key: cord-0795028-o9r868my authors: Cucchiari, David; Egri, Natalia; Bodro, Marta; Herrera, Sabina; Del Risco‐Zevallos, Jimena; Casals‐Urquiza, Joaquim; Cofan, Frederic; Moreno, Asunción; Rovira, Jordi; Banon‐Maneus, Elisenda; Ramirez‐Bajo, Maria J; Ventura‐Aguiar, Pedro; Pérez‐Olmos, Anna; Garcia‐Pascual, Marta; Pascal, Mariona; Vilella, Anna; Trilla, Antoni; Ríos, José; Palou, Eduard; Juan, Manel; Bayés, Beatriu; Diekmann, Fritz title: Cellular and humoral response after mRNA‐1273 SARS‐CoV‐2 vaccine in kidney transplant recipients date: 2021-05-26 journal: Am J Transplant DOI: 10.1111/ajt.16701 sha: 8a5450405e540df1f97c3d58d729fca006fe4ec4 doc_id: 795028 cord_uid: o9r868my According to preliminary data, seroconversion after mRNA SARS‐CoV‐2 vaccination might be unsatisfactory in Kidney Transplant Recipients (KTRs). However, it is unknown if seronegative patients develop at least a cellular response that could offer a certain grade of protection against SARS‐CoV‐2. To answer this question, we prospectively studied 148 recipients of either kidney (133) or kidney‐pancreas (15) grafts with assessment of IgM/IgG spike (S) antibodies and ELISpot against the nucleocapside (N) and the S protein at baseline and two weeks after receiving the second dose of the mRNA‐1273 (Moderna) vaccine. At baseline, 31 patients (20.9%) had either IgM/IgG or ELISpot positivity and were considered to be SARS‐CoV‐2‐pre‐immunized, while 117 (79.1%) patients had no signs of either cellular or humoral response and were considered SARS‐CoV‐2‐naïve. After vaccination, naïve patients who developed either humoral or cellular response were finally 65.0%, of which 29.9% developed either IgG or IgM and 35.0% S‐ELISpot positivity. Factors associated with vaccine unresponsiveness were diabetes and treatment with anti‐thymocytes globulins during the last year. Side effects were consistent with that of the pivotal trial and no DSAs developed after vaccination. In conclusion, mRNA‐1273 SARS‐CoV‐2 vaccine elicits either cellular or humoral response in almost two thirds of KTRs. Since the beginning of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic there have been over 130 million reported cases, and over 3 million deaths [1] . The speed of the SARS-CoV-2 expansion causing a world pandemic has led to the rapid development of numerous vaccines, several of which are already approved for emergency use in humans in the U.S. and Europe [2] . Among the mRNA vaccines, the BNT162b2 (Comirnaty) This article is protected by copyright. All rights reserved [Pfizer/BioNTech] [3] and mRNA-1273 [Moderna] [4] mRNA vaccines have data on immunocompetent people showing 94.1-95% efficacy in preventing Coronavirus Disease 2019 . Kidney transplant recipients (KTR) are among the most vulnerable groups of patients to develop severe COVID-19 with higher reported morbidity and mortality compared to the general population [5] . There were neither solid-organ transplant (SOT) recipients nor immunosuppressive patients in the phase 3 trials of the Moderna and Pfizer vaccines. Despite the lack of information on the safety and immunogenicity of new mRNA vaccines against in this population and considering that the potential benefits of the vaccine likely outweigh the risks, both the European Society for Organ Transplantation and the American Society for Transplantation recommend vaccination of SOT recipients. Previous studies in the setting of influenza vaccination have shown that the influenza vaccine efficacy is not optimal in KTRs [6, 7] , and further studies have shown that additional doses or higher doses are needed to increase immunogenicity [8, 9] . The authors of a recent study of solid organ transplant recipients receiving mRNA vaccines (48% received the mRNA-1273 vaccine) could show that only 17% of the patients developed a humoral response (anti-S1 or antireceptor-binding domain) at a median of 20 days after the first vaccine dose [10] . Our hypothesis is that the elicited humoral and cellular immune responses to mRNA-1273 SARS-CoV-2 vaccination in kidney transplant recipients could be lower than the reported response in the general population due to both immunosuppressive therapy and primary underlying co-morbid conditions . The primary objective of our study was to evaluate cellular and humoral responses in KTRs who received the mRNA-1273 (Moderna) vaccine. One-hundred and sixty-six (166) patients who were actively followed-up at the Hospital Clínic of Barcelona after kidney transplantation were initially screened. 148 of these patients were recipients of a kidney graft and 18 recipients of both pancreas and kidney grafts. Exclusion criteria for receiving the vaccine and entering the study included age < 18 years, transplantation within the last three months, having received anti-thymocyte globulins (ATG) or Rituximab in the last three months for rejection and active SARS-CoV-2 infection. History of previous was not an exclusion criterion and patients were considered for vaccination three months after the infection episode. Finally 162 patients received the first dose of the mRNA-1273 SARS-CoV-2 vaccine (Moderna, Cambridge, MA, U.S.), as 1 patient received another vaccine and 3 refused to participate in the study. Thirteen patients out of this population were excluded from the final Accepted Article analysis, since data was incomplete in 12 cases and one patient was excluded due to COVID-19 three weeks after the first dose. So, the final population included 148 patients (133 recipients of a kidney and 15 recipients of kidney-pancreas grafts). All of these patients received the 2 doses of the vaccine and complete data was available. A study flow-chart is depicted in Figure S1 . After signing the informed consent, blood samples were withdrawn from patients at baseline and 2 weeks after the second dose of the mRNA-1273 SARS-CoV-2 vaccine (100mcg administered in the deltoid region, 4 weeks apart from the first dose). In patients who tested to be IgG positive, another blood sample was withdrawn at 2 to 3 weeks after the first dose. The choice of the timepoints was based on the previous experience of the phase-1 trial (11) . The Institutional Ethics Committee approved the study (code HCB/2021/0222). At all the time-points, we studied the antibody response against the S protein (IgM/IgG) and the cellular response to both the nucleocapside (N) and spike (S) proteins of SARS-CoV-2 virus by means of the ELISpot technique. Patients were further categorized as either SARS-CoV-2-naïve or SARS-CoV-2-pre-immunized according to the baseline status before receiving the vaccine. If patients proved to have either cellular or humoral response at baseline they were defined as "pre-immunized", considering this baseline immunity to derive from previous exposure to SARS-CoV-2.In all the other cases patients were defined as "naïve". The objective of the study was to determine the biological response to the vaccine in SARS-CoV-2 naïve patients, defined as positive if patients developed two weeks after the second dose either antibodies (IgM or IgG) or cellular response to the S protein, assessed through the ELISpot technique. No-response to vaccine was defined as negativity of both antibodies and ELISpot assay two weeks after the second dose of the vaccine. Results on patients who proved to be preimmunized to SARS-CoV-2 are presented apart. Secondary outcomes included the analysis of all the baseline factors associated with noresponse to the vaccine for either cellular or humoral response or both. Safety analysis included phone interview with patients 48-72 hours after each dose in order to assess the patients' reported short-term side-effects, defined on a semiquantitative scale as none/mild/moderate/severe. As a safety measure, also donor-specific antibodies were assessed at baseline and 2 weeks after the second dose by Luminex technique; an allele was considered positive if the MFI was greater than 1500 and 4 times higher than the Lowest Reactive Antigen (LRA) of the same locus [12] . This article is protected by copyright. All rights reserved In order to establish seroprevalence, we used a serological assay based on the Luminex technique that has the benefit of a higher dynamic range than other assays, favoring the quantification of immunoglobulin levels. We measured antibodies against the Receptor-Binding Domain (RBD) of the spike glycoprotein of SARS-CoV-2 by Luminex [13] . Crude median fluorescent intensities (MFI) were exported using the xPONENT software. Assay cutoff was calculated as the mean plus 2 standard deviations of log10-transformed MFIs of a donor pool of 30 negative samples obtained before the COVID-19 pandemic. The data used for the calculations were the ratio of the raw MFI of the particular individual with the raw MFI obtained from the donor pool and a value >=1 was considered to be positive. Sensitivity of the assay using samples from participants previously diagnosed with COVID-19 and with more than 10 days since the onset of symptoms was 97% for IgG and 75% for IgM, with specificities of 100% for IgG and IgM. Stimulation was conducted with 2x10 5 PBMCs in X-VIVO TM To quantify positive peptide-specific responses, spots of the unstimulated wells were subtracted from the peptide-stimulated wells, and the results expressed as Spot forming units SFU/2x10 5 This article is protected by copyright. All rights reserved PBMCs. We determined SARS-CoV-2-specific spots by spot increment, defined as stimulated spot numbers >6 SFU/2x10 5 PBMCs. This cut-off was defined calculating the mean ± 2 standard deviations in a group of healthy donors obtained prior to the start of the pandemic of SARS-CoV-2. Spot counting was done automatically and re-evaluated manually in all cases. Description of baseline characteristics was tabulated by groups defined as pre-immunized and Of the final population including 148 patients, 31 were SARS-CoV-2-pre-immunized (n=31) at baseline, for presenting either positive S-protein antibodies (IgM or IgG, n=16), or a N/S protein positive ELISpot (n=15). Of these patients, only 5 had a history of proven infection assessed by PCR swab and 2 more had close contact with positive cases in the family. The other 117 patients were negative for both S-protein antibodies and ELISpot tests, and were therefore considered as SARS-CoV-2 naïve and none of them had history of clinically evident COVID-19 ( Figure 1 ). Patients who were IgM positive but IgG negative (n=4, all ELISpot negative) were tested with a PCR 48 hours after the analysis in order to rule out acute infection, and in all cases the PCR was negative. Mean age of the studied population was 57.62 ± 14.32, being significantly higher in the SARS-CoV-2-naïve group, with a predominance of male sex (70.9%). There was also a higher proportion of kidney-pancreas recipients in the SARS-CoV-2-pre-immunized group. In all the other baseline parameters no differences were observed between the two groups ( (Figure 3a-b) . Of the final population of 117 SARS- This article is protected by copyright. All rights reserved In This article is protected by copyright. All rights reserved positivity in 20/26 cases (76.9%, P=0.553). Patient-reported side effects on a semiquantitative scale (none/mild/moderate/severe) were consistent with the pivotal trial ( Figure 4) , with pain in the injection site being the most commonly reported affecting 86% and 75% of the included population after the first and the second dose respectively. The second most commonly reported side effect was fatigue that affected 25% and 27% of population after the first and the second dose, respectively. There were no significant differences between the first and the second dose for all the reported side effects. Patients took analgesics in 19.3% and 26.8% of cases after the first and the second dose, respectively. DSAs were present in 5 cases at baseline (3.4% of the entire population) and no cases of denovo DSAs were observed after the second dose. Kidney transplant recipients are at especially high risk of unfavourable outcome in case of infection with SARS-CoV-2. The reported mortality rate is up to 25% in these patients (14) . Since treatment alternatives are still scarce, as of yet the only possible strategy beyond masks and social distancing is an effective and safe vaccine. Although the current vaccine strategies -with the exception of attenuated virus -seem to be safe, there are little data available in kidney transplant recipients in terms of both safety and effectiveness. We show herein that the mRNA-1273 SARS-Cov-2 vaccine is safe in kidney transplant recipients and that the SARS-CoV-2 vaccine is associated with the same side effect profile as in the pivotal study (15) . Main side effects were pain at the injection side and fatigue. Importantly, no de-novo DSAs appeared after receiving the second dose. In SARS-CoV-2 mRNA vaccine studies in the general population seroconversion was observed in practically all patients (11, 16) . However, as expected, in our cohort the response rate was lower than in the general population, a finding that is coherent with the available data in the field. This article is protected by copyright. All rights reserved seroconversion of 54% in patients receiving a mRNA-vaccine, either mRNA-1273 (Moderna) or BNT162b2 (Pfizer) (18) . A point of novelty in our study is the assessment of cellular response through the ELISpot technique that, to our knowledge, is currently unknown in KTRs. A strong T cell response is part of the consequences of coronavirus infections and seems to play an important role in terms of long-term immunological memory (19) . Especially, in a population with a reduced antibody response information about the T cell response should be part of the assessment and furthermore it could be part of an individualized management strategy (20) . Taking It is well known that the response rate to viral vaccines is less intensive in patients with immunosuppression [6, 7] . For example, the Hepatitis B vaccine response rate is 40% in liver transplant recipients (21) , while in stem-cell transplanted patients only 51.9% achieve a response (22) . Further studies are necessary in order to evaluate if a third vaccine dose could increase the level of protection from the vaccine in the SOT population. Moreover, at this point it seems especially reasonable to vaccinate the family members and caregivers of solid organ transplant recipients as part of a cocoon strategy. Cocooning is a well-known principle for vaccinations if the target population cannot be vaccinated or are at risk of having a low response rate (23) . In any case, these results highlight the need to reach herd immunity as fast as possible in order to protect the SOT population. This article is protected by copyright. All rights reserved To our knowledge this is the first study that identifies diabetes mellitus in solid organ transplant recipients as a "risk factor" for not developing an immunogenic response to the vaccine of SARS-CoV-2. In the setting of the hepatitis B virus vaccine Schillie et al. observed that diabetes mellitus patients seemed to have a reduced response to the hepatitis B vaccines. These authors stated that diabetic patients showed an appropriate humoral response to vaccination in general, but impaired cellular response may account for less robust antibody production after hepatitis B vaccination (24). These authors propose as possible causes of this phenomenon less circulating helper T cells, an alteration of the CD4-to-CD8 lymphocyte ratio, and reduced lymphocyte blastogenesis as well as impaired antigen presentation. Maintenance immunosuppression did not seem to have any influence on the immunological response, with the exception of mTOR inhibition associated with a more favorable humoral response. A preliminary study observed that mycophenolate was associated with less humoral response (10) (Table 4 ). It has to be highlighted that also lymphopenia, independently of ATG, was associated with S-ELISpot non-response (Table 3, right). One may argue that the two variables are associated with each other, as ATG typically causes profound lymphopenia early after transplantation and is associated with immunesenescence at the long-term (25) . However, the percentage of patients at baseline with lymphopenia (28.6%) was higher than that of patients who received ATG during the last year (11.6%); this highlights that different mechanism apart from ATG are implicated after kidney transplantation, including maintenance immunosuppression and comorbidities, and that lymphopenia per se represents a risk factor for not developing a cellular response. In our multivariate analysis, a glomerular filtration rate below 30 mL/min barely missed statistical significance as an independent risk factor for no immunological response. In a recently published study with a mRNA-vaccine in dialysis patients, Grupper et al observed a robust, although less intense, antibody response in 96% of cases. Therefore, it could be speculated that in our pharmacologically immunosuppressed patients the most important factor is the immunosuppression and not impaired renal function (26) . The limitations of our study include a low number of patients in order to draw solid conclusions about the real protective effect of the vaccine. However, a low rate of seroconversion or of cellular response might be surrogate parameters for less efficacy. Moreover, our study lacks a healthy This article is protected by copyright. All rights reserved control group. However, we figured that the already published data on healthy individuals are convincing enough in order to get relevant results without a control group, especially considering that in a situation of scarcity a control group would be difficult if it consists of individuals who do not belong to risk populations. Another possible limitation of our study is the absence of serial measurements after vaccination. Long-term data on safety are also needed and will be followedup. In conclusion, the mRNA SARS-CoV-2 vaccine provoked an immune response in 65% of patients who received immunosuppression due to a kidney or kidney-pancreas transplant. This is a lower response rate than in the general population. New strategies need to be developed in order to adequately protect this vulnerable group. Age ( This article is protected by copyright. All rights reserved This article is protected by copyright. All rights reserved Table 3 -Univariable and multivariable analysis on factors associated with vaccine no-response according to antibodies or ELISpot results two weeks after the 2 nd dose of mRNA-1273 SARS-CoV-2 vaccine. This article is protected by copyright. All rights reserved Table 4 -Univariable and multivariable analysis on factors associated with global vaccine no-response, defined as the negativity of both antibodies and ELISpot assay two weeks after the 2 nd dose of mRNA-1273 SARS-CoV-2 vaccine. This article is protected by copyright. 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