Development and Validation of Multivariable Prediction Models of Serological Response to SARS-CoV-2 Vaccination in Kidney Transplant Recipients (preprint)

Background Repeated vaccination against SARS-CoV-2 increases serological response in kidney transplant recipients (KTR) with high interindividual variability. No decision support tool exists to predict SARS-CoV-2 vaccination response in KTR. Methods We developed, internally and externally validated five different multivariable prediction models of serological response after the third and fourth vaccine dose against SARS-CoV-2 in KTR. Using 27 candidate predictor variables, we applied statistical and machine learning approaches including logistic regression (LR), LASSO-regularized LR, random forest, and gradient boosted regression trees. For development and internal validation, data from 585 vaccinations were used. External validation was performed in four independent, international validation datasets comprising 191, 184, 254, and 323 vaccinations, respectively. Findings LASSO-regularized LR performed on the whole development dataset yielded a 23- and 11- variable model, respectively. External validation showed AUC-ROC of 0.855, 0.749, 0.828, and 0.787 for the sparser 11-variable model, yielding an overall performance 0.819. Interpretation An 11-variable LASSO-regularized LR model predicts vaccination response in KTR with good overall accuracy. Implemented as an online tool, it can guide decisions when choosing between different immunization strategies to improve protection against COVID-19 in KTR.

Declining Course of Humoral Immune Response in Initially Responding Kidney Transplant Recipients after Repeated SARS-CoV-2 Vaccination (preprint)

Immunogenicity of SARS-CoV-2 vaccines in kidney transplant recipients is limited, resulting in inadequately low serological response rates and low immunoglobulin (Ig) levels, correlating with reduced protection against death and hospitalization from COVID-19. We retrospectively examined the time course of anti-SARS-CoV-2 Ig antibody levels after up to five repeated vac-cinations in 644 previously nonresponding kidney transplant recipients. Using anti SARS-CoV-2 IgG/IgA ELISA and the total Ig ECLIA assays, we compare antibody levels at 1 month with levels at 2 and 4 months, respectively. Additionally, we correlate the measurements of the used assays. Between 1 and 2 months, and between 1 and 4 months, mean anti-SARS-CoV-2 Ig levels in re-sponders decreased by 14% and 25%, respectively, depending on the assay. Absolute Ig values and time course of antibody levels and showed high interindividual variability. Ig levels de-creased by at least 20% in 77 of 148 paired samples with loss of sufficient serological protection over time occurring in 18 out of 148 (12.2%). IgG ELISA and total Ig ECLIA assays showed a strong positive correlation (Kendall's tau=0.78), yet the two assays determined divergent results in 99 of 751 (13.2%) measurements. IgG and IgA assays showed overall strong correlation but divergent results in 270 of 1.173 (23.0%) cases and only weak correlation of antibody levels in positive samples. Large interindividual variability and significant loss of serological protection after 4 months supports repeated serological sampling and consideration of shorter vaccination intervals in kidney transplant recipients.

Serological Response to Three, Four and Five Doses of SARS-CoV-2 Vaccine in Kidney Transplant Recipients (preprint)

Background Mortality from COVID-19 among kidney transplant recipients (KTR) is high, and their response to three vaccinations against SARS-CoV-2 is strongly impaired. Methods We retrospectively analyzed serological response of up to five doses of SARS-CoV-2 vaccine in KTR from December 27, 2020, until December 31, 2021. Particularly, the influence of different dose adjustment regimens for mycophenolic acid (MPA) on serological response to fourth vaccination was analyzed. Results In total, 4.277 vaccinations against SARS-CoV-2 in 1.478 patients were analyzed. Serological response was 19.5% after 1.203 basic immunizations, and increased to 29.4%, 55.6%, and 57.5% in response to 603 third, 250 fourth and 40 fifth vaccinations, resulting in a cumulative response rate of 88.7%. In patients with calcineurin inhibitor and MPA maintenance immunosuppression, pausing MPA and adding 5 mg prednisolone equivalent before the fourth vaccination increased serological response rate to 75% in comparison to no dose adjustment (52%) or dose reduction (46%). Belatacept-treated patients had a response rate of 8.7% (4/46) after three vaccinations and 12.5% (3/25) after four vaccinations. Conclusion Except for belatacept-treated patients, repeated SARS-CoV-2 vaccination of up to five times effectively induces serological response in kidney transplant recipients. It can be enhanced by pausing MPA at the time of vaccination.

Three-month follow-up of heterologous vs homologous third vaccination in kidney transplant recipients (preprint)

Importance Response to SARS-CoV-2 vaccines in kidney transplant recipients (KTR) is severely reduced. Heterologous 3rd vaccination combining mRNA and vector vaccines did not increase seroconversion at four weeks after vaccination but evolution of antibody levels beyond the first month remain unknown. Objective To assess changes in antibody response following a 3rd vaccination with mRNA or vector vaccine in KTR from month one to month three after vaccination. Design, Setting and Participants Three-month follow-up (pre-specified secondary endpoint) of a single-center, single-blinded, 1:1 randomized, controlled trial on 3rd vaccination against SARS-CoV-2 in 201 KTR who did not develop SARS-CoV-2 spike protein antibodies following two doses of an mRNA vaccine. Intervention(s) mRNA (BNT162b2 or mRNA-1273) or vector (Ad26COVS1) as third SARS-CoV-2 vaccine Main Outcomes and Measures Main outcome was seroconversion at the second follow-up between 60-120 days after the 3rd vaccination. Subsequently, higher cut-off levels associated with neutralizing capacity and protective immunity were applied (i.e. >15, >100, >141 and >264 BAU/mL). In addition, trajectories of antibody levels from month one to month three were analyzed. Finally, SARS-CoV-2 specific CD4 and CD8 T-cells at four weeks were compared among the 18 top responders in both groups. Results A total of 169 patients were available for the three-month follow-up. Overall, seroconversion at three months was similar between both groups (45% versus 50% for mRNA and vector group, respectively; OR=1.24, 95%CI=[0.65, 2.37], p=0.539). However, when applying higher cut-off levels, a significantly larger number of individual in the vector group reached antibody levels > 141 and > 264 BAU/mL at the three-month follow-up (141 BAU/mL: 4% vs. 15% OR=4.96, 95%CI=[1.29, 28.21], p=0.009 and 264 BAU/mL: 1% vs. 10% OR=8.75, 95%CI=[1.13, 396.17], p=0.018 for mRNA vs. vector vaccine group, respectively). In line, antibody levels in seroconverted patients further increased from month one to month three in the vector group while remaining unchanged in the mRNA group (median increase: mRNA= 1.35 U/mL and vector = 27.6 U/mL, p = 0.004). Of particular note, there was no difference in the CD4 and CD8 T-cell response between the mRNA and vector vaccine group at month one. Conclusions and Relevance: Despite a similar overall seroconversion rate at three months following third vaccination in KTR, a heterologous third booster vaccination with Ad26COVS1 resulted in significantly higher antibody levels in responders. Trial Registration: EurdraCT: 2021-002927-39

Temporary hold of mycophenolate boosts SARS-CoV-2 vaccination-specific humoral and cellular immunity in kidney transplant recipients (preprint)

Transplant recipients exhibit an impaired protective immunity after SARS-CoV-2 vaccination, potentially caused by mycophenolate (MPA) immunosuppression. Recent data from autoimmune patients suggest that temporary MPA hold might significantly improve booster vaccination outcomes. We applied a fourth dose of SARS-CoV-2 vaccine during temporary (5 weeks) MPA hold to 29 kidney transplant recipients, who had not mounted a humoral immune-response to previous vaccinations. Seroconversion until day 32 after vaccination was observed in 76% of patients, associated with acquisition of virus neutralizing capacity. Interestingly, 21/25 (84%) CNI-treated patients responded, but only 1/4 Belatacept-treated patients. In line with humoral responses, counts and relative frequencies of spike receptor binding domain (RBD) specific B cells were significantly increased on day 7 after vaccination, with an increase in RBD specific CD27++CD38+ plasmablasts. Whereas overall proportions of spike-reactive CD4+ T cells remained unaltered after the fourth dose, frequencies were positively correlated with specific IgG levels. Importantly, antigen-specific proliferating Ki67+ and in vivo activated PD1+ T cells significantly increased after re-vaccination during MPA hold, whereas cytokine production and memory differentiation remained unaffected. In summary, MPA hold was safe and augmented all arms of immunity during booster vaccination, suggesting its implementation in vaccination protocols for clinically stable transplant recipients.

Cytosorb® Rescue for COVID-19 Patients with Vasoplegic Shock (CytoResc): A Prospective, Open-Label, Randomised Controlled Pilot Study (preprint)

Background: Several observations indicate a hyperinflammatory state in severely ill COVID-19 patients. The aim of this study was to investigate the effect of extracorporeal cytokine elimination by CytoSorb® on COVID-19 associated vasoplegic shock.Methods: In this prospective randomised pilot study COVID-19 patients with vasoplegic shock requiring norepinephrine >0·2 µg/kg/min, CRP >100 mg/L and indication for hemodialysis were randomised 1:1 to receive CytoSorb® treatment for 3-7 days or standard of care. The primary endpoint was time until resolution of vasoplegic shock, estimated by a Cox-regression model. Secondary endpoints included mortality, serum interleukin-6 concentrations, and catecholamine requirements. The study was registered in the German Registry of Clinical Trials (DRKS00021447).Findings: From November 2020 to March 2021, 50 patients were enrolled of which 23 patients were randomised to receive CytoSorb® treatment and 26 patients to receive standard of care. One patient randomised to cytokine adsorption was excluded due to withdrawal of informed consent. Resolution of vasoplegic shock was observed in 13 (56·5%) of 23 patients in the CytoSorb® and 12 (46·2%) of 26 patients in the control group after a median of 5 (IQR 4-5) and 4 (IQR 3-5) days, respectively. The hazard ratio (HR) for the primary endpoint, adjusted for the predefined variables age, gender, ECMO-therapy, or time from shock onset to study inclusion was HR 1·23 (95%CI: 0·54-2·79), p=0·63). The mortality rate was 78% in the CytoSorb® and 73% in the control group (unadjusted HR 1·17 (95%CI: 0·61-2.23), p=0·64). The effects on inflammatory markers and catecholamine requirements and the type and rates of adverse events were similar in the two groups.Interpretation: In this pilot trial in severely ill COVID-19 patients CytoSorb® treatment did not improve resolution of vasoplegic shock as compared to standard treatment. Mortality rates, catecholamine requirements, inflammatory markers and adverse events did not differ between the two groups.Trial Registration: The study was registered in the German Registry of Clinical Trials (DRKS00021447Funding: Internal university fundsDeclaration of Interest: HS, LJL, MP, TK, PT, FS, KUE, SK, JVK, MO, AKrü, A Kra, KB declare no conflicts of interest. PE received honoraria from GSK and AstraZeneca and filed two patents for novel urinary biomarkers outside the submitted work. ST received research funding and honoraria for workshops and lectures from Orionpharma. He additionally received honoraria for workshops and lectures from Edwards and honoraria for lectures from Amomed and Smith&Nephews. CS received grants from: Drägerwerk AG& Co.KGaA; German Reseach Society; German Aerospace Center; Einstein Foundation Berlin; Federal Joint Committee (G-BA); Inner University grants; Project Management Agency; Non-Profit Promoting Science and Education; European Society of Anesthesiology and Intensive Care; Baxter Deutschland GmbH; Cytosorbents Europe GmbH; Edwards Lifsciences Germany GmbH; Fresenius Medical Care; Grünenthal GmbH; Massimo Europe Ltd.; Pfizer Pharma PFE GmbH; Georg Thieme Verlag, Dr. F Köhler Chemie GmbH; Sintetica GmbH; Stifterverband für die deutsche Wissenschaft e.V./Philips; Stiftung Charié; AGUETTANT Deutschland GmbH; AbbVie Deutschland GmbH & Co.KG; Amomed Pharma GmbH; InTouch Health; Copra System GmbH; Correvio GmbH; Max Plank Gesellschaft zur Förderung der Wissenschaften e.V.; Deutsche Gesellschaft für Anästhesiologie & Intensivmedizin (DGAI); Stifterverband für die Deutsche Wissenschaft e.V./Medtronic; Philipps ElectronicsNederland BV; BMG, BMBF, German Research Society all outside the submitted work. In addition, CS has different patents. DK received fees for speaking at a symposia organized on behalf of Fresenius Medical Care AG, Germany.Ethical Approval: The original protocol and the changes were approved by the local ethicscommittee (EA1/069/20).

B and T cell responses after a third dose of SARS-CoV-2 vaccine in Kidney Transplant Recipients (preprint)

BackgroundAccumulating evidence suggests that solid organ transplant recipients, as opposed to the general population, show strongly impaired responsiveness towards standard SARS-CoV-2 mRNA-based vaccination, demanding alternative strategies for protection of this vulnerable group. MethodsIn line with recent recommendations, a third dose of either heterologous ChAdOx1 (AstraZeneca) or homologous BNT162b2 (BioNTech) was administered to 25 kidney transplant recipients (KTR) without humoral response after 2 doses of BNT162b2, followed by analysis of serological responses and vaccine-specific B- and T-cell immunity. Results9/25 (36%) KTR under standard immunosuppressive treatment seroconverted until day 27 after the third vaccination, while one patient developed severe COVID-19 infection immediately after vaccination. Cellular analysis seven days after the third dose showed significantly elevated frequencies of viral spike protein receptor binding domain specific B cells in humoral responders as compared to non-responders. Likewise, portions of spike-reactive CD4+ T helper cells were significantly elevated in seroconverting patients. Furthermore, overall frequencies of IL-2+, IL-4+ and polyfunctional CD4+ T cells significantly increased after the third dose, whereas memory/effector differentiation remained unaffected. ConclusionsOur data suggest that a fraction of transplant recipients benefits from triple vaccination, where seroconversion is associated with quantitative and qualitative changes of cellular immunity. At the same time, the study highlights that modified vaccination approaches for immunosuppressed patients still remain an urgent medical need. Significance statementProtection of solid organ transplant recipients against SARS-Cov-2 by vaccination remains an unmet need given the low immunogenicity of available vaccines in the presence of immunosuppression. Administration of a third dose to 25 kidney transplant recipients (KTR) resulted in seroconversion in 36% of patients, associated with significant quantitative and functional changes within the spike-antigen-specific B-cell- and CD4+ T-helper cell compartment. Our data support the need for individual humoral monitoring of immunosuppressed individuals after vaccination as well as continued efforts to adapt vaccination protocols for this at-risk group.

Diagnostic accuracy of a novel SARS-CoV-2 antigen-detecting rapid diagnostic test from standardized self-collected anterior nasal swabs (preprint)

Background Antigen-detecting rapid diagnostic tests (Ag-RDT) for SARS-CoV-2 offer new opportunities for the quick and laboratory-independent identification of infected individuals for control of the SARS-CoV-2 pandemic. Despite the potential benefits, nasopharyngeal sample collection is frequently perceived as uncomfortable by patients and requires trained healthcare personnel with protective equipment. Therefore, anterior nasal self-sampling is increasingly recognized as a valuable alternative. Methods We performed a prospective, single-center, point of care validation of an Ag-RDT using a polypropylene absorbent collector for standardized self-collected anterior nasal swabs. Real-Time Polymerase Chain Reaction (RT-PCR) from combined oropharyngeal/nasopharyngeal swabs served as a comparator. Primary endpoint was sensitivity of the standardized Ag-RDT in symptomatic patients with medium or high viral concentration ([≥] 1 million RNA copies on RT-PCR for SARS-CoV-2). Results Between February 12 and March 22, 2021, 388 participants were enrolled. After exclusion of 9 patients for which no PCR result could be obtained, the novel Ag-RDT was evaluated based on 379 participants, of which 273 were symptomatic and 106 asymptomatic. In 61 samples from symptomatic patients with medium or high viral load ([≥] 1 million RNA copies), the sensitivity of the standardized Ag-RDT was 96.7% (59/61; 95%CI: 88.7-99.6%) for the primary endpoint. In total, 62 positive Ag-RDT results were detected out of 70 RT-PCR positive individuals, yielding an overall sensitivity of 88.6% (95%CI: 78.7-94.9%). Specificity was 99.7% (95%CI: 98.2-100%) in 309 RT-PCR negative individuals. Conclusion Here, we present a validation of a novel Ag-RDT with a standardized sampling process for anterior nasal self-collection, which meets WHO criteria of [≥]80% sensitivity and [≥]97% specificity. Although less sensitive than RT-PCR, this assay could be beneficial due to its rapid results, ease of use, and suitability for standardized self-testing. (Funded by Dragerwerk AG & Co. KGaA, Lubeck, Germany; ClinicalTrials.gov number NCT04698993)

Impaired antigen-specific memory B cell and plasma cell responses including lack of specific IgG upon SARS-CoV-2 BNT162b2 vaccination among Kidney Transplant and Dialysis patients (preprint)

Patients with kidney failure are at increased risk during the COVID-19 pandemic and effective vaccinations are needed. It is not known how efficient mRNA vaccines mount B and plasma cell responses in dialysis patients (DP) or kidney transplant recipients (KTR) compared to healthy controls (HC). We studied humoral and B cell responses of 25 HC, 44 DP and 40 KTR. Markedly impaired anti-BNT162b2 responses were identified among KTR and DP compared to 100% seroconversion in HC. In DP, the response was delayed (3-4 weeks after boost) and reduced with anti-S1 IgG positivity in 31 (70.5%) and anti-S1 IgA in 30 (68.2%) of 44, respectively. In contrast, KTR did not develop IgG response except one patient who had prior unrecognized infection and developed anti-S1 IgG. The majority of antigen-specific B cells (RBD+) were identified in the plasmablast or post-switch memory B cell compartments in HC, whereas these RBD+ B cells were enriched among pre-switch and naive B cells from DP and KTR. Single cell transcriptome and CITE-seq analyses found reduced frequencies of plasmablasts, TCF7+CD27+GZMK+ T cells and proliferating MKI67-expressing lymphocytes among KTR non-responders. Importantly, the frequency and absolute number of antigen-specific circulating plasmablasts in the whole cohort correlated with the Ig response, a characteristic not reported for other vaccinations. In conclusion, this data indicate that lack of T cell help related to immunosuppression results in impaired germinal center differentiation of B and plasma cell memory. There is an urgent need to improve vaccination protocols in patients after kidney transplantation or on chronic dialysis. One Sentence SummaryKidney transplant recipients and dialysis patients show a markedly diminished humoral response and impaired molecular B cell memory formation upon vaccination with BNT162b2.

Impaired Humoral and Cellular Immunity after SARS-CoV2 BNT162b2 (Tozinameran) Prime-Boost Vaccination in Kidney Transplant Recipients (preprint)

Novel mRNA-based vaccines have been proven powerful tools to combat the global pandemic caused by SARS-CoV2 with BNT162b2 efficiently protecting individuals from COVID-19 across a broad age range. Still, it remains largely unknown how renal insufficiency and immunosuppressive medication affect development of vaccine induced immunity. We therefore comprehensively analyzed humoral and cellular responses in kidney transplant recipients after prime-boost vaccination with BNT162b2. As opposed to all healthy vaccinees and the majority of hemodialysis patients, only 4/39 and 1/39 transplanted individuals showed IgA and IgG seroconversion at day 8{+/-}1 after booster immunization with minor changes until day 23{+/-}5, respectively. Although most transplanted patients mounted spike-specific T helper cell responses, frequencies were significantly reduced compared to controls and dialysis patients, accompanied by a broad impairment in effector cytokine production, memory differentiation and activation-related signatures. Spike-specific CD8+ T cell responses were less abundant than their CD4+ counterparts in healthy controls and hemodialysis patients and almost undetectable in transplant patients. Signs of alloreactivity promoted by BNT162b2 were not documented within the observation period. In summary, our data strongly suggest revised vaccination approaches in immunosuppressed patients, including individual immune monitoring for protection of this vulnerable group at risk to develop severe COVID-19.

Immunogenicity of COVID-19 Tozinameran Vaccination in Patients on Chronic Dialysis (preprint)

ObjectivePatients with kidney failure have notoriously weak responses to common vaccines. Thus, immunogenicity of novel SARS-CoV-2 vaccines might be impaired in this group. To determine immunogenicity of SARS-CoV-2 vaccination in patients with chronic dialysis, we analyzed the humoral and T-cell response after two doses of mRNA vaccine Tozinameran (BNT162b2 BioNTech/Pfizer). Design, Settings, and ParticipantsThis observational study included 43 patients on dialysis before vaccination with two doses of Tozinameran 21 days apart. Overall, 36 patients completed the observation period. Serum samples were analyzed by SARS-CoV-2 specific antibodies [~]1 and [~]3-4 weeks after the second vaccination. In addition, SARS-CoV-2-specific T-cell responses were assessed at the later time point by an interferon-gamma release assay (IGRA). Outcomes at later timepoints were compared to a group of 44 elderly patients with no dialysis after immunization with Tozinameran. ExposuresBlood drawings during regular laboratory routine assessment right before start of dialysis therapy or at the time of vaccination and at follow-up study visits. Main Outcomes and MeasuresAssessment of immunogenicity after vaccination against SARS-CoV-2 in patients on and without dialysis. ResultsMedian age of patients on chronic dialysis was 74.0 years (IQR 66.0, 82.0). The proportion of males was higher (69.4%) than females. Only 20/36 patients (55.6%, 95%CI: 38.29-71.67) developed SARS-CoV-2-IgG antibodies at first sampling, whereas 32/36 patients (88.9%, 95%CI:73.00-96.38) demonstrated seropositivity at the second sampling. Seroconversion rates and antibody titers were significantly lower compared to a cohort of vaccinees with similar age but no chronic dialysis (>90% seropositivity). SARS-CoV-2-specific T-cell responses 3 weeks after second vaccination were detected in 21/31 vaccinated dialysis patients (67.7%, 95%CI: 48.53-82.68) compared to 42/44 (93.3%, 95%CI: 76.49-98.84) in controls of similar age. Conclusion and RelevancePatients on dialysis demonstrate a delayed, but robust immune response three weeks after the second dose, which indicates effective vaccination of this vulnerable group. However, the lower immunogenicity of Tozinameran in these patients needs further attention to develop potential countermeasures such as an additional booster vaccination.

Efficacy and Complications of Regional Citrate Anticoagulation During Continuous Renal Replacement Therapy in Critically Ill Patients with COVID-19 (preprint)

Background: Enhanced coagulation in coronavirus disease 2019 (COVID-19) patients is considered a major obstacle for continuous renal replacement therapy (CRRT), but systematic analyses are sparse. We compared filter survival and citrate-induced complications during CRRT with regional citrate anticoagulation (RCA) in COVID-19 and Non-COVID-19 patients. Methods: : In this retrospective study we included all consecutive adult patients (n=97) with acute kidney injury (AKI) treated with RCA-CRRT at seven ICUs of a tertiary university hospital over the tree month period. Medical data were collected to compare the efficacy and complications of RCA-CRRT between COVID-19 (n=44) and Non-COVID-19 patients (n=53). Results: : There was no significant difference in the number of CRRT filters used per patient in COVID-19 vs. Non-COVID-19 patients (median 5 vs 3 filters, p=0.103). Mean filter run-time was significantly higher in COVID-19 patients compared to Non-COVID-19 patients (68.4 (95%CI 67.0-69.9) vs. 65.2 (95%CI 63.2-67.2) hours, respectively; log-rank 0.014). COVID-19 patients showed significantly higher activated partial thromboplastin time (aPTT) throughout the CRRT due to systemic anticoagulation compared to Non-COVID-19 patients (54 (IQR 45 – 61) vs. 47 (IQR 41 - 58) seconds, respectively; p<0.001). A significantly higher incidence of combined metabolic disturbances (metabolic alkalosis, hypercalcemia and hypernatremia), consistent with reduced filter patency and citrate overload during RCA, was observed in COVID-19 patients compared to Non-COVID-19 patients (19.1% vs. 12.7%, respectively; p=0.04). These metabolic disarrangements were resistant to per-protocol adjustments and disappeared after replacement of the CRRT-filter. Conclusions: : In contrast to initial concerns, adequate filter life-span can be achieved with RCA during CRRT in COVID-19 patients. However, close monitoring of the acid-base balance appears warranted, as these patients tend to develop reduced filter patency leading to a higher incidence of citrate overload and metabolic disturbances. Trial Registration (local authority): EA1/285/20 (Ethikkommission der Charité - Universitätsmedizin Berlin); date of registration 08.10.2020.