Leslie R. Bisset

Reference values for peripheral blood lymphocyte phenotypes applicable to the healthy adult population in Switzerland

Objectives:  Use of domestic reference values is known to improve the accuracy of flow cytometric analysis by integrating local variation due to race, gender, and age. In the absence of previously published estimates, we now report establishment of reference values for a wide range of peripheral blood lymphocyte phenotypes applicable to the healthy adult population in Switzerland and other regions with similar demographic characteristics.

A Randomized Trial of Simplified Maintenance Therapy with Abacavir, Lamivudine, and Zidovudine in Human Immunodeficiency Virus Infection

This randomized study evaluated the efficacy and tolerability of continued treatment with protease inhibitor plus nucleoside-analogue combination regimens (n=79) or a change to the simplified regimen of abacavir-lamivudine-zidovudine (n=84) in patients with suppressed human immunodeficiency virus type 1 (HIV-1) RNA for > or = 6 months who did not have the reverse transcriptase 215 mutation. After a median follow-up of 84 weeks, virologic failure was 6% in the continuation and 15% in the simplified group (P=.081). Previous zidovudine monotherapy or dual therapy and archived reverse transcriptase resistance mutations in HIV-1 DNA at baseline were significant predictors of failure. Study treatment was discontinued because of adverse events in 20% of the continuation and 7% of the simplified group (P=.021). Simplification to abacavir-lamivudine-zidovudine significantly decreased nonfasting cholesterol and triglyceride levels; however, this switch strategy carries a risk of virologic failure when treatment history or resistance testing suggest the presence of archived resistance mutations to the simplified regimen.

Highly active antiretroviral therapy during early HIV infection reverses T-cell activation and maturation abnormalities.

Objectives:To evaluate the impact of early initiation of highly active antiretroviral therapy (HAART) on disease-induced T-cell activation and maturation abnormalities during asymptomatic HIV infection. Design:A prospective open-label trial of zidovudine, lamivudine and ritonavir in treatment-naive asymptomatic HIV-infected individuals with CD4 cells ≥ 400 × 106/l. Methods:Peripheral blood CD4+ and CD8+ T cells derived from 15 asymptomatic HIV-infected individuals (median baseline CD4+ cells, 608 × 106/l; CD8+ cells, 894 × 106/l; plasma HIV RNA, 3.93 log10 copies/ml) undergoing therapy with zidovudine (300 mg twice daily), lamivudine (150 mg twice daily), and ritonavir (600 mg twice daily) were assessed for changes in expression of phenotypic markers of T-cell activation (HLA-DR and CD38) and maturation (CD45RA and CD45RO). At weeks 0, 2, 4, 8, 12, 16, 20 and 24, T-cell subsets were quantified by flow cytometry and plasma HIV viral loads determined using reverse transcription PCR. Results:HAART-induced decrease in plasma HIV RNA levels coincided with a significant reduction in numbers of activated CD4+/HLA-DR+ (maximum change, −36%; P ≤ 0.05), CD8+/HLA-DR+ (maximum change, −66%; P ≤ 0.005) and CD8+/CD38+ (maximum change, −51%; P ≤ 0.01) T cells. A concomitant significant increase in numbers of naive CD4+/CD45RA+ (maximum change, +12%; P ≤ 0.005) and memory CD4+/CD45RO+ (maximum change, +6%; P ≤ 0.05) T cells was also evident, which contrasted with a significant decrease in memory CD8+/CD45RO+ cells (maximum change, −42%; P ≤ 0.005). Conclusion:The observed ability of HAART during early asymptomatic HIV infection to initiate rapid reversal of disease-induced T-cell activation and maturation abnormalities, while preserving pretherapy levels of immune function, supports the concept that therapeutic advantage is to be gained by commencing early aggressive antiretroviral therapy.

Change in circulating levels of the chemokines macrophage inflammatory proteins 1α and 1β, RANTES, monocyte chemotactic protein-1 and interleukin-16 following treatment of severely immunodeficient HIV-infected individuals with indinavir

Objective:To evaluate the in vivo relationship between HIV replication and circulating levels of the chemokines macrophage inflammatory protein (MIP)-1α, MIP-1β, RANTES (acronym for Regulated upon Activation, Normal T-cell Expressed and presumably Secreted), interleukin (IL)-16 and monocyte chemotactic protein (MCP)-1, which have recently been characterized as factors capable of regulating in vitro HIV replication. Design and methods:We have compared changes in plasma HIV-RNA levels and circulating levels of MIP-1α, MIP-1β, RANTES, IL-16 and MCP-1 in 20 severely immunodeficient HIV-infected individuals (CD4+ T cells = 14 ± 3 cells × 106/l; plasma HIV RNA = 4.45 ± 0.27 log10 copies/ml) undergoing treatment with the HIV protease inhibitor indinavir that was added to pre-existing nucleoside-based therapy. At weeks 0, 2, 6 and 12, viral load was quantified using a commercial reverse- transcription polymerase chain reaction assay, peripheral blood T-cell sub- populations assessed by flow cytometry, and chemokine levels quantified using commercial sandwich enzyme-linked immunosorbent assay kits. Results:Following initiation of indinavir-based therapy, significant decreases in plasma HIV-RNA levels (change = 2.0 ± 0.75 log10 copies/ml) were observed in conjunction with significant increases in absolute CD4+ (change = 83 ± 19 cells × 106/l) and CD8+ (change = 293 ± 96 cells × 106/l) T-cell numbers. Concomitantly, significant increases in MIP-1α (19% increase), MIP-1β (14% increase), RANTES (15% increase) and IL-16 (1213% increase) levels occurred. In contrast, MCP-1 levels decreased significantly (47% decrease). Conclusion:The in vivo demonstration of an association between diminishing plasma HIV-RNA levels and the emergence of a circulating chemokine profile capable of inhibiting HIV replication corroborates recent in vitro observations and provides evidence for the restoration of chemokine capacity by HIV protease inhibitor-based therapy.

Simple monitoring of antiretroviral therapy with a signal-amplification-boosted HIV-1 p24 antigen assay with heat-denatured plasma.

Objective:Virus load determination has become indispensable for the management of HIV patients, but depends on expensive assays of a low throughput. We evaluated whether a highly improved HIV-1 p24 antigen detection procedure which involves heat-mediated immune complex dissociation and signal-amplification-boosted enzyme-linked immunosorbent assay (ELISA) was suitable for antiretroviral treatment monitoring. Design and methods:Virus load in plasma was determined for 127 plasma samples taken at 0, 2, 6, 12, 18, 24, 30 and 36 weeks from 23 patients with CD4+ T cells <50 × 106/l who received indinavir 800 mg three times daily in addition to prior antiretroviral treatment. Tests included polymerase chain reaction (PCR) for viral RNA, measured prospectively with the Roche Amplicor kit, and retrospective batch testing of heat-denatured samples for p24 antigen by the DuPont HIV-1 p24 Core Profile ELISA linked with a tyramide signal amplification step. Particle-associated reverse transcriptase (RT) by the product-enhanced RT (PERT) assay was determined as an independent third-opinion viral load marker. Results:p24 antigen was detected as sensitively as viral RNA. Overall detection during a median observation time of 25 weeks (range, 0–39) amounted to 75.6% for antigen and 73.6% for RNA. The antigen detection limit was 0.2 pg/ml. Antigen was detectable in all 23 baseline samples, whereas RNA was undetectable in one. Antigen and RNA levels in 79 samples positive for both markers correlated with r = 0.714 (P < 0.0001). Average changes in levels of p24 antigen and RNA at eight timepoints correlated with r = 0.982 (P < 0.0001). In individual patients, the two parameters behaved similarly, and in certain cases virtually identically. RT activity was measurable in all samples. Conclusions:The performance of this antigen detection procedure is comparable to RNA PCR, thus providing a simple, high throughput alternative in monitoring the efficacy of antiretroviral treatment.

Residual HIV-RNA levels persist for up to 2.5 years in peripheral blood mononuclear cells of patients on potent antiretroviral therapy.

The long-term response of 10 asymptomatic, antiretroviral therapy-naive HIV-1-infected patients to potent combination antiretroviral therapy was characterized by monitoring levels of HIV-1 RNA in plasma, peripheral blood mononuclear cells (PBMC), and lymphoid tissue using highly sensitive HIV-1 RNA assays. Although plasma viral loads were continuously suppressed to levels below 50 HIV-1 RNA copies/ml for up to 2.5 years (60-128 weeks), HIV-1 RNA was still detectable at very low levels (1 to 49 HIV-1 RNA copies/ml) in 25% of the samples. In corresponding PBMC specimens, residual HIV-RNA was detectable in as much as 91% of samples tested (1 to 420 HIV-1 RNA copies/microg total RNA). Similarly, HIV-1 RNA levels in lymphoid tissue also remained detectable at a high frequency (86%). A highly significant correlation was demonstrated between therapy-induced change in PBMC HIV-1 RNA levels and change in plasma HIV-1 RNA levels (r2 = 0.69; p = 0.003). These findings support the concept that measurement of HIV-1 RNA in the easily accessible PBMC compartment is relevant for evaluating the potency of current and future antiretroviral therapies.

HIV-1 p24 antigen is a significant inverse correlate of CD4 T-cell change in patients with suppressed viremia under long-term antiretroviral therapy

An HIV-1 p24 antigen test involving signal amplification-boosted ELISA of heat-denatured plasma was evaluated prospectively in 55 patients whose viral RNA in plasma had previously been suppressed for at least 6 months under antiretroviral combination therapy. During a median follow-up of 504 days, CD4 counts increased by a median of 62 cells per year. By univariate and multivariate linear regression analysis, the level of p24 antigen as expressed by the absorbance/cutoff ratio was a significant inverse correlate of both the CD4 count in a sample (p =.013) and its annual change in a patient (p <.0001). The p24 antigen retained significance even among 48 individuals whose HIV-1 RNA, apart from occasional blips, remained below 400 copies/mL. Batch-wise retesting of 70 samples from 5 such patients with a further improved procedure showed measurable p24 antigen in all but 1 sample and an inverse correlation with both the CD4 count (p =.0331) and percentage (p <.0001), thus confirming the prospectively generated data. Comparison of p24 antigen and HIV-1 RNA concentrations indicate that the p24 antigen detected in these samples is not associated with viral RNA-containing particles and may originate from other compartments of virus expression.

Effects of early antiretroviral treatment on HIV-1 RNA in blood and lymphoid tissue: a randomized trial of double versus triple therapy.

Summary: To assess the effects of early initiation of antiretroviral therapy on cell‐free and cell‐associated viral load in blood and lymphoid tissue, we performed a randomized, open‐label, multicenter trial comparing a double (zidovudine + lamivudine) and triple (zidovudine + lamivudine + ritonavir) drug combination in treatment‐naive, asymptomatic patients with CD4 counts >400 cells/&mgr;l. HIV‐1 RNA was measured in plasma, peripheral blood mononuclear cells, and sequential tonsil or lymph node biopsies (27 patients); the study follow‐up was 2 years. Among 42 randomized patients, the proportion with plasma HIV‐1 RNA <50 copies/ml was 16% and 74% at week 24 (p < .001) in those randomized to double and triple therapy, respectively, necessitating frequent treatment intensification in the double arm. After a rapid decline within 4 weeks in both arms, cell‐associated HIV‐1 RNA decreased further only in those patients with sustained suppression of plasma viral load, but remained almost always detectable at low levels, indicating persisting transcription of viral RNA. CD4 counts increased by 200 to 250 cells/&mgr;l at week 96 in both arms without significant differences (intent‐to‐treat analyses). Thus, even if treatment is initiated early in asymptomatic patients with preserved CD4 counts, three drugs are necessary to achieve sustained decreases of HIV load in blood and lymphoid tissue.

Combined effect of zidovudine (ZDV), lamivudine (3TC) and abacavir (ABC) antiretroviral therapy in suppressing in vitro FIV replication

In view of close similarities at the molecular and clinical levels, feline immunodeficiency virus (FIV) infection of the domestic cat is subject of increasing attention as an animal model for human immunodeficiency virus (HIV) infection. A range of reverse transcriptase inhibitors effective against HIV are also active against FIV, allowing successful use of the cat model to investigate drug interactions and resistance development. Nevertheless, while combined nucleoside analog and protease inhibitor usage has proven remarkably effective in treating HIV infection, combination antiretroviral therapy of FIV infection has been hampered by lack of protease inhibitors specific for FIV. In an attempt to circumvent this problem, we have examined the feasibility of applying in the FIV system combination protocols lacking a protease inhibitor. We now report that, as observed during HIV infection, the nucleoside analog abacavir (ABC or 1592U89) is able to effectively block in vitro FIV-replication. Furthermore, we demonstrate that combined usage of ABC with the nucleoside analogs zidovudine (ZDV or AZT) and lamivudine (3TC) also blocks in vitro FIV replication in a synergistic manner. However, in contrast to its effect on HIV replication, the ribonucleotide reductase inhibitor hydroxyurea (HU) is unable to effectively control in vitro FIV replication.

Quantification of in vitro retroviral replication using a one-tube real-time RT-PCR system incorporating direct RNA preparation.

The methodological and logistic benefits gained from assessing in vitro antiretroviral replication using one-tube real-time RT-PCR procedures are currently diminished by a continuing need for prior RNA isolation. We now report a simple and inexpensive modification of a commercially available one-tube RT-PCR assay, consisting of detergent-based virus lysis in the presence of a ribonuclease inhibitor, which can be used to directly quantify retroviral RNA levels in culture supernatant. This approach circumvents the potential loss of RNA inherent to RNA-isolation procedures based on prior extraction and demonstrates a dynamic range of at least 4 logs. Using in vitro culture systems incorporating either HIV-1 or FIV, we show that this ability to isolate retroviral RNA directly during the RT-PCR process can provide an equivalent alternative to one of the more time and resource-consuming steps in quantifying in vitro retroviral RNA levels.