key: cord-0740200-sl18wl19 authors: Lindemann, M.; Krawczyk, A.; Dolff, S.; Konik, M.; Rohn, H.; Platte, M.; Thuemmler, L.; Schwarzkopf, S.; Schipper, L.; Bormann, M.; van de Sand, L.; Breyer, M.; Klump, H.; Knop, D.; Lenz, V.; Temme, C.; Dittmer, U.; Horn, P. A.; Witzke, O. title: SARS-CoV-2-specific humoral and cellular immunity in renal transplant and haemodialysis patients treated with convalescent plasma date: 2020-12-11 journal: nan DOI: 10.1101/2020.12.09.20239673 sha: 55f279ae6ba024e359f60d34f711e91632101a04 doc_id: 740200 cord_uid: sl18wl19 Background. When patients with chronic kidney disease are infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) they can face two specific problems: Virus-specific immune responses may be impaired and remdesivir, an antiviral drug described to shorten the time to recovery, is contraindicated. Antiviral treatment with convalescent plasma could be an alternative treatment option. Methods. In this case series we present two kidney transplant recipients and two patients dependent on haemodialysis who were infected with SARS-CoV-2 and received convalescent plasma. Antibodies against the spike 1 protein of SARS-CoV-2 were determined sequentially by IgG ELISA and neutralization assay and specific T cell responses by interferon-gamma ELISpot. Results. Prior to treatment, in three patients antibodies were undetectable by ELISA (ratio < 1.1), corresponding to low neutralizing antibody titers ([≤] 1:40). One patient was also negative to the ELISpot and two showed weak responses. After convalescent plasma treatment we observed an increase of SARS-CoV-2-specific antibodies (IgG ratio and neutralization titer) and of specific T cell responses. After intermittent clinical improvement one kidney transplant recipient again developed typical symptoms at day 12 after treatment and received a second cycle of convalescent plasma treatment. Altogether, three patients clinically improved and could be discharged from hospital. However, one multimorbid female in her early eighties deceased. Conclusions. Our data suggest that the success of convalescent plasma therapy may only be temporary in patients with chronic kidney disease; which requires an adaptation of the treatment regimen. Close monitoring after treatment is needed for this patient group. Background. When patients with chronic kidney disease are infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) they can face two specific problems: Virus-specific immune responses may be impaired and remdesivir, an antiviral drug described to shorten the time to recovery, is contraindicated. Antiviral treatment with convalescent plasma could be an alternative treatment option. Methods. In this case series we present two kidney transplant recipients and two patients dependent on haemodialysis who were infected with SARS-CoV-2 and received convalescent plasma. Antibodies against the spike 1 protein of SARS-CoV-2 were determined sequentially by IgG ELISA and neutralization assay and specific T cell responses by interferon-gamma ELISpot. Results. Prior to treatment, in three patients antibodies were undetectable by ELISA (ratio < 1.1), corresponding to low neutralizing antibody titers (≤ 1:40). One patient was also negative to the ELISpot and two showed weak responses. After convalescent plasma treatment we observed an increase of SARS-CoV-2-specific antibodies (IgG ratio and neutralization titer) and of specific T cell responses. After intermittent clinical improvement one kidney transplant recipient again developed typical symptoms at day 12 after treatment and received a second cycle of convalescent plasma treatment. Altogether, three patients clinically improved and could be discharged from hospital. However, one multimorbid female in her early eighties deceased. Our data suggest that the success of convalescent plasma therapy may only be temporary in patients with chronic kidney disease; which requires an adaptation of the treatment regimen. Close monitoring after treatment is needed for this patient group. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint In patients with chronic kidney disease and infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) treatment can be complicated because their immune function is suppressed due to medication to prevent allograft rejection and/or the underlying kidney disease. Thereby, the formation of specific antibodies and of T cell immunity is impaired; which can result in a prolonged persistence of SARS-CoV-2 (for up to two months 1 ). Furthermore, remdesivir, an antiviral nucleoside analogue which shortened the time to recovery in adults hospitalized with coronavirus 2019 (COVID-19) disease 2 , is contraindicated in this special cohort. Antiviral treatment with convalescent plasma (CP) could be an alternative treatment option. Data on patients with chronic kidney disease infected with SARS-CoV-2 and receiving CP treatment are still limited. We are aware of only 12 described kidney transplant recipients who received CP. [3] [4] [5] [6] Whereas clinical improvement after CP has been shown for all six kidney transplant recipients included in three studies [3] [4] [5] , in the fourth study 6 a mortality rate for solid organ recipients (including six with kidney allograft) in the range of recipients without CP treatment 7-9 was reported (23% 6 vs. 24-32% 7-9 , respectively). However, the previous reports did not present data on the course of SARS-CoV-2-specific antibodies or T cells in the patients. It was the aim of the current study to follow-up up virus-specific humoral and cellular immunity in patients with chronic kidney disease who were infected with SARS-CoV-2 and received CP therapy. We functionally analysed the antibodies (by neutralisation assay) and measured T cell responses by the highly sensitive ELISpot method, using . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101/2020.12.09.20239673 doi: medRxiv preprint various protein antigens of SARS-CoV-2 as specific stimuli. Finally, in one transplant recipient who again developed typical COVID-19 symptoms after initial clinical improvement we had the chance to modify the treatment regimen and to apply a second cycle of CP therapy. The current case series includes two renal transplant recipients and two haemodialysis patients (Table 1) and their respective CP donors. One patient suffered from moderate and three from severe COVID-19 disease. 10 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101/2020.12.09.20239673 doi: medRxiv preprint To assess SARS-CoV-2-specific humoral immunity, IgG antibodies were determined by a CE marked Anti-SARS-CoV-2 IgG semi-quantitative ELISA (Euroimmun, Lübeck, Germany), according to the manufacturer's instructions. The ELISA plates were coated with recombinant SARS-CoV-2 spike (S) 1 protein (receptor binding domain). Serum samples were analysed automatically at a 1:100 dilution, using the Immunomat TM (Virion\Serion, Würzburg, Germany). Results are given as ratio (patient sample / control sample). An antibody ratio of The function of specific antibodies was measured by a cell-culture based neutralization assay, using Vero E6 cells (ATCC® CRL-1586™, Manassas, Virginia, USA) and a clinical isolate of SARS-CoV-2. 11,12 Neutralization capacity was determined by endpoint dilution assay, expressed as 50% tissue culture infective dose (TCID 50 )/mL. Serial dilutions (1:20 to 1:1280) of the respective sera were pre-incubated with 100 TCID 50 of SARS-CoV-2 for one hour at 37°C and added afterwards to confluent Vero E6 cells cultured in 96-well microtiter plates. On day 3 after infection, the cells were stained with crystal violet (Roth, Karlsruhe, Germany) solved in 20 % methanol (Merck, Darmstadt, Germany) and the appearance of cytopathic effects (CPE) was analysed by light . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101/2020.12.09.20239673 doi: medRxiv preprint microscopy. The neutralizing titer was defined as the reciprocal of the highest serum dilution at which no CPE breakthrough in any of the triplicate cultures was observed. To assess SARS-CoV-2-specific cellular immunity, we performed ELISpot assays, using peptide pools of the S1/S2 protein, of the S1 protein and of the membrane (M) protein CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101/2020.12.09.20239673 doi: medRxiv preprint In three patients with undetectable SARS-CoV-2-specific IgG (ratio <1.1) and low neutralizing antibody titers (≤ 1:40) we observed an increase of antibody titers ( Figure 1A -C). One patient who was transplanted twice (RTX01) initially showed a clinical response to CP therapy, but at day 12 again developed typical symptoms of COVID-19 disease (fever and shortage of air). Therefore, she received another cycle of CP therapy Figure 1D ). SARS-CoV-2-specific antibodies in the CP donor of the fourth patient were lower (ratio 3.39, neutralizing titer 1:320). Cellular immunity could be followed-up by IFN-γ ELISpot in three patients, using four different SARS-CoV-2-specific antigens (peptide pools of the S1/S2, S1 and M protein and an S1 protein antigen). In all three patients IFN-γ production to the ELISpot intermittently increased after CP therapy, reaching a maximum at day 6 to 14 after therapy. These three patients clinically improved and could be discharged from hospital. However, one multimorbid female in her early . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101/2020.12.09.20239673 doi: medRxiv preprint eighties (HD01) deceased due to COVID-19 pneumonia on day 4 after initiation of CP therapy. Our data indicate that CP therapy led to an increase of specific humoral and cellular immunity in two kidney transplant recipients and two haemodialysis patients with SARS-CoV-2 infection. CP therapy could bridge the phase of acute COVID-19 disease. However, presumably due to drug-induced immunosuppression or impaired kidney function, the immune responses were not as long-acting as expected. In one patient with two prior kidney transplantations (RTX01) two cycles of therapy were necessary for successful treatment. It can be supposed that the patient herself was unable to mount an adequate antibody response and that the passively transferred antibodies partly bound the virus that resides in the affected organs and in the respective lymphoid tissue. 13 Theoretically, it is possible that CP therapy mitigates the native humoral immune response and leaves an individual vulnerable to subsequent reinfection with SARS-CoV-2. 3, 14 This phenomenon appears more likely in immunosuppressed vs. otherwise healthy individuals. Concerning ELISpot data, we observed a maximum of IFN-γ responses shortly after completion of the CP cycle. Of note, cellular immunity is regarded as important for recovery from SARS-CoV-2 infection 15 and appears as shortlived in the current cohort. As CP therapy is a form of passive immunization, an increase of cellular responses is not expected at first glance. It can be speculated that due to the reduction of the viral load the patients were able to generate specific T cell responses by themselves. It was previously described that high viral load (of the . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101/2020.12.09.20239673 doi: medRxiv preprint hepatitis B virus) caused antigen-specific immune tolerance. 16 Possibly, a similar imbalance between viral load and control by the immune system occurred in the current cohort. After an initial increase IFN-γ production decreased again, which could reflect the fact that due to the decrease of antibodies antigen-specific immune tolerance reappeared or that pro-inflammatory immune responses shifted to anti-inflammatory responses. 17 Moreover, treatment with tacrolimus, mycophenolate mofetil and prednisone (in both transplant recipients) and toxic metabolites due to chronic kidney disease suppressed T cell function, which could impede long-term protection against reinfection. 3, 18 Both kidney transplant recipients showed clinical improvement and three out of four patients with chronic kidney disease; which is in the range of previous reports 3-6 . However, due to the low patient number, it was beyond the aim of our study to answer the question if CP therapy was effective. This answer can only be given by large randomized clinical studies such as the Randomised Evaluation of COVID-19 Therapy (RECOVERY) trial 19 ; which are currently underway. In conclusion, our data suggest that despite an increase of SARS-CoV-2-specific immunity the success of CP therapy may only be temporary in a subset of patients with chronic kidney disease. Thus, short-term treatment control appears mandatory for this patient group. If necessary, the the treatment regimen has to be adapted. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101/2020.12.09.20239673 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101/2020.12.09.20239673 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101/2020.12.09.20239673 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101/2020.12.09.20239673 doi: medRxiv preprint Figure 1 . Course of specific humoral and cellular immunity in four patients with chronic kidney disease infected with SARS-CoV-2 and receiving convalescent plasma treatment. Antibodies were determined by an S1 specific IgG ELISA (Euroimmun) and by cell-culture based neutralization assay (NT titer). T cell responses were analysed by an interferon-gamma (IFN-γ) ELISpot assay, using peptide pools of the S1/S2, S1 and M protein and an S1 protein antigen as specific stimuli (depicted as S1/S2, S1, M and S ELI). We here present data on two kidney transplant recipients (RTX01, RTX02) and two patients on haemodialysis (HD01, HD02) and compared their immune responses with those of the corresponding donors of convalescent plasma (CP, shaded area). SARS-CoV-2-specific antibody data (IgG ratio and NT titer) are given on the left Y axis and ELISpot data on the right one. Vertical dotted lines indicate the time points of convalescent plasma applications (CP1, CP2 and CP3). Related data points are connected. Persistently positive severe acute respiratory syndrome coronavirus 2 (SARS-COV2) nasopharyngeal PCR in a kidney transplant recipient Remdesivir for the Treatment of Covid-19 -Preliminary Report Successful recovery from COVID-19 in three kidney transplant recipients who received convalescent plasma therapy Convalescent plasma therapy: Helpful treatment of COVID-19 in a kidney transplant recipient presenting with serve clinical manifestation and complex complications Treatment of Immunocompromised COVID-19 patients with Convalescent Plasma Treatment with convalescent plasma in solid organ transplant recipients with COVID-19: Experience at large transplant center Covid-19 and Kidney Transplantation COVID-19 in solid organ transplant recipients: Initial report from the US epicenter COVID-19 and Kidney Transplantation: Results from the TANGO International Transplant Consortium severe-acute-respiratory-infection-when-novel-coronavirus-(ncov)-infection-is-suspected> Published 2020 Susceptibility of SARS-CoV-2 to UV irradiation Cellular Immunity in COVID-19 Convalescents with PCR-Confirmed Infection but with Undetectable SARS-CoV-2-Specific IgG Overlapping and discrete aspects of the pathology and pathogenesis of the emerging human pathogenic coronaviruses SARS-CoV, MERS-CoV, and 2019-nCoV Passively acquired antibodies suppress humoral but not cell-mediated immunity in mice immunized with live attenuated respiratory syncytial virus vaccines SARS-CoV-2-reactive T cells in healthy donors and patients with COVID-19 Overcoming immune tolerance in chronic hepatitis B by therapeutic vaccination Longitudinal analyses reveal immunological misfiring in severe COVID-19 COVID-19: implications for immunosuppression in kidney disease and transplantation Randomised Evaluation of COVID-19 Therapy (RECOVERY) The authors would like to thank Babette Große-Rhode and Martina Filipovic for their excellent technical assistance. We furthermore thank all volunteers for their participation and the donation of blood samples.