RESUMO
Immediate ß2-integrin activation upon T cell receptor stimulation is critical for effective interaction between T cells and their targets and may therefore be used for the rapid identification and isolation of functional T cells. We present a simple and sensitive flow cytometry-based assay to assess antigen-specific T cells using fluorescent intercellular adhesion molecule (ICAM)-1 multimers that specifically bind to activated ß2-integrins. The method is compatible with surface and intracellular staining; it is applicable for monitoring of a broad range of virus-, tumor-, and vaccine-specific CD8+ T cells, and for isolating viable antigen-reacting cells. ICAM-1 binding correlates with peptide-MHC multimer binding but, notably, it identifies the fraction of antigen-specific CD8+ T cells with immediate and high functional capability (i.e., expressing high levels of cytotoxic markers and cytokines). Compared with the currently available methods, staining of activated ß2-integrins presents the unique advantage of requiring activation times of only several minutes, therefore delivering functional information nearly reflecting the in vivo situation. Hence, the ICAM-1 assay is most suitable for rapid and precise monitoring of functional antigen-specific T cell responses, including for patient samples in a variety of clinical settings, as well as for the isolation of functional T cells for adoptive cell-transfer immunotherapies.
Assuntos
Antígenos/imunologia , Antígenos CD18/imunologia , Linfócitos T CD8-Positivos/imunologia , Adolescente , Transferência Adotiva/métodos , Adulto , Humanos , Imunoterapia Adotiva/métodos , Molécula 1 de Adesão Intercelular/imunologia , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Adulto JovemRESUMO
Antigenic variation of the Plasmodium falciparum multicopy var gene family enables parasite evasion of immune destruction by host antibodies. Expression of a particular var subgroup, termed upsA, is linked to the obstruction of blood vessels in the brain and to the pathogenesis of human cerebral malaria. The mechanism determining upsA activation remains unknown. Here we show that an entirely new type of gene silencing mechanism involving an exonuclease-mediated degradation of nascent RNA controls the silencing of genes linked to severe malaria. We identify a novel chromatin-associated exoribonuclease, termed PfRNase II, that controls the silencing of upsA var genes by marking their transcription start site and intron-promoter regions leading to short-lived cryptic RNA. Parasites carrying a deficient PfRNase II gene produce full-length upsA var transcripts and intron-derived antisense long non-coding RNA. The presence of stable upsA var transcripts overcomes monoallelic expression, resulting in the simultaneous expression of both upsA and upsC type PfEMP1 proteins on the surface of individual infected red blood cells. In addition, we observe an inverse relationship between transcript levels of PfRNase II and upsA-type var genes in parasites from severe malaria patients, implying a crucial role of PfRNase II in severe malaria. Our results uncover a previously unknown type of post-transcriptional gene silencing mechanism in malaria parasites with repercussions for other organisms. Additionally, the identification of RNase II as a parasite protein controlling the expression of virulence genes involved in pathogenesis in patients with severe malaria may provide new strategies for reducing malaria mortality.
Assuntos
Exorribonucleases/metabolismo , Inativação Gênica , Genes de Protozoários/genética , Malária Cerebral/parasitologia , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , RNA de Protozoário/metabolismo , Alelos , Variação Antigênica/genética , Cromatina/enzimologia , Regulação para Baixo/genética , Eritrócitos/parasitologia , Exorribonucleases/deficiência , Exorribonucleases/genética , Humanos , Íntrons/genética , Malária Falciparum/parasitologia , Plasmodium falciparum/patogenicidade , Regiões Promotoras Genéticas/genética , Proteínas de Protozoários/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Protozoário/genética , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Sítio de Iniciação de Transcrição , Virulência/genética , Fatores de Virulência/genéticaRESUMO
Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is an important malaria virulence factor. The protein family can be divided into clinically relevant subfamilies. ICAM-1-binding group A PfEMP1 proteins also bind endothelial protein C receptor and have been associated with cerebral malaria in children. IgG to these PfEMP1 proteins is acquired later in life than that to group A PfEMP1 not binding ICAM-1. The kinetics of acquisition of IgG to group B and C PfEMP1 proteins binding ICAM-1 is unclear and was studied here. Gene sequences encoding group B and C PfEMP1 with DBLß domains known to bind ICAM-1 were used to identify additional binders. Levels of IgG specific for DBLß domains from group A, B, and C PfEMP1 binding or not binding ICAM-1 were measured in plasma from Ghanaian children with or without malaria. Seven new ICAM-1-binding DBLß domains from group B and C PfEMP1 were identified. Healthy children had higher levels of IgG specific for ICAM-1-binding DBLß domains from group A than from groups B and C. However, the opposite pattern was found in children with malaria, particularly among young patients. Acquisition of IgG specific for DBLß domains binding ICAM-1 differs between PfEMP1 groups.
Assuntos
Anticorpos Antiprotozoários/biossíntese , Imunoglobulina G/biossíntese , Molécula 1 de Adesão Intercelular/genética , Malária Cerebral/imunologia , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/genética , Criança , Pré-Escolar , Eritrócitos/imunologia , Eritrócitos/parasitologia , Feminino , Expressão Gênica , Gana , Humanos , Lactente , Molécula 1 de Adesão Intercelular/imunologia , Malária Cerebral/genética , Malária Cerebral/parasitologia , Malária Cerebral/patologia , Malária Falciparum/genética , Malária Falciparum/parasitologia , Malária Falciparum/patologia , Masculino , Plasmodium falciparum/patogenicidade , Polimorfismo Genético , Ligação Proteica , Domínios Proteicos , Proteínas de Protozoários/classificação , Proteínas de Protozoários/imunologia , Estações do Ano , Índice de Gravidade de DoençaRESUMO
Cerebral malaria (CM) is a potentially deadly outcome of Plasmodium falciparum malaria that is precipitated by sequestration of infected erythrocytes (IEs) in the brain. The adhesion of IEs to brain endothelial cells is mediated by a subtype of parasite-encoded erythrocyte membrane protein 1 (PfEMP1) that facilitates dual binding to host intercellular adhesion molecule 1 (ICAM-1) and endothelial protein receptor C (EPCR). The PfEMP1 subtype is characterized by the presence of a particular motif (DBLß_motif) in the constituent ICAM-1-binding DBLß domain. The rate of natural acquisition of DBLß_motif-specific IgG antibodies and the ability to induce such antibodies by vaccination are unknown, and the aim of this study was to provide such data. We used an enzyme-linked immunosorbent assay (ELISA) to measure DBLß-specific IgG in plasma from Ghanaian children with malaria. The ability of human immune plasma and DBLß-specific rat antisera to inhibit the interaction between ICAM-1 and DBLß was assessed using ELISA and in vitro assays of IE adhesion under flow. The acquisition of DBLß_motif-specific IgG coincided with age-specific susceptibility to CM. Broadly cross-reactive antibodies inhibiting the interaction between ICAM-1 and DBLß_motif domains were detectable in immune plasma and in sera of rats immunized with specific DBLß_motif antigens. Importantly, antibodies against the DBLß_motif inhibited ICAM-1-specific in vitro adhesion of erythrocytes infected by four of five P. falciparum isolates from cerebral malaria patients. We conclude that natural exposure to P. falciparum as well as immunization with specific DBLß_motif antigens can induce cross-reactive antibodies that inhibit the interaction between ICAM-1 and a broad range of DBLß_motif domains. These findings raise hope that a vaccine designed specifically to prevent CM is feasible.
Assuntos
Imunoglobulina G/imunologia , Molécula 1 de Adesão Intercelular/metabolismo , Malária Cerebral/imunologia , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Adolescente , Motivos de Aminoácidos , Anticorpos Neutralizantes/imunologia , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Sítios de Ligação , Criança , Pré-Escolar , Reações Cruzadas/imunologia , Gana , Humanos , Imunoglobulina G/metabolismo , Lactente , Vacinas Antimaláricas/imunologia , Malária Cerebral/metabolismo , Malária Cerebral/parasitologia , Malária Falciparum/metabolismo , Malária Falciparum/parasitologia , Ligação Proteica/imunologia , Domínios e Motivos de Interação entre Proteínas , Proteínas de Protozoários/química , TanzâniaRESUMO
Background: Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) mediates the binding and accumulation of infected erythrocytes (IE) to blood vessels and tissues. Specific interactions have been described between PfEMP1 and human endothelial proteins CD36, intercellular adhesion molecule-1 (ICAM-1), and endothelial protein C receptor (EPCR); however, cytoadhesion patterns typical for pediatric malaria syndromes and the associated PfEMP1 members are still undefined. Methods: In a cohort of 94 hospitalized children with malaria, we characterized the binding properties of IE collected on admission, and var gene transcription using quantitative polymerase chain reaction. Results: IE from patients with cerebral malaria were more likely to bind EPCR and ICAM-1 than IE from children with uncomplicated malaria (P = .007). The level of transcripts encoding CIDRα1.4 and CIDRα1.5 domain subclasses was higher in patients with severe disease (P < .05). IE populations exhibiting binding to all 3 receptors had higher levels of transcripts encoding PfEMP1 with CIDRα1.4 and Duffy binding-like (DBL)-ß3 domains than parasites, which only bound CD36. Conclusions: These results underpin the significance of EPCR binding in pediatric malaria patients that require hospital admission, and support the notion that complementary receptor interactions of EPCR binding PfEMP1with ICAM-1 amplifies development of severe malaria symptoms.
Assuntos
Antígenos CD/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Malária Cerebral/parasitologia , Malária Falciparum/parasitologia , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Receptores de Superfície Celular/metabolismo , Adesão Celular , Pré-Escolar , Células Endoteliais/metabolismo , Receptor de Proteína C Endotelial , Humanos , Lactente , Ligação Proteica , Transcrição GênicaRESUMO
The virulence of Plasmodium falciparum is linked to the ability of infected erythrocytes (IE) to adhere to the vascular endothelium, mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1). In this article, we report the functional characterization of an mAb that recognizes a panel of PfEMP1s and inhibits ICAM-1 binding. The 24E9 mouse mAb was raised against PFD1235w DBLß3_D4, a domain from the group A PfEMP1s associated with severe malaria. 24E9 recognizes native PfEMP1 expressed on the IE surface and shows cross-reactivity with and cross-inhibition of the ICAM-1 binding capacity of domain cassette 4 PfEMP1s. 24E9 Fab fragments bind DBLß3_D4 with nanomolar affinity and inhibit ICAM-1 binding of domain cassette 4-expressing IE. The antigenic regions targeted by 24E9 Fab were identified by hydrogen/deuterium exchange mass spectrometry and revealed three discrete peptides that are solvent protected in the complex. When mapped onto a homology model of DBLß3_D4, these cluster to a defined, surface-exposed region on the convex surface of DBLß3_D4. Mutagenesis confirmed that the site most strongly protected is necessary for 24E9 binding, which is consistent with a low-resolution structure of the DBLß3_D4::24E9 Fab complex derived from small-angle x-ray scattering. The convex surface of DBLß3_D4 has previously been shown to contain the ICAM-1 binding site of DBLß domains, suggesting that the mAb acts by occluding the ICAM-1 binding surface. Conserved epitopes, such as those targeted by 24E9, are promising candidates for the inclusion in a vaccine interfering with ICAM-1-specific adhesion of group A PfEMP1 expressed by P. falciparum IE during severe malaria.
Assuntos
Anticorpos Monoclonais/imunologia , Sítios de Ligação de Anticorpos/imunologia , Molécula 1 de Adesão Intercelular/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Adesão Celular , Células Cultivadas , Endotélio Vascular/metabolismo , Endotélio Vascular/parasitologia , Epitopos/imunologia , Membrana Eritrocítica/imunologia , Eritrócitos/parasitologia , Hibridomas , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Camundongos , Dados de Sequência Molecular , Estrutura Terciária de ProteínaRESUMO
BACKGROUND: Malawi experienced prolonged use of sulfadoxine/pyrimethamine (SP) as the front-line anti-malarial drug, with early replacement of chloroquine and delayed introduction of artemisinin-based combination therapy. Extended use of SP, and its continued application in pregnancy is impacting the genomic variation of the Plasmodium falciparum population. METHODS: Whole genome sequence data of P. falciparum isolates covering 2 years of transmission within Malawi, alongside global datasets, were used. More than 745,000 SNPs were identified, and differences in allele frequencies between countries assessed, as well as genetic regions under positive selection determined. RESULTS: Positive selection signals were identified within dhps, dhfr and gch1, all components of the parasite folate pathway associated with SP resistance. Sitting predominantly on a dhfr triple mutation background, a novel copy number increase of ~twofold was identified in the gch1 promoter. This copy number was almost fixed (96.8% frequency) in Malawi samples, but found at less than 45% frequency in other African populations, and distinct from a whole gene duplication previously reported in Southeast Asian parasites. CONCLUSIONS: SP resistance selection pressures have been retained in the Malawian population, with known resistance dhfr mutations at fixation, complemented by a novel gch1 promoter duplication. The effects of the duplication on the fitness costs of SP variants and resistance need to be elucidated.
Assuntos
Antimaláricos/uso terapêutico , Variação Genética , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/classificação , Plasmodium falciparum/efeitos dos fármacos , Pirimetamina/uso terapêutico , Sulfadoxina/uso terapêutico , Pré-Escolar , Combinação de Medicamentos , Resistência a Medicamentos , Feminino , Frequência do Gene , Genoma de Protozoário , Genótipo , Humanos , Lactente , Malaui , Masculino , Mutação , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , Polimorfismo de Nucleotídeo Único , Seleção Genética , Análise de Sequência de DNARESUMO
Cerebral Plasmodium falciparum malaria is characterized by adhesion of infected erythrocytes (IEs) to the cerebral microvasculature. This has been linked to parasites expressing the structurally related group A subset of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family of IE adhesion ligands and to IEs with affinity for ICAM-1. However, recent evidence has cast doubt on both these associations, tempering hopes of the feasibility of developing a vaccine based on ICAM-1-binding PfEMP1. In this study, we report the identification of a domain cassette (DC) present in group A var genes from six genetically distinct P. falciparum parasites. The three domains in the cassette, which we call DC4, had a high level of sequence identity and cluster together phylogenetically. Erythrocytes infected by these parasites and selected in vitro for expression of DC4 adhered specifically to ICAM-1. The ICAM-1-binding capacity of DC4 was mapped to the C-terminal third of its Duffy-binding-like ß3 domain. DC4 was the target of broadly cross-reactive and adhesion-inhibitory IgG Abs, and levels of DC4-specific and adhesion-inhibitory IgG increased with age among P. falciparum-exposed children. Our study challenges earlier conclusions that group A PfEMP1 proteins are not central to ICAM-1-specific IE adhesion and support the feasibility of developing a vaccine preventing cerebral malaria by inhibiting cerebral IE sequestration.
Assuntos
Anticorpos Bloqueadores/metabolismo , Antígenos de Protozoários/metabolismo , Membrana Eritrocítica/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Mutagênese Insercional/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/metabolismo , Animais , Anticorpos Bloqueadores/genética , Antígenos de Protozoários/classificação , Antígenos de Protozoários/genética , Sítios de Ligação de Anticorpos/genética , Adesão Celular/genética , Adesão Celular/imunologia , Sequência Conservada/genética , Sequência Conservada/imunologia , Reações Cruzadas/imunologia , Membrana Eritrocítica/genética , Membrana Eritrocítica/imunologia , Genômica/métodos , Células HEK293 , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/imunologia , Mutagênese Insercional/genética , Plasmodium falciparum/genética , Ligação Proteica/genética , Ligação Proteica/imunologia , Estrutura Terciária de Proteína/genética , Proteínas de Protozoários/classificação , Proteínas de Protozoários/genética , RatosRESUMO
BACKGROUND: Selection by host immunity and antimalarial drugs has driven extensive adaptive evolution in Plasmodium falciparum and continues to produce ever-changing landscapes of genetic variation. METHODS: We performed whole-genome sequencing of 69 P. falciparum isolates from Malawi and used population genetics approaches to investigate genetic diversity and population structure and identify loci under selection. RESULTS: High genetic diversity (π = 2.4 × 10(-4)), moderately high multiplicity of infection (2.7), and low linkage disequilibrium (500-bp) were observed in Chikhwawa District, Malawi, an area of high malaria transmission. Allele frequency-based tests provided evidence of recent population growth in Malawi and detected potential targets of host immunity and candidate vaccine antigens. Comparison of the sequence variation between isolates from Malawi and those from 5 geographically dispersed countries (Kenya, Burkina Faso, Mali, Cambodia, and Thailand) detected population genetic differences between Africa and Asia, within Southeast Asia, and within Africa. Haplotype-based tests of selection to sequence data from all 6 populations identified signals of directional selection at known drug-resistance loci, including pfcrt, pfdhps, pfmdr1, and pfgch1. CONCLUSIONS: The sequence variations observed at drug-resistance loci reflect differences in each country's historical use of antimalarial drugs and may be useful in formulating local malaria treatment guidelines.
Assuntos
Adaptação Biológica , Evolução Molecular , Genoma de Protozoário , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Antimaláricos/uso terapêutico , Criança , Pré-Escolar , Feminino , Variação Genética , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/epidemiologia , Malaui , Masculino , Epidemiologia Molecular , Plasmodium falciparum/isolamento & purificação , Seleção Genética , Análise de Sequência de DNARESUMO
The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigens play a major role in cytoadhesion of infected erythrocytes (IE), antigenic variation, and immunity to malaria. The current consensus on control of variant surface antigen expression is that only one PfEMP1 encoded by one var gene is expressed per cell at a time. We measured var mRNA transcript levels by real-time Q-PCR, analysed var gene transcripts by single-cell FISH and directly compared these with PfEMP1 antigen surface expression and cytoadhesion in three different antibody-selected P. falciparum 3D7 sub-lines using live confocal microscopy, flow cytometry and in vitro adhesion assays. We found that one selected parasite sub-line simultaneously expressed two different var genes as surface antigens, on single IE. Importantly, and of physiological relevance to adhesion and malaria pathogenesis, this parasite sub-line was found to bind both CD31/PECAM1 and CD54/ICAM1 and to adhere twice as efficiently to human endothelial cells, compared to infected cells having only one PfEMP1 variant on the surface. These new results on PfEMP1 antigen expression indicate that a re-evaluation of the molecular mechanisms involved in P. falciparum adhesion and of the accepted paradigm of absolutely mutually exclusive var gene transcription is required.
Assuntos
Eritrócitos/metabolismo , Eritrócitos/parasitologia , Molécula 1 de Adesão Intercelular/metabolismo , Malária Falciparum/metabolismo , Plasmodium falciparum/patogenicidade , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Proteínas de Protozoários/metabolismo , Variação Antigênica , Antígenos de Superfície/metabolismo , Northern Blotting , Western Blotting , Adesão Celular , Movimento Celular , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Hibridização in Situ Fluorescente , Molécula 1 de Adesão Intercelular/genética , Malária Falciparum/genética , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
Background: Plasmodium falciparum variant surface antigens (VSAs) contribute to malaria pathogenesis by mediating cytoadhesion of infected red blood cells to the microvasculature endothelium. In this study, we investigated the association between anti-VSA antibodies and clinical outcome in a controlled human malaria infection (CHMI) study. Method: We used flow cytometry and ELISA to measure levels of IgG antibodies to VSAs of five heterologous and one homologous P. falciparum parasite isolates, and to two PfEMP1 DBLß domains in blood samples collected a day before the challenge and 14 days after infection. We also measured the ability of an individual's plasma to inhibit the interaction between PfEMP1 and ICAM1 using competition ELISA. We then assessed the association between the antibody levels, function, and CHMI defined clinical outcome during a 21-day follow-up period post infection using Cox proportional hazards regression. Results: Antibody levels to the individual isolate VSAs, or to two ICAM1-binding DBLß domains of PfEMP1, were not associated with a significantly reduced risk of developing parasitemia or of meeting treatment criteria after the challenge after adjusting for exposure. However, anti-VSA antibody breadth (i.e., cumulative response to all the isolates) was a significant predictor of reduced risk of requiring treatment [HR 0.23 (0.10-0.50) p= 0.0002]. Conclusion: The breadth of IgG antibodies to VSAs, but not to individual isolate VSAs, is associated with protection in CHMI.
Assuntos
Malária Falciparum , Malária , Anticorpos Antiprotozoários , Antígenos de Protozoários , Antígenos de Superfície , Humanos , Imunoglobulina G , Plasmodium falciparumRESUMO
How immunity to malaria develops remains one of the great unresolved issues in bio-medicine and resolution of its various paradoxes is likely to be the key to developing effective malaria vaccines. The basic epidemiological observations are; under conditions of intense natural transmission, humans do become immune to P. falciparum malaria, but this is a slow process requiring multiple disease episodes which many, particularly young children, do not survive. Adult survivors are immune to the symptoms of malaria, and unless pregnant, can control the growth of most or all new inoculations. Sterile immunity is not achieved and chronic parasitization of apparently healthy adults is the norm. In this article, we analyse the best understood malaria "antigenic variation" system, that based on Plasmodium falciparum's PfEMP1-type cytoadhesion antigens, and critically review recent literature on the function and control of this multi-gene family of parasite variable surface antigens.
Assuntos
Variação Antigênica , Plasmodium falciparum , Animais , Antígenos de Protozoários , Antígenos de Superfície , Eritrócitos , Humanos , Malária Falciparum , Proteínas de ProtozoáriosRESUMO
The binding of erythrocytes infected with mature blood stage parasites to the vascular bed is key to the pathogenesis of malignant malaria. The binding is mediated by members of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family. PfEMP1s can be divided into groups, and it has previously been suggested that parasites expressing group A or B/A PfEMP1s are most pathogenic. To test the hypothesis that the first malaria infections in infants and young children are dominated by parasites expressing A and B/A PfEMP1s, we measured the plasma Ab level against 48 recombinant PfEMP1 domains of different groupings in 1342 individuals living in five African villages characterized by markedly different malaria transmission. We show that children progressively acquire a broader repertoire of anti-PfEMP1 Abs, but that the rate of expansion is governed by transmission intensity. However, independently of transmission intensity, Abs are first acquired to particular Duffy binding ligand-like domains belonging to group A or B/A PfEMP1s. The results support the view that anti-PfEMP1 Ab responses effectively structure the expenditure of the repertoire of PfEMP1 maintained by the parasite. Parasites expressing certain group A and B/A PfEMP1s are responded to first by individuals with limited previous exposure, and the resulting Abs reduce the fitness and pathogenicity of these parasites during subsequent infections. This allows parasites expressing less pathogenic PFEMP1s to dominate during later infections. The identification of PfEMP1 domains expressed by parasites causing disease in infants and young children is important for development of vaccines protecting against severe malaria.
Assuntos
Anticorpos Antiprotozoários/biossíntese , Membrana Eritrocítica/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Adolescente , Adulto , Fatores Etários , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/sangue , Antígenos de Protozoários/imunologia , Criança , Pré-Escolar , Estudos Transversais , Membrana Eritrocítica/parasitologia , Humanos , Lactente , Recém-Nascido , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Malária Falciparum/transmissão , Pessoa de Meia-Idade , Parasitemia/sangue , Parasitemia/imunologia , Parasitemia/parasitologia , Plasmodium falciparum/patogenicidade , Prevalência , Estrutura Terciária de Proteína , Proteínas de Protozoários/sangue , Adulto JovemRESUMO
BACKGROUND: Sickle cell trait (HbAS) protects against severe Plasmodium falciparum malaria but not against placental malaria (PM). In this study, P falciparum erythrocyte membrane protein (PfEMP1)-specific antibodies were measured in HbAA and HbAS Beninese pregnant women as a proxy of exposure to specific PfEMP1 variants. METHODS: Plasma samples collected at delivery from 338 HbAA and 63 HbAS women were used to measure immunoglobulin (Ig)G levels to 6 recombinant PfEMP1 proteins and 3 corresponding native proteins expressed on the infected erythrocyte (IE) surface. Immunoglobulin G-mediated inhibition of VAR2CSA+ IEs adhesion to chondroitin sulfate A (CSA) was also tested. RESULTS: Levels of PfEMP1-specific IgG were similar in the 2 groups, except for native IT4VAR09 on IEs, where IgG levels were significantly higher in HbAS women. Adjusted odds ratios for women with positive IgG to HB3VAR06 and PFD1235w suggest a lower risk of infection with these virulent variants among HbAS individuals. The percentage of IEs binding to CSA did not differ between HbAA and HbAS women, but it correlated positively with levels of anti-VAR2CSA and parity. Women with PM had lower levels of anti-VAR2CSA-specific IgG and lower IgG-mediated inhibition of IE adhesion to CSA. CONCLUSIONS: The findings support similar malaria exposure in HbAA and HbAS women and a lack of HbAS-dependent protection against placental infection among pregnant women.
RESUMO
We have previously shown that conformational change in the ß2-integrin is a very early activation marker that can be detected with fluorescent multimers of its ligand intercellular adhesion molecule (ICAM)-1 for rapid assessment of antigen-specific CD8+ T cells. In this study, we describe a modified protocol of this assay for sensitive detection of functional antigen-specific CD4+ T cells using a monoclonal antibody (clone m24 Ab) specific for the open, high-affinity conformation of the ß2-integrin. The kinetics of ß2-integrin activation was different on CD4+ and CD8+ T cells (several hours vs. few minutes, respectively); however, m24 Ab readily stained both cell types 4-6 h after antigen stimulation. With this protocol, we were able to monitor ex vivo effector and memory CD4+ and CD8+ T cells specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), cytomegalovirus (CMV), Epstein-Barr virus (EBV), and hepatitis B virus (HBV) in whole blood or cryopreserved peripheral blood mononuclear cells (PBMCs) of infected or vaccinated individuals. By costaining ß2-integrin with m24 and CD154 Abs, we assessed extremely low frequencies of polyfunctional CD4+ T cell responses. The novel assay used in this study allows very sensitive and simultaneous screening of both CD4+ and CD8+ T cell reactivities, with versatile applicability in clinical and vaccination studies.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Integrinas/metabolismo , Adulto , Idoso , Sequência de Aminoácidos , Sítios de Ligação , COVID-19/genética , COVID-19/imunologia , COVID-19/metabolismo , COVID-19/virologia , Proteínas de Transporte/química , Citocinas/metabolismo , Citomegalovirus/imunologia , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Feminino , Antígenos HLA/química , Antígenos HLA/imunologia , Interações Hospedeiro-Patógeno/genética , Humanos , Imuno-Histoquímica , Imunofenotipagem , Integrinas/genética , Molécula 1 de Adesão Intercelular/química , Molécula 1 de Adesão Intercelular/metabolismo , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Ligação Proteica , Multimerização Proteica , SARS-CoV-2/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Cerebral malaria (CM) is caused by the binding of Plasmodium falciparum-infected erythrocytes (IEs) to the brain microvasculature, leading to inflammation, vessel occlusion, and cerebral swelling. We have previously linked dual intercellular adhesion molecule-1 (ICAM-1)- and endothelial protein C receptor (EPCR)-binding P. falciparum parasites to these symptoms, but the mechanism driving the pathogenesis has not been identified. Here, we used a 3D spheroid model of the blood-brain barrier (BBB) to determine unexpected new features of IEs expressing the dual-receptor binding PfEMP1 parasite proteins. Analysis of multiple parasite lines shows that IEs are taken up by brain endothelial cells in an ICAM-1-dependent manner, resulting in breakdown of the BBB and swelling of the endothelial cells. Via ex vivo analysis of postmortem tissue samples from CM patients, we confirmed the presence of parasites within brain endothelial cells. Importantly, this discovery points to parasite ingress into the brain endothelium as a contributing factor to the pathology of human CM.
Assuntos
Barreira Hematoencefálica/patologia , Malária Cerebral/patologia , Malária Cerebral/parasitologia , Proteínas de Protozoários/genética , Adulto , Animais , Endocitose , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Receptor de Proteína C Endotelial/metabolismo , Eritrócitos/parasitologia , Eritrócitos/patologia , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Microvilosidades/metabolismo , Modelos Biológicos , Simulação de Acoplamento Molecular , Parasitos/metabolismo , Plasmodium falciparum/isolamento & purificação , Plasmodium falciparum/ultraestrutura , Ligação Proteica , Isoformas de Proteínas/metabolismo , Ratos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologiaRESUMO
Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a variant antigen expressed on the surface of malaria-infected erythrocytes. PfEMP1 attaches to the vascular lining and allows infected erythrocytes to avoid filtration through the spleen. Each parasite genome encodes about 60 different PfEMP1 variants, each PfEMP1 comprises several domains in its extracellular region, and the PfEMP1 repertoire in different parasites contains domain types that are serologically cross-reactive. In this longitudinal study, we followed 672 children living in an area of high malaria transmission during the first years of life and compared the acquisitions of antibodies to 32 Duffy-binding ligand-like (DBL) domains representing different types. For each child, we determined whether an antibody response to each domain was acquired before, after, or at the same time as responses to each of the other domains. We next used this information to calculate population-level odds ratios to measure the odds that antibodies to a given domain were acquired before antibodies to other domains. Odds ratios for 269 of the 496 possible domain combinations were statistically significant. Thus, the sequence in which individuals acquire antibodies to different PfEMP1 domains is ordered, and children in areas of endemicity first acquire antibodies to particular PfEMP1 domains encoded by the so-called group A and B/A var genes. The results imply that anti-PfEMP1 antibodies effectively structure PfEMP1 expression and play a major role in limiting parasite multiplication in the blood.
Assuntos
Anticorpos Antiprotozoários/sangue , Variação Genética , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/química , Proteínas de Protozoários/imunologia , Adolescente , Adulto , Animais , Eritrócitos/parasitologia , Feminino , Humanos , Lactente , Recém-Nascido , Malária Falciparum/parasitologia , Pessoa de Meia-Idade , Plasmodium falciparum/metabolismo , Estrutura Terciária de Proteína , Proteínas de Protozoários/genética , Tanzânia , Adulto JovemRESUMO
In Plasmodium falciparum-endemic areas, pregnancy-associated malaria (PAM) is an important health problem. The condition is precipitated by accumulation of parasite-infected erythrocytes (IEs) in the placenta, and this process is mediated by parasite-encoded variant surface antigens (VSA) binding to chondroitin sulfate A (CSA). Parasites causing PAM express unique VSA types, VSAPAM, which can be serologically classified as sex specific and parity dependent. It is sex specific because men from malaria-endemic areas do not develop VSAPAM antibodies; it is parity dependent because women acquire anti-VSAPAM immunoglobulin (Ig) G as a function of parity. Previously, it was shown that transcription of var2csa is up-regulated in placental parasites and parasites selected for CSA binding. Here, we show the following: (a) that VAR2CSA is expressed on the surface of CSA-selected IEs; (b) that VAR2CSA is recognized by endemic plasma in a sex-specific and parity-dependent manner; (c) that high anti-VAR2CSA IgG levels can be found in pregnant women from both West and East Africa; and (d) that women with high plasma levels of anti-VAR2CSA IgG give birth to markedly heavier babies and have a much lower risk of delivering low birth weight children than women with low levels.
Assuntos
Peso ao Nascer/imunologia , Sulfatos de Condroitina/metabolismo , Eritrócitos/parasitologia , Malária Falciparum/imunologia , Placenta/parasitologia , Proteínas de Protozoários/metabolismo , África , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Eritrócitos/metabolismo , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Malária Falciparum/metabolismo , Masculino , Microscopia Confocal , Gravidez , Proteínas Recombinantes/metabolismo , Fatores SexuaisRESUMO
Parasite-encoded variant surface antigens (VSAs) like the var gene-encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family are responsible for antigenic variation and infected red blood cell (RBC) cytoadhesion in P. falciparum malaria. Parasites causing severe malaria in nonimmune patients tend to express a restricted subset of VSA (VSA(SM)) that differs from VSA associated with uncomplicated malaria and asymptomatic infection (VSA(UM)). We compared var gene transcription in unselected P. falciparum clone 3D7 expressing VSA(UM) to in vitro-selected sublines expressing VSA(SM) to identify PfEMP1 responsible for the VSA(SM) phenotype. Expression of VSA(SM) was accompanied by up-regulation of Group A var genes. The most prominently up-regulated Group A gene (PFD1235w/MAL7P1.1) was translated into a protein expressed on the infected RBC surface. The proteins encoded by Group A var genes, such as PFD1235w/MAL7P1.1, appear to be involved in the pathogenesis of severe disease and are thus attractive candidates for a vaccine against life-threatening P. falciparum malaria.
Assuntos
Antígenos de Protozoários/genética , Genes de Protozoários/genética , Malária Falciparum/fisiopatologia , Plasmodium falciparum/genética , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/sangue , Sequência de Bases , Criança , Clonagem Molecular , Primers do DNA , Membrana Eritrocítica/parasitologia , Regulação da Expressão Gênica/genética , Humanos , Malária Falciparum/sangue , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Gênica/genéticaRESUMO
BACKGROUND: The PFD1235w Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigen is associated with severe malaria in children and can be expressed on the surface of infected erythrocytes (IE) adhering to ICAM1. However, the exact three-dimensional structure of this PfEMP1 and its surface-exposed epitopes are unknown. An insect cell and Escherichia coli based system was used to express single and double domains encoded by the pfd1235w var gene. The resulting recombinant proteins have been evaluated for yield and purity and their ability to induce rat antibodies, which react with the native PFD1235w PfEMP1 antigen expressed on 3D7PFD1235w-IE. Their recognition by human anti-malaria antibodies from previously infected Tanzanian donors was also analysed. METHODS: The recombinant proteins were run on SDS-PAGE and Western blots for quantification and size estimation. Insect cell and E. coli-produced recombinant proteins were coupled to a bead-based Luminex assay to measure the plasma antibody reactivity of 180 samples collected from Tanzanian individuals. The recombinant proteins used for immunization of rats and antisera were also tested by flow cytometry for their ability to surface label 3D7PFD1235w-IE. RESULTS: All seven pAcGP67A constructs were successfully expressed as recombinant protein in baculovirus-infected insect cells and subsequently produced to a purity of 60-97% and a yield of 2-15 mg/L. By comparison, only three of seven pET101/D-TOPO constructs expressed in the E. coli system could be produced at all with purity and yield ranging from 3-95% and 6-11 mg/L. All seven insect cell, but only two of the E. coli produced proteins induced antibodies reactive with native PFD1235w expressed on 3D7PFD1235w-IE. The recombinant proteins were recognized in an age- and transmission intensity-dependent manner by antibodies from 180 Tanzanian individuals in a bead-based Luminex assay. CONCLUSIONS: The baculovirus based insect cell system was distinctly superior to the E. coli expression system in producing a larger number of different recombinant PFD1235w protein domains and these were significantly easier to purify at a useful yield. However, proteins produced in both systems were able to induce antibodies in rats, which can recognize the native PFD1235w on the surface of IE.