Glycan shifting on hepatitis C virus (HCV) E2 glycoprotein is a mechanism for escape from broadly neutralizing antibodies

Hepatitis C virus (HCV) infection is a major cause of liver disease and hepatocellular carcinoma. Glycan shielding has been proposed to be a mechanism by which HCV masks broadly neutralizing epitopes on its viral glycoproteins. However, the role of altered glycosylation in HCV resistance to broadly neutralizing antibodies is not fully understood. Here, we have generated potent HCV neutralizing antibodies hu5B3.v3 and MRCT10.v362 that, similar to the previously described AP33 and HCV1, bind to a highly conserved linear epitope on E2. We utilize a combination of in vitro resistance selections using the cell culture infectious HCV and structural analyses to identify mechanisms of HCV resistance to hu5B3.v3 and MRCT10.v362. Ultra deep sequencing from in vitro HCV resistance selection studies identified resistance mutations at asparagine N417 (N417S, N417T and N417G) as early as 5 days post treatment. Comparison of the glycosylation status of soluble versions of the E2 glycoprotein containing the respective resistance mutations revealed a glycosylation shift from N417 to N415 in the N417S and N417T E2 proteins. The N417G E2 variant was glycosylated neither at residue 415 nor at residue 417 and remained sensitive to MRCT10.v362. Structural analyses of the E2 epitope bound to hu5B3.v3 Fab and MRCT10.v362 Fab using X-ray crystallography confirmed that residue N415 is buried within the antibody–peptide interface. Thus, in addition to previously described mutations at N415 that abrogate the β-hairpin structure of this E2 linear epitope, we identify a second escape mechanism, termed glycan shifting, that decreases the efficacy of broadly neutralizing HCV antibodies

Different Angiogenic Potentials of Mesenchymal Stem Cells Derived from Umbilical Artery, Umbilical Vein, and Wharton's Jelly

Human mesenchymal stem cells derived from the umbilical cord (UC) are a favorable source for allogeneic cell therapy. Here, we successfully isolated the stem cells derived from three different compartments of the human UC, including perivascular stem cells derived from umbilical arteries (UCA-PSCs), perivascular stem cells derived from umbilical vein (UCV-PSCs), and mesenchymal stem cells derived from Wharton’s jelly (WJ-MSCs). These cells had the similar phenotype and differentiation potential toward adipocytes, osteoblasts, and neuron-like cells. However, UCA-PSCs and UCV-PSCs had more CD146+ cells than WJ-MSCs (P<0.05). Tube formation assay in vitro showed the largest number of tube-like structures and branch points in UCA-PSCs among the three stem cells. Additionally, the total tube length in UCA-PSCs and UCV-PSCs was significantly longer than in WJ-MSCs (P<0.01). Microarray, qRT-PCR, and Western blot analysis showed that UCA-PSCs had the highest expression of the Notch ligand Jagged1 (JAG1), which is crucial for blood vessel maturation. Knockdown of Jagged1 significantly impaired the angiogenesis in UCA-PSCs. In summary, UCA-PSCs are promising cell populations for clinical use in ischemic diseases

Pathogenesis of liver injury : virus-induced autoantibody response against asialoglycoprotein receptor in woodchuck (Marmota Monax) viral hepatitis

The purpose of this research was to recognize the induction and pathogenic potential of autoantibodies against a liver-specific asialoglycoprotein receptor (ASGPR) in the course of hepadnavirus infection. Three sections of research work by using a woodchuck model of hepatitis B as an experimental system are presented in this thesis. -- First, woodchuck hepatic ASGPR (wASGPR) was purified and its polypeptide subunit composition, ligand binding properties and antigenicity were characterized. The isolated receptor is a hetero-oligomeric complex constituted of two subunits with molecular masses of 40- and 47-kDa. Antisera were generated in guinea pigs by challenge with the affinity-purified, bioactive wASGPR and rabbit ASGPR (rASGPR) preparations and in woodchucks by immunization with purified rASGPR. With the help of these antisera, a strong antigenic cross-reactivity of the wASGPR polypeptides with the ASGPRs from other species was demonstrated. Further, using antisera raised against isolated rASGPR polypeptides, the distinct antigenic specificity of each of the wASGPR subunits was revealed. Surface expression of wASGPR on the woodchuck hepatocytes was demonstrated by immunofluorescent staining of the isolated cells. Detailed analyses of the purified woodchuck hepatic plasma membranes (HPMs) showed that both subunits of wASGPR were present in the HPM preparations, although the membrane-bound 47-kDa subunit was identifiable only after exposure of HPMs to the ligand of the ASGPR. -- Second, the induction, dynamic patterns and specificity of autoantibodies to hepatic wASGPR were studied in animals with experimental woodchuck hepatitis virus (WHV) infection. Results showed that WHV infection induced anti-ASGPR autoantibodies or led to the rise in their levels in almost all animals experimentally infected with WHV (12 of 13). This demonstrated that the induction of this hepatocyte-specific autoreactivity was a common consequence of WHV infection. The relationship between anti-ASGPR reactivity existing prior to WHV inoculation and the outcome of experimental WHV hepatitis was also investigated. The results revealed that the occurrence of chronic hepatitis was significantly greater in the group of animals with pre-existing anti-ASGPR autoantibodies (54.6%) than that among woodchucks without autoantibodies in preinoculation sera (15.6%, P < 0.05), suggesting that the outcome of WHV infection might be influenced by the status of anti-ASGPR autoreactivity. Western blot analyses showed that WHV-induced anti-ASGPR autoantibodies recognized both 40- and 47-kDa subunits of wASGPR and rASGPR, implying that both receptor polypeptides may serve as targets for the pathogenic immune reactions. Furthermore, no antigenic cross-reactivity between wASGPR and WHV antigens could be identified by using antibodies against ASGPR or WHV envelope, suggesting that mechanisms other than viral protein mimicry could be involved in the induction of anti-ASGPR autoantibody in the course of WHV infection. -- Third, in vitro investigations of the pathogenic effects of the WHV-induced anti-ASGPR autoantibodies showed that these antibodies had the ability to inhibit specific ligand binding to the hepatocyte surface ASGPR. Also, Ig fractions from the same anti-ASGPR reactive sera were hepatocytotoxic in the presence of active complement. These findings indicate that virus-induced autoantibodies against hepatic ASGPR have the potential to directly contribute to liver injury and to the distortion of the hepatocyte clearance of asialoglycoproteins in viral hepatitis. Further careful studies are required to determine the significance of anti-ASGPR autoimmunity in the pathogenesis of liver injury in viral hepatitis. The woodchuck model of virus-induced ASGPR-specific autoimmunity could play an important role in these investigations

A Genetically Modified Protein-Based Hydrogel for 3D Culture of AD293 Cells

<div><p>Hydrogels have strong application prospects for drug delivery, tissue engineering and cell therapy because of their excellent biocompatibility and abundant availability as scaffolds for drugs and cells. In this study, we created hybrid hydrogels based on a genetically modified tax interactive protein-1 (TIP1) by introducing two or four cysteine residues in the primary structure of TIP1. The introduced cysteine residues were crosslinked with a four-armed poly (ethylene glycol) having their arm ends capped with maleimide residues (4-armed-PEG-Mal) to form hydrogels. In one form of the genetically modification, we incorporated a peptide sequence ‘GRGDSP’ to introduce bioactivity to the protein, and the resultant hydrogel could provide an excellent environment for a three dimensional cell culture of AD293 cells. The AD293 cells continued to divide and displayed a polyhedron or spindle-shape during the 3-day culture period. Besides, AD293 cells could be easily separated from the cell-gel constructs for future large-scale culture after being cultured for 3 days and treating hydrogel with trypsinase. This work significantly expands the toolbox of recombinant proteins for hydrogel formation, and we believe that our hydrogel will be of considerable interest to those working in cell therapy and controlled drug delivery.</p></div

A schematic diagram of hydrogel formation.

<p>The Michael addition between maleimides of 4-armed-PEG-Mal and thiols in each mutant protein leads to the formation of 3D networks for hydrogelations. The blue balls represent TIP1 protein. The yellow balls represent amino acids that were replaced by cysteine in TIP1. The pink lines represent 4-armed-PEG-Mal.</p

Hydrogel formation and stability.

<p>Optical images of three resulting hydrogels formed by 2.0 wt% of 4-armed-PEG-Mal and 2.0 wt% of the corresponding protein. A, TIP1 4C gel; B, TIP1 2C gel; and C, TIP1 2C RGD gel. Both TIP1 2C gel and TIP1 2C RGD gel remained transparent after 24 hours.</p

Hydrogel characterization.

<p>A, A rheological measurement in dynamic frequency sweep mode at the strain of 1% for each gel containing 2.0 wt% of 4-armed-PEG-Mal and 2.0 wt% of the protein. Closed symbols: elasticity (G’) values and open symbols: viscosity (G”) values. Circles: TIP1 2C gel and triangles: TIP1 2C RGD gel. B, An SEM image of the TIP1 2C gel. C, An SEM image of the TIP1 2C RGD gel.</p

Determining cell viability.

<p>A live-dead assay of AD293 cells cultured in TIP1 2C RGD (top) and TIP1 2C (down) hydrogels at different time points. A and D, day 1; B and E, day 2; and C and F, day 3. Live cells were stained green and dead cells were stained red. Magnification: 20×.</p

The purification of TIP1 2C.

<p>The size-exclusion chromatography of TIP1 2C was performed in a Superose 12 10/300 at two concentrations. Inset: A 20% SDS-PAGE gel result for each fraction.</p