Tarek Elbeik

The Relation of Virologic and Immunologic Markers to Clinical Outcomes after Nucleoside Therapy in HIV-Infected Adults with 200 to 500 CD4 Cells per Cubic Millimeter

BACKGROUND We studied measures of human immunodeficiency virus (HIV) replication, the viral phenotype, and immune function (CD4 cell counts) and the relation of changes in these indicators to clinical outcomes in a subgroup of patients in a controlled trial of early antiretroviral treatment for HIV, the AIDS Clinical Trials Group Study 175. METHODS The 391 subjects, each of whom entered the study with a single screening CD4 cell count of 200 to 500 per cubic millimeter, were randomly assigned to receive zidovudine alone, didanosine alone, zidovudine plus didanosine, or zidovudine plus zalcitabine. Plasma concentrations of HIV RNA were assessed in 366 subjects, and viral isolates from 332 subjects were assayed for the presence of the syncytium-inducing phenotype. RESULTS After eight weeks, the mean (+/-SE) decrease from base line in the concentration of HIV RNA, expressed as the change in the base 10 log of the number of copies per milliliter, was 0.26+/-0.06 for patients treated with zidovudine alone, 0.65+/-0.07 for didanosine alone, 0.93+/-0.10 for zidovudine plus didanosine, and 0.89+/-0.06 for zidovudine plus zalcitabine (P<0.001 for each of the pairwise comparisons with zidovudine alone). Multivariate proportional-hazards models showed that higher base-line concentrations of plasma HIV RNA, less suppression of plasma HIV RNA by treatment, and the presence of the syncytium-inducing phenotype were significantly associated with an increased risk of progression to the acquired immunodeficiency syndrome and death. After adjustment for these measures of viral replication and for the viral phenotype, CD4 cell counts were not significant predictors of clinical outcome. CONCLUSIONS Both the risk of the progression of HIV disease and the efficacy of antiretroviral therapy are strongly associated with the plasma level of HIV RNA and with the viral phenotype. The changes in the plasma concentration of HIV RNA predict the changes in CD4 cell counts and survival after treatment with reverse-transcriptase inhibitors.

Monitoring plasma HIV-1 RNA levels in addition to CD4+ lymphocyte count improves assessment of antiretroviral therapeutic response

The duration of disease-free survival after infection with human immunodeficiency virus type 1 (HIV-1) varies considerably during antiretroviral therapy. Patients with similar CD4+ lymphocyte counts progress at different rates when they are given the same antiretroviral therapy. Better prediction of risk for progression and its association with viral suppression may help improve antiretroviral management for individual patients and speed the development of new drugs. Higher plasma HIV-1 RNA levels are associated with poorer clinical status and lower CD4+ lymphocyte counts [1-3] and predict subsequent outcome [4-11]. The biological variability of plasma HIV-1 RNA levels in patients receiving stable therapeutic regimens must be quantified to define the magnitude of an antiviral effect that can be reliably detected after antiretroviral treatment is initiated. Determination of infectious HIV-1 titers in mononuclear cells of peripheral blood by quantitative microculture [12, 13] or syncytium-inducing phenotype of an HIV-1 isolate may provide information that is different from or complementary to the information gleaned from measuring plasma HIV-1 RNA levels [14-16]. However, studies have not yet conclusively determined whether measurements of CD4+ lymphocytes in conjunction with any or all of these virological variables should be recommended to optimize prediction or guide antiretroviral treatment more effectively. In this report, we quantify the relative roles of CD4+ lymphocyte counts, plasma HIV-1 RNA levels, infectious HIV-1 titers in mononuclear cells of peripheral blood, and the syncytium-inducing viral phenotype as predictors of disease progression during a clinical trial of combination therapy [17]. Our approach was to assess the value of plasma HIV-1 RNA levels and CD4+ lymphocyte count, both of which are readily available to clinicians, and then to assess the additional value of the infectious HIV-1 titer in mononuclear cells of peripheral blood and the syncytium-inducing viral phenotype. We also quantify the variability of plasma HIV-1 RNA levels. Our results suggest guidelines for using these measures in clinical practice for predicting the effectiveness of antiretroviral therapy over 1 year. Methods Study Design We prospectively evaluated virological, immunologic, and clinical data from patients who participated in the intensive virology substudy of ACTG (AIDS Clinical Trials Group) Protocol 241; ACTG Protocol 241 was a multicenter, randomized, double-blind, placebo-controlled trial of 398 patients receiving nevirapine, zidovudine, and didanosine compared with zidovudine and didanosine [17]. All patients at 8 of the 16 AIDS Clinical Trials Units who participated in the main study were enrolled in the substudy (n = 198). For 48 weeks, all 198 patients received open-label zidovudine (600 mg/d) and didanosine (400 mg/d for patients weighing 60 kg and 250 mg/d for patients weighing <60 kg). One hundred of the substudy patients were randomly assigned to receive nevirapine (200 mg/d for the first 2 weeks and 400 mg/d thereafter), and 98 were assigned to receive matching placebo. Participants gave written informed consent, and the protocol was approved by the institutional review board at each participating AIDS Clinical Trials Unit. The study was funded by the ACTG of the National Institute of Allergy and Infectious Diseases; supplemental funding for virology was provided by Boehringer Ingelheim Pharmaceuticals (Ridgefield, Connecticut). Study drugs were provided by Glaxo Wellcome (Research Triangle Park, North Carolina), Bristol-Myers Squibb (Princeton, New Jersey), and Boehringer Ingelheim Pharmaceuticals. However, all data were gathered by members of the ACTG and were analyzed and interpreted by the authors, who had sole responsibility for the decision to submit the manuscript for publication. Evaluation of Patients Stable therapy at baseline was defined as the absence of reported change in antiretroviral therapy from 30 days before the preentry visit until the entry visit. All patients were followed prospectively for progression of HIV-related disease. Progression was defined as the development of a new acquired immunodeficiency syndrome (AIDS)-defining event [18]; a newly diagnosed, deep-seated bacterial infection or bacteremia that was not related to injection drug use or an intravascular catheter; pulmonary or extrapulmonary tuberculosis; recurrent Pneumocystis carinii pneumonia; recurrent toxoplasmosis of the central nervous system; or death. Reports of disease progression were reviewed by the study chair; only events that could be confirmed were used in the analysis. We measured CD4+ lymphocyte counts, plasma HIV-1 RNA levels, and infectious HIV-1 titers in mononuclear cells of peripheral blood at the preentry visit (within 14 days of starting study treatment), at the entry visit (before starting study treatment and at least 72 hours after the preentry visit), and at the visits 8 and 48 weeks after the start of study treatment. Specimens could be obtained at any time of day. We used the geometric mean of preentry and entry measurements as the baseline value for each variable. The presence of the syncytium-inducing viral phenotype was determined at the entry visit. Standardized assays were used to determine CD4+ lymphocyte counts [19, 20], infectious HIV-1 titer in mononuclear cells of peripheral blood (in infectious units per million cells) using real-time testing [13, 21], and syncytium-inducing viral phenotype of a virus isolated from mononuclear cells of peripheral blood using MT-2 cells [22]. Plasma samples were frozen at 70C; HIV-1 RNA levels were measured by quantitative reverse transcription polymerase chain reaction assay (Roche Molecular Systems, Alameda, California, and Branchburg, New Jersey) [23]. The lower limit of detection for this assay was 200 HIV-1 RNA copies/mL. Levels of HIV-1 RNA in plasma samples collected from the same patient at the preentry, entry, week 8, and week 48 visits were determined in a single laboratory assay. Statistical Analysis Analysis of plasma HIV-1 RNA levels and infectious HIV-1 titers in mononuclear cells of peripheral blood was done after log10 transformation. Plasma levels of HIV-1 RNA that were below the detectable limit were assigned the value of 200 copies/mL. Infectious HIV-1 titers in mononuclear cells of peripheral blood outside the measurable range (0.22 to 7493 infectious units per million cells) were assigned the value of 0.22 infectious units per million cells if they were below the range and 7493 infectious units per million cells if they were above the range. Linear regression analysis [24] was used to compare the mean plasma HIV-1 RNA levels, infectious HIV-1 titers in mononuclear cells of peripheral blood, and CD4+ lymphocyte counts according to patient characteristics at baseline and to assess factors associated with the long-term change (from baseline to week 48) in CD4+ lymphocyte counts. Logistic regression analysis [25] was used to assess the association at baseline of the percentage of patients who had AIDS with virological measures and CD4+ lymphocyte counts. The intrapatient SD of plasma HIV-1 RNA levels was estimated using the method of moments for variance components [26]. Spearman correlation coefficients were used to assess the association between preentry and entry measurements. Proportional hazards models [27] were used to assess the association between the risk for disease progression or death and baseline levels and early changes (from baseline to week 8) in plasma HIV-1 RNA levels, infectious HIV-1 titers in mononuclear cells of peripheral blood, and log-transformed CD4+ lymphocyte counts as well as baseline syncytium-inducing viral phenotype. These models were stratified by study treatment to control for any differential effects of the two study regimens. Results Patient Characteristics at Study Entry The mean CD4+ lymphocyte count of the 198 patients before treatment was 145 cells/mm3 (range, 1 to 443 cells/mm3). Patients were a median of 39 years of age, predominantly male (81%), predominantly white (76%), and predominantly free of a previous AIDS-defining diagnosis (86%). All but 3 patients had taken zidovudine before study entry, 44% had taken didanosine, and 35% had taken zalcitabine. The median duration of cumulative previous nucleoside therapy was 25 months, and 34% of patients had received therapy for longer than 36 months. Virological Measures at Baseline by Patient Characteristics Table 1 shows the mean plasma HIV-1 RNA levels, infectious HIV-1 titers in mononuclear cells of peripheral blood, and CD4+ lymphocyte counts at baseline for patients stratified by characteristics that were significantly associated with viral load. We also assessed the associations with age, sex, racial or ethnic group, self-reported homosexuality, and duration of previous nucleoside therapy, but these associations were not significant. Table 1. Plasma HIV-1 RNA Level, Infectious HIV-1 Titer in Mononuclear Cells of Peripheral Blood, and CD4+ Lymphocyte Count at Baseline* Patients with a history of AIDS had a significantly higher mean baseline level of HIV-1 RNA in plasma and a significantly lower mean CD4+ lymphocyte count than did those without such a history (Table 1). More patients with a history of AIDS than those without had baseline HIV-1 isolates with the syncytium-inducing viral phenotype (58% compared with 36%; P = 0.015). However, in a multivariate analysis, only the CD4+ lymphocyte count at baseline was significantly associated with a history of AIDS. Thus, disease status at baseline was explained by CD4+ lymphocyte counts and not by any of the virological measures that were considered. Variability of Virological Measures in Patients Receiving Stable Treatment Variation in plasma HIV-1 RNA levels was evaluated by comparing the preentry and entry measures from the 167 patients who reported no changes in treatment from 30 days

Correlation between response to acyclovir and foscarnet therapy and in vitro susceptibility result for isolates of herpes simplex virus from human immunodeficiency virus-infected patients.

In vitro susceptibility testing of herpes simplex virus (HSV) isolates will play an increasingly important role in guiding the clinical management of immunocompromised hosts who have lesions that are poorly responsive to therapy with standard antiviral agents. We assessed the correlation between the in vitro susceptibility result using a plaque reduction assay in Vero cells and the response to antiviral therapy with acyclovir or foscarnet for 243 clinical isolates of HSV collected from 115 human immunodeficiency virus-infected patients. The in vitro results and clinical responses were highly associated for both acyclovir and foscarnet (P < 0.001 and P < 0.001, respectively). The predictive values of a susceptible result (50% effective concentrations, < 2 micrograms/ml for acyclovir and < 100 micrograms/ml for foscarnet) for complete healing of lesions were 62% for acyclovir and 82% for foscarnet; the predictive values of a resistant result for failure to heal were 95% for acyclovir and 88% for foscarnet. Thus, in vitro testing has clinical utility in guiding therapy, although the 1 to 2 weeks required to derive a definitive result by the plaque reduction assay is a major limitation.

Thalidomide stimulates T cell responses and interleukin 12 production in HIV-infected patients.

We performed a placebo-controlled study to evaluate the effects of immunomodulatory treatment with thalidomide on HIV levels, TNF-alpha levels, and immune status of 31 HIV-infected individuals, after temporary suppression of viral replication with antiretroviral drugs. Treatment with a combination of zidovudine and lamivudine (ZDV/LMV) for 14 days resulted in a median decline in plasma viremia of 1.94 log10 RNA equivalents/ml. After discontinuation of ZDV/LMV, thalidomide therapy (200 mg/day for 4 weeks) did not retard the prompt return of HIV titers to the pretreatment levels, and had no effect on plasma levels of TNF-alpha. In contrast, thalidomide treatment resulted in significant immune stimulation. We observed increased levels of plasma soluble IL-2 receptor, soluble CD8 antigen, and IL-12 (p < 0.01 for all parameters), as well as increased cutaneous delayed-type hypersensitivity reactions to recall antigens (p < 0.01) in thalidomide-treated patients. These changes were associated with a median increase in HIV titer of 0.2 log10 RNA equivalents/ml in the thalidomide-treated group (p < 0.05), which resolved after stopping the drug. Further studies were performed in vitro to elucidate the mechanism of thalidomide-induced immune stimulation. When purified T cells from HIV-infected individuals were stimulated by immobilized anti-CD3 in the presence of thalidomide, a costimulatory effect of the drug was observed, resulting in increased production of IL-2 and IFN-gamma, and increased T cell-proliferative responses. Further experiments showed that thalidomide increased IL-12 production by antigen-presenting cells in a T cell-dependent manner. Our findings suggest a potential application for thalidomide as a novel immune adjuvant in HIV disease.

Multicenter Evaluation of the Performance Characteristics of the Bayer VERSANT HCV RNA 3.0 Assay (bDNA)

ABSTRACT In this multicenter evaluation, the VERSANT HCV RNA 3.0 Assay (bDNA) (Bayer Diagnostics, Tarrytown, N.Y.) was shown to have excellent reproducibility, linearity, and analytical sensitivity across specimen collection matrices (serum, EDTA, ACD-A), and hepatitis C virus (HCV) genotypes 1 to 6. The VERSANT HCV bDNA Assay has a reportable range of 615 to 7,690,000 (7.69 × 106) IU/ml. The total coefficient of variation (CV) ranged from 32.4% at 615 IU/ml to 17% at 6.8 × 106 IU/ml. The assay was linear across the reportable range. Analytical specificity of 98.8% was determined by testing 999 specimens from volunteer blood donors. Evaluation of HCV genotypes using RNA transcripts of representative clones of 1a, 1b, 2a, 2b, 2c, 3a, 4a, 5a, and 6a and patient specimens showed that the largest difference between genotype 1, upon which the assay is standardized, and non-1 genotypes was within 1.5-fold. Testing of potentially interfering endogenous substances and exogenous substances and conditions found no interference in HCV-positive or HCV-negative specimens except for unconjugated bilirubin at concentrations of ≥20 mg/dl and protein at concentrations of ≥9 g/dl. Biological variability was estimated from 29 clinically stable individuals not on HCV therapy who were tested weekly over an 8-week period. The combined estimate of total (biologic plus assay) variability was 0.15 log10 standard deviation (CV, 36.1%), a fold change of 2.6. Thus, the observed fold change between any two consecutive HCV RNA measures is expected to be less than 2.6-fold (equivalent to 0.41 log10 IU/ml) 95% of the time in clinically stable individuals.

Diminished Spontaneous Apoptosis in Lymphocytes from Human Immunodeficiency Virus-Infected Long-Term Nonprogressors

The relationship between peripheral lymphocyte apoptosis and human immunodeficiency virus disease progression was studied in infected subgroups with distinct profiles of progression. Long-term nonprogressors (LTNP) and seronegative controls had levels of spontaneous apoptosis significantly lower than those for recent seroconverters who had CD4 cell counts similar to those of nonprogressors but with a high likelihood of disease progression. Lymphocytes from nonprogressors and seronegative controls also showed negligible spontaneous caspase-3 activity, a biochemical indicator for apoptosis, whereas early progressors exhibited substantial activity. In contrast, when activated with mitogens, the lymphocytes from both LTNP and progressors displayed indistinguishable levels of heightened apoptosis. Spontaneous apoptosis and plasma viremia levels correlated positively in progressors, but not in LTNP. These findings demonstrate that increased lymphocyte apoptosis is evident prior to CD4 T cell decline and that LTNP are relatively resistant to the factors that induce accentuated levels of spontaneous but not mitogen-induced cell death.

Comparative analysis of HIV-1 viral load assays on subtype quantification: Bayer Versant HIV-1 RNA 3.0 versus Roche Amplicor HIV-1 Monitor version 1.5

Summary: Quantification of HIV‐1 subtypes is essential for appropriate clinical management. Whereas viral load assays were initially developed to accurately quantify subtype B, the recent worldwide spread of non‐B subtypes and the introduction of treatment programs in regions with non‐B subtypes have prompted adaptations of these assays. The Bayer Versant HIV‐1 RNA 3.0 Assay (branched DNA [bDNA] 3.0) and the Roche Amplicor HIV‐1 Monitor version 1.5 (Amplicor 1.5) assays are reported to quantify all subtypes in group M; however, evaluation of performance characteristics remains limited. In this study, we evaluated the accuracy and reliability of bDNA 3.0 and Amplicor 1.5 on multiple serially diluted viral isolates from HIV‐1 group M, subtypes A through F. Testing was conducted on both assay systems in two independent laboratories. Comparative pansubtype quantification from regression analysis showed that quantification by bDNA 3.0 was approximately 0.3 log‐fold lower than that by Amplicor 1.5. Comparative pansubtype accuracy analysis showed data points more closely distributed about their respective regression lines and thus showing greater reliability by bDNA 3.0 than by Amplicor 1.5.

Variability and Prognostic Values of Virologic and CD4 Cell Measures in Human Immunodeficiency Virus Type 1-Infected Patients with 200–500 CD4 Cells/mm3 (ACTG 175)

Virologic measurements are increasingly used to evaluate prognosis and treatment responses in human immunodeficiency virus (HIV) type 1 infection. Markers of HIV-1 replication, including infectious HIV-1 titer from peripheral blood mononuclear cells, serum HIV-1 p24 antigen, plasma HIV-1 RNA, CD4 cell numbers, and viral syncytium-inducing (SI) phenotype, were determined in 391 virology substudy participants in AIDS Clinical Trials Group study 175. The subjects had 200-500 CD4 cells/mm3. All markers of viral replication significantly correlated with one another and were inversely related to CD4 cell number. Disease progression to an AIDS-defining event or death or loss of >50% of CD4 cells was associated with infectious HIV-1 titer (P < .001), HIV-1 RNA (P < .001), and HIV-1 p24 antigen (P = .007). In multivariate proportional hazards models, p24 antigen was never significant when HIV-1 RNA level was included. In a model containing infectious HIV-1 titer (P = .038), HIV-1 RNA (P < .001), SI phenotype (P < .001), and CD4 cell number (P = .18), only the virologic parameters remained significantly associated with progression.

Simultaneous Runs of the Bayer VERSANT HIV-1 Version 3.0 and HCV bDNA Version 3.0 Quantitative Assays on the System 340 Platform Provide Reliable Quantitation and Improved Work Flow

ABSTRACT Branched DNA (bDNA) assays to quantify human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) consist of three distinct steps, including sample processing, hybridization, and detection, and utilize the System 340 platform for plate incubation and washing. Sample processing differs: HIV-1 from 1 ml of plasma is concentrated by high-speed centrifugation, whereas HCV plasma or serum samples are used without concentration. The first step of hybridization involves viral lysis at 63°C: HIV-1 is performed in a heat block, whereas HCV is performed in System 340. The remaining hybridization and detection steps are similar for HIV-1 and HCV and executed on System 340. In the present study, the HIV-1 bDNA assay was adapted for viral lysis in the System 340 platform. The adaptation, test method 2, includes a 20-s vortex of concentrated viral pellet and lysis working solution, transfer of viral lysate to the 96-well capture plate, and transfer to System 340 programmed for HCV assay specifications. With test method 2, specificity and quantification were within assay specifications. HCV bDNA methodology remains unchanged. Hence, an HIV-1 and an HCV bDNA can be run simultaneously on System 340. With simultaneous testing, laboratories can run full plates, as well as combinations of full and partial plates. Also, simultaneous HIV-1 and HCV bDNA permits labor consolidation and improved workflow while maintaining multitasking and rapid patient result turnaround.

Short Communication Antibody and Cellular Immune Responses in Breakthrough Infection Subjects after HIV Type 1 Glycoprotein 120 Vaccination

HIV-specific antibodies and CD8+ T cell antiviral responses were evaluated in three human immunodeficiency virus 1 (HIV-1) gp120 vaccine recipients who later became infected with HIV-1. Titers of neutralizing antibody to the HIV-1(SF2) vaccine isolate were boosted, but titers of antibody to the autologous infecting viruses were never high and required at least 6 months after HIV infection to develop. Similarly, a marginal noncytotoxic CD8+ T cell antiviral response was observed only in one of the three vaccinees 3 months after HIV-1 infection. The infecting virus isolates had several amino acid substitutions in the HIV-1 envelope V3 region but were similar to other regional HIV-1 clade B isolates. Viral loads were similar to those of other HIV-1-infected individuals who had not been vaccinated and transient CD4+ T cell declines were observed in each person, suggesting that the vaccine was not effective at controlling these prognostic markers early in infection.