Erythrocyte Binding Research Articles

Estradiol prodrugs (EP) for efficient oral estrogen treatment and abolished effects on estrogen modulated liver functions

Oral compared to parenteral estrogen administration is characterized by reduced systemic but prominent hepatic estrogenic effects on lipids, hemostatic factors, GH-/IGF I axis, angiotensinogen. In order to avoid such adverse metabolic effects of oral treatment, estradiol (E2) prodrugs (EP) were designed which bypass the liver tissue as inactive molecules. Carbone17-OH sulfonamide [-O2–NH2] substituted esters of E2 (EC508, others) were synthesized and tested for carbonic anhydrase II (CA-II) binding. CA II in erythrocytes is thought to oppose extraction of EP from portal vein blood during liver passage. Ovariectomized (OVX, day minus 14) rats were orally treated once daily from day 1–3. Sacrifice day 4. Uteri were dissected and weighed. Cholesterol fractions and angiotensinogen were determined in plasma. Oral E2 and ethinyl estradiol (EE) generated dose related uterine growth and important hepatic estrogenic effects. EP induced uterine growth at about hundred-fold lower doses. This was possible with almost absent effects on plasma cholesterol or angiotensinogen. Preliminary pharmacokinetic studies with EC508 used intravenous and oral administration in male rats. Resulting blood levels revealed complete oral bioavailability. Further high blood- but low plasma concentrations indicated erythrocyte binding of EC508 in vivo as potential mechanism of low extraction at liver passage. Very high systemic estrogenicity combined with markedly lower or absent adverse hepatic estrogenic effects is evidence for a systemic release of E2 from sulfonamide EP. In conclusion, tested oral EP bypass the liver in erythrocytes furnishing systemic estradiol at hydrolysis. This mechanism avoids the hepatic estrogenic impact of conventional oral estrogen therapy.

Baculovirus-expressed Plasmodium reichenowi EBA-140 merozoite ligand is host specific

Plasmodium reichenowi, an ape malaria parasite is morphologically identical and genetically similar to Plasmodium falciparum, infects chimpanzees but not humans. Genomic studies revealed that all primate malaria parasites belong to Laverania subgenus. Laverania parasites exhibit strict host specificity, but the molecular mechanisms underlying these host restrictions remain unexplained.Plasmodium merozoites express multiple binding ligands that recognize specific receptors on erythrocytes, including micronemal proteins belonging to P. falciparum EBL family. It was shown that erythrocyte binding antigen-175 (EBA-175), erythrocyte binding ligand-1 (EBL-1), erythrocyte binding antigen-140 (EBA-140) recognize erythrocyte surface sialoglycoproteins - glycophorins A, B, C, respectively. EBA-140 merozoite ligand hijacks glycophorin C (GPC), a minor erythrocyte sialoglycoprotein, to invade the erythrocyte through an alternative invasion pathway.A homolog of P. falciparum EBA-140 protein was identified in P. reichenowi. The amino acid sequences of both EBA-140 ligands are very similar, especially in the conservative erythrocyte binding region (Region II). It has been suggested that evolutionary changes in the sequence of EBL proteins may be associated with Plasmodium host restriction.In this study we obtained, for the first time, the recombinant P. reichenowi EBA-140 ligand Region II using baculovirus expression vector system. We show that the ape EBA-140 Region II is host specific and binds to chimpanzee erythrocytes in the dose and sialic acid dependent manner. Further identification of the erythrocyte receptor for this ape ligand is of great interests, since it may reveal the molecular basis of host restriction of both P. reichenowi and its deadliest human counterpart, P. falciparum.

Interaction between Endothelial Protein C Receptor and Intercellular Adhesion Molecule 1 to Mediate Binding of Plasmodium falciparum-Infected Erythrocytes to Endothelial Cells

ABSTRACTIntercellular adhesion molecule 1 (ICAM-1) and the endothelial protein C receptor (EPCR) are candidate receptors for the deadly complication cerebral malaria. However, it remains unclear if Plasmodium falciparum parasites with dual binding specificity are involved in cytoadhesion or different parasite subpopulations bind in brain microvessels. Here, we investigated this issue by studying different subtypes of ICAM-1-binding parasite lines. We show that two parasite lines expressing domain cassette 13 (DC13) of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family have dual binding specificity for EPCR and ICAM-1 and further mapped ICAM-1 binding to the first DBLβ domain following the PfEMP1 head structure in both proteins. As PfEMP1 head structures have diverged between group A (EPCR binders) and groups B and C (CD36 binders), we also investigated how ICAM-1-binding parasites with different coreceptor binding traits influence P. falciparum-infected erythrocyte binding to endothelial cells. Whereas levels of binding to tumor necrosis factor alpha (TNF-α)-stimulated endothelial cells from the lung and brain by all ICAM-1-binding parasite lines increased, group A (EPCR and ICAM-1) was less dependent than group B (CD36 and ICAM-1) on ICAM-1 upregulation. Furthermore, both group A DC13 parasite lines had higher binding levels to brain endothelial cells (a microvascular niche with limited CD36 expression). This study shows that ICAM-1 is a coreceptor for a subset of EPCR-binding parasites and provides the first evidence of how EPCR and ICAM-1 interact to mediate parasite binding to both resting and TNF-α-activated primary brain and lung endothelial cells.

Real-time measurement of Plasmodium falciparum-infected erythrocyte cytoadhesion with a quartz crystal microbalance.

BackgroundAn important virulence mechanism of the malaria parasite Plasmodium falciparum is cytoadhesion, the binding of infected erythrocytes to endothelial cells in the second half of asexual blood stage development. Conventional methods to investigate adhesion of infected erythrocytes are mostly performed under static conditions, many are based on manual or semi-automated read-outs and are, therefore, difficult to standardize. Quartz crystal microbalances (QCM) are sensitive to nanogram-scale changes in mass and biomechanical properties and are increasingly used in biomedical research. Here, the ability of QCM is explored to measure binding of P. falciparum-infected erythrocytes to two receptors: CD36 and chondroitin sulfate A (CSA) under flow conditions.MethodsBinding of late stage P. falciparum parasites is measured in comparison to uninfected erythrocytes to CD36- and CSA-coated quartzes by QCM observing frequency shifts. CD36-expressing cell membrane fragments and CSA polysaccharide were coated via poly-l-lysine to the quartz. The method was validated by microscopic counting of attached parasites and of erythrocytes to the coated quartzes.ResultsFrequency shifts indicating binding of infected erythrocytes could be observed for both receptors CD36 and CSA. The frequency shifts seen for infected and uninfected erythrocytes were strongly correlated to the microscopically counted numbers of attached cells.ConclusionsIn this proof-of-concept experiment it is shown that QCM is a promising tool to measure binding kinetics and specificity of ligand-receptor interactions using viable, parasite-infected erythrocytes. The method can improve the understanding of the virulence of P. falciparum and might be used to cross-validate other methods.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1374-7) contains supplementary material, which is available to authorized users.

Receptor specific binding regions of Plasmodium vivax tryptophan rich antigens and parasite growth inhibition activity of PvTRAg35.2

Plasmodium tryptophan rich proteins play important role in host–parasite interaction. Earlier, we have described that one of the merozoite expressed Plasmodium vivax tryptophan-rich antigen PvTRAg35.2 binds to the host erythrocytes, have conserved sequences in parasite population, and generates humoral as well as cellular immune responses in humans during this parasitic infection. Here, we show that PvTRAg35.2 interferes with the parasite growth in a heterologous Plasmodium falciparum culture system. This probably suggests the recognition of the common erythrocyte receptor(s) by certain merozoite ligands of these two parasite species. We have mapped the erythrocyte binding activity of PvTRAg35.2 to its two different regions positioned at amino acid residues 155–190 and 263–283. Binding of these peptide domains to the erythrocytes was inhibited by anti-PvTRAg35.2 antibodies either raised in rabbit or produced by the P. vivax patients. The cross-competition between peptides of PvTRAg35.2 and PvTRAg33.5 or PvTRAg38 during erythrocyte binding assay suggested sharing of host cell receptors by these PvTRAgs. Further studies on these receptor–ligand interactions may lead to the development of therapeutic agents for P. vivax malaria.

Strain-Transcending Inhibitory Antibodies against Homologous and Heterologous Strains of Duffy Binding Protein region II.

Duffy binding protein region II (DBPII) is a promising vaccine candidate against vivax malaria. However, polymorphisms of DBPII are the major obstacle to designing a successful vaccine. Here, we examined whether anti-DBPII antibodies from individual P. vivax exposures provide strain-transcending immunity and whether their presence is associated with DBPII haplotypes found in patients with acute P. vivax. The ability of antibodies to inhibit DBL-TH-erythrocyte binding was tested by COS7 erythrocyte binding inhibition assay. Seven samples of high responders (HR) were identified from screening anti-DBPII levels. HR no.3 and HR no.6 highly inhibited all DBL-TH binding to erythrocytes, by >80%. Antibodies from these two patients’ plasma had the potential to be broadly inhibitory against DBL-TH1, -TH2, -TH6, -TH7, -TH8 and -TH9 haplotypes when plasma was serially diluted from 1:500 to 1:2000. To further examine the association of DBPII haplotypes and the ability of antibodies to broadly inhibit DBL-TH variants, the individual samples underwent sequencing analysis and the inhibitory function of the anti-DBPII antibodies was tested. The patterns of DBPII polymorphisms in acute patients were classified into two groups, DBPII Sal I (55%) and DBL-TH variants (45%). Plasma from Sal I and DBPII-TH patients who had the highest inhibition against Sal I or DBL-TH4 and -TH5 was serially diluted from 1:500 to 1:2000 and their inhibitory capacity was tested against a panel of DBL-TH haplotypes. Results provided evidence of both strain-transcending inhibition as well as strain-specific inhibition by antibodies that blocked erythrocyte binding against some DBL-TH variants and against homologous alleles. This study demonstrated broad inhibition by anti-DBPII antibodies against DBL-TH haplotypes in natural P. vivax exposed individuals. The identification of conserved epitopes among DBL-TH may have implications for vaccine development of a DBPII-based vaccine against diverse P. vivax infections.

Age-dependent carriage of alleles and haplotypes of Plasmodium falciparum sera5, eba-175, and csp in a region of intense malaria transmission in Uganda

Background: The development of malaria vaccines is constrained by genetic polymorphisms exhibited by Plasmodium falciparum antigens. We investigated the age-dependent distribution of alleles or haplotypes of three P. falciparum malaria vaccine candidates, circumsporozoite protein (csp), erythrocyte binding antigen 175 (eba-175) and serine repeat antigen 5 (sera5) in a region of intense malaria transmission in Uganda. Methods & Materials: A cross sectional study was carried out between August and November 2009. Blood samples were collected after informed consent from 250 individuals below 5 years, 5-10 years and above 10 years olds. P. falciparum DNA was extracted from all samples. Alleles of sera5 and eba-175 were determined by polymerase chain reaction (PCR) amplification followed by resolution of PCR products by agarose gel electrophoresis and allele calling using photographs of ethidium bromide-stained gels. Haplotypes of CSP were identified by sequencing 63 PCR products and using P.falciparum 7G8 strain sequence as a reference. Results: Both eba-175 FCR3 (48/178) and CAMP (16/178) alleles were observed with the FCR3 (24/67) allele being predominant among children aged below 5 years old while the CAMP (12/67) allele was predominant among older individuals. Both sera5 alleles ORI (6/204) and ORII (103/204) were observed in the population but ORII was more prevalent. SERA5 ORII allele was significantly associated with age (P values < 0.0001), parasite density (P value < 0.0001) and clinical outcomes (P value = 0.018). There was marked CSP diversity in the Th2/Th3 region. Out of 63 sequences, 16 conformed to the reference strain and one (1/16) was similar with a West African haplotype and the majority (14/16) of the haplotypes were unique to this study region. Conclusion: There was an age-dependent distribution of CSP haplotypes with more haplotypes being harbored by < 5-year olds, (10/16) compared to adults (2/16). Interestingly, the CSP haplotype corresponding to 3D7 whose prototypical sequence is identical to the sequence of the leading malaria vaccine candidate RTS, S was not observed. Our data suggest that eba-175 FCR3 allele, sera5 ORII allele, and CSP haplotypes are targets of host immunity and under immune selection pressure in Apac District.

Structurally conserved erythrocyte-binding domain in Plasmodium provides a versatile scaffold for alternate receptor engagement

Understanding how malaria parasites gain entry into human red blood cells is essential for developing strategies to stop blood stage infection. Plasmodium vivax preferentially invades reticulocytes, which are immature red blood cells. The organism has two erythrocyte-binding protein families: namely, the Duffy-binding protein (PvDBP) and the reticulocyte-binding protein (PvRBP) families. Several members of the PvRBP family bind reticulocytes, specifically suggesting a role in mediating host cell selectivity of P. vivax. Here, we present, to our knowledge, the first high-resolution crystal structure of an erythrocyte-binding domain from PvRBP2a, solved at 2.12 Å resolution. The monomeric molecule consists of 10 α-helices and one short β-hairpin, and, although the structural fold is similar to that of PfRh5--the essential invasion ligand in Plasmodium falciparum--its surface properties are distinct and provide a possible mechanism for recognition of alternate receptors. Sequence alignments of the crystallized fragment of PvRBP2a with other PvRBPs highlight the conserved placement of disulfide bonds. PvRBP2a binds mature red blood cells through recognition of an erythrocyte receptor that is neuraminidase- and chymotrypsin-resistant but trypsin-sensitive. By examining the patterns of sequence diversity within field isolates, we have identified and mapped polymorphic residues to the PvRBP2a structure. Using mutagenesis, we have also defined the critical residues required for erythrocyte binding. Characterization of the structural features that govern functional erythrocyte binding for the PvRBP family provides a framework for generating new tools that block P. vivax blood stage infection.

Kaempferol inhibits Entamoeba histolytica growth by altering cytoskeletal functions

The flavonoid kaempferol obtained from Helianthemum glomeratum, an endemic Mexican medicinal herb used to treat gastrointestinal disorders, has been shown to inhibit growth of Entamoeba histolytica trophozoites in vitro; however, the mechanisms associated with this activity have not been documented. Several works reported that kaempferol affects cytoskeleton in mammalian cells. In order to gain insights into the action mechanisms involved in the anti-amoebic effect of kaempferol, here we evaluated the effect of this compound on the pathogenic events driven by the cytoskeleton during E. histolytica infection. We also carried out a two dimensional gel-based proteomic analysis to evidence modulated proteins that could explain the phenotypical changes observed in trophozoites. Our results showed that kaempferol produces a dose-dependent effect on trophozoites growth and viability with optimal concentration being 27.7μM.Kaempferol also decreased adhesion, it increased migration and phagocytic activity, but it did not affect erythrocyte binding nor cytolytic capacity of E. histolytica. Congruently, proteomic analysis revealed that the cytoskeleton proteins actin, myosin II heavy chain and cortexillin II were up-regulated in response to kaempferol treatment. In conclusion, kaempferol anti-amoebic effects were associated with deregulation of proteins related with cytoskeleton, which altered invasion mechanisms.

Studies on Immunogenicity and Antigenicity of Baculovirus-Expressed Binding Region of Plasmodium falciparum EBA-140 Merozoite Ligand.

The erythrocyte binding ligand 140 (EBA-140) is a member of the Plasmodium falciparum erythrocyte binding antigens (EBA) family, which are considered as prospective candidates for malaria vaccine development. EBA proteins were identified as important targets for naturally acquired inhibitory antibodies. Natural antibody response against EBA-140 ligand was found in individuals living in malaria-endemic areas. The EBA-140 ligand is a paralogue of the well-characterized P. falciparum EBA-175 protein. They both share homology of domain structure, including the binding region (Region II), which consists of two homologous F1 and F2 domains and is responsible for ligand–erythrocyte receptor interaction during merozoite invasion. It was shown that the erythrocyte receptor for EBA-140 ligand is glycophorin C-a minor human erythrocyte sialoglycoprotein. In studies on the immunogenicity of P. falciparum EBA ligands, the recombinant proteins are of great importance. In this report, we have demonstrated that the recombinant baculovirus-obtained EBA-140 Region II is immunogenic and antigenic. It can raise specific antibodies in rabbits, and it is recognized by natural antibodies present in sera of patients with malaria, and thus, it may be considered for inclusion in multicomponent blood-stage vaccines.

Novel and cost-effective refolding of a recombinant receptor binding domain of Plasmodium falciparum EBA-175

Plasmodium falciparum erythrocyte binding antigen (EBA-175), which is a 175kDa protein, binds with sialic acid residues on glycophorin A during invasion of human erythrocytes. Receptor-binding residues of EBA-175 lie in a conserved, amino-terminal, cysteine-rich region, region F2 of EBA-175 (PfF2), that is homologous to the binding domains of other erythrocyte binding proteins. Recombinant PfF2 was expressed in Escherichia coli, purified from inclusion bodies, renatured by rapid dilution in presence of proline and purified to homogeneity by ion-exchange chromatography. Recombinant PfF2 was refolded at higher concentrations with improved refolding efficiency and yield. The refolded protein was characterized using biochemical methods and shown to be pure, homogenous and functional as it binds human erythrocytes with specificity. Immunization of mice and rabbits with recombinant PfF2 formulated with Freund’s and Montanide ISA720 adjuvants, elicited high and potent antibody responses. These observations support the development of a novel and cost-effective process for production of recombinant PfF2, a blood stage vaccine candidate for P. falciparum malaria.

Recognition of Human Erythrocyte Receptors by the Tryptophan-Rich Antigens of Monkey Malaria Parasite Plasmodium knowlesi.

BackgroundThe monkey malaria parasite Plasmodium knowlesi also infect humans. There is a lack of information on the molecular mechanisms that take place between this simian parasite and its heterologous human host erythrocytes leading to this zoonotic disease. Therefore, we investigated here the binding ability of P. knowlesi tryptophan-rich antigens (PkTRAgs) to the human erythrocytes and sharing of the erythrocyte receptors between them as well as with other commonly occurring human malaria parasites.MethodsSix PkTRAgs were cloned and expressed in E.coli as well as in mammalian CHO-K1 cell to determine their human erythrocyte binding activity by cell-ELISA, and in-vitro rosetting assay, respectively.ResultsThree of six PkTRAgs (PkTRAg38.3, PkTRAg40.1, and PkTRAg67.1) showed binding to human erythrocytes. Two of them (PkTRAg40.1 and PkTRAg38.3) showed cross-competition with each other as well as with the previously described P.vivax tryptophan-rich antigens (PvTRAgs) for human erythrocyte receptors. However, the third protein (PkTRAg67.1) utilized the additional but different human erythrocyte receptor(s) as it did not cross-compete for erythrocyte binding with either of these two PkTRAgs as well as with any of the PvTRAgs. These three PkTRAgs also inhibited the P.falciparum parasite growth in in-vitro culture, further indicating the sharing of human erythrocyte receptors by these parasite species and the biological significance of this receptor-ligand interaction between heterologous host and simian parasite.ConclusionsRecognition and sharing of human erythrocyte receptor(s) by PkTRAgs with human parasite ligands could be part of the strategy adopted by the monkey malaria parasite to establish inside the heterologous human host.

Correlating quantitative real-time PCR to rapid diagnostic test and RNA transcript expression in isolated gametocytemia and asexual parasitemia of Plasmodium falciparum malaria.

BackgroundAt present, only microscopic examination of stained thick and thin blood smears for malaria can differentiate clinically relevant asexual parasitemia from clinically irrelevant isolated gametocytemia. Microscopy is time consuming, labour intensive, and requires significant technical expertise to perform. Simple and rapid tests that can distinguish asexual from isolated sexual parasitemia are needed.MethodsTo determine if parasitemia and cycle threshold (CT) values on Plasmodium genus and P. falciparum-specific quantitative polymerase chain reaction (qPCR) assays correlate to positivity of rapid diagnostic test (RDT), and 18S rRNA gene copy number, we analyzed blood samples from Ontario patients with isolated P. falciparum gametocytemia or asexual stages. RNA transcripts were evaluated to determine whether there is correlation of expression to different life cycle stages of P. falciparum.Results45 specimens containing isolated P. falciparum gametocytes, and 40 specimens containing isolated asexual stages by microscopy were identified and analyzed. By RDT, 40 of 45 (88.9 %) isolated gametocytemia specimens and 40 of 40 (100 %) asexual-stage specimens were positive for Plasmodium falciparum-specific histidine rich protein-2 (HRP-2). Fourteen of 45 (31.1 %) isolated gametocytemia specimens, and 36 of 40 (90 %) asexual-stage specimens were positive for Plasmodium genus aldolase T2 band. Positivity of the aldolase T2 band was associated with lower mean Plasmodium genus and P. falciparum-specific CT values, and to higher mean 18S rRNA gene copy by qPCR for both isolated gametocytemia and asexual-stage specimens. There was also a negative correlation of asexual parasitemia to both CT values, and positive correlation to 18S rRNA gene copy number. Analysis of asexual stage-specific erythrocyte binding antigen (eba-175) transcripts on 25 isolated gametocytemia and 20 asexual-stage specimens gave a positive predictive value of 62.5 % and negative predictive value of 100 % for asexual parasitemia. Thus, an absence of eba-175 transcripts excluded the presence of asexual (clinically relevant) parasitemia.ConclusionsPositivity of the aldolase T2 band of BinaxNow RDT correlated to higher parasite load in both isolated gametocytemia and asexual-stage specimens. Asexual stage-specific eba-175 RNA transcript expression provided reasonable negative predictive value for exclusion of asexual parasitemia in clinical samples, but was present in both isolated gametocytemia and asexual stage specimens.

Natural acquired inhibitory antibodies to Plasmodium vivax Duffy binding protein (PvDBP-II) equally block erythrocyte binding of homologous and heterologous expressed PvDBP-II on the surface of COS-7 cells.

The binding domain of Plasmodium vivax Duffy binding protein (PvDBP-II) is a promising blood-stage vaccine candidate for vivax malaria. For the development of a successful vivax malaria vaccine based on DBP-II, the antigenic diversity and also naturally occurring functional antibodies to different PvDBP-II variant types in the various populations must be determined. However, similar to other blood-stage antigens, allelic variation within the PvDBP-II is a fundamental challenge for the development of a broadly efficient vaccine. The present study was performed to define whether the polymorphisms in PvDBP-II influence the nature of functional inhibitory activity of naturally acquired or induced anti-DBP-II antibodies in mice. In this investigation, five genetically distinct variants of PvDBP-II were transiently expressed on the COS-7 cell surface. Erythrocyte-binding inhibition assay (EBIA) was performed using human sera infected with corresponding and non-corresponding P. vivax variants as well as by the use of mice sera immunized with different expressed recombinant PvDBP-IIs. EBIA results showed that the inhibitory percentage varied between 50 and 63 % by using sera from infected individuals, and in case of mouse antisera, inhibition was in the range of 76-86 %. Interestingly, no significant difference was detected in red blood cell binding inhibition when different PvDBP-II variants on the COS-7 cell surfaces were incubated with heterologous and homologous sera infected with PvDBP-II variants. This suggests that the detected polymorphisms in all five forms of PvDBP-II may not affect functional activity of anti-DBP-II antibodies. In conclusion, our results revealed that there are functional cross-reactive antibody responses to heterologous PvDBP-II variants that might provide a broader inhibitory response against all, or at least the majority of strains compared to single allele of this protein that should be considered in development of PvDBP-II-based vaccine.

Abstract 4271: Lack of CD47 membrane mobility contributes to the poor erythrocyte binding of SIRPαFc, a novel CD47-blocking cancer immunotherapeutic

Abstract CD47 binds to SIRPα on the surface of macrophages and delivers a “do not eat” signal that suppresses phagocytosis. There is strong evidence that many liquid and solid tumors exploit the CD47-SIRPα pathway to escape macrophage-mediated destruction. Blockade of CD47 using a soluble SIRPα-Fc fusion protein (SIRPαFc) has emerged as a promising strategy to neutralize the suppressive effects of CD47 and promote the eradication of tumor cells. We have previously reported data demonstrating that human SIRPαFc binds strongly to tumor cells but very poorly to human red blood cells (RBCs), despite abundant surface expression of CD47 on RBCs and strong reactivity with CD47-specific antibodies. Here we expand upon these early findings and assess inter-species differences in RBC binding. Our results, based on a panel of 43 human donors, clearly show that SIRPαFc binds very poorly to human RBCs regardless of gender, ABO blood group or Rh antigen status. Consistent with this finding, SIRPαFc was unable to induce agglutination of RBCs in vitro, although hemagglutination was triggered by CD47-blocking antibodies. Curiously, although the binding affinity of human SIRPαFc to cynomolgus macaque CD47 is approximately 10-fold lower than the binding to the human target, it binds strongly to cyno RBCs. This indicates that affinity alone does not predict the ability to bind erythrocytes. Instead, we hypothesized that the mobility of CD47 in the RBC membrane is a key determinant of SIRPαFc binding and thus compared the detergent solubilization profile of CD47 in human and cyno RBCs. CD47 was observed to segregate largely into the detergent-soluble fraction in monkey erythrocytes but was localized primarily to the insoluble pellet fraction in human RBCs, suggesting greater membrane mobility in cyno compared to human RBCs. This finding is consistent with a model in which CD47 mobility is required to form high affinity clusters with SIRPαFc, and indeed we have previously observed that pre-clustering CD47 with a non-blocking antibody converts human RBCs into strong SIRPαFc binders. Finally, the consequences of cyno RBC binding were assessed in vivo. Significant depletion of RBCs was evident following intravenous infusions of SIRPαFc in cyno monkeys. We speculate that similar anemia is likely to occur in humans treated with CD47-blocking antibodies that bind to human erythrocytes, but not with a low RBC-binding SIRPαFc therapeutic. In conclusion, human SIRPαFc binds very poorly to human RBCs but is highly reactive with cyno erythrocytes. This unusual pattern of species cross-reactivity may result from species-specific differences in CD47 membrane mobility and is consistent with a model in which SIRPαFc binding requires mobile CD47 to form high affinity clusters. We predict that anemia, which occurs in monkeys following SIRPαFc administration, is not likely to occur in human patients where significant RBC binding is absent. Citation Format: Penka S. Petrova, Karen Dodge, Tanya Prasolava, Vien Chai, Xinli Pang, Robert A. Uger. Lack of CD47 membrane mobility contributes to the poor erythrocyte binding of SIRPαFc, a novel CD47-blocking cancer immunotherapeutic. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4271. doi:10.1158/1538-7445.AM2015-4271

Genetic evidence for contribution of human dispersal to the genetic diversity of EBA-175 in Plasmodium falciparum.

BackgroundThe 175-kDa erythrocyte binding antigen (EBA-175) of Plasmodium falciparum plays a crucial role in merozoite invasion into human erythrocytes. EBA-175 is believed to have been under diversifying selection; however, there have been no studies investigating the effect of dispersal of humans out of Africa on the genetic variation of EBA-175 in P. falciparum.MethodsThe PCR-direct sequencing was performed for a part of the eba-175 gene (regions II and III) using DNA samples obtained from Thai patients infected with P. falciparum. The divergence times for the P. falciparum eba-175 alleles were estimated assuming that P. falciparum/Plasmodium reichenowi divergence occurred 6 million years ago (MYA). To examine the possibility of diversifying selection, nonsynonymous and synonymous substitution rates for Plasmodium species were also estimated.ResultsA total of 32 eba-175 alleles were identified from 131 Thai P. falciparum isolates. Their estimated divergence time was 0.13–0.14 MYA, before the exodus of humans from Africa. A phylogenetic tree for a large sequence dataset of P. falciparumeba-175 alleles from across the world showed the presence of a basal Asian-specific cluster for all P. falciparum sequences. A markedly more nonsynonymous substitutions than synonymous substitutions in region II in P. falciparum was also detected, but not within Plasmodium species parasitizing African apes, suggesting that diversifying selection has acted specifically on P. falciparumeba-175.ConclusionsPlasmodium falciparumeba-175 genetic diversity appeared to increase following the exodus of Asian ancestors from Africa. Diversifying selection may have played an important role in the diversification of eba-175 allelic lineages. The present results suggest that the dispersals of humans out of Africa influenced significantly the molecular evolution of P. falciparum EBA-175.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0820-2) contains supplementary material, which is available to authorized users.

Immunization with the MAEBL M2 Domain Protects against Lethal Plasmodium yoelii Infection

Malaria remains a world-threatening disease largely because of the lack of a long-lasting and fully effective vaccine. MAEBL is a type 1 transmembrane molecule with a chimeric cysteine-rich ectodomain homologous to regions of the Duffy binding-like erythrocyte binding protein and apical membrane antigen 1 (AMA1) antigens. Although MAEBL does not appear to be essential for the survival of blood-stage forms, ectodomains M1 and M2, homologous to AMA1, seem to be involved in parasite attachment to erythrocytes, especially M2. MAEBL is necessary for sporozoite infection of mosquito salivary glands and is expressed in liver stages. Here, the Plasmodium yoelii MAEBL-M2 domain was expressed in a prokaryotic vector. C57BL/6J mice were immunized with doses of P. yoelii recombinant protein rPyM2-MAEBL. High levels of antibodies, with balanced IgG1 and IgG2c subclasses, were achieved. rPyM2-MAEBL antisera were capable of recognizing the native antigen. Anti-MAEBL antibodies recognized different MAEBL fragments expressed in CHO cells, showing stronger IgM and IgG responses to the M2 domain and repeat region, respectively. After a challenge with P. yoelii YM (lethal strain)-infected erythrocytes (IE), up to 90% of the immunized animals survived and a reduction of parasitemia was observed. Moreover, splenocytes harvested from immunized animals proliferated in a dose-dependent manner in the presence of rPyM2-MAEBL. Protection was highly dependent on CD4(+), but not CD8(+), T cells toward Th1. rPyM2-MAEBL antisera were also able to significantly inhibit parasite development, as observed in ex vivo P. yoelii erythrocyte invasion assays. Collectively, these findings support the use of MAEBL as a vaccine candidate and open perspectives to understand the mechanisms involved in protection.

Engineered binding to erythrocytes induces immunological tolerance to E. coli asparaginase.

Antigen-specific immune responses to protein drugs can hinder efficacy and compromise safety because of drug neutralization and secondary clinical complications. We report a tolerance induction strategy to prevent antigen-specific humoral immune responses to therapeutic proteins. Our modular, biomolecular approach involves engineering tolerizing variants of proteins such that they bind erythrocytes in vivo upon injection, on the basis of the premise that aged erythrocytes and the payloads they carry are cleared tolerogenically, driving the deletion of antigen-specific T cells. We demonstrate that binding the clinical therapeutic enzyme Escherichia coli l-asparaginase to erythrocytes in situ antigen-specifically abrogates development of antibody titers by >1000-fold and extends the pharmacodynamic effect of the drug 10-fold in mice. Additionally, a single pretreatment dose of erythrocyte-binding asparaginase tolerized mice to multiple subsequent doses of the wild-type enzyme. This strategy for reducing antigen-specific humoral responses may enable more effective and safer treatment with therapeutic proteins and drug candidates that are hampered by immunogenicity.

Rifins, rosetting, and red blood cells

The binding of multiple uninfected erythrocytes to a central malaria parasite-infected erythrocyte (IE) is called rosetting. Rosetting has been associated with severe disease, but its functional significance,and the host receptors and parasite ligands involved are only partially known. A recent study, which describes yet another piece in this already complex puzzle, provides a welcome boost and a broadening of an important malaria research field.

Variation in Plasmodium falciparum erythrocyte invasion phenotypes and merozoite ligand gene expression across different populations in areas of malaria endemicity.

Plasmodium falciparum merozoites use diverse alternative erythrocyte receptors for invasion and variably express cognate ligands encoded by the erythrocyte binding antigen (eba) and reticulocyte binding-like homologue (Rh) gene families. Previous analyses conducted on parasites from single populations in areas of endemicity revealed a wide spectrum of invasion phenotypes and expression profiles, although comparisons across studies have been limited by the use of different protocols. For direct comparisons within and among populations, clinical isolates from three different West African sites of endemicity (in Ghana, Guinea, and Senegal) were cryopreserved and cultured ex vivo after thawing in a single laboratory to assay invasion of target erythrocytes pretreated with enzymes affecting receptor subsets. Complete invasion assay data from 67 isolates showed no differences among the populations in the broad range of phenotypes measured by neuraminidase treatment (overall mean, 40.6% inhibition) or trypsin treatment (overall mean, 83.3% inhibition). The effects of chymotrypsin treatment (overall mean, 79.2% inhibition) showed heterogeneity across populations (Kruskall-Wallis P = 0.023), although the full phenotypic range was seen in each. Schizont-stage transcript data for a panel of 8 invasion ligand genes (eba175, eba140, eba181, Rh1, Rh2a, Rh2b, Rh4, and Rh5) were obtained for 37 isolates, showing similar ranges of variation in each population except that eba175 levels tended to be higher in parasites from Ghana than in those from Senegal (whereas levels of eba181 and Rh2b were lower in parasites from Ghana). The broad diversity in invasion phenotypes and gene expression seen within each local population, with minimal differences among them, is consistent with a hypothesis of immune selection maintaining parasite variation.