Ann J. Melvin

Evidence that low-level viremias during effective highly active antiretroviral therapy result from two processes : Expression of archival virus and replication of virus

ABSTRACT Episodes of low-level viremia (LLV), with plasma human immunodeficiency virus type 1 (HIV-1) RNA levels ranging from 50 to 400 copies (c)/ml, occur commonly during highly active antiretroviral therapy (HAART). LLV has been associated with virologic failure of HAART in some studies, while in others LLV did not appear to affect the clinical outcome. To understand the processes leading to LLV, genetic analyses were used to determine whether plasma virions emanated from archived or from newly evolved viral genomes. Episodes of LLV (plasma HIV-1 RNA, 50 to 379 [median, 77] c/ml) were detected in 21/37 (57%) HIV-1-infected children with median plasma HIV-1 RNA levels of <50 c/ml during 79 patient years of HAART. Viral sequences were derived by direct sequencing of PCR products from 21 plasma specimens diluted to end point. In phylogenetic analysis, LLV viral sequences grouped with virus from early in the course of infection in 8/11 subjects. Six specimens had multiple identical viral sequences, suggesting origin from clonally expanded infected cells. LLV plasma virus evolved over time, indicating viral replication, in 3/11 subjects. Two of these had frequent LLV, including the selection of drug-resistant mutants. In summary, plasma virus from episodes of LLV during effective HAART appeared to originate from two distinct processes, (i) clonal outgrowth from long-lived HIV-1-infected cells, presumably following activation and proliferation of these cells, and (ii) ongoing viral replication that included the selection of new drug-resistant mutants. These observations provide a plausible explanation for the divergent clinical outcomes previously associated with LLV.

Simple, Sensitive, and Specific Detection of Human Immunodeficiency Virus Type 1 Subtype B DNA in Dried Blood Samples for Diagnosis in Infants in the Field

ABSTRACT The detection of virus is used to diagnose human immunodeficiency virus type 1 (HIV-1) infection in infants due to the persistence of maternal antibodies for a year or more. An HIV-1 DNA PCR assay with simple specimen collection and processing was developed and evaluated. Whole blood was collected on filter paper that lysed cells and bound the DNA, eliminating specimen centrifugation and extraction procedures. The DNA remained bound to the filter paper during PCR amplification. Assays of copy number standards showed reproducible detection of 5 to 10 copies of HIV-1 in 5 μl of whole blood. The sensitivity of the assay did not decrease after storage of the standards on filter paper for 3 months at room temperature or after incubation at 37 or 45°C for 20 h. The primers used for nested PCR of the HIV-1pol gene amplified templates from a reference panel of multiple HIV-1 subtypes but did not amplify a subtype A or a subtype C virus from children living in Seattle. The assay had a sensitivity of 98.4% and a specificity of 98.3% for testing of 122 specimens from 35 HIV-1-infected and 16 uninfected children and 43 seronegative adults living in Washington. The assay had a sensitivity of 99% and a specificity of 100% for testing of 102 HIV-1-positive (as determined by enzyme immunoassay) Peruvian women and 6 seropositive and 34 seronegative infants. This assay, with adsorption of whole blood to filter paper and no specimen processing, provides a practical, economical, sensitive, and specific method for the diagnosis of HIV-1 subtype B infection in infants.

Clinical, virologic and immunologic responses of children with advanced human immunodeficiency virus type 1 disease treated with protease inhibitors

OBJECTIVE To determine the effects of combination antiretroviral therapy including a protease inhibitor (PI combination therapy) in children with advanced HIV-1 disease. STUDY DESIGN An observational study of HIV-1 plasma RNA, lymphocyte subsets, delayed type hypersensitivity and physical growth after initiation of PI combination therapy. RESULTS In nine children the median HIV-1 plasma RNA decreased 1.7 log10 (mean, 1.57; range, 0.7 to 2.2) following PI combination therapy and CD4 cells increased a median of 499 (mean, 528; range, 9 to 1088) cells/microl. A rebound of RNA, associated with the development of resistance to the PI, occurred in three subjects. Three of six children were no longer anergic and all nine achieved normal weight-growth velocities. Ritonavir was well-tolerated, despite its bitter taste; however, four of five children treated with indinavir developed renal complications. CONCLUSIONS PI combination therapy in children with advanced HIV-1 disease was associated with a decrease in HIV-1 RNA, improved immunologic measures and normal or better weight gain. Of concern was the rebound in plasma HIV-1 associated with resistance to the PI observed in one-third of patients. This emphasizes the need for larger studies to define optimal PI containing regimens with long term efficacy in children.

First-line antiretroviral therapy with a protease inhibitor versus non-nucleoside reverse transcriptase inhibitor and switch at higher versus low viral load in HIV-infected children: an open-label, randomised phase 2/3 trial

BACKGROUND Children with HIV will be on antiretroviral therapy (ART) longer than adults, and therefore the durability of first-line ART and timing of switch to second-line are key questions. We assess the long-term outcome of protease inhibitor and non-nucleoside reverse transcriptase inhibitor (NNRTI) first-line ART and viral load switch criteria in children. METHODS In a randomised open-label factorial trial, we compared effectiveness of two nucleoside reverse transcriptase inhibitors (NRTIs) plus a protease inhibitor versus two NRTIs plus an NNRTI and of switch to second-line ART at a viral load of 1000 copies per mL versus 30,000 copies per mL in previously untreated children infected with HIV from Europe and North and South America. Random assignment was by computer-generated sequentially numbered lists stratified by age, region, and by exposure to perinatal ART. Primary outcome was change in viral load between baseline and 4 years. Analysis was by intention to treat, which we defined as all patients that started treatment. This study is registered with ISRCTN, number ISRCTN73318385. FINDINGS Between Sept 25, 2002, and Sept 7, 2005, 266 children (median age 6.5 years; IQR 2.8-12.9) were randomly assigned treatment regimens: 66 to receive protease inhibitor and switch to second-line at 1000 copies per mL (PI-low), 65 protease inhibitor and switch at 30,000 copies per mL (PI-higher), 68 NNRTI and switch at 1000 copies per mL (NNRTI-low), and 67 NNRTI and switch at 30,000 copies per mL (NNRTI-higher). Median follow-up was 5.0 years (IQR 4.2-6.0) and 188 (71%) children were on first-line ART at trial end. At 4 years, mean reductions in viral load were -3.16 log(10) copies per mL for protease inhibitors versus -3.31 log(10) copies per mL for NNRTIs (difference -0.15 log(10) copies per mL, 95% CI -0.41 to 0.11; p=0.26), and -3.26 log(10) copies per mL for switching at the low versus -3.20 log(10) copies per mL for switching at the higher threshold (difference 0.06 log(10) copies per mL, 95% CI -0.20 to 0.32; p=0.56). Protease inhibitor resistance was uncommon and there was no increase in NRTI resistance in the PI-higher compared with the PI-low group. NNRTI resistance was selected early, and about 10% more children accumulated NRTI mutations in the NNRTI-higher than the NNRTI-low group. Nine children had new CDC stage-C events and 60 had grade 3/4 adverse events; both were balanced across randomised groups. INTERPRETATION Good long-term outcomes were achieved with all treatments strategies. Delayed switching of protease-inhibitor-based ART might be reasonable where future drug options are limited, because the risk of selecting for NRTI and protease-inhibitor resistance is low. FUNDING Paediatric European Network for Treatment of AIDS (PENTA) and Pediatric AIDS Clinical Trials Group (PACTG/IMPAACT).

Multiple Viral Genetic Analyses Detect Low-Level Human Immunodeficiency Virus Type 1 Replication during Effective Highly Active Antiretroviral Therapy

ABSTRACT To evaluate human immunodeficiency virus type 1 (HIV-1) replication and selection of drug-resistant viruses during seemingly effective highly active antiretroviral therapy (HAART), multiple HIV-1 env and pol sequences were analyzed and viral DNA levels were quantified from nucleoside analog-experienced children prior to and during a median of 5.1 (range, 1.8 to 6.4) years of HAART. Viral replication was detected at different rates, with apparently increasing sensitivity: 1 of 10 by phylogenetic analysis; 2 of 10 by viral evolution with increasing genetic distances from the most recent common ancestor (MRCA) of infection; 3 of 10 by selection of drug-resistant mutants; and 6 of 10 by maintenance of genetic distances from the MRCA. When four- or five-drug antiretroviral regimens were given to these children, persistent plasma viral rebound did not occur despite the accumulation of highly drug-resistant genotypes. Among the four children without genetic evidence of viral replication, a statistically significant decrease in the genetic distance to the MRCA was detected in three, indicating the persistence of a greater number of early compared to recent viruses, and their HIV-1 DNA decreased by ≥0.9 log10, resulting in lower absolute DNA levels (P = 0.007). This study demonstrates the variable rates of viral replication when HAART has suppressed plasma HIV-1 RNA for years to a median of <50 copies/ml and that combinations of four or five antiretroviral drugs suppress viral replication even after short-term virologic failure of three-drug HAART and despite ongoing accumulation of drug-resistant mutants. Furthermore, the decrease of cellular HIV-1 DNA to low absolute levels in those without genetic evidence of viral replication suggests that monitoring viral DNA during HAART may gauge low-level replication.

Virologic and Immunologic Outcomes after 24 Weeks in HIV Type 1-Infected Adolescents Receiving Highly Active Antiretroviral Therapy

BACKGROUND Adolescents represent the fastest growing demographic group of new human immunodeficiency virus (HIV) infections in the United States. At present, there is little information available about their response to therapy. METHODS We studied 120 adolescents infected via high-risk behaviors who began receiving highly active antiretroviral therapy (HAART), to determine their virologic and immunologic response to therapy. RESULTS Subjects were enrolled at 28 sites of the Pediatric Acquired Immunodeficiency Syndrome Clinical Trials Group. After 16-24 weeks of HAART, 59% of subjects had reproducible undetectable virus loads, according to repeat measurements (virologic success). As enumerated by flow-cytometric analysis, increases in levels of CD4 helper cells (both naive and memory) and decreases in levels of CD8 suppressor cells were observed. Partial restoration of some immunologic parameters for patients who did not achieve virologic success was also observed, but to a more limited extent than for adolescents with virologic success. Adherence to HAART was the only predictor of achieving undetectable virus loads. CONCLUSIONS Adolescents have the capacity to improve their immunologic status with HAART. Lower than expected success in virologic control is related to lack of adherence, and efforts to improve treatment outcome must stress measures to assure adherence to medication.

Metabolic abnormalities in HIV type 1-infected children treated and not treated with protease inhibitors

Our objective was to determine whether HIV-infected children treated with protease inhibitors (PIs) have different blood lipid, insulin, and glucose levels and body composition than HIV-infected children not treated with PIs. A cross-sectional cohort study was performed; in which 23 children were treated with combination antiretroviral therapy including a PI for at least 6 months and 12 children were treated with nucleoside reverse transcriptase inhibitors only (no-PI group). Levels of lipids, apolipoprotein B (apoB), insulin, and glucose were determined in the fasting state. Body composition and fat distribution were determined by anthropometric measurements and dual energy X-ray absorptiometry (DEXA) scan. Total cholesterol levels were higher in the PI-treated children (5.33 +/- 0.87 mM) than in the no-PI children (3.69 +/- 0.59 mM) (p < 0.0001). Similarly, low-density lipoprotein (LDL) levels were also elevated in the PI-treated children (3.27 +/- 0.76 vs. 2.14 +/- 0.51 mM) (p < 0.0001). ApoB and high-density lipoprotein (HDL), and to a lesser degree triglyceride levels, were also increased in the PI-treated children. Apart from percent arm fat as measured by DEXA, there were no differences between the two groups in measures of body composition or in their fasting glucose and insulin levels. The results from this cross-sectional cohort study suggest that the predominant lipid abnormalities associated with treatment with combination antiretroviral therapy including a PI in HIV-1-infected children are elevated total and LDL cholesterol.

Effect of Pregnancy and Zidovudine Therapy on Viral Load in HIV-1-Infected Women

The objective of this study was to determine the effect of pregnancy and zidovudine (ZDV) on viral load in HIV-1 infected women. A prospective nonrandomized cohort study was conducted at a university medical center and affiliated clinic and included 44 HIV-1-seropositive pregnant women seen between June 1991 and September 1995. Twenty-three women initiated ZDV therapy during their pregnancy. Seventeen women did not take antiretrovirals, and four women took ZDV prior to and throughout pregnancy. HIV-1 viral load as determined by quantitative peripheral blood mononuclear cell (PBMC) culture and quantitative plasma RNA levels was measured at various times during pregnancy and in the postpartum period. HIV-1 load, by both infectivity and RNA levels, was relatively low and remained stable during pregnancy and through 6 weeks post partum. Initiation of ZDV therapy during pregnancy did not result in a significant decrease in viral load at delivery when controlling for the effect of pregnancy. In those women who received ZDV therapy only during pregnancy, there was a trend toward an increase in viral load measured by PBMC infectivity 6 months post partum compared with the levels before the initiation of ZDV. Mother-to-child transmission of HIV-1 occurred in one of 27 (4%) ZDV-treated women and in two of 16 (12.5%) untreated women. Among HIV-1-infected pregnant women with low viral levels, HIV-1 plasma RNA and infectivity remained stable during and after gestation. Although these results are based on a relatively small number of women and should be considered preliminary, the lack of significant ZDV-associated diminution in viral levels suggests that the protective effect of ZDV on the mother-to-child transmission of HIV-1 may not be due to the reduction in maternal viral levels but, by inference, may be due to the prevention of HIV-1 reverse transcription in the newborn.

Ontogeny of T Lymphocyte Function in the Neonate

ABSTRACT: T cell precursors are first detected in the thymus at eight weeks of gestation. By 15 to 20 weeks of gestation, T‐cell precursors expressing αβ and γδ T‐cell receptors are present in the thymus in numbers relatively similar to those found in postnatal life. However, recent data suggest that T‐cell receptor diversity is more limited during fetal and neonatal life than in adults. Additionally, the functional capacity of T cells in the fetus and neonate is immature, in that neonatal T cells express a limited repertoire of lymphokines in response to activation. Specifically, the production of the lymphokines, interferon‐γ and interleukin‐4, which participate in the maturation of cytotoxic cells, activation of macrophages, and the maturation and modulation of B cell function and isotype expression, is reduced more than tenfold compared to cells from adults. This appears to result primarily from the lack of memory T cells in the fetus and neonate, reflecting their antigenic naivete. The difference in lymphokine expression is due to diminished transcription of these genes in neonatal T cells in response to activation. Preliminary data indicate that differences in essential promoter elements regulating transcription of these lymphokine genes plays a role in their differential expression in T cells.

Safety and Immunogenicity of an HIV-1 Recombinant Canarypox Vaccine in Newborns and Infants of HIV-1–Infected Women

Pediatric AIDS Clinical Trials Group protocol 326 is a study of 2 formulations of recombinant canarypox ALVAC vaccine (vCP205) against human immunodeficiency virus type 1 (HIV-1). HIV-1-exposed infants were randomized to receive 1 of 2 formulations of vCP205 or placebo at birth and 4, 8, and 12 weeks. The vaccines were safe. Lymphoproliferative responses were detected at > or =2 time points in 44%-56% of vaccinees and none of the placebo recipients. A cytotoxic T lymphocyte response on at least 1 occasion was detected in 62.5% of infants in cohort 1 (10(6.08) median tissue culture dose [TCID(50)] vaccine formulation) and 44% of infants in cohort 2 (10(6.33) TCID(50) vaccine formulation). Rare mucosal immunoglobulin A responses and no measurable vaccine-elicited serum antibodies were detected. In children, vCP205 appeared to be safe and immunogenic.