Structure of HCMV glycoprotein B in the postfusion conformation bound to a neutralizing human antibody

Human cytomegalovirus (HCMV) poses a significant threat to immunocompromised individuals and neonates infected in utero. Glycoprotein B (gB), the herpesvirus fusion protein, is a target for neutralizing antibodies and a vaccine candidate due to its indispensable role in infection. Here we show the crystal structure of the HCMV gB ectodomain bound to the Fab fragment of 1G2, a neutralizing human monoclonal antibody isolated from a seropositive subject. The gB/1G2 interaction is dominated by aromatic residues in the 1G2 heavy chain CDR3 protruding into a hydrophobic cleft in the gB antigenic domain 5 (AD-5). Structural analysis and comparison with HSV gB suggest the location of additional neutralizing antibody binding sites on HCMV gB. Finally, immunoprecipitation experiments reveal that 1G2 can bind to HCMV virion gB suggesting that its epitope is exposed and accessible on the virus surface. Our data will support the development of vaccines and therapeutic antibodies against HCMV infection

Expression of the Human Cytomegalovirus Pentamer Complex for vaccine use in a CHO system

Human cytomegalovirus (HCMV) causes significant disease worldwide. Multiple HCMV vaccines have been tested in man but only partial protection has been achieved. The HCMV gH/gL/UL128/UL130/UL131A complex (Pentamer) is the main target of neutralizing antibodies in HCMV seropositive individuals and immunization with Pentamer raises high titers of neutralizing antibodies in small animals and non-human primates (NHP). Thus, Pentamer is a promising candidate for a future HCMV vaccine. Development of a Pentamer-based subunit vaccine requires expression of high amounts of a functional and stable complex. We describe here the development of a mammalian expression system for large scale Pentamer production. Several approaches comprising three different CHO-originated cell lines and multiple vector as well as selection strategies were tested. Stable cell pools expressed the HCMV Pentamer at a titer of approximately 60 mg/L at laboratory scale. A FACS-based single cell sorting approach allowed selection of a high expressing clone producing Pentamer at levels of approximately 400 mg/L in a laboratory scale fed-batch culture. Expression in a 50L bioreactor led to the production of HCMV Pentamer at comparable titers indicating the feasibility of further scale-up to production scale. The CHO produced HCMV Pentamer complex bound to a panel of human neutralizing antibodies and raised potently neutralizing immune response in mice. Thus, we have generated an expression system for the large scale production of functional HCMV Pentamer at high titers suitable for future subunit vaccine production

Structural and biochemical studies of HCMV gH/gL/gO and pentamer reveal mutually exclusive cell entry complexes

Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and the leading viral cause of birth defects after congenital infection. The glycoprotein complexes gH/gL/gO and gH/gL/UL128/UL130/UL131A (Pentamer) are key targets of the human humoral response against HCMV and are required for HCMV entry into fibroblasts and endothelial/epithelial cells, respectively. We expressed and characterized soluble forms of gH/gL, gH/gL/gO, and Pentamer. Mass spectrometry and mutagenesis analysis revealed that gL-Cys144 forms disulfide bonds with gO-Cys351 in gH/gL/gO and with UL128-Cys162 in the Pentamer. Notably, Pentamer harboring the UL128-Cys162Ser/gL-Cys144Ser mutations had impaired syncytia formation and reduced interference of HCMV entry into epithelial cells. Electron microscopy analysis showed that HCMV gH/gL resembles HSV gH/gL and that gO and UL128/UL130/UL131A bind to the same site at the gH/gL N terminus. These data are consistent with gH/gL/gO and Pentamer forming mutually exclusive cell entry complexes and reveal the overall location of gH/gL-, gH/gL/ gO-, and Pentamer-specific neutralizing antibody binding sites. Our results provide, to our knowledge, the first structural view of gH/gL/ gO and Pentamer supporting the development of vaccines and antibody therapeutics against HCMV

The human cytomegalovirus UL116 gene encodes an envelope glycoprotein forming a complex with gH independently from gL

Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and is the leading viral cause of birth defects after congenital infection. HCMV infection relies on the recognition of cell-specific receptors by one of the viral envelope glycoprotein complexes. Either the gH/gL/gO or the gH/gL/UL128/UL130/UL131A (Pentamer) complex has been found to fulfill this role, accounting for HCMV entry into almost all cell types. We have studied the UL116 gene product, a putative open reading frame identified by in silico analysis and predicted to code for a secreted protein. Virus infection experiments in mammalian cells demonstrated that UL116 is expressed late in the HCMV replication cycle and is a heavily glycosylated protein that first localizes to the cellular site of virus assembly and then inserts into the virion envelope. Transient-transfection studies revealed that UL116 is efficiently transported to the plasma membrane when coexpressed with gH and that gL competes with UL116 for gH binding. Further evidence for gH/UL116 complex formation was obtained by coimmunoprecipitation experiments on both transfected and infected cells and biochemical characterization of the purified complex. In summary, our results show that the product of the UL116 gene is an HCMV envelope glycoprotein that forms a novel gH-based complex alternative to gH/gL. Remarkably, the gH/UL116 complex is the first herpesvirus gH-based gL-less complex

Structural and biochemical studies of HCMV gH/gL/gO and Pentamer reveal mutually exclusive cell entry complexes

Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and the leading viral cause of birth defects after congenital infection. The glycoprotein complexes gH/gL/gO and gH/gL/UL128/UL130/UL131A (Pentamer) are key targets of the human humoral response against HCMV and are required for HCMV entry into fibroblasts and endothelial/epithelial cells, respectively. We expressed and characterized soluble forms of gH/gL, gH/gL/gO, and Pentamer. Mass spectrometry and mutagenesis analysis revealed that gL-Cys144 forms disulfide bonds with gO-Cys351 in gH/gL/gO and with UL128-Cys162 in the Pentamer. Notably, Pentamer harboring the UL128-Cys162Ser/gL-Cys144Ser mutations had impaired syncytia formation and reduced interference of HCMV entry into epithelial cells. Electron microscopy analysis showed that HCMV gH/gL resembles HSV gH/gL and that gO and UL128/UL130/UL131A bind to the same site at the gH/gL N terminus. These data are consistent with gH/gL/gO and Pentamer forming mutually exclusive cell entry complexes and reveal the overall location of gH/gL-, gH/gL/gO-, and Pentamer-specific neutralizing antibody binding sites. Our results provide, to our knowledge, the first structural view of gH/gL/gO and Pentamer supporting the development of vaccines and antibody therapeutics against HCMV

Antigenic Characterization of the HCMV gH/gL/gO and Pentamer Cell Entry Complexes Reveals Binding Sites for Potently Neutralizing Human Antibodies

<div><p>Human Cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and in fetuses following congenital infection. The glycoprotein complexes gH/gL/gO and gH/gL/UL128/UL130/UL131A (Pentamer) are required for HCMV entry in fibroblasts and endothelial/epithelial cells, respectively, and are targeted by potently neutralizing antibodies in the infected host. Using purified soluble forms of gH/gL/gO and Pentamer as well as a panel of naturally elicited human monoclonal antibodies, we determined the location of key neutralizing epitopes on the gH/gL/gO and Pentamer surfaces. Mass Spectrometry (MS) coupled to Chemical Crosslinking or to Hydrogen Deuterium Exchange was used to define residues that are either in proximity or part of neutralizing epitopes on the glycoprotein complexes. We also determined the molecular architecture of the gH/gL/gO- and Pentamer-antibody complexes by Electron Microscopy (EM) and 3D reconstructions. The EM analysis revealed that the Pentamer specific neutralizing antibodies bind to two opposite surfaces of the complex, suggesting that they may neutralize infection by different mechanisms. Together, our data identify the location of neutralizing antibodies binding sites on the gH/gL/gO and Pentamer complexes and provide a framework for the development of antibodies and vaccines against HCMV.</p></div

Additional file 7: Figure S6. of The beagle dog MicroRNA tissue atlas: identifying translatable biomarkers of organ toxicity

Top 20 expressed kidney miRNA. Top 20 expressed kidney miRNA found in the dog miRNA tissue atlas compared to previously published miRNA expression data from macro-dissection studies of the cortex and medulla of the cat and dog (Ichii et al). Shaded boxes indicate that the dog miRNA was among the top 10 expressed kidney miRNAs in the dog atlas; bolded miRNAs indicate that the miRNA was among the top 20. Comparison between dog whole kidney (dog altas) and data from dog cortex and medulla (Ichii et al) show good correlation with 7/10 miRNAs and 8/10 miRNAs expressed in the top 10 miRNAs, respectively, when compared to kidney expression observed in the dog atlas. Similarly, a comparison of dog whole kidney data (dog atlas) to cat cortex and medulla (Ichii et al) show good correlation as well with 7/10 and 6/10 miRNAs expressed in the top 10 miRNAs, respectively, when compared to dog whole kidney data (dog atlas). (PDF 41 kb

Chemical crosslinking and MS analysis of Pentamer and Pentamer/Fab complexes.

<p>Chemical crosslinking coupled to MS was used to identify exposed lysines that are in proximity within the Pentamer complex or between the Pentamer and neutralizing Fabs bound to it. <b>(A)</b> UL128 contains many exposed lysines able to crosslink inter-molecularly and intra-molecularly with gH (K283) and UL130 (K108-K131). The central portion of UL130 (K108-K154) crosslinks gH (K283) and UL131 (K103). The disulfide bridge between gL (C144) and UL128 (C162) is also indicated. <b>(B)</b> Crosslinking sites between neutralizing antibodies and Pentamer components are indicated. In agreement with the EM data, the majority of the interaction occurs within the UL128 and UL130 subunits.</p