David Katzenstein

A Trial Comparing Nucleoside Monotherapy with Combination Therapy in HIV-Infected Adults with CD4 Cell Counts from 200 to 500 per Cubic Millimeter

BACKGROUND This double-blind study evaluated treatment with either a single nucleoside or two nucleosides in adults infected with human immunodeficiency virus type 1 (HIV-1) whose CD4 cell counts were from 200 to 500 per cubic millimeter. METHODS We randomly assigned 2467 HIV-1--infected patients (43 percent without prior antiretroviral treatment) to one of four daily regimens: 600 mg of zidovudine; 600 mg of zidovudine plus 400 mg of didanosine; 600 mg of zidovudine plus 2.25 mg of zalcitabine; or 400 mg of didanosine. The primary end point was a > or = 50 percent decline in the CD4 cell count, development of the acquired immunodeficiency syndrome (AIDS), or death. RESULTS Progression to the primary end point was more frequent with zidovudine alone (32 percent) than with zidovudine plus didanosine (18 percent; relative hazard ratio, 0.50; P<0.001), zidovudine plus zalcitabine (20 percent; relative hazard ratio, 0.54; P<0.001), or didanosine alone (22 percent; relative hazard ratio, 0.61; P<0.001). The relative hazard ratios for progression to an AIDS-defining event or death were 0.64 (P=0.005) for zidovudine plus didanosine, as compared with zidovudine alone, 0.77 (P=0.085) for zidovudine plus zalcitabine, and 0.69 (P=0.019) for didanosine alone. The relative hazard ratios for death were 0.55 (P=0.008), 0.71 (P=0.10), and 0.51 (P=0.003), respectively. For zidovudine plus zalcitabine, the benefits were limited to those without previous treatment. CONCLUSIONS Treatment with zidovudine plus didanosine, zidovudine plus zalcitabine, or didanosine alone slows the progression of HIV disease and is superior to treatment with zidovudine alone. Antiretroviral therapy can improve survival in patients with 200 to 500 CD4 cells per cubic millimeter.

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.

Reduced intraepidermal nerve fiber density in HIV-associated sensory neuropathy

Objective: To explore the relationship between intraepidermal nerve fiber (IENF) density in HIV-associated sensory neuropathy (HIV-SN) to measurements of neuropathy severity and progression of HIV disease. Background: SN affects 30% of individuals with AIDS, and treatment is often ineffective. Recombinant human nerve growth factor (rhNGF) has been proposed as a trophic factor for unmyelinated nerve fibers injured in HIV-SN, and a clinical trial has recently concluded. Skin biopsy with IENF density determination has emerged as a diagnostic test for patients with small-fiber sensory neuropathy. Methods: Sixty-two of the 270 patients with HIV-SN who participated in the trial of rhNGF were included in a substudy examining epidermal nerve fibers. IENF density was compared with neuropathic pain intensity (measured with the Gracely Pain Scale), patient and physician global pain assessments, quantitative sensory testing, CD4 counts, and plasma HIV RNA levels both at baseline and at conclusion of the placebo-controlled phase. Results: IENF density was inversely correlated with neuropathic pain as measured by patient (p = 0.004) and physician (p = 0.05) global pain assessments, but not using the Gracely Pain Scale. Decreased IENF density at the distal leg was associated with lower CD4 counts and higher plasma HIV RNA levels. IENF density measurements were stable over time. Conclusions: IENF loss at the distal leg is associated with increased neuropathic pain, lower CD4 counts, and higher plasma viral load in HIV-SN. The robustness of the longitudinal measurement of IENF density supports its use in future longitudinal studies and clinical trials.

Abacavir–Lamivudine versus Tenofovir–Emtricitabine for Initial HIV-1 Therapy

BACKGROUND The use of fixed-dose combination nucleoside reverse-transcriptase inhibitors (NRTIs) with a nonnucleoside reverse-transcriptase inhibitor or a ritonavir-boosted protease inhibitor is recommended as initial therapy in patients with human immunodeficiency virus type 1 (HIV-1) infection, but which NRTI combination has greater efficacy and safety is not known. METHODS In a randomized, blinded equivalence study involving 1858 eligible patients, we compared four once-daily antiretroviral regimens as initial therapy for HIV-1 infection: abacavir-lamivudine or tenofovir disoproxil fumarate (DF)-emtricitabine plus efavirenz or ritonavir-boosted atazanavir. The primary efficacy end point was the time from randomization to virologic failure (defined as a confirmed HIV-1 RNA level > or = 1000 copies per milliliter at or after 16 weeks and before 24 weeks, or > or = 200 copies per milliliter at or after 24 weeks). RESULTS A scheduled interim review by an independent data and safety monitoring board showed significant differences in virologic efficacy, according to the NRTI combination, among patients with screening HIV-1 RNA levels of 100,000 copies per milliliter or more. At a median follow-up of 60 weeks, among the 797 patients with screening HIV-1 RNA levels of 100,000 copies per milliliter or more, the time to virologic failure was significantly shorter in the abacavir-lamivudine group than in the tenofovir DF-emtricitabine group (hazard ratio, 2.33; 95% confidence interval, 1.46 to 3.72; P<0.001), with 57 virologic failures (14%) in the abacavir-lamivudine group versus 26 (7%) in the tenofovir DF-emtricitabine group. The time to the first adverse event was also shorter in the abacavir-lamivudine group (P<0.001). There was no significant difference between the study groups in the change from the baseline CD4 cell count at week 48. CONCLUSIONS In patients with screening HIV-1 RNA levels of 100,000 copies per milliliter or more, the times to virologic failure and the first adverse event were both significantly shorter in patients randomly assigned to abacavir-lamivudine than in those assigned to tenofovir DF-emtricitabine. (ClinicalTrials.gov number, NCT00118898.)

A phase II trial of nerve growth factor for sensory neuropathy associated with HIV infection

Objective: To evaluate the safety and efficacy of recombinant human nerve growth factor (rhNGF) in HIV-associated sensory neuropathy (SN) within a multicenter, placebo-controlled, randomized trial (ACTG 291). Background: SN affects 30% of individuals with AIDS, is worsened by neurotoxic antiretrovirals, and its treatment is often ineffective. NGF is trophic for small sensory neurons and stimulates the regeneration of damaged nerve fibers. Methods: A total of 270 patients with HIV-associated SN were randomized to receive placebo, 0.1 μg/kg rhNGF, or 0.3 μg/kg rhNGF by double-blinded subcutaneous injection twice weekly for 18 weeks. The primary outcome was change in self-reported neuropathic pain intensity (Gracely Pain Scale). Secondary outcomes included an assessment of global improvement in neuropathy by patients and investigators, neurologic examination, use of prescription analgesics, and quantitative sensory testing. In a subset, epidermal nerve fiber densities were determined in punch skin biopsies. Results: Both doses of NGF produced significant improvements in average and maximum daily pain compared with placebo. Positive treatment effects were also observed for global pain assessments (p = 0.001) and for pin sensitivity (p = 0.019). No treatment differences were found with respect to mood, analgesic use, or epidermal nerve fiber densities. Injection site pain was the most frequent adverse event, and resulted in unblinding in 39% of subjects. Severe transient myalgic pain occurred in eight patients, usually from accidental overdosing. There were no changes in HIV RNA levels or other laboratory indices. Conclusions: We found a positive effect of recombinant human nerve growth factor on neuropathic pain and pin sensitivity in HIV-associated sensory neuropathy. rhNGF was safe and well tolerated, but injection site pain was frequent.

HIV-1 genotypic resistance patterns predict response to saquinavir-ritonavir therapy in patients in whom previous protease inhibitor therapy had failed.

Combination antiretroviral therapy for HIV-1 infection has resulted in profound control of HIV replication in vivo, improved immune function, and significant decreases in AIDS-related morbidity and mortality (1-9). For many persons, however, this therapy does not provide sustained viral suppression or durable clinical benefit (10, 11). Potential reasons for the loss of viral suppression include host immune defects, poor adherence to therapy, pharmacologic factors, and drug resistance (10-17). However, HIV-1 resistance to drug therapy is probably the central factor in the loss of viral suppression (18-22). Mutations that result in reduced drug susceptibility have been demonstrated in vitro for all currently available antiretroviral agents, and some of these mutations have been associated with increasing plasma HIV-1 RNA levels and disease progression in clinical trials (19-30). Genotypic and phenotypic methods of measuring drug resistance are increasingly available to clinicians (31-37). However, the role of these tests in clinical practice has not been fully assessed. Many experts have been skeptical of resistance testing, although a recent consensus statement provides cautious support for testing in certain clinical circumstances (38-42). Our objective was to determine the genotypic predictors of virologic response to saquinavirritonavir combination therapy in patients in whom therapy with at least one protease inhibitor-containing antiretroviral regimen had failed. We investigated whether HIV-1 reverse transcriptase and protease genotype predicts virologic response to saquinavirritonavir by week 12 and week 26 and compared those data with predictors from clinical and antiretroviral treatment history. Methods Patients Two of the investigators treated patients in a university-based clinic that provides primary care for 500 HIV-infected adults. We identified 54 patients who received saquinavirritonavir between October 1996 and February 1998 after therapy with at least one protease inhibitor-containing antiretroviral regimen had failed. Treatment failure was defined as a greater than 0.5 log10 copies/mL (more than threefold) increase in plasma HIV RNA level from a nadir value, an HIV RNA level greater than 10 000 copies/mL, or detectable HIV RNA after the level had been below the threshold of detection (<500 copies/mL) during a therapeutic regimen for more than 12 weeks. Study patients received 400 to 600 mg of saquinavir in a hard-gel formulation (Invirase, Roche Laboratories, Nutley, New Jersey) and 300 to 400 mg of ritonavir in capsule form (Norvir, Abbott Laboratories, Abbott Park, Illinois) twice daily. In addition to the two protease inhibitors, 47 patients (87%) received two nucleoside reverse transcriptase inhibitors, 4 received three nucleoside reverse transcriptase inhibitors, 2 received two nucleoside reverse transcriptase inhibitors and either nevirapine or delavirdine, and one patient received lamivudine. Clinical and demographic variables were abstracted from the medical records. Adherence, as recorded in the patients record, was categorized by the self-reported number of missed doses in the month before evaluation and was classified as none, one to two, three to seven, or eight or more. Plasma levels of HIV-1 RNA were monitored on average every 4 to 6 weeks, and samples were stored at 70 C. The Stanford University Panel on Medical Human Subjects approved this study (#M1272). HIV Genotyping Baseline HIV-1 genotype was evaluated in plasma specimens that were stored within 1 month before initiation of saquinavirritonavir therapy and were obtained while patients were still receiving an ineffective antiretroviral regimen. Plasma HIV-1 RNA was extracted, and nested polymerase chain reaction (PCR) amplification generated a 1.3-kb fragment encompassing protease and the first 750 nucleotides of reverse transcriptase (43, 44). Direct bidirectional dideoxynucleotide terminator cycle sequencing of the PCR product was performed as described elsewhere (44). Sequencing reactions were analyzed by using an ABI 377 instrument (Perkin-Elmer, Foster City, California) and were manually proofread and edited. Sequences were compared to the HIV-1 clade B consensus sequence (Los Alamos database), and differences in amino acid sequence, including positions that contained a mixture of wild-type and mutant residues, were classified as mutations (45). Phylogenetic analysis of HIV-1 RNA sequence verified lack of cross-contamination (data not shown). A priori, we decided to assess reverse transcriptase codons 41, 67, 69, 70, 74, 75, 151, 184, 210, 215, and 219 as predictors of virologic response. Mutations at these codons are known to be associated with resistance to one of the nucleoside reverse transcriptase inhibitors (25, 45). In the protease gene, mutations at codons 30, 46, 48, 54, 82, 84, and 90 were evaluated as potential predictors. We chose these major mutations a priori because they are associated with in vitro resistance to a protease inhibitor or occur commonly in patients in whom therapy with currently licensed protease inhibitors is failing. We also evaluated all other protease codons as predictors of response. Virologic Outcomes Virologic response to saquinavirritonavir was measured at two time points between 3 and 18 weeks and again around week 24 (range, 22 to 36 weeks); the median time points of the three follow-up evaluations were 4, 12, and 26 weeks. Levels of HIV-1 RNA were measured by using the Amplicor HIV Monitor Assay (Roche Molecular Systems, Alameda, California). Specimens with HIV RNA below the level of detection (<500 copies/mL) on this assay were retested by using the ultrasensitive modification with a lower limit of detection of less than 50 copies/mL (43). Virologic response to saquinavirritonavir therapy was categorized on the basis of the larger response from baseline to the first or second evaluation [median, 4 and 12 weeks]. The ordinal categories were 1) complete response if the plasma HIV-1 RNA level was less than 500 copies/mL, 2) partial response if the reduction from baseline RNA level was 0.5 log10 copies/mL or more but was not less than 500 copies/mL, and 3) nonresponse if reduction from baseline values was less than 0.5 log10 copies/mL. Statistical Analysis Demographic, clinical, and genotypic variables were analyzed as potential predictors of virologic response by using bivariate linear regression and multivariable linear regression. In the multivariable models, we included a subset of the reverse transcriptase mutations (listed above) identified through stepwise regression as significant (P<0.05) predictors. This subset of mutations was included in the model as a signed-sum variable. For the protease mutations, we included the signed sum of the seven major mutations listed above and the signed sum of three additional mutations at codons 10, 19, and 71, which were found to be statistically significant bivariate predictors. The signed-sum variable is derived by a summation of the relevant mutations identified in the baseline sequence. A separate sum is derived from the seven major protease mutations, the three additional protease mutations, and the subset of statistically significant reverse transcriptase mutations (codons 69 and 210). In the signed-sum variable, mutations that are positively associated with virologic outcome (such as protease mutation D30N) are assigned a positive sign (+1) and mutations negatively associated with outcome are assigned a negative sign (1). We used the Cook distance to assess skewing of the ordinal outcome variable in the final multivariable model (model 5, Table 3). The value of 0.11 indicated no significant skewing; this result supports the use of linear regression models (46). We also evaluated the multivariate models for bias that would result from overfitting of the data. We used a bootstrap technique to estimate bias (optimism) in the explained variance values (R 2) for the models presented and found minimal upward bias; for example, model 3 in Table 3 has a bias of approximately one fifth of the SE (data for other models not shown) (47). A bootstrap technique was used to provide the 95% CI estimates for the R 2 values in Table 3. We selected 25 bootstrap samples of 54 with replacement from the original 54 patients to estimate the 95% CIs. The Wilcoxon rank test was used for comparisons between specific previous protease inhibitors in Table 1, and the F test was used for comparisons between models in Table 3. Two-sided P values are reported for all analyses. All analyses were conducted by using S-PLUS, version 4.0 (MathSoft, Seattle, Washington). Table 1. Baseline Demographics, Clinical Characteristics, and Antiretroviral Treatment History as Predictors of Virologic Response to saquinavirritonavir Therapy Table 2. Protease Mutation Patterns at Baseline and Response to saquinavirritonavir Combination Therapy Table 3. Multivariable Linear Regression Models of Clinical, Antiretroviral Treatment History, and HIV-1 Genotypic Predictors of Virologic Response by Week 12 of saquinavirritonavir Therapy Results Virologic Response to saquinavirritonavir Therapy Of the 54 study patients, 22 (41%) achieved a complete response, with plasma HIV-1 RNA levels less than 500 copies/mL by the second follow-up evaluation (at a median of 12 weeks). Of these 22 patients, 10 (18.5% of the entire cohort) achieved a plasma HIV-1 RNA level less than 50 copies/mL. Fourteen patients (26%) had a partial response to saquinavirritonavir, and 18 (33%) were nonresponders (Table 1). The virologic response to saquinavirritonavir is shown by initial response category in Figure 1. The response waned somewhat in the partial and complete response groups by week 26: The HIV RNA level remained below 500 copies/mL in 15 patients (28%) and below 50 copies/mL in 10 patients (19%). Figure 1. Virologic response to saquinavir plus ritonavir through week 26 based on response by week 12. Pred

Hierarchical Targeting of Subtype C Human Immunodeficiency Virus Type 1 Proteins by CD8+ T Cells: Correlation with Viral Load

ABSTRACT An understanding of the relationship between the breadth and magnitude of T-cell epitope responses and viral loads is important for the design of effective vaccines. For this study, we screened a cohort of 46 subtype C human immunodeficiency virus type 1 (HIV-1)-infected individuals for T-cell responses against a panel of peptides corresponding to the complete subtype C genome. We used a gamma interferon ELISPOT assay to explore the hypothesis that patterns of T-cell responses across the expressed HIV-1 genome correlate with viral control. The estimated median time from seroconversion to response for the cohort was 13 months, and the order of cumulative T-cell responses against HIV proteins was as follows: Nef > Gag > Pol > Env > Vif > Rev > Vpr > Tat > Vpu. Nef was the most intensely targeted protein, with 97.5% of the epitopes being clustered within 119 amino acids, constituting almost one-third of the responses across the expressed genome. The second most targeted region was p24, comprising 17% of the responses. There was no correlation between viral load and the breadth of responses, but there was a weak positive correlation (r = 0.297; P = 0.034) between viral load and the total magnitude of responses, implying that the magnitude of T-cell recognition did not contribute to viral control. When hierarchical patterns of recognition were correlated with the viral load, preferential targeting of Gag was significantly (r = 0.445; P = 0.0025) associated with viral control. These data suggest that preferential targeting of Gag epitopes, rather than the breadth or magnitude of the response across the genome, may be an important marker of immune efficacy. These data have significance for the design of vaccines and for interpretation of vaccine-induced responses.

Use of Changes in Plasma Levels of Human Immunodeficiency Virus Type 1 RNA to Assess the Clinical Benefit of Antiretroviral Therapy

Data from 1330 human immunodeficiency virus type 1 (HIV-1)-infected patients enrolled in seven antiretroviral treatment trials were analyzed to characterize the clinical benefit of treatment-mediated reductions in plasma HIV-1 RNA levels. The risk of a new AIDS-defining event or death was reduced proportionally to the magnitude of the reduction of the HIV-1 RNA level during the first 6 months of therapy. Pretherapy HIV-1 RNA levels were prognostic independently of on-therapy levels. In addition, the reduction in risk associated with any given reduction of the level of HIV-1 RNA did not vary by pretherapy level. Having either a reduction in HIV-1 RNA level or an increase in CD4+ lymphocyte count, or both, was associated with a delay in clinical disease progression. This indicates that patient prognosis should be assessed using both HIV-1 RNA and CD4+ lymphocyte responses to therapy.

Atazanavir Plus Ritonavir or Efavirenz as Part of a 3-Drug Regimen for Initial Treatment of HIV-1: A Randomized Trial

BACKGROUND Limited data compare once-daily options for initial therapy for HIV-1. OBJECTIVE To compare time to virologic failure; first grade-3 or -4 sign, symptom, or laboratory abnormality (safety); and change or discontinuation of regimen (tolerability) for atazanavir plus ritonavir with efavirenz-containing initial therapy for HIV-1. DESIGN A randomized equivalence trial accrued from September 2005 to November 2007, with median follow-up of 138 weeks. Regimens were assigned by using a central computer, stratified by screening HIV-1 RNA level less than 100 000 copies/mL or 100 000 copies/mL or greater; blinding was known only to the site pharmacist. (ClinicalTrials.gov registration number: NCT00118898) SETTING 59 AIDS Clinical Trials Group sites in the United States and Puerto Rico. PATIENTS Antiretroviral-naive patients. INTERVENTION Open-label atazanavir plus ritonavir or efavirenz, each given with with placebo-controlled abacavir-lamivudine or tenofovir disoproxil fumarate (DF)-emtricitabine. MEASUREMENTS Primary outcomes were time to virologic failure, safety, and tolerability events. Secondary end points included proportion of patients with HIV-1 RNA level less than 50 copies/mL, emergence of drug resistance, changes in CD4 cell counts, calculated creatinine clearance, and lipid levels. RESULTS 463 eligible patients were randomly assigned to receive atazanavir plus ritonavir and 465 were assigned to receive efavirenz, both with abacavir-lamivudine; 322 (70%) and 324 (70%), respectively, completed follow-up. The respective numbers of participants in each group who received tenofovir DF-emtricitabine were 465 and 464; 342 (74%) and 343 (74%) completed follow-up. Primary efficacy was similar in the group that received atazanavir plus ritonavir and and the group that received efavirenz and did not differ according to whether abacavir-lamivudine or tenofovir DF-emtricitabine was also given. Hazard ratios for time to virologic failure were 1.13 (95% CI, 0.82 to 1.56) and 1.01 (CI, 0.70 to 1.46), respectively, although CIs did not meet prespecified criteria for equivalence. The time to safety (P = 0.048) and tolerability (P < 0.001) events was longer in persons given atazanavir plus ritonavir than in those given efavirenz with abacavir-lamivudine but not with tenofovir DF-emtricitabine. LIMITATIONS Neither HLA-B*5701 nor resistance testing was the standard of care when A5202 enrolled patients. The third drugs, atazanavir plus ritonavir and efavirenz, were open-label; the nucleoside reverse transcriptase inhibitors were prematurely unblinded in the high viral load stratum; and 32% of patients modified or discontinued treatment with their third drug. CONCLUSION Atazanavir plus ritonavir and efavirenz have similar antiviral activity when used with abacavir-lamivudine or tenofovir DF-emtricitabine. PRIMARY FUNDING SOURCE National Institutes of Health.

Detection and Qualification of Human Immunodeficiency Virus RNA in Patient Serum by Use of the Polymerase Chain Reaction

Human immunodeficiency virus (HIV) RNA was detected and quantified in the serum of HIV-seropositive individuals using the polymerase chain reaction (PCR) and a nonisotopic enzyme-linked affinity assay. Of 55 HIV-infected patients who were not receiving therapy, serum HIV RNA was detected in 9 of 19 who were asymptomatic, 11 of 16 with AIDS-related complex (ARC), and 18 of 20 with AIDS, with copy numbers ranging from 10(2) to greater than or equal to 5 x 10(4) 200 microliters of serum based on a relationship between absorbance and known copy number of gag gene RNA. Linear regression analysis demonstrated a correlation between infectious titer in 42 patient sera cocultured with donor peripheral blood mononuclear cells (PBMC) and PCR product absorbance (r = .70, P less than .01). Serum HIV RNA detected by PCR also correlated with serum p24 antigen positivity, CD4 counts less than 400/mm3, and the presence of HIV-related symptoms or disease. Quantification of infectious HIV RNA in cell-free serum by PCR may be useful as a marker for for disease progression or in monitoring antiviral therapy.