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Summary: Background: Human immune responses to infection and vaccination are heterogenous, driven by multiple factors including genetics, environmental exposures and personal infection histories. For malaria caused by Plasmodium falciparum parasites, host factors that impact on humoral immunity are poorly understood. Methods: We investigated the role of latent cytomegalovirus (CMV) on the host immune response to malaria using samples obtained from individuals in previously conducted Phase 1 trials of blood stage P. falciparum Controlled Human Malaria Infection (CHMI) and in a MSP1 vaccine clinical trial. Induced antibody and functions of antibodies, as well as CD4 T cell responses were quantified. Findings: CMV seropositivity was associated with reduced induction of parasite specific antibodies following malaria infection and vaccination. During infection, reduced antibody induction was associated with modifications to the T -follicular helper (Tfh) cell compartment. CMV seropositivity was associated with a skew towards Tfh1 cell subsets before and after malaria infection, and reduced activation of Tfh2 cells. Protective Tfh2 cell activation was only associated with antibody development in individuals who were CMV seronegative, and a higher proportion of Tfh1 cells was associated with lower antibody development in individuals who were CMV seropositive. During MSP1 vaccination, reduced antibody induction in individuals who were CMV seropositive was associated with CD4 T cell expression of terminal differentiation marker CD57. Interpretation: These findings suggest that CMV seropositivity may be negatively associated with malaria antibody development. Further studies in larger cohorts, particularly in malaria endemic regions are required to investigate whether CMV infection may modify immunity to malaria gained during infection or vaccination in children. Funding: Work was funded by National Health and Medical Research Council of Australia, CSL Australia and Snow Medical Foundation. Funders had no role in data generation, writing of manuscript of decision to submit for publication.

Abstract Plasmodium falciparum malaria drives immunoregulatory responses across multiple cell subsets, which protects from immunopathogenesis, but also hampers the development of effective anti-parasitic immunity. Understanding malaria induced tolerogenic responses in specific cell subsets may inform development of strategies to boost protective immunity during drug treatment and vaccination. Here, we analyse the immune landscape with single cell RNA sequencing during P. falciparum malaria. We identify cell type specific responses in sub-clustered major immune cell types. Malaria is associated with an increase in immunosuppressive monocytes, alongside NK and γδ T cells which up-regulate tolerogenic markers. IL-10-producing Tr1 CD4 T cells and IL-10-producing regulatory B cells are also induced. Type I interferon responses are identified across all cell types, suggesting Type I interferon signalling may be linked to induction of immunoregulatory networks during malaria. These findings provide insights into cell-specific and shared immunoregulatory changes during malaria and provide a data resource for further analysis.

The development of highly effective malaria vaccines and improvement of drug-treatment protocols to boost antiparasitic immunity are critical for malaria elimination. However, the rapid establishment of parasite-specific immune regulatory networks following exposure to malaria parasites hampers these efforts. Here, we identified stimulator of interferon genes (STING) as a critical mediator of type I interferon production by CD4+ T cells during blood-stage Plasmodium falciparum infection. The activation of STING in CD4+ T cells by cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) stimulated IFNB gene transcription, which promoted development of IL-10– and IFN-γ–coproducing CD4+ T (type I regulatory [Tr1]) cells. The critical role for type I IFN signaling for Tr1 cell development was confirmed in vivo using a preclinical malaria model. CD4+ T cell sensitivity to STING phosphorylation was increased in healthy volunteers following P. falciparum infection, particularly in Tr1 cells. These findings identified STING expressed by CD4+ T cells as an important mediator of type I IFN production and Tr1 cell development and activation during malaria.

Despite being key drivers of protective antibodies against malaria, little is known regarding the host and parasite factors that influence CD4 T-follicular helper cell and antibody development. Authors utilise samples from a study of children living in an area of high malaria transmission in Uganda, to characterize Tfh cells and functional antibodies to multiple parasites stages.

Control of intracellular parasites responsible for malaria requires host IFN-γ+T-bet+CD4+ T cells (Th1 cells) with IL-10 produced by Th1 cells to mitigate the pathology induced by this inflammatory response. However, these IL-10–producing Th1 (induced type I regulatory [Tr1]) cells can also promote parasite persistence or impair immunity to reinfection or vaccination. Here, we identified molecular and phenotypic signatures that distinguished IL-10–Th1 cells from IL-10+Tr1 cells in Plasmodium falciparum–infected people who participated in controlled human malaria infection studies, as well as C57BL/6 mice with experimental malaria caused by P. berghei ANKA. We also identified a conserved Tr1 cell molecular signature shared between patients with malaria, dengue, and graft-versus-host disease. Genetic manipulation of primary human CD4+ T cells showed that the transcription factor cMAF played an important role in the induction of IL-10, while BLIMP-1 promoted the development of human CD4+ T cells expressing multiple coinhibitory receptors. We also describe heterogeneity of Tr1 cell coinhibitory receptor expression that has implications for targeting these molecules for clinical advantage during infection. Overall, this work provides insights into CD4+ T cell development during malaria that offer opportunities for creation of strategies to modulate CD4+ T cell functions and improve antiparasitic immunity.

Abstract 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).

Abstract 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.

Antibodies plays critical roles in the adaptive immune response to infectious agents including malaria. Here the authors defined antibody interactions with -Fcγ-receptors expressed on immune cells with sporozoites of Plasmodium falciparum, and identified specific target epitopes of antibodies.

Abstract Background Plasmodium falciparum malaria increases plasma levels of the cytokine Fms-like tyrosine kinase 3 ligand (Flt3L), a haematopoietic factor associated with dendritic cell (DC) expansion. It is unknown if the zoonotic parasite Plasmodium knowlesi impacts Flt3L or DC in human malaria. This study investigated circulating DC and Flt3L associations in adult malaria and in submicroscopic experimental infection. Methods Plasma Flt3L concentration and blood CD141+ DC, CD1c+ DC and plasmacytoid DC (pDC) numbers were assessed in (i) volunteers experimentally infected with P. falciparum and in Malaysian patients with uncomplicated (ii) P. falciparum or (iii) P. knowlesi malaria. Results Plasmodium knowlesi caused a decline in all circulating DC subsets in adults with malaria. Plasma Flt3L was elevated in acute P. falciparum and P. knowlesi malaria with no increase in a subclinical experimental infection. Circulating CD141+ DCs, CD1c+ DCs and pDCs declined in all adults tested, for the first time extending the finding of DC subset decline in acute malaria to the zoonotic parasite P. knowlesi. Conclusions In adults, submicroscopic Plasmodium infection causes no change in plasma Flt3L but does reduce circulating DCs. Plasma Flt3L concentrations increase in acute malaria, yet this increase is insufficient to restore or expand circulating CD141+ DCs, CD1c+ DCs or pDCs. These data imply that haematopoietic factors, yet to be identified and not Flt3L, involved in the sensing/maintenance of circulating DC are impacted by malaria and a submicroscopic infection. The zoonotic P. knowlesi is similar to other Plasmodium spp in compromising DC in adult malaria.

Summary: 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.

Abstract Background Anaemia is a major consequence of malaria, caused by the removal of both infected and uninfected red blood cells (RBCs) from the circulation. Complement activation and reduced expression of complement regulatory proteins (CRPs) on RBCs are an important pathogenic mechanism in severe malarial anaemia in both Plasmodium falciparum and Plasmodium vivax infection. However, little is known about loss of CRPs on RBCs during mild malarial anaemia and in low-density infection. Methods The expression of CRP CR1, CD55, CD59, and the phagocytic regulator CD47, on uninfected normocytes and reticulocytes were assessed in individuals from two study populations: (1) P. falciparum and P. vivax-infected patients from a low transmission setting in Sabah, Malaysia; and, (2) malaria-naïve volunteers undergoing P. falciparum induced blood-stage malaria (IBSM). For clinical infections, individuals were categorized into anaemia severity categories based on haemoglobin levels. For IBSM, associations between CRPs and haemoglobin level were investigated. Results CRP expression on RBC was lower in Malaysian individuals with P. falciparum and P. vivax mild malarial anaemia compared to healthy controls. CRP expression was also reduced on RBCs from volunteers during IBSM. Reduction occurred on normocytes and reticulocytes. However, there was no significant association between reduced CRPs and haemoglobin during IBSM. Conclusions Removal of CRPs occurs on both RBCs and reticulocytes during Plasmodium infection even in mild malarial anaemia and at low levels of parasitaemia.

Antibodies against Plasmodium falciparum merozoites that fix complement can inhibit blood-stage replication. Here, Reiling et al. show that complement-fixing antibodies strongly correlate with protective immunity in children, identify the merozoite targets, and predict antigen combinations that should result in strong protection.

Vaccines that target Plasmodium falciparum gametocytes have the potential to reduce malaria transmission and are thus attractive targets for malaria control. However, very little is known about human immune responses to gametocytes present in human hosts. We evaluated naturally-acquired antibodies to gametocyte-infected erythrocytes (gametocyte-IEs) of different developmental stages compared to other asexual parasite stages among naturally-exposed Kenyan residents. We found that acquired antibodies strongly recognized the surface of mature asexual-IEs, but there was limited reactivity to the surface of gametocyte-IEs of different stages. We used genetically-modified P. falciparum with suppressed expression of PfEMP1, the major surface antigen of asexual-stage IEs, to demonstrate that PfEMP1 is a dominant target of antibodies to asexual-IEs, in contrast to gametocyte-IEs. Antibody reactivity to gametocyte-IEs was similar to asexual-IEs lacking PfEMP1. Significant antibody reactivity to the surface of gametocytes was observed when outside of the host erythrocyte, including recognition of the major gametocyte antigen, Pfs230. This indicates that there is a deficiency of acquired antibodies to gametocyte-IEs despite the acquisition of antibodies to gametocyte antigens and asexual IEs. Our findings suggest that the acquisition of substantial immunity to the surface of gametocyte-IEs is limited, which may facilitate immune evasion to enable malaria transmission even in the face of substantial host immunity to malaria. Further studies are needed to understand the basis for the limited acquisition of antibodies to gametocytes and whether vaccine strategies can generate substantial immunity.

Cytokine-producing CD4 T cells have important roles in immunity against Plasmodium falciparum (Pf) malaria. However, the factors influencing functional differentiation of Pf-specific CD4 T cells in naturally exposed children are not well understood. Moreover, it is not known which CD4 T-cell cytokine-producing subsets are most critical for protection. We measured Pf-specific IFNγ-, IL10-, and TNFα-producing CD4 T-cell responses by multi-parametric flow cytometry in 265 children aged 6 months to 10 years enrolled in a longitudinal observational cohort in a high malaria transmission site in Uganda. We found that both age and parasite burden were independently associated with cytokine production by CD4 T cells. IL10 production by IFNγ+ CD4 T cells was higher in younger children and in those with high-parasite burden during recent infection. To investigate the role of CD4 T cells in immunity to malaria, we measured associations of Pf-specific CD4 cytokine-producing cells with the prospective risk of Pf infection and clinical malaria, adjusting for household exposure to Pf-infected mosquitos. Overall, the prospective risk of infection was not associated with the total frequency of Pf-specific CD4 T cells, nor of any cytokine-producing CD4 subset. However, the frequency of CD4 cells producing IL10 but not inflammatory cytokines (IFNγ and TNFα) was associated with a decreased risk of clinical malaria once infected. These data suggest that functional polarization of the CD4 T-cell response may modulate the clinical manifestations of malaria and play a role in naturally acquired immunity.

Abstract Objectives Malaria, caused by Plasmodium infection, remains a major global health problem. Monocytes are integral to the immune response, yet their transcriptional and functional responses in primary Plasmodium falciparum infection and in clinical malaria are poorly understood. Methods The transcriptional and functional profiles of monocytes were examined in controlled human malaria infection with P. falciparum blood stages and in children and adults with acute malaria. Monocyte gene expression and functional phenotypes were examined by RNA sequencing and flow cytometry at peak infection and compared to pre‐infection or at convalescence in acute malaria. Results In subpatent primary infection, the monocyte transcriptional profile was dominated by an interferon (IFN) molecular signature. Pathways enriched included type I IFN signalling, innate immune response and cytokine‐mediated signalling. Monocytes increased TNF and IL‐12 production upon in vitro toll‐like receptor stimulation and increased IL‐10 production upon in vitro parasite restimulation. Longitudinal phenotypic analyses revealed sustained significant changes in the composition of monocytes following infection, with increased CD14+CD16− and decreased CD14−CD16+ subsets. In acute malaria, monocyte CD64/FcγRI expression was significantly increased in children and adults, while HLA‐DR remained stable. Although children and adults showed a similar pattern of differentially expressed genes, the number and magnitude of gene expression change were greater in children. Conclusions Monocyte activation during subpatent malaria is driven by an IFN molecular signature with robust activation of genes enriched in pathogen detection, phagocytosis, antimicrobial activity and antigen presentation. The greater magnitude of transcriptional changes in children with acute malaria suggests monocyte phenotypes may change with age or exposure.

The development of effective malaria vaccines remains a global health priority. Currently, the most advanced vaccine, known as RTS,S, has only shown modest efficacy in clinical trials. Thus, the development of more efficacious vaccines by improving the formulation of RTS,S for increased efficacy or to interrupt malaria transmission are urgently needed. The RTS,S vaccine is based on the presentation of a fragment of the sporozoite antigen on the surface of virus-like particles (VLPs) based on human hepatitis B virus (HBV). In this study, we have developed and evaluated a novel VLP platform based on duck HBV (known as Metavax) for malaria vaccine development. This platform can incorporate large and complex proteins into VLPs and is produced in a Hansenula cell line compatible with cGMP vaccine production. Here, we have established the expression of leading P. falciparum malaria vaccine candidates as VLPs. This includes Pfs230 and Pfs25, which are candidate transmission-blocking vaccine antigens. We demonstrated that the VLPs effectively induce antibodies to malaria vaccine candidates with minimal induction of antibodies to the duck-HBV scaffold antigen. Antibodies to Pfs230 also recognised native protein on the surface of gametocytes, and antibodies to both Pfs230 and Pfs25 demonstrated transmission-reducing activity in standard membrane feeding assays. These results establish the potential utility of this VLP platform for malaria vaccines, which may be suitable for the development of multi-component vaccines that achieve high vaccine efficacy and transmission-blocking immunity.

In humans, immunity to Plasmodium sp. generally takes the form of protection from symptomatic malaria (i.e., 'clinical immunity') rather than infection ('sterilizing immunity'). In contrast, mice infected with Plasmodium develop sterilizing immunity, hindering progress in understanding the mechanistic basis of clinical immunity. Here we present a novel model in which mice persistently infected with P. chabaudi exhibit limited clinical symptoms despite sustaining patent parasite burdens for many months. Characterization of immune responses in persistently infected mice revealed development of CD4+ T cell exhaustion, increased production of IL-10, and expansion of B cells with an atypical surface phenotype. Additionally, persistently infected mice displayed a dramatic increase in circulating nonclassical monocytes, a phenomenon that we also observed in humans with both chronic Plasmodium exposure and asymptomatic infection. Following pharmacological clearance of infection, previously persistently infected mice could not control a secondary challenge, indicating that persistent infection disrupts the sterilizing immunity that typically develops in mouse models of acute infection. This study establishes an animal model of asymptomatic, persistent Plasmodium infection that recapitulates several central aspects of the immune response in chronically exposed humans. As such, it provides a novel tool for dissection of immune responses that may prevent development of sterilizing immunity and limit pathology during infection.

FoxP3+ regulatory CD4 T cells (Tregs) help to maintain the delicate balance between pathogen-specific immunity and immune-mediated pathology. Prior studies suggest that Tregs are induced by P. falciparum both in vivo and in vitro; however, the factors influencing Treg homeostasis during acute and chronic infections, and their role in malaria immunopathogenesis, remain unclear. We assessed the frequency and phenotype of Tregs in well-characterized cohorts of children residing in a region of high malaria endemicity in Uganda. We found that both the frequency and absolute numbers of FoxP3+ Tregs in peripheral blood declined markedly with increasing prior malaria incidence. Longitudinal measurements confirmed that this decline occurred only among highly malaria-exposed children. The decline of Tregs from peripheral blood was accompanied by reduced in vitro induction of Tregs by parasite antigen and decreased expression of TNFR2 on Tregs among children who had intense prior exposure to malaria. While Treg frequencies were not associated with protection from malaria, there was a trend toward reduced risk of symptomatic malaria once infected with P. falciparum among children with lower Treg frequencies. These data demonstrate that chronic malaria exposure results in altered Treg homeostasis, which may impact the development of antimalarial immunity in naturally exposed populations.

Exposure to Plasmodium falciparum is associated with circulating "atypical" memory B cells (atMBCs), which appear similar to dysfunctional B cells found in HIV-infected individuals. Functional analysis of atMBCs has been limited, with one report suggesting these cells are not dysfunctional but produce protective antibodies. To better understand the function of malaria-associated atMBCs, we performed global transcriptome analysis of these cells, obtained from individuals living in an area of high malaria endemicity in Uganda. Comparison of gene expression data suggested down-modulation of B cell receptor signaling and apoptosis in atMBCs compared to classical MBCs. Additionally, in contrast to previous reports, we found upregulation of Fc receptor-like 5 (FCRL5), but not FCRL4, on atMBCs. Atypical MBCs were poor spontaneous producers of antibody ex vivo, and higher surface expression of FCRL5 defined a distinct subset of atMBCs compromised in its ability to produce antibody upon stimulation. Moreover, higher levels of P. falciparum exposure were associated with increased frequencies of FCRL5+ atMBCs. Together, our findings suggest that FCLR5+ identifies a functionally distinct, and perhaps dysfunctional, subset of MBCs in individuals exposed to P. falciparum.

In a previous Phase 1/2b malaria vaccine trial testing the 3D7 isoform of the malaria vaccine candidate Merozoite surface protein 2 (MSP2), parasite densities in children were reduced by 62%. However, breakthrough parasitemias were disproportionately of the alternate dimorphic form of MSP2, the FC27 genotype. We therefore undertook a dose-escalating, double-blinded, placebo-controlled Phase 1 trial in healthy, malaria-naïve adults of MSP2-C1, a vaccine containing recombinant forms of the two families of msp2 alleles, 3D7 and FC27 (EcMSP2-3D7 and EcMSP2-FC27), formulated in equal amounts with Montanide® ISA 720 as a water-in-oil emulsion.The trial was designed to include three dose cohorts (10, 40, and 80 µg), each with twelve subjects receiving the vaccine and three control subjects receiving Montanide® ISA 720 adjuvant emulsion alone, in a schedule of three doses at 12-week intervals. Due to unexpected local reactogenicity and concern regarding vaccine stability, the trial was terminated after the second immunisation of the cohort receiving the 40 µg dose; no subjects received the 80 µg dose. Immunization induced significant IgG responses to both isoforms of MSP2 in the 10 µg and 40 µg dose cohorts, with antibody levels by ELISA higher in the 40 µg cohort. Vaccine-induced antibodies recognised native protein by Western blots of parasite protein extracts and by immunofluorescence microscopy. Although the induced anti-MSP2 antibodies did not directly inhibit parasite growth in vitro, IgG from the majority of individuals tested caused significant antibody-dependent cellular inhibition (ADCI) of parasite growth.As the majority of subjects vaccinated with MSP2-C1 developed an antibody responses to both forms of MSP2, and that these antibodies mediated ADCI provide further support for MSP2 as a malaria vaccine candidate. However, in view of the reactogenicity of this formulation, further clinical development of MSP2-C1 will require formulation of MSP2 in an alternative adjuvant.Australian New Zealand Clinical Trials Registry 12607000552482.