Antibodies from malaria-exposed pregnant women recognize trypsin resistant epitopes on the surface of Plasmodium falciparum-infected erythrocytes selected for adhesion to chondroitin sulphate A

BACKGROUND: The ability of Plasmodium falciparum-infected erythrocytes to adhere to the microvasculature endothelium is thought to play a causal role in malaria pathogenesis. Cytoadhesion to endothelial receptors is generally found to be highly sensitive to trypsinization of the infected erythrocyte surface. However, several studies have found that parasite adhesion to placental receptors can be markedly less sensitive to trypsin. This study investigates whether chondroitin sulphate A (CSA) binding parasites express trypsin-resistant variant surface antigens (VSA) that bind female-specific antibodies induced as a result of pregnancy associated malaria (PAM). METHODS: Fluorescence activated cell sorting (FACS) was used to measure the levels of adult Scottish and Ghanaian male, and Ghanaian pregnant female plasma immunoglobulin G (IgG) that bind to the surface of infected erythrocytes. P. falciparum clone FCR3 cultures were used to assay surface IgG binding before and after selection of the parasite for adhesion to CSA. The effect of proteolytic digestion of parasite erythrocyte surface antigens on surface IgG binding and adhesion to CSA and hyaluronic acid (HA) was also studied. RESULTS: P. falciparum infected erythrocytes selected for adhesion to CSA were found to express trypsin-resistant VSA that are the target of naturally acquired antibodies from pregnant women living in a malaria endemic region of Ghana. However in vitro adhesion to CSA and HA was relatively trypsin sensitive. An improved labelling technique for the detection of VSA expressed by CSA binding isolates has also been described. CONCLUSION: The VSA expressed by CSA binding P. falciparum isolates are currently considered potential targets for a vaccine against PAM. This study identifies discordance between the trypsin sensitivity of CSA binding and surface recognition of CSA selected parasites by serum IgG from malaria exposed pregnant women. Thus, the complete molecular definition of an antigenic P. falciparum erythrocyte surface protein that can be used as a malaria in pregnancy vaccine has not yet been achieved

Glucagon-like peptide-1 analogue, liraglutide, in experimental cerebral malaria: implications for the role of oxidative stress in cerebral malaria

BACKGROUND: Cerebral malaria from Plasmodium falciparum infection is major cause of death in the tropics. The pathogenesis of the disease is complex and the contribution of reactive oxygen and nitrogen species (ROS/RNS) in the brain is incompletely understood. Insulinotropic glucagon-like peptide-1 (GLP-1) mimetics have potent neuroprotective effects in animal models of neuropathology associated with ROS/RNS dysfunction. This study investigates the effect of the GLP-1 analogue, liraglutide against the clinical outcome of experimental cerebral malaria (ECM) and Plasmodium falciparum growth. Furthermore the role of oxidative stress on ECM pathogenesis is evaluated. METHODS: ECM was induced in Plasmodium berghei ANKA-infected C57Bl/6j mice. Infected Balb/c (non-cerebral malaria) and uninfected C57Bl/6j mice were included as controls. Mice were treated twice-daily with vehicle or liraglutide (200 μg/kg). ROS/RNS were quantified with in vivo imaging and further analyzed ex vivo. Brains were assayed for cAMP, activation of cAMP response element binding protein (CREB) and nitrate/nitrite. Plasmodium falciparum was cultivated in vitro with increasing doses of liraglutide and growth and metabolism were quantified. RESULTS: The development and progression of ECM was not affected by liraglutide. Indeed, although ROS/RNS were increased in peripheral organs, ROS/RNS generation was not present in the brain. Interestingly, CREB was activated in the ECM brain and may protect against ROS/RNS stress. Parasite growth was not adversely affected by liraglutide in mice or in P. falciparum cultures indicating safety should not be a concern in type-II diabetics in endemic regions. CONCLUSIONS: Despite the breadth of models where GLP-1 is neuroprotective, ECM was not affected by liraglutide providing important insight into the pathogenesis of ECM. Furthermore, ECM does not induce excess ROS/RNS in the brain potentially associated with activation of the CREB system

Analysis of IgG with specificity for variant surface antigens expressed by placental Plasmodium falciparum isolates

BACKGROUND: Pregnancy-associated malaria (PAM) is caused by Plasmodium falciparum-infected erythrocytes that can sequester in placental intervillous space by expressing particular variant surface antigens (VSA) that can mediate adhesion to chondroitin sulfate A (CSA) in vitro. IgG antibodies with specificity for the VSA expressed by these parasites (VSA(PAM)) are associated with protection from maternal anaemia, prematurity and low birth weight, which is the greatest risk factor for death in the first month of life. METHODS: In this study, the development of anti-VSA(PAM )antibodies in a group of 151 women who presented to the maternity ward of Albert Schweitzer Hospital in Lambaréné, Gabon for delivery was analysed using flow cytometry assays. Plasma samples from placenta infected primiparous women were also investigated for their capacity to inhibit parasite binding to CSA in vitro. RESULTS: In the study cohort, primiparous as well as secundiparous women had the greatest risk of infection at delivery as well as during pregnancy. Primiparous women with infected placentas at delivery showed higher levels of VSA(PAM)-specific IgG compared to women who had no malaria infections at delivery. Placental isolates of Gabonese and Senegalese origin tested on plasma samples from Gabon showed parity dependency and gender specificity patterns. There was a significant correlation of plasma reactivity as measured by flow cytometry between different placental isolates. In the plasma of infected primiparous women, VSA(PAM)-specific IgG measured by flow cytometry could be correlated with anti-adhesion antibodies measured by the inhibition of CSA binding. CONCLUSION: Recognition of placental parasites shows a parity- and sex- dependent pattern, like that previously observed in laboratory strains selected to bind to CSA. Placental infections at delivery in primiparous women appear to be sufficient to induce functional antibodies which can both recognize the surface of the infected erythrocytes as well as block their binding to CSA. The correlation between serum reactivities of placental field isolates from different geographic locations and collected at different times is indicative of the conserved nature of the antigen(s) mediating PAM

Expression of Plasmodium falciparum erythrocyte membrane protein 1 in experimentally infected humans

BACKGROUND: Parasites causing severe malaria in non-immune patients express a restricted subset of variant surface antigens (VSA), which are better recognized by immune sera than VSA expressed during non-severe disease in semi-immune individuals. The most prominent VSA are the var gene-encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family, which is expressed on the surface of infected erythrocytes where it mediates binding to endothelial receptors. Thus, severe malaria may be caused by parasites expressing PfEMP1 variants that afford parasites optimal sequestration in immunologically naïve individuals and high effective multiplication rates. METHODS: var gene transcription was analysed using real time PCR and PfEMP1 expression by western blots as well as immune plasma recognition of parasite cultures established from non-immune volunteers shortly after infection with NF54 sporozoites. RESULTS: In cultures representing the first generation of parasites after hepatic release, all var genes were transcribed, but GroupA var genes were transcribed at the lowest levels. In cultures established from second or third generation blood stage parasites of volunteers with high in vivo parasite multiplication rates, the var gene transcription pattern differed markedly from the transcription pattern of the cultures representing first generation parasites. This indicated that parasites expressing specific var genes, mainly belonging to group A and B, had expanded more effectively in vivo compared to parasites expressing other var genes. The differential expression of PfEMP1 was confirmed at the protein level by immunoblot analysis. In addition, serological typing showed that immune sera more often recognized second and third generation parasites than first generation parasites. CONCLUSION: In conclusion, the results presented here support the hypothesis that parasites causing severe malaria express a subset of PfEMP1, which bestows high parasite growth rates in individuals with limited pre-existing immunity

Plasmodium falciparum associated with severe childhood malaria preferentially expresses PfEMP1 encoded by group A var genes.

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

CD36 selection of 3D7 Plasmodium falciparum associated with severe childhood malaria results in reduced VAR4 expression

<p>Abstract</p> <p>Background</p> <p>A subset of the <it>Plasmodium falciparum </it>erythrocyte membrane protein 1 (PfEMP1_SM) is involved in the cytoadherence of <it>P. falciparum</it>-infected red blood cells (iRBC) contributing to the pathogenesis of severe disease among young children in malaria endemic areas. The PfEMP1_SMare encoded by group A <it>var </it>genes that are composed of a more constrained range of amino acid sequences than groups B and C <it>var </it>genes encoding PfEMP1_UMassociated with uncomplicated malaria. Also, unlike <it>var </it>genes from groups B and C, those from group A do not have sequences consistent with CD36 binding – a major cytoadhesion phenotype of <it>P. falciparum </it>isolates.</p> <p>Methods</p> <p>A 3D7 PfEMP1_SMsub-line (3D7_SM) expressing VAR4 (PFD1235w/MAL8P1.207) was selected for binding to CD36. The protein expression of this parasite line was monitored by surface staining of iRBC using VAR4-specific antibodies. The serological phenotype of the 3D7_SMparasites was determined by flow cytometry using malaria semi-immune and immune plasma and transcription of the 59 <it>var </it>genes in 3D7 were analysed by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) using <it>var</it>-specific primers.</p> <p>Results</p> <p>A selection-induced increased adhesion of 3D7_SMiRBC to CD36 resulted in a reduced <it>var4 </it>transcription and VAR4 surface expression.</p> <p>Conclusion</p> <p>VAR4 is not involved in CD36 adhesion. The current findings are consistent with the notion that CD36 adhesion is not associated with particular virulent parasite phenotypes, such as those believed to be exhibited by VAR4 expressing parasites.</p

Potential impact of host immunity on malaria treatment outcome in Tanzanian children infected with Plasmodium falciparum

<p>Abstract</p> <p>Background</p> <p>In malaria endemic areas children may recover from malaria after chemotherapy in spite of harbouring genotypically drug-resistant <it>Plasmodium falciparum</it>. This phenomenon suggests that there is a synergy between drug treatment and acquired immunity. This hypothesis was examined in an area of moderately intense transmission of <it>P. falciparum </it>in Tanzania during a drug trail with sulphadoxine-pyrimethamine (SP) or amodiaquine (AQ).</p> <p>Methods</p> <p>One hundred children with uncomplicated malaria were treated with either SP or AQ and followed for 28 days. Mutations in parasite genes related to SP and AQ-resistance as well as human sickle cell trait and alpha-thalassaemia were determined using PCR and sequence-specific oligonucleotide probes and enzyme-linked immunosorbent assay (SSOP-ELISA), and IgG antibody responses to a panel of <it>P. falciparum </it>antigens were assessed and related to treatment outcome.</p> <p>Results</p> <p>Parasitological or clinical treatment failure (TF) was observed in 68% and 38% of children receiving SP or AQ, respectively. In those with adequate clinical and parasitological response (ACPR) compared to children with TF, and for both treatment regimens, prevalence and levels of anti-Glutamate-rich Protein (GLURP)-specific IgG antibodies were significantly higher (P < 0.001), while prevalence of parasite haplotypes associated with SP and AQ resistance was lower (P = 0.02 and P = 0.07, respectively). Interestingly, anti-GLURP-IgG antibodies were more strongly associated with treatment outcome than parasite resistant haplotypes, while the IgG responses to none of the other 11 malaria antigens were not significantly associated with ACPR.</p> <p>Conclusion</p> <p>These findings suggest that GLURP-specific IgG antibodies in this setting contribute to clearance of drug-resistant infections and support the hypothesis that acquired immunity enhances the clinical efficacy of drug therapy. The results should be confirmed in larger scale with greater sample size and with variation in transmission intensity.</p