Low incidence of de novo HLA antibodies after COVID-19 vaccination: A cohort study of patients awaiting kidney transplantation.

BACKGROUND: Antibodies against human leukocyte antigen (anti-HLA Abs) are associated with an increased risk of allograft loss. Herein, we report the prospective follow-up for anti-HLA Abs formation in 103 patients with end-stage kidney disease on the waiting list for transplantation who underwent COVID-19 vaccination. PATIENTS AND METHODS: Sera were tested before and after vaccination using Luminex technology. The cohort comprised of 62 males and 41 females with a mean age of 56 ± 14 years. The patients received BNT162b2 (80.4%), mRNA-1273 (18.5%), AZD1222 (0.40%), or ChAdOx1-S (0.80%) vaccine. Patients were tested before and within 119 ± 50, 95 ± 46 and 25 ± 26 days after the first, second, and third dose of the vaccine, respectively. RESULTS: No significant change in calculated panel reactive antibody (cPRA) after vaccination was seen. Although 98.1% of patients had no change in anti-HLA Abs profile or cPRA after vaccination, two patients (1.9%) developed de novo anti-HLA Abs against class I or II HLA antigens. In those two patients, the cPRA changed from 0% and 63% at baseline to 9% and 90% after vaccination, respectively. Both patients received the BNT162b2 mRNA-based vaccine. The earliest detected anti-HLA Abs was 18 days after the first dose. CONCLUSION: In rare cases, new anti-HLA antibodies were observed after COVID-19 vaccination, with potential implications for transplantation. The low incidence of this phenomenon is outweighed by the clinical benefits of vaccination.

Discovery of three novel HLA-DPA1 alleles, HLA-DPA1*01:147N, 01:03:47, and 02:106 using next-generation sequencing.

Characterization of three novel HLA-DPA1 alleles bearing null, synonymous, and non-synonymous mutations.

Identification of four novel HLA-DQ alleles, HLA-DQA1*01:106, -DQA1*01:107, -DQA1*05:74 and -DQB1*05:01:48.

Characterization of new alleles, three HLA-DQA1 and one HLA-DQB1, bearing non-synonymous and synonymous mutations, respectively.

Two novel HLA class I alleles, HLA-C*04:493 and -A*26:01:78, identified using next-generation sequencing.

Two novel HLA class I alleles bearing point mutations, HLA-C*04:493 and HLA- A*26:01:78, were identified.

Identification of a novel allele with a frameshift mutation, HLA-DRB4*01:165N, using next-generation sequencing.

HLA-DRB4*01:165N exhibits deletion of a nucleotide in exon 3, producing a premature stop codon.

Donor Genetic Predisposition to High Interleukin-10 Production Appears Protective against Acute Graft-Versus-Host Disease.

The persistence of graft-versus-host disease (GVHD) as the principal complication of allogeneic hematopoietic cell transplantation (HCT) demonstrates that HLA matching alone is insufficient to prevent alloreactivity. We performed molecular and functional characterization of 22 candidate cytokine genes for their potential to improve matching in 315 myeloablative, 10/10 HLA-matched donor−recipient pairs. Recipients of a graft carrying the -1082GG IL10 gene promoter region variant had a three-fold lower incidence of grade II−IV acute GVHD compared to IL10-1082AA graft recipients (SHR = 0.25, p = 0.005). This was most evident in matched unrelated donor (MUD) transplants, where the greatest alloreactivity is expected. IL10-1082GG transplants did not experience an increased incidence of relapse, and, consequently, overall survival was two-fold higher in IL10-1082GG MUD transplants (HR = 0.17, p = 0.023). Longitudinal post-transplant measurements demonstrated that -1082GG is a high-IL10-producing and -expressing genotype with attenuated CD8+ T-cell reconstitution. High post-transplant donor chimerism in T- and myeloid-cells (>95%) confirmed a predominant donor, rather than recipient, genotype effect on immune function and aGVHD. To date, this is the first study to report corroborating genome-to-cellular evidence for a non-HLA donor immunogenetic variant that appears to be protective against GVHD. The incorporation of IL10 variants in donor selection criteria and clinical-management decisions has the potential to improve patient outcomes.

Next-generation sequencing identifies two novel HLA class II alleles, HLA-DRB1*01:115 and HLA-DRB1*14:224.

We identified two novel HLA class II alleles by NGS, HLA-DRB1*01:115 and HLA-DRB1*14:224.

The novel HLA class II allele, DPB1*1284:01, identified using next-generation sequencing.

Our laboratory has identified DPB1*1284:01 as a novel HLA Class II allele by NGS.

An Integrated Clinical and Genetic Prediction Model for Tacrolimus Levels in Pediatric Solid Organ Transplant Recipients.

BACKGROUND: There are challenges in achieving and maintaining therapeutic tacrolimus levels after solid organ transplantation (SOT). The purpose of this genome-wide association study was to generate an integrated clinical and genetic prediction model for tacrolimus levels in pediatric SOT. METHODS: In a multicenter prospective observational cohort study (2015-2018), children <18 years old at their first SOT receiving tacrolimus as maintenance immunosuppression were included (455 as discovery cohort; 322 as validation cohort). Genotyping was performed using a genome-wide single nucleotide polymorphism (SNP) array and analyzed for association with tacrolimus trough levels during 1-y follow-up. RESULTS: Genome-wide association study adjusted for clinical factors identified 25 SNPs associated with tacrolimus levels; 8 were significant at a genome-wide level (P < 1.025 × 10-7). Nineteen SNPs were replicated in the validation cohort. After removing SNPs in strong linkage disequilibrium, 14 SNPs remained independently associated with tacrolimus levels. Both traditional and machine learning approaches selected organ type, age at transplant, rs776746, rs12333983, and rs12957142 SNPs as the top predictor variables for dose-adjusted 36- to 48-h posttacrolimus initiation (T1) levels. There was a significant interaction between age and organ type with rs776476*1 SNP (P < 0.05). The combined clinical and genetic model had lower prediction error and explained 30% of the variation in dose-adjusted T1 levels compared with 18% by the clinical and 12% by the genetic only model. CONCLUSIONS: Our study highlights the importance of incorporating age, organ type, and genotype in predicting tacrolimus levels and lays the groundwork for developing an individualized age and organ-specific genotype-guided tacrolimus dosing algorithm.

SARS Cov-2 vaccination induces de novo donor-specific HLA antibodies in a renal transplant patient on waiting list: A case report.

The ability of COVID-19 vaccination to induce anti-HLA antibodies (Abs) formation in renal transplant candidates is not well studied. A 42-year-old man on a renal transplant waitlist, with no sensitization history, was tested for DSA before and after COVID-19 vaccination. Patient has consistently tested negative for COVID-19 virus. Eighteen days after receiving first dose of mRNA-based vaccine, flow cytometry crossmatch (FCXM) was strongly positive with de novo donor-specific Ab (dnDSA) against B57 and de novo non-DSA against B58. Before vaccination, preliminary FCXM was negative with no anti-HLA Abs. This event prompted the transplant team to cancel the surgery. COVID-19 vaccination could be associated with anti-HLA Abs formation in renal patients on waitlists that could affect future transplantability.

Relevant SARS-CoV-2 Genome Variation through Six Months of Worldwide Monitoring.

Real-time genome monitoring of the SARS-CoV-2 pandemic outbreak is of utmost importance for designing diagnostic tools, guiding antiviral treatment and vaccination strategies. In this study, we present an accurate method for temporal and geographical comparison of mutational events based on GISAID database genome sequencing. Among 42523 SARS-CoV-2 genomes analyzed, we found 23202 variants compared to the reference genome. The Ti/Tv (transition/transversion) ratio was used to filter out possible false-positive errors. Transition mutations generally occurred more frequently than transversions. Our clustering analysis revealed remarkable hotspot mutation patterns for SARS-CoV-2. Mutations were clustered based on how their frequencies changed over time according to each geographical location. We observed some clusters showing a clear variation in mutation frequency and continuously evolving in the world. However, many mutations appeared in specific periods without a clear pattern over time. Various important nonsynonymous mutations were observed, mainly in Oceania and Asia. More than half of these mutations were observed only once. Four hotspot mutations were found in all geographical locations at least once: T265I (NSP2), P314L (NSP12), D614G (S), and Q57H (ORF3a). The current analysis of SARS-CoV-2 genomes provides valuable information on the geographical and temporal mutational evolution of SARS-CoV-2.

Four novel HLA class II alleles identified using next-generation sequencing.

We describe here four novel HLA class II alleles, DPA1*01:33, DPB1*1082:01, DPB1*1144:01, and DQA1*01:45, identified in our laboratory following the implementation of next generation sequencing (NGS) for routine high resolution HLA typing of donors and recipients for solid organ transplant and hematopoetic stem cell transplant. The NGS technology has improved our ability to match at all clinically HLA loci and to facilitate the interpretation of allele specific antibody and eplet matching.

Comparison of abacavir-specific effector and proliferating functions of CD8 T cells in abacavir-treated HIV-1 patients.

Susceptibility to abacavir hypersensitivity (ABH) in HIV-1-positive patients is strongly linked to the carriage of HLA-B*57:01 and the potential mechanism includes drug-specific activation of cytokine producing CD8 T cells exclusively in individuals carrying HLA-B*57:01. Here, we report a detailed characterization of abacavir-induced functional response of CD8 T cells in HLA-B*57:01pos individuals. Peripheral blood mononuclear cells (PBMNCs) from HLA-B*57:01pos ABHpos and HLA-B*57:01neg ABHneg individuals were stimulated with abacavir. Multicolor flow cytometry was performed to assess the cytokine (IFNγ) production and degranulation (CD107a expression) after 6-18 hr culture and to enumerate proliferating CD4/CD8 T cells by culturing carboxyfluorescein diacetate succinimidyl ester-loaded PBMNCs for 7 days. CD8 T cells from HLA-B*57:01pos ABHpos individuals were multifunctional: proliferating, IFNγ producing, degranulating (CD107apos ), and both degranulating and IFNγ producing (CD107apos IFNγpos ). Degranulating CD8 T cells in general and both degranulating and IFNγ producing CD8 T cells in particular dominated abacavir-specific immune response. All functional responses were partially blocked by addition of HLA-B*57:01-reactive Bw4 mAb, but not by non-HLA-B*57:01-reactive Bw6 mAb. In conclusion, the study demonstrates that abacavir-specific CD8 T-cell-restricted immune response in HLA-B*57:01pos ABHpos HIV-1 patients has multiple effector and proliferating functions, where the primary effector response appears to be the release of cytolytic granules. The findings have implications for immunotherapy of HLA-related drug hypersensitivities.

Comparison of sequence-specific oligonucleotide probe vs next generation sequencing for HLA-A, B, C, DRB1, DRB3/B4/B5, DQA1, DQB1, DPA1, and DPB1 typing: Toward single-pass high-resolution HLA typing in support of solid organ and hematopoietic cell transplant programs.

Many clinical laboratories supporting solid organ transplant programs use multiple HLA genotyping technologies, depending on individual laboratory needs. Sequence-specific primers and quantitative polymerase chain reaction (qPCR) serve the rapid turnaround necessary for deceased donor workup, while sequence-specific oligonucleotide probe (SSOP) technology is widely employed for higher volumes. When clinical need mandates high-resolution data, Sanger sequencing-based typing (SBT) has been the "gold standard." However, all those methods commonly yield ambiguous typing results that utilize valuable laboratory resources when resolution is required. In solid organ transplantation, high-resolution typing may provide critical information for highly sensitized patients with donor-specific anti-HLA antibodies (DSA), particularly when DSA involve HLA alleles not discriminated by SSOP typing. Arguments against routine use of SBT include assay complexity, long turnaround times (TAT), and increased costs. Here, we compare a next generation sequencing (NGS) technology with SSOP for accuracy, effort, turnaround time, and level of resolution for genotyping of 11 HLA loci among 289 specimens from five clinical laboratories. Results were concordant except for SSOP misassignments in eight specimens and 21 novel sequences uniquely identified by NGS. With few exceptions, SSOP generated ambiguous results while NGS provided unambiguous three-field allele assignments. For complete HLA genotyping of up to 24 samples by either SSOP or NGS, bench work was completed on day 1 and typing results were available on day 2. This study provides compelling evidence that, although not viable for STAT typing of deceased donors, a single-pass NGS HLA typing method has direct application for solid organ transplantation.

CD-34+ and VE-cadherin+ endothelial progenitor cells in preeclampsia and normotensive pregnancies.

OBJECTIVE: The objective of our study was to determine levels of endothelial progenitor cells (EPCs) in preeclampsia and normotensive pregnant women. STUDY DESIGN: Prospective cohort study of women with preeclampsia and normotensive pregnancies. EPCs were estimated by flow cytometry. Multiple linear regression was used to assess the association of EPCs with preeclampsia adjusting for maternal age, body mass index (BMI), gestation and ethnicity. MAIN OUTCOME MEASURE: Levels of EPCs in preeclampsia and normotensive pregnancies, with CD-34 and vascular endothelial (VE)-cadherin as markers of EPCs. VE-cadherin is an endothelial cell adhesion molecule used to delineate endothelial lineage of EPCs. RESULTS: There were thirty women in the preeclampsia group and thirty-three in the normotensive group. The two groups were similar except for the BMI and blood pressures, which were higher in preeclampsia. On multiple linear regression, EPCs numbers were significantly higher by 29 (95% confidence interval 11.7-46.6, p = 0.001) in preeclampsia compared to the normotensive group. There was significant positive correlation between EPCs and systolic blood pressure in preeclampsia (Spearman correlation coefficient 0.39, p = 0.03). CONCLUSION: Although widely used in cardiovascular disease other than preeclampsia, this is the first study using VE-cadherin as a marker of endothelial lineage to define EPCs in preeclampsia. Our results suggest the higher number of EPCs in preeclampsia may be a response of the bone marrow to endothelial injury.

The identification of a novel HLA-B allele, HLA-B*27:05:38.

HLA-B*27:05:38 differs from HLA-B*27:05:02:01 by 2 mismatches in exon 3, resulting in 2 synonymous mutations.

Identification of a novel HLA-C allele, HLA-C*07:778, in an unrelated hematopoietic stem cell donor.

HLA-C*07:778 differs from HLA-C*07:01:01:01 by a single nonsynonymous nucleotide substitution at codon 263 (CAC→CAG).

Genetic Polymorphisms of TLR4 and MICA are Associated with Severity of Trachoma Disease in Tanzania.

AIM: To examine the association of TLR4 Asp299Gly and MICA exon 5 microsatellites polymorphisms with severity of trachoma in a sub-Saharan East Africa population of Tanzanian villagers. METHODS: The samples were genotyped for MICA exon 5 microsatellites and the TLR4 299 A/G polymorphism by Restriction Fragment Length Polymorphism (RFLP), and GeneScan®, respectively. The association of TLR4 Asp299Gly and MICA exon 5 microsatellites with inflammatory trachoma (TI) and trichiasis (TI) were examined. RESULTS: The results showed an association between TLR4 and MICA polymorphisms and trachoma disease severity, as well as with protection. TLR4 an allele was significantly associated with inflammatory trachoma (p=0.0410), while the G allele (p=0.0410) was associated with protection. CONCLUSION: TLR4 and MICA may modulate the risk of severity to trachoma disease by modulating the immune response to Ct. In addition; the increased frequency of MICA-A9 heterozygote in controls may suggest a positive selection of these alleles in adaptation to environments where Ct is endemic.

Donor-Recipient Matching for KIR Genotypes Reduces Chronic GVHD and Missing Inhibitory KIR Ligands Protect against Relapse after Myeloablative, HLA Matched Hematopoietic Cell Transplantation.

BACKGROUND: Allogeneic hematopoietic cell transplantation (HCT) can be curative for many hematologic diseases. However, complications such as graft-versus-host disease (GVHD) and relapse of primary malignancy remain significant and are the leading causes of morbidity and mortality. Effects of killer Ig-like receptors (KIR)-influenced NK cells on HCT outcomes have been extensively pursued over the last decade. However, the relevance of the reported algorithms on HLA matched myeloablative HCT with rabbit antithymocyte globulin (ATG) is used for GVHD prophylaxis remains elusive. Here we examined the role of KIR and KIR-ligands of donor-recipient pairs in modifying the outcomes of ATG conditioned HLA matched sibling and unrelated donor HCT. METHODS AND FINDINGS: The study cohort consisted of 281 HLA matched sibling and unrelated donor-recipient pairs of first allogeneic marrow or blood stem cell transplantation allocated into 'discovery' (135 pairs) and 'validation' (146 pairs) cohorts. High resolution HLA typing was obtained from the medical charts and KIR gene repertoires were obtained by a Luminex® based SSO method. All surviving patients were followed-up for a minimum of two years. KIR and HLA class I distributions of HCT pairs were stratified as per applicable definitions and were tested for their association with cause specific outcomes [acute GVHD grade II-IV (aGVHD), chronic GVHD needing systemic therapy (cGVHD) and relapse] using a multivariate competing risks regression model as well as with survival outcomes [relapse-free survival (RFS), cGVHD & relapse free survival (cGRFS) and overall survival (OS)] by multivariate Cox proportional hazards regression model. A significant association between KIR genotype mismatching (KIR-B/x donor into KIR-AA recipient or vice versa) and cGVHD was found in both discovery (p = 0.001; SHR = 2.78; 95%CI: 1.50-5.17) and validation cohorts (p = 0.005; SHR = 2.61; 95%CI: 1.33-5.11). High incidence of cGVHD associated with KIR genotype mismatching was applicable to both sibling and unrelated donors and was specific to recipients who had one or two C1 bearing HLA-C epitopes (HLA-C1/x, p = 0.001; SHR = 2.40; 95%CI: 1.42-4.06). When compared with KIR genotype mismatched transplants, HLA-C1/x patients receiving grafts from KIR genotype matched donors had a significantly improved cGRFS (p = 0.013; HR = 1.62; 95%CI: 1.11-2.39). Although there was no effect of KIR genotype matching on survival outcomes, a significantly reduced incidence of relapse (p = 0.001; SHR = 0.22; 95%CI: 0.10-0.54) and improved relapse-free survival (p = 0.038; HR = 0.40; 95%CI: 0.17-0.95) was observed with one or more missing ligands for donor inhibitory KIR among the recipients of unrelated donor transplants. CONCLUSIONS: The present study for the first time presents the beneficial effects of KIR genotype matching in reducing cGVHD in myeloablative transplant setting using HLA matched (sibling and unrelated) donors. The findings offer a clinically applicable donor selection strategy that can help control cGVHD without affecting the risk of relapse and/or identify patients at a high risk of developing cGVHD as potential candidates for preemptive therapy. The findings also affirm the beneficial effect of one or more missing inhibitory KIR ligands in the recipient in reducing relapse and improving a relapse free survival in unrelated donor transplants.