Florence Komurian-Pradel

Quantitation of HCV RNA using real-time PCR and fluorimetry

Real-time PCR technology may provide an accurate and sensitive method to quantify hepatitis C virus (HCV) RNA. So far, studies have been carried out using the Taqman technology with the ABI Prism 7700 sequence detector. An alternative and simple real-time PCR assay is described with no probe requirement, based on the SYBR Green I dye and LightCycler fluorimeter. Amplicon synthesis was monitored continuously by SYBR Green I dye binding to double stranded DNA during PCR of the 5 HCV non-coding (NC) region. Specificity was verified by amplicon melting temperatures. An external standard curve was constructed with serial 10 fold dilutions of a modified synthetic HCV 5 NC RNA. A wide range linear relationship (up to 3.7x10(9) copies/ml) was observed between number of PCR cycle needed to detect a fluorescent signal and number of RNA copy. Intra- and inter-assay coefficients of variation were 0.7 to 2.1 and 3.7% respectively, indicating good reproducibility of the method. Thirty-three HCV positive sera of different genotypes were quantified by this method and gave similar but more sensitive results compared to the branched DNA (bDNA) technology.

Vaccine-induced early control of hepatitis c virus infection in chimpanzees fails to impact on hepatic PD-1 and chronicity

Broad T cell and B cell responses to multiple HCV antigens are observed early in individuals who control or clear HCV infection. The prevailing hypothesis has been that similar immune responses induced by prophylactic immunization would reduce acute virus replication and protect exposed individuals from chronic infection. Here, we demonstrate that immunization of naïve chimpanzees with a multicomponent HCV vaccine induced robust HCV‐specific immune responses, and that all vaccinees exposed to heterologous chimpanzee‐adapted HCV 1b J4 significantly reduced viral RNA in serum by 84%, and in liver by 99% as compared to controls (P = 0.024 and 0.028, respectively). However, despite control of HCV in plasma and liver in the acute period, in the chronic phase, 3 of 4 vaccinated animals developed persistent infection. Analysis of expression levels of proinflammatory cytokines in serial hepatic biopsies failed to reveal an association with vaccine outcome. However, expression of IDO, CTLA‐4 (1) and PD‐1 levels in liver correlated with clearance or chronicity. Conclusion: Despite early control of virus load, a virus‐associated tolerogenic‐like state can develop in certain individuals independent of vaccination history. (HEPATOLOGY 2007;45:602–613.)