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Background Chemoprophylaxis vaccination with sporozoites (CVac) with chloroquine induces protection against a homologous Plasmodium falciparum sporozoite (PfSPZ) challenge, but whether blood-stage parasite exposure is required for protection remains unclear. Chloroquine suppresses and clears blood-stage parasitemia, while other antimalarial drugs, such as primaquine, act against liver-stage parasites. Here, we evaluated CVac regimens using primaquine and/or chloroquine as the partner drug to discern whether blood-stage parasite exposure impacts protection against homologous controlled human malaria infection. Methods In a Phase I, randomized, partial double-blind, placebo-controlled study of 36 malaria-naive adults, all CVac subjects received chloroquine prophylaxis and bites from 12–15 P. falciparum–infected mosquitoes (CVac-chloroquine arm) at 3 monthly iterations, and some received postexposure primaquine (CVac-primaquine/chloroquine arm). Drug control subjects received primaquine, chloroquine, and uninfected mosquito bites. After a chloroquine washout, subjects, including treatment-naive infectivity controls, underwent homologous, PfSPZ controlled human malaria infection and were monitored for parasitemia for 21 days. Results No serious adverse events occurred. During CVac, all but 1 subject in the study remained blood-smear negative, while only 1 subject (primaquine/chloroquine arm) remained polymerase chain reaction–negative. Upon challenge, compared to infectivity controls, 3/3 chloroquine arm subjects displayed delayed patent parasitemia (P = .01) but not sterile protection, while 3/11 primaquine/chloroquine subjects remained blood-smear negative. Conclusions CVac-primaquine/chloroquine is safe and induces sterile immunity to P. falciparum in some recipients, but a single 45 mg dose of primaquine postexposure does not completely prevent blood-stage parasitemia. Unlike previous studies, CVac-chloroquine did not produce sterile immunity. Clinical Trials Registration NCT01500980.

Malaria remains one of the most prevalent and lethal human infectious diseases worldwide. A comprehensive characterization of antibody responses to blood stage malaria is essential to support the development of future vaccines, sero-diagnostic tests, and sero-surveillance methods. We constructed a proteome array containing 4441 recombinant proteins expressed by the blood stages of the two most common human malaria parasites, P. falciparum (Pf) and P. vivax (Pv), and used this array to screen sera of Papua New Guinea children infected with Pf, Pv, or both (Pf/Pv) that were either symptomatic (febrile), or asymptomatic but had parasitemia detectable via microscopy or PCR. We hypothesized that asymptomatic children would develop antigen-specific antibody profiles associated with antidisease immunity, as compared with symptomatic children. The sera from these children recognized hundreds of the arrayed recombinant Pf and Pv proteins. In general, responses in asymptomatic children were highest in those with high parasitemia, suggesting that antibody levels are associated with parasite burden. In contrast, symptomatic children carried fewer antibodies than asymptomatic children with infections detectable by microscopy, particularly in Pv and Pf/Pv groups, suggesting that antibody production may be impaired during symptomatic infections. We used machine-learning algorithms to investigate the relationship between antibody responses and symptoms, and we identified antibody responses to sets of Plasmodium proteins that could predict clinical status of the donors. Several of these antibody responses were identified by multiple comparisons, including those against members of the serine enriched repeat antigen family and merozoite protein 4. Interestingly, both P. falciparum serine enriched repeat antigen-5 and merozoite protein 4 have been previously investigated for use in vaccines. This machine learning approach, never previously applied to proteome arrays, can be used to generate a list of potential seroprotective and/or diagnostic antigens candidates that can be further evaluated in longitudinal studies.

Background Immunization with genetically engineered, attenuated malaria parasites (GAP) that arrest during liver infection confers sterile protection in mouse malaria models. A first generation Plasmodium falciparum GAP ( Pf p52 − /p36 − GAP) was previously generated by deletion of two pre-erythrocytic stage-expressed genes (P52 and P36) in the NF54 strain. Methods A first-in-human, proof-of-concept, safety and immunogenicity clinical trial in six human volunteers was conducted. Exposure consisted of delivery of Pf p52 − /p36 − GAP sporozoites via infected Anopheles mosquito bite with a five-bite/volunteer exposure followed by an approximately 200-bite exposure/volunteer one month later. Results The exposures were well tolerated with mild to moderate local and systemic reactions. All volunteers remained blood stage negative after low dose exposure. Five volunteers remained blood stage negative after high dose exposure. One volunteer developed peripheral parasitemia twelve days after high dose exposure. Together the findings indicate that Pf p52 − /p36 − GAP was severely but not completely attenuated. All six volunteers developed antibodies to CSP. Furthermore, IFN-γ responses to whole sporozoites and multiple antigens were elicited in 5 of 6 volunteers, with both CD4 and CD8 cell cytokine production detected. Conclusion Severe attenuation and favorable immune responses following administration of a first generation Pf p52 − /p36 − GAP suggests that further development of live-attenuated strains using genetic engineering should be pursued.

The development of pre-erythrocytic Plasmodium vivax vaccines is hindered by the lack of in vitro culture systems or experimental rodent models. To help bypass these roadblocks, we exploited the fact that naturally exposed Fy- individuals who lack the Duffy blood antigen (Fy) receptor are less likely to develop blood-stage infections; therefore, they preferentially develop immune responses to pre-erythrocytic-stage parasites, whereas Fy+ individuals experience both liver- and blood-stage infections and develop immune responses to both pre-erythrocytic and erythrocytic parasites. We screened 60 endemic sera from P. vivax-exposed Fy+ or Fy- donors against a protein microarray containing 91 P. vivax proteins with P. falciparum orthologs that were up-regulated in sporozoites. Antibodies against 10 P. vivax antigens were identified in sera from P. vivax-exposed individuals but not unexposed controls. This technology has promising implications in the discovery of potential vaccine candidates against P. vivax malaria.

In addition to naturally occurring regulatory T (nTreg) cells derived from the thymus, functionally competent Treg cells can be induced in vitro from peripheral blood lymphocytes in response to TCR stimulation with cytokine costimulation. Using these artificial stimulation conditions, both naive as well as memory CD4+ T cells can be converted into induced Treg (iTreg) cells, but the cellular origin of such iTreg cells in vivo or in response to more physiologic stimulation with pathogen-derived antigens is less clear. Here, we demonstrate that a freeze/thaw lysate of Plasmodium falciparum schizont extract (PfSE) can induce functionally competent Treg cells from peripheral lymphocytes in a time- and dose-dependent manner without the addition of exogenous costimulatory factors. The PfSE-mediated induction of Treg cells required the presence of nTreg cells in the starting culture. Further experiments mixing either memory or naive T cells with antigen presenting cells and CFSE-labeled Treg cells identified CD4+CD45RO+CD25− memory T cells rather than Treg cells as the primary source of PfSE-induced Treg cells. Taken together, these data suggest that in the presence of nTreg cells, PfSE induces memory T cells to convert into iTreg cells that subsequently expand alongside PfSE-induced effector T cells.

Scientists who gathered at the most recent Keystone Symposia meeting on malaria described several advances, including applications of new technologies as well as new insights into pathogenesis and immunology. Enhanced tools to visualize the immune response to the Plasmodium pathogen, and systems biology approaches to interrogate host–parasite interactions, are altering our understanding of immunity and disease. At the same time, a partially effective subunit vaccine in human trials, and new models of highly protective human immunity, are raising expectations for immunological control of this ancient pandemic.

Regulatory T cells (Treg) play crucial roles in regulating autoimmune responses and immunity to tumors and infectious diseases. However, numerous subpopulations of Treg are now being described and the utility of various Treg markers is being reassessed. Here we report the results of a detailed phenotypic comparison of two supposedly regulatory human T-cell populations, namely CD4+FOXP3+ T cells and CD4+CD25hi T cells. We find that CD4+FOXP3+ cells are extremely heterogeneous with respect to CD25 expression and that FOXP3+ and CD25hi CD4+ T cells differ in their expression of chemokine receptors (CCR), CD95 and Bcl-2, suggestive of distinct migration characteristics and susceptibility to apoptosis. Further, we propose that CD25 expression should be regarded as an activation marker rather than as a defining marker of Treg. Lastly, CD4+FOXP3+ T cells activated in vitro with malaria antigen expressed the highest levels of CCR4 and CD95, and the lowest levels of CCR7, indicating that they are most likely generated from effector memory cells during an immune response and rapidly succumb to apoptosis at the end of the response.

Natural killer (NK) cells are lymphoid cells that mediate significant cytotoxic activity and produce high levels of pro-inflammatory cytokines in response to infection. During viral infection, NK cell cytotoxicity and cytokine production is induced principally by monocyte-macrophage- and dendritic cell-derived cytokines but virally encoded ligands for NK cells are also beginning to be described. NK derived interferon-gamma (IFN-gamma) production is also essential for control of several protozoal infections including toxoplasmosis, trypanosomiasis, leishmaniasis and malaria. The activation of NK cells by protozoan pathogens is also believed to be cytokine-mediated although some recent studies suggest that direct recognition of parasites by NK cells also occurs. Both indirect signalling via accessory cell-derived cytokines and direct signalling, presumably through NK receptors, are needed in order for human malaria parasites (Plasmodium falciparum) to optimally stimulate NK activity.

Background Controlled human malaria infection (CHMI) studies which recapitulate mosquito-borne infection are a critical tool to identify protective vaccine and drug candidates for advancement to field trials. In partnership with the Walter Reed Army Institute of Research, the CHMI model was established at the Seattle Biomedical Research Institute's Malaria Clinical Trials Center (MCTC). Activities and reagents at both centers were aligned to ensure comparability and continued safety of the model. To demonstrate successful implementation, CHMI was performed in six healthy malaria-naïve volunteers. Methods All volunteers received NF54 strain Plasmodium falciparum by the bite of five infected Anopheles stephensi mosquitoes under controlled conditions and were monitored for signs and symptoms of malaria and for parasitemia by peripheral blood smear. Subjects were treated upon diagnosis with chloroquine by directly observed therapy. Immunological (T cell and antibody) and molecular diagnostic (real-time quantitative reverse transcriptase polymerase chain reaction [qRT-PCR]) assessments were also performed. Results All six volunteers developed patent parasitemia and clinical malaria. No serious adverse events occurred during the study period or for six months post-infection. The mean prepatent period was 11.2 days (range 9–14 days), and geometric mean parasitemia upon diagnosis was 10.8 parasites/µL (range 2–69) by microscopy. qRT-PCR detected parasites an average of 3.7 days (range 2–4 days) earlier than blood smears. All volunteers developed antibodies to the blood-stage antigen merozoite surface protein 1 (MSP-1), which persisted up to six months. Humoral and cellular responses to pre-erythrocytic antigens circumsporozoite protein (CSP) and liver-stage antigen 1 (LSA-1) were limited. Conclusion The CHMI model was safe, well tolerated and characterized by consistent prepatent periods, pre-symptomatic diagnosis in 3/6 subjects and adverse event profiles as reported at established centers. The MCTC can now evaluate candidates in the increasingly diverse vaccine and drug pipeline using the CHMI model. Trial Registration ClinicalTrials.gov NCT01058226

Whole-parasite malaria vaccines have shown promise in clinical trials. We recently reported the first human trial of a malaria vaccine based on Plasmodium falciparum genetically attenuated parasites (PfGAP). Herein we report for the first time that PfGAP induces prolonged functional humoral responses in humans. Six volunteers were exposed to 5 bites of PfGAP-infected mosquitoes followed by approximately 200 bites. Plasma collected from all volunteers 3 months after the last exposure efficiently inhibits invasion of hepatocytes by P. falciparum sporozoites. The level of inhibition observed is comparable to that attained using plasma collected after 4–5 intravenously administrations of high numbers of irradiated sporozoites, validating the potential of PfGAP malaria vaccines. Our data highlight the role of antibody responses in pre-erythrocytic stages of human malaria, and suggests that to be protective, malaria vaccines might need to elicit long-lasting functional antibodies in addition to cellular responses.

Measurable residual disease detection (MRD) is an important tool for early detection and monitoring relapse. Traditionally, bone marrow is collected and analyzed by flow cytometry (FC) to evaluate MRD status in acute lymphoblastic leukemia patients (ALL). More recently, highly sensitive molecular methods such as deep sequencing allow for higher sensitivity detection of MRD. Although the bone marrow is considered to be more representative of tumor burden than sampling the blood, bone marrow collection is more invasive, disruptive, and resource intensive than peripheral blood draws. ALL patients enrolled in the Seattle Children's Pediatric Leukemia Adoptive Therapy (PLAT-02) trial undergo MRD assessment pre and post CD19 CAR T cell infusion using FC analysis of a bone marrow sample. We have used high throughput sequencing (HTS) in paired blood and bone marrow samples from these patients to evaluate the complete B cell repertoire and assess MRD up to 63 days post-infusion. To-date, we have obtained 38 paired samples. Using HTS alone we have observed a significant correlation in both the frequency of total reads and the number of malignant cells per million in paired peripheral blood and bone marrow samples. 26/38 of the bone marrow samples were determined either aparticulate or hypocellular. Importantly, 8/14 of patients that were MRD negative in the bone marrow by FC were MRD positive in the blood by HTS. These data suggest, that, in cases where the bone marrow is hypocellular or aparticulate, peripheral blood samples evaluated by HTS are more sensitive than traditional flow cytometric analysis in the bone marrow for detection of MRD. The impact of B cell aplasia may be important in increasing the sensitivity of HTS in the detection of MRD. The ability to detect an ALL B cell clone in the blood through HTS has many clinical implications; blood can be sampled more often, is less of a burden to patients, and can provide earlier prediction of relapse. Citation Format: Hannah Smithers, Olivia Finney, Ilan Kirsch, Beryl Crossley, Michael Jensen, Rebecca Gardner. Molecular detection of ALL in the peripheral blood is more sensitive than flow cytometric analysis of the bone marrow in patients with treatment-related hypocellularity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4893.

3048Background: CD19 CAR-T cell has proven efficacy for treatment of aggressive ALL. However, limited CAR-T cell persistence and/orCD19 loss are barriers to therapeutic success. Methods: A Phase I ...

Co-infection with HIV and P. falciparum worsens the prognosis of both infections; however, the mechanisms driving this adverse interaction are not fully delineated. To evaluate this, we studied HIV-1 and P. falciparum interactions in vitro using peripheral blood mononuclear cells (PBMCs) from human malaria naïve volunteers experimentally infected with P. falciparum in a malaria challenge trial. PBMCs collected before the malaria challenge and at several time points post-infection were infected with HIV-1 and co-cultured with either P. falciparum infected (iRBCs) or uninfected (uRBCs) red blood cells. HIV p24Ag and TNF-α, IFN-γ, IL-4, IL-6, IL-10, IL-17, and MIP-1α were quantified in the co-culture supernatants. In general, iRBCs stimulated more HIV p24Ag production by PBMCs than did uRBCs. HIV p24Ag production by PBMCs in the presence of iRBCs (but not uRBCs) further increased during convalescence (days 35, 56, and 90 post-challenge). In parallel, iRBCs induced higher secretion of pro-inflammatory cytokines (TNF-α, IFN-γ, and MIP-1α) than uRBCs, and production increased further during convalescence. Because the increase in p24Ag production occurred after parasitemia and generalized immune activation had resolved, our results suggest that enhanced HIV production is related to the development of anti-malaria immunity and may be mediated by pro-inflammatory cytokines.

T cell-mediated inflammatory immune responses contribute to both the clearance and pathology of malaria infections; the host's ability to down-regulate inflammation once parasitemia is controlled is crucial to avoid immune-mediated pathology but remains poorly understood. Various regulatory populations of T lymphocytes can modulate inflammatory immune responses and there has been considerable recent interest in the potential for regulatory T cells to modify the outcome of both murine and human malaria infections. Here, we review these studies, focussing in particular on recent studies in humans, propose a model by which different regulatory T cell populations might contribute to the control of inflammation at different stages of infection and discuss the implications for the design of safe and effective malaria vaccines.

An important aspect of clinical immunity to malaria is the ability to down-regulate inflammatory responses, once parasitaemia is under control, in order to avoid immune-mediated pathology. The role of classical (CD4(+)CD25(+)CD127(lo/-)FOXP3(+)) Treg in this process, however, remains controversial. Thus, we have characterized the frequency, phenotype and function of Treg populations, over time, in healthy individuals in The Gambia. We observed that both the percentage and the absolute number of CD4(+)FOXP3(+)CD127(lo/-) T cells were higher among individuals living in a rural village with highly seasonal malaria transmission than among individuals living in an urban area where malaria rarely occurs. These CD4(+)FOXP3(+)CD127(lo/-) T cells exhibited an effector memory and apoptosis-prone phenotype and suppressed cytokine production in response to malaria antigen. Cells from individuals exposed to malaria expressed significantly higher levels of mRNA for forkhead box P3 and T-box 21 (T-BET) at the end of the malaria transmission season than at the end of the non-transmission season. Importantly, the ratio of T-BET to forkhead box P3 was remarkably consistent between populations and over time, indicating that in healthy individuals, a transient increase in Th1 responses during the malaria transmission season is balanced by a commensurate Treg response, ensuring that immune homeostasis is maintained.

Failure to establish an appropriate balance between pro- and anti-inflammatory immune responses is believed to contribute to pathogenesis of severe malaria. To determine whether this balance is maintained by classical regulatory T cells (CD4+ FOXP3+ CD127−/low; Tregs) we compared cellular responses between Gambian children (n = 124) with severe Plasmodium falciparum malaria or uncomplicated malaria infections. Although no significant differences in Treg numbers or function were observed between the groups, Treg activity during acute disease was inversely correlated with malaria-specific memory responses detectable 28 days later. Thus, while Tregs may not regulate acute malarial inflammation, they may limit memory responses to levels that subsequently facilitate parasite clearance without causing immunopathology. Importantly, we identified a population of FOXP3−, CD45RO+ CD4+ T cells which coproduce IL-10 and IFN-γ. These cells are more prevalent in children with uncomplicated malaria than in those with severe disease, suggesting that they may be the regulators of acute malarial inflammation., Author Summary While Tregs have been implicated in regulation of the immune response to chronic infections, their potential in determining disease outcome in acute infections is unclear. In this study we have found that Tregs are unable to control the florid inflammation during acute, severe P. falciparum malaria infections, suggesting that this component of the immunoregulatory arsenal may be rapidly overwhelmed by virulent infections. Further, we identified, for the first time in an acute human infection, a population of IL-10-producing Th-1 effector cells and found that IL-10-producing Th-1 cells were associated with development of uncomplicated as opposed to severe malaria, leading us to suggest that such “self-regulating” Th-1 cells may contribute to clearing malaria infections without inducing immune-mediated pathology. In addition, we found evidence that malaria-induced Tregs may limit the magnitude of malaria-specific memory responses detectable 28 days later, which may reduce the risk of immune-mediated pathology upon reinfection and may explain how immunity to severe disease can be gained after as little as one or two infections. We conclude that vaccines designed to induce cell-mediated responses should be assessed for their ability to induce IL-10 producing Th-1 cells and Tregs.

It is well documented that the density of Plasmodium in its vertebrate host modulates the physiological response induced; this in turn regulates parasite survival and transmission. It is less clear that parasite density in the mosquito regulates survival and transmission of this important pathogen. Numerous studies have described conversion rates of Plasmodium from one life stage to the next within the mosquito, yet few have considered that these rates might vary with parasite density. Here we establish infections with defined numbers of the rodent malaria parasite Plasmodium berghei to examine how parasite density at each stage of development (gametocytes; ookinetes; oocysts and sporozoites) influences development to the ensuing stage in Anopheles stephensi, and thus the delivery of infectious sporozoites to the vertebrate host. We show that every developmental transition exhibits strong density dependence, with numbers of the ensuing stages saturating at high density. We further show that when fed ookinetes at very low densities, oocyst development is facilitated by increasing ookinete number (i.e., the efficiency of ookinete–oocyst transformation follows a sigmoid relationship). We discuss how observations on this model system generate important hypotheses for the understanding of malaria biology, and how these might guide the rational analysis of interventions against the transmission of the malaria parasites of humans by their diverse vector species., Author Summary Malaria, one of the world's most devastating parasitic diseases, is caused by protozoan parasites of the genus Plasmodium and is transmitted between mammalian hosts by Anopheles mosquitoes. Within the mosquito, the parasite undergoes four sequential developmental transformations as it passes from the bloodmeal through the mosquito's midgut epithelium to the salivary glands, from where the parasite is inoculated when the mosquito bites the vertebrate host. This study demonstrates, in a laboratory model, that parasite input density at every developmental stage in the mosquito regulates output to the ensuing form. Statistical models were fitted to experimental data to identify and describe the most appropriate functional relationships. In all cases, the relationships between two consecutive parasite stages can saturate at high parasite densities, suggesting that at high parasite densities parasite numbers may have to be reduced substantially to effect an appreciable decrease in parasite transmission. These results may help establish a rational basis for new studies on species of medical importance and further our understanding of how interventions designed to reduce parasite survival within the mosquito might be expected to impact upon transmission.