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R F Anders

Publications and source records attributed to R F Anders.

At least 37 records · Page 2Linked to original sources
PubMed1996

The disulfide bond structure of Plasmodium apical membrane antigen-1.

Apical membrane antigen-1 (AMA-1) of Plasmodium falciparum is one of the leading asexual blood stage antigens being considered for inclusion in a malaria vaccine. The ability of this molecule to induce a protective immune response has been shown to be dependent upon a conformation stabilized by disulfide bonds. In this study we have utilized the reversed-phase high performance liquid chromatography of dithiothreitol-reduced and nonreduced tryptic digests of Plasmodium chabaudi AMA-1 secreted from baculovirus-infected insect cells, in conjunction with N-terminal sequencing and electrospray-ionization mass spectrometry, to identify and assign disulfide-linked peptides. All 16 cysteine residues that are conserved in all known sequences of AMA-1 are incorporated into intramolecular disulfide bonds. Six of the eight bonds have been assigned unequivocally, whereas the two unassigned disulfide bonds connect two Cys-Xaa-Cys sequences separated by 14 residues. The eight disulfide bonds fall into three nonoverlapping groups that define three possible subdomains within the AMA-1 ectodomain. Although the pattern of disulfide bonds within subdomain III has not been fully elucidated, one of only two possible linkage patterns closely resembles the cystine knot motif found in growth factors. Sites of amino acid substitutions in AMA-1 that are well separated in the primary sequence are clustered by the disulfide bonds in subdomains II and III. These findings are consistent with the conclusion that these amino acid substitutions are defining conformational disulfide bond-dependent epitopes that are recognized by protective immune responses.

Amino Acid Sequence↗
PubMed1996

Protective immune responses to apical membrane antigen 1 of Plasmodium chabaudi involve recognition of strain-specific epitopes.

Apical membrane antigen 1 (AMA-1), an asexual blood-stage antigen of Plasmodium falciparum, is an important candidate for testing as a component of a malaria vaccine. This study investigates the nature of diversity in the Plasmodium chabaudi adami homolog of AMA-1 and the impact of that diversity on the efficacy of the recombinant antigen as a vaccine against challenge with a heterologous strain of P. chabaudi. The nucleotide sequence of the AMA-1 gene from strain DS differs from the published 556KA sequence at 79 sites. The large number of mutations, the nonrandom distribution of both synonymous and nonsynonymous mutations, and the nature of both the codon changes and the resulting amino acid substitutions suggest that positive selection operates on the AMA-1 gene in regions coding for antigenic sites. Protective immune responses induced by AMA-1 were strain specific. Immunization of mice with the refolded ectodomain of DS AMA-1 provided partial protection against challenge with virulent DS (homologous) parasites but failed to protect against challenge with avirulent 556KA (heterologous) parasites. Passive immunization of mice with rabbit antibodies raised against the same antigen had little effect on heterologous challenge but provided significant protection against the homologous DS parasites.

Amino Acid Sequence↗
PubMed1995

Solution structure of a polypeptide containing four heptad repeat units from a merozoite surface antigen of Plasmodium falciparum.

The Plasmodium falciparum antigen SPAM (secreted polymorphic antigen associated with merozoites) contains an unusual set of heptad repeat units with alanine at the a and d positions. Twelve heptads with the consensus sequence AXXAXXX occur in three blocks of four, linked by short nonrepetitive sequences. A 38-residue polypeptide comprising the first block of four heptad units and five flanking residues at either end, SPAM-H1, has been synthesized and its structure in aqueous solution determined from 1H NMR data. Sedimentation equilibrium showed the peptide to be monomeric in aqueous solution. Its structure was determined from 1H NMR-derived distance and dihedral angle constraints by using distance geometry calculations, restrained simulated annealing, and conjugate gradient energy minimization in the CHARMm force field. The polypeptide contains an alpha-helix extending from Ser10 (position e of the first heptad) to at least Lys32 (position f of the fourth heptad) and possibly as far as Val35. The helix is bent, partly as result of a kink around residues 19-20. The conformations of the nine N-terminal residues and the six C-terminal residues are not well defined by the NMR data. The rms deviation from the average of the 20 best structures over the well-defined region (residues 11-31, which have backbone angular order parameters > 0.8) was 1.56 A for backbone heavy atoms (N, C alpha, and C) and 2.12 A for all heavy atoms. 2H2O exchange experiments identified slowly exchanging amide protons near the C-terminus and the last two turns of the helix. The unusual stability of the C-terminus reflects the presence of a new C-capping motif, which may involve the side chain of an asparagine in a position external to the C-cap residue. Possible interactions of the H1 sequence with the other two heptad repeat units in the intact merozoite antigen are discussed.

Amino Acid Sequence↗
PubMed1995

Humoral and cell-mediated immunity to the Plasmodium falciparum ring-infected erythrocyte surface antigen in an adult population exposed to highly endemic malaria.

A parasitological and immunological survey was carried out in an area in Papua New Guinea highly endemic for malaria. Two hundred fourteen adult individuals were selected for studies to assess their immune responses against the malaria vaccine candidate ring-infected erythrocyte surface antigen (RESA). Total immunoglobulin G (IgG) antibodies directed against RESA as well as specific IgG1, IgG2, and IgG3 antibodies were determined. Humoral responses directed against RESA were frequent in all IgG subclasses. Only IgG3 responses were found to be age dependent. Total anti-RESA IgG antibodies were not correlated with protection against malaria as measured by parasite prevalence, parasite density, or health center attendance. In contrast, cytophilic antibodies (IgG1 and IgG3) were associated with reduced Plasmodium falciparum prevalence and reduced health center attendance. T-cell proliferation in general was low and very infrequent. No correlation between humoral and cellular immune responses could be found. Parasite density, parasite prevalence, and health center visits tended to be reduced in individuals with good humoral and cell-mediated immune responses.

Animals↗
PubMed1995

Humoral response to Plasmodium falciparum ring-infected erythrocyte surface antigen in a highly endemic area of Papua New Guinea.

The prevalence and concentration of antibodies to ring-infected erythrocyte surface antigen (RESA) were measured in blood samples collected during a cross-sectional survey conducted in Papua New Guinea. Antibodies were measured by enzyme-linked immunosorbent assay to the recombinant RESA protein in 1,398 subjects and to RESA 8 and RESA 11 synthetic peptides in a subsample of 200 adults. Overall, the seropositivity rate to recombinant RESA was 66% and the geometric mean antibody concentration was 28 micrograms/ml. There was a slow increase in antibody prevalence and concentration with age that continued to occur even after 40 years of age. In children less than 10 years of age, there was a significant positive correlation between both RESA antibody prevalence and concentration and concurrent infection with Plasmodium falciparum. The opposite was true in adults more than 20 years of age, with those having a high antibody concentration to RESA being less likely to be parasitemic at the time of the survey. This observation was consistent with the finding of a weak but significant negative correlation between log antibody concentration and log P. falciparum density, which was mainly found in adults. No consistent correlation was found between humoral immune response to RESA and morbidity indicators.

Adolescent↗
PubMed1994

Plasmodium falciparum: a region of polymorphism in the 3' end of the gene for the ring-infected erythrocyte surface antigen.

A clone expressing the 3' end of the RESA gene of the Palo Alto isolate of Plasmodium falciparum was isolated by screening a lambda expression library of Mung bean nuclease-digested genomic DNA fragments with antibodies from malaria patients. The DNA sequence of the RESA gene of this isolate has seven single-base changes compared to that seen in the Papua New Guinean isolate FC27. Six of the seven mutations cause amino acid substitutions. The same amino acid sequence as the Palo Alto sequence is found in the Ghanaian isolate NF7. To determine whether these are the only changes in this region of the molecule, we performed PCR analyses on 12 blood samples from malaria patients from Indonesia. The Palo Alto form of the RESA gene was detected in 10 of the samples and the FC27 form of the gene was detected in the other 2 samples. No other forms of the RESA gene were found, suggesting that RESA exists in two alleles for the examined region of the gene. These results indicate that there are two major allelic forms of RESA present in this part of Indonesia.

Amino Acid Sequence↗
PubMed1994

Molecular variation in a novel polymorphic antigen associated with Plasmodium falciparum merozoites.

A cDNA clone encoding part of a novel polymorphic merozoite antigen from Plasmodium falciparum was isolated by screening a cDNA library with human immune serum from Papua New Guinea. Immunofluorescence microscopy and immunoblotting with affinity-purified antibodies recognized a highly polymorphic antigen, Ag956, present in schizonts and merozoites. Biosynthetic labeling and immunoprecipitation experiments demonstrated that Ag956 is proteolytically cleaved during merozoite maturation. The complete genomic sequence of Ag956 from the D10 clone of P. falciparum isolate FC27 encodes a secreted protein of calculated molecular mass 43,243 that is very hydrophilic and contains a region of unusual heptad repeats of the general structure AXXAXXX. This antigen has been named the secreted polymorphic antigen associated with merozoites (SPAM). The sequence of a second SPAM allele from the 3D7 clone of isolate NF54 reveals that the alanine heptad repeats and the hydrophilic C-terminal half of the protein are conserved. Variation among SPAM alleles is the result of deletions and amino acid substitutions in non-repetitive sequences within and flanking the alanine heptad-repeat domain. Heptad repeats in which the a and d position contain hydrophobic residues generate amphipathic alpha-helices which give rise to helical bundles or coiled-coil structures in proteins. Thus, SPAM is the first example of a P. falciparum antigen in which a repetitive sequence has features characteristic of a well-defined structural element.

Amino Acid Sequence↗
PubMed1994

Allelic variants of the Plasmodium falciparum merozoite surface antigen 2 (MSA-2) in a geographically restricted area of Irian Jaya.

Blood samples were collected from 12 residents of 4 villages in the Oksibil area of Irian Jaya. Eleven patients were positive for Plasmodium falciparum infection as evidenced by successful amplification of the MSA-2 gene by the polymerase chain reaction. Two patients showed evidence of infection by 2 strains of Plasmodium falciparum. All MSA-2 genes were completely sequenced and all could be assigned to one of the two major allelic families of MSA-2, however all MSA-2 gene sequences differed from previously described alleles. Five new allelic forms were identified, one of which was present in 8 of the 11 patients. Within small natural populations of P. falciparum, it appears that variation in MSA-2 approximates that seen world-wide. All samples were also analysed by hybridisation of amplified DNA to family specific probes and all samples hybridised to known probes. Our results demonstrate that there is a degree of microheterogeneity of MSA-2 that is undetectable by hybridisation studies alone.

Alleles↗
PubMed1994

The Plasmodium falciparum protein RESA interacts with the erythrocyte cytoskeleton and modifies erythrocyte thermal stability.

The ring-infected erythrocyte surface antigen (RESA) associates with spectrin in the erythrocyte membrane (Foley, M., Tilley, L., Sawyer, W. H. and Anders, R. F. (1991) Mol. Biochem. Parasitol., 46, 137-148). A fragment of the RESA protein, which was expressed in Escherichia coli, was found to bind to inside-out vesicles of erythrocyte membranes in an apparently saturable manner. Upon extraction of inside-out vesicles with Triton X-100, the RESA fragment remained associated with the erythrocyte cytoskeleton. Using the technique of steady-state fluorescence polarisation, we have studied the thermal denaturation of fluorescein-labelled spectrin in the presence of recombinant RESA. We found that the RESA fragment partially protected spectrin against heat-induced conformational changes. Furthermore, erythrocytes infected with a RESA (-) laboratory strain (FCR3) were shown to be more susceptible to heat-induced fragmentation than erythrocytes infected with a RESA (+) strain of the parasite. RESA does not, however, appear to play an essential role in the invasion process per se as erythrocytes resealed to contain anti-RESA antibodies were efficiently invaded.

Animals↗
PubMed1994

Diversity of agglutinating phenotype, cytoadherence, and rosette-forming characteristics of Plasmodium falciparum isolates from Papua New Guinean children.

The relationship between antigenic variation, cytoadherence, rosette formation, and the pathogenesis of malaria has led to great interest in the diversity of these properties in Plasmodium falciparum isolates from different communities. In this study, we extend previous investigations by delineating the spectrum of agglutinating phenotypes, adherence to C32 melanoma cells, human umbilical vein endothelial cells (HUVEC), CD36, and intracellular adhesion molecule-1 (ICAM-1), and rosette-forming ability of a group of 20 P. falciparum isolates from Papua New Guinean children. Agglutination phenotypes were determined by using both the children's convalescent serum and a panel of adult immune sera. The wide range of variant antigenic types in the community was demonstrated by the failure of the agglutination assays to identify any two isolates with the same agglutinating phenotype in this, the largest study of its kind. Comparison of agglutination profiles from fresh and cryopreserved isolates demonstrated the general acceptability of cryopreservation before testing, but cautioned that some isolates may undergo selection and phenotypic change during the process. Nineteen isolates were able to bind to at least one of the four ligands studied and showed marked variation in both avidity and specificity of binding. The purified proteins ICAM-1 and CD36 proved to be the most useful assay ligands for investigating field isolates, with 18 isolates binding to at least one protein and 14 to both. No correlation was found between the binding of isolates to any two ligands nor between the binding of a standardized inoculum and the level of the patient's presenting parasitemia. All isolates from the study group were found to form rosettes (at a mean rate of 14.6% of cultured trophozoites involved in rosettes). A lack of correlation between rosette formation and CD36 binding suggests that the previously reported role of CD36 as a rosette formation receptor may not be important for isolates from Papua New Guinea.

Agglutination Tests↗
PubMed1994

Relationship between humoral response to Plasmodium falciparum merozoite surface antigen-2 and malaria morbidity in a highly endemic area of Papua New Guinea.

The prevalence and concentration of antibodies to merozoite surface antigen-2 (MSA-2) were measured in blood samples collected during a cross-sectional survey. Antibodies were measured by enzyme-linked immunosorbent assay using two recombinant proteins that closely approximated the full-length mature MSA-2 polypeptides expressed by the Plasmodium falciparum isolate FC27 and the cloned line 3D7 and that were representative of the dimorphic forms of MSA-2. Antibodies were also measured to a form of the 3D7 MSA-2 lacking the central repetitive sequences (d3D7). High antibody prevalence was observed to all three antigens: the overall prevalence of IgG to FC27, 3D7, and d3D7 was 91%, 90%, and 90%, respectively. The majority of individuals > or = 5 years of age had antibodies to both forms of MSA-2. The geometric mean antibody units increased with age with a plateau being reached by 15-20 years of age. There was a significant positive association of antibody prevalence with both the presence of the parasite and an enlarged spleen in children. This study provides the first evidence that antibodies against nonrepeat regions of MSA-2 are associated with fewer fever episodes and less anemia, both known to be indicators of malaria morbidity.

Adolescent↗
PubMed1994

Plasmodium falciparum infection of splenectomized and intact Guyanan Saimiri monkeys.

Spleen-intact and splenectomized Saimiri monkeys of Guyanan origin were examined for their potential suitability for Plasmodium falciparum protection studies. The animals could be readily infected with adapted strains of P. falciparum (Indochina 1/CDC and Uganda Palo Alto FUP strains), but spontaneously recovered without drug treatment and without development of severe clinical disease. In intact animals, peak parasitemia prior to recovery generally ranged from 0.1% to 10%, whereas in splenectomized animals the peak parasitemia was generally higher so that some animals were given drug treatment to assist in recovery from infection. In reinfection studies, previously infected spleen-intact monkeys demonstrated sterile immunity to the homologous parasite strain but not to a heterologous strain. However, in monkeys infected with the heterologous strain, the peak parasitemia was less than in the first infection and of shorter duration. Splenectomized animals did not demonstrate sterile immunity although the peak parasitemia achieved was less than in the previous infection of each of these monkeys. While the lack of major clinical disease indicated that these monkeys did not provide a good animal model for human malaria, the development of protective immunity was consistent with a useful role in evaluating candidate vaccine antigens.

Animals↗
PubMed1993

Molecular variation in Plasmodium falciparum: polymorphic antigens of asexual erythrocytic stages.

Numerous polymorphic antigens of the asexual erythrocytic stages of P. falciparum are now well characterized. Diversity in some of these antigens, including MSA-1, MSA-2 and the S-antigen is associated with changes in the repeat sequences which are frequently a prominent structural feature of malaria antigens. It is not known whether the variation in repeats causes allelic gene products to adopt different conformations but variation in and around the repeats in SPAM, a newly characterized secreted antigen, preserve the unusual alanine-heptad repeats which we assume generate a helical bundle in this protein. Mutations in non-repetitive regions of the S-antigen and in AMA-1, an antigen lacking repeats, are strongly biased towards those which alter the amino acid sequence. This and other evidence indicates the operation of biological selection but the role of immune responses as a selection pressure operating on these diverse antigens remains to be established.

Amino Acid Sequence↗
PubMed1993

The S-antigen of Plasmodium falciparum: repertoire and origin of diversity.

S-antigens are heat-stable, highly polymorphic proteins released by Plasmodium falciparum at the time of schizont rupture. Previously determined S-antigen sequences allowed the proposal of a general gene structure consisting of 5 sequence blocks. The sequence of the central block of tandem repeats provides a useful means of distinguishing the S-antigen allele and also its serotype, whereas the amino and carboxy terminal sequences defined the S-antigen family, 4 of which have been described. We present the sequence of 3 new S-antigen alleles, for the isolates HB3, KF1916 and KF1917. The allele-defining repeat sequence is ETGPGKAGEQG for HB3, GDQTEGS(S/A)GGK for KF1917 and AGSNE(E/K) for KF1916. The sequences of these newly described S-antigens are consistent with the proposed general gene structure and all belong to defined families, although carboxy-terminal sequences appear to be much more variable within a family than previously realised.

Alleles↗
PubMed1993

A Plasmodium chabaudi antigen located in the parasitophorous vacuole membrane.

In order to study antigens from the rodent malaria Plasmodium chabaudi, clones reacting with mouse hyperimmune serum were selected from an expression library of blood-stage P. chabaudi cDNAs in the vector pGEX-2T. Sixty-four such clones were shown to derive from 19 different P. chabaudi antigens. One of these, Antigen 3008 (Ag3008), has a predicted size of 17.5 kDa and an observed size of 24 kDa. It is located in the parasitophorous vacuole membrane of maturing parasites and in the dense granules of merozoites. The cDNA sequence predicts a highly charged molecule with an N-terminal signal sequence and a central transmembrane domain, but no tandem repeats. The sequence reported for Pc24, a previously identified but uncharacterized P. chabaudi protein, corresponds to part of the 3' untranslated region in the Ag3008 mRNA. Ag3008 has many features in common with the P. falciparum circumsporozoite protein-related antigen (CRA).

Amino Acid Sequence↗
PubMed1992

Adherence of infected erythrocytes to venular endothelium selects for antigenic variants of Plasmodium falciparum.

Erythrocytes (E) infected with asexual forms of malaria parasites exhibit surface antigenic variation. In Plasmodium falciparum infections, the variant Ag is the P. falciparum E membrane protein 1 (PfEMP1). This molecule may also mediate the adherence of infected E to host venular endothelium. We show here that parasite lines selected for increased adherence to endothelial cells have undergone antigenic variation. Three adherent lines selected from the same P. falciparum clone reacted with the same agglutinating antiserum that failed to agglutinate the parental clone. Immunoprecipitation experiments with the agglutinating anti-serum demonstrated that the selected lines expressed cross-reactive forms of PfEMP1 that were of higher m.w. and antigenically distinct from PfEMP1 of the parental clone. When one of the adherent lines was cloned in the absence of selection, a range of variant antigenic types emerged with differing cytoadherence phenotypes. These findings show that selection for cytoadherence in vitro favors the emergence of antigenic variants of P. falciparum and suggest that the requirement for cytoadherence in vivo may restrict the range of antigenic variants of P. falciparum in natural infections.

Animals↗