Durability of Humoral and Cellular Immunity after an Extended Primary Series with Heterologous Inactivated SARS-CoV-2 Prime-Boost and ChAdOx1 nCoV-19 in Dialysis Patients (ICON3).

The durability of a three-dose extended primary series of COVID-19 vaccine in dialysis patients remains unknown. Here, we assessed dynamic changes in SARS-CoV-2-specific humoral and cell-mediated immunity at baseline, 3 months, and 6 months after the extended primary series in 29 hemodialyzed (HD), 28 peritoneal dialyzed (PD) patients, and 14 healthy controls. Participants received two doses of inactivated SARS-CoV-2 vaccine followed by a dose of ChAdOx1 nCoV-19 vaccine. At 6 months, median anti-RBD IgG titers (IQR) significantly declined from baseline in the HD (1741 (1136−3083) BAU/mL vs. 373 (188−607) BAU/mL) and PD (1093 (617−1911) BAU/mL vs. 180 (126−320) BAU/mL) groups, as did the mean percent inhibition of neutralizing antibodies (HD: 96% vs. 81%; PD: 95% vs. 73%) (all p < 0.01). Age and post-vaccination serological response intensity were predictors of early humoral seroprotection loss. In contrast, cell-mediated immunity remained unchanged. In conclusion, humoral immunity declined substantially in dialysis patients, while cell-mediated immunity remained stable 6 months after the extended heterologous primary series of two inactivated SARS-CoV-2/ChAdOx1 nCoV-19 vaccine. A booster dose could be considered in dialysis patients 3 months after this unique regimen, particularly in the elderly or those with a modest initial humoral response.

The Detection of SARS-CoV2 Antigen in Wastewater Using an Automated Chemiluminescence Enzyme Immunoassay.

The SARS-CoV-2 virus, which is driving the current COVID-19 epidemic, has been detected in wastewater and is being utilized as a surveillance tool to establish an early warning system to aid in the management and prevention of future pandemics. qPCR is the method usually used to detect SARS-CoV-2 in wastewater. There has been no study using an immunoassay that is less laboratory-intensive than qPCR with a shorter turnaround time. Therefore, we aimed to evaluate the performance of an automated chemiluminescence enzyme immunoassay (CLEIA) for SARS-CoV-2 antigen in wastewater. The CLEIA assay achieved 100% sensitivity and 66.7% specificity in a field-captured wastewater sample compared to the gold standard RT-qPCR. Our early findings suggest that the SARS-CoV-2 antigen can be identified in wastewater samples using an automated CLEIA, reducing the turnaround time and improving the performance of SARS-CoV-2 wastewater monitoring during the pandemic.

An additional dose of viral vector COVID-19 vaccine and mRNA COVID-19 vaccine in kidney transplant recipients: A randomized controlled trial (CVIM 4 study).

Immunogenicity following an additional dose of Coronavirus disease 2019 (COVID-19) vaccine was investigated in an extended primary series among kidney transplant (KT) recipients. Eighty-five KT participants were randomized to receive either an mRNA (M group; n = 43) or viral vector (V group; n = 42) vaccine. Among them, 62% were male, with a median (IQR) age of 50 (43-59) years and post-transplantation duration of 46 (26-82) months. At 2 weeks post-additional dose, there was no difference in the seroconversion rate between the M and V groups (70% vs. 65%, p = .63). A median (IQR) of anti-RBD antibody level was not statistically different between the M group compared with the V group (51.8 [5.1-591] vs. 28.5 [2.9-119.3] BAU/ml, p = .18). Furthermore, the percentage of participants with positive SARS-CoV-2 surrogate virus neutralization test results was not statistically different between groups (20% vs. 15%, p = .40). S1-specific T cell and RBD-specific B cell responses were also comparable between the M and V groups (230 [41-420] vs. 268 [118-510], p = .65 and 2 [0-10] vs. 2 [0-13] spot-forming units/106 peripheral blood mononuclear cells, p = .60). In conclusion, compared with an additional dose of viral vector COVID-19 vaccine, a dose of mRNA COVID-19 vaccine did not elicit significantly different responses in KT recipients, regarding either humoral or cell-mediated immunity. (TCTR20211102003).

A Simple Method to Detect SARS-CoV-2 in Wastewater at Low Virus Concentration.

Background: Since its initial appearance in December 2019, coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread globally. Wastewater surveillance has been demonstrated as capable of identifying infection clusters early. The purpose of this study was to investigate a quick and simple method to detect SARS-CoV-2 in wastewater in Thailand during the early stages of the second outbreak wave when the prevalence of the disease and the virus concentration in wastewater were low. Methods: Wastewater samples were collected from a hospital caring for patients with COVID-19 and from 35 markets, two of which were associated with recently reported COVID-19 cases. Then, samples were concentrated by membrane filtering prior to SARS-CoV-2 detection by RT-qPCR. Results: SARS-CoV-2 RNA was detected in the wastewater samples from the hospital; the Ct values for the N, ORF1ab, and S genes progressively increased as the number of patients admitted to the treatment floor decreased. Notably, the ORF1ab and S genes were still detectable in wastewater even when only one patient with COVID-19 remained at the hospital. SARS-CoV-2 RNA was detected in the wastewater samples from fresh market where COVID-19 cases were reported. Conclusions: Our findings suggest that wastewater surveillance for SARS-CoV-2 is sensitive and can detect the virus even in places with a high ambient temperature and relatively low prevalence of COVID-19.

Associations of lymphocyte subpopulations with clinical phenotypes and long-term outcomes in juvenile-onset systemic lupus erythematosus.

OBJECTIVE: Juvenile-onset systemic lupus erythematosus (JSLE) is a complex and heterogeneous immune-mediated disease. Cellular components have crucial roles in disease phenotypes and outcomes. We aimed to determine the associations of lymphocyte subsets with clinical manifestations and long-term outcomes in JSLE patients. METHODS: A cohort of 60 JSLE patients provided blood samples during active disease, of whom 34 provided further samples during inactive disease. In a longitudinal study, blood samples were obtained from 49 of the JSLE patients at 0, 3, and 6 months. The healthy control (HC) group consisted of 42 age-matched children. Lymphocyte subsets were analyzed by flow cytometry. RESULTS: The percentages of CD4+ T, γδ T, and NK cells were significantly decreased in JSLE patients compared with HC, while the percentages of CD8+ T, NKT, and CD19+ B cells were significantly increased. The percentage of regulatory T cells (Tregs) was significantly lower in JSLE patients with lupus nephritis (LN) than in non-LN JSLE patients and HC. The patients were stratified into high and low groups by the median frequency of each lymphocyte subset. The γδ T cells high group and NK cells high group were significantly related to mucosal ulcer. The CD4+ T cells high group was significantly associated with arthritis, and the NKT cells high group was substantially linked with autoimmune hemolytic anemia. The CD8+ T cells low group was mainly related to vasculitis, and the Tregs low group was significantly associated with LN. The percentage of Tregs was significantly increased at 6 months of follow-up, and the LN JSLE group had a lower Treg percentage than the non-LN JSLE group. Predictors of remission on therapy were high Tregs, high absolute lymphocyte count, direct Coombs test positivity, and LN absence at enrollment. CONCLUSION: JSLE patients exhibited altered lymphocyte subsets, which were strongly associated with clinical phenotypes and long-term outcomes.

Effects of Desflurane and Sevoflurane anesthesia on regulatory T cells in patients undergoing living donor kidney transplantation: a randomized intervention trial.

BACKGROUND: Volatile anesthetic agents used during surgery have immunomodulatory effects which could affect postoperative outcomes. Recognizing that regulatory T cells (Tregs) plays crucial roles in transplant tolerance and high peripheral blood Tregs associated with stable kidney graft function, knowing which volatile anesthetic agents can induce peripheral blood Tregs increment would have clinical implications. This study aimed to compare effects of desflurane and sevoflurane anesthesia on peripheral blood Tregs induction in patients undergoing living donor kidney transplantation. METHODS: A prospective, randomized, double-blind trial in living donor kidney transplant recipients was conducted at a single center, tertiary-care, academic university hospital in Thailand during August 2015 - June 2017. Sixty-six patients were assessed for eligibility and 40 patients who fulfilled the study requirement were equally randomized and allocated to desflurane versus sevoflurane anesthesia during transplant surgery. The primary outcome included absolute changes of peripheral blood CD4+CD25+FoxP3+Tregs which measured by flow cytometry and expressed as the percentage of the total population of CD4+ T lymphocytes at pre-exposure (0-h) and post-exposure (2-h and 24-h) to anesthetic gas. P-value < 0.05 denoted statistical significance. RESULTS: Demographic data were comparable between groups. No statistical difference of peripheral blood Tregs between desflurane and sevoflurane groups observed at the baseline pre-exposure (3.6 ± 0.4% vs. 3.1 ± 0.4%; p = 0.371) and 2-h post-exposure (3.0 ± 0.3% vs. 3.5 ± 0.4%; p = 0.319). At 24-h post-exposure, peripheral blood Tregs was significantly higher in desflurane group (5.8 ± 0.5% vs. 4.1 ± 0.3%; p = 0.008). Within group analysis showed patients receiving desflurane, but not sevoflurane, had 2.7% increase in peripheral blood Treg over 24-h period (p < 0.001). CONCLUSION: This study provides the clinical trial-based evidence that desflurane induced peripheral blood Tregs increment after 24-h exposure, which could be beneficial in the context of kidney transplantation. Mechanisms of action and clinical advantages of desflurane anesthesia based on Treg immunomodulation should be investigated in the future. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02559297 . Registered 22 September 2015 - retrospectively registered.

CMV-Reactive NK Cells in Pediatric Post-Hematopoietic Stem Cell Transplant.

BACKGROUND: Immune reconstitution of T cells has been proven to be a protective factor against cytomegalovirus (CMV) reactivation in patients post-hematopoietic stem cell transplant. Recently, more evidence has suggested that natural killer (NK) cells also play role in the protection against CMV reactivation in these patients. METHODS: CMV-specific T cells and CMV-reactive NK cells from pediatric patients undergoing hematopoietic stem cell transplant were examined by flow cytometry. These cells were defined as cells producing interferon gamma (IFNγ) upon stimulation with CMV antigens. RESULTS: This study demonstrated that NK cells reactive to CMV do exist in pediatric patients after stem cell transplant. These cells vigorously responded to stimulation with CMV peptides (pp65 and IE1) and to a lesser extent to CMV whole lysate by secretion of IFNγ. Patients with CMV reactivation tended to have less CMV-reactive NK cells than those without. CONCLUSION: Reconstitution of CMV-reactive NK cells, together with CMV-specific T cells, may play a role in the control of CMV infections in patients after stem cell transplant.

Capillary blood as an alternative specimen for enumeration of percentages of lymphocyte subsets.

OBJECTIVE: Capillary blood has been increasingly used in point-of-care setting for clinical monitoring in immunology and infectious diseases. We explored whether percentages of lymphocyte subsets (T-cells; CD3+, helper T-cells; CD4+, cytotoxic T-cells; CD8+, B-cells; CD19+, NK cells; CD56+, gamma delta T-cells, and regulatory T-cells) with regard to total lymphocyte count from capillary and venous blood of healthy volunteers were in good agreement. RESULTS: All percentages of lymphocyte subsets with regard to total lymphocyte count from capillary blood were significantly correlated with those from venous blood (r ≥ 0.9 for every cell type). However, Bland-Altman plots showed high agreement between capillary and venous samples only in those of CD3+, CD4+, and CD8+ cells (limit of agreement percentages from mean venous blood < 20%). However, the agreement of percentages of other lymphocyte subsets from venous and capillary blood was mediocre. We concluded that capillary blood could be used as an alternative for venous blood to determine percentages of CD3+, CD4+, and CD8+ cells with regard to total lymphocyte count.

Suppressive Characteristics of Umbilical Cord Blood-derived Regulatory T Cells After Ex Vivo Expansion on Autologous and Allogeneic T Effectors and Various Lymphoblastic Cells.

The third-party umbilical cord blood (UCB)-derived regulatory T cells (Treg) are an alternative to donor-derived Treg as cellular therapy of graft-versus-host disease following hematopoietic stem cell transplantation. However, their suppressive characteristics against autologous and allogeneic T effector cells (Teff) have rarely been documented. The exact role of UCB-Treg in hematologic malignancies is also uncertain. Here, we investigated the direct effects of UCB-Treg on the proliferation of autologous Teff, as compared with allogeneic Teff, and also determined cellular fates of lymphoblasts after UCB-Treg co-culture. UCB-Treg were isolated from 8 UCB samples using 2-step immunomagnetic bead sorting. After 10-day ex vivo expansion, up to 60-fold increase in cell number with 76.7%±4.9% of CD4CD25CD127FoxP UCB-Treg was obtained. Further characterization showed that ex vivo-expanded UCB-Treg contained a higher proportion of CD95CD45RACCR4Treg-B subpopulation compared with the CD95CD45RACCR4Treg-A subpopulation (13.0%±4.8% vs. 0.8%±0.7%; P<0.05), along with the detecting of substantial amounts of secretory IL-10 (57.7±17.8 pg/mL) and TGF-ß1 (196.5±29.7 pg/mL) in culture supernatants. After 4 days co-culture with UCB-Treg (at the ratio of 1:1), the proliferation of autologous and allogeneic Teff was decreased comparably (43.6%±17.5% vs. 37.6±17.7%; P=0.437). Suppression was independent of HLA-A, B, and DRB1 compatibility between UCB-Treg and Teff. UCB-Treg co-culture with various lymphoblasts showed proliferative suppression of Jurkat T lymphoblasts (45.4%±20.5% at the ratio of 1:1), but not Namalwa and Raji B lymphoblasts. All lymphoblasts had no significant cell apoptosis or death after co-culture. In conclusion, the ex vivo-expanded UCB-Treg had no difference in autologous and allogeneic Teff suppression. UCB-Treg therapy in patients with graft-versus-host disease who have a primary disease of T-cell leukemia may have additional benefits in the prevention of relapsed disease.

Viral-specific T-cell response in hemorrhagic cystitis after haploidentical donor stem cell transplantation.

Viral hemorrhagic cystitis (HC) after hematopoietic stem cell transplantation (HSCT) can be devastating. Standard treatment modalities have not been well established, but immune reconstitution may be necessary for sustained viral clearance. We studied five pediatric patients who developed viral HC after haplo-identical HSCT. All patients developed virus-specific CD4- and CD8-positive T cells, and the emergence of these viral-specific T cells was temporally associated with successful viral clearance.

In vitro cytokine changes after pediatric liver transplantation.

BACKGROUND: Patients with chronic liver disease have been shown to have impaired immune statuses. Liver transplantation (LT) is the standard treatment for end-stage liver disease patients and immunosuppressive drugs are commonly used to prevent graft rejection. There is an increasing evidence of de novo food allergies post LT. OBJECTIVE: To investigate the cytokine response of peripheral blood mononuclear cells (PBMCs) of pediatric LT recipients before and six months after transplantation. METHOD: PBMCs collected before and six months after LT were stimulated with phytohemagglutinin (PHA), beta-lactoglobulin (BLG), tacrolimus (Tac), dexamethasone (Dex), and a combination of BLG and Dex (B+D), BLG and Tac (B+T), BLG and Tac plus Dex (B+T+D). Culture supernatants were measured for IL-5, IFN-γ and IL-10. Blood for liver function tests, complete blood counts, total IgE and specific IgE (sIgE) to cow's milk were recorded. RESULTS: A total of five pediatric LT recipients were enrolled in the study. There were no food allergy cases. Total IgE and sIgE to cow's milk decreased significantly after LT. After transplantation, there was a significant increase in IL-5, IFN-γ and IL-10 in culture supernatants of PHA-stimulated PBMCs. Among different stimulations in post transplantation's PBMCs, the level of IL-5 significantly increased in B+D was suppressed with the combination of B+T+D. The level of IL-10 significantly increased in all conditions containing BLG both before and after transplantation. CONCLUSION: There was an improvement of the in vitro-cytokine responses after liver transplantations. Immunosuppressive drugs used in post transplantation had an effect on the cytokine responses.

Pretransplant immunosuppression followed by reduced-toxicity conditioning and stem cell transplantation in high-risk thalassemia: a safe approach to disease control.

Patients with class 3 thalassemia with high-risk features for adverse events after high-dose chemotherapy with hematopoietic stem cell transplantation (HSCT) are difficult to treat, tending to either suffer serious toxicity or fail to establish stable graft function. We performed HSCT in 18 such patients age ≥7 years and hepatomegaly using a novel approach with pretransplant immunosuppression followed by a myeloablative reduced-toxicity conditioning regimen (fludarabine and i.v. busulfan [Flu-IV Bu]) and then HSCT. The median patient age was 14 years (range, 10 to 18 years). Before the Flu-IV Bu + antithymocyte globulin conditioning regimen, all patients received 1 to 2 cycles of pretransplant immunosuppression with fludarabine and dexamethasone. Thirteen patients received a related donor graft, and 5 received an unrelated donor graft. An initial prompt engraftment of donor cells with full donor chimerism was observed in all 18 patients, but 2 patients developed secondary mixed chimerism that necessitated withdrawal of immunosuppression to achieve full donor chimerism. Two patients (11%) had acute grade III-IV graft-versus-host disease, and 5 patients had limited chronic graft-versus-host disease. The only treatment-related mortality was from infection, and with a median follow-up of 42 months (range, 4 to 75), the 5-year overall survival and thalassemia-free survival were 89%. We conclude that this novel sequential immunoablative pretransplantation conditioning program is safe and effective for patients with high-risk class 3 thalassemia exhibiting additional comorbidities.