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R F Anders

Publications and source records attributed to R F Anders.

At least 55 records · Page 3Linked to original sources
PubMed1992

Cloning and analysis of the RESA-2 gene: a DNA homologue of the ring-infected erythrocyte surface antigen gene of Plasmodium falciparum.

We have cloned and sequenced a homologue of the ring-infected erythrocyte surface antigen (RESA) gene from Plasmodium falciparum designated RESA-2. Two reading frames with high homology to exon 1 and exon 2 of RESA at both the nucleotide and amino acid levels were identified in the RESA-2 sequence. However, RESA-2 does not contain either of the blocks of tandem repeats present in RESA. The lack of an RNA transcript in either asexual or sexual stage parasites and the presence of an in-frame stop codon in the second reading frame suggests RESA-2 could be a pseudogene. Its lack of expression in asexual stages demonstrates that it does not complement the RESA deletion in isolate FCR3.

Amino Acid Sequence↗
PubMed1991

Structural diversity in the Plasmodium falciparum merozoite surface antigen 2.

Antigens associated with the surface of merozoites of the malaria parasite Plasmodium falciparum are directly accessible to immune attack and therefore are prime vaccine candidates. We have previously shown that one of the two known merozoite surface antigens (merozoite surface antigen 2; MSA-2) exhibits considerable sequence and antigenic diversity in different isolates. The sequences of MSA-2 from three isolates revealed a central domain composed of repeats that vary in number, length, and sequence, flanked in turn by nonrepetitive variable sequences and by conserved N- and C-terminal domains. We report here the sequences of a further four MSA-2 alleles, containing repetitive sequences that are related but not identical to each other. The seven alleles of MSA-2 can be divided into two distinct allele families on the basis of nonrepetitive sequences. Hybridization studies with repeat probes indicated that all of the 44 P. falciparum isolates examined contained repeat regions similar to those defined in known MSA-2 sequences.

Amino Acid Sequence↗
PubMed1991

The ring-infected erythrocyte surface antigen of Plasmodium falciparum associates with spectrin in the erythrocyte membrane.

The malaria parasite Plasmodium falciparum synthesises a protein, RESA, which associates with the membrane of newly invaded erythrocytes. Using spent supernatants from P. falciparum growing in culture as a source of soluble RESA we have developed an assay to examine the characteristics of RESA binding to the erythrocyte membrane in vitro. RESA associated with the Triton X-100 insoluble proteins on the inner face of the host erythrocyte membrane but did not bind to the outer surface of intact erythrocytes. Other proteins present in culture supernatants did not bind to the erythrocyte membrane. RESA was co-sedimented with the ternary complex formed between actin, spectrin and band 4.1 and co-precipitated with spectrin precipitated with anti-spectrin antibodies. The extent of association between RESA and the inner face of the erythrocyte membrane was reduced by the inclusion of excess purified spectrin in the assay. Thus, RESA appears to be associated with spectrin in the erythrocyte membrane skeleton.

Animals↗
PubMed1991

Plasmodium falciparum ring-infected erythrocyte surface antigen is released from merozoite dense granules after erythrocyte invasion.

Electron microscopy was used to study the fate of Plasmodium falciparum ring-infected erythrocyte surface antigen after merozoite invasion by using postembedding immunolabeling. The antigen was localized to small dense granules located centrally or laterally in free merozoites. In newly invaded erythrocytes, labeling was found in pockets of the parasitophorous vacuole space or in aggregates closely associated with the parasitophorous vacuole. These patterns indicate that ring-infected erythrocyte surface antigen is contained in merozoite dense granules that are released after merozoite invasion and not via apical rhoptry ducts at the time of merozoite attachment.

Animals↗
PubMed1991

Failure of recombinant vaccinia viruses expressing Plasmodium falciparum antigens to protect Saimiri monkeys against malaria.

Saimiri sciurus monkeys were immunized at multiple sites with recombinant vaccinia viruses expressing Plasmodium falciparum antigen genes and boosted 4 weeks later. Control monkeys were immunized with a thymidine kinase-negative vaccinia virus mutant. Two weeks later, all of the monkeys were challenged by intravenous inoculation of P. falciparum (Indochina strain) parasites. A group of unimmunized monkeys was challenged in parallel. All of the monkeys that received vaccinia virus recombinants or the control virus produced good anti-vaccinia virus antibody responses. However, those that received a single construct containing ring-infected erythrocyte surface antigen (RESA) given at eight sites did not produce significant antibody to any of the three major RESA repeat epitopes after immunization but were primed for an enhanced antibody response after challenge infection with P. falciparum. Most of the monkeys produced detectable antibodies to the RESA epitopes after challenge infection. One group of monkeys was immunized with four constructs (expressing RESA, two merozoite surface antigens [MSA-1 and MSA-2], and a rhoptry protein [AMA-1]), each given at two sites. While these monkeys failed to produce significant antibody against MSA-2 or AMA-1 after immunization, they produced enhanced responses against these antigens after challenge infection. Immunization involved an allelic form of MSA-2 different from that present in the parasite challenge strain, so that the enhanced responses seen after challenge infection indicated the presence of T-cell epitopes common to both allelic forms. No groups of monkeys showed any evidence of protection against challenge, as determined by examination of the resulting parasitemias.

Animals↗
PubMed1991

Immunization of owl monkeys with the ring-infected erythrocyte surface antigen of Plasmodium falciparum.

Aotus nancymai were immunized with the 4-mer, 8-mer, and 11-mer repeat peptides of the ring-infected erythrocyte surface antigen molecule of Plasmodium falciparum conjugated to diphtheria toxoid with muramyl dipeptide (MDP) as adjuvant. Immunization failed to induce protective immunity against the Uganda Palo Alto strain of P. falciparum as judged by maximum levels of parasitemia of immunized monkeys relative to those of controls. The fused polypeptide FPAg632, when combined with MDP, also failed to induce protective immunity. However, the maximum level of parasitemia and serologic response to the 11-mer peptide were inversely correlated. The safety of the use of MDP was evident.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1991

Antigenic diversity in Plasmodium falciparum.

There are several mechanisms responsible for the extensive antigenic diversity found in the asexual blood stages of Plasmodium falciparum. Failure to express antigens is a feature of many isolates cultured in vitro but probably is not a major cause of antigenic diversity in vivo. Numerous point mutations occur in allelic forms of asexual blood stage antigens and are assumed to contribute to antigenic diversity but as yet few such mutations have been mapped to antigenic epitopes. A major cause of antigenic diversity is the expression of different repetitive sequences in allelic forms of several antigens including the S-antigen and the two merozoite surface antigens, MSA-1 and MSA-2. The sequencing data indicates that S-antigen genes fall into many allelic families whereas both MSA-1 and MSA-2 are dimorphic. Further diversity has arisen as a result of intragenic recombinations between the dimorphic forms of both MSA-1 and MSA-2. In addition to this diversity reflecting the expression of different allelic genes, asexual blood stages of malaria parasites undergo antigenic variation in that clonal parasite populations can vary the form of an antigen on the surface of infected erythrocytes. Antibodies or DNA probes directed against variable repeat sequences can be used to distinguish different isolates of P. falciparum. The use of antibodies to S-antigen repeats has been particularly useful for typing the parasites causing infections. The application of S-antigen typing to field studies in Papua New Guinea has demonstrated marked diversity in the parasites causing infections in one area.

Animals↗
PubMed1990

Rotational dynamics of the integral membrane protein, band 3, as a probe of the membrane events associated with Plasmodium falciparum infections of human erythrocytes.

Time-resolved phosphorescence anisotropy was used to study the molecular organisation of band 3 in the erythrocyte membrane. Three different rotational relaxation regimes of mobile band 3 were resolved. These populations may represent different aggregation states of band 3 within the membrane, or they may result from association of band 3 with other proteins at the cytoplasmic surface. The polycation spermine decreases the apparent mobility of band 3 by a mechanism that does not involve the underlying cytoskeleton. A monoclonal antibody directed against the cytoplasmic portion of band 3 can also cause an increase in the immobile fraction of band 3 molecules. This monoclonal antibody will inhibit invasion of erythrocytes by malaria parasites. Membranes prepared from erythrocytes infected with mature stages of the malaria parasite, Plasmodium falciparum, show altered dynamic properties corresponding to a marked restriction of band 3 mobility.

Animals↗
PubMed1990

The mature-parasite-infected erythrocyte surface antigen (MESA) of Plasmodium falciparum associates with the erythrocyte membrane skeletal protein, band 4.1.

Several proteins synthesized by mature asexual stages of Plasmodium falciparum interact with the erythrocyte membrane skeleton. One of these is the mature-parasite-infected erythrocyte surface antigen (MESA; also called PfEMP2), a phosphoprotein of 250-300 kDa, which is found on the internal face of the erythrocyte membrane. When MESA is precipitated with anti-MESA antibodies, another phosphoprotein of 80 kDa is co-precipitated. This 80-kDa phosphoprotein was identified by peptide mapping as the erythrocyte membrane component band 4.1. Thus, MESA is apparently anchored at the erythrocyte membrane through an association with band 4.1. Band 4.1 is more intensely phosphorylated in infected erythrocytes and is increased in relative molecular mass in erythrocytes infected by isolates of P. falciparum that cytoadhere.

Animals↗
PubMed1990

The ring-infected erythrocyte surface antigen protein of Plasmodium falciparum is phosphorylated upon association with the host cell membrane.

The ring-infected erythrocyte surface antigen (RESA) is a 155-kDa malarial polypeptide which is released from merozoites and becomes associated with the erythrocyte membrane at the time of invasion. Inside-out vesicles (IOVs) prepared from Plasmodium falciparum-infected erythrocytes contain RESA, presumably bound to the membrane skeleton, as it is largely insoluble in Triton X-100. When these IOVs were incubated with [gamma-32P]ATP, a 155-kDa polypeptide was labeled in IOVs from infected, but not from uninfected erythrocytes. Immunoprecipitation using specific rabbit antisera confirmed that RESA is indeed a phosphoprotein. Phosphoamino acid analysis revealed phosphoserine and a small amount of phosphothreonine, but no phosphotyrosine. Labeling of intact parasitized erythrocytes with inorganic [32P]phosphate for several hours in culture resulted in RESA in Triton-insoluble extracts being phosphorylated. Labeling of synchronized parasites showed that RESA was phosphorylated only when it became associated with the erythrocyte membrane, and although RESA was abundant in mature parasites, it was not phosphorylated. RESA, released into the culture supernatants during the growth of P. falciparum, bound to IOVs prepared from normal uninfected erythrocytes, and subsequent labeling with [gamma-32P]ATP resulted in the phosphorylation of RESA. The evidence suggests that RESA is phosphorylated by an erythrocyte membrane kinase and probably not by a parasite-encoded enzyme.

Adenosine Triphosphate↗
PubMed1990

Plasmodium falciparum: two antigens of similar size are located in different compartments of the rhoptry.

Two previously described antigens, AMA-1 and QF3, which are located in the rhoptries of Plasmodium falciparum merozoites have polypeptides of similar relative molecular masses. On immunoblots, antibodies to both antigens recognized polypeptides of relative molecular mass 80,000 and 62,000 in all isolates tested. Two-dimensional electrophoresis showed that the isoelectric points of the two antigens were different. QF3 being more basic than AMA-1. AMA-1 was soluble in Triton X-114 whereas QF3 partitioned into the aqueous phase after temperature-dependent phase separation. In immunoelectron microscopic studies. QF3 was found in the body of the rhoptry whereas AMA-1 was consistently found in the neck of the rhoptry. Both antigens gave a punctate double-dot pattern in mature schizonts and merozoites when visualized by fluorescence microscopy, but AMA-1 antibodies also appeared to label the merozoite surface. QF3 was also detected in ring-infected erythrocytes whereas AMA-1 was not. Synthesis of both antigens was first observed in mature trophozoites and immature schizonts. Pulse-chase experiments showed that the Mr 80,000 polypeptide of the AMA-1 gene was subject to immediate processing to the Mr 62,000 product. This cleavage pattern was not stage specific. The Mr 80,000 polypeptide of QF3 was derived from a short-lived Mr 84,000 precursor polypeptide. Processing of the Mr 80,000 polypeptide to an Mr 62,000 polypeptide was restricted to the period of merozoite maturation and reinvasion. Hence AMA-1 and QF3 are different antigens with polypeptides of similar size but located in different compartments of the merozoite rhoptries.

Animals↗
PubMed1990

Structural diversity in the 45-kilodalton merozoite surface antigen of Plasmodium falciparum.

An integral membrane protein associated with the merozoite surface of Plasmodium falciparum termed merozoite surface antigen 2 (the 45-kDa merozoite surface antigen), occurs in antigenically diverse forms. Here we report the sequences of the MSA 2 gene from two other isolates of P. falciparum. The 43 N-terminal residues and the 74 C-terminal residues of all three MSA 2 sequences are highly conserved, but between these conserved regions there are dramatic differences among the alleles. Instead of the two copies of a 32-amino-acid repeat present in the MSA 2 of isolate FC27, MSA 2 from clone 3D7 and isolate Indochina 1 contain 5 and 12 copies respectively of the four amino acid sequence Gly Gly Ser Ala. The sequences flanking the repeats also differ among the three antigens. The repeats in MSA 2 appear to be immunodominant during natural infection, and antibodies to the repeat regions of different alleles react with a restricted number of parasite isolates.

Amino Acid Sequence↗
PubMed1990

Clonal repertoire analysis of murine B cells specific for repeat sequence antigens of Plasmodium falciparum.

Clonal analysis of the murine B-cell repertoire has been used to investigate the possible role of tandem repeat sequence epitopes of Plasmodium falciparum in immune evasion. A limiting dilution culture system was used whereby murine spleen cells were stimulated with the B-cell mitogen lipopolysaccharide (LPS) in the presence of 3T3 fibroblast filler cells. One in three B cells were shown to produce clones secreting immunoglobulin measurable by an ELISA. The frequency of antibody forming cell precursors (AFCp) specific for the 3' repeat epitopes of the ring injected erythrocyte surface antigen (RESA) was estimated in non-primed mice and found to be low. However, an accurate frequency determination was not possible using this method since the detection of the few positive cultures was found to depend on the presence of more than one AFCp or its products. Limiting dilution analysis was used to assess the frequency and repertoire of splenic AFCp at various times after immunization with a synthetic peptide of the RESA 3' repeat epitope (8 x 4-mer), presented in various ways. There was no marked increase in LPS-responsive AFCp specific for this antigen at the level of either IgM or IgG secretion. This was in marked contrast to the antibody response in vivo, where moderate IgG antibody titres, normally indicative of a secondary response, were seen in the serum of the same mice used for AFCp assay. This discrepancy between serum titre and AFCp frequency following immunization was not apparent with a non-malarial antigen, keyhole limpet haemocyanin (KLH). It was concluded that the LPS-stimulated limiting dilution culture system was not registering RESA-specific memory AFCp. These results raise the possibility that the malarial antigens are deficient in memory B-cell generation, or that secondary responses to these determinants may arise from a distinct B-cell progenitor which is non-responsive to LPS in vitro.

Adjuvants, Immunologic↗
PubMed1990

Anti-idiotypic antibodies counteract the invasion inhibition capacity of antibodies to major epitopes of the Plasmodium falciparum antigen Pf155/RESA.

Rabbits were immunized with the synthetic peptide EENVEHDA or (EENV)2, corresponding to a tandemly repeated sequence in the C-terminal part of the Plasmodium falciparum antigen Pf155/RESA, or with Escherichia coli-derived fusion proteins containing the corresponding repeats. For all sera, the capacity of the total immunoglobulin G fractions to inhibit P. falciparum merozoite invasion in vitro was similar and relatively low. Affinity purification of Pf155/RESA-specific antibodies on parasite-infected erythrocyte monolayers or on peptide columns increased the inhibitory capacity 50 to 5,000 times, whereas the immunofluorescence titers were increased only 10 times. The addition of small amounts of total immunoglobulin G to the affinity-purified antibodies gave a marked and dose-dependent reduction of the inhibitory capacity of the purified antibodies. However, this reduction was only seen in combinations where the immunoglobulin G fraction was from the same serum as the affinity-purified antibodies, suggesting that it was mediated by anti-idiotypic antibodies reacting with non-cross-reacting idiotopes of the invasion-inhibiting antibodies.

Amino Acid Sequence↗