TCR repertoire analysis by next generation sequencing allows complex differential diagnosis of T cell-related pathology.

Clonotype analysis is essential for complete characterization of antigen-specific T cells. Moreover, knowledge on clonal identity allows tracking of antigen-specific T cells in whole blood and tissue infiltrates and can provide information on antigenic specificity. Here, we developed a next generation sequencing (NGS)-based platform for the highly quantitative clonotype characterization of T cells and determined requirements for the unbiased characterization of the input material (DNA, RNA, ex vivo derived or cell culture expanded T cells). Thereafter we performed T cell receptor (TCR) repertoire analysis of various specimens in clinical settings including cytomegalovirus (CMV), polyomavirus BK (BKV) reactivation and acute cellular allograft rejection. Our results revealed dynamic nature of virus-specific T cell clonotypes; CMV reactivation was linked to appearance of new highly abundant antigen-specific clonalities. Moreover, analysis of clonotype overlap between BKV-, alloantigen-specific T cell-, kidney allograft- and urine-derived lymphocytes provided hints for the differential diagnosis of allograft dysfunction and enabled appropriate therapy adjustment. We believe that the established approach will provide insights into the regulation of virus-specific/anti-tumor immunity and has high diagnostic potential in the clinical routine.

The role of positive feedback loops involving anti-dsDNA and anti-anti-dsDNA antibodies in autoimmune glomerulonephritis.

Autoimmune glomerulonephritis (GN) is a potentially life-threatening renal inflammation occurring in a significant percentage of systemic lupus erythematosus (SLE) patients. It has been suggested that GN develops and persists due to a positive feedback loop, in which inflammation is promoted by the deposition in the kidney of immune complexes (IC) containing double-stranded DNA (dsDNA) and autoantibodies specific to it, leading to cellular death, additional release to circulation of dsDNA, continuous activation of dsDNA-specific autoreactive B cells and further formation of IC. We have recently presented a generic model exploring the dynamics of IC-mediated autoimmune inflammatory diseases, applicable also to GN. Here we extend this model by incorporating into it a specific B cell response targeting anti-dsDNA antibodies-a phenomenon whose occurrence in SLE patients is well-supported empirically. We show that this model retains the main results found for the original model studied, particularly with regard to the sensitivity of the steady state properties to changes in parameter values, while capturing some disease-specific observations found in GN patients which are unaccountable using our previous model. In particular, the extended model explains the findings that this inflammation can be ameliorated by treatment without lowering the level of anti-dsDNA antibodies. Moreover, it can account for the inverse oscillations of anti-dsDNA and anti-anti-dsDNA antibodies, previously reported in lupus patients. Finally, it can be used to suggest a possible explanation to the so-called regulatory role of TLR9, found in murine models of lupus; i.e., the fact that the knockdown of this DNA-sensing receptor leads, as expected, to a decrease in the level of anti-dsDNA antibodies, but at the same time results in a counter-intuitive amplification of the autoreactive immune response and an exacerbated inflammation. Several predictions can be derived from the analysis of the presented model, allowing its experimental verification.

The impact of fibrosis and steatosis on early viral kinetics in HCV genotype 1-infected patients treated with Peg-IFN-alfa-2a and ribavirin.

Hepatitis C viral (HCV) kinetics after initiation of interferon-based therapy provide valuable insights for understanding virus pathogenesis, evaluating treatment antiviral effectiveness and predicting treatment outcome. Adverse effects of liver fibrosis and steatosis on sustained virological response have been frequently reported, yet their impacts on the early viral kinetics remain unclear. In this study, associations between histology status and early viral kinetics were assessed in 149 HCV genotype 1-infected patients treated with pegylated interferon alfa-2a and ribavirin (DITTO trial). In multivariate analyses adjusted for critical factors such as IL28B genotype and baseline viral load, presence of significant fibrosis (Ishak stage > 2) was found to independently reduce the odds of achieving an initial reduction (calculated from day 0 to day 4) in HCV RNA of ≥2 logIU/mL (adjusted OR 0.03, P = 0.004) but was not associated with the second-phase slope of viral decline (calculated from day 8 to day 29). On the contrary, presence of liver steatosis was an independent risk factor for not having a rapid second-phase slope, that is, ≥0.3 logIU/mL/week (adjusted OR 0.22, P = 0.012) but was not associated with the first-phase decline. Viral kinetic modelling theory suggests that significant fibrosis primarily impairs the treatment antiviral effectiveness in blocking viral production by infected cells, whereas the presence of steatosis is associated with a lower net loss of infected cells. Further studies will be necessary to identify the biological mechanisms underlain by these findings.

IL28B polymorphisms predict reduction of HCV RNA from the first day of therapy in chronic hepatitis C.

BACKGROUND & AIMS: Single nucleotide polymorphisms (SNPs) associated with IL28B influence the outcome of peginterferon-α/ribavirin therapy of chronic hepatitis C virus (HCV) infection. We analyzed the kinetics of HCV RNA during therapy as a function of IL28B SNPs. METHODS: IL28B SNPs rs8099917, rs12979860, and rs12980275 were genotyped in 242 HCV treatment-naïve Caucasian patients (67% genotype 1, 28% genotype 2 or 3) receiving peginterferon-α2a (180 µg weekly) and ribavirin (1000-1200 mg daily) with serial HCV-RNA quantifications. Associations between IL28B polymorphisms and early viral kinetics were assessed, accounting for relevant covariates. RESULTS: In the multivariate analyses for genotype 1 patients, the T allele of rs12979860 (T(rs12979860)) was an independent risk factor for a less pronounced first phase HCV RNA decline (log(10) 0.89IU/ml among T carriers vs. 2.06 among others, adjusted p < 0.001) and lower rapid (15% vs. 38%, adjusted p = 0.007) and sustained viral response rates (48% vs. 66%, adjusted p < 0.001). In univariate analyses, T(rs12979860) was also associated with a reduced second phase decline (p = 0.002), but this association was no longer significant after adjustment for the first phase decline (adjusted p = 0.8). In genotype 2/3 patients, T(rs12979860) was associated with a reduced first phase decline (adjusted p = 0.04), but not with a second phase decline. CONCLUSIONS: Polymorphisms in IL28B are strongly associated with the first phase viral decline during peginterferon-α/ribavirin therapy of chronic HCV infection, irrespective of HCV genotype.

Very early prediction of response to HCV treatment with PEG-IFN-alfa-2a and ribavirin in HIV/HCV-coinfected patients.

The objective of this study was to find very early viral kinetic markers to predict nonresponse to hepatitis C virus (HCV) therapy in a group of human immunodeficiency virus (HIV)/HCV-coinfected patients. Twenty-six patients (15 HCV genotype-1 and 11 genotype-3) were treated with a 48-week regimen of peginterferon-alfa-2a (PEG-IFN) (180 µg/week) and weight-based ribavirin (11 mg/kg/day). Samples were collected at baseline; 4, 8, 12, 18, 24, 30, 36 and 42 h; days 2, 3, 4, 7, 8, 15, 22, 29, 43 and 57 then weekly and monthly. Five patients discontinued treatment. Seven patients (27%) achieved a sustained virological response (SVR). Nadir HCV RNA levels were observed 1.6 ± 0.3 days after initiation of therapy, followed by a 0.3- to 12.9-fold viral rebound until the administration of the second dose of PEG-IFN, which were not associated with SVR or HCV genotype. A viral decline <1.19 log for genotype-1 and <0.97 log for genotype-3, 2 days after starting therapy, had a negative predictive value (NPV) of 100% for SVR. The day 2 virological response had a similar positive predictive value for SVR as a rapid virological response at week 4. In addition, a second-phase viral decline slope (i.e., measured from day 2 to 29) <0.3 log/week had a NPV = 100% for SVR. We conclude that first-phase viral decline at day 2 and second-phase viral decline slope (<0.3 log/week) are excellent predictors of nonresponse. Further studies are needed to validate these viral kinetic parameters as early on-treatment prognosticators of nonresponse in patients with HCV and HIV.

Modeling immune complex-mediated autoimmune inflammation.

A number of autoimmune diseases are thought to feature a particular type of self-sustaining inflammation, caused by the deposition of immune complexes (IC) in the inflamed tissue and a consequent activation of local effector cells. The persistence of this inflammation is due to a positive feedback loop, where autoantigen particles released as part of the tissue damage caused by the inflammation stimulate autoreactive B cells, leading to the formation of further immune complexes and their subsequent deposition. We present a mathematical model for the exploration of IC-mediated autoimmune inflammation and its clinical implications. We characterize the possible differences between normal individuals and those susceptible to such inflammation, and show that both random perturbations and bifurcations can lead to disease onset. Our model explains how defects in the mechanisms responsible for cellular debris clearance contribute to the development of disease, in agreement with empirical evidence. Moreover, we show that parameters governing the dynamics of immune complexes, such as their clearance rate, have an even stronger effect in determining the behavior of the system. We demonstrate the existence of hysteresis, implying that once IC-mediated autoimmune inflammation is triggered, its long-term suppression may be difficult to achieve. Our results can serve to guide the development of novel therapies to autoimmune diseases involving this type of inflammation.

Understanding hepatitis C viral dynamics with direct-acting antiviral agents due to the interplay between intracellular replication and cellular infection dynamics.

The current paradigm for modeling viral kinetics and resistance evolution after treatment initiation considers only the level of circulating virus and cellular infection (CI model), while the intra-cellular level is disregarded. This model was successfully used to explain HIV dynamics and Hepatitis C virus (HCV) dynamics during interferon-based therapy. However, in the new era of direct-acting antiviral agents (DAAs) against HCV, viral kinetics is characterized by a more rapid decline of the wild-type virus as well as an early emergence of resistant strains that jeopardize the treatment outcome. Although the CI model can be extended to describe these new kinetic patterns, this approach has qualitative and quantitative limitations. Instead, we suggest that a more appropriate approach would consider viral dynamics at the cell infection level, as done currently, as well as at the intracellular level. Indeed, whereas in HIV integrated DNA serves as a static replication unit and mutations occur only once per infected cell, HCV replication is deeply affected by DAAs and furthermore processes of resistance evolution can occur at the intra-cellular level with a faster time-scale. We propose a comprehensive model of HCV dynamics that considers both extracellular and intracellular levels of infection (ICCI model). Intracellular viral genomic units are used to form replication units, which in turn synthesize genomic units that are packaged and secreted as virions infecting more target cells. Resistance evolution is modeled intra-cellularly, by different genomic- and replication-unit strains with particular relative-fitness and drug sensitivity properties, allowing for a rapid resistance takeover. Using the ICCI model, we show that the rapid decline of wild-type virus results from the ability of DAAs to destabilize the intracellular replication. On the other hand, this ability also favors the rapid emergence, intracellularly, of resistant virus. By considering the interaction between intracellular and extracellular infection we show that resistant virus, able to maintain a high level of intracellular replication, may nevertheless be unable to maintain rapid enough de novo infection rate at the extracellular level. Hence this model predicts that in HCV, and contrary to our experience with HIV, the emergence of productively resistant virus may not systematically prevent from a viral decline in the long-term. Thus, the ICCI model can explain the transient viral rebounds observed with DAA treatment as well as the viral resistance found in most patients with viral relapse at the end of DAA combination therapy.

Low- and standard-dose peginterferon alfa-2a for chronic hepatitis C, genotype 2 or 3: efficacy, tolerability, viral kinetics and cytokine response.

BACKGROUND: Chronic infection with hepatitis C, genotype 2/3, responds better than other genotypes to peginterferon and ribavirin treatment. We hypothesized that a lower dose of peginterferon would be as effective, but less toxic than standard doses. AIM: To test the hypothesis that a lower dose of peginterferon would be as effective as, but less toxic than, standard doses. METHODS: A total of 30 patients were treated with low-dose peginterferon alfa-2a (90 microg/week) and 27 patients with standard doses (180 microg/week) for 24 weeks in combination with 800 mg/day of ribavirin. Patients who failed treatment were offered 48 weeks of standard-dose treatment. Viral and serum inducible protein 10 (IP-10) levels were measured and early viral kinetic parameters were calculated. RESULTS: Sustained virological response was achieved in 68% of the low-dose and 87% of the standard-dose patients (per protocol, P = 0.79 for non-inferiority). Re-treatment was successful in all patients who tolerated full dose and duration. The standard-dose group had greater first-phase declines of viral levels and faster time to negativity. The second-phase slope was not dose-dependent. IP-10 induction was significantly greater with the standard dose. Although fatigue and general feeling during treatment were worse for standard dose, haematological toxicity and depression did not differ between groups. CONCLUSION: A lower dose of peginterferon is associated with some symptomatic benefit, but the response is not equivalent to standard dosing.

Prediction of nonSVR to therapy with pegylated interferon-alpha2a and ribavirin in chronic hepatitis C genotype 1 patients after 4, 8 and 12 weeks of treatment.

In patients with chronic hepatitis C genotype 1, the current algorithm for treatment discontinuation is based on no early virological response (<2 log decline in hepatitis C virus (HCV)-RNA) at 12 weeks. It is important to determine whether prediction of nonsustained virological response (NR) before 12 weeks can be robustly obtained by statistical methods. We used longitudinal discriminant analysis (LDA) to build and cross-validate models including baseline patient characteristics and measurements of serum HCV-RNA in the first 4, 8 or 12 weeks of treatment. The performance of each model was evaluated by the partial AUC (PA) index, exploring the accuracy of prediction in the range of high negative predictive values. Models were compared by computing 95% confidence intervals for the difference in PA indices. NR was best predicted before week 12 by a single HCV-RNA measurement at week 8 taken together with gender, BMI and age (W8 model, PA index = 0.857). This model was not inferior to models that included a measurement at week 12 (PA index = 0.831). The best model obtained with LDA within the first 4 weeks, which included measurements at days 4, 8 and at week 4, was found to be inferior to the week 8 model (PA index = 0.796). These results indicate that lack of sustained viral response is best predicted after 8 weeks of treatment and that waiting until 12 weeks does not improve the prediction.

Impact of hepatic steatosis on viral kinetics and treatment outcome during antiviral treatment of chronic HCV infection.

Liver steatosis is highly prevalent in chronic hepatitis C virus (HCV) infection, especially in patients infected with genotype 3 virus, but its significance for the outcome of antiviral treatment is not fully understood. We have monitored steatosis in liver biopsies from 231 patients with chronic HCV infection who received pegylated recombinant interferon-alpha and ribavirin in a phase III study (DITTO trial). The degree of steatosis, along with relevant metabolic parameters, was correlated with the early disappearance of virus and with the final outcome of treatment. Our data suggest that the presence of steatosis impairs the early reduction of viral load during treatment in patients infected with HCV genotype 3 and non-3. Steatosis negatively affected the final outcome of treatment mainly in patients infected with HCV genotype non-3 virus. Based on these findings, we propose that interventions aiming at reducing hepatic steatosis prior to the onset of antiviral therapy may be of benefit to patients infected with HCV of the non-3 genotypes. Patients infected with genotype 3, on the other hand, should be offered early antiviral treatment.

Autoimmunity as an immune defense against degenerative processes: a primary mathematical model illustrating the bright side of autoimmunity.

Self-tolerance, or the ability of the immune system to refrain from destroying the organism's own tissues, is a prerequisite for proper immune system operation. How such self-tolerance is achieved is still a subject of debate. The belief that autoimmunity poses a continuous threat to the organism was challenged by data demonstrating that autoimmunity has a protective function after traumatic injury to the central nervous system. This finding led us to suggest the 'comprehensive immunity' approach by which autoimmunity is viewed as a special case of immunity, namely as a defense mechanism that operates by fighting against the threat of potential destructive activity originated or mediated within the organism, similarly to the immune defense that operates against the threat from exogenous pathogens. We present a primary mathematical spatio-temporal model that supports this concept. The numerical solutions of this model illustrate the beneficial operation of a well-controlled immune response specific to self-antigens residing in the site of lesion. The model also explains how the response to self might be tolerated on a day-to-day basis. In addition, we demonstrate that the same autoimmune response, operating at different levels of regulation, can lead to either an autoimmune disease or a degenerative disorder. This preliminary qualitative model supports our contention that the way autoimmunity is perceived should be revised.

Slow viral dynamics of hepatitis C virus genotype 4.

Patients infected with hepatitis C virus genotype 4 (HCV-4) respond to interferon alpha (IFN) as poorly as those infected with genotype 1. However, there is no information on the viral dynamics of HCV-4. Interferon-ribavirin treatment was administered to untreated patients infected with HCV-4. Viral load was assessed with Versant 3.0. Viral dynamics parameters were estimated based on the bi-phasic model for HCV during IFN treatment. Viral kinetics of HCV-4 follow a bi-phasic decline pattern also. The mean effectiveness of IFN in blocking production of HCV-4 was a decline of 77.8% during the first day of treatment. The half-life of free virions was estimated at 3.5 h and that of infected cells from 1.9 to over 70 days. The viral dynamics parameters of HCV-4 appear similar to those of HCV-1 and slower than those of HCV-2. HCV-4 infected patients should be grouped with those with HCV-1 when therapeutic schemes are considered in relation to genotype.

First phase hepatitis c viral kinetics in previous nonresponders patients.

A large proportion of patients fails to respond to treatment for hepatitis C. Initiation of interferon therapy is associated with a rapid first phase decline in viremia, reflecting inhibition of viral production or release from infected cells. We characterized first phase viral kinetics in previous nonresponder patients and compared the antiviral efficacy of interferon in nonresponders to that observed in naive patients. Twenty nonresponders with genotype 1 infection were evaluated. Ten received a single 15 mcg dose of interferon alfacon-1 and ten were given a 30 mcg dose. Viral kinetic data from previously untreated historical control patients with genotype 1 infection who received 9 mcg (n = 12) or 15 mcg (n = 13) of interferon alfacon-1 provided a basis for comparison. Antiviral efficacy was evaluated by calculating the reduction in HCV RNA levels during the first 24 h after interferon administration (log effectiveness). Hepatitis C virus levels decreased exponentially in previous nonresponder patients. Non-responders treated with 30 mcg of interferon alfacon-1 exhibited a greater log drop than non-responders receiving 15 mcg (P = 0.01). The log effectiveness of 15 mcg of interferon alfacon-1 in nonresponders was similar to 9 mcg in naives and was significantly < 15 mcg (P = 0.04) in naïve patients. The 30 mcg dose provided similar log effectiveness in nonresponders compared with 15 mcg in naive patients and exceeded the log effectiveness of 9 mcg in previously untreated patients (P = 0.035). Nonresponders who had greater than a 50% decrease in HCV RNA level from baseline at the end of previous treatment had a larger reduction in viral load at 24 h compared with those who had not achieved that level of response with prior therapy (P = 0.04). In conclusion, the log effectiveness of interferon was lower in nonresponders compared with treatment naive patients. The difference in antiviral effectiveness in previous nonresponders was overcome by higher interferon doses.

RANTES production from CD4+ lymphocytes correlates with host genotype and rates of human immunodeficiency virus type 1 disease progression.

Several chemokine and chemokine receptor parameters were measured in peripheral blood mononuclear cells obtained from patients before they became infected with human immunodeficiency virus type 1 (HIV-1). After HIV-1 infection, the parameters were compared with plasma HIV-1 RNA levels and with rates of CD4(+) lymphocyte decline. Patients who were heterozygous for the Delta32CCR5 allele had significantly higher levels of RANTES production from their CD4(+) lymphocytes than did patients who did not carry the Delta32CCR5 allele (P=.01). Higher RANTES production levels from ex vivo-activated CD4(+)-enriched lymphocytes, but not CD8(+) lymphocytes, correlated with lower plasma HIV-1 RNA levels 9-12 months after infection (P= .01) and with slower rates of CD4(+) lymphocyte decline (P= .002). CCR5 expression levels on ex vivo-activated CD4(+) lymphocytes did not correlate with markers of disease progression. These results further support the hypothesis that chemokine production levels are associated with HIV-1 replication in vivo.

Viral kinetics in patients with chronic hepatitis C treated with standard or peginterferon alpha2a.

BACKGROUND & AIMS: Covalent attachment of a 40-kilodalton polyethylene glycol moiety to interferon alpha2a (peginterferon alpha2a) results in sustained delivery and reduced clearance compared with standard interferon alpha2a. The aim of the study was to compare viral kinetics in patients treated with standard or peginterferon alpha2a. METHODS: Patients with chronic hepatitis C were randomly assigned to receive either standard interferon alpha2a thrice weekly (n = 16) or 180 microg peginterferon alpha2a once weekly (n = 17) for 48 weeks. HCV RNA was quantitated before and frequently during treatment. RESULTS: The extent of the second-phase decline of HCV RNA, representing the degradation rate of infected cells during therapy for responding patients, was 0.02 +/- 0.03 day(-1) (HCV-1), 0.88 +/- 0.64 day(-1) (HCV non-1), 0.06 +/- 0.08 day(-1) (HCV-1), and 0.44 +/- 0.33 day(-1) (HCV non-1) in patients treated with standard or peginterferon alpha2a, respectively. The second-phase decline was low (<0.05 day(-1)) in most patients without a virological end-of-treatment response, and the second-phase decline was high (>0.25 day(-1)) in all patients with sustained virological response. CONCLUSIONS: The degradation rate of infected cells is HCV genotype dependent. Treatment with peginterferon alpha2a may reinforce the death rate of infected cells (particularly in HCV-1-infected patients) or stabilize the therapeutic effect on viral production. The second-phase decline of HCV RNA is predictive of virological sustained response.

Down-regulation of CD8+ T-cell expansions in patients with human immunodeficiency virus infection receiving highly active combination therapy.

Analysis of T-cell receptor (TCR) repertoire usage made by peripheral T lymphocytes during the chronic phase of HIV-1 infection has revealed the presence of clonal expansions of CD8 T cells that are also shown to be largely HIV-specific. Yet, it remains unclear whether the global repertoire perturbation observed during the chronic phase of the infection is also HIV-related and reversible in the long term with the application of highly active antiretroviral therapy. Furthermore, the diversity and the stability of repertoire usage after a relapse of viral replication were never examined. Eight patients were observed longitudinally up to 31 months under triple-association therapy. When viral replication was steadily suppressed, CD8 repertoires were significantly stabilized. Conversely, in situations of incomplete or only transient viral suppression, persistence or rebound in repertoire perturbation was observed. Finally, a T-cell response remarkably different from baseline, as reflected by a repertoire switch, was generated after the discontinuation of highly active therapy. In conclusion, a sustained control of HIV replication correlated with profound modifications of the CD8 repertoire usage. These data also suggested that autovaccination by the withdrawal of antiviral drugs would result in the selection and expansion of T-cell clones that were not necessarily dominant before the onset of treatment.

Changes in anti-viral effectiveness of interferon after dose reduction in chronic hepatitis C patients: a case control study.

BACKGROUND: High dose interferon induction treatment of hepatitis C viral infection blocks viral production over 95%. Since dose reduction is often performed due to clinical considerations, the effect of dose reduction on hepatitis C virus kinetics was studied. METHODS: A new model that allowed longitudinal changes in the parameters of viral dynamics was used in a group of genotype-1 patients (N = 15) with dose reduction from 10 to 3 million units of interferon daily in combination with ribavirin, in comparison to a control group (N = 9) with no dose reduction. RESULTS: Dose reduction gave rise to a complex viral kinetic pattern, which could be only explained by a decrease in interferon effectiveness in blocking virion production. The benefit of the rapid initial viral decline following the high induction dose is lost after dose reduction. In addition, in some patients also the second phase viral decline slope, which is highly predictive of success of treatment, was impaired by the dose reduction resulting in smaller percentage of viral clearance in the dose reduction group. CONCLUSIONS: These findings, while explaining the failure of many induction schedules, suggest that for genotype-1 patients induction therapy should be continued till HCVRNA negativity in serum in order to increase the sustained response rate for chronic hepatitis C.

The stage-structured predator-prey model and optimal harvesting policy.

In this paper, we establish a mathematical model of two species with stage structure and the relation of predator-prey, to obtain the necessary and sufficient condition for the permanence of two species and the extinction of one species or two species. We also obtain the optimal harvesting policy and the threshold of the harvesting for sustainable development.

Hepatitis C virus kinetics.

The balance of virus production and clearance for untreated patients with chronic hepatitis C virus (HCV) results in a decline of viraemia when initiating active antiviral treatment. During the first phase of interferon-alpha therapy, after a delay of about 8-9 h, the kinetics of the viral load is characterized by a rapid dose-dependent decline. This early response can be observed for almost all patients treated with interferon-alpha. After about 24-48 h, the viral decline enters a second phase of relatively slow exponential decay during the following weeks of therapy. Non-responding patients, however, show constant viraemia or even a rebound during this second phase. The rate of the exponential decline of the viral load in responding patients in this second phase is less sensitive to the dose of interferon-alpha and varies considerably among patients. Furthermore, combination therapy with interferon-alpha plus ribavirin does not significantly improve the initial viral decay, although it may prevent more patients from rebounding. Mathematical modelling of viral dynamics reveals high turnover rates of pre-treatment viral production and clearance, and permits the estimation of in vivo half-lives of a few hours for free HCV virions and of 1-70 days for productively infected cells. Infected cell death rate, which determines the second phase decline slope, is predictive of response to treatment. Current models indicate that the early biphasic viral decline is explained if interferon-alpha partially blocks virion production from infected cells, yet they do not rule out additional antiviral or immunological effects. Therapeutic implications are the advisability of use of frequent (daily) and comparatively high initial doses. In conclusion, kinetic analysis of the viral decay during the first weeks of treatment permits the prediction of response at the end-of-therapy and might help to evaluate new drugs and to optimize therapy.