key: cord-0301033-5oiwc1wg authors: Schrezenmeier, E.; Rincon-Arevalo, H.; Jens, A.; Stefanski, A.-L.; Hammett, C.; Osmanodja, B.; Koch, N.; Zukunft, B.; Beck, J.; Oellerich, M.; Pross, V.; Stahl, C.; Choi, M.; Bachmann, F.; Liefeldt, L.; Glander, P.; Schuetz, E.; Bornemann-Kolatzki, K.; Lopez del Moral, C.; Schrezenmeier, H.; Ludwig, C.; Jahrdoerfer, B.; Eckardt, K. U.; Lachmann, N.; Kotsch, K.; Doerner, T.; Halleck, F.; Sattler, A.; Budde, K. title: Temporary hold of mycophenolate boosts SARS-CoV-2 vaccination-specific humoral and cellular immunity in kidney transplant recipients date: 2022-01-08 journal: nan DOI: 10.1101/2022.01.05.21268478 sha: 6a59f11b345f39cf65807b966a5af07f0b123b73 doc_id: 301033 cord_uid: 5oiwc1wg 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. 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. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted January 8, 2022. ; https://doi.org/10.1101/2022.01.05.21268478 doi: medRxiv preprint Protection of kidney transplant recipients (KTR) from coronavirus disease 2019 , caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has not been sufficiently achieved by conventional vaccination protocols. Mortality of fully vaccinated KTR after infection remains unacceptably high with almost 8% in a registry analysis from the United Kingdom (1) and up to 20% in other cohorts (2, 3) , despite the presence of vaccine-specific T cells. Our previous studies revealed a strong impairment of both humoral and cellular immunity in transplant recipients after two doses of BNT162b2, with antigen-specific B and T cell responses being both quantitatively and functionally affected (4, 5) . Since the majority of KTR does not benefit from a third dose (6) , modified vaccination protocols are required to achieve protection of this at-risk population. Analysis of larger transplant patient cohorts indicated that mycophenolate (MPA) based treatment constitutes a major risk factor for impairment of vaccine-induced humoral immunity (7, 8) . In line with the aforementioned, a case series with patients suffering from rheumatic and musculoskeletal diseases demonstrated that temporary hold of MPA leads to augmented humoral responses to SARS-CoV-2 vaccination (9). According to European guidelines, the majority of kidney transplanted individuals receive triple immunosuppressive medication including calcineurin inhibitors (CNI), corticosteroids (CS) and MPA. Withdrawal of steroids or MPA in a tacrolimus-based treatment protocol for up to three years has been shown to be safe in a large multicenter study, with no increase in acute rejections or impaired kidney function (10) . Similar data were obtained from other trials (11) (12) (13) . Furthermore, hold of MPA is routinely recommended during pregnancy (14) , underlining the feasibility of this approach. To examine the impact of short-term MPA withdrawal on vaccination outcome, 29 KTR, being seronegative after triple SARS-CoV-2 vaccination, were converted to an MPA-free immunosuppressive regimen. Patients were closely monitored for clinical parameters, including kidney function, anti-All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted January 8, 2022. ; https://doi.org/10.1101/2022.01.05.21268478 doi: medRxiv preprint human leucocyte antigen (HLA) antibodies and donor-derived cell-free DNA (dd-cfDNA) (15, 16) ; assessment of vaccine-specific immunity encompassed in-depth analysis of specific B and T cell analyses, IgG and IgA levels and neutralization capacity. The study cohort included 29 KTR with a lack of serological response after a three-dose vaccine protocol. Fourteen patients were homogeneously vaccinated (3x mRNA vaccine), 15 patients were vaccinated heterologously (mixed mRNA-and vector-based). All patients received BNT162b2 (BioNTech/Pfizer) as a fourth vaccine. Mean time interval between the third and fourth vaccination was 59.1 (±12.6) days. All patients were initially on antimetabolite treatment, 28/29 on MPA and 1/29 on azathioprine (Aza). 25/29 received CNI based medication while 4 patients received Belatacept. All patients stopped MPA or Aza 4-7 days before the fourth vaccination, based on the assumption that pharmacodynamic drug effects wean after 3-4 days (17) . Treatment was paused until day 28-35 (mean depicted as "day 32" in all figures; second time point for serological response analysis). In patients with no or low corticosteroid (CS) medication, CS was restarted or increased to 5 mg prednisone equivalent together with MPA hold. In the four patients on Belatacept, two stopped MPA and one Aza, while it was replaced by CS in only one patient. One Belatacept treated patient was switched to tacrolimus and CS. Demographics are summarized in Table 1 . Seroconversion (OD ratio>1.1) for anti-S1 domain IgG occurred in 10/29 (34.5%) individuals until day 7 after the fourth vaccination, while anti-S1 domain IgA was positive in 7/29 KTR (24.1%). Neutralization capacity above 30% was achieved in 11/29 (37.9%) patients ( Figure 1A -C). On day 32 after vaccination, 22/29 (76%) patients showed anti-S1 domain IgG levels above the threshold for positivity. Anti-S1 domain IgA and neutralization capacity levels were unavailable for 8 individuals. For the remaining individuals, IgA was positive in 11/21 patients (52.2%) and neutralization capacity was above threshold in 15/21 (71.4%) patients ( Figure 1A -C). In patients with CNI treatment before All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted January 8, 2022. ; https://doi.org/10.1101/2022.01.05.21268478 doi: medRxiv preprint vaccination, anti-S1 domain IgG seroconversion occurred in 21/25 (84%) patients, while 3/4 patients on Belatacept remained negative; only one became weakly positive just above the threshold on day 32 (Supplementary Figure 1B) . Anti-S1 domain specific IgG on day 32 did not differ between patients upon heterologous or homologous vaccination (Supplementary Figure 1A) . Antigen-specific B cells were identified by fluorescent double-labelling of reactive cells with recombinant receptor binding domain (RBD) (5) (Supplementary Figure 1D ). Frequencies and absolute counts of RBD + B cells increased 7 days after vaccination compared to baseline ( Figure 1D ). Interestingly, especially the frequency of RBD + plasmablasts, which have been shown to be an early sign of vaccine response (5), increased after four vaccinations ( Figure 1E ). The inosine monophosphate dehydrogenase (IMPDH) activity in erythrocytes has recently been described as a useful pharmacodynamic marker for MPA exposure that reflects the MPA exposure after 8 weeks of constant dosing (17) . High IMPDH levels were found in patients with MPA toxicity and low levels in patients with biopsy-proven acute rejections (17) . In the current cohort, the mean IMPDH activity before MPA hold at a time of steady state was 1192.73 pmol XMP/h/mg Hb (±474.24), and IMPDH activity did not negatively correlate with anti-S1 domain IgG on day 32 (Supplementary Figure 1C ). SARS-CoV-2 spike protein reactive CD4 + T helper cells were detected within PBMC based on activationinduced co-expression of CD154 and CD137 after stimulation with 15-mer peptides (overlapping by 11 amino acids, respectively) covering the complete spike glycoprotein sequence, as previously reported (4, 18) . The gating strategy, including subset identification, is depicted in Supplementary Figure 2 . A positive T cell response was defined when stimulated PBMC contained more than threefold higher frequencies of CD154 + CD137 + CD4 + T cells as compared to the unstimulated control (stimulation index of 3) with at least twenty events, being in accordance with comparable studies (19) . The prevalence of cellular responders was similar (>85%) after the third and fourth vaccination with no significant All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted January 8, 2022. ; https://doi.org/10.1101/2022.01.05.21268478 doi: medRxiv preprint differences in relative and absolute frequencies of antigen-reactive T cells. Of note, levels of anti-SARS-CoV-2 spike S1 domain specific IgG were positively correlated with frequencies of spike-specific T cells ( Figure 2A ). Spike-specific T cells of individuals after the fourth vaccination contained significantly higher portions of cells expressing the proliferation marker Ki67; the same applied to expression of PD-1, indicating recent in vivo activation ( Figure 2B ). Interestingly, we did not detect significantly elevated frequencies of antigen-specific T cells expressing IFNγ, TNFα, IL-2 or IL-4 after the fourth dose ( Figure 2C ); this also applied to proportions of specific polyfunctional IFNγ + TNFα + IL-2 + T cells or cells secreting none of the three cytokines ( Figure 2D ). IL-4 was excluded from polyfunctionality analyses due to low frequencies of positive cells. Antigen-reactive T cells from individuals after the third and fourth dose showed similar frequencies of CD45RO + CD62Leffector/memory and CD45RO -CD62Leffector-type T cells, respectively ( Figure 2E ). Since conversion of an established immunosuppressive regimen bears the risk of adverse events such as rejection or the generation of de-novo HLA antibodies, we performed anti-HLA antibody testing before and after vaccination in 27/29 study participants. No patient developed de-novo HLA antibodies, and antibody pattern and strength remained unchanged in 3 patients with preexisting donor-specific HLA antibodies before vaccination. Kidney function remained stable after day 32. Dd-cfDNA, a novel marker for subclinical allograft injury and rejection (16, 20) was available for 16/29 KTR and did not increase. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted January 8, 2022. ; https://doi.org/10.1101/2022.01.05.21268478 doi: medRxiv preprint The current study investigates the immunological impact and clinical safety of a fourth dose of a SARS-CoV-2 vaccine during short-term MPA hold in KTR who did not seroconvert after three vaccine doses. We found a significant increase in humoral responders at day 32 (76% irrespective of previous treatment, 84% in individuals on standard CNI regimen), together with an increase in neutralizing antibodies and occurrences of vaccine-specific B cells and plasmablasts. We further observed higher ex vivo activation of spike-specific T cells that quantitatively correlate with spike S1 specific IgG at day 32. So far, the response to SARS-CoV-2 mRNA-and vector-based vaccines in KTR has been disappointing (4, 21) , resulting in high infection and hospitalization rates in fully vaccinated individuals (3). The early recommendation of a third vaccination for solid organ recipients, that has meanwhile been extended to the general population (22) , entailed IgG seroconversion rates of up to 68 % (23), a feature that was, however, not reproducible for patients receiving triple immunosuppressive therapy and being seronegative before the third vaccination (6) . Already positive but low pre-vaccination IgG levels were associated with superior outcomes after a fourth dose on stable immunosuppression, leading to seroconversion rates of 42-50% (24, 25) . Still, seronegative patients after repeated re-vaccination clearly represent the most vulnerable subgroup with higher risk of severe COVID19 (1, 3) . This aspect is becoming increasingly important with the emergence of new viral variants, resulting in reduced neutralization capacity even in healthy individuals (26) . Our approach to withdraw MPA, followed by re-vaccination, obviously impacted all arms of immunity with a strong impact on B cell activation and differentiation, thereby boosting spike-specific antibody production. Interestingly, seroconversion was already detectable on day 7 in 34.4% of patients, as compared to only 12% of individuals receiving a third dose under MPA treatment (6), highlighting enhanced immune kinetics in the absence of anti-metabolites. Importantly, seroconversion did not All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted January 8, 2022. ; https://doi.org/10.1101/2022.01.05.21268478 doi: medRxiv preprint depend on the type of previous vaccines since we did not observe differences between patients who received a heterologous or homologous vaccination regimen. So far, anti-metabolites including MPA and Aza have been primarily demonstrated to impair B cell proliferation and plasmablast formation in autoimmunity (27) , but to also block expansion and activation of naïve and memory B cells isolated from healthy individuals (28) (29) (30) . Mechanistically, MPA inhibits IL-6-mediated STAT3-signalling, a prerequisite for plasma cell differentiation (31) and critical for their survival and immunoglobulin secretion in the bone marrow (32) . To the best of our knowledge, our data for the first time verify MPA effects on B cells in an antigen-specific context, including impairment of spike-specific CD27 ++ CD38 + plasmablast formation. With respect to the T cell compartment, our data are in line with recent studies showing that production of IFNγ, TNFα or IL-2 by polyoma-virus BK specific memory T cells or bulk mucosal-associated invariant T cells remains unaffected by MPA (33, 34) . This might be related to the fact that the IL-6/STAT3 axis is selectively involved in differentiation of IL-17 secreting Th17 cells (35). As opposed to cytokine production, in vivo activation, as evidenced by Ki67 and PD-1 expression, significantly increased in vaccine-specific CD4 + T cells after MPA hold, suggesting that initial activation/proliferation is more sensitive to antimetabolites as has been demonstrated for purified naïve human T cells (36) . Interestingly, our finding that higher frequencies of spike-reactive T cells correlate with specific IgG levels mirrors early analyses of healthy SARS-CoV-2 vaccinees (37). As an obvious limitation of our approach, a subgroup of patients with previous or ongoing Belatacept treatment did not benefit from MPA hold. Although patient numbers were small, our data support the notion that Belatacept efficiently inhibits vaccine responses (38) irrespective of the presence of antimetabolites, suggesting that other approaches are needed for Belatacept-treated patients. In summary our data provide evidence that temporal hold of MPA for 5 weeks in patients under previous CNI, MPA ± CS is a viable option to accelerate and increase vaccine efficacy in KTR, particularly given that graft function remained stable and no rejection episodes or increases in anti-HLA antibodies All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted January 8, 2022. ; https://doi.org/10.1101/2022.01.05.21268478 doi: medRxiv preprint and dd-cfDNA plasma concentrations were observed. Our study thus highlights a potential rapid vaccination strategy for at-risk patients under standard CNI-based immunosuppression that warrants testing in larger cohorts. All participants gave written informed consent for sample collection according to the approval of the ethics committees of the Charité-Universitätsmedizin Berlin (EA2/010/21, EA4/188/20). Patient demographics are summarized in Table 1 . Peripheral blood and serum samples were collected immediately before and 7±2 days after the fourth vaccination (humoral, B-and T-cell analyses) and 28-35 days (mean "day 32") after the fourth dose (humoral analyses, HLA antibodies, assessment of dd-cfDNA). Serological assessment was performed as previously reported (5, 6, 39) . In brief, SARS-CoV-2 S1 domain specific IgG and IgA was determined by ELISA (Euroimmun). Previous or current SARS-CoV-2 infection was excluded based on medical history in combination with negativity in a SARS-CoV-2 nucleoprotein specific ELISA (Euroimmun). Samples were considered positive with OD ratios of ≥1.1 as per manufacturer´s guidelines. An OD ratio value was determined by calculating the ratio of the OD of the respective test sample over the OD of the internal calibrator provided with the ELISA kit. Virus neutralization capacity of sera was analyzed using a surrogate SARS-CoV-2 neutralization test (GenScript) with more than 30% being defined as a positive response as described previously (40, 41) . Clinical parameters were extracted from our patient database (42) . All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted January 8, 2022. ; https://doi.org/10.1101/2022.01.05.21268478 doi: medRxiv preprint Erythrocyte IMPDH activity was measured as described recently (17) as a part of clinical routine. The last available IMPDH in a steady state condition before change in MPA dose is reported in Table 1 . The measurement of dd-cfDNA was performed as described previously (15, 43) . In brief, for each patient, 4 informative independent single nucleotide polymorphism (SNP), assays are used for which the recipient has a homozygous allelic state and the graft carries at least 1 heterozygous allele. These are selected from a predefined set of 40 SNPs. These 4 SNP assays were used to quantify the dd-cfDNA (%) concentration, defined as donor alleles/(donor alleles + recipient alleles). Results for SNPs with heterozygous graft genotypes were corrected by a factor 2. Total cfDNA was extracted from up to 8 mL of plasma collected in certified blood collection tubes (Streck Corp, Omaha, Nebraska). The measurement was performed using droplet-digital PCR. Results were corrected for extraction efficiency and cfDNA fragmentation in absolute quantification, as described previously (20) . The absolute concentration of dd-cfDNA per mL of plasma was calculated by multiplying total cfDNA (copies/mL) and dd-cfDNA (%). Time dependent changes for total cfDNA and dd-cfDNA fraction (%) in the posttransplant course were assessed in a cohort of 300 KTR, as described previously (44) . All experiments have been performed as previously described (5, 18, 39) . In brief, peripheral blood Where applicable, graphs show means ± SD. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted January 8, 2022. ; https://doi.org/10.1101/2022.01.05.21268478 doi: medRxiv preprint All rights reserved. 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