Genetic therapies against HIV

Highly active antiretroviral therapy prolongs the life of HIV-infected individuals, but it requires lifelong treatment and results in cumulative toxicities and viral-escape mutants. Gene therapy offers the promise of preventing progressive HIV infection by sustained interference with viral replication in the absence of chronic chemotherapy. Gene-targeting strategies are being developed with RNA-based agents, such as ribozymes, antisense, RNA aptamers and small interfering RNA, and protein-based agents, such as the mutant HIV Rev protein M10, fusion inhibitors and zinc-finger nucleases. Recent advances in T-cell-based strategies include gene-modified HIV-resistant T cells, lentiviral gene delivery, CD8(+) T cells, T bodies and engineered T-cell receptors. HIV-resistant hematopoietic stem cells have the potential to protect all cell types susceptible to HIV infection. The emergence of viral resistance can be addressed by therapies that use combinations of genetic agents and that inhibit both viral and host targets. Many of these strategies are being tested in ongoing and planned clinical trials.     

A quantitative basis for antiretroviral therapy for HIV-1 infection

Highly active antiretroviral therapy (HAART) has dramatically decreased mortality from HIV-1 infection and is a major achievement of modern medicine. However, there is no fundamental theory of HAART. Elegant models describe the dynamics of viral replication, but a metric for the antiviral activity of drug combinations relative to a target value needed for control of replication is lacking. Treatment guidelines are based on empirical results of clinical trials in which other factors such as regimen tolerability also affect outcome. Why only certain drug combinations control viral replication remains unclear. Here we quantify the intrinsic antiviral activity of antiretroviral drug combinations. We show that most single antiretroviral drugs show previously unappreciated complex nonlinear pharmacodynamics that determine their inhibitory potential at clinical concentrations. We demonstrate that neither of the major theories for drug combinations accurately predicts the combined effects of multiple antiretrovirals. However, the combined effects can be understood with a new approach that considers the degree of independence of drug effects. This analysis allows a direct comparison of the inhibitory potential of different drug combinations under clinical concentrations, reconciles the results of clinical trials, defines a target level of inhibition associated with treatment success and provides a rational basis for treatment simplification and optimization.     

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.     

Drugs of abuse and HIV infection/replication: implications for mother-fetus transmission

Human immunodeficiency virus (HIV) infection and progression of acquired immunodeficiency syndrome (AIDS) can be modulated by a number of cofactors, including drugs of abuse. Opioids, cocaine, cannabinoids, methamphetamine (METH), alcohol, and other substances of abuse have been implicated as risk factors for HIV infection, as they all have the potential to compromise host immunity and facilitate viral replication. Although epidemiologic evidence regarding the impact of drugs of abuse on HIV disease progression is mixed, in vitro studies as well as studies using in vivo animal models have indicated that drugs of abuse have the ability to enhance HIV infection/replication. Drugs of abuse may also be a risk factor for perinatal transmission of HIV. Because high levels of viral load in maternal blood are associated with increased risk of HIV vertical transmission, it is likely that drugs of abuse play an important role in promoting mother-fetus transmission. Furthermore, because the neonatal immune system differs qualitatively from the adult system, it is possible that maternal exposure to drugs of abuse would exacerbate neonatal immunity defects, facilitating HIV infection of neonate immune cells and promoting HIV vertical transmission. The availability and use of antiretroviral therapy for women infected with HIV increase, there is an increasing interest in determining the impact of drug abuse on efficacy of AIDS Clinical Trials Group (ACTG)-standardized treatment regimens for woman infected with HIV in the context of HIV vertical transmission.     

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.     

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.     

Laser‐enhanced drug delivery of antiretroviral drugs into human immunodeficiency virus‐1 infected TZMbl cells

The introduction of highly active antiretroviral therapy (HAART) has significantly increased life expectancy and improved management of the human immunodeficiency virus‐1 (HIV‐1) disease globally. This well‐established treatment regime has shown to reduce viral capacity to undetectable limits when using traditional clinical assays. The establishment of viral reservoirs during the early stages of infection are the major contributors to failure of the current regimens to eradicate HIV‐1 infection since the reservoirs are not affected by antiretroviral drugs (ARVs). Therefore, advanced modification of the present treatment and investigation of novel antiretroviral drug delivery system are needed. The aim of this study was to use femtosecond (fs) laser pulses to deliver ARVs into HIV‐1 infected TZMbl cells. Different ARVs were translocated into TZMbl cells using fs pulsed laser (800 nm) with optimum power of 4 μW and 10 ms laser to cell exposure time. Changes in cellular processes were evaluated using cellular morphology, viability, cytotoxicity and luciferase activity assays. Cells treated with the laser in the presence of ARVs showed a significant reduction in viral infectivity, cell viability and an increase in cytotoxicity. This study demonstrated that fs laser pulses were highly effective in delivering ARVs into HIV‐1 infected TZMbl cells, causing a significant reduction in HIV‐1 infection.     

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.     

A few specialized issues that should be focused on anti-HIV drug evaluation in vitro

Since the introduction of antiretroviral therapy(ART),the lifespan and quality of life of patients infected with HIV have been significantly improved.But treatment efficacy was compromised eventually by the development of resistance to antiretroviral drugs,and more new anti-HIV drugs with lower toxicity and higher activity were needed.Based on the experience and lessons learned from the treatment in the developed countries,US FDA suggested that more pharmacodynamical researches should be considered ahead of the clinical trials.To facilitate the anti-HIV drug research and development,we reviewed a few specialized issues that should be focused on drug evaluations in vitro,including: 1)Mechanism of action studies,demonstrating the candidate drug's efficacy to specifically inhibit viral replication or a virus-specific function and confirm the drug target.2)Drug resistance studies,selecting the drug-resistant variants in vitro and determining the activities inhibiting HIV isolates resistant to approved antiretroviral drugs of the same class.3)Antiviral activity in vitro in the presence of serum proteins,ascertaining whether an investigational product is significantly bound by serum proteins.4)Combination activity analysis,evaluating in vitro antiviral activity of an investigational product in two-drug combinations with other drugs approved.     

Synergistic inhibition of cell-to-cell HIV-1 infection by combinations of single chain variable fragments and fusion inhibitors

Cell-to-cell spread of HIV permits ongoing viral replication in the presence of antiretroviral therapy and is suggested to be a major contributor to sexual transmission by mucosal routes. Fusion inhibitors that prevent viral entry have been developed, but their clinical applications have been limited by weak antiviral activity, short half-life, and the low genetic barrier to development of resistance. We examined the inhibitory activities of a series of single-chain variable fragments (scFvs) targeting the V3 and CD4i epitopes against both cell-free and cell-to-cell HIV infection. We found that all anti-V3 scFvs, including two newly constructed scFvs, showed broad neutralization activity against a panel of subtype B viruses compared with the corresponding IgGs. All scFvs neutralized cell-free infection by HIV-1_{JR-FL WT} and fusion inhibitor-resistant mutants. In addition, all anti-V3 scFvs and some CD4i scFvs significantly inhibited cell fusion, while their IgG counterparts did not. Furthermore, scFvs-fusion inhibitors combinations, such as C34 and SC34, showed synergistic inhibition of cell fusion by both HIV-1_{JR-FL WT} and fusion inhibitor-resistant mutants. The most prominent combinational effect was observed for 916B2 CD4i scFv with SC34. The delayed fusion kinetics of fusion inhibitor-resistant mutants partly explain their synergistic inhibition by such combinations. Our data demonstrate the advantages of using scFvs over their parent IgGs for inhibiting both cell-free and cell-to-cell infection. High synergistic inhibition of cell fusion by using scFvs-fusion inhibitors combinations suggests the possibility of intensification therapy adding this combination to current anti-HIV treatment regimens.     

Modulation of apoptosis by HIV protease inhibitors

Advances in treatment have transformed the Human Immunodeficiency Virus (HIV) infection from a progressive and ultimately fatal disease to one that can be managed effectively by chronic suppressive antiretroviral therapy. The drugs now used to treat HIV infection not only inhibit viral replication but also have effects on cellular metabolism and homeostasis. Of particular interest to cellular immunologists, members of the HIV Protease Inhibitor (PI) class of antiretroviral agents possess intrinsic immunomodulatory and antiapoptotic properties. This review focuses on the development and use of PI together with their impact on HIV disease, immunity, and apoptosis.