Image_1_Predicting the Effectiveness of Hepatitis C Virus Neutralizing Antibodies by Bioinformatic Analysis of Conserved Epitope Residues Using Public Sequence Data.tiff

<p>Hepatitis C virus (HCV) is a global health issue. Although direct-acting antivirals are available to target HCV, there is currently no vaccine. The diversity of the virus is a major obstacle to HCV vaccine development. One approach toward a vaccine is to utilize a strategy to elicit broadly neutralizing antibodies (bNAbs) that target highly-conserved epitopes. The conserved epitopes of bNAbs have been mapped almost exclusively to the E2 glycoprotein. In this study, we have used HCV-GLUE, a bioinformatics resource for HCV sequence data, to investigate the major epitopes targeted by well-characterized bNAbs. Here, we analyze the level of conservation of each epitope by genotype and subtype and consider the most promising bNAbs identified to date for further study as potential vaccine leads. For the most conserved epitopes, we also identify the most prevalent sequence variants in the circulating HCV population. We examine the distribution of E2 sequence data from across the globe and highlight regions with no coverage. Genotype 1 is the most prevalent genotype worldwide, but in many regions, it is not the dominant genotype. We find that the sequence conservation data is very encouraging; several bNAbs have a high level of conservation across all genotypes suggesting that it may be unnecessary to tailor vaccines according to the geographical distribution of genotypes.</p

Table_2_Predicting the Effectiveness of Hepatitis C Virus Neutralizing Antibodies by Bioinformatic Analysis of Conserved Epitope Residues Using Public Sequence Data.xlsx

<p>Hepatitis C virus (HCV) is a global health issue. Although direct-acting antivirals are available to target HCV, there is currently no vaccine. The diversity of the virus is a major obstacle to HCV vaccine development. One approach toward a vaccine is to utilize a strategy to elicit broadly neutralizing antibodies (bNAbs) that target highly-conserved epitopes. The conserved epitopes of bNAbs have been mapped almost exclusively to the E2 glycoprotein. In this study, we have used HCV-GLUE, a bioinformatics resource for HCV sequence data, to investigate the major epitopes targeted by well-characterized bNAbs. Here, we analyze the level of conservation of each epitope by genotype and subtype and consider the most promising bNAbs identified to date for further study as potential vaccine leads. For the most conserved epitopes, we also identify the most prevalent sequence variants in the circulating HCV population. We examine the distribution of E2 sequence data from across the globe and highlight regions with no coverage. Genotype 1 is the most prevalent genotype worldwide, but in many regions, it is not the dominant genotype. We find that the sequence conservation data is very encouraging; several bNAbs have a high level of conservation across all genotypes suggesting that it may be unnecessary to tailor vaccines according to the geographical distribution of genotypes.</p

Table_3_Predicting the Effectiveness of Hepatitis C Virus Neutralizing Antibodies by Bioinformatic Analysis of Conserved Epitope Residues Using Public Sequence Data.xlsx

<p>Hepatitis C virus (HCV) is a global health issue. Although direct-acting antivirals are available to target HCV, there is currently no vaccine. The diversity of the virus is a major obstacle to HCV vaccine development. One approach toward a vaccine is to utilize a strategy to elicit broadly neutralizing antibodies (bNAbs) that target highly-conserved epitopes. The conserved epitopes of bNAbs have been mapped almost exclusively to the E2 glycoprotein. In this study, we have used HCV-GLUE, a bioinformatics resource for HCV sequence data, to investigate the major epitopes targeted by well-characterized bNAbs. Here, we analyze the level of conservation of each epitope by genotype and subtype and consider the most promising bNAbs identified to date for further study as potential vaccine leads. For the most conserved epitopes, we also identify the most prevalent sequence variants in the circulating HCV population. We examine the distribution of E2 sequence data from across the globe and highlight regions with no coverage. Genotype 1 is the most prevalent genotype worldwide, but in many regions, it is not the dominant genotype. We find that the sequence conservation data is very encouraging; several bNAbs have a high level of conservation across all genotypes suggesting that it may be unnecessary to tailor vaccines according to the geographical distribution of genotypes.</p

Table_1_Predicting the Effectiveness of Hepatitis C Virus Neutralizing Antibodies by Bioinformatic Analysis of Conserved Epitope Residues Using Public Sequence Data.xlsx

<p>Hepatitis C virus (HCV) is a global health issue. Although direct-acting antivirals are available to target HCV, there is currently no vaccine. The diversity of the virus is a major obstacle to HCV vaccine development. One approach toward a vaccine is to utilize a strategy to elicit broadly neutralizing antibodies (bNAbs) that target highly-conserved epitopes. The conserved epitopes of bNAbs have been mapped almost exclusively to the E2 glycoprotein. In this study, we have used HCV-GLUE, a bioinformatics resource for HCV sequence data, to investigate the major epitopes targeted by well-characterized bNAbs. Here, we analyze the level of conservation of each epitope by genotype and subtype and consider the most promising bNAbs identified to date for further study as potential vaccine leads. For the most conserved epitopes, we also identify the most prevalent sequence variants in the circulating HCV population. We examine the distribution of E2 sequence data from across the globe and highlight regions with no coverage. Genotype 1 is the most prevalent genotype worldwide, but in many regions, it is not the dominant genotype. We find that the sequence conservation data is very encouraging; several bNAbs have a high level of conservation across all genotypes suggesting that it may be unnecessary to tailor vaccines according to the geographical distribution of genotypes.</p

Effect of hypoxic conditions on the expression of the DDX3 gene in breast epithelial cells.

<p>MCF 10A (a & c) and MCF 7 (b & d) cells were cultured under normoxic conditions (20% O₂) or subjected to 200 µM CoCl₂ or 1% O₂ for 8, 12 and 24 h (a & b) qRT-PCR analysis was performed using specific primers for human DDX3 and HPRT as an internal normalization control. The expression level under normoxia was set to 1. DDX3 protein levels (c & d) in cell lysates of MCF 10A and MCF 7 cells were determined by immunoblots using anti-DDX3 antibodies. In these cases, HIF-1α and GAPDH served as controls indicating hypoxic conditions and equal protein loading respectively. Error bars represent ±SD.</p

ChIP assay: <i>in vivo</i> binding of HIF-1 to the DDX3 promoter in MCF 10A cells.

<p>At the top of the gel is a schematic representation of the DDX3 promoter. Arrows flank the region (-423 to -115) amplified by PCR with DDX3 promoter specific primers. Gel shows: lane 1- molecular weight (MW) marker, lane 2- total input chromatin, lane 3-acetyl histone H3 precipitation, lane 4-anti-HIF-1α precipitation under hypoxic conditions, lane 5-anti-HIF-1α under normoxic conditions, and lane 6-anti-actin precipitation. Identical volumes from each final precipitation were used for PCR (except for the input chromatin, which was diluted 100x).</p

Characterization of the putative HRE containing sequences of the human DDX3 promoter.

<p>(a) Schematic representation of putative core HRE sequences in the promoter region of the DDX3 gene. (b) Linear representations of the DDX3 promoter-reporter constructs and the relative firefly luciferase activity of each in transient transfection assays in MCF 7 cells with pRRL-CMV (renilla luciferase expressing vector) as an internal control. After transfection cells were incubated in normoxia for 24 h Relative firefly luciferase activities are presented as a histogram at the right. (c) Effects of over-expression of HIF-1α in MCF 7 cells under normoxia. Cells were co-transfected with the DDX3 promoter-reporter constructs shown in (b), with pCDNA-HIF-1α (constitutive HIF-1α expressing vector), and pRRL-CMV as the internal control. Relative firefly luciferase activities are presented. Mean values from three independent transfections are shown. Error bars represent ±SD.</p

Specificity of HIF-1α dependent activation of the DDX3 gene.

<p>Stable MCF 10A and MCF 7-HIF-1α shRNA clones were generated. (a) MCF 10A-shHIF-1α and (b) MCF 7-shHIF-1α cells were subjected to 200 µM CoCl₂ for the indicated times. qRT-PCR analysis was performed using specific primers for human DDX3, HIF-1α and HIF-2α. DDX3, HIF-1α, and HIF-2α protein levels in cell lysates of MCF 10A-shHIF-1α (c) and MCF 7-shHIF-1α (d) cells were examined by immunoblots using protein specific antibodies. GAPDH served as a loading control. Error bars represent ±SD.</p

Correlation between HIF-1α and DDX3 protein expression in MDA-MB-231 breast cancer cells.

<p>(a) Immunoblot showing expression levels of HIF-1α following CoCl₂ treatment in HIF-1α knockdown cells. (b) Histogram showing relative fold expression of DDX3 mRNA protein levels of MDA-MB-231 versus MDA-MB-231-shHIF-1α cells. Expression of DDX3 was downregulated in MDA-MB-231-shHIF-1α relative to MDA-MB-231 cells at both the mRNA and protein levels. (c) Immunohistochemical staining for HIF-1α and DDX3 in sequential slices of a MDA-MB-231 tumor xenograft. Tumor sections were stained with antibodies that are specific for HIF-1α and DDX3. Samples were counterstained with hematoxylin to reveal morphology. Error bars represent ±SD.</p

Viral evolution in the presence of increasing HMAb HC33.1 concentrations.

<p>(<b>A</b>) Dual antibody immunofluorescence staining of Huh7.5 cells infected with JFH1 2a HCVcc during multiple passages in increasing concentration of HC33.1. IFA is shown for P0, P18, P27 and P31. HCV E2 glycoprotein was stained with HC33.1 under which viral escape variants were selected (green, upper set of panels), or with CBH-5, a neutralizing domain B HMAb that does not share the same epitope with HC33.1 on E2 (green, lower set of panels). Total virus-infected cells were stained with anti-NS3 antibody labeled with Alexa-594 (red). The cells were counterstained with Hoechst nuclear stain H33342 (blue). The captured images were superimposed (merge). (<b>B and C</b>) Sequence analysis of escape variants in increasing concentrations of HC33.1. Circle graph represents the change in composition of variants from selected passages, as indicated on the top, P0–P34. Specific variants are color coded as indicated in the legend. The table presents the corresponding HC33.1 concentration and the relative intensity in IFA binding by HC33.1. The arrow line divides the viral evolution into four phases (Phase I–IV), based on the appearance in each phase of a variant(s) bearing a specific mutation that became dominant. Viral RNA in the corresponding cell culture supernatants were analyzed by single colony sequencing following RT-PCR amplifications.</p