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Absence of Detectable HIV-1 Viremia after Treatment Cessation in an Infant
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Download the PDF here
Baby Steps on the Road to HIV Eradication - Editorial
Scott M. Hammer, M.D.
NEJM October 23, 2013
The durable suppression of plasma human immunodeficiency virus type 1 (HIV-1) RNA that is conferred by potent antiretroviral therapy (ART) has been associated with dramatic improvements in immunologic and clinical well-being and reductions in morbidity and mortality from opportunistic complications. It has also brought the restoration of normal life expectancies within our grasp, has effectively eliminated the vertical transmission of HIV in resource-unconstrained settings in women who receive antenatal care, and can substantially reduce horizontal transmission.1
Concomitantly, the ability to drive plasma HIV-1 RNA to levels below the limits of quantification of sensitive clinical assays has unveiled the limitations of our current knowledge and therapies. Residual viremia, defined as less than 20 copies of plasma HIV-1 RNA per milliliter, can be detected in most persons receiving effective therapy, and replication-competent virus can be isolated from peripheral-blood CD4+ T cells cultured ex vivo from such patients. The best-described cellular reservoir is the resting, memory CD4+ T cell, but other cell types probably contribute.2 The persistence of the virus in cell reservoirs is thought to feed the residual viremia by means of intermittent release of infectious virus rather than by rounds of ongoing viral replication, although this point is still debated.2 The persistence of HIV in reservoirs has at least two major implications: it may be responsible for the ongoing immune activation and inflammatory state seen in virologically suppressed persons, which, in turn, may be linked to the nonopportunistic complications of cardiovascular, renal, hepatic, and malignant disease; and, with integrated HIV proviral DNA, this reservoir presents the biggest challenge to achieving the complete eradication of replication-competent HIV-1 from an infected person.
There has been one well-documented case of an adult in whom the eradication of replication-competent HIV-1 (i.e., a cure) has been achieved.3This person required a hematopoietic stem-cell transplant for acute myelogenous leukemia, with the donor being homozygous for the 32-base-pair deletion in the chemokine (C-C motif) receptor 5 gene (CCR5 delta32), making the patient's engrafted lymphocytes resistant to infection with the R5 (CCR5-tropic) HIV-1 strain. Bone marrow ablation and graft-versus-host disease also probably contributed to the cure in this patient, which has now been documented for more than 5 years.4 Other reports of reductions of the viral reservoir after hematopoietic stem-cell transplantation for the treatment of lymphoma or early ART have subsequently appeared that support the hypothesis that the HIV reservoir is a viable therapeutic target.5-7
Persaud and colleagues8 now describe in the Journal a case of a child who was born at 35 weeks of gestation to an HIV-infected mother who had not received prenatal care and thus was not receiving ART at the time of delivery. The child began receiving combination ART 30 hours after birth, and the subsequent early course was notable for the decreasing but detectable viremia through 19 days of age. After a 5-month absence from care, during which ART was not administered, viral rebound was not detected when the child presented again at 23 months of age.
Challenging questions have been raised by this case. Was infection established in the infant? Are the early virologic findings a result of maternal-fetal circulatory exchange? Could this be a case of postexposure prophylaxis? The positive HIV-1 DNA polymerase chain reaction drawn at 30 hours and the plasma HIV-1 RNA level of 19,812 copies per milliliter drawn separately at 31 hours taken together meet the criteria for neonatal infection, and the early test positivity is compatible with in utero, rather than intrapartum, infection. The transfer of maternal cells and virions would probably not result in an HIV-1 RNA level that was eight times as high in the neonate as it was in the mother (maternal viral load at 24 hours, 2423 copies per milliliter), nor would it result in 19 days of viremia. These data, along with the classic trajectory of viremic decline during ART, support the authors' perspective that the infant was truly infected.
The big question, of course, is, "Is the child cured of HIV infection?" The best answer at this moment is a definitive "maybe." This uncertainty is due to the need for long-term follow-up while the child is not receiving ART and the imprecision with which the viral reservoir can be measured. Assays for proviral DNA in the child at 24 and 26 months of age were inconclusive, but the failure to isolate replication-competent HIV-1 from resting CD4+ T cells and the absence of rebound viremia months after ART was discontinued are compelling.
The eradication of HIV-1 is now the goal of a formidable research effort. One line of investigation is focused on reducing, destabilizing, or eliminating the latent cell reservoir. Drugs such as the histone deacetylase inhibitors (e.g., vorinostat) can disrupt the epigenetic control of integrated provirus, resulting in viral RNA transcription.9 However, this may not result in reliable killing of the cell, which suggests that the addition of an immune effector mechanism may be necessary to ensure the death of these cells.10 Destabilization of the viral reservoir can also be achieved by engaging the programmed death 1 receptor on the surface of latently infected CD4+ T cells. Another line of investigation being pursued is gene therapy directed at interrupting the expression of CCR5 coreceptors.11
The child described by Persaud et al. may be unique, and thus we have to exercise caution before inferring general principles from this case report. This said, we are at the stage at which individual case reports can provide proofs of principle, stimulate hypotheses, and lead to carefully designed experimental therapeutic studies involving both adults and children that, we hope, will lead us down the road to the reduction or eradication of the HIV-1 reservoir. To paraphrase the Chinese philosopher, Lao-tzu, "A journey of a thousand miles begins with a single step." In the case of HIV infection, this may turn out to be a baby step.
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Absence of Detectable HIV-1 Viremia after Treatment Cessation in an Infant
Deborah Persaud, M.D., Hannah Gay, M.D., Carrie Ziemniak, M.S., Ya Hui Chen, B.A., Michael Piatak, Ph.D., Tae-Wook Chun, Ph.D., Matthew Strain, M.D., Ph.D., Douglas Richman, M.D., and Katherine Luzuriaga, M.D.
NEJM October 23, 2013
Nearly 70 million persons have acquired HIV-1 infection since the epidemic was recognized,1 but a "cure" has been documented in one person, known as "the Berlin Patient."2,3 A cure for HIV-1 infection occurred in this person after he underwent treatment for acute myelogenous leukemia with total ablative chemotherapy, radiation therapy, and stem-cell transplantation with donor cells homozygous for chemokine receptor 5 (CCR5) delta32, with associated graft-versus-host disease. The case of the Berlin Patient shows that long-lived, replication-competent HIV-1 reservoirs can be reduced or cleared sufficiently to permit the discontinuation of ART without subsequent viral rebound. We report data from a 30-month-old child who had met the standard diagnostic criteria for HIV-1 infection and who now has undetectable levels of circulating HIV-1 RNA, proviral DNA, and HIV-1 antibodies, as assessed by means of clinical assays, after combination ART was administered between 30 hours and 18 months of age.
Case Report
An infant was born by spontaneous vaginal delivery at 35 weeks of gestation to a woman who had received no prenatal care. Rapid HIV-1 testing in the mother was positive during labor. Delivery occurred before antiretroviral prophylaxis was administered. Maternal HIV-1 infection was confirmed by means of Western blot testing. The mother's plasma viral load, CD4+ T-cell count, and HIV-1 subtype and genotype are summarized in Table 1.
ART was initiated in the infant at 30 hours of age. A three-drug regimen of zidovudine (at a dose of 2 mg per kilogram of body weight every 6 hours), lamivudine (at a dose of 4 mg per kilogram twice daily), and nevirapine (at a dose of 2 mg per kilogram twice daily) was selected to provide prophylaxis for high-risk HIV-1 exposure and to minimize the likelihood of generating resistant viral variants in the event that the infant had been infected in utero.
The detection of HIV-1 DNA in peripheral-blood mononuclear cells (PBMCs) in blood obtained at 30 hours of age (Table 1) and the detection of HIV-1 RNA (19,812 copies per milliliter) in a separate blood sample collected at 31 hours of age met the standard diagnostic criteria for HIV-1 infection in the infant.4 Therefore, ART was continued. When the infant was 1 week of age, ritonavir-boosted lopinavir was substituted for nevirapine to reduce the risk of antiretroviral drug resistance in case there was incomplete adherence to the prescribed ART. The decision to implement this regimen preceded warnings from the Food and Drug Administration against the use of ritonavir-boosted lopinavir in infants younger than 14 days of age.5
While the infant was receiving ART, the HIV-1 RNA level remained detectable in plasma at three additional time points (2617 copies per milliliter at 6 days of age, 516 copies per milliliter at 11 days of age, and 265 copies per milliliter at 19 days of age) (Table 1 and Figure 1) before declining below assay-detection limits when the infant was 29 days of age. The decline in the plasma viral load was biphasic and similar to that described previously in HIV-1 infected infants7,8 and adults receiving ART.9
In the infant's first year of life, during which the infant was not breast-fed, adherence to ART was assessed as adequate on the basis of pharmacy records indicating timely prescription refills, undetectable plasma viral loads, and red-cell macrocytosis during zidovudine treatment. Concern about medication adherence was raised when the child was 18 months of age, when the red-cell mean corpuscular volume, which had been more than 101 femtoliters (fl) before 15 months of age, decreased to 95 fl, although the plasma level of HIV-1 RNA remained undetectable (<20 copies per milliliter).
Between 18 and 23 months of age, the child missed several clinic visits. When the child was brought back for care at 23 months of age, the mother reported that ART had been discontinued when the child was 18 months of age, although pharmacy records indicated that the prescription was last refilled when the child was 15 months of age. In blood samples obtained at 23 and 24 months of age, the plasma level of HIV-1 RNA was undetectable. A repeat HIV-1 DNA polymerase-chain-reaction (PCR) test at 24 months of age was negative, as was an HIV-1 antibody test.
At the time of this report, the child had not received any antiretroviral drugs through 30 months of age, and the plasma level of HIV-1 RNA had remained undetectable by routine clinical assays (Figure 1). Circulating HIV-1 antibodies have also not been detected (Figure S1 in the Supplementary Appendix, available with the full text of this article at NEJM.org). The CD4+ T-cell percentages were within or exceeded the normal range for age at all time points tested (Figure 1C).6 Growth and development have been normal. Detailed virologic and immunologic studies were performed to characterize the persistence of HIV-1 infection in this unique case.
Methods
Diagnosis and Monitoring of HIV-1 Infection
We performed standard HIV-1 DNA PCR testing with the use of the Amplicor HIV-1 DNA assay (Roche Molecular Systems) and HIV-1 RNA testing with the use of the COBAS AmpliPrep/COBAS Taqman HIV-1 test (Roche Molecular Systems). The testing was performed at laboratories that were certified by the Clinical Laboratory Improvement Amendments (CLIA) program: Focus Diagnostics and Mayo Medical Laboratories for HIV-1 DNA testing and University of Mississippi Medical Center for HIV-1 RNA testing. The COBAS AmpliPrep/COBAS Taqman HIV-1 test, version 1.0 (limit of detection [LOD], <48 copies per milliliter), was used for samples tested before the child was 5 months of age, and version 2.0 (LOD, <20 copies per milliliter) was used thereafter. CD4+ T cells were enumerated with the use of flow cytometry.
HIV-1-Specific Immune Responses, HLA Typing, and CCR5 Genotyping
HIV-1 antibody testing was performed with the use of enzyme-linked immunosorbent assays and Western blot testing (Cambridge Biotech HIV-1 Western Blot Kit, Maxim Biomedical). Intracellular cytokine assays were used to detect HIV-1-specific CD4+ and CD8+ T-cell responses to pooled gag and nef peptides. Cell-surface markers for immune activation were evaluated with the use of flow cytometry. HLA typing was performed with the use of the Bio-Rad HLA Sequence Specific Primers kit (Biotest). CCR5 genotyping for the delta32 mutation was performed with the use of previously published methods.10
Virologic Assays to Quantify HIV-1 Persistence
The presence of replication-competent virus in maternal and infant samples was determined with the use of a limiting-dilution quantitative coculture assay of PBMCs enriched for resting CD4+ T cells.11 Replication efficiencies of the maternal isolates were compared with an HIV-1 reference strain to assess viral fitness.12 Low-level viremia was quantified by means of two assays: a single-copy viral-load assay13 and a modified COBAS AmpliPrep/COBAS Taqman HIV-1 test, version 2.0.14 We tested for the presence of proviral DNA and 2-long terminal repeat circles in PBMCs, resting CD4+ T cells, and cellular fractions enriched for monocytes and activated cells with the use of a highly sensitive droplet digital PCR assay (LOD, <3 copies per 106 cells).15
Results
Immunologic and Virologic Testing
Maternal HIV-1 antibody tests that were performed 24 months after delivery showed reactivity to all nine HIV-1 proteins tested (Figure S1 in the Supplementary Appendix). The mother did not begin receiving ART. At 26 months after delivery, the maternal HIV-1 RNA level was 6763 copies per milliliter. At 28 months after delivery, fully replication-competent virus was recovered from a maternal blood sample at a frequency of 137 infectious units per million resting CD4+ T cells; viral isolates showed replication kinetics similar to the IIIB laboratory strain of HIV-1. HLA typing of the mother and infant revealed shared haplotypes, confirming their relationship (Table 1). Neither the mother nor the infant had HLA class I alleles that are associated with spontaneous control of HIV-1 infection (HLA-B27 or HLA-B*57); both were nonmutated for CCR5.
HIV-1 antibodies were not detected in the child at 24, 26, and 28 months of age (Figure S1 in the Supplementary Appendix). T-cell activation markers were within normal limits at 24 and 30 months of age (at 24 months, 2% of the T cells were CD4+/HLA-DR+; at 30 months, 6% of the T cells were CD8+/CD38+/HLA-DR+). HIV-1-specific CD4+ and CD8+ T-cell responses were not detected at 28 months of age.
Ultrasensitive virologic tests were performed to evaluate the persistence of HIV-1 infection in the child (Table 2). Viral RNA was detected at a single-copy level in plasma obtained at 24 months of age but was not detected at 26 months of age. HIV-1 proviral DNA was detected near the limits of detection at both time points but not in resting CD4+ T cells (Table 2). A culture of 22 million resting CD4+ T cells obtained at 24 months of age, during a period in which the child was not receiving ART, did not yield replication-competent HIV-1.
Discussion
We report data from a 30-month-old child who met the standard diagnostic criteria for HIV-1 infection, received combination ART between 30 hours and 18 months of age, and subsequently had controlled HIV-1 viremia for 12 months while not receiving ART. The absence of rebound viremia, the undetectable replication-competent virus, the almost-complete disappearance of cell-associated HIV-1 DNA, and the absence of HIV-1-specific immune responses while the child was not receiving ART suggest that replication-competent HIV-1 reservoirs may not have been established or were markedly abated, if not extinguished.16
Rare cases of transient HIV infection in infants have been reported, on the basis of intermittent detection of HIV-1 p24 antigen or DNA in peripheral blood.17-20 One cohort study showed that 6.4% of 188 infants had one or more positive tests for HIV that were followed by negative tests.20However, forensic and phylogenetic studies could not rule out laboratory contamination or sample mislabeling in reported cases of spontaneous HIV-1 clearance.21 In the present case report, the five positive tests indicating HIV-1 infection were performed in CLIA-certified laboratories with the use of separate blood specimens and standard clinical-assay protocols. Under this scenario, repeated sample mix-up or contamination was unlikely. Unfortunately, clinical specimens are not routinely saved, which precluded further assessment of specimens from those time points.
The current standard definition of HIV-1 infection in an infant requires the detection of HIV-1 nucleic acids in at least two separate clinical samples.4 An infant in whom HIV-1 DNA is detected in blood obtained within the first 48 hours after birth is considered to have acquired the infection in utero.22 The detection of HIV-1 proviral DNA in this infant's peripheral blood at 30 hours of age, along with a plasma viral load of 19,812 copies per milliliter detected at 31 hours of age, is consistent with in utero infection. In two separate studies, the median viral loads at birth in infants who had been infected in utero were 10,800 copies per milliliter23 and 26,940 copies per milliliter,24 as compared with undetectable levels in infants who were infected peripartum.
We believe that infection with replicating virus was likely, given the sequential detection of RNA in three serial blood samples obtained during the first few weeks of life while the infant was receiving combination ART with reverse-transcriptase inhibitors. The biphasic decline in viremia after the initiation of ART, with the rapid clearance (steep decline) of short-lived virus-producing cells (e.g., activated CD4+ T cells), followed by a slower decline, is consistent with the inhibition of active infection rather than with the clearance of passively transferred HIV-1-infected maternal cells without infection of the infant's cells. With these findings, the infant met the standard diagnostic criteria for HIV-1 infection (most likely acquired in utero) as well as the criteria for initiation of ART and for participation in clinical trials sponsored by the National Institutes of Health.22-25
We evaluated whether maternal-fetal blood transfusion could account for the detection of HIV-1 nucleic acid in the infant. An initial plasma viral load of approximately 20,000 copies per milliliter in a 2.5-kg neonate is equivalent to a total body count of 5 million HIV-1 RNA copies or approximately 2.5 million virions. Such a result would have required the transfer of approximately 2 liters of maternal blood into this infant (whose total blood volume is only 250 ml). Likewise, the possibility that persistent detection of HIV-1 RNA during the first 3 weeks of life represented the passive transfer of virions from maternal blood is not consistent with the early-infection events associated with postexposure prophylaxis in maternal-fetal transmission or neonatal HIV-1 infection, considering that the half-life of virions is 1 to 2 hours at most. Thus, the repeated detection of HIV-1 RNA in plasma through the infant's first 19 days of life, while the infant was receiving ART, shows that there were virus-producing cells in the infant.
It is also conceivable that maternal microchimerism could have resulted in the detection of HIV-1 nucleic acid in the infant. Microchimerism has been detected in infants born to HIV-positive women but has not been associated with an increased risk of HIV-1 transmission.26 Although we were not able to directly assess maternal cells in the infant's circulation, transfused maternal cells at the levels reported previously (0.3 to 0.6% of neonatal cells26) are not likely to have resulted in the levels of plasma HIV-1 RNA that were detected shortly after birth in this infant.
A curious finding in this infant is the persistence of traces of HIV-1 nucleic acid. The traces of viral nucleic acid may represent the persistence of replication-defective genomes or false positive signals at the threshold of detection of the assays, as was recently reported for the Berlin Patient.27Attempts to amplify HIV-1 from infant samples obtained for viral sequencing at 24 and 26 months of age were unsuccessful, as were efforts to locate the screening samples obtained when the infant was a newborn to perform viral sequencing. The latter situation also precluded linkage analysis to maternal viral variants, which represents a limitation of this case report.
ART that is initiated between 1 and 3 months of age has been shown to modify HIV-1 persistence in that infants who receive early treatment are often HIV-1 seronegative and do not have circulating viremia while they are receiving ART, but viral rebound has been observed when ART is discontinued.28-30 Approximately 5 to 10% of adults who receive early ART have sustained control of HIV-1 replication after the discontinuation of therapy, although evidence for ongoing HIV-1 infection remains.31,32 The current case differs from those described in children29 and adults32who receive early treatment in that circulating proviral HIV-1 DNA and plasma HIV-1 RNA were only intermittently detected at levels just above the limits of detection of the assays, replication-competent virus could not be recovered, and the child remains HIV-1 seronegative even though ART was discontinued. This situation closely mimics the virologic and immunologic biomarkers of the Berlin Patient at 5 years of follow-up.27
Viral and host factors could have contributed to the reported outcome in this child. The maternal HIV-1 RNA levels were relatively low at delivery, although fully replication-competent virus, with replication kinetics that were similar to a laboratory strain, was readily cultured from maternal cells 26 months post partum, from a blood sample showing a plasma viral load of nearly 7000 copies per milliliter (Figure S2 in the Supplementary Appendix). Although neither the mother nor the infant had HLA class I alleles that have been associated with control of HIV-1 infection,33 it is possible that strong maternal immune responses or other unknown host factors were responsible for the relatively low viral load seen in the mother, although maternal samples were not available for additional testing. The child remains in care, and at the age of 36 months, at least 18 months after the cessation of ART, the child has no detectable level of HIV-1 RNA.
This case suggests that very early ART may interfere with either the quantities or qualities of persistent reservoirs of replication-competent virus. Antiretroviral prophylaxis is routinely recommended for infants who have been exposed to HIV-1, with multidrug regimens recommended in high-risk cases. Furthermore, the initiation of ART in infected infants markedly reduces HIV-related mortality among infants25 and is recommended by the World Health Organization. This global standard may facilitate planned proof-of-concept studies of very early ART to modify persistent HIV-1 infection in infants, with an aim toward sparing them a lifetime of therapy.
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