite-specific cytokine production and protection remained unaltered. Replacement of CQ by MQ for CPS immunization resulted in significantly reduced percentages of IFNγ producing memory T cells in the liver (p = 0.01), but similar protection. CONCLUSIONS This study does not provide evidence for a direct beneficial effect of CQ on the induction of sporozoite-induced immune responses and protection in P. berghei malaria models. Alternatively, the higher efficiency of CPS compared to RAS might be explained by an indirect effect of CQ through limiting blood-stage exposure after immunization or to increased antigen exposure and, therefore, improved breadth of the immune response.EMB was supported by Top Institute Pharma (grant T4-102) and KN was supported by the NWO Mozaiek (grant no. 017.005.011)

Host-Based Biomarkers in Saliva for the Diagnosis of Pulmonary Tuberculosis in Children: A Mini-Review

The diagnosis of pulmonary tuberculosis (TB) in children remains a significant challenge due to its paucibacillary nature, non-specificity of symptoms and suboptimal sensitivity of available diagnostic methods. In young children particularly, it is difficult to obtain high-quality sputum specimens for testing, with this group the least likely to be diagnosed, while most at risk of severe disease. The World Health Organization (WHO) has prioritized research into rapid biomarker-based tests for TB using easily obtainable non-sputum samples, such as saliva. However, the role of biomarkers in saliva for diagnosing TB in children has not been fully explored. In this mini-review, we discuss the value of saliva as a diagnostic specimen in children given its ready availability and non-invasive nature of collection, and review the literature on the use of host-based biomarkers in saliva for diagnosing active pulmonary TB in adults and children. Based on available data from adult studies, we highlight that combinations of cytokines and other proteins show promise in reaching WHO-endorsed target product profiles for new TB triage tests. Given the lack of pediatric research on host biomarkers in saliva and the differing immune response to TB infection between children and adults, we recommend that pediatric studies are now performed to discover and validate salivary host biosignatures for diagnosing pulmonary TB in children. Future directions for pediatric saliva studies are discussed, with suggestions for technologies that can be applied for salivary biomarker discovery and point-of-care test development

Whole-blood transcriptomic signatures induced during immunization by chloroquine prophylaxis and Plasmodium falciparum sporozoites

A highly effective vaccine that confers sterile protection to malaria is urgently needed. Immunization under chemoprophylaxis with sporozoites (CPS) consistently confers high levels of protection in the Controlled Human Malaria infection (CHMI) model. To provide a broad, unbiased assessment of the composition and kinetics of direct ex vivo human immune responses to CPS, we profiled whole-blood transcriptomes by RNA-seq before and during CPS immunization and following CHMI challenge. Differential expression of genes enriched in modules related to T cells, NK cells, protein synthesis, and mitochondrial processes were detected in fully protected individuals four weeks after the first immunization. Non-protected individuals demonstrated transcriptomic changes after the third immunization and the day of treatment, with upregulation of interferon and innate inflammatory genes and downregulation of B-cell signatures. Protected individuals demonstrated more significant interactions between blood transcription modules compared to non-protected individuals several weeks after the second and third immunizations. These data provide insight into the molecular and cellular basis of CPS-induced immune protection from P. falciparum infection

Novel approaches to whole sporozoite vaccination against malaria

AbstractThe parasitic disease malaria threatens more than 3 billion people worldwide, resulting in more than 200 million clinical cases and almost 600,000 deaths annually. Vaccines remain crucial for prevention and ultimately eradication of infectious diseases and, for malaria, whole sporozoite based immunization has been shown to be the most effective in experimental settings. In addition to immunization with radiation-attenuated sporozoites, chemoprophylaxis and sporozoites (CPS) is a highly efficient strategy to induce sterile protection in humans. Genetically attenuated parasites (GAP) have demonstrated significant protection in rodent studies, and are now being advanced into clinical testing. This review describes the existing pre-clinical and clinical data on CPS and GAP, discusses recent developments and examines how to transform these immunization approaches into vaccine candidates for clinical development

Evaluation of immunity against malaria using luciferase-expressing Plasmodium berghei parasites

<p>Abstract</p> <p>Background</p> <p>Measurement of liver stage development is of key interest in malaria biology and vaccine studies. Parasite development in liver cells can be visualized in real-time, both in culture and in live mice, using a transgenic <it>Plasmodium berghei </it>parasite, <it>Pb</it>GFP-Luc_con, expressing the bioluminescent reporter luciferase. This study explores the benefit of using these parasites for the evaluation of immunity against malaria, compared to qRT-PCR techniques <it>in vivo </it>and <it>in vitro</it>.</p> <p>Methods</p> <p>Mice were immunized with either radiation attenuated sporozoites (RAS) or wildtype sporozoites under chloroquine prophylaxis (CPS) and challenged with <it>Pb</it>GFP-Luc_con.The <it>in vitro </it>transgenic sporozoites neutralization assay (TSNA) was adapted by replacing <it>Pb</it>CS(Pf) parasites for <it>Pb</it>GFP-Luc_conparasites.</p> <p>Results</p> <p>Application of <it>Pb</it>GFP-Luc_contransgenic parasites provides live quantitative visual information about the relation between parasite liver load and protection. Moreover, fast and reproducible results are obtained by using these parasites in the transgenic sporozoites neutralization assay, measuring functional antibody-mediated immune responses.</p> <p>Conclusions</p> <p><it>Pb</it>GFP-Luc_conparasites are a straightforward and valuable tool for comprehension of the biological and immunological principles underlying protection against malaria.</p

Uncertainty in tuberculosis clinical decision-making: An umbrella review with systematic methods and thematic analysis

Tuberculosis is a major infectious disease worldwide, but currently available diagnostics have suboptimal accuracy, particularly in patients unable to expectorate, and are often unavailable at the point-of-care in resource-limited settings. Test/treatment decision are, therefore, often made on clinical grounds. We hypothesized that contextual factors beyond disease probability may influence clinical decisions about when to test and when to treat for tuberculosis. This umbrella review aimed to identify such factors, and to develop a framework for uncertainty in tuberculosis clinical decision-making. Systematic reviews were searched in seven databases (MEDLINE, CINAHL Complete, Embase, Scopus, Cochrane, PROSPERO, Epistemonikos) using predetermined search criteria. Findings were classified as barriers and facilitators for testing or treatment decisions, and thematically analysed based on a multi-level model of uncertainty in health care. We included 27 reviews. Study designs and primary aims were heterogeneous, with seven meta-analyses and three qualitative evidence syntheses. Facilitators for decisions to test included providers’ advanced professional qualification and confidence in tests results, availability of automated diagnostics with quick turnaround times. Common barriers for requesting a diagnostic test included: poor provider tuberculosis knowledge, fear of acquiring tuberculosis through respiratory sampling, scarcity of healthcare resources, and complexity of specimen collection. Facilitators for empiric treatment included patients’ young age, severe sickness, and test inaccessibility. Main barriers to treatment included communication obstacles, providers’ high confidence in negative test results (irrespective of negative predictive value). Multiple sources of uncertainty were identified at the patient, provider, diagnostic test, and healthcare system levels. Complex determinants of uncertainty influenced decision-making. This could result in delayed or missed diagnosis and treatment opportunities. It is important to understand the variability associated with patient-provider clinical encounters and healthcare settings, clinicians’ attitudes, and experiences, as well as diagnostic test characteristics, to improve clinical practices, and allow an impactful introduction of novel diagnostics

Transcriptomic evidence for modulation of host inflammatory responses during febrile Plasmodium falciparum malaria

Identifying molecular predictors and mechanisms of malaria disease is important for understanding how Plasmodium falciparum malaria is controlled. Transcriptomic studies in humans have so far been limited to retrospective analysis of blood samples from clinical cases. In this prospective, proof-of-principle study, we compared whole-blood RNA-seq profiles at pre-and post-infection time points from Malian adults who were either asymptomatic (n = 5) or febrile (n = 3) during their first seasonal PCR-positive P. falciparum infection with those from malaria-naïve Dutch adults after a single controlled human malaria infection (n = 5). Our data show a graded activation of pathways downstream of pro-inflammatory cytokines, with the highest activation in malaria-naïve Dutch individuals and significantly reduced activation in malaria-experienced Malians. Newly febrile and asymptomatic infections in Malians were statistically indistinguishable except for genes activated by pro-inflammatory cytokines. The combined data provide a molecular basis for the development of a pyrogenic threshold as individuals acquire immunity to clinical malaria

Modest heterologous protection after Plasmodium falciparum sporozoite immunization: a double-blind randomized controlled clinical trial.

BACKGROUND: A highly efficacious vaccine is needed for malaria control and eradication. Immunization with Plasmodium falciparum NF54 parasites under chemoprophylaxis (chemoprophylaxis and sporozoite (CPS)-immunization) induces the most efficient long-lasting protection against a homologous parasite. However, parasite genetic diversity is a major hurdle for protection against heterologous strains. METHODS: We conducted a double-blind, randomized controlled trial in 39 healthy participants of NF54-CPS immunization by bites of 45 NF54-infected (n = 24 volunteers) or uninfected mosquitoes (placebo; n = 15 volunteers) against a controlled human malaria infection with the homologous NF54 or the genetically distinct NF135.C10 and NF166.C8 clones. Cellular and humoral immune assays were performed as well as genetic characterization of the parasite clones. RESULTS: NF54-CPS immunization induced complete protection in 5/5 volunteers against NF54 challenge infection at 14 weeks post-immunization, but sterilely protected only 2/10 and 1/9 volunteers against NF135.C10 and NF166.C8 challenge infection, respectively. Post-immunization plasma showed a significantly lower capacity to block heterologous parasite development in primary human hepatocytes compared to NF54. Whole genome sequencing showed that NF135.C10 and NF166.C8 have amino acid changes in multiple antigens targeted by CPS-induced antibodies. Volunteers protected against heterologous challenge were among the stronger immune responders to in vitro parasite stimulation. CONCLUSIONS: Although highly protective against homologous parasites, NF54-CPS-induced immunity is less effective against heterologous parasite clones both in vivo and in vitro. Our data indicate that whole sporozoite-based vaccine approaches require more potent immune responses for heterologous protection. TRIAL REGISTRATION: This trial is registered in clinicaltrials.gov, under identifier NCT02098590

Safety, Immunogenicity, and Protective Efficacy of Intradermal Immunization with Aseptic, Purified, Cryopreserved Plasmodium falciparum Sporozoites in Volunteers Under Chloroquine Prophylaxis

Immunization of volunteers under chloroquine prophylaxis by bites of *Plasmodium falciparum* sporozoite (PfSPZ)–infected mosquitoes induces > 90% protection against controlled human malaria infection (CHMI). We studied intradermal immunization with cryopreserved, infectious PfSPZ in volunteers taking chloroquine (PfSPZ chemoprophylaxis vaccine [CVac]). Vaccine groups 1 and 3 received 3x monthly immunizations with 7.5 x 10^4 PfSPZ. Control groups 2 and 4 received normal saline. Groups 1 and 2 underwent CHMI (#1) by mosquito bite 60 days after the third immunization. Groups 3 and 4 were boosted 168 days after the third immunization and underwent CHMI (#2) 137 days later. Vaccinees (11/20, 55%) and controls (6/10, 60%) had the same percentage of mild to moderate solicited adverse events. After CHMI #1, 8/10 vaccinees (group 1) and 5/5 controls (group 2) became parasitemic by microscopy; the two negatives were positive by quantitative real-time polymerase chain reaction (qPCR). After CHMI #2, all vaccinees in group 3 and controls in group 4 were parasitemic by qPCR. Vaccinees showed weak antibody and no detectable cellular immune responses. Intradermal immunization with up to 3 x 10^5 PfSPZ-CVac was safe, but induced only minimal immune responses and no sterile protection against Pf CHMI. INTRODUCTIO

Controlled human malaria infection with graded numbers of Plasmodium falciparum NF135.C10- or NF166.C8-infected mosquitoes

Controlled human malaria infections (CHMIs) with Plasmodium falciparum (Pf) parasites are well established. Exposure to five Pf (NF54)-infected Anopheles mosquitoes results in 100% infection rates in malaria-näive volunteers. Recently Pf clones NF135.C10 and NF166.C8 were generated for application in CHMIs. Here, we tested the clinical infection rates of these clones, using graded numbers of Pf-infected mosquitoes. In a double-blind randomized trial, we exposed 24 malaria-näive volunteers to bites from one, two, or five mosquitoes infected with NF135.C10 or NF166.C8. The primary endpoint was parasitemia by quantitative polymerase chain reaction. For both strains, bites by five infected mosquitoes resulted in parasitemiain4/4 volunteers; 3/4 volunteers developed parasitemia after exposure to one or two infected mosquitoes infected with either clone. The prepatent period was 7.25 ± 4.0 days (median ± range). There were no serious adverse events and comparable clinical symptoms between all groups. These data confirm the eligibility of NF135.C10 and NF166.C8 for use in CHMI studies