Molecular profiling reveals features of clinical immunity and immunosuppression in asymptomatic P. falciparum malaria.

Clinical immunity to P. falciparum malaria is non-sterilizing, with adults often experiencing asymptomatic infection. Historically, asymptomatic malaria has been viewed as beneficial and required to help maintain clinical immunity. Emerging views suggest that these infections are detrimental and constitute a parasite reservoir that perpetuates transmission. To define the impact of asymptomatic malaria, we pursued a systems approach integrating antibody responses, mass cytometry, and transcriptional profiling of individuals experiencing symptomatic and asymptomatic P. falciparum infection. Defined populations of classical and atypical memory B cells and a TH2 cell bias were associated with reduced risk of clinical malaria. Despite these protective responses, asymptomatic malaria featured an immunosuppressive transcriptional signature with upregulation of pathways involved in the inhibition of T-cell function, and CTLA-4 as a predicted regulator in these processes. As proof of concept, we demonstrated a role for CTLA-4 in the development of asymptomatic parasitemia in infection models. The results suggest that asymptomatic malaria is not innocuous and might not support the induction of immune processes to fully control parasitemia or efficiently respond to malaria vaccines.

Integrated systems immunology approach identifies impaired effector T cell memory responses as a feature of progression to severe dengue fever.

BACKGROUND: Typical symptoms of uncomplicated dengue fever (DF) include headache, muscle pains, rash, cough, and vomiting. A proportion of cases progress to severe dengue hemorrhagic fever (DHF), associated with increased vascular permeability, thrombocytopenia, and hemorrhages. Progression to severe dengue is difficult to diagnose at the onset of fever, which complicates patient triage, posing a socio-economic burden on health systems. METHODS: To identify parameters associated with protection and susceptibility to DHF, we pursued a systems immunology approach integrating plasma chemokine profiling, high-dimensional mass cytometry and peripheral blood mononuclear cell (PBMC) transcriptomic analysis at the onset of fever in a prospective study conducted in Indonesia. RESULTS: After a secondary infection, progression to uncomplicated dengue featured transcriptional profiles associated with increased cell proliferation and metabolism, and an expansion of ICOS+CD4+ and CD8+ effector memory T cells. These responses were virtually absent in cases progressing to severe DHF, that instead mounted an innate-like response, characterised by inflammatory transcriptional profiles, high circulating levels of inflammatory chemokines and with high frequencies of CD4low non-classical monocytes predicting increased odds of severe disease. CONCLUSIONS: Our results suggests that effector memory T cell activation might play an important role ameliorating severe disease symptoms during a secondary dengue infection, and in the absence of that response, a strong innate inflammatory response is required to control viral replication. Our research also identified discrete cell populations predicting increased odds of severe disease, with potential diagnostic value.

IgM+ memory B cells induced in response to Plasmodium berghei adopt a germinal centre B cell phenotype during secondary infection.

Emerging evidence started to delineate multiple layers of memory B cells, with distinct effector functions during recall responses. Whereas most studies examining long-lived memory B cell responses have focussed on the IgG+ memory B cell compartment, IgM+ memory B cells have only recently started to receive attention. It has been proposed that unlike IgG+ memory B cells, which differentiate into antibody-secreting plasma cells upon antigen re-encounter, IgM+ memory B cells might have the additional capacity to establish secondary germinal centre (GC) responses. The precise function of IgM+ memory B cells in the humoral immune response to malaria has not been fully defined. Using a murine model of severe malaria infection and adoptive transfer strategies we found that IgM+ memory B cells induced in responses to P. berghei ANKA readily proliferate upon re-infection and adopt a GC B cell-like phenotype. The results suggest that that IgM+ memory B cells might play an important role in populating secondary GCs after re-infection with Plasmodium, thereby initiating the induction of B cell clones with enhanced affinity for antigen, at faster rates than naive B cells.

CD14+ monocytes are the main leucocytic sources of CXCL10 in response to Plasmodium falciparum.

The CXCR3 chemokine CXCL10 or IFN-γ inducible protein 10 (IP-10) has been identified as an important biomarker of cerebral malaria (CM) mortality in children. Studies in mouse malaria infection models have shown that CXCL10 blockade alleviates brain intravascular inflammation and protects infected mice from CM. Despite the key role that CXCL10 plays in the development of CM, the leucocytic sources of CXCL10 in response to human malaria are not known. Here we investigated CXCL10 responses to Plasmodium falciparum in peripheral blood mononuclear cells (PBMCs). We found that PBMCs from malaria-unexposed donors produce CXCL10 in response to P. falciparum and that this response is IFN-γ-dependent. Moreover, CD14+ monocytes were identified as the main leucocytic sources of CXCL10 in peripheral blood, suggesting an important role for innate immune responses in the activation of this pathway involved in the development of symptomatic malaria.

Antibodies to Intercellular Adhesion Molecule 1-Binding Plasmodium falciparum Erythrocyte Membrane Protein 1-DBLß Are Biomarkers of Protective Immunity to Malaria in a Cohort of Young Children from Papua New Guinea.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) mediates parasite sequestration to the cerebral microvasculature via binding of DBLß domains to intercellular adhesion molecule 1 (ICAM1) and is associated with severe cerebral malaria. In a cohort of 187 young children from Papua New Guinea (PNG), we examined baseline levels of antibody to the ICAM1-binding PfEMP1 domain, DBLß3PF11_0521, in comparison to four control antigens, including NTS-DBLα and CIDR1 domains from another group A variant and a group B/C variant. Antibody levels for the group A antigens were strongly associated with age and exposure. Antibody responses to DBLß3PF11_0521 were associated with a 37% reduced risk of high-density clinical malaria in the follow-up period (adjusted incidence risk ratio [aIRR] = 0.63 [95% confidence interval {CI}, 0.45 to 0.88; P = 0.007]) and a 25% reduction in risk of low-density clinical malaria (aIRR = 0.75 [95% CI, 0.55 to 1.01; P = 0.06]), while there was no such association for other variants. Children who experienced severe malaria also had significantly lower levels of antibody to DBLß3PF11_0521 and the other group A domains than those that experienced nonsevere malaria. Furthermore, a subset of PNG DBLß sequences had ICAM1-binding motifs, formed a distinct phylogenetic cluster, and were similar to sequences from other areas of endemicity. PfEMP1 variants associated with these DBLß domains were enriched for DC4 and DC13 head structures implicated in endothelial protein C receptor (EPCR) binding and severe malaria, suggesting conservation of dual binding specificities. These results provide further support for the development of specific classes of PfEMP1 as vaccine candidates and as biomarkers for protective immunity against clinical P. falciparum malaria.

Monocyte- and Neutrophil-Derived CXCL10 Impairs Efficient Control of Blood-Stage Malaria Infection and Promotes Severe Disease.

CXCL10, or IFN-γ-inducible protein 10, is a biomarker associated with increased risk for Plasmodium falciparum-mediated cerebral malaria (CM). Consistent with this, we have previously shown that CXCL10 neutralization or genetic deletion alleviates brain intravascular inflammation and protects Plasmodium berghei ANKA-infected mice from CM. In addition to organ-specific effects, the absence of CXCL10 during infection was also found to reduce parasite biomass. To identify the cellular sources of CXCL10 responsible for these processes, we irradiated and reconstituted wild-type (WT) and CXCL10(-/-) mice with bone marrow from either WT or CXCL10(-/-) mice. Similar to CXCL10(-/-) mice, chimeras unable to express CXCL10 in hematopoietic-derived cells controlled infection more efficiently than WT controls. In contrast, expression of CXCL10 in knockout mice reconstituted with WT bone marrow resulted in high parasite biomass levels, higher brain parasite and leukocyte sequestration rates, and increased susceptibility to CM. Neutrophils and inflammatory monocytes were identified as the main cellular sources of CXCL10 responsible for the induction of these processes. The improved control of parasitemia observed in the absence of CXCL10-mediated trafficking was associated with a preferential accumulation of CXCR3(+)CD4(+) T follicular helper cells in the spleen and enhanced Ab responses to infection. These results are consistent with the notion that some inflammatory responses elicited in response to malaria infection contribute to the development of high parasite densities involved in the induction of severe disease in target organs.

Mechanisms of naturally acquired immunity to P. falciparum and approaches to identify merozoite antigen targets.

Malaria is one the most serious infectious diseases with over 200 million clinical cases annually. Most cases of the severe disease are caused by Plasmodium falciparum. The blood stage of Plasmodium parasite is entirely responsible for malaria-associated pathology. The population most susceptible to severe malaria are children under the age of 5, with low levels of immunity. It is only after many years of repeated exposure that individuals living in endemic areas develop clinical immunity. This form of protection prevents clinical episodes by substantially reducing parasite burden. Naturally acquired immunity predominantly targets blood-stage parasites with antibody responses being the main mediators of protection. The targets of clinical immunity are the extracellular merozoite and the infected erythrocyte surface, with the extremely diverse PfEMP1 proteins the main target here. This observation provides a strong rationale that an effective anti-malaria vaccine targeting blood-stage parasites is achievable. Thus the identification of antigenic targets of naturally acquired immunity remains an important step towards the formulation of novel vaccine combinations before testing their efficacy in clinical trials. This review summarizes the main findings to date defining antigenic targets present on the extracellular merozoite associated with naturally acquired immunity to P. falciparum malaria.

Synergistic effect of IL-12 and IL-18 induces TIM3 regulation of γδ T cell function and decreases the risk of clinical malaria in children living in Papua New Guinea.

BACKGROUND: γδ T cells are important for both protective immunity and immunopathogenesis during malaria infection. However, the immunological processes determining beneficial or detrimental effects on disease outcome remain elusive. The aim of this study was to examine expression and regulatory effect of the inhibitory receptor T-cell immunoglobulin domain and mucin domain 3 (TIM3) on γδ T cells. While TIM3 expression and function on conventional αß T cells have been clearly defined, the equivalent characterization on γδ T cells and associations with disease outcomes is limited. This study investigated the functional capacity of TIM3+ γδ T cells and the underlying mechanisms contributing to TIM3 upregulation and established an association with malaria disease outcomes. METHODS: We analyzed TIM3 expression on γδ T cells in 132 children aged 5-10 years living in malaria endemic areas of Papua New Guinea. TIM3 upregulation and effector functions of TIM3+ γδ T cells were assessed following in vitro stimulation with parasite-infected erythrocytes, phosphoantigen and/or cytokines. Associations between the proportion of TIM3-expressing cells and the molecular force of infection were tested using negative binomial regression and in a Cox proportional hazards model for time to first clinical episode. Multivariable analyses to determine the association of TIM3 and IL-18 levels were conducted using general linear models. Malaria infection mouse models were utilized to experimentally investigate the relationship between repeated exposure and TIM3 upregulation. RESULTS: This study demonstrates that even in the absence of an active malaria infection, children of malaria endemic areas have an atypical population of TIM3-expressing γδ T cells (mean frequency TIM3+ of total γδ T cells 15.2% ± 12). Crucial factors required for γδ T cell TIM3 upregulation include IL-12/IL-18, and plasma IL-18 was associated with TIM3 expression (P = 0.002). Additionally, we show a relationship between TIM3 expression and infection with distinct parasite clones during repeated exposure. TIM3+ γδ T cells were functionally impaired and were associated with asymptomatic malaria infection (hazard ratio 0.54, P = 0.032). CONCLUSIONS: Collectively our data demonstrate a novel role for IL-12/IL-18 in shaping the innate immune response and provide fundamental insight into aspects of γδ T cell immunoregulation. Furthermore, we show that TIM3 represents an important γδ T cell regulatory component involved in minimizing malaria symptoms.

Different Regions of Plasmodium falciparum Erythrocyte-Binding Antigen 175 Induce Antibody Responses to Infection of Varied Efficacy.

BACKGROUND: Increasing evidence suggests that antibodies against merozoite proteins involved in Plasmodium falciparum invasion into the red blood cell play an important role in clinical immunity to malaria. Erythrocyte-binding antigen 175 (EBA-175) is the best-characterized P. falciparum invasion ligand, reported to recognize glycophorin A on the surface of erythrocytes. Its protein structure comprises 6 extracellular regions. Whereas region II contains Duffy binding-like domains involved in the binding to glycophorin A, the functional role of regions III-V is less clear. METHODS: We developed a novel cytometric bead array for assessment of antigen-specific antibody concentration in plasma to evaluate the efficacy of immune responses to different regions of EBA-175 and associations between antibody levels with protection from symptomatic malaria in a treatment-reinfection cohort study. RESULTS: We found that while antibodies to region II are highly abundant, circulating levels as low as 5-10 µg/mL of antibodies specific for region III or the highly conserved regions IV-V predict strong protection from clinical malaria. CONCLUSIONS: These results lend support for the development of conserved regions of EBA-175 as components in a combination of a malaria vaccine.

Merozoite Antigens of Plasmodium falciparum Elicit Strain-Transcending Opsonizing Immunity.

It is unclear whether naturally acquired immunity to Plasmodium falciparum results from the acquisition of antibodies to multiple, diverse antigens or to fewer, highly conserved antigens. Moreover, the specific antibody functions required for malaria immunity are unknown, and hence informative immunological assays are urgently needed to address these knowledge gaps and guide vaccine development. In this study, we investigated whether merozoite-opsonizing antibodies are associated with protection from malaria in a strain-specific or strain-transcending manner by using a novel field isolate and an immune plasma-matched cohort from Papua New Guinea with our validated assay of merozoite phagocytosis. Highly correlated opsonization responses were observed across the 15 parasite strains tested, as were strong associations with protection (composite phagocytosis score across all strains in children uninfected at baseline: hazard ratio of 0.15, 95% confidence interval of 0.04 to 0.63). Opsonizing antibodies had a strong strain-transcending component, and the opsonization of transgenic parasites deficient for MSP3, MSP6, MSPDBL1, or P. falciparum MSP1-19 (PfMSP1-19) was similar to that of wild-type parasites. We have provided the first evidence that merozoite opsonization is predominantly strain transcending, and the highly consistent associations with protection against diverse parasite strains strongly supports the use of merozoite opsonization as a correlate of immunity for field studies and vaccine trials. These results demonstrate that conserved domains within merozoite antigens targeted by opsonization generate strain-transcending immune responses and represent promising vaccine candidates.

CD8+ T cells from a novel T cell receptor transgenic mouse induce liver-stage immunity that can be boosted by blood-stage infection in rodent malaria.

To follow the fate of CD8+ T cells responsive to Plasmodium berghei ANKA (PbA) infection, we generated an MHC I-restricted TCR transgenic mouse line against this pathogen. T cells from this line, termed PbT-I T cells, were able to respond to blood-stage infection by PbA and two other rodent malaria species, P. yoelii XNL and P. chabaudi AS. These PbT-I T cells were also able to respond to sporozoites and to protect mice from liver-stage infection. Examination of the requirements for priming after intravenous administration of irradiated sporozoites, an effective vaccination approach, showed that the spleen rather than the liver was the main site of priming and that responses depended on CD8α+ dendritic cells. Importantly, sequential exposure to irradiated sporozoites followed two days later by blood-stage infection led to augmented PbT-I T cell expansion. These findings indicate that PbT-I T cells are a highly versatile tool for studying multiple stages and species of rodent malaria and suggest that cross-stage reactive CD8+ T cells may be utilized in liver-stage vaccine design to enable boosting by blood-stage infections.

GM-CSF-responsive monocyte-derived dendritic cells are pivotal in Th17 pathogenesis.

Although multiple dendritic cell (DC) subsets have the potential to induce Th17 differentiation in vitro, the key DC that is critical in Th17 induction and Th17-mediated disease remains moot. In this study, we revealed that CCR2(+) monocyte-derived DCs (moDCs), but not conventional DCs, were critical for in vivo Th17 induction and autoimmune inflammation. Functional comparison in vitro indicated that moDCs are the most potent type of Th17-inducing DCs compared with conventional DCs and plasmacytoid DCs. Furthermore, we demonstrated that the importance of GM-CSF in Th17 induction and Th17-mediated disease is its endowment of moDCs to induce Th17 differentiation in vivo, although it has little effect on moDC numbers. Our findings identify the in vivo cellular targets that can be selectively manipulated to ameliorate Th17-mediated inflammatory diseases, as well as the mechanism of GM-CSF antagonism in such diseases.

Immunological processes underlying the slow acquisition of humoral immunity to malaria.

Malaria is one of the most serious infectious diseases with ~250 million clinical cases annually. Most cases of severe disease are caused by Plasmodium falciparum. The blood stage of Plasmodium parasite is entirely responsible for malaria-associated pathology. Disease syndromes range from fever to more severe complications, including respiratory distress, metabolic acidosis, renal failure, pulmonary oedema and cerebral malaria. The most susceptible population to severe malaria is children under the age of 5, with low levels of immunity. It is only after many years of repeated exposure, that individuals living in endemic areas develop clinical immunity. This form of protection does not result in sterilizing immunity but prevents clinical episodes by substantially reducing parasite burden. Naturally acquired immunity predominantly targets blood-stage parasites and it is known to require antibody responses. A large body of epidemiological evidence suggests that antibodies to Plasmodium antigens are inefficiently generated and rapidly lost in the absence of ongoing exposure, which suggests a defect in the development of B cell immunological memory. This review summarizes the main findings to date contributing to our understanding on cellular processes underlying the slow acquisition of humoral immunity to malaria. Some of the key outstanding questions in the field are discussed.

Antibodies to the Plasmodium falciparum Proteins MSPDBL1 and MSPDBL2 Opsonize Merozoites, Inhibit Parasite Growth, and Predict Protection From Clinical Malaria.

Increasing evidence suggests that antibodies against merozoite surface proteins (MSPs) play an important role in clinical immunity to malaria. Two unusual members of the MSP-3 family, merozoite surface protein duffy binding-like (MSPDBL)1 and MSPDBL2, have been shown to be extrinsically associated to MSP-1 on the parasite surface. In addition to a secreted polymorphic antigen associated with merozoite (SPAM) domain characteristic of MSP-3 family members, they also contain Duffy binding-like (DBL) domain and were found to bind to erythrocytes, suggesting that they play a role in parasite invasion. Antibody responses to these proteins were investigated in a treatment-reinfection study conducted in an endemic area of Papua New Guinea to determine their contribution to naturally acquired immunity. Antibodies to the SPAM domains of MSPDBL1 and MSPDBL2 as well as the DBL domain of MSPDBL1 were found to be associated with protection from Plasmodium falciparum clinical episodes. Moreover, affinity-purified anti-MSPDBL1 and MSPDBL2 were found to inhibit in vitro parasite growth and had strong merozoite opsonizing capacity, suggesting that protection targeting these antigens results from ≥2 distinct effector mechanisms. Together these results indicate that MSPDBL1 and MSPDBL2 are important targets of naturally acquired immunity and might constitute potential vaccine candidates.

The role of chemokines in severe malaria: more than meets the eye.

Plasmodium falciparum malaria is responsible for over 250 million clinical cases every year worldwide. Severe malaria cases might present with a range of disease syndromes including acute respiratory distress, metabolic acidosis, hypoglycaemia, renal failure, anaemia, pulmonary oedema, cerebral malaria (CM) and placental malaria (PM) in pregnant women. Two main determinants of severe malaria have been identified: sequestration of parasitized red blood cells and strong pro-inflammatory responses. Increasing evidence from human studies and malaria infection animal models revealed the presence of host leucocytes at the site of parasite sequestration in brain blood vessels as well as placental tissue in complicated malaria cases. These observations suggested that apart from secreting cytokines, leucocytes might also contribute to disease by migrating to the site of parasite sequestration thereby exacerbating organ-specific inflammation. This evidence attracted substantial interest in identifying trafficking pathways by which inflammatory leucocytes are recruited to target organs during severe malaria syndromes. Chemo-attractant cytokines or chemokines are the key regulators of leucocyte trafficking and their potential contribution to disease has recently received considerable attention. This review summarizes the main findings to date, investigating the role of chemokines in severe malaria and the implication of these responses for the induction of pathogenesis and immunity to infection.

An immunohistochemical atlas of necroptotic pathway expression.

Necroptosis is a lytic form of regulated cell death reported to contribute to inflammatory diseases of the gut, skin and lung, as well as ischemic-reperfusion injuries of the kidney, heart and brain. However, precise identification of the cells and tissues that undergo necroptotic cell death in vivo has proven challenging in the absence of robust protocols for immunohistochemical detection. Here, we provide automated immunohistochemistry protocols to detect core necroptosis regulators - Caspase-8, RIPK1, RIPK3 and MLKL - in formalin-fixed mouse and human tissues. We observed surprising heterogeneity in protein expression within tissues, whereby short-lived immune barrier cells were replete with necroptotic effectors, whereas long-lived cells lacked RIPK3 or MLKL expression. Local changes in the expression of necroptotic effectors occurred in response to insults such as inflammation, dysbiosis or immune challenge, consistent with necroptosis being dysregulated in disease contexts. These methods will facilitate the precise localisation and evaluation of necroptotic signaling in vivo.

Advances in infection and immunity: from bench to bedside.

This report summarizes recent advances on host-pathogen interactions, innate and adaptive responses to infection, as well as novel strategies for the control of infectious diseases.

Natural regulatory T cells in malaria: host or parasite allies?

Plasmodium falciparum malaria causes 500 million clinical cases with approximately one million deaths each year. After many years of exposure, individuals living in endemic areas develop a form of clinical immunity to disease known as premunition, which is characterised by low parasite burdens rather than sterilising immunity. The reason why malaria parasites persist under a state of premunition is unknown but it has been suggested that suppression of protective immunity might be a mechanism leading to parasite persistence. Although acquired immunity limits the clinical impact of infection and provides protection against parasite replication, experimental evidence indicates that cell-mediated immune responses also result in detrimental inflammation and contribute to the aetiology of severe disease. Thus, an appropriate regulatory balance between protective immune responses and immune-mediated pathology is required for a favourable outcome of infection. As natural regulatory T (T(reg)) cells are identified as an immunosuppressive lineage able to modulate the magnitude of effector responses, several studies have investigated whether this cell population plays a role in balancing protective immunity and pathogenesis during malaria. The main findings to date are summarised in this review and the implication for the induction of pathogenesis and immunity to malaria is discussed.

CyTOF mass cytometry analysis of human memory CD4+ T cells and memory B cells.

High-dimensional mass cytometry provides unparalleled insight into the cellular composition of the immune system. Here, we describe a mass-cytometry-based protocol to examine memory CD4+ T cell and memory B cell (MBC) responses in human peripheral blood. This approach allows for the identification of >50 distinct memory CD4+ T cell and MBC populations from a single clinical sample. This highly reproducible protocol has been successfully applied to multiple infectious disease settings to identify correlates of susceptibility or protection from infection. For complete details on the use and execution of this protocol, please refer to Ioannidis et al. (2021).

Bezlotoxumab prevents extraintestinal organ damage induced by Clostridioides difficile infection.

Clostridioides difficile is the most common cause of infectious antibiotic-associated diarrhea, with disease mediated by two major toxins TcdA and TcdB. In severe cases, systemic disease complications may arise, resulting in fatal disease. Systemic disease in animal models has been described, with thymic damage an observable consequence of severe disease in mice. Using a mouse model of C. difficile infection, we examined this disease phenotype, focussing on the thymus and serum markers of systemic disease. The efficacy of bezlotoxumab, a monoclonal TcdB therapeutic, to prevent toxin mediated systemic disease complications was also examined. C. difficile infection causes toxin-dependent thymic damage and CD4+CD8+ thymocyte depletion in mice. These systemic complications coincide with changes in biochemical markers of liver and kidney function, including increased serum urea and creatinine, and hypoglycemia. Administration of bezlotoxumab during C. difficile infection prevents systemic disease and thymic atrophy, without blocking gut damage, suggesting the leakage of gut contents into circulation may influence systemic disease. As the thymus has such a crucial role in T cell production and immune system development, these findings may have important implications in relapse of C. difficile disease and impaired immunity during C. difficile infection. The prevention of thymic atrophy and reduced systemic response following bezlotoxumab treatment, without altering colonic damage, highlights the importance of systemic disease in C. difficile infection, and provides new insights into the mechanism of action for this therapeutic.Abbreviations: Acute kidney injury (AKI); Alanine Transaminase (ALT); Aspartate Aminotransferase (AST); C. difficile infection (CDI); chronic kidney disease (CKD); combined repetitive oligo-peptides (CROPS); cardiovascular disease (CVD); Double positive (DP); hematoxylin and eosin (H&E); immunohistochemical (IHC); multiple organ dysfunction syndrome (MODS); phosphate buffered saline (PBS); standard error of the mean (SEM); surface layer proteins (SLP); Single positive (SP); wild-type (WT).