HIV coreceptor phenotyping in the clinical setting

The introduction of CCR5 antagonists increases the options available for constructing antiretroviral regimens. However, this option is coupled with the caveat that patients should be tested for HIV coreceptor tropism prior to initiating CCR5 antagonist-based therapy. Failure to screen for CXCR4 usage increases the risk of using an ineffective drug, thus reducing the likelihood of viral suppression and increasing their risk for developing antiretroviral resistance. This review discusses current and future methods of determining HIV tropism, with a focus on their utility in the clinical setting for screening purposes. Some of these methods include recombinant phenotypic tests, such as the Monogram Trofile assay, as well as genotype-based predictors, heteroduplex tracking assays, and flow cytometry based methods. Currently, the best evidence supports the use of phenotypic methods, although other methods of screening for HIV coreceptor usage prior to the administration of CCR5 antagonists may reduce costs and increase turnaround time over phenotypic methods. The presence of low levels of X4 virus is a challenge to all assay methods, resulting in reduced sensitivity in clinical, patient-derived samples when compared to clonally derived samples. Gaining a better understanding of the output of these assays and correlating them with clinical progression and therapy response will provide some indication on how both genotype-based, and phenotypic assays for determining HIV coreceptor usage can be improved. In addition, leveraging new technologies capable of detecting low-level minority species may provide the most significant advances in ensuring that individuals with low levels of dual/mixed tropic virus are not inadvertently prescribed CCR5 antagonists.     

International perspectives on antiretroviral resistance. Phenotypic and genotypic resistance assays: methodology, reliability, and interpretations

Phenotypic and genotypic resistance assays-methods to measure the susceptibility of HIV to drug therapy-are becoming widely available to clinicians for use in aiding long-term antiretroviral treatment planning. Phenotypic tests assess the actual response of a patient's viral isolate to individual antiretroviral drugs in culture, and genotypic tests sequence viral DNA, providing an inferred measure of resistance based on the evaluator's knowledge of established HIV-1 genetic mutational patterns for resistance to antiretroviral drugs. These tests, when used as intended, can provide information useful in planning successful antiretroviral regimens. Both types of assays have been shown to provide reliable and reproducible measures of resistance, with certain caveats: accuracy depends on the experience of the interpreter and laboratory; results from the available tests are not interchangeable, and clinically relevant thresholds of resistance have not been fully defined. Because of pharmacokinetic and cross-resistance issues, the extrapolation of single-drug data to combination regimens is difficult to accomplish and must be pooled with treatment and viral load history to best identify effective subsequent treatment regimens. This article describes the experience to date establishing the efficacy, accuracy, reproducibility, and limitations of the phenotypic and genotypic resistance assays used today.     

Current management challenges in HIV: antiretroviral resistance

Emergence of drug-resistant viral variants is a major reason why HIV-infected patients experience viral rebound during antiretroviral therapy. Although combination antiretroviral therapy substantially inhibits viral replication, replication-competent mutant virus remains. In addition, it is now clear that virologic failure is not necessarily caused by failure of all drugs in a regimen. The use of resistance-testing data can assist in understanding the reasons for failure of antiretroviral therapy. However, there is a need for additional trials to better define the role resistance testing may play in developing management approaches to mitigate or minimize emergence of resistant HIV.     

Antiretroviral drug resistance testing

While antiretroviral drugs, those approved for clinical use and others under evaluation, attempt in lowering viral load and boost the host immune system, antiretroviral drug resistance acts as a major impediment in the management of human immune deficiency virus type-1 (HIV-1) infection. Antiretroviral drug resistance testing has become an important tool in the therapeutic management protocol of HIV-1 infection. The reliability and clinical utilities of genotypic and phenotypic assays have been demonstrated. Understanding of complexities of interpretation of genotyping assay, along with updating of lists of mutation and algorithms and determination of clinically relevant cut-offs for phenotypic assays are of paramount importance. The assay results are to be interpreted and applied by experienced HIV practitioners, after taking into consideration the clinical profile of the patient. This review sums up the methods of assay currently available for measuring resistance to antiretroviral drugs and outlines the clinical utility and limitations of these assays.     

Therapeutic vaccines against HIV infection

Resistance to medication, adverse effects in the medium-to-long-term and cost all place important limitations on lifelong adherence to combined antiretroviral therapy (cART). In this context, new therapeutic alternatives to 'cART for life' in HIV-infected patients merit investigation. Some data suggest that strong T cell-mediated immunity to HIV can indeed limit virus replication and protect against CD4 depletion and disease progression. The combination of cART with immune therapy to restore and/or boost immune-specific responses to HIV has been proposed, the ultimate aim being to achieve a 'functional cure'. In this scenario, new, induced, HIV-specific immune responses would be able to control viral replication to undetectable levels, mimicking the situation of the minority of patients who control viral replication without treatment and do not progress to AIDS. Classical approaches such as whole inactivated virus or recombinant protein initially proved useful as therapeutic vaccines. Overall, however, the ability of these early vaccines to increase HIV-specific responses was very limited and study results were discouraging, as no consistent immunogenicity was demonstrated and there was no clear impact on viral load. Recent years have seen the development of new approaches based on more innovative vectors such as DNA, recombinant virus or dendritic cells. Most clinical trials of these new vectors have demonstrated their ability to induce HIV-specific immune responses, although they show very limited efficacy in terms of controlling viral replication. However, some preliminary results suggest that dendritic cell-based vaccines are the most promising candidates. To improve the effectiveness of these vaccines, a better understanding of the mechanisms of protection, virological control and immune deterioration is required; without this knowledge, an efficacious therapeutic vaccine will remain elusive.     

Recent and future therapeutic advances in the management of HIV infection

Human immunodeficiency virus (HIV) infection is a worldwide pandemic that remains a major health and socio-epidemiological problem at the beginning of the 21st century, including in developed countries. In 2006, 10 years after the introduction of highly active antiretroviral therapy (HAART), therapeutic needs in HIV are different from what they were a decade ago. Physicians now handle more than 20 different molecules, all active against HIV virus. However, they still need safe, tolerable and simple regimens in order to improve patient's compliance to therapy, as well as more potent drugs in cases of multi-resistant viral strains. Our understanding of HIV infection and its medical management has improved and will certainly continue to evolve. We are currently taking benefit from the elaboration and evaluation of various new therapeutic concepts and promising future medical approaches based on already 20 years experience in the field of HIV management, as well as on the rapidly expanding pharmaceutical development and researches in this domain. Nevertheless, the majority of these new therapeutic strategies are still to be further evaluated, and their practical application is awaiting the results of numerous ongoing clinical trials.     

A physician's primer to antiretroviral drug resistance testing

Evaluating the genetic composition of HIV has evolved from a traditional epidemiologic research tool into a widely used clinical asset in the management of HIV infection. Genotypic and phenotypic testing is designed to identify drugs less likely to be therapeutically effective. Genotypic assays identify specific "gene mutations" or nucleotide substitutions known to confer drug resistance, whereas phenotypic assays measure the amount of drug necessary to inhibit viral replication in vitro. Prospective studies of antiretroviral drug resistance testing have shown its value as an independent predictor of optimal virologic response to drug therapy. Current guidelines recommend use of these tests following treatment failure and during pregnancy; considerations for testing include primary infection and treatment-naive patients. The identification of drug resistance can help the clinician individualize treatment regimens to attain maximal viral suppression and patient longevity.     

Antiretroviral resistance testing for clinical management

The advent of highly active antiretroviral therapy (HAART) has dramatically changed the management of HIV disease. These powerful regimens of drugs have reduced morbidity and mortality as well as number of hospitalizations among individuals with HIV. Nevertheless, many patients fail to achieve viral suppression. Various factors underlie this problem, but resistance to current medications has been increasingly recognized as an important element. Several methods of assessing antiretroviral resistance in HIV-1 are currently available. Phenotype assays directly assess the level of susceptibility of a virus to specific drugs. Genotype assays determine the presence of viral mutations that may affect viral susceptibility to a particular drug or class of drugs. An increasing amount of data indicates that antiretroviral resistance testing may improve response to therapy and increase the likelihood of achieving viral suppression. In this paper we review the available data regarding the role of antiretroviral resistance testing and compare the two different assays. We also discuss limitations of the current assays, interpretation of the data, and current consensus guidelines. Although many questions remain, antiretroviral resistance testing along with expert opinion can aid in the management of HIV disease.     

The role of HIV and antiretroviral therapy in bone disease

Current knowledge suggests that both HIV and antiretroviral drugs are likely to contribute to bone disorders in patients with HIV infection. This article includes a review and update on the part played by the virus and the drugs in the low bone mineral density of HIV-infected patients, and a discussion about their implications in clinical practice. HIV viral proteins may affect osteoblast and osteoclast function, and many clinical studies have shown that during antiretroviral therapy, especially at the beginning, there is an accelerated bone mineral loss associated with bone resorption markers, which may be of differing intensity depending on the HIV drugs used. Vitamin D insufficiency/deficiency is highly prevalent and in some investigations it has been associated with antiretroviral therapy, more often with regimens based on efavirenz. Recent data suggest that immune reconstitution may play a major role in early antiretroviral therapy-related bone loss. Given the complex interaction between HIV and drugs in causing low bone mineral density, optimization of antiretroviral therapy and preemptive strategies aimed to prevent bone loss during therapy may be of paramount importance.     

The Alphabet Soup of HIV Reservoir Markers

Purpose of Review

Despite the success of antiretroviral therapy in suppressing HIV, life-long therapy is required to avoid HIV reactivation from long-lived viral reservoirs. Currently, there is intense interest in searching for therapeutic interventions that can purge the viral reservoir to achieve complete remission in HIV patients off antiretroviral therapy. The evaluation of such interventions relies on our ability to accurately and precisely measure the true size of the viral reservoir. In this review, we assess the most commonly used HIV reservoir assays, as a clear understanding of the strengths and weaknesses of each is vital for the accurate interpretation of results and for the development of improved assays.

Recent Findings

The quantification of intracellular or plasma HIV RNA or DNA levels remains the most commonly used tests for the characterization of the viral reservoir. While cost-effective and high-throughput, these assays are not able to differentiate between replication-competent or defective fractions or quantify the number of infected cells. Viral outgrowth assays provide a lower bound for the fraction of cells that can produce infectious virus, but these assays are laborious, expensive and substantially underestimate the potential reservoir of replication-competent provirus. Newer assays are now available that seek to overcome some of these problems, including full-length proviral sequencing, inducible HIV RNA assays, ultrasensitive p24 assays and murine adoptive transfer techniques.

Summary

The development and evaluation of strategies for HIV remission rely upon our ability to accurately and precisely quantify the size of the remaining viral reservoir. At this time, all current HIV reservoir assays have drawbacks such that combinations of assays are generally needed to gain a more comprehensive view of the viral reservoir. The development of novel, rapid, high-throughput assays that can sensitively quantify the levels of the replication-competent HIV reservoir is still needed.

     

Drug-resistant human immunodefiency virus

The development of antiretroviral therapy has led to a major reduction in human immunodeficiency virus (HIV)-related mortality. There are now six antiretroviral drug classes, with more than 20 unique antiretroviral drugs. However, HIV drug resistance occurs with all antiretroviral agents. Drug resistance can affect the response to antiretroviral therapy and is associated with increased mortality. The emergence of resistance in persons on antiretroviral therapy and the transmission of drug-resistant HIV strains to newly infected persons are now major public health concerns. Resistant variants that make up as little as 1% of the viral population in an HIV-infected person are clinically important, as they can rapidly grow under drug selection pressure and lead to therapy failure. However, current resistance assays used in the clinic reliably detect resistant variants only if they make up at least 20% of the circulating viral population. Recently, antiretroviral drugs have been developed that can inhibit HIV replication at new sites within the viral life cycle. These new drugs may improve clinical outcomes in persons infected with multidrug-resistant HIV. This review addresses the epidemiology and biological mechanisms of HIV drug resistance and the new approaches to detect and combat HIV drug resistance.     

The role of CD8+ T-cell response in HIV infection

CD8+ T-cells with cytotoxic (CTL) activity play a pivotal role in controlling viral infections. Although most patients chronically infected with HIV have CTL response against the virus, for reasons that are not well understood this response is not able to successfully control viral replication. The crucial role of this type of response has been clearly demonstrated in the setting of acute infection using the simian model of AIDS, in which a strong CTL response develops, supporting its role in humans. This approach has been possible due to the development of new assays to quantify CTL activity with great sensitivity and specificity. The interaction of CTL response and HIV during this acute stage of infection is crucial, since it most probably determines the viral set-point and thus the rate of HIV disease progression. In the setting of chronic HIV infection, the use of tetrameric complexes and IFN-gamma production assays have made it possible to investigate the different functional aspects of these cells and have also facilitated the evaluation of this response in large patient populations. Defects in cytokine production and in perforin expression have been found, as well as alterations in phenotypic maturation and a low proliferation of these cells. All these findings have been cited to explain the inability of CTLs to efficiently control virus replication. Nonetheless, accumulating evidence points toward an important role for CTL response in the partial containment of HIV replication in chronic infection. An especially strong support for this observation derives from studies analyzing the selective pressure exerted by the immune response over viral evolution. Very recently, longitudinal and cross-sectional studies in large populations of patients have demonstrated that viral evolution is in part driven by HIV-specific T-cell responses.     

Pathogenesis of HIV in the gastrointestinal tract

Gut-associated lymphoid tissue (GALT) is an important site for early HIV replication and severe CD4+ T-cell depletion. Initiation of highly active antiretroviral therapy leads to incomplete suppression of viral replication and substantially delayed and only partial restoration of CD4+ T cells in GALT compared with peripheral blood. Persistent viral replication in GALT leads to replenishment and maintenance of viral reservoirs. Increased levels of inflammation, immune activation, and decreased levels of mucosal repair and regeneration contribute to enteropathy. Assessment of gut mucosal immune system will provide better insights into the efficacy of highly active antiretroviral therapy in immune restoration and suppression of viral reservoirs.     

International perspectives on antiretroviral resistance. Clinical utility of resistance testing: retrospective and prospective data supporting use and current recommendations

Data from retrospective and prospective studies support use of genotypic and phenotypic resistance assays to guide treatment changes when initial or subsequent antiretroviral regimens fail. Several retrospective studies have shown that response to antiretroviral therapy can be predicted based on genotypic analysis of HIV, with baseline genotypic evidence of resistance predicting virologic failure. Other retrospective analyses demonstrated that phenotypic drug sensitivity correlates with increased viral load suppression, particularly when virus remains sensitive to two or three drugs at initiation of the regimen. Furthermore, prospective studies such as VIRADAPT and Genotype-Assisted Antiretroviral Resistance Testing (GART) have substantiated that drug selection based on genotypic assay results yield superior viral suppression compared with empiric treatment assignment. One additional study suggested significant improvement in short-term virologic outcome when phenotypic testing was used to guide treatment selection. Based on these findings, resistance testing is currently recommended for patients with acute HIV infection, those who have failed one or more antiretroviral regimens, and pregnant women. Although these tests move toward becoming a standard of care, several research questions remain: the long-term benefit of resistance testing is not yet certain, the interpretation of specific genotypic resistance patterns needs to be better defined, and clinical cut-off points for phenotypic resistance need to be established. As these issues continue to be studied, resistance testing likely will prove a reliable tool to help plan successful ART strategies.     

Replication capacity,biological phenotype,and drug resistance of HIV strains isolated from patients failing antiretroviral therapy

The fitness of human immunodeficiency virus (HIV) in vivo depends on the interaction of a multitude of viral and host factors. The aim of this study was to analyze the biological phenotype and the intrinsic capacity of the HIV isolates with drug-resistance mutations to replicate efficiently in the absence of drugs. An open label multicenter cross-sectional study was undertaken on 28 HIV-infected patients failing antiretroviral treatment. The subjects were studied for CD4+ cell count, HIV viral load, syncytium-inducing phenotype, genotypic drug-resistance assay, and replication capacity of HIV isolates assessed by co-culture assay. All HIV isolates showed a decreased replication capacity compared with wild-type strains. The lowest replication capacity was detected in HIV strains with more than five drug-resistance mutations. The highest replication capacity was observed in strains carrying the K103N and Y181C primary mutations that emerged after treatment with non-nucleoside analogue inhibitors. Isolates with R5 biological phenotype had a higher number of resistant mutations than X4 isolates (P = 0.004). Particularly, the R5 phenotype was detected in all 6 isolates with more than 14 drug-resistance mutations. Patients with R5 strains had plasma viral load similar to patients with X4 strains, but marginally higher CD4+ cell counts, and their HIV isolates had significantly lower replication capacity of HIV isolates (P = 0.008). No patient carrying HIV with a maintained replication capacity had a viral load less than 30,000 copies/ml. In patients failing HAART, the detection of HIV isolates with the R5 biological phenotype correlates with CD4+ cell count, an impaired replication capacity, and a high number of drug-resistance mutations.     

A vaccinia-based elispot assay for detection of CD8+ T cells from HIV-1 infected children

HIV-specific CD8+ T lymphocytes participate in the control of viral replication in infected patients. These responses are of low intensity in young infants and are decreased by antiretroviral therapy. In the present study, we report on a recombinant Vaccinia virus (rVV)-based Elispot assay for the detection of HIV-specific CD8+ T cells immediately after isolation of peripheral blood mononuclear cells (PBMC). The rVV-based assay was highly sensitive; 48 out of 50 children had a positive response against the rVV encoding HIV Env-Gag-Pol antigen. Interferon-gamma was produced by CD8+ T cells, and CD14+/15+ cells were the main cell subset presenting antigens expressed by rVV. We observed that the cell input per well had a critical influence on the sensitivity of the assay. Results from the ex vivo Elispot assay correlated poorly with those of the 51Cr release assay performed after expansion of PBMC in vitro; thus, both assays gave information on different subsets and/or functions of the HIV-specific T cell response.     

Inhibition of vesicular stomatitis virus replication in the course of HIV infection in patients with different stages of immunodeficiency

The replication of vesicular stomatitis virus (VSV) in isolated human leukocytes has been used to measure the level of nonspecific antiviral immunity. However, during infection with some pathogens, the main effect observed is caused by interaction between the pathogen and VSV. This was also noted in advanced stages of HIV infection, when an inverse association between HIV viral load and VSV replication was found. The mutual effect was markedly stronger than the correlation between the VSV replication level and CD4(+) T-cell count. Since successful antiretroviral therapy is associated with a decrease in HIV viremia to undetectable levels, the effect of such therapy on VSV replication was expected and confirmed in this investigation. In fact, increased VSV titers were observed together with decreased HIV viral load, particularly in the case of efficient therapeutic schemes, for example those including lopinavir/ritonavir. The results showed that VSV replication capacity reflected the progression of HIV infection. Moreover, the presence of interferon in the plasma of AIDS patients was found to be only partially responsible for the inhibition of VSV replication. The results suggest a specific HIV-VSV interaction, whether direct or indirect. Thus the VSV replication assay may be applied in evaluating the stage of HIV infection.