Anne C. Teirlinck

Long-term protection against malaria after experimental sporozoite inoculation: an open-label follow-up study

BACKGROUND We have shown that immunity to infection with Plasmodium falciparum can be induced experimentally in malaria-naive volunteers through immunisation by bites of infected mosquitoes while simultaneously preventing disease with chloroquine prophylaxis. This immunity was associated with parasite-specific production of interferon γ and interleukin 2 by pluripotent effector memory cells in vitro. We aim to explore the persistence of protection and immune responses in the same volunteers. METHODS In an open-label study at the Radboud University Nijmegen Medical Centre (Nijmegen, Netherlands), from November to December, 2009, we rechallenged previously immune volunteers (28 months after immunisation) with the bites of five mosquitoes infected with P falciparum. Newly recruited malaria-naive volunteers served as infection controls. Our primary outcome was the detection of blood-stage parasitaemia by microscopy. We assessed the kinetics of parasitaemia with real-time quantitative PCR (rtPCR) and recorded clinical signs and symptoms. In-vitro production of interferon γ and interleukin 2 by effector memory T cells was studied after stimulation with sporozoites and red blood cells infected with P falciparum. Differences in cellular immune responses between the study groups were assessed with the Mann-Whitney test. This study is registered with ClinicalTrials.gov, number NCT00757887. FINDINGS Four of six immune volunteers were microscopically negative after rechallenge. rtPCR-based detection of blood-stage parasites in these individuals was negative throughout follow-up. Patent parasitaemia was delayed in the remaining two immunised volunteers. In-vitro assays showed the long-term persistence of parasite-specific pluripotent effector memory T-cell responses in protected volunteers. The four protected volunteers reported several mild to moderate adverse events, of which the most commonly reported symptom was headache (one to three episodes per volunteer). The two patients with delayed patency had adverse events similar to those in the control group. INTERPRETATION Artificially induced immunity lasts longer than generally recorded after natural exposure; providing a new avenue of research into the mechanisms of malaria immunity. FUNDING Dioraphte Foundation.

Protection against malaria after immunization by chloroquine prophylaxis and sporozoites is mediated by preerythrocytic immunity

Volunteers immunized under chloroquine chemoprophylaxis with Plasmodium falciparum sporozoites (CPS) develop complete, long-lasting protection against homologous sporozoite challenge. Chloroquine affects neither sporozoites nor liver-stages, but kills only asexual forms in erythrocytes once released from the liver into the circulation. Consequently, CPS immunization exposes the host to antigens from both preerythrocytic and blood stages, and induced immunity might target either of these stages. We therefore explored the life cycle stage specificity of CPS-induced protection. Twenty-five malaria-naïve volunteers were enrolled in a clinical trial, 15 of whom received CPS immunization. Five immunized subjects and five controls received a sporozoite challenge by mosquito bites, whereas nine immunized and five control subjects received an i.v. challenge with P. falciparum-infected erythrocytes. The latter approach completely bypasses preerythrocytic stages, enabling a direct comparison of protection against either life cycle stage. CPS-immunized subjects (13 of 14) developed anticircumsporozoite antibodies, whereas only one volunteer generated minimal titers against typical blood-stage antigens. IgG from CPS-immunized volunteers did not inhibit asexual blood-stage growth in vitro. All CPS-immunized subjects (5 of 5) were protected against sporozoite challenge. In contrast, nine of nine CPS-immunized subjects developed parasitemia after blood-stage challenge, with identical prepatent periods and blood-stage multiplication rates compared with controls. Intravenously challenged CPS-immunized subjects showed earlier fever and increased plasma concentrations of inflammatory markers D-dimer, IFN-γ, and monokine induced by IFN-γ than i.v. challenged controls. The complete lack of protection against blood-stage challenge indicates that CPS-induced protection is mediated by immunity against preerythrocytic stages. However, evidence is presented for immune recognition of P. falciparum-infected erythrocytes, suggesting memory responses unable to generate functional immunity.

Longevity and Composition of Cellular Immune Responses Following Experimental Plasmodium falciparum Malaria Infection in Humans

Cellular responses to Plasmodium falciparum parasites, in particular interferon-gamma (IFNγ) production, play an important role in anti-malarial immunity. However, clinical immunity to malaria develops slowly amongst naturally exposed populations, the dynamics of cellular responses in relation to exposure are difficult to study and data about the persistence of such responses are controversial. Here we assess the longevity and composition of cellular immune responses following experimental malaria infection in human volunteers. We conducted a longitudinal study of cellular immunological responses to sporozoites (PfSpz) and asexual blood-stage (PfRBC) malaria parasites in naïve human volunteers undergoing single (n = 5) or multiple (n = 10) experimental P. falciparum infections under highly controlled conditions. IFNγ and interleukin-2 (IL-2) responses following in vitro re-stimulation were measured by flow-cytometry prior to, during and more than one year post infection. We show that cellular responses to both PfSpz and PfRBC are induced and remain almost undiminished up to 14 months after even a single malaria episode. Remarkably, not only ‘adaptive’ but also ‘innate’ lymphocyte subsets contribute to the increased IFNγ response, including αβT cells, γδT cells and NK cells. Furthermore, results from depletion and autologous recombination experiments of lymphocyte subsets suggest that immunological memory for PfRBC is carried within both the αβT cells and γδT compartments. Indeed, the majority of cytokine producing T lymphocytes express an CD45RO+ CD62L- effector memory (EM) phenotype both early and late post infection. Finally, we demonstrate that malaria infection induces and maintains polyfunctional (IFNγ+IL-2+) EM responses against both PfRBC and PfSpz, previously found to be associated with protection. These data demonstrate that cellular responses can be readily induced and are long-lived following infection with P. falciparum, with a persisting contribution by not only adaptive but also (semi-)innate lymphocyte subsets. The implications hereof are positive for malaria vaccine development, but focus attention on those factors potentially inhibiting such responses in the field.

Controlled Human Malaria Infections by Intradermal Injection of Cryopreserved Plasmodium falciparum Sporozoites

Controlled human malaria infection with sporozoites is a standardized and powerful tool for evaluation of malaria vaccine and drug efficacy but so far only applied by exposure to bites of Plasmodium falciparum (Pf)-infected mosquitoes. We assessed in an open label Phase 1 trial, infection after intradermal injection of respectively 2,500, 10,000, or 25,000 aseptic, purified, vialed, cryopreserved Pf sporozoites (PfSPZ) in three groups (N = 6/group) of healthy Dutch volunteers. Infection was safe and parasitemia developed in 15 of 18 volunteers (84%), 5 of 6 volunteers in each group. There were no differences between groups in time until parasitemia by microscopy or quantitative polymerase chain reaction, parasite kinetics, clinical symptoms, or laboratory values. This is the first successful infection by needle and syringe with PfSPZ manufactured in compliance with regulatory standards. After further optimization, the use of such PfSPZ may facilitate and accelerate clinical development of novel malaria drugs and vaccines.

Mycobacterium tuberculosis induces IL-17A responses through TLR4 and dectin-1 and is critically dependent on endogenous IL-1

In the present study, we dissected the pathways that trigger the IL‐17A responses by MTB. Dectin‐1 and TLR4 were shown to be involved in MTB‐induced IL‐17A production, and blockade of the NOD2, TLR2, or MR had no effect on IL‐17A. The MAPK Erk, known to mediate transcription of IL‐1β mRNA, was strongly involved in the IL‐17A production induced by MTB. The intracellular enzymes caspase‐1 and serine proteases, which process pro‐IL‐1β into the active IL‐1β, were also crucial for the induction of IL‐17A. Lastly, the MTB‐induced IL‐17A response was strongly dependent on signaling through the IL‐1R but not the IL‐6R pathway. In conclusion, the MTB‐induced IL‐17A response relies strongly on the endogenous IL‐1 pathway and IL‐1R signaling. TLR4 and dectin‐1 are the main receptors responsible for mediating the signals responsible for IL‐17A production by MTB. These findings contribute to a better understanding of the host response to mycobacteria and provide the opportunity to explore potential, novel, therapeutic strategies against TB.

Cytotoxic Markers Associate With Protection Against Malaria in Human Volunteers Immunized With Plasmodium falciparum Sporozoites

Background. Immunization of healthy volunteers by bites from Plasmodium falciparum–infected mosquitoes during chloroquine chemoprophylaxis (hereafter, chemoprophylaxis and sporozoites [CPS] immunization) induces sterile protection against malaria. CPS-induced protection is mediated by immunity against pre-erythrocytic stages, presumably at least partially by cytotoxic cellular responses. We therefore aimed to investigate the association of CPS-induced cytotoxic T-cell markers with protection. Methods. In a double-blind randomized controlled trial, we performed dose titration of CPS immunization followed by homologous challenge infection in 29 subjects. Immune responses were assessed by in vitro restimulation of peripheral blood mononuclear cells and flow cytometry. Results. Dose-dependent complete protection was obtained in 4 of 5 volunteers after immunization with bites from 45 P. falciparum–infected mosquitoes, in 8 of 9 volunteers with bites from 30, and in 5 of 10 volunteers with bites from 15 (odds ratio [OR], 5.0; 95% confidence interval [CI], 1.5–17). Completely protected subjects had significantly higher proportions of CD4 T cells expressing the degranulation marker CD107a (OR, 8.4; 95% CI, 1.5–123; P = .011) and CD8 cells producing granzyme B (OR, 11; 95% CI, 1.9–212; P = .004) after P. falciparum restimulation. Conclusions. These data underline the efficiency of CPS immunization to induce sterile protection and support a possible role for cytotoxic CD4 and CD8 T-cell responses in pre-erythrocytic immunity. Clinical Trials Registration. NCT01218893.

NF135.C10: a new Plasmodium falciparum clone for controlled human malaria infections

We established a new field clone of Plasmodium falciparum for use in controlled human malaria infections and vaccine studies to complement the current small portfolio of P. falciparum strains, primarily based on NF54. The Cambodian clone NF135.C10 consistently produced gametocytes and generated substantial numbers of sporozoites in Anopheles mosquitoes and diverged from NF54 parasites by genetic markers. In a controlled human malaria infection trial, 3 of 5 volunteers challenged by mosquitoes infected with NF135.C10 and 4 of 5 challenged with NF54 developed parasitemia as detected with microscopy. The 2 strains induced similar clinical signs and symptoms as well as cellular immunological responses. Clinical Trials Registration NCT01002833.

Memory-like IFN-γ response by NK cells following malaria infection reveals the crucial role of T cells in NK cell activation by P. falciparum.

NK cells are rapid IFN‐γ responders to Plasmodium falciparum‐infected erythrocytes (PfRBC) in vitro and are involved in controlling early parasitaemia in murine models, yet little is known about their contribution to immune responses following malaria infection in humans. Here, we studied the dynamics of and requirements for in vitro NK responses to PfRBC in malaria‐naïve volunteers undergoing a single experimental malaria infection under highly controlled circumstances, and in naturally exposed individuals. NK‐specific IFN‐γ responses to PfRBC following exposure resembled an immunological recall pattern and were tightly correlated with T‐cell responses. However, although PBMC depleted of CD56+ cells retained 20–55% of their total IFN‐γ response to PfRBC, depletion of CD3+ cells completely abrogated the ability of remaining PBMC, including NK cells, to produce IFN‐γ. Although NK responses to PfRBC were partially dependent on endogenous IL‐2 signaling and could be augmented by exogenous IL‐2 in whole PBMC populations, this factor alone was insufficient to rescue NK responses in the absence of T cells. Thus, NK cells make a significant contribution to total IFN‐γ production in response to PfRBC as a consequence of their dependency on (memory) T‐cell help, with likely positive implications for malaria vaccine development.

Memory B-Cell and Antibody Responses Induced by Plasmodium falciparum Sporozoite Immunization

Background Immunization of healthy volunteers during receipt of chemoprophylaxis with Plasmodium falciparum sporozoites (CPS-immunization) induces sterile protection from malaria. Antibody responses have long been known to contribute to naturally acquired immunity against malaria, but their association with sterile protection after whole sporozoite immunization is not well established. We therefore studied the induction and kinetics of malaria parasite antigen-specific antibodies and memory B-cells (MBCs) during CPS-immunization and their correlation with protection from challenge infection. Methods We assessed humoral reactivity to 9 antigens representing different stages of the life cycle of P. falciparum by performing standardized MBC enzyme-linked immunospot and enzyme-linked immunosorbent assays on peripheral blood mononuclear cells and plasma samples from 38 Dutch volunteers enrolled in 2 randomized controlled clinical trials. Results MBCs and antibodies recognizing pre-erythrocytic and cross-stage antigens were gradually acquired during CPS-immunization. The magnitude of these humoral responses did not correlate with protection but directly reflected parasite exposure in CPS-immunization and challenge. Conclusions Humoral responses to the malarial antigens circumsporozoite protein, liver-stage antigen-1, apical membrane antigen-1, and merozoite surface protein-1 do not to predict protection from challenge infection but can be used as sensitive marker of recent parasite exposure. Clinical Trials Registration NCT01236612 and NCT01218893.

BAFF and BAFF Receptor Levels Correlate with B Cell Subset Activation and Redistribution in Controlled Human Malaria Infection

Characteristic features of Plasmodium falciparum malaria are polyclonal B cell activation and an altered composition of the blood B cell compartment, including expansion of CD21−CD27− atypical memory B cells. BAFF is a key cytokine in B cell homeostasis, but its potential contribution to the modulation of the blood B cell pool during malaria remains elusive. In the controlled human malaria model (CHMI) in malaria-naive Dutch volunteers, we therefore examined the dynamics of BAFF induction and B cell subset activation and composition, to investigate whether these changes are linked to malaria-induced immune activation and, in particular, induction of BAFF. Alterations in B cell composition after CHMI closely resembled those observed in endemic areas. We further found distinct kinetics of proliferation for individual B cell subsets across all developmental stages. Proliferation peaked either immediately after blood-stage infection or at convalescence, and for most subsets was directly associated with the peak parasitemia. Concomitantly, plasma BAFF levels during CHMI were increased and correlated with membrane-expressed BAFF on monocytes and dendritic cells, as well as blood-stage parasitemia and parasite-induced IFN-γ. Correlating with increased plasma BAFF and IFN-γ levels, IgD−CD38lowCD21−CD27− atypical B cells showed the strongest proliferative response of all memory B cell subsets. This provides unique evidence for a link between malaria-induced immune activation and temporary expansion of this B cell subset. Finally, baseline BAFF-R levels before CHMI were predictive of subsequent changes in proportions of individual B cell subsets. These findings suggest an important role of BAFF in facilitating B cell subset proliferation and redistribution as a consequence of malaria-induced immune activation.