Human Monoclonal Antibody HCV1 Effectively Prevents and Treats HCV Infection in Chimpanzees

Hepatitis C virus (HCV) infection is a leading cause of liver transplantation and there is an urgent need to develop therapies to reduce rates of HCV infection of transplanted livers. Approved therapeutics for HCV are poorly tolerated and are of limited efficacy in this patient population. Human monoclonal antibody HCV1 recognizes a highly-conserved linear epitope of the HCV E2 envelope glycoprotein (amino acids 412–423) and neutralizes a broad range of HCV genotypes. In a chimpanzee model, a single dose of 250 mg/kg HCV1 delivered 30 minutes prior to infusion with genotype 1a H77 HCV provided complete protection from HCV infection, whereas a dose of 50 mg/kg HCV1 did not protect. In addition, an acutely-infected chimpanzee given 250 mg/kg HCV1 42 days following exposure to virus had a rapid reduction in viral load to below the limit of detection before rebounding 14 days later. The emergent virus displayed an E2 mutation (N415K/D) conferring resistance to HCV1 neutralization. Finally, three chronically HCV-infected chimpanzees were treated with a single dose of 40 mg/kg HCV1 and viral load was reduced to below the limit of detection for 21 days in one chimpanzee with rebounding virus displaying a resistance mutation (N417S). The other two chimpanzees had 0.5–1.0 log₁₀ reductions in viral load without evidence of viral resistance to HCV1. In vitro testing using HCV pseudovirus (HCVpp) demonstrated that the sera from the poorly-responding chimpanzees inhibited the ability of HCV1 to neutralize HCVpp. Measurement of antibody responses in the chronically-infected chimpanzees implicated endogenous antibody to E2 and interference with HCV1 neutralization although other factors may also be responsible. These data suggest that human monoclonal antibody HCV1 may be an effective therapeutic for the prevention of graft infection in HCV-infected patients undergoing liver transplantation.     

Impact of IL28B-related single nucleotide polymorphisms on liver histopathology in chronic hepatitis C genotype 2 and 3

Background and Aims

Recently, several genome-wide association studies have revealed that single nucleotide polymorphisms (SNPs) in proximity to IL28B predict spontaneous clearance of HCV infection as well as outcome following peginterferon and ribavirin therapy among HCV genotype 1 infected patients. The present study aimed to evaluate the impact of IL28B SNP variability on liver histology in the context of a phase III treatment trial (NORDynamIC) for treatment-naïve patients with chronic HCV genotype 2 or 3 infection, where pretreatment liver biopsies were mandatory.

Methods

Three hundred and thirty-nine Caucasian patients had samples available for IL28B genotyping (rs12979860) of whom 314 had pretreatment liver biopsies that were evaluated using the Ishak protocol, allowing for detailed grading and staging of liver histopathology.

Results

IL28B CC_rs12979860 genotype in HCV genotype 3 infected patients was associated with higher ALT levels (p<0.0001), higher AST to platelet ratio index (APRI; p = 0.001), and higher baseline viral load (p<0.0001) as compared to patients with the CT or TT genotypes. Additionally the CC_rs12979860 genotype entailed more pronounced portal inflammation (p = 0.02) and steatosis (p = 0.03). None of these associations were noted among HCV genotype 2 infected patients.

Conclusion

This study shows that the CC_rs12979860 SNP is associated with more pronounced liver histopathology in patients chronically infected with HCV genotype 3, which may be secondary to higher viral load. The finding that IL28B variability did not impact on liver pathology or viral load among genotype 2 infected patients implies that IL28B may differentially regulate the course of genotype 2 and 3 infection.     

Protection from the 2009 H1N1 Pandemic Influenza by an Antibody from Combinatorial Survivor-Based Libraries

Influenza viruses elude immune responses and antiviral chemotherapeutics through genetic drift and reassortment. As a result, the development of new strategies that attack a highly conserved viral function to prevent and/or treat influenza infection is being pursued. Such novel broadly acting antiviral therapies would be less susceptible to virus escape and provide a long lasting solution to the evolving virus challenge. Here we report the in vitro and in vivo activity of a human monoclonal antibody (A06) against two isolates of the 2009 H1N1 pandemic influenza virus. This antibody, which was obtained from a combinatorial library derived from a survivor of highly pathogenic H5N1 infection, neutralizes H5N1, seasonal H1N1 and 2009 “Swine” H1N1 pandemic influenza in vitro with similar potency and is capable of preventing and treating 2009 H1N1 influenza infection in murine models of disease. These results demonstrate broad activity of the A06 antibody and its utility as an anti-influenza treatment option, even against newly evolved influenza strains to which there is limited immunity in the general population.     

A Gaussia Luciferase Cell-Based System to Assess the Infection of Cell Culture- and Serum-Derived Hepatitis C Virus

Robust replication of hepatitis C virus (HCV) in cell culture occurs only with the JFH-1 (genotype 2a) recombinant genome. The aim of this study was to develop a system for HCV infection quantification analysis and apply it for the selection of patient sera that may contain cell culture infectious viruses, particularly of the most clinically important genotype 1. Initially, a hepatoma cell line (designated Huh-7.5/EG(4A/4B)GLuc) was generated that stably expressed the enhanced green fluorescent protein (EGFP) fused in-frame to the secreted Gaussia luciferase via a recognition sequence of the viral NS3/4A protease. Upon HCV infection, NS3/4A cleaved at its signal and the Gaussia was secreted to the culture medium, thus facilitating the infection quantification. The Huh-7.5/EG(4A/4B)GLuc cell line provided a rapid and highly sensitive quantification of HCV infection in cell culture using JFH-1-derived viruses. Furthermore, the Huh-7.5/EG(4A/4B)GLuc cells were also shown to be a suitable host for the discovery of anti-HCV inhibitors by using known compounds that target distinct stages of the HCV life cycle; the Ź-factor of this assay ranged from 0.72 to 0.75. Additionally, eighty-six sera derived from HCV genotype 1b infected liver transplant recipients were screened for their in vitro infection and replication potential. Approximately 12% of the sera contained in vitro replication-competent viruses, as deduced by the Gaussia signal, real time quantitative PCR, immunofluorescence and capsid protein secretion. We conclude that the Huh-7.5/EG(4A/4B)GLuc cell line is an excellent system not only for the screening of in vitro replication-competent serum-derived viruses, but also for the subsequent cloning of recombinant isolates. Additionally, it can be utilized for high-throughput screening of antiviral compounds.     

Hepatitis C Virus Driven AXL Expression Suppresses the Hepatic Type I Interferon Response

Treatment of chronic hepatitis C virus (HCV) infection is evolving rapidly with the development of novel direct acting antivirals (DAAs), however viral clearance remains intimately linked to the hepatic innate immune system. Patients demonstrating a high baseline activation of interferon stimulated genes (ISGs), termed interferon refractoriness, are less likely to mount a strong antiviral response and achieve viral clearance when placed on treatment. As a result, suppressor of cytokine signalling (SOCS) 3 and other regulators of the IFN response have been identified as key candidates for the IFN refractory phenotype due to their regulatory role on the IFN response. AXL is a receptor tyrosine kinase that has been identified as a key regulator of interferon (IFN) signalling in myeloid cells of the immune system, but has not been examined in the context of chronic HCV infection. Here, we show that AXL is up-regulated following HCV infection, both in vitro and in vivo and is likely induced by type I/III IFNs and inflammatory signalling pathways. AXL inhibited type IFNα mediated ISG expression resulting in a decrease in its antiviral efficacy against HCV in vitro. Furthermore, patients possessing the favourable IFNL3 rs12979860 genotype associated with treatment response, showed lower AXL expression in the liver and a stronger induction of AXL in the blood, following their first dose of IFN. Together, these data suggest that elevated AXL expression in the liver may mediate an IFN-refractory phenotype characteristic of patients possessing the unfavourable rs12979860 genotype, which is associated with lower rates of viral clearance.     

HLA-DQB1*03 Confers Susceptibility to Chronic Hepatitis C in Japanese: A Genome-Wide Association Study

Hepatitis C virus (HCV) establishes a chronic infection in 70-80% of infected individuals. Many researchers have examined the effect of human leukocyte antigen (HLA) on viral persistence because of its critical role in the immune response against exposure to HCV, but almost all studies have proven to be inconclusive. To identify genetic risk factors for chronic HCV infection, we analyzed 458,207 single nucleotide polymorphisms (SNPs) in 481 chronic HCV patients and 2,963 controls in a Japanese cohort. Next, we performed a replication study with an independent panel of 4,358 cases and 1,114 controls. We further confirmed the association in 1,379 cases and 25,817 controls. In the GWAS phase, we found 17 SNPs that showed suggestive association (P < 1 × 10^-5). After the first replication study, we found one intronic SNP in the HLA-DQ locus associated with chronic HCV infection, and when we combined the two studies, the association reached the level of genome-wide significance. In the second replication study, we again confirmed the association (P _combined = 3.59 × 10⁻¹⁶, odds ratio [OR] = 0.79). Subsequent analysis revealed another SNP, rs1130380, with a stronger association (OR=0.72). This nucleotide substitution causes an amino acid substitution (R55P) in the HLA-DQB1 protein specific to the DQB1*03 allele, which is common worldwide. In addition, we confirmed an association with the previously reported IFNL3-IFNL4 locus and propose that the effect of DQB1*03 on HCV persistence might be affected by the IFNL4 polymorphism. Our findings suggest that a common amino acid substitution in HLA-DQB1 affects susceptibility to chronic infection with HCV in the Japanese population and may not be independent of the IFNL4 genotype.     

Rapid,Sensitive, and Accurate Evaluation of Drug Resistant Mutant (NS5A-Y93H) Strain Frequency in Genotype 1b HCV by Invader Assay

Daclatasvir and asunaprevir dual oral therapy is expected to achieve high sustained virological response (SVR) rates in patients with HCV genotype 1b infection. However, presence of the NS5A-Y93H substitution at baseline has been shown to be an independent predictor of treatment failure for this regimen. By using the Invader assay, we developed a system to rapidly and accurately detect the presence of mutant strains and evaluate the proportion of patients harboring a pre-treatment Y93H mutation. This assay system, consisting of nested PCR followed by Invader reaction with well-designed primers and probes, attained a high overall assay success rate of 98.9% among a total of 702 Japanese HCV genotype 1b patients. Even in serum samples with low HCV titers, more than half of the samples could be successfully assayed. Our assay system showed a better lower detection limit of Y93H proportion than using direct sequencing, and Y93H frequencies obtained by this method correlated well with those of deep-sequencing analysis (r = 0.85, P <0.001). The proportion of the patients with the mutant strain estimated by this assay was 23.6% (164/694). Interestingly, patients with the Y93H mutant strain showed significantly lower ALT levels (p=8.8 x 10^-4), higher serum HCV RNA levels (p=4.3 x 10^-7), and lower HCC risk (p=6.9 x 10^-3) than those with the wild type strain. Because the method is both sensitive and rapid, the NS5A-Y93H mutant strain detection system established in this study may provide important pre-treatment information valuable not only for treatment decisions but also for prediction of disease progression in HCV genotype 1b patients.     

Sex-specific association between X-linked Toll-like receptor 7 with the outcomes of hepatitis C virus infection

Toll-like receptor 7 (TLR7) senses hepatitis C virus (HCV) infection and drives the host specific innate and adaptive immune response. The aim of this study was to estimate the distributions of TLR7 single nucleotide polymorphisms (SNPs), including rs179019 and rs3853839, as well as the effect of TLR7 gene variants on TLR7 mRNA expression and cytokine production in response to TLR7 agonist in vitro. TLR7 SNP genotyping was performed among a Chinese sample population of 418 patients with persistent HCV infection, 317 patients with HCV spontaneous clearance, and 989 healthy controls. TLR7 mRNA expression and TLR7-specific IFN-α and IL-6 secretion in peripheral blood mononuclear cells, derived from 60 healthy individuals in vitro, were then quantified. We identified the association of TLR7 rs3853839C allele, haplotype CC and haplotype AC (rs179019/rs3853839) with protection against HCV persistence in Chinese females (OR = 0.49, 95% CI = 0.29–0.81, P = 0.01 for rs3853839 GC; OR = 0.29, 95% CI = 0.11–0.75, P = 0.01 for rs3853839 CC; OR = 0.51, 95% CI = 0.38–0.77, P < 0.01 for haplotype CC; OR = 0.29, 95% CI = 0.10–0.88, P = 0.03 for haplotype AC). In addition, the rs3853839 CC genotype among female carriers had significantly low TLR7 mRNA expression (P = 0.006 for GG vs. CC, P = 0.021 for GC vs. CC), along with decreased IFN-α (P = 0.002 for GG vs. CC, P = 0.021 for GC vs. CC) and increased antiviral IL-6 production (P = 0.002 for GG vs. CC, P = 0.030 for GC vs. CC), after treatment with Imiquimod in vitro. The cytokine profile among rs3853839 CC genotype female carriers may indicate a pronounced protective effect against persistent HCV infection. The functional polymorphism of TLR7 rs3853839C allele was found to be sex-specific and associated with protection against HCV persistence among Chinese females, which may be due to specific IFN-α and IL-6 secretion profiles.     

Previously infected chimpanzees are not consistently protected against reinfection or persistent infection after reexposure to the identical hepatitis C virus strain

Protective immunity after resolved hepatitis C virus (HCV) infection has been reported. However, the breadth of this immunity has remained controversial, and the role of neutralizing antibodies has not been well-defined. In the present study, two chimpanzees (CH96A008 and CH1494) with resolved monoclonal H77C (genotype 1a) infection were rechallenged with low-dose homologous H77C virus about 12 months after viral clearance; CH96A008 became persistently infected, and CH1494 had transient viremia lasting 2 weeks. CH1494 was subsequently either partially or completely protected following five homologous rechallenges with monoclonal H77C or polyclonal H77 and after six heterologous rechallenges with HC-J4 (genotype 1b) or HC-J6 (genotype 2a) viruses. Subsequently, a final challenge with H77C resulted in persistent HCV infection. In both chimpanzees, serum neutralizing antibodies against retroviral pseudoparticles bearing the H77C envelope proteins were not detected during the initial infection or during rechallenge. However, anamnestic cellular immune responses developed during the initial homologous rechallenge, in particular in CH96A008, which developed a persistent infection. Polyprotein sequences of viruses recovered from CH1494 after the two homologous rechallenges that resulted in transient viremia were identical with the H77C virus. In contrast, the polyprotein sequences of viruses recovered from both chimpanzees after homologous rechallenge resulting in persistent infection had numerous changes. These findings have important implications for our understanding of immunity against HCV; even in the best-case scenario with autologous rechallenge, low-level viral persistence was seen in the presence of primed T-cell responses.     

Antibody responses induced by SHIV infection are more focused than those induced by soluble native HIV-1 envelope trimers in non-human primates

The development of an effective human immunodeficiency virus (HIV-1) vaccine is a high global health priority. Soluble native-like HIV-1 envelope glycoprotein trimers (Env), including those based on the SOSIP design, have shown promise as vaccine candidates by inducing neutralizing antibody responses against the autologous virus in animal models. However, to overcome HIV-1’s extreme diversity a vaccine needs to induce broadly neutralizing antibodies (bNAbs). Such bNAbs can protect non-human primates (NHPs) and humans from infection. The prototypic BG505 SOSIP.664 immunogen is based on the BG505 env sequence isolated from an HIV-1-infected infant from Kenya who developed a bNAb response. Studying bNAb development during natural HIV-1 infection can inform vaccine design, however, it is unclear to what extent vaccine-induced antibody responses to Env are comparable to those induced by natural infection. Here, we compared Env antibody responses in BG505 SOSIP-immunized NHPs with those in BG505 SHIV-infected NHPs, by analyzing monoclonal antibodies (mAbs). We observed three major differences between BG505 SOSIP immunization and BG505 SHIV infection. First, SHIV infection resulted in more clonal expansion and less antibody diversity compared to SOSIP immunization, likely because of higher and/or prolonged antigenic stimulation and increased antigen diversity during infection. Second, while we retrieved comparatively fewer neutralizing mAbs (NAbs) from SOSIP-immunized animals, these NAbs targeted more diverse epitopes compared to NAbs from SHIV-infected animals. However, none of the NAbs, either elicited by vaccination or infection, showed any breadth. Finally, SOSIP immunization elicited antibodies against the base of the trimer, while infection did not, consistent with the base being placed onto the virus membrane in the latter setting. Together these data provide new insights into the antibody response against BG505 Env during infection and immunization and limitations that need to be overcome to induce better responses after vaccination.     

Impact of prior Dengue immunity on Zika vaccine protection in rhesus macaques and mice

Pre-existing immunity to flaviviruses can influence the outcome of subsequent flavivirus infections. Therefore, it is critical to determine whether baseline DENV immunity may influence subsequent ZIKV infection and the protective efficacy of ZIKV vaccines. In this study, we investigated the impact of pre-existing DENV immunity induced by vaccination on ZIKV infection and the protective efficacy of an inactivated ZIKV vaccine. Rhesus macaques and mice inoculated with a live attenuated DENV vaccine developed neutralizing antibodies (NAbs) to multiple DENV serotypes but no cross-reactive NAbs responses to ZIKV. Animals with baseline DENV NAbs did not exhibit enhanced ZIKV infection and showed no overall reduction in ZIKV vaccine protection. Moreover, passive transfer of purified DENV-specific IgG from convalescent human donors did not augment ZIKV infection in STAT2 ^-/- and BALB/c mice. In summary, these results suggest that baseline DENV immunity induced by vaccination does not significantly enhance ZIKV infection or impair the protective efficacy of candidate ZIKV vaccines in these models. These data can help inform immunization strategies in regions of the world with multiple circulating pathogenic flaviviruses.     

Patterns of Hepatitis C Virus RNA Levels during Acute Infection: The InC3 Study

Background

Understanding the patterns of HCV RNA levels during acute hepatitis C virus (HCV) infection provides insights into immunopathogenesis and is important for vaccine design. This study evaluated patterns of HCV RNA levels and associated factors among individuals with acute infection.

Methods

Data were from an international collaboration of nine prospective cohorts of acute HCV (InC³ Study). Participants with well-characterized acute HCV infection (detected within three months post-infection and interval between the peak and subsequent HCV RNA levels≤120 days) were categorised by a priori-defined patterns of HCV RNA levels: i) spontaneous clearance, ii) partial viral control with persistence (≥1 log IU/mL decline in HCV RNA levels following peak) and iii) viral plateau with persistence (increase or <1 log IU/mL decline in HCV RNA levels following peak). Factors associated with HCV RNA patterns were assessed using multinomial logistic regression.

Results

Among 643 individuals with acute HCV, 162 with well-characterized acute HCV were identified: spontaneous clearance (32%), partial viral control with persistence (27%), and viral plateau with persistence (41%). HCV RNA levels reached a high viraemic phase within two months following infection, with higher levels in the spontaneous clearance and partial viral control groups, compared to the viral plateau group (median: 6.0, 6.2, 5.3 log IU/mL, respectively; P=0.018). In the two groups with persistence, Interferon lambda 3 (IFNL3) CC genotype was independently associated with partial viral control compared to viral plateau (adjusted odds ratio [AOR]: 2.75; 95%CI: 1.08, 7.02). In the two groups with viral control, female sex was independently associated with spontaneous clearance compared to partial viral control (AOR: 2.86; 95%CI: 1.04, 7.83).

Conclusions

Among individuals with acute HCV, a spectrum of HCV RNA patterns is evident. IFNL3 CC genotype is associated with initial viral control, while female sex is associated with ultimate spontaneous clearance.     

Hepatitis C Virus Core-Derived Peptides Inhibit Genotype 1b Viral Genome Replication via Interaction with DDX3X

The protein DDX3X is a DEAD-box RNA helicase that is essential for the hepatitis C virus (HCV) life cycle. The HCV core protein has been shown to bind to DDX3X both in vitro and in vivo. However, the specific interactions between these two proteins and the functional importance of these interactions for the HCV viral life cycle remain unclear. We show that amino acids 16–36 near the N-terminus of the HCV core protein interact specifically with DDX3X both in vitro and in vivo. Replication of HCV replicon NNeo/C-5B RNA (genotype 1b) is significantly suppressed in HuH-7-derived cells expressing green fluorescent protein (GFP) fusions to HCV core protein residues 16–36, but not by GFP fusions to core protein residues 16–35 or 16–34. Notably, the inhibition of HCV replication due to expression of the GFP fusion to HCV core protein residues 16–36 can be reversed by overexpression of DDX3X. These results suggest that the protein interface on DDX3X that binds the HCV core protein is important for replicon maintenance. However, infection of HuH-7 cells by HCV viruses of genotype 2a (JFH1) was not affected by expression of the GFP fusion protein. These results suggest that the role of DDX3X in HCV infection involves aspects of the viral life cycle that vary in importance between HCV genotypes.     

TNF,IL6, and IL1B Polymorphisms Are Associated with Severe Influenza A (H1N1) Virus Infection in the Mexican Population

Background

Hypercytokinemia is the main immunopathological mechanism contributing to a more severe clinical course in influenza A (H1N1) virus infections. Most patients infected with the influenza A (H1N1) pdm09 virus had increased systemic levels of pro-inflammatory cytokines; including interleukin IL-6, IL-8, and tumor necrosis factor-alpha (TNF-α). We propose that single-nucleotide polymorphisms (SNPs) in the promoter regions of pro-inflammatory genes are associated with the severity of influenza A (H1N1) pdm09 virus infection.

Methods

145 patients with influenza A (H1N1) (pA/H1N1), 133 patients with influenza-like illness (ILI), and 360 asymptomatic healthy contacts (AHCs) were included. Eleven SNPs were genotyped in six genes (TNF, LT, IL1B, IL6, CCL1, and IL8) using real-time PCR; the ancestral genotype was used for comparison. Genotypes were correlated with 27 clinical severity variables. Ten cytokines (GM-CSF, TNF-α, IL-2, IL-1β, IL-6, IL-8, IFN-γ, IL-10, IL-5, and IL-4) were measured on a Luminex 100.

Results

The IL6 rs1818879 (GA) heterozygous genotype was associated with severe influenza A (H1N1) virus infection (odds ratio [OR] = 5.94, 95% confidence interval [CI] 3.05–11.56), and two IL1B SNPs, rs16944 AG and rs3136558 TC, were associated with a decreased risk of infection (OR = 0.52 and OR = 0.51, respectively). Genetic susceptibility was determined (pA/H1N1 vs. AHC): the LTA rs909253 TC heterozygous genotype conferred greater risk (OR = 1.9), and a similar association was observed with the IL1B rs3136558 CC genotype (OR = 1.89). Additionally, severely ill patients were compared with moderately ill patients. The TNF-238 GA genotype was associated with an increased risk of disease severity (OR = 16.06, p = 0.007). Compared with ILIs, patients with severe pA/H1N1 infections exhibited increased serum IL-5 (p <0.001) and IL-6 (p  =  0.007) levels.

Conclusions

The TNF gene was associated with disease severity, whereas IL1B and IL6 SNPs were associated with influenza A (H1N1) virus infection.     

Identification of Alpha Interferon-Induced Envelope Mutations of Hepatitis C Virus In Vitro Associated with Increased Viral Fitness and Interferon Resistance

Alpha interferon (IFN-α) is an essential component of innate antiviral immunity and of treatment regimens for chronic hepatitis C virus (HCV) infection. Resistance to IFN might be important for HCV persistence and failure of IFN-based therapies. Evidence for HCV genetic correlates of IFN resistance is limited. Experimental studies were hampered by lack of HCV culture systems. Using genotype (strain) 1a(H77) and 3a(S52) Core-NS2 JFH1-based recombinants, we aimed at identifying viral correlates of IFN-α resistance in vitro. Long-term culture with IFN-α2b in Huh7.5 cells resulted in viral spread with acquisition of putative escape mutations in HCV structural and nonstructural proteins. Reverse genetic studies showed that primarily amino acid changes I348T in 1a(H77) E1 and F345V/V414A in 3a(S52) E1/E2 increased viral fitness. Single-cycle assays revealed that I348T and F345V/V414A enhanced viral entry and release, respectively. In assays allowing viral spread, these mutations conferred a level of IFN-α resistance exceeding the observed fitness effect. The identified mutations acted in a subtype-specific manner but were not found in genotype 1a and 3a patients, who failed IFN-α therapy. Studies with HCV recombinants with different degrees of culture adaptation confirmed the correlation between viral fitness and IFN-α resistance. In conclusion, in vitro escape experiments led to identification of HCV envelope mutations resulting in increased viral fitness and conferring IFN-α resistance. While we established a close link between viral fitness and IFN-α resistance, identified mutations acted via different mechanisms and appeared to be relatively specific to the infecting virus, possibly explaining difficulties in identifying signature mutations for IFN resistance.     

Anti-HCV activity of the Chinese medicinal fungus Cordyceps militaris

Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. Although the sustained virologic response rate in the treatment of genotype 1 using new triple therapy (pegylated-interferon, ribavirin, and telaprevir/boceprevir) has been improved by more than 70%, several severe side effects such as skin rash/ageusia and advanced anemia have become a problem. Under these circumstances, a new type of anti-HCV oral drug with few side effects is needed. Our recently developed HCV drug assay systems, including the HuH-7 cell line-derived OR6 and AH1R, and the Li23 cell line-derived ORL8 and ORL11, allow genome-length HCV RNAs (several strains of genotype 1b) encoding renilla luciferase to replicate efficiently. Using these systems as anti-HCV candidates, we have identified numerous existing medicines that can be used against HCV with few side effects, such as statins and teprenon. To obtain additional anti-HCV candidates, we evaluated a number of oral health supplements, and found that the capsule but not the liquid form of Cordyceps militaris (CM) (Ascomycotinanorth, North Chinese caterpillar fungus), which is used as a Chinese herbal medicine, exhibited moderate anti-HCV activity. In combination with interferon-α or ribavirin, CM exhibited an additive inhibitory effect. Among the main components of CM, cordycepin, but not ergosterol, contributed to the anti-HCV activity of CM. In consideration of all these results, we suggest that CM would be useful as an oral anti-HCV agent in combination with interferon-α and/or ribavirin.     

Ligand design,synthesis and biological anti-HCV evaluations for genotypes 1b and 4a of certain 4-(3- & 4-[3-(3,5-dibromo-4-hydroxyphenyl)-propylamino]phenyl) butyric acids and 3-(3,5-dibromo-4-hydroxyphenyl)-propylamino-acetamidobenzoic acid esters

4-(4-[N-1-carboxy-3-(3,5-dibromo-4-hydroxyphenyl)-3-oxo-propylamino]phenyl)-4-oxo-butyric acid (V), 4-(3- & 4-[N-1-carboxy-3-(3,5-dibromo-4-hydroxyphenyl)-3-oxo-propylaminophenyl]-2-aryl-4-oxo-butyric acids (Xa–e) and 4-(2-alkyl-2-[N-3-(3,5-dibromo-4-hydroxyphenyl)-1-carboxy-3-oxo-propylamino]acetamido) benzoate esters (XVa–e) were designed, synthesized and biologically evaluated as anti-HCV for genotypes 1b and 4a. The design was based on their docking scores with HCV NS3/4A protease-binding site of the genotype 1b (1W3C), which is conserved in the genotype 4a structure. The docking scores predicted that most of these molecules have higher affinity to the HCV NS3/4A enzyme more than Indoline lead. These compounds were synthesized and evaluated for their cytopathic inhibitory activity against RAW HCV cell cultures of genotype 4a and also examined against Huh 5–2 HCV cell culture of genotype 1b, utilizing Luciferase and MTS assays. Compounds Xa and Xb have 95 and 80% of the activity of Ribavirin against genotype 4a and compounds XVa, XVb and XVd exerted high percentage inhibitory activity against genotype 1b equal 87.7, 84.3 and 82.8%, respectively, with low EC₅₀ doses.     

Response prediction in chronic hepatitis C by assessment of IP-10 and IL28B-related single nucleotide polymorphisms

Background

High baseline levels of IP-10 predict a slower first phase decline in HCV RNA and a poor outcome following interferon/ribavirin therapy in patients with chronic hepatitis C. Several recent studies report that single nucleotide polymorphisms (SNPs) adjacent to IL28B predict spontaneous resolution of HCV infection and outcome of treatment among HCV genotype 1 infected patients.

Methods and Findings

In the present study, we correlated the occurrence of variants at three such SNPs (rs12979860, rs12980275, and rs8099917) with pretreatment plasma IP-10 and HCV RNA throughout therapy within a phase III treatment trial (HCV-DITTO) involving 253 Caucasian patients. The favorable SNP variants (CC, AA, and TT, respectively) were associated with lower baseline IP-10 (P = 0.02, P = 0.01, P = 0.04) and were less common among HCV genotype 1 infected patients than genotype 2/3 (P<0.0001, P<0.0001, and P = 0.01). Patients carrying favorable SNP genotypes had higher baseline viral load than those carrying unfavorable variants (P = 0.0013, P = 0.029, P = 0.0004 respectively). Among HCV genotype 1 infected carriers of the favorable C, A, or T alleles, IP-10 below 150 pg/mL significantly predicted a more pronounced reduction of HCV RNA from day 0 to 4 (first phase decline), which translated into increased rates of RVR (62%, 53%, and 39%) and SVR (85%, 76%, and 75% respectively) among homozygous carriers with baseline IP-10 below 150 pg/mL. In multivariate analyses of genotype 1-infected patients, baseline IP-10 and C genotype at rs12979860 independently predicted the first phase viral decline and RVR, which in turn independently predicted SVR.

Conclusions

Concomitant assessment of pretreatment IP-10 and IL28B-related SNPs augments the prediction of the first phase decline in HCV RNA, RVR, and final therapeutic outcome.     

Different HCV Genotype Distributions of HIV-Infected Individuals in Henan and Guangxi,China

Background

Due to shared transmission routes, hepatitis C virus (HCV) infection is highly prevalent among people infected with human immunodeficiency virus (HIV). Highly active antiretroviral therapy (HAART) is associated with hepatotoxicity, leading to the negative effects on patients with HIV/HCV co-infection. In order to provide valuable information for HCV management in this particular population, we investigated the HCV genotypes in HIV-infected individuals from Henan and Guangxi, the two provinces with the most HIV-infected cases in China.

Methods

Individuals, who acquired HIV infection through various risk routes, were recruited from Henan and Guangxi. Test of antibodies against HCV (anti-HCV) was conducted, and detection of HCV RNA was performed by PCR amplification. HCV subtypes were determined by direct sequencing of amplicons, followed by phylogenetic analysis.

Results

We recruited a total of 1,112 HIV-infected people in this present study. Anti-HCV was detected from 218 (50.1%) patients from Henan and 81 (12.0%) patients from Guangxi, respectively. The highest prevalence of HIV/HCV co-infection was observed from FBDs (former blood donors) (87.2%) in Henan and IDUs (intravenous drug users) (81.8%) in Guangxi, respectively. The seroprevalence rate of HCV among people with sexual contact was significantly higher in Henan than in Guangxi (18.7% vs. 3.5%, P<0.05). The positive rate of HCV RNA in Henan and Guangxi was 30.6% (133/435) and 11.2% (76/677), respectively. Moreover, we found that 20 anti-HCV negative samples were HCV positive by PCR amplification. HCV subtype 1b (52.7%) was predominant in Henan, followed by subtype 2a (41.9%). The most frequently detected subtypes in Guangxi were 6a (35.6%) and 3b (32.9%).

Conclusion

The HCV genotype distributions were different in HIV-infected people from Henan and Guangxi. HIV/HCV co-infection was not only linked to the transmission routes, but also associated with the geographic position.     

Protection against Helicobacter pylori infection by a trivalent fusion vaccine based on a fragment of urease B-UreB414

A multivalent fusion vaccine is a promising option for protection against Helicobacter pylori infection. In this study, UreB414 was identified as an antigenic fragment of urease B subunit (UreB) and it induced an antibody inhibiting urease activity. Immunization with UreB414 partially protected mice from H. pylori infection. Furthermore, a trivalent fusion vaccine was constructed by genetically linking heat shock protein A (HspA), H. pylori adhesin A (HpaA), and UreB414, resulting in recombinant HspA-HpaA-UreB414 (rHHU). Its protective effect against H. pylori infection was tested in BALB/c mice. Oral administration of rHHU significantly protected mice from H. pylori infection, which was associated with H. pylori-specific antibody production and Th1/Th2-type immune responses. The results show that a trivalent fusion vaccine efficiently combats H. pylori infection, and that an antigenic fragment of the protein can be used instead of the whole protein to construct a multivalent vaccine.