Rabbit IgA Hinges That Resist IgA1 Protease Action Provide Options for Improved IgA-Based Therapeutic Agents.

Immunoglobulin A provides a major line of defence against pathogens and plays a key role in the maintenance of the commensal microbiota in the intestinal tract. Having been shown to be more effective at tumour cell killing than IgG and strongly active against pathogens present in the mucosae, IgA antibodies have been attracting significant attention in recent years for use as therapeutic antibodies. To improve their therapeutic potential, bioengineered IgA forms with increased serum half-life and neutralizing abilities have been developed but the IgA hinge, which impacts susceptibility to bacterial proteases and ability to bridge between target and effector cells, has not yet been explored. The European rabbit has 15 IgA subclasses with exclusive hinge region motifs and varying lengths, constituting a unique model to evaluate the functional capabilities offered by incorporation of longer IgA hinges into immunoglobulins. Hinge regions from rabbit IgAs, featuring different lengths and sequences, were inserted into human IgA1 heavy chain to substitute the IgA1 hinge. These hinges did not appear to affect antigen binding nor the ability of the engineered chimeric IgA1 to bind and trigger FcαRI, as detected by IgA-mediated cell agglutination and release of superoxide by neutrophils. All rabbit hinge-human IgA1 hybrids were resistant to Clostridrum ramosum IgA protease enzyme digestion, as predicted by the lack of the cleavage site in the rabbit hinges. Some IgA1s featuring long rabbit hinges were cleaved by Neisseria meningitidis IgA1 protease cleavage type 1 or 2 enzymes, despite the lack of the predicted cleavage sites. More interestingly, the hybrid featuring the rabbit IgA15 hinge was not affected by any of the IgA proteases. The IgA15 hinge is longer than that found in human IgA1 and is composed by a unique motif with a stretch of nine consecutive Ser residues. These characteristics allow the preservation of a long hinge, with associated ability to bridge distantly spaced antigens and provide higher avidity binding, while remaining resistant to IgA protease degradation. The data suggest that the rabbit Cα15 hinge represents an interesting alternative hinge sequence for therapeutic human IgA antibodies that remains resistant to proteolytic cleavage.

IgA: Structure, Function, and Developability.

Immunoglobulin A (IgA) plays a key role in defending mucosal surfaces against attack by infectious microorganisms. Such sites present a major site of susceptibility due to their vast surface area and their constant exposure to ingested and inhaled material. The importance of IgA to effective immune defence is signalled by the fact that more IgA is produced than all the other immunoglobulin classes combined. Indeed, IgA is not just the most prevalent antibody class at mucosal sites, but is also present at significant concentrations in serum. The unique structural features of the IgA heavy chain allow IgA to polymerise, resulting in mainly dimeric forms, along with some higher polymers, in secretions. Both serum IgA, which is principally monomeric, and secretory forms of IgA are capable of neutralising and removing pathogens through a range of mechanisms, including triggering the IgA Fc receptor known as FcαRI or CD89 on phagocytes. The effectiveness of these elimination processes is highlighted by the fact that various pathogens have evolved mechanisms to thwart such IgA-mediated clearance. As the structure-function relationships governing the varied capabilities of this immunoglobulin class come into increasingly clear focus, and means to circumvent any inherent limitations are developed, IgA-based monoclonal antibodies are set to emerge as new and potent options in the therapeutic arena.

Identification of a new European rabbit IgA with a serine-rich hinge region.

In mammals, the most striking IgA system belongs to Lagomorpha. Indeed, 14 IgA subclasses have been identified in European rabbits, 11 of which are expressed. In contrast, most other mammals have only one IgA, or in the case of hominoids, two IgA subclasses. Characteristic features of the mammalian IgA subclasses are the length and amino acid sequence of their hinge regions, which are often rich in Pro, Ser and Thr residues and may also carry Cys residues. Here, we describe a new IgA that was expressed in New Zealand White domestic rabbits of IGHVa1 allotype. This IgA has an extended hinge region containing an intriguing stretch of nine consecutive Ser residues and no Pro or Thr residues, a motif exclusive to this new rabbit IgA. Considering the amino acid properties, this hinge motif may present some advantage over the common IgA hinge by affording novel functional capabilities. We also sequenced for the first time the IgA14 CH2 and CH3 domains and showed that IgA14 and IgA3 are expressed.

Correction: Novel IgG-Degrading Enzymes of the IgdE Protease Family Link Substrate Specificity to Host Tropism of Streptococcus Species.

[This corrects the article DOI: 10.1371/journal.pone.0164809.].

Immunoglobulins and their receptors, and subversion of their protective roles by bacterial pathogens.

Immunoglobulins (Igs) play critical roles in immune defence against infectious disease. They elicit potent elimination processes such as triggering complement activation and engaging specific Fc receptors present on immune cells, resulting in phagocytosis and other killing mechanisms. Many important pathogens have evolved mechanisms to subvert or evade Ig-mediated defence. One such mechanism used by several pathogenic bacteria features proteins that bind the Ig Fc region and compromise engagement of host effector molecules. Examples include different IgA-binding proteins produced by Staphylococcus aureus, Streptococcus pyogenes, and group B streptococci, all of which interact with the same interdomain region on IgA Fc. Since this region also forms the interaction site for the major human IgA-specific Fc receptor CD89, the bacteria are able to evade CD89-mediated clearance mechanisms. Similar disruption of Ig effector function by pathogen Ig-binding proteins is evident in other species. Remarkably, all the Ig-binding proteins studied in detail to date are seen to target the CH2-CH3 domain interface in the Ig Fc region, suggesting a common mode of immune evasion. A second Ig subversion mechanism that has evolved independently in numerous pathogens involves proteases that cleave Ig molecules within their hinge regions, uncoupling the antigen recognition capability of the Fab region from clearance mechanisms elicited by the Fc region. The emerging understanding of the structural basis for the recognition of Igs as substrates for these proteases and as interaction partners for Ig-binding proteins may open up new avenues for treatment or vaccination.

Novel IgG-Degrading Enzymes of the IgdE Protease Family Link Substrate Specificity to Host Tropism of Streptococcus Species.

Recently we have discovered an IgG degrading enzyme of the endemic pig pathogen S. suis designated IgdE that is highly specific for porcine IgG. This protease is the founding member of a novel cysteine protease family assigned C113 in the MEROPS peptidase database. Bioinformatical analyses revealed putative members of the IgdE protease family in eight other Streptococcus species. The genes of the putative IgdE family proteases of S. agalactiae, S. porcinus, S. pseudoporcinus and S. equi subsp. zooepidemicus were cloned for production of recombinant protein into expression vectors. Recombinant proteins of all four IgdE family proteases were proteolytically active against IgG of the respective Streptococcus species hosts, but not against IgG from other tested species or other classes of immunoglobulins, thereby linking the substrate specificity to the known host tropism. The novel IgdE family proteases of S. agalactiae, S. pseudoporcinus and S. equi showed IgG subtype specificity, i.e. IgdE from S. agalactiae and S. pseudoporcinus cleaved human IgG1, while IgdE from S. equi was subtype specific for equine IgG7. Porcine IgG subtype specificities of the IgdE family proteases of S. porcinus and S. pseudoporcinus remain to be determined. Cleavage of porcine IgG by IgdE of S. pseudoporcinus is suggested to be an evolutionary remaining activity reflecting ancestry of the human pathogen to the porcine pathogen S. porcinus. The IgG subtype specificity of bacterial proteases indicates the special importance of these IgG subtypes in counteracting infection or colonization and opportunistic streptococci neutralize such antibodies through expression of IgdE family proteases as putative immune evasion factors. We suggest that IgdE family proteases might be valid vaccine targets against streptococci of both human and veterinary medical concerns and could also be of therapeutic as well as biotechnological use.

Leporid immunoglobulin G shows evidence of strong selective pressure on the hinge and CH3 domains.

Immunoglobulin G (IgG) is the predominant serum immunoglobulin and has the longest serum half-life of all the antibody classes. The European rabbit IgG has been of significant importance in immunological research, and is therefore well characterized. However, the IgG of other leporids has been disregarded. To evaluate the evolution of this gene in leporids, we sequenced the complete IGHG for six other genera: Bunolagus, Brachylagus, Lepus, Pentalagus, Romerolagus and Sylvilagus. The newly sequenced leporid IGHG gene has an organization and structure similar to that of the European rabbit IgG. A gradient in leporid IgG constant domain diversity was observed, with the CH1 being the most conserved and the CH3 the most variable domain. Positive selection was found to be acting on all constant domains, but with a greater incidence in the CH3 domain, where a cluster of three positively selected sites was identified. In the hinge region, only three polymorphic positions were observed. The same hinge length was observed for all leporids. Unlike the variation observed for the European rabbit, all 11 Lepus species studied share exactly the same hinge motif, suggesting its maintenance as a result of an advantageous structure or conformation.

Computational analyses of an evolutionary arms race between mammalian immunity mediated by immunoglobulin A and its subversion by bacterial pathogens.

IgA is the predominant immunoglobulin isotype in mucosal tissues and external secretions, playing important roles both in defense against pathogens and in maintenance of commensal microbiota. Considering the complexity of its interactions with the surrounding environment, IgA is a likely target for diversifying or positive selection. To investigate this possibility, the action of natural selection on IgA was examined in depth with six different methods: CODEML from the PAML package and the SLAC, FEL, REL, MEME and FUBAR methods implemented in the Datamonkey webserver. In considering just primate IgA, these analyses show that diversifying selection targeted five positions of the Cα1 and Cα2 domains of IgA. Extending the analysis to include other mammals identified 18 positively selected sites: ten in Cα1, five in Cα2 and three in Cα3. All but one of these positions display variation in polarity and charge. Their structural locations suggest they indirectly influence the conformation of sites on IgA that are critical for interaction with host IgA receptors and also with proteins produced by mucosal pathogens that prevent their elimination by IgA-mediated effector mechanisms. Demonstrating the plasticity of IgA in the evolution of different groups of mammals, only two of the eighteen selected positions in all mammals are included in the five selected positions in primates. That IgA residues subject to positive selection impact sites targeted both by host receptors and subversive pathogen ligands highlights the evolutionary arms race playing out between mammals and pathogens, and further emphasizes the importance of IgA in protection against mucosal pathogens.

Insights from Fc receptor biology: a route to improved antibody reagents.

Fc receptors and their interaction with antibodies will be a major theme at the forthcoming FASEB Science Research Conference on Immunoreceptors to be held in Snowmass this July (details available at www.faseb.org/src/home.aspx, follow the tabs for Immunoreceptors). Since its inception in the mid 1980s, this meeting series has maintained a focus on Fc receptors, and this year's meeting will be no exception.

The generation and evaluation of recombinant human IgA specific for Plasmodium falciparum merozoite surface protein 1-19 (PfMSP1 19).

BACKGROUND: Human immunoglobulin G (IgG) plays an important role in mediating protective immune responses to malaria. Although human serum immunoglobulin A (IgA) is the second most abundant class of antibody in the circulation, its contribution, if any, to protective responses against malaria is not clear. RESULTS: To explore the mechanism(s) by which IgA may mediate a protective effect, we generated fully human IgA specific for the C-terminal 19-kDa region of Plasmodium falciparum merozoite surface protein 1 (PfMSP1 19), a major target of protective immune responses. This novel human IgA bound antigen with an affinity comparable to that seen for an epitope-matched protective human IgG1. Furthermore, the human IgA induced significantly higher NADPH-mediated oxidative bursts and degranulation from human neutrophils than the epitope-matched human IgG1 from which it was derived. Despite showing efficacy in in vitro functional assays, the human IgA failed to protect against parasite challenge in vivo in mice transgenic for the human Fcα receptor (FcαRI/CD89). A minority of the animals treated with IgA, irrespective of FcαRI expression, showed elevated serum TNF-α levels and concomitant mouse anti-human antibody (MAHA) responses. CONCLUSIONS: The lack of protection afforded by MSP1 19-specific IgA against parasite challenge in mice transgenic for human FcαRI suggests that this antibody class does not play a major role in control of infection. However, we cannot exclude the possibility that protective capacity may have been compromised in this model due to rapid clearance and inappropriate bio-distribution of IgA, and differences in FcαRI expression profile between humans and transgenic mice.

A novel human IgA monoclonal antibody protects against tuberculosis.

Abs have been shown to be protective in passive immunotherapy of tuberculous infection using mouse experimental models. In this study, we report on the properties of a novel human IgA1, constructed using a single-chain variable fragment clone (2E9), selected from an Ab phage library. The purified Ab monomer revealed high binding affinities for the mycobacterial α-crystallin Ag and for the human FcαRI (CD89) IgA receptor. Intranasal inoculations with 2E9IgA1 and recombinant mouse IFN-γ significantly inhibited pulmonary H37Rv infection in mice transgenic for human CD89 but not in CD89-negative littermate controls, suggesting that binding to CD89 was necessary for the IgA-imparted passive protection. 2E9IgA1 added to human whole-blood or monocyte cultures inhibited luciferase-tagged H37Rv infection although not for all tested blood donors. Inhibition by 2E9IgA1 was synergistic with human rIFN-γ in cultures of purified human monocytes but not in whole-blood cultures. The demonstration of the mandatory role of FcαRI (CD89) for human IgA-mediated protection is important for understanding of the mechanisms involved and also for translation of this approach toward development of passive immunotherapy of tuberculosis.

Localization of the equine IgG-binding domain in the fibrinogen-binding protein (FgBP) of Streptococcus equi subsp. equi.

Fibrinogen-binding protein (FgBP, also termed SeM) is a cell-wall-associated anti-phagocytic M-like protein of the equine pathogen Streptococcus equi subsp. equi, and binds fibrinogen (Fg) and IgG. FgBP binds Fg avidly through residues located at the extreme N terminus of the molecule, whereas the IgG-binding site is more centrally located between the A and B repeats. FgBP binds equine IgG4 and IgG7 subclasses through interaction with the CH2-CH3 interdomain region of IgG-Fc, and possesses overlapping Fc-binding sites with protein A and protein G. In this study, FgBP truncates containing defined internal deletions were used to identify a stretch of 14 aa (residues 335-348) critical for IgG binding. Protein chimeras consisting of the non-IgG-binding alpha-helical coiled-coil M5 protein fused to FgBP sequences were used to identify a minimal equine IgG-binding domain consisting of residues 329-360. Competition ELISA tests suggested that IgG does not compromise Fg binding and vice versa.

Structural requirements for the interaction of human IgM and IgA with the human Fcalpha/mu receptor.

Here we unravel the structural features of human IgM and IgA that govern their interaction with the human Fcalpha/mu receptor (hFcalpha/muR). Ligand polymerization status was crucial for the interaction, because hFcalpha/muR binding did not occur with monomeric Ab of either class. hFcalpha/muR 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 hFcalpha/muR binding. IgM binding required contributions from both Cmu3 and Cmu4 Fc domains, whereas for dIgA, an exposed loop in the Calpha3 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 FcalphaRI 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 hFcalpha/muR binding. Furthermore, secretory component (SC, the extracellular portion of pIgR) and bacterial IgA-binding proteins were shown to inhibit the dIgA-hFcalpha/muR interaction. Therefore, we have identified a motif in the IgA-Fc inter-domain region critical for hFcalpha/muR interaction, and highlighted the multi-functional nature of a key site for protein-protein interaction at the IgA Fc domain interface.

A common theme in interaction of bacterial immunoglobulin-binding proteins with immunoglobulins illustrated in the equine system.

The M protein of Streptococcus equi subsp. equi known as fibrinogen-binding protein (FgBP) is a cell wall-associated protein with antiphagocytic activity that binds IgG. Recombinant versions of the seven equine IgG subclasses were used to investigate the subclass specificity of FgBP. FgBP bound predominantly to equine IgG4 and IgG7, with little or no binding to the other subclasses. Competitive binding experiments revealed that FgBP could inhibit the binding of staphylococcal protein A and streptococcal protein G to both IgG4 and IgG7, implicating the Fc interdomain region in binding to FgBP. To identify which of the two IgG Fc domains contributed to the interaction with FgBP, we tested two human IgG1/IgA1 domain swap mutants and found that both domains are required for full binding, with the CH3 domain playing a critical role. The binding site for FgBP was further localized using recombinant equine IgG7 antibodies with single or double point mutations to residues lying at the CH2-CH3 interface. We found that interaction of FgBP with equine IgG4 and IgG7 was able to disrupt C1q binding and antibody-mediated activation of the classical complement pathway, demonstrating an effective means by which S. equi may evade the immune response. The mode of interaction of FgBP with IgG fits a common theme for bacterial Ig-binding proteins. Remarkably, for those interactions studied in detail, it emerges that all the Ig-binding proteins target the CH2-CH3 domain interface, regardless of specificity for IgG or IgA, streptococcal or staphylococcal origin, or host species (equine or human).

The different effector function capabilities of the seven equine IgG subclasses have implications for vaccine strategies.

Recombinant versions of the seven equine IgG subclasses were expressed in CHO cells. All assembled into intact immunoglobulins stabilised by disulphide bridges, although, reminiscent of human IgG4, a small proportion of equine IgG4 and IgG7 were held together by non-covalent bonds alone. All seven IgGs were N-glycosylated. In addition IgG3 appeared to be O-glycosylated and could bind the lectin jacalin. Staphylococcal protein A displayed weak binding for the equine IgGs in the order: IgG1>IgG3>IgG4>IgG7>IgG2=IgG5>IgG6. Streptococcal protein G bound strongly to IgG1, IgG4 and IgG7, moderately to IgG3, weakly to IgG2 and IgG6, and not at all to IgG5. Analysis of antibody effector functions revealed that IgG1, IgG3, IgG4, IgG5 and IgG7, but not IgG2 and IgG6, were able to elicit a strong respiratory burst from equine peripheral blood leukocytes, predicting that the former five IgG subclasses are able to interact with Fc receptors on effector cells. IgG1, IgG3, IgG4 and IgG7, but not IgG2, IgG5 and IgG6, were able to bind complement C1q and activate complement via the classical pathway. The differential effector function capabilities of the subclasses suggest that, for maximum efficacy, equine vaccine strategies should seek to elicit antibody responses of the IgG1, IgG3, IgG4, and IgG7 subclasses.

Conformation-specific antibodies targeting the trimer-of-hairpins motif of the human T-cell leukemia virus type 1 transmembrane glycoprotein recognize the viral envelope but fail to neutralize viral entry.

Human T-cell leukemia virus type 1 (HTLV-1) entry into cells is dependent upon the viral envelope glycoprotein-catalyzed fusion of the viral and cellular membranes. Following receptor activation of the envelope, the transmembrane glycoprotein (TM) is thought to undergo a series of fusogenic conformational transitions through a rod-like prehairpin intermediate to a compact trimer-of-hairpins structure. Importantly, synthetic peptides that interfere with the conformational changes of TM are potent inhibitors of membrane fusion and HTLV-1 entry, suggesting that TM is a valid target for antiviral therapy. To assess the utility of TM as a vaccine target and to explore further the function of TM in HTLV-1 pathogenesis, we have begun to examine the immunological properties of TM. Here we demonstrate that a recombinant trimer-of-hairpins form of the TM ectodomain is strongly immunogenic. Monoclonal antibodies raised against the TM immunogen specifically bind to trimeric forms of TM, including structures thought to be important for membrane fusion. Importantly, these antibodies recognize the envelope on virally infected cells but, surprisingly, fail to neutralize envelope-mediated membrane fusion or infection by pseudotyped viral particles. Our data imply that, even in the absence of overt membrane fusion, there are multiple forms of TM on virally infected cells and that some of these display fusion-associated structures. Finally, we demonstrate that many of the antibodies possess the ability to recruit complement to TM, suggesting that envelope-derived immunogens capable of eliciting a combination of neutralizing and complement-fixing antibodies would be of value as subunit vaccines for intervention in HTLV infections.

IgA is a more potent inducer of NADPH oxidase activation and degranulation in blood eosinophils than IgE.

Human eosinophils can mediate both beneficial and detrimental responses in parasitic and allergic diseases. Binding of aggregated immunoglobulin to Fc receptors on eosinophils mediates important defence processes, including generation of activated oxygen species resulting from NADPH oxidase activation, and eosinophil peroxidase release following degranulation. The abilities of a matched set of IgA, IgG and IgE antibodies to elicit such responses in blood-derived eosinophils were compared using a chemiluminescence assay. IgA and IgG, but not IgE, were found to trigger NADPH oxidase activation and degranulation in eosinophils. This non-responsiveness to IgE did not result from receptor blockade by endogenous IgE since no blood-derived IgE was detectable on freshly isolated eosinophils. Moreover, while cross-linking of FcalphaRI by specific mAbs triggered NADPH oxidase activation and degranulation in blood-derived eosinophils, equivalent cross-linking of FcvarepsilonRI or FcvarepsilonRII did not elicit such responses. Therefore IgA is more potent at eliciting activated oxygen species release and degranulation in eosinophils than IgE, suggesting that the importance of IgA in eosinophil activation in immune defence and allergy may have been underestimated.

Sites in the CH3 domain of human IgA1 that influence sensitivity to bacterial IgA1 proteases.

The influence of regions, other than the hinge, on the susceptibility of human IgA1 to cleavage by diverse bacterial IgA1 proteases, was examined using IgA1 mutants bearing amino acid deletions, substitutions, and domain swaps. IgA1 lacking the tailpiece retained its susceptibility to cleavage by all of the IgA1 proteases. The domain swap molecule alpha1alpha2gamma3, in which the CH3 domain of IgA1 was exchanged for that of human IgG1, was resistant to cleavage with the type 1 and 2 serine IgA1 proteases of Neisseria meningitidis, Neisseria gonorrhoeae, and Haemophilus influenzae, but remained sensitive to cleavage with the metallo-IgA1 proteases of Streptococcus pneumoniae, Streptococcus oralis, Streptococcus sanguis, and Streptococcus mitis. Substitution of the IgA1 Calpha3 domain motif Pro440 -Phe443 into the corresponding position in the Cgamma3 domain of alpha1alpha2gamma3 resulted now in sensitivity to the type 2 IgA1 protease of N. meningitidis, indicating the possible requirement of these amino acids for sensitivity to this protease. For the H. influenzae type 2 protease, resistance of an IgA1 mutant in which the CH3 domain residues 399-409 were exchanged with those from IgG1, but sensitivity of mutant HuBovalpha3 in which the Calpha3 domain of bovine IgA replaces that of human IgA1, suggests that CH3 domain residues Glu403, Gln406, and Thr409 influence sensitivity to this enzyme. Hence, unlike the situation with the metallo-IgA1 proteases of Streptococcus spp., the sensitivity of human IgA1 to cleavage with the serine IgA1 proteases of Neisseria and Haemophilus involves their binding to different sites specifically in the CH3 domain.

The function of immunoglobulin A in immunity.

The vast surfaces of the gastrointestinal, respiratory, and genitourinary tracts represent major sites of potential attack by invading micro-organisms. Immunoglobulin A (IgA), as the principal antibody class in the secretions that bathe these mucosal surfaces, acts as an important first line of defence. IgA, also an important serum immunoglobulin, mediates a variety of protective functions through interaction with specific receptors and immune mediators. The importance of such protection is underlined by the fact that certain pathogens have evolved mechanisms to compromise IgA-mediated defence, providing an opportunity for more effective invasion. IgA function may also be perturbed in certain disease states, some of which are characterized by deposition of IgA in specific tissues. This review details current understanding of the roles played by IgA in both health and disease.