BACKGROUND Treatment of Plasmodium vivax malaria requires the clearing of asexual parasites, but relapse can be prevented only if dormant hypnozoites are cleared from the liver (a treatment termed “radical cure”). Tafenoquine is a single-dose 8-aminoquinoline that has recently been registered for the radical cure of P. vivax . METHODS This multicenter, double-blind, double-dummy, parallel group, randomized, placebo-controlled trial was conducted in Ethiopia, Peru, Brazil, Cambodia, Thailand, and the Philippines. We enrolled 522 patients with microscopically confirmed P. vivax infection (>100 to <100,000 parasites per microliter) and normal glucose-6-phosphate dehydrogenase (G6PD) activity (with normal activity defined as ≥70% of the median value determined at each trial site among 36 healthy male volunteers who were otherwise not involved in the trial). All patients received a 3-day course of chloroquine (total dose of 1500 mg). In addition, patients were assigned to receive a single 300-mg dose of tafenoquine on day 1 or 2 (260 patients), placebo (133 patients), or a 15-mg dose of prima-quine once daily for 14 days (129 patients). The primary outcome was the Kaplan– Meier estimated percentage of patients who were free from recurrence at 6 months, defined as P. vivax clearance without recurrent parasitemia. RESULTS In the intention-to-treat population, the percentage of patients who were free from recurrence at 6 months was 62.4% in the tafenoquine group (95% confidence interval [CI], 54.9 to 69.0), 27.7% in the placebo group (95% CI, 19.6 to 36.6), and 69.6% in the primaquine group (95% CI, 60.2 to 77.1). The hazard ratio for the risk of recurrence was 0.30 (95% CI, 0.22 to 0.40) with tafenoquine as compared with placebo (P<0.001) and 0.26 (95% CI, 0.18 to 0.39) with primaquine as compared with placebo (P<0.001). Tafenoquine was associated with asymptomatic declines in hemoglobin levels, which resolved without intervention. CONCLUSIONS Single-dose tafenoquine resulted in a significantly lower risk of P. vivax recurrence than placebo in patients with phenotypically normal G6PD activity. (Funded by GlaxoSmith-Kline and Medicines for Malaria Venture; DETECTIVE ClinicalTrials.gov number, NCT01376167.)

Cyclic paroxysm and high fever are hallmarks of malaria and are associated with high levels of pyrogenic cytokines, including IL-1β. In this report, we describe a signature for the expression of inflammasome-related genes and caspase-1 activation in malaria. Indeed, when we infected mice, Plasmodium infection was sufficient to promote MyD88-mediated caspase-1 activation, dependent on IFN-γ-priming and the expression of inflammasome components ASC, P2X7R, NLRP3 and/or NLRP12. Pro-IL-1β expression required a second stimulation with LPS and was also dependent on IFN-γ-priming and functional TNFR1. As a consequence of Plasmodium-induced caspase-1 activation, mice produced extremely high levels of IL-1β upon a second microbial stimulus, and became hypersensitive to septic shock. Therapeutic intervention with IL-1 receptor antagonist prevented bacterial-induced lethality in rodents. Similar to mice, we observed a significantly increased frequency of circulating CD14+CD16−Caspase-1+ and CD14dimCD16+Caspase-1+ monocytes in peripheral blood mononuclear cells from febrile malaria patients. These cells readily produced large amounts of IL-1β after stimulation with LPS. Furthermore, we observed the presence of inflammasome complexes in monocytes from malaria patients containing either NLRP3 or NLRP12 pyroptosomes. We conclude that NLRP12/NLRP3-dependent activation of caspase-1 is likely to be a key event in mediating systemic production of IL-1β and hypersensitivity to secondary bacterial infection during malaria.

SUMMARY Neutrophils are the most abundant leukocyte population in the bloodstream, the primary compartment of Plasmodium sp. infection. Yet, the role of these polymorphonuclear cells in mediating either resistance or pathogenesis of malaria is poorly understood. We report that circulating neutrophils from malaria patients are highly activated, as indicated by a strong type I interferon transcriptional signature, increased expression of surface activation markers, the enhanced release of reactive oxygen species and myeloperoxidase, as well as the high frequency of low-density granulocytes. The activation of neutrophils was associated with increased levels of serum alanine and aspartate aminotransferases, indicating liver damage. In a rodent malaria model, we observed an intense recruitment of neutrophils to liver sinusoids. Neutrophil migration, IL-1β and chemokine expression as well as liver damage were all dependent on type I interferon signaling. The data suggests that type I interferon signaling have a central role in neutrophil activation and malaria pathogenesis.

SummaryBackgroundChloroquine remains the mainstay of treatment for Plasmodium vivax malaria despite increasing reports of treatment failure. We did a systematic review and meta-analysis to investigate the effect of chloroquine dose and the addition of primaquine on the risk of recurrent vivax malaria across different settings.MethodsA systematic review done in MEDLINE, Web of Science, Embase, and Cochrane Database of Systematic Reviews identified P vivax clinical trials published between Jan 1, 2000, and March 22, 2017. Principal investigators were invited to share individual patient data, which were pooled using standardised methods. Cox regression analyses with random effects for study site were used to investigate the roles of chloroquine dose and primaquine use on rate of recurrence between day 7 and day 42 (primary outcome). The review protocol is registered in PROSPERO, number CRD42016053310.FindingsOf 134 identified chloroquine studies, 37 studies (from 17 countries) and 5240 patients were included. 2990 patients were treated with chloroquine alone, of whom 1041 (34·8%) received a dose below the target 25 mg/kg. The risk of recurrence was 32·4% (95% CI 29·8–35·1) by day 42. After controlling for confounders, a 5 mg/kg higher chloroquine dose reduced the rate of recurrence overall (adjusted hazard ratio [AHR] 0·82, 95% CI 0·69–0·97; p=0·021) and in children younger than 5 years (0·59, 0·41–0·86; p=0·0058). Adding primaquine reduced the risk of recurrence to 4·9% (95% CI 3·1–7·7) by day 42, which is lower than with chloroquine alone (AHR 0·10, 0·05–0·17; p<0·0001).InterpretationChloroquine is commonly under-dosed in the treatment of vivax malaria. Increasing the recommended dose to 30 mg/kg in children younger than 5 years could reduce substantially the risk of early recurrence when primaquine is not given. Radical cure with primaquine was highly effective in preventing early recurrence and may also improve blood schizontocidal efficacy against chloroquine-resistant P vivax.FundingWellcome Trust, Australian National Health and Medical Research Council, and Bill & Melinda Gates Foundation.

Although the importance of humoral immunity to malaria has been established, factors that control antibody production are poorly understood. Follicular helper T cells (Tfh cells) are pivotal for generating high-affinity, long-lived antibody responses. While it has been proposed that expansion of antigen-specific Tfh cells, interleukin (IL) 21 production and robust germinal center formation are associated with protection against malaria in mice, whether Tfh cells are found during Plasmodium vivax (P. vivax) infection and if they play a role during disease remains unknown. Our goal was to define the role of Tfh cells during P. vivax malaria. We demonstrate that P. vivax infection triggers IL-21 production and an increase in Tfh cells (PD-1+ICOS+CXCR5+CD45RO+CD4+CD3+). As expected, FACS-sorted Tfh cells, the primary source of IL-21, induced immunoglobulin production by purified naïve B cells. Furthermore, we found that P. vivax infection alters the B cell compartment and these alterations were dependent on the number of previous infections. First exposure leads to increased proportions of activated and atypical memory B cells and decreased frequencies of classical memory B cells, whereas patients that experienced multiple episodes displayed lower proportions of atypical B cells and higher frequencies of classical memory B cells. Despite the limited sample size, but consistent with the latter finding, the data suggest that patients who had more than five infections harbored more Tfh cells and produce more specific antibodies. P. vivax infection triggers IL-21 production by Tfh that impact B cell responses in humans.

Infection with Plasmodium vivax results in strong activation of monocytes, which are important components of both the systemic inflammatory response and parasite control. The overall goal of this study was to define the role of monocytes during P. vivax malaria. Here, we demonstrate that P. vivax–infected patients display significant increase in circulating monocytes, which were defined as CD14+CD16− (classical), CD14+CD16+ (inflammatory), and CD14loCD16+ (patrolling) cells. While the classical and inflammatory monocytes were found to be the primary source of pro-inflammatory cytokines, the CD16+ cells, in particular the CD14+CD16+ monocytes, expressed the highest levels of activation markers, which included chemokine receptors and adhesion molecules. Morphologically, CD14+ were distinguished from CD14lo monocytes by displaying larger and more active mitochondria. CD14+CD16+ monocytes were more efficient in phagocytizing P. vivax-infected reticulocytes, which induced them to produce high levels of intracellular TNF-α and reactive oxygen species. Importantly, antibodies specific for ICAM-1, PECAM-1 or LFA-1 efficiently blocked the phagocytosis of infected reticulocytes by monocytes. Hence, our results provide key information on the mechanism by which CD14+CD16+ cells control parasite burden, supporting the hypothesis that they play a role in resistance to P. vivax infection.

Background The activation of innate immune responses by Plasmodium vivax results in activation of effector cells and an excessive production of pro-inflammatory cytokines that may culminate in deleterious effects. Here, we examined the activation and function of neutrophils during acute episodes of malaria. Materials and Methods Blood samples were collected from P. vivax -infected patients at admission (day 0) and 30–45 days after treatment with chloroquine and primaquine. Expression of activation markers and cytokine levels produced by highly purified monocytes and neutrophils were measured by the Cytometric Bead Assay. Phagocytic activity, superoxide production, chemotaxis and the presence of G protein-coupled receptor (GRK2) were also evaluated in neutrophils from malaria patients. Principal Findings Both monocytes and neutrophils from P. vivax -infected patients were highly activated. While monocytes were found to be the main source of cytokines in response to TLR ligands, neutrophils showed enhanced phagocytic activity and superoxide production. Interestingly, neutrophils from the malaria patients expressed high levels of GRK2, low levels of CXCR2, and displayed impaired chemotaxis towards IL-8 (CXCL8). Conclusion Activated neutrophils from malaria patients are a poor source of pro-inflammatory cytokines and display reduced chemotactic activity, suggesting a possible mechanism for an enhanced susceptibility to secondary bacterial infection during malaria.

Plasmodium vivax invasion into human reticulocytes is a complex process. The Duffy binding protein (DBP) dimerization with its cognate receptor is vital for junction formation in the invasion process. Due to its functional importance, DBP is considered a prime vaccine candidate, but variation in B-cell epitopes at the dimer interface of DBP leads to induction of strain-limited immunity. We believe that the polymorphic residues tend to divert immune responses away from functionally conserved epitopes important for receptor binding or DBP dimerization. As a proof of concept, we engineered the vaccine DEKnull to ablate the dominant Bc epitope to partially overcome strain-specific immune antibody responses. Additional surface engineering on the next generation immunogen, DEKnull-2, provides an immunogenicity breakthrough to conserved protective epitopes. DEKnull-2 elicits a stronger broadly neutralizing response and reactivity with long-term persistent antibody responses of acquired natural immunity. By using novel engineered DBP immunogens, we validate that the prime targets of protective immunity are conformational epitopes at the dimer interface. These successful results indicate a potential approach that can be used generally to improve efficacy of other malaria vaccine candidates.