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.

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.

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.

Infrequent detection of HIV-1-specific, but not cytomegalovirus-specific, CD8(+) T cell responses in young HIV-1-infected infants.

Early potent combination antiretroviral therapies (ART) for HIV-1 infection can preserve or restore immune function, but control of viral replication early in infection may interfere with the development of HIV-1-specific immune responses. Using an IFN-gamma ELISPOT assay, we evaluated the breadth and intensity of HIV-1-specific CD8(+) T cell responses in 17 vertically infected infants who began ART at 1-23 mo of age. CMV-specific responses were also characterized in three infants coinfected with HIV-1 and CMV. Before ART, HIV-1-specific CD8(+) T cell responses were detected in two of 13 (15%) infants <6 mo of age. HIV-1-specific CD8(+) T cells became undetectable in these two infants after the control of viral replication. Intermittent HIV-1-specific responses were noted in six infants who did not experience durable control of viral replication. In contrast, HIV-1-specific responses were detected before ART in four of four infants >6 mo of age and became persistently undetectable in only one child. CMV-specific CD8(+) T cell responses were persistently detected in all HIV-1 and CMV coinfected infants. In conclusion, HIV-1-specific CD8(+) T cell responses were less commonly detected before therapy in young infants than in older infants. Suppression of viral replication appeared to interfere with the development and maintenance of HIV-1-specific CD8(+) T cell responses. The detection of CMV-specific responses in HIV-1 and CMV coinfected infants suggests a selective defect in the generation or maintenance of HIV-1-specific CD8(+) T cell responses. Therapeutic HIV-1 vaccine strategies in young infants may prolong the clinical benefit of ART by expanding the HIV-1-specific CD8(+) T cell pool.

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.

Human immunodeficiency virus type 1 (HIV-1) gp120-specific antibodies in neonates receiving an HIV-1 recombinant gp120 vaccine.

Infants born to human immunodeficiency virus type 1 (HIV-1)-infected mothers were immunized at birth and at ages 4, 12, and 20 weeks with low-, medium-, or high-dose recombinant gp120 vaccine with MF59 adjuvant (HIV-1(SF-2); n=52) or with MF59 alone as a placebo (n=9). An accelerated schedule (birth and ages 2, 8, and 20 weeks) was used for an additional 10 infants receiving the defined optimal dose and for 3 infants receiving placebo. At 24 weeks, anti-gp120 ELISA titers were greater for vaccine-immunized than for placebo-immunized infants on both schedules, and 87% of vaccinees had a vaccine-induced antibody response. At 12 weeks, antibody titers of infants on the accelerated vaccine schedule exceeded those of infants receiving placebo (4949 vs. 551; P=.01), and 63% of the vaccinees met the response criteria. Thus, an accelerated schedule of gp120 vaccinations generated an antibody response to HIV-1 envelope distinct from transplacental maternal antibody by age 12 weeks. These results provide support for further studies of vaccine strategies to prevent mother-to-infant HIV-1 transmission.

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.

Early therapy of vertical human immunodeficiency virus type 1 (HIV-1) infection: control of viral replication and absence of persistent HIV-1-specific immune responses.

Studies of potent antiretroviral combination regimens were undertaken in young infants to evaluate the potential for long-term suppression of viral replication and to evaluate the immune consequences of such therapies. Early combination antiretroviral therapy led to a loss of plasma viremia, cultivable virus, and labile extrachromosomal replication intermediates. Despite preservation of immune function, persistent human immunodeficiency type 1 (HIV-1)-specific immune responses were not detected in most infants. The absence of detectable, persisting immune responses in most HIV-1-infected infants treated early contrasts with what is typically seen in adults who are treated early. These results are consistent with the notion that early combination antiretroviral therapy of HIV-1-infected infants allows the long-term suppression of viral replication.

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.

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.

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.

Long-term nonprogressive infection with human immunodeficiency virus type 1 in a hemophilia cohort.

Seven long-term nonprogressors (LTNPs) have been identified in a cohort of 128 human immunodeficiency virus (HIV)-1 infected individuals with hemophilia. Studies included quantitation of virus by polymerase chain reaction, characterization of primary virus isolates in vitro, analysis of lymphocyte surface markers, and measurement of virus-specific cytotoxic T lymphocytes (CTLs). Viruses of LTNPs exhibited slow growth in vivo and in vitro. LTNPs had expansion of CD8 T cells with increased expression of HLA-DR. Intermittent HIV-1-specific CTL effector activity was detected in freshly isolated peripheral blood mononuclear cells of most LTNPs. CTL precursor frequencies were higher in LTNPs than in patients with progressive disease. Virus antigen-specific lymphoproliferation was vigorous in some LTNPs. Thus, LTNPs in this cohort have maintained remarkably low virus burdens and vigorous HIV-1-specific cell-mediated immunity over a 15-year period. The presence of expanded, activated CD8 T cells with cytotoxic effector function in the peripheral blood suggests ongoing viral replication.

A polymorphism in the regulatory region of the CC-chemokine receptor 5 gene influences perinatal transmission of human immunodeficiency virus type 1 to African-American infants.

There are natural mutations in the coding and noncoding regions of the human immunodeficiency virus type 1 (HIV-1) CC-chemokine coreceptor 5 (CCR5) and in the related CCR2 protein (the CCR2-64I mutation). Individuals homozygous for the CCR5-Delta32 allele, which prevents CCR5 expression, strongly resist HIV-1 infection. Several genetic polymorphisms have been identified within the CCR5 5' regulatory region, some of which influence the rate of disease progression in adult AIDS study cohorts. We genotyped 1,442 infants (1,235 uninfected and 207 HIV-1 infected) for five CCR5 and CCR2 polymorphisms: CCR5-59353-T/C, CCR5-59356-C/T CCR5-59402-A/G, CCR5-Delta32, and CCR2-64I. The clinical significance of each genotype was assessed by measuring whether it influenced the rate of perinatal HIV-1 transmission among 667 AZT-untreated mother-infant pairs (554 uninfected and 113 HIV-1 infected). We found that the mutant CCR5-59356-T allele is relatively common in African-Americans (20.6% allele frequency among 552 infants) and rare in Caucasians and Hispanics (3.4 and 5.6% of 174 and 458 infants, respectively; P < 0.001). There were 38 infants homozygous for CCR5-59356-T, of whom 35 were African-Americans. Among the African-American infants in the AZT-untreated group, there was a highly significant increase in HIV-1 transmission to infants with two mutant CCR5-59356-T alleles (47.6% of 21), compared to those with no or one mutant allele (13.4 to 14.1% of 187 and 71, respectively; P < 0.001). The increased relative risk was 5.9 (95% confidence interval, 2.3 to 15.3; P < 0.001). The frequency of the CCR5-59356-T mutation varies between population groups in the United States, a low frequency occurring in Caucasians and a higher frequency occurring in African-Americans. Homozygosity for CCR5-59356-T is strongly associated with an increased rate of perinatal HIV-1 transmission.

Treatment of human immunodeficiency virus 1-infected infants and children with the protease inhibitor nelfinavir mesylate.

An open-label study was conducted of nelfinavir mesylate, given with reverse transcriptase inhibitors to human immunodeficiency virus 1 (HIV-1)-infected infants and children 3 months to 13 years of age. Doses of nelfinavir mesylate of 20-30 mg/kg yielded drug exposures comparable to those seen in adults. The drug was well tolerated; mild diarrhea was the primary toxic effect observed. Seventy-one percent (39) of the 55 evaluable subjects had an initial decrease in plasma HIV-1 RNA, of at least 0.7 log10 copies/mL; suppression of plasma HIV-1 RNA levels to < 400 copies/mL was observed in 15. Children who began taking at least one new reverse transcriptase inhibitor near the time when nelfinavir mesylate was started, and those with a > or = 24% proportion of CD4 lymphocytes, had a greater chance of achieving and maintaining a decline in plasma HIV-1 RNA to < 400 copies/mL. Suppression of viremia was achieved in children as young as 3 months of age.

Persistent HIV-1-specific CTL clonal expansion despite high viral burden post in utero HIV-1 infection.

To address the issue of clonal exhaustion in humans, we monitored HLA class I-restricted, epitope-specific CTL responses in an in utero HIV-1-infected infant from 3 mo through 5 years of age. Serial functional CTL precursor assays demonstrated persistent, vigorous, and broadly directed HIV-1 specific CTL activity with a dominant response against an epitope in HIV-1 Gag-p17 (SLYNTVATL, aa 77-85). A clonal CTL response directed against the immunodominant, HLA-A*0201-restricted epitope was found to persist over the entire observation period, as shown by TCR analysis of cDNA libraries generated from PBMC. The analysis of autologous viral sequences did not reveal any escape mutations within the targeted epitope, and viral load measurement indicated ongoing viral replication. Furthermore, inhibition of viral replication assays indicated that the epitope was properly processed from autologous viral protein. These data demonstrate that persistent exposure to high levels of viral Ag does not necessarily lead to clonal exhaustion and that epitope-specific clonal CTL responses induced within the first weeks of life can persist for years without inducing detectable viral escape variants.

Frequent detection of escape from cytotoxic T-lymphocyte recognition in perinatal human immunodeficiency virus (HIV) type 1 transmission: the ariel project for the prevention of transmission of HIV from mother to infant.

Host immunologic factors, including human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL), are thought to contribute to the control of HIV type 1 (HIV-1) replication and thus delay disease progression in infected individuals. Host immunologic factors are also likely to influence perinatal transmission of HIV-1 from infected mother to infant. In this study, the potential role of CTL in modulating HIV-1 transmission from mother to infant was examined in 11 HIV-1-infected mothers, 3 of whom transmitted virus to their offspring. Frequencies of HIV-1-specific human leukocyte antigen class I-restricted CTL responses and viral epitope amino acid sequence variation were determined in the mothers and their infected infants. Maternal HIV-1-specific CTL clones were derived from each of the HIV-1-infected pregnant women. Amino acid substitutions within the targeted CTL epitopes were more frequently identified in transmitting mothers than in nontransmitting mothers, and immune escape from CTL recognition was detected in all three transmitting mothers but in only one of eight nontransmitting mothers. The majority of viral sequences obtained from the HIV-1-infected infant blood samples were susceptible to maternal CTL. These findings demonstrate that epitope amino acid sequence variation and escape from CTL recognition occur more frequently in mothers that transmit HIV-1 to their infants than in those who do not. However, the transmitted virus can be a CTL susceptible form, suggesting inadequate in vivo immune control.

The Ariel Project: A prospective cohort study of maternal-child transmission of human immunodeficiency virus type 1 in the era of maternal antiretroviral therapy.

In a prospective cohort study, clinical and biologic factors that contribute to maternal-child transmission of human immunodeficiency virus type 1 (HIV-1) were studied. HIV-infected pregnant women and their infants were evaluated prospectively according to a standardized protocol. Of 204 evaluable women, 81% received zidovudine during their pregnancy. The infection rate among the 209 evaluable infants was 9.1%. By univariate analysis, histologic chorioamnionitis, prolonged rupture of membranes, and a history of genital warts were significantly associated with transmission. Additional factors associated with transmission that approached significance included a higher maternal virus load at delivery and the presence of cocaine in the urine. In a logistic regression model, histologic chorioamnionitis was the only independent predictor of transmission. Despite a significantly higher transmission rate at one site, no unique viral genotype was found at any site. Thus, chorioamnionitis was found to be the major risk factor for transmission among women receiving zidovudine.

Dynamics of human immunodeficiency virus type 1 replication in vertically infected infants.

Plasma human immunodeficiency virus type 1 (HIV-1) turnover and kinetics were studied in children aged 15 days to 2 years following the initiation of a triple antiretroviral drug regimen consisting of zidovudine, lamivudine, and nevirapine. HIV-1 turnover was at least as rapid as that previously described in adults; turnover rates were more rapid in infants and children aged 3 months to 2 years than in infants less than 3 months of age. These data confirm the central role of HIV-1 replication in the pathogenesis of vertical HIV-1 infection and reinforce the importance of early, potent combination therapies for the long-term control of HIV-1 replication.