Although both assays measure the functionality of inhibitory antibodies, they measure immune responses in different ways. functions of the broadly neutralizing anti-influenza antibody. This phenomenon CID5721353 presumably represents one of the mechanisms by which SIgAs present in human respiratory mucosa prevent infection by antigen-drifted influenza viruses. Understanding the mechanisms involved in cross neutralization of viruses by SIgAs might facilitate the development of vaccine strategies against viral infection of mucosal tissues. == Author summary == SIgAs exist as mainly dimers and tetramers and play critical roles in mucosal immune responses against influenza. Detailed characterization of these anti-viral SIgA is important for better understanding of the mechanisms underlying anti-viral immunity. Here, we describe a means of generating a recombinant tetrameric monoclonal SIgA to enable exhaustive characterization of tetrameric SIgAs. The tetrameric monoclonal SIgA possessing variable regions of anti-influenza viruses broadly neutralizing antibody show that tetramerization of SIgA improves target breadth, but not the peak potency, of their anti-viral functions. These results broaden our knowledge about the fundamental role of SIgA tetramerization in CID5721353 anti-viral humoral response at the human respiratory mucosa. == Introduction == Secretory IgA antibodies (SIgAs) play an important role as a first line Rabbit polyclonal to DFFA of defense by inactivating pathogens on mucosal surfaces; this is especially true in the case of viruses such as influenza [1,2]. Recently, extensive efforts were made to develop novel vaccines that induce immunity via the mucosal route. SIgA is the major contributor CID5721353 to humoral mucosal immunity and is a key molecule that underpins the action of mucosal vaccines [3,4]. Therefore, understanding how SIgA works is important if we are to accelerate development of mucosal vaccines. IgA is the major immunoglobulin isotype in humans; indeed, its production exceeds that of CID5721353 all other immunoglobulin classes combined [2]. In addition, IgA displays a number of features that make it unique among the immunoglobulin classes; the most characteristic of these is its quaternary structure [5]. Most of the IgA in human serum is monomeric (comprising two heavy (H) and two light (L) chains). IgA present in external secretions is highly heterogeneous, although the majority is present in the form of polymers in which the heavy chains are covalently linked by a J chain. Moreover, these polymeric IgA antibodies are associated with the extracellular portion of the polymeric immunoglobulin receptor (pIgR), called the secretory component (SC), resulting in SIgA [5]. SIgA is composed primarily of dimers, although some larger polymeric forms, particularly tetramers, are present at low levels [510]. These tetrameric SIgA antibodies display greater neutralizing activity against influenza A viruses in the nasal mucosa than monomers or dimers [8,9]. However, the molecular mechanisms that underlie these characteristics of tetrameric SIgA remain largely unknown. Therefore, to elucidate these molecular mechanisms and evaluate the impact of SIgA polymerization on protection against viral infections, it is essential to obtain IgA antibodies as monomers, dimers, and tetramers that display identical variable regions; only in this way can we make a fairly accurate comparison of their functions. Although severalin vitromethods of generating recombinant polymeric IgA have been reported, they focus mainly on producing dimeric IgA [1113] rather than tetramers. No one has yet developed a method of generating trimeric or tetrameric IgA molecules. Here, we developed a method of generating recombinant monoclonal human tetrameric SIgAs by co-expressing human H, L, and J chains plus the SC in mammalian cells. This simple method enabled us to examine the effects of SIgA polymerization on its anti-viral activity against influenza A viruses. We compared the reactivity and functionality of generated broadly neutralizing antibodies (bnAb) comprising monomeric IgA, dimeric SIgA, or tetrameric SIgA and found that SIgA polymerization led to a marked increase in activity against viruses to which the antibodies bind with low-affinity, but not with high-affinity at monomeric state. Taken together, the results suggest that SIgA polymerization improves target breadth, but not the peak potency of anti-viral functions of bnAbs against influenza A viruses. == Results == == Co-expression of the SC along with human H, L, and J chains in mammalian cells enables generation of recombinant tetrameric monoclonal SIgAsin vitro == Previous studies report that co-expression of H, L, and J chains in mammalian cells results in formation of dimeric IgA [1114]. However, production of polymeric IgA antibodies.