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Intravenous immunoglobulin in the treatment of paediatric cerebral malaria
Abstract
Hyperimmune globulin can inhibit and reverse the cytoadherence between Piasmodium falciparum- infected erythrocytes and melanoma cells in vitro. Cytoadherence is believed to mediate disease in cerebral malaria. Therefore we studied the efficacy of i.v. immunoglobulin, purified from the plasma of local semi-immune blood donors, as an adjunct to standard treatment for cerebral malaria in Malawian children.
The immunoglobulin preparation (IFAT antimalarial antibody titre 1:5120) recognized erythrocyte-associated antigens of each of 22 Malawian P. falciparum isolates studied, and reversed binding of Malawian isolates to melanoma cells. Immunoglobulin did not reverse binding to human monocytes or to cells of the human hystiocytic lymphoma cell line U937.
Thirty-one children with P. falciparum parasitaemia and unrousable coma were enrolled. All were treated with i.v. quinine dihydrochloride; in addition patients were randomized to receive either immunoglobulin (400 mg/kg by i.v. infusion over 3 h) or placebo (albumen and sucrose by similar infusion) in a double blind trial with sequential analysis. Of 16 patients receiving immunoglobulin, five (31%) died and five survivors had neurological sequelae. Of 15 patients receiving placebo, one (7%) died and two had sequelae. Parasite clearance, fever clearance and coma resolution times in survivors were similar in the two groups.
Although the difference in outcome between the two groups was not significant, the trial was stopped because immunoglobulin was demonstrated not to be superior to placebo.
... Collection of sera from Malawian adults that were previously used to determine its effectiveness as an adjunct therapy to treat cerebral malaria was approved by the National Health Sciences Research Committee of Malawi [19]. Plasma from adult travellers, microscopy-diagnosed with malaria and species further confirmed by multiplex PCR for all species except P. knowlesi [20] after returning from visits to malaria-endemic regions were obtained from the Karolinska University Hospital, Stockholm, Sweden (n = 81). ...
... A smaller subset within the expanded panel of recombinant proteins which were immunoreactive needed to be identified as suitable serological markers of infection. To determine which of the P. ovale and P. malariae proteins were immunoreactive, the responses to IgG, IgM and IgA immunoglobulins were tested using sera pooled from over 800 adults living in Malawi [19] where there is active transmission for both species [35]. For P. malariae, P41, MSP5, and MSP10 were selected as they were the most immunoreactive and expressed at acceptable . ...
Background:
Malaria remains a global health problem and accurate surveillance of Plasmodium parasites that are responsible for this disease is required to guide the most effective distribution of control measures. Serological surveillance will be particularly important in areas of low or periodic transmission because patient antibody responses can provide a measure of historical exposure. While methods for detecting host antibody responses to Plasmodium falciparum and Plasmodium vivax are well established, development of serological assays for Plasmodium knowlesi, Plasmodium ovale and Plasmodium malariae have been inhibited by a lack of immunodiagnostic candidates due to the limited availability of genomic information.
Methods:
Using the recently completed genome sequences from P. malariae, P. ovale and P. knowlesi, a set of 33 candidate cell surface and secreted blood-stage antigens was selected and expressed in a recombinant form using a mammalian expression system. These proteins were added to an existing panel of antigens from P. falciparum and P. vivax and the immunoreactivity of IgG, IgM and IgA immunoglobulins from individuals diagnosed with infections to each of the five different Plasmodium species was evaluated by ELISA. Logistic regression modelling was used to quantify the ability of the responses to determine prior exposure to the different Plasmodium species.
Results:
Using sera from European travellers with diagnosed Plasmodium infections, antigens showing species-specific immunoreactivity were identified to select a panel of 22 proteins from five Plasmodium species for serological profiling. The immunoreactivity to the antigens in the panel of sera taken from travellers and individuals living in malaria-endemic regions with diagnosed infections showed moderate power to predict infections by each species, including P. ovale, P. malariae and P. knowlesi. Using a larger set of patient samples and logistic regression modelling it was shown that exposure to P. knowlesi could be accurately detected (AUC = 91%) using an antigen panel consisting of the P. knowlesi orthologues of MSP10, P12 and P38.
Conclusions:
Using the recent availability of genome sequences to all human-infective Plasmodium spp. parasites and a method of expressing Plasmodium proteins in a secreted functional form, an antigen panel has been compiled that will be useful to determine exposure to these parasites.
... Hyperimmune IgG and naïve sera were probed on the array as controls. Hyperimmune IgG was pooled from 834 HIV and HBsAg seronegative Malawian adults [34], and the naïve control was pooled from 30 malaria-naïve adults from the United States. Both controls were probed at 1:100 (20% Escherichia coli lysate; GenScript). ...
... Children appear to be at risk for symptomatic malaria until they are older than the children described here. Pooled immune IgG from Malawian adults [34] (see Additional file 6) was seroreactive across all PfEMP1 binding groups. Multiple exposures are likely required to develop significant cross-reactive antibody to hypervariable extracellular PfEMP1 domains. ...
Background:
Antibody immunity is thought to be essential to prevent severe Plasmodium falciparum infection, but the exact correlates of protection are unknown. Over time, children in endemic areas acquire non-sterile immunity to malaria that correlates with development of antibodies to merozoite invasion proteins and parasite proteins expressed on the surface of infected erythrocytes.
Results:
A 1000 feature P. falciparum 3D7 protein microarray was used to compare P. falciparum-specific seroreactivity during acute infection and 30 days after infection in 23 children with uncomplicated malaria (UM) and 25 children with retinopathy-positive cerebral malaria (CM). All children had broad P. falciparum antibody reactivity during acute disease. IgM reactivity decreased and IgG reactivity increased in convalescence. Antibody reactivity to CIDR domains of "virulent" PfEMP1 proteins was low with robust reactivity to the highly conserved, intracellular ATS domain of PfEMP1 in both groups. Although children with UM and CM differed markedly in parasite burden and PfEMP1 exposure during acute disease, neither acute nor convalescent PfEMP1 seroreactivity differed between groups. Greater seroprevalence to a conserved Group A-associated ICAM binding extracellular domain was observed relative to linked extracellular CIDRα1 domains in both case groups. Pooled immune IgG from Malawian adults revealed greater reactivity to PfEMP1 than observed in children.
Conclusions:
Children with uncomplicated and cerebral malaria have similar breadth and magnitude of P. falciparum antibody reactivity. The utility of protein microarrays to measure serological recognition of polymorphic PfEMP1 antigens needs to be studied further, but the study findings support the hypothesis that conserved domains of PfEMP1 are more prominent targets of cross reactive antibodies than variable domains in children with symptomatic malaria. Protein microarrays represent an additional tool to identify cross-reactive Plasmodium antigens including PfEMP1 domains that can be investigated as strain-transcendent vaccine candidates.
... Three hundred milligrams of a human anti-malaria antibody pool [72,73] was used to affinity purify human IgG antibodies against rPfPMT as described for other proteins [74]. ...
... A human anti-malaria antibody pool [72,73] was passed consecutively over four affinity resins with recombinant P. falciparum LDH, GAPDH, Cox17 and PMT coupled to the resins. Approximately 0.41 mg of affinity purified human antibodies detecting rPfPMT was eluted from the rPfPMT affinity resin ( Table 3). ...
Background:
Plasmodium knowlesi is recognised as the main cause of human malaria in Southeast Asia. The disease is often misdiagnosed as P. falciparum or P. malariae infections by microscopy, and the disease is difficult to eliminate due to its presence in both humans and monkeys. P. knowlesi infections can rapidly cause severe disease and require prompt diagnosis and treatment. No protein biomarker exists for the rapid diagnostic test (RDT) detection of P. knowlesi infections. Plasmodium knowlesi infections can be diagnosed by PCR.
Methods and principal findings:
Phosphoethanolamine-N-methyltransferase (PMT) is involved in malaria lipid biosynthesis and is not found in the human host. The P. falciparum, P. vivax and P. knowlesi PMT proteins were recombinantly expressed in BL21(DE3) Escherichia coli host cells, affinity purified and used to raise antibodies in chickens. Antibodies against each recombinant PMT protein all detected all three recombinant proteins and the native 29 kDa P. falciparum PMT protein on western blots and in ELISA. Antibodies against a PMT epitope (PLENNQYTDEGVKC) common to all three PMT orthologues detected all three proteins. Antibodies against unique peptides from each orthologue of PMT, PfCEVEHKYLHENKE, PvVYSIKEYNSLKDC, PkLYPTDEYNSLKDC detected only the parent protein in western blots and P. falciparum infected red blood cell lysates or blood lysates spiked with the respective proteins. Similar concentrations of PfPMT and the control, PfLDH, were detected in the same parasite lysate. The recombinant PfPMT protein was detected by a human anti-malaria antibody pool.
Conclusion:
PMT, like the pan-specific LDH biomarker used in RDT tests, is both soluble, present at comparable concentrations in the parasite and constitutes a promising antimalarial drug target. PMT is absent from the human proteome. PMT has the potential as a biomarker for human malaria and in particular as the first P. knowlesi specific protein with diagnostic potential for the identification of a P. knowlesi infection.
... Similarly to what occurred with corticosteroids, treatment with intravenous immunoglobulin was associated with increased deleterious outcomes compared to the placebo group, including higher mortality and more neurological sequelae in children [35]. The clinical failure of this therapy may reflect the lack of success to reverse cytoadherence and sequestration [35]. ...
... Similarly to what occurred with corticosteroids, treatment with intravenous immunoglobulin was associated with increased deleterious outcomes compared to the placebo group, including higher mortality and more neurological sequelae in children [35]. The clinical failure of this therapy may reflect the lack of success to reverse cytoadherence and sequestration [35]. ...
Background:
Despite recent efforts and successes in reducing the malaria burden globally, this infection still accounts for an estimated 212 million clinical cases, 2 million severe malaria cases, and approximately 429,000 deaths annually. Even with the routine use of effective anti-malarial drugs, the case fatality rate for severe malaria remains unacceptably high, with cerebral malaria being one of the most life-threatening complications. Up to one-third of cerebral malaria survivors are left with long-term cognitive and neurological deficits. From a population point of view, the decrease of malaria transmission may jeopardize the development of naturally acquired immunity against the infection, leading to fewer total cases, but potentially an increase in severe cases. The pathophysiology of severe and cerebral malaria is not completely understood, but both parasite and host determinants contribute to its onset and outcomes. Adjunctive therapy, based on modulating the host response to infection, could help to improve the outcomes achieved with specific anti-malarial therapy.
Results and conclusions:
In the last decades, several interventions targeting different pathways have been tested. However, none of these strategies have demonstrated clear beneficial effects, and some have shown deleterious outcomes. This review aims to summarize evidence from clinical trials testing different adjunctive therapy for severe and cerebral malaria in humans. It also highlights some preclinical studies which have evaluated novel strategies and other candidate therapeutics that may be evaluated in future clinical trials.
... Several published experiments have addressed inhibition of adhesion of pRBC by mAb, focussing on the prevention of de-novo adhesion [20], but there have been very few studies looking at the potential to reverse existing pRBC cytoadherence [21]. Reversing pRBC sequestration has been considered as an attractive contributing strategy for the management of SM [22], as an adjunct to standard anti-parasite treatment. The rationale for reversing sequestration was based on the beneficial effects of administration of anti-malaria immunoglobulins from adults with malaria to children with mild malaria in Thailand [23]. ...
... While the reversal of cytoadherence by human serum containing relevant antibodies, both in vitro and in vivo, suggests that a pool of high-titre malarial antibodies, shown to have reactivity with the surface of infected erythrocytes [25] could reverse adhesion in vivo, a double blind, placebo-controlled administration of the antibodies as an adjunct to quinine (the best available anti-malarial at the time) had no measurable observed effect on adhesion, and did not affect patient recovery [22]. The inherent difficulty with this approach is, at least in part (but see also [33]), the variable nature of PfEMP1 and, therefore, the complexity of the antibody pool to interfere with cytoadherence. ...
Background
Sequestration of parasitized red blood cells from the peripheral circulation during an infection with Plasmodium falciparum is caused by an interaction between the parasite protein PfEMP1 and receptors on the surface of host endothelial cells, known as cytoadherence. Several lines of evidence point to a link between the pathology of severe malaria and cytoadherence, therefore blocking adhesion receptors involved in this process could be a good target to inhibit pRBC sequestration and prevent disease. In a malaria endemic setting this is likely to be used as an adjunct therapy by reversing existing cytoadherence. Two well-characterized parasite lines plus three recently derived patient isolates were tested for their cytoadherence to purified receptors (CD36 and ICAM-1) as well as endothelial cells. Monoclonal antibodies against human CD36 and ICAM-1 were used to inhibit and reverse infected erythrocyte binding in static and flow-based adhesion assays.
Results
Anti-ICAM-1 and CD36 monoclonal antibodies were able to inhibit and reverse P. falciparum binding of lab and recently adapted patient isolates in vitro. However, reversal of binding was incomplete and varied in its efficiency between parasite isolates.
Conclusions
The results show that, as a proof of concept, disturbing existing ligand–receptor interactions is possible and could have potential therapeutic value for severe malaria. The variation seen in the degree of reversing existing binding with different parasite isolates and the incomplete nature of reversal, despite the use of high affinity inhibitors, suggest that anti-adhesion approaches as adjunct therapies for severe malaria may not be effective, and the focus may need to be on inhibitory approaches such as vaccines.
... Binding of human IgM to their DBL domains masks DBLMSP and DBLMSP2 from host antibodies -The fact that the DBL domains of DBLMSP and DBLMSP2 retain IgM binding despite their extraordinary sequence diversity suggests that DBLMSP and DBLMSP2 are targeted by the host immune system and that IgM binding promotes parasite survival. To determine whether individuals regularly exposed to P. falciparum mount an immune response to DBLMSP and DBLMSP2, we compared the immunoreactivity of the full-length proteins and DBL domains of both DBLMSP and DBLMSP2 from the 3D7 strain to purified IgG from Malawian immune adults (33). As comparators, we used AMA1 and RH5, which have previously been shown to be highly and weakly immunoreactive, respectively, to antibodies in sera from Kenyan, Malian and Senegalese immune adults (20,(34)(35)(36). ...
... Immunoreactivity analysis -Pentamerised, normalised β-lactamasetagged parasite proteins were incubated with serial dilutions of purified human IgM for 90 minutes before being transferred to 20 μg/mL purified IgG from Malawian adults (33), immobilised on ProteinG-coated microtitre plates (Pierce). After 60 minutes, the plates were washed and incubated with nitrocefin at 125 μg/mL. ...
Diversity at pathogen genetic loci can be driven by host adaptive immune selection pressure and may reveal proteins important
for parasite biology. Population-based genome sequencing of Plasmodium falciparum - the parasite responsible for the most
severe form of malaria - has highlighted two related polymorphic genes called dblmsp and dblmsp2, which encode Duffy binding-like
(DBL) domain-containing proteins located on the merozoite surface but whose function remains unknown. Using recombinant proteins
and transgenic parasites, we show DBLMSP and DBLMSP2 directly and avidly bind human IgM via their DBL domains. We used whole
genome sequence data from over 400 African and Asian P. falciparum isolates to show that dblmsp and dblmsp2 exhibit extreme
protein polymorphism in their DBL domain, with multiple variants of two major allelic classes present in every population
tested. Despite this variability, the IgM-binding function was retained across diverse sequence representatives. While this
interaction did not seem to have an effect on the ability of the parasite to invade red blood cells, binding of DBLMSP and
DBLMSP2 to IgM inhibited the overall immunoreactivity of these proteins to IgG from patients who had been exposed to the parasite.
This suggests that IgM binding might mask these proteins from the host humoral immune system.
... A variety of other adjunctive treatments aimed at reducing mortality from severe malaria have been evaluated in clinical trials without showing evidence of benefit, including adrenaline [35], anti-tumor necrosis factor antibody [36], aspirin [37], dexamethasone [38], heparin [37], human albumin [39], intravenous immunoglobulin [40], iron chelators [41,42], low-molecularweight dextran [43], mannitol [44,45], N-acetylcysteine [46], phenobarbitone [47], and ursodeoxycholic acid [48]. ...
... It is possible that the potent and rapid onset of the parasiticidal effect of artesunate obscures the inhibiting effects on sequestration of levamisole. (36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54) .889 ...
Background. Cytoadherence and sequestration of erythrocytes containing mature stages of Plasmodium falciparum are central to the pathogenesis of severe malaria. The oral anthelminthic drug levamisole inhibits cytoadherence in vitro and reduces sequestration of late-stage parasites in uncomplicated falciparum malaria treated with quinine.
Methods. Fifty-six adult patients with severe malaria and high parasitemia admitted to a referral hospital in Bangladesh were randomized to receive a single dose of levamisole hydrochloride (150 mg) or no adjuvant to antimalarial treatment with intravenous artesunate.
Results. Circulating late-stage parasites measured as the median area under the parasite clearance curves were 2150 (interquartile range [IQR], 0–28 025) parasites/µL × hour in patients treated with levamisole and 5489 (IQR, 192–25 848) parasites/µL × hour in controls (P = .25). The “sequestration ratios” at 6 and 12 hours for all parasite stages and changes in microvascular blood flow did not differ between treatment groups (all P > .40). The median time to normalization of plasma lactate (<2 mmol/L) was 24 (IQR, 12–30) hours with levamisole vs 28 (IQR, 12–36) hours without levamisole (P = .15).
Conclusions. There was no benefit of a single-dose of levamisole hydrochloride as adjuvant to intravenous artesunate in the treatment of adults with severe falciparum malaria. Rapid parasite killing by intravenous artesunate might obscure the effects of levamisole.
... Additionally , a purified IgG standard was incorporated into the assay and allowed for the extrapolation of relative antibody concentrations . Eleven two-fold serial dilutions of a reference Malaria Immune Globulin (MIG) reagent (Central Laboratory Blood Transfusion Service SRC, Switzerland) [30] were included for every antigen to generate a standard ELISA curve. This preparation contains 50 mg/ml of immunoglobulins (98% IgG) purified from a pool of healthy Malawian adult plasma and was originally manufactured to test its potential use as an adjunct therapy to quinine in the treatment of cerebral malaria [30] . ...
... Eleven two-fold serial dilutions of a reference Malaria Immune Globulin (MIG) reagent (Central Laboratory Blood Transfusion Service SRC, Switzerland) [30] were included for every antigen to generate a standard ELISA curve. This preparation contains 50 mg/ml of immunoglobulins (98% IgG) purified from a pool of healthy Malawian adult plasma and was originally manufactured to test its potential use as an adjunct therapy to quinine in the treatment of cerebral malaria [30] . The four-parameter logistic function was used to fit the standard curve in GraphPad Prism version 4.0 (GraphPad Software, San Diego, CA). ...
Antibodies to selected Plasmodium falciparum merozoite antigens are often reported to be associated with protection from malaria in one epidemiological cohort, but not in another. Here, we sought to understand this paradox by exploring the hypothesis that a threshold concentration of antibodies is necessary for protection. We analyzed data from two independent cohorts along the Kenyan coast, one in which antibodies to AMA1, MSP-2 and MSP-3 were associated with protection from malaria (Chonyi) and another in which this association was not observed (Junju). We used a malaria reference reagent to standardize antibody measurements across both cohorts, and applied statistical methods to derive the threshold concentration of antibodies against each antigen that best correlated with a reduced risk of malaria (the protective threshold), in the Chonyi cohort. We then tested whether antibodies in Junju reached the protective threshold concentrations observed in the Chonyi cohort. Except for children under 3 years, the age-matched proportions of children achieving protective threshold concentrations of antibodies against AMA1 and MSP-2 were significantly lower in Junju compared to Chonyi (Fishers exact test, P<0.01). For MSP-3, this difference was significant only among 4-5 year olds. We conclude that although antibodies are commonly detected in malaria endemic populations, they may be present in concentrations that are insufficient for protection. Our results have implications for the analysis and interpretation of similar data from immuno-epidemiological studies.
... However, in another trial conducted with falciparum malaria patients, treatment with pentoxifylline did not have any effect on clinical outcome and patients reported mild adverse side effects, while in one trial with a small sample size of ten pediatric CM patients, the mortality rate was unusually high in the pentoxifyllinetreated group compared to the control group (Hemmer et al., 1997;Lell et al., 2010). Intravenous administration of immunoglobulin was also shown not to be superior to placebo in a trial with pediatric CM patients and may have been associated with deleterious effects (Taylor et al., 1992). ...
Cerebral malaria (CM) is one of the most severe complications of malaria infection characterized by coma and neurological effects. Despite standardized treatment of malaria infection with artemisinin-based combination therapies (ACT), the mortality rate is still high, and it primarily affects pediatric patients. ACT reduces parasitemia but fails to adequately target the pathogenic mechanisms underlying CM, including blood-brain-barrier (BBB) disruption, endothelial activation/dysfunction, and hyperinflammation. The need for adjunctive therapies to specifically treat this form of severe malaria is critical as hundreds of thousands of people continue to die each year from this disease. Here we present a summary of some potential promising therapeutic targets and treatments for CM, as well as some that have been tested and deemed ineffective or, in some cases, even deleterious. Further exploration into these therapeutic agents is warranted to assess the effectiveness of these potential treatments for CM patients.
... The observation of heavily infected red blood cell (iRBC) sequestration in the cerebral vasculature of children who died of CM, often accompanied by intra-and peri-vascular pathology including ring hemorrhages, led to the generally accepted hypothesis that iRBC sequestration in the cerebral vasculature and the resulting mechanical obstruction lead to inflammation, impaired vasoregulation, and blood brain barrier (BBB) dysfunction thus causing this severe progression of malaria [4]. Therapies targeting the purposed downstream effects of sequestration were investigated in multiple clinical trials [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19], but none improved clinical outcomes. These failures suggested that the downstream effects of iRBC sequestration were poor targets of therapies. ...
Background
Despite treatment with highly effective antimalarial drugs, malaria annually claims the lives of over half a million children under 5-years of age in sub-Saharan Africa. Cerebral malaria (CM), defined as Plasmodium falciparum infection with coma, is the severe malaria syndrome with the highest mortality. Studies in the CM mouse model suggest that a T cell-mediated response underlies CM pathology, opening a new target for therapy in humans. This trial aims to establish the preliminary safety of one such novel therapy, the glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON).
Methods
In this phase I/IIa dose-escalation clinical trial, a single dose of intravenous (IV) DON is administered to three participants groups—healthy adults and adults with uncomplicated malaria, then pediatric participants with CM—to primarily assess safety. The secondary objective of this trial is to assess pharmacokinetics of DON over a range of doses. The open-label adult portion of the trial enrolls 40 healthy adults concurrently with 40 adults with uncomplicated malaria. Cohorts of 10 participants receive a single IV dose of DON with doses escalating between cohorts from 0.1 mg/kg, 1.0 mg/kg, 5.0 mg/kg, to 10 mg/kg. Following subsequent safety review, a randomized, double-blind, and placebo-controlled pediatric study enrolls 72 participants aged 6 months to 14 years with CM. The pediatric portion of the study minimally spans three malaria seasons including a planned interim analysis after 50% of pediatric enrollments. The first half of pediatric participants receive DON 0.1 mg/kg, 1.0 mg/kg, or placebo. Dosing for the second half of pediatric participants is informed by the safety and preliminary efficacy results of those previously enrolled. The pediatric portion of the study has an exploratory outcome evaluating the preliminary efficacy of DON. Efficacy is assessed by diagnostics predictive of CM outcome: electroencephalography (EEG), magnetic resonance imaging (MRI), and transcranial doppler (TCD), measured before and after DON administration. All participants with malaria receive standard of care antimalarials in accordance with local guidelines, regardless of study drug dose group.
Discussion
This preliminary safety and efficacy study evaluates DON, a candidate adjunctive therapy for pediatric CM. If results support DON preliminary safety and efficacy, follow-up phase II and III clinical trials will be indicated.
Trial registration
This trial was registered on ClinicalTrials.gov on 28 July 2022 (NCT05478720).
... iRBCs with trophozoite stage parasites from cycle 1, treated with DMSO or rapamycin in cycle 0 to disrupt the first peak of pfap2-p expression, were washed thrice with PBS supplemented with 0.1% BSA. iRBCs were either untreated or treated with pooled human immune serum 21 . When untreated, the same volume of 0.1% BSA in PBS was added and incubated for 30 min at room temperature. ...
Malaria-associated pathogenesis such as parasite invasion, egress, host cell remodelling and antigenic variation requires concerted action by many proteins, but the molecular regulation is poorly understood. Here we have characterized an essential Plasmodium -specific Apicomplexan AP2 transcription factor in Plasmodium falciparum (PfAP2-P; pathogenesis) during the blood-stage development with two peaks of expression. An inducible knockout of gene function showed that PfAP2-P is essential for trophozoite development, and critical for var gene regulation, merozoite development and parasite egress. Chromatin immunoprecipitation sequencing data collected at timepoints matching the two peaks of pfap2-p expression demonstrate PfAP2-P binding to promoters of genes controlling trophozoite development, host cell remodelling, antigenic variation and pathogenicity. Single-cell RNA sequencing and fluorescence-activated cell sorting revealed de-repression of most var genes in Δpfap2-p parasites. Δpfap2-p parasites also overexpress early gametocyte marker genes, indicating a regulatory role in sexual stage conversion. We conclude that PfAP2-P is an essential upstream transcriptional regulator at two distinct stages of the intra-erythrocytic development cycle.
... La réduction de l'inflammation systémique grâce à l'administration chez l'homme d'Immunoglobulines IV n'a pas permis de diminuer la mortalité, et des séquelles neurologiques ont notamment été décrites dans certaines études (405). ...
En 2020, le paludisme reste un problème majeur de santé publique. Malgré des avancées scientifiques capitales, il est encore responsable de plus de 400 000 décès chaque année, principalement chez les enfants en Afrique Sub-saharienne. Le neuropaludisme est l'une des formes les plus sévères du paludisme. Sa physiopathologie est complexe, impliquant l'adhésion des érythrocytes infectés par Plasmodium falciparum à l'endothélium vasculaire dans le cerveau. Ce mécanisme conduit à une obstruction du flux sanguin, une inflammation locale, une altération de la barrière hémato-encéphalique et à un éventail de réponses cellulaires visant à résoudre le processus de neuroinflammation. Parmi ces cellules, les monocytes/macrophages jouent un rôle clé de par leur capacité à s'adapter à leur microenvironnement en fonction des différents signaux qu'ils perçoivent, modulant ainsi les balances pro/anti-inflammatoire et pro/antioxydante, essentielles à la résolution du neuropaludisme. Les monocytes sont composés de trois sous-populations dont le rôle dans la physiopathologie du neuropaludisme a été peu étudié et reste à éclaircir. Pour compléter ces connaissances, une approche combinant modèle murin et études chez des patients béninois a été menée. Une première étude a été conduite chez des enfants béninois présentant un paludisme simple, une anémie sévère palustre, ou un neuropaludisme dans le but de déterminer si les paramètres monocytaires constituent des facteurs de sévérité et/ou de risque de décès au cours d'un paludisme grave. Les pourcentages et phénotypes des sous-populations monocytaires obtenus chez les trois groupes d'enfants lors de leur admission dans les centres de santé (J0) ont été comparés. Les résultats ont permis de mettre en lumière l'implication des monocytes non classiques dans la protection vis-à-vis des formes sévères de paludisme et de confirmer l'importance du récepteur CD36 des monocytes comme facteur protecteur de sévérité et de survenue de décès. Pour aller plus loin, un travail a ensuite été mené sur un modèle murin de neuropaludisme mis en place pour se rapprocher des conditions réelles, à savoir un traitement tardif, et débouchant sur une résolution de l'infection. Des souris C57BL/6 ont été infectées par P. berghei ANKA (J0) et traitées tardivement à la chloroquine. La cinétique d'évolution des sous-populations monocytaires a été suivie en termes de pourcentage et d'expression protéique et génique, à la fois dans le sang, la rate et le cerveau, jusqu'à la résolution de l'infection (J12). Les résultats suggèrent une implication des monocytes classiques et intermédiaires dans la neuroinflammation, au contraire des monocytes non classiques. A J12 était observé un nouvel afflux de monocytes intermédiaires vers le cerveau, suggérant également un rôle de cette sous-population dans la résolution tardive de la neuroinflammation. Les analyses protéique et génique ont mis en évidence une implication favorable de Nrf2, CD36, HO-1, CMH-II, COX-2 et de la 12/15-LOX dans la résolution de l'infection. Une deuxième étude a ensuite été menée chez des enfants béninois, avec un plus grand nombre de patients et un suivi à J3 et J30. Deux groupes de sujets ont été inclus, présentant un paludisme simple (n = 94) ou un neuropaludisme (n = 65). Dans l'ensemble, les résultats indiquent que les monocytes non classiques et intermédiaires étaient respectivement impliqués dans l'amélioration clinique et la survie des patients. L'expression de CD14, CD16, CD36 et HLA-DR était un facteur protecteur vis-à-vis du neuropaludisme. De manière intéressante, nos résultats montrent également une association entre une réponse pro-oxydante moins active, la sévérité du paludisme, et la survenue de décès. L'ensemble de ces résultats apporte un nouvel éclairage sur l'implication des monocytes/macrophages au cours du neuropaludisme.
... The MIG was prepared from purified immunoglobulin G (98% IgG) obtained from a pool of 834 Malawian adult sera (Taylor et al., 1992). For positive controls, a characterized pool of hyper-immune sera (PHIS) was obtained from a random selection of adult residents of Junju location, Kilifi (Osier et al., 2014;Murungi et al., 2016), and a pool of sera from gametocyte positive individuals from the AFIRM cohort with high gametocytaemia were also included. ...
Introduction
Naturally acquired immune responses against antigens expressed on the surface of mature gametocytes develop in individuals living in malaria-endemic areas. Evidence suggests that such anti-gametocyte immunity can block the development of the parasite in the mosquito, thus playing a role in interrupting transmission. A better comprehension of naturally acquired immunity to these gametocyte antigens can aid the development of transmission-blocking vaccines and improve our understanding of the human infectious reservoir.
Methods
Antigens expressed on the surface of mature gametocytes that had not previously been widely studied for evidence of naturally acquired immunity were identified for protein expression alongside Pfs230-C using either the mammalian HEK293E or the wheat germ cell-free expression systems. Where there was sequence variation in the candidate antigens (3D7 vs a clinical isolate PfKE04), both variants were expressed. ELISA was used to assess antibody responses against these antigens, as well as against crude stage V gametocyte extract (GE) and AMA1 using archived plasma samples from individuals recruited to participate in malaria cohort studies. We analyzed antibody levels (estimated from optical density units using a standardized ELISA) and seroprevalence (defined as antibody levels greater than three standard deviations above the mean levels of a pool of malaria naïve sera). We described the dynamics of antibody responses to these antigens by identifying factors predictive of antibody levels using linear regression models.
Results
Of the 25 antigens selected, seven antigens were produced successfully as recombinant proteins, with one variant antigen, giving a total of eight proteins for evaluation. Antibodies to the candidate antigens were detectable in the study population (N = 216), with seroprevalence ranging from 37.0% (95% CI: 30.6%, 43.9%) for PSOP1 to 77.8% (95% CI: 71.6%, 83.1%) for G377 (3D7 variant). Responses to AMA1 and GE were more prevalent than those to the gametocyte proteins at 87.9% (95% CI: 82.8%, 91.9%) and 88.3% (95% CI: 83.1%, 92.4%), respectively. Additionally, both antibody levels and breadth of antibody responses were associated with age and concurrent parasitaemia.
Conclusion
Age and concurrent parasitaemia remain important determinants of naturally acquired immunity to gametocyte antigens. Furthermore, we identify novel candidates for transmission-blocking activity evaluation.
... Entre tales tratamientos se encuentran los esteroides, los cuales, sin embargo, mostraron aumento de efectos adversos y no hubo disminución de la mortalidad (25). Similares resultados se obtuvieron en estudios con inmunoglobulina intravenosa en población pediátrica (26), y también se han estudiado Cartagena Y., Monsalve C, Toro ME. terapias dirigidas al factor de necrosis tumoral (anti TNF) como los anticuerpos monoclonales, sin encontrar efectividad (27). Otros posibles tratamientos adyuvantes que han mostrado ser seguros, como el sulfato de curdlan, un glucano 1 → 3-β-d sulfatado (28), la pentoxifilina, un inhibidor de fosfodiesterasa, (29), aún no han mostrado ser efectivos en disminuir la mortalidad y las secuelas neurológicas. ...
La malaria sigue siendo un problema de salud pública que afecta especialmente a las regiones tropicales y los países en vía de desarrollo, y Latinoamérica es una región endémica para la enfermedad. A pesar de que se ha demostrado una disminución de los casos de malaria en general, los casos de malaria complicada se mantienen estables. Entre las complicaciones graves de esta infección parasitaria está la malaria cerebral, que si bien se considera infrecuente, está asociada con una mortalidad de hasta el 20 %, especialmente en niños, y además produce altas tasas de discapacidad: alrededor del 11 % de los niños y el 25 % de los adultos que la padecen. De ahí la relevancia de conocerla y hacer la detección temprana de esta complicación. En este escrito se presenta la definición de malaria cerebral y su mecanismos fisiopatológicos, desde la obstrucción microvascular, la tormenta de citoquinas, hasta la alteración endotelial. Se llama la atención sobre el cuadro de signos y síntomas, la importancia de mantener esta sospecha clínica y la necesidad de considerar los principales diagnósticos diferenciales; se menciona la utilidad de cada una de las ayudas diagnósticas y la limitación por su poca disponibilidad en muchas áreas geográficas. Se deja el mensaje a todo el equipo de salud de estar atentos a detectar oportunamente las complicaciones sistémicas. Se presentan las bases del tratamiento actual y hacia dónde va la investigación en vacunas. Esta revisión es también una invitación a reflexionar sobre el enfoque de esta patología y la necesaria inclusión de otros factores que considerar, como las condiciones culturales, socioeconómicas y de educación que inciden en el comportamiento de la enfermedad en las comunidades afectadas.
... Some authors promote the use of intravenous immunoglobulins (IV-Ig) in refractory cases as a second line therapy. Indeed, IV-Ig have been demonstrated to exert beneficial effects by inhibiting and reverting the cytoadherence of infected erythrocytes in vitro [29]. Stangel et al. were the first group to report the ability of polyclonal immunoglobulins in vitro to modulate nitric oxygen production and microglial function in vitro. ...
Background
Malaria still represents a major health threat, in terms of both morbidity and mortality. Complications of malaria present a diversified clinical spectrum, with neurological involvement leading to the most serious related-conditions. The authors recently encountered a case of a 60-year old Italian man presenting with confusion, language disturbances and Parkinson-like syndrome 3 weeks after complete remission from severe Plasmodium falciparum cerebral malaria. Chemical and microbiological analysis revealed aseptic meningitis, diffuse encephalitis and abnormal immune-activation. Re-infection and recrudescence of infection were excluded. Further analysis excluded paraneoplastic and autoimmune causes of encephalitis. A diagnosis of Post-Malaria Neurological Syndrome (PMNS) was finally formulated and successfully treated with high dose of steroids.
Methods
A systematic research of current literature related to PMNS was performed.
Results
151 cases of PMNS were included, the majority of which occurred after severe P. falciparum infections. Four main clinical pattern were identified: 37% of the cases presented as “classical” PMNS, 36% presented as delayed cerebellar ataxia (DCA), 18% resembled acute inflammatory demyelinating polyneuropathy (AIDP), and 8% presented as acute disseminated encephalomyelitis (ADEM)-like form. Differentiation between different forms was not always simple, as clinical and radiological findings frequently overlap. Overall, in almost all of the tested cases, cerebrospinal fluid was found pathological; EEG revealed nonspecific encephalopathy in 30% of classical PMNS and 67% ADEM; imaging tests were found abnormal in 92% of ADEM-like forms. Pathogenesis remains unclear. An autoimmune mechanism is the most corroborated pathogenic hypothesis. Overall, the majority of PMNS cases revert without specific treatment. In most severe forms, high dose steroids, intravenous immunoglobulins, and plasmapheresis have been shown to improve symptoms.
Conclusions
PMNS is a disabling complication of malaria. The overall incidence is not known, due to frequent misdiagnosis and under-reporting. Pathogenesis is not also fully understood, but rapid response to immune-modulating treatment along with similarities to auto-immune neurological disease, strongly support a dysregulated immunological genesis of this condition. The lack of randomized controlled studies regarding therapeutic approaches is a major unmet need in this setting. A systematic collection of all the PMNS cases would be desirable, in order to increase awareness of this rare condition and to prospectively investigate the most appropriate management.
... To mitigate the deleterious effects of HCM, various clinical trials involving erythropoietin (EPO) 30 , amodiaquine-artesunate 31 , dihydroartemisinin (DHA)-piperaquine 32 , curdlan sulfate 33 , pentoxifylline 34 and intravenous immunoglobulin 35 have been conducted with mixed results and various side effects. These failures may have been due to the lack of a suitable ex vivo model for pre-clinical screening of these drugs. ...
Human cerebral malaria (HCM), a severe encephalopathy associated with Plasmodium falciparum infection, has a 20–30% mortality rate and predominantly affects African children. The mechanisms mediating HCM-associated brain injury are difficult to study in human subjects, highlighting the urgent need for non-invasive ex vivo human models. HCM elevates the systemic levels of free heme, which damages the blood-brain barrier and neurons in distinct regions of the brain. We determined the effects of heme on induced pluripotent stem cells (iPSCs) and a three-dimensional cortical organoid system and assessed apoptosis and differentiation. We evaluated biomarkers associated with heme-induced brain injury, including a pro-inflammatory chemokine, CXCL-10, and its receptor, CXCR3, brain-derived neurotrophic factor (BDNF) and a receptor tyrosine-protein kinase, ERBB4, in the organoids. We then tested the neuroprotective effect of neuregulin-1 (NRG-1) against heme treatment in organoids. Neural stem and mature cells differentially expressed CXCL-10, CXCR3, BDNF and ERBB4 in the developing organoids and in response to heme-induced neuronal injury. The organoids underwent apoptosis and structural changes that were attenuated by NRG-1. Thus, cortical organoids can be used to model heme-induced cortical brain injury associated with HCM pathogenesis as well as for testing agents that reduce brain injury and neurological sequelae.
... These observations led to the generally accepted hypothesis that iRBC sequestration in the cerebrovasculature and the resulting sequelae, including mechanical obstruction, inflammation, impaired vasoregulation, and BBB dysregulation, causes this clinical syndrome (4). Clinical trials of various therapies attempting to target the downstream effects of this mechanism have been carried out (13), including trials of dexamethasone to decrease cerebral edema (14,15), intravenous immunoglobulin to reverse iRBC sequestration (16), TNF-specific monoclonal antibodies to decrease immune activation (17), pentoxifylline to decrease TNF production and improve RBC deformability (18,19), nitric oxide (NO) inhalation to modulate endothelial activation (20,21), heparin or aspirin to counter a prothrombotic state (22), osmotic agents to decrease cerebral edema and reduce intracranial pressure (ICP) (23)(24)(25), and therapies to expand intravascular volume to ameliorate acidosis (26,27). Unfortunately, none of these trials improved clinical outcomes (13,20,28). ...
Background:
Cerebral malaria (CM) accounts for nearly 400,000 deaths annually in African children. Current dogma suggests that CM results from infected RBC (iRBC) sequestration in the brain microvasculature and resulting sequelae. Therapies targeting these events have been unsuccessful; findings in experimental models suggest that CD8+ T cells drive disease pathogenesis. However, these data have largely been ignored because corroborating evidence in humans is lacking. This work fills a critical gap in our understanding of CM pathogenesis that is impeding development of therapeutics.
Methods:
Using multiplex immunohistochemistry, we characterized cerebrovascular immune cells in brain sections from 34 children who died from CM or other causes. Children were grouped by clinical diagnosis (CM+ or -), iRBC sequestration (Seqhi, lo, or 0) and HIV status (HIV+ or -).
Results:
We identified effector CD3+CD8+ T cells engaged on the cerebrovasculature in 69% of CM+ HIV- children. The number of intravascular CD3+CD8+ T cells was influenced by CM status (CM+ vs -, P = 0.004) and sequestration level (Seqhi > lo, P = 0.010). HIV co-infection significantly increased T cell numbers and shifted cells from an intravascular (P = 0.004) to perivascular (P < 0.0001) distribution.
Conclusion:
Within the studied cohort, CM is associated with cerebrovascular engagement of CD3+CD8+ T cells, which is exacerbated by HIV coinfection. Thus, CD3+CD8+ T cells are highly promising targets for CM adjunctive therapy, opening new avenues for the treatment of this deadly disease.
Funding:
This research was supported by the Intramural Research Program of the National Institutes of Health.
... Research on critically ill children has been ongoing at the Queen Elizabeth Central Hospital (QECH) in Blantyre, Malawi, since 1986, and has included observational studies (Grau et al., 1989;M. E. Molyneux, Taylor, Wirima, & Borgstein, 1989;Taylor, Borgstein, & Molyneux, 1993;Taylor, Molyneux, Wirima, Fletcher, & Morris, 1988), clinical trials (Lell et al., 2010;Taylor et al., 1992& Kremsner et al., 2012, autopsy-based analyses, and, most recently, observational studies incorporating magnetic resonance imaging (MRI). These studies on severe malaria have taken place in a pediatric research ward (PRW) that has three patient bays-one for admission and two for ongoing medical care (M. ...
Informed consent is an ethical requirement in clinical research. Obtaining informed consent is challenging in resource-constrained settings. We report results of a formative qualitative study that examined factors that facilitate and hinder informed consent for clinical research among critically ill children in Malawi. We argue that truly informed consent in a pediatric intensive care unit (PICU) is challenged by parental distress, time constraints when balancing care for critically ill patients with research-related tasks, and social hierarchies and community mistrust toward certain research procedures. We interviewed health care providers and parents of children attending a critical care unit to identify potential challenges and solicit strategies for addressing them. Providers and caregivers suggested practical solutions to enhance research participant understanding of clinical trial research, including the use of visual materials, community engagement strategies, and using patients as advocates in promoting understanding of research procedures.
... To ensure success, it will be important to determine if key PfEMP1-receptor interactions are common between different virulent PfEMP1 forms. While immune sera mediated desequestration in a P. falciparum squirrel monkey model of malaria [123], it was ineffective in pediatric CM patients [124]. More research is needed to determine what makes an effective desequestration therapy. ...
Despite the recent successes of artemisinin-based antimalarial drugs, many still die from severe malaria, and eradication efforts are hindered by the limited drugs currently available to target transmissible gametocyte parasites and liver-resident dormant Plasmodium vivax hypnozoites. Host-targeted therapy is a new direction for infectious disease drug development and aims to interfere with host molecules, pathways, or networks that are required for infection or that contribute to disease. Recent advances in our understanding of host pathways involved in parasite development and pathogenic mechanisms in severe malaria could facilitate the development of host-targeted interventions against Plasmodium infection and malaria disease. This review discusses new opportunities for host-targeted therapeutics for malaria and the potential to harness drug polypharmacology to simultaneously target multiple host pathways using a single drug intervention.
... Historically, adjunct therapies have been found to be of benefit to animals in models of severe malaria [2][3][4]. However, the effects have not been possible to reproduce in humans infected with P. falciparum likely because the hosts, the parasites and the pathology that bring about severe malaria are only partially alike in animals and man [5][6][7][8]. Indeed, no adjunct treatment with acceptable safety profile has been shown to be of benefit to humans with severe malaria [9]. ...
Severe malaria
Even with the best available treatment, the mortality from severe Plasmodium falciparum malaria remains high. Typical features at death are high parasite loads and obstructed micro- vasculature. Infected erythrocytes (IE) containing mature parasites bind to the host receptor heparan sulfate, which is also an important receptor for merozoite invasion. To block merozoite invasion has not previously been proposed as an adjunctive therapeutic approach but it may preclude the early expansion of an infection that else leads to exacerbated sequestration and death.
Sevuparin in phase I study
The drug sevuparin was developed from heparin because heparan sulfate and heparin are nearly identical, so the rationale was that sevuparin would act as a decoy receptor during malaria infection. A phase I study was performed in healthy male volunteers and sevuparin was found safe and well tolerated.
Sevuparin in phase I/II clinical study
A phase I/II clinical study was performed in which sevuparin was administered via short intravenous infusions to malaria patients with uncomplicated malaria who were also receiving atovaquone/proguanil treatment. This was a Phase I/II, randomized, open label, active control, parallel assignment study.
Sevuparin was safe and well tolerated in the malaria patients. The mean relative numbers of ring-stage IEs decreased after a single sevuparin infusion and mature parasite IEs appeared transiently in the circulation. The effects observed on numbers of merozoites and throphozoites in the circulation, were detected already one hour after the first sevuparin injection. Here we report the development of a candidate drug named sevuparin that both blocks merozoite invasion and transiently de-sequesters IE in humans with P. falciparum malaria.
Trial registration
ClinicalTrials.gov NCT01442168
... Adjunctive therapies, including the desequestration of pRBCs with IgG, neutralization of tumour necrosis factor (TNF) and treatment with nitric oxide, are beneficial in animal models of severe malaria 16,28,167 . However, these results have not been reproduced in humans that are infected with P. falciparum [168][169][170] , probably because the host-parasite interactions and pathogenic processes that produce severe malaria differ between species 28 . Still, some success has been seen in a phase II clinical trial that used sulfated, negatively charged curdlan sulfate in addition to conventional therapy (artesunate) in patients with severe malaria 171 . ...
Proliferation and differentiation inside erythrocytes are important steps in the life cycle of Plasmodium spp. To achieve these, the parasites export polypeptides to the surface of infected erythrocytes; for example, to import nutrients and to bind to other erythrocytes and the host microvasculature. Binding is mediated by the adhesive polypeptides Plasmodium falciparum-encoded repetitive interspersed families of polypeptides (RIFINs), subtelomeric variant open reading frame (STEVOR) and P. falciparum erythrocyte membrane protein 1 (PfEMP1), which are encoded by multigene families to ensure antigenic variation and evasion of host immunity. These variant surface antigens are suggested to mediate the sequestration of infected erythrocytes in the microvasculature and block the blood flow when binding is excessive. In this Review, we discuss the multigene families of surface variant polypeptides and highlight their roles in causing severe malaria.
... We also measured IgG to parasite schizont lysate (3D7) using a standard ELISA protocol . Eleven serial dilutions of a purified IgG preparation obtained from Malawian adults (Taylor et al., 1992) were included for every antigen tested to obtain a standard dilution curve that allowed the conversion of median fluorescence intensity (MFI) readings to arbitrary antibody concentrations. A pool of plasma obtained from Kilifi adults was included in a single well on each plate as a positive control to allow for standardisation of day-to-day and plate-to-plate variation. ...
Young infants are less susceptible to severe episodes of malaria but the targets and mechanisms of protection are not clear. Cord blood antibodies may play an important role in mediating protection but many studies have examined their association with the outcome of infection or non-severe malaria. Here, we investigated whether cord blood IgG to Plasmodium falciparum merozoite antigens and antibody-mediated effector functions were associated with reduced odds of developing severe malaria at different time points during the first year of life. We conducted a case-control study of well-defined severe falciparum malaria nested within a longitudinal birth cohort of Kenyan children. We measured cord blood total IgG levels against five recombinant merozoite antigens and antibody function in the growth inhibition activity (GIA) and neutrophil antibody-dependent respiratory burst (ADRB) assays. We also assessed the decay of maternal antibodies during the first 6 months of life. The mean antibody half-life range was 2.51 months (95% confidence interval (CI): 2.19 – 2.92) to 4.91 months (95% CI: 4.47 – 6.07). The rate of decline of maternal antibodies was inversely proportional to the starting concentration. The functional assay of ADRB activity predicted significantly reduced odds of developing severe malaria during the first 6 months of life (Odds ratio (OR) 0.07, 95% CI: 0.007 – 0.74, P =0.007). Identification of the targets of antibodies mediating ADRB activity could contribute to the development of malaria vaccines that protect against severe episodes of malaria in early infancy.
... Plasma immunoglobulin G (IgG) to the recombinant antigens was measured using a previously described multiplex bead-based assay [15]. Serially-diluted malariaimmune globulin (MIG) [23] was included in each plate as a standard positive control, allowing for the conversion of mean fluorescent intensities to relative antibody concentrations in arbitrary units (AUs) and correction of inter-plate variation. Negative controls, consisting of pooled plasma from adult P. falciparum unexposed donors residing in the United Kingdom, were included in each plate to allow for the determination of seropositivity cut-offs. ...
Epidemiological studies indicate that some children experience many more episodes of clinical malaria than their age mates in a given location. Whether this is as a result of the micro-heterogeneity of malaria transmission with some children effectively getting more exposure to infectious mosquitoes than others, or reflects a failure in the acquisition of immunity needs to be elucidated. Here, we investigated the determinants of increased susceptibility to clinical malaria by comparing the intensity of exposure to Plasmodium falciparum and the acquisition of immunity in children at the extreme ends of the over-dispersed distribution of the incidence of clinical malaria.
The study was nested within a larger cohort in an area where the intensity of malaria transmission was low. We identified children who over a five-year period experienced 5 to 16 clinical malaria episodes (children at the tail-end of the over-dispersed distribution, n = 35), remained malaria-free (n = 12) or had a single episode (n = 26). We quantified antibodies against seven Plasmodium falciparum merozoite antigens in plasma obtained at six cross-sectional surveys spanning these five years. We analyzed the antibody responses to identify temporal dynamics that associate with disease susceptibility.
Children experiencing multiple episodes of malaria were more likely to be parasite positive by microscopy at cross-sectional surveys (X (2) test for trend 14.72 P = 0.001) and had a significantly higher malaria exposure index, than those in the malaria-free or single episode groups (Kruskal-Wallis test P = 0.009). In contrast, the five-year temporal dynamics of anti-merozoite antibodies were similar in the three groups. Importantly in all groups, antibody levels were below the threshold concentrations previously observed to be correlated with protective immunity.
We conclude that in the context of a low malaria transmission setting, susceptibility to clinical malaria is not accounted for by anti-merozoite antibodies but appears to be a consequence of increased parasite exposure. We hypothesize that intensive exposure is a prerequisite for protective antibody concentrations, while little to modest exposure may manifest as multiple clinical infections with low levels of antibodies. These findings have implications for interventions that effectively lower malaria transmission intensity.
... The authors suggested that whilst there was evidence that antibody acts to retain and prolong TNF within the circulation (leading to fever reduction) this may inadvertently increase its harmful effects on the vascular endothelium and, in turn, aggravate neurologic complications. A trial investigating pooled intravenous immunoglobulin (IVIG) from blood bank adult donors and, thus, presumptively immune to malaria in Malawian children with cerebral malaria was stopped early after enrolling 31 children for futility (in accordance to a pre-planned stopping rule) [123]. For those receiving IVIG 10/16 (62.5%) died or developed neurological sequelae compared to 3/15 (20%) in the placebo arm. ...
Over 90% of the world’s severe and fatal Plasmodium falciparum malaria is estimated to affect young children in sub-Sahara Africa, where it remains a common cause of hospital admission and inpatient mortality. Few children will ever be managed on high dependency or intensive care units and, therefore, rely on simple supportive treatments and parenteral anti-malarials. There has been some progress on defining best practice for antimalarial treatment with the publication of the AQUAMAT trial in 2010, involving 5,425 children at 11 centres across 9 African countries, showing that in artesunate-treated children, the relative risk of death was 22.5% (95% confidence interval (CI) 8.1 to 36.9) lower than in those receiving quinine. Human trials of supportive therapies carried out on the basis of pathophysiology studies, have so far made little progress on reducing mortality; despite appearing to reduce morbidity endpoints, more often than not they have led to an excess of adverse outcomes. This review highlights the spectrum of complications in African children with severe malaria, the therapeutic challenges of managing these in resource-poor settings and examines in-depth the results from clinical trials with a view to identifying the treatment priorities and a future research agenda.
... Having now honed in on the group of retinopathy-positive CM patients at highest risk of dying (Figure 4), identifying adjunctive therapy targeting increased brain volume becomes a high priority. A number of clinical trials targeting various putative mechanisms (cerebral edema 3,42 , sequestration 43 , inflammation 44 , decreased red cell deformability 45,46 , coagulation abormalities 47 vasogenic edema 39,48 have been carried out, but none improved clinical outcomes. There are several possible explanations for the trial failures: the trials were underpowered (there were probably high rates of misclassification, especially because these studies were conducted prior to the appreciation of the malarial retinopathy criterion 31 ; the putative mechanism was not critical to the pathogenesis of fatal malaria; the mechanism was relevant, but the intervention was timed ineffectively; and/or the primary outcome was death, and only a very large study can detect a small but statistically significant decrease in case fatality 49 . ...
Several advances in our understanding of pediatric cerebral malaria (CM) have been made over the past 25 years. Accurate clinical diagnosis is enhanced by the identification of a characteristic retinopathy, visible by direct or indirect ophthalmoscopy, the retinal changes (retinal whitening, vessel color changes, white-centered hemorrhages) being consistently associated with intracerebral sequestration of parasites in autopsy studies. Autopsies have yielded information at tissue levels in fatal CM, but new insights into critical pathogenetic processes have emerged from neuroimaging studies, which, unlike autopsy-based studies, permit serial observations over time and allow comparisons between fatal cases and survivors. Brain swelling has emerged as the major risk factor for death, and, among survivors, brain volume diminishes spontaneously over 24-48 hours. Studies of life-threatening and fatal malaria are suggesting new approaches to identifying and caring for those at highest risk; potential adjuvants should be evaluated and implemented where they are most needed.
© 2015 New York Academy of Sciences.
... The ability of patient plasma to disrupt rosettes was assayed as described before [4]. As a control a purified IgG fraction of a Malawian hyper-immune plasma pool was used [51]. Malaria naïve Swedish plasma was analyzed as negative control. ...
Plasmodium falciparum is the most lethal of the human malaria parasites. The virulence is associated with the capacity of the infected red blood cell (iRBC) to sequester inside the deep microvasculature where it may cause obstruction of the blood-flow when binding is excessive. Rosetting, the adherence of the iRBC to uninfected erythrocytes, has been found associated with severe malaria and found to be mediated by the NTS-DBL1α-domain of Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1). Here we show that the reactivity of plasma of Cameroonian children with the surface of the FCR3S1.2-iRBC correlated with the capacity to disrupt rosettes and with the antibody reactivity with a recombinant PfEMP1 (NTS-DBL1α of IT4var60) expressed by parasite FCR3S1.2. The plasma-reactivity in a microarray, consisting of 96 overlapping 15-mer long peptides covering the NTS-DBL1α domain from IT4var60 sequence, was compared with their capacity to disrupt rosettes and we identified five peptides where the reactivity were correlated. Three of the peptides were localized in subdomain-1 and 2. The other two peptide-sequences were localized in the NTS-domain and in subdomain-3. Further, principal component analysis and orthogonal partial least square analysis generated a model that supported these findings. In conclusion, human antibody reactivity with short linear-peptides of NTS-DBL1α of PfEMP1 suggests subdomains 1 and 2 to hold anti-rosetting epitopes recognized by anti-rosetting antibodies. The data suggest rosetting to be mediated by the variable areas of PfEMP1 but also to involve structurally relatively conserved areas of the molecule that may induce biologically active antibodies.
... Pooled serum from 20 adult blood donors from the same village was used to affinity-purify antigen-specific antibodies. A reference Malaria Immune Globulin (MIG) reagent (Central Laboratory of the Swiss Red Cross Blood Transfusion Service, Berne Switzerland) [31] was used for validation experiments and as a positive control for the cohort assays. This preparation contains 50 mg/ml of immunoglobulins (98% IgG) purified from a pool of healthy Malawian adult plasma and was originally manufactured to test its potential use as an adjunct therapy to quinine in the treatment of cerebral malaria. ...
Background
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.
Methods
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.
Results
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.
Conclusions
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.
... In nature, protective antibodies largely recognize proteins in their native conformation; therefore, to examine whether the recombinant merozoite library proteins were correctly folded, their immunoreactivity against hyperimmune sera from adults living in malaria-endemic regions was tested. All 20 proteins from the library expressed at useable levels were arrayed on a streptavidin-coated, microtitre plate and their relative immunoreactivity to pooled sera from Malawian adults was compared to that from malaria-naïve individuals [52]. All but one (MSRP4) of the proteins were immunoreactive ( Figure 2). ...
Plasmodium falciparum is the aetiological agent for malaria, a deadly infectious disease for which no vaccine has yet been licensed. The proteins displayed on the merozoite cell surface have long been considered attractive vaccine targets because of their direct exposure to host antibodies; however, progress in understanding the functional role of these targets has been hindered by technical challenges associated with expressing these proteins in a functionally active recombinant form. To address this, a method that enables the systematic expression of functional extracellular Plasmodium proteins was previously developed, and used to create a library of 42 merozoite proteins.
To compile a more comprehensive library of recombinant proteins representing the repertoire of P. falciparum merozoite extracellular proteins for systematic vaccine and functional studies, genome-wide expression profiling was used to identify additional candidates. Candidate proteins were recombinantly produced and their integrity and expression levels were tested by Western blotting and ELISA.
Twenty-five additional genes that were upregulated during late schizogony, and predicted to encode secreted and cell surface proteins, were identified and expressed as soluble recombinant proteins. A band consistent with the entire ectodomain was observed by immunoblotting for the majority of the proteins and their expression levels were quantified. By using sera from malaria-exposed immune adults, the immunoreactivity of 20 recombinant proteins was assessed, and most of the merozoite ligands were found to carry heat-labile epitopes. To facilitate systematic comparative studies across the entire library, multiple Plasmodium proteins were simultaneously purified using a custom-made platform.
A library of recombinant P. falciparum secreted and cell surface proteins was expanded by 20 additional proteins, which were shown to express at usable levels and contain conformational epitopes. This resource of extracellular P. falciparum merozoite proteins, which now contains 62 full-length ectodomains, will be a valuable tool in elucidating the function of these proteins during the blood stages of infection, and facilitate the comparative assessment of blood stage vaccine candidates.
... Fluorescence from 100 beads/antigen was read and reported as mean fluorescence intensity (MFI) and background responses to GST and MBP tags were subtracted. Serially diluted malaria immunoglobulin [25] added to each plate as a standard positive control corrected for plate-to-plate variations and allowed for conversion of MFIs to relative antibody concentrations in arbitrary units. Sera from P. falciparum– naive donors were added to each plate to correct for background nonspecific antibody binding. ...
Background:
Elucidating the mechanisms of naturally acquired immunity to Plasmodium falciparum infections would be highly valuable for malaria vaccine development. Asymptomatic multiclonal infections have been shown to predict protection from clinical malaria in a transmission-dependent manner, but the mechanisms underlying this are unclear. We assessed the breadth of antibody responses to several vaccine candidate merozoite antigens in relation to the infecting parasite population and clinical immunity.
Methods:
In a cohort study in Tanzania, 320 children aged 1-16 years who were asymptomatic at baseline were included. We genotyped P. falciparum infections by targeting the msp2 gene using polymerase chain reaction and capillary electrophoresis and measured antibodies to 7 merozoite antigens using a multiplex assay. We assessed the correlation between the number of clones and the breadth of the antibody response, and examined their effects on the risk of malaria during 40 weeks of follow-up using age-adjusted multivariate regression models.
Results:
The antibody breadth was positively correlated with the number of clones (RR [risk ratio], 1.63; 95% confidence interval [CI], 1.32-2.02). Multiclonal infections were associated with a nonsignificant reduction in the risk of malaria in the absence of antibodies (RR, 0.83; 95% CI, .29-2.34). The breadth of the antibody response was significantly associated with a reduced risk of malaria in the absence of infections (RR, 0.25; 95% CI, .09-.66). In combination, these factors were associated with a lower risk of malaria than they were individually (RR, 0.14; 95% CI, .04-.48).
Conclusions:
These data suggest that malaria vaccines mimicking naturally acquired immunity should ideally induce antibody responses that can be boosted by natural infections.
Chronic, asymptomatic malaria infections contribute substantially to disease transmission and likely represent the most significant impediment preventing malaria elimination and eradication. Plasmodium falciparum parasites evade antibody recognition through transcriptional switching between members of the var gene family, which encodes the major virulence factor and surface antigen on infected red blood cells. This process can extend infections for up to a year; however, infections have been documented to last for over a decade, constituting an unseen reservoir of parasites that undermine eradication and control efforts. How parasites remain immunologically invisible for such lengthy periods is entirely unknown. Here we show that in addition to the accepted paradigm of mono-allelic var gene expression, individual parasites can simultaneously express multiple var genes or enter a state in which little or no var gene expression is detectable. This unappreciated flexibility provides parasites with greater adaptive capacity than previously understood and challenges the dogma of mutually exclusive var gene expression. It also provides an explanation for the antigenically invisible parasites observed in chronic asymptomatic infections.
Natural killer (NK) cells are potent immune effectors that can be activated via antibody-mediated Fc receptor engagement. Using multiparameter flow cytometry, we found that NK cells degranulate and release IFN-γ upon stimulation with antibody-opsonized Plasmodium falciparum merozoites. Antibody-dependent NK (Ab-NK) activity was largely strain transcending and enhanced invasion inhibition into erythrocytes. Ab-NK was associated with the successful control of parasitemia after experimental malaria challenge in African adults. In an independent cohort study in children, Ab-NK increased with age, was boosted by concurrent P. falciparum infections, and was associated with a lower risk of clinical episodes of malaria. Nine of the 14 vaccine candidates tested induced Ab-NK, including some less well-characterized antigens: P41, P113, MSP11, RHOPH3, and Pf_11363200. These data highlight an important role of Ab-NK activity in immunity against malaria and provide a potential mechanism for evaluating vaccine candidates.
Reorganization of host red blood cells by the malaria parasite Plasmodium falciparum enables their sequestration via attachment to the microvasculature. This artificially increases the dwelling time of the infected red blood cells within inner organs such as the brain, which can lead to cerebral malaria. Cerebral malaria is the deadliest complication patients infected with P. falciparum can experience and still remains a major public health concern despite effective antimalarial therapies. Here, the current understanding of the effect of P. falciparum cytoadherence and their secreted proteins on structural features of the human blood‐brain barrier and their involvement in the pathogenesis of cerebral malaria are highlighted. Advanced 2D and 3D in vitro models are further assessed to study this devastating interaction between parasite and host. A better understanding of the molecular mechanisms leading to neuronal and cognitive deficits in cerebral malaria will be pivotal in devising new strategies to treat and prevent blood‐brain barrier dysfunction and subsequent neurological damage in patients with cerebral malaria. Cerebral malaria is the deadliest complication patients infected with the protozoan parasite Plasmodium falciparum (P. falciparum) can experience. Herein the current understanding of the effect an infection with P. falciparum has on structural features of the human brain is detailed. Advanced 2D and 3D in vitro models are further assessed to study this devastating interaction between parasite and host.
Neurocritical care (NCC) is an emerging field within critical care medicine, reflecting the widespread prevalence of neurologic injury in critically ill patients. Morbidity and mortality from neurocritical illness (NCI) have been reduced substantially in resource-rich settings (RRS), owing to the development of advanced technologies, neuro-specific units, and subspecialized medical training. Despite shouldering much of the burden of NCI worldwide, resource-limited settings (RLS) face immense hurdles when implementing guidelines generated in RRS. This review summarizes the current epidemiology, management, and outcomes of the most common NCIs in RLS and offers commentary on future directions in NCC practiced in RLS.
Ring-infected erythrocytes are the predominant asexual stage in the peripheral circulation but are rarely investigated in the context of acquired immunity against Plasmodium falciparum malaria. Here we compare antibody-dependent phagocytosis of ring-infected parasite cultures in samples from a controlled human malaria infection (CHMI) study (NCT02739763). Protected volunteers did not develop clinical symptoms, maintained parasitaemia below a predefined threshold of 500 parasites/μl and were not treated until the end of the study. Antibody-dependent phagocytosis of both ring-infected and uninfected erythrocytes from parasite cultures was strongly correlated with protection. A surface proteomic analysis revealed the presence of merozoite proteins including erythrocyte binding antigen-175 and −140 on ring-infected and uninfected erythrocytes, providing an additional antibody-mediated protective mechanism for their activity beyond invasion-inhibition. Competition phagocytosis assays support the hypothesis that merozoite antigens are the key mediators of this functional activity. Targeting ring-stage parasites may contribute to the control of parasitaemia and prevention of clinical malaria.
Background: Studies of long-term malaria cohorts have provided essential insights into how Plasmodium falciparum interacts with humans, and influences the development of antimalarial immunity. Immunity to malaria is acquired gradually after multiple infections, some of which present with clinical symptoms. However, there is considerable variation in the number of clinical episodes experienced by children of the same age within the same cohort. Understanding this variation in clinical symptoms and how it relates to the development of naturally acquired immunity is crucial in identifying how and when some children stop experiencing further malaria episodes. Where variability in clinical episodes may result from different rates of acquisition of immunity, or from variable exposure to the parasite.
Methods: Using data from a longitudinal cohort of children residing in an area of moderate P. falciparum transmission in Kilifi district, Kenya, we fitted cumulative episode curves as monotonic-increasing splines, to 56 children under surveillance for malaria from the age of 5 to 15.
Results: There was large variability in the accumulation of numbers of clinical malaria episodes experienced by the children, despite being of similar age and living in the same general location. One group of children from a particular sub-region of the cohort stopped accumulating clinical malaria episodes earlier than other children in the study. Despite lack of further clinical episodes of malaria, these children had higher asymptomatic parasite densities and higher antibody titres to a panel of P. falciparum blood-stage antigens.
Conclusions: This suggests development of clinical immunity rather than lack of exposure to the parasite, and supports the view that this immunity to malaria disease is maintained by a greater exposure to P. falciparum , and thus higher parasite burdens. Our study illustrates the complexity of anti-malaria immunity and underscores the need for analyses which can sufficiently reflect the heterogeneity within endemic populations.
Natural killer cells are potent immune effectors that can be activated via antibody mediated Fc receptor engagement. Using multiparameter flow cytometry, we found that natural killer (NK) cells degranulate and release IFNγ upon stimulation with antibody-opsonized Plasmodium falciparum merozoites. Antibody-dependent NK activity (Ab-NK) was largely strain-transcending and enhanced the inhibition of invasion into erythrocytes. Ab-NK was associated with the successful control of parasitemia following experimental malaria challenge in African adults. In an independent cohort study in children, Ab-NK increased with age, was boosted by concurrent falciparum infections and associated with a lower risk of clinical episodes of malaria. Nine of 14 vaccine candidates tested induced Ab-NK including some less well-studied antigens - P41, P113, MSP11, RHOPH3, and Pf _11363200. These data highlight an important role for ab-NK in immunity against malaria and provide a new mechanism for the evaluation of vaccine candidates
One Sentence Summary
Antibody-dependent natural killer activation is induced by merozoites and associated with immunity against malaria
Background: Studies of long-term malaria cohorts have provided essential insights into how Plasmodium falciparum interacts with humans, and influences the development of antimalarial immunity. Immunity to malaria is acquired gradually after multiple infections, some of which present with clinical symptoms. However, there is considerable variation in the number of clinical episodes experienced by children of the same age within the same cohort. Understanding this variation in clinical symptoms and how it relates to the development of naturally acquired immunity is crucial in identifying how and when some children stop experiencing further malaria episodes. Where variability in clinical episodes may result from different rates of acquisition of immunity, or from variable exposure to the parasite.
Methods: Using data from a longitudinal cohort of children residing in an area of moderate P. falciparum transmission in Kilifi district, Kenya, we fitted cumulative episode curves as monotonic-increasing splines, to 56 children under surveillance for malaria from the age of 5 to 15.
Results: There was large variability in the accumulation of numbers of clinical malaria episodes experienced by the children, despite being of similar age and living in the same general location. One group of children from a particular sub-region of the cohort stopped accumulating clinical malaria episodes earlier than other children in the study. Despite lack of further clinical episodes of malaria, these children had higher asymptomatic parasite densities and higher antibody titres to a panel of P. falciparum blood-stage antigens.
Conclusions: This suggests development of clinical immunity rather than lack of exposure to the parasite, and supports the view that this immunity to malaria disease is maintained by a greater exposure to P. falciparum , and thus higher parasite burdens. Our study illustrates the complexity of anti-malaria immunity and underscores the need for analyses which can sufficiently reflect the heterogeneity within endemic populations.
Cerebral malaria (CM) is a major life-threatening disease caused by Plasmodium falciparum infection in humans. The complex pathogenic mechanisms underlying the fatal neurological complications of the disease are still not completely elucidated. The autopsy studies in fatal cases of human CM and advances in knowledge from various animal models have offered insight into the precise mechanism of the disease. The parasite sequestration in the brain microvascular endothelial cells and dysregulated host immune system together determine the pathophysiology of CM. Despite optimal treatment with antimalarials, 25% of the patients suffer from post-treatment neurological and cognitive deficits. In this review, we have discussed the components of the pathogenic mechanisms of CM and the current scenario of treatment.
Introduction: Pooled human immunoglobulins (IGs) are prepared from plasma obtained from healthy donors as a concentrated antibody-containing solution. In addition, high-titer IGs (hyperimmune) against a specific pathogen can be obtained from vaccinated or convalescing donors. Currently, IGs can be used for the treatment of a variety of infections for which no specific therapy exists or that remain difficult to treat. Moreover, the recent pathogen outbreaks for which there is no approved treatment have renewed attention to the role of convalescent plasma and IGs.
Areas covered: In this review, a historical perspective of the use of sera and IGs in humans as anti-infective agents (any viral, bacterial, parasitic infection), excluding immunodeficient patients, is presented from early development to the latest clinical studies. A Medline search was conducted to examine the peer-reviewed literature, with no date limits.
Expert commentary: Human pooled plasma-derived IG products benefit from the polyclonal response of every individual donor and from the interindividual variability in such response. The trend to increased availability of vaccines for infectious diseases also opens new potential applications of hyperimmune IGs for emerging or re-emerging infectious diseases (e.g.: Ebola, Zika, Dengue), for the prevention and treatment in the general population, healthcare personnel and caregivers.
In most fields of medicine, experimentation starts with studies in vitro, moves to animal models and eventually proceeds to research on humans. Malaria provides a good example of the limits of this progression. The most important malarial parasite of man, Plasmodium falciparum, only infects man. The specificity of this relationship accounts for the many differences which exist between artificial models of falciparum malaria and natural infections. Ultimately, human infections appear to be the sole, relevant ‘model’ for the study of human-Plasmodium interactions. Immunoglobulin-transfer experiments, for example, clearly indicated that antibodies mediated the state of acquired immunity called premunition. However, further studies in vitro or in animal models led to conflicting results about how the antibodies acted. Transfer experiments in human volunteers, appropriately coupled to in-vitro studies, seemed the only way to help solve this issue. The design of these investigations, with its technical and ethical aspects, is reviewed here, along with the main published and unpublished results. The identification of a monocyte-mediated, antibody-dependent (ADCI) mechanism led to a new merozoite-surface antigen (MSP-3) being identified and provided an explanation for the long delay in the acquisition of protection. It appears that experiments in humans not only help to confirm indications obtained using animal models, but can also have a truly exploratory role, since they can both raise completely new issues and provide answers to them.
Severe malaria (SM) is a life-threatening complication of infection with Plasmodium 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 0-2 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 two years of life. We quantified immunological parameters in serum collected before the SM event in cases and their individually matched controls to evaluate the prospective odds of developing SM in the first two years of life.Anti-AMA1 antibodies were associated with a significant reduction in the odds of developing SM (OR 0.37, 95% CI 0.15 - 0.90, P=0.029) after adjustment for responses to all other merozoite antigens tested, whilst those against MSP-2, MSP-3, PfRh2, MSP-119 and the infected red blood cell surface 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 - 0.82, P=0.03). Assays of these two functional mechanisms were poorly correlated (rs=0.12, P=0.07). Our data provides 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.
Plasmodium falciparum and other human malaria parasites can cause a variety of life-threatening syndromes. Diagnosis is difficult because none of these syndromes is unique to malaria, and parasitemia may be incidental rather than responsible for the illness. Malaria must be considered in the differential diagnosis of each of the severe syndromes it can cause - severe anemia, coma and convulsions, acidosis, hypoglycemia, shock, acute renal failure, intravascular hemolysis, acute respiratory distress syndrome and disseminated intravascular coagulation. The possibility of an alternative or additional infection must be considered and, in many circumstances, covered by initial emergency treatment. In the comatose patient the presence of a distinctive retinopathy is suggestive that malaria is the cause of the disease. Management must begin with emergency measures to ensure vital functions, followed by prompt provision of supportive measures (airway protection, glucose, fluids, oxygen, blood, antipyresis and anticonvulsants), and supervised referral to an appropriate available facility offering optimal clinical management. In a remote site, specific antimalarial therapy may be started with artesunate by suppository to cover the journey to hospital, where treatment can continue with intravenous or intramuscular quinine or with intravenous artesunate, followed by full artemisinin combination therapy as soon as oral treatment is possible. Identifying and managing severe disease events remains crucially important as malaria control measures successfully reduce the burden of the infection, with the possibility of waning of both immunity of hosts to the infection and familiarity of clinicians with the disease.
Severe malaria is a medical emergency requiring early intervention to prevent death. This article highlights key aspects of the management of severe malaria syndromes in the intensive care unit, with a focus on individual case management of imported malaria. Key differences in the presentation, management, and outcomes of severe malaria by endemicity and by age group are emphasized. In all groups with severe malaria, intravenous artesunate is the antimalarial agent of choice. This article discusses specific antimalarial therapies, optimal supportive management strategies, differences from strategies for bacterial sepsis, and trials of adjunctive therapy for severe malaria in humans.
A vaccine against Plasmodium falciparum is needed to augment currently available malaria control tools. A handful of parasite antigens are at various stages of pre-clinical and clinical development. Amongst these is P. falciparum apical membrane antigen 1 (PfAMA1), an antigen expressed by asexual stage parasites and believed to be important for the parasite’s invasion of both red blood cells and liver cells. The immune response to PfAMA1 is mediated mainly by antibodies that prevent parasite invasion of host cells. PfAMA1 however shows allelic polymorphism, with anti-PfAMA1 antibody responses exhibiting strain-specificity.
This thesis investigated multi-allele vaccine formulation strategies that would overcome the strain-specificity of antibody responses to PfAMA1. The main findings of this thesis are that i) different PfAMA1 alleles share epitopes to which functional cross-strain antibodies can be induced, ii) a three-allele PfAMA1 formulation yields the greatest proportion of functional cross-strain antibodies, and iii) three PfAMA1 alleles, irrespective of the adjuvant used for formulation and whether they are administrated as a multi-allele formulation or sequentially, induce similar proportions of cross-strain antibodies. Overall, a multi-allele formulation with three in silico-designed PfAMA1 candidates yields antibodies that inhibit several parasites in vitro and warrant their development as a human blood stage vaccine.
Forty-four children were studied to compare the pathogenesis of fever in cerebral malaria, uncomplicated malaria and measles at the Eldoret District Hospital (EDH). A control group of normal children was used. The three patient groups were studied for three consecutive days measuring skin and core temperature three-times a day using the Liquid Crystal Device (LCD) thermometer. A statistical analysis of the results within and between the groups was carried out for core and skin temperature over the study period. No statistical differences were found between the groups for either the skin or the core temperature, but a significant statistical difference was demonstrated between the core and the skin temperature for all of the groups for each of the three days. No statistical difference was found when the differences between the core and skin temperature were compared between cerebral malaria and uncomplicated malaria. The possible roles of fever in morbidity and mortality are discussed, with special reference to cerebral malaria.
Ten variant populations derived from the Indochina-1 strain of Plasmodium falciparum were analyzed by using (i) hyperimmune serum raised against some of these populations in squirrel monkeys and (ii) an oligonucleotide probe based on the rep-20 sequence, which had previously been shown to be a useful marker of diversity. Although all 10 subpopulations had an identical fingerprint pattern on Southern blots probed with the oligonucleotide, thus demonstrating a homogeneous genetic makeup, they all had a different phenotype for erythrocyte-associated antigens, thus confirming serological variant-specific differences. Antibodies to erythrocyte-associated antigens were measured with a new technique including immunogold and silver enhancement. The results of this study indicate that antigenic variation can occur without major genomic reorganization.
Sequestration, the adherence of infected erythrocytes containing late developmental stages of the parasite (trophozoites and schizonts) to the endothelium of capillaries and venules, is characteristic of Plasmodium falciparum infections. We have studied two host factors, the spleen and antibody, that influence sequestration of P. falciparum in the squirrel monkey. Sequestration of trophozoite/schizont-infected erythrocytes that occurs in intact animals is reduced in splenectomized animals; in vitro, when infected blood is incubated with monolayers of human melanoma cells, trophozoite/schizont-infected erythrocytes from intact animals but not from splenectomized animals bind to the melanoma cells. The switch in cytoadherence characteristics of the infected erythrocytes from nonbinding to binding occurs with a cloned parasite. Immune serum can inhibit and reverse in vitro binding to melanoma cells of infected erythrocytes from intact animals. Similarly, antibody can reverse in vivo sequestration as shown by the appearance of trophozoite/schizont-infected erythrocytes in the peripheral blood of an intact animal after inoculation with immune serum. These results indicate that the spleen modulates the expression of parasite alterations of the infected erythrocyte membrane responsible for sequestration and suggest that the prevention and reversal of sequestration could be one of the effector mechanisms involved in antibody-mediated protection against P. falciparum malaria.
We studied the inactivation of the etiologic agent of the acquired immunodeficiency syndrome, human immunodeficiency virus, in the course of the Kistler and Nitschmann cold ethanol fractionation of human blood plasma. By measuring reverse transcriptase activity and viral infectivity, we have shown that the virus load is reduced by a factor of 10^4 during the initial and at least a factor of 10^6 during the subsequent steps of the fractionation procedure. This loss of virus may be observed in the absence or in the presence of antibodies against human immunodeficiency virus and is due to a combination of chemical inactivation, physical partition, and injury caused by repeated freezing and thawing. The laboratory data therefore further confirm epidemiological studies which indicate that immunoglobulin preparations obtained by ethanol fractionation do not transmit human immunodeficiency virus.
Antigenic variation and sequestration in organ capillary systems are two of the ways by which malaria parasites escape immune effector mechanisms. Here Marcel Hommel discusses the presence of variant antigens and endothelial-binding molecules on the surface of infected erythrocytes in the context of malaria immunity.
The effect of pepsin treatment at pH 4 on the infectivity of several enveloped viruses was assessed under the conditions used during the production of intravenous immunoglobulins. It was shown that the prototypes of four virus families–human immunodeficiency virus (Lentivirinae), herpes simplex virus type 1 and human cytomegalovirus (Herpesviridae), Semliki Forest virus (Togaviridae), and vesicular stomatitis virus (Rhabdoviridae)–were inactivated by this procedure. With vesicular stomatitis virus as a model, the contributions of both low pH and pepsin were demonstrated, and pepsin had a synergistic or additive action.
• 1.1) Gamma globulin from adult West Africans which is known to have a consistent anti-malarial action in West African children was administered to East African subjects with severe P. falciparum infections. Nine children aged 6–20 months with an initial parasitaemia of 10,900–375,000 per c.m., each received 1.4 to 2.2 grammes γ-globulin.
• 2.2) In all cases γ-globulin therapy was followed by an abrupt reduction in parasite density and recovery from clinical illness similar to that observed in treated West African children. By the fourth day the average parasite count was less than 1 per cent. of the mean initial count; in five out of nine cases, parasite levels remained within the range of the West African children during the remaining 5 days of observation. These results indicate that West African γ-globulin contains antibody active against East African malaria, so that P. falciparum parasites of East and West Africa must be antigenically similar.
• 3.3) Three East African children showed an undoubted recrudescence of parasitaemia after the fourth day, so that the occurrence of a serologically distinct strain of P. falciparum resistant to West African immune γ-globulin cannot at present be excluded.
Plasmodium falciparum-parasitized erythrocytes obtained from infected owl monkeys (Aotus trivirgatus) and stored in the frozen state retain both the capacity to incorporate 14C isoleucine into protein and to infect animals. The cryopreservation method involves the use of glycerol and the reconstitution of isotonicity after thawing. Animals have been infected from material held for up to 273 days and protein synthesis has been demonstrated in vitro for up to 180 days after freezing. The specificity of protein synthesis as an activity of the parasites was shown by the inactivity of control uninfected erythrocytes stored by the same method. Additional evidence for the specificity of the reaction was obtained from inhibition studies with chloroquine; a 7 X 10(-5) M concentration of the drug resulted in 50% inhibition of the initial rate of protein synthesis.
We observed considerable diversity in the cytoadherence of Plasmodium falciparum isolates from Malawi to melanoma cells, U937 cells and human peripheral monocytes. Each isolate exhibited a unique cytoadherence profile for the three human cell types. These isolates generally adhered well to U937 cells and fresh monocytes, moderately to melanoma cells and poorly to TE 671, MIA-Pa-Ca, WI 38, PLC/PRF/5 and HeLa cells. An antimalarial immunoglobulin pool inhibited binding to melanoma cells by 50% or more and to U937 cells by 25% or less. There was no correlation between in vitro cytoadherence to the three cells and clinical disease. These results suggest that malarial adherence ligands exposed on the surface of infected erythrocytes vary from one isolate to another.
The effect of pepsin treatment at pH 4 on the infectivity of several enveloped viruses was assessed under the conditions used during the production of intravenous immunoglobulins. It was shown that the prototypes of four virus families--human immunodeficiency virus (Lentivirinae), herpes simplex virus type 1 and human cytomegalovirus (Herpesviridae), Semliki Forest virus (Togaviridae), and vesicular stomatitis virus (Rhabdoviridae)--were inactivated by this procedure. With vesicular stomatitis virus as a model, the contributions of both low pH and pepsin were demonstrated, and pepsin had a synergistic or additive action.
The protective effect of African IgG antibodies against Plasmodium falciparum malaria was investigated by passive transfer in Thai patients. Sera from 333 African adults were collected in the Cote d'Ivoire and subjected to extensive screening. One hundred fifty-three samples were discarded for safety reasons, and IgG was extracted from those remaining under conditions allowing their use by the intravenous (iv) route. Eight Thai patients with P. falciparum parasitemia were treated by iv inoculation of the IgG: six with a 100 mg/kg dose given over three days, one with a single 20 mg/kg dose, and one with a single 200 mg/kg dose. To ensure a safety margin of at least 48 hours, subjects were chosen among patients having a recrudescent parasitemia following quinine treatment failure at the RI level. At that stage, symptoms were mild or absent and parasitemia was low but increasing (range 4, 200-9,000/microliters). The IgG pool exerted a profound, stage-specific, but non-sterilizing effect on each of the strains tested, and proved to be safe. Asexual parasitemia decreased by a mean 728-fold (range 46-1,086), while gametocytes were unaffected. Clearance of parasites and symptoms was as fast or faster than with drugs, and was consistent in the eight patients treated, suggesting that target antigens were equally expressed in geographically remote isolates. In peripheral blood smears, no mature forms were seen at any time during the followup, which does not support the hypothesis that reversal of cytoadherence occurred. After the disappearance of the transferred antibodies, recrudescent parasites from three patients were found to be susceptible to the same extent (mean decrease of 1,310-fold) to the same IgG preparation, indicating that selection of parasites able to escape the effect of antibodies had not occurred. No adverse side-effects were detected during the followup, which lasted one year.
We studied the inactivation of the etiologic agent of the acquired immunodeficiency syndrome, human immunodeficiency virus, in the course of the Kistler and Nitschmann cold ethanol fractionation of human blood plasma. By measuring reverse transcriptase activity and viral infectivity, we have shown that the virus load is reduced by a factor of 10(4) during the initial and at least a factor of 10(6) during the subsequent steps of the fractionation procedure. This loss of virus may be observed in the absence or in the presence of antibodies against human immunodeficiency virus and is due to a combination of chemical inactivation, physical partition, and injury caused by repeated freezing and thawing. The laboratory data therefore further confirm epidemiological studies which indicate that immunoglobulin preparations obtained by ethanol fractionation do not transmit human immunodeficiency virus.
Red blood cells infected with mature stages of the malaria parasite Plasmodium falciparum bind to the endothelial lining of capillaries and venules. This sequestration is important for the survival of the parasite but may have severe consequences for the host. For example, it is involved in the causation of cerebral malaria which carries 25% mortality. Knob-like protrusions present on the surface of infected erythrocytes have been considered necessary but not sufficient for this cytoadherence. Here we describe the adhesion to endothelial cells of infected erythrocytes which do not have knobs. A human monoclonal antibody (33G2) which was specific for an epitope containing regularly spaced dimers of glutamic acid present in the repeated amino-acid sequences of some defined P. falciparum antigens was found to inhibit cyto-adherence and may therefore be an important reagent for elucidating the molecular basis of parasite sequestration.
We studied the relationship between presenting features and outcome in 131 Malawian children admitted with cerebral malaria (P. falciparum malaria and unrousable coma). A method was devised for the measurement of depth of coma in children too young to speak. Twenty patients (15 per cent) died and 12 (9 per cent) recovered with residual neurological sequelae. Presenting clinical signs significantly associated with adverse outcome (death or sequelae) were profound coma, signs of decerebration, absence of corneal reflexes, convulsions at the time of admission and age under three years. Laboratory findings of prognostic significance were hypoglycaemia, leucocytosis, hyperparasitaemia, elevated plasma concentrations of alanine and 5'-nucleotidase, and elevated plasma or cerebrospinal fluid lactate. A prognostic index based on eight of these risk factors that can readily be ascertained at the bedside or in a ward sideroom, was more accurately predictive of outcome than any single feature. Such an index may be valuable as a measure of severity of illness for establishing the comparability of study groups, and for evaluating the role of other factors in the pathogenesis of cerebral malaria.
The currently used methods for assessing the therapeutic response to antimalarial drugs are relatively imprecise and insensitive.
These methods are inadequate in severe malaria when the objectives of treatment are to save life and prevent complications.
Very large studies are needed to demonstrate significant differences in mortality, but measurement of the rates of clinical,
biochemical, and parasitological response may provide useful comparative information. Definitions, assessment criteria, procedures,
and data collection forms should be standardized and evaluated prospectively. Antimalarial drug treatment in different clinical
situations should be assessed in terms of the balance between the risks of drug toxicity and the benefits of the antimalarial
drug action. This balance is considerably different in severe falciparum malaria compared with uncomplicated malaria infections.
Hypoglycaemia, defined as a plasma glucose concentration below 2.2 mmol/l, developed in 15 of 47 prospectively studied Gambian children with severe chloroquine-sensitive falciparum malaria. 5 of these hypoglycaemic children died compared with 1 in the normoglycaemic group (p = 0.02). In contrast to previous observations in quinine-treated adults, in whom hypoglycaemia was associated with hyperinsulinaemia, plasma concentrations of insulin were appropriately low and plasma ketones were high. Raised plasma concentrations of lactate and alanine suggested impairment of hepatic gluconeogenesis. In African children, hypoglycaemia is an important and treatable manifestation of severe malaria and is unrelated to antimalarial treatment.
Because of concern about the safety of immune globulins prepared for injection, we studied the effects of ethanol fractionation of human plasma on human lymphotropic virus, type III, (HTLV-III) by spiking the products of various fractionation steps with HTLV-III. Tests of inactivation and removal indicated that the ratio of residual live virus in plasma fractions/live virus in starting plasma was about 1 X 10(-15) for precipitate II from which immune globulin for injection is manufactured. The results are reassuring regarding the potential safety of immune globulin.
Hypoglycemia may develop in patients with severe untreated malaria and can complicate the course of treatment with parenteral quinine as a result of quinine-induced hyperinsulinemia. Intravenous quinine is used increasingly as the therapy of choice in patients with severe malaria, most of whom are children. To assess the importance of both pretreatment and quinine-related hypoglycemia in children in an area in which the disease is endemic, we prospectively studied 95 Malawian children with falciparum malaria and altered consciousness who were treated with intravenous quinine. Nineteen patients had hypoglycemia before treatment. Seven (37 percent) died, and five of the survivors (26 percent) had neurologic sequelae. The corresponding values for patients who were initially normoglycemic were 4 percent and 4 percent, respectively (P less than 0.0001). Hypoglycemia was associated with low plasma insulin concentrations and with elevated plasma concentrations of lactate, alanine, and 5'-nucleotidase--a finding that suggests that impaired hepatic gluconeogenesis but not hyperinsulinemia contributes to the pathogenesis of pretreatment hypoglycemia. All patients were given quinine dihydrochloride in a 5 percent dextrose infusion, and those with hypoglycemia received 50 percent dextrose. Hypoglycemia recurred in seven of the patients with pretreatment hypoglycemia, but these episodes were also not associated with hyperinsulinemia. Of the 76 children who were initially normoglycemic, none became hypoglycemic during the course of treatment with intravenous quinine. We conclude that hypoglycemia is a frequent complication of falciparum malaria in children and that it reflects severe disease and is associated with a poor prognosis. We did not find it to be a complication of quinine treatment.
An indirect fluorescent antibody test for glutaraldehyde-fixed, ring-infected erythrocyte surface antigen was performed on
admission sera from 45 patients with complicated cerebral Plasmodium falciparum malaria, 33 with uncomplicated cerebral malaria, 91 non-cerebral malaria patients, and 53 blood donors from a non-malarious
area. 14 (31%), 28 (85%), 64 (70%), and 1 (2%), respectively, had titres ⩾1/25, considered as positive. The seropositive rate
and the geometric mean reciprocal titre of patients with complicated cerebral malaria were significantly lower than those
of uncomplicated and non-cerebral patients, particularly in the 6–14 and 15–29 year age groups. Compared with non-cerebral
patients, lower seropositive rates for patients with complicated cerebral malaria were demonstrated only in those who had
been ill 4 or more days before admission; whereas lower rates for patients with complications, when compared with rates in
those with uncomplicated cerebral malaria, occurred irrespective of the duration of illness.
The effect of sera on the cytoadherence in vitro of Plasmodium falciparum-infected erythrocytes to melanoma cells was examined. Sera from 19 healthy individuals living in endemic malarious areas in Thailand and 24 patients with P. falciparum malaria were tested against four local P. falciparum isolates. Out of 57 sera examined, 12 (21%) showed significant inhibition (greater than 50%) of cytoadherence for at least one isolate. Anti-malarial IgG antibody titres were determined for all 57 sera and although 11 of the 12 inhibitory sera had relatively high titres, 36 out of 47 sera with similarly high titres showed no significant inhibitory activity. Convalescent sera were no more effective than corresponding acute sera in inhibiting the cytoadherence of erythrocytes infected with any of the four heterologous isolates examined. Sera which significantly inhibited cytoadherence were also capable of reversing cytoadherence, and pooled plasma, from healthy individuals living in malarious areas, was effective in significantly reversing the in vitro cytoadherence of all the five parasite isolates examined. The results confirm the antibody mediated strain-specific nature of the inhibition of cytoadherence and stress the difficulty in selecting immune sera potentially useful for the immunotherapy of cerebral malaria patients in Thailand.
It has been previously shown that endothelial cells (EC) can modulate T-cell responsiveness by mimicking monocyte (AC) function in several different in vitro systems. We now report that EC and AC differ quantitatively in their ability to provide help for IL-2 generation and T-cell induced B-cell differentiation into immunoglobulin secreting cells (ISC). Equal numbers of EC were deficient when compared to AC for promoting ISC generation, but exceeded AC for IL-2 production. Adding optimal numbers of EC drive non-adherent cell cultures to produce more than twice as much IL-2 as adding any number of AC. Furthermore, small numbers of EC were capable of modulating ongoing immune responses when added to cultures containing AC. IL-2 production by PBM was doubled by the addition of enough EC to comprise only 3% of the total culture. EC do not just mimic monocytes in immune responses, but modulate these responses in unique ways.
For investigation of the pathogenesis of cerebral malaria, immediate postmortem samples from brain and other tissues of patients dying with Plasmodium falciparum malaria, with (CM) or without (NCM) cerebral malaria, were processed for electron microscopy. Counts of parasitized erythrocytes (PRBCs) in cerebral and other vessels showed that the proportion of PRBCs was higher in CM than in NCM, and also that the proportion of PRBCs was higher in the brain than in other organs examined in both CM and NCM. Cerebral vessels from CM patients were more tightly packed with RBCs than those from NCM patients, but there was no significant difference in the amount or degree of endothelial damage or numbers of vessels with endothelial pseudopodia. Fibrillar (fibrin) deposits were present in a small proportion of vessels, but no thrombosis was present. There was neither acute nor chronic inflammation, and leukocytes were absent within or outside cerebral vessels. There was no immune complex deposition in cerebral vessels. Parasites in cerebral vessels were mainly trophozoites or schizonts. Occasional RBC remnants following parasite release were seen. Some parasites were degenerate, resembling crisis forms. PRBCs adhered to endothelium via surface knobs. It is concluded that there is no evidence for an inflammatory or immune pathogenesis for human cerebral malaria and that the clinical effects probably relate to anoxia and the metabolic activities of the parasites.
Low complement C3 levels were found in the sera of most children with acute Plasmodium falciparum malaria. C4 and Clq levels were very low whilst glycine rich β glycoprotein (GBG) levels were only slightly depressed, suggesting that complement activation was occurring via the classical pathway. It is suggested that complement activation may play a part in initiating the complex series of changes that leads to the vascular damage seen in this infection.
An important feature of Plasmodium falciparum malaria which differentiates it from other human malarias is that erythrocytes infected with trophozoites and schizonts are not present in the peripheral blood but are sequestered along capillary and venular endothelium. Infected erythrocytes attach via parasite-induced ultrastructural modifications on the surface of the infected cells, called 'knobs'. This sequestration may be important for parasite survival because it prevents infected erythrocytes from circulating through the spleen where they could be eliminated. We have established an in vitro correlate of sequestration and used it to demonstrate that immune sera from repeatedly infected Aotus monkeys inhibit binding of infected erythrocytes to endothelial cells. We have investigated whether antiserum that blocks binding of one isolate of P. falciparum to target cells can block or reverse binding of other isolates. We report here that sera which block or reverse binding are strain-specific, indicating that the corresponding antigens on the surface of the infected erythrocytes are strain (isolate)-specific.
The pathophysiology of malaria infection is presented from animal studies and the various manifestations occurring in human cases. Maegraith (1974) proposed the concept of a chain reaction of physiological processes that leads to the disease following malarial infection. It may be seen that the malaria parasites first damage the infected red blood cells directly and then initiate a chain reaction of nonspecific inflammatory processes and later on immunological responses aggravating further the inflammatory reactions. Because of ther interdependence in nature of these changes as suggested by Maegraith in 1977 it is usually difficult to clearly identify these three mechanisms.
We measured the acid-base status of children with falciparum malaria in order to determine the prognostic significance and rate of resolution of acidaemia in patients with severe disease. We prospectively studied 141 Malawian children who were admitted to Hospital, with falciparum malaria, 60 of whom had cerebral malaria (unrousable coma, unable to localize a painful stimulus). Of the 60 patients with cerebral malaria 25 (42%) were acidaemic (capillary blood pH < 7.3); of 81 children with uncomplicated malaria 4 (5%) were acidaemic (p < 0.0001). Eleven patients died; of these, eight presented with cerebral malaria, eight with acidaemia and seven with both. The strong association of altered acid-base status with disease severity and mortality was independent of other previously identified predictors of illness and death in malaria. Acidaemia was not associated with shock, bacteraemia or hypoxaemia. Acidaemic patients had a slower mean respiratory rate and a higher incidence of respiratory rhythm abnormalities than other patients, suggesting that acidaemia is in part the result of inadequate respiratory compensation for metabolic acidosis. Although acidaemia is quickly corrected by fluids and antimalarial drugs, specific therapy to correct acidaemia needs evaluation in children with severe malaria.
Chemical and physical inactivation of human T lymphotropic virus, Type III (HTLV-III)
- Ma Wells
- A Wittek
- C Marcus-Sekura
13 Wells MA, Wittek A, Marcus-Sekura C et al. Chemical and physical
inactivation of human T lymphotropic virus, Type III (HTLV-III).
Transfusion 1986; 26:110-30.
PEST 2. 0 computer package and operating manual
- J Whitehead
- H Brunier
Whitehead J, Brunier H. PEST 2.0 computer package and operating
manual. University of Reading, 1989.
Antigenic and genomic diversity of Piasmodium falciparum isolates
- L Bansil
Bansil L. Antigenic and genomic diversity of Plasmodiumfalciparum
isolates. PhD Thesis, University of Liverpool, June 1992.
Pathophysiology of malaria In: StricklandGT, ed. Clinics in tropical medicine and communicable diseases
- Nj White
Safety of therapeutic immune globulin preparations with respect to transmission of human T-lymphotropic virus type III/ lymphadenopathy-associated virus infection. Morbidity and Mortality
- Editorial
Editorial. Safety of therapeutic immune globulin preparations with
respect to transmission of human T-lymphotropic virus type III/
lymphadenopathy-associated virus infection. Morbidity and Mortality Weekly Report 1986; 35:231-3.
Chemical and physical inactivation of human T lymphotropic virus. Type III (HTLV-III)
- Wells
Safety of therapeutic immune globulin preparations with respect to transmission of human T-lymphotropic virus type 111/ lymphadenopathy-associated virus infection
- Editorial
14 Editorial. Safety of therapeutic immune globulin preparations with
respect to transmission of human T-lymphotropic virus type III/
lymphadenopathy-associated virus infection. Morbidity and Mortality Weekly Report 1986; 35:231-3.