Aktivierung von Endothelzellen durch Plasmodium falciparum-infizierte Erythrocyten und Charakterisierung potentieller Adhäsionsrezeptoren

Während einer Infektion mit dem Malariaerreger Plasmodium falciparum treten oft schwerwiegende Komplikationen auf, da parasitierte Erythrocyten an Endothelzellen adhärieren und in der Mikrovaskulatur akkumulieren. Cytoadhäsion wird vermittelt durch die Interaktion von Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP-1) auf der Oberfläche der Erythrocyten mit Wirtsrezeptoren auf den Endothelzellen. Die Expression einiger Adhäsionsrezeptoren des Wirts u.a. von ICAM-1 und VCAM-1 erfolgt nur in einer entzündlichen Umgebung und erfordert eine Aktivierung der Endothelzellen. In einem in vitro-Modellsystem konnte die Aktivierung primärer humaner Endothelzellen durch direkte Interaktion mit P. falciparum-infizierten Erythrocyten nachgewiesen werden, lösliche Mediatoren sind für eine Aktivierung nicht ausreichend. Die Fähigkeit zur Stimulation der Endothelzellen ist abhängig von der Parasitämie, der Parasitenanzahl und der Expositionsdauer, jedoch unabhängig vom Adhäsionsphänotyp der Plasmodien. Die Endothelzellen reagieren auf den Stimulus mit einer veränderten Regulation der Genexpression auf Transkriptionsebene, wie durch Microarray-Analysen gezeigt werden konnte. Es entsteht ein proadhäsiver Phänotyp der Endothelzellen, der u.a. durch die verstärkte Expression der Adhäsionsmoleküle ICAM-1 und CD44, sowie durch die Sekretion der Cytokine und Chemokine MIP-3α, IL-8, MCP-1 und IL-6 gekennzeichnet ist. Somit kann eine Aktivierung der Endothelzellen durch Kontakt mit P. falciparum-infizierten Erythrocyten erfolgen, ohne daß eine Aktivierung der Makrophagen oder Monocyten vorausgehen muß. Durch die Expression der unterschiedlichen Chemokine durch die stimulierten Endothelzellen könnten Leukocyten angelockt werden, die die Organe infiltrieren und ebenfalls aktiviert werden. Sequestrierung von Monocyten, Makrophagen, Blutplättchen und P. falciparuminfizierten Erythrocyten bewirken u.a. den Verschluß der Blutgefäße sowie eine weitere Sekretion von Cytokinen, die zur Entstehung einer komplizierten Malaria beitragen und damit zum Tod des Patienten führen können. Eine verstärkte Sequestrierung P. falciparuminfizierter Erythrocyten, sowie von Leukocyten an die aktivierten Endothelzellen bleibt allerdings zu untersuchen. Cytoadhäsion-vermittelnde Moleküle auf humanen Endothelzellen konnten bisher nur in einigen Organen identifiziert werden, ein spezifischer Rezeptor auf sinusoidalen Endothelzellen der Leber wurde bisher noch nicht charakterisiert. Durch initiale Adhäsion P. falciparum-infizierter Erythrocyten an einen leberspezifischen Rezeptor auf sinusoidalen Endothelzellen könnte die anti-parasitäre Immunantwort so verändert werden, daß die Plasmodien einen Überlebensvorteil im Wirt erhalten, z.B. durch Entstehung von Immuntoleranz in der Leber. Liver/lymph node specific ICAM-3 grabbing nonintegrin (LSIGN), ein Homolog zu dem C-Typ Lektin Dendritic cell specific ICAM-3 grabbing nonintegrin (DC-SIGN), wird in der Leber auf sinusoidalen Endothelzellen exprimiert. Der Oberflächenrezeptor L-SIGN vermittelt die Endocytose von mannosehaltigen Liganden, was vermutlich zu der Präsentation von Antigenen auf MHC-Molekülen an T-Lymphocyten führt. Es konnte weder eine L-SIGN-spezifische Invasion der CHOL-SIGN Zellen durch P. berghei Sporozoiten, noch eine Cytoadhäsion P. falciparum-infizierter Erythrocyten an das auf CHO Zellen exprimierte L-SIGN, nachgewiesen werden. Obwohl eine L-SIGN-spezifische Cytoadhäsion der selektionierten P. falciparum-Klone nicht nachgewiesen werden konnte, so hatten diese Parasiten die Fähigkeit erworben Chondroitinsulfat-unabhängig an CHO Zellen zu cytoadhärieren. Da CHO-745 Zellen keinen der bisher bekannten Adhäsionsrezeptoren exprimieren, muß noch mindestens ein weiterer Cytoadhäsion-vermittelnder Rezeptor auf CHO Zellen zu finden sein. Erythrocyten, die mit den P. falciparum-Klonen FCR3 und 3D7 infiziert waren, ließen sich auf einen CHO-745 spezifischen Bindungsphänotyp selektionieren (3D7745 und FCR3745). Eine Charakterisierung der PfEMP-1 codierenden Transkripte (var) in diesen Parasitenpopulationen lieferten vier stabile vollständige var Transkripte für den P. falciparum Klon 3D7 und ein stabiles vollständiges var Transkript für FCR3. Dies lässt darauf schließen, daß die Interaktion parasitierter Erythrocyten mit den CHO-745 Zellen durch PfEMP-1 vermittelt wird

Disruption of Var2csa Gene Impairs Placental Malaria Associated Adhesion Phenotype

Infection with Plasmodium falciparum during pregnancy is one of the major causes of malaria related morbidity and mortality in newborn and mothers. The complications of pregnancy-associated malaria result mainly from massive adhesion of Plasmodium falciparum-infected erythrocytes (IE) to chondroitin sulfate A (CSA) present in the placental intervillous blood spaces. Var2CSA, a member of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family is the predominant parasite ligand mediating CSA binding. However, experimental evidence suggests that other host receptors, such as hyaluronic acid (HA) and the neonatal Fc receptor, may also support placental binding. Here we used parasites in which var2csa was genetically disrupted to evaluate the contribution of these receptors to placental sequestration and to identify additional adhesion receptors that may be involved in pregnancy-associated malaria. By comparison to the wild-type parasites, the FCR3Δvar2csa mutants could not be selected for HA adhesion, indicating that var2csa is not only essential for IE cytoadhesion to the placental receptor CSA, but also to HA. However, further studies using different pure sources of HA revealed that the previously observed binding results from CSA contamination in the bovine vitreous humor HA preparation. To identify CSA-independent placental interactions, FCR3Δvar2csa mutant parasites were selected for adhesion to the human placental trophoblastic BeWo cell line. BeWo selected parasites revealed a multi-phenotypic adhesion population expressing multiple var genes. However, these parasites did not cytoadhere specifically to the syncytiotrophoblast lining of placental cryosections and were not recognized by sera from malaria-exposed women in a parity dependent manner, indicating that the surface molecules present on the surface of the BeWo selected population are not specifically expressed during the course of pregnancy-associated malaria. Taken together, these results demonstrate that the placental malaria associated phenotype can not be restored in FCR3Δvar2csa mutant parasites and highlight the key role of var2CSA in pregnancy malaria pathogenesis and for vaccine development

Clinical development of placental malaria vaccines and immunoassays harmonization:a workshop report

International audiencePlacental malaria caused by Plasmodium falciparum infection constitutes a major health problem manifesting as severe disease and anaemia in the mother, impaired fetal development, low birth weight or spontaneous abortion. Prevention of placental malaria currently relies on two key strategies that are losing efficacy due to spread of resistance: long-lasting insecticide-treated nets and intermittent preventive treatment during pregnancy. A placental malaria vaccine would be an attractive, cost-effective complement to the existing control tools. Two placental malaria vaccine candidates are currently in Phase Ia/b clinical trials. During two workshops hosted by the European Vaccine Initiative, one in Paris in April 2014 and the other in Brussels in November 2014, the main actors in placental malaria vaccine research discussed the harmonization of clinical development plans and of the immunoassays with a goal to define standards that will allow comparative assessment of different placental malaria vaccine candidates. The recommendations of these workshops should guide researchers and clinicians in the further development of placental malaria vaccines

A Dendritic Cell–Specific Intercellular Adhesion Molecule 3–Grabbing Nonintegrin (Dc-Sign)–Related Protein Is Highly Expressed on Human Liver Sinusoidal Endothelial Cells and Promotes HIV-1 Infection

The discovery of dendritic cell (DC)-specific intercellular adhesion molecule (ICAM)-3–grabbing nonintegrin (DC-SIGN) as a DC-specific ICAM-3 binding receptor that enhances HIV-1 infection of T cells in trans has indicated a potentially important role for adhesion molecules in AIDS pathogenesis. A related molecule called DC-SIGNR exhibits 77% amino acid sequence identity with DC-SIGN. The DC-SIGN and DC-SIGNR genes map within a 30-kb region on chromosome 19p13.2-3. Their strong homology and close physical location indicate a recent duplication of the original gene. Messenger RNA and protein expression patterns demonstrate that the DC-SIGN–related molecule is highly expressed on liver sinusoidal cells and in the lymph node but not on DCs, in contrast to DC-SIGN. Therefore, we suggest that a more appropriate name for the DC-SIGN–related molecule is L-SIGN, liver/lymph node–specific ICAM-3–grabbing nonintegrin. We show that in the liver, L-SIGN is expressed by sinusoidal endothelial cells. Functional studies indicate that L-SIGN behaves similarly to DC-SIGN in that it has a high affinity for ICAM-3, captures HIV-1 through gp120 binding, and enhances HIV-1 infection of T cells in trans. We propose that L-SIGN may play an important role in the interaction between liver sinusoidal endothelium and trafficking lymphocytes, as well as function in the pathogenesis of HIV-1

Safety, Immunogenicity and Efficacy of Prime-Boost Vaccination with ChAd63 and MVA Encoding ME-TRAP against Plasmodium falciparum Infection in Adults in Senegal.

Malaria transmission is in decline in some parts of Africa, partly due to the scaling up of control measures. If the goal of elimination is to be achieved, additional control measures including an effective and durable vaccine will be required. Studies utilising the prime-boost approach to deliver viral vectors encoding the pre-erythrocytic antigen ME-TRAP (multiple epitope thrombospondin-related adhesion protein) have shown promising safety, immunogenicity and efficacy in sporozoite challenge studies. More recently, a study in Kenyan adults, similar to that reported here, showed substantial efficacy against P. falciparum infection. One hundred and twenty healthy male volunteers, living in a malaria endemic area of Senegal were randomised to receive either the Chimpanzee adenovirus (ChAd63) ME-TRAP as prime vaccination, followed eight weeks later by modified vaccinia Ankara (MVA) also encoding ME-TRAP as booster, or two doses of anti-rabies vaccine as a comparator. Prior to follow-up, antimalarials were administered to clear parasitaemia and then participants were monitored by PCR for malaria infection for eight weeks. The primary endpoint was time-to-infection with P. falciparum malaria, determined by two consecutive positive PCR results. Secondary endpoints included adverse event reporting, measures of cellular and humoral immunogenicity and a meta-analysis of combined vaccine efficacy with the parallel study in Kenyan adults.We show that this pre-erythrocytic malaria vaccine is safe and induces significant immunogenicity, with a peak T-cell response at seven days after boosting of 932 Spot Forming Cells (SFC)/106 Peripheral Blood Mononuclear Cells(PBMC) compared to 57 SFC/ 106 PBMCs in the control group. However, a vaccine efficacy was not observed: 12 of 57 ME-TRAP vaccinees became PCR positive during the intensive monitoring period as compared to 13 of the 58 controls (P = 0.80). This trial confirms that vaccine efficacy against malaria infection in adults may be rapidly assessed using this efficient and cost-effective clinical trial design. Further efficacy evaluation of this vectored candidate vaccine approach in other malaria transmission settings and age-de-escalation into the main target age groups for a malaria vaccine is in progress

Dissection of the Role of PfEMP1 and ICAM-1 in the Sensing of Plasmodium falciparum-Infected Erythrocytes by Natural Killer Cells

BACKGROUND: Host innate immunity contributes to malaria clinical outcome by providing protective inflammatory cytokines such as interferon-γ, and by shaping the adaptive immune response. Plasmodium falciparum (Pf) is the etiologic agent of the most severe forms of human malaria. Natural Killer (NK) cells are lymphocytes of the innate immune system that are the first effectors to produce interferon-γ in response to Pf. However, the molecular bases of Pf-NK cell recognition events are unknown. Our study focuses on the role of Pf erythrocyte membrane protein 1 (PfEMP1), a major Pf virulence factor. PfEMP1 is expressed on parasitized-erythrocytes and participates to vascular obstruction through the binding to several host receptors. PfEMP1 is also a pivotal target for host antibody response to Pf infection. METHODOLOGY/PRINCIPAL FINDINGS: Using genetically-engineered parasite mutant strains, a human genetic deficiency, and blocking antibodies, we identified two receptor-ligand pairs involved in two uncoupled events occurring during the sensing of Pf infection by NK cells. First, PfEMP1 interaction with one of its host receptor, chondroitin sulfate A, mediates the cytoadhesion of Pf-infected erythrocytes to human NK cell lines, but is not required for primary NK cell activation. Second, intercellular adhesion molecule-1 (ICAM-1), another host receptor for PfEMP1, is mandatory for NK cell interferon-γ response. In this case, ICAM-1 acts via its engagement with its host ligand, LFA-1, and not with PfEMP1, consistent with the obligatory cross-talk of NK cells with macrophages for their production of interferon-γ. CONCLUSION/SIGNIFICANCE: PfEMP1-independent but ICAM-1/LFA-1-dependent events occurring during NK cell activation by Pf highlight the fundamental role of cellular cooperation during innate immune response to malaria

Safety and Immunogenicity of Malaria Vectored Vaccines Given with Routine Expanded Program on Immunization Vaccines in Gambian Infants and Neonates: A Randomized Controlled Trial.

BACKGROUND: Heterologous prime-boost vaccination with chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) encoding multiple epitope string thrombospondin-related adhesion protein (ME-TRAP) has shown acceptable safety and promising immunogenicity in African adult and pediatric populations. If licensed, this vaccine could be given to infants receiving routine childhood immunizations. We therefore evaluated responses to ChAd63 MVA ME-TRAP when co-administered with routine Expanded Program on Immunization (EPI) vaccines. METHODS: We enrolled 65 Gambian infants and neonates, aged 16, 8, or 1 week at first vaccination and randomized them to receive either ME-TRAP and EPI vaccines or EPI vaccines only. Safety was assessed by the description of vaccine-related adverse events (AEs). Immunogenicity was evaluated using IFNγ enzyme-linked immunospot, whole-blood flow cytometry, and anti-TRAP IgG ELISA. Serology was performed to confirm all infants achieved protective titers to EPI vaccines. RESULTS: The vaccines were well tolerated in all age groups with no vaccine-related serious AEs. High-level TRAP-specific IgG and T cell responses were generated after boosting with MVA. CD8+ T cell responses, previously found to correlate with protection, were induced in all groups. Antibody responses to EPI vaccines were not altered significantly. CONCLUSION: Malaria vectored prime-boost vaccines co-administered with routine childhood immunizations were well tolerated. Potent humoral and cellular immunity induced by ChAd63 MVA ME-TRAP did not reduce the immunogenicity of co-administered EPI vaccines, supporting further evaluation of this regimen in infant populations. CLINICAL TRIAL REGISTRATION: The clinical trial was registered on http://Clinicaltrials.gov (NCT02083887) and the Pan-African Clinical Trials Registry (PACTR201402000749217)

Safety and Immunogenicity of ChAd63 and MVA ME-TRAP in West African Children and Infants.

Malaria remains a significant global health burden and a vaccine would make a substantial contribution to malaria control. Chimpanzee Adenovirus 63 Modified Vaccinia Ankara Multiple epitope thrombospondin adhesion protein (ME-TRAP) and vaccination has shown significant efficacy against malaria sporozoite challenge in malaria-naive European volunteers and against malaria infection in Kenyan adults. Infants are the target age group for malaria vaccination; however, no studies have yet assessed T-cell responses in children and infants. We enrolled 138 Gambian and Burkinabe children in four different age-groups: 2-6 years old in The Gambia; 5-17 months old in Burkina Faso; 5-12 months old, and also 10 weeks old, in The Gambia; and evaluated the safety and immunogenicity of Chimpanzee Adenovirus 63 Modified Vaccinia Ankara ME-TRAP heterologous prime-boost immunization. The vaccines were well tolerated in all age groups with no vaccine-related serious adverse events. T-cell responses to vaccination peaked 7 days after boosting with Modified Vaccinia Ankara, with T-cell responses highest in 10 week-old infants. Heterologous prime-boost immunization with Chimpanzee Adenovirus 63 and Modified Vaccinia Ankara ME-TRAP was well tolerated in infants and children, inducing strong T-cell responses. We identify an approach that induces potent T-cell responses in infants, which may be useful for preventing other infectious diseases requiring cellular immunity

Viral Vector Malaria Vaccines Induce High-Level T Cell and Antibody Responses in West African Children and Infants.

Heterologous prime-boosting with viral vectors encoding the pre-erythrocytic antigen thrombospondin-related adhesion protein fused to a multiple epitope string (ME-TRAP) induces CD8+ T cell-mediated immunity to malaria sporozoite challenge in European malaria-naive and Kenyan semi-immune adults. This approach has yet to be evaluated in children and infants. We assessed this vaccine strategy among 138 Gambian and Burkinabe children in four cohorts: 2- to 6-year olds in The Gambia, 5- to 17-month-olds in Burkina Faso, and 5- to 12-month-olds and 10-week-olds in The Gambia. We assessed induction of cellular immunity, taking into account the distinctive hematological status of young infants, and characterized the antibody response to vaccination. T cell responses peaked 7 days after boosting with modified vaccinia virus Ankara (MVA), with highest responses in infants aged 10 weeks at priming. Incorporating lymphocyte count into the calculation of T cell responses facilitated a more physiologically relevant comparison of cellular immunity across different age groups. Both CD8+ and CD4+ T cells secreted cytokines. Induced antibodies were up to 20-fold higher in all groups compared with Gambian and United Kingdom (UK) adults, with comparable or higher avidity. This immunization regimen elicited strong immune responses, particularly in young infants, supporting future evaluation of efficacy in this key target age group for a malaria vaccine