Despite the major contribution that ribavirin provides in improving treatment outcomes in chronic HCV infection, the mechanism by which it acts has been hard to tease apart. The poor understanding of its mechanism of action has made development of ribavirin analogues difficult. A close examination of early viral kinetics, mutagenesis and cytokine levels during peginterferon therapy with and without ribavirin offers some clues to understanding the action of this important adjunctive agent.
Ribavirin had no effect on first phase viral decline. The initial drop in viral load after starting therapy is thought to be due to inhibition of viral replication and clearance of free circulating virus by interferon, which is unlikely to be affected by any of the proposed mechanisms of ribavirin action14
. In this study, the major effect of ribavirin was seen in improvement of the second phase viral decline, which is believed to be due to clearance of infected hepatocytes. Importantly, the effect of ribavirin was not observed in all patients, but only in those with an adequate initial response to peginterferon. Patients with an adequate first phase decline who received ribavirin had a significantly improved second phase slope. No difference in second phase slope was seen between treatment groups in patients with a poor first phase decline. If the first phase decline is primarily due to the effect of peginterferon, these data suggest that the addition of ribavirin leads to an acceleration in viral decline, but a prerequisite of this effect is adequate interferon responsiveness. This observation may shed some light on understanding how ribavirin improves outcomes of therapy.
Of the proposed mechanisms of action for ribavirin, those suggesting direct antiviral activity such as polymerase or IMPDH inhibition, would be expected to act independently of interferon, producing similar results in those with and without an adequate first phase decline. Similarly, promotion of a Th1 phenotype and improvement in antiviral immune responsiveness would likely also be independent of the initial efficacy of interferon16
. In addition, previous studies indicate that T cell responses decline during peginterferon therapy and in this study, Th1 cytokines were low or undetectable in serum25
. An immunomodulatory effect may be more important in late responses as proposed by Hermann et al who suggested that this may account for the triphasic decline seen in some patients17
The other two proposed mechanisms of ribavirin action, mutagenesis and interferon-related gene induction, are both compatible with the kinetics data observed in this study. Dixit and colleagues suggested that the mutagenic effect of ribavirin would be more pronounced when given with interferon than as monotherapy16
. They argued that during interferon treatment the production of new viral genomes is decreased, thereby increasing the concentration of ribavirin per new genome. If incorporation of ribavirin increases mutational frequency in a dose-per-remaining-genome-dependent manner, the mutagenic effect of ribavirin would be enhanced with greater efficacy of interferon. Although early viral kinetics in this study were improved with ribavirin in patients who responded to interferon, there was no increase in mutational frequency as detected by direct sequencing. It is possible that reliance on consensus sequencing rather than clonal analysis may have missed a mutagenic effect, however previous studies of the effect of ribavirin monotherapy have shown correlation between the two approaches9
We have previously reported that hepatic ISG induction is greater in patients treated with ribavirin and peginterferon than in those treated with peginterferon alone12
. In vitro
data also show enhanced activation of interferon signaling with adjunctive ribavirin therapy10, 11
. If ribavirin functions primarily by enhancing ISG induction, the greatest benefit would be in patients who respond to interferon, as observed in this study. In support of this mechanism, evaluation of interferon-inducible serum cytokines (IP10, MIG and MCP-1) showed greater induction in patients receiving ribavirin who had an adequate initial response to interferon. The degree of induction of IP10 correlated strongly with first and second phase decline, suggesting that the degree of ISG activation was driving viral clearance. Together these data support the concept that ribavirin acts, at least partially, through augmentation of interferon signaling.
Sensitivity analysis around the threshold for interferon responsiveness showed that the results were unchanged for a range of values. At very high thresholds for interferon responsiveness (>1.0 log10
IU/mL), the improvement in second phase slope in patients receiving ribavirin was no longer significant. This is in keeping with previous kinetic modeling suggesting that when interferon efficacy is very high, ribavirin has a negligible effect on second phase. Although Dixit et al modeled ribavirin kinetics based on the mutagenesis hypothesis16
, their findings are also consistent with ISG induction. Patients with maximal interferon efficacy would presumably have maximal ISG induction, as has been shown with ISG expression in PBMCs during therapy26
. Further gene induction by the addition of ribavirin would be of little benefit. However, in patients with no clinical response to interferon, there is likely a significant impairment in interferon signaling, which may also affect the ability of ribavirin to induce ISGs. Ribavirin has the greatest effect in patients with an adequate, but not overwhelming, response to interferon.
If ribavirin enhances the effect of interferon, it might also then be expected to improve the initial first-phase kinetics, however this has not been observed in any studies to date. Because of the profound drop in viral load in the first 48 hours, a small additional effect of ribavirin may be difficult to observe. In the second phase, as viral levels decline more slowly, the additional benefit of ribavirin may become more apparent. An additional consideration is that ISGs involved in initial viral clearance may differ significantly from those involved in clearance of infected cells. To date, there is a little understanding of which ISGs are responsible for the antiviral effects of interferon. In this study, IP10, MIG and MCP1 were selected as convenient markers of ISG activation because they are interferon inducible and abundant in serum23, 24
, although not necessarily involved in HCV clearance directly. The observation that induction of all three of these genes correlated with first and second phase decline as well as with ribavirin serum concentration, suggests that ribavirin is enhancing ISG induction broadly, which ultimately improves viral clearance despite the fact that the precise genes involved, their mechanisms and timing of action remain unknown.
Previous studies have shown that high baseline IP10 concentration correlates with non-response to therapy27, 28
. In this cohort, a similar, albeit not statistically significant, pattern was observed (p=0.14). IP10 serum concentrations may be a good surrogate for intrahepatic ISG activation with high pretreatment levels of both predicting treatment non-response. Hepatic ISG induction after the first dose of interferon has been reported to be a marker of treatment responsiveness12, 29
and similarly we found that serum IP10 induction correlated with early viral kinetics. Thus, IP10 may serve as a reliable surrogate marker for hepatic ISG expression.
In summary, analysis of early viral kinetics revealed that ribavirin improved the second phase of viral decline, but only in patients with an adequate initial response to peginterferon. Ribavirin treatment enhanced interferon-related cytokine induction, which correlated with viral decline. Ribavirin showed no significant effect on mutational frequency. Although there may be multiple mechanisms by which ribavirin enhances HCV therapy, these data support the notion that ribavirin augments the activity of interferon resulting in an acceleration of viral decline.