Persistence of episomal HIV-1 infection intermediates in patients on highly active anti-retroviral therapy.

Treatment of HIV-1-infected individuals with a combination of anti-retroviral agents results in sustained suppression of HIV-1 replication, as evidenced by a reduction in plasma viral RNA to levels below the limit of detection of available assays. However, even in patients whose plasma viral RNA levels have been suppressed to below detectable levels for up to 30 months, replication-competent virus can routinely be recovered from patient peripheral blood mononuclear cells and from semen. A reservoir of latently infected cells established early in infection may be involved in the maintenance of viral persistence despite highly active anti-retroviral therapy. However, whether virus replication persists in such patients is unknown. HIV-1 cDNA episomes are labile products of virus infection and indicative of recent infection events. Using episome-specific PCR, we demonstrate here ongoing virus replication in a large percentage of infected individuals on highly active anti-retroviral therapy, despite sustained undetectable levels of plasma viral RNA. The presence of a reservoir of 'covert' virus replication in patients on highly active anti-retroviral therapy has important implications for the clinical management of HIV-1-infected individuals and for the development of virus eradication strategies.

Early HIV-1 envelope-specific cytotoxic T lymphocyte responses in vertically infected infants.

High frequencies of cytotoxic T lymphocyte precursors (CTLp) recognizing HIV-1 laboratory strain gene products have been detected in adults within weeks of primary infection. In contrast, HIV-1-specific CTLp are uncommonly detected in infants younger than 6 mo. To address the hypothesis that the use of target cells expressing laboratory strain env gene products might limit the detection of HIV-1 env-specific CTLp in early infancy, recombinant vaccinia vectors (vv) expressing HIV-1 env genes from early isolates of four vertically infected infants were generated. The frequencies of CTLp recognizing target cells infected with vv-expressing env gene products from early isolates and HIV-1 IIIB were serially measured using limiting dilution followed by in vitro stimulation with mAb to CD3. In one infant, the detection of early isolate env-specific CTLp preceded the detection of IIIB-specific CTLp. CTLp recognizing HIV-1 IIIB and infant isolate env were detected by 6 mo of age in two infants. In a fourth infant, HIV-1 IIIB env and early isolate env-specific CTLp were simultaneously detected at 12 mo of age. These results provide evidence that young infants can generate HIV-1-specific CTL responses and provide support for the concept of neonatal vaccination to prevent HIV-1 transmission. However, the early predominance of type-specific CTL detected in some young infants suggests that the use of vaccines based on laboratory strains of HIV-1 may not protect against vertical infection.

Limiting dilution analysis of cytotoxic T lymphocytes to human immunodeficiency virus gag antigens in infected persons: in vitro quantitation of effector cell populations with p17 and p24 specificities.

The presence of cytotoxic T lymphocytes (CTL) to the gag antigens of human immunodeficiency virus (HIV) has been described in infected populations. We found that the majority of this immune response as measured in bulk CTL assays of unstimulated peripheral blood mononuclear cells (PBMC) is directed against the p24 component of the p55 gag precursor protein. Using limiting dilution analysis of this effector cell population we confirm that the majority of activated gag-specific CTL circulating in the PBMC of infected hemophilic patients are directed at p24 determinants and are present at frequencies of 1/36,000 to 1/86,000 lymphocytes. By performing in vitro stimulation after limiting dilution, the precursor population of gag-specific CTL are characterized and quantitated. HIV gag-specific CTL precursors are identified at frequencies of 1/1700 to 1/17,000 lymphocytes and are made up of cells with both p17 and p24 specificities. No HIV gag-specific CTL precursor cells are identified in the PBMC of HIV-uninfected individuals. These studies demonstrate that CTL directed at both p17 and p24 determinants make up the cellular immune repertoire in HIV-infected individuals but that only the p24-specific CTL are routinely found in an activated state in the circulation.

The EPIICAL project: an emerging global collaboration to investigate immunotherapeutic strategies in HIV-infected children.

The EPIICAL (Early-treated Perinatally HIV-infected Individuals: Improving Children's Actual Life with Novel Immunotherapeutic Strategies) project arises from the firm belief that perinatally infected children treated with suppressive antiretroviral therapy (ART) from early infancy represent the optimal population model in which to study novel immunotherapeutic strategies aimed at achieving ART-free remission. This is because HIV-infected infants treated within 2-3 months of life have a much reduced viral reservoir size, and rarely show HIV-specific immunity but preserve normal immune development. The goal of EPIICAL is the establishment of an international collaboration to develop a predictive platform using this model to select promising HIV therapeutic vaccine candidates, leading to prioritisation or deprioritisation of novel immunotherapeutic strategies. To establish this platform, the EPIICAL Consortium aims to: develop predictive models of virological and immunological dynamics associated with response to early ART and to treatment interruption using available data from existing cohorts/studies of early-treated perinatally HIV-infected children; optimise methodologies to better characterise immunological, virological and genomic correlates/profiles associated with viral control; test novel immunotherapeutic strategies using in vivo proof-of-concept (PoC) studies with the aim of inducing virological, immunological and transcriptomic correlates/profiles equivalent to those defined by the predictive model. This approach will strengthen the capacity for discovery, development and initial testing of new therapeutic vaccine strategies through the integrated efforts of leading international scientific groups, with the aim of improving the health of HIV-infected individuals.

Quantitation of HIV-1 in whole blood of infected children.

OBJECTIVE: To validate the technique of HIV-1 culture from whole blood for the quantitation of viral load in infected children. PATIENTS: Forty-three HIV-1-infected children were followed in two paediatric centres. METHODS: Quantitative HIV-1 cultures from unfractionated whole blood using an end-point dilution technique were compared with simultaneous quantitative cultures of peripheral blood mononuclear cells (PBMC) and plasma. RESULTS: Good sensitivity (93%) of the methods used was confirmed. A close correlation (r = 0.80) was observed between HIV-1 titres measured directly from whole blood and those expected from PBMC and plasma titres. The mean whole blood viral load was higher in patients with more severe signs of disease, but the difference did not reach statistical significance. The whole blood viral titres measured sequentially at monthly intervals remained within one dilution of each other in 16 of the 22 patients studied. CONCLUSION: In this study, the quantitation of HIV-1 in unfractionated blood allowed for a reliable and sensitive measurement of the whole blood viral load in infected children.

Immunogenicity and efficacy of childhood vaccines in HIV-1-infected children.

Children infected by HIV-1 are particularly vulnerable to severe, recurrent, or unusual infections by vaccine-preventable pathogens. Routine immunisations seem to be generally safe for HIV-1-infected children, but responses may be suboptimal. Early HIV-1-induced immune attrition associated with viral replication may particularly interfere with the development of memory responses. In high HIV-1 prevalence regions, the accumulation of susceptible hosts may compromise disease-control efforts. Although early control of viral replication through treatment with highly active therapy may preserve immune function and responses to routine childhood vaccines, availability is limited in the areas most affected. In this review, we provide an overview of the immunogenicity and efficacy of childhood vaccines in HIV-1-infected children. The possible immunological bases for defective responses are discussed; unanswered questions and the need for further research are delineated.

Pharmacokinetics of nelfinavir in human immunodeficiency virus-infected infants.

BACKGROUND: Nelfinavir dosed at approximately 20 to 30 mg/kg three times a day (TID) in older children provides exposure similar to 750 mg TID in adults. However, the pharmacokinetics (PK) of nelfinavir in infants who are < 2 years of age is not well-described. The objective of this study was to determine the pharmacokinetics of nelfinavir in infants < 2 years of age. METHODS: Nelfinavir concentrations were evaluated in 22 HIV-infected infants between 15 days and 2 years of age receiving nelfinavir as part of Pediatric ACTG Study 356. Nelfinavir therapy was initiated at approximately 25 mg/kg TID (n = 18) or approximately 55 mg/kg twice a day (n = 4) and given in combination with nevirapine, stavudine and lamivudine. PK samples were obtained predose and 1.5 and 4 h postdose at approximately 6-month intervals. Eight infants (all < or = 3 months of age) also had intensive PK samples collected at Week 1. RESULTS: The median apparent clearance in the infants with intensive pharmacokinetic sampling was 2.7 liters/h/kg (range, 1.8 to > or = 10) and was similar between twice a day and TID dosing cohorts. Overall nelfinavir concentrations at all collection times were lower in these infants than previously reported in older pediatric patients. CONCLUSIONS: Nelfinavir concentrations in infants are highly variable and lower than those seen in adult or older pediatric populations receiving labeled dosing. Therefore it is necessary to further evaluate nelfinavir safety, effectiveness and pharmacokinetics at higher doses than used among other pediatric populations.

Viral and immunopathogenesis of vertical HIV-1 infection.

High-level viral replication is the primary determinant of CD4 depletion or disease development in HIV-1--infected children. The developing immune system of infants might allow for more efficient viral replication and less efficient immune containment of viral replication. Advances in the understanding of the pathogenesis of vertical HIV-1 infection suggest that the use of potent combination regimens to control HIV-1 replication offers the best opportunity to prevent or reverse the sequelae of HIV-1 infection.

Lack of alternative coreceptor use by pediatric HIV-1 R5 isolates for infection of primary cord or adult peripheral blood mononuclear cells.

HIV-1 infection of neonates results in an extended acute period of virus replication, frequent neurological problems and reduced survival compared to adults. In adults, R5 viruses mainly infect CCR5(+) CD4(+) memory T-cells. In neonates, CCR5(+) memory T-cells form a substantially smaller fraction of total lymphocytes. We therefore tested whether alternative coreceptors confer infection of lymphocytes by pediatric isolates. Pediatric HIV-1 R5 isolates failed to replicate in Delta32/Delta32 CCR5 PBMCs or in cord PBMCs treated with a CCR5 inhibitor. These results do not indicate a role for alternative coreceptors and provide support for CCR5 inhibitors in the therapy of HIV-1(+) neonates.

Pathogenesis of pediatric human immunodeficiency virus type 1 infection.

This discussion of the pathogenesis of infant human immunodeficiency virus (HIV) type 1 infection emphasizes features unique to pediatric disease and provides general insights into HIV illness in all populations. Therapies designed to interrupt transmission of HIV can be most efficiently addressed through studies of maternal-infant transmission. The rapid progression of disease in children constantly emphasizes the urgent need for continued progress in treatment of HIV infection and presents a unique opportunity optimally to assess effects of early intervention.

Quantitation of human immunodeficiency virus in vertically infected infants and children.

We used a quantitative human immunodeficiency virus, type 1 (HIV-1) culture method to determine whether there is a relationship between the amount of replicating virus in the blood of vertically infected children and the relatively short latency period before development of symptomatic disease in these children. HIV-1 titers were determined by end point dilution in the peripheral blood mononuclear cells and the plasma of 30 infected (CDC class P1 and P2), 36 indeterminate (CDC class PO), and 19 uninfected (CDC class P3) infants and children born to HIV-1 seropositive mothers. HIV-1 was recovered from 35 (90%) of 39 PBMC cultures and 23 (60%) of 38 plasma cultures of infected patients not receiving antiretroviral therapy. The mean HIV-1 titers tended to be higher in patients with more advanced disease (P2, D, E, or F: 1760 TCID/10(6) PBMC, 460 TCID/ml plasma) than in asymptomatic or mildly symptomatic patients (P1; P2, A or C: 90 TCID/10(6) PBMC; 60 TCID/ml plasma). A poor correlation between HIV-1 titers and serum p24 antigen levels was found. No correlation was observed between viral titers and relative or absolute numbers of CD4 lymphocytes. Plasma virus titers were lower in 9 patients receiving zidovudine (ZDV) therapy (mean 2 TCID/ml) than in untreated patients of similar clinical status. The viral titers measured in the blood of vertically infected infants and children were on the same order of magnitude as the viral titers measured in HIV-infected adults. We conclude that the relatively rapid progression to symptomatic disease of the majority of vertically infected patients is not due to a higher load of replicating virus in blood.

Deficient human immunodeficiency virus type 1-specific cytotoxic T cell responses in vertically infected children.

Cytotoxic T lymphocyte (CTL) responses to human immunodeficiency virus type 1 (HIV-1) gag proteins were studied prospectively in 17 children (12 infected) born of mothers with HIV-1 seropositivity and in five pediatric patients with hemophilia infected by transfusion of HIV-1-contaminated factor VIII concentrate. B lymphoblastoid cells infected with vaccinia virus vectors expressing HIV-1 gag gene products were combined with autologous peripheral blood mononuclear cells to detect circulating CTLs. Effector cells were defined by monoclonal antibody-mediated, complement-dependent cytolysis. Circulating HIV-1 gag-specific cytotoxic responses were detectable in 4 of 5 HIV-1-infected pediatric hemophilic patients, and were similar in magnitude to those previously described in adults. In contrast, circulating HIV-1 gag-specific cytolysis was detectible in only 3 of 12 vertically infected children. Depletion data revealed that the majority of detectible gag-specific cytolysis was CD8 T cell-mediated. No apparent relationships between CD4 T cell counts, CD8 T cells counts, or serum p24 antigen levels and CTL responses were seen. Deficient CTL development may, in part, explain the more rapid onset of symptomatic disease following vertical HIV infection.

Effect of HIV on thymic function before and after antiretroviral therapy in children.

Studies were undertaken to investigate the role of the thymus in T cell reconstitution in human immunodeficiency virus (HIV)-infected children treated with antiretroviral therapy. Nine pediatric patients who acquired HIV perinatally were treated with multidrug combinations of antiretroviral agents. Plasma virus load and CD4+ and CD8+ T cell subsets were measured, and thymus function was measured by quantifying T cell receptor rearrangement excision circles in peripheral blood. Patients with virus loads remaining >400 RNA copies/mL plasma were classified as virologic nonresponders. Thymus function was initially decreased in all subjects. After antiretrovirus therapy, peripheral CD4+ T cells increased in all subjects. Thymus function was restored in 4 of 5 virologic responders but in only 1 of 4 virologic nonresponders. This suggests that HIV has an adverse effect upon thymic function in pediatric HIV infection. Potent antiretroviral therapy restores thymic function but is affected by the degree to which virus suppression is achieved.

HIV-1-specific cytotoxic T lymphocyte responses in the first year of life.

HIV-1-specific CTL responses were prospectively evaluated in infants born to HIV-1-seropositive women to assess the capability of the young infant to generate HIV-1-specific CTL and to examine the potential role of HIV-1-specific CTL in the pathogenesis of vertical infection. Our results indicate that some young infants, and even the fetus, seem to be capable of generating virus-specific CTL responses. The detection of HIV-1-specific CTL responses varied among infants, however, with respect to timing, HIV-1 gene product recognition, and the magnitude of detectable responses; HIV-1-specific CTL responses were uncommonly detected in the first few months of life. The less consistent detection of HIV-specific CTL in early infancy contrasts with reports of the detection of HIV-1-specific CTL soon after primary infection in adults. HIV-1-specific CTL were not detected in any uninfected infants born to HIV-1-seropositive women. This description of HIV-1-specific CTL in infants may have important implications for understanding the pathogenesis of vertical HIV-1 infection and for the development of a vaccine to interrupt vertical infection.

Early viremia and immune responses in vertical human immunodeficiency virus type 1 infection.

Thirty-three infants born to human immunodeficiency virus type 1 (HIV-1)-seropositive women were evaluated from birth using plasma and peripheral blood mononuclear cell (PBMC) cultures, polymerase chain reaction, and serum p24 antigen assays. Five children were identified as infected. Evidence of infection was found in cord blood and subsequent samples from 2 infected children, suggesting in utero infection. Virologic studies on cord blood and early neonatal specimens from the 3 other infected children were negative but became positive by 8 weeks of age, suggesting either intrapartum transmission or sequestration of virus with subsequent detection. An increase in blood (plasma and PBMC) virus titers consistent with primary viremia was observed in 4 infants between 3 and 16 weeks of age. Blood virus titers subsequently declined in the absence of antiretroviral therapy and in the absence of activated HIV-1-specific cytotoxic T lymphocyte responses or broadly neutralizing antibodies. Confirmation of these results in larger studies may be helpful in the design of clinical trials to interrupt vertical transmission or to modify the course of infection.

Diagnosis of vertical human immunodeficiency virus type 1 infection by whole blood culture.

The qualitative and quantitative performance of a human immunodeficiency virus type 1 (HIV-1) whole blood culture method was assessed for use in the diagnosis of vertical HIV-1 infection. This method requires < 1 mL of whole blood and allows for the quantification of blood virus load. HIV-1 was isolated from 36 (95%) of 38 whole blood specimens from 27 HIV-1-infected children compared with 0 of 16 whole blood specimens from 16 uninfected children. HIV-1 titers were significantly higher in severely symptomatic children (2450 TCID/mL) than in less symptomatic children (179 TCID/mL). This simple quantitative culture assay may be useful for the early diagnosis of vertical HIV-1 infection, the assessment of blood virus load, and the evaluation of antiretroviral therapies.

Differential evolution and stability of epitope-specific CD8(+) T cell responses in EBV infection.

Murine models of lymphocytic choriomeningitis virus infection suggest that the memory CD8(+) T cell repertoire is reflective of the CD8(+) T cell repertoire generated during acute infection. Less is known regarding the evolution of CD8(+) T cell repertoires during human viral infections. We therefore examined epitope-specific CD8(+) T cell responses in a large cohort of individuals with acute through latent Epstein-Barr virus infection. Using 16 of 20 published EBV epitopes restricted by HLA-A2, HLA-A3 or HLA-B7, we showed that lytic cycle-specific CD8(+) T cell responses predominated during acute EBV infection. However, whereas HLA-A2(+)-restricted BMLF-1-specific CD8(+) T cell responses were maintained through latency, HLA-A2(+)- and HLA-B7(+)-restricted BZLF-1, as well as HLA-A3(+)-restricted BRLF-1 CD8(+) T cell responses, were generated but not readily maintained. Analyses of CD8(+) T cell responses to EBV latent cycle Ags showed delayed detection and lower frequencies of latent epitope-specific CD8(+) T cell responses during acute EBV infection, with maintenance of these responses 1 yr post-EBV infection. Early BMLF-1 and EBNA-3A epitope-specific CD8(+) T cell frequencies did not correlate with their frequencies at 1 yr postinfection. Interestingly, populations of EBV-specific CD8(+) T cells were stable during 20 mo in our long term EBV-seropositive populations, suggesting homeostasis between virus and the host immune system. This study demonstrates that CD8(+) T cell repertoires generated during persistent viral infections are not simply reflective of the initial pool of CD8(+) T cells and provides evidence that the generation of CD8(+) T cell responses to a persistent infection is a dynamic process.