SARS-CoV-2-specific CD8+ T cells from people with long COVID establish and maintain effector phenotype and key TCR signatures over 2 years.

Long COVID occurs in a small but important minority of patients following COVID-19, reducing quality of life and contributing to healthcare burden. Although research into underlying mechanisms is evolving, immunity is understudied. SARS-CoV-2-specific T cell responses are of key importance for viral clearance and COVID-19 recovery. However, in long COVID, the establishment and persistence of SARS-CoV-2-specific T cells are far from clear, especially beyond 12 mo postinfection and postvaccination. We defined ex vivo antigen-specific B cell and T cell responses and their T cell receptors (TCR) repertoires across 2 y postinfection in people with long COVID. Using 13 SARS-CoV-2 peptide-HLA tetramers, spanning 11 HLA allotypes, as well as spike and nucleocapsid probes, we tracked SARS-CoV-2-specific CD8+ and CD4+ T cells and B-cells in individuals from their first SARS-CoV-2 infection through primary vaccination over 24 mo. The frequencies of ORF1a- and nucleocapsid-specific T cells and B cells remained stable over 24 mo. Spike-specific CD8+ and CD4+ T cells and B cells were boosted by SARS-CoV-2 vaccination, indicating immunization, in fully recovered and people with long COVID, altered the immunodominance hierarchy of SARS-CoV-2 T cell epitopes. Meanwhile, influenza-specific CD8+ T cells were stable across 24 mo, suggesting no bystander-activation. Compared to total T cell populations, SARS-CoV-2-specific T cells were enriched for central memory phenotype, although the proportion of central memory T cells decreased following acute illness. Importantly, TCR repertoire composition was maintained throughout long COVID, including postvaccination, to 2 y postinfection. Overall, we defined ex vivo SARS-CoV-2-specific B cells and T cells to understand primary and recall responses, providing key insights into antigen-specific responses in people with long COVID.

Repeated CD45RA-depleted DLI successfully increases donor chimerism in a patient with beta-thalassemia major after haploidentical stem cell transplant.

Allogeneic hematopoietic stem cell transplantation is curative for transfusion-dependent thalassemia, but mixed chimerism (MC) may herald graft rejection. We report a child who failed bone marrow transplant (BMT) from matched unrelated donor (MUD) successfully salvaged with haploidentical peripheral blood stem cell transplant (PBSCT), but had MC in T-lymphocyte compartment despite near-complete donor chimerism in myeloid compartment. MC was successfully improved by repeated CD45RA-depleted donor lymphocyte infusion (DLI). A 2-year-old Chinese girl with beta-thalassemia major underwent 12/12-MUD BMT with HU/AZA/Cy/Flu/Bu/TT conditioning resulted in graft rejection. As donor refused second donation, rescue haploidentical PBSCT was performed with alemtuzumab/fludarabine/treosulfan conditioning. Harvest product was CD3/CD45RA depleted with extra products cryopreserved. Split cell chimerism performed 1-month after haplo-transplant showed 97% mother, 3% MUD, and 0% host for granulocytes but 38% mother, 62% MUD, and 0% host for CD3 + T cells. In view of low haploidentical donor chimerism in T-lymphocyte compartment, CD45RA-depleted DLI using cryopreserved product was performed on day + 38, after thymoglobulin 3 mg/kg given as T-cell depletion 3 days beforehand. T-cell chimerism improved to 51% mother and 49% MUD post-DLI. Second cryopreserved CD45RA-depleted DLI was given 17 days after the first DLI (day + 55), and 100% full chimerism of mother's T cells was gradually established without significant graft-versus-host disease (GVHD) or viral reactivation. To conclude, split lineage chimerism determination is beneficial to guide management strategy. For MC in T-cell compartment, CD45RA-depleted DLI is a potential alternative to unselected T cells as it carries lower risk of GVHD and infection.

Selective T cell-depleted haploidentical hematopoietic stem cell transplantation for relapsed/refractory neuroblastoma.

Relapsed/refractory NB carries a bleak outcome, warranting novel treatment options. HaploHSCT induces a graft-versus-NB effect via natural killer cell alloreactivity. Review of patients with relapsed/refractory NB who underwent haploHSCT with ex vivo T-cell depletion in our unit from 2013 through 2018. Ten patients were identified (male=5; median age at haploHSCT=6.45 y, range: 3.49-11.02 y). Indications were relapsed in 7 and refractoriness in 3; disease status at haploHSCT was CR in 2, PR in 6, and PD in 2. All patients received peripheral blood stem cell grafts after ex vivo T-cell depletion (CD3/CD19-depletion=1; TCR-αß/CD19-depletion=4; CD3/CD45RA-depletion=4; and TCR-αß/CD45RA-depletion=1). Conditioning regimens were fludarabine-based. Neutrophils engrafted on median D + 10 (range: D + 9 to +13), and platelets engrafted (≥20 × 109 /L) on median D + 8 (range: D + 5 to D + 14). Early T- and NK-cell recovery were evident. Of the 10 patients, acute rejection developed in 1 (who died of PD despite rescue HSCT), and 1 died of sepsis before engraftment; 8 experienced full donor-chimerism post-HSCT. Among the 8, 6 experienced CR, 1 died of PD, and 1 died of pulmonary hypertensive crisis before evaluation. At publication, 4 were in remission (2.8, 7.4, 28.5, and 58.9 months). No significant GvHD occurred. HaploHSCT with selective ex vivo T-cell depletion may be a safe and useful salvage strategy for relapsed/refractory NB.

A simplified method of calculating cPRA for kidney allocation application in Hong Kong: a retrospective study.

Calculated panel reactive antibody (cPRA) represents possibility of encountering an incompatible donor for organ transplant candidates and has gradually replaced traditional PRA as a measurement of sensitization level. We tested two cPRA calculation methods on a cohort of renal candidate (n = 613). HLA typing of 563 Chinese deceased renal donors was used to estimate allele and haplotype frequencies of Hong Kong donor pool. The OPTN formula was adopted to generate cPRA (cPRA (freq)). We also incorporated a computer script to compare unacceptable antigens of patients against HLA phenotype of donors. The cPRA based on historical donor filtering was the percentage of filter out count over total number of donors (cPRA (filter)). Values of cPRA (freq) and cPRA (filter) showed almost perfect agreement with Lin's correlation coefficient equal to 1.000. SD of bias was 0.6 cPRA point. Limit of agreement was 0.9 to -1.5 points difference. Furthermore, the poor agreement between our in-house cPRA and values from other online calculators indicated the necessity to use local population data for accurate cPRA calculation. Built-in donor filtering method was more practicable for Hong Kong due to factors such as cost and flexibility. An on-going donor pool can reflect population allele frequencies and permits efficient periodic update of cPRA.

ABO-adjusted cPRA metric for kidney allocation in an Asian-predominant population.

Recent studies showed that ABO-adjusted calculated panel reactive antibody (ABO-cPRA) may better reflect the histocompatibility level in a multi-ethnic population, but such data in Asians is not available. We developed an ABO-adjusted cPRA metric on a cohort of waitlist kidney transplant patients (n = 647, 99% Chinese) in Hong Kong, based on HLA alleles and ABO frequencies of local donors. The concordance between the web-based ABO-cPRA calculator and the impact on kidney allocation were evaluated. The blood group distribution for A, B, O and AB among waitlist kidney candidates were 26.2%, 27.5%, 40.1%, and 6.1%, and their chances of encountering incompatible blood group donors were 32.6%, 32.4%, 57.6%, and 0%, respectively. There is poor agreement between web-based ABO-cPRA calculator and our locally developed metrics. Over 90% of patients showed an increase in cPRA after ABO adjustment, most notably in those with cPRA between 70% and 79%. Blood group O patients had a much greater increase in cPRA scores after adjustment while patients of blood group A and B had similar increment. 10.6% of non-AB blood group waitlist patients had ABO-cPRA elevated to ≥80%. A local ABO-adjusted cPRA metric is required for Asian populations and may improve equity in kidney distribution for patients with disadvantageous blood groups. The result from the current study potentially helps other countries/localities in establishing their own unified ABO-cPRA metrics and predict the impact on kidney allocation.

Identification of 58 novel HLA alleles identified in Chinese individuals by next-generation sequencing.

HLA genes are the most polymorphic in the human genome. High resolution HLA typing from 13,870 bone marrow donors in Hong Kong was obtained using Next-generation sequencing (NGS) technology. Among the 67 novel alleles identified, official HLA allele names of 50 novel class I alleles (HLA-A, -B, -C) and 8 novel class II alleles (HLA-DRB1, -DQB1) were assigned by the World Health Organization (WHO) Nomenclature Committee for Factors of the HLA System.

HLA-B*15:11 status and carbamazepine-induced severe cutaneous adverse drug reactions in HLA-B*15:02 negative Chinese.

BACKGROUND: HLA-B*15:11 is associated with carbamazepine (CBZ)-induced severe cutaneous adverse drug reactions (SCARs) in Japanese and some Asian populations, but such data remains relatively limited in Chinese. Routine HLA-B*15:02 screening is mandatory before CBZ commencement, however, SCARs related to CBZ were still observed in non-HLA*B-15:02 carriers. OBJECTIVE: We aimed to find out the prevalence of HLA-B*15:11 in Chinese patients and its associations with CBZ-induced SCARs. METHOD: We screened 8,328 blood samples collected for HLA allele typing before CBZ commencement during the period of January 2014 to December 2019. In HLA-B*15:02 negative Chinese patients, HLA-B*15:11 status were further screened, and the incidence of SCARs in the CBZ group was compared with the control group without CBZ use. RESULT: In this cohort, 1416 out of 8328 patients (17%) tested HLA-B*15:02 positive and were advised to avoid CBZ, while 80 (0.96%) were found to be HLA-B*15:11 positive. In 6911 (83%) patients who tested HLA-B*15:02 negative, 70 (1.01%) were HLA-B*15:11 positive. Five out of 70 (7.14%) patients had SCARs. The incidence of SCARs in HLA-B*15:11 carriers who received CBZ was significantly higher than those without CBZ (17.4% [4/23] vs. 2.13% [1/47], P = 0.037*). The odds ratio was 9.68 (95% CI 1.02-92.4, P = 0.048*). These included: one Stevens-Johnson syndrome (SJS), two DRESS, and one MPE after CBZ use, while one developed MPE after phenytoin use in control. CONCLUSION: HLA-B*15:11 is a potential risk factor of CBZ-induced SCARs in HLA-B*15:02 negative Chinese patients. Further screening of HLA-B*15:11 status in those HLA-B*15:02 negative patients is recommended to avoid undesirable SCARs.

Safety and immunogenicity of 3 doses of BNT162b2 and CoronaVac in children and adults with inborn errors of immunity.

Our study (NCT04800133) aimed to determine the safety and immunogenicity in patients with IEIs receiving a 3-dose primary series of mRNA vaccine BNT162b2 (age 12+) or inactivated whole-virion vaccine CoronaVac (age 3+) in Hong Kong, including Omicron BA.1 neutralization, in a nonrandomized manner. Intradermal vaccination was also studied. Thirty-nine patients were vaccinated, including 16 with homologous intramuscular 0.3ml BNT162b2 and 17 with homologous intramuscular 0.5ml CoronaVac. Two patients received 3 doses of intradermal 0.5ml CoronaVac, and 4 patients received 2 doses of intramuscular BNT162b2 and the third dose with intradermal BNT162b2. No safety concerns were identified. Inadequate S-RBD IgG and surrogate virus neutralization responses were found after 2 doses in patients with humoral immunodeficiencies and especially so against BA.1. Dose 3 of either vaccine increased S-RBD IgG response. T cell responses against SARS-CoV-2 antigens were detected in vaccinated IEI patients by intracellular cytokine staining on flow cytometry. Intradermal third dose vaccine led to high antibody response in 4 patients. The primary vaccination series of BNT162b2 and CoronaVac in adults and children with IEIs should include 3 doses for optimal immunogenicity.

Assessment of SARS-CoV-2 Immunity in Convalescent Children and Adolescents.

Persistence of protective immunity for SARS-CoV-2 is important against reinfection. Knowledge on SARS-CoV-2 immunity in pediatric patients is currently lacking. We opted to assess the SARS-CoV-2 adaptive immunity in recovered children and adolescents, addressing the pediatrics specific immunity towards COVID-19. Two independent assays were performed to investigate humoral and cellular immunological memory in pediatric convalescent COVID-19 patients. Specifically, RBD IgG, CD4+, and CD8+ T cell responses were identified and quantified in recovered children and adolescents. SARS-CoV-2-specific RBD IgG detected in recovered patients had a half-life of 121.6 days and estimated duration of 7.9 months compared with baseline levels in controls. The specific T cell response was shown to be independent of days after diagnosis. Both CD4+ and CD8+ T cells showed robust responses not only to spike (S) peptides (a main target of vaccine platforms) but were also similarly activated when stimulated by membrane (M) and nuclear (N) peptides. Importantly, we found the differences in the adaptive responses were correlated with the age of the recovered patients. The CD4+ T cell response to SARS-CoV-2 S peptide in children aged <12 years correlated with higher SARS-CoV-2 RBD IgG levels, suggesting the importance of a T cell-dependent humoral response in younger children under 12 years. Both cellular and humoral immunity against SARS-CoV-2 infections can be induced in pediatric patients. Our important findings provide fundamental knowledge on the immune memory responses to SARS-CoV-2 in recovered pediatric patients.

Establishing Simultaneous T Cell Receptor Excision Circles (TREC) and K-Deleting Recombination Excision Circles (KREC) Quantification Assays and Laboratory Reference Intervals in Healthy Individuals of Different Age Groups in Hong Kong.

The clinical experience gathered throughout the years has raised awareness of primary immunodeficiency diseases (PIDD). T cell receptor excision circles (TREC) and kappa-deleting recombination excision circles (KREC) assays for thymic and bone marrow outputs measurement have been widely implemented in newborn screening (NBS) programs for Severe Combined Immunodeficiency. The potential applications of combined TREC and KREC assay in PIDD diagnosis and immune reconstitution monitoring in non-neonatal patients have been suggested. Given that ethnicity, gender, and age can contribute to variations in immunity, defining the reference intervals of TREC and KREC levels in the local population is crucial for setting up cut-offs for PIDD diagnosis. In this retrospective study, 479 healthy Chinese sibling donors (240 males and 239 females; age range: 1 month-74 years) from Hong Kong were tested for TREC and KREC levels using a simultaneous quantitative real-time PCR assay. Age-specific 5th-95th percentile reference intervals of TREC and KREC levels (expressed in copies per µL blood and copies per 106 cells) were established in both pediatric and adult age groups. Significant inverse correlations between age and both TREC and KREC levels were observed in the pediatric age group. A significant higher KREC level was observed in females than males after 9-12 years of age but not for TREC. Low TREC or KREC levels were detected in patients diagnosed with mild or severe PIDD. This assay with the established local reference intervals would allow accurate diagnosis of PIDD, and potentially monitoring immune reconstitution following haematopoietic stem cell transplantation or highly active anti-retroviral therapy in the future.

Life-threatening cryoglobulinemia in HCV-negative Southern Chinese and a novel association with structural aortic abnormalities.

Cryoglobulinemia is uncommon in Southern Chinese in Hong Kong, with tropical climates and low incidence of hepatitis C virus (HCV) infection. Eight positive cases were detected among 481 patients screened for cryoglobulins over a 10-year period. Three HCV carriers (38%) ran benign courses. The others included two carriers of hepatitis B virus (HBV), two patients with adenocarcinoma, and one with chronic lymphocytic lymphoma. Four of them (except one HBV carrier) ran fulminant courses and died of cryoglobulin-related complications. Interestingly, all four cases also had structural aortic abnormalities, including multiple dissections, aneurysms, and congenital aortic arch abnormalities, which were often life threatening. The association of aortic abnormality and cryoglobulinemia is hitherto unreported and may be peculiar to our ethnic group. Aggressive control of the cryoglobulin may help to reduce aortic intima damage.

Protease inhibitor phenotypes and serum alpha-1-antitrypsin levels in patients with COPD: a study from Hong Kong.

OBJECTIVE: Many studies have suggested that an imbalance of protease activation and inhibition might result in COPD with emphysema. Levels of alpha-1-antitrypsin (alpha1-AT), the key protease inhibitor, are genetically determined by alleles that present in many phenotypes/subtypes, some of which are associated with deficiency of the protein. We prospectively evaluated the prevalence of the protease inhibitor (Pi) alleles and phenotypes together with the serum alpha1-AT levels in Chinese patients with COPD. METHODOLOGY: The study population comprised 356 patients with COPD. The male-to-female ratio was 4 : 1 with a mean age of 72.4 years (range 44-93 years). Isoelectric focusing was used for Pi phenotyping/subtyping. The frequencies of Pi alleles and phenotypes were compared with the frequencies in 1085 healthy unrelated Chinese control subjects. The serum alpha1-AT levels were measured by the Cobas Fara assay. RESULTS: PiZ was not detected. No significant difference in distribution of PiM phenotypes/subtypes between patients with COPD and healthy controls was observed, except for M1M3 and M2M3. There was also a significant difference in the proportion of variant S and F alleles between the disease group and the control population. CONCLUSION: The low prevalence of deficiency Pi phenotypes/subtypes suggests a lack of contribution of alpha1-AT deficiency to the pathogenesis of COPD in Chinese patients. The strategy of launching an alpha1-AT deficiency detection program among COPD patients, based on the recommendation of the World Health Organization, may not be readily applicable in our local setting.