Flaviviruses are a group of human pathogens causing severe encephalitic or hemorrhagic diseases that include West Nile, dengue and yellow fever viruses. Here, using X-ray crystallography we have defined the structure of the flavivirus cross-reactive antibody E53 that engages the highly conserved fusion loop of the West Nile virus envelope glycoprotein. Using cryo-electron microscopy, we also determined that E53 Fab binds preferentially to spikes in noninfectious, immature flavivirions but is unable to bind significantly to mature virions, consistent with the limited solvent exposure of the epitope. We conclude that the neutralizing impact of E53 and likely similar fusion-loop-specific antibodies depends on its binding to the frequently observed immature component of flavivirus particles. Our results elucidate how fusion-loop antibodies, which comprise a significant fraction of the humoral response against flaviviruses, can function to control infection without appreciably recognizing mature virions. As these highly cross-reactive antibodies are often weakly neutralizing they also may contribute to antibodydependent enhancement and flavi virus pathogenesis thereby complicating development of safe and effective vaccines.

SUMMARY Osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B (RANK) are members of the TNFR superfamily that regulate osteoclast formation and function by competing for RANK ligand (RANKL). RANKL promotes osteoclast development through RANK activation, while OPG inhibits this process by sequestering RANKL. For comparison, we solved crystal structures of RANKL with RANK, and RANKL with OPG. Complementary biochemical and functional studies reveal that the monomeric cytokine-binding region of OPG binds RANKL with ~500 fold higher affinity than RANK, and inhibits RANKL-stimulated osteoclastogenesis ~150 times more effectively, in part because the binding cleft of RANKL makes unique contacts with OPG. Several side chains as well as the C-D and D-E loops of RANKL occupy different orientations when bound to OPG versus RANK. High affinity OPG binding requires a 90s-loop Phe residue that is mutated in juvenile Paget’s disease. These results suggest cytokine plasticity may help to fine tune specific TNF-family cytokine/receptor pair selectivity.

Estrogen-deficient osteoporosis may be an inflammatory disorder and we therefore asked if IL-17 participates in its pathogenesis. Deletion of the principal IL-17 receptor (IL-17RA) protects mice from ovariectomy (OVX)-induced bone loss. Further supporting a central role of IL-17 in its pathogenesis, OVX-induced osteoporosis is prevented by a blocking antibody targeting the cytokine. IL-17 promotes osteoclastogenesis by stimulating RANK ligand (RANKL) expression by osteoblastic cells, mediated by the IL-17RA SEFIR/TILL domain. Estrogen deprivation, however does not enhance IL-17RA mRNA expression by osteoblasts or in bone, but augments that of Act1, an IL17RA-interacting protein and signaling mediator. Similar to IL-17RA−/− mice, those lacking Act1 are protected from OVX-induced bone loss. Also mirroring IL-17RA-deficiency, absence of Act1 in osteoblasts, but not osteoclasts, impairs osteoclastogenesis via dampened RANKL expression. Transduction of WT Act1 into Act1−/− osteoblasts substantially rescues their osteoclastogenic capacity. The same construct, however, lacking its E3 ligase U-box or its SEFIR domain, which interacts with its counterpart in IL-17RA, fails to do so. Estrogen deprivation, therefore, promotes RANKL expression and bone resorption in association with upregualtion of the IL-17 effector, Act1, supporting the concept that post-menopausal osteoporosis is a disorder of innate immunity.

Clonal hematopoiesis is common in older persons and is associated with an increased risk of hematologic cancer. Here, we review studies establishing an association between clonal hematopoiesis and hematopoietic malignancy, discuss features of clonal hematopoiesis that are predictive of leukemic progression, and explore the role of hematopoietic stressors in the evolution of clonal hematopoiesis to acute myeloid leukemia or myelodysplastic syndrome. Clonal hematopoiesis due to point mutations or structural variants, such as copy number alterations, are associated with an approximately 10-fold increased risk of hematopoietic malignancy. Although the absolute risk of hematopoietic malignancy is low, certain features of clonal hematopoiesis may confer a higher risk of transformation, including the presence of TP53 or splicesome gene mutations, a variant allele fraction greater than 10%, the presence of multiple mutations, and altered red blood indices. Clonal hematopoiesis in the setting of peripheral blood cytopenias carries a very high risk of progression to a myeloid malignancy and merits close observation. There is emerging evidence to suggest the hematopoietic stressors contribute both to the development of clonal hematopoiesis and progression to hematopoietic malignancy. Specifically, there is evidence that genotoxic stress from chemotherapy or radiation therapy, ribosome biogenesis stress, and possibly inflammation may increase the risk of transformation from clonal hematopoiesis to a myeloid malignancy. Models that incorporate features of clonal hematopoiesis along with an assessment of hematopoietic stressors may eventually help predict and prevent the development of hematopoietic malignancies.

Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis. Approximately one-third of cases do not have a known genetic cause. Exome sequencing of 104 persons with congenital neutropenia identified heterozygous missense variants of CLPB (caseinolytic peptidase B) in 5 SCN cases, with 5 more cases identified through additional sequencing efforts or clinical sequencing. CLPB encodes an adenosine triphosphatase (ATPase) implicated in protein folding and mitochondrial function. Prior studies showed that biallelic mutations of CLPB are associated with a syndrome of 3-methylglutaconic aciduria, cataracts, neurologic disease, and variable neutropenia. However, 3-methylglutaconic aciduria was not observed and, other than neutropenia, these clinical features were uncommon in our series. Moreover, the CLPB variants are distinct, consisting of heterozygous variants that cluster near the ATP-binding pocket. Both genetic loss of CLPB and expression of CLPB variants results in impaired granulocytic differentiation of human hematopoietic progenitors and increased apoptosis. These CLPB variants associate with wildtype CLPB and inhibit its ATPase and disaggregase activity in a dominant-negative fashion. Finally, expression of CLPB variants is associated with impaired mitochondrial function but does not render cells more sensitive to endoplasmic reticulum stress. Together, these data show that heterozygous CLPB variants are a new and relatively common cause of congenital neutropenia and should be considered in the evaluation of patients with congenital neutropenia.