Ruth Dickover

Clearance of HIV Infection in a Perinatally Infected Infant

BACKGROUND We describe a child who was identified shortly after birth as infected with the human immunodeficiency virus type 1 (HIV-1), but whose infection appears to have completely cleared. Asymptomatic HIV-1 infection was diagnosed in the mother during the fourth month of pregnancy. The infant was delivered vaginally at 36 weeks, received no blood products, and was not breast-fed. METHODS AND RESULTS HIV-1 was detected by culture of the infants peripheral-blood mononuclear cells at 19 and 51 days of age. Plasma from the infant was also culture-positive for HIV-1 at 51 days of age by DNA polymerase chain reaction (PCR). Nucleotide-sequence analysis of HIV-1 DNA showed extremely close homology of the cultures obtained 32 days apart, and forensic markers of genetic identity for the two cultures were identical. Hence, inadvertent viral contamination or error in the collection of specimens was highly unlikely. At 12 months of age the infant was seronegative for HIV-1, and numerous subsequent cultures and tests by PCR have also been negative for HIV-1. The child is five years of age at this writing, is HIV-seronegative, and remains well, with normal growth and development and no laboratory or clinical evidence of HIV-1 infection. CONCLUSIONS The infant we describe was infected perinatally with HIV-1, but the infection subsequently cleared and the infant remained without detectable HIV-1 infection five years later.

Multicenter Evaluation of Use of Dried Blood and Plasma Spot Specimens in Quantitative Assays for Human Immunodeficiency Virus RNA: Measurement, Precision, and RNA Stability

ABSTRACT Eleven laboratories evaluated the use of dried blood and plasma spots for quantitation of human immunodeficiency virus (HIV) RNA by two commercially available RNA assays, the Roche Amplicor HIV-1 Monitor and the bioMerieux NucliSens HIV-1 QT assays. The recovery of HIV RNA was linear over a dynamic range extending from 4,000 to 500,000 HIV type 1 RNA copies/ml. The Monitor assay appeared to have a broader dynamic range and seemed more sensitive at lower concentrations. However, the NucliSens assay gave more consistent results and could be performed without modification of the kit. HIV RNA was stable in dried whole blood or plasma stored at room temperature or at −70°C for up to 1 year. Dried blood and dried plasma spots can be used as an easy and inexpensive means for the collection and storage of specimens under field conditions for the diagnosis of HIV infection and the monitoring of antiretroviral therapy.

Optimization and Limitations of Use of Cryopreserved Peripheral Blood Mononuclear Cells for Functional and Phenotypic T-Cell Characterization

ABSTRACT The goals of this study were to optimize processing methods of cryopreserved peripheral blood mononuclear cells (PBMC) for immunological assays, identify acceptance parameters for the use of cryopreserved PBMC for functional and phenotypic assays, and to define limitations of the information obtainable with cryopreserved PBMC. Blood samples from 104 volunteers (49 human immunodeficiency virus-infected and 55 uninfected) were used to assess lymphocyte proliferation in response to tetanus, candida, and pokeweed-mitogen stimulation and to enumerate CD4+ and CD8+ T cells and T-cell subpopulations by flow cytometry. We determined that slowly diluting the thawed PBMC significantly improved viable cell recovery, whereas the use of benzonase improved cell recovery only sometimes. Cell storage in liquid nitrogen for up to 15 months did not affect cell viability, recovery, or the results of lymphocyte proliferation assays (LPA) and flow cytometry assays. Storage at −70°C for ≤3 weeks versus storage in liquid nitrogen before shipment on dry ice did not affect cell viability, recovery, or flow cytometric results. Storage at −70°C was associated with slightly higher LPA results with pokeweed-mitogen but not with microbial antigens. Cell viability of 75% was the acceptance parameter for LPA. No other acceptance parameters were found for LPA or flow cytometry assay results for cryopreserved PBMC. Under optimized conditions, LPA and flow cytometry assay results for cryopreserved and fresh PBMC were highly correlated, with the exception of phenotypic assays that used CD45RO or CD62L markers, which seemed labile to freezing and thawing.

Role of Maternal Autologous Neutralizing Antibody in Selective Perinatal Transmission of Human Immunodeficiency Virus Type 1 Escape Variants

ABSTRACT Perinatal human immunodeficiency virus type 1 (HIV-1) transmission is characterized by acquisition of a homogeneous viral quasispecies, yet the selective factors responsible for this genetic bottleneck are unclear. We examined the role of maternal autologous neutralizing antibody (aNAB) in selective transmission of HIV-1 escape variants to infants. Maternal sera from 38 infected mothers at the time of delivery were assayed for autologous neutralizing antibody activity against maternal time-of-delivery HIV-1 isolates in vitro. Maternal sera were also tested for cross-neutralization of infected-infant-first-positive-time-point viral isolates. Heteroduplex and DNA sequence analyses were then performed to identify the initial infecting virus as a neutralization-sensitive or escape HIV-1 variant. In utero transmitters (n = 14) were significantly less likely to have aNAB to their own HIV-1 strains at delivery than nontransmitting mothers (n = 17, 14.3% versus 76.5%, P = 0.003). Cross-neutralization assays of infected-infant-first-positive-time-point HIV-1 isolates indicated that while 14/21 HIV-1-infected infant first positive time point isolates were resistant to their own mothers aNAB, no infant isolate was inherently resistant to antibody neutralization by all sera tested. Furthermore, both heteroduplex (n = 21) and phylogenetic (n = 9) analyses showed that selective perinatal transmission and/or outgrowth of maternal autologous neutralization escape HIV-1 variants occurs in utero and intrapartum. These data indicate that maternal autologous neutralizing antibody can exert powerful protective and selective effects in perinatal HIV-1 transmission and therefore has important implications for vaccine development.

Early Prognostic Indicators in Primary Perinatal Human Immunodeficiency Virus Type 1 Infection: Importance of Viral RNA and the Timing of Transmission on Long-Term Outcome

The time of perinatal human immunodeficiency virus type 1 (HIV-1) transmission and the pattern of early plasma viremia as predictors of disease progression were evaluated in infected infants followed from birth. Cox proportional hazards modeling demonstrated that a 1-log higher HIV-1 RNA copy number at birth was associated with a 40% increase in the relative hazard (RH) of developing CDC class A or B symptoms (P = .004), a 60% increase in developing AIDS (P = .01), and an 80% increase in the of risk death (P = .023) over the follow-up period of up to 8 years. The peak HIV-1 RNA copy number for infants during primary viremia was also predictive of progression to AIDS (RH, 9.9; 95% confidence interval [95% CI], 1.8-54.1; P = .008) and death (RH, 6.9; 95% CI, 1.1-43.8; P = .04). The results indicate that high levels of HIV-1 RNA at birth and during primary viremia are associated with early onset of symptoms and rapid disease progression to AIDS and death in perinatally infected children.

Long-term safety and efficacy of a once-daily regimen of emtricitabine, didanosine, and efavirenz in HIV-infected, therapy-naive children and adolescents: Pediatric AIDS clinical trials group protocol P1021

BACKGROUND. Compliance with complex antiretroviral therapy regimens is a problem for HIV-1–infected children and their families. Simple, safe, and effective regimens are important for long-term therapeutic success. METHODS. A novel, once-daily dosing regimen of 3 antiretroviral drugs, emtricitabine, didanosine, and efavirenz, was tested in 37 therapy-naive HIV-infected children and adolescents between 3 and 21 years of age (inclusive). Subjects were followed for ≥96 weeks on an intention-to-treat basis. Signs, symptoms, plasma HIV-1 RNA viral load, CD4 counts, and safety laboratories were followed regularly. End points were the proportion of subjects with plasma HIV <400 or 50 HIV copies per mL and safety and tolerability of the regimen. RESULTS. Thirty-seven subjects enrolled at 16 sites. Two subjects with rashes during the first 2 weeks of therapy were the only adverse events leading to study-drug discontinuation. Other early (before protocol-scheduled conclusion) study discontinuations included 3 viral failures on treatment and 5 patients who stopped therapy for apparently nonmedical reasons. Possible drug-related adverse events included 1 grade 4 low-glucose and 5 varied grade 3 events. There were no deaths. Virologic outcomes demonstrated that 32 (85%) of 37 subjects achieved viral suppression to <400 RNA copies per mL, and 26 (72%) of 37 subjects maintained sustained suppression at <50 copies per mL through week 96. The median baseline CD4 count was 310 per μL (17%), which increased at week 96 by a median of +329 cells per μL (by +18% CD4). Pharmacokinetic results were as predicted for emtricitabine, didanosine, and efavirenz capsules, whereas efavirenz concentrations in children receiving efavirenz oral solution were lower than anticipated, requiring a dose escalation after the planned assessment point. CONCLUSIONS. A once-daily regimen of emtricitabine, didanosine, and efavirenz proved to be safe and tolerable and demonstrated good immunologic and virologic efficacy in this 2-year study.

Continuous improvement in the immune system of HIV-infected children on prolonged antiretroviral therapy

Background: The goal of HAART is to promote reconstitution of CD4+ T cells and other immune responses. We evaluated the extent and the kinetics of immune reconstitution in HIV-infected children over 144 weeks of successful HAART. Methods: Thirty-seven children receiving their first HAART regimen had plasma HIV RNA; T cells and subpopulations; T-cell rearrangement excision circles (TREC) DNA; candida, HIVCD4 and HIVCD8 enzyme-linked immunospot measured at regular intervals. Results: Plasma HIV RNA became undetectable in 81% of patients at 24 weeks and remained undetectable in 77% at 144 weeks. In contrast, CD4+% continuously increased. Distribution of T-cell subpopulations changed rapidly during the first 48 weeks of HAART and more slowly thereafter. At 144 weeks, total, naive and activated CD4+% and naive CD8+% of HIV-infected children were not significantly different from those of healthy age-matched controls, whereas total and activated CD8+% remained elevated. CD4+ and CD8+ TREC content increased only during the first 48 weeks of HAART. They positively correlated with each other and with total CD4+%, naive CD4+% and naive CD8+%. Candida and HIVCD4 enzyme-linked immunospot increased over time reaching peak values at 48 weeks and 144 weeks, respectively. HIVCD8 enzyme-linked immunospot decreased in magnitude over 144 weeks of HAART but retained its breadth. Baseline CD4+% positively correlated with CD4+% and with functional immune reconstitution at week 144, whereas baseline TREC correlated with TREC at week 144. Conclusion: HIV-infected children acquired normal distribution of CD4+ T cells and other subpopulations and recovered CD4-mediated HIV immunity after 144 weeks of HAART.

Decreased CD8 Cell-Mediated Viral Suppression and Other Immunologic Characteristics of Women Who Transmit Human Immunodeficiency Virus to Their Infants

CD8 T cell function, lymphocyte surface phenotype, serum markers of immunologic activation, and viral burden were assessed in 75 human immunodeficiency virus (HIV)-infected pregnant women, including 9 who transmitted infection to their infants. Serial studies during and after pregnancy showed no significant differences in levels of cell-surface or serum activation molecules in transmitting compared to nontransmitting mothers, with the exception of a postpartum increase in tumor necrosis factor alpha in transmitting women. The transmitting women had a median plasma viral load of 65,516 RNA copies/mL at delivery versus 5139 in nontransmitting women. During the third trimester, the CD8 cells of 81% of the nontransmitting and 44% of the transmitting mothers suppressed HIV production in vitro by >50%. Women with <50% suppression had a 3.4 times greater risk of transmitting HIV to their infants. CD8 suppression and viral load were interrelated, but when either CD4 percent or AZT use was controlled for, suppression was still significant.

Correlation of Immune Activation during Late Pregnancy and Early Postpartum with Increases in Plasma HIV RNA, CD4/CD8 T Cells, and Serum Activation Markers

ABSTRACT A previously observed rise in the plasma viral load postpartum in both treated and untreated HIV-positive women remains unexplained. Virological and immunological markers were evaluated in HIV-negative controls and HIV-positive pregnant women with and without antiretroviral treatment. Plasma HIV RNA, CD4/CD8 T cells, and serum activation markers were sequentially measured during the third trimester, at delivery, and 2 to 8 weeks postpartum in a cohort of HIV-positive pregnant women (n = 96) enrolled in a maternal-fetal HIV transmission study and a control group of HIV-negative pregnant women (n = 28). Mean plasma HIV RNA (P = 0.003) increased from delivery to postpartum, and mean CD4 T cells (P = 0.002) and serum β2-microglobulin (P < 0.0001) increased from the third trimester through postpartum among the HIV-positive women. Mean CD8 T cells increased from the third trimester through postpartum in women receiving zidovudine (ZDV) and in those not treated (P < 0.05) but remained stable in those on highly active antiretroviral therapy (HAART) and the HIV-negative controls. Increases in serum β2-microglobulin were correlated with increases in HIV RNA (P = 0.01). HIV-positive pregnant women showed postpartum increases in plasma HIV RNA, CD4 T cells, and serum β2-microglobulin regardless of the treatment regimen. The rise in CD4 T cells and β2-microglobulin was also observed in HIV-negative pregnant women, suggesting hormonal changes and/or labor-induced cytokines may contribute to immune activation. Immune activation correlated with increased plasma HIV RNA in postpartum women despite treatment, although HAART appeared to blunt the effect. The observed rise in plasma HIV RNA postpartum, which correlated with markers of immune activation, may have implications for enhanced transmission to infants through early breast-feeding and to sexual partners.

Combined evaluation of sexually transmitted infections in HIV-infected pregnant women and infant HIV transmission

Background Sexually transmitted infections (STIs) including Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Treponema pallidum (TP), and cytomegalovirus (CMV) may lead to adverse pregnancy and infant outcomes. The role of combined maternal STIs in HIV mother-to-child transmission (MTCT) was evaluated in mother-infant pairs from NICHD HPTN 040. Methodology Urine samples from HIV-infected pregnant women during labor were tested by polymerase chain reaction (PCR) for CT, NG, and CMV. Infant HIV infection was determined by serial HIV DNA PCR testing. Maternal syphilis was tested by VDRL and confirmatory treponemal antibodies. Results A total of 899 mother-infant pairs were evaluated. Over 30% had at least one of the following infections (TP, CT, NG, and/or CMV) detected at the time of delivery. High rates of TP (8.7%), CT (17.8%), NG (4%), and CMV (6.3%) were observed. HIV MTCT was 9.1% (n = 82 infants). HIV MTCT was 12.5%, 10.3%, 11.1%, and 26.3% among infants born to women with CT, TP, NG or CMV respectively. Forty-two percent of HIV-infected infants were born to women with at least one of these 4 infections. Women with these infections were nearly twice as likely to have an HIV-infected infant (aOR 1.9, 95% CI 1.1–3.0), particularly those with 2 STIs (aOR 3.4, 95% CI 1.5–7.7). Individually, maternal CMV (aOR 4.4 1.5–13.0) and infant congenital CMV (OR 4.1, 95% CI 2.2–7.8) but not other STIs (TP, CT, or NG) were associated with an increased risk of HIV MTCT. Conclusion HIV-infected pregnant women identified during labor are at high risk for STIs. Co-infection with STIs including CMV nearly doubles HIV MTCT risk. CMV infection appears to confer the largest risk of HIV MTCT. Trial registration NCT00099359.