假病毒技术用于抗HIV-1药物筛选及抗药性分析

HIV抗药性的产生严重阻碍了HIV疾病的治疗进程,因此开发新的抗HIV药物以及对病毒进行抗药性分析对于提高HIV疾病的治疗效果非常重要。将利用HIV假病毒构建的抗HIV药物评价系统及病毒抗药性分析系统应用于药物筛选及耐药性分析具有常规方法无法替代的优点。介绍了目前常用的几种类型HIV假病毒的特点和构建方法,同时介绍了假病毒感染细胞的系统及其在细胞水平筛选抗HIV药物和对HIV临床分离株进行耐药性分析这两方面的应用,并通过与常规方法进行比较来分析利用假病毒技术进行研究的优势及局限性, 还通过总结大量学者的研究成果证明了利用假病毒技术进行药物筛选和抗药性分析具有准确、安全和高效的特点。     

Antiretroviral drug resistance mutations sustain or enhance CTL recognition of common HIV-1 Pol epitopes

Antiretroviral drug resistance and escape from CTL are major obstacles to effective control of HIV replication. To investigate the possibility of combining drug and immune-based selective pressures against HIV, we studied the effects of antiretroviral drug resistance mutations on CTL recognition of five HIV-1 Pol epitopes presented by common HLA molecules. We found that these common drug resistance mutations sustain or even enhance the antigenicity and immunogenicity of HIV-1 Pol CTL epitopes. Variable patterns of cross-reactive and selective recognition of wild-type and corresponding variant epitopes demonstrate a relatively diverse population of CD8(+) T cells reactive against these epitopes. Variant peptides with multiple drug resistance mutations still sustained CTL recognition, and some HIV-infected individuals demonstrated strong CD8(+) T cell responses against multiple CTL epitopes incorporating drug resistance mutations. Selective reactivity against variant peptides with drug resistance mutations reflected ongoing or previous exposure to the indicated drug, but was not dependent upon the predominance of the mutated sequence in endogenous virus. The frequency and diversity of CTL reactivity against the variant peptides incorporating drug resistance mutations and the ability of these peptides to activate and expand CTL precursors in vitro indicate a significant functional interface between the immune system and antiretroviral therapy. Thus, drug-resistant variants of HIV are susceptible to immune selective pressure that could be applied to combat transmission or emergence of antiretroviral drug-resistant HIV strains and to enhance the immune response against HIV.     

Bioinformatics-assisted anti-HIV therapy

Highly active antiretroviral therapy (HAART), in which three or more drugs are given in combination, has substantially improved the clinical management of HIV-1 infection. Still, the emergence of drug-resistant variants eventually leads to therapy failure in most patients. In such a scenario, the high diversity of resistance-associated mutational patterns complicates the choice of an optimal follow-up regimen. To support physicians in this task, a range of bioinformatics tools for predicting drug resistance or response to combination therapy from the viral genotype have been developed. With several free and commercial software services available, computational advice is rapidly gaining acceptance as an important element of rational decision-making in the treatment of HIV infection.     

Evolution of phenotypic drug susceptibility and viral replication capacity during long-term virologic failure of protease inhibitor therapy in human immunodeficiency virus-infected adults

Continued use of antiretroviral therapy despite the emergence of drug-resistant human immunodeficiency virus (HIV) has been associated with the durable maintenance of plasma HIV RNA levels below pretherapy levels. The factors that may account for this partial control of viral replication were assessed in a longitudinal observational study of 20 HIV-infected adults who remained on a stable protease inhibitor-based regimen despite ongoing viral replication (plasma HIV RNA levels consistently >500 copies/ml). Longitudinal plasma samples (n = 248) were assayed for drug susceptibility and viral replication capacity (measured by using a single-cycle recombinant-virus assay). The initial treatment-mediated decrease in plasma viremia was directly proportional to the reduction in replicative capacity (P = 0.01). Early virologic rebound was associated the emergence of a virus population exhibiting increased protease inhibitor phenotypic resistance, while replicative capacity remained low. During long-term virologic failure, plasma HIV RNA levels often remained stable or increased slowly, while phenotypic resistance continued to increase and replicative capacity decreased slowly. The emergence of primary genotypic mutations within protease (particularly V82A, I84V, and L90M) was temporally associated with increasing phenotypic resistance and decreasing replicative capacity, while the emergence of secondary mutations within protease was associated with more-gradual changes in both phenotypic resistance and replicative capacity. We conclude that HIV may be constrained in its ability to become both highly resistant and highly fit and that this may contribute to the continued partial suppression of plasma HIV RNA levels that is observed in some patients with drug-resistant viremia.     

Hidden Drug Resistant HIV to Emerge in the Era of Universal Treatment Access in Southeast Asia

Background

Universal access to first-line antiretroviral therapy (ART) for HIV infection is becoming more of a reality in most low and middle income countries in Asia. However, second-line therapies are relatively scarce.

Methods and Findings

We developed a mathematical model of an HIV epidemic in a Southeast Asian setting and used it to forecast the impact of treatment plans, without second-line options, on the potential degree of acquisition and transmission of drug resistant HIV strains. We show that after 10 years of universal treatment access, up to 20% of treatment-naïve individuals with HIV may have drug-resistant strains but it depends on the relative fitness of viral strains.

Conclusions

If viral load testing of people on ART is carried out on a yearly basis and virological failure leads to effective second-line therapy, then transmitted drug resistance could be reduced by 80%. Greater efforts are required for minimizing first-line failure, to detect virological failure earlier, and to procure access to second-line therapies.     

Targeted therapy of human immunodeficiency virus-related disease.

Since the discovery of human immunodeficiency virus (HIV) as a pathogenic retrovirus linked to acquired immunodeficiency syndrome (AIDS), a number of potentially useful strategies for antiretroviral therapy of AIDS and its related diseases have emerged. One such strategy involves use of the broad family of 2',3'-dideoxynucleosides, to which 3'-azido-2',3'-dideoxythymidine (AZT) belongs. AZT has been shown to reduce the replication of HIV in vivo and to confer significant clinical benefits in patients in both early and advanced stages of infection. Other members of the family, 2',3'-dideoxycytidine (ddC), 2',3'-dideoxyinosine (ddI), and 2',3'-didehydro-2',3'-dideoxythymidine (d4T), have also been reported to be active against HIV in short-term clinical trials. The armamentarium of antiretroviral agents is rapidly growing. Various nonnucleoside agents have recently been identified to be active against HIV in vitro. HIV-1 protease inhibitors are notable as possible new therapies for HIV-1-related diseases. However, we have faced several new challenges in the antiretroviral therapy in AIDS. These include long-term drug-related toxicities; emergence of drug-resistant HIV variants; and development of various cancers, particularly as effective therapies prolong survival. Progress in understanding structure-activity relations and clinical effectiveness will continue with dideoxynucleoside analogs. However, it seems certain that a variety of nonnucleoside analogs affecting multiple steps in viral replication will become available before long, and combination therapies using multiple antiretroviral drugs will be available. Such therapies will exert major effects against the moribidity and mortality caused by HIV.     

Retention of antigen on follicular dendritic cells and B lymphocytes through complement-mediated multivalent ligand-receptor interactions: theory and application to HIV treatment

In HIV-infected patients, large quantities of HIV are associated with follicular dendritic cells (FDCs) in lymphoid tissue. During antiretroviral therapy, most of this virus disappears after six months of treatment, suggesting that FDC-associated virus has little influence on the eventual outcome of long-term therapy. However, a recent theoretical study using a stochastic model for the interaction of HIV with FDCs indicated that some virus may be retained on FDCs for years, where it can potentially reignite infection if treatment is interrupted. In that study, an approximate expression was used to estimate the time an individual virion remains on FDCs during therapy. Here, we determine the conditions under which this approximation is valid, and we develop expressions for the time a virion spends in any bound state and for the effect of rebinding on retention. We find that rebinding, which is influenced by diffusion, may play a major role in retention of HIV on FDCs. We also consider the possibility that HIV is retained on B cells during therapy, which like FDCs also interact with HIV. We find that virus associated with B cells is unlikely to persist during therapy.     

Treatment-mediated alterations in HIV fitness preserve CD4+ T cell counts but have minimal effects on viral load

For most HIV-infected patients, antiretroviral therapy controls viral replication. However, in some patients drug resistance can cause therapy to fail. Nonetheless, continued therapy with a failing regimen can preserve or even lead to increases in CD4+ T cell counts. To understand the biological basis of these observations, we used mathematical models to explain observations made in patients with drug-resistant HIV treated with enfuvirtide (ENF/T-20), an HIV-1 fusion inhibitor. Due to resistance emergence, ENF was removed from the drug regimen, drug-sensitive virus regrown, and ENF was re-administered. We used our model to study the dynamics of plasma-viral RNA and CD4+ T cell levels, and the competition between drug-sensitive and resistant viruses during therapy interruption and re-administration. Focusing on resistant viruses carrying the V38A mutation in gp41, we found ENF-resistant virus to be 17±3% less fit than ENF-sensitive virus in the absence of the drug, and that the loss of resistant virus during therapy interruption was primarily due to this fitness cost. Using viral dynamic parameters estimated from these patients, we show that although re-administration of ENF cannot suppress viral load, it can, in the presence of resistant virus, increase CD4+ T cell counts, which should yield clinical benefits. This study provides a framework to investigate HIV and T cell dynamics in patients who develop drug resistance to other antiretroviral agents and may help to develop more effective strategies for treatment.     

Helper-dependent vs. helper-independent CTL responses in HIV infection: implications for drug therapy and resistance.

Clinical data from HIV-infected patients, as well as theoretical studies, suggest that CTL responses in the presence and absence of CD4 cell help are qualitatively different. In the presence of help, CTL responses are maintained despite very low antigenic loads and control the infection in the long term. In the absence of specific helper cell responses, CTL require high antigenic loads to be maintained, are short lived at low levels of antigen, and do not control the infection in the long term. This paper describes mathematical models analysing the dynamics of helper-dependent and helper-independent CTL in HIV infection with special focus on the dynamics during drug therapy in chronic infection. Theory suggests that a fast rate of virus spread results in high degrees of helper cell impairment which promotes the development of helper-independent CTL responses and compromised immunological control. In agreement with clinical findings, the model suggests that upon start of therapy, there is a transient increase in the level of CTL, followed by a decline to low levels once virus load has been significantly suppressed. According to the model, the presence of helper-independent CTL can promote the establishment of a helper-dependent memory response. Interestingly, this gives rise to the prediction that a relatively early stop of therapy, before the level of CTL has fallen below a threshold, can promote improved immunological control. Issues concerning the timing and duration of treatment are discussed. The CTL kinetics during drug therapy also provide new insights into the principles underlying the emergence of drug-resistant strains during the course of treatment.     

Mutation and control of the human immunodeficiency virus

We examine the dynamics of infection by the human immunodeficiency virus (HIV), as well as therapies that minimize viral load, restore adaptive immunity, and use minimal dosage of anti-HIV drugs. Virtual therapies for wild-type infections are demonstrated; however, the HIV infection is never cured, requiring continued treatment to keep the condition in remission. With high viral turnover and mutation rates, drug-resistant strains of HIV evolve quickly. The ability of optimal therapy to contain drug-resistant strains is shown to depend upon the relative fitness of mutant strains.     

Modeling the role of macrophages in HIV persistence during antiretroviral therapy

HIV preferentially infects activated CD4+ T cells. Current antiretroviral therapy cannot eradicate the virus. Viral infection of other cells such as macrophages may contribute to viral persistence during antiretroviral therapy. In addition to cell-free virus infection, macrophages can also get infected when engulfing infected CD4+ T cells as innate immune sentinels. How macrophages affect the dynamics of HIV infection remains unclear. In this paper, we develop an HIV model that includes the infection of CD4+ T cells and macrophages via cell-free virus infection and cell-to-cell viral transmission. We derive the basic reproduction number and obtain the local and global stability of the steady states. Sensitivity and viral dynamics simulations show that even when the infection of CD4+ T cells is completely blocked by therapy, virus can still persist and the steady-state viral load is not sensitive to the change of treatment efficacy. Analysis of the relative contributions to viral replication shows that cell-free virus infection leads to the majority of macrophage infection. Viral transmission from infected CD4+ T cells to macrophages during engulfment accounts for a small fraction of the macrophage infection and has a negligible effect on the total viral production. These results suggest that macrophage infection can be a source contributing to HIV persistence during suppressive therapy. Improving drug efficacies in heterogeneous target cells is crucial for achieving HIV eradication in infected individuals.

     

Stochastic interplay between mutation and recombination during the acquisition of drug resistance mutations in human immunodeficiency virus type 1

The emergence of drug resistance mutations in human immunodeficiency virus (HIV) has been a major setback in the treatment of infected patients. Besides the high mutation rate, recombination has been conjectured to have an important impact on the emergence of drug resistance. Population genetic theory suggests that in populations limited in size recombination may facilitate the acquisition of beneficial mutations. The viral population in an infected patient may indeed represent such a population limited in size, since current estimates of the effective population size range from 500 to 10(5). To address the effects of limited population size, we therefore expand a previously described deterministic population genetic model of HIV replication by incorporating the stochastic processes that occur in finite populations of infected cells. Using parameter estimates from the literature, we simulate the evolution of drug-resistant viral strains. The simulations show that recombination has only a minor effect on the rate of acquisition of drug resistance mutations in populations with effective population sizes as small as 1,000, since in these populations, viral strains typically fix beneficial mutations sequentially. However, for intermediate effective population sizes (10(4) to 10(5)), recombination can accelerate the evolution of drug resistance by up to 25%. Furthermore, a reduction in population size caused by drug therapy can be overcome by a higher viral mutation rate, leading to a faster evolution of drug resistance.     

HIV-1 pharmacoresistance: clinical and therapeutic implications

The primary objective of antiretroviral therapy is to suppress viral replication as soon as possible, as much as possible and for as long as possible, a concept so clearly emphasized by David Ho in 1995: "Treat HIV early and hard!". That, however, seems an ideal objective by a number of reasons, recently recognized as fundamental: unavailability of treatments able to eradicate the infection, difficulty to reach compliance to HAART (Highly Active Antiretroviral Therapy), emergence of drug resistance and cross-resistance. (Cross)-resistance in particular has the potential both to waste future therapeutic options and to be transmitted during HIV infection. Therefore, HIV pharmacoresistance has to be considered one of the most challenging focal point in research on antiretroviral therapy. Understanding of causes, evolutionary patterns and consequences of resistance in terms of viroimmunological and clinical response appears inescapable to strategically plan and monitor treatment. Rather than to eradicate the infection with regimens more and more hard but more and more difficult to comply with, the realistic approach is to construct a strategic therapeutic itinerary tailored to the bio-psycho-social patient conditions and to the saving of therapeutic options. The latter means the rational sequencing of the drug employment for a long-term therapy, potentially life-long.     

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.     

Persistence of transmitted drug resistance among subjects with primary human immunodeficiency virus infection

Following interruption of antiretroviral therapy among individuals with acquired drug resistance, preexisting drug-sensitive virus emerges relatively rapidly. In contrast, wild-type virus is not archived in individuals infected with drug-resistant human immunodeficiency virus (HIV) and thus cannot emerge rapidly in the absence of selective drug pressure. Fourteen recently HIV-infected patients with transmitted drug-resistant virus were followed for a median of 2.1 years after the estimated date of infection (EDI) without receiving antiretroviral therapy. HIV drug resistance and pol replication capacity (RC) in longitudinal plasma samples were assayed. Resistance mutations were characterized as pure populations or mixtures. The mean time to first detection of a mixture of wild-type and drug-resistant viruses was 96 weeks (1.8 years) (95% confidence interval, 48 to 192 weeks) after the EDI. The median time to loss of detectable drug resistance using population-based assays ranged from 4.1 years (conservative estimate) to longer than the lifetime of the individual (less conservative estimate). The transmission of drug-resistant virus was not associated with virus with reduced RC. Sexual transmission of HIV selects for highly fit drug-resistant variants that persist for years. The prolonged persistence of transmitted drug resistance strongly supports the routine use of HIV resistance genotyping for all newly diagnosed individuals.     

Prevalence and evolution of drug resistance HIV-1 variants in Henan, China

To understand the prevalence and evolution of drug resistant HIV strains in Henan China after the implementation of free antiretroviral therapy for AIDS patients. 45 drug na?ve AIDS patients, 118 AIDS patients who received three months antiretroviral therapy and 124 AIDS patients who received six months antiretroviral treatment were recruited in the southern part of Henan province. Information on general condition, antiretroviral medicines, adherence and clinical syndromes were collected by face to face interview. Meanwhile, 14 ml EDTA anticoagulant blood was drawn. CD4/CD8 T cell count, viral load and genotypic drug resistance were tested. The rates of clinical improvement were 55.1% and 50.8% respectively three months and six months after antiretroviral therapy. The mean CD4 cell count after antiretroviral therapy was significantly higher than in drug na?ve patients. The prevalence rate of drug resistant HIV strains were 13.9%, 45.4% and 62.7% in drug na?ve patients, three month treatment patients and six month treatment patients, respectively. The number of resistance mutation codons and the frequency of mutations increased significantly with continued antiretroviral therapy. The mutation sites were primarily at the 103, 106 and 215 codons in the three-month treatment group and they increased to 15 codon mutations in the six-month treatment group. From this result, the evolution of drug resistant strains was inferred to begin with the high level NNRTI resistant strain, and then develop low level resistant strains to NRTIs. The HIV strains with high level resistance to NVP and low level resistance to AZT and DDI were highly prevalent because of the AZT+DDI+NVP combination therapy. These HIV strains were also cross resistant to DLV, EFV, DDC and D4T. Poor adherence to therapy was believed to be the main reason for the emergence and prevalence of drug resistant HIV strains. The prevalence of drug resistant HIV strains was increased with the continuation of antiretroviral therapy in the southern part of Henan province. Measures, that could promote high level adherence, provide new drugs and change ART regimens in failing patients, should be implemented as soon as possible.     

Prevalence and evolution of drug resistance HIV-1 variants in Henan, China

To understand the prevalence and evolution of drug resistant HIV strains in Henan China after the implementation of free antiretroviral therapy for AIDS patients. 45 drug naive AIDS patients, 118 AIDS patients who received three months antiretroviral therapy and 124 AIDS patients who received six months antiretroviral treatment were recruited in the southern part of Henan province. Information on general condition, antiretroviral medicines, adherence and clinical syndromes were collected by face to face interview. Meanwhile, 14ml EDTA anticoagulant blood was drawn. CD4/CD8 T cell count, viral load and genotypic drug resistance were tested. The rates of clinical improvement were 55.1% and 50.8% respectively three months and six months after antiretroviral therapy. The mean CD4 cell count after antiretroviral therapy was significantly higher than in drug naive patients. The prevalence rate of drug resistant HIV strains were 13.9%, 45.4% and 62.7% in drug naive patients, three month treatment patients and six month treatment patients, respectively.The number of resistance mutation codons and the frequency of mutations increased significantly with continued antiretroviral therapy. The mutation sites were primarily at the 103, 106 and 215 codons in the three-month treatment group and they increased to 15 codon mutations in the six-month treatment group. From this result, the evolution of drug resistant strains was inferred to begin with the high level NNRTI resistant strain, and then develop low level resistant strains to NRTIs. The HIV strains with high level resistance to NVP and low level resistance to AZT and DDI were highly prevalent because of the AZT DDI NVP combination therapy. These HIV strains were also cross resistant to DLV, EFV, DDC and D4T. Poor adherence to therapy was believed to be the main reason for the emergence and prevalence of drug resistant HIV strains. The prevalence of drug resistant HIV strains was increased with the continuation of antiretroviral therapy in the southern part of Henan province. Measures, that could promote high level adherence,provide new drugs and change ART regimens in failing patients, should be implemented as soon as possible.