Ranveig Braathen

The J Chain Is Essential for Polymeric Ig Receptor-Mediated Epithelial Transport of IgA

Local production of secretory (S)IgA provides adaptive immunologic protection of mucosal surfaces, but SIgA is also protective when administered passively, such as in breast milk. Therefore, SIgA is a potential candidate for therapeutic administration, but its complex structure with four different polypeptide chains produced by two distinct cell types complicates recombinant production. The J chain is critical in the structure of SIgA because it is required for efficient polymerization of IgA and for the affinity of such polymers to the secretory component (SC)/polymeric (p)IgR. To better understand the role of the J chain in SIgA production, we have generated various mutant forms of the human J chain and analyzed the function of these mutants when coexpressed with IgA. We found that the C terminus of the J chain was not required for the formation of IgA polymers, but was essential for the binding of pIgA to SC. Likewise, we found that two of the intrachain disulfide bridges (Cys13:Cys101 and Cys109:Cys134) were also required for the binding of pIgA to SC but, interestingly, not for IgA polymerization. Conversely, the last intrachain disulfide bridge (Cys72:Cys92) was not essential for either of these two J chain functions. Finally, we demonstrated that the presence of only Cys15 or Cys69 was sufficient to support polymerization of IgA, but that these polymers were mostly noncovalently stabilized. Nevertheless, these polymers bound free SC with nearly the same affinity as pIgA containing wild-type J chain, but were transcytosed by pIgR-expressing polarized epithelial cells at a reduced efficiency.

Secretory Antibody Formation: Conserved Binding Interactions between J Chain and Polymeric Ig Receptor from Humans and Amphibians

Abs of the secretory Ig (SIg) system reinforce numerous innate defense mechanisms to protect the mucosal surfaces against microbial penetration. SIgs are generated by a unique cooperation between two distinct cell types: plasma cells that produce polymers of IgA or IgM (collectively called pIgs) and polymeric Ig receptor (pIgR)-expressing secretory epithelial cells that mediate export of the pIgs to the lumen. Apical delivery of SIgs occurs by cleavage of the pIgR to release its extracellular part as a pIg-bound secretory component, whereas free secretory components are derived from an unoccupied receptor. The joining chain (J chain) is crucial in pIg/SIg formation because it serves to polymerize Igs and endows them with a binding site for the pIgR. In this study, we show that the J chain from divergent tetrapods including mammals, birds, and amphibians efficiently induced polymerization of human IgA, whereas the J chain from nurse shark (a lower vertebrate) did not. Correctly assembled polymers showed high affinity to human pIgR. Sequence analysis of the J chain identified two regions, conserved only in tetrapods, which by mutational analysis were found essential for pIgA-pIgR complexing. Furthermore, we isolated and characterized pIgR from the amphibian Xenopus laevis and demonstrated that its pIg binding domain showed high affinity to human pIgA. These results showed that the functional site of interaction between pIgR, J chain and Ig H chains is conserved in these species and suggests that SIgs originated in an ancestor common to tetrapods.

Identification of residues in the Cmu4 domain of polymeric IgM essential for interaction with Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1).

The binding of nonspecific 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. Herein 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 Abs, IgM mutants, and anti-IgM mAbs showed that PfEMP1 binds to the Fc portion of the human IgM H 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 recognized by Fc-binding proteins from bacteria, suggesting that this region of Ig 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 that it shows functional similarities with Fc-binding proteins from pathogenic bacteria.

Structural requirements for the interaction of human IgM and IgA with the human Fcα/μ receptor

Here we unravel the structural features of human IgM and IgA that govern their interaction with the human Fcα/μ receptor (hFcα/μR). Ligand polymerization status was crucial for the interaction, because hFcα/μR binding did not occur with monomeric Ab of either class. hFcα/μR 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 hFcα/μR binding. IgM binding required contributions from both Cμ3 and Cμ4 Fc domains, whereas for dIgA, an exposed loop in the Cα3 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 FcαRI 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 hFcα/μR binding. Furthermore, secretory component (SC, the extracellular portion of pIgR) and bacterial IgA‐binding proteins were shown to inhibit the dIgA–hFcα/μR interaction. Therefore, we have identified a motif in the IgA–Fc inter‐domain region critical for hFcα/μR interaction, and highlighted the multi‐functional nature of a key site for protein–protein interaction at the IgA Fc domain interface.

Identification of a Polymeric Ig Receptor Binding Phage-displayed Peptide That Exploits Epithelial Transcytosis without Dimeric IgA Competition

The polymeric Ig receptor (pIgR), also called membrane secretory component (SC), mediates epithelial transcytosis of polymeric immunoglobulins (pIgs). J Chain-containing polymeric IgA (pIgA) and pentameric IgM bind pIgR at the basolateral epithelial surface. After transcytosis, the extracellular portion of the pIgR is cleaved at the apical side, either complexed with pIgs as bound SC or unoccupied as free SC. This transport pathway may be exploited to target bioactive molecules to the mucosal surface. To identify small peptide motifs with specific affinity to human pIgR, we used purified free SC and selection from randomized, cysteine-flanked 6- and 9-mer phage-display libraries. One of the selected phages, called C9A, displaying the peptide CVVWMGFQQVC, showed binding both to human free SC and SC complexed with pIgs. However, the pneumococcal surface protein SpsA (Streptococcus pneumoniae secretory IgA-binding protein), which binds human SC at a site distinct from the pIg binding site, competed with the C9A phage for binding to SC. The C9A phage showed greatly increased transport through polarized Madin-Darby canine kidney cells transfected with human pIgR. This transport was not affected by pIgA nor did it inhibit pIgR-mediated pIgA transcytosis. A free peptide of identical amino acid sequence as that displayed by the C9A phage inhibited phage interaction with SC. This implied that the C9A peptide sequence may be exploited for pIgR-mediated epithelial transport without interfering with secretory immunity.

Heterodimeric Barnase-Barstar Vaccine Molecules: Influence of One versus Two Targeting Units Specific for Antigen Presenting Cells

It is known that targeting of antigen to antigen presenting cells (APC) increases immune responses. However, it is unclear if more than one APC-specific targeting unit in the antigenic molecule will increase responses. To address this issue, we have here made heterodimeric vaccine molecules that each express four different fusion subunits. The bacterial ribonuclease barnase and its inhibitor barstar interact with high affinity, and the barnase-barstar complex was therefore used as a dimerization unit. Barnase and barstar were fused N-terminally with single chain fragment variable (scFv)s targeting units specific for either MHC class II molecules on APC or the hapten 5-iodo-4-hydroxy-3-nitrophenylacetyl (NIP). C-terminal antigenic fusions were either the fluorescent protein mCherry or scFv315 derived from myeloma protein M315. The heterodimeric vaccine molecules were formed both in vitro and in vivo. Moreover, the four different fused moieties appeared to fold correctly since they retained their specificity and function. DNA vaccination with MHC class II-targeted vaccine induced higher mCherry-specific IgG1 responses compared to non-targeted control. Since mCherry and MHC class II are in trans in this heterodimer, this suggests that heterodimeric proteins are formed in vivo without prior protein purification. Surprisingly, one targeting moiety was sufficient for the increased IgG1 response, and addition of a second targeting moiety did not increase responses. Similar results were found in in vitro T cell assays; vaccine molecules with one targeting unit were as potent as those with two. In combination with the easy cloning strategy, the heterodimeric barnase-barstar vaccine molecule could provide a flexible platform for development of novel DNA vaccines with increased potency.

Simultaneous Targeting of Multiple Hemagglutinins to APCs for Induction of Broad Immunity against Influenza

There is a need for vaccines that can confer broad immunity against highly diverse pathogens, such as influenza. The efficacy of conventional influenza vaccines is dependent on accurate matching of vaccines to circulating strains, but slow and limited production capacities increase the probability of vaccine mismatches. In contrast, DNA vaccination allows for rapid production of vaccines encoding novel influenza Ags. The efficacy of DNA vaccination is greatly improved if the DNA-encoded vaccine proteins target APCs. In this study, we have used hemagglutinin (HA) genes from each of six group 1 influenza viruses (H5, H6, H8, H9, H11, and H13), and inserted these into a DNA vaccine format that induces delivery of the HA protein Ags to MHC class II molecules on APCs. Each of the targeted DNA vaccines induced high titers of strain-specific anti-HA Abs. Importantly, when the six HA vaccines were mixed and injected simultaneously, the strain-specific Ab titers were maintained. In addition, the vaccine mixture induced Abs that cross-reacted with strains not included in the vaccine mixture (H1) and could protect mice against a heterosubtypic challenge with the H1 viruses A/Puerto Rico/8/1934 (H1N1) and A/California/07/2009 (H1N1). The data suggest that vaccination with a mixture of HAs could be useful for induction of strain-specific immunity against strains represented in the mixture and, in addition, confer some degree of cross-protection against unrelated influenza strains.

The Magnitude and IgG Subclass of Antibodies Elicited by Targeted DNA Vaccines Are Influenced by Specificity for APC Surface Molecules

Upon APC-targeted DNA vaccination, transfected cells secrete fusion proteins with targeting units specific for surface molecules on APC. In this study, we have tested several different targeting units for their ability to influence the magnitude and subclass of Ab responses to hemagglutinin from influenza A virus. The experiments employed bivalent homodimeric Ig-based molecules (vaccibodies). The overall efficiency in BALB/c mice depended on the targeting units in the following order: αMHC class II > αCD11c > αCD40 > Xcl-1 = MIP-1α > FliC > GM-CSF > Flt-3L > αDEC205. GM-CSF induced mainly IgG1, whereas Xcl1, MIP-1α, αCD40, and αDEC205 induced predominantly IgG2a. A more balanced mixture of IgG1 and IgG2a was observed with αCD11c, αMHC class II, Flt-3L, and FliC. Similar results of IgG subclass–skewing were obtained in Th1-prone C57BL/6 mice with a more limited panel of vaccines. IgG1 responses in BALB/c occurred early after immunization but declined relatively rapidly over time. IgG2a responses appeared later but lasted longer (>252 d) than IgG1 responses. The most efficient targeting units elicited short- and long-term protection against PR8 influenza (H1N1) virus in BALB/c mice. The results suggest that targeting of Xcr1+ conventional type 1 dendritic cells preferentially induces IgG2a responses, whereas simultaneous targeting of several dendritic cell subtypes also induces IgG1 responses. The induction of distinct subclass profiles by different surface molecules supports the APC–B cell synapse hypothesis. The results may contribute to generation of more potent DNA vaccines that elicit high levels of Abs with desired biologic effector functions.