Background SARS-CoV2 can induce a strong host immune response. Many studies have evaluated antibody response following SARS-CoV2 infections. This study investigated the immune response and T cell receptor diversity in people who had recovered from SARS-CoV2 infection (COVID-19). Methods Using the nCounter platform, we compared transcriptomic profiles of 162 COVID-19 convalescent donors (CCD) and 40 healthy donors (HD). 69 of the 162 CCDs had two or more time points sampled. Results After eliminating the effects of demographic factors, we found extensive differential gene expression up to 241 days into the convalescent period. The differentially expressed genes were involved in several pathways, including virus-host interaction, interleukin and JAK-STAT signaling, T-cell co-stimulation, and immune exhaustion. A subset of 21 CCD samples was found to be highly “perturbed,” characterized by overexpression of PLAU, IL1B, NFKB1, PLEK, LCP2, IRF3, MTOR, IL18BP, RACK1, TGFB1, and others. In addition, one of the clusters, P1 (n = 8) CCD samples, showed enhanced TCR diversity in 7 VJ pairs (TRAV9.1_TCRVA_014.1, TRBV6.8_TCRVB_016.1, TRAV7_TCRVA_008.1, TRGV9_ENST00000444775.1, TRAV18_TCRVA_026.1, TRGV4_ENST00000390345.1, TRAV11_TCRVA_017.1). Multiplexed cytokine analysis revealed anomalies in SCF, SCGF-b, and MCP-1 expression in this subset. Conclusions Persistent alterations in inflammatory pathways and T-cell activation/exhaustion markers for months after active infection may help shed light on the pathophysiology of a prolonged post-viral syndrome observed following recovery from COVID-19 infection. Future studies may inform the ability to identify druggable targets involving these pathways to mitigate the long-term effects of COVID-19 infection. Trial Registration: https://clinicaltrials.gov/ct2/show/NCT04360278 Registered April 24, 2020.
Background Since the beginning of the COVID‐19 pandemic, cryopreservation of hematopoietic progenitor cell (HPC) products has been increasingly used to ensure allogeneic donor graft availability prior to recipient conditioning for transplantation. However, in addition to variables such as graft transport duration and storage conditions, the cryopreservation process itself may adversely affect graft quality. Furthermore, the optimal methods to assess graft quality have not yet been determined. Study Design and Methods A retrospective review was performed on all cryopreserved HPCs processed and thawed at our facility from 2007 to 2020, including both those collected onsite and by the National Marrow Donor Program (NMDP). HPC viability studies were also performed on fresh products, retention vials, and corresponding final thawed products by staining for 7‐AAD (flow cytometry), AO/PI (Cellometer), and trypan blue (manual microscopy). Comparisons were made using the Mann–Whitney test. Results For HPC products collected by apheresis (HPC(A)), pre‐cryopreservation and post‐thaw viabilities, as well as total nucleated cell recoveries were lower for products collected by the NMDP compared to those collected onsite. However, there were no differences seen in CD34+ cell recoveries. Greater variation in viability testing was observed using image‐based assays compared to flow‐based assays, and on cryo‐thawed versus fresh samples. No significant differences were observed between viability measurements obtained on retention vials versus corresponding final thawed product bags. Discussion Our studies suggest extended transport may contribute to lower post‐thaw viabilities, but without affecting CD34+ cell recoveries. To assess HPC viability prior to thaw, testing of retention vials offers predictive utility, particularly when automated analyzers are used.
BackgroundCoronavirus disease 2019 (COVID-19) results in robust but dysregulated acute immune response characterized by pro-inflammatory cytokine production and T-cell exhaustion, but little is known concerning immune response following recovery. We assessed immune function in convalescent plasma donors (CCD) who had recovered from COVID-19.MethodsThe cellular immune response and T-cell receptor (TCR) diversity in CCD was investigated using the nCounter host response and TCR diversity panels. 270 CCD and 40 healthy donor (HD) blood samples collected 11 to 193 days after diagnosis were analyzed. The CCD samples were from 162 donors, 69 donated more than once. All HD donated only once.ResultsMany genes were differentially expressed for months following infection. Analysis of samples collected 0 to 90 days post-diagnosis found that 19 of 773 genes were differentially expressed among CCD and HD (FDR < 0.05) (figure 1a). At 90 to 120 days, 120 to 150 and >150 post-diagnosis, 13, 58 and 4 genes were differentially expressed respectively (FDR < 0.05) (figures 1b-d). At 120 to 150 days the differentially expressed genes included those in Treg differentiation, type III interferon signaling and chemokine signaling pathways. 76 genes were differently expressed at least once during the time windows described above. (Figure 1e). Among CCD, the expression of CTLA-4, ICOS, ICOSLG, OSM and CXCR4 were initially elevated but fell to HD levels at the end of the study period. The expression of LILRA6, CCR2 and CX3CR1 increased or remained elevated throughout (figure 1f).A subset of samples departed notably from the average trend. The transcriptome of each CCD sample was scored by its similarity to the mean transcriptome of HD samples. This analysis revealed 21 CCD samples from 19 unique donors were highly perturbed from HD samples (figure 2a). Among these highly perturbed samples 80% were collected > 90 days post-diagnosis. The perturbed samples clustered into two groups, labelled P1 and P2 (figure 2b) and displayed dysregulation of distinct gene sets (figures 2c, 2d). The P1 were characterized by increased expression of genes in myeloid inflammation, type 1 interferon and innate immune signaling pathways, lower COVID antibody levels and increased T-cell receptor diversity. P2 were characterized by highly up-regulated CD44, BCL2, TGFB1, IL18BP, IL27RA, and IL11RA.Abstract 953 Figure 1Longitudinal trends in CCD gene expression. a-d: Differential expression results in HD vs. 4 time windows of CCD. Genes with FDR <0.1 are labeled; e: average CCD log2 fold-changes from HD over time. Color is only given for times where the Loess regression is different from the mean HD with p < 0.05; f: longitudinal results for selected genes. Orange lines connect CCD samples over time. Blue lines show inner 95% quantiles of HD samplesAbstract 953 Figure 2CCD with more severe departure from HD gene expression. a: CCD samples (in orange) were scored for perturbation from the mean HD (in blue), and 21 highly perturbed sample subsets emerged; b: clustering of the 21 highly perturbed patients. The dendrogram was cut to define two groups. c: volcano plots comparing expression in P1 (left) and P2 (right) vs. CCD; d: longitudinal trends of selected genes perturbed in P1 and P2ConclusionsImmune dysregulation in CCD continues at least 6 months post-infection. Some CCDs experienced marked transcriptional changes which may be the result of COVID-19 reactivation and could be responsible for long-haul syndrome.AcknowledgementsN/ATrial RegistrationNCT04360278ReferencesN/A Ethics ApprovalN/AConsentN/A
BackgroundWith the explosive growth of cancer immunotherapies, cancer vaccines have been in the spotlight for their ability to turn cold tumors hot. Particularly, dendritic cell vaccines (DCV) are capable of harnessing the immune system to recognize single or multiple epitopes as they are professional antigen presenting cells. However, DCVs have not been recognized as the platform of choice in many studies due to relatively high cost, difficulty in standardizing manufacturing methods and risk of product inconsistency. We have been using monocyte-derived DCs transduced with an adenovirus vector expressing HER2/neu in a clinical trial to treat HER2-expressing cancers. The vaccine was administered on weeks 0, 4, 8, 16 and 24 at 4 different dose-levels; 5 × 10E6, 10 × 10E6, 20 × 10E6 and 40 × 10E6 viable cells. The clinical outcome of the study is under analysis.1 To further optimize the safety and consistency of DCV, we reviewed the issues encountered in a first-in-human clinical trial during the manufacture of these cells at the NIH Clinical Center.MethodsManufacturing records of NCT01730118 A Phase I Study of an Autologous DCV Targeting HER2 in Solid Tumors were reviewed to identify any complications or deviations encountered during manufacturing from apheresis to delivery of the fresh DCVs (figure 1).ResultsBetween April 2013 and October 2019, 134 vaccines were manufactured for 33 patients. A total of 113 (84%) DCVs were administered, with 103 (91%) of those meeting release criteria, and the remaining administered under authorized medical exception (AME). All patients underwent a single apheresis collection with 18 (median, range 15–20) liters processed and a goal of 6 aliquots (333 × 10E6 monocytes/vial). Dual lumen catheterization was required in 23 (70%) patients, and all procedural reactions required no or minimal intervention. Summaries enumerate aberrancies encountered during the manufacturing process (table 1). Overall, interpatient variabilities may have contributed to 92 (78%) events, while 26 (22%) events arose in a ‘controllable’, patient-unrelated environment.Abstract 610 Figure 1Autologous DC vaccine manufacturing at the NIH clinical centerAbstract 610 Table 1ConclusionsIn spite of the variable events encountered during the manufacturing process, the majority of products were administered successfully. Patient-related variabilities were linked to most of the events. Future studies should be designed to minimize the impact of such variabilities on DCVs to provide high-quality personalized therapies. Manufacturing one large lot of DCs and cryopreserving enough aliquots for the entire study and the incorporation of an automated, closed cell culture system may reduce the aforementioned incidents and improve product quality.AcknowledgementsThis study was supported by the NIH Clinical Center and Center for Cancer Research, the National Cancer Institute. The authors are indebted to the staffs at NIH Clinical Center and the patients.Ethics ApprovalThe study was approved by NCI/NIH Institutional Review Board (#534360, 13C0016).ReferencesMaeng, H.M., et al., Preliminary results of a phase I clinical trial using an autologous dendritic cell cancer vaccine targeting HER2 in patients with metastatic cancer or operated high-risk bladder cancer (NCT01730118). Journal of Clinical Oncology 2019. 37(15_suppl): p. 2639–2639.Jin, P., et al., Plasma from some cancer patients inhibits adenoviral Ad5f35 vector transduction of dendritic cells. Cytotherapy 2018;20(5): p. 728–739.
Introduction Severe acute respiratory syndrome coronavirus-2 (SARS-CoV2) can induce a strong host immune response. Several groups have investigated the course of antibody responses in patients recovering from SARS-CoV-2 infections but little is known about the recovery of cellular immunity. This study investigated the cellular immune response in people who had recovered from SARS-CoV2 infection. Methods 162 coronavirus disease 2019 (COVID-19) convalescent plasma donors (CCD) and 40 healthy donor (HD) controls were enrolled prospectively in an IRB-approved protocol (Clinical Trials Number: NCT04360278) and provided written informed consent to participate in the study. Using the nCounter platform and host response panel with 785 genes across more than 50 pathways, we compared transcriptomic profiles on RNA samples obtained from the peripheral blood leukocytes of these 162 CCD and 40 HD. Additionally, in 69 of the 162 CCD samples, we evaluated transcriptomic trends at more than one-time point during the convalescent period. Results Age, sex, ethnicity, and body mass index distributions were similar among the CCD and HD. With respect to baseline complete blood counts, hemoglobin, platelets, and absolute basophil and eosinophil counts, all were similar among CCD and HD (Table 1). However, despite sample collections occurring several days after convalescence, mean counts for absolute neutrophil counts, absolute monocyte counts, and absolute lymphocyte counts were significantly higher among CCD compared to HD. 30-90 days after diagnosis, 19 of 773 genes differed (FDR < 0.05) between the average CCD and HD samples. Up-regulated genes included MAFB, CTLA4, PTGS2, and the chemokine signaling genes CXCR4, CXCL5, CXCL2 and CCR4. Down-regulated genes included PTGER2, CASP8, and the interleukins IL36A, IL31, IL20 and IL21 (Figure 1 a,b). Differential gene expression persisted for months. At 90-120 days, 13 genes were differentially regulated, including again MAFB CXCR4, PTGS2, CXCL2 and PTGER2, plus SMAD4. At 120-150 days post-diagnosis, 58 genes were differentially expressed (FDR < 0.05) compared to HD. Pathways with up-regulated genes included Treg differentiation, type III interferon signaling and chemokine signaling. 150-360 days post-diagnosis, 4 genes remained up-regulated on average (FDR < 0.05): PTGS2, PIK3CR, CXCL1 and SMAD4 (Figure 1 c,d). Individual patients varied considerably from the mean trend. Scoring samples by their similarity to the gene expression profile of the mean HD sample, 21 CCD samples from 20 unique patients (12%) were identified as highly perturbed from HD. 84% of these highly perturbed samples were collected > 90 days post-diagnosis. Of these 21 samples, 6 were distinguished by > 2-fold up-regulation of a cluster of interleukin and type-1 interferon genes (Figure 2). Conclusions Overall, our study identified important gene expression trends in CCD compared to HD in the post-acute period. The changes varied with time and among donors. As the expression of T-cell inhibitory molecule CTLA4 fell, the number of differentially expressed increased with the most marked changes occurring 120 to 150 days post-diagnosis in genes in chemokine signaling, type III interferon signaling and Treg pathways. Persistent alterations in inflammatory pathways and T-cell activation/exhaustion markers for months after active infection may help shed light on the pathophysiology of a prolonged post-viral syndrome observed in individuals following recovery from COVID-19 infection. Our data may serve as the basis for risk modification strategies in the period of active infection. Future studies may inform the ability to identify druggable targets involving these pathways to mitigate the long-term effects of COVID-19 infection. Figure 1 Figure 1. Disclosures Danaher: NanoString Technologies: Current Employment, Current holder of individual stocks in a privately-held company.
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