Exposure of an occluded hemagglutinin epitope drives selection of a class of cross-protective influenza antibodies.

Germinal center (GC) B cells at viral replication sites acquire specificity to poorly immunogenic but conserved influenza hemagglutinin (HA) epitopes. Here, high-throughput epitope mapping of local GC B cells is used to identify conserved HA epitope selecting cross-reactive antibodies that mediate heterosubtypic protection. A distinct feature of this epitope is an occlusion in the naive trimeric HA structure that is exposed in the post-fusion HA structure to occur under low pH conditions during viral replication. Importantly, systemic immunization by the post-fusion HA antigen results in GC B cells targeting the occluded epitope, and induces a class of protective antibodies that have cross-group specificity and afford protection independent of virus neutralization activity. Furthermore, this class of broadly protective antibodies develops at late time points and persists. Our results identify a class of cross-protective antibodies that are selected at the viral replication site, and provide insights into vaccine strategies using the occluded epitope.

Glycosphingolipids in the regulation of the nervous system.

The highest expression of gangliosides, sialic acid-containing glycosphingolipids (GSLs), is found in the nervous tissue of vertebrates. Changes in the profiles of gangliosides during the development of nervous tissues indicate that they are involved in the regulation of neurogenesis and synaptogenesis. Their distinct distribution patterns support the suggestion that they are involved in both the differentiation and function of neural cells. In addition to results of studies of GSLs done using biochemical, histopathological, and cell biological approaches, recent progress in the genetic engineering of glycosyltransferase genes has resulted in novel findings and concepts about their roles in the nervous system. Roles of GSLs in the regulation of signaling that determine cell fates in membrane microdomains such as lipid rafts have been extensively studied. In particular, gene targeting of glycosyltransferases in mice has enabled investigation of the in vivo functions of GSLs. The majority of abnormal phenotypes exhibited by knockout (KO) mice may reflect an abnormal structure and a resultant altered function of lipid rafts caused by alterations in their GSL composition. Generally speaking, abnormal phenotypes found in most KO mice were milder than expected, suggesting that the remaining GSLs compensate for the functions of those lost. There are also functions that cannot be replaced by the remaining GSLs. Thus, there may be two modes of function of GSLs: one is nonspecific and can be carried out by multiple GSLs, the second mode is that in which the function of the missing GSL(s) cannot be compensated by others. Identification of natural ligands for individual GSLs is crucial in order to clarify the functions of each structure.

Disruption of GM2/GD2 synthase gene resulted in overt expression of 9-O-acetyl GD3 irrespective of Tis21.

GM2/GD2 synthase gene knockout mice lack all complex gangliosides, which are abundantly expressed in the nervous systems of vertebrates. In turn, they have increased precursor structures GM3 and GD3, probably replacing the roles of the depleted complex gangliosides. In this study, we found that 9-O-acetyl GD3 is also highly expressed as one of the major glycosphingolipids accumulating in the nervous tissues of the mutant mice. The identity of the novel component was confirmed by neuraminidase treatment, thin layer chromatography-immunostaining, two-dimensional thin layer chromatography with base treatment, and mass spectrometry. All candidate factors reported to be possible inducer of 9-O- acetylation, such as bitamine D binding protein, acetyl CoA transporter, or O-acetyl ganglioside synthase were not up-regulated. Tis21 which had been reported to be a 9-O-acetylation inducer was partially down-regulated in the null mutants, suggesting that Tis21 is not involved in the induction of 9-O-acetyl-GD3 and that accumulated high amount of GD3 might be the main factor for the dramatic increase of 9-O-acetyl GD3. The ability to acetylate exogenously added GD3 in the normal mouse astrocytes was examined, showing that the wild-type brain might be able to synthesize very low levels of 9-O-acetyl GD3. Increased 9-O-acetyl GD3, in addition to GM3 and GD3, may play an important role in the compensation for deleted complex gangliosides in the mutant mice.