PhD student, The University Of Melbourne
To understand which anti-malarial antibodies are predominantly acquired in children
Malaria is a deadly parasitic disease causing nearly 200,000 neonatal and 10,000 maternal deaths each year. The aim of this project is to understand which anti-malarial antibodies are predominantly acquired in children and how these antibodies provide protection from severe disease. One of the aims of the study was to discover whether nutrient supplementation improved the development of malarial antibody immunity in children up to 18 months of age. Antibody levels against merozoite surface protein 1 (MSP1 19kD) and MSP2, erythrocyte binding antigen 175 (EBA175), reticulocyte binding protein homologue 2A (Rh2A9), schizont extract and variant antigens expressed on the surface of infected erythrocytes were measured using paired samples collected at 6 and 18 months from 432 Malawian children in a randomised controlled trial of prenatal nutrient supplementation with iron & folic acid or pre- and postnatal multiple micronutrients or lipid-based nutrient. In another part of the study, the aim was to use PfEMP1 antibody profiles defined on admission and at follow-up in severe or mild malaria to identify crucial responses lacking in children in severe malaria from Papua New Guinea. The results showed that pre- and postnatal nutrient supplementation did not alter malaria antibody acquisition during infancy. The prevalence and levels of antibodies to malaria antigens increased following infection in early childhood. Neither the age at first malaria infection nor the number of episodes in early life materially affected the acquisition of antibodies. The results suggested that some merozoite antigens are useful biomarkers of malaria exposure in infancy.
Abstract: The capacity of Plasmodium falciparum-infected erythrocytes to bind uninfected erythrocytes (rosetting) is associated with severe malaria in African children. Rosetting is mediated by a subset of the variant surface antigens PfEMP1 targeted by protective antibody responses. Analysis of the response to rosette-forming parasites and their PfEMP1 adhesive domains is essential for understanding the acquisition of protection against severe malaria. To this end, the antibody response to a rosetting variant was analysed in children recruited with severe or uncomplicated malaria or asymptomatic P. falciparum infection.Serum was collected from Beninese children with severe malaria, uncomplicated malaria or P. falciparum asymptomatic infection (N = 65, 37 and 52, respectively) and from immune adults (N = 30) living in the area. Infected erythrocyte surface-reactive IgG, rosette disrupting antibodies and IgG to the parasite crude extract were analysed using the single variant Palo Alto VarO-infected line. IgG, IgG1 and IgG3 to PfEMP1-varO-derived NTS-DBL1α1, CIDRγ and DBL2βC2 recombinant domains were analysed by ELISA. Antibody responses were compared in the clinical groups. Stability of the response was studied using a blood sampling collected 14 months later from asymptomatic children.Seroprevalence of erythrocyte surface-reactive IgG was high in adults (100%) and asymptomatic children (92.3%) but low in children with severe or uncomplicated malaria (26.1% and 37.8%, respectively). The IgG, IgG1 and IgG3 antibody responses to the varO-derived PfEMP1 domains were significantly higher in asymptomatic children than in children with clinical malaria in a multivariate analysis correcting for age and parasite density at enrolment. They were essentially stable, although levels tended to decrease with time. VarO-surface reactivity correlated positively with IgG reactivity to the rosetting domain varO-NTS-DBL1α1. None of the children sera, including those with surface-reactive antibodies possessed anti-VarO-rosetting activity, and few adults had rosette-disrupting antibodies.Children with severe and uncomplicated malaria had similar responses. The higher prevalence and level of VarO-reactive antibodies in asymptomatic children compared to children with malaria is consistent with a protective role for anti-VarO antibodies against clinical falciparum malaria. The mechanism of such protection seems independent of rosette-disruption, suggesting that the cytophilic properties of antibodies come into play.
Pub.: 07 Oct '10, Pinned: 17 Jun '17
Abstract: Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a variant antigen expressed on the surface of infected erythrocytes. Each parasite genome contains about 40 PfEMP1 genes, but only 1 PfEMP1 gene is expressed at a given time. PfEMP1 serves as a parasite-sequestering ligand to endothelial cells and enables the parasites to avoid splenic passage. PfEMP1 antibodies may protect from disease by inhibiting sequestration, thus facilitating the destruction of infected erythrocytes in the spleen. In this study, we have measured antibodies in Ghanaian children to a conserved region of PfEMP1 by enzyme-linked immunosorbent assay and antibodies to variant molecules on erythrocytes infected with field isolates of P. falciparum by flow cytometry. Based on close clinical monitoring, the children were grouped into those who did (susceptible) and those who did not (protected) have malaria during the season. The prevalences of antibodies to both the conserved PfEMP1 peptide and the variant epitopes were greater than 50%, and the levels of immunoglobulin G (IgG) correlated with age. The levels of antibodies to both the conserved peptide and the variant epitopes were higher in protected than in susceptible children. After correcting for the effect of age, the levels of IgG to variant antigens on a Sudanese and a Ghanaian parasite isolate remained significantly higher in protected than in susceptible children. Thus, the levels of IgG to variant antigens expressed on the surface of infected erythrocytes correlated with protection from clinical malaria. In contrast, the levels of IgG to a peptide derived from a conserved part of PfEMP1 did not correlate with protection from malaria.
Pub.: 12 May '01, Pinned: 17 Jun '17
Abstract: Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), expressed on P. falciparum-infected erythrocytes, is a major family of clonally variant targets of naturally acquired immunity to malaria. Previous studies have demonstrated that in areas where malaria is endemic, antibodies to infected erythrocytes from children with severe malaria tend to be more seroprevalent than antibodies to infected erythrocytes from children with nonsevere malaria. These data have led to a working hypothesis that PfEMP1 variants associated with parasite virulence are relatively conserved in structure. However, the longevity of such serologically conserved variants in the parasite population is unknown. Here, using infected erythrocytes from recently sampled clinical P. falciparum samples, we measured serological conservation using pools of antibodies in sera that had been sampled 10 to 12 years earlier. The serological conservation of infected erythrocytes strongly correlated with the expression of specific PfEMP1 subsets previously found to be associated with severe malaria. However, we found no association between serological conservation per se and disease severity within these data. This contrasts with the simple hypothesis that P. falciparum isolates with a serologically conserved group of PfEMP1 variants cause severe malaria. The data are instead consistent with periodic turnover of the immunodominant epitopes of PfEMP1 associated with severe malaria.
Pub.: 18 Feb '16, Pinned: 17 Jun '17
Abstract: 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 immune plasma matched cohort from Papua New Guinea and 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 0.15, 95% confidence interval 0.04-0.63). Opsonizing antibodies had a strong strain-transcending component, and the opsonization of transgenic parasites deficient for MSP3, MSP6, MSPDBL1 or PfMSP1-19 was similar to wild-type parasites. We have provided the first evidence that merozoite opzonisation 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.
Pub.: 18 May '16, Pinned: 17 Jun '17
Abstract: Plasmodium falciparum blood-stage antigens such as merozoite surface protein 1 (MSP-1), apical membrane antigen 1 (AMA-1), and the 175-kDa erythrocyte binding antigen (EBA-175) are considered important targets of naturally acquired immunity to malaria. However, it is not clear whether antibodies to these antigens are effectors in protection against clinical disease or mere markers of exposure. In the context of a randomized, placebo-controlled trial of intermittent preventive treatment in infants conducted between 2002 and 2004, antibody responses to Plasmodium falciparum blood-stage antigens in a cohort of 302 Mozambican children were evaluated by immunofluorescence antibody test and enzyme-linked immunosorbent assay at 5, 9, 12, and 24 months of age. We found that IgG subclass responses to EBA-175 were differentially associated with the incidence of malaria in the follow-up period. A double amount of cytophilic IgG1 or IgG3 was associated with a significant decrease in the incidence of malaria (incidence rate ratio [IRR] = 0.49, 95% confidence interval [CI] = 0.25 to 0.97, and P = 0.026 and IRR = 0.44, CI = 0.19 to 0.98, and P = 0.037, respectively), while a double amount of noncytophilic IgG4 was significantly correlated with an increased incidence of malaria (IRR = 3.07, CI = 1.08 to 8.78, P = 0.020). No significant associations between antibodies to the 19-kDa fragment of MSP-1 (MSP-1(19)) or AMA-1 and incidence of malaria were found. Age, previous episodes of malaria, present infection, and neighborhood of residence were the main factors influencing levels of antibodies to all merozoite antigens. Deeper understanding of the acquisition of antibodies against vaccine target antigens in early infancy is crucial for the rational development and deployment of malaria control tools in this vulnerable population.
Pub.: 16 Dec '11, Pinned: 17 Jun '17
Abstract: Individuals living in areas where malaria is endemic are repeatedly exposed to many different malaria parasite antigens. Studies on naturally acquired antibody-mediated immunity to clinical malaria have largely focused on the presence of responses to individual antigens and their associations with decreased morbidity. We hypothesized that the breadth (number of important targets to which antibodies were made) and magnitude (antibody level measured in a random serum sample) of the antibody response were important predictors of protection from clinical malaria. We analyzed naturally acquired antibodies to five leading Plasmodium falciparum merozoite-stage vaccine candidate antigens, and schizont extract, in Kenyan children monitored for uncomplicated malaria for 6 months (n = 119). Serum antibody levels to apical membrane antigen 1 (AMA1) and merozoite surface protein antigens (MSP-1 block 2, MSP-2, and MSP-3) were inversely related to the probability of developing malaria, but levels to MSP-1(19) and erythrocyte binding antigen (EBA-175) were not. The risk of malaria was also inversely associated with increasing breadth of antibody specificities, with none of the children who simultaneously had high antibody levels to five or more antigens experiencing a clinical episode (17/119; 15%; P = 0.0006). Particular combinations of antibodies (AMA1, MSP-2, and MSP-3) were more strongly predictive of protection than others. The results were validated in a larger, separate case-control study whose end point was malaria severe enough to warrant hospital admission (n = 387). These findings suggest that under natural exposure, immunity to malaria may result from high titers antibodies to multiple antigenic targets and support the idea of testing combination blood-stage vaccines optimized to induce similar antibody profiles.
Pub.: 05 Mar '08, Pinned: 17 Jun '17
Abstract: Naturally acquired immunity to clinical malaria is thought to be mainly antibody-mediated, but reports on antigen targets are contradictory. Recognition of multiple antigens may be crucial for protection. In this study, the magnitude of antibody responses and their temporal stability was assessed for a panel of malaria antigens in relation to protection against clinical Plasmodium falciparum malaria.Malian children aged two to 14 years were enrolled in a longitudinal study and followed up by passive and active case detection for seven months. Plasma was collected at enrolment and at the beginning, in the middle and after the end of the transmission season. Antibody titres to the P. falciparum-antigens apical membrane protein (AMA)-1, merozoite surface protein (MSP)-1₁₉, MSP-3, glutamine-rich protein (GLURP-R0) and circumsporozoite antigen (CSP) were assessed by enzyme-linked immunosorbent assay (ELISA) for 99 children with plasma available at all time points. Parasite carriage was determined by microscopy and nested PCR.Antibody titres to all antigens, except MSP-1₁₉, and the number of antigens recognized increased with age. After malaria exposure, antibody titres increased in children that had low titres at baseline, but decreased in those with high baseline responses. No significant differences were found between antibody titers for individual antigens between children remaining symptomatic or asymptomatic after exposure, after adjustment for age. Instead, children remaining asymptomatic following parasite exposure had a broader repertoire of antigen recognition.The present study provides immune-epidemiological evidence from a limited cohort of Malian children that strong recognition of multiple antigens, rather than antibody titres for individual antigens, is associated with protection from clinical malaria.
Pub.: 06 Feb '15, Pinned: 17 Jun '17
Abstract: An understanding of the mechanisms mediating protective immunity against malaria in humans is currently lacking, but critically important to advance the development of highly efficacious vaccines. Antibodies play a key role in acquired immunity, but the functional basis for their protective effect remains unclear. Furthermore, there is a strong need for immune correlates of protection against malaria to guide vaccine development.Using a validated assay to measure opsonic phagocytosis of Plasmodium falciparum merozoites, we investigated the potential role of this functional activity in human immunity against clinical episodes of malaria in two independent cohorts (n = 109 and n = 287) experiencing differing levels of malaria transmission and evaluated its potential as a correlate of protection.Antibodies promoting opsonic phagocytosis of merozoites were cytophilic immunoglobulins (IgG1 and IgG3), induced monocyte activation and production of pro-inflammatory cytokines, and were directed against major merozoite surface proteins (MSPs). Consistent with protective immunity in humans, opsonizing antibodies were acquired with increasing age and malaria exposure, were boosted on re-infection, and levels were related to malaria transmission intensity. Opsonic phagocytosis was strongly associated with a reduced risk of clinical malaria in longitudinal studies in children with current or recent infections. In contrast, antibodies to the merozoite surface in standard immunoassays, or growth-inhibitory antibodies, were not significantly associated with protection. In multivariate analyses including several antibody responses, opsonic phagocytosis remained significantly associated with protection against malaria, highlighting its potential as a correlate of immunity. Furthermore, we demonstrate that human antibodies against MSP2 and MSP3 that are strongly associated with protection in this population are effective in opsonic phagocytosis of merozoites, providing a functional link between these antigen-specific responses and protection for the first time.Opsonic phagocytosis of merozoites appears to be an important mechanism contributing to protective immunity in humans. The opsonic phagocytosis assay appears to be a strong correlate of protection against malaria, a valuable biomarker of immunity, and provides a much-needed new tool for assessing responses to blood-stage malaria vaccines and measuring immunity in populations.
Pub.: 02 Jul '14, Pinned: 17 Jun '17
Abstract: Severe malaria (SM) is a life-threatening complication of infection withPlasmodium falciparum Epidemiological observations have long indicated that immunity against SM is acquired relatively rapidly, but prospective studies to investigate its immunological basis are logistically challenging and have rarely been undertaken. We investigated the merozoite targets and antibody-mediated mechanisms associated with protection against SM in Kenyan children aged 0 to 2 years. We designed a unique prospective matched case-control study of well-characterized SM clinical phenotypes nested within a longitudinal birth cohort of children (n= 5,949) monitored over the first 2 years of life. We quantified immunological parameters in sera collected before the SM event in cases and their individually matched controls to evaluate the prospective odds of developing SM in the first 2 years of life. Anti-AMA1 antibodies were associated with a significant reduction in the odds of developing SM (odds ratio [OR] = 0.37; 95% confidence interval [CI] = 0.15 to 0.90;P= 0.029) after adjustment for responses to all other merozoite antigens tested, while those against MSP-2, MSP-3,Plasmodium falciparumRh2 [PfRh2], MSP-119, and the infected red blood cell surface antigens were not. The combined ability of total IgG to inhibit parasite growth and mediate the release of reactive oxygen species from neutrophils was associated with a marked reduction in the odds of developing SM (OR = 0.07; 95% CI = 0.006 to 0.82;P= 0.03). Assays of these two functional mechanisms were poorly correlated (Spearman rank correlation coefficient [rs] = 0.12;P= 0.07). Our data provide epidemiological evidence that multiple antibody-dependent mechanisms contribute to protective immunity via distinct targets whose identification could accelerate the development of vaccines to protect against SM.
Pub.: 21 Jan '16, Pinned: 17 Jun '17
Abstract: Plasmodium falciparum is the most virulent human malaria parasite due to its ability to cytoadhere in the microvasculature. Non-human primate studies demonstrated a relationship between 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 characterised. Knob and KAHRP expression, CD36 adhesion and antibody recognition of parasitised erythrocytes (PE) 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 characterisation. All parasites were infective in vivo However, infectivity of NF54 was dramatically reduced. In vitro characterisation revealed that unlike other cell bank parasites, NF54 PE lacked knobs and did not cytoadhere. Recognition of NF54 PE by immune sera was observed, suggesting PfEMP1 expression. Subsequent recovery of knob-expression and CD36-mediated adhesion was observed in PE derived from participants infected with NF54. Knobless cell bank parasites have a dramatic reduction in their infectivity and ability to adhere to CD36. Subsequent infection of malaria-naïve volunteers restores knob-expression and CD36-mediated cytoadherence, thereby showing that the human environment can modulate virulence.
Pub.: 07 Jul '16, Pinned: 17 Jun '17
Abstract: Most severe Plasmodium falciparum infections are experienced by young children. Severe symptoms are precipitated by vascular sequestration of parasites expressing a particular subset of the polymorphic P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion molecules. Parasites binding human endothelial protein C receptor (EPCR) through the CIDRα1 domain of certain PfEMP1 were recently associated with severe malaria in children. However, it has remained unclear to which extend the EPCR‐binding CIDRα1 domains epitomize PfEMP1 expressed in severe malaria. Here, we characterized the near full‐length transcripts dominating the var transcriptome in children with severe malaria and found that the only common feature of the encoded PfEMP1 was CIDRα1 domains. Such genes were highly and dominantly expressed in both children with severe malarial anaemia and cerebral malaria. These observations support the hypothesis that the CIDRα1‐EPCR interaction is key to the pathogenesis of severe malaria and strengthen the rationale for pursuing a vaccine or adjunctive treatment aiming at inhibiting or reducing the damaging effects of this interaction.
Pub.: 27 Jun '16, Pinned: 17 Jun '17
Abstract: Sequestration of Plasmodium falciparum-infected erythrocytes in host blood vessels is a key triggering event in the pathogenesis of severe childhood malaria, which is responsible for about one million deaths every year. Sequestration is mediated by specific interactions between members of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family and receptors on the endothelial lining. Severe childhood malaria is associated with expression of specific PfEMP1 subtypes containing domain cassettes (DCs) 8 and 13 (ref. 3), but the endothelial receptor for parasites expressing these proteins was unknown. Here we identify endothelial protein C receptor (EPCR), which mediates the cytoprotective effects of activated protein C, as the endothelial receptor for DC8 and DC13 PfEMP1. We show that EPCR binding is mediated through the amino-terminal cysteine-rich interdomain region (CIDRα1) of DC8 and group A PfEMP1 subfamilies, and that CIDRα1 interferes with protein C binding to EPCR. This PfEMP1 adhesive property links P. falciparum cytoadhesion to a host receptor involved in anticoagulation and endothelial cytoprotective pathways, and has implications for understanding malaria pathology and the development of new malaria interventions.
Pub.: 07 Jun '13, Pinned: 17 Jun '17
Abstract: Severe malaria syndromes are precipitated by Plasmodium falciparum parasites binding to endothelial receptors on the vascular lining. This binding is mediated by members of the highly variant P. falciparum erythrocyte membrane protein 1 (PfEMP1) family. We have previously identified a subset of PfEMP1 proteins associated with severe malaria and found that the receptor for these PfEMP1 variants is endothelial protein C receptor (EPCR). The binding is mediated through the amino-terminal cysteine-rich interdomain region (CIDR) of the subtypes α1.1 and α1.4 to α1.8. In this study, we investigated the acquisition of anti-CIDR antibodies using plasma samples collected in four study villages with different malaria transmission intensities in northeastern Tanzania during a period with a decline in malaria transmission. We show that individuals exposed to high levels of malaria transmission acquire antibodies to EPCR-binding CIDR domains early in life and that these antibodies are acquired more rapidly than antibodies to other CIDR domains. The rate by which antibodies to EPCR-binding CIDR domains are acquired in populations in areas where malaria is endemic is determined by the malaria transmission intensity, and on a population level, the antibodies are rapidly lost if transmission is interrupted. This indicates that sustained exposure is required to maintain the production of the antibodies.
Pub.: 28 May '15, Pinned: 17 Jun '17
Abstract: By attaching infected erythrocytes to the vascular lining, Plasmodium falciparum parasites leave blood circulation and avoid splenic clearance. This sequestration is central to pathogenesis. Severe malaria is associated with parasites expressing an antigenically distinct subset of P. falciparum erythrocyte membrane protein 1 (PfEMP1) mediating the binding to endothelial receptors. Previous studies indicate that PfEMP1, with so-called CIDRα1 domains capable of binding endothelial protein C receptor (EPCR), constitute the PfEMP1 subset associated with severe paediatric malaria. To analyse the relative importance of different subtypes of CIDRα1 domains, we compared pfemp1 transcripts in children with severe malaria (including 9 fatal and 114 surviving cases), children hospitalised with uncomplicated malaria (N=42), children with mild malaria not requiring hospitalisation (N=10) and children with parasiteamia and no ongoing fever (N=12). High levels of transcripts encoding EPCR-binding PfEMP1 were found in patients with symptomatic infections and the abundance of these transcripts increased with disease severity. The CIDRα1 subtype transcript composition varied markedly between patients, and none of the subtypes were dominant. Transcript level analyses targeting other domain types indicated that subtypes of DBLβ or DBLζ domains might mediate binding phenomena that, in conjunction with EPCR binding, could contribute to pathogenesis. The observations strengthen the rationale for targeting PfEMP1-EPCR interaction by vaccines and adjunctive therapies. Interventions should target the EPCR binding of all CIDRα1 subtypes.
Pub.: 01 Feb '17, Pinned: 17 Jun '17