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.

Perturbation of CD4+ and CD8+ T-cell repertoires during progression to AIDS and regulation of the CD4+ repertoire during antiviral therapy.

The T-cell antigen receptor (TCR) repertoire was studied longitudinally by analyzing the varying lengths of the beta chain CDR3 hypervariable region during the course of HIV-1 infection and following combination antiretroviral therapy. Drastic restrictions in CD8+ T-cell repertoire usage were found at all stages of natural progression and persisted during the first six months of treatment. In contrast, significant CD4+ T-cell repertoire perturbations were not found in early stages of infection but correlated with progression to AIDS. Out of ten patients presenting with pretreatment perturbations, normalization of the CD4+ repertoire was observed in eight good responders, but not in two cases of unsuccessful therapy. These results indicate that, besides CD4+ cell count rise, an efficient control of HIV replication may allow qualitative modifications of the CD4+ repertoire balance.

A chemokine receptor CCR2 allele delays HIV-1 disease progression and is associated with a CCR5 promoter mutation.

Viral and host factors influence the rate of HIV-1 disease progression. For HIV-1 to fuse, a CD4+ cell must express a co-receptor that the virus can use. The chemokine receptors CCR5 and CXCR4 are used by R5 and X4 viruses, respectively. Most new infections involve transmission of R5 viruses, but variants can arise later that also use CXCR4 (R5-X4 or X4 viruses). This is associated with an increased rate of CD4+ T-cell loss and poor prognosis. The ability of host cells to support HIV-1 entry also influences progression. The absence of CCR5 in approximately 1% of the Caucasian population, due to homozygosity for a 32-nucleotide deletion in the coding region (delta32-CCR5 allele), very strongly protects against HIV-1 transmission. Heterozygosity for the delta32-CCR5 allele delays progression typically by 2 years. A recent study showed that a conservative substitution (V64I) in the coding region of CCR2 also has a significant impact on disease progression, but not on HIV-1 transmission. This was unexpected, since CCR2 is rarely used as a co-receptor in vitro and the V64I change is in a transmembrane region. Because a subsequent study did not confirm this effect on progression to disease, we analyzed CCR2-V64I using subjects in the Chicago MACS. We show that CCR2-V64I is indeed protective against disease progression and go on to show that the CCR2-V64I allele is in complete linkage disequilibrium with a point mutation in the CCR5 regulatory region.

The role of a mutant CCR5 allele in HIV-1 transmission and disease progression.

A 32-nucleotide deletion (delta 32) within the beta-chemokine receptor 5 (CCR5) gene has been described in subjects who remain uninfected despite extensive exposure to HIV-1. This allele was found to be common in the Caucasian population with a frequency of 0.0808, but was not found in people of African or Asian ancestry. To determine its role in HIV-1 transmission and disease progression, we analyzed the CCRS genotype of 1252 homosexual men enrolled in the Chicago component of the Multicenter AIDS Cohort Study (MACS). No infected participant was found to be homozygous for the delta 32 allele, whereas 3.6% of at-risk but uninfected Caucasian participants were homozygous, showing the highly protective role of this genotype against sexual acquisition of HIV-1. No evidence was found to suggest that heterozygotes were protected against HIV-1 infection, but a limited protective role against disease progression was noted. The delta 32 allele of CCR5 is therefore an important host factor in HIV-1 transmission and pathogenesis.

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.

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.

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.

HIV-1 dynamics in vivo: virion clearance rate, infected cell life-span, and viral generation time.

A new mathematical model was used to analyze a detailed set of human immunodeficiency virus-type 1 (HIV-1) viral load data collected from five infected individuals after the administration of a potent inhibitor of HIV-1 protease. Productively infected cells were estimated to have, on average, a life-span of 2.2 days (half-life t 1/2 = 1.6 days), and plasma virions were estimated to have a mean life-span of 0.3 days (t 1/2 = 0.24 days). The estimated average total HIV-1 production was 10.3 x 10(9) virions per day, which is substantially greater than previous minimum estimates. The results also suggest that the minimum duration of the HIV-1 life cycle in vivo is 1.2 days on average, and that the average HIV-1 generation time--defined as the time from release of a virion until it infects another cell and causes the release of a new generation of viral particles--is 2.6 days. These findings on viral dynamics provide not only a kinetic picture of HIV-1 pathogenesis, but also theoretical principles to guide the development of treatment strategies.

Hepatitis C viral dynamics in vivo and the antiviral efficacy of interferon-alpha therapy.

To better understand the dynamics of hepatitis C virus and the antiviral effect of interferon-alpha-2b (IFN), viral decline in 23 patients during therapy was analyzed with a mathematical model. The analysis indicates that the major initial effect of IFN is to block virion production or release, with blocking efficacies of 81, 95, and 96% for daily doses of 5, 10, and 15 million international units, respectively. The estimated virion half-life (t1/2) was, on average, 2.7 hours, with pretreatment production and clearance of 10(12) virions per day. The estimated infected cell death rate exhibited large interpatient variation (corresponding t1/2 = 1.7 to 70 days), was inversely correlated with baseline viral load, and was positively correlated with alanine aminotransferase levels. Fast death rates were predictive of virus being undetectable by polymerase chain reaction at 3 months. These findings show that infection with hepatitis C virus is highly dynamic and that early monitoring of viral load can help guide therapy.

Adaptive evolution of human immunodeficiency virus-type 1 during the natural course of infection.

The rate of progression to disease varies considerably among individuals infected with human immunodeficiency virus-type 1 (HIV-1). Analyses of semiannual blood samples obtained from six infected men showed that a rapid rate of CD4 T cell loss was associated with relative evolutionary stasis of the HIV-1 quasispecies virus population. More moderate rates of CD4 T cell loss correlated with genetic evolution within three of four subjects. Consistent with selection by the immune constraints of these subjects, amino acid changes were apparent within the appropriate epitopes of human leukocyte antigen class I-restricted cytotoxic T lymphocytes. Thus, the evolutionary dynamics exhibited by the HIV-1 quasispecies virus populations under natural selection are compatible with adaptive evolution.

HIV-1 rebound during interruption of highly active antiretroviral therapy has no deleterious effect on reinitiated treatment. Comet Study Group.

BACKGROUND: Potent antiretroviral therapy (ART) with a protease inhibitor-based regimen is commonly used to treat HIV-1-infected patients. Transient treatment interruptions because of drug intolerance or other reasons are not uncommon. HIV-1 dynamics during therapy interruption and its consequences for the subsequent reinitiation of therapy have not been properly studied. METHODS: Ten antiretroviral-naive, HIV-1-infected subjects (mean baseline CD4 cell count of 414 cells/mm3 and plasma viral load of 4.8 log10 copies/ml) were treated with the triple drug ART regimen indinavir/zidovudine/lamivudine for 28 days. Therapy was then interrupted for 28 days, after which the same ART regimen was re-started. RESULTS: HIV-1 in plasma declined during the first 7 days of therapy with T1/2 of 1.5 days, and during days 7-28 with T1/2 of 8.9 days. Once therapy was interrupted, a delay of 4-7 days was observed in all subjects, preceding a rapid viral rebound with a mean doubling time of 1.6 days. Mean viral load after 28 days of interruption was 96% of baseline. Upon reinitiation of the same ART regimen, viral load declined at rates similar to those observed during the initial therapy (T1/2 of 1.6 and 8.0 days, respectively). No resistance-conferring mutations were observed in the HIV-1 reverse transcriptase (RT) and protease regions after the interruption of therapy. Plasma viral loads were maintained below 200 copies/ml in subjects continuing therapy for 4 (n = 9) to 12 (n = 5) months, with a mean CD4 cell count increase of 145 cells/mm3. CONCLUSIONS: The reintroduction of efficient ART therapy after a 1 month interruption shows viral kinetics similar to that of naive patients, and is not associated with the development of resistance. No deleterious effect on the reinitiated therapy was observed in patients who temporarily discontinued ART therapy. Nevertheless, because viral load rebounds back to baseline during treatment interruption, viral suppression is in effect put off by that period of time.

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.

Analysis of a biallelic polymorphism in the tumor necrosis factor alpha promoter and HIV type 1 disease progression.

The relevance of a TNF-alpha promoter polymorphism, a G-to-A polymorphic sequence at position-308, was examined to test whether variant alleles of TNF-alpha affect susceptibility to infection with HIV-1 and progression to AIDS. Analysis of specimens from cohorts of HIV-1 positive homosexual men demonstrated that 3 of the 32 (9.4%) HIV-1-infected long-term nonprogressors (LTNPs) were homozygous for the uncommon TNF-2 allele compared with 3 of the 196 (1.5%) HIV-1-seronegative blood donors and uninfected homosexual men (p < 0.05). There was no difference in heterozygosity among HIV-1-seropositive or -seronegative groups, although some of the seropositive men heterozygous for the TNF2 genotype were also heterozygous for CCR5delta32. However, no significant association was found between TNF genotypes and time of survival, CD4 slopes, or viral loads when seroincident (n = 109) and seroprevalent cases (n = 442) from the Chicago MACS were analyzed. Functional analysis of lymphocytes from the seronegative group revealed no difference in endogenous or mitogen-induced TNF-alpha production, as well as susceptibility to in vitro HIV-1 infection between different TNF-genotype donors. These data suggest that TNF genotypes do not play a direct role in HIV-1 disease progression; however, they could potentially be part of a multigenic linkage that may be involved in delaying progression to AIDS.