Efficacy of 3D screens for sustainable mosquito control: a semi-field experimental hut evaluation in northeastern Tanzania.

BACKGROUND: A three-dimensional window screen (3D-Screen) has been developed to create a window double-screen trap (3D-WDST), effectively capturing and preventing the escape of mosquitoes. A 2015 laboratory study demonstrated the 3D-Screen's efficacy, capturing 92% of mosquitoes in a double-screen setup during wind tunnel assays. To further evaluate its effectiveness, phase II experimental hut trials were conducted in Muheza, Tanzania. METHODS: Three experimental hut trials were carried out between 2016 and 2017. Trial I tested two versions of the 3D-WDST in huts with open or closed eaves, with one version using a single 3D-Screen and the other using two 3D-Screens. Trial II examined the 3D-WDST with two 3D-Screens in huts with or without baffles, while Trial III compared handmade and machine-made 3D structures. Mosquito capturing efficacy of the 3D-WDST was measured by comparing the number of mosquitoes collected in the test hut to a control hut with standard exit traps. RESULTS: Trial I showed that the 3D-WDST with two 3D-Screens used in huts with open eaves achieved the highest mosquito-capturing efficacy. This treatment captured 33.11% (CI 7.40-58.81) of female anophelines relative to the total collected in this hut (3D-WDST and room collections) and 27.27% (CI 4.23-50.31) of female anophelines relative to the total collected in the control hut (exit traps, room, and verandahs collections). In Trial II, the two 3D-Screens version of the 3D-WDST captured 70.32% (CI 56.87-83.77) and 51.07% (CI 21.72-80.41) of female anophelines in huts with and without baffles, respectively. Compared to the control hut, the capturing efficacy for female anophelines was 138.6% (37.23-239.9) and 42.41% (14.77-70.05) for huts with and without baffles, respectively. Trial III demonstrated similar performance between hand- and machine-made 3D structures. CONCLUSIONS: The 3D-WDST proved effective in capturing malaria vectors under semi-field experimental hut conditions. Using 3D-Screens on both sides of the window openings was more effective than using a single-sided 3D-Screen. Additionally, both hand- and machine-made 3D structures exhibited equally effective performance, supporting the production of durable cones on an industrial scale for future large-scale studies evaluating the 3D-WDST at the community level.

Intranasal administration of adenoviral vaccines expressing SARS-CoV-2 spike protein improves vaccine immunity in mouse models.

The ongoing SARS-CoV-2 pandemic is controlled but not halted by public health measures and mass vaccination strategies which have exclusively relied on intramuscular vaccines. Intranasal vaccines can prime or recruit to the respiratory epithelium mucosal immune cells capable of preventing infection. Here we report a comprehensive series of studies on this concept using various mouse models, including HLA class II-humanized transgenic strains. We found that a single intranasal (i.n.) dose of serotype-5 adenoviral vectors expressing either the receptor binding domain (Ad5-RBD) or the complete ectodomain (Ad5-S) of the SARS-CoV-2 spike protein was effective in inducing i) serum and bronchoalveolar lavage (BAL) anti-spike IgA and IgG, ii) robust SARS-CoV-2-neutralizing activity in the serum and BAL, iii) rigorous spike-directed T helper 1 cell/cytotoxic T cell immunity, and iv) protection of mice from a challenge with the SARS-CoV-2 beta variant. Intramuscular (i.m.) Ad5-RBD or Ad5-S administration did not induce serum or BAL IgA, and resulted in lower neutralizing titers in the serum. Moreover, prior immunity induced by an intramuscular mRNA vaccine could be potently enhanced and modulated towards a mucosal IgA response by an i.n. Ad5-S booster. Notably, Ad5 DNA was found in the liver or spleen after i.m. but not i.n. administration, indicating a lack of systemic spread of the vaccine vector, which has been associated with a risk of thrombotic thrombocytopenia. Unlike in otherwise genetically identical HLA-DQ6 mice, in HLA-DQ8 mice Ad5-RBD vaccine was inferior to Ad5-S, suggesting that the RBD fragment does not contain a sufficient collection of helper-T cell epitopes to constitute an optimal vaccine antigen. Our data add to previous promising preclinical results on intranasal SARS-CoV-2 vaccination and support the potential of this approach to elicit mucosal immunity for preventing transmission of SARS-CoV-2.

Hijacking the human complement inhibitor C4b-binding protein by the sporozoite stage of the Plasmodium falciparum parasite.

The complement system is considered the first line of defense against pathogens. Hijacking complement regulators from blood is a common evasion tactic of pathogens to inhibit complement activation on their surfaces. Here, we report hijacking of the complement C4b-binding protein (C4bp), the regulator of the classical and lectin pathways of complement activation, by the sporozoite (SPZ) stage of the Plasmodium falciparum parasite. This was shown by direct binding of radiolabeled purified C4bp to live SPZs as well as by binding of C4bp from human serum to SPZs in indirect immunofluorescence assays. Using a membrane-bound peptide array, peptides from the N-terminal domain (NTD) of P. falciparum circumsporozoite protein (CSP) were found to bind C4bp. Soluble biotinylated peptide covering the same region on the NTD and a recombinantly expressed NTD also bound C4bp in a dose-dependent manner. NTD-binding site on C4bp was mapped to the CCP1-2 of the C4bp α-chain, a common binding site for many pathogens. Native CSP was also co-immunoprecipitated with C4bp from human serum. Preventing C4bp binding to the SPZ surface negatively affected the SPZs gliding motility in the presence of functional complement and malaria hyperimmune IgG confirming the protective role of C4bp in controlling complement activation through the classical pathway on the SPZ surface. Incorporating the CSP-C4bp binding region into a CSP-based vaccine formulation could induce vaccine-mediated immunity that neutralizes this immune evasion region and increases the vaccine efficacy.

CHANDELIER-ASSISTED VERSUS STANDARD SCLERAL BUCKLING FOR PRIMARY RHEGMATOGENOUS RETINAL DETACHMENT: A Randomized Clinical Study.

PURPOSE: To compare anatomical outcomes, functional outcomes, and rate of complications of standard scleral buckling (SSB) versus chandelier-assisted scleral buckling (CSB) in phakic eyes with rhegmatogenous retinal detachment. METHODS: Patients were randomly assigned to either SSB or CSB. Surgical success/failure rate, corrected distance visual acuity, surgical operating time, and rate of intraoperative and postoperative complications including epiretinal membranes by spectral domain optical coherence tomography were compared between groups. RESULTS: A total of 50 eyes of 49 patients were included. At 6 months, there was no statistically significant difference between groups in primary success, or final anatomical success ( P > 0.9); mean corrected distance visual acuity at any visit ( P values >0.05); or mean surgical time: 120.3 ± 39.05 and 102.48 ± 43.76 minutes for the SSB and CSB, respectively ( P = 0.1). The CSB had a higher rate of postoperative complications (34.8%) compared with the SSB (3.8%) ( P < 0.05). On spectral domain optical coherence tomography, CSB had a statistically significant higher rate of epiretinal membranes compared with SSB (44% vs. 19% [ P < 0.05]) and showed vitreous entrapment in the chandelier sclerotomy site on the ultrasonic biomicroscopy. CONCLUSION: Chandelier-assisted scleral buckling surgery does not offer encouraging advantages over SSB. On the contrary, we detected a higher rate of complications with CSB especially epiretinal membranes development.

Long-term changes in ocular rigidity following scleral buckling for rhegmatogenous retinal detachment.

PURPOSE: To investigate the long-term effect of scleral buckling on corneal biomechanics and the effect of change of scleral properties on intraocular pressure (IOP) measurements. METHODS: This is a prospective case series, patients with rhegmatogenous retinal detachment prepared for scleral buckling were included. Goldmann applanation tonometry was used to measure IOP (GAT IOP). Ocular Response Analyzer (ORA) was used to measure corneal hysteresis (CH), corneal resistance factor (CRF), goldmann-corrected IOP (IOPg), and corneal-compensated IOP (IOPcc) preoperatively, and 1, 3, and 6 months postoperatively. RESULTS: Thirty-three eyes included in the final analysis, with an average age 38.4 ± 16.2 years. CH and CRF decreased significantly at first, third, sixth months post-scleral buckling; however, this effect decreased with time as follows; preoperative: 8.9 ± 1.5 and 8.5 ± 2.1, first month: 6.8 ± 1.6 and 7.1 ± 1.8 (P value = 0.00, 0.002), third month: 7.8 ± 1.5 and 7.6 ± 1.6 (P value = 0.001, 0.008), and sixth month: 7.7 ± 1.3 and 7.6 ± 1.7 (P value = 0.002, 0.055). IOP cc was 19.3 ± 3.6, 17.1 ± 4, and 17.6 ± 2.9 at 1, 3, and 6 months, and these readings were significantly higher than GAT (13.6 ± 7.6, 12.4 ± 5.1, and 12.1 ± 2.9, P values = 0.00) and IOPg (14.9 ± 3.6, 13.5 ± 4.1, and 13.9 ± 3.5, P values = 0.00). The change in CH at each visit is correlated with the difference between the IOPcc and GAT measurements. CONCLUSION: The conventional Goldmann applanation tonometry underestimates post buckle IOP measurements due corneal biomechanics changes. ORA might be an alternative and accurate method of measurement; however, further investigation is warranted.

Exploration of Pyrazolo[1,5-a]pyrimidines as Membrane-Bound Pyrophosphatase Inhibitors.

Inhibition of membrane-bound pyrophosphatase (mPPase) with small molecules offer a new approach in the fight against pathogenic protozoan parasites. mPPases are absent in humans, but essential for many protists as they couple pyrophosphate hydrolysis to the active transport of protons or sodium ions across acidocalcisomal membranes. So far, only few nonphosphorus inhibitors have been reported. Here, we explore the chemical space around previous hits using a combination of screening and synthetic medicinal chemistry, identifying compounds with low micromolar inhibitory activities in the Thermotoga maritima mPPase test system. We furthermore provide early structure-activity relationships around a new scaffold having a pyrazolo[1,5-a]pyrimidine core. The most promising pyrazolo[1,5-a]pyrimidine congener was further investigated and found to inhibit Plasmodium falciparum mPPase in membranes as well as the growth of P. falciparum in an ex vivo survival assay.

Safety and immunogenicity evaluation of inactivated whole-virus-SARS-COV-2 as emerging vaccine development in Egypt.

BACKGROUND: Current worldwide pandemic coronavirus disease 2019 (COVID-19) with high numbers of mortality rates and huge economic problems require an urgent demand for safe and effective vaccine development. Inactivated SARS-CoV2 vaccine with alum. Hydroxide can play an important role in reducing the impacts of the COVID-19 pandemic. In this study, vaccine efficacy was evaluated through the detection of the neutralizing antibodies that protect mice from challenge with SARS-CoV 2 3 weeks after the second dose. We conclude that the vaccine described here has safety and desirable properties, and our data support further development and plans for clinical trials. METHODS: Characterized SARS-COV-2 strain, severe acute respiratory syndrome coronavirus 2 isolates (SARS-CoV-2/human/EGY/Egy-SERVAC/2020) with accession numbers; MT981440; MT981439; MT981441; MT974071; MT974069; and MW250352 at GenBank were isolated from Egyptian patients SARS-CoV-2-positive. Development of inactivated vaccine was carried out in a BSL-3 facilities and the immunogenicity was determined in mice at two doses (55 and 100 µg per dose). RESULTS: The distinct cytopathic effect induced by SARS-COV-2 propagation on Vero cell monolayers and the viral particles were identified as Coronaviridae by transmission electron microscopy and RT-PCR on infected cells cultures. Immunogenicity of the developed vaccine indicated the high antigen-binding and neutralizing antibody titers, regardless of the dose concentration, with excellent safety profiles and no deaths or clinical symptoms in mice groups. The efficacy of the inactivated vaccine formulation was tested by the wild virus challenge of the vaccinated mice and viral replication detection in lung tissues. CONCLUSIONS: Vaccinated mice recorded complete protection from challenge infection via inhibition of SARS-COV-2 replication in the lung tissues of mice following virus challenge, regardless of the level of serum neutralizing antibodies. This finding will support future trials for the evaluation of an applicable SARS-CoV-2 vaccine candidate.

Genomic characterization of SARS-CoV-2 in Egypt.

Introduction: The novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread throughout the globe, causing a pandemic. In Egypt over 115,000 individuals were infected so far. Objective: In the present study, the objective is to perform a complete genome sequence of SAR-CoV2 isolated from Egyptian coronavirus disease (COVID-19) patients. Methods: Nasopharyngeal swabs were collected from 61 COVID-19 patients who attended at National Cancer Institute, Kasr Al-Aini Hospital and the army hospital. Viral RNA was extracted and whole genomic sequencing was conducted using Next Generation Sequencing. Results: In all cases, the sequenced virus has at least 99% identity to the reference Wuhan 1. The sequence analysis showed 204 distinct genome variations including 114 missense mutations, 72 synonymous mutations, 1 disruptive in-frame deletion, 7 downstream gene mutations, 6 upstream gene mutations, 3 frame-shift deletions, and 1 in-frame deletion. The most dominant clades were G/GH/GR/O and the dominant type is B. Conclusion: The whole genomic sequence of SARS-CoV2 showed 204 variations in the genomes of the Egyptian isolates, where the Asp614Gly (D614G) substitution is the most common among the samples (60/61). So far, there were no strikingly variations specific to the Egyptian population, at least for this set of samples.

Discovery of Membrane-Bound Pyrophosphatase Inhibitors Derived from an Isoxazole Fragment.

Membrane-bound pyrophosphatases (mPPases) regulate energy homeostasis in pathogenic protozoan parasites and lack human homologues, which makes them promising targets in e.g. malaria. Yet only few nonphosphorus inhibitors have been reported so far. Here, we explore an isoxazole fragment hit, leading to the discovery of small mPPase inhibitors with 6-10 µM IC50 values in the Thermotoga maritima test system. Promisingly, the compounds retained activity against Plasmodium falciparum mPPase in membranes and inhibited parasite growth.

Complement in malaria: immune evasion strategies and role in protective immunity.

The malaria parasite has for long been thought to escape host complement attack as a survival strategy. However, it was only recently that complement evasion mechanisms of the parasite were described. Simultaneously, the role of complement in antibody-mediated naturally acquired and vaccine-induced protection against malaria has also been reported. Such findings should be considered in future vaccine design, given the current need to develop more efficacious vaccines against malaria. Parasite antigens derived from molecules mediating functions crucial for parasite survival, such as complement evasion, or parasite antigens against which antibody responses lead to an efficient complement attack could present new candidates for vaccines. In this review, we discuss recent findings on complement evasion by the malaria parasites and the emerging role of complement in antibody-mediated protection against malaria. We emphasize that immune responses to vaccines based on complement inhibitors should not only induce complement-activating antibodies but also neutralize the escape mechanisms of the parasite.

Asymmetry in catalysis by Thermotoga maritima membrane-bound pyrophosphatase demonstrated by a nonphosphorus allosteric inhibitor.

Membrane-bound pyrophosphatases are homodimeric integral membrane proteins that hydrolyze pyrophosphate into orthophosphates, coupled to the active transport of protons or sodium ions across membranes. They are important in the life cycle of bacteria, archaea, plants, and parasitic protists, but no homologous proteins exist in vertebrates, making them a promising drug target. Here, we report the first nonphosphorus allosteric inhibitor of the thermophilic bacterium Thermotoga maritima membrane-bound pyrophosphatase and its bound structure together with the substrate analog imidodiphosphate. The unit cell contains two protein homodimers, each binding a single inhibitor dimer near the exit channel, creating a hydrophobic clamp that inhibits the movement of ß-strand 1-2 during pumping, and thus prevents the hydrophobic gate from opening. This asymmetry of inhibitor binding with respect to each homodimer provides the first clear structural demonstration of asymmetry in the catalytic cycle of membrane-bound pyrophosphatases.

3D mosquito screens to create window double screen traps for mosquito control.

BACKGROUND: Mosquitoes are vectors for many diseases such as malaria. Insecticide-treated bed nets and indoor residual spraying of insecticides are the principal malaria vector control tools used to prevent malaria in the tropics. Other interventions aim at reducing man-vector contact. For example, house screening provides additive or synergistic effects to other implemented measures. We used commercial screen materials made of polyester, polyethylene or polypropylene to design novel mosquito screens that provide remarkable additional benefits to those commonly used in house screening. The novel design is based on a double screen setup made of a screen with 3D geometric structures parallel to a commercial mosquito screen creating a trap between the two screens. Owing to the design of the 3D screen, mosquitoes can penetrate the 3D screen from one side but cannot return through the other side, making it a unidirectional mosquito screen. Therefore, the mosquitoes are trapped inside the double screen system. The permissiveness of both sides of the 3D screens for mosquitoes to pass through was tested in a wind tunnel using the insectary strain of Anopheles stephensi. RESULTS: Among twenty-five tested 3D screen designs, three designs from the cone, prism, or cylinder design groups were the most efficient in acting as unidirectional mosquito screens. The three cone-, prism-, and cylinder-based screens allowed, on average, 92, 75 and 64% of Anopheles stephensi mosquitoes released into the wind tunnel to penetrate the permissive side and 0, 0 and 6% of mosquitoes to escape through the non-permissive side, respectively. CONCLUSIONS: A cone-based 3D screen fulfilled the study objective. It allowed capturing 92% of mosquitoes within the double screen setup inside the wind tunnel and blocked 100% from escaping. Thus, the cone-based screen effectively acted as a unidirectional mosquito screen. This 3D screen-based trap design could therefore be used in house screening as a means of avoiding infective bites and reducing mosquito population size.

Anopheles midgut epithelium evades human complement activity by capturing factor H from the blood meal.

Hematophagous vectors strictly require ingesting blood from their hosts to complete their life cycles. Exposure of the alimentary canal of these vectors to the host immune effectors necessitates efficient counteractive measures by hematophagous vectors. The Anopheles mosquito transmitting the malaria parasite is an example of hematophagous vectors that within seconds can ingest human blood double its weight. The innate immune defense mechanisms, like the complement system, in the human blood should thereby immediately react against foreign cells in the mosquito midgut. A prerequisite for complement activation is that the target cells lack complement regulators on their surfaces. In this work, we analyzed whether human complement is active in the mosquito midgut, and how the mosquito midgut cells protect themselves against complement attack. We found that complement remained active for a considerable time and was able to kill microbes within the mosquito midgut. However, the Anopheles mosquito midgut cells were not injured. These cells were found to protect themselves by capturing factor H, the main soluble inhibitor of the alternative complement pathway. Factor H inhibited complement on the midgut cells by promoting inactivation of C3b to iC3b and preventing the activity of the alternative pathway amplification C3 convertase enzyme. An interference of the FH regulatory activity by monoclonal antibodies, carried to the midgut via blood, resulted in increased mosquito mortality and reduced fecundity. By using a ligand blotting assay, a putative mosquito midgut FH receptor could be detected. Thereby, we have identified a novel mechanism whereby mosquitoes can tolerate human blood.

A switch in infected erythrocyte deformability at the maturation and blood circulation of Plasmodium falciparum transmission stages.

Achievement of malaria elimination requires development of novel strategies interfering with parasite transmission, including targeting the parasite sexual stages (gametocytes). The formation of Plasmodium falciparum gametocytes in the human host takes several days during which immature gametocyte-infected erythrocytes (GIEs) sequester in host tissues. Only mature stage GIEs circulate in the peripheral blood, available to uptake by the Anopheles vector. Mechanisms underlying GIE sequestration and release in circulation are virtually unknown. We show here that mature GIEs are more deformable than immature stages using ektacytometry and microsphiltration methods, and that a switch in cellular deformability in the transition from immature to mature gametocytes is accompanied by the deassociation of parasite-derived STEVOR proteins from the infected erythrocyte membrane. We hypothesize that mechanical retention contributes to sequestration of immature GIEs and that regained deformability of mature gametocytes is associated with their release in the bloodstream and ability to circulate. These processes are proposed to play a key role in P falciparum gametocyte development in the host and to represent novel and unconventional targets for interfering with parasite transmission.

Exposure of the Plasmodium falciparum clonally variant STEVOR proteins on the merozoite surface.

BACKGROUND: Plasmodium falciparum merozoites are free invasive forms that invade host erythrocytes in iterative cycles in the presence of different arms of the immune system. Variant antigens are known to play a role in immune evasion and several gene families coding for variant antigens have been identified in P. falciparum. However, none of them have been reported to be expressed on the surface of merozoites. METHODS: Flow cytometry, immunofluorescence microscopy, and immunoblotting assays were performed to assess surface exposure, membrane association and stage specific expression of the STEVOR family of variants proteins, respectively. RESULTS: Using a polyclonal antibody (anti-PFL2610w) with a broad specificity towards different STEVOR variants, the STEVOR proteins were identified on the surface of non-permeabilized/non-fixed merozoites in flow cytometry assays. Anti-PFL2610w antibody showed that several STEVORs were expressed in the trophozoite stage of the parasite but only one variant was integrated into the merozoite membrane. Moreover, this antibody failed to identify STEVORs on the surface of the parent schizont infected erythrocytes (IE) although they were readily identified when schizont IE were permeabilized. CONCLUSIONS: These data suggest for a role for STEVOR in immune evasion by P. falciparum merozoites to allow successful invasion of erythrocytes. Additionally, the expression of STEVORs in the schizont stage may only represent a step in the biogenesis process of the merozoite surface coat.

Plasmodium falciparum variant STEVOR antigens are expressed in merozoites and possibly associated with erythrocyte invasion.

BACKGROUND: Plasmodium falciparum STEVOR proteins, encoded by the multicopy stevor gene family have no known biological functions. Their expression and unique locations in different parasite life cycle stages evoke multiple functionalities. Their abundance and hypervariability support a role in antigenic variation. METHODS: Immunoblotting of total parasite proteins with an anti-STEVOR antibody was used to identify variant antigens of this gene family and to follow changes in STEVOR expression in parasite populations panned on CSA or CD36 receptors. Immunofluorescence assays and immunoelectron microscopy were performed to study the subcellular localization of STEVOR proteins in different parasite stages. The capacity of the antibody to inhibit merozoite invasion of erythrocytes was assessed to determine whether STEVOR variants were involved in the invasion process. RESULTS: Antigenic variation of STEVORs at the protein level was observed in blood stage parasites. STEVOR variants were found to be present on the merozoite surface and in rhoptries. An insight into a participation in erythrocyte invasion was gained through an immunofluorescence analysis of a sequence of thin slides representing progressive steps in erythrocyte invasion. An interesting feature of the staining pattern was what appeared to be the release of STEVORs around the invading merozoites. Because the anti-STEVOR antibody did not inhibit invasion, the role of STEVORs in this process remains unknown. CONCLUSION: The localization of STEVOR proteins to the merozoite surface and the rhoptries together with its prevalence as a released component in the invading merozoite suggest a role of these antigens in adhesion and/or immune evasion in the erythrocyte invasion process. These observations would also justify STEVORs for undergoing antigenic variation. Even though a role in erythrocyte invasion remains speculative, an association of members of the STEVOR protein family with invasion-related events has been shown.

Variant proteins of the Plasmodium falciparum RIFIN family show distinct subcellular localization and developmental expression patterns.

In order to avoid immune recognition in favor of a chronic infection, the malaria parasite Plasmodium falciparum has developed means to express clonally variant antigens at the surface of the infected erythrocyte (IE). Proteins of the var and rif multicopy gene families, encoding PfEMP1 and RIFINs, respectively, have been implicated in these processes. Here, we studied members of the latter family and present data revealing different subcellular localization patterns for RIFIN variants belonging to two distinct subgroups, which have been designated A- and B-type RIFINs. While A-type RIFINs were found to be associated with the parasite and transported to the surface of infected erythrocytes via Maurer's clefts, B-type RIFINs appeared to be mostly retained inside the parasite. However, expression of both subtypes does not seem to be mutually exclusive. Moreover, both A- and B-type variants were also expressed in the merozoite, present either in the apical region (A-type) or in the cytosol (B-type). The presence of RIFINs in merozoites suggests that antigenic variation in P. falciparum is not only restricted to parasite-derived proteins at the IE surface, but the phenomenon also prevails in other life cycle stages. Interestingly, some RIFIN variants were detected only in intracellular stages and not in merozoites, pointing to differential developmental expression patterns for distinct members of this large protein family.

The impact of IgG antibodies to recombinant Plasmodium falciparum 732var CIDR-1alpha domain in mothers and their newborn babies.

Different domains of a novel full-length var gene (termed 732var) isolated from a placenta of a malaria-infected woman were expressed in Escherichia coli as recombinant proteins and analysed biochemically and immunologically. Two of these, the Duffy binding-like (DBL)-3gamma domain and the cysteine-rich interdomain region (CIDR)-1alpha were able to bind chondroitin sulfate A and CD36, respectively. The DBL-3gamma domain was investigated in a previous study and confirmed here to exhibit anti-disease characteristics related to pregnancy-associated malaria. Mothers with high anti-DBL-3gamma antibody levels were protected from placental infection. The novel finding in this study is that babies born to mothers carrying anti-CIDR-1alpha antibodies had a delayed time to the first infection.

Receptor-binding studies of the DBLgamma domain of Plasmodium falciparum erythrocyte membrane protein 1 from a placental isolate.

We have previously identified a number of DBLgamma domains in Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) transcripts obtained from placental parasite isolates, showing that they bind specifically to chondroitin sulfate A (CSA) (Khattab A, Kun J, Deloron P, Kremsner PG, Klinkert MQ. Variants of Plasmodium falciparum erythrocyte membrane protein 1 expressed by different placental parasites are closely related and adhere to chondroitin sulfate A. J Infect Dis 2001;183:1165-9). Here we give a more detailed physico-chemical and binding characterisation of the soluble, recombinant DBLgamma domain derived from one of these isolates. Results from circular dichroism and limited proteolysis experiments are consistent with the recombinant domain being expressed with the native fold. Specific binding of DBLgamma to placental cryosections was demonstrated by labeling with antibodies raised against the recombinant domain; binding was diminished after treatment of the cryosections with chondroitinase or by blocking with anti-CSA antibody, showing that CSA mediates the interaction. Binding of the DBLgamma domain to purified placental chondroitin sulfate proteoglycan (CSPG) was also studied using surface plasmon resonance techniques, with DBLgamma as analyte and CSPG immobilised on the sensor chip; these quantitative measurements gave an affinity constant in the mu-molar range under the conditions used. The native conformation of the DBLgamma domain is essential for CSPG recognition since binding to the sensor chip is abolished when the protein is irreversibly reduced. As with the placental cryosections, association was significantly reduced after treating the immobilised CSPG with chondroitinase. Together, these results demonstrate specific interaction between the DBLgamma domain and the placental receptor.

Maurer's clefts-restricted localization, orientation and export of a Plasmodium falciparum RIFIN.

RIFINs are clonally variant antigens expressed in Plasmodium falciparum. Transfection and the green fluorescence protein (GFP) tagged either internally or C-terminally to the 3D7 PFI0050c RIFIN gene product were used to investigate protein localization, orientation and trafficking. Green fluorescence pattern emerging from live transfectant parasites expressing each of the RIFIN-GFP chimera was different. The internally GFP-tagged protein was exported to Maurer's clefts (MC) in the erythrocyte cytosol, whereas the C-terminally GFP-tagged full-length RIFIN chimera was not trafficked out of the parasite. Interestingly, when some RIFIN-specific C-terminal amino acid sequences were removed, the resulting truncated molecule reached the MC. Using anti-RIFIN and anti-GFP antibodies to probe both live and fixed transfectants, staining was confined to MC and was not detected on the erythrocyte surface, a location previously suggested for this protein family. From selective permeabilization experiments, the highly variable portion of the RIFIN-GFP-insertion chimera appeared to be exposed to the erythrocyte cytosol, presumably anchored in the MC membrane via the two transmembrane domains. Trafficking of both chimeras in young ring stages was sensitive to Brefeldin A (BFA), although older rings showed differential sensitivity to BFA.