The impact of genetic variations in sofosbuvir metabolizing enzymes and innate immunity mediators on treatment outcome in HCV-infected patients.

Hepatitis C virus (HCV) is the leading cause of liver diseases worldwide. At present, combinations of different classes of direct-acting antiviral agents (DAAs) are used as treatment options for HCV, in which sofosbuvir (SOF) is the common DAA among different therapeutic regimes. In Egypt, SOF plus daclatasvir (DCV) is the widely used anti-HCV treatment protocol. Herein, we aimed to assess the association between 3 single-nucleotide polymorphisms (SNPs) at the genes coding for 2 SOF metabolizing enzymes: histidine triad nucleotide-binding protein 1 (HINT1) rs4696/rs7728773 and nucleoside diphosphate kinase 1 (NME1) rs3760468, together with the most potent anti-HCV innate molecule, i.e., interferon lambda 3 (IFNL3) rs12979860 and the response to SOF/DCV in Egyptian patients chronically infected with genotype 4 (GT4). SNPs were genotyped using real-time PCR in DNA from patients who achieved sustained virological response (SVR) at 12 weeks post-SOF/DCV treatment (i.e., responders; n = 188), patients who failed to achieve SVR12 (i.e., non-responders; n = 109), and healthy controls (n = 62). Our results demonstrated that patients bearing HINT1 rs7728773 CT/TT (odds ratio 2.119, 95% CI 1.263-3.559, p = 0.005) and IFNL3 rs12979860 CC (odds ratio 3.995, 95% CI 2.126-7.740, p = 0.0001) were more likely to achieve SVR12. However, neither HINT1 rs4696 nor NME1 rs3760468 seems to contribute to the responsiveness to SOF/DCV. Binary regression analysis defined 5 predictor factors independently associated with SVR12: age, bilirubin, hemoglobin, early stages of fibrosis, and combined HINT1 rs7728773 and IFNL3 rs12979860 favorable and mixed genotypes (odds ratio 3.134, 95% CI 1.518-6.47, p = 0.002), and that was confirmed by the combined ROC curve for the 5 predictor factors (AUC = 0.91, 95% CI 0.869-0.95, P = 0.0001). In conclusion, these data suggest that the two SNPs have the potential in predicting the response rate to SOF/DCV treatment in patients infected with HCV GT4. This study is the first to investigate the pharmacogenetics of SOF metabolizing enzyme and introduce HINT1 rs7728773 as a novel SNP that predicts the treatment efficacy.

Reactivation of human cytomegalovirus inhibits expression of liver fibrosis related cytokines in patients chronically infected with hepatitis C virus genotype 4a.

BACKGROUND: The impact of human cytomegalovirus (HCMV) reactivation on the expression pattern of matrix metalloproteinases, their inhibitors and related cytokines during HCV infection poorly understood. METHODS: Reactivation of CMV in 95 subjects (75 chronically infected HCV patients and 20 healthy subjects) was examined. All studied subjects had detectable IgG antibodies for CMV, but only 35/75 of HCV patients (46.7%) had detectable CMV DNA. The expressions of 11 fibrosis related genes by quantitative real-time PCR were analyzed in subjects' PBMCs. The serum levels of TGFß2 and PDGFα have been measured by ELISA. RESULTS: Chronically infected HCV patients with reactivated CMV had less expression of TGF-ß1, TGF-ß2, PDGFα and STAT1 transcripts than HCV patients with latent CMV (p = 0.037, 0.006, 0.001 and 0.009; respectively) and normal controls (TGF-ß2, p = 0.008). Moreover the expression of (TGFß2 and PDGFα) genes decreased significantly in CMV-reactivated patients during the early stage of fibrosis relative to the comparable stage of HCV infection (p = 0.004 and 0.008; respectively). Besides, the mRNA abundance of STAT1 gene in CMV-reactivated patients decreased dramatically as compared to HCV infections during the late stage of fibrosis (p = 0.014). The TGFß2 protein level has been declined dramatically in CMV-reactivated patients compared to HCV infected patients and control group (p = 0.001 and 0.033; respectively). Our results suggest that CMV reactivation disrupts the expression of several cytokines as compared to solitary infection with HCV. Noticeably, the expressions of matrix metalloproteinases genes and their inhibitors have not been significantly influenced by reactivation of CMV. CONCLUSION: The current data reveal that reactivation of CMV partially blocks the upregulation of 2 important pro-inflammatory cytokines i.e. TGFß 2 and PDGFα at early stages of fibrosis, moreover this CMV mediated blockage of the STAT1 shows statistical significance at late stage of fibrosis.

Development of a gene signature for predicting cirrhosis risk score of chronic liver disease associated with HCV infection in Egyptians.

BACKGROUND: Complex diseases such as fibrosis are likely polygenic. Lately, cirrhosis risk score (CRS) clearly discriminated Chronic HCV patients with high-risk versus those with low-risk for cirrhosis better than clinical factors. METHODS: Herein, the CRS was assessed via genotyping by allelic discrimination assays in 243 HCV Egyptian patients categorized into 164 patients didn't develop HCC (93 mild, 71 advanced fibrosis); and 79 patients developed HCC. APRI and FIB-4 scores were calculated, compared with CRS and correlated with degree of fibrosis progression. RESULTS: Median of the three CRS, APRI and FIB-4 scores were significantly elevated in late fibrotic and HCC patients (p < 0.001); however CRS displayed proper discrimination (mild fibrosis (0.59; 0.4-0.75), advanced fibrosis (0.75; 0.7-0.86) and HCC (0.73; 0.57-0.77); (p < 0.001)). The ROC analysis of CRS score displayed modest accuracy to discriminate between mild and advanced fibrotic patient; AUC was 0.73; p < 0.0001), while AUC was only 0.57 (p = 0.05) for the discrimination between HCC and no HCC. Moreover, the combination of CRS, APRI and FIB4 lessened the power of correlation (AUC, 0.63 (p < 0.0001)) in fibrosis prognosis. In HCC prognosis, the combination of CRS, APRI and FIB4 in HCC patients showed modest accuracy with AUC, 0.59 (p = 0.0001). CONCLUSION: The diagnostic accuracy of FIB-4 for predicting liver fibrosis was nearly identical to that of CRS, however the strength of CRS score stemmed from that it is built on 7 SNPs host genetic factor. Our study validates non invasive algorithms for fibrosis prognosis purposes which may aid in decision making for therapeutic intervention.

A multiepitope peptide vaccine against HCV stimulates neutralizing humoral and persistent cellular responses in mice.

BACKGROUND: Although DAAs hold promise to significantly reduce rates of chronic HCV infections, its eradication still requires development of an effective vaccine. Prolonged T cell responses and cross neutralizing antibodies are ideal for vaccination against the infection. We aimed to design and synthesize a 6 multi epitope peptide vaccine candidate and provide evidence for production of extended cellular and neutralizing Abs in mice. METHODS: Six peptides derived from conserved epitopes in E1, E2 (n = 2),NS4B, NS5A and NS5B were designed, synthesized in a multiple antigenic peptide (MAP) form and administered w/o adjuvant to BALB/c mice as HCVp6-MAP at doses ranging from 800 ng to 16 µg. Humoral responses to structural epitopes were assayed by ELISA at different times after injection. ELISpot assay was used to evaluate IFN É£ producing CD4+/ CD8+ T- lymphocytes at extended durations i.e. > 20 weeks. Viral neutralization by mice sera was tested for genotypes 2a (JFH1) and a chimeric 2a/4a virus (ED43/JFH1) in HCVcc culture. RESULTS: HCVp6-MAP confers potent viral neutralization and specific cellular responses at > 1600 ng/ animal for at least 20 weeks. CONCLUSION: We report on a promising anti HCV vaccine for future studies on permissive hosts and in clinical trials.

Level of Human Antibodies Targeting HCV E1/E2 Peptides and Spontaneous Clearance of HCV in Blood Donors.

BACKGROUND: Hepatitis C virus (HCV) is a major health problem worldwide particularly in Egypt. The humoral immune response has an important function in the control of HCV infection. The aim of this study was to investigate the role of neutralizing antibodies in Hepatitis C Virus (HCV) clearance in infected individuals. METHODS: This study was carried out on apparently healthy blood donors (n = 200). Detectable HCV antibodies were assessed by commercial ELISA and specific human immunoglobulins targeting peptides derived from HCV E1/E2 glycoproteins were measured in donors' blood using an in house optimized ELISA. Human IgG purification was carried out from positive HCV RNA and negative HCV RNA samples in order to evaluate its neutralizing activity in vitro using Huh 7 cells. RESULTS: The studied cohort included 96/200 subjects who tested positive for HCV antibodies, among which 56/96 (58%) samples were positive for HCV RNA (Group 1) and 40/96 (42%) samples had undetectable HCV RNA (Group 2). ELISA results showed that Human HCV immunoglobulin (HHI) targeting HCV E1 synthetic peptide (a.a. 315 - 323) was detectable in 63/96 (66%) and HHI targeting HCV E2 (a.a. 412 - 419) tested positive in 14/96 (15%) while 19/96 (20%) were positive for HCV E2 (a.a. 517 - 531). HHI higher than the cutoff level against peptide HCV E1 (a.a. 315 - 323) was detected in 22/63 (35%) in Group 2 and positive in 41/63 (65%) in Group 1. HHI against peptide HCV E2 (a.a. 412 - 419) was positive in 7 (50%) blood donors in Group 2 and also positive in 7 (50%) of Group 1. While HHI targeting HCV E2 (a.a. 517 - 531) was positive in 11 (60%) in Group 2 compared with 8 cases (40%) in Group 1. Purified human antibodies from cases positive for HCV antibodies and negative for HCV RNA showed in vitro neutralization at concentrations 30 and 10 µg/mL while the same concentration of purified human IgG from cases positive for HCV RNA showed no viral neutralization. CONCLUSIONS: The tested epitope(s) derived from HCV envelope E1 and E2 are important for viral clearance and hence can be used for HCV vaccine development.

Low Molecular Mass Polypeptide 7 Single Nucleotide Polymorphism is Associated with the Progression of Liver Fibrosis in Patients Infected with Hepatitis C Virus Genotype 4.

BACKGROUND: The hepatitis C virus (HCV) is a leading cause of liver disease and the consequent complications of cirrhosis. However, there is no precise biomarker to predict the patients at high risk of developing progressive disease. We showed previously the implication of low molecular mass polypeptide-7 (LMP-7) single nucleotide varia- tions in the response to combined pegylated IFN and ribavirin therapy in patients infected with HCV genotype 4. In this study, we examined the possible relationship between LMP-7 genotypes and both the degree of liver fibrosis and the transition from end stage of fibrosis to hepatocellular carcinoma (HCC). METHODS: LMP-7 single nucleotide variation at codon 49 (substitution from A to C) was determined using restriction fragment length polymorphism analysis in leucocyte DNA from healthy subjects (n = 36) and HCV-chronically infected patients of genotype 4 either with different grades of liver fibrosis (n = 77) or with hepatocellular carci- noma (n = 25). Chronic HCV-infected patients having liver fibrosis were categorized into two groups based on the degree of fibrosis, early fibrosis (F0-F2, n = 37) and late fibrosis (F3-F4, n = 40). RESULTS: Our results demonstrated that patients with the LMP-7 CA/AA genotypes were more likely to have advanced fibrosis scores than those bearing the CC genotype, although LMP-7 polymorphism does not seem to contribute to the progression from the late stage of fibrosis to hepatocellular carcinoma. CONCLUSIONS: These results suggest that LMP-7 polymorphism is a candidate prognostic marker in mathematical models designed for predicting the progression of HCV-related liver disease. Nevertheless, the mechanism whereby LMP-7 leads to the progression of liver fibrosis remains to be determined.

Alteration of the Total Nuclear DNA Ploidy in Different Histopathological Liver Tissues Negative and Positive for HCV RNA.

BACKGROUND: Hepatitis C virus (HCV) infection is associated with the development of hepatocellular carcinoma (HCC). The molecular mechanisms of HCV-associated carcinogenesis are unknown. We aim to investigate the alteration of the total nuclear DNA content (ploidy) in different histopathological liver tissues infected with HCV and their relation to the seropositivity of HCV RNA. METHODS: Blood and liver tissues were collected from 26 patients. Diagnosis was carried out according to clinical and pathological examinations by specialized physicians. HCV RNA was detected in patients' sera and tissue samples by RT-PCR. To examine nuclear DNA ploidy, liver tissues were stained with blue Fulgen using the image analysis techniques. Finally, the patients' DNA content was examined by histochemical analysis depending on the optical density of DNA from liver biopsies using the grey image menu in each specimen. RESULTS: The HCV RT-PCR results demonstrated that 13/26 (50%) patients had detectable HCV RNA in their sera samples while 18/26 (69%) had detectable HCV RNA in liver tissues. The DNA content from those patients measured by image cytometry showed a high level of alteration of nuclear DNA ploidy and proliferation in liver tissues with HCC, less alteration of nuclear DNA ploidy in cirrhotic patients, and least proliferation nearly normal in liver fibrosis patients. Moreover, the results of histochemical analysis confirmed the DNA image cytometry results and showed that positive HCV RNA liver tissues had more DNA ploidy than negative HCV RNA liver tissues with statistical significance (p-value < 0.05). CONCLUSIONS: HCV positive liver tissue had alterations in DNA content (ploidy) which may lead to liver disease progression, malignant transformation of the liver cells and development of hepatocellular carcinoma.

Mouse monoclonal antibody towards e1 specific epitope blocks viral entry and intracellular viral replication in vitro.

We characterized viral neutralization by a murine monoclonal antibody (mAb315) developed against conserved E1 specific epitope aa 315-323 at pre- and post-binding steps of infection into Huh7 cells. Detection of native virus in infected Huh7 cells by mAb315 were demonstrated by immunostaining. Inhibitions of viral entry by three different concentrations of mAb315 were measured by intracellular amplification of HCV RNA post infection. HCV RNA positive sera from 24 patients were used to infect Huh7 cell line in absence or presence of mouse monoclonal antibody produced in Balb/c mice or culture supernatant of mouse hybrid cells. Monoclonal Ab mAb315 could detect synthetic peptide p315 adsorbed on peripheral human lymphocytes by flow cytometry and showed high immuno reactivity to E1 viral antigen in infected Huh7 cells by immunostaining. Antibody-mediated neutralization assays demonstrated the ability of mAb315 to block HCV binding/entry to target cells at 0.73 mg/mL ascitic fluid or 250 µg/mL culture supernatant of mouse hybrid cells. Sixteen of 24 infected sera could infect Huh7 cells (67%). Binding/entry of HCV was completely blocked by mAb315 in 11/16 cases (69%). These findings suggest that mAb315 can induce HCV neutralization in vitro, which makes it a candidate for developing HCV therapeutic antibodies.

In vitro neutralization of HCV by goat antibodies against peptides encompassing regions downstream of HVR-1 of E2 glycoprotein.

This article aims at testing several in vitro systems with various viral sources and cell lines for propagation of HCV to evaluate goat antibodies raised against three E2 epitopes in viral neutralization experiments. Four human cell lines (Huh-7, Huh-7.5, HepG2, and CaCo2) were tested using two different HCV viral sources; Genotype 4 infected sera and J6/JFH HCV cc particles. Neutralization capacity of goat Abs against conserved E2 epitopes; p412 (a.a 412-419), p517 (a.a 517-531), and p430 (a.a 430-447) were examined in the above mentioned in vitro systems. Although infection with patients' sera seems to mimic the in vitro situation, it has limited replication rates as compared with HCV cc particularly in Huh7.5 cells. Non-HCV adapted Huh-7 cells were also found susceptible for transfection with J6/JFH virus but at much slower kinetics. The results of the neutralization assay showed that anti p412 and anti p517 were highly neutralizing to HCVcc. Our data demonstrate that antibodies directed against the viral surface glycoprotein E2 reduced the infectivity of the J6/JFH virus and are promising agents for immunotherapy and HCV vaccine development.

HCV treatment outcome depends on SNPs of IFNL3-Gene polymorphisms (rs12979860) and cirrhotic changes in liver parenchyma.

The allelic discrimination of IFNL3-(rs12979860 C > T) polymorphism reveals ambiguous associations with the effectiveness of oral HCV treatment. Solitary intra peripheral-blood-mononuclear-cells (PBMCs) HCV-RNA antisense-strands are independently detected in naïve and experienced cases regardless of viremia or hepatic-parenchymal alterations. We examined the frequencies of IFNL3-genetic variants with chronic-HCV-induced liver changes during the sustained virologic response (SVR) by evaluating the PBMCs- HCV-PCR after oral antiviral therapy. Methods: Twelve weeks after finishing oral antiviral therapy, the effects of IFNL3-genetic variants were evaluated in three groups of patients: Group-I (n = 25) showed HCV-RNA negativity in both serum and PBMCs-, group II (n = 52) showed positivity of HCV-RNA in PBMCs, and group-III (n = 25) had positive HCV-RNA in serum. The genetic variants of the IFNL3-gene were estimated for all the enrolled cases and correlated with their hepatic image changes. Results: IFNL3-(rs12979860) genotyping in post-direct acting antivirals (DAAs) SVR and HCV-relapse revealed: a) high frequency of CC-genotype and C-allele in group I compared to group II (P < 0.005) and group III(P ≤ 0.05) when hepatic-parenchyma looks normal by ultrasound b) frequent CT-genotype and T-allele in group II compared with I(P < 0.01) and III(P < 0.05) when liver tissues are bright (early cirrhotic-changes) c) frequent TT-genotype and T-allele in group III relative to I (P < 0.05) and II (P ≤ 0.08) when liver-tissues appear coarse by ultrasound. Conclusion: Outcomes of HCV treatment depend on host IFNL3-gene polymorphism and hepatic-parenchymal changes. A high frequency of wild-CC-genotype and C-allele is observed in patients with normal hepatic parenchyma and that achieved SVR. Solitary relapse in PBMCs occurs on increasing CT-genotype frequency when liver tissues are bright. Serologic relapse is detected when TT-genotype and T-allele are dominant in association with the cirrhotic liver. Therefore, IFNL3-gene-SNP analysis as a genetic predictor in relation to ultra-sonographic hepatic-parenchymal changes could be valuable for selecting the patients with the highest priority for treatment.

The Impact of Direct-Acting Antiviral Agents on Cytomegalovirus Reactivation in Chronic Hepatitis C Infection.

OBJECTIVE: The co-infection of HCV/CMV may accelerate the progression of liver diseases and worsen responsiveness to IFN treatment. The Direct-acting antiviral agents (DAAs), currently approved therapy for HCV, may cause a transient change in immune status, favoring the reactivation of other viruses. The current study aims to evaluate the impact of DAAs treatment on the reactivation of latent CMV in HCV patients. METHODS: The serological IgG, IgM Abs against CMV were detected by ELISA on192 HCV patients. The seronegative CMV IgM patients received (sofosbuvir/daclatasvir) regimen, then the CMV reactivation was examined by measuring the CMV IgM by ELISA and CMV DNA by real-time PCR. RESULTS: The serological data revealed that all patients were positive for CMV IgG (100%) while (64%) patients were positive for CMV IgM. The seronegative CMV IgM (36%) received the DAAs protocol. The sustained virological response was monitored by measuring the HCV RNA viremia in the patient sera. The serological data revealed that 28.6% of patients had a reactivation of CMV, while 18.5% of patients had detectable CMV DNA viremia. Moreover, there was a significant improvement in liver function as well as a decrease in FIB-4 and APRI scores at EOT. SVR was reached 97.4% among the total studied patients (N= 192). CONCLUSION: CMV co-infection has no impact on the response rate to DAAs. However, the CMV reactivation might have occurred after the complete eradication of HCV by DAAs.

Highly Sensitive Serum miRNA Panel for the Diagnosis of Hepatocellular Carcinoma in Egyptian Patients with HCV-Related HCC.

OBJECTIVE: This study aimed at exploring the potential role of a panel of serum micro-RNA (miRNA) markers in liver fibrosis and hepatocellular carcinoma (HCC) diagnosis in patients with chronic hepatitis C virus (HCV) infection. METHODS: The study included 157 chronic HCV patients and 62 HCC patients who presented to the Cairo University Center for Hepatic Fibrosis, Endemic Medicine Department, from 2015 to 2017. Relevant clinical and laboratory data were collected and sera were subjected to miRNA expression profiling. Eleven miRNA markers were studied and receiver operating characteristic curves were constructed to investigate the best cutoff values of the miRNAs that showed altered expression in HCC compared to HCV-associated advanced fibrosis. RESULTS: miRNA expression profiling revealed 5 miRNAs (miR-124, miR-141, miR-205, miR-208a, miR-499a) were significantly upregulated and 2 miRNAs were significantly downregulated (miR-103a, miR-15a) in HCC compared to advanced fibrosis patients. No significant difference was observed in miRNA expression between advanced fibrosis and early hepatic fibrosis apart from a significant downregulation of miR-155-5p in advanced fibrosis. CONCLUSION: Serum miRNAs could serve as potential diagnostic tools for the diagnosis of HCC.

Assessment of human cytomegalovirus co-infection in Egyptian chronic HCV patients.

Human cytomegalovirus (HCMV) is the most common cause of severe morbidity and mortality in immune- compromised individuals. This study was conducted to determine the incidence of HCMV infection in HCV patients who either spontaneously cleared the virus or progressed to chronic HCV infection. The study included a total of eighty four cases (48 females and 36 males) that were referred to blood banks for blood donation with an age range of 18-64 years (mean age 37.62 ± 10.03 years). Hepatitis C virus RNA and HCMV DNA were detected in sera by RT-nested PCR and nested PCR respectively in all subjects. Immunoglobulin G levels for HCV and HCMV were determined. Besides, IgM antibodies for HCMV infection were also determined in subjects' sera. Fifty three out of 84 cases (63%) were positive for HCV-RNA while 31 (37%) cases had negative HCV RNA. Forty six (87%) and 13 (25%) cases out of 53 HCV RNA positive patients were positive for HCMV IgG and IgM antibodies respectively. While 20 of 53 cases (38%) had detectable HCMV DNA. To examine the role of HCMV infection in HCV spontaneous resolution, two groups of HCV patients, group 1) chronic HCV infection (positive HCV RNA and positive IgG antibodies) vs group 2) spontaneous resolution (negative HCV RNA and positive IgG antibodies) were compared. The percentages of positive CMV IgG and IgM results is higher in chronic HCV patient than those in spontaneously cleared HCV patients and the difference is highly statistically significant (P value < 0.001). Also, there is a general trend towards elevated levels of CMV IgG antibodies in HCV chronic patients than those in spontaneously cleared HCV patients (P value < 0.02). HCMV DNA detection in group 1 was more than twice the value observed in group 2 (38% vs 14.3%, P value < 0.001). Moreover, levels of liver enzymes were significantly higher in HCV RNA positive cases co-infected with HCMV DNA than HCMV negative cases (P value < 0.001). The results indicate the role of HCMV in the liver pathogenesis. We conclude that chronic HCV patients co-infected with HCMV infection can be regarded as high risk groups for liver disease progression where they should be monitored for the long term outcome of the disease.

Neutralizing activities of caprine antibodies towards conserved regions of the HCV envelope glycoprotein E2.

Anti HCV vaccine is not currently available and the present antiviral therapies fail to cure approximately half of the treated HCV patients. This study was designed to assess the immunogenic properties of genetically conserved peptides derived from the C-terminal region of HVR-1 and test their neutralizing activities in a step towards developing therapeutic and/or prophylactic immunogens against HCV infection. Antibodies were generated by vaccination of goats with synthetic peptides derived from HCV E2. Viral neutralizing capacity of the generated anti E2 antibodies was tested using in vitro assays. Goats immunized with E2 synthetic peptides termed p412 [a.a 412-419], p430 [a.a 430-447] and p517 [a.a 517-531] generated high titers of antibody responses 2 to 4.5 fold higher than comparable titers of antibodies to the same epitopes in chronic HCV patients. In post infection experiments of native HCV into cultured Huh7.5 cells anti p412 and anti p 517 were proven to be neutralizing to HCV genotype 4a from patients' sera (87.5% and 75% respectively). On the contrary anti p430 exhibited weak viral neutralization capacity on the same samples (31.25%). Furthermore Ab mixes containing anti p430 exhibited reduced viral neutralization properties. From these experiments one could predict that neutralization by Abs towards different E2-epitopes varies considerably and success in the enrichment of neutralization epitope-specific antibodies may be accompanied by favorable results in combating HCV infection. Also, E2 conserved peptides p517 and p412 represent potential components of a candidate peptide vaccine against HCV infection.

Human cytomegalovirus infection inhibits response of chronic hepatitis-C-virus-infected patients to interferon-based therapy.

BACKGROUND AND AIM: Cytomegalovirus (CMV) is a ubiquitous pathogen that infects the majority of humans. Co-infection of CMV and hepatitis C virus (HCV) may deteriorate the prognosis of HCV-infected patients. This study was conducted to examine the role of CMV reactivation in determining the response rate to treatment with interferon and ribavirin therapy in chronic HCV patients. METHODS: Viral loads and genotyping were assessed using reverse transcription polymerase chain reaction and Innolipa systems, respectively. Reactivation of CMV in HCV patients who were all positive for CMV immunoglobulin G antibodies was tested by amplification of the gB1 gene using the end-point dilution quantitative-nested polymerase chain reaction method. RESULTS: CMV DNA was detected in 89.7% of non-responders and in 34.6% of sustained virological responders. Patients with reactivated CMV had significantly higher fibrosis scores (72.7%) than those with undetectable CMV DNA (23.8%, P=0.002). Patients with positive CMV had higher rates of non-response and relapse (79.5%) than those with negative CMV DNA (19%). Chronic HCV patients with latent CMV had higher rates of response (81%) to treatment than those with reactivated CMV (20.5%, P<0.001). Therefore, HCV patients with reactivated CMV and advanced fibrosis were least likely to achieve a sustained virological response following interferon therapy. This possibility is reduced to 50% of its original value in patients with reactivated CMV without fibrosis. CONCLUSIONS: Besides the staging of liver fibrosis, CMV co-infection should be considered as an extremely important factor when designing predictive models for HCV response to interferon treatment.

Single nucleotide polymorphism at exon 7 splice acceptor site of OAS1 gene determines response of hepatitis C virus patients to interferon therapy.

BACKGROUND AND AIM: Response to interferon therapy and disease progression in hepatitis C virus (HCV) infected patients differs among individuals, suggesting a possibility of a contribution of host genetic factors. 2'-5'-oligoadenylate synthetase 1 (OAS1), an important component of the innate immune system with a proven antiviral function, may therefore have a relationship with the response to interferon therapy and clinical course of HCV disease. Our aim was to determine the frequency of single nucleotide polymorphism (SNP) at exon 7 splice acceptor site (SAS) of the OAS1 gene in relation to the interferon response and status of HCV infection. METHODS: A 203 bp fragment containing exon 7 SAS was amplified in 70 HCV chronic patients and 50 healthy controls. SNP was examined using restriction fragment length polymorphism (RFLP) genotyping method. Correlations of SNP genotypes with response to interferon and clinical status of patients were statistically analyzed. RESULTS: There was an increasing trend of response from AA to AG to GG genotypes (P = 0.007). Genotype AA was associated with non-response to interferon and higher degree of liver fibrosis (P = 0.05). Multivariate analysis showed this SNP as independent and a significant determinant of the outcome of interferon therapy (odds ratio 4.913 [95% confidence interval 1.365-8.2], P = 0.006). CONCLUSIONS: This is the first study to show a significant association between the functional SNP at exon 7 SAS of OAS1 gene and the viral response to interferon in chronic HCV patients. Patients with AA genotype were associated with progressive HCV disease and viral resistance to interferon therapy. This OAS SNP is a potential bio-marker to predict IFN response in chronic hepatitis C patients.

Bioinformatics prediction of B and T cell epitopes within the spike and nucleocapsid proteins of SARS-CoV2.

BACKGROUND: The striking difference in severity of SARS CoV2 infection among global population is partly attributed to viral factors. With the spike (S) and nucleocapsid (N) are the most immunogenic subunits, genetic diversity and antigenicity of S and N are key players in virulence and in vaccine development. AIM: This paper aims at identifying immunogenic targets for better vaccine development and/or immunotherapy of COVID 19 pandemic. METHODS: 18 complete genomes of SARS CoV2 (n=14), SARS CoV (n=2) and MERS CoV (n=2) were examined. Bioinformatics of viral genetics and protein folding allowed functional tuning of NH2 Terminal Domain (NTD) of S protein and development of epitope maps for B and T cell responses. CONCLUSION: A deletion of amino acid residues Y144 and G107 were discovered in NTD of S protein derived from Indian and French isolates resulting in altered pocket structure exclusively located in NTD and reduced affinity of NTD binding to endogenous nAbs and disrupted NTD mediated cell entry. We therefore, proposed a set of B and T cell epitopes based on Immune Epitope Database, homologous epitopes for nAbs in convalescent plasma post SARS CoV infection and functional domains of S (NTD, Receptor Binding domain and the unique polybasic Furin cleavage site at S1/S2 junction). Nevertheless, laboratory data are required to develop vaccine and immunotherapeutics.

Key Players of Hepatic Fibrosis.

Hepatic fibrosis is a complex mechanism defined by the net deposition of the extracellular matrix (ECM) owing to liver injury caused by multiple etiologies such as viral hepatitis and nonalcoholic fatty liver disease. Many cell types are implicated in liver fibrosis development and progression. In general, liver fibrosis starts with the recruitment of inflammatory immune cells to generate cytokines, growth factors, and other activator molecules. Such chemical mediators drive the hepatic stellate cells (HSCs) to activate the production of the ECM component. The activation of HSC is thus a crucial event in the fibrosis initiation, and a significant contributor to collagen deposition (specifically type I). This review explores the causes and mechanisms of hepatic fibrosis and focuses on the roles of key molecules involved in liver fibro genesis, some of which are potential targets for therapeutics to hamper liver fibro genesis.

Correlation between IL28B/TLR4 genetic variants and HCC development with/without DAAs treatment in chronic HCV patients.

In Egypt, Sofosbuvir (SOF) in combination with Dataclasvir (DCV) is the broadly used DAAs with excellent therapeutic profile. This study is designed to explore the relation between IL28B/TLR4 genetic variants and each of the followings; HCC development post SOF/DCV treatment, progression to HCC in naïve patients and SOF/DCV therapy outcome. A total of 493 blood samples were collected (controls (n = 70); HCV patients treated with SOF/DCV (n = 252) of whom 65 patients developed HCC, 187 patients didn't develop HCC (125 responders, 62 relapsers); naïve HCV patients (n = 171) had early (n = 48), late liver fibrosis (n = 21) and HCC (n = 102)). Both SNPs were genotyped using a TaqMan 5' allelic discrimination assay. At IL28B rs12979860 SNP, the C allele was significantly correlating with the response rate more than T allele (OR 1.9, 95% CI 1.29-2.9, p = 0.004), while at TLR4 rs4986791 SNP, no association was found (OR 6.5, 95% 0.57-75.28, p = 0.09). Both SNPs couldn't detect the probability for HCC emergence after treatment. In naïve patients, the protective alleles were detected in their lowest frequency in HCC patients (p = 0.1, for rs12979860 and, p = 0.001 for rs4986791). SOF/DCV combination improved SVR rates in HCV genotype 4a infected patients regardless of IL28B genotype, with the best rates in those lacking the T allele.

Recipient interleukin 6 gene polymorphism and expression predict HCV recurrence post liver transplantation.

BACKGROUND: Liver transplantation (LTX)is a lifesaving- effective protocol for patients suffering end stage liver disease (ESLD) and its complications post HCV infection. Recurrence of disease is a frequent clinical complication that is observed in patients undergoing LTX. Cytokines play a central role in the immunological events occurring after the surgery. METHODS: Using Allelic Discrimination PCR, the allelic variation G174C of IL-6 gene was investigated. The abundance of IL6- mRNA and plasma IL6 cytokine levels were evaluated by using qRT-PCR in peripheral blood mononuclear cells (PBMCs) and enzyme-linked immunosorbent assay (ELISA) respectively in 76 liver transplant recipients recruited from Al Sahel teaching hospital, Ministry of Health and Population Cairo Egypt within the period between June 2015 and October 2017. RESULTS: The frequencies of IL-6 GG genotype and the G allele were significantly detected more in LTX recipients who experienced HCV recurrence versus those who did not suffer recurrence when compared to healthy controls (P = 0.001) and (P = 0.006), respectively. On the contrary, levels of IL-6 related transcripts in PBMC's of recurrent patients were indifferent from non-recurrent patients and healthy controls (P ≥ 0.124). Interestingly, the circulating IL-6 protein in plasma was significantly elevated in recurrent as compared to the non-recurrent recipients (P = 0.002). CONCLUSION: HCV recurrence post liver transplantation occur more frequently in patients with -174 G/G IL-6 genotype and elevated plasma IL-6 levels.