Characterization of a Plasmodium chabaudi gene encoding a protein with glutamate-rich tandem repeats.

Several highly antigenic proteins containing tandem repeats rich in glutamic acid residues have been described in Plasmodium falciparum. However, relatively little information is available about analogous genes in rodent parasites. This report describes a 4.2-kb genomic DNA fragment from P. chabaudi with a deduced amino acid sequence that is predominantly glutamate-rich tandem repeats. Several different monoclonal antibodies raised against a 93-kDa P. chabaudi protein, which does not correspond to the cloned DNA fragment, recognize a recombinant protein expressed from the 4.2-kb DNA fragment. The only sequence similarities between these two genes are tandem repeats with a predominance of glutamate pairs followed by a hydrophobic residue. This repetitious-sequence motif may be the basis for the observed cross-reactivity. A similar motif has been demonstrated to be the basis for antibody cross-reactivity between glutamate-rich proteins of P. falciparum. The expression of multiple glutamate-rich proteins with cross-reacting epitopes may be a general phenomenon in Plasmodium species.

Phosphorylation of infected erythrocyte membrane proteins in Plasmodium chabaudi.

We identify phosphoproteins that are associated to the infected erythrocyte membrane of Plasmodium chabaudi. The specific antibodies that recognize the erythrocyte membrane-associated antigens, namely PcEMA1 and PcLEMA, are studied. According to sequencing analysis, neither the gene structure nor the predicted properties of these two antigens are completely distinct. The PcEMA1 is a 50 kDa acidic protein, with a pI of about 4.4, that contains 25 phosphorylation sites principally located in the repetitive sequence. Almost all of this molecule is hydrophilic. The predicted (predictment ability) of this protein is, hence, associated to the host's erythrocyte cytoskeleton and it is synthesized through an entire erythrocyte cycle. PcLEMA, instead, is a 74 kDa basic protein, with a pI of about 9.8, that contains 11 possible phosphorylation sites. This gene contains two exons: exon 1 shows transmembrane characteristics while exon 2 contains part of the hydrophilic repetitive sequences. Thus this protein is predicted as a transmembrane-associated protein and is synthesized only at the last stages of the erythrocytic cycle. In in-vitro phosphorylation experiments, both PcEMA1 and PcLEMA are phosphorylated by endogenous kinases activities. However, the degree of phosphorylation differs between the two in that PcEMA1 reveals a higher phosphorylation intensity than PcLEMA.

Naturally occurring antibodies devoid of light chains.

Random association of VL and VH repertoires contributes considerably to antibody diversity. The diversity and the affinity are then increased by hypermutation in B cells located in germinal centres. Except in the case of 'heavy chain' disease, naturally occurring heavy-chain antibodies have not been described, although antigen binding has been demonstrated for separated heavy chains or cloned VH domains. Here we investigate the presence of considerable amounts of IgG-like material of M(r) 100K in the serum of the camel (Camelus dromedarius). These molecules are composed of heavy-chain dimers and are devoid of light chains, but nevertheless have an extensive antigen-binding repertoire, a finding that calls into question the role of light chains in the camel. Camel heavy-chain IgGs lack CH1, which in one IgG class might be structurally replaced by an extended hinge. Heavy-chain IgGs are a feature of all camelids. These findings open new perspectives in the engineering of antibodies.

Structure of a Plasmodium chabaudi acidic phosphoprotein that is associated with the host erythrocyte membrane.

We have characterized by molecular cloning and sequencing a Plasmodium chabaudi antigen that is associated with the membrane of the infected erythrocyte throughout the entire intraerythrocytic cycle. The protein (PcEMA1) has a predicted size of 50 kDa and contains a major tandem repeat array of 16 octapeptides that constitutes almost 30% of the protein. At its amino-terminus, PcEMA1 has a string of hydrophobic residues characteristic of a secreted protein, but does not contain a hydrophobic membrane-spanning segment. The antigen appears to reside on the cytoplasmic face of the erythrocytic membrane. PcEMA1 has a predicted pI of 4.4 and is a potential phosphoprotein.

Molecular cloning and sequence analysis of the gene encoding the major merozoite surface antigen of Plasmodium chabaudi chabaudi IP-PC1.

The complete nucleotide sequence of the gene encoding the precursor to the major merozoite surface antigens of Plasmodium chabaudi chabaudi strain IP-PC1 has been determined. A single open reading frame was detected, that coded for a protein of 199 kDa. The encoded protein (p199) contains putative signal and membrane anchor sequences and shows a clustering of Cys residues in the last 120 amino acids. Incompletely conserved tandem repeat oligopeptides are present at different positions in the molecule. P199 shows 69% overall homology to the analogous antigen in Plasmodium yoelii yoelii strain YM. The divergence between these antigens is largely confined to 4 areas where a number of insertions and/or deletions have occurred. All repeats occur in these divergent regions. The overall homology with both alleles of Plasmodium falciparum PMMSA is 33%.

T cell-mediated immunity in murine malaria. II. Induction of protective immunity to P. chabaudi by antigen-fed macrophages and antigen-educated lymphocytes.

We previously described assay systems for generating antigen specific proliferating T cells to P. chabaudi antigens. In the present study we examine whether the various sensitization approaches confer immunity against a cloned virulent strain IP-PCI of P. chabaudi. We present data indicating that effective specific protective immunity can be induced through P. chabaudi antigen fed macrophages and antigen educated spleen cells (initiator lymphocytes). The expression of this protective immunity is proposed to depend on (a) antigen presentation and/or accessory function of macrophages and (b) the subsequent activation of T cell functions related to protection. Indeed analysis of different macrophage populations revealed a correlation between the expression of Ia molecules and IL-1 secretion with their capacity to induce antigen specific T cells in vivo and subsequent protective immune mechanisms. Thus these results emphasize the critical functions of accessory cells in determining the outcome of malaria infections.

T-cell mediated immunity in murine malaria. I. Induction of T-cell dependent proliferative responses to Plasmodium chabaudi.

To investigate the mechanisms of cell mediated immunity to malaria, we studied different systems to measure specific activation of T lymphocytes by P. chabaudi antigens. Mice were primed by subcutaneous administration of parasite antigens followed by co-cultivation of lymphocytes taken from the draining lymph nodes in the presence of the priming antigen. A marked proliferative response was observed which was shown to be antigen specific, T-cell mediated and accessory cell dependent. Continuous T-cell lines were propagated in culture by repetitive restimulation in the presence of antigen and accessory cells, followed by expansion in a conditioned medium containing T-cell growth factors. These lines could be induced to proliferate to the priming antigen only in the presence of syngeneic accessory cells thus indicating that H-2 restriction operates in the recognition of plasmodium antigens by T cells. We also induced parasite specific T cells by the use of an in vitro primary 'education' system. Lymphocytes from unprimed mice were sensitized on parasite-fed macrophages and were then injected subcutaneously into each hind foot pad of syngeneic animals. This led to recruitment of antigen-reactive cells which were assayed in vitro by the ability of lymphocytes taken from the draining popliteal lymph nodes to proliferate in response to the sensitizing antigen. In vivo immunization with Plasmodium antigen fed macrophages also signalled antigen specific T cells that recruited reactive T cells in the draining lymph nodes.

Rabbit heavy chain haplotypes--allotypic determinants expressed by VH-CH recombinants.

This report summarizes our current understanding of the heavy chain haplotypes found in our laboratories' rabbits. Independently derived data from several laboratories have been synthesizes into a consistent picture of the linked inheritance of allotypic markers found on the different heavy chain classes and subclasses of rabbit immunoglobulins in pedigreed rabbits, including the families of three apparent VH-CH recombinants. In one recombinant, the entire group of CH markers (C mu, C gamma, and C alpha) recombined with the set of VH. Although in the other two recombinants all CH markers may also have recombined as a group, in one of these only IgG and IgA CH genes were informative; in the other recombinant, only the IgG allotypes were informative. Some allotypic determinants found on IgM molecules ("conformational") appear only when a specific variable region allotype (VHa) is combined with a specific mu constant region allotype (C mu). New combinations of VHa and C mu allotypes were generated in two of the genetic recombinants and led to new "conformational" determinants. The gains and losses observed lend support to the hypothesis that the determinants result from conformations generated by the combination of allotype-specific VH and C mu protein sequences. Conceivably, DNA events that join VH to diversity (D)- and joining (J)-coding sequences or mRNA processing events that splice J to C mu could be involved in generating the sequences that form allotype-specific determinants.

Suppression of VH, C gamma and C mu gene products in rabbits by maternal immunization against IgM allotype and detection of somatic recombinant molecules in these animals.

Suppression of the mu-chain allotype Ms16 was obtained in young heterozygous Ms16/Ms17 rabbits by immunizing the Ms17 mother against the paternal Ms16 allotype. Examination of the concentration of the VH and CH allotypes of paternal origin (a2,Ms16 and e14) in the offspring, revealed not only Ms16 suppression but also VH and C gamma allotypic suppression. The degree of suppression for the C gamma markers (4-25-fold decrease) was much less than that for the C mu or VH markers (50-150-fold decrease). The excess C gamma marker (e14) was found to be present for the major part on molecules possessing the VH allotype derived from the homologous allelic chromosome. The pattern of persistence of molecules with the suppressed C gamma marker in the serum is consistent with the idea that these molecules arise by somatic recombination and that the gene order is VH, C mu and C gamma.

Phylogeny of the rabbit gamma-chain determinants: a d12-like antigenic determinant in Pronolagus rupestris.

A survey for constant region gamma-chain allotypes (de locus) was undertaken in different lagomorph genera. As yet only Pronolagus rupestris, a paleolaginae, showed the presence of a determinant similar to rabbit d12 although it lacked the widespread e15 determinant. All seven individuals possessed the d12 like determinant which was studied by immunodiffusion, haemagglutination inhibition, radiobinding and binding inhibition assays. In addition, a new enzymic method for typing for d12, based on the presence of the asymmetric rabbit hinge carbohydrate linked to the d12 characteristic threonine, is presented. This method suggests, however, that, unlike the d12 rabbit, Pronolagus does not seem to have a majority of IgG molecules with a glycosylated hinge.

Double line phenotypes in rabbit IgG allotypes and their relation to the cis/trans configuration of the IgG markers.

Rabbit antiallotype sera raised against heavy chain markers sometimes show double precipitin lines with all or some of the corresponding antigens (double and single line phenotypes). In a number of cases the double line phenotypes behave as alleles of the single line phenotypes and this feature allows a genetic and immunochemical analysis of these systems. In three cases that have been analysed, the double line phenotype arise when a precipitating a locus allotype and a non-precipitating d or e locus allotype are present on the same molecule (a1 and d14), (a1 and d11), (a3 and d11). This only happens when the corresponding genes are present on the same chromosome (cis configuration) of the diploid pair. These sera are therefore useful for determining directly the genotype of the animals.

Restriction of the anti-bovine serum albumin response in rabbits immunized with Micrococcus lysodeikticus.

Rabbits capable of producing antibodies of restricted heterogeneity in response to Micrococcus lysodeikticus are equally capable of producing antibodies of restricted heterogeneity to bovine serum albumin. These antibodies are produced when animals are simultaneously injected with micrococcus and BSA and their specificity is restricted to a small number of epitopes. These results suggest that micrococcal vaccines can induce the restriction of heterogeneity in antibodies raised against totally unrelated antigens.

Evidence for quasi-silent germline genes coding for phylogenetically ancient determinants of the rabbit a locus allotypes.

Anti-a2 sera raised in a3 rabbits are shown to detect determinants on a2 molecules which are different from those detected by anti-a2 sera raised in a1 animals. The former determinants are occasionally observed at a low level in rabbits of the a1 allotype and at a high level in sera of Leporidae of different genera. The two types of anti-a2 sera are shown to compete for the same sterical region of the a2 molecules. All homogeneous a2 molecules which have been tested show both types of determinants.