Treatment intensification does not reduce residual HIV-1 viremia in patients on highly active antiretroviral therapy

In HIV-1-infected individuals on currently recommended antiretroviral therapy (ART), viremia is reduced to <50 copies of HIV-1 RNA per milliliter, but low-level residual viremia appears to persist over the lifetimes of most infected individuals. There is controversy over whether the residual viremia results from ongoing cycles of viral replication. To address this question, we conducted 2 prospective studies to assess the effect of ART intensification with an additional potent drug on residual viremia in 9 HIV-1-infected individuals on successful ART. By using an HIV-1 RNA assay with single-copy sensitivity, we found that levels of viremia were not reduced by ART intensification with any of 3 different antiretroviral drugs (efavirenz, lopinavir/ritonavir, or atazanavir/ritonavir). The lack of response was not associated with the presence of drug-resistant virus or suboptimal drug concentrations. Our results suggest that residual viremia is not the product of ongoing, complete cycles of viral replication, but rather of virus output from stable reservoirs of infection.     

Role of steroid priming in the treatment of chronic hepatitis B

Chronic hepatitis B is the result of the immunological response of the host to persistently replicating hepatitis B virus. Steroids can modulate this response; after steroid administration, viral replication increases and after drug withdrawal, it decreases in conjunction with a temporal increase in the transaminase level. Long‐term decrease of the viral level is observed in some patients, but the beneficial effect of steroid withdrawal alone has not been confirmed. Interferon and other anti‐viral agents can suppress virus replication, but the effect of these agents is still unsatisfactory. The combination of steroid priming and anti‐viral treatment may be beneficial. Recent large‐scale controlled trials indicate the utility of the combination therapy, but other randomized trials show no significant difference between therapy with or without steroid priming. Although steroid priming in combination with antiviral agents may be useful in some patients, it should be carefully performed due to the potential risk of liver failure.     

Effects of adenine arabinoside on serum and intrahepatic replicative forms of duck hepatitis B virus in chronic infection

Adenine arabinoside is an antiviral agent which has been used in a number of clinical studies for the treatment of chronic infections with hepatitis B virus. In order to better understand its effects and mode of action, we treated ducks chronically infected with duck hepatitis B virus with a 2-week course and monitored the effects of the drug on viral replication by studying duck hepatitis B virus DNA in liver and serum using molecular biological techniques. We found the drug to be effective in ducks only at much higher doses than those used in humans. At high doses, adenine arabinoside had a dose-related inhibitory effect on viral replication during treatment, but there was a rapid return toward baseline values soon after the cessation of treatment. The supercoiled form of viral DNA was found to be most resistant to adenine arabinoside therapy, and the drug had a disproportionate inhibitory effect on viral plus (noncoding) strand synthesis. We conclude that adenine arabinoside likely exerts its effect in hepadna virus infections predominantly through inhibition of viral DNA polymerase. On the basis of our current study and previous trials in hepatitis B virus-infected patients, we predict that adenine arabinoside will not efficiently eliminate viral replication in chronic hepadna virus infection, when used as the sole therapeutic modality. Adenine arabinoside may have a role to play as an adjunct to immunomodulation or interferon therapy in chronic hepatitis B virus infection in man.     

Modelling the kinetics of hepatitis C virus RNA decline over 4 weeks of treatment with pegylated interferon alpha-2b

Summary.  Viral kinetic models for hepatitis C virus (HCV) have generally assumed that the effectiveness of therapy in blocking virion production, ε, is constant. However, with pegylated interferon α-2b (PEG-IFN) given weekly, there are significant changes in drug concentration between doses that may lead to changes in drug effectiveness and viral rebounds towards the end of the dosing interval. Here we investigate the effects of using a model that assumes a constant effectiveness for studies involving PEG-IFN. We simulated PEG-IFN treatment in a population of 294 computer simulated 'patients', each characterized by a different set of pharmacokinetic and pharmacodynamic parameters. We then sampled the simulated treatment data over 4 weeks with a schedule similar to that used in viral kinetic studies, and fitted a viral kinetic model assuming constant drug effectiveness, the CE model, to that data. Although the CE model was able to fit to the data well in most cases, the parameter estimates obtained scattered widely both above and below the true values. Thus, this model is less useful to analyse HCV RNA data during therapy with PEG-IFN than with standard IFN given daily. With PEG-IFN accurate estimation of viral dynamic parameters necessitates concomitant measurements of serum viral load and drug concentration.     

Treatment of chronic hepatitis c

Since the discovering of the hepatitis C virus in 1989, the treatment of hepatitis C has considerably improved. Initially, with interferon alpha used as a single drug, the sustained virological response rate was below 20%. Then, with the use of combination therapy of interferon a with ribavirin, the response rate increased to 41%. More recently, combination of pegylated interferons with ribavirin give a response rate of about 54-63%. The long-term follow-up studies showed that sustained virological response is generally associated with clinical and histological improvement. The indication of therapy is mainly based on the results of the liver biopsy which is the best way to assess the prognosis of the liver disease. Therefore, treatment is indicated in patients with moderate or severe necroinflammation or fibrosis. The tolerability of combination therapy is relatively poor with a frequent flu-like syndrome and an impaired quality of life. Factors associated with a poor response to treatment are essentially genotype 1 and high viral load. To further improve the efficacy of therapy, different new drugs are under investigation (amantadine, cytokines). These drugs may be candidates for new combinations. In addition, intensive research is currently done for the development of inhibitors of viral enzymes (helicase, protease or polymerase) and anti-sense oligonucleotides, ribozymes and therapeutic vaccine.     

Resistance to HIV protease inhibitors

Summary. Resistance to the HIV-1 protease inhibitor indinavir involves the accumulation of multiple amino acid substitutions in the viral protease. A minimum of 11 amino acid positions have been identified as potential contributors to phenotypic resistance. Three or more amino acid substitutions in the protease are required before resistance becomes measurable (≥four-fold). Further losses in susceptibility follow the stepwise accumulation of additional amino acid substitutions, indicating that antiviral activity (selective pressure) is maintained despite the appearance of multiple amino acid substitutions in the viral protease. Importantly, the sequential nature of these changes indicates that the effects of these substitutions are additive, and that the evolution of resistance is driven by viral replication. This result has significant implications for therapy. It predicts that viral variants resistant to indinavir are unlikely to pre-exist in protease inhibitor-naive patients, and further, that high-level resistance can only develop if the virus is allowed to replicate in the presence of the drug. The use of indinavir in combination with other antiretroviral agents has been demonstrated to dramatically reduce the incidence of resistance mutations, suggesting that with maximal suppression of viral replication, long-term control of HIV-1 infection may be achievable. Thus, the goal of therapy must be to never to allow the virus to replicate. This can be best accomplished by initiating therapy with a maximally suppressive regimen, to reduce viral replication as much as possible, and by imposing a high genetic barrier to resistance. Previous use of other protease inhibitors or inadequate adherence to therapy may compromise the long-term benefit of indinavir by allowing the virus to gain a foothold through the development of resistance. An understanding of these issues will be critical in realizing the full potential of this potent new drug for the control of HIV-1 infection.     

Molecular virology of hepatitis B virus and the development of antiviral drug resistance

Abstract: The active replication phases of the hepatitis B virus (HBV) are marked by a high frequency of mutational events resulting from an enormous daily viral turnover rate and an error‐prone polymerase. Because HBV is not directly cytopathic, it is the host's immune response that accounts for most of the liver disease associated with persistent infection. HBV escape mutants can be found at the transitional phases of chronic hepatitis B, such as hepatitis B e antigen (HBeAg)‐positive versus HBeAg‐negative infection, and these mutants are selected out from the diverse quasispecies pool comprising the HBV population at that time. Not surprisingly, the introduction of nucleoside and nucleotide analog therapy has also seen the emergence of drug resistance, which has become the major factor limiting the long‐term application of antiviral agents for patients with chronic hepatitis B. Thus, the prevention of resistance requires the adoption of strategies that effectively control virus replication and exploit a detailed understanding of the mechanisms and processes that drive the emergence of drug resistance.     

Kinetics of hepatitis B viral load during 48 weeks of treatment with 600 mg vs 100 mg of lamivudine daily

Oral therapy for chronic hepatitis B remains suboptimal. Mathematical modelling of viral decay kinetics to rapidly assess potential antiviral regimens has proved valuable for human immunodeficiency virus and cytomegalovirus. We defined the kinetics of viral replication in 10 chronic hepatitis B patients randomized to lamivudine 100 mg vs 600 mg for 48 weeks. Viral decay kinetics conformed to a biphasic pattern in nine of 10 subjects. Persons receiving 600 mg daily of lamivudine exhibited a 1.6-fold faster decay rate in the infected cell compartment (0.028/day vs 0.017/day, P = 0.06) and a greater overall change in serum viral load when compared with those receiving 100 mg (4.06 vs 1.52 log(10) copies/mL, P = 0.08). More potent therapy appeared to result in more rapid decrease in the infected cell population. Studies using mathematical modelling of viral decay may be a useful method to evaluate single or combination therapy for HBV infection in vivo.     

Palivizumab is highly effective in suppressing respiratory syncytial virus in an immunosuppressed animal model

Respiratory syncytial virus (RSV) is widely recognized as a leading cause of pneumonia, with substantial mortality, in bone marrow transplant recipients. We tested the efficacy of a systemic monoclonal antibody (MAB) preparation possessing a high titer of anti-RSV neutralizing antibody, palivizumab (Synagis) for prophylaxis and therapy of RSV infection in cytoxan (CY) immunosuppressed cotton rats, a model in which the efficacy of a polyclonal anti-RSV product (Respigam) has been demonstrated. Both prophylaxis and therapy with this MAB were highly effective in reducing pulmonary viral replication. However, multiple sequential therapeutic doses of MAB were necessary to control rebound viral replication in continually suppressed animals.     

Quantification of intrinsic residual viral replication in treated HIV-infected patients

The intrinsic rate of viral replication in HIV-infected patients treated with antiretroviral combination therapy is estimated by using a mathematical model of viral dynamics. This intrinsic replication is found to be episodic, varying considerably in quantity between patients (even among those achieving long-term undetectable levels of viremia) and is always reduced by increasing the potency of the antiviral drug regimen. The analysis reveals that even in conditions of perfect patient adherence and drug penetration a substantial level of residual viral replication is expected. The rate of evolution in the viral quasispecies, and thus also the probability of new drug-resistant viral strains being created, is proportional to the total amount of residual viral replication. Under most circumstances, the viral population continues to turn over rapidly during therapy, albeit at a much reduced level.     

Selection of multiresistant hepatitis B virus during sequential nucleoside-analogue therapy

Hepatitis B virus (HBV) drug resistance to lamivudine is always accompanied by mutations in the viral polymerase gene at position 550, termed group 1 (M550V with L526M) or group 2 (M550I) mutations. The latter mutation has not been associated with famciclovir resistance. Thus, the addition of famciclovir to lamivudine therapy in persons with group 2 lamivudine resistance may lead to virus suppression. The effect of lamivudine/famciclovir combination therapy on HBV infection was monitored in 5 lamivudine-resistant patients by quantitative polymerase chain reaction and polymerase gene sequencing of serum virus. No patients treated with combination therapy had a decline in HBV load >1 log10. Continual evolution of the viral polymerase was detected in association with virologic resistance to both drugs. Cloning experiments identified the preexistence of these multidrug-resistant virus variants as minority species prior to addition of famciclovir therapy. HBV resistance to lamivudine monotherapy is associated with a complex mixture of variants that limit the efficacy of second-line nucleoside-analogue therapy. First-line potent combination therapy may reduce the emergence of HBV drug resistance.     

Hepatitis C advances in antiviral therapy: What is accepted treatment now?

The vast number of patients with hepatitis C represent a huge medical and economic burden. While 20-30% of these patients progress and develop advanced liver disease, the majority do not. Thus, it is crucial to identify patients suitable for treatment and those who may benefit most from therapy. Anti-viral therapy is recommended for those patients with chronic hepatitis C who also have elevated liver tests, detectable hepatitis C virus ribonucleic acid and significant inflammation and/or fibrosis on liver biopsy. Currently, the most effective initial therapy is the combination of interferon plus ribavirin. The sustained viral response rate (SVR) is 36-41% following a 24- or 48-week course of therapy. In general, patients with the genotype 1 infection should receive 48 weeks of therapy, and those with genotypes 2 or 3 infection only 24 weeks. Viral load estimations are problematic because of normal fluctuations (up to 0.5-10 log), assay variability and lack of a universally accepted standard; thus, viral load testing is not recommended routinely at present. The sustained viral response rate produces improvements in quality of life and liver histology (including reversal of bridging fibrosis and cirrhosis is some), and durable responses lasting 5-11 years in 95-97% of cases. While the optimal dose of ribavirin is currently unknown, available data suggest that higher doses increase efficacy (albeit with a greater degree of anemia). The dose of 800 mg/day may be the most appropriate lower dose for those patients who require dosage modification for anemia or other side-effects. Patients who have relapsed after interferon monotherapy can be successfully retreated with higher doses of interferon for 1 year or the combination of interferon and ribavarin for 24-48 weeks. Preliminary data suggest that patients with an unfavorable profile, including those with genotype 1 infection, should probably be retreated with interferon and ribavirin for 48 rather than 24 weeks. With our current best therapies, the majority of patients still do not achieve the benefits of a sustained response. Re-treatment with interferon and ribavirin may achieve a sustained response in approximately 10-25% of these patients. In the immediate future, once-weekly pegylated interferons will replace standard interferons. Initial data suggest that SVR achieved with these drugs in combination with ribavirin is increased to 54-61%, but dose modifications and side-effects are more frequent. They will thus provide an incremental benefit in terms of efficacy, particularly for genotype 1-infected patients.     

Theoretical rationale for the use of sequential single-drug antiretroviral therapy for treatment of HIV infection

BACKGROUND: Subpopulations of HIV with mutations associated with resistance to antiretroviral drugs often have reduced replicative capacity, so virus with resistance mutations for all existing and new antiretroviral drugs is likely to be appreciably impaired. Issues of toxicity, quality of life and economics mean that the simultaneous use of all these drugs in combination is unrealistic. We aimed to explore the use of sequential monotherapy regimens using a mathematical model of quasi-species dynamics, to see if these could take advantage of the poor replicative capacity of highly resistant virus. METHODS: We assume for each of seven drugs that a single mutation is associated with the ability to replicate (effective reproductive ratio, R > 1) in the presence of that drug as monotherapy. Parameters included were drug efficacy, the cost of resistance mutations and the number of new target cells arising daily. RESULTS: The use of seven drugs in a daily/weekly sequential monotherapy cycle led to substantial viral suppression (in the presence of all resistant viral subpopulations) for a wider range of parameter values than a continuous five-drug regimen. Although on any one day/week there is a viral subpopulation with R > 1 (e.g. that with resistance only to the current drug), this subpopulation does not have time to grow sufficiently during the short period when that drug is being taken. CONCLUSION: These results provide a rationale for trials of sequential regimens, using as wide a number of drugs with different resistance-associated mutations as possible, as a potential 'resistance-proof' strategy for achieving significant viral load suppression.     

Disentangling the lifespans of hepatitis C virus‐infected cells and intracellular vRNA replication‐complexes during direct‐acting anti‐viral therapy

The decay rate of hepatitis C virus (HCV)‐infected cells during therapy has been used to determine the duration of treatment needed to attain a sustained virologic response, but with direct‐acting anti‐virals (DAA), this rate has been difficult to estimate. Here, we show that it is possible to estimate it, by simultaneously analysing the viral load and alanine aminotransferase (ALT) kinetics during combination DAA therapy. We modelled the HCV RNA and ALT serum kinetics in 26 patients with chronic HCV genotype 1b infection, under four different sofosbuvir‐based combination treatments. In all patients, ALT decayed exponentially to a set point in the normal range by 1‐3 weeks after initiation of therapy. The model indicates that the ALT decay rate during the first few weeks after initiation of therapy reflects the death rate of infected cells, with an estimated median half‐life of 2.5 days in this patient population. This information allows independent estimation of the rate of loss of intracellular replication complexes during therapy. Our model also predicts that the final ALT set point is not related to the release of ALT by dying HCV‐infected cells. Using ALT data, one can separately obtain information about the rate of ‘cure’ of HCV‐infected cells versus their rate of death, something not possible when analysing only HCV RNA data. This information can be used to compare the effects of different DAA combinations and to rationally evaluate their anti‐viral effects.     

Suppression of cytokine storm with a sphingosine analog provides protection against pathogenic influenza virus

Human pandemic H1N1 2009 influenza virus rapidly infected millions worldwide and was associated with significant mortality. Antiviral drugs that inhibit influenza virus replication are the primary therapy used to diminish disease; however, there are two significant limitations to their effective use: (i) antiviral drugs exert selective pressure on the virus, resulting in the generation of more fit viral progeny that are resistant to treatment; and (ii) antiviral drugs do not directly inhibit immune-mediated pulmonary injury that is a significant component of disease. Here we show that dampening the host's immune response against influenza virus using an immunomodulatory drug, AAL-R, provides significant protection from mortality (82%) over that of the neuraminidase inhibitor oseltamivir alone (50%). AAL-R combined with oseltamivir provided maximum protection against a lethal challenge of influenza virus (96%). Mechanistically, AAL-R inhibits cellular and cytokine/chemokine responses to limit immunopathologic damage, while maintaining host control of virus replication. With cytokine storm playing a role in the pathogenesis of a wide assortment of viral, bacterial, and immunologic diseases, a therapeutic approach using sphingosine analogs is of particular interest.     

Human cytomegalovirus in a SCID-hu mouse: thymic epithelial cells are prominent targets of viral replication.

Animal models of human cytomegalovirus (CMV) infections have not been available to study pathogenesis or to evaluate antiviral drugs. Severe combined immunodeficient mice implanted with human fetal tissues (SCID-hu) were found to support CMV replication and may provide a model for this species-specific virus. When conjoint implants of human fetal thymus and liver were inoculated with a low-passage-number isolate of CMV, strain Toledo, consistent high-level viral replication was detected 5, 12, 15, 28, and 35 days after inoculation and virus replication continued for up to 9 months. Other human tissue implants, including lung and colon, were also found to support viral growth but with greater variability in levels and for a shorter duration. As expected, the species specificity of human CMV was preserved in this model such that virus was detected in the human conjoint thymus/liver implant but not in surrounding mouse tissues. The majority of virus-infected cells were localized in the thymic medulla rather than cortical region of the implant and immunofluorescence analysis identified epithelial cells rather than any hematopoietic cell population as the principal hosts for viral replication. Finally, treatment of infected animals with ganciclovir reduced viral replication, thereby demonstrating the value of this system for evaluating antiviral therapies. This animal model opens the way for a range of investigations not previously possible with human CMV.     

Hepatitis B virus-specific T cell response in chronic hepatitis B patients treated with lamivudine and interferon-alpha.

AIMS: The goal of the present study was to assess the impact combination antiviral therapy has on immune responses in chronic hepatitis B. MATERIALS AND METHODS: T cell responses were studied in 16 chronically hepatitis B virus (HBV)-infected patients treated with sequential, partially overlapping, lamivudine-interferon (IFN)-alpha combination therapy. RESULTS: HBcAg-specific lymphoproliferative response (LPR) was transiently detected in four of five patients who achieved virus suppression (HBV DNA < 10(4) genome equivalents/ml) at end of dual therapy, and then reverted to pre-treatment viral load after therapy discontinuation. In contrast, no significant HBcAg-specific LPR was detected in 8 patients who did not attain profound HBV suppression, as well as in three patients who experienced no HBV DNA rebound after therapy discontinuation. CONCLUSIONS: This pilot study suggests that restored viral replication after pharmacological suppression drives the immune response to HBV in chronically infected patients. Further characterization of the adaptive immunity and its regulatory mechanisms at time of therapy discontinuation appears therefore necessary in controlled trials.     

New targets for antiviral therapy of chronic hepatitis C

Until recently, chronic hepatitis C caused by persistent infection with the hepatitis C virus (HCV) has been treated with a combination of pegylated interferon-alpha (PEG-IFNα) and ribavirin (RBV). This situation has changed with the development of two drugs targeting the NS3/4A protease, approved for combination therapy with PEG-IFNα/RBV for patients infected with genotype 1 viruses. Moreover, two additional viral proteins, the RNA-dependent RNA polymerase (residing in NS5B) and the NS5A protein have emerged as promising drug targets and a large number of antivirals targeting these proteins are at different stages of clinical development. Although this progress is very promising, it is not clear whether these new compounds will suffice to eradicate the virus in an infected individual, ideally by using a PEG-IFNα/RBV-free regimen, or whether additional compounds targeting other factors that promote HCV replication are required. In this respect, host cell factors have emerged as a promising alternative. They reduce the risk of development of antiviral resistance and they increase the chance for broad-spectrum activity, ideally covering all HCV genotypes. Work in the last few years has identified several host cell factors used by HCV for productive replication. These include, amongst others, cyclophilins, especially cyclophilinA (cypA), microRNA-122 (miR-122) or phosphatidylinositol-4-kinase III alpha. For instance, cypA inhibitors have shown to be effective in combination therapy with PEG-IFN/RBV in increasing the sustained viral response (SVR) rate significantly compared to PEG-IFN/RBV. This review briefly summarizes recent advances in the development of novel antivirals against HCV.