The binding of non-specific human IgM to the surface of infected erythrocytes is important in rosetting, a major virulence factor in the pathogenesis of severe malaria due to Plasmodium falciparum, and IgM binding has also been implicated in placental malaria. Here we have identified the IgM-binding parasite ligand from a virulent P. falciparum strain as PfEMP1 (TM284var1 variant), and localized the region within this PfEMP1 variant that binds IgM (DBL4β domain). We have used this parasite IgM-binding protein to investigate the interaction with human IgM. Interaction studies with domain-swapped antibodies, IgM mutants and anti-IgM mAbs showed that PfEMP1 binds to the Fc portion of the human IgM heavy chain and requires the IgM Cμ4 domain. Polymerization of IgM was shown to be crucial for the interaction because PfEMP1 binding did not occur with mutant monomeric IgM molecules. These results with PfEMP1 protein have physiological relevance because infected erythrocytes from strain TM284 and four other IgM-binding P. falciparum strains showed analogous results to those seen with the DBL4β domain. Detailed investigation of the PfEMP1 binding site on IgM showed that some of the critical amino acids in the IgM Cμ4 domain are equivalent to those regions of IgG and IgA recognised by Fc-binding proteins from bacteria, suggesting that this region of immunoglobulin molecules may be of major functional significance in host-microbe interactions. We have therefore shown that PfEMP1 is an Fc-binding protein of malaria parasites specific for polymeric human IgM, and shows functional similarities with Fc-binding proteins from pathogenic bacteria.

J chain is associated with pentameric IgM and dimeric IgA via disulfide bonds involving the penultimate cysteine residue in the secretory tailpiece of the mu or the alpha heavy chain. We have investigated the structural basis for incorporation of J chain by analyzing several IgM mutants, IgA mutants and IgG/IgM hybrid molecules. IgM mutants with the mu secretory tailpiece replaced by the alpha secretory tailpiece and/or Cys414 replaced by serine incorporated J chain, although in reduced amounts correlating with reduced pentamer/polymer formation. In addition to pentamers, tetramers of IgMC414S contained J chain, while no J chain was associated with smaller polymers or hexamers of IgM. An IgA/IgM hybrid tailpiece abolished J chain incorporation to pentameric IgM. Analysis of IgG molecules that have added a secretory tailpiece and/or have IgM domain replacements showed that J chain incorporation depends on regions of the C(mu)4 domain in addition to the tailpiece. Features of the C(mu)3 domain other than Cys414 also play a role in efficient formation of pentamers and J chain incorporation, while the C(mu)2 domain is not specifically required. By analysis of two IgA mutants that formed larger polymers than IgAwt, we found J chain equally incorporated into dimers, trimers, tetramers and pentamers. Thus, the results show that J chain incorporation into IgA does not depend on the polymeric structure, while J chain incorporation into IgM is restricted to certain polymeric conformations.

Here we unravel the structural features of human IgM and IgA that govern their interaction with the human Fca/l receptor (hFca/lR). Ligand polymerization status was crucial for the interaction, because hFca/lR binding did not occur with monomeric Ab of either class. hFca/lR bound IgM with an affinity in the nanomolar range, whereas the affinity for dimeric IgA (dIgA) was tenfold lower. Panels of mutant IgM and dIgA were used to identify regions critical for hFca/lR binding. IgM binding required contributions from both Cl3 and Cl4 Fc domains, whereas for dIgA, an exposed loop in the Ca3 domain was crucial. This loop, comprising residues Pro440-Phe443, lies at the Fc domain interface and has been implicated in the binding of host receptors FcaRI and polymeric Ig receptor (pIgR), as well as IgA-binding proteins produced by certain pathogenic bacteria. Substitutions within the Pro440-Phe443 loop resulted in loss of hFca/lR binding. Furthermore, secretory component (SC, the extracellular portion of pIgR) and bacterial IgA-binding proteins were shown to inhibit the dIgA-hFca/lR interaction. Therefore, we have identified a motif in the IgA-Fc inter-domain region critical for hFca/lR interaction, and highlighted the multi-functional nature of a key site for protein-protein interaction at the IgA Fc domain interface.Key words: Human Fca/m receptor . IgA . IgM IntroductionA receptor for IgM and IgA, termed Fca/m receptor (Fca/mR), was first described in the mouse where it is constitutively expressed on the majority of B cells and macrophages [1]. Mouse Fca/mR (mFca/mR) can mediate endocytosis of IgM-coated targets, and may contribute to the primary stages of the immune response to microbes. It has been suggested that mFca/mR, due to its high affinity for IgM and intermediate affinity for IgA, may assist also in the maintenance of plasma concentrations of IgM and IgA [2]. Although there is no significant homology between Fca/mR and other known proteins, the N-terminal Ig-like domain of Fca/mR Ã These authors contributed equally to this work. Certain mAb raised against mFca/mR have been found to bind to a peptide encompassing the region from mFca/mR's Ig-like domain that corresponds to the Ig-binding motif in domain 1 of pIgR [6]. Interestingly, these mAb can block binding of IgA and IgM to mFca/mR. The fact that these same mAb cross-react with hFca/mR, which carries the conserved motif in its Ig-like domain, suggests that the site for IgM and IgA on hFca/mR lies in this same region. In contrast, the site(s) on human IgM and IgA that interacts with hFca/mR is unknown. To gain a better understanding of the structural requirements for the interaction of human IgA and IgM with hFca/mR, we have produced recombinant dIgA and pentameric IgM and have adopted a mutational approach to identify individual residues on the Fc involved in interaction with hFca/mR. We demonstrate that both the Cm3 and Cm4 domains of IgM are required for binding to the receptor, and that residues at the Fc inter-domain region of dIgA are critical for hFca...

A major objective in vaccine development is the design of reagents that give strong, specific T cell responses. We have constructed a series of rAb with specificity for MHC class II (I-E). Each has one of four different class II-restricted T cell epitopes genetically introduced into the first C domain of the H chain. These four epitopes are: 91–101 λ2315, which is presented by I-Ed; 110–120 hemagglutinin (I-Ed); 323–339 OVA (I-Ad); and 46–61 hen egg lysozyme (I-Ak). We denote such APC-specific, epitope-containing Ab “Troybodies.” When mixed with APC, all four class II-specific Troybodies were ∼1,000 times more efficient at inducing specific T cell activation in vitro compared with nontargeting peptide Ab. Furthermore, they were 1,000–10,000 times more efficient than synthetic peptide or native protein. Conventional intracellular processing of the Troybodies was required to load the epitopes onto MHC class II. Different types of professional APC, such as purified B cells, dendritic cells, and macrophages, were equally efficient at processing and presenting the Troybodies. In vivo, class II-specific Troybodies were at least 100 times more efficient at targeting APC and activating TCR-transgenic T cells than were the nontargeting peptide Ab. Furthermore, they were 100–100,000 times more efficient than synthetic peptide or native protein. The study shows that class II-specific Troybodies can deliver a variety of T cell epitopes to professional APC for efficient presentation, in vitro as well as in vivo. Thus, Troybodies may be useful as tools in vaccine development.

Targeting of antigens to antigen-presenting cells (APCs) increases CD4 ؉ T cell activation, and this observation can be exploited in the development of new vaccines. We have chosen an antigen-targeting approach in which we make recombinant antibodies (Abs) with T cell epitopes in their constant region and APC-specific variable regions. Three commonly used model epitopes, amino acids 110 -120 of hemagglutinin, 323-339 of ovalbumin, and 46 -61 of hen egg lysozyme, were introduced as loops in the C H1 domain of human IgG3. For all three epitopes, we show that the recombinant molecules are secreted from transfected cells. The epitopes are presented to specific T cells, and targeting to IgD on B cells in vitro enhances the presentation efficiency by 10 4 to 10 5 compared with the free peptide. After i.v. injection, the epitopes targeted to IgD are presented by splenic APCs to activate specific T cells, whereas little or no activation could be detected without targeting, even after the amount of antigen injected was increased 100-fold or more. Because a wide variety of T cell epitopes, in terms of both length and secondary structure, can be tolerated in loops in constant domains of Abs, the Ab constant region seems to have the intrinsic stability that is needed for this fusion molecule strategy. It might thus be possible to load the Ab with several different epitopes in loops in different domains and thereby make a targeted multisubunit vaccine.O ver recent years, considerable efforts have been directed toward the development of efficient vaccines aimed to stimulate T cells. Effector CD4 ϩ T cells are central in this regard because they regulate B cells and are essential for induction of CD8 ϩ CTL responses through both cytokine secretion (1) and dendritic cell sensitization (2). CD4 ϩ T cells mainly recognize peptides derived from the processing of extracellular antigens by antigen-presenting cells (APCs), presented in association with MHC class II molecules. Targeting of antigen to APCs by coupling to APC-specific Abs increases CD4 ϩ T cell activation, probably because of increased uptake of antigen (3, 4).Several new prospects for preparation of T cell vaccines have been suggested based on the identification and characterization of MHC-associated peptides. Synthetic peptides that correspond to these T cell epitopes may represent ideal subunits for safe vaccines. However, synthetic peptides are poor immunogens because of their small molecular size and very short serum half-life. To circumvent these disadvantages, a variety of recombinant proteins that carry short immunogenic epitopes have been described, and these so-called antigenized molecules represent a new generation of subunit vaccines (5-9). Genetically engineered Ab molecules can function as such delivery systems for T cell peptides (10, 11) and have the advantage of being self-proteins devoid of the side effects sometimes associated with microbial vaccines and, moreover, have a long half-life compared with synthetic peptides.Abs are processed by APCs, and s...