The Quantum Cosmological Wavefunction at Very Early Times for a Quadratic Gravity Theory

The quantum cosmological wavefunction for a quadratic gravity theory derived from the heterotic string effective action is obtained near the inflationary epoch and during the initial Planck era. Neglecting derivatives with respect to the scalar field, the wavefunction would satisfy a third-order differential equation near the inflationary epoch which has a solution that is singular in the scale factor limit $a(t)\to 0$. When scalar field derivatives are included, a sixth-order differential equation is obtained for the wavefunction and the solution by Mellin transform is regular in the $a\to 0$ limit. It follows that inclusion of the scalar field in the quadratic gravity action is necessary for consistency of the quantum cosmology of the theory at very early times.Comment: Tex, 13 page

Sub-grouping of Plasmodium falciparum 3D7 var genes based on sequence analysis of coding and non-coding regions

BACKGROUND: The variant surface antigen family Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) is an important target for protective immunity and is implicated in the pathology of malaria through its ability to adhere to host endothelial receptors. The sequence diversity and organization of the 3D7 PfEMP1 repertoire was investigated on the basis of the complete genome sequence. METHODS: Using two tree-building methods we analysed the coding and non-coding sequences of 3D7 var and rif genes as well as var genes of other parasite strains. RESULTS: var genes can be sub-grouped into three major groups (group A, B and C) and two intermediate groups B/A and B/C representing transitions between the three major groups. The best defined var group, group A, comprises telomeric genes transcribed towards the telomere encoding PfEMP1s with complex domain structures different from the 4-domain type dominant of groups B and C. Two sequences belonging to the var1 and var2 subfamilies formed independent groups. A rif subgroup transcribed towards the centromere was found neighbouring var genes of group A such that the rif and var 5' regions merged. This organization appeared to be unique for the group A var genes CONCLUSION: The grouping of var genes implies that var gene recombination preferentially occurs within var gene groups and it is speculated that the groups reflect a functional diversification evolved to cope with the varying conditions of transmission and host immune response met by the parasite

A versatile, high through-put, bead-based phagocytosis assay for Plasmodium falciparum

Abstract Antibody-mediated phagocytosis is an important immune effector mechanism against Plasmodium falciparum-infected erythrocytes (IE); however, current phagocytosis assays use IE collected from infected individuals or from in vitro cultures of P. falciparum, making them prone to high variation. A simple, high-throughput flow cytometric assay was developed that uses THP-1 cells and fluorescent beads covalently-coupled with the malarial antigen VAR2CSA. The assay is highly repeatable, provides both the overall percent phagocytosis and semi-quantitates the number of antigen-coupled beads internalized

A method for visualizing surface-exposed and internal PfEMP1 adhesion antigens in Plasmodium falciparum infected erythrocytes

<p>Abstract</p> <p>Background</p> <p>The insertion of parasite antigens into the host erythrocyte membrane and the structure and distribution of <it>Plasmodium falciparum </it>adhesion receptors on that membrane are poorly understood. Laser scanning confocal microscopy (LSCM) and a novel labelling and fixation method have been used to obtain high resolution immuno-fluorescent images of erythrocyte surface PfEMP1 and internal antigens which allow analysis of the accumulation of PfEMP1 on the erythrocyte membrane during asexual development.</p> <p>Methods</p> <p>A novel staining technique has been developed which permits distinction between erythrocyte surface PfEMP1 and intracellular PfEMP1, in parasites whose nuclear material is exceptionally well resolved. Primary antibody detection by fluorescence is carried out on the live parasitized erythrocyte. The surface labelled cells are then fixed using paraformaldehyde and permeabilized with a non-ionic detergent to permit access of antibodies to internal parasite antigens. Differentiation between surface and internal antigens is achieved using antibodies labelled with different fluorochromes and confocal microscopy</p> <p>Results</p> <p>Surface exposed PfEMP1 is first detectable by antibodies at the trophozoite stage of intracellular parasite development although the improved detection method indicates that there are differences between different laboratory isolates in the kinetics of accumulation of surface-exposed PfEMP1.</p> <p>Conclusion</p> <p>A sensitive method for labelling surface and internal PfEMP1 with up to three different fluorochromes has been developed for laser scanning confocal optical microscopy and the analysis of the developmental expression of malaria adhesion antigens.</p

Structural Insight into Epitopes in the Pregnancy-Associated Malaria Protein VAR2CSA

Pregnancy-associated malaria is caused by Plasmodium falciparum malaria parasites binding specifically to chondroitin sulfate A in the placenta. This sequestration of parasites is a major cause of low birth weight in infants and anemia in the mothers. VAR2CSA, a polymorphic multi-domain protein of the PfEMP1 family, is the main parasite ligand for CSA binding, and identification of protective antibody epitopes is essential for VAR2CSA vaccine development. Attempts to determine the crystallographic structures of VAR2CSA or its domains have not been successful yet. In this study, we propose 3D models for each of the VAR2CSA DBL domains and we show that regions in the fold of VAR2CSA inter-domain 2 and a PfEMP1 CIDR domain seem to be homologous to the EBA-175 and Pkα-DBL fold. This suggests that ID2 could be a functional domain. We also identify regions of VAR2CSA present on the surface of native VAR2CSA by comparing reactivity of plasma containing anti-VAR2CSA antibodies in peptide array experiments before and after incubation with native VAR2CSA. By this method we identify conserved VAR2CSA regions targeted by antibodies that react with the native molecule expressed on infected erythrocytes. By mapping the data onto the DBL models we present evidence suggesting that the S1+S2 DBL sub-domains are generally surface-exposed in most domains, whereas the S3 sub-domains are less exposed in native VAR2CSA. These results comprise an important step towards understanding the structure of VAR2CSA on the surface of CSA-binding infected erythrocytes

Antibodies to full-length and the DBL5 domain of VAR2CSA in pregnant women after long-term implementation of intermittent preventive treatment in Etoudi, Cameroon

In high malaria transmission settings, the use of sulfadoxine-pyrimethamine-based intermittent preventive treatment during pregnancy (IPTp-SP) has resulted in decreased antibody (Ab) levels to VAR2CSA. However, information of Ab levels in areas of low or intermediate malaria transmission after long-term implementation of IPTp-SP is still lacking. The present study sought to evaluate antibody prevalence and levels in women at delivery in Etoudi, a peri-urban area in the capital of Yaoundé, Cameroon, that is a relatively low-malaria transmission area. Peripheral plasma samples from 130 pregnant women were collected at delivery and tested for IgG to the full-length recombinant VAR2CSA (FV2) and its most immunogenic subdomain, DBL5. The study was conducted between 2013 and 2015, approximately ten years after implementation of IPTp-SP in Cameroon. About 8.6% of the women attending the clinic had placental malaria (PM). One, two or 3 doses of SP did not impact significantly on either the percentage of women with Ab to FV2 and DBL5 or Ab levels in Ab-positive women compared to women not taking SP. The prevalence of Ab to FV2 and DBL5 was only 36.9% and 36.1%, respectively. Surprisingly, among women who had PM at delivery, only 61.5% and 57.7% had Ab to FV2 and DBL5, respectively, with only 52.9% and 47.1% in PM-positive paucigravidae and 77.7% of multigravidae having Ab to both antigens. These results suggest that long-term implementation of IPTp-SP in a low-malaria transmission area results in few women having Ab to VAR2CSA

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

Identification of glycosaminoglycan binding regions in the Plasmodium falciparum encoded placental sequestration ligand, VAR2CSA

<p>Abstract</p> <p>Background</p> <p>Pregnancy malaria is caused by <it>Plasmodium falciparum</it>-infected erythrocytes binding the placental receptor chondroitin sulfate A (CSA). This results in accumulation of parasites in the placenta with severe clinical consequences for the mother and her unborn child. Women become resistant to placental malaria as antibodies are acquired which specifically target the surface of infected erythrocytes binding in the placenta. VAR2CSA is most likely the parasite-encoded protein which mediates binding to the placental receptor CSA. Several domains have been shown to bind CSA <it>in vitro</it>; and it is apparent that a VAR2CSA-based vaccine cannot accommodate all the CSA binding domains and serovariants. It is thus of high priority to define minimal ligand binding regions throughout the VAR2CSA molecule.</p> <p>Methods</p> <p>To define minimal CSA-binding regions/peptides of VAR2CSA, a phage display library based on the entire <it>var2csa </it>coding region was constructed. This library was screened on immobilized CSA and cells expressing CSA resulting in a limited number of CSA-binding phages. Antibodies against these peptides were affinity purified and tested for reactivity against CSA-binding infected erythrocytes.</p> <p>Results</p> <p>The most frequently identified phages expressed peptides residing in the parts of VAR2CSA previously defined as CSA binding. In addition, most of the binding regions mapped to surface-exposed parts of VAR2CSA. The binding of a DBL2X peptide to CSA was confirmed with a synthetic peptide. Antibodies against a CSA-binding DBL2X peptide reacted with the surface of infected erythrocytes indicating that this epitope is accessible for antibodies on native VAR2CSA on infected erythrocytes.</p> <p>Conclusion</p> <p>Short continuous regions of VAR2CSA with affinity for multiple types of CSA were defined. A number of these regions localize to CSA-binding domains and to surface-exposed regions within these domains and a synthetic peptide corresponding to a peptide sequence in DBL2 was shown to bind to CSA and not to CSC. It is likely that some of these epitopes are involved in native parasite CSA adhesion. However, antibodies directed against single epitopes did not inhibit parasite adhesion. This study supports phage display as a technique to identify CSA-binding regions of large proteins such as VAR2CSA.</p

A clinically validated Drosophila S2 based vaccine platform for production of malaria vaccines

Drosophila S2 insect cell expression is less known than the extensively used Spodoptera or Trichoplusia ni (Hi-5) insect cell based Baculovirus expression system (BEVS). Nevertheless it has been used in research for almost 40 years. The cell line was derived from late stage Drosophila melanogaster (Fruit fly) embryos by Schneider in the 1970s, who named the cell line Drosophila Schneider line 2 (synonyms: S2, SL2, D.mel. 2). The system has been widely applied to fundamental research, where the availability of the whole genome sequence of Drosophila melanogaster (1, 2) and the S2 cells’ susceptibility to RNA interference methods (3, 4) have enabled genome wide RNAi screening and whole genome expression analysis techniques to be used to great effect. S2 cells have proved to be highly effective for the production of proteins from a great variety of protein classes (5), such as: viral proteins, toxins, membrane proteins, enzyme, etc. Recent publications have also shown the strength of the S2 system in expression of Virus Like Particles (VLPs) (6). ExpreS2ion has developed the ExpreS2, Drosophila S2 platform to achieve improved yields for difficult to express proteins. Furthermore, several technologies have been developed to improve the ease of use of the system, as well as enable fast and efficient screening of multiple constructs. S2 based production processes for two malaria vaccine clinical trails with The Jenner Institute, Oxford University (Rh5 (7,8), blood-stage malaria) and Copenhagen University (VAR2CSA (9) pregnancy associated malaria) have been developed. The placental malaria vaccine is currently in a phase Ia trail in Germany, and a Phase Ib trial in Benin. The blood-stage malaria vaccine is currently in Phase IIa trial and is expecting results by the end of 2018. Several transmission-blocking candidates have been identified over the years with some of the most prominent being pfs48/45, Pfs230C and Pfs25(10). Other vaccine targets focus on blood-stage malaria such as Rh5, PfRIPR and CyrPA. We will present data on the development of a high producing Pfs25 monoclonal cell line and the purification from said cell line,as well as expression data on a range of other malaria vaccine targets. This present the clinically validated ExpreS2 platform as a complete system for a wide range of malaria targeting vaccines. (1) Adams M.D. et al. Science 2000 287:2185-2195 (2) Ashburner M, et al. Genome Res. 2005 Dec;15(12):1661-7 (3) Neumüller RA, et al. Wiley Interdiscip Rev Syst Biol Med. 2011 Jul-Aug; 3(4):471-8 (4) D’Ambrosio M.V. et al. J. Cell Biol. Vol. 191 No. 3 471–478 (5) Schetz J.A. et al. Protein Expression in the Drosophila Schneider 2 Cell System, Current Protocols in Neuroscience, 2004 (6) Yang L. et al. J Virol. 2012, Jul;86(14):7662-76. (7) Wright K.E. et al. Nature, 2014 Nov 20;515(7527):427-30 (8) Hjerrild K.A. et al. Sci Rep. 2016 Jul 26;6:30357 (9) Nielsen M.A. et al. PLoS One. 2015 Sep 1;10(9):e0135406 (10) Chaturvedi N et al. Indian J Med Res. 2016 Jun;143(6):696-71