Cross-reacting epitopes shared between Plasmodium falciparum and its host: the origin of autoreactive antibodies?
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Amino Acid Sequence↗
Biomedical subjects
A Scherf
Publications and source records attributed to A Scherf.
Genetic diversity in the major merozoite surface antigen of Plasmodium falciparum: high prevalence of a third polymorphic form detected in strains derived from malaria patients.
We studied the diversity of the polymorphic 195-kDa antigen (p190) of Plasmodium from infected individuals. Genomic parasite DNA was extracted from the blood of 30 donors from different endemic areas of Brazil. The 5' region, encoding the polymorphic N-terminal part of p190 was analysed following polymerase chain reaction (PCR). Multiple infections of genetically distinct parasites could be detected within infected malaria patients. Sequence analysis and oligodeoxyribonucleotide typing of the PCR products demonstrated the prevalence of a third polymorphic form of p190.
Animals↗
Parasite antigens expressed in Escherichia coli. A refined approach for epidemiological analysis.
A simple method is described to generate carrier-free recombinant antigens following their expression in Escherichia coli. A plasmid, called pMSgt11, has been constructed such that the cleavage site for the protease factor Xa separates the recombinant antigen from an enzymatically active beta-galactosidase. Thus, rapid purification of the active beta-galactosidase recombinant protein, followed by digestion with factor Xa, releases the antigen of interest. The pMSgt11 plasmid is compatible with the phage expression vector, lambda gt11 and the feasibility of applying this system has been demonstrated using malarial recombinant antigens. Inserts from lambda gt11 recombinant Plasmodium falciparum clones have been recloned into the EcoRI site of pMSgt11 and the expressed soluble fusion proteins have been purified from crude extracts using a one step affinity chromatography. After protease digestion, the fusion protein cleavage products were analysed by immunoblot with a panel of different human immune sera. We were able to successfully demonstrate specific antibody titers to the parasite-derived carrier-free antigen, without interference from anti-Escherichia coli-specific antibodies. The general application of this approach to epidemiological analysis is discussed.
Animals↗
The major merozoite surface antigen (MSAI) of plasmodium falciparum.
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Letter↗
Immunogenicity and antigenicity in rabbits of a repeated sequence of Plasmodium falciparum antigen Pf155/RESA fused to two immunoglobulin G-binding domains of staphylococcal protein A.
A synthetic gene encoding a tetramer of the repeated subunit EENVEHDA of the Plasmodium falciparum antigen Pf155/RESA was expressed in a dual-expression system. The resulting fusion proteins, designated ZZ-M1 and BB-M1, comprised the EENVEHDA repeats and either two immunoglobulin G-binding domains from staphylococcal protein A or the human serum albumin-binding domains from streptococcal protein G, respectively. The soluble fusion proteins were affinity purified to homogeneity in one-step procedures. ZZ-M1 was used for immunization of rabbits. The rabbit antisera reacted with BB-M1 in an enzyme-linked immunosorbent assay and with Pf155/RESA in immunofluorescence of infected erythrocytes and immunoblotting. Inhibition studies revealed that the antibodies mainly recognized epitopes formed by two or more EENVEHDA subunits and were remarkably specific for Pf155/RESA. Importantly, the antibodies also inhibited P. falciparum merozoite reinvasion in vitro efficiently, indicating that they reacted with biologically important epitopes exposed on the native antigen. Immunization with Freund complete adjuvant resulted in high levels of specific immunoglobulin G antibodies over a 1-year period, whereas the antibody response obtained after immunization without adjuvant was generally weaker, immunoglobulin G and M mediated, and not sustained for longer periods. However, these titers were restored after booster injection. Taken together, the results support the usefulness of recombinant gene constructs of this type as immunogens for malaria vaccines.
Amino Acid Sequence↗
Intra- and extracellular routing in P. falciparum.
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Amino Acid Sequence↗
Reactivity of the human monoclonal antibody 33G2 with repeated sequences of three distinct Plasmodium falciparum antigens.
The human mAb 33G2 has high capacity to inhibit in vitro invasion of erythrocytes by Plasmodium falciparum merozoites and, thus, is of special interest with regard to protective immunity against the parasite. In order to obtain more information about asexual blood stage Ag of P. falciparum that are seen by this antibody, material from synchronized P. falciparum cultures was studied by immunofluorescence, immunoelectron microscopy, and immunoblotting. Reactivity was mainly confined to the membrane of infected erythrocytes. Soon after merozoite invasion the antibody stained the erythrocyte membrane. This membrane-associated staining faded during intracellular development of the parasites. Beginning about 18 h after invasion, a dotted pattern appeared which increased in strength with time and persisted to schizont rupture. Pf155/RESA was the major Ag recognized in immunoblots of parasites collected throughout the entire erythrocytic cycle, although other polypeptides also bound the antibody. Among these was a 260-kDa polypeptide found in late trophozoites and schizonts. The specificity of the antibody was analyzed with synthetic peptides corresponding to repeated sequences in the P. falciparum Ag Pf155/RESA, Pf11.1, and Ag332. Synthetic peptides related to Ag332 were the most efficient inhibitors of antibody binding in immunofluorescence studies and cell ELISA. A beta-galactosidase-Ag332 fusion protein was also efficient in reversing reinvasion inhibition caused by 33G2. These results define a family of cross-reactive P. falciparum Ag recognized by mAb 33G2 and suggest that Ag332 was its original target.
Amino Acid Sequence↗
Sequence and length polymorphism of a major malaria vaccine candidate analysed following DNA amplification.
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Animals↗
A versatile phage lambda expression vector system for cloning in Escherichia coli.
By integrating fragments from the expression plasmids pJK2 and pJK4 into a derivative of the bacteriophage lambda, we constructed the phage expression vectors lambda JK2 and lambda JK4, which allow efficient cloning of genomic or cDNA either into the 5' end or the 3' end of the lacZ gene of Escherichia coli. Expression of barrier-free DNA in phase may lead to fusion proteins consisting of active beta-galactosidase (beta Gal) plus an additional polypeptide encoded by the inserted DNA. Analysis of distinct recombinant clones is quick and easy, due to the reversible integration of the plasmid into the genome. As an example, we constructed an expression library of genomic Plasmodium falciparum DNA in lambda JK2. We polymerised (amplified) and expressed a synthetic DNA fragment, which codes for a potential antigenic determinant of the 11-1 gene of Plasmodium falciparum as a fusion to the N terminus of active beta Gal. We demonstrate that such chimeric molecules can be affinity-purified and that polypeptides can be separated from the beta Gal part by cleavage with the protease factor Xa.
Amino Acid Sequence↗
A heat shock-like protein from the human malaria parasite Plasmodium falciparum induces autoantibodies.
The humoral immune response to a 72-kDa heat shock-like protein of Plasmodium falciparum has been analyzed using mouse monoclonal antibodies (mAb) and human immune sera. Three regions of the molecule containing B cell epitopes were identified by screening a sublibrary encoding the COOH-terminal half of the antigen with the mAb. One B cell epitope mapped to a region poorly conserved between the parasite 72-kDa polypeptide and mammalian heat-shock proteins (Hsp 70). Another mAb, G10C9, reacted with an amino acid region that has a high degree of homology with mouse (87.5%) and human (81.2%) Hsp 70. Both mouse and human cells were recognized by this mAb when analyzed by indirect immunofluorescence and by two-dimensional immunoblots. Sera from humans infected with malaria also recognized the human Hsp 70. Thus, our results indicate that autoantibodies directed against host Hsp 70 can be induced by the homologous parasite protein.
Amino Acid Sequence↗
Cross-reactive antigenic determinants present on different Plasmodium falciparum blood-stage antigens.
A gene encoding a previously undescribed antigen of Plasmodium falciparum has been isolated from a genomic expression library by use of a pool of human immune sera. Northern blot analysis indicated that the gene is expressed at the late stages of the intra-erythrocytic cycle. This antigen, 332, contains a series of degenerated amino acid repeats. Human antibodies affinity-purified on the 332 recombinant antigen reacted with a family of parasite proteins that are products of different genes. We identified antigens 11.1 and Pf155-RESA as members of this family and confirmed, using a human monoclonal antibody, the presence of cross-reacting determinants. The sequences of these antigens also share some structural homologies. The significance of this family of blood-stage antigens is discussed.
Amino Acid Sequence↗
The 11-1 gene of Plasmodium falciparum codes for distinct fast evolving repeats.
The 11-1 gene of Plasmodium falciparum has been investigated by DNA sequence analysis. It begins at the 5' end with a putative miniexon coding for a polypeptide which has the characteristics of a signal sequence. The miniexon is followed by a small intron. This again is followed by a large exon consisting of 9-, 18- and 27-bp repeats embedded in unique DNA. Specific antibodies isolated by affinity chromatography on a purified recombinant fusion protein expressing the three- and six-amino acid repeats were used to identify the product of the 11-1 gene. In exhibits size variations from 260 to 350 kd in different strains. Southern blot analysis with synthetic DNA as probe demonstrates that the 18-bp repeat is absent or drastically altered in two strains whereas the other repeats are present in all seven strains investigated. The unusual preference for G in the third position of some codons of the repeats but not in the unique sequences indicates rapid evolution of the repeats. Slippage during replication, unequal crossing over and selection are discussed as possible mechanisms leading rapidly to extreme diversity.
Amino Acid Sequence↗
Specific and nonspecific immunodiagnostic properties of recombinant and synthetic Plasmodium falciparum antigens.
Six Plasmodium falciparum/beta-galactosidase fusion proteins produced by a genomic DNA expression library, and two synthetic Plasmodium falciparum antigens were applied to ELISA and tested for their immunodiagnostic properties. Results were compared to reference methods, i.e. fluorescence antibody test with whole cell antigen and ELISA with detergent-soluble crude schizont antigen. Anti-Plasmodium falciparum antibodies could be detected by these molecular antigens to varying extents in human sera. Undesired reactivity to fusion proteins due to the high prevalence of antibodies to beta-galactosidase in human sera and undesired reactivity to one of the synthetic antigens (P12) frequently occurred. The antibodies responsible for the nonspecific reactivity could not be identified. It was concluded that the application of molecular Plasmodium falciparum antigens to ELISA represents a practicable approach to immunodiagnosis of malaria if the construction of epitopes that bind antibodies other than Plasmodium falciparum antibodies can be avoided.
Animals↗
The polymorphic 11.1 locus of Plasmodium falciparum.
Clone pPF11.1 encodes a Plasmodium falciparum antigen expressed during the intraerythrocytic cycle and containing tandem repeats of a 9 amino acid unit. We report here an analysis of the genomic region specific for 11.1, which extends over 30 kb. It contains two blocks of repeats, spanning 13 kb and 9 kb. The restriction map suggests that the locus may result from a gene duplication. The 11.1 region is present in all P. falciparum strains examined so far. Southern analysis of 8 distinct isolates indicates that the locus is highly polymorphic. Thus the pPF11.1 repeats constitute a sensitive and discriminating probe to type P. falciparum strains.
Alleles↗
Characterisation of P. falciparum antigenic determinants isolated from a genomic expression library by differential antibody screening.
A genomic expression library of P.falciparum has been differentially screened with a number of immune sera. The response of 9 clones to the various sera is presented, together with the DNA sequence encoding the epitopes. All but one clone are extremely A+T rich and unlike the other P.falciparum epitopes described, are not composed of amino acid repeats. One clone, which responds specifically with a protective serum, has been analysed in detail. The epitope is carried on a 160kd antigen which is transcribed from a single gene to give a protein expressed in all of the erythrocytic forms. DNA sequence of this clone reveals it to have more than one open reading frame, only one of which is transcribed in the blood stages. The possible significance of the other open readings frames is discussed.
Amino Acid Sequence↗
The gene product of the Plasmodium falciparum 11.1 locus is a protein larger than one megadalton.
The Plasmodium falciparum 11.1 gene locus on chromosome 10 extends over 30 kb and contains approximately 22 kb of a tandemly repeated 27-bp sequence. Biochemical and size similarities have been noted between the reported 11.1 antigen and a variable-Mr, surface-radioiodinatable protein which appears to be involved in the cytoadherence of red blood cells infected with mature intraerythrocytic parasites to venular endothelium. We attempted to determine if these proteins were identical. Using rabbit antibody and affinity purified human immune sera specific for peptides encoded by the 27-bp repeat and a flanking 5' region, we have shown that the 11.1 locus encodes a protein of more than 1000 kDa. This protein cross-reacts with an uncharacterized 260-kDa protein, previously identified as the gene product of the 11.1 locus, and Pf155-RESA, but not with the radioiodinatable protein. The 11.1 protein that we have identified is a malarial protein of unprecedented size.
Animals↗
Cloning and expression of genomic DNA sequences coding for putative erythrocyte membrane-associated antigens of Plasmodium falciparum.
Genomic DNA fragments of Plasmodium falciparum generated by mung bean nuclease digestion were cloned in the lambda expression vector lambda JK2. The resulting library was screened with a rabbit antiserum raised against purified membranes of P. falciparum-infected erythrocytes and with a serum pool from immune humans from an endemic area of Liberia. Positive clones were rescreened with a series of human and monkey sera. Twelve selected clones were analysed in detail. Four of them corresponded to already described membrane-associated P. falciparum antigens. The other positive clones contained inserts which, according to the nucleotide sequence, Southern blot analysis and immunological characteristics, correspond to so far unknown antigens.
Amino Acid Sequence↗
Chondroitin-4-sulphate (proteoglycan), a receptor for Plasmodium falciparum-infected erythrocyte adherence on brain microvascular endothelial cells.
Adherence of Plasmodium falciparum parasitized erythrocytes to the microvascular endothelium is mediated by different receptors expressed by endothelial cells. The study of the adherence of P. falciparum-infected erythrocytes to Saimiri monkey brain microvascular endothelial cells revealed the presence of an additional receptor, which was identified and further characterized. This receptor was also found on the surface of primary human lung endothelial cells (HLEC). We developed two mAbs to this receptor which very efficiently blocked the adherence of parasite strains to Saimiri brain endothelial cells (SBEC). The ability of these mAb to bind to SBEC was partially blocked by chondroitin-4-sulphate (CSA). Competitive inhibition assays on adherence of parasitized red blood cells (PRBC) showed that CSA, but not hyaluronic acid, chondroitin-6-sulphate, dermatan sulphate, keratane sulphate, heparan sulphate or chondroitin-4S-disaccharide, was able to almost completely inhibit PRBC adherence. The same effect was obtained with chondroitinase ABC and AC, but not B, hyaluronidase or heparinase. These results strongly suggest that a member of the chondroitin-glycosaminoglycan family, CSA, represents an additional receptor used by P. falciparum PRBC to cytoadhere to microvascular endothelial cells.
Animals↗