Chapter1: Immune Responses To SARS-CoV-2 Infection And Vaccination in Dialysis Patients And Kidney Transplant Recipients

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Abstract: Dialysis patients and kidney transplant (KTX)recipients suffer from an impaired immune system and show a decreased response to the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2)vaccination. We performed a retrospective analysis of 1505 serological SARS-CoV-2 measurements obtained from 887 dialysis patients and 86 KTX recipients. The results were separated by patient subgroups (dialysis/KTX) as well as SARS-CoV-2 status. The latter criterion included SARS-CoV-2-naive patients with or without COVID-19 vaccination and convalescent patients receiving a booster shot. Serologies of 27 vaccinated healthy individuals served as the reference group. Vaccine-induced cellular immune response was quantified by an interferon-γy release assay in 32 KTX recipients. We determined seroconversion rates of 92.6%, 93.4%, and 71.4% in dialysis patients vaccinated with either BNT162b2, mRNA-1273, or AZD1222, respectively. Vaccination-induced anti-SARS-CoV-2 antibody titers were lower in dialysis patients compared to healthy individuals, and vaccination with mRNA-1273 induced higher titers than BNT162b2. The initial seroconversion rate was 39.5% in KTX recipients vaccinated with BNT162b2. A linear regression model identified medication with mycophenolate-mofetil/mycophenolic acid as an independent risk factor for missing seroconversion. Within a cohort of 32 KTX recipients, cellular and humoral immune reactivity to SARS-CoV-2 was detectable in three patients only. Conclusively, vaccine-induced seroconversion rates were similar in dialysis patients compared to healthy individuals but were strongly impaired in KTX recipients. Anti-SARS-CoV-2 lgG titers elicited by double active immunization were significantly lower in both cohorts compared to healthy individuals, and immune responses to vaccination vanished quickly.

Keywords: COVID-19;immunosuppression; protection; titer; antibodies; kidney disease

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1. Introduction

Hemodialysis (HD) patients and kidney transplant (KTX) recipients are at risk for severe coronavirus disease-19 (COVID-19)[1,2]. Surprisingly, although limited in number, observational studies have revealed no difference in the immune response to the severe acute respiratory syndrome coronavirus type 2(SARS-CoV-2) following natural infection in chronically immunosuppressed patients and immunocompetent individuals [3]. Apparently, age and frailty as well as the number and severity of pre-existing comorbidities—-more than pharmacological immunosuppression—are prognostic factors for a severe COVID-19 disease course in both immunocompromised and immunocompetent patients [4,5]. Notwithstanding, European public health data indicate chronic kidney disease(CKD) and dialysis as critical prognostic comorbidities associated with increased mortality upon SARS-CoV-2 infection [6].In addition, impaired seroconversion and longevity of SARS-CoV-2 immune responses after infection further threatens HD and KTX patients [4,7,8].

Active immunization against SARS-CoV-2, in particular with messenger ribonucleic acid (mRNA)vaccines, is considered the most effective preventive strategy to protect vulnerable individuals from infection and severe COVID-19 courses [9]. However, both the intensity and duration of the immune response after vaccination in specific patient groups have not been fully characterized. Compared to the general population, HD patients are considered to present diminished SARS-CoV-2 antibody titers and have a delayed immune response [10-12]. After the second injection, the seroconversion rate may be as high as 90% and a cellular immune response can be detected in approximately 80% of vaccinated individuals[13-15]. The increasing available data from different studies on the efficacy of SARS-CoV-2 vaccination in solid organ transplant (SOT)recipients revealed a strongly impaired humoral and cellular immune response compared to the general population, with a reported seroconversion of below 45% and additionally an impaired cellular response [16-19].

Thus far, the definition of an antibody titer conferring protection is still being intensively investigated and the determination of antibody levels is not routinely performed [20]. We systematically analyzed the seroconversion rate and antibody titer in 681 HD patients and 86 KTX recipients to characterize the humoral response to both PCR-confirmed SARS-CoV-2 infection and vaccination(BNT162b2(BioNTech/Pfizer, Mainz, Germany/New York, NY, USA), mRNA-1273 (Moderna, CA, USA)and AZD1222(AstraZeneca/University of Oxford, Cambridge/Oxford, UK)) in a retrospective manner. Serologies of 27 mRNA-vaccinated healthy individuals served as a reference group. Furthermore, the cellular immune response was prospectively quantified in 32 KTX recipients to fully characterize mRNA vaccine-induced protection.

2. Materials and Methods

2.1. Study Design, Patients, and Ethical Statement

To characterize the humoral response to both infection and vaccination, we retrospectively analyzed SARS-CoV-2 serologies from dialysis and KTX patients performed at the University Hospital Cologne, Germany, starting at the beginning of the COVID-19 pandemic in April 2020 until June 2021. This cohort contained 206 SARS-CoV-2-naive dialysis patients in which anti-SARS-CoV-2 antibodies were screened every three months for one year from April 2020 to April 2021. Regarding vaccination, antibody measurements were obtained between March 2021 and June 2021 from 627 SARS-CoV-2-naive dialysis patients, of whom 475 received BNT162b2(BNT162b2(BioNTech/Pfizer, Mainz, Ger-many/New York, NY, USA), 138 mRNA-1273(Moderna, CA, USA), and 14 AZD1222 (AstraZeneca/University of Oxford, Cambridge/Oxford, UK), respectively, were available. In addition, the serologies of 54 dialysis patients who had recovered from SARS-CoV-2

were obtained, of whom 38 received a booster shot (all BNT162b2). Regarding SARS-CoV-2-naive KTX recipients, sera were obtained from 86 patients to monitor BNT162b2 vaccination. Serologies of 27 healthy individuals vaccinated with mRNA-1273 served as a control group. All vaccines were administered in clinical routine at the discretion of the treating physician and as advised by the manufacturers. All of the measurements were performed in clinical routine and analyzed retrospectively after approval from the local institutional review board (Ethics Committee of the Medical Faculty of the University of Cologne, Germany, EK21-1398). Additionally, humoral and cellular immune responses were examined prospectively in 32 SARS-CoV-2-naive KTX recipients between June and September 2021. This study was also approved by the Ethics Committee of the Medical Faculty of the University of Cologne, Germany (EK21-1112)and was conducted in line with the Declaration of Helsinki.

2.2.Serological Assays for the Detection of Anti-SARS-CoV-2 Antibodies

Anti-SARS-CoV-2 IgG targeting the spike (S) protein was detected by the semiquantitative Euroimmun anti-SARS-CoV-2 IgG ELISA using the Euroimmun Analvzer I (Eu-roimmun Diagnostik, Lübeck, Germany), and by the quantitative IDKeanti-SARS-CoV-2 IgG ELISA (Immundiagnostik AG, Bensheim, Germany)using the DYNEX DSX(Dynex Technologies, Chantilly, VA, USA), both using the recombinant S1 antigen of the spike protein, or the chemiluminescent microparticle immunoassay by Abbott for the quantitative detection of anti-spike RBD (receptor binding domain)IgG (SARS-CoV-2 IgG II Quant)using the automated system Alinity i(Abbott, Abbott Park, IL, USA). The results of the quantitative IgG assays targeting S1 or RBD were expressed in BAU/mL(binding antibody unit)using the conversion factor generated from a World Health Organization (WHO)internal standard and provided by each manufacturer. Additionally, 29 patients from the hemodialysis group were parallel examined using these two quantitative SARS-CoV-2 IgG assays to determine if they delivered comparable IgG titers. The nucleocapsid (NC) protein-targeting antibodies were detected by the pan-immunoglobulin immunoassay Elecsys anti-SARS-CoV-2 electrochemiluminescence immunoassay(ECLIA)using the Cobas 8000 (Roche Diagnostics, Mannheim, Germany). All assays were interpreted according to the manufacturer's recommendations.

2.3.Determination of T-Cell Response to SARS-CoV-2

The CD4+ and CD8+ T-cell-mediated response to SARS-CoV-2 was measured by the commercial whole blood interferon-γ （IFN-γ）release assay （IGRA） QuantiFERON⑧ SARS-CoV-2 RUO(QIAGEN GmbH, Hilden, Germany). T-cells were stimulated with epitopes of S1 and S2 subunits of SARS-CoV-2 spike protein (antigen 1 and 2). IFN-y concentration was measured by a chemiluminescence immunoassay(CLIA), as previously described [21]. An IFN-y level > 0.15 IU/mL was scored as reactive [22].

2.4.Statistic

Statistical analyses were performed using GraphPad Prism software version 5 (Graph-Pad Prism Software Inc.San Diego, CA, USA) and SPSSversion 27.0(SPSS Inc., Chicago, IL, USA). Non-normal distributions of antibody titers were expressed as median ± interquartile range and compared using the Kruskal-Wallis/Mann-Whitney U tests and Spearman Correlation. Regarding patient characteristics, categorical variables were summarized by absolute numbers and frequencies, and numerical variables by median and interquartile range, respectively. Categorical variables were analyzed using a chi-square test, and numerical variables using a Mann-Whitney U test in a descriptive manner. Correlations—-adjusted for used assay, age, and time between event and blood sample—-were calculated using a linear regression model with Spearman analysis. p<0.05 was considered statistically significant. To examine the comparability of the two immunoassays, a Passing-Bablok regression was carried out using the software Analyze-it v5.50(Analyse-it Software, Ltd. Leeds, UK).