Infection-induced plasmablasts are a nutrient sink that impairs humoral immunity to malaria.

Plasmodium parasite-specific antibodies are critical for protection against malaria, yet the development of long-lived and effective humoral immunity against Plasmodium takes many years and multiple rounds of infection and cure. Here, we report that the rapid development of short-lived plasmablasts during experimental malaria unexpectedly hindered parasite control by impeding germinal center responses. Metabolic hyperactivity of plasmablasts resulted in nutrient deprivation of the germinal center reaction, limiting the generation of memory B cell and long-lived plasma cell responses. Therapeutic administration of a single amino acid to experimentally infected mice was sufficient to overcome the metabolic constraints imposed by plasmablasts and enhanced parasite clearance and the formation of protective humoral immune memory responses. Thus, our studies not only challenge the current model describing the role and function of blood-stage Plasmodium-induced plasmablasts but they also reveal new targets and strategies to improve anti-Plasmodium humoral immunity.

Complement in malaria immunity and vaccines.

Developing efficacious vaccines for human malaria caused by Plasmodium falciparum is a major global health priority, although this has proven to be immensely challenging over the decades. One major hindrance is the incomplete understanding of specific immune responses that confer protection against disease and/or infection. While antibodies to play a crucial role in malaria immunity, the functional mechanisms of these antibodies remain unclear as most research has primarily focused on the direct inhibitory or neutralizing activity of antibodies. Recently, there is a growing body of evidence that antibodies can also mediate effector functions through activating the complement system against multiple developmental stages of the parasite life cycle. These antibody-complement interactions can have detrimental consequences to parasite function and viability, and have been significantly associated with protection against clinical malaria in naturally acquired immunity, and emerging findings suggest these mechanisms could contribute to vaccine-induced immunity. In order to develop highly efficacious vaccines, strategies are needed that prioritize the induction of antibodies with enhanced functional activity, including the ability to activate complement. Here we review the role of complement in acquired immunity to malaria, and provide insights into how this knowledge could be used to harness complement in malaria vaccine development.

The Plasmodium falciparum parasitophorous vacuole protein P113 interacts with the parasite protein export machinery and maintains normal vacuole architecture.

Infection with Plasmodium falciparum parasites results in approximately 627,000 deaths from malaria annually. Key to the parasite's success is their ability to invade and subsequently grow within human erythrocytes. Parasite proteins involved in parasite invasion and proliferation are therefore intrinsically of great interest, as targeting these proteins could provide novel means of therapeutic intervention. One such protein is P113 which has been reported to be both an invasion protein and an intracellular protein located within the parasitophorous vacuole (PV). The PV is delimited by a membrane (PVM) across which a plethora of parasite-specific proteins are exported via the Plasmodium Translocon of Exported proteins (PTEX) into the erythrocyte to enact various immune evasion functions. To better understand the role of P113 we isolated its binding partners from in vitro cultures of P. falciparum. We detected interactions with the protein export machinery (PTEX and exported protein-interacting complex) and a variety of proteins that either transit through the PV or reside on the parasite plasma membrane. Genetic knockdown or partial deletion of P113 did not significantly reduce parasite growth or protein export but did disrupt the morphology of the PVM, suggesting that P113 may play a role in maintaining normal PVM architecture.

Comparison of Antibody Responses and Parasite Clearance in Artemisinin Therapeutic Efficacy Studies in the Democratic Republic of Congo and Asia.

BACKGROUND: Understanding the effect of immunity on Plasmodium falciparum clearance is essential for interpreting therapeutic efficacy studies designed to monitor emergence of artemisinin drug resistance. In low-transmission areas of Southeast Asia, where resistance has emerged, P. falciparum antibodies confound parasite clearance measures. However, variation in naturally acquired antibodies across Asian and sub-Saharan African epidemiological contexts and their impact on parasite clearance re yet to be quantified. METHODS: In an artemisinin therapeutic efficacy study, antibodies to 12 pre-erythrocytic and erythrocytic P. falciparum antigens were measured in 118 children with uncomplicated P. falciparum malaria in the Democratic Republic of Congo (DRC) and compared with responses in patients from Asian sites, described elsewhere. RESULTS: Parasite clearance half-life was shorter in DRC patients (median, 2 hours) compared with most Asian sites (median, 2-7 hours), but P. falciparum antibody levels and seroprevalences were similar. There was no evidence for an association between antibody seropositivity and parasite clearance half-life (mean difference between seronegative and seropositive, -0.14 to +0.40 hour) in DRC patients. CONCLUSIONS: In DRC, where artemisinin remains highly effective, the substantially shorter parasite clearance time compared with Asia was not explained by differences in the P. falciparum antibody responses studied.

Multifunctional Antibodies Are Induced by the RTS,S Malaria Vaccine and Associated With Protection in a Phase 1/2a Trial.

BACKGROUND: RTS,S is the leading malaria vaccine candidate but only confers partial efficacy against malaria in children. RTS,S is based on the major Plasmodium falciparum sporozoite surface antigen, circumsporozoite protein (CSP). The induction of anti-CSP antibodies is important for protection; however, it is unclear how these protective antibodies function. METHODS: We quantified the induction of functional anti-CSP antibody responses in healthy malaria-naive adults (N = 45) vaccinated with RTS,S/AS01. This included the ability to mediate effector functions via the fragment crystallizable (Fc) region, such as interacting with human complement proteins and Fcγ-receptors (FcγRs) that are expressed on immune cells, which promote various immunological functions. RESULTS: Our major findings were (1) RTS,S-induced antibodies mediated Fc-dependent effector functions, (2) functional antibodies were generally highest after the second vaccine dose, (3) functional antibodies targeted multiple regions of CSP, (4) participants with higher levels of functional antibodies had a reduced probability of developing parasitemia following homologous challenge (P < .05), and (5) nonprotected subjects had higher levels of anti-CSP IgM. CONCLUSIONS: Our data suggest a role for Fc-dependent antibody effector functions in RTS,S-induced immunity. Enhancing the induction of these functional activities may be a strategy to improve the protective efficacy of RTS,S or other malaria vaccines. CLINICAL TRIALS REGISTRATION: NCT00075049.

Reduced risk of placental parasitemia associated with complement fixation on Plasmodium falciparum by antibodies among pregnant women.

BACKGROUND: The pathogenesis of malaria in pregnancy (MiP) involves accumulation of P. falciparum-infected red blood cells (pRBCs) in the placenta, contributing to poor pregnancy outcomes. Parasite accumulation is primarily mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1). Magnitude of IgG to pRBCs has been associated with reduced risk of MiP in some studies, but associations have been inconsistent. Further, antibody effector mechanisms are poorly understood, and the role of antibody complement interactions is unknown. METHODS: Studying a longitudinal cohort of pregnant women (n=302) from a malaria-endemic province in Papua New Guinea (PNG), we measured the ability of antibodies to fix and activate complement using placental binding pRBCs and PfEMP1 recombinant domains. We determined antibody-mediated complement inhibition of pRBC binding to the placental receptor, chondroitin sulfate A (CSA), and associations with protection against placental parasitemia. RESULTS: Some women acquired antibodies that effectively promoted complement fixation on placental-binding pRBCs. Complement fixation correlated with IgG1 and IgG3 antibodies, which dominated the response. There was, however, limited evidence for membrane attack complex activity or pRBC lysis or killing. Importantly, a higher magnitude of complement fixing antibodies was prospectively associated with reduced odds of placental infection at delivery. Using genetically modified P. falciparum and recombinant PfEMP1 domains, we found that complement-fixing antibodies primarily targeted a specific variant of PfEMP1 (known as VAR2CSA). Furthermore, complement enhanced the ability of antibodies to inhibit pRBC binding to CSA, which was primarily mediated by complement C1q protein. CONCLUSIONS: These findings provide new insights into mechanisms mediating immunity to MiP and reveal potential new strategies for developing malaria vaccines that harness antibody-complement interactions.

Safety, infectivity and immunogenicity of a genetically attenuated blood-stage malaria vaccine.

BACKGROUND: There is a clear need for novel approaches to malaria vaccine development. We aimed to develop a genetically attenuated blood-stage vaccine and test its safety, infectivity, and immunogenicity in healthy volunteers. Our approach was to target the gene encoding the knob-associated histidine-rich protein (KAHRP), which is responsible for the assembly of knob structures at the infected erythrocyte surface. Knobs are required for correct display of the polymorphic adhesion ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1), a key virulence determinant encoded by a repertoire of var genes. METHODS: The gene encoding KAHRP was deleted from P. falciparum 3D7 and a master cell bank was produced in accordance with Good Manufacturing Practice. Eight malaria naïve males were intravenously inoculated (day 0) with 1800 (2 subjects), 1.8 × 105 (2 subjects), or 3 × 106 viable parasites (4 subjects). Parasitemia was measured using qPCR; immunogenicity was determined using standard assays. Parasites were rescued into culture for in vitro analyses (genome sequencing, cytoadhesion assays, scanning electron microscopy, var gene expression). RESULTS: None of the subjects who were administered with 1800 or 1.8 × 105 parasites developed parasitemia; 3/4 subjects administered 3× 106 parasites developed significant parasitemia, first detected on days 13, 18, and 22. One of these three subjects developed symptoms of malaria simultaneously with influenza B (day 17; 14,022 parasites/mL); one subject developed mild symptoms on day 28 (19,956 parasites/mL); and one subject remained asymptomatic up to day 35 (5046 parasites/mL). Parasitemia rapidly cleared with artemether/lumefantrine. Parasitemia induced a parasite-specific antibody and cell-mediated immune response. Parasites cultured ex vivo exhibited genotypic and phenotypic properties similar to inoculated parasites, although the var gene expression profile changed during growth in vivo. CONCLUSIONS: This study represents the first clinical investigation of a genetically attenuated blood-stage human malaria vaccine. A P. falciparum 3D7 kahrp- strain was tested in vivo and found to be immunogenic but can lead to patent parasitemia at high doses. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (number: ACTRN12617000824369 ; date: 06 June 2017).

PTEX is an essential nexus for protein export in malaria parasites.

During the blood stages of malaria, several hundred parasite-encoded proteins are exported beyond the double-membrane barrier that separates the parasite from the host cell cytosol. These proteins have a variety of roles that are essential to virulence or parasite growth. There is keen interest in understanding how proteins are exported and whether common machineries are involved in trafficking the different classes of exported proteins. One potential trafficking machine is a protein complex known as the Plasmodium translocon of exported proteins (PTEX). Although PTEX has been linked to the export of one class of exported proteins, there has been no direct evidence for its role and scope in protein translocation. Here we show, through the generation of two parasite lines defective for essential PTEX components (HSP101 or PTEX150), and analysis of a line lacking the non-essential component TRX2 (ref. 12), greatly reduced trafficking of all classes of exported proteins beyond the double membrane barrier enveloping the parasite. This includes proteins containing the PEXEL motif (RxLxE/Q/D) and PEXEL-negative exported proteins (PNEPs). Moreover, the export of proteins destined for expression on the infected erythrocyte surface, including the major virulence factor PfEMP1 in Plasmodium falciparum, was significantly reduced in PTEX knockdown parasites. PTEX function was also essential for blood-stage growth, because even a modest knockdown of PTEX components had a strong effect on the parasite's capacity to complete the erythrocytic cycle both in vitro and in vivo. Hence, as the only known nexus for protein export in Plasmodium parasites, and an essential enzymic machine, PTEX is a prime drug target.

Host immunity to Plasmodium falciparum and the assessment of emerging artemisinin resistance in a multinational cohort.

Artemisinin-resistant falciparum malaria, defined by a slow-clearance phenotype and the presence of kelch13 mutants, has emerged in the Greater Mekong Subregion. Naturally acquired immunity to malaria clears parasites independent of antimalarial drugs. We hypothesized that between- and within-population variations in host immunity influence parasite clearance after artemisinin treatment and the interpretation of emerging artemisinin resistance. Antibodies specific to 12 Plasmodium falciparum sporozoite and blood-stage antigens were determined in 959 patients (from 11 sites in Southeast Asia) participating in a multinational cohort study assessing parasite clearance half-life (PCt1/2) after artesunate treatment and kelch13 mutations. Linear mixed-effects modeling of pooled individual patient data assessed the association between antibody responses and PCt1/2.P. falciparum antibodies were lowest in areas where the prevalence of kelch13 mutations and slow PCt1/2 were highest [Spearman ρ = -0.90 (95% confidence interval, -0.97, -0.65), and Spearman ρ = -0.94 (95% confidence interval, -0.98, -0.77), respectively]. P. falciparum antibodies were associated with faster PCt1/2 (mean difference in PCt1/2 according to seropositivity, -0.16 to -0.65 h, depending on antigen); antibodies have a greater effect on the clearance of kelch13 mutant compared with wild-type parasites (mean difference in PCt1/2 according to seropositivity, -0.22 to -0.61 h faster in kelch13 mutants compared with wild-type parasites). Naturally acquired immunity accelerates the clearance of artemisinin-resistant parasites in patients with falciparum malaria and may confound the current working definition of artemisinin resistance. Immunity may also play an important role in the emergence and transmission potential of artemisinin-resistant parasites.

Antibody Targets on the Surface of Plasmodium falciparum-Infected Erythrocytes That Are Associated With Immunity to Severe Malaria in Young Children.

BACKGROUND: Sequestration of Plasmodium falciparum-infected erythrocytes (IEs) in the microvasculature contributes to pathogenesis of severe malaria in children. This mechanism is mediated by antigens expressed on the IE surface. However, knowledge of specific targets and functions of antibodies to IE surface antigens that protect against severe malaria is limited. METHODS: Antibodies to IE surface antigens were examined in a case-control study of young children in Papua New Guinea presenting with severe or uncomplicated malaria (n = 448), using isolates with a virulent phenotype associated with severe malaria, and functional opsonic phagocytosis assays. We used genetically modified isolates and recombinant P. falciparum erythrocyte membrane protein 1 (PfEMP1) domains to quantify PfEMP1 as a target of antibodies associated with disease severity. RESULTS: Antibodies to the IE surface and recombinant PfEMP1 domains were significantly higher in uncomplicated vs severe malaria and were boosted following infection. The use of genetically modified P. falciparum revealed that PfEMP1 was a major target of antibodies and that PfEMP1-specific antibodies were associated with reduced odds of severe malaria. Furthermore, antibodies promoting the opsonic phagocytosis of IEs by monocytes were lower in those with severe malaria. CONCLUSIONS: Findings suggest that PfEMP1 is a dominant target of antibodies associated with reduced risk of severe malaria, and function in part by promoting opsonic phagocytosis.

Human Immunization With a Polymorphic Malaria Vaccine Candidate Induced Antibodies to Conserved Epitopes That Promote Functional Antibodies to Multiple Parasite Strains.

Background: Overcoming antigenic diversity is a key challenge in the development of effective Plasmodium falciparum malaria vaccines. Strategies that promote the generation of antibodies targeting conserved epitopes of vaccine antigens may provide protection against diverse parasites strains. Understanding differences between vaccine-induced and naturally acquired immunity is important to achieving this goal. Methods: We analyzed antibodies generated in a phase 1 human vaccine trial, MSP2-C1, which included 2 allelic forms of MSP2, an abundant vaccine antigen on the merozoite surface. Vaccine-induced responses were assessed for functional activity against multiple parasite strains, and cross-reactivity of antibodies was determined using competition ELISA and epitope mapping approaches. Results: Vaccination induced cytophilic antibody responses with strain-transcending opsonic phagocytosis and complement-fixing function. In contrast to antibodies acquired via natural infection, vaccine-induced antibodies were directed towards conserved epitopes at the C-terminus of MSP2, whereas naturally acquired antibodies mainly targeted polymorphic epitopes. Functional activity of C-terminal-targeted antibodies was confirmed using monoclonal antibodies that promoted opsonic phagocytosis against multiple parasite strains. Conclusion: Vaccination generated markedly different responses to polymorphic antigens than naturally acquired immunity and targeted conserved functional epitopes. Induction of antibodies targeting conserved regions of malaria antigens provides a promising vaccine strategy to overcome antigenic diversity for developing effective malaria vaccines.

Patterns of protective associations differ for antibodies to P. falciparum-infected erythrocytes and merozoites in immunity against malaria in children.

Acquired antibodies play an important role in immunity to P. falciparum malaria and are typically directed towards surface antigens expressed by merozoites and infected erythrocytes (IEs). The importance of specific IE surface antigens as immune targets remains unclear. We evaluated antibodies and protective associations in two cohorts of children in Papua New Guinea. We used genetically-modified P. falciparum to evaluate the importance of PfEMP1 and a P. falciparum isolate with a virulent phenotype. Our findings suggested that PfEMP1 was the dominant target of antibodies to the IE surface, including functional antibodies that promoted opsonic phagocytosis by monocytes. Antibodies were associated with increasing age and concurrent parasitemia, and were higher among children exposed to a higher force-of-infection as determined using molecular detection. Antibodies to IE surface antigens were consistently associated with reduced risk of malaria in both younger and older children. However, protective associations for antibodies to merozoite surface antigens were only observed in older children. This suggests that antibodies to IE surface antigens, particularly PfEMP1, play an earlier role in acquired immunity to malaria, whereas greater exposure is required for protective antibodies to merozoite antigens. These findings have implications for vaccine design and serosurveillance of malaria transmission and immunity.

Establishment of a yeast-based VLP platform for antigen presentation.

BACKGROUND: Chimeric virus-like particles (VLP) allow the display of foreign antigens on their surface and have proved valuable in the development of safe subunit vaccines or drug delivery. However, finding an inexpensive production system and a VLP scaffold that allows stable incorporation of diverse, large foreign antigens are major challenges in this field. RESULTS: In this study, a versatile and cost-effective platform for chimeric VLP development was established. The membrane integral small surface protein (dS) of the duck hepatitis B virus was chosen as VLP scaffold and the industrially applied and safe yeast Hansenula polymorpha (syn. Pichia angusta, Ogataea polymorpha) as the heterologous expression host. Eight different, large molecular weight antigens of up to 412 amino acids derived from four animal-infecting viruses were genetically fused to the dS and recombinant production strains were isolated. In all cases, the fusion protein was well expressed and upon co-production with dS, chimeric VLP containing both proteins could be generated. Purification was accomplished by a downstream process adapted from the production of a recombinant hepatitis B VLP vaccine. Chimeric VLP were up to 95% pure on protein level and contained up to 33% fusion protein. Immunological data supported surface exposure of the foreign antigens on the native VLP. Approximately 40 mg of chimeric VLP per 100 g dry cell weight could be isolated. This is highly comparable to values reported for the optimized production of human hepatitis B VLP. Purified chimeric VLP were shown to be essentially stable for 6 months at 4 °C. CONCLUSIONS: The dS-based VLP scaffold tolerates the incorporation of a variety of large molecular weight foreign protein sequences. It is applicable for the display of highly immunogenic antigens originating from a variety of pathogens. The yeast-based production system allows cost-effective production that is not limited to small-scale fundamental research. Thus, the dS-based VLP platform is highly efficient for antigen presentation and should be considered in the development of future vaccines.

A single point in protein trafficking by Plasmodium falciparum determines the expression of major antigens on the surface of infected erythrocytes targeted by human antibodies.

Antibodies to blood-stage antigens of Plasmodium falciparum play a pivotal role in human immunity to malaria. During parasite development, multiple proteins are trafficked from the intracellular parasite to the surface of P. falciparum-infected erythrocytes (IEs). However, the relative importance of different proteins as targets of acquired antibodies, and key pathways involved in trafficking major antigens remain to be clearly defined. We quantified antibodies to surface antigens among children, adults, and pregnant women from different malaria-exposed regions. We quantified the importance of antigens as antibody targets using genetically engineered P. falciparum with modified surface antigen expression. Genetic deletion of the trafficking protein skeleton-binding protein-1 (SBP1), which is involved in trafficking the surface antigen PfEMP1, led to a dramatic reduction in antibody recognition of IEs and the ability of human antibodies to promote opsonic phagocytosis of IEs, a key mechanism of parasite clearance. The great majority of antibody epitopes on the IE surface were SBP1-dependent. This was demonstrated using parasite isolates with different genetic or phenotypic backgrounds, and among antibodies from children, adults, and pregnant women in different populations. Comparisons of antibody reactivity to parasite isolates with SBP1 deletion or inhibited PfEMP1 expression suggest that PfEMP1 is the dominant target of acquired human antibodies, and that other P. falciparum IE surface proteins are minor targets. These results establish SBP1 as part of a critical pathway for the trafficking of major surface antigens targeted by human immunity, and have key implications for vaccine development, and quantifying immunity in populations.

Infectivity of Plasmodium falciparum in Malaria-Naive Individuals Is Related to Knob Expression and Cytoadherence of the Parasite.

Plasmodium falciparum is the most virulent human malaria parasite because of its ability to cytoadhere in the microvasculature. Nonhuman primate studies demonstrated relationships among knob expression, cytoadherence, and infectivity. This has not been examined in humans. Cultured clinical-grade P. falciparum parasites (NF54, 7G8, and 3D7B) and ex vivo-derived cell banks were characterized. Knob and knob-associated histidine-rich protein expression, CD36 adhesion, and antibody recognition of parasitized erythrocytes (PEs) were evaluated. Parasites from the cell banks were administered to malaria-naive human volunteers to explore infectivity. For the NF54 and 3D7B cell banks, blood was collected from the study participants for in vitro characterization. All parasites were infective in vivo However, infectivity of NF54 was dramatically reduced. In vitro characterization revealed that unlike other cell bank parasites, NF54 PEs lacked knobs and did not cytoadhere. Recognition of NF54 PEs by immune sera was observed, suggesting P. falciparum erythrocyte membrane protein 1 expression. Subsequent recovery of knob expression and CD36-mediated adhesion were observed in PEs derived from participants infected with NF54. Knobless cell bank parasites have a dramatic reduction in infectivity and the ability to adhere to CD36. Subsequent infection of malaria-naive volunteers restored knob expression and CD36-mediated cytoadherence, thereby showing that the human environment can modulate virulence.

Surface antigens of Plasmodium falciparum-infected erythrocytes as immune targets and malaria vaccine candidates.

Understanding the targets and mechanisms of human immunity to malaria caused by Plasmodium falciparum is crucial for advancing effective vaccines and developing tools for measuring immunity and exposure in populations. Acquired immunity to malaria predominantly targets the blood stage of infection when merozoites of Plasmodium spp. infect erythrocytes and replicate within them. During the intra-erythrocytic development of P. falciparum, numerous parasite-derived antigens are expressed on the surface of infected erythrocytes (IEs). These antigens enable P. falciparum-IEs to adhere in the vasculature and accumulate in multiple organs, which is a key process in the pathogenesis of disease. IE surface antigens, often referred to as variant surface antigens, are important targets of acquired protective immunity and include PfEMP1, RIFIN, STEVOR and SURFIN. These antigens are highly polymorphic and encoded by multigene families, which generate substantial antigenic diversity to mediate immune evasion. The most important immune target appears to be PfEMP1, which is a major ligand for vascular adhesion and sequestration of IEs. Studies are beginning to identify specific variants of PfEMP1 linked to disease pathogenesis that may be suitable for vaccine development, but overcoming antigenic diversity in PfEMP1 remains a major challenge. Much less is known about other surface antigens, or antigens on the surface of gametocyte-IEs, the effector mechanisms that mediate immunity, and how immunity is acquired and maintained over time; these are important topics for future research.

Sequential processing of merozoite surface proteins during and after erythrocyte invasion by Plasmodium falciparum.

Plasmodium falciparum causes malaria disease during the asexual blood stages of infection when merozoites invade erythrocytes and replicate. Merozoite surface proteins (MSPs) are proposed to play a role in the initial binding of merozoites to erythrocytes, but precise roles remain undefined. Based on electron microscopy studies of invading Plasmodium merozoites, it is proposed that the majority of MSPs are cleaved and shed from the surface during invasion, perhaps to release receptor-ligand interactions. In this study, we demonstrate that there is not universal cleavage of MSPs during invasion. Instead, there is sequential and coordinated cleavage and shedding of proteins, indicating a diversity of roles for surface proteins during and after invasion. While MSP1 and peripheral surface proteins such as MSP3, MSP7, serine repeat antigen 4 (SERA4), and SERA5 are cleaved and shed at the tight junction between the invading merozoite and erythrocyte, the glycosylphosphatidylinositol (GPI)-anchored proteins MSP2 and MSP4 are carried into the erythrocyte without detectable processing. Following invasion, MSP2 rapidly degrades within 10 min, whereas MSP4 is maintained for hours. This suggests that while some proteins that are shed upon invasion may have roles in initial contact steps, others function during invasion and are then rapidly degraded, whereas others are internalized for roles during intraerythrocytic development. Interestingly, anti-MSP2 antibodies did not inhibit invasion and instead were carried into erythrocytes and maintained for approximately 20 h without inhibiting parasite development. These findings provide new insights into the mechanisms of invasion and knowledge to advance the development of new drugs and vaccines against malaria.

Adults with Plasmodium falciparum malaria have higher magnitude and quality of circulating T-follicular helper cells compared to children.

BACKGROUND: Protective malarial antibodies are acquired more rapidly in adults than children, independently of cumulative exposure, however the cellular responses mediating these differences are unknown. CD4 T-follicular helper (Tfh) cells have key roles in inducing antibodies, with Th2-Tfh cell activation associated with antibody development in malaria. Whether Tfh cell activation in malaria is age dependent is unknown and no studies have compared Tfh cell activation in children and adults with malaria. METHODS: We undertook a comprehensive study of Tfh cells, along with B cells and antibody induction in children and adults with malaria. Activation and proliferation of circulating Tfh (cTfh) cell subsets was measured ex vivo and parasite-specific Tfh cell frequencies and functions studied with Activation Induced Marker (AIM) assays and intracellular cytokine staining. FINDINGS: During acute malaria, the magnitude of cTfh cell activation was higher in adults than in children and occurred across all cTfh cell subsets in adults but was restricted only to the Th1-cTfh subset in children. Further, adults had higher levels of parasite-specific cTfh cells, and cTfh cells which produced more Th2-Tfh associated cytokine IL-4. Consistent with a role of higher Tfh cell activation in rapid immune development in adults, adults had higher activation of B cells during infection and higher induction of antibodies 7 and 28 days after malaria compared to children. INTERPRETATION: Our data provide evidence that age impacts Tfh cell activation during malaria, and that these differences may influence antibody induction after treatment. Findings have important implications for vaccine development in children. FUNDING: This word was supported by the National Health and Medical Research Council of Australia, Wellcome Trust, Charles Darwin University Menzies School of Health Research, Channel 7 Children's Research Foundation, and National Health Institute.

Age-dependent changes in circulating Tfh cells influence development of functional malaria antibodies in children.

T-follicular helper (Tfh) cells are key drivers of antibodies that protect from malaria. However, little is known regarding the host and parasite factors that influence Tfh and functional antibody development. Here, we use samples from a large cross-sectional study of children residing in an area of high malaria transmission in Uganda to characterize Tfh cells and functional antibodies to multiple parasites stages. We identify a dramatic re-distribution of the Tfh cell compartment with age that is independent of malaria exposure, with Th2-Tfh cells predominating in early childhood, while Th1-Tfh cell gradually increase to adult levels over the first decade of life. Functional antibody acquisition is age-dependent and hierarchical acquired based on parasite stage, with merozoite responses followed by sporozoite and gametocyte antibodies. Antibodies are boosted in children with current infection, and are higher in females. The children with the very highest antibody levels have increased Tfh cell activation and proliferation, consistent with a key role of Tfh cells in antibody development. Together, these data reveal a complex relationship between the circulating Tfh compartment, antibody development and protection from malaria.

Anti-Gametocyte Antigen Humoral Immunity and Gametocytemia During Treatment of Uncomplicated Falciparum Malaria: A Multi-National Study.

Introduction: Understanding the human immune response to Plasmodium falciparum gametocytes and its association with gametocytemia is essential for understanding the transmission of malaria as well as progressing transmission blocking vaccine candidates. Methods: In a multi-national clinical efficacy trial of artemisinin therapies (13 sites of varying transmission over South-East Asia and Africa), we measured Immunoglobulin G (IgG) responses to recombinant P. falciparum gametocyte antigens expressed on the gametocyte plasma membrane and leading transmission blocking vaccine candidates Pfs230 (Pfs230c and Pfs230D1M) and Pfs48/45 at enrolment in 1,114 participants with clinical falciparum malaria. Mixed effects linear and logistic regression were used to determine the association between gametocyte measures (gametocytemia and gametocyte density) and antibody outcomes at enrolment. Results: Microscopy detectable gametocytemia was observed in 11% (127/1,114) of participants at enrolment, and an additional 9% (95/1,114) over the follow-up period (up to day 42) (total 20% of participants [222/1,114]). IgG levels in response to Pfs230c, Pfs48/45 and Pfs230D1M varied across study sites at enrolment (p < 0.001), as did IgG seroprevalence for anti-Pfs230c and D1M IgG (p < 0.001), but not for anti-Pfs48/45 IgG (p = 0.159). In adjusted analyses, microscopy detectable gametocytemia at enrolment was associated with an increase in the odds of IgG seropositivity to the three gametocyte antigens (Pfs230c OR [95% CI], p: 1.70 [1.10, 2.62], 0.017; Pfs48/45: 1.45 [0.85, 2.46], 0.174; Pfs230D1M: 1.70 [1.03, 2.80], 0.037), as was higher gametocyte density at enrolment (per two-fold change in gametocyte density Pfs230c OR [95% CI], p: 1.09 [1.02, 1.17], 0.008; Pfs48/45: 1.05 [0.98, 1.13], 0.185; Pfs230D1M: 1.07 [0.99, 1.14], 0.071). Conclusion: Pfs230 and Pfs48/45 antibodies are naturally immunogenic targets associated with patent gametocytemia and increasing gametocyte density across multiple malaria endemic settings, including regions with emerging artemisinin-resistant P. falciparum.