Monoclonal antibodies to a synthetic peptide corresponding to a repeated sequence in the Plasmodium falciparum antigen Pf155.

Mouse monoclonal antibodies were prepared against a synthetic peptide (EENVEHDA) corresponding to a tandemly repeated sequence in the C-terminus of the Plasmodium falciparum antigen Pf155. One antibody (IgG1) producing hybridoma was studied in detail. The specificity of the antibody was determined by enzyme-linked immunosorbent assays using bovine serum albumin-conjugated or free peptides as solid phase antigens and various synthetic peptides for inhibition. The antibody reacted with Pf155 as detected by immunofluorescence and immunoblotting. It was also an efficient inhibitor of merozoite invasion in P. falciparum in vitro cultures indicating that it defines a biologically important epitope present on the native Pf155 molecule.

Ultrastructural analysis of fresh Plasmodium falciparum-infected erythrocytes and their cytoadherence to human leukocytes.

Sixty fresh Plasmodium falciparum isolates obtained from Gambian children with mild or cerebral malaria were investigated by transmission electron microscopy for the expression of knob-like protrusions (K+) on the surface of the infected erythrocytes. More than six-hundred infected erythrocytes were analyzed. Knob-forming parasites were present in all 60 isolates. Although knobless parasites (K-) were found in 25 (42%) of the isolates, only 39 were K-, while 577 were K+. Nine of the 39 K- infected erythrocytes that were studied in greater detail appeared to be asexual parasites because they were either segmented or they lacked mitochondrial DNA-like filaments and cristae, which are abundant in immature gametocytes. No difference was observed in the relative frequency of K+K- infected erythrocytes in isolates from patients with mild or cerebral malaria. Binding of both knobby and knobless infected erythrocytes to autologous leukocytes including monocytes, neutrophils, lymphocytes and plasma cells was found in some of the primary in vitro cultures. By using P. falciparum laboratory strains of known phenotypes and leukocytes from healthy blood bank donors, it was established that this novel adherence phenomenon was related to that of cytoadherence to certain melanoma or endothelial cells. Cytoadherent infected erythrocytes that bind to leukocytes enhance antibody-independent phagocytosis and induce cellular aggregation, while non-cytoadherent or rosetting infected erythrocytes do not.(ABSTRACT TRUNCATED AT 250 WORDS)

Geographical distribution of Plasmodium falciparum erythrocyte rosetting and frequency of rosetting antibodies in human sera.

Uninfected erythrocytes bind spontaneously to those infected with certain strains of Plasmodium falciparum. This is known as spontaneous erythrocyte rosetting. We have studied the occurrence and frequency of rosetting in 75 fresh patient isolates and have identified rosetting strains from Africa, South America, and Asia. Rosetting was present in 49% of the isolates tested; the frequency of rosetting red blood cells (RBC) in individual isolates was 0-75% when scored during the first cycle of in vitro growth. Rosetting antibodies were found in 15 out of 73 (21%) Liberian sera as measured by disruption of rosettes in vitro. However, antibodies able to inhibit CD36 dependent cytoadherence of P. falciparum-infected RBC were not detected in these sera. Erythrocyte rosetting is a geographically widespread phenomenon. Rosetting antibodies seem to be induced by natural infection and the molecular mechanism of rosette formation seems distinct from that of endothelial cytoadherence.

Ultrastructural and immunohistochemical analysis of cholangiocarcinoma in immunized Syrian golden hamsters infected with Opisthorchis viverrini and administered with dimethylnitrosamine.

Utilizing the experimental model in Syrian golden hamsters, we explored the role of immunization in carcinogenesis. The animals, which were infected with liver flukes (Opisthorchis viverrini), and administered a subcarcinogenic dose of dimethylnitrosamine, developed cancer. Pre-immunizing with a crude somatic antigen did not reduce cancer development, but accelerated carcinogenesis. Histopathological analysis of the cancer tissues was done once at week 30 and again at week 39 using H and E staining, immunostaining for the p53 tumor suppressor phosphoprotein, and electron microscopy. Thirty weeks after immunization, the immunized hamsters developed tubular adenocarcinoma at a higher rate (71.43%) than the non-immunized group (20.00%). This rate (20.00%) increased to 63.64% by week 39. The small foci cancer in the non-immunized group decreased in frequency from 80.00% (at week 30) to 36.36% (by week 39), suggesting the small foci cancer progressed to tubular adenocarcinoma during the 9-week interval. Most of the observed tubular adenocarcinoma was well differentiated. Nearly all hamsters that tested positive for cancer also tested positive for p53 immunostaining in the epithelia of the small bile ducts. The positive reaction for p53-immunostaining was localized in the rough endoplasmic reticulum, Golgi apparatus and perinuclear membranes. The electron micrographs of these positive p53-immunostained cells showed characteristics of early cancer. The detection of p53 in early cancer development makes it a candidate as a tumor marker.

Circulating immune complexes in serum from patients with dengue haemorrhagic fever.

Circulating immune complexes were detectable in 80% of serum from patients with dengue haemorrhagic fever. The immune complexes were detected for the first time on day two after the onset of the fever. The amount of complexes reached the maximum value on day 4 or 5 after onset, or when the patients developed shock or subsidence of fever, after which the complexes decreased in number. The number of complexes also correlated well with the clinical grading (severity) of the disease, i.e. the maximum amount was shown in grade III. These complexes may play a part in the pathogenesis of this disease.

Plasmodium falciparum: analysis of the interaction of antigen Pf155/RESA with the erythrocyte membrane.

The location of the Plasmodium falciparum vaccine candidate antigen Pf155/RESA in the membrane of infected erythrocytes was analzyed by means of selective surface radioiodination and immunofluorescence of surface-modified cells. The lack of radiolabel in Pf155/RESA as well as its localization by immunofluorescence similar to that of the N-terminal region of erythrocyte band 3 suggests that the antigen is associated with the cytoplasmic phase of the erythrocyte membrane. In concordance with this, Pf155/RESA was detected by immunofluorescence on the surface of inside out membrane vesicles from P. falciparum-infected erythrocytes. Pf155/RESA from spent culture medium also bound to inside out membrane vesicles of normal erythrocytes as well as to cytoskeletal shells of such vesicles, but failed to bind to sealed right-side out membrane vesicles. Depletion of spectrin from the vesicles abolished antigen binding, suggesting that Pf155/RESA association with the erythrocyte cytoskeleton is mediated by spectrin.

Cytoadherence of knobby and knobless Plasmodium falciparum-infected erythrocytes.

Cytoadherence of Plasmodium falciparum-infected erythrocytes to melanoma cells was analysed using strains or isolates of parasites expressing or not expressing knobs (K+ or K- phenotype) on the erythrocyte surface. Both K+ and K- parasites had the capacity to cytoadhere to melanoma cells. Using a panel of melanoma cell lines with different surface expression of the cytoadherence receptors CD36, thrombospondin and ICAM-1 indicated that CD36 was the major receptor for parasites of both K+ and K- phenotypes. Binding competition experiments between K+ and K- -infected erythrocytes suggested that K+ cytoadherence is of higher affinity than that of K- parasites. However, some K- cytoadherence was also found in isolates containing mixed populations of K+ and K- parasites. The interaction of the two types of infected erythrocytes with melanoma cells also differed ultrastructurally, erythrocytes of K+ phenotype showing intimate interdigitations with microvilli on the melanoma cells, while erythrocytes of K- phenotype displayed more separated interactions with fewer sites of contact and involving only a few melanoma cell microvilli. One and the same infected erythrocyte may co-express the ligand for CD36-mediated cytoadherence and the structures mediating binding of uninfected erythrocytes to form rosettes.

Absence of antigenic diversity in Pf155, a major parasite antigen in membranes of erythrocytes infected with Plasmodium falciparum.

Pf155 is a merozoite-derived polypeptide antigen which the parasite Plasmodium falciparum deposits in the membranes of erythrocytes at invasion. Eleven laboratory strains or clones of P. falciparum and a large number of isolates obtained from patients from different parts of the world were studied for antigenic diversity in Pf155. Immunoglobulin G antibodies from different serum samples from P. falciparum-infected donors were affinity purified on monolayers of glutaraldehyde-fixed and air-dried erythrocytes infected with P. falciparum of different origins and tested in different combinations by immunoblotting, reinvasion inhibition, and a modified immunofluorescence procedure in which the membranes of recently infected erythrocytes were stained. Similar experiments were performed with monoclonal and oligoclonal antibodies specific for different epitopes in the C-terminal region of Pf155. No strain- or isolate-associated antigenic diversity or size variation of Pf155 was detected, indicating that the immunodominant regions of this antigen are highly conserved throughout the world.

Synthetic gene construct expressing a repeated and highly immunogenic epitope of the Plasmodium falciparum antigen Pf155.

The Plasmodium falciparum-derived antigen Pf155 contains two blocks of tandemly repeated amino acid sequences. A pair of complementary oligonucleotides, encoding the C-terminally located repeat Val-Glu-His-Asp-Ala-Glu-Glu-Asn, were synthesized. The oligonucleotides were polymerized by ligation, and the resulting multimers were cloned into an expression vector. One construct that contained four copies of the repeat was expressed in Escherichia coli. The product, a fusion protein, was soluble and produced in high amounts. It reacted in immunoblotting with a monoclonal antibody to a synthetic octapeptide (Glu-Glu-Asn-Val-Glu-His-Asp-Ala). Rabbits immunized with partially purified fusion protein, either with or without adjuvant, formed antibodies against this octapeptide. These antibodies reacted with Pf155 both in parasite extracts and when deposited in the membrane of infected erythrocytes. Furthermore, these antibodies inhibited merozoite reinvasion in vitro as efficiently as human antibodies to the octapeptide sequence in Pf155, induced by natural infection. The results suggest that products of synthetic gene constructs may be a suitable basis for an anti-merozoite vaccine.