Characterization of a modified red cell membrane protein expressed on erythrocytes infected with the human malaria parasite Plasmodium falciparum: possible role as a cytoadherent mediating protein

Infections with the human malaria Plasmodium falciparum are characterized by the retention of parasitized erythrocytes in tissue capillaries and venules. Erythrocytes containing trophozoites and schizonts attach to the endothelial cells that line these vessels by means of structurally identifiable excrescences present on the surface of the infected cell. Such excrescences, commonly called knobs, are visible by means of scanning or transmission electron microscopy. The biochemical mechanisms responsible for erythrocyte adherence to the endothelial cell are still undefined. In an attempt to identify the cytoadhesive molecule on the surface of the infected cell, we have prepared monoclonal antibodies to knob-bearing erythrocytes infected with the FCR-3 strain of P. falciparum. One of these monoclonal antibodies, designed 4A3, is an IgM that reacts (by means of immunofluorescence) with the surface of unfixed erythrocytes bearing mature parasites of the knobby line; it does not react with knobless lines or uninfected erythrocytes. By immunoelectron microscopy the monoclonal antibody 4A3 was localized to the knob region. In an in vitro cytoadherence assay, the monoclonal antibody partially blocked the binding of knob-bearing cells (FCR-3 strain) to formalin-fixed amelanotic melanoma cells. The monoclonal antibody was used to immunoprecipitate a protein from extracts of knobby erythrocytes that had been previously surface iodinated. By a two-dimensional peptide mapping technique, the antigen recognized by the monoclonal antibody was found to be structurally related to band 3 protein, the human erythrocyte anion transporter.     

Plasmodium falciparum: identification and localization of a knob protein antigen expressed by a cDNA clone

Differential screening of cDNA libraries constructed from knobby and predominantly knobless Plasmodium falciparum isolates, identified the sequence SD17. Chromosome blotting experiments have shown that this sequence, which is located on chromosome 2 of most isolates, was deleted in the cloned parasite line E12 of the FCQ27/PNG isolate. Here we show that erythrocytes infected with the SD17-containing cloned line D10 have typical knob structures on their surfaces, whereas those infected with the line E12 lack knobs. An expression clone was constructed from SD17 and used to affinity purify antibodies from the sera of individuals living in areas of Papua New Guinea where malaria is endemic. The antibodies reacted in immunoblotting experiments with a single polypeptide that varied in Mr from 85,000 to 105,000 among different isolates. The antigen was not expressed in the knobless clone E12. Postembedding immunoelectron microscopy showed localization of the antigen over the knobs of FC27 and two other isolates, largely on the cytoplasmic side. We conclude that the parasite antigen corresponding to clone SD17 is a knob protein.     

Antigenic variation in malaria: a 3' genomic alteration associated with the expression of a P. knowlesi variant antigen

Antigenic variation of malaria parasites was discovered in P. knowlesi, using a schizont-infected cell agglutination (SICA) assay to detect variant antigens expressed at the surface of infected erythrocytes. Later studies utilizing stable clones, Pk1(A+) and its direct derivative, Pk1(B+)1+, showed that SICA[+] clones express distinct parasite-encoded antigens of approximately 200 kDa. Here we identify a P. knowlesi variant antigen gene and cDNA and demonstrate that it encodes the 205 kDa variant antigen expressed by B+ parasites. This gene belongs to a multigene family, which we term SICAvar. Its ten-exon structure with seven cysteine-rich coding modules is unique compared to P. falciparum var genes. Further, we highlight a 3' genomic alteration that we predict is related to SICAvar gene switching.     

The reactivities of human erythrocyte autoantibodies anti-Pr2, anti-Gd, Fl and Sa with gangliosides in a chromatogram binding assay.

The thin layer chromatogram binding assay was used to study the reaction of several natural-monoclonal autoantibodies which recognize sialic acid-dependent antigens of human erythrocytes. Immunostaining of gangliosides derived from human and bovine erythrocytes was achieved with four autoantibodies designated anti-Pr2, anti-Gd, Sa and Fl, each of which has a different haemagglutination pattern with untreated and proteinase-treated erythrocytes and with cells of I and i antigen types. From the chromatogram binding patterns of anti-Pr2 with gangliosides of the neolacto and the ganglio series, it is deduced that this antibody reacts best with N-acetylneuraminic acid when it is alpha 2-3- or alpha 2-6-linked to a terminal Gal(beta 1-4)Glc/GlcNAc GlcNAc sequence and to a lesser extent when it is alpha 2-3-linked to a terminal Gal(beta 1-3)GalNAc sequence or to an internal galactose and when it is alpha 2-8-linked to another, internal N-acetylneuraminic acid residue. The other three antibodies differ from anti-Pr2 in their lack of reaction with glycolipids of the ganglio series. They react with the NeuAc(alpha 2-3)Gal(beta 1-4)Glc/GlcNAc sequence as found in GM3 and in glycolipids of the neolacto series, but show a preference for the latter, longer sequences. Thus all four antibodies react with sialylated oligosaccharides containing i type (linear) and I type (branched) neolacto backbones. Fl antibody differs from the other three in its stronger reaction with branched neolacto sequences in accordance with its stronger agglutination of erythrocytes of I rather than i type. The four antibodies show a specificity for N-acetyl- rather than N-glycolyl-neuraminic acid.     

Plasmodium falciparum merozoites: isolation by density gradient centrifugation using Percoll and antigenic analysis

Merozoites of Plasmodium falciparum were isolated and immunocytochemically analyzed. Mature parasites from knobby (K+) and knobless (K-) strains were incubated for 4 to 5 hr in RPMI 1640 with 10% serum and 10% RBC extract. About 12 to 14% of the merozoites released were recovered by density gradient centrifugation using Percoll. From 1 to 3 X 10(9) merozoites were obtained per collection. The merozoite preparations were contaminated with 10% residual bodies, about 0.1% infected and uninfected erythrocytes, about 0.1% RBC-free trophozoites and schizonts, and numerous small (less than 0.5 microns) membrane vesicles. Merozoites from the K+ and K- strains were morphologically and, by an indirect, ferritin-labeled antibody assay using serum from immune Aotus, antigenically indistinguishable. Although the residual body coats reacted with the immune Aotus serum, the membrane vesicles, some of which were seen to be blebbing from merozoites, did not react with this serum or a serum against erythrocytes. This paper describes a procedure that can be used to obtain large numbers of merozoites with little contamination by host erythrocytes.     

Towards a vaccine against pregnancy-associated malaria

The consequences of pregnancy-associated malaria on pregnant women (anaemia), their babies (birth weight reduction), and infants (increased morbidity and mortality) are well documented. Field observations during the last decade have underlined the key role of the interactions between P. falciparum variable surface antigens expressed on infected erythrocytes and a novel receptor: chondroitin sulfate A (CSA) for the placental sequestration of infected erythrocytes. Identification of a distinct P. folciparum erythrocyte membrane protein 1 (PfEMP1) variant, VAR2CSA, as the dominant variant surface antigen and as a clinically important target for protective immune response to pregnancyassociated malaria has raised hope for developing a new preventive strategy based on inducing these immune responses by vaccination. However, despite particular structure and interclonal conservation of VAR2CSA among other PfEMP1, significant challenges still exist concerning the development of a VAR2CSA-based vaccine with profound efficacy.     

ABO Blood Groups Influence Macrophage-mediated Phagocytosis of Plasmodium falciparum-infected Erythrocytes

Erythrocyte polymorphisms associated with a survival advantage to Plasmodium falciparum infection have undergone positive selection. There is a predominance of blood group O in malaria-endemic regions, and several lines of evidence suggest that ABO blood groups may influence the outcome of P. falciparum infection. Based on the hypothesis that enhanced innate clearance of infected polymorphic erythrocytes is associated with protection from severe malaria, we investigated whether P. falciparum-infected O erythrocytes are more efficiently cleared by macrophages than infected A and B erythrocytes. We show that human macrophages in vitro and mouse monocytes in vivo phagocytose P. falciparum-infected O erythrocytes more avidly than infected A and B erythrocytes and that uptake is associated with increased hemichrome deposition and high molecular weight band 3 aggregates in infected O erythrocytes. Using infected A₁, A₂, and O erythrocytes, we demonstrate an inverse association of phagocytic capacity with the amount of A antigen on the surface of infected erythrocytes. Finally, we report that enzymatic conversion of B erythrocytes to type as O before infection significantly enhances their uptake by macrophages to observed level comparable to that with infected O wild-type erythrocytes. These data provide the first evidence that ABO blood group antigens influence macrophage clearance of P. falciparum-infected erythrocytes and suggest an additional mechanism by which blood group O may confer resistance to severe malaria.     

Antibody formation to autologous erythrocytes following the immunization with rat erythrocytes of normal animals and mice tolerant to the immunizing antigen

(CBA X C57B1/6)F1 mice immunized three times with rat erythrocytes produced antibodies both to this antigen and to autologous erythrocytes. Most of the antibodies to rat erythrocytes belonged to IgM isotype while antibodies to autologous red cells were of IgG isotype. Combined injection of thymectomized (CBA X C57B1/6)F1 mice with a massive dose of rat spleen cells and cyclophosphamide induced in animals stable tolerance to rat cells. Inducibility of antibodies to autologous red cells in tolerant mice injected 3-5 times with rat erythrocytes was drastically reduced. Nonspecific suppression (thymectomy and cyclophosphamide) did not prevent production of autoantibodies.     

The hemagglutinating properties and adsorption on erythrocytes of Coxsackie B-3 viruses and their selected bentonite variants

The paper is devoted to the study of the Coxsackie B-3 viruses and their bentonite variants passaged on the primary and transplanted cell cultures for their hemagglutinating (HA) properties and a degree of adsorption on erythrocytes. It is shown possible to detect the HA activity of the Coxsackie B-3 viruses which are passaged on the transplanted cell cultures: the variant Abent has the hemagglutinating activity relative to human and rabbit erythrocytes. The variant Abent+ of the Coxsackie B-3 virus passaged on the cell culture Vero is established not to be absorbed on erythrocytes. The variant Abent and the initial population of the Coxsackie B-3 virus are adsorbed on the human erythrocytes by 90-99%. Coxsackie B-3 virus and its bentonite variants reproduced on the primary cell culture of human embryonic fibroblasts and possessing the hemagglutinating activity are 100-1000 times better adsorbed on human erythrocytes than the viruses passaged in the transplanted culture.     

Immunocytogenetics. V. A highly conserved NOR antigen (He) is facultatively associated with nucleolar or nucleoplasmic granules

A new protein antigen of the nucleolus organizer region (NOR), designated He, was recognized by human autoantibodies obtained from a patient with Raynaud phenomenon. In mitotic cells of all vertebrate species tested. He serum selectively immunostained the chromosomal NORs. A completely unexpected characteristic of the He antigen was its location during interphase. In mammalian cell substrates, it was concentrated in numerous nucleoplasmic granules, with minor amounts of the antigen uniformly distributed throughout the entire nucleus. In interphase nuclei of lower vertebrate cells, however, the antigen was preferentially located in the nucleolus. The antigenicity of He is not dependent on RNA or DNA; its cytochemical properties operationally classify it as a nonhistone component of the chromosome scaffold. The He antigen was present in the residual nucleolar structures of cells that were not at all active in rRNA synthesis, such as mammalian late spermatids and amphibian erythrocytes.     

Haemoglobin C and S role in acquired immunity against Plasmodium falciparum malaria

A recently proposed mechanism of protection for haemoglobin C (HbC; beta6Glu-->Lys) links an abnormal display of PfEMP1, an antigen involved in malaria pathogenesis, on the surface of HbC infected erythrocytes together with the observation of reduced cytoadhesion of parasitized erythrocytes and impaired rosetting in vitro. We investigated the impact of this hypothesis on the development of acquired immunity against Plasmodium falciparum variant surface antigens (VSA) encoding PfEMP1 in HbC in comparison with HbA and HbS carriers of Burkina Faso. We measured: i) total IgG against a single VSA, A4U, and against a panel of VSA from severe malaria cases in human sera from urban and rural areas of Burkina Faso of different haemoglobin genotypes (CC, AC, AS, SC, SS); ii) total IgG against recombinant proteins of P. falciparum asexual sporozoite, blood stage antigens, and parasite schizont extract; iii) total IgG against tetanus toxoid. Results showed that the reported abnormal cell-surface display of PfEMP1 on HbC infected erythrocytes observed in vitro is not associated to lower anti- PfEMP1 response in vivo. Higher immune response against the VSA panel and malaria antigens were observed in all adaptive genotypes containing at least one allelic variant HbC or HbS in the low transmission urban area whereas no differences were detected in the high transmission rural area. In both contexts the response against tetanus toxoid was not influenced by the beta-globin genotype. These findings suggest that both HbC and HbS affect the early development of naturally acquired immunity against malaria. The enhanced immune reactivity in both HbC and HbS carriers supports the hypothesis that the protection against malaria of these adaptive genotypes might be at least partially mediated by acquired immunity against malaria.     

Studies on immune responses to parasite antigens in mice: VII. Cells secreting antibodies to modified mouse erythrocytes in Babesia rodhaini-infected mice

Increased numbers of plaque-forming cells (PFC) secreting antibodies to bromelain-treated mouse erythrocytes were detected in the spleens of BALB/c·nu/+ mice heavily infected with Babesia rodhaini but not in the spleens of infected hypothymic BALB/ c·nu/nu mice. Whether the products of these cells, antierythrocyte autoantibodies, play any role in the destruction of intact erythrocytes in parasitized mice is unknown, but it is noteworthy that only low numbers of PFC were detected, and the modification of mouse erythrocytes was obligatory for detection of PFC.     

Evidence that autoimmunity in NZB mice is caused by a defect in immune specificity rather than a generalized defect in tolerance of self-antigens

NZB mice which were already producing anti-erythrocyte autoantibodies were not able to respond to their own liver F antigen, thus providing evidence that their autoimmunity is not caused by a generalized breakdown in self-tolerance mechanisms. The specificity of autoantibodies produced in the spontaneous hemolytic anemia was different from that of antierythrocyte antibodies induced in normal mice and in young NZB mice by injections of rat erythrocytes. This indicates that the B-cell clones which can be triggered by heterologous antigen are different from those responsible for the NZB disease; the latter clones may not exist in normal mice.     

Structural alteration of the membrane of erythrocytes infected with Plasmodium falciparum

Human erythrocytes infected with five strains of Plasmodium falciparum and Aotus erythrocytes infected with three strains of P. falciparum were studied by thin-section and freeze-fracture electron microscopy. All strains of P. falciparum we studied induced electron-dense conical knobs, measuring 30-40 nm in height and 90-100 nm in diameter on erythrocyte membranes. Freeze-fracture demonstrated that the knobs were distributed over the membrane of both human and Aotus erythrocytes. A distinct difference was seen between the intramembrane particle (IMP) distribution over the knobs of human and Aotus erythrocyte membranes. There was no change in IMP distribution in infected human erythrocyte membranes, but infected Aotus erythrocytes showed an aggregation of IMP over the P face of the knobs with a clear zone at the base. This difference in IMP distribution was related only to the host species and not to parasite strains. Biochemical analysis demonstrated that a higher proportion of band 3 was bound to the cytoskeleton of uninfected Aotus erythrocytes than uninfected human erythrocytes after Triton X-100 extraction. This may account for the different effects of P. falciparum infection on IMP distribution in the two different cell types.     

The detergent solubility properties of a malarial (Plasmodium knowlesi) variant antigen expressed on the surface of infected erythrocytes

Four detergents have been compared for identification of the Plasmodium knowlesi variant antigen on infected erythrocytes by immunoprecipitation analysis. Erythrocytes infected with late trophozoite and schizont forms of cloned asexual parasites were labeled by lactoperoxidase-catalyzed radioiodination and extracted either with the anionic detergents sodium dodecyl sulfate (SDS) or cholate, the neutral detergent Triton X-100, or the zwitterion 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS). After addition of Triton X-100 to SDS and cholate extracts, parallel immunoprecipitations of the four extracts were performed using rhesus monkey antisera of defined agglutinability. Identical results were obtained with clone Pk1(A+), which has 125I-variant antigens of Mr 210,000 and 190,000, and with clone Pk1(B+)1+, which has variant antigens of Mr 200,000-205,000. SDS yielded maximal levels of immunoprecipitated 125I-variant antigens. Variant-specific immunoprecipitation was detected in some experiments with Triton X-100 and cholic acid but with significantly lower recovery than with SDS. CHAPS extraction did not yield the variant antigens on immunoprecipitation. The variant antigens could also be identified in Triton X-100-insoluble material by subsequent extraction with SDS, indicating that failure to recover these proteins in the Triton X-100-soluble fraction is due to failure of this detergent to extract the variant antigens rather than to degradation during extraction. We suggest that the 125I-variant antigens either have a structure that renders them intrinsically insoluble in Triton X-100, cholate, or CHAPS, or that they are associated in some way with host cell membrane components that also resist solubilization by these detergents.     

Association of autoantibodies with anemia, splenomegaly, and glomerulonephritis in experimental African trypanosomiasis.

Rats experimentally infected with Trypanosoma brucei rhodesiense developed a syndrome characterized by anemia, splenomegaly, and glomerulonephritis. Serologic evaluation revealed that the syndrome was accompanied by the presence of 3 autoantibodies--cold-active hemagglutinin, immunoconglutinin, and antibody to fibrinogen/fibrin products. Fluorescein isothiocyanate conjugated antibody tests showed the presence of fixed complement and fibrinogen on both trypanosomes and erythrocytes. All infected rats died by the ninth day of the infection with 5 animals showing signs of pulmonary involvement and shock. From these observations it is suggested that autoantigens, autoantibodies, and complement may have been causal in this syndrome.     

Altered cell surface antigen expression in bladder carcinoma detected by a new hemagglutinating monoclonal antibody

A hemagglutinating monoclonal IgM antibody (MoAb145) was produced against a high incidence red blood cell membrane antigen. By the specific red cell adherence test, the antibody also reacted with human bladder epithelium; in addition, expression of the MoAb145 antigen was lost in some cases of transitional cell carcinoma of the bladder, in a manner similar to the ABH blood group. Hemagglutination studies with a panel of erythrocytes lacking specific high incidence red blood cell membrane antigens indicated that MoAb145 did not recognize ABH specificity but rather a determinant absent from rare MN variant erythrocytes, including En(a-) erythrocytes, which lack glycophorin-alpha. Failure of MoAb145 to stain, by indirect immunofluorescence, the erythroleukemia cell line K562, which expresses glycophorin-alpha and the MN blood group, and failure to inhibit MoAb145 hemagglutination with an erythrocyte sialoglycoprotein fraction that contained MN blood group activity suggests that MoAb145 does not recognize either glycophorin-alpha or the MN blood group, but rather another membrane determinant, which is altered in En(a-) erythrocytes. This study demonstrates a new epitope detected by MoAb145 that is shared between human erythrocyte membranes and bladder epithelia, and is affected by neoplastic transformation in transitional cell carcinoma of the bladder.     

Trypanosoma cruzi: Effect on B-cell-responsive and -responding clones

During the course of Trypanosoma cruzi infection in C57BL/6 mice, which are relatively resistant to the parasite, the hosts developed antibody activity against previously unencountered antigens. The anti-sheep erythrocyte and antitrinitrophenyl antibody levels increased rapidly from Day 7 of infection, reached a peak by the 21st day, and were maintained at this level through 120 days postinfection in these mice. In contrast, highly susceptible C3H(He) mice did not have demonstrable antibody responses to SRBC or TNP during the 24-day infection period. Autoantibody activity against the selfantigens presented on isologous erythrocytes or thymocytes, however, were reduced in infected C57BL/6 mice. No significant reduction in autoreactivity to the self-antigens on erythrocytes or thymocytes was observed in C3H(He) mice infected with T. cruzi although a trend of reduced autoresponsiveness toward erythrocytes appeared to be developing by the time of death. C57BL/6 mice immunized with sheep erythrocytes as neonates and infected with T. cruzi as adults, or adult mice primed with low doses of sheep erythrocytes prior to infection, had elevated antibody responses to sheep erythrocytes unless the mice were immunized with sheep erythrocytes during the course of infection, in which case suppression of the response against sheep erythrocytes resulted. The nonspecific synthesis of immunoglobulins in infected C57BL/6 mice was, in part, a result of the lymphocyteactivating properties of T. cruzi-associated antigens. The T. cruzi-associated antigens induced proliferative and differentiative responses in spleen cells in vitro. It is proposed that the T. cruzi-associated antigens differentially affect lymphocytes capable of responding to antigen and those lymphocytes previously stimulated by antigen.