Children with sickle cell disease (SCD) suffer life-threatening transient aplastic crisis (TAC) when infected with parvovirus B19. In utero, infection of healthy fetuses may result in anemia, ...hydrops, and death. Unfortunately, although promising vaccine candidates exist, no product has yet been licensed. One barrier to vaccine development has been the lack of a cost-effective, standardized parvovirus B19 neutralization assay. To fill this void, we evaluated the unique region of VP1 (VP1u), which contains prominent targets of neutralizing antibodies. We discovered an antigenic cross-reactivity between VP1 and VP2 that, at first, thwarted the development of a surrogate neutralization assay. We overcame the cross-reactivity by designing a mutated VP1u (VP1uAT) fragment. A new VP1uAT ELISA yielded results well correlated with neutralization (Spearman’s correlation coefficient = 0.581; p = 0.001), superior to results from a standard clinical diagnostic ELISA or an ELISA with virus-like particles. Virus-specific antibodies from children with TAC, measured by the VP1uAT and neutralization assays, but not other assays, gradually increased from days 0 to 120 post-hospitalization. We propose that this novel and technically simple VP1uAT ELISA might now serve as a surrogate for the neutralization assay to support rapid development of a parvovirus B19 vaccine.
Allogeneic hematopoietic stem-cell transplantation for X-linked severe combined immunodeficiency (SCID-X1) often fails to reconstitute immunity associated with T cells, B cells, and natural killer ...(NK) cells when matched sibling donors are unavailable unless high-dose chemotherapy is given. In previous studies, autologous gene therapy with γ-retroviral vectors failed to reconstitute B-cell and NK-cell immunity and was complicated by vector-related leukemia.
We performed a dual-center, phase 1-2 safety and efficacy study of a lentiviral vector to transfer
complementary DNA to bone marrow stem cells after low-exposure, targeted busulfan conditioning in eight infants with newly diagnosed SCID-X1.
Eight infants with SCID-X1 were followed for a median of 16.4 months. Bone marrow harvest, busulfan conditioning, and cell infusion had no unexpected side effects. In seven infants, the numbers of CD3+, CD4+, and naive CD4+ T cells and NK cells normalized by 3 to 4 months after infusion and were accompanied by vector marking in T cells, B cells, NK cells, myeloid cells, and bone marrow progenitors. The eighth infant had an insufficient T-cell count initially, but T cells developed in this infant after a boost of gene-corrected cells without busulfan conditioning. Previous infections cleared in all infants, and all continued to grow normally. IgM levels normalized in seven of the eight infants, of whom four discontinued intravenous immune globulin supplementation; three of these four infants had a response to vaccines. Vector insertion-site analysis was performed in seven infants and showed polyclonal patterns without clonal dominance in all seven.
Lentiviral vector gene therapy combined with low-exposure, targeted busulfan conditioning in infants with newly diagnosed SCID-X1 had low-grade acute toxic effects and resulted in multilineage engraftment of transduced cells, reconstitution of functional T cells and B cells, and normalization of NK-cell counts during a median follow-up of 16 months. (Funded by the American Lebanese Syrian Associated Charities and others; LVXSCID-ND ClinicalTrials.gov number, NCT01512888.).
Elevated tricuspid valve regurgitation jet velocity (TRV ≥ 2.5 m/s) is associated with mortality among adults with sickle cell disease (SCD), but correlative biomarkers are not studied according to ...treatment exposure or genotypes. To investigate the associations between biomarkers and TRV elevation, we examined the relationship between TRV and hemolytic, inflammatory, and cardiac biomarkers, stratified by disease‐modifying treatments and SCD genotype. In total, 294 participants with SCD (mean age, 11.0 ± 3.7 years) and 49 hereditary spherocytosis (HS; mean age, 22.9 ± 19.75 years) were included for comparison and enrolled. TRV was elevated in 30.7% of children with SCD overall: 18.8% in HbSC/HbSβ+‐thalassemia, 28.9% in untreated HbSS/HbSβ0‐thalassemia, 34.2% in HbSS/HbSβ0‐thalassemia hydroxyurea‐treated, and 57% in HbSS/HbSβ0‐thalassemia chronic transfusion treated. TRV was elevated in 10.7% and 27.8% in HS children and adults, respectively. In children with SCD, elevated TRV was correlated with hemoglobin (odds ratio OR = 0.78, P = 0.004), lactate dehydrogenase (LDH; OR = 2.52, P = 0.005), and N‐terminal pro‐brain natriuretic peptide (NT‐pro BNP; OR = 1.003, P = 0.004). In multivariable logistic regression, adjusting for genotype, sex, hemolytic index, and treatment, hemoglobin concentration remained the only significant variable associated with elevated TRV in untreated HbSS/HbSβ0‐thalassemia participants. TRV was not associated with inflammatory markers, other markers of hemolysis, or NT‐pro BNP in untreated HbSS/HbSβ0‐thalassemia. Neither hemoglobin nor LDH was associated with TRV in HbSC/HbSβ+‐thalassemia. These results suggest that elevated TRV is influenced by the degree of anemia, possibly reflecting sickling as part of the disease pathophysiology. Prospective studies should monitor hemoglobin concentration as children with SCD age into adulthood, prompting initiation of TRV screening and monitoring.
Abstract
Sickle cell disease (SCD) is caused by a mutant β-globin gene and affects more than 100,000 individuals in the USA alone. Sickled RBCs and ischemic events cause tissue damage, altered ...splenic architecture, and inflammation. When children are infected with parvovirus B19, they can develop life-threatening aplastic crisis. Previous reports have described immune defects among patients with SCD, and their potential correlations with splenic dysfunction or inflammation. In a multi-center study involving St. Jude, NIH, Novartis, and GlaxoSmithKline, we examined (i) inflammatory cytokines/chemokines in patients with SCD, treated or untreated with hydroxyurea (HU); (ii) immune responses toward parvovirus B19 infections in children with SCD; and (iii) immune responses in a mouse model for SCD against a candidate Saccharomyces cerevisiae-derived parvovirus B19 VLP vaccine. Results showed that HU treatment reduced serum cytokines/chemokines in patients with SCD (e.g. TGFα, TNFα, and sVCAM-1). Both HU-treated and untreated children, regardless of their cytokine/chemokine profile, responded to parvovirus B19. Binding antibodies were apparent in the nasal cavity (a common point-of entry for the pathogen) and sera by day 7 post-infection, and virus-specific neutralizing antibodies were induced. In transgenic mice with SCD (expressing human α, βS, and γ globin genes), we observed normal antibody activities toward the VLP vaccine. Together, data show that parvovirus B19 -specific B cells withstand the aberrant SCD environment better than their previously-described Streptococcus pneumonia-specific B cell counterparts. Data encourage rapid development of the VLP vaccine to prevent aplastic crises in children with SCD.
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Early trials of gene therapy for X-linked Severe Combined Immunodeficiency (XSCID) restored T cell immunity in most cases, but did not correct B cell function and carried a high risk of iatrogenic ...leukemia. The subsequent development of self-inactivating γ-retroviral vectors has enhanced safety, but has not restored B cell function to date. We developed a new approach for XSCID gene therapy that utilizes a safety modified lentiviral (LV) vector (CL20-i4-EF1α-hGC-OPT) together with reduced exposure busulfan (Bu) conditioning for newly diagnosed infants with XSCID. Recently, we reported that the combination of reduced dose Bu used together with our LV vector restored T and B cell function in older XSCID children with declining immune function following haploidentical transplantation (De Ravin SS et al, Sci Transl Med, 2016). Here we report initial results of LVXSCID-ND; a multi-center, phase I/ II safety and efficacy study using our LV vector and dose-adjusted Bu for the first time in newly diagnosed XSCID infants. We enrolled six subjects over the last 12 months (median age of 4.5 months, range: 2-12 months). Purified bone marrow (BM) CD34+ cells were transduced with the CL20-i4-EF1α-hGC-OPT vector generated by a stable producer cell line and then cryopreserved to facilitate central manufacturing for multiple study sites and evaluation of release criteria prior to conditioning. Busulfan was given as two single daily doses to target a total cumulative area-under-the-curve (cAUC) of 22 mg*hr/L (60-90 mg*hr/L = myeloablative cAUC). The median dose of transduced CD34+ cells was 8.06 x 106 cells/kg (range: 4.6 - 11 x 106) and the median vector copy number (VCN) in graft CFU-C was 0.40 copies /cell (range: 0.16 - 0.97). An average Bu cAUC of 22.9 mg*hr/L (range: 20.0 to 24.2) was achieved, which was within 10% of the intended cAUC in all patients. As of July 2017, no severe adverse events related to BM harvest, Bu exposure, or cell infusion have been observed. In the first 5 evaluable cases, complete hematopoietic recovery occurred by 3-4 weeks without any blood product support. Follow-up data from the oldest patient who presented with high levels of maternal T cell engraftment, severe neutropenia requiring G-CSF therapy, CMV viral infection, with a graft VCN of 0.16, and who is now 12 months post therapy, demonstrated delayed and partial T cell reconstitution. Cases 2 and 3 have been followed for nine and six months, respectively and have significantly higher VCNs in peripheral blood (PB) subsets (CD14/15+myeloid cells 0.65, 0.30 copies/cell: CD3+ T cells 2.78, 2.71; CD19+ B cells 1.01, 0.78; and CD56+ NK cells 2.72, 2.11 respectively). Bone marrow aspirates on week 16 yielded VCNs in sorted CD34+ cells of 0.56, 0.50; and BM myeloid CFU-GM of 0.61, 0.24. Rapid T cell reconstitution in cases 2 and 3 resulted in normal numbers of CD3+, CD4+, CD4 naïve, and CD8+ cells with TRECs 626 and 1170 copies per ug DNA 16-20 weeks post therapy. In both cases at 16 weeks, T cell proliferation was 86% and 81% of control, and V-β spectratype scores were normal at 194 and 155. At nine months, case 2 had a normal isohemagglutinin anti-A titer of 1:32 and normal 4-week trough serum immunoglobulins levels (IgG 713 mg/dl, IgM 54 mg/dl, and IgA 16 mg/dl). IVIG supplementation has been discontinued for approximately 3 months before vaccination responses are assessed. Case 4 has been followed for 12 weeks with PB VCNs for CD3+ cells of 1.36 copies/cell, CD19+ 0.58, CD56+ 1.38 and CD14/15+ 0.02. Flow cytometry analysis of PB at 12 weeks shows 4.5% of PB leukocytes are CD3+, with 61% CD4+, 11% CD8+, and a significant fraction expressing a CD45RA+, RO- naive phenotype at this early time point. Cases 5 and 6 are still too early to evaluate for immune phenotype and function. Preliminary vector insertion site analysis shows highly polyclonal marking patterns in case 2 with 11,000 insertion sites in CD3+ cells, 5049 sites in CD19+ cells, 756 sites in BM myeloid CFU-C without any evidence of clonal dominance. In summary, gene therapy for newly diagnosed XSCID patients using a LV vector with targeted reduced exposure Bu conditioning is well tolerated and results in rapid T cell reconstitution in most cases. Efficient vector marking in bone marrow CD34+ cells, myeloid cells, and B cells indicate that this approach will likely provide broad immune reconstitution rather than restricted T cell correction seen in past trials using γ-retroviral vectors with no Bu.
Long-Boyle:InsightRX: Consultancy. Puck:UpToDate: Patents & Royalties: Recieve royalties to write and edit entries on primary immune disorders; InVitae, a clinical DNA sequencing company: Other: Spouse employment and stock options. Cowan:Homology Medicine: Consultancy.
Background: Patients with sickle cell disease (SCD) have an increased risk of developing elevated tricuspid regurgitation jet velocity (TRV), which is associated with some markers of hemolysis and ...increased morbidity and mortality among affected adults. Elevated TRV also occurs in children with SCD, but with unclear clinical consequences. To date, most studies have not investigated elevated TRV by genotypes or the effects of disease-modifying therapies (e.g., hydroxyurea or chronic transfusions). Furthermore, there is paucity of data in non-sickling hemolytic anemias (HAs), which represents another group with anemia, chronic hemolysis and potential risk for elevated TRV. To better understand the prevalence of TRV elevation and its correlates, we prospectively studied a large cohort of individuals with sickling and non-sickling HAs.
Methods: We enrolled children 5 to 18 years with SCD and other forms of HA in the Long Term Effects of Erythrocyte Lysis trial (ELYSIS, NCT 00842621). Adult parents or grandparents of children with non-sickling HA with the same diagnosis were also eligible. Prospective measurement of TRV by 2-D echocardiogram with left ventricular function (M-mode) and color flow Doppler was performed. Biomarkers and markers of hemolysis were obtained concurrently. All assessments were obtained ≥ 4 weeks from illness, transfusion, or hospitalization. All echocardiograms were reviewed by a single cardiologist. Our goals were three-fold: 1) to validate the association between TRV and lactate dehydrogenase (LDH) in sickling and non-sickling HAs, 2) to investigate the effect of disease-modifying therapies on TRV elevation in sickling HAs, and 3) to investigate the role of other biomarkers of hemolysis and vasculopathy on TRV elevation in sickling and non-sickling HAs.
Results: 355 children and adults with sickling or non-sickling HAs were enrolled (Table). The proportion of TRV ≥2.5 m/sec as well as median LDH, N-terminal pro-brain natriuretic peptide (NTproBNP), and argninine/ornithine (arg/orn) ratio are shown for each cohort studied. Children with severe SCD receiving chronic transfusions (CTXFN) had considerably higher TRV values than all other cohorts. LDH was significantly associated with TRV in children with severe untreated SCD regardless of cut-off value (2.5, 2.8, or 3 m/sec), but not in any of the other cohorts. No significant association was noted between TRV and NTproBNP, whether evaluated as a continuous variable or dichotomous variable (using 160 pg/mL as the threshold) or between TRV and arg/orn ratio. LDH and Hb were associated with elevated TRV in children with severe untreated SCD, but only hemoglobin (Hb) remained significant in multivariate analysis including other markers for hemolysis (total bilirubin, aspartate aminotransferase, and absolute reticulocyte count), NTproBNP, and arg/orn ratio. TableCohortNAge (years)TRV ≥ 2.5 m/s (%)LDH (U/L)NTproBNP (pg/mL)Arg/Orn ratioSevere SCD, untreated(HbSS/HbSβ0-thal)1178.1(5-18.5)29.6582 (214-1315)86 (8-804)1.1 (0.4-7.5)Severe SCD, CTXFN2912.9 (5.7-18.3)57.1*491 (211-1150)50 (6-260)1 (0.6-1.8)Severe SCD, Hydroxyurea7613.2 (5.1-18)34.2450 (214-1354)65 (9-316)0.9 (0.2-1.8)Non-severe SCD(HbSC, HbSβ+-thal)7210.4 (5.2-17.8)21.7304 (157-811)36 (6-266)1.2 (0.1-1.8)Pediatric non-sickling HAs(hereditary spherocytosis, pyruvate kinase deficiency, unstable Hb variants)4010.5 (5.1-17.5)18259 (154-2622)75 (6-293)1.2 (0.3-2.7)Adult non-sickling HAs(hereditary spherocytosis, unstable Hb variants)2139 (27.9-59.8)27.8185 (136-263)42 (8-183)1.3 (0.9-2.2)
Note: * Significantly higher than reference group (severe SCD untreated)
Conclusions: Prevalence of elevated TRV in children with untreated SCD in our study is similar to previous literature, but our other cohorts varied by diagnosis and/or treatment received. The highest prevalence was found in children with severe SCD on CTXFN, possibly reflecting severity of disease. Our study validates the association between TRV and LDH in severe untreated SCD; however this association was not present in non-severe sickle genotypes or non-sickling HAs. The lack of association between TRV and NTproBNP suggests that this is not a useful marker to predict elevated TRV in these patients. The degree of anemia was an important determinant of TRV elevation, but contrary to prior literature, LDH was not.
No relevant conflicts of interest to declare.
Background: Up to 40% of children with sickle cell anemia (SCA) will have abnormalities on brain imaging due to their hematologic disorder, much of which is subclinical. Common abnormalities on brain ...magnetic resonance imaging/magnetic resonance angiography (MRI/MRA) include leukoencephalopathy from microvascular ischemic insult and vascular stenosis from endothelial damage. Silent cerebral ischemic insults are progressive; further neurologic abnormalities, including overt stroke, are more common among children with ischemic findings on MRI compared to children without them. Furthermore, poor performance on neuropsychological testing, lower IQ, and higher rates of grade retention are common in children with SCA and cerebral ischemic disease. The effect of hydroxyurea treatment on the development and progression of vascular stenosis and leukoencephalopathy is unclear. This study aimed to longitudinally evaluate the development of intracerebral abnormalities through serial MRI/MRA in children with SCA (HbSS and HbSβ0-thalassemia) who receive long-term therapy with hydroxyurea.
Methods: Children with SCA and no prior history of overt stroke enrolled in the Hydroxyurea Study of Long-Term Effects (HUSTLE NCT00305175) underwent brain MRI/MRA, transcranial doppler (TCD) examinations, and laboratory evaluations immediately before hydroxyurea initiation and after 3 and 6 years of treatment to maximum tolerated dose. MRI/MRAs were reviewed for the presence or absence of vascular stenosis and leukoencephalopathy. Leukoencephalopathy was defined as white matter T2 hyperintensity. Proportions of abnormal MRI/MRA findings were compared between baseline and 3 years and baseline and 6 years using McNemar’s test and the Wilcoxon-Mann-Whitney exact test was used to explore associations with laboratory parameters.
Results: Forty-six children with SCA, mean age 9.4 years (range 1-17.3), had an MRI/MRA at baseline and 3 years post-initiation of hydroxyurea. Ten children had an additional MRI/MRA after 6-years of hydroxyurea therapy. Frequencies of leukoencephalopathy and vascular stenosis are shown in the table. Prevalence of leukoencephalopathy before hydroxyurea therapy was higher than that reported in the literature for untreated children with SCA of similar age. There were no significant differences between baseline imaging findings and those at 3 and 6 years.
Table:Prevalence of leukoencephalopathy and vascular stenosis at baseline, 3 and 6 yearsBaseline (n=47) Median age 9.6 years (range 1.0 to 17.3)3 years (n=46) Median age 13.1 years (range 4.4 to 21.1)6 years (n=10) Median age 14.5 years (range 8.3 to 18.3)Leukoencephalopathy27 (57.4%)28 (60.9%)*5 (50%)*Stenosis3 (6.4%)1 (2.2%)*0*all p>0.05
Children with leukoencephalopathy at baseline and 3 years were older than those without (mean 10.7 vs. 7.7 years, p=0.01; 10.5 vs. 7.5 years, p=0.02 respectively). Lower HbF at baseline was associated with the presence of leukoencephalopathy at year 3 (median HbF 4.6% vs. 12.4%, p=0.008), but there was no association between HbF at 3-years and the presence of leukoencephalopathy at 3-years (median HbF 16.4% vs. 13.2%, p=0.55). When stratified by age, these findings were similar. TCD velocities and other hematologic parameters were not associated with MRI/MRA abnormalities. The small number of vascular stenosis cases precluded further analyses of this outcome.
Conclusions: In this longitudinal study, children treated with hydroxyurea for 3-6 years did not demonstrate an increased frequency of vascular stenosis or leukoencephalopathy on brain MRI/MRA during treatment. Older age at hydroxyurea initiation and lower pre-hydroxyurea HbF percentage were associated with the presence of leukoencephalopathy at baseline and 3-years. These findings suggest that hydroxyurea may mitigate the expected progression of vascular stenosis and leukoencephalopathy in children with SCA, and that this therapy should be initiated early on, before the development of cerebrovascular disease, particularly among those with a low HbF percentage.
Off Label Use: hydroxyurea for children with sickle cell disease.
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