Antigenicity of the infected-erythrocyte and merozoite surfaces in Falciparum malaria.

The antigenicity of altered structures induced by Plasmodium falciparum in the membranes of infected Aotus monkey and human erythrocytes was examined. Antisera were obtained from monkeys made immune to malaria. Bound antibodies were shown to be localized on the knob protrusions of infected erythrocytes of both human and monkey origin and from both in vitro and in vivo infections. Therefore, P. falciparum infection has produced similar antigenic changes in the erythrocyte surfaces of both man and monkey. Uninfected erythrocytes and all knobless-infected erythrocytes bound no antibody from immune sera. Strains of P. falciparum from widely different geographic areas that were cultured in vitro in human erythrocytes induced structures (knobs) which have common antigenicity. Merozoites were agglutinated by cross-linking of their cell coats when incubated with immune sera. The binding of ferritin-labeled antibody was heavy on the coats of both homologous and heterologous strains of the parasite, indicating that the merozoite surfaces of these strains share common antigens.

Reversal of knob formation on Plasmodium falciparum-infected erythrocytes.

The human malarial parasite Plasmodium falciparum can produce surface protrusions (knobs) on infected erythrocytes; however, long-term culturing of the parasite results in the appearance of knobless cells. In this study it was found that a knob-producing clone lost the ability to produce knobs in vitro. Furthermore, a clone not producing knobs derived from the knob-producing clone regained the capacity to produce knobby cells in vitro. Certain parasite proteins were associated with the knobby phenotype but not with the knobless type. These results indicate that the parasites change in vitro in a spontaneous and reversible manner independent of immunological selection.

Complement activation by the surface of Plasmodium falciparum infected erythrocytes.

The surface of trophozoite-stage Plasmodium falciparum infected erythrocytes will, in the presence of immune human or owl monkey serum, activate the classical complement pathway. This was demonstrated with a sensitive, enzyme-linked immunosorbent assay which detects the complex, C1s-C1 inhibitor, which is only generated when the classical pathway is activated. A second enzyme-linked immunosorbent assay, as well as Covaspheres coated with affinity-purified anti-C3, showed that immune activation of the classical pathway by infected erythrocytes resulted in the accumulation of significant amounts of C3b on the erythrocyte surface. During the development of the parasite to the trophozoite stage, the erythrocyte membrane is also transformed from a non-activator into a surface capable of activating complement by the alternative pathway. Erythrocytes infected with trophozoite-stage parasites directly activated the alternative complement pathway. This activation led to the specific binding of an average of 15,000 C3b molecules per infected cell. Alternative pathway activation was augmented by anti-parasite antibody. Such conditions mediated the accumulation of an average of 36,000 C3b molecules per infected erythrocyte. The amounts of C3b on the infected erythrocyte surface did not lead to cellular lysis. They are, however, likely to have a major impact on the total in vivo response to this parasite.

Conservation and antigenicity of N-terminal sequences of GP185 from different Plasmodium falciparum isolates.

Complementary DNA (cDNA) clones for GP185, a major antigenically diverse glycoprotein of Plasmodium falciparum, were isolated from a cDNA library of the Honduras I/CDC (Honduras I) isolate, and 1052 bp were sequenced. The expression of cDNA fragments in Escherichia coli using the vector pCQV2 allowed verification of the reading frame. This GP185 cDNA sequence, like the cDNA sequence for a homologous gene of the K1 isolate [Hall et al., Nature 311 (1984) 379-382], codes for a polypeptide which is truncated due to multiple, in-frame stop codons. This polypeptide corresponds to the N-terminal 15% of the proposed coding region of the GP185 gene [Holder et al., Nature 317 (1985) 270-273]. Comparison of the nucleotide sequences for the GP185 gene of Honduras I and five other isolates indicated that there are two areas of conserved DNA sequence, one of 310 bp (beginning 181 bp upstream from the proposed initiation codon) and the other of greater than or equal to 360 bp (located entirely within the coding region), separated by a region encoding isolate-specific tandem amino acid repeats. Rat antiserum was raised to a fusion protein derived from the conserved regions and the intervening repeat region of this Honduras I protein. This antiserum bound GP185 on immunoblots of the homologous Honduras I isolate and the heterologous K1 isolate, which has different tandem repeats. Serum from owl monkeys and humans previously infected with P. falciparum reacted with the fusion protein on immunoblots demonstrating that determinants in the N-terminal 15% of GP185 were immunogenic in infected individuals and suggesting that some of these sites are conserved among isolates.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a high-molecular-weight phosphoprotein synthesized by the human malarial parasite Plasmodium falciparum.

During its intra-erythrocytic cycle, Plasmodium falciparum synthesizes a protein of apparent Mr 250,000-300,000. Its precise size is dependent on the P. falciparum isolate examined. This protein contains phosphate covalently bound to one or more serine residues and hence is termed PP300. Monoclonal antibody, McAb4-1F, binds to PP300 on immunoblots of protein extracts from all parasite isolates tested, both those exhibiting and those lacking the knob phenotype. Using McAb4-1F, the polypeptide was shown to be physically associated with the plasma membrane in a membrane-isolation procedure. However, in an indirect immunofluorescence assay the McAb appeared to bind to antigen associated with the erythrocyte plasma membrane in parasitized cells. However, it reacted only to fixed, not unfixed, parasitized erythrocytes indicating that the epitope is not normally exposed to extracellular antibodies. Clone 29-2 was isolated by a McAb4-1F immunoscreen of a P. falciparum complementary DNA (cDNA) expression library created in pUC8. Rat anti-clone serum which was raised to the purified protein encoded by the lacZ-29-2 fusion in pUC8 reacted with PP300 in immunoblots of parasite antigen. In Southern-blot analyses of parasite DNA digested with EcoRI, HindIII, or EcoRV, the 29-2 DNA insert hybridized to more than one fragment even though the insert lacked internal sites for these enzymes. In addition, hybridization studies were conducted using two oligodeoxy-nucleotides which were constructed based on the sequence of a cDNA clone which encoded part of a similar high-molecular-weight P. falciparum protein [Coppel et al., Mol. Biochem. Parasitol. 20 (1986) 265-277]. Analysis of these results indicates that the two cDNA sequences are parts of the same gene or a family of related genes.

Terminal galactose residues and the antigenicity of Plasmodium falciparum glycoproteins.

The presence of terminal alpha-D-galactosyl residues in the carbohydrate chains of glycoprotein antigens from the asexual blood stages of Plasmodium falciparum is demonstrated by the alpha-D-galactosidase sensitivity of particular parasite antigens, the use of specific glycosidases to cleave sugars from parasite glycoproteins radiolabeled with [3H]glucosamine, and the ability of Bandeirea simplicifolia lectin, which has a specificity for terminal alpha-galactosyl residues, to bind to the parasite. The carbohydrate side chains, and in particular the terminal alpha-galactosyl residues, are shown to have an important role in determining the binding of antibodies to parasite glycoproteins.

Synthesis of merozoite proteins and glycoproteins during the schizogony of Plasmodium falciparum.

We have investigated the protein and glycoprotein content of Plasmodium falciparum merozoites by metabolically labeling cultures of schizont-stage parasites with [35S]methionine or with [3H]glucosamine followed by incubation in nonradioactive medium to allow the schizonts to mature into merozoites, infect new erythrocytes, and develop into ring-stage parasites. The ring stages were separated from schizonts by sedimentation through Percoll. Labeled proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by fluorography. Using [35S]methionine, four major proteins (p) with apparent relative molecular weights (Mr) = 202k , 136k , 82k , and 46k and two proteins of intermediate labeling (Mr = 185k and 142k ) were observed in the schizont-labeled ring-stage parasites. Because corresponding proteins were also observed in the schizont stage, we conclude that they had been present in the invading merozoite. In contrast, prominent proteins which were generally labeled during the ring stage and some major schizont-stage proteins were virtually absent in the schizont-labeled ring-stage. By labeling the parasite proteins with [3H]glucosamine followed by separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, five major glycoproteins (gp) of apparent Mr = 185k , 88k , 56k , 46k , and 34k were identified. Their presence in both the schizont and the schizont-labeled ring stage demonstrated that the merozoite contains glycoproteins. Immune owl monkey serum recognized all five glycoproteins. A comparison of proteins by two-dimensional gel electrophoresis (isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis) suggested that p185 and gp185 were identical, as were p46 and gp46 .

Plasmodium falciparum antigens associated with membrane structures in the host erythrocyte cytoplasm.

Hybridomas were made from mice immunized with plasma membranes from erythrocytes infected with Plasmodium falciparum. Among the monoclonal antibodies produced, a series reacted with antigens in the host cell cytoplasm. Immunoelectron microscopy, along with indirect fluorescent antibody double labeling experiments, were used to further localize the antigens to membrane structures (presumably Maurer's clefts) in the erythrocyte cytoplasm. The epitopes thus localized are found on three parasite proteins (20 kDa, 29 kDa, and 45 kDa) and one parasite glycoprotein (45 kDa). They are likely to be part of a transport system for the parasite.

Cross-reactive asparagine-rich determinants shared between several blood-stage antigens of Plasmodium falciparum and the circumsporozoite protein.

From a Plasmodium falciparum cDNA expression library derived from mRNA of the asexual blood stages, we isolated and sequenced five different cDNA clones whose predicted protein products were unusually rich in asparagine (Asn). Two of the clones, R5 and G5, contain tandem imperfectly repeated sequences based on Asn-Asn-Thr (NNT) and Asn-Asn-Met (NNM) respectively. The other three, E4, C5 and R13, as well as G5, contain stretches of polyasparagine varying in length from 2 to 26 residues. Results of DNA blotting experiments with the individual cDNA sequences as probes suggest that each of the five clones corresponds to a different P. falciparum gene. The fragments of P. falciparum proteins expressed by the cDNA clones shared cross-reactive antigenic determinants which were present on multiple P. falciparum proteins. In immunoblotting experiments, owl monkey antibodies selected for binding to the polypeptide expressed by clone E4, C5 or G5 reacted with the expressed proteins from all 5 clones, and with at least 10 proteins from schizont infected erythrocytes. The cross-reactive epitopes could be modeled by two Asn-rich peptide structures: (1) (NNT)8, whose sequence was based on the R5 repeat; and (2) (NPNA)6, whose sequence was based on the Asn-rich repeat of the P. falciparum circumsporozoite protein (CSP). Antibodies that bound to each peptide were selected from sera of immune monkeys that had never been exposed to sporozoites. The selected antibodies bound all 5 expressed proteins in immunoblotting assays and also bound to several proteins from parasitized erythrocytes. Such cross reactivity between the CSP repeating unit and several blood-stage antigens has not been previously reported.

Two Plasmodium falciparum merozoite surface polypeptides share epitopes with a single Mr 185 000 parasite glycoprotein.

The malarial parasite Plasmodium falciparum synthesizes a major glycoprotein (gp) of Mr 185 000 during its asexual blood cycle. Immunoprecipitation of [35S]methionine- or [3H]glucosamine-labeled schizont antigens indicated that two groups of polypeptides were distinguished with anti-gp 185 mouse monoclonal antibodies: group A was composed of glycosylated molecules of Mr 185 000, 120 000, 90 000, 88 000, 46 000, and 40 000 while group B contained, in addition to gp 185, polypeptides of Mr 152 000, 106 000 and 83 000. The latter polypeptides lacked detectable amounts of radiolabeled saccharide. The smaller Mr polypeptides were specifically immunoprecipitated and not merely coprecipitated with gp 185. Our results suggest that gp 185 contains at least two structurally distinct domains which may be processed independently into either the group A or group B polypeptides. Although gp 185 may not be a merozoite surface protein, representative group A and group B-specific monoclonal antibodies bound to surface antigens of the merozoite as demonstrated by immunolabeling followed by electron microscopy. Therefore, at least one group A antigen and one group B antigen appeared to be on the extracellular surface of the merozoite. The proteins found in immunoprecipitates after both (1) sonication in aqueous medium and ultracentrifugation and (2) solubilization and phase separation of parasite molecules with Triton X-114 suggested that the group A and group B polypeptides and glycoproteins are either soluble or peripheral membrane proteins. Some of these, therefore, may be components of the surface coat of the merozoite.

Plasmodium falciparum: comparison of in vitro growth of knobby and knobless isolates.

Variants (K-) of three strains of Plasmodium falciparum which do not produce the erythrocyte surface alterations that have been called knobs have been compared with their wildtype knobby (K+) parents. The K- variants achieve higher parasitemias, incorporate radiolabeled isoleucine more rapidly, and produce a higher percentage of multiply-infected cells than do their K+ parents. Nevertheless, immune owl monkey sera cause approximately the same percentage inhibition of growth of both K+ and K- organisms when included in the growth medium at a 1% concentration.

In vitro inhibition of intracellular growth of Plasmodium falciparum by immune sera.

Beginning at the ring stage, synchronized cultures of Plasmodium falciparum were grown in suspension for 22-32 hours. Intracellular growth was assayed by measuring cellular uptake and incorporation into protein of 35S-methionine. Low concentrations (2%) of serum from immune humans and Aotus monkeys were found to inhibit the uptake of the 35S-methionine. The amount of inhibition for a given serum was often inversely related to its indirect fluorescent antibody test titer. Inhibition occurred during the trophozoite stage and was not obtained with a clone lacking the erythrocyte modifications referred to as knobs. Thus, a sensitive new assay is described which allows detection of factors in immune primate sera which can affect maturation of P. falciparum within the erythrocyte. These serum factors are likely to be antibodies which react with antigens expressed at the trophozoite stage on the surface of K+-infected erythrocytes.

Reaction of immune sera with components of the human malarial parasite, Plasmodium falciparum.

Human and monkey sera from individuals exposed to Plasmodium falciparum were characterized by indirect immunofluorescence, in vitro parasite growth inhibition, and immunoprecipitation of 125I-labeled parasite antigens followed by analytical sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In general there was a good correlation between fluorescence titer and the ability of a serum to inhibit parasite growth in vitro. Exceptions were found, however. Some variance was seen in the ability of a given serum to inhibit different strains of the parasite. The significance of this is unknown. The proteins bound by human sera with high and low in vitro inhibitory capacities were compared by SDS-PAGE. The human sera which did not inhibit parasite growth in vitro well differed from those which did by failing to efficiently bind certain parasite components having molecular weights in the range of 200,000, 70,000-85,000, and 45,000.

Proteins responsible for a punctate fluorescence pattern in Plasmodium falciparum merozoites.

Mouse monoclonal antibodies (McAbs) have been used to characterize the proteins of the asexual erythrocytic cycle of Plasmodium falciparum. Three different McAbs react with antigens of the schizont and extracellular merozoite to give a punctate fluorescence pattern. In many cases, such areas of fluorescence were composed of two adjacent, fluorescent bodies; these were distinct from the nuclei. In contrast, McAbs which bound to the ring-stage parasite were not localized, but were diffusely distributed within or around the ring-stage parasite. These McAbs immunoprecipitated five prominent, 35S-methionine-labeled schizont proteins (p) of Mr 82K, 70K, 67K, 39K, and 37K. Only p82, p39, and p37 were immunoprecipitated from schizont-labeled ring-stage parasites; thus, it appears that p70 and p67 are modified, degraded, or secreted some time between intracellular merozoite maturation and erythrocyte invasion.

Translation in vitro of RNA from the human malarial parasite Plasmodium falciparum.

RNA has been isolated from the human malarial parasite, Plasmodium falciparum, and translated in vitro using two systems: rabbit-reticulocyte lysate and Xenopus laevis oocytes. Polypeptides are synthesized that are characteristic of the parasite isolate and the developmental stage from which the RNA was purified. Many of these polypeptides are precipitated by antibody from an immune monkey. Analysis of the primary structure of these polypeptides should give the information necessary to produce a vaccine against this parasite.

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.

Heat shock-like polypeptides of the sporozoites and merozoites of Eimeria bovis.

Heat shock proteins (hsps) are a group of highly conserved polypeptides found in a wide variety of organisms. Polypeptides of sporozoites and merozoites of Eimeria bovis, blotted onto nitrocellulose, were probed with antibodies to artificially constructed peptides representing portions of the cDNA-generated fragment of pf75, the 75K hsp of merozoites of Plasmodium falciparum. Polypeptide antigens of sporozoites and merozoites of E. bovis with molecular weights of 46K, 71-72K, and 75K reacted with antibodies against pf75, indicating that they are hsp70 (the 70K family of hsps) or hsp70 cognates (noninducible proteins homologous to hsps). Radiolabeling with 125I and treatment with antibodies against pf75 detected a 71K antigen on the merozoite surface. Hsps in sporozoites of E. bovis are either constitutive or evoked by treatment at 37 C for in vitro excystation. If hsp70 is mandatory for parasite survival, it may prove to be an appropriate antigen for a vaccine against bovine coccidiosis.

Inhibition of the in vitro growth of Plasmodium falciparum. I. The effects of immune serum and purified immunoglobulin from owl monkeys.

Sera from Aotus sp. monkeys (karyotypes II, III, and IV) which were immune to Plasmodium falciparum have been used to inhibit the in vitro growth of this human malaria parasite. Culture conditions used for the assays allowed 50- to 100-fold increases in the number of A+ erythrocytes infected in a 96-hr period in control cultures. Although normal monkey serum did not support growth as well as normal human serum, mixtures of normal monkey and human serum were found that did. Compared to such controls, as little as 3.5% immune monkey serum was found to cause approximately 56% inhibition in 4 days (2 replicative cycles). Purified globulin from immune monkeys inhibited 40% at 2 mg/ml and 75% at 7 mg/ml after a single replicative cycle. These data suggest that serum antibody is likely to play a major role in providing Aotus monkeys with protective immunity to P. falciparum.

Plasmodium falciparum polypeptides associated with the infected erythrocyte plasma membrane.

Plasmodium falciparum proteins associated with plasma membranes of infected erythrocytes were identified by using three techniques: isolated plasma membranes from infected and uninfected erythrocytes were compared by gel electrophoresis and silver staining; isolated plasma membranes from cells metabolically labeled with [35S]methionine were assayed by gel electrophoresis; and uninfected and infected intact erythrocytes were surface-labeled by lactoperoxidase iodination, and the labeled polypeptides were compared by gel electrophoresis. The results from these experiments indicate that at least six parasite-derived polypeptides (Mr = greater than 240,000, 150,000, 55,000, 45,000, 35,000, and 20,000) are associated with the infected erythrocyte plasma membrane. At least four of these peptides (Mr = 55,000, 45,000, 35,000, and 20,000) may be exposed on the surface of the infected erythrocytes.